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Sample records for cellulose-specific molecular probes

  1. Microscopic Analysis of Corn Fiber Using Corn Starch- and Cellulose-Specific Molecular Probes

    SciTech Connect

    Porter, S. E.; Donohoe, B. S.; Beery, K. E.; Xu, Q.; Ding, S.-Y.; Vinzant, T. B.; Abbas, C. A.; Himmel, M. E.

    2007-09-01

    Ethanol is the primary liquid transportation fuel produced from renewable feedstocks in the United States today. The majority of corn grain, the primary feedstock for ethanol production, has been historically processed in wet mills yielding products such as gluten feed, gluten meal, starch, and germ. Starch extracted from the grain is used to produce ethanol in saccharification and fermentation steps; however the extraction of starch is not 100% efficient. To better understand starch extraction during the wet milling process, we have developed fluorescent probes that can be used to visually localize starch and cellulose in samples using confocal microscopy. These probes are based on the binding specificities of two types of carbohydrate binding modules (CBMs), which are small substrate-specific protein domains derived from carbohydrate degrading enzymes. CBMs were fused, using molecular cloning techniques, to a green fluorescent protein (GFP) or to the red fluorescent protein DsRed (RFP). Using these engineered probes, we found that the binding of the starch-specific probe correlates with starch content in corn fiber samples. We also demonstrate that there is starch internally localized in the endosperm that may contribute to the high starch content in corn fiber. We also surprisingly found that the cellulose-specific probe did not bind to most corn fiber samples, but only to corn fiber that had been hydrolyzed using a thermochemical process that removes the residual starch and much of the hemicellulose. Our findings should be of interest to those working to increase the efficiency of the corn grain to ethanol process.

  2. Fabrication of molecular tension probes.

    PubMed

    Kim, Sung Bae; Fujii, Rika

    2016-01-01

    A unique bioluminescent imaging probe is introduced for illuminating molecular tension appended by protein-protein interactions (PPIs) of interest. A full-length luciferase is sandwiched between two proteins of interest via minimal flexible linkers. The ligand-activated PPIs append intramolecular tension to the sandwiched luciferase, boosting or dropping the enzymatic activity in a quantitative manner. This method guides construction of a new lineage of bioassays for determining molecular tension appended by ligand-activated PPIs. The summary of the method is: •Molecular tension appended by protein-protein interactions (PPI) is visualized with a luciferase.•Estrogen activities are quantitatively illuminated with the molecular tension probes.•Full-length Renilla luciferase enhances the optical intensities after bending by PPI.

  3. Fabrication of molecular tension probes

    PubMed Central

    Kim, Sung Bae; Fujii, Rika

    2016-01-01

    A unique bioluminescent imaging probe is introduced for illuminating molecular tension appended by protein–protein interactions (PPIs) of interest. A full-length luciferase is sandwiched between two proteins of interest via minimal flexible linkers. The ligand-activated PPIs append intramolecular tension to the sandwiched luciferase, boosting or dropping the enzymatic activity in a quantitative manner. This method guides construction of a new lineage of bioassays for determining molecular tension appended by ligand-activated PPIs. The summary of the method is: • Molecular tension appended by protein–protein interactions (PPI) is visualized with a luciferase. • Estrogen activities are quantitatively illuminated with the molecular tension probes. • Full-length Renilla luciferase enhances the optical intensities after bending by PPI. PMID:27222821

  4. Design and Development of Molecular Imaging Probes

    PubMed Central

    Chen, Kai; Chen, Xiaoyuan

    2013-01-01

    Molecular imaging, the visualization, characterization and measurement of biological processes at the cellular, subcellular level, or even molecular level in living subjects, has rapidly gained importance in the dawning era of personalized medicine. Molecular imaging takes advantage of the traditional diagnostic imaging techniques and introduces molecular imaging probes to determine the expression of indicative molecular markers at different stages of diseases and disorders. As a key component of molecular imaging, molecular imaging probe must be able to specifically reach the target of interest in vivo while retaining long enough to be detected. A desirable molecular imaging probe with clinical translation potential is expected to have unique characteristics. Therefore, design and development of molecular imaging probe is frequently a challenging endeavor for medicinal chemists. This review summarizes the general principles of molecular imaging probe design and some fundamental strategies of molecular imaging probe development with a number of illustrative examples. PMID:20388106

  5. Techniques for Molecular Imaging Probe Design

    PubMed Central

    Reynolds, Fred; Kelly, Kimberly A.

    2011-01-01

    Molecular imaging allows clinicians to visualize disease specific molecules, thereby providing relevant information in the diagnosis and treatment of patients. With advances in genomics and proteomics and underlying mechanisms of disease pathology, the number of targets identified has significantly outpaced the number of developed molecular imaging probes. There has been a concerted effort to bridge this gap with multidisciplinary efforts in chemistry, proteomics, physics, material science, and biology; all essential to progress in molecular imaging probe development. In this review, we will discuss target selection, screening techniques and probe optimization with the aim of developing clinically relevant molecularly targeted imaging agents. PMID:22201532

  6. Molecular Imaging Probe Development using Microfluidics

    PubMed Central

    Liu, Kan; Wang, Ming-Wei; Lin, Wei-Yu; Phung, Duy Linh; Girgis, Mark D.; Wu, Anna M.; Tomlinson, James S.; Shen, Clifton K.-F.

    2012-01-01

    In this manuscript, we review the latest advancement of microfluidics in molecular imaging probe development. Due to increasing needs for medical imaging, high demand for many types of molecular imaging probes will have to be met by exploiting novel chemistry/radiochemistry and engineering technologies to improve the production and development of suitable probes. The microfluidic-based probe synthesis is currently attracting a great deal of interest because of their potential to deliver many advantages over conventional systems. Numerous chemical reactions have been successfully performed in micro-reactors and the results convincingly demonstrate with great benefits to aid synthetic procedures, such as purer products, higher yields, shorter reaction times compared to the corresponding batch/macroscale reactions, and more benign reaction conditions. Several ‘proof-of-principle’ examples of molecular imaging probe syntheses using microfluidics, along with basics of device architecture and operation, and their potential limitations are discussed here. PMID:22977436

  7. Molecular beacons: an optimal multifunctional biological probe.

    PubMed

    Li, Yongsheng; Zhou, Xiaoyan; Ye, Duyun

    2008-09-01

    Molecular beacon technology is set up based on fluorescence resonance energy transfer (FRET) and the complementary pairing principles. These fluorescent molecular probes, which are very highly specific and sensitive, have now become one important tool in medical and biological researches. This review introduces the molecular beacons structure, principle, the main impact factors, the labeling of the molecular beacons, and research progress on molecular beacons fluorescent-label in the polymerase chain reaction (PCR), DNA sequence analysis, gene dynamic detection in living cells, protein (enzyme)-nucleic acid interactions and applications in clinical medicine.

  8. Protein-based tumor molecular imaging probes

    PubMed Central

    Lin, Xin; Xie, Jin

    2013-01-01

    Molecular imaging is an emerging discipline which plays critical roles in diagnosis and therapeutics. It visualizes and quantifies markers that are aberrantly expressed during the disease origin and development. Protein molecules remain to be one major class of imaging probes, and the option has been widely diversified due to the recent advances in protein engineering techniques. Antibodies are part of the immunosystem which interact with target antigens with high specificity and affinity. They have long been investigated as imaging probes and were coupled with imaging motifs such as radioisotopes for that purpose. However, the relatively large size of antibodies leads to a half-life that is too long for common imaging purposes. Besides, it may also cause a poor tissue penetration rate and thus compromise some medical applications. It is under this context that various engineered protein probes, essentially antibody fragments, protein scaffolds, and natural ligands have been developed. Compared to intact antibodies, they possess more compact size, shorter clearance time, and better tumor penetration. One major challenge of using protein probes in molecular imaging is the affected biological activity resulted from random labeling. Site-specific modification, however, allows conjugation happening in a stoichiometric fashion with little perturbation of protein activity. The present review will discuss protein-based probes with focus on their application and related site-specific conjugation strategies in tumor imaging. PMID:20232092

  9. Design of Targeted Cardiovascular Molecular Imaging Probes

    PubMed Central

    Anderson, Carolyn J.; Bulte, Jeff W.M.; Chen, Kai; Chen, Xiaoyuan; Khaw, Ban-An; Shokeen, Monica; Wooley, Karen L.; VanBrocklin, Henry F.

    2013-01-01

    Molecular imaging relies on the development of sensitive and specific probes coupled with imaging hardware and software to provide information about the molecular status of a disease and its response to therapy, which are important aspects of disease management. As genomic and proteomic information from a variety of cardiovascular diseases becomes available, new cellular and molecular targets will provide an imaging readout of fundamental disease processes. A review of the development and application of several cardiovascular probes is presented here. Strategies for labeling cells with superparamagnetic iron oxide nanoparticles enable monitoring of the delivery of stem cell therapies. Small molecules and biologics (e.g., proteins and antibodies) with high affinity and specificity for cell surface receptors or cellular proteins as well as enzyme substrates or inhibitors may be labeled with single-photon–emitting or positron-emitting isotopes for nuclear molecular imaging applications. Labeling of bispecific antibodies with single-photon–emitting isotopes coupled with a pretargeting strategy may be used to enhance signal accumulation in small lesions. Emerging nanomaterials will provide platforms that have various sizes and structures and that may be used to develop multimeric, multimodal molecular imaging agents to probe one or more targets simultaneously. These platforms may be chemically manipulated to afford molecules with specific targeting and clearance properties. These examples of molecular imaging probes are characteristic of the multidisciplinary nature of the extraction of advanced biochemical information that will enhance diagnostic evaluation and drug development and predict clinical outcomes, fulfilling the promise of personalized medicine and improved patient care. PMID:20395345

  10. Molecular imaging probe development: a chemistry perspective

    PubMed Central

    Nolting, Donald D; Nickels, Michael L; Guo, Ning; Pham, Wellington

    2012-01-01

    Molecular imaging is an attractive modality that has been widely employed in many aspects of biomedical research; especially those aimed at the early detection of diseases such as cancer, inflammation and neurodegenerative disorders. The field emerged in response to a new research paradigm in healthcare that seeks to integrate detection capabilities for the prediction and prevention of diseases. This approach made a distinct impact in biomedical research as it enabled researchers to leverage the capabilities of molecular imaging probes to visualize a targeted molecular event non-invasively, repeatedly and continuously in a living system. In addition, since such probes are inherently compact, robust, and amenable to high-throughput production, these probes could potentially facilitate screening of preclinical drug discovery, therapeutic assessment and validation of disease biomarkers. They could also be useful in drug discovery and safety evaluations. In this review, major trends in the chemical synthesis and development of positron emission tomography (PET), optical and magnetic resonance imaging (MRI) probes are discussed. PMID:22943038

  11. Molecular Probes for Fluorescence Lifetime Imaging

    PubMed Central

    Sarder, Pinaki; Maji, Dolonchampa; Achilefu, Samuel

    2015-01-01

    Visualization of biological processes and pathologic conditions at the cellular and tissue levels largely rely on the use of fluorescence intensity signals from fluorophores or their bioconjugates. To overcome the concentration dependency of intensity measurements, evaluate subtle molecular interactions, and determine biochemical status of intracellular or extracellular microenvironments, fluorescence lifetime (FLT) imaging has emerged as a reliable imaging method complementary to intensity measurements. Driven by a wide variety of dyes exhibiting stable or environment-responsive FLTs, information multiplexing can be readily accomplished without the need for ratiometric spectral imaging. With knowledge of the fluorescent states of the molecules, it is entirely possible to predict the functional status of biomolecules or microevironment of cells. Whereas the use of FLT spectroscopy and microscopy in biological studies is now well established, in vivo imaging of biological processes based on FLT imaging techniques is still evolving. This review summarizes recent advances in the application of the FLT of molecular probes for imaging cells and small animal models of human diseases. It also highlights some challenges that continue to limit the full realization of the potential of using FLT molecular probes to address diverse biological problems, and outlines areas of potential high impact in the future. PMID:25961514

  12. A quantum spin-probe molecular microscope

    NASA Astrophysics Data System (ADS)

    Perunicic, V. S.; Hill, C. D.; Hall, L. T.; Hollenberg, L. C. L.

    2016-10-01

    Imaging the atomic structure of a single biomolecule is an important challenge in the physical biosciences. Whilst existing techniques all rely on averaging over large ensembles of molecules, the single-molecule realm remains unsolved. Here we present a protocol for 3D magnetic resonance imaging of a single molecule using a quantum spin probe acting simultaneously as the magnetic resonance sensor and source of magnetic field gradient. Signals corresponding to specific regions of the molecule's nuclear spin density are encoded on the quantum state of the probe, which is used to produce a 3D image of the molecular structure. Quantum simulations of the protocol applied to the rapamycin molecule (C51H79NO13) show that the hydrogen and carbon substructure can be imaged at the angstrom level using current spin-probe technology. With prospects for scaling to large molecules and/or fast dynamic conformation mapping using spin labels, this method provides a realistic pathway for single-molecule microscopy.

  13. A quantum spin-probe molecular microscope

    PubMed Central

    Perunicic, V. S.; Hill, C. D.; Hall, L. T.; Hollenberg, L.C.L.

    2016-01-01

    Imaging the atomic structure of a single biomolecule is an important challenge in the physical biosciences. Whilst existing techniques all rely on averaging over large ensembles of molecules, the single-molecule realm remains unsolved. Here we present a protocol for 3D magnetic resonance imaging of a single molecule using a quantum spin probe acting simultaneously as the magnetic resonance sensor and source of magnetic field gradient. Signals corresponding to specific regions of the molecule's nuclear spin density are encoded on the quantum state of the probe, which is used to produce a 3D image of the molecular structure. Quantum simulations of the protocol applied to the rapamycin molecule (C51H79NO13) show that the hydrogen and carbon substructure can be imaged at the angstrom level using current spin-probe technology. With prospects for scaling to large molecules and/or fast dynamic conformation mapping using spin labels, this method provides a realistic pathway for single-molecule microscopy. PMID:27725630

  14. Probing adhesion forces at the molecular scale

    SciTech Connect

    Thomas, R.C.; Houston, J.E.; Michalske, T.A.

    1996-12-31

    Measurements of adhesion forces at the molecular scale, such as those discussed here, are necessary to understand macroscopic boundary-layer behavior such as adhesion, friction, wear, lubrication, and many other important phenomena. The authors` recent interfacial force microscopy (IFM) studies have provided detailed information about the mechanical response of both self-assembled monolayer (SAM) films and the underlying substrates. In addition, they recently demonstrated that the IFM is useful for studying the chemical nature of such films. In this talk, the authors discuss a new method for studying surface interactions and chemical reactions using the IFM. To quantitatively measure the work of adhesion and bond energies between two organic thin films, they modify both a Au substrate and a Au probe with self-assembling organomercaptan molecules having either the same or different end groups (-CH{sub 3}, -NH{sub 2}, and -COOH), and then analyze the force-versus-displacement curves (force profiles) that result from the approach to contact of the two surfaces. Their results show that the magnitude of the adhesive forces measured between methyl-methyl interactions are in excellent agreement with van der Waals calculations using Lifshitz theory and previous experimentally determined values. Moreover, the measured peak adhesive forces scale as expected for van der Waals, hydrogen-bonding, and acid-base interactions.

  15. Molecular Crowding Effects on Microgel-Tethered Oligonucleotide Probes.

    PubMed

    Ma, Youlong; Libera, Matthew

    2016-06-28

    Microgel tethering is a nontraditional method with which to bind oligonucleotide hybridization probes to a solid surface. Microgel-tethering physically positions the probes away from the underlying hard substrate and maintains them in a highly waterlike environment. This paper addresses the question of whether molecular crowding affects the performance of microgel-tethered molecular beacon probes. The density of probe-tethering sites is controlled experimentally using thin-film blends of biotin-terminated [PEG-B] and hydroxyl-terminated [PEG-OH] poly(ethylene glycol) from which microgels are synthesized and patterned by electron beam lithography. Fluorescence measurements indicate that the number of streptavidins, linear DNA probes, hairpin probes, and molecular beacon probes bound to the microgels increases linearly with increasing PEG-B/PEG-OH ratio. For a given tethering-site concentration, more linear probes can bind than structured probes. Crowding effects emerge during the hybridization of microgel-tethered molecular beacons but not during the hybridization of linear probes, as the tethering density increases. Crowding during hybridization is associated with conformational constraints imposed by the close proximity of closed and hybridized structured probes. The signal-to-background ratio (SBR) of hybridized beacons is highest and roughly constant for low tethering densities and decreases at the highest tethering densities. Despite differences between microgel tethering and traditional oligonucleotide surface-immobilization approaches, these results show that crowding defines an optimum tethering density for molecular beacon probes that is less than the maximum possible, which is consistent with previous studies involving various linear and structured oligonucleotide probes. PMID:27253904

  16. Molecular Crowding Effects on Microgel-Tethered Oligonucleotide Probes.

    PubMed

    Ma, Youlong; Libera, Matthew

    2016-06-28

    Microgel tethering is a nontraditional method with which to bind oligonucleotide hybridization probes to a solid surface. Microgel-tethering physically positions the probes away from the underlying hard substrate and maintains them in a highly waterlike environment. This paper addresses the question of whether molecular crowding affects the performance of microgel-tethered molecular beacon probes. The density of probe-tethering sites is controlled experimentally using thin-film blends of biotin-terminated [PEG-B] and hydroxyl-terminated [PEG-OH] poly(ethylene glycol) from which microgels are synthesized and patterned by electron beam lithography. Fluorescence measurements indicate that the number of streptavidins, linear DNA probes, hairpin probes, and molecular beacon probes bound to the microgels increases linearly with increasing PEG-B/PEG-OH ratio. For a given tethering-site concentration, more linear probes can bind than structured probes. Crowding effects emerge during the hybridization of microgel-tethered molecular beacons but not during the hybridization of linear probes, as the tethering density increases. Crowding during hybridization is associated with conformational constraints imposed by the close proximity of closed and hybridized structured probes. The signal-to-background ratio (SBR) of hybridized beacons is highest and roughly constant for low tethering densities and decreases at the highest tethering densities. Despite differences between microgel tethering and traditional oligonucleotide surface-immobilization approaches, these results show that crowding defines an optimum tethering density for molecular beacon probes that is less than the maximum possible, which is consistent with previous studies involving various linear and structured oligonucleotide probes.

  17. MOLECULAR PROBES FOR EXTRACELLULAR ADENOSINE RECEPTORS

    PubMed Central

    Jacobson, Kenneth A.; Ukena, Dieter; Padgett, William; Kirk, Kenneth L.; Daly, John W.

    2012-01-01

    Derivatives of adenosine receptor agonists (N6-phenyladenosines) and antagonists (1,3-dialkyl-8-phenylxanthines) bearing functionalized chains suitable for attachment to other molecules have been reported [Jacobson et al., J. med. Chem. 28, 1334 and 1341 (1985)]. The “functionalized congener” approach has been extended to the synthesis of spectroscopic and other probes for adenosine receptors that retain high affinity (Ki ~ 10−9 −10−8 M) in A1-receptor binding. The probes have been synthesized from an antagonist xanthine amine congener (XAC) and an adenosine amine congener (ADAC). [3H]ADAC has been synthesized and found to bind highly specifically to A1-adenosine receptors of rat and calf cerebral cortical membranes with KD values of 1.4 and 0.34 nM respectively. The higher affinity in the bovine brain, seen also with many of the probes derived from ADAC and XAC, is associated with phenyl substituents. The spectroscopic probes contain a reporter group attached at a distal site of the functionalized chain. These bifunctional ligands may contain a spin label (e.g. the nitroxyl radical TEMPO) for electron spin resonance spectroscopy, or a fluorescent dye, including fluorescein and 4-nitrobenz-2-oxa-1,3-diazole (NBD), or labels for 19F nuclear magnetic resonance spectroscopy. Potential applications of the spectroscopic probes in characterization of adenosine receptors are discussed. PMID:3036153

  18. Molecular probes for the in vivo imaging of cancer

    PubMed Central

    Alford, Raphael; Ogawa, Mikako; Choyke, Peter L.

    2012-01-01

    Advancements in medical imaging have brought about unprecedented changes in the in vivo assessment of cancer. Positron emission tomography, single photon emission computed tomography, optical imaging, and magnetic resonance imaging are the primary tools being developed for oncologic imaging. These techniques may still be in their infancy, as recently developed chemical molecular probes for each modality have improved in vivo characterization of physiologic and molecular characteristics. Herein, we discuss advances in these imaging techniques, and focus on the major design strategies with which molecular probes are being developed. PMID:19823742

  19. Molecular Probes for Thermometry in Microfluidic Devices

    NASA Astrophysics Data System (ADS)

    Gosse, Charlie; Bergaud, Christian; Löw, Peter

    The temperature is an important parameter with regard to chemical reactivity. It is therefore essential to ensure good thermal control within microsystems designed to carry out biological analysis. We begin by reviewing temperature measurement in the context of the lab-on-a-chip, and outlining the various generic strategies available. We then turnmore specifically to luminescentmolecular probes.We shall show that they all exploit the effect of temperature on a chemical reaction (in the broad sense of the term). More precisely, these probes can be divided in three main categories depending on whether one relies on a phase transition, the modification of a reaction rate, or a shift in an equilibrium. We shall also discuss the main experimental strategies used to transform the image obtained by fluorescence microscopy into a thermal map. Finally, we shall extend the discussion to a few other spectroscopic techniques and examine the prospects for this particular area of microfluidics.

  20. Ionization probes of molecular structure and chemistry

    SciTech Connect

    Johnson, P.M.

    1993-12-01

    Various photoionization processes provide very sensitive probes for the detection and understanding of the spectra of molecules relevant to combustion processes. The detection of ionization can be selective by using resonant multiphoton ionization or by exploiting the fact that different molecules have different sets of ionization potentials. Therefore, the structure and dynamics of individual molecules can be studied even in a mixed sample. The authors are continuing to develop methods for the selective spectroscopic detection of molecules by ionization, and to use these methods for the study of some molecules of combustion interest.

  1. Nanoparticle Probes for Structural and Functional Photoacoustic Molecular Tomography

    PubMed Central

    Chen, Haobin; Yuan, Zhen; Wu, Changfeng

    2015-01-01

    Nowadays, nanoparticle probes have received extensive attention largely due to its potential biomedical applications in structural, functional, and molecular imaging. In addition, photoacoustic tomography (PAT), a method based on the photoacoustic effect, is widely recognized as a robust modality to evaluate the structure and function of biological tissues with high optical contrast and high acoustic resolution. The combination of PAT with nanoparticle probes holds promises for detecting and imaging diseased tissues or monitoring their treatments with high sensitivity. This review will introduce the recent advances in the emerging field of nanoparticle probes and their preclinical applications in PAT, as well as relevant perspectives on future development. PMID:26609534

  2. Silicon cantilever functionalization for cellulose-specific chemical force imaging of switchgrass

    DOE PAGESBeta

    Lee, Ida; Evans, Barbara R.; Foston, Marcus B.; Ragauskas, Arthur J.

    2015-05-08

    A method for direct functionalization of silicon and silicon nitride cantilevers with bifunctional silanes was tested with model surfaces to determine adhesive forces for different hydrogen-bonding chemistries. Application for biomass surface characterization was tested by mapping switchgrass and isolated switchgrass cellulose in topographic and force-volume mode using a cellulose-specific cantilever.

  3. Molecular beacons: fluorogenic probes for living cell study.

    PubMed

    Fang, Xiaohong; Mi, Yanming; Li, Jianwei Jeffery; Beck, Terry; Schuster, Sheldon; Tan, Weihong

    2002-01-01

    Molecular beacons are a new class of fluorescent probes that can report the presence of specific nucleic acids with high sensitivity and excellent specificity. In addition to their current wide applications in monitoring the progress of polymerase chain reactions, their unique properties make them promising probes for the detection and visualization of target biomolecules in living cells. This article is focused on our recent research in exploring the potential of using molecular beacon for living-cell studies in three important areas: the monitoring of mRNA in living cells, the development of ultrasmall DNA/RNA biosensors, and the novel approach of combining molecular beacon's signal transduction mechanism with aptamer's specificity for real-time protein detection. These applications demonstrate molecular beacon's unique properties in bioanalysis and bioassay development.

  4. Probing Radiation Damage at the Molecular Level

    NASA Astrophysics Data System (ADS)

    Mason, N. J.; Smialek, M. A.; Moore, S. A.; Folkard, M.; Hoffmann, S. V.

    2006-12-01

    Radiation damage of DNA and other cellular components has traditionally been attributed to ionisation via direct impact of high-energy quanta or by complex radical chemistry. However recent research has shown that strand breaks in DNA may be initiated by secondary electrons and is strongly dependent upon the target DNA base identity. Such research provides the fascinating perspective that it is possible that radiation damage may be described and understood at an individual molecular level introducing new possibilites for therapy and perhaps providing an insight into the origins of life.

  5. An elegant biosensor molecular beacon probe: challenges and recent solutions.

    PubMed

    Kolpashchikov, Dmitry M

    2012-01-01

    Molecular beacon (MB) probes are fluorophore- and quencher-labeled short synthetic DNAs folded in a stem-loop shape. Since the first report by Tyagi and Kramer, it has become a widely accepted tool for nucleic acid analysis and triggered a cascade of related developments in the field of molecular sensing. The unprecedented success of MB probes stems from their ability to detect specific DNA or RNA sequences immediately after hybridization with no need to wash out the unbound probe (instantaneous format). Importantly, the hairpin structure of the probe is responsible for both the low fluorescent background and improved selectivity. Furthermore, the signal is generated in a reversible manner; thus, if the analyte is removed, the signal is reduced to the background. This paper highlights the advantages of MB probes and discusses the approaches that address the challenges in MB probe design. Variations of MB-based assays tackle the problem of stem invasion, improve SNP genotyping and signal-to-noise ratio, as well as address the challenges of detecting folded RNA and DNA.

  6. An Elegant Biosensor Molecular Beacon Probe: Challenges and Recent Solutions

    PubMed Central

    Kolpashchikov, Dmitry M.

    2012-01-01

    Molecular beacon (MB) probes are fluorophore- and quencher-labeled short synthetic DNAs folded in a stem-loop shape. Since the first report by Tyagi and Kramer, it has become a widely accepted tool for nucleic acid analysis and triggered a cascade of related developments in the field of molecular sensing. The unprecedented success of MB probes stems from their ability to detect specific DNA or RNA sequences immediately after hybridization with no need to wash out the unbound probe (instantaneous format). Importantly, the hairpin structure of the probe is responsible for both the low fluorescent background and improved selectivity. Furthermore, the signal is generated in a reversible manner; thus, if the analyte is removed, the signal is reduced to the background. This paper highlights the advantages of MB probes and discusses the approaches that address the challenges in MB probe design. Variations of MB-based assays tackle the problem of stem invasion, improve SNP genotyping and signal-to-noise ratio, as well as address the challenges of detecting folded RNA and DNA. PMID:24278758

  7. Hybrid molecular probe for nucleic acid analysis in biological samples.

    PubMed

    Yang, Chaoyong James; Martinez, Karen; Lin, Hui; Tan, Weihong

    2006-08-01

    The ability to detect changes in gene expression, especially in real-time and with sensitivity sufficient enough to monitor small variations in a single-cell, will have considerable value in biomedical research and applications. Out of the many available molecular probes for intracellular monitoring of nucleic acids, molecular beacon (MB) is the most frequently used probe with the advantages of high sensitivity and selectivity. However, any processes in which the MB stem-loop structure is broken will result in a restoration of the fluorescence in MB. This brings in a few possibilities for false positive signal such as nuclease degradation, protein binding, thermodynamic fluctuation, solution composition variations (such as pH, salt concentration) and sticky-end pairing. These unwanted processes do exist inside living cells, making nucleic acid monitoring inside living cells difficult. We have designed and synthesized a hybrid molecular probe (HMP) for intracellular nucleic acid monitoring to overcome these problems. HMP has two DNA probes, one labeled with a donor and the other an acceptor. The two DNA probes are linked by a poly(ethylene glycol) (PEG) linker, with each DNA being complementary to adjacent areas of a target sequence. Target binding event brings the donor and acceptor in proximity, resulting in quenching of the donor fluorescence and enhancement of the acceptor emission. The newly designed HMP has high sensitivity, selectivity, and fast hybridization kinetics. The probe is easy to design and synthesize. HMP does not generate any false positive signal upon digestion by nuclease, binding by proteins, forming complexes by sticky-end pairing, or by other molecular interaction processes. HMP is capable of selectively detecting nucleic acid targets from cellular samples.

  8. Investigation of the hybrid molecular probe for intracellular studies

    PubMed Central

    Martinez, Karen; Medley, Colin D.; Yang, Chaoyong James; Tan, Weihong

    2009-01-01

    Monitoring gene expression in vivo is essential to the advancement of biological studies, medical diagnostics, and drug discovery. Adding to major efforts in developing molecular probes for mRNA monitoring, we have recently developed an alternative tool, the hybrid molecular probe (HMP). To optimize the probe, a series of experiments were performed to study the properties of HMP hybridization kinetics and stability. The results demonstrated the potential of the HMP as a prospective tool for use in both hybridization studies and in vitro and in vivo analyses. The HMP has shown no tendency to produce false positive signals, which is a major concern for living cell studies. Moreover, HMP has shown the ability to detect the mRNA expression of different genes inside single cells from both basal and stimulated genes. As an effective alternative to conventional molecular probes, the proven sensitivity, simplicity, and stability of HMPs show promise for their use in monitoring mRNA expression in living cells. PMID:18421445

  9. Detection of toxoplasma gondii with a DNA molecular beacon probe

    NASA Astrophysics Data System (ADS)

    Xu, Shichao; Yao, Cuicui; Wei, Shuoming; Zhang, Jimei; Sun, Bo; Zheng, Guo; Han, Qing; Hu, Fei; Zhou, Hongming

    2008-12-01

    Toxoplasma gondii is a microscopic parasite that may infect humans, so there is an increasing concern on the early detection of latent Toxoplasma gondii infection in recent years. We currently report a rapid and sensitive method for Toxoplasma gondii based on molecular beacon (MB) probe. The probe based on fluorescence resonance energy transfer (FRET) with a stem-loop DNA oligonucleotide was labeled with CdTe/ZnS quantum dots (energy donor) at 5' end and BHQ-2 (energy acceptor) at 3' end, respectively. The probe was synthesized in PBS buffer at pH 8.2, room temperature for 24 h. Then target DNA was injected under the condition of 37°C, hybridization for 2 h, in Tris-HCl buffer. The data from fluorescence spectrum (FS) showed that ca 65% of emitted fluorescence was quenched, and about 50% recovery of fluorescence intensity was observed after adding target DNA, which indicated that the target DNA was successfully detected by MB probe. The detecting limitation was determined as ca 5 nM. Moreover, specificity of the probe was investigated by adding target DNA with one-base-pair mismatch, the low fluorescence recovery indicated the high specificity. The results showed that the current sensing probe will be a useful and convenient tool in Toxoplasma gondii early detection.

  10. Engineering imaging probes and molecular machines for nanomedicine.

    PubMed

    Tong, Sheng; Cradick, Thomas J; Ma, Yan; Dai, Zhifei; Bao, Gang

    2012-10-01

    Nanomedicine is an emerging field that integrates nanotechnology, biomolecular engineering, life sciences and medicine; it is expected to produce major breakthroughs in medical diagnostics and therapeutics. Due to the size-compatibility of nano-scale structures and devices with proteins and nucleic acids, the design, synthesis and application of nanoprobes, nanocarriers and nanomachines provide unprecedented opportunities for achieving a better control of biological processes, and drastic improvements in disease detection, therapy, and prevention. Recent advances in nanomedicine include the development of functional nanoparticle based molecular imaging probes, nano-structured materials as drug/gene carriers for in vivo delivery, and engineered molecular machines for treating single-gene disorders. This review focuses on the development of molecular imaging probes and engineered nucleases for nanomedicine, including quantum dot bioconjugates, quantum dot-fluorescent protein FRET probes, molecular beacons, magnetic and gold nanoparticle based imaging contrast agents, and the design and validation of zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs) for gene targeting. The challenges in translating nanomedicine approaches to clinical applications are discussed.

  11. Probing ultrafast molecular dynamics in O2 using XUV/IR pump-probe studies

    NASA Astrophysics Data System (ADS)

    Ray, D.; Sturm, F. P.; Wright, T. W.; Ranitovic, P.; Shivaram, N. H.; Bocharova, I.; Belkacem, A.; Weber, Th.

    2015-05-01

    We investigate the molecular dynamics via different dissociative and autoionizing pathways in molecular oxygen using a pump-probe scheme with ultrashort extreme ultraviolet (XUV) laser pulses. Our primary focus is to study the molecular dynamics in the superexcited Rydberg states in a time-resolved manner. The O2 molecules are pumped by 20.2 eV and 23.1 eV XUV pulses (13th and 15th harmonics). Probing the relaxation dynamics with an infrared (IR) pulse at very long delays (100s of fs) enables us to measure the lifetimes of these Rydberg states. We also observe an enhancement and suppression of vibrational levels of the O2+ion due to the presence of IR. The high flux XUV pulses used for this experiment are generated in an Ar gas by IR pulses from our state-of-the-art 30 mJ, 50 Hz laser system. The pulses are overlapped with the supersonic jet in our Momentum Imaging for TimE Resolved Studies (MISTERS) setup. The cold target in our setup, combined with a very tight focussing geometry and a 3D momentum detection capability gives a high kinetic energy resolution. Molecular dynamics in other polyatomic molecules are also under investigation. Chemical Sciences Division, Lawrence Berkeley National Laboratory.

  12. Pyrene excimer signaling molecular beacons for probing nucleic acids.

    PubMed

    Conlon, Patrick; Yang, Chaoyong James; Wu, Yanrong; Chen, Yan; Martinez, Karen; Kim, Youngmi; Stevens, Nathan; Marti, Angel A; Jockusch, Steffen; Turro, Nicholas J; Tan, Weihong

    2008-01-01

    Molecular beacon DNA probes, containing 1-4 pyrene monomers on the 5' end and the quencher DABCYL on the 3' end, were engineered and employed for real-time probing of DNA sequences. In the absence of a target sequence, the multiple-pyrene labeled molecular beacons (MBs) assumed a stem-closed conformation resulting in quenching of the pyrene excimer fluorescence. In the presence of target, the beacons switched to a stem-open conformation, which separated the pyrene label from the quencher molecule and generated an excimer emission signal proportional to the target concentration. Steady-state fluorescence assays resulted in a subnanomolar limit of detection in buffer, whereas time-resolved signaling enabled low-nanomolar target detection in cell-growth media. It was found that the excimer emission intensity could be scaled by increasing the number of pyrene monomers conjugated to the 5' terminal. Each additional pyrene monomer resulted in substantial increases in the excimer emission intensities, quantum yields, and excited-state lifetimes of the hybridized MBs. The long fluorescence lifetime ( approximately 40 ns), large Stokes shift (130 nm), and tunable intensity of the excimer make this multiple-pyrene moiety a useful alternative to traditional fluorophore labeling in nucleic acid probes.

  13. Pyrene Excimer Signaling Molecular Beacons for Probing Nucleic Acids

    PubMed Central

    Conlon, Patrick; Yang, Chaoyong James; Wu, Yanrong; Chen, Yan; Martinez, Karen; Kim, Youngmi; Stevens, Nathan; Marti, Angel A.; Jockusch, Steffen

    2008-01-01

    Molecular beacon DNA probes, containing one to four pyrene monomers on the 5′ end and the quencher DABCYL on the 3′ end, were engineered and employed for real-time probing of DNA sequences. In the absence of a target sequence, the multiple-pyrene labeled molecular beacons (MBs) assumed a stem-closed conformation resulting in quenching of the pyrene excimer fluorescence. In the presence of target, the beacons switched to a stem-open conformation which separated the pyrene label from the quencher molecule and generated an excimer emission signal proportional to the target concentration. Steady-state fluorescence assays resulted in a sub-nanomolar limit of detection in buffer, while time-resolved signaling enabled low-nanomolar target detection in cell growth media. It was found that the excimer emission intensity could be scaled by increasing the number of pyrene monomers conjugated to the 5′ terminal. Each additional pyrene monomer resulted in substantial increases in the excimer emission intensities, quantum yields, and excited-state lifetimes of the hybridized MBs. The long fluorescence lifetime (~40 ns), large Stokes shift (130 nm), and tunable intensity of the excimer make this multiple-pyrene moiety a useful alternative to traditional fluorophore labeling in nucleic acid probes. In addition, this excimer complex serves as an efficient FRET donor for red-emitting fluorophores, such as TMR, for further extending the Stokes shift of the fluorescent complex. PMID:18078339

  14. Aptamers: versatile molecular recognition probes for cancer detection

    PubMed Central

    Sun, Hongguang; Tan, Weihong; Zu, Youli

    2015-01-01

    In the past two decades, aptamers have emerged as a novel class of molecular recognition probes comprising uniquely-folded short RNA or single-stranded DNA oligonucleotides that bind to their cognate targets with high specificity and affinity. Aptamers, often referred to as “chemical antibodies”, possess several highly desirable features for clinical use. They can be chemically synthesized and are easily conjugated to a wide range of reporters for different applications, and are able to rapidly penetrate tissues. These advantages significantly enhance their clinical applicability, and render them excellent alternatives to antibody-based probes in cancer diagnostics and therapeutics. Aptamer probes based on fluorescence, colorimetry, magnetism, electrochemistry, and in conjunction with nanomaterials (e.g., nanoparticles, quantum dots, single-walled carbon nanotubes, and magnetic nanoparticles) have provided novel ultrasensitive cancer diagnostic strategies and assays. Furthermore, promising aptamer targeted-multimodal tumor imaging probes have been recently developed in conjunction with fluorescence, positron emission tomography (PET), single-photon emission computed tomography (SPECT), and magnetic resonance imaging (MRI). The capabilities of the aptamer-based platforms described herein underscore the great potential they hold for the future of cancer detection. In this review, we highlight the most prominent recent developments in this rapidly advancing field. PMID:26618445

  15. Detection of Toxoplasma gondii with a DNA molecular beacon probe

    NASA Astrophysics Data System (ADS)

    Zhou, Cun; Xu, Shichao; Yang, Juan; Zhang, Jimei; Dai, Zhao; Zheng, Guo; Sun, Bo; Sun, Shuqing; Feng, Teilin; Zi, Yan; Liang, Chu; Luo, Hao

    2009-07-01

    Toxoplasma gondii is a kind of microscopic parasite that may infect humans, and there are increasing concerns on the early detection of latent Toxoplasma gondii infection in recent years. This research highlights a new type of molecular beacon (MB) fluorescent probe for Toxoplasma DNA testing. We combined high-efficiency fluorescent inorganic core-shell quantum dots-CdTe/ZnS (as fluorescent energy donor) and BHQ-2 (energy acceptor) to the single-strand DNA of Toxoplasma gondii, and a molecular beacon sensing system based on fluorescence resonance energy transfer (FRET) was achieved. Core-shell quantum dots CdTe/ZnS was firstly prepared in aqueous solution, and the influencing factor of its fluorescent properties, including CdTe/Na2S/Zn(CH3COO)2 (v/v), dependence of reaction time, temperature, and pH, is investigated systematically. The synthesized quantum dots and molecular beacon were characterized by transmission electron microscopy (TEM), ultraviolet-visible spectrophotometer (UV-vis), fluorescent spectrophotometer (FS), respectively. The TEM results showed that CdTe/ZnS core-shell quantum dots is ~11nm in size, and the quantum dots is water-soluble well. The sensing ability of target DNA of assembled MB was investigated, and results showed that the target Toxoplasma gonddi DNA can be successfully detected by measuring the change of fluorescence intensity. The results showed that the current sensing probe will be a useful and convenient tool in Toxoplasma gondii early detection.

  16. Ultrafast Molecular Dynamics probed by Vacuum Ultraviolet Pulses

    NASA Astrophysics Data System (ADS)

    Cryan, James; Champenois, Elio; Shivaram, Niranjan; Wright, Travis; Yang, Chan-Shan; Falcone, Roger; Belkacem, Ali

    2014-05-01

    We present time-resolved measurements of the relaxation dynamics in small molecular systems (CO2 and C2H4) following ultraviolet (UV) photo-excitation. We probe these excitations through photoionization and velocity map imaging (VMI) spectroscopy. Vacuum and extreme ultraviolet (VUV/XUV) pump and probe pulses are created by exploiting strong-field high harmonic generation (HHG) from our state-of-the-art 30 mJ, 1 kHz laser system. Three dimensional photoelectron and photoion momentum images recorded with our VMI spectrometer reveal non-Born Oppenheimer dynamics in the vicinity of a conical intersection, and allow us track the state of the system as a function of time. We also present initial experiments with the goal of controlling the dynamics near a conical intersection using a strong-field IR pulse. Finally, we will show progress towards measurements of time-resolved molecular frame photoelectron angular distributions (TRMFPADs) by applying our VUV/XUV pulse sequence to an aligned molecular ensemble. Supported by Chemical Sciences, Geosciences and Biosciences division of BES/DOE.

  17. Plant sentinels and molecular probes that monitor environmental munitions contaminants

    SciTech Connect

    Jackson, P.J.; DeWitt, J.G.; Hill, K.K.; Kuske, C.R.; Kim, D.Y.

    1994-08-01

    Plants accumulate TNT and similar compounds from soil. Their sessile nature requires that plants adapt to environmental changes by biochemical and molecular means. In principle, it is possible to develop a monitoring capability based on expression of any gene that is activated by specific environmental conditions. The authors have identified plant genes activated upon exposure to TNT. Partial gene sequences allow design of DNA probes that measure TNT-induced gene activity. These will be used to develop sensitive assays that monitor gene expression in plants growing in environments possibly contaminated with explosives.

  18. Frequency Domain Fluorescent Molecular Tomography and Molecular Probes for Small Animal Imaging

    NASA Astrophysics Data System (ADS)

    Kujala, Naresh Gandhi

    Fluorescent molecular tomography (FMT) is a noninvasive biomedical optical imaging that enables 3-dimensional quantitative determination of fluorochromes distributed in biological tissues. There are three methods for imaging large volume tissues based on different light sources: (a) using a light source of constant intensity, through a continuous or constant wave, (b) using a light source that is intensity modulated with a radio frequency (RF), and (c) using ultrafast pulses in the femtosecond range. In this study, we have developed a frequency domain fluorescent molecular tomographic system based on the heterodyne technique, using a single source and detector pair that can be used for small animal imaging. In our system, the intensity of the laser source is modulated with a RF frequency to produce a diffuse photon density wave in the tissue. The phase of the diffuse photon density wave is measured by comparing the reference signal with the signal from the tissue using a phasemeter. The data acquisition was performed by using a Labview program. The results suggest that we can measure the phase change from the heterogeneous inside tissue. Combined with fiber optics and filter sets, the system can be used to sensitively image the targeted fluorescent molecular probes, allowing the detection of cancer at an early stage. We used the system to detect the tumor-targeting molecular probe Alexa Fluor 680 and Alexa Fluor 750 bombesin peptide conjugates in phantoms as well as mouse tissues. We also developed and evaluated fluorescent Bombesin (BBN) probes to target gastrin-releasing peptide (GRP) receptors for optical molecular imaging. GRP receptors are over-expressed in several types of human cancer cells, including breast, prostate, small cell lung, and pancreatic cancers. BBN is a 14 amino acid peptide that is an analogue to human gastrin-releasing peptide that binds specifically to GRPr receptors. BBN conjugates are significant in cancer detection and therapy. The

  19. Design, synthesis, and biological evaluation of potent discodermolide fluorescent and photoaffinity molecular probes.

    PubMed

    Smith, Amos B; Rucker, Paul V; Brouard, Ignacio; Freeze, B Scott; Xia, Shujun; Horwitz, Susan Band

    2005-11-10

    [structure: see text] The design, synthesis, and biological evaluation of a series of (+)-discodermolide molecular probes possessing photoaffinity and fluorescent appendages has been achieved. Stereoselective olefin cross-metathesis comprised a key tactic for construction of two of the molecular probes. Three photoaffinity probes were radiolabeled with tritium.

  20. E × B probe measurements in molecular and electronegative plasmas.

    PubMed

    Renaud, D; Gerst, D; Mazouffre, S; Aanesland, A

    2015-12-01

    This paper reports on the design, the building, the calibration, and the use of a compact E × B probe that acts as a velocity filter or a mass filter for ion species. A series of measurements has been performed in the discharge and in the beam of the PEGASES (Plasma Propulsion with Electronegative GASES) ion source. PEGASES is a unique inductively coupled radio-frequency source able to generate a beam of positive and negative ions when operated with an electronegative gas. In this study, experiments have been carried out with SF6. Calibrated E × B probe spectra indicate that the diagnostic tool can be used to determine the ion velocity and the plasma composition even when many molecular fragments are present. In addition, the probe is able to detect both positive and negative ions. Measurements show a large variety of positively charged ions coming from SF6. Conversely, the beam is solely composed of F(-) and SF6(-) negative ions in compliance with computer simulations. PMID:26724027

  1. E × B probe measurements in molecular and electronegative plasmas

    NASA Astrophysics Data System (ADS)

    Renaud, D.; Gerst, D.; Mazouffre, S.; Aanesland, A.

    2015-12-01

    This paper reports on the design, the building, the calibration, and the use of a compact E × B probe that acts as a velocity filter or a mass filter for ion species. A series of measurements has been performed in the discharge and in the beam of the PEGASES (Plasma Propulsion with Electronegative GASES) ion source. PEGASES is a unique inductively coupled radio-frequency source able to generate a beam of positive and negative ions when operated with an electronegative gas. In this study, experiments have been carried out with SF6. Calibrated E × B probe spectra indicate that the diagnostic tool can be used to determine the ion velocity and the plasma composition even when many molecular fragments are present. In addition, the probe is able to detect both positive and negative ions. Measurements show a large variety of positively charged ions coming from SF6. Conversely, the beam is solely composed of F- and SF 6- negative ions in compliance with computer simulations.

  2. Probing the Molecular Mechanisms of the Fracture of Semicrystalline Polyethylene

    NASA Astrophysics Data System (ADS)

    Benkoski, J. J.; Flores, P.; Kramer, E. J.

    2003-03-01

    The effects of molecular architecture on the fracture properties of semicrystalline polymers were probed at diblock copolymer-reinforced interfaces between polystyrene (PS) and polyethylene (PE). The PE used for this study was a model ethylene-butene copolymer which was chosen for its compatibility with hydrogenated poly(styrene-b-1,4-tetradeuteriobutadiene). For a series of these diblock copolymers, the areal chain density (Σ) and the molecular weight of the PE block (M_n) were varied systematically to observe their effects on the interfacial fracture energy (G_c). At low Σ, Gc stayed relatively constant, and was roughly 1 J/m^2. Above a critical value of Σ, the fracture energy climbed rapidly. This critical value decreased with increasing M_n. The detection of deuterium on the fracture surfaces indicated that pullout of the PE block was the predominant failure mechanism when Mn <= 30 kg/mol. Since the entanglement molecular weight of PE is approximately 1 kg/mol, interfacial reinforcement does not appear to depend on the formation of entanglements for this system. The critical Mn coincides instead with the point at which the root-mean-square end-to-end length of the PE block exceeds the long period of the PE crystal lamellae (L).

  3. Sensitive quantification of somatic mutations using molecular inversion probes.

    PubMed

    Hirani, Rena; Connolly, Ashley R; Putral, Lisa; Dobrovic, Alexander; Trau, Matt

    2011-11-01

    Somatic mutations in DNA can serve as cancer specific biomarkers and are increasingly being used to direct treatment. However, they can be difficult to detect in tissue biopsies because there is often only a minimal amount of sample and the mutations are often masked by the presence of wild type alleles from nontumor material in the sample. To facilitate the sensitive and specific analysis of DNA mutations in tissues, a multiplex assay capable of detecting nucleotide changes in less than 150 cells was developed. The assay extends the application of molecular inversion probes to enable sensitive discrimination and quantification of nucleotide mutations that are present in less than 0.1% of a cell population. The assay was characterized by detecting selected mutations in the KRAS gene, which has been implicated in up to 25% of all cancers. These mutations were detected in a single multiplex assay by incorporating the rapid flow cytometric readout of multiplexable DNA biosensors.

  4. Luminescent chiral lanthanide(III) complexes as potential molecular probes

    PubMed Central

    Muller, Gilles

    2009-01-01

    This perspective gives an introduction into the design of luminescent lanthanide(III)-containing complexes possessing chiral properties and used to probe biological materials. The first part briefly describes general principles, focusing on the optical aspect (i.e. lanthanide luminescence, sensitization processes) of the most emissive trivalent lanthanide ions, europium and terbium, incorporated into molecular luminescent edifices. This is followed by a short discussion on the importance of chirality in the biological and pharmaceutical fields. The second part is devoted to the assessment of the chiroptical spectroscopic tools available (typically circular dichroism and circularly polarized luminescence) and the strategies used to introduce a chiral feature into luminescent lanthanide(III) complexes (chiral structure resulting from a chiral arrangement of the ligand molecules surrounding the luminescent center or presence of chiral centers in the ligand molecules). Finally, the last part illustrates these fundamental principles with recent selected examples of such chiral luminescent lanthanide-based compounds used as potential probes of biomolecular substrates. PMID:19885510

  5. Molecular beacon probes of oligonucleotides photodamaged by psoralen.

    PubMed

    Shire, Zahra J; Loppnow, Glen R

    2012-01-01

    Ultraviolet A (UVA)-irradiated 4'-hydroxymethyl-4,5',8-trimethyl psoralen (HMT) in the presence of a poly-dT(17) and dA(7) TTA(8) oligonucleotides produces HMT-dT(17) and HMT-dA(7) TTA(8) adducts in aqueous solution. In this article, we determine whether these HMT-dT(17) and HMT-dA(7) TTA(8) adducts can be detected with a molecular beacon (MB) probe. We measure the degree of damage in dT(17) and dA(7) TTA(8) solutions containing UVA-activated HMT via monitoring the decrease in MB fluorescence. Photoproduct formation is confirmed by MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-fight mass spectrometry measurements) and absorption spectroscopy. The MB fluorescence decreases upon UVA irradiation in the presence of HMT with a single-exponential time constants of 114.2 ± 6.5 min for HMT-dT(17) adducts and 677.8 ± 181.8 min for HMT-dA(7) TTA(8) adducts. Our results show that fluorescent MB probes are a selective, robust and accurate tool for detecting UVA-activated HMT-induced DNA damage.

  6. Molecular beacon-metal nanowire interface: effect of probe sequence and surface coverage on sensor performance.

    PubMed

    Cederquist, Kristin B; Stoermer Golightly, Rebecca; Keating, Christine D

    2008-08-19

    We report the effect of surface coverage and sequence on the performance of 5' thiolated, 3' fluorophore-labeled DNA hairpin probes bound to Au/Ag striped ("barcoded") metal nanowires. Coverage was controlled by varying probe concentration, buffer ionic strength, and by addition of short hydroxy-terminated alkanethiol diluent molecules during probe assembly onto the nanowire surface. Surface dilution of the surface-bound probes with a omega-hydroxyl alkanethiol, a commonly accepted practice in the surface-bound DNA literature, did not appreciably improve sensor performance as compared to similar probe coverages without hydroxyalkanethiol diluents; this finding underscores the differences between the molecular beacon probes used here and more traditional nonfluorescent, random coil probes. We found that intermediate probe coverage of approximately 10 (12) molecules/cm (2) gave the best discrimination between presence and absence of a target sequence. Because we are interested in multiplexed assays, we also compared several beacon probe sequences having different stabilities for secondary structure formation in solution; we found that both probe surface coverage and sensor performance varied for different probe sequences. When five different molecular beacon probes, each bound to barcoded nanowires, were used in a multiplexed, wash-free assay for target oligonucleotides corresponding to viral nucleic acid sequences, these differences in probe performance did not prevent accurate target identification. We anticipate that the findings described here will also be relevant to other applications involving molecular beacons or other structured nucleic acid probes immobilized on metal surfaces.

  7. Conductive probe AFM measurements of conjugated molecular wires.

    PubMed

    Ishida, Takao; Mizutani, Wataru; Liang, Tien-Tzu; Azehara, Hiroaki; Miyake, Koji; Sasaki, Shinya; Tokumoto, Hiroshi

    2003-12-01

    The electrical conduction of self-assembled monolayers (SAMs) made from conjugated molecules was measured using conductive probe atomic force microscopy (CP-AFM), with a focus on the molecular structural effect on conduction. First, the electrical conduction of SAMs made from phenylene oligomer SAMs was measured. The resistances through the monolayers increased exponentially with an increase in molecular length and the decay constants of transconductance beta were about 0.45 to 0.61 A(-1) measured at lower bias region. We further investigated the influence of applied load on the resistances. The resistances through terphenyl SAMs increased with an increase in the applied load up to 14 nN. Second, using an insertion technique into insulating alkanethiol SAMs, the electrical conduction of single conjugated terphenyl methanethiol and oligo(para-phenylenevinylene) (OPV) molecules embedded into insulating alkanethiol SAMs were measured. Electrical currents through these single molecules of OPVs were estimated to be larger than those through single terphenyl molecules, suggesting that the OPV structure can increase the electrical conduction of single molecules. Third, apparent negative differential resistance (NDR) was observed at higher bias measurements of SAMs. The appearance of NDR might be related to roughness of SAM surface, because apparent NDR was often observed on rough surfaces. In any case, the tip-molecule contact condition strongly affected carrier transport through metal tip/SAM/metal junction.

  8. Molecular electrostatics for probing lone pair-π interactions.

    PubMed

    Mohan, Neetha; Suresh, Cherumuttathu H; Kumar, Anmol; Gadre, Shridhar R

    2013-11-14

    An electrostatics-based approach has been proposed for probing the weak interactions between lone pair containing molecules and π deficient molecular systems. For electron-rich molecules, the negative minima in molecular electrostatic potential (MESP) topography give the location of electron localization and the MESP value at the minimum (Vmin) quantifies the electron-rich character of that region. Interactive behavior of a lone pair bearing molecule with electron deficient π-systems, such as hexafluorobenzene, 1,3,5-trinitrobenzene, 2,4,6-trifluoro-1,3,5-triazine and 1,2,4,5-tetracyanobenzene explored within DFT brings out good correlation of the lone pair-π interaction energy (E(int)) with the Vmin value of the electron-rich system. Such interaction is found to be portrayed well with the Electrostatic Potential for Intermolecular Complexation (EPIC) model. On the basis of the precise location of MESP minimum, a prediction for the orientation of a lone pair bearing molecule with an electron deficient π-system is possible in the majority of the cases studied.

  9. Toxoplasma gondii DNA detection with a magnetic molecular beacon probe

    NASA Astrophysics Data System (ADS)

    Xu, Shichao; Yao, Cuicui; Wei, Shuoming; Zhang, Jimei; Dai, Zhao; Zheng, Guo; Sun, Bo; Han, Qing; Hu, Fei; Zhou, Hongming

    2008-12-01

    Toxoplasma Gondii infection is widespread in humans worldwide and reported infection rates range from 3%-70%, depending on the populations or geographic areas, and it has been recognized as a potential food safety hazard in our daily life. A magnetic molecular beacon probe (mMBP), based on theory of fluorescence resonance energy transfer (FRET), was currently reported to detect Toxoplasma Gondii DNA. Nano-sized Fe3O4 were primarily prepared by coprecipitation method in aqueous phase with NaOH as precipitator, and was used as magnetic core. The qualified coreshell magnetic quantum dots (mQDs), i.e. CdTe(symbol)Fe3O4, were then achieved by layer-by-layer method when mol ratio of Fe3O4/CdTe is 1/3, pH at 6.0, 30 °C, and reactant solution was refluxed for 30 min, the size of mQDs were determined to be 12-15 nm via transmission electron microscopy (TEM). Over 70% overlap between emission spectrum of mQDs and absorbance spectrum of BHQ-2 was observed, this result suggests the synthesized mQDs and BHQ-2 can be utilized as energy donor and energy acceptor, respectively. The sensing probe was fabricated and a stem-loop Toxoplasma Gondii DNA oligonucleotide was labeled with mQDs at the 5' end and BHQ-2 at 3' end, respectively. Target Toxoplasma gondii DNA was detected under conditions of 37 °C, hybridization for 2h, at pH8.0 in Tris-HCl buffer. About 30% recovery of fluorescence intensity was observed via fluorescence spectrum (FS) after the Toxoplasma gondii DNA was added, which suggested that the Toxoplasma Gondii DNA was successfully detected. Specificity investigation of the mMBP indicated that relative low recovery of fluorescence intensity was obtained when the target DNA with one-base pair mismatch was added, this result indicated the high specificity of the sensing probe. Our research simultaneously indicated that mMBP can be conveniently separated from the unhybridized stem-loop DNA and target DNA, which will be meaningful in DNA sensing and purification process.

  10. Probing Molecular Organization and Electronic Dynamics at Buried Organic Interfaces

    NASA Astrophysics Data System (ADS)

    Roberts, Sean

    2015-03-01

    Organic semiconductors are a promising class of materials due to their ability to meld the charge transport capabilities of semiconductors with many of the processing advantages of plastics. In thin film organic devices, interfacial charge transfer often comprises a crucial step in device operation. As molecular materials, the density of states within organic semiconductors often reflect their intermolecular organization. Truncation of the bulk structure of an organic semiconductor at an interface with another material can lead to substantial changes in the density of states near the interface that can significantly impact rates for interfacial charge and energy transfer. Here, we will present the results of experiments that utilize electronic sum frequency generation (ESFG) to probe buried interfaces in these materials. Within the electric dipole approximation, ESFG is only sensitive to regions of a sample that experience a breakage of symmetry, which occurs naturally at material interfaces. Through modeling of signals measured for thin organic films using a transfer matrix-based formalism, signals from buried interfaces between two materials can be isolated and used to uncover the interfacial density of states.

  11. Agonist Derived Molecular Probes for A2A Adenosine Receptors

    PubMed Central

    Jacobson, Kenneth A.; Pannell, Lewis K.; Ji, Xiao-duo; Jarvis, Michael F.; Williams, Michael; Hutchison, Alan J.; Barrington, William W.; Stiles, Gary L.

    2011-01-01

    The adenosine agonist 2-(4-(2-carboxyethyl)phenylethylamino)-5′-N-ethylcarboxamidoadenosine (CGS21680) was recently reported to be selective for the A2A adenosine receptor subtype, which mediates its hypotensive action. To investigate structurelactivity relationships at a distal site, CGS21680 was derivatized using a functionalized congener approach. The carboxylic group of CGS21680 has been esterified to form a methyl ester, which was then treated with ethylenediamine to produce an amine congener. The amine congener was an intermediate for acylation reactions, in which the reactive acyl species contained a reported group, or the precursor for such. For radioiodination, derivatives of p-hydroxyphenylpropionic, 2-thiophenylacetic, and p-aminophenylacetic acids were prepared. The latter derivative (PAPA-APEC) was iodinated electrophilically using [125I]iodide resulting in a radioligand which was used for studies of competition of binding to striatal A, adenosine receptors in bovine brain. A biotin conjugate and an aryl sulfonate were at least 350-fold selective for A, receptors. For spectroscopic detection, a derivative of the stable free radical tetramethyl-1-piperidinyloxy (TEMPO) was prepared. For irreversible inhibition of receptors, meta- and para-phenylenediisothiocyanate groups were incorporated in the analogs. We have demonstrated that binding at A2A receptors is relatively insensitive to distal structural changes at the 2-position, and we report high affinity molecular probes for receptor characterization by radioactive, spectroscopic and affinity labelling methodology. PMID:2561548

  12. Using molecular beacons to probe molecular interactions between lactate dehydrogenase and single-stranded DNA.

    PubMed

    Fang, X; Li, J J; Tan, W

    2000-07-15

    The interactions between two key macromolecular species, nucleic acids and proteins, control many important biological processes. There have been limited effective methodologies to study these interactions in real time. In this work, we have applied a newly developed molecular beacon (MB) DNA probe for the analysis of an enzyme, lactate dehydrogenase (LDH), and for the investigation of its properties of binding with single-stranded DNA. Molecular beacons are single-stranded oligonucleotide probes designed to report the presence of specific complementary nucleic acids by fluorescence detection. The interaction between LDH and MB has resulted in a significant fluorescence signal enhancement, which is used for the elucidation of MB/LDH binding properties. The processes of binding between MB and different isoenzymes of LDH have been studied. The results show that the stoichiometry of LDH-5/MB binding is 1:1, and the binding constant is 1.9 x 10(-7) M(-1). We have also studied salt effects, binding sites, temperature effects, pH effects, and the binding specificities for different isoenzymes. Our results demonstrate that MB can be effectively used for sensitive protein quantitation and for efficient protein-DNA interaction studies. MB has a signal transduction mechanism built within the molecule and can thus be used for the development of rapid protein assays and for real-time measurements.

  13. Monitoring molecular beacon DNA probe hybridization at the single-molecule level.

    PubMed

    Yao, Gang; Fang, Xiaohong; Yokota, Hiroaki; Yanagida, Toshio; Tan, Weihong

    2003-11-21

    We have monitored the reaction dynamics of the DNA hybridization process on a liquid/solid interface at the single-molecule level by using a hairpin-type molecular beacon DNA probe. Fluorescence images of single DNA probes were recorded by using total internal reflection fluorescence microscopy. The fluorescence signal of single DNA probes during the hybridization to individual complementary DNA probes was monitored over time. Among 400 molecular beacon DNA probes that we tracked, 349 molecular beacons (87.5 %) were hybridized quickly and showed an abrupt fluorescence increase, while 51 probes (12.5 %) reacted slowly, resulting in a gradual fluorescence increase. This ratio stayed about the same when varying the concentrations of cDNA in MB hybridization on the liquid/surface interface. Statistical data of the 51 single-molecule hybridization images showed that there was a multistep hybridization process. Our results also showed that photostability for the dye molecules associated with the double-stranded hybrids was better than that for those with the single-stranded molecular beacon DNA probes. Our results demonstrate the ability to obtain a better understanding of DNA hybridization processes using single-molecule techniques, which will improve biosensor and biochip development where surface-immobilized molecular beacon DNA probes provide unique advantages in signal transduction.

  14. Optical probes of atomic and molecular decay processes.

    SciTech Connect

    Pratt, S. T.

    2008-01-01

    characterize both the photoexcitation and subsequent decay processes. These techniques include mass spectrometry, dispersive and threshold photoelectron spectroscopy, laser-induced fluorescence, fluorescence-dip spectroscopy, and laser-induced grating spectroscopy. While the instrumentation is currently available for each of these techniques, two instruments deserve special mention. First, a high-resolution magnetic-bottle electron spectrometer has been developed that is equipped with a pulsed, skimmed molecular beam source. This instrument is capable of {approx}3-4 meV resolution in the electron kinetic energy while providing a collection efficiency of {approx}50%. Second, a time-of-flight mass spectrometer has been constructed with a similar molecular beam source. This instrument is currently being adapted to allow both ion- and electron-imaging studies. A typical experimental study is performed in three steps. First, resonant one-color multiphoton ionization is used to map out the transition between the ground state of the molecule of interest and the low-lying excited state to be used as an intermediate in the double-resonance process. The lasers used in these studies are Nd:YAG-pumped dye lasers with {approx}5 ns pulse durations. In general, this pump transition corresponds to a one- or two-photon process, and the laser output is frequency doubled, tripled, or mixed to generate light in the region of interest. In molecules such as ammonia and aniline, the pump transitions of interest are well characterized, allowing the unambiguous choice of pump transitions that access levels with the rovibronic character of interest. In other cases, the spectroscopy of the pump transition must be analyzed before it can be useful for the double-resonance experiments. In the second step, the pump laser is fixed on the pump transition of interest and a second laser is used to probe transitions from the upper state of the pump transition to the autoionizing or predissociating resonances in

  15. An ALuc-Based Molecular Tension Probe for Sensing Intramolecular Protein-Protein Interactions.

    PubMed

    Kim, Sung-Bae; Nishihara, Ryo; Suzuki, Koji

    2016-01-01

    Optical imaging of protein-protein interactions (PPIs) facilitates comprehensive elucidation of intracellular molecular events. The present protocol demonstrates an optical measure for visualizing molecular tension triggered by any PPI in mammalian cells. A unique design of single-chain probes was fabricated, in which a full-length artificial luciferase (ALuc(®)) was sandwiched between two model proteins of interest, e.g., FKBP and FRB. A molecular tension probe comprising ALuc23 greatly enhances the bioluminescence in response to varying concentrations of rapamycin, and named "tension probe (TP)." The basic probe design can be further modified towards eliminating the C-terminal end of ALuc and was found to improve signal-to-background ratios, named "combinational probe." TPs may become an important addition to the tool box of bioassays in the determination of protein dynamics of interest in mammalian cells. PMID:27424905

  16. Pump-Probe Noise Spectroscopy of Molecular Junctions.

    PubMed

    Ochoa, Maicol A; Selzer, Yoram; Peskin, Uri; Galperin, Michael

    2015-02-01

    The slow response of electronic components in junctions limits the direct applicability of pump-probe type spectroscopy in assessing the intramolecular dynamics. Recently the possibility of getting information on a sub-picosecond time scale from dc current measurements was proposed. We revisit the idea of picosecond resolution by pump-probe spectroscopy from dc measurements and show that any intramolecular dynamics not directly related to charge transfer in the current direction is missed by current measurements. We propose a pump-probe dc shot noise spectroscopy as a suitable alternative. Numerical examples of time-dependent and average responses of junctions are presented for generic models. PMID:26261965

  17. NIH workshop on clinical translation of molecular imaging probes and technology--meeting report.

    PubMed

    Liu, Christina H; Sastre, Antonio; Conroy, Richard; Seto, Belinda; Pettigrew, Roderic I

    2014-10-01

    A workshop on "Clinical Translation of Molecular Imaging Probes and Technology" was held August 2, 2013 in Bethesda, Maryland, organized and supported by the National Institute of Biomedical Imaging and Bioengineering (NIBIB). This workshop brought together researchers, clinicians, representatives from pharmaceutical companies, molecular probe developers, and regulatory science experts. Attendees met to talk over current challenges in the discovery, validation, and translation of molecular imaging (MI) probes for key clinical applications. Participants also discussed potential strategies to address these challenges. The workshop consisted of 4 sessions, with 14 presentations and 2 panel discussions. Topics of discussion included (1) challenges and opportunities for clinical research and patient care, (2) advances in molecular probe design, (3) current approaches used by industry and pharmaceutical companies, and (4) clinical translation of MI probes. In the presentations and discussions, there were general agreement that while the barriers for validation and translation of MI probes remain high, there are pressing clinical needs and development opportunities for targets in cardiovascular, cancer, endocrine, neurological, and inflammatory diseases. The strengths of different imaging modalities, and the synergy of multimodality imaging, were highlighted. Participants also underscored the continuing need for close interactions and collaborations between academic and industrial partners, and federal agencies in the imaging probe development process.

  18. Probing flexible conformations in molecular junctions by inelastic electron tunneling spectroscopy

    SciTech Connect

    Deng, Mingsen; Ye, Gui; Jiang, Jun; Cai, Shaohong; Sun, Guangyu

    2015-01-15

    The probe of flexible molecular conformation is crucial for the electric application of molecular systems. We have developed a theoretical procedure to analyze the couplings of molecular local vibrations with the electron transportation process, which enables us to evaluate the structural fingerprints of some vibrational modes in the inelastic electron tunneling spectroscopy (IETS). Based on a model molecule of Bis-(4-mercaptophenyl)-ether with a flexible center angle, we have revealed and validated a simple mathematical relationship between IETS signals and molecular angles. Our results might open a route to quantitatively measure key geometrical parameters of molecular junctions, which helps to achieve precise control of molecular devices.

  19. Use of sloppy molecular beacon probes for identification of mycobacterial species.

    PubMed

    El-Hajj, Hiyam H; Marras, Salvatore A E; Tyagi, Sanjay; Shashkina, Elena; Kamboj, Mini; Kiehn, Timothy E; Glickman, Michael S; Kramer, Fred Russell; Alland, David

    2009-04-01

    We report here the use of novel "sloppy" molecular beacon probes in homogeneous PCR screening assays in which thermal denaturation of the resulting probe-amplicon hybrids provides a characteristic set of amplicon melting temperature (T(m)) values that identify which species is present in a sample. Sloppy molecular beacons possess relatively long probe sequences, enabling them to form hybrids with amplicons from many different species despite the presence of mismatched base pairs. By using four sloppy molecular beacons, each possessing a different probe sequence and each labeled with a differently colored fluorophore, four different T(m) values can be determined simultaneously. We tested this technique with 27 different species of mycobacteria and found that each species generates a unique, highly reproducible signature that is unaffected by the initial bacterial DNA concentration. Utilizing this general paradigm, screening assays can be designed for the identification of a wide range of species.

  20. Laser-cooled atomic ions as probes of molecular ions

    SciTech Connect

    Brown, Kenneth R.; Viteri, C. Ricardo; Clark, Craig R.; Goeders, James E.; Khanyile, Ncamiso B.; Vittorini, Grahame D.

    2015-01-22

    Trapped laser-cooled atomic ions are a new tool for understanding cold molecular ions. The atomic ions not only sympathetically cool the molecular ions to millikelvin temperatures, but the bright atomic ion fluorescence can also serve as a detector of both molecular reactions and molecular spectra. We are working towards the detection of single molecular ion spectra by sympathetic heating spectroscopy. Sympathetic heating spectroscopy uses the coupled motion of two trapped ions to measure the spectra of one ion by observing changes in the fluorescence of the other ion. Sympathetic heating spectroscopy is a generalization of quantum logic spectroscopy, but does not require ions in the motional ground state or coherent control of the ion internal states. We have recently demonstrated this technique using two isotopes of Ca{sup +} [Phys. Rev. A, 81, 043428 (2010)]. Limits of the method and potential applications for molecular spectroscopy are discussed.

  1. A Molecular Imaging Approach to Mercury Sensing Based on Hyperpolarized (129)Xe Molecular Clamp Probe.

    PubMed

    Guo, Qianni; Zeng, Qingbin; Jiang, Weiping; Zhang, Xiaoxiao; Luo, Qing; Zhang, Xu; Bouchard, Louis-S; Liu, Maili; Zhou, Xin

    2016-03-14

    Mercury pollution, in the form of mercury ions (Hg(2+)), is a major health and environmental hazard. Commonly used sensors are invasive and limited to point measurements. Fluorescence-based sensors do not provide depth resolution needed to image spatial distributions. Herein we report a novel sensor capable of yielding spatial distributions by MRI using hyperpolarized (129)Xe. A molecular clamp probe was developed consisting of dipyrrolylquinoxaline (DPQ) derivatives and twocryptophane-A cages. The DPQ derivatives act as cation receptors whereas cryptophane-A acts as a suitable host molecule for xenon. When the DPQ moiety interacts with mercury ions, the molecular clamp closes on the ion. Due to overlap of the electron clouds of the two cryptophane-A cages, the shielding effect on the encapsulated Xe becomes important. This leads to an upfield change of the chemical shift of the encapsulated Xe. This sensor exhibits good selectivity and sensitivity toward the mercury ion. This mercury-activated hyperpolarized (129)Xe-based chemosensor is a new concept method for monitoring Hg(2+) ion distributions by MRI.

  2. Semiconducting Polymer Nanoparticles as Photoacoustic Molecular Imaging Probes in Living Mice

    PubMed Central

    Pu, Kanyi; Shuhendler, Adam J.; Jokerst, Jesse V.; Mei, Jianguo; Gambhir, Sanjiv S.; Bao, Zhenan; Rao, Jianghong

    2014-01-01

    Photoacoustic (PA) imaging holds great promise for the visualization of physiology and pathology at the molecular level with deep tissue penetration and fine spatial resolution. To fully utilize this potential, PA molecular imaging probes have to be developed. Herein we introduce near infrared (NIR) light absorbing semiconducting polymer nanoparticles (SPNs) as a new class of contrast agents for PA molecular imaging. SPNs can produce stronger signal than commonly used single-wall carbon nanotubes and gold nanorods on a per mass basis, permitting whole-body lymph node PA mapping in living mice at a low systematic injection mass. Furthermore, SPNs possess high structural flexibility, narrow PA spectral profiles, and strong resistance to photodegradation and oxidation, which enables development of the first NIR ratiometric PA probe for in vivo real-time imaging of reactive oxygen species—vital chemical mediators of many diseases. These results demonstrate SPNs an ideal nanoplatform for developing PA molecular probes. PMID:24463363

  3. Near-infrared Molecular Probes for In Vivo Imaging

    PubMed Central

    Zhang, Xuan; Bloch, Sharon; Akers, Walter; Achilefu, Samuel

    2012-01-01

    Cellular and tissue imaging in the near-infrared (NIR) wavelengths between 700 and 900 nm is advantageous for in vivo because of the low absorption of biological molecules in this region. This Unit presents protocols for small animal imaging using planar and fluorescence lifetime imaging techniques. Included is an overview of NIR fluorescence imaging of cells and small animals using NIR organic fluorophores, nanoparticles, and multimodal imaging probes. The development, advantages, and application of NIR fluorescent probes that have been used for in vivo imaging are also summarized. The use of NIR agents in conjunction with visible dyes and considerations in selecting imaging agents are discussed. We conclude with practical considerations for the use of these dyes in cell and small animal imaging applications. PMID:22470154

  4. Probing Interactions in Complex Molecular Systems through Ordered Assembly

    SciTech Connect

    De Yoreo, J J; Bartelt, M C; Orme, C A; Villacampa, A; Weeks, B L; Miller, A E

    2002-01-31

    Emerging from the machinery of epitaxial science and chemical synthesis, is a growing emphasis on development of self-organized systems of complex molecular species. The nature of self-organization in these systems spans the continuum from simple crystallization of large molecules such as dendrimers and proteins, to assembly into large organized networks of nanometer-scale structures such as quantum dots or nanoparticles. In truth, self-organization in complex molecular systems has always been a central feature of many scientific disciplines including fields as diverse as structural biology, polymer science and geochemistry. But over the past decade, changes in those fields have often been marked by the degree to which researchers are using molecular-scale approaches to understand the hierarchy of structures and processes driven by this ordered assembly. At the same time, physical scientists have begun to use their knowledge of simple atomic and molecular systems to fabricate synthetic self-organized systems. This increasing activity in the field of self-organization is testament to the success of the physical and chemical sciences in building a detailed understanding of crystallization and epitaxy in simple atomic and molecular systems, one that is soundly rooted in thermodynamics and chemical kinetics. One of the fundamental challenges of chemistry and materials science in the coming decades is to develop a similarly well-founded physical understanding of assembly processes in complex molecular systems. Over the past five years, we have successfully used in situ atomic force microscopy (AFM) to investigate the physical controls on single crystal epitaxy from solutions for a wide range of molecular species. More recently, we have combined this method with grazing incidence X-ray diffraction and kinetic Monte Carlo modeling in order to relate morphology to surface atomic structure and processes. The purpose of this proposal was to extend this approach to assemblies

  5. Molecular modeling of lipid probes and their influence on the membrane.

    PubMed

    Faller, Roland

    2016-10-01

    In this review a number of Molecular Dynamics simulation studies are discussed which focus on the understanding of the behavior of lipid probes in biomembranes. Experiments often use specialized probe moieties or molecules to report on the behavior of a membrane and try to gain information on the membrane as a whole from the probe lipids as these probes are the only things an experiment sees. Probes can be used to make NMR, EPR and fluorescence accessible to the membrane and use fluorescent or spin-active moieties for this purpose. Clearly membranes with and without probes are not identical which makes it worthwhile to elucidate the differences between them with detailed atomistic simulations. In almost all cases these differences are confined to the local neighborhood of the probe molecules which are sparsely used and generally present as single molecules. In general, the behavior of the bulk membrane lipids can be qualitatively understood from the probes but in most cases their properties cannot be directly quantitatively deduced from the probe behavior. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.

  6. A single molecular beacon probe is sufficient for the analysis of multiple nucleic acid sequences.

    PubMed

    Gerasimova, Yulia V; Hayson, Aaron; Ballantyne, Jack; Kolpashchikov, Dmitry M

    2010-08-16

    Molecular beacon (MB) probes are dual-labeled hairpin-shaped oligodeoxyribonucleotides that are extensively used for real-time detection of specific RNA/DNA analytes. In the MB probe, the loop fragment is complementary to the analyte: therefore, a unique probe is required for the analysis of each new analyte sequence. The conjugation of an oligonucleotide with two dyes and subsequent purification procedures add to the cost of MB probes, thus reducing their application in multiplex formats. Here we demonstrate how one MB probe can be used for the analysis of an arbitrary nucleic acid. The approach takes advantage of two oligonucleotide adaptor strands, each of which contains a fragment complementary to the analyte and a fragment complementary to an MB probe. The presence of the analyte leads to association of MB probe and the two DNA strands in quadripartite complex. The MB probe fluorescently reports the formation of this complex. In this design, the MB does not bind the analyte directly; therefore, the MB sequence is independent of the analyte. In this study one universal MB probe was used to genotype three human polymorphic sites. This approach promises to reduce the cost of multiplex real-time assays and improve the accuracy of single-nucleotide polymorphism genotyping.

  7. Molecular Cloning of MAPK Gene Family Using Synthetic Oligonucleotide Probe.

    PubMed

    Zhou, Song; Wang, Qin; Chen, Jing; Chen, Jiang-Ye

    1999-01-01

    MAPK(mitogen activated protein kinase) is a kind of Ser/Thr protein kinase. The MAPKs play an important role in several different signal transduction pathways. The MAPKs may also have a role in morphorgenesis of Candida albicans. An oligonucleotide probe was used to screen novel MAPKs in C. albicans. All MAPKs shared high homogeneity in their eleven kinase subdomains, especially subdoman VII and VIII. In subdomain VII, nearly all MAPKs have the same KIDFGLAR sequence, and the two known MAPKs in C. albicans CEK1 and MKC1 have only one different nucleotide in that DNA sequence. This probe was hybridized with C. albicans genomic DNA. Under stringent conditions, the probe could only hybridize with CEK1 and MKC1 gene fragment. But when hybridized at 40 degrees in non-SDS solution, two novel bands appeared. This condition was used to screen SC5314 DNA library, and many positive clones with different hybridization density were obtained. The strongest hybridization clones were identified to contain CEK1 and MKC1 gene. From the stronger positive hybridization clones, two novel genes were identified. The first gene, named CRK1(CDC2-related protein kinase 1), shared high homogeneity to MAPKs, but was not of them. It is closest to SGV1 from S. cerevisiae (with homology 47%) and PITALRE from human (with homology 41%), both of which are CDC2-related protein kinases. The second gene called CEK2(Candida albicans extracelluar signal-regulated kinase 2) is a novel MAPK of Candida albicans, which shares the highest identity with CEK1 and its S. cerevisiae homologs, FUS3 and KSS1, two redundant MAPKs in yeast pheromone response and morphogenesis. PMID:12114967

  8. Probing - and - Molecular Interactions via Irmpd Experiments and Computational Chemistry

    NASA Astrophysics Data System (ADS)

    Hopkins, Scott; McMahon, Terry

    2015-06-01

    Experiments carried out at the CLIO Free Electron Facility have been used to probe a range of novel bonding motifs and dissociation dynamics in a variety of chemical systems. Among these are species which exhibit anion-pi interactions in complexes of halide ions with aromatic ring systems with electron withdrawing substituents; charge solvated and zwitterionic clusters of protonated methylamines with phenylalanines; hydrogen bonded dimers of nucleic acid analogues and Pd complexes potentially involving agnostic hydrogen bond interactions. Accompanying DFT computational work is used to assist in identifying the most probable structure(s) present in the IRMPD experiments.

  9. Molecular beacons as probes of RNA unfolding under native conditions.

    PubMed

    Hopkins, Julia F; Woodson, Sarah A

    2005-01-01

    Hybridization of fluorescent molecular beacons provides real-time detection of RNA secondary structure with high specificity. We used molecular beacons to measure folding and unfolding rates of the Tetrahymena group I ribozyme under native conditions. A molecular beacon targeted against 15 nt in the 5' strand of the P3 helix specifically hybridized with misfolded forms of the ribozyme, without invading the native tertiary structure. The beacon associated with the misfolded ribozyme 300 times more slowly than with an unstructured oligonucleotide containing the same target sequence, suggesting that the misfolded ribozyme core remains structured in the absence of Mg2+. The rate of beacon hybridization under native conditions revealed a linear relationship between the free energy of unfolding and Mg2+ concentration. A small fraction of the RNA population unfolded very rapidly, suggesting parallel unfolding in one step or through misfolded intermediates.

  10. Molecular probes for nonlinear optical imaging of biological membranes

    NASA Astrophysics Data System (ADS)

    Blanchard-Desce, Mireille H.; Ventelon, Lionel; Charier, Sandrine; Moreaux, Laurent; Mertz, Jerome

    2001-12-01

    Second-harmonic generation (SHG) and two-photon excited fluorescence (TPEF) are nonlinear optical (NLO) phenomena that scale with excitation intensity squared, and hence give rise to an intrinsic 3-dimensional resolution when used in microscopic imaging. TPEF microscopy has gained widespread popularity in the biology community whereas SHG microscopy promises to be a powerful tool because of its sensitivity to local asymmetry. We have implemented an approach toward the design of NLO-probes specifically adapted for SHG and/or TPEF imaging of biological membranes. Our strategy is based on the design of nanoscale amphiphilic NLO-phores. We have prepared symmetrical bolaamphiphilic fluorophores combining very high two-photon absorption (TPA) cross-sections in the visible red region and affinity for cellular membranes. Their incorporation and orientation in lipid membranes can be monitored via TPEF anisotropy. We have also prepared amphiphilic push-pull chromophores exhibiting both large TPA cross-sections and very large first hyperpolarizabilities in the near-IR region. These NLO-probes have proved to be particularly useful for imaging of biological membranes by simultaneous SHG and TPEF microscopy and offer attractive prospects for real-time imaging of fundamental biological processes such as adhesion, fusion or reporting of membrane potentials.

  11. Ferroelectric Polymer Matrix for Probing Molecular Organization in Perylene Diimides.

    PubMed

    Chellappan, Kishore V; Kandappa, Sunil Kumar; Rajaram, Sridhar; Narayan, K S

    2015-01-15

    Ferroelectric films of poly(vinylidene fluoride-co-trifluoroethylene) (PVDF-TrFE) provide a controlled environment to study the aggregation tendency of functional molecules such as perylene diimides (PDIs). The local electric field and free volume confinement parameters offered by the matrix are tailored to study the organizational and assembly characteritics of molecular acceptors. The optical properties of planar and nonplanar PDIs in the ferroelectric polymer matrix were studied systematically over a wide range that encompassed the ferroelectric transition temperature. This approach provides valuable insight into the properties of molecular materials used in applications ranging from bulk heterostructure-based photovoltaics to nonlinear optical materials.

  12. A specific molecular beacon probe for the detection of human prostate cancer cells.

    PubMed

    Jiang, Yu Lin; McGoldrick, Christopher A; Yin, Deling; Zhao, Jing; Patel, Vini; Brannon, Marianne F; Lightner, Janet W; Krishnan, Koyamangalath; Stone, William L

    2012-06-01

    The small-molecule, water-soluble molecular beacon probe 1 is hydrolyzed by the lysate and living cells of human prostate cancer cell lines (LNCaP), resulting in strong green fluorescence. In contrast, probe 1 does not undergo significant hydrolysis in either the lysate or living cells of human nontumorigenic prostate cells (RWPE-1). These results, corroborated by UV-Vis spectroscopy and fluorescent microscopy, reveal that probe 1 is a sensitive and specific fluorogenic and chromogenic sensor for the detection of human prostate cancer cells among nontumorigenic prostate cells and that carboxylesterase activity is a specific biomarker for human prostate cancer cells.

  13. Integrating molecular dynamics simulations with chemical probing experiments using SHAPE-FIT

    PubMed Central

    Kirmizialtin, Serdal; Hennelly, Scott P.; Schug, Alexander; Onuchic, Jose N.; Sanbonmatsu, Karissa Y.

    2016-01-01

    Integration and calibration of molecular dynamics simulations with experimental data remains a challenging endeavor. We have developed a novel method to integrate chemical probing experiments with molecular simulations of RNA molecules by using a native structure-based model. Selective 2’-hydroxyl acylation by primer extension (SHAPE) characterizes the mobility of each residue in the RNA. Our method, SHAPE-FIT, automatically optimizes the potential parameters of the forcefield according to measured reactivities from SHAPE. The optimized parameter set allows simulations of dynamics highly consistent with SHAPE probing experiments. Such atomistic simulations, thoroughly grounded in experiment, can open a new window on RNA structure-function relations. PMID:25726467

  14. Molecular Probes for Imaging Myelinated White Matter in CNS

    PubMed Central

    Wu, Chunying; Wei, Jinjun; Tian, Donghua; Feng, Yue; Miller, Robert H.; Wang, Yanming

    2009-01-01

    Abnormalities and changes in myelination in the brain are seen in many neurodegenerative disorders such as multiple sclerosis (MS). Direct detection and quantification of myelin content in vivo is desired to facilitate diagnosis and therapeutic treatments of myelin-related diseases. The imaging studies require use of myelin-imaging agents that readily enter the brain and selectively bind to myelinated regions. For this purpose, we have systematically evaluated a series of stilbene derivatives as myelin imaging agents. Spectrophotometry-based and radioligand-based binding assays showed that these stilbene derivatives exhibited relatively high myelin-binding affinities. In vitro myelin staining exhibited that the compounds selectively stained intact myelinated regions in wild type mouse brain. In situ tissue staining demonstrated that the compounds readily entered the mouse brain and selectively labeled myelinated white matter regions. These studies suggested that these stilbene derivatives can be used as myelin-imaging probes to monitor myelin pathology in vivo. PMID:18844339

  15. Optimizing the sensitivity of photoluminescent probes using time-resolved spectroscopy: a molecular beacon case study.

    PubMed

    Huang, Kewei; Martí, Angel A

    2012-09-18

    Improving probes so that they can perform more sensitive and accurate detections is at the heart of much fundamental and applied research. Within the past few years a considerable amount of effort has been devoted to the study of photoluminescent probes in combination with time-resolved photoluminescence spectroscopy (TRPS). Although TRPS is a powerful and important technique for improving the sensitivity of long-lived probes, there is a lack of a general methodology that would allow one to unambiguously optimize the parameter affecting this technique. In this manuscript it will be shown how parameters that are probe- and technique-specific can affect the effectiveness of TRPS in improving sensitivity. Furthermore, it will be demonstrated that, when TRPS is used, the sensitivity of the probe is strongly dependent on the time window used to generate the time-resolved emission spectra (TRES). A method will be described that will allow one to remove the uncertainty in the selection of the time window that would yield the optimum improvement in probe performance, as well as the experimental parameters that need to be considered. Molecular beacon probes (MBs) were used to demonstrate these points. These probes show signal-to-background ratios (S/B) of less than 9 when SSPS is used, which can be easily enhanced to 17 using TRPS. The detection limits were also improved when TRPS is used allowing detecting target DNA with concentrations as low as 13.6 nM.

  16. Molecular-beacon-based tricomponent probe for SNP analysis in folded nucleic acids.

    PubMed

    Nguyen, Camha; Grimes, Jeffrey; Gerasimova, Yulia V; Kolpashchikov, Dmitry M

    2011-11-11

    Hybridization probes are often inefficient in the analysis of single-stranded DNA or RNA that are folded in stable secondary structures. A molecular beacon (MB) probe is a short DNA hairpin with a fluorophore and a quencher attached to opposite sides of the oligonucleotide. The probe is widely used in real-time analysis of specific DNA and RNA sequences. This study demonstrates how a conventional MB probe can be used for the analysis of nucleic acids that form very stable (T(m) > 80 °C) hairpin structures. Here we demonstrate that the MB probe is not efficient in direct analysis of secondary structure-folded analytes, whereas a MB-based tricomponent probe is suitable for these purposes. The tricomponent probe takes advantage of two oligonucleotide adaptor strands f and m. Each adaptor strand contains a fragment complementary to the analyte and a fragment complementary to a MB probe. In the presence of a specific analyte, the two adaptor strands hybridize to the analyte and the MB probe, thus forming a quadripartite complex. DNA strand f binds to the analyte with high affinity and unwinds its secondary structure. Strand m forms a stable complex only with the fully complementary analyte. The MB probe fluorescently reports the formation of the quadripartite associate. It was demonstrated that the DNA analytes folded in hairpin structures with stems containing 5, 6, 7, 8, 9, 11, or 13 base pairs can be detected in real time with the limit of detection (LOD) lying in the nanomolar range. The stability of the stem region in the DNA analyte did not affect the LOD. Analytes containing single base substitutions in the stem or in the loop positions were discriminated from the fully complementary DNA at room temperature. The tricomponent probe promises to simplify nucleic acid analysis at ambient temperatures in such applications as in vivo RNA monitoring, detection of pathogens, and single nucleotide polymorphism (SNP) genotyping by DNA microarrays.

  17. Pre-Assembly of Near-Infrared Fluorescent Multivalent Molecular Probes for Biological Imaging.

    PubMed

    Peck, Evan M; Battles, Paul M; Rice, Douglas R; Roland, Felicia M; Norquest, Kathryn A; Smith, Bradley D

    2016-05-18

    A programmable pre-assembly method is described and shown to produce near-infrared fluorescent molecular probes with tunable multivalent binding properties. The modular assembly process threads one or two copies of a tetralactam macrocycle onto a fluorescent PEGylated squaraine scaffold containing a complementary number of docking stations. Appended to the macrocycle periphery are multiple copies of a ligand that is known to target a biomarker. The structure and high purity of each threaded complex was determined by independent spectrometric methods and also by gel electrophoresis. Especially helpful were diagnostic red-shift and energy transfer features in the absorption and fluorescence spectra. The threaded complexes were found to be effective multivalent molecular probes for fluorescence microscopy and in vivo fluorescence imaging of living subjects. Two multivalent probes were prepared and tested for targeting of bone in mice. A pre-assembled probe with 12 bone-targeting iminodiacetate ligands produced more bone accumulation than an analogous pre-assembled probe with six iminodiacetate ligands. Notably, there was no loss in probe fluorescence at the bone target site after 24 h in the living animal, indicating that the pre-assembled fluorescent probe maintained very high mechanical and chemical stability on the skeletal surface. The study shows how this versatile pre-assembly method can be used in a parallel combinatorial manner to produce libraries of near-infrared fluorescent multivalent molecular probes for different types of imaging and diagnostic applications, with incremental structural changes in the number of targeting groups, linker lengths, linker flexibility, and degree of PEGylation.

  18. Probing molecular choreography through single-molecule biochemistry.

    PubMed

    van Oijen, Antoine M; Dixon, Nicholas E

    2015-12-01

    Single-molecule approaches are having a dramatic impact on views of how proteins work. The ability to observe molecular properties at the single-molecule level allows characterization of subpopulations and acquisition of detailed kinetic information that would otherwise be hidden in the averaging over an ensemble of molecules. In this Perspective, we discuss how such approaches have successfully been applied to in vitro-reconstituted systems of increasing complexity.

  19. Ultrafast X-ray Auger probing of photoexcited molecular dynamics.

    PubMed

    McFarland, B K; Farrell, J P; Miyabe, S; Tarantelli, F; Aguilar, A; Berrah, N; Bostedt, C; Bozek, J D; Bucksbaum, P H; Castagna, J C; Coffee, R N; Cryan, J P; Fang, L; Feifel, R; Gaffney, K J; Glownia, J M; Martinez, T J; Mucke, M; Murphy, B; Natan, A; Osipov, T; Petrović, V S; Schorb, S; Schultz, Th; Spector, L S; Swiggers, M; Tenney, I; Wang, S; White, J L; White, W; Gühr, M

    2014-01-01

    Molecules can efficiently and selectively convert light energy into other degrees of freedom. Disentangling the underlying ultrafast motion of electrons and nuclei of the photoexcited molecule presents a challenge to current spectroscopic approaches. Here we explore the photoexcited dynamics of molecules by an interaction with an ultrafast X-ray pulse creating a highly localized core hole that decays via Auger emission. We discover that the Auger spectrum as a function of photoexcitation--X-ray-probe delay contains valuable information about the nuclear and electronic degrees of freedom from an element-specific point of view. For the nucleobase thymine, the oxygen Auger spectrum shifts towards high kinetic energies, resulting from a particular C-O bond stretch in the ππ* photoexcited state. A subsequent shift of the Auger spectrum towards lower kinetic energies displays the electronic relaxation of the initial photoexcited state within 200 fs. Ab-initio simulations reinforce our interpretation and indicate an electronic decay to the nπ* state.

  20. Probing Molecular Dynamics by Laser-Induced Backscattering Holography.

    PubMed

    Haertelt, Marko; Bian, Xue-Bin; Spanner, Michael; Staudte, André; Corkum, Paul B

    2016-04-01

    We use differential holography to overcome the forward scattering problem in strong-field photoelectron holography. Our differential holograms of H_{2} and D_{2} molecules exhibit a fishbonelike structure, which arises from the backscattered part of the recolliding photoelectron wave packet. We demonstrate that the backscattering hologram can resolve the different nuclear dynamics between H_{2} and D_{2} with subangstrom spatial and subcycle temporal resolution. In addition, we show that attosecond electron dynamics can be resolved. These results open a new avenue for ultrafast studies of molecular dynamics in small molecules. PMID:27081975

  1. Probing Molecular Dynamics by Laser-Induced Backscattering Holography

    NASA Astrophysics Data System (ADS)

    Haertelt, Marko; Bian, Xue-Bin; Spanner, Michael; Staudte, André; Corkum, Paul B.

    2016-04-01

    We use differential holography to overcome the forward scattering problem in strong-field photoelectron holography. Our differential holograms of H2 and D2 molecules exhibit a fishbonelike structure, which arises from the backscattered part of the recolliding photoelectron wave packet. We demonstrate that the backscattering hologram can resolve the different nuclear dynamics between H2 and D2 with subangstrom spatial and subcycle temporal resolution. In addition, we show that attosecond electron dynamics can be resolved. These results open a new avenue for ultrafast studies of molecular dynamics in small molecules.

  2. Quantitative analysis of localized surface plasmons based on molecular probing.

    PubMed

    Deeb, Claire; Bachelot, Renaud; Plain, Jérôme; Baudrion, Anne-Laure; Jradi, Safi; Bouhelier, Alexandre; Soppera, Olivier; Jain, Prashant K; Huang, Libai; Ecoffet, Carole; Balan, Lavinia; Royer, Pascal

    2010-08-24

    We report on the quantitative characterization of the plasmonic optical near-field of a single silver nanoparticle. Our approach relies on nanoscale molecular molding of the confined electromagnetic field by photoactivated molecules. We were able to directly image the dipolar profile of the near-field distribution with a resolution better than 10 nm and to quantify the near-field depth and its enhancement factor. A single nanoparticle spectral signature was also assessed. This quantitative characterization constitutes a prerequisite for developing nanophotonic applications.

  3. Fiber-optic Raman sensing of cell proliferation probes and molecular vibrations: Brain-imaging perspective

    NASA Astrophysics Data System (ADS)

    Doronina-Amitonova, Lyubov V.; Fedotov, Il'ya V.; Ivashkina, Olga I.; Zots, Marina A.; Fedotov, Andrei B.; Anokhin, Konstantin V.; Zheltikov, Aleksei M.

    2012-09-01

    Optical fibers are employed to sense fingerprint molecular vibrations in ex vivo experiments on the whole brain and detect cell proliferation probes in a model study on a quantitatively controlled solution. A specifically adapted spectral filtering procedure is shown to allow the Raman signal from molecular vibrations of interest to be discriminated against the background from the fiber, allowing a highly sensitive Raman detection of the recently demonstrated EdU (5-ethynyl-2'-deoxyuridine) labels of DNA synthesis in cells.

  4. Disease-specific target gene expression profiling of molecular imaging probes: database development and clinical validation.

    PubMed

    Chan, Lawrence Wing-Chi; Ngo, Connie Hiu-Ching; Wang, Fengfeng; Zhao, Moss Y; Zhao, Mengying; Law, Helen Ka-Wai; Wong, Sze Chuen Cesar; Yung, Benjamin Yat-Ming

    2014-01-01

    Molecular imaging probes can target abnormal gene expression patterns in patients and allow early diagnosis of disease. For selecting a suitable imaging probe, the current Molecular Imaging and Contrast Agent Database (MICAD) provides descriptive and qualitative information on imaging probe characteristics and properties. However, MICAD does not support linkage with the expression profiles of target genes. The proposed Disease-specific Imaging Probe Profiling (DIPP) database quantitatively archives and presents the gene expression profiles of targets across different diseases, anatomic regions, and subcellular locations, providing an objective reference for selecting imaging probes. The DIPP database was validated with a clinical positron emission tomography (PET) study on lung cancer and an in vitro study on neuroendocrine cancer. The retrieved records show that choline kinase beta and glucose transporters were positively and significantly associated with lung cancer among the targets of 11C-choline and [18F]fluoro-2-deoxy-2-d-glucose (FDG), respectively. Their significant overexpressions corresponded to the findings that the uptake rate of FDG increased with tumor size but that of 11C-choline remained constant. Validated with the in vitro study, the expression profiles of disease-associated targets can indicate the eligibility of patients for clinical trials of the treatment probe. A Web search tool of the DIPP database is available at http://www.polyu.edu.hk/bmi/dipp/. PMID:25022454

  5. Co-encapsulating the fusogenic peptide INF7 and molecular imaging probes in liposomes increases intracellular signal and probe retention.

    PubMed

    Burks, Scott R; Legenzov, Eric A; Martin, Erik W; Li, Changqing; Lu, Wuyuan; Kao, Joseph P Y

    2015-01-01

    Liposomes are promising vehicles to deliver diagnostic and therapeutic agents to cells in vivo. After uptake into cells by endocytosis, liposomes are degraded in the endolysosomal system. Consequently, the encapsulated cargo molecules frequently remain sequestered in endosomal compartments; this limits their usefulness in many applications (e.g. gene delivery). To overcome this, various fusogenic peptides have been developed to facilitate delivery of liposomally-encapsulated molecules into the cytosol. One such peptide is the pH-sensitive influenza-derived peptide INF7. Liposomal delivery of imaging agents is an attractive approach for enabling cell imaging and cell tracking in vivo, but can be hampered by inadequate intracellular accumulation and retention of probes caused by exocytosis (and possible degradation) of endosome-entrapped probes. Such signal loss could be minimized by facilitating escape of probe molecules from endolysosomal compartments into the cytosol. We investigated the ability of co-encapsulated INF7 to release liposomally-delivered rhodamine fluorophores into the cytosol after endosomal acidification/maturation. We co-encapsulated INF7 and fluorescent rhodamine derivatives having vastly different transport properties to show that after endocytosis by CV1 cells, the INF7 peptide is activated by acidic endosomal pH and facilitates efficient release of the fluorescent tracers into the cytosol. Furthermore, we show that INF7-facilitated escape from endosomes markedly enhanced retention of tracers that cannot be actively extruded from the cytosol. Minimizing loss of intracellular probes improves cellular imaging by increasing the signal-to-noise ratio of images and lengthening the time window that imaging can be performed. In particular, this will enhance in vivo electron paramagnetic resonance imaging, an emergent magnetic resonance imaging modality requires exogenous paramagnetic imaging agents and is highly promising for cellular and molecular

  6. Molecular Dynamic Simulations of Interaction of an AFM Probe with the Surface of an SCN Sample

    NASA Technical Reports Server (NTRS)

    Bune, Adris; Kaukler, William; Rose, M. Franklin (Technical Monitor)

    2001-01-01

    Molecular dynamic (MD) simulations is conducted in order to estimate forces of probe-substrate interaction in the Atomic Force Microscope (AFM). First a review of available molecular dynamic techniques is given. Implementation of MD simulation is based on an object-oriented code developed at the University of Delft. Modeling of the sample material - succinonitrile (SCN) - is based on the Lennard-Jones potentials. For the polystyrene probe an atomic interaction potential is used. Due to object-oriented structure of the code modification of an atomic interaction potential is straight forward. Calculation of melting temperature is used for validation of the code and of the interaction potentials. Various fitting parameters of the probe-substrate interaction potentials are considered, as potentials fitted to certain properties and temperature ranges may not be reliable for the others. This research provides theoretical foundation for an interpretation of actual measurements of an interaction forces using AFM.

  7. A Bridge Not Too Far: Linking Disciplines Through Molecular Imaging Probes.

    PubMed

    Valliant, John F

    2016-09-01

    The field of nuclear medicine will rely increasingly on the discovery, proper evaluation, and clinical use of molecular imaging probes and on collaborations. Collaborations will include new initiatives among experts already involved in the field and with researchers, technologists, and clinicians from different areas of science and medicine. This article serves to highlight some of the opportunities in which molecular imaging and nuclear medicine in conjunction with probe development, new imaging technologies, and multidisciplinary collaborations can have a significant impact on health care and basic science from the perspective of a person involved in probe development. The article emphasizes breast cancer, but the concepts are readily applied to other areas of medicine and medical research. PMID:27601414

  8. Ratiometric and near-infrared molecular probes for the detection and imaging of zinc ions.

    PubMed

    Carol, Priya; Sreejith, Sivaramapanicker; Ajayaghosh, Ayyappanpillai

    2007-03-01

    The detection and imaging of Zn2+ in biological samples are of paramount interest owing to the role of this cation in physiological functions. This is possible only with molecular probes that specifically bind to Zn2+ and result in changes in emission properties. A "turn-on" emission or shift in the emission color upon binding to Zn2+ should be ideal for in vivo imaging. In this context, ratiometric and near-IR probes are of particular interest. Therefore, in the area of chemosensors or molecular probes, the design of fluorophores that allow ratiometric sensing or imaging in the near-IR region is attracting the attention of chemists. The purpose of this Focus Review is to highlight recent developments in this area and stress the importance of further research for future applications.

  9. A Molecularly Targeted Theranostic Probe for Ovarian Cancer

    PubMed Central

    Chen, Wenxue; Bardhan, Rizia; Bartels, Marc; Perez-Torres, Carlos; Pautler, Robia G.; Halas, Naomi J.; Joshi, Amit

    2014-01-01

    Overexpression of the human epidermal growth factor receptor (HER) family has been implicated in ovarian cancer because of its participation in signaling pathway regulating cellular proliferation, differentiation, motility, and survival. Currently, effective diagnostic and therapeutic schemes are lacking for treating ovarian cancer and consequently ovarian cancer has a high mortality rate. While HER2 receptor expression does not usually affect the survival rates of ovarian cancer to the same extent as in breast cancer, it can be employed as a docking site for directed nanotherapies in cases with de novo or acquired chemotherapy resistance. In this study, we have exploited a novel gold nanoshell-based complex (nanocomplex) for targeting, dual modal imaging, and photothermal therapy of HER2 overexpressing and drug resistant ovarian cancer OVCAR3 cells in vitro. The nanocomplexes are engineered to simultaneously provide contrast as fluorescence optical imaging probe and a magnetic resonance imaging (MRI) agent. Both immunofluorescence staining and MRI successfully demonstrate that nanocomplex-anti-HER2 conjugates specifically bind to OVCAR3 cells as opposed to the control, MDA-MB-231 cells, which have low HER2 expression. In addition, nanocomplexes targeted to OVCAR3 cells, when irradiated with near infrared (NIR) laser result in selective destruction of cancer cells through photothermal ablation. We also demonstrate that NIR light therapy and the nanocomplexes by themselves are non-cytotoxic in vitro. To the best of our knowledge, this is the first demonstration of a successful integration of dual modal bioimaging with photothermal cancer therapy for treatment of ovarian cancer. Based on their efficacy in vitro, these nanocomplexes are highly promising for image guided photo-thermal therapy of ovarian cancer as well as other HER2 overexpressing cancers. PMID:20371708

  10. Molecular detection of bacterial pathogens using microparticle enhanced double-stranded DNA probes.

    PubMed

    Riahi, Reza; Mach, Kathleen E; Mohan, Ruchika; Liao, Joseph C; Wong, Pak Kin

    2011-08-15

    Rapid, specific, and sensitive detection of bacterial pathogens is essential toward clinical management of infectious diseases. Traditional approaches for pathogen detection, however, often require time-intensive bacterial culture and amplification procedures. Herein, a microparticle enhanced double-stranded DNA probe is demonstrated for rapid species-specific detection of bacterial 16S rRNA. In this molecular assay, the binding of the target sequence to the fluorophore conjugated probe thermodynamically displaces the quencher probe and allows the fluorophore to fluoresce. By incorporation of streptavidin-coated microparticles to localize the biotinylated probes, the sensitivity of the assay can be improved by 3 orders of magnitude. The limit of detection of the assay is as few as eight bacteria without target amplification and is highly specific against other common pathogens. Its applicability toward clinical diagnostics is demonstrated by directly identifying bacterial pathogens in urine samples from patients with urinary tract infections.

  11. Relaxation dynamics of the Fe₈ molecular nanomagnet as probed by nuclear magnetic resonance.

    PubMed

    Carretta, S; Bianchi, A; Santini, P; Amoretti, G

    2010-05-28

    The relaxation dynamics in molecular nanomagnets can be probed by measurements of NMR 1/T(1). By modelling magnetoelastic interactions, we theoretically investigate the behaviour of the prototype Fe(8) nanomagnet. The results of our model are in agreement with AC susceptibility and recent NMR measurements.

  12. Highly sensitive detection of alkaline phosphatase using molecular beacon probes based on enzymatic polymerization.

    PubMed

    Ma, Changbei

    2012-06-01

    We have developed a new methodology for highly sensitive alkaline phosphatase assay using molecular beacon probes. No incubation step is needed to obtain a limit of detection for ALP of 2×10(-16) M. Furthermore, ALP inhibition by the inhibitor okadaic acid is shown, demonstrating the potential for high-throughput screening for inhibitors.

  13. NEAR-INFRARED DYES: Probe Development and Applications in Optical Molecular Imaging

    PubMed Central

    Nolting, Donald D.; Gore, John C.; Pham, Wellington

    2010-01-01

    The recent emergence of optical imaging has brought forth a unique challenge for chemists: development of new biocompatible dyes that fluoresce in the near-infrared (NIR) region for optimal use in biomedical applications. This review describes the synthesis of NIR dyes and the design of probes capable of noninvasively imaging molecular events in small animal models. PMID:21822405

  14. Probing the chemistry of molecular heterojunctions using thermoelectricity.

    PubMed

    Baheti, Kanhayalal; Malen, Jonathan A; Doak, Peter; Reddy, Pramod; Jang, Sung-Yeon; Tilley, T Don; Majumdar, Arun; Segalman, Rachel A

    2008-02-01

    Thermopower measurements offer an alternative transport measurement that can characterize the dominant transport orbital and is independent of the number of molecules in the junction. This method is now used to explore the effect of chemical structure on the electronic structure and charge transport. We interrogate junctions, using a modified scanning tunneling microscope break junction technique, where: (i) the 1,4-benzenedithiol (BDT) molecule has been modified by the addition of electron-withdrawing or -donating groups such as fluorine, chlorine, and methyl on the benzene ring; and (ii) the thiol end groups on BDT have been replaced by the cyanide end groups. Cyanide end groups were found to radically change transport relative to BDT such that transport is dominated by the lowest unoccupied molecular orbital in 1,4-benzenedicyanide, while substituents on BDT generated small and predictable changes in transmission. PMID:18269258

  15. Progeroid syndromes: probing the molecular basis of aging?

    PubMed Central

    Kipling, D; Faragher, R G

    1997-01-01

    A valid method of studying age related degenerative pathologies is to study human genetic diseases that appear to accelerate many, though not necessarily all, features of the aging process. Such diseases are described as progeroid syndromes because of their possible relevance to many aspects of aging and age related disease. This article describes the recent progress made at the cellular and molecular levels in understanding the pathogenesis of one of the best characterised of these disorders, Werner's syndrome. These observations are related to some of the less well characterised progeroid syndromes within the context of the cell senescence hypothesis of aging, a theory formulated to explain the aging of regenerative tissue in normal individuals. Images PMID:9497912

  16. Molecular Imaging Probes for Positron Emission Tomography and Optical Imaging of Sentinel Lymph Node and Tumor

    NASA Astrophysics Data System (ADS)

    Qin, Zhengtao

    Molecular imaging is visualizations and measurements of in vivo biological processes at the molecular or cellular level using specific imaging probes. As an emerging technology, biocompatible macromolecular or nanoparticle based targeted imaging probes have gained increasing popularities. Those complexes consist of a carrier, an imaging reporter, and a targeting ligand. The active targeting ability dramatically increases the specificity. And the multivalency effect may further reduce the dose while providing a decent signal. In this thesis, sentinel lymph node (SLN) mapping and cancer imaging are two research topics. The focus is to develop molecular imaging probes with high specificity and sensitivity, for Positron Emission Tomography (PET) and optical imaging. The objective of this thesis is to explore dextran radiopharmaceuticals and porous silicon nanoparticles based molecular imaging agents. Dextran polymers are excellent carriers to deliver imaging reporters or therapeutic agents due to its well established safety profile and oligosaccharide conjugation chemistry. There is also a wide selection of dextran polymers with different lengths. On the other hand, Silicon nanoparticles represent another class of biodegradable materials for imaging and drug delivery. The success in fluorescence lifetime imaging and enhancements of the immune activation potency was briefly discussed. Chapter 1 begins with an overview on current molecular imaging techniques and imaging probes. Chapter 2 presents a near-IR dye conjugated probe, IRDye 800CW-tilmanocept. Fluorophore density was optimized to generate the maximum brightness. It was labeled with 68Ga and 99mTc and in vivo SLN mapping was successfully performed in different animals, such as mice, rabbits, dogs and pigs. With 99mTc labeled IRDye 800CW-tilmanocept, chapter 3 introduces a two-day imaging protocol with a hand-held imager. Chapter 4 proposed a method to dual radiolabel the IRDye 800CW-tilmanocept with both 68Ga and

  17. A targeted molecular probe for colorectal cancer imaging

    NASA Astrophysics Data System (ADS)

    Attramadal, T.; Bjerke, R.; Indrevoll, B.; Moestue, S.; Rogstad, A.; Bendiksen, R.; Healey, A.; Johannesen, E.

    2008-02-01

    Colorectal cancer is a major cause of cancer death. Morbidity, mortality and healthcare costs can be reduced if the disease can be detected at an early stage. Screening is a viable approach as there is a clear link to risk factors such as age. We have developed a fluorescent contrast agent for use during colonoscopy. The agent is administered intravenously and is targeted to an early stage molecular marker for colorectal cancer. The agent consists of a targeting section comprising a peptide, and a fluorescent reporter molecule. Clinical imaging of the agent is to be performed with a far red fluorescence imaging channel (635 nm excitation/660-700 nm emission) as an adjunct to white light colonoscopy. Preclinical proof of mechanism results are presented. The compound has a K d of ~3nM. Two human xenograft tumour models were used. Tumour cells were implanted and grown subcutaneously in nude mice. Imaging using a fluorescence reflectance imaging system and quantitative biodistribution studies were performed. Substances tested include the targeted agent, and a scrambled sequence of the peptide (no binding) used as a negative control. Competition studies were also performed by co-administration of 180 times excess unlabelled peptide. Positive imaging contrast was shown in the tumours, with a clear relationship to expression levels (confirmed with quantitative biodistribution data). There was a significant difference between the positive and negative control substances, and a significant reduction in contrast in the competition experiment.

  18. Molecular probes for imaging of hypoxia in the retina.

    PubMed

    Evans, Stephanie M; Kim, Kwangho; Moore, Chauca E; Uddin, Md Imam; Capozzi, Megan E; Craft, Jason R; Sulikowski, Gary A; Jayagopal, Ashwath

    2014-11-19

    Hypoxia has been associated with retinal diseases which lead the causes of irreversible vision loss, including diabetic retinopathy, retinopathy of prematurity, and age-related macular degeneration. Therefore, technologies for imaging hypoxia in the retina are needed for early disease detection, monitoring of disease progression, and assessment of therapeutic responses in the patient. Toward this goal, we developed two hypoxia-sensitive imaging agents based on nitroimidazoles which are capable of accumulating in hypoxic cells in vivo. 2-nitroimidazole or Pimonidazole was conjugated to fluorescent dyes to yield the imaging agents HYPOX-1 and HYPOX-2. Imaging agents were characterized in cell culture and animal models of retinal vascular diseases which exhibit hypoxia. Both HYPOX-1 and -2 were capable of detecting hypoxia in cell culture models with >10:1 signal-to-noise ratios without acute toxicity. Furthermore, intraocular administration of contrast agents in mouse models of retinal hypoxia enabled ex vivo detection of hypoxic tissue. These imaging agents are a promising step toward translation of hypoxia-sensitive molecular imaging agents in preclinical animal models and patients.

  19. Molecular beacon probes of photodamage in thymine and uracil oligonucleotides.

    PubMed

    Yarasi, Soujanya; McConachie, Cheryl; Loppnow, Glen R

    2005-01-01

    Molecular beacons (MB) are becoming more common as sequence-selective detectors of nucleic acids. Although they can easily detect single-base mismatches, they have never been used to directly detect DNA or RNA damage. To measure the degree of ultraviolet (UV) light damage in oligonucleotides, we report a novel MB approach for general detection of photoproducts in UV-irradiated rU17 and dT17 oligonucleotides. With monochromatic UV light irradiation at ca 280 nm under anoxic conditions, the oligonucleotide absorption decays with a single-exponential time constant of 123+/-1 min for rU17 and with double-exponential time constants of 78+/-0.5 min (99%) and 180+/-5 min (0.05%) for dT17 oligonucleotides. Under the same conditions, the MB fluorescence decays more quickly, with single-exponential time constants of 19+/-2 and 26+/-3 min for rU17 and dT17, respectively. Similar kinetics were observed with broadband UV light irradiation of oligonucleotides. The differences in the UV damage kinetics of dT17 and rU17 and their detection by absorption and fluorescence techniques will be discussed in the context of differential instabilities introduced in the nucleic acid-MB duplex by the different photoproducts formed.

  20. Design and synthesis of fluorescent and biotin tagged probes for the study of molecular actions of FAF1 inhibitor.

    PubMed

    Yoo, Sung-eun; Yu, Changsun; Jung, SeoHee; Kim, Eunhee; Kang, Nam Sook

    2016-02-15

    To study the molecular action of ischemic Fas-mediated cell death inhibitor, we prepared fluorescent-tagged and biotin-tagged probes of the potent inhibitor, KR-33494, of ischemic cell death. We used the molecular modeling technique to find the proper position for attaching those probes with minimum interference in the binding process of probes with Fas-mediated cell death target, FAF1.

  1. Charge transport in molecular junctions: From tunneling to hopping with the probe technique

    SciTech Connect

    Kilgour, Michael; Segal, Dvira

    2015-07-14

    We demonstrate that a simple phenomenological approach can be used to simulate electronic conduction in molecular wires under thermal effects induced by the surrounding environment. This “Landauer-Büttiker’s probe technique” can properly replicate different transport mechanisms, phase coherent nonresonant tunneling, ballistic behavior, and hopping conduction. Specifically, our simulations with the probe method recover the following central characteristics of charge transfer in molecular wires: (i) the electrical conductance of short wires falls off exponentially with molecular length, a manifestation of the tunneling (superexchange) mechanism. Hopping dynamics overtakes superexchange in long wires demonstrating an ohmic-like behavior. (ii) In off-resonance situations, weak dephasing effects facilitate charge transfer, but under large dephasing, the electrical conductance is suppressed. (iii) At high enough temperatures, k{sub B}T/ϵ{sub B} > 1/25, with ϵ{sub B} as the molecular-barrier height, the current is enhanced by a thermal activation (Arrhenius) factor. However, this enhancement takes place for both coherent and incoherent electrons and it does not readily indicate on the underlying mechanism. (iv) At finite-bias, dephasing effects may impede conduction in resonant situations. We further show that memory (non-Markovian) effects can be implemented within the Landauer-Büttiker’s probe technique to model the interaction of electrons with a structured environment. Finally, we examine experimental results of electron transfer in conjugated molecular wires and show that our computational approach can reasonably reproduce reported values to provide mechanistic information.

  2. Exogenous Molecular Probes for Targeted Imaging in Cancer: Focus on Multi-modal Imaging

    PubMed Central

    Joshi, Bishnu P.; Wang, Thomas D.

    2010-01-01

    Cancer is one of the major causes of mortality and morbidity in our healthcare system. Molecular imaging is an emerging methodology for the early detection of cancer, guidance of therapy, and monitoring of response. The development of new instruments and exogenous molecular probes that can be labeled for multi-modality imaging is critical to this process. Today, molecular imaging is at a crossroad, and new targeted imaging agents are expected to broadly expand our ability to detect and manage cancer. This integrated imaging strategy will permit clinicians to not only localize lesions within the body but also to manage their therapy by visualizing the expression and activity of specific molecules. This information is expected to have a major impact on drug development and understanding of basic cancer biology. At this time, a number of molecular probes have been developed by conjugating various labels to affinity ligands for targeting in different imaging modalities. This review will describe the current status of exogenous molecular probes for optical, scintigraphic, MRI and ultrasound imaging platforms. Furthermore, we will also shed light on how these techniques can be used synergistically in multi-modal platforms and how these techniques are being employed in current research. PMID:22180839

  3. Simple and universal platform for logic gate operations based on molecular beacon probes.

    PubMed

    Park, Ki Soo; Seo, Myung Wan; Jung, Cheulhee; Lee, Joon Young; Park, Hyun Gyu

    2012-07-23

    A new platform technology is herein described with which to construct molecular logic gates by employing the hairpin-structured molecular beacon probe as a basic work unit. In this logic gate operation system, single-stranded DNA is used as the input to induce a conformational change in a molecular beacon probe through a sequence-specific interaction. The fluorescent signal resulting from the opening of the molecular beacon probe is then used as the output readout. Importantly, because the logic gates are based on DNA, thus permitting input/output homogeneity to be preserved, their wiring into multi-level circuits can be achieved by combining separately operated logic gates or by designing the DNA output of one gate as the input to the other. With this novel strategy, a complete set of two-input logic gates is successfully constructed at the molecular level, including OR, AND, XOR, INHIBIT, NOR, NAND, XNOR, and IMPLICATION. The logic gates developed herein can be reversibly operated to perform the set-reset function by applying an additional input or a removal strand. Together, these results introduce a new platform technology for logic gate operation that enables the higher-order circuits required for complex communication between various computational elements.

  4. Raman probing of molecular interactions of alginate biopolymers with cells

    NASA Astrophysics Data System (ADS)

    Chourpa, Igor; Carpentier, Philippe; Maingault, Philippe; Fetissoff, Franck; Dubois, Pierre

    2000-05-01

    The biological polymers extracted from brown algae, alginates, are novel materials in biotechnology and biomedicine. Their ability to form viscous gels is used to immobilize or encapsulate yeast, enzymes, living cells and drugs. Calcium-alginate fibers are extensively used in wound dressings since exhibit antihaemostatic and healing properties. The problem with alginate-made dressings in surgery is their slow biodegradability: if entrapped within tissues, they can induce a local cellular recruitment with an inflammatory response contemporaneous to the resorption phase. In part, this problem is a consequence of poor solubility of the calcium alginates in water. Although calcium alginate fibers can exchange calcium ions with sodium ions from the wound exudate to create a calcium/sodium alginate fibers, the residual alginates are thought to be not totally degradable in vivo. Rapid and non- destructive characterization of series of the crude alginates and calcium alginate fibers has been performed using Raman spectroscopy with near IR excitation. Study of structural organization of the polymeric chains within calcium alginate fibers have been previously reported as made by confocal Raman multispectral imaging (CRMSI) in visible. Here, the Raman approach has been used to monitor the ion exchange reactions for different types of alginates and their salts in vitro. For in vivo evaluation, histological sections of alginate-treated rat tissue have been analyzed by light microscopy and CRMSI. The in vitro Raman modeling and the histochemical mapping were a necessary precursor for application of the Raman microprobe to follow in a non-invasive way the alginate-cell molecular interactions in rat tissue.

  5. Molecularly resolved label-free sensing of single nucleobase mismatches by interfacial LNA probes

    PubMed Central

    Mishra, Sourav; Lahiri, Hiya; Banerjee, Siddhartha; Mukhopadhyay, Rupa

    2016-01-01

    So far, there has been no report on molecularly resolved discrimination of single nucleobase mismatches using surface-confined single stranded locked nucleic acid (ssLNA) probes. Herein, it is exemplified using a label-independent force-sensing approach that an optimal coverage of 12-mer ssLNA sensor probes formed onto gold(111) surface allows recognition of ssDNA targets with twice stronger force sensitivity than 12-mer ssDNA sensor probes. The force distributions are reproducible and the molecule-by-molecule force measurements are largely in agreement with ensemble on-surface melting temperature data. Importantly, the molecularly resolved detection is responsive to the presence of single nucleobase mismatches in target sequences. Since the labelling steps can be eliminated from protocol, and each force-based detection event occurs within milliseconds' time scale, the force-sensing assay is potentially capable of rapid detection. The LNA probe performance is indicative of versatility in terms of substrate choice - be it gold (for basic research and array-based applications) or silicon (for ‘lab-on-a-chip’ type devices). The nucleic acid microarray technologies could therefore be generally benefited by adopting the LNA films, in place of DNA. Since LNA is nuclease-resistant, unlike DNA, and the LNA-based assay is sensitive to single nucleobase mismatches, the possibilities for label-free in vitro rapid diagnostics based on the LNA probes may be explored. PMID:27025649

  6. Probing the molecular structure of interfacial films and crystals

    NASA Astrophysics Data System (ADS)

    Wang, Anfeng

    The properties of outside surfaces were found to play an important role in the nucleation and crystallization processes. Thus controlling the surface properties would provide an effective means for crystal engineering. Hydrophobic surface is prepared by self-assembled monolayer (SAM) formation of octadecyltrichlorosilane (OTS) on silicon surface, with the hydrophobicity adjusted by the monolayer coverage. Silicon wafer treated by RCA method is hydrophilic, so are SAMs formed by two amine-terminated organosilanes on silicon. However these three hydrophilic surfaces are unstable, due to contamination of the amine-terminated SAMs and hydrolysis of RCA treated silicon. Polymethine dyes, BDH+Cl- and BDH +ClO4-, are synthesized and characterized by UV spectra and crystal morphology. They have identical UV spectrum in dilute solutions due to the same chromophore, and J-aggregation happens at much higher concentrations. IR spectra are analyzed to monitor the crystallization process of BDH+Cl- OTS SAM surface and the crystallization process of BDH+Cl- on substrates with varying hydrophobicity was monitored by optical microscopy and compared. Due to the extreme flexibility of polysiloxane, silicone surfactants can arrange themselves at the interfaces quickly to adopt configurations with minimum free energy. Polysiloxane is hydrophobic but not oleophilic, which makes them effective emulsifiers and stabilizers in aqueous and nonaqueous media. The interaction between an AFM Si3N4 tip and a hydrophobic surface in silicone polyether (SPE) solution in the presence of ethanol was investigated by Atomic Force Microscopy (AFM) force measurement. ABA triblock type and comb-type SPE surfactants, adsorbed at the liquid-solid interface, provide steric barriers, even with significant addition of ethanol. On the contrary, conventional low-molecular weight and polymeric alkyl surfactants display no steric barrier even in the presence of moderate amount of ethanol. This unique property makes

  7. Boronate Affinity-Molecularly Imprinted Biocompatible Probe: An Alternative for Specific Glucose Monitoring.

    PubMed

    Chen, Guosheng; Qiu, Junlang; Fang, Xu'an; Xu, Jianqiao; Cai, Siying; Chen, Qing; Liu, Yan; Zhu, Fang; Ouyang, Gangfeng

    2016-08-19

    A biocompatible probe for specific glucose recognition is based on photoinitiated boronate affinity-molecular imprinted polymers (BA-MIPs). The unique pre-self-assembly between glucose and boronic acids creates glucose-specific memory cavities in the BA-MIPs coating. As a result, the binding constant toward glucose was enhanced by three orders of magnitude. The BA-MIPs probe was applied to glucose determination in serum and urine and implanted into plant tissues for low-destructive and long-term in vivo continuous glucose monitoring. PMID:27411946

  8. Picosecond Transient Photoconductivity in Functionalized Pentacene Molecular Crystals Probed by Terahertz Pulse Spectroscopy

    NASA Astrophysics Data System (ADS)

    Hegmann, F. A.; Tykwinski, R. R.; Lui, K. P.; Bullock, J. E.; Anthony, J. E.

    2002-11-01

    We have measured transient photoconductivity in functionalized pentacene molecular crystals using ultrafast optical pump-terahertz probe techniques. The single crystal samples were excited using 800nm, 100fs pulses, and the change in transmission of time-delayed, subpicosecond terahertz pulses was used to probe the photoconducting state over a temperature range from 10 to 300K. A subpicosecond rise in photoconductivity is observed, suggesting that mobile carriers are a primary photoexcitation. At times longer than 4ps, a power-law decay is observed consistent with dispersive transport.

  9. New hairpin-structured DNA probes: alternatives to classical molecular beacons

    NASA Astrophysics Data System (ADS)

    Friedrich, Achim; Habl, Gregor; Sauer, Markus; Wolfrum, Jürgen; Hoheisel, Jörg; Marmé, Nicole; Knemeyer, Jens-Peter

    2007-02-01

    In this article we report on two different classes of self-quenching hairpin-structured DNA probes that can be used as alternatives to Molecular Beacons. Compared to other hairpin-structured DNA probes, the so-called smart probes are labeled with only one extrinsic dye. The fluorescence of this dye is efficiently quenched by intrinsic guanine bases via a photo-induced electron transfer reaction in the closed hairpin. After hybridization to a target DNA, the distance between dye and the guanines is enlarged and the fluorescence is restored. The working mechanism of the second class of hairpin DNA probes is similar, but the probe oligonucleotide is labeled at both ends with an identical chromophore and thus the fluorescence of the closed hairpin is reduced due to formation of non-fluorescent dye dimers. Both types of probes are appropriate for the identification of single nucleotide polymorphisms and in combination with confocal single-molecule spectroscopy sensitivities in the picomolar range can be achieved.

  10. Bioengineered Probes for Molecular Magnetic Resonance Imaging in the Nervous System

    PubMed Central

    2012-01-01

    The development of molecular imaging probes has changed the nature of neurobiological research. Some of the most notable successes have involved the use of biological engineering techniques for the creation of fluorescent protein derivatives for optical imaging, but recent work has also led to a number of bioengineered probes for magnetic resonance imaging (MRI), the preeminent technique for noninvasive investigation of brain structure and function. Molecular MRI agents are beginning to be applied for experiments in the nervous system, where they have the potential to bridge from molecular to systems or organismic levels of analysis. Compared with canonical synthetic small molecule agents, biomolecular or semibiosynthetic MRI contrast agents offer special advantages due to their amenability to molecular engineering approaches, their properties in some cases as catalysts, and their specificity in targeting and ligand binding. Here, we discuss an expanding list of instances where biological engineering techniques have aided in the design of MRI contrast agents and reporter systems, examining both advantages and limitations of these types of probes for studies in the central nervous system. PMID:22896803

  11. Universal Molecular Scaffold for Facile Construction of Multivalent and Multimodal Imaging Probes.

    PubMed

    Gai, Yongkang; Xiang, Guangya; Ma, Xiang; Hui, Wenqi; Ouyang, Qin; Sun, Lingyi; Ding, Jiule; Sheng, Jing; Zeng, Dexing

    2016-03-16

    Multivalent and multimodal imaging probes are rapidly emerging as powerful chemical tools for visualizing various biochemical processes. Herein, we described a bifunctional chelator (BFC)-based scaffold that can be used to construct such promising probes concisely. Compared to other reported similar scaffolds, this new BFC scaffold demonstrated two major advantages: (1) significantly simplified synthesis due to the use of this new BFC that can serve as chelator and linker simultaneously; (2) highly efficient synthesis rendered by using either click chemistry and/or total solid-phase synthesis. In addition, the versatile utility of this molecular scaffold has been demonstrated by constructing several multivalent/multimodal imaging probes labeled with various radioisotopes, and the resulting radiotracers demonstrated substantially improved in vivo performance compared to the two individual monomeric counterparts.

  12. Molecular beacon probes combined with amplification by NASBA enable homogeneous, real-time detection of RNA.

    PubMed

    Leone, G; van Schijndel, H; van Gemen, B; Kramer, F R; Schoen, C D

    1998-05-01

    Molecular beacon probes can be employed in a NASBA amplicon detection system to generate a specific fluorescent signal concomitantly with amplification. A molecular beacon, designed to hybridize within the target sequence, was introduced into NASBA reactions that amplify the genomic RNA of potato leafroll virus (PLRV). During amplification, the probe anneals to the antisense RNA amplicon generated by NASBA, producing a specific fluorescent signal that can be monitored in real-time. The assay is rapid, sensitive and specific. As RNA amplification and detection can be carried out in unopened vessels, it minimizes the risk of carry-over contaminations. Robustness has been verified on real-world samples. This homogeneous assay, called AmpliDet RNA, is a significant improvement over current detection methods for NASBA amplicons and is suitable for one-tube applications ranging from high-throughput diagnostics to in vivo studies of biological activities.

  13. A robust molecular probe for Ångstrom-scale analytics in liquids.

    PubMed

    Nirmalraj, Peter; Thompson, Damien; Dimitrakopoulos, Christos; Gotsmann, Bernd; Dumcenco, Dumitru; Kis, Andras; Riel, Heike

    2016-01-01

    Traditionally, nanomaterial profiling using a single-molecule-terminated scanning probe is performed at the vacuum-solid interface often at a few Kelvin, but is not a notion immediately associated with liquid-solid interface at room temperature. Here, using a scanning tunnelling probe functionalized with a single C60 molecule stabilized in a high-density liquid, we resolve low-dimensional surface defects, atomic interfaces and capture Ångstrom-level bond-length variations in single-layer graphene and MoS2. Atom-by-atom controllable imaging contrast is demonstrated at room temperature and the electronic structure of the C60-metal probe complex within the encompassing liquid molecules is clarified using density functional theory. Our findings demonstrates that operating a robust single-molecular probe is not restricted to ultra-high vacuum and cryogenic settings. Hence the scope of high-precision analytics can be extended towards resolving sub-molecular features of organic elements and gauging ambient compatibility of emerging layered materials with atomic-scale sensitivity under experimentally less stringent conditions. PMID:27516157

  14. Advanced electric-field scanning probe lithography on molecular resist using active cantilever

    NASA Astrophysics Data System (ADS)

    Kaestner, Marcus; Aydogan, Cemal; Lipowicz, Hubert-Seweryn; Ivanov, Tzvetan; Lenk, Steve; Ahmad, Ahmad; Angelov, Tihomir; Reum, Alexander; Ishchuk, Valentyn; Atanasov, Ivaylo; Krivoshapkina, Yana; Hofer, Manuel; Holz, Mathias; Rangelow, Ivo W.

    2015-03-01

    The routine "on demand" fabrication of features smaller than 10 nm opens up new possibilities for the realization of many novel nanoelectronic, NEMS, optical and bio-nanotechnology-based devices. Based on the thermally actuated, piezoresistive cantilever technology we have developed a first prototype of a scanning probe lithography (SPL) platform able to image, inspect, align and pattern features down to single digit nano regime. The direct, mask-less patterning of molecular resists using active scanning probes represents a promising path circumventing the problems in today's radiation-based lithography. Here, we present examples of practical applications of the previously published electric field based, current-controlled scanning probe lithography on molecular glass resist calixarene by using the developed tabletop SPL system. We demonstrate the application of a step-and-repeat scanning probe lithography scheme including optical as well as AFM based alignment and navigation. In addition, sequential read-write cycle patterning combining positive and negative tone lithography is shown. We are presenting patterning over larger areas (80 x 80 μm) and feature the practical applicability of the lithographic processes.

  15. A robust molecular probe for Ångstrom-scale analytics in liquids

    NASA Astrophysics Data System (ADS)

    Nirmalraj, Peter; Thompson, Damien; Dimitrakopoulos, Christos; Gotsmann, Bernd; Dumcenco, Dumitru; Kis, Andras; Riel, Heike

    2016-08-01

    Traditionally, nanomaterial profiling using a single-molecule-terminated scanning probe is performed at the vacuum-solid interface often at a few Kelvin, but is not a notion immediately associated with liquid-solid interface at room temperature. Here, using a scanning tunnelling probe functionalized with a single C60 molecule stabilized in a high-density liquid, we resolve low-dimensional surface defects, atomic interfaces and capture Ångstrom-level bond-length variations in single-layer graphene and MoS2. Atom-by-atom controllable imaging contrast is demonstrated at room temperature and the electronic structure of the C60-metal probe complex within the encompassing liquid molecules is clarified using density functional theory. Our findings demonstrates that operating a robust single-molecular probe is not restricted to ultra-high vacuum and cryogenic settings. Hence the scope of high-precision analytics can be extended towards resolving sub-molecular features of organic elements and gauging ambient compatibility of emerging layered materials with atomic-scale sensitivity under experimentally less stringent conditions.

  16. A robust molecular probe for Ångstrom-scale analytics in liquids

    PubMed Central

    Nirmalraj, Peter; Thompson, Damien; Dimitrakopoulos, Christos; Gotsmann, Bernd; Dumcenco, Dumitru; Kis, Andras; Riel, Heike

    2016-01-01

    Traditionally, nanomaterial profiling using a single-molecule-terminated scanning probe is performed at the vacuum–solid interface often at a few Kelvin, but is not a notion immediately associated with liquid–solid interface at room temperature. Here, using a scanning tunnelling probe functionalized with a single C60 molecule stabilized in a high-density liquid, we resolve low-dimensional surface defects, atomic interfaces and capture Ångstrom-level bond-length variations in single-layer graphene and MoS2. Atom-by-atom controllable imaging contrast is demonstrated at room temperature and the electronic structure of the C60–metal probe complex within the encompassing liquid molecules is clarified using density functional theory. Our findings demonstrates that operating a robust single-molecular probe is not restricted to ultra-high vacuum and cryogenic settings. Hence the scope of high-precision analytics can be extended towards resolving sub-molecular features of organic elements and gauging ambient compatibility of emerging layered materials with atomic-scale sensitivity under experimentally less stringent conditions. PMID:27516157

  17. Fabrication of Molecular Strain Probes for Illuminating Protein-Protein Interactions.

    PubMed

    Kim, Sung-Bae; Fujii, Rika

    2016-01-01

    A unique bioluminescent imaging probe is introduced for illuminating molecular tension appended by protein-protein interactions (PPIs) of interest. A full-length luciferase is sandwiched between two proteins of interest via minimal flexible linkers. The ligand-activated PPIs append intramolecular tension to the sandwiched luciferase, boosting or dropping the enzymatic activity in a quantitative manner. This method guides construction of a new lineage of bioassays for ligand-activated PPIs. PMID:27424904

  18. Performance of Molecular Inversion Probes (MIP) in Allele CopyNumber Determination

    SciTech Connect

    Wang, Yuker; Moorhead, Martin; Karlin-Neumann, George; Wang,Nicolas J.; Ireland, James; Lin, Steven; Chen, Chunnuan; Heiser, LauraM.; Chin, Koei; Esserman, Laura; Gray, Joe W.; Spellman, Paul T.; Faham,Malek

    2007-05-14

    We have developed a new protocol for using MolecularInversion Probes (MIP) to accurately and specifically measure allele copynumber (ACN). The new protocol provides for significant improvementsincluding the reduction of input DNA (from 2?g) by more than 25 fold (to75ng total genomic DNA), higher overall precision resulting in one orderof magnitude lower false positive rate, and greater dynamic range withaccurate absolute copy number up to 60 copies.

  19. Probing ultrafast electronic and molecular dynamics with free-electron lasers

    NASA Astrophysics Data System (ADS)

    Fang, L.; Osipov, T.; Murphy, B. F.; Rudenko, A.; Rolles, D.; Petrovic, V. S.; Bostedt, C.; Bozek, J. D.; Bucksbaum, P. H.; Berrah, N.

    2014-06-01

    Molecular dynamics is an active area of research, focusing on revealing fundamental information on molecular structures and photon-molecule interaction and with broad impacts in chemical and biological sciences. Experimental investigation of molecular dynamics has been advanced by the development of new light sources and techniques, deepening our understanding of natural processes and enabling possible control and modification of chemical and biomolecular processes. Free-electron lasers (FELs) deliver unprecedented intense and short photon pulses in the vacuum ultraviolet and x-ray spectral ranges, opening a new era for the study of electronic and nuclear dynamics in molecules. This review focuses on recent molecular dynamics investigations using FELs. We present recent work concerning dynamics of molecular interaction with FELs using an intrinsic clock within a single x-ray pulse as well as using an external clock in a pump-probe scheme. We review the latest developments on correlated and coincident spectroscopy in FEL-based research and recent results revealing photo-induced interaction dynamics using these techniques. We also describe new instrumentations to conduct x-ray pump-x-ray probe experiments with spectroscopy and imaging detectors.

  20. Tuning Cellular Uptake of Molecular Probes by Rational Design of Their Assembly into Supramolecular Nanoprobes.

    PubMed

    Lock, Lye Lin; Reyes, Claudia D; Zhang, Pengcheng; Cui, Honggang

    2016-03-16

    Intracellular sensing of pathologically relevant biomolecules could provide essential information for accurate evaluation of disease staging and progression, yet the poor cellular uptake of water-soluble molecular probes limits their use as protease sensors. In other cases such as extracellular sensing, cellular uptake should be effectively inhibited. Self-assembly of molecular probes into supramolecular nanoprobes presents a potential strategy to alter their interaction mechanisms with cells to promote or reduce their cellular uptake. Here, we report on the design, synthesis, and assembly of peptide-based molecular beacons into supramolecular protease sensors of either spherical or filamentous shapes. We found that positively charged spherical nanobeacons demonstrate much higher cellular uptake efficiency than its monomeric form, thus making them most suitable for intracellular sensing of the lysosomal protease cathepsin B. Our results also suggest that assembly into filamentous nanobeacons significantly reduces their internalization by cancer cells, an important property that can be utilized for probing extracellular protease activities. These studies provide important guiding principles for rational design of supramolecular nanoprobes with tunable cellular uptake characteristics.

  1. Probing vacuum-induced coherence via magneto-optical rotation in molecular systems

    NASA Astrophysics Data System (ADS)

    Kumar, Pardeep; Deb, Bimalendu; Dasgupta, Shubhrangshu

    2016-05-01

    Vacuum-induced coherence (VIC) arises due to the quantum interference between the spontaneous emission pathways from the degenerate excited states to a common ground state. The stringent requirement for the VIC to occur is the nonorthogonality of the transition dipole matrix elements. Unlike atoms, molecules are the promising systems for exploration of VIC, as it is possible to identify the non-orthogonal transitions due to the coupling of the rotation of molecular axis with molecular electronic angular momentum. Usually, the possible signatures of VIC are obtained by manipulating the absorption of the probe field. In this paper, we show how the dispersion of the probe field can be manipulated to obtain a measurable signature of VIC. Precisely speaking, we explore a way to probe VIC in molecules by observing its influence on magneto-optical rotation (MOR). We show that VIC in the presence of a control laser and a magnetic field can lead to large enhancement in the rotation of the plane of polarization of a linearly polarized weak laser with vanishing circular dichroism. This effect can be realized in cold molecular gases. Such a large MOR angle may be used as a tool for optical magnetometry to detect weak magnetic field with large measurement sensitivity.

  2. Constitutional Dynamic Chemistry-based New Concept of Molecular Beacons for High Efficient Development of Fluorescent Probes.

    PubMed

    Chang, Xingmao; Yu, Chunmeng; Wang, Gang; Fan, Jiayun; Zhang, Jianyun; Qi, Yanyu; Liu, Kaiqiang; Fang, Yu

    2015-06-01

    Inspired by the concept of constitutional dynamic chemistry, we propose a new and well-adaptable strategy for developing molecular beacon (MB)-like fluorescent probes. To demonstrate the strategy, we synthesized and used an amino group containing pyrenyl derivative of cholesterol (CP) for the construction of new fluorescent probes with EDTA and sulfuric acid. The probes as created were successfully used for n-hexane purity checking and Ba(2+)and Pb(2+)sensing, respectively.

  3. Real-time PCR detection of telomerase activity using specific molecular beacon probes.

    PubMed

    Kong, Deming; Jin, Yawei; Yin, Yuji; Mi, Huaifeng; Shen, Hanxi

    2007-06-01

    Telomerase is a potentially important biomarker and a prognostic indicator of cancer. Several techniques for assessing telomerase activity, including the telomeric repeat amplification protocol (TRAP) and its modified versions, have been developed. Of these methods, real-time quantitative TRAP (RTQ-TRAP) is considered the most promising. In this work, a novel RTQ-TRAP method is developed in which a telomeric repeats-specific molecular beacon is used. The use of the molecular beacon can improve the specificity of the RTQ-TRAP assay, making the method suitable for studying the overall processivity results and the turnover rate of telomerase. In addition, the real-time, closed-tube protocol used obviates the need for post-amplification procedures, reduces the risk of carryover contamination, and supports high throughput. Its performance in synthetic telomerase products and cell extracts suggests that the developed molecular beacon assay can further enhance the clinical utility of telomerase activity as a biomarker/indicator in cancer diagnosis and prognosis. The method also provides a novel approach to the specific detection of some particular gene sequences to which sequence-specific fluorogenic probes cannot be applied directly. Figure Real-time PCR detection of telomerase activity using specific molecular beacon probes.

  4. [The Ion Identification and Molecular Logic Gate of a Thiacalix[4]arene Fluorescent Probe].

    PubMed

    Wu, Fu-yong; Yu, Mei; Mu, Lan; Zeng, Xi; Wang, Rui-xiao; Takehiko Yamato

    2016-01-01

    A disubstituted phthalimide-based thiacalix[4] arene derivative (probe s1) was synthesized from cone 1, 3-thiacalix[4] arene and hydroxyethyl phthalimide, with benzyl appended the lower edge of thiacalix[4]-arene by triazole ring in the 2,4 position. The relative fluorescence quantum yield of probe s1 is 0.43 in CH3CN solvent. The strong fluorescence emission of probe s1 at 390 nm wavelength can be selectively quenched by Fe3+ in DMF/H2O solution. Similarly, the presence of I- also induced a significant fluorescence quenching of probe s1 at 310 nm wavelength in CH3CN solution. Spectral titration and isothermal titration calorimetry were showed that probe s1 with Fe3+ or I- both form 1 : 1 complexes, the binding constants up to 10(5) and coordinate process were spontaneous. The linear ranges of fluorescence detect Fe3+ or I- were 1.0 x 10(-7) - 1.6 x 10(-4) mol x L(-1) and 1.0 x 10(-7) - 8.5 x 10(-5) mol x L(-1), detection limits were up to 2.30 x 10(-8) mol x L(-1) and 1.17 x 10(-8) mol x L(-1), respectively. Meanwhile, take advantage of identification and coordination action, a logic circuit constructed at the molecular level by controlling two input signals of Fe3+ and F-, which causing probe s1 cycling of fluorescence emission or quenching. IR spectrum speculated that the nitrogen atoms of triazole groups are involved in the complexation with Fe3+, while the hydrogen atoms of triazole groups were complexed with I- by hydrogen bonding. PMID:27228760

  5. Design of molecular beacons: 3' couple quenchers improve fluorogenic properties of a probe in real-time PCR assay.

    PubMed

    Ryazantsev, Dmitry Y; Kvach, Maksim V; Tsybulsky, Dmitry A; Prokhorenko, Igor A; Stepanova, Irina A; Martynenko, Yury V; Gontarev, Sergey V; Shmanai, Vadim V; Zavriev, Sergey K; Korshun, Vladimir A

    2014-06-01

    Convenient preparation of fluorogenic hairpin DNA probes (molecular beacons) carrying a pair of FAM fluorophores (located close to 5'-terminus of the probe) or a pair of BHQ1 quenchers on 3'-terminus (with (BHQ1)2 or BHQ1-BHQ1 composition) is reported. These probes were used for the first time in a real-time PCR assay and showed considerable improvements in fluorogenic properties (the total fluorescence increase or signal-to-background ratio) in assay conditions vs. conventional one-FAM-one-BHQ1 molecular beacon probes as well as vs. hydrolyzable one-FAM-one-BHQ1 TaqMan probes. At the same time, such multiple modifications of the probe do not influence its Cq (a fractional PCR cycle used for quantification). The probe MB14 containing a BHQ1-BHQ1 pair showed a PCR fluorescence/background value of 9.6 which is more than two times higher than that of a regular probe MB2 (4.6). This study demonstrates prospects for the design of highly fluorogenic molecular beacon probes suitable for quantitative real-time PCR and for other potential applications (e.g. intracellular RNA detection and SNP/mutation analysis).

  6. Effect of nuclear motion on molecular high order harmonic pump probe spectroscopy.

    PubMed

    Bredtmann, Timm; Chelkowski, Szczepan; Bandrauk, André D

    2012-11-26

    We study pump-probe schemes for the real time observation of electronic motion on attosecond time scale in the molecular ion H(2)(+) and its heavier isotope T(2)(+) while these molecules dissociate on femtosecond time scale by solving numerically the non-Born-Oppenheimer time-dependent Schrödinger equation. The UV pump laser pulse prepares a coherent superposition of the three lowest lying quantum states and the time-delayed mid-infrared, intense few-femtosecond probe pulse subsequently generates molecular high-order harmonics (MHOHG) from this coherent electron-nuclear wavepacket (CENWP). Varying the pump-probe time delay by a few hundreds of attoseconds, the MHOHG signal intensity is shown to vary by orders of magnitude. Due to nuclear movement, the coherence of these two upper states and the ground state is lost after a few femtoseconds and the MHOHG intensity variations as function of pump-probe delay time are shown to be equal to the period of electron oscillation in the coherent superposition of the two upper dissociative quantum states. Although this electron oscillation period and hence the periodicity of the harmonic spectra are quite constant over a wide range of internuclear distances, a strong signature of nuclear motion is seen in the actual shapes and ways in which these spectra change as a function of pump-probe delay time, which is illustrated by comparison of the MHOHG spectra generated by the two isotopes H(2)(+) and T(2)(+). Two different regimes corresponding roughly to internuclear distances R < 4a(0) and R > 4a(0) are identified: For R < 4a(0), the intensity of a whole range of frequencies in the plateau region is decreased by orders of magnitude when the delay time is changed by a few hundred attoseconds whereas in the cutoff region the peaks in the MHOHG spectra are red-shifted with increasing pump-probe time delay. For R > 4a(0), on the other hand, the peaks both in the cutoff and plateau region are red-shifted with increasing delay times

  7. Probing electron-phonon excitations in molecular junctions by quantum interference.

    PubMed

    Bessis, C; Della Rocca, M L; Barraud, C; Martin, P; Lacroix, J C; Markussen, T; Lafarge, P

    2016-02-11

    Electron-phonon coupling is a fundamental inelastic interaction in condensed matter and in molecules. Here we probe phonon excitations using quantum interference in electron transport occurring in short chains of anthraquinone based molecular junctions. By studying the dependence of molecular junction's conductance as a function of bias voltage and temperature, we show that inelastic scattering of electrons by phonons can be detected as features in conductance resulting from quenching of quantum interference. Our results are in agreement with density functional theory calculations and are well described by a generic two-site model in the framework of non-equilibrium Green's functions formalism. The importance of the observed inelastic contribution to the current opens up new ways for exploring coherent electron transport through molecular devices.

  8. Functional second harmonic generation microscopy probes molecular dynamics with high temporal resolution

    PubMed Central

    Förderer, Moritz; Georgiev, Tihomir; Mosqueira, Matias; Fink, Rainer H. A.; Vogel, Martin

    2016-01-01

    Second harmonic generation (SHG) microscopy is a powerful tool for label free ex vivo or in vivo imaging, widely used to investigate structure and organization of endogenous SHG emitting proteins such as myosin or collagen. Polarization resolved SHG microscopy renders supplementary information and is used to probe different molecular states. This development towards functional SHG microscopy is calling for new methods for high speed functional imaging of dynamic processes. In this work we present two approaches with linear polarized light and demonstrate high speed line scan measurements of the molecular dynamics of the motor protein myosin with a time resolution of 1 ms in mammalian muscle cells. Such a high speed functional SHG microscopy has high potential to deliver new insights into structural and temporal molecular dynamics under ex vivo or in vivo conditions. PMID:26977360

  9. Bordetella pertussis detection by spectrofluorometry using polymerase chain reaction (PCR) and a molecular beacon probe.

    PubMed

    Poddar, S K; Le, C T

    2001-06-01

    Bordetella pertussis was detected by spectrofluorometry following PCR incorporating a molecular beacon probe in the reaction. A DNA fragment from the tandem repeat sequence region (IS 481) of the genome of B. pertussis was amplified in presence of the probe complementary to an internal segment of the amplified DNA fragment. Fluorescein (FAM) and DABCYL were used as the fluorophore and quencher in the probe. The probe was characterized for its signal to noise ratio by homogeneous solution hybridization with a complementary oligonucleotide. Measurement of fluorescent signal at the emission maxima of FAM, immediately after a PCR was used to detect the B. pertussis target, with no additional steps. Presence of B. pertussis in a sample was also examined by agarose gel electrophoresis of the PCR product. A serial diluted stock of B. pertussis (ATCC strain #9797) and fourteen clinical isolates of B. pertussis were examined. The sensitivity of detection by fluorescent measurement was found to be at least in the range of 0.01-0.1 CFU per 10 microl of the sample and was equal to or better than that detected by agarose gel analysis.

  10. Probing the Physical Conditions of Dense Molecular Gas in ULIRGs with LVG modelling

    NASA Astrophysics Data System (ADS)

    Leonidaki, Ioanna; Zhang, Zhi-Yu; Greve, Thomas; Xilouris, Manolis

    2015-08-01

    The gas-rich content of Ultra Luminous Infrared Galaxies (ULIRGs) constitutes a great laboratory in characterising the physical processes occuring in molecular gas and hence probing star formation properties. In particular, molecules with large dipole moments such as CS, HCN, HCO+, which are the fuel of star formation, can reveal the physical/excitation conditions of molecular gas phases in galaxies. For that reason, we compiled the aforementioned dense gas tracers in a sample of local (U)LIRGs in order to investigate the physical properties of the gas while at the same time put constrains on their excitation conditions. The sample in use consists of 26 galaxies all observed within the framework of the Herschel Comprehensive (U)LIRG Emission Survey (HerCULES). For all galaxies, we compiled our ground-based spectral line observations as well as all available data from the literature. Using Large Velocity Gradient (LVG) radiative transfer models in these spectral lines and in a wide parameter space [n(H2), Tkin, Nmol], and combining multiple molecules and multiple excitation components, it is possible to break the degeneracy between different parameters and to probe molecular gas physical conditions ranging from the cold and low-density average states in giant molecular clouds all the way up to the state of the gas found only near their star-forming regions. We then analyse the best LVG solution ranges to match the observed SLEDs (using more than one excitation components where necessary) in order to disentangle different molecular gas phases and possibly different molecular gas heating mechanisms.

  11. Molecular recognition of α-cyclodextrin (CD) to choral amino acids based on methyl orange as a molecular probe

    NASA Astrophysics Data System (ADS)

    Yuexian, Fan; Yu, Yang; Shaomin, Shuang; Chuan, Dong

    2005-03-01

    The molecular recognition interaction of α-CD to chiral amino acids was investigated by using spectrophotometry based on methyl orange as a molecular probe. The molecular recognition ability depended on the inclusion formation constants. The molecular recognition of α-CD to aromatic amino acids was the order: DL-tryptophan > L-tryptophan > L-phenylalanine > L-tyrosine ≈ DL-β-3,4-dihydroxy-phenylalanine; whereas for aliphatic amino acids, the order was: L- iso-leucine > L-leucine ≈ L-methionine ≈ DL-mehtionine > D-leucine. The effect of temperature on the inclusion interaction was examined and the thermodynamic parameters of inclusion process, Δ G, Δ H, Δ S, were determined. The experimental results indicated that the inclusion process was an exothermic and enthalpy-driven process accompanied with a negative or minor positive entropic contribution. The inclusion interaction between α-CD and amino acids satisfied the law of enthalpy-entropy compensation. The compensation temperature was 291 K.

  12. Micro-CT molecular imaging of tumor angiogenesis using a magnetite nano-cluster probe.

    PubMed

    Liu, Ping; Li, Jing; Zhang, Chunfu; Xu, Lisa X

    2013-06-01

    Due to its high resolution, micro-CT is desirable for molecular imaging of tumor angiogenesis. However, the sensitivity of micro-CT to contrast agents is relatively low. Therefore, the purpose of this study is to develop high micro-CT sensitive molecular imaging probes for direct visualization and dynamic monitoring of tumor angiogenesis. To this end, Arg-Gly-Asp (RGD) peptides conjugated magnetite nano clusters (RGD-MNCs) were developed by assembling individual magnetite nano particles into clusters with amphiphilic (maleimide) methoxypoly(ethylene glycol)-b-poly(lactic acid) ((Mal)mPEG-PLA) copolymer and subsequently encoding RGD peptides onto the clusters for specific targeting alpha(v)beta3 integrin. The hydrodynamic size of RGD-MNCs was about 85 nm. To test its specificity, alpha(v)beta3 positive cells (H1299) were incubated with magnetite nano clusters (MNCs), RGD-MNCs or RGD-MNCs competition with free RGD peptides. Prussian Blue staining and inductively coupled plasma optical emission spectrometer (ICP-OES) measurements indicated that the cell uptake of RGD-MNCs was significantly more than that of MNCs, which could be inhibited by free RGD peptides. For detection of tumor angiogenesis, mice bearing H1299 tumors were injected intravenously with RGD-MNCs at the dose of 400 micro mol Fe/kg. Tumor angiogenic hot spots as well as individual angiogenic vessels could be clearly manifested by micro-CT imaging 12 h post injection, which was dynamically monitored with the extension of probe circulation time. Subsequent histological studies of tumor tissues verified that RGD-MNCs registered tumor angiogenic vessels. Our study demonstrated that RGD-MNC probes fabricated in this study could be used to effectively target alpha(v)beta3 integrin. Using high resolution micro-CT in combination with the probes, tumor angiogenesis could be studied dynamically.

  13. Exploring Ultrafast Molecular Dynamics using Photoelectron Spectra from UV/XUV Pump-Probe Experiments

    NASA Astrophysics Data System (ADS)

    Champenois, Elio; Cryan, James; Shivaram, Niranjan; Wright, Travis; Belkacem, Ali

    2015-05-01

    The motion of atoms in molecules can drive electron dynamics via non-adiabatic couplings. In small molecules such as Ethylene, Carbon Dioxide, and Nitrophenol, this can lead to isomerization, electronic relaxation, or other time-dependent effects following excitation from a bonding to an anti-bonding molecular orbital. To study these mechanisms, we use ultraviolet photons of various energies from a bright High Harmonic Generation source to first initiate dynamics and subsequently probe the system through ionization. We record the kinetic energy and angular distribution of the resultant photoelectrons using a Velocity Map Imaging spectrometer, allowing us to track the evolution of the electronic state.

  14. Molecular Adsorption on Nano Colloidal Particles Probed by Second Harmonic Generation

    NASA Astrophysics Data System (ADS)

    Jen, Shih-Hui; Dai, Hai-Lung

    2006-03-01

    It is shown that second-harmonic generation (SHG), detected at 90 degree angle from the fundamental beam propagation direction, can be used to probe molecular adsorption on spherical nano colloidal particles with diameter as small as 50 nm. Measurements done with the malachite green dye adsorbed on polystyrene particles with diameters ranging from 50 to 250 nm show that the SHG signal from these surface adsorbed molecules tilts toward larger scattering angles when the particle size becomes smaller. This phenomenon can be rigorously described by the nonlinear Rayleigh-Gans-Debye theory and used for measuring the density and adsorption free energy of molecules adsorbed on nanometer size colloidal particles.

  15. Bis-pyrene-labeled molecular beacon: a monomer-excimer switching probe for the detection of DNA base alteration.

    PubMed

    Yamana, Kazushige; Ohshita, Yoshikazu; Fukunaga, Yudai; Nakamura, Mitsunobu; Maruyama, Atsushi

    2008-01-01

    A new bis-pyrene-labeled oligonucleotide probe (BP-probe) has been designed for the detection of a single base mismatch in single strand (ss) DNA as a target. The sequence of BP-probe was chosen to form stem-loop structure similar to a molecular beacon (MB-probe), yielding bis-pyrene-labeled molecular beacon (BP-MB-probe). Partially double stranded (ds) BP-MB-probes were prepared by complexation with oligonucleotides whose sequences are complementary to the loop segment but not to the stem and exchangeable with the target DNA. The partially ds BP-MB-probes were shown to exhibit monomer fluorescence as major fluorescence, while the ss BP-MB-probe in the stem-loop form displays strong excimer fluorescence. The strand exchange reactions between partially ds BP-MB-probe and target ss DNA in the presence of cationic comb-type copolymer as a catalyst were monitored by the excimer fluorescence changes. The existence of a mismatched base can be determined by the slower PASE rates compared with fully matched DNA.

  16. Molecular Zipper: a fluorescent probe for real-time isothermal DNA amplification.

    PubMed

    Yi, Jizu; Zhang, Wandi; Zhang, David Y

    2006-01-01

    Rolling-circle amplification (RCA) and ramification amplification (RAM, also known as hyperbranched RCA) are isothermal nucleic acid amplification technologies that have gained a great application in in situ signal amplification, DNA and protein microarray assays, single nucleotide polymorphism detection, as well as clinical diagnosis. Real-time detection of RCA or RAM products has been a challenge because of most real-time detection systems, including Taqman and Molecular Beacon, are designed for thermal cycling-based DNA amplification technology. In the present study, we describe a novel fluorescent probe construct, termed molecular zipper, which is specially designed for quantifying target DNA by real-time monitoring RAM reactions. Our results showed that the molecular zipper has very low background fluorescence due to the strong interaction between two strands. Once it is incorporated into the RAM products its double strand region is opened by displacement, therefore, its fluorophore releases a fluorescent signal. Applying the molecular zipper in RAM assay, we were able to detect as few as 10 molecules within 90 min reaction. A linear relationship was observed between initial input of targets and threshold time (R2 = 0.985). These results indicate that molecular zipper can be applied to real-time monitoring and qualification of RAM reaction, implying an amenable method for automatic RAM-based diagnostic assays.

  17. Molecular Zipper: a fluorescent probe for real-time isothermal DNA amplification

    PubMed Central

    Yi, Jizu; Zhang, Wandi; Zhang, David Y.

    2006-01-01

    Rolling-circle amplification (RCA) and ramification amplification (RAM, also known as hyperbranched RCA) are isothermal nucleic acid amplification technologies that have gained a great application in in situ signal amplification, DNA and protein microarray assays, single nucleotide polymorphism detection, as well as clinical diagnosis. Real-time detection of RCA or RAM products has been a challenge because of most real-time detection systems, including Taqman and Molecular Beacon, are designed for thermal cycling-based DNA amplification technology. In the present study, we describe a novel fluorescent probe construct, termed molecular zipper, which is specially designed for quantifying target DNA by real-time monitoring RAM reactions. Our results showed that the molecular zipper has very low background fluorescence due to the strong interaction between two strands. Once it is incorporated into the RAM products its double strand region is opened by displacement, therefore, its fluorophore releases a fluorescent signal. Applying the molecular zipper in RAM assay, we were able to detect as few as 10 molecules within 90 min reaction. A linear relationship was observed between initial input of targets and threshold time (R2 = 0.985). These results indicate that molecular zipper can be applied to real-time monitoring and qualification of RAM reaction, implying an amenable method for automatic RAM-based diagnostic assays. PMID:16822854

  18. Molecular gas in the Galactic center region. III. Probing shocks in molecular cores

    NASA Astrophysics Data System (ADS)

    Huettemeister, S.; Dahmen, G.; Mauersberger, R.; Henkel, C.; Wilson, T. L.; Martin-Pintado, J.

    1998-06-01

    Multiline observations of C(18) O and SiO isotopomers toward 33 molecular peaks in the Galactic center region, taken at the SEST, JCMT and HHT telescopes, are presented. The C(18) O presumably traces the total H_2 column density, while the SiO traces gas affected by shocks and high temperature chemistry. The J =2-> 1 line of SiO is seen only in few regions of the Galactic disk. This line is easily detected in all Galactic center sources observed. A comparison of the strength of the rare isotopomers (29) SiO and (30) SiO to the strength of the main isotopomer (28) SiO implies that the J = 2 -> 1 transition of (28) SiO is optically thick. The (29) Si/(30) Si isotope ratio of 1.6 in the Galactic center clouds is consistent with the terrestrial value. Large Velocity Gradient models show that the dense component (n_H_2 >= 10(4) \\percc) in typical molecular cores in the Galactic center is cool (\\TKIN ~ 25 K), contrary to what is usually found in Giant Molecular Clouds in the disk, where the densest cores are the hottest. High kinetic temperatures, > 100 K, known to exist from NH_3 studies, are only present at lower gas densities of a few 10(3) cm(-3) , where SiO is highly subthermally excited. Assuming that \\CEIO\\ traces all of the molecular gas, it is found that in all cases but one, SiO emission is compatible with arising in gas at higher density that is (presently) relatively cool. The relative abundance of SiO is typically 10(-9) , but differs significantly between individual sources. It shows a dependence on the position of the source within the Galactic center region. High abundances are found in those regions for which bar potential models predict a high likelihood for cloud-cloud collisions. These results can be used to relate the amount of gas that has encountered shocks within the last ~ 10(6) years to the large scale kinematics in the inner ~ 500 pc of the Galaxy. Based on observations obtained at the Swedish-ESO Submillimeter Telescope (SEST, Project C

  19. Screening for the breast cancer gene (BRCA1) using a biochip system and molecular beacon probes immobilized on solid surfaces.

    PubMed

    Culha, Mustafa; Stokes, David L; Griffin, Guy D; Vo-Dinh, Tuan

    2004-01-01

    We describe the use of a biochip based on complementary metal oxide semiconductor (CMOS) technology for detection of specific genetic sequences using molecular beacons (MB) immobilized on solid surfaces as probes. The applicability of this miniature detection system for screening for the BRCA1 gene is evaluated using MB probes, designed especially for the BRCA1 gene. MB probes are immobilized on a zeta-probe membrane by biotin-streptavidin immobilization. Two immobilization strategies are investigated to obtain optimal assay sensitivity. The MB is immobilized by manual spotting on zeta-probe membrane surfaces with the use of a custom-made stamping system. The detection of the BRCA1 gene using an MB probe is successfully demonstrated and expands the use of the CMOS biochip for medical applications.

  20. Quantitative degenerate four-wave mixing spectroscopy: Probes for molecular species

    SciTech Connect

    Farrow, R.; Rakestraw, D.; Paul, P.; Lucht, R.; Danehy, P.; Friedman-Hill, E.; Germann, G.

    1993-12-01

    Resonant degenerate four-wave mixing (DFWM) is currently the subject of intensive investigation as a sensitive diagnostic tool for molecular species. DFWM has the advantage of generating a coherent (beam-like) signal which results in null-background detection and provides excellent immunity to background-light interference. Since multiple one-photon resonances are involved in the signal generation process, the DFWM technique can allow sensitive detection of molecules via electronic, vibrational or rotational transitions. These properties combine to make DFWM a widely applicable diagnostic technique for the probing of molecular species. The authors are conducting fundamental and applied investigations of DFWM for quantitative measurements of trace species in reacting gases. During the past year, efforts have been focussed in two areas: (1) understanding the effects of collisional processes on the DFWM signal generation process, and (2) exploring the applicability of infrared DFWM to detect polyatomic molecules via rovibrational transitions.

  1. Bifunctional colorimetric oligonucleotide probe based on a G-quadruplex DNAzyme molecular beacon.

    PubMed

    Zhang, Libing; Zhu, Jinbo; Li, Tao; Wang, Erkang

    2011-12-01

    A label-free bifunctional colorimetric oligonucleotide probe for DNA and protein detection has been developed on the basis of a novel catalytic molecular beacon consisting of two hairpin structures and a split G-quadruplex DNAzyme in the middle. The two loops of this molecular beacon consist of thrombin aptamer sequence and the complementary sequence of target DNA, which are utilized to sense single-stranded DNA and thrombin. The G-quadruplex DNAzyme can effectively catalyze the H(2)O(2)-mediated oxidation of 3,3',5,5'-tetramethylbenzidine sulfate to generate colorimetric signal. Upon addition of the target, the DNA or protein combines with one loop of the hairpin structures, and meanwhile drives the middle G-quadruplex DNAzyme to dissociate. This results in a decrease of catalytic activity, enabling the separate analysis of DNA and thrombin.

  2. Molecular dynamics in rod-like liquid crystals probed by muon spin resonance spectroscopy.

    PubMed

    McKenzie, Iain; Scheuermann, Robert; Sedlak, Kamil; Stoykov, Alexey

    2011-08-01

    Muoniated spin probes were produced by the addition of muonium (Mu) to two rod-like liquid crystals: N-(4-methoxybenzylidene)-4'-n-butylaniline (MBBA) and cholesteryl nonanoate (CN). Avoided level crossing muon spin resonance spectroscopy was used to characterize the muoniated spin probes and to probe dynamics at the molecular level. In MBBA Mu adds predominantly to the carbon of the bridging imine group and the muon and methylene proton hyperfine coupling constants (hfccs) of the resulting radical shift in the nematic phase due to the dipolar hyperfine coupling, the ordering of the molecules along the applied magnetic field and fluctuations about the local director. The amplitude of these fluctuations in in the nematic phase of MBBA is determined from the temperature dependence of the methylene proton hfcc. Mu adds to the double bond of the steroidal ring system of CN and the temperature dependence of the Δ(1) line width provides information about the amplitude of the fluctuations about the local director in the chiral nematic phase and the slow isotropic reorientation in the isotropic phase.

  3. Microfluidic means of achieving attomolar detection limits with molecular beacon probes.

    PubMed

    Puleo, Christopher M; Wang, Tza-Huei

    2009-04-21

    We used inline, micro-evaporators to concentrate and transport DNA targets to a nanoliter single molecule fluorescence detection chamber for subsequent molecular beacon probe hybridization and analysis. This use of solvent removal as a unique means of target transport in a microanalytical platform led to a greater than 5000-fold concentration enhancement and detection limits that pushed below the femtomolar barrier commonly reported using confocal fluorescence detection. This simple microliter-to-nanoliter interconnect for single molecule counting analysis resolved several common limitations, including the need for excessive fluorescent probe concentrations at low target levels and inefficiencies in direct handling of highly dilute biological samples. In this report, the hundreds of bacteria-specific DNA molecules contained in approximately 25 microliters of a 50 aM sample were shuttled to a four nanoliter detection chamber through micro-evaporation. Here, the previously undetectable targets were enhanced to the pM regime and underwent probe hybridization and highly-efficient fluorescent event analysis via microfluidic recirculation through the confocal detection volume. This use of microfluidics in a single molecule detection (SMD) platform delivered unmatched sensitivity and introduced compliment technologies that may serve to bring SMD to more widespread use in replacing conventional methodologies for detecting rare target biomolecules in both research and clinical labs.

  4. Noninvasive imaging of multiple myeloma using near infrared fluorescent molecular probe

    NASA Astrophysics Data System (ADS)

    Hathi, Deep; Zhou, Haiying; Bollerman-Nowlis, Alex; Shokeen, Monica; Akers, Walter J.

    2016-03-01

    Multiple myeloma is a plasma cell malignancy characterized by monoclonal gammopathy and osteolytic bone lesions. Multiple myeloma is most commonly diagnosed in late disease stages, presenting with pathologic fracture. Early diagnosis and monitoring of disease status may improve quality of life and long-term survival for multiple myeloma patients from what is now a devastating and fatal disease. We have developed a near-infrared targeted fluorescent molecular probe with high affinity to the α4β1 integrin receptor (VLA-4)overexpressed by a majority of multiple myeloma cells as a non-radioactive analog to PET/CT tracer currently being developed for human diagnostics. A near-infrared dye that emits about 700 nm was conjugated to a high affinity peptidomimmetic. Binding affinity and specificity for multiple myeloma cells was investigated in vitro by tissue staining and flow cytometry. After demonstration of sensitivity and specificity, preclinical optical imaging studies were performed to evaluate tumor specificity in murine subcutaneous and metastatic multiple myeloma models. The VLA-4-targeted molecular probe showed high affinity for subcutaneous MM tumor xenografts. Importantly, tumor cells specific accumulation in the bone marrow of metastatic multiple myeloma correlated with GFP signal from transfected cells. Ex vivo flow cytometry of tumor tissue and bone marrow further corroborated in vivo imaging data, demonstrating the specificity of the novel agent and potential for quantitative imaging of multiple myeloma burden in these models.

  5. NIR fluorescence lifetime sensing through a multimode fiber for intravascular molecular probing

    NASA Astrophysics Data System (ADS)

    Ingelberts, H.; Hernot, S.; Debie, P.; Lahoutte, T.; Kuijk, M.

    2016-04-01

    Coronary artery disease (CAD) contributes to millions of deaths each year. The identification of vulnerable plaques is essential to the diagnosis of CAD but is challenging. Molecular probes can improve the detection of these plaques using intravascular imaging methods. Fluorescence lifetime sensing is a safe and robust method to image these molecular probes. We present two variations of an optical system for intravascular near-infrared (NIR) fluorescence lifetime sensing through a multimode fiber. Both systems are built around a recently developed fast and efficient CMOS detector, the current-assisted photonic sampler (CAPS) that is optimized for sub-nanosecond NIR fluorescence lifetime sensing. One system mimics the optical setup of an epifluorescence microscope while the other uses a practical fiber optic coupler to separate fluorescence excitation and emission. We test both systems by measuring the lifetime of several NIR dyes in DMSO solutions and we show that these systems are capable of detecting lifetimes of solutions with concentrations down to 370 nM and this with short acquisition times. These results are compared with time-correlated single photon counting (TCSPC) measurements for reference.

  6. Real-time observation of DNA repair: 2-aminopurine as a molecular probe

    NASA Astrophysics Data System (ADS)

    Krishnan, Rajagopal; Butcher, Christina E.; Oh, Dennis H.

    2008-02-01

    Triplex forming oligos (TFOs) that target psoralen photoadducts to specific DNA sequences have generated interest as a potential agent in gene therapy. TFOs also offer an opportunity to study the mechanism of DNA repair in detail. In an effort to understand the mechanism of DNA repair at a specific DNA sequence in real-time, we have designed a plasmid containing a psoralen reaction site adjacent to a TFO binding site corresponding to a sequence within the human interstitial collagenase gene. Two 2-aminopurine residues incorporated into the purine-rich strand of the TFO binding site and located within six nucleotides of the psoralen reaction site serve as molecular probes for excision repair events involving the psoralen photoadducts on that DNA strand. In duplex DNA, the 2-aminopurine fluorescence is quenched. However, upon thermal or formamide-induced denaturation of duplex DNA to single stranded DNA, the 2-aminopurine fluorescence increases by eight fold. These results suggest that monitoring 2-aminopurine fluorescence from plasmids damaged by psoralen TFOs may be a method for measuring excision of single-stranded damaged DNA from the plasmid in cells. A fluorescence-based molecular probe to the plasmid may significantly simplify the real-time observation of DNA repair in both populations of cells as well as single cells.

  7. A low molecular weight artificial RNA of unique size with multiple probe target regions

    NASA Technical Reports Server (NTRS)

    Pitulle, C.; Dsouza, L.; Fox, G. E.

    1997-01-01

    Artificial RNAs (aRNAs) containing novel sequence segments embedded in a deletion mutant of Vibrio proteolyticus 5S rRNA have previously been shown to be expressed from a plasmid borne growth rate regulated promoter in E. coli. These aRNAs accumulate to high levels and their detection is a promising tool for studies in molecular microbial ecology and in environmental monitoring. Herein a new construct is described which illustrates the versatility of detection that is possible with aRNAs. This 3xPen aRNA construct carries a 72 nucleotide insert with three copies of a unique 17 base probe target sequence. This aRNA is 160 nucleotides in length and again accumulates to high levels in the E. coli cytoplasm without incorporating into ribosomes. The 3xPen aRNA illustrates two improvements in detection. First, by appropriate selection of insert size, we obtained an aRNA which provides a unique and hence, easily quantifiable peak, on a high resolution gel profile of low molecular weight RNAs. Second, the existence of multiple probe targets results in a nearly commensurate increase in signal when detection is by hybridization. These aRNAs are naturally amplified and carry sequence segments that are not found in known rRNA sequences. It thus may be possible to detect them directly. An experimental step involving RT-PCR or PCR amplification of the gene could therefore be avoided.

  8. 08-ERD-071 Final Report: New Molecular Probes and Catalysts for Bioenergy Research

    SciTech Connect

    Thelen, M P; Rowe, A A; Siebers, A K; Jiao, Y

    2011-03-07

    A major thrust in bioenergy research is to develop innovative methods for deconstructing plant cell wall polymers, such as cellulose and lignin, into simple monomers that can be biologically converted to ethanol and other fuels. Current techniques for monitoring a broad array of cell wall materials and specific degradation products are expensive and time consuming. To monitor various polymers and assay their breakdown products, molecular probes for detecting specific carbohydrates and lignins are urgently needed. These new probes would extend the limited biochemical techniques available, and enable realtime imaging of ultrastructural changes in plant cells. Furthermore, degradation of plant biomass could be greatly accelerated by the development of catalysts that can hydrolyze key cell wall polysaccharides and lignin. The objective of this project was to develop cheap and efficient DNA reagents (aptamers) used to detect and quantify polysaccharides, lignin, and relevant products of their breakdown. A practical goal of the research was to develop electrochemical aptamer biosensors, which could be integrated into microfluidic devices and used for high-throughput screening of enzymes or biological systems that degrade biomass. Several important model plant cell wall polymers and compounds were targeted for specific binding and purification of aptamers, which were then tested by microscopic imaging, circular dichroism, surface plasmon resonance, fluorescence anisotropy, and electrochemical biosensors. Using this approach, it was anticiated that we could provide a basis for more efficient and economically viable biofuels, and the technologies established could be used to design molecular tools that recognize targets sought in medicine or chemical and biological defense projects.

  9. Single cell molecular recognition of migrating and invading tumor cells using a targeted fluorescent probe to receptor PTPmu.

    PubMed

    Burden-Gulley, Susan M; Qutaish, Mohammed Q; Sullivant, Kristin E; Tan, Mingqian; Craig, Sonya E L; Basilion, James P; Lu, Zheng-Rong; Wilson, David L; Brady-Kalnay, Susann M

    2013-04-01

    Detection of an extracellular cleaved fragment of a cell-cell adhesion molecule represents a new paradigm in molecular recognition and imaging of tumors. We previously demonstrated that probes that recognize the cleaved extracellular domain of receptor protein tyrosine phosphatase mu (PTPmu) label human glioblastoma brain tumor sections and the main tumor mass of intracranial xenograft gliomas. In this article, we examine whether one of these probes, SBK2, can label dispersed glioma cells that are no longer connected to the main tumor mass. Live mice with highly dispersive glioma tumors were injected intravenously with the fluorescent PTPmu probe to test the ability of the probe to label the dispersive glioma cells in vivo. Analysis was performed using a unique three-dimensional (3D) cryo-imaging technique to reveal highly migratory and invasive glioma cell dispersal within the brain and the extent of colabeling by the PTPmu probe. The PTPmu probe labeled the main tumor site and dispersed cells up to 3.5 mm away. The cryo-images of tumors labeled with the PTPmu probe provide a novel, high-resolution view of molecular tumor recognition, with excellent 3D detail regarding the pathways of tumor cell migration. Our data demonstrate that the PTPmu probe recognizes distant tumor cells even in parts of the brain where the blood-brain barrier is likely intact. The PTPmu probe has potential translational significance for recognizing tumor cells to facilitate molecular imaging, a more complete tumor resection and to serve as a molecular targeting agent to deliver chemotherapeutics to the main tumor mass and distant dispersive tumor cells.

  10. Single cell molecular recognition of migrating and invading tumor cells using a targeted fluorescent probe to receptor PTPmu.

    PubMed

    Burden-Gulley, Susan M; Qutaish, Mohammed Q; Sullivant, Kristin E; Tan, Mingqian; Craig, Sonya E L; Basilion, James P; Lu, Zheng-Rong; Wilson, David L; Brady-Kalnay, Susann M

    2013-04-01

    Detection of an extracellular cleaved fragment of a cell-cell adhesion molecule represents a new paradigm in molecular recognition and imaging of tumors. We previously demonstrated that probes that recognize the cleaved extracellular domain of receptor protein tyrosine phosphatase mu (PTPmu) label human glioblastoma brain tumor sections and the main tumor mass of intracranial xenograft gliomas. In this article, we examine whether one of these probes, SBK2, can label dispersed glioma cells that are no longer connected to the main tumor mass. Live mice with highly dispersive glioma tumors were injected intravenously with the fluorescent PTPmu probe to test the ability of the probe to label the dispersive glioma cells in vivo. Analysis was performed using a unique three-dimensional (3D) cryo-imaging technique to reveal highly migratory and invasive glioma cell dispersal within the brain and the extent of colabeling by the PTPmu probe. The PTPmu probe labeled the main tumor site and dispersed cells up to 3.5 mm away. The cryo-images of tumors labeled with the PTPmu probe provide a novel, high-resolution view of molecular tumor recognition, with excellent 3D detail regarding the pathways of tumor cell migration. Our data demonstrate that the PTPmu probe recognizes distant tumor cells even in parts of the brain where the blood-brain barrier is likely intact. The PTPmu probe has potential translational significance for recognizing tumor cells to facilitate molecular imaging, a more complete tumor resection and to serve as a molecular targeting agent to deliver chemotherapeutics to the main tumor mass and distant dispersive tumor cells. PMID:22987116

  11. Rapid detection and quantitation of Bluetongue virus (BTV) using a Molecular Beacon fluorescent probe assay.

    PubMed

    Orrù, Germano; Ferrando, Maria Laura; Meloni, Mauro; Liciardi, Manuele; Savini, Giovanni; De Santis, Paola

    2006-10-01

    Bluetongue virus (BTV) is the causative agent of Bluetongue (BT) disease in ruminant livestock and occurs almost worldwide between latitudes 35 degrees S and 50 degrees N; 24 serotypes of BTV are known of which 8 circulate periodically within parts of the Mediterranean Region. A fast (about 3.5 h) and versatile diagnostic procedure able to detect and quantify BTV-RNA, has been developed using a Molecular Beacon (MB) fluorescent probe; PCR primers were designed to target 91 bp within the NS3 conserved region of the viral RNA segment 10 (S10) and bracketed the MB fluorescence probe hybridisation site. The MB fluorescent probe was used to develop two Bluetongue serogroup-specific assays: a quantitative real time reverse transcriptase polymerase chain reaction (RT-PCR) and a traditional RT-PCR. These were tested using BTV-RNAs extracted from the blood and organs of BT-affected animals, and from virus isolate suspensions. The samples included ten serotypes (BTV-1-BTV-9 and BTV-16); of these, BTV serotypes -1, -2, -4, -9 and -16 have since 1998 been involved in the extensive outbreaks of BT across the Mediterranean Region. To evaluate the specificity and sensitivity of the MB probe, all positive samples (and negative controls) were tested using the developed quantitative real time RT-PCR and traditional RT-PCR assays. The former test had a detection limit of 10(3) cDNA molecules per reaction with a log-linear quantification range of up to 10(11) (R2 = 0.98), while the latter test was able to detect 500 cDNA-BTV molecules/PCR. The results show that the MB fluorescent probe is both rapid and versatile for the laboratory diagnosis of Bluetongue and for quantifying levels of viraemia in BTV-affected animals. An "in silico" comparison of the primers and MB fluorescent probe used in this study showed that it is possible to detect all 24 serotypes of BTV.

  12. Dip-pen microarraying of molecular beacon probes on microgel thin-film substrates.

    PubMed

    Dai, Xiaoguang; Libera, Matthew

    2014-11-01

    The integration of microarray-based nucleic acid detection technologies and microfluidics is attractive, because the combination of small sample volumes, relatively short diffusion distances, and solid-phase detection enhances the development of multiplexed assays with improved sensitivity and minimal sample size. However, traditional microarray spotting methods typically create probe spot sizes of ∼50-100 μm diameter, comparable to the dimensions of many microfluidic channels. In addition, detection of hybridization events typically requires a post-hybridization labeling step. We address both issues by exploring the use of dip-pen nanolithography (DPN) to pattern linear oligonucleotides and self-reporting molecular beacon (MB) probes on streptavidin-functionalized poly(ethylene glycol) microgel thin-film substrates. In contrast to many systems involving DPN deposition, the fluorescence of the labeled probes enables their amount and spatial distribution to be characterized by optical microscopy. Their deposition rate decreases with increasing DPN dwell time, consistent with a Langmuir adsorption model, but the linear relationship between spot diameter and time(1/2) indicates that spot size is diffusion controlled. We then use DPN to pattern MB probes for the mecA and spa genes in Staphylococcus aureus as a 2-column array with 1 μm spot sizes and 5 μm spot spacings, and we use this array to differentiate targets characteristic of methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus. This duplexed self-reporting gel-tethered MB microarray not only shows high specificity but also a high signal-to-background ratio.

  13. 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

  14. Multiscale diffusion of a molecular probe in a crowded environment: a concept

    NASA Astrophysics Data System (ADS)

    Currie, Megan; Thao, Chang; Timerman, Randi; Welty, Robb; Berry, Brenden; Sheets, Erin D.; Heikal, Ahmed A.

    2015-08-01

    Living cells are crowded with macromolecules and organelles. Yet, it is not fully understood how macromolecular crowding affects the myriad of biochemical reactions, transport and the structural stability of biomolecules that are essential to cellular function and survival. These molecular processes, with or without electrostatic interactions, in living cells are therefore expected to be distinct from those carried out in test tube in dilute solutions where excluded volumes are absent. Thus there is an urgent need to understand the macromolecular crowding effects on cellular and molecular biophysics towards quantitative cell biology. In this report, we investigated how biomimetic crowding affects both the rotational and translation diffusion of a small probe (rhodamine green, RhG). For biomimetic crowding agents, we used Ficoll-70 (synthetic polymer), bovine serum albumin and ovalbumin (proteins) at various concentrations in a buffer at room temperature. As a control, we carried out similar measurements on glycerolenriched buffer as an environment with homogeneous viscosity as a function of glycerol concentration. The corresponding bulk viscosity was measured independently to test the validity of the Stokes-Einstein model of a diffusing species undergoing a random walk. For rotational diffusion (ps-ns time scale), we used time-resolved anisotropy measurements to examine potential binding of RhG as a function of the crowding agents (surface structure and size). For translational diffusion (μs-s time scale), we used fluorescence correlation spectroscopy for single-molecule fluctuation analysis. Our results allow us to examine the diffusion model of a molecular probe in crowded environments as a function of concentration, length scale, homogeneous versus heterogeneous viscosity, size and surface structures. These biomimetic crowding studies, using non-invasive fluorescence spectroscopy methods, represent an important step towards understanding cellular biophysics and

  15. Intraoperative Assessment of Breast Cancer Margins ex vivo using Aqueous Quantum Dot-Functionalized Molecular Probes

    NASA Astrophysics Data System (ADS)

    Au, Giang Hoang Thuy

    Breast cancer is increasingly diagnosed at an early stage, allowing the diseased breast to be removed only partially or breast conserving surgery (BCS). Current BCS procedures have no rapid methods during surgery to assess if the surgical margin is clear of cancer, often resulting in re-excision. The current breast cancer re-excision rate is estimated to be 15% to as high as 60%. It would be desirable if there is a rapid and reliable breast cancer margin assessment tool in the operating room to help assess if the surgical margin is clean to minimize unnecessary re-excisions. In this research, we seek to develop an intraoperative, molecular probe-based breast cancer surgical margin assessment tool using aqueous quantum dots (AQDs) coupled with cancer specific biomarkers. Quantum dots (QDs) are photoluminescent semiconductor nanoparticles that do not photobleach and are brighter than organic fluorescent dyes. Aqueous quantum dots (AQDs) such as CdSe and near infrared (NIR) CdPbS developed in Shih's lab emit light longer than 600 nm. We have examined conjugating AQDs with antibodies to cancer specific biomarkers such as Tn antigen, a cancer-associated glycan antigen for epithelial cancers. We showed that AQDs could achieve ~80% antibody conjugation efficiency, i.e., 100 times less antibodies than required by commercial, making such AQD molecular probe surgical margin evaluation economically feasible. By conjugating AQDs with anti-Tn-antigen antibody, the AQDs molecular probe exhibited 94% sensitivity and 92% specificity in identifying breast cancer against normal breast tissues as well as benign breast tumors in 480 tissue blocks from 126 patients. Furthermore, mice model and clinical human studies indicated that AQDs imaging did not interfere with the following pathological staining. More interestingly, we showed that it it possible to directly conjugate one antibody to multiple AQDs, further reduces the required amount of antibodies needed, a feat that could not be

  16. Molecular Cooling as a Probe of Star Formation: Spitzer Looking Forward to Herschel

    NASA Technical Reports Server (NTRS)

    Bergin, Edwin A.; Maret, Sebastien; Yuan, Yuan; Sonnentrucker, Paule; Green, Joel D.; Watson, Dan M.; Harwit, Martin O.; Kristensen, Lars E.; Melnick, Gary J.; Tolls, Volker; Werner, Michael W.; Willacy, Karen

    2009-01-01

    We explore here the question of how cloud physics can be more directly probed when one observes the majority of cooling emissions from molecular gas. For this purpose we use results from a recent Spitzer Space Telescope study of the young cluster of embedded objects in NGC1333. For this study we mapped the emission from eight pure H2 rotational lines, from S(0) to S(7). The H2 emission appears to be associated with the warm gas shocked by the multiple outflows present in the region. The H2 lines are found to contribute to 25 - 50% of the total outflow luminosity, and can be used to more directly ascertain the importance of star formation feedback on the natal cloud. From these lines, we determine the outflow mass loss rate and, indirectly, the stellar infall rate, the outflow momentum and the kinetic energy injected into the cloud over the embedded phase. The latter is found to exceed the binding energy of individual cores, suggesting that outflows could be the main mechanism for cores disruption. Given the recent launch of Herschel and the upcoming operational lifetime of SOFIA we discuss how studies of molecular cooling can take a step beyond understanding thermal balance to exploring the origin, receipt, and transfer of energy in atomic and molecular gas in a wide range of physical situations.

  17. Symmetric vs asymmetric PCR and molecular beacon probe in the detection of a target gene of adenovirus.

    PubMed

    Poddar, S K

    2000-02-01

    A DNA fragment (307 bp) from the conserved region of an adenovirus gene (hexon) was amplified by symmetric and by asymmetric polymerase chain reaction (PCR). Two amplifications, one in the absence other in the presence of a molecular beacon probe were conducted by both symmetric and asymmetric PCR. The probe sequence was complementary to an internal segment of the amplified fragment. The product amplified in the absence and presence of the probe was detected by agarose gel and fluorescence analysis, respectively. A symmetric PCR results in exponentially grown double stranded DNA. An asymmetric PCR generates one of the strands by linear ampIlification and a fraction of its total product as double-stranded DNA limited by the concentration ratio of the primers used. Thus asymmetric PCR provided lower intensity signal hence less sensitivity than symmetric PCR by agarose gel analysis as expected. However, signal from a beacon probe based PCR assay is generated only from the probe fraction that hybridizes successfully competing against the strand complementary to the target strand of the product generated by PCR. The symmetric PCR has so far been used for the molecular beacon based fluorescent signal detection. The present study compared the level of fluorescent signal detectable from a symmetric PCR with that from an asymmetric PCR. The fluorescent data analysis demonstrated that a significant higher level of fluorescent signal hence higher sensitivity of detection is obtainable using asymmetric PCR than symmetric PCR performed in presence of the molecular beacon probe.

  18. Probing the permeability of polyelectrolyte multilayer capsules via a molecular beacon approach.

    PubMed

    Angelatos, Alexandra S; Johnston, Angus P R; Wang, Yajun; Caruso, Frank

    2007-04-10

    Application of polyelectrolyte multilayer (PEM) capsules as vehicles for the controlled delivery of substances, such as drugs, genes, pesticides, cosmetics, and foodstuffs, requires a sound understanding of the permeability of the capsules. We report the results of a detailed investigation into probing capsule permeability via a molecular beacon (MB) approach. This method involves preparing MB-functionalized bimodal mesoporous silica (BMSMB) particles, encapsulating the BMSMB particles within the PEM film to be probed, and then incubating the encapsulated BMSMB particles with DNA target sequences of different lengths. Permeation of the DNA targets through the capsule shell causes the immobilized MBs to open due to hybridization of the DNA targets with the complementary loop region of the MBs, resulting in an increase in the MB fluorescence. The assay conditions (BMSMB particle concentration, MB loading within the BMS particles, DNA target concentration, DNA target size, pH, sodium chloride concentration) where the MB-DNA sensing process is effective were first examined. The permeability of DNA through poly(sodium 4-styrenesulfonate) (PSS)/poly(allylamine hydrochloride) (PAH) multilayer films, with and without a poly(ethyleneimine) (PEI) precursor layer, was then investigated. The permeation of the DNA targets decreases considerably as the thickness of the PEM film encapsulating the BMSMB particles increases. Furthermore, the presence of a PEI precursor layer gives rise to less permeable PSS/PAH multilayers. The diffusion coefficients calculated for the DNA targets through the PEM capsules range from 10-19 to 10-18 m2 s-1. This investigation demonstrates that the MB approach to measuring permeability is an important new tool for the characterization of PEM capsules and is expected to be applicable for probing the permeability of other systems, such as membranes, liposomes, and emulsions.

  19. Motion of Molecular Probes and Viscosity Scaling in Polyelectrolyte Solutions at Physiological Ionic Strength

    PubMed Central

    Sozanski, Krzysztof; Wisniewska, Agnieszka; Kalwarczyk, Tomasz; Sznajder, Anna; Holyst, Robert

    2016-01-01

    We investigate transport properties of model polyelectrolyte systems at physiological ionic strength (0.154 M). Covering a broad range of flow length scales—from diffusion of molecular probes to macroscopic viscous flow—we establish a single, continuous function describing the scale dependent viscosity of high-salt polyelectrolyte solutions. The data are consistent with the model developed previously for electrically neutral polymers in a good solvent. The presented approach merges the power-law scaling concepts of de Gennes with the idea of exponential length scale dependence of effective viscosity in complex liquids. The result is a simple and applicable description of transport properties of high-salt polyelectrolyte solutions at all length scales, valid for motion of single molecules as well as macroscopic flow of the complex liquid. PMID:27536866

  20. 2-Carbamido-1,3-indandione - a Fluorescent Molecular Probe and Sunscreen Candidate.

    PubMed

    Enchev, Venelin; Angelov, Ivan; Mantareva, Vanya; Markova, Nadezhda

    2015-11-01

    The present work reports theoretical and experimental studies on the photophysical properties of two tautomeric forms of 2-carbamido-1,3-indandione (CAID). By means of UV-vis, steady-state and time-dependent fluorescence spectroscopy it is shown that both enol forms, 2-(hydroxylaminomethylidene)-indan-1,3-dione and 2-carboamide-1-hydroxy-3-oxo-indan, coexist in solution. On the base of spectroscopic studies of CAID interaction with human serum albumin and DNA sequences, it was shown that the compound has potential and it is suitable for use as fluorescent molecular probe for investigation of different biomolecules. CAID shows relatively high photostability within 3 h irradiation period. Such behavior of the investigated compound supposes possibilities for using of the CAID molecule as sunscreen because of strong absorption in UVA, UVB and UVC light spectra. PMID:26342735

  1. PALS: A unique probe for the molecular organisation of biopolymer matrices

    NASA Astrophysics Data System (ADS)

    Roussenova, M.; Alam, M. A.

    2013-06-01

    This short review aims to illustrate the versatility of Positron Annihilation Lifetime Spectroscopy (PALS) when utilized for the characterization of biopolymers (e.g.: starch, fractionated maltooligomers, gelatin and cellulose derivatives) commonly used for the formulation of pharmaceutical encapsulants. By showing examples from a number of recent PALS studies, we illustrate that this technique can be used to probe the changes in thermodynamic state and molecular packing for a wide range of biopolymer matrices as a function of temperature, matrix composition and water content. This provides a basis for establishing composition-structure-property relationships for these materials, which would eventually enable the rational control of their macroscopic properties and the design of optimal encapsulating matrices and intelligent drug delivery systems.

  2. Development of Functional Fluorescent Molecular Probes for the Detection of Biological Substances

    PubMed Central

    Suzuki, Yoshio; Yokoyama, Kenji

    2015-01-01

    This review is confined to sensors that use fluorescence to transmit biochemical information. Fluorescence is, by far, the most frequently exploited phenomenon for chemical sensors and biosensors. Parameters that define the application of such sensors include intensity, decay time, anisotropy, quenching efficiency, and luminescence energy transfer. To achieve selective (bio)molecular recognition based on these fluorescence phenomena, various fluorescent elements such as small organic molecules, enzymes, antibodies, and oligonucleotides have been designed and synthesized over the past decades. This review describes the immense variety of fluorescent probes that have been designed for the recognitions of ions, small and large molecules, and their biological applications in terms of intracellular fluorescent imaging techniques. PMID:26095660

  3. Molecular beacon probes for the detection of cisplatin-induced DNA damage.

    PubMed

    Shire, Zahra J; Loppnow, Glen R

    2012-04-01

    Cisplatin (cis-diamminedichloroplatinum(II)) causes crosslinking of DNA at AG and GG sites in cellular DNA, inhibiting replication, and making it a useful anti-cancer drug. Several techniques have been used previously to detect nucleic acid damage but most of these tools are labour-intensive, time-consuming, and/or expensive. Here, we describe a sensitive, robust, and quantitative tool for detecting cisplatin-induced DNA damage by using fluorescent molecular beacon probes (MB). Our results show a decrease of fluorescence in the presence of cisplatin-induced DNA damage, confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The decrease in fluorescence upon damage scales with the number of AG and GG sites, indicating the ability of MB to quantitatively detect DNA damage by cisplatin.

  4. Synthesis and Bioconjugation of Gold Nanoparticles as Potential Molecular Probes for Light-Based Imaging Techniques

    PubMed Central

    Rayavarapu, Raja Gopal; Petersen, Wilma; Ungureanu, Constantin; Post, Janine N.; van Leeuwen, Ton G.; Manohar, Srirang

    2007-01-01

    We have synthesized and characterized gold nanoparticles (spheres and rods) with optical extinction bands within the “optical imaging window.” The intense plasmon resonant driven absorption and scattering peaks of these nanoparticles make them suitable as contrast agents for optical imaging techniques. Further, we have conjugated these gold nanoparticles to a mouse monoclonal antibody specific to HER2 overexpressing SKBR3 breast carcinoma cells. The bioconjugation protocol uses noncovalent modes of binding based on a combination of electrostatic and hydrophobic interactions of the antibody and the gold surface. We discuss various aspects of the synthesis and bioconjugation protocols and the characterization results of the functionalized nanoparticles. Some proposed applications of these potential molecular probes in the field of biomedical imaging are also discussed. PMID:18354723

  5. A screen of approved drugs and molecular probes identifies therapeutics with anti-Ebola virus activity.

    PubMed

    Johansen, Lisa M; DeWald, Lisa Evans; Shoemaker, Charles J; Hoffstrom, Benjamin G; Lear-Rooney, Calli M; Stossel, Andrea; Nelson, Elizabeth; Delos, Sue E; Simmons, James A; Grenier, Jill M; Pierce, Laura T; Pajouhesh, Hassan; Lehár, Joseph; Hensley, Lisa E; Glass, Pamela J; White, Judith M; Olinger, Gene G

    2015-06-01

    Currently, no approved therapeutics exist to treat or prevent infections induced by Ebola viruses, and recent events have demonstrated an urgent need for rapid discovery of new treatments. Repurposing approved drugs for emerging infections remains a critical resource for potential antiviral therapies. We tested ~2600 approved drugs and molecular probes in an in vitro infection assay using the type species, Zaire ebolavirus. Selective antiviral activity was found for 80 U.S. Food and Drug Administration-approved drugs spanning multiple mechanistic classes, including selective estrogen receptor modulators, antihistamines, calcium channel blockers, and antidepressants. Results using an in vivo murine Ebola virus infection model confirmed the protective ability of several drugs, such as bepridil and sertraline. Viral entry assays indicated that most of these antiviral drugs block a late stage of viral entry. By nature of their approved status, these drugs have the potential to be rapidly advanced to clinical settings and used as therapeutic countermeasures for Ebola virus infections. PMID:26041706

  6. Nanolithography by scanning probes on calixarene molecular glass resist using mix-and-match lithography

    NASA Astrophysics Data System (ADS)

    Kaestner, Marcus; Hofer, Manuel; Rangelow, Ivo W.

    2013-07-01

    Going "beyond the CMOS information-processing era," taking advantage of quantum effects occurring at sub-10-nm level, requires novel device concepts and associated fabrication technologies able to produce promising features at acceptable cost levels. Herein, the challenge affecting the lithographic technologies comprises the marriage of down-scaling the device-relevant feature size towards single-nanometer resolution with a simultaneous increase of the throughput capabilities. Mix-and-match lithographic strategies are one promising path to break through this trade-off. Proof-of-concept combining electron beam lithography (EBL) with the outstanding capabilities of closed-loop electric field current-controlled scanning probe nanolithography (SPL) is demonstrated. This combination, whereby also extreme ultraviolet lithography (EUVL) is possible instead of EBL, enables more: improved patterning resolution and reproducibility in combination with excellent overlay and placement accuracy. Furthermore, the symbiosis between EBL (EUVL) and SPL expands the process window of EBL (EUVL) beyond the state of the art, allowing SPL-based pre- and post-patterning of EBL (EUVL) written features at critical dimension levels with scanning probe microscopy-based pattern overlay alignment capability. Moreover, we are able to modify the EBL (EUVL) pattern even after the development step. The ultra-high resolution mix-and-match lithography experiments are performed on the molecular glass resist calixarene using a Gaussian e-beam lithography system operating at 10 keV and a home-developed SPL setup.

  7. Benzene Probes in Molecular Dynamics Simulations Reveal Novel Binding Sites for Ligand Design.

    PubMed

    Tan, Yaw Sing; Reeks, Judith; Brown, Christopher J; Thean, Dawn; Ferrer Gago, Fernando Jose; Yuen, Tsz Ying; Goh, Eunice Tze Leng; Lee, Xue Er Cheryl; Jennings, Claire E; Joseph, Thomas L; Lakshminarayanan, Rajamani; Lane, David P; Noble, Martin E M; Verma, Chandra S

    2016-09-01

    Protein flexibility poses a major challenge in binding site identification. Several computational pocket detection methods that utilize small-molecule probes in molecular dynamics (MD) simulations have been developed to address this issue. Although they have proven hugely successful at reproducing experimental structural data, their ability to predict new binding sites that are yet to be identified and characterized has not been demonstrated. Here, we report the use of benzenes as probe molecules in ligand-mapping MD (LMMD) simulations to predict the existence of two novel binding sites on the surface of the oncoprotein MDM2. One of them was serendipitously confirmed by biophysical assays and X-ray crystallography to be important for the binding of a new family of hydrocarbon stapled peptides that were specifically designed to target the other putative site. These results highlight the predictive power of LMMD and suggest that predictions derived from LMMD simulations can serve as a reliable basis for the identification of novel ligand binding sites in structure-based drug design. PMID:27532490

  8. [Study on molecular hybridization with biotin-labelled HPV 16 DNA probe in human cervical carcinoma].

    PubMed

    Li, Y; Huang, G Q; Mao, T; Huang, Y F; Xiao, H Y; Liu, B L

    1989-09-01

    Biotin-labelled human papillomavirus (HPV) 16 type DNA probe was prepared by the techniques of molecular biology. And dot hybridization technique was used to detect the HPV 16 homologous sequences in the tissues DNA of human cervical carcinoma. The results indicated that 16 cases out of 28 of the human cervical carcinoma tissues were positive. The positive rate was 57%. The other 4 cases of normal uterine cervix tissues were negative. Only 1 in 4 chronic cervicitis tissues showed positive. The HPV 16 plasmid DNA, as the positive control group, showed strong positive, while lambda-phage DNA was negative. The results have shown that the genome of the HPV actually exists in the tissue of the cervical carcinoma and that there is a close relationship between the cervical carcinoma and HPV infection. This experiment adopted the Biotin-labelled HPV 16 DNA probe. And it may provide us with a quick and sensitive method for investigation of the infection of HPV and its role in the carcinogenesis of cervical carcinoma. PMID:2560458

  9. Rupture mechanism of aromatic systems from graphite probed with molecular dynamics simulations.

    PubMed

    Leng, Yumin; Chen, Jian; Zhou, Beifei; Gräter, Frauke

    2010-07-01

    Intermolecular interactions involving aromatic rings are of pivotal importance in many areas of chemistry, biology and materials science. Mimicking recent atomic force microscopy (AFM) experiments that measured the adhesion forces of single pi-pi complexes, here interactions between pyrene/coronene and graphite have been probed by force-probe molecular dynamics (FPMD) simulations. The pyrene or coronene molecule was connected to a virtual spring through a flexible poly(ethylene glycol) (PEG) linker and was pulled away from graphite in water under constant velocity. Pyrene and coronene showed similar unbinding pathways featuring four states, with a transition and an intermediate state connecting the bound and unbound states in terms of distance and interplanar angles. Transient conformations with tilted orientations (approximately 40 degrees) and with one side of the aromatic structure still in contact with the graphite surface (approximately 70 degrees) were identified as the transition and intermediate states, respectively, similar to previously observed perpendicularly stacked benzene dimers. The distance to transition state x(tr) was determined to be 0.23 +/- 0.03 nm both for pyrene/graphite and coronene/graphite. The complexes share similar unbinding pathways, but coronene binds to graphite more strongly than to pyrene.

  10. Probing Solvation Dynamics around Aromatic and Biological Molecules at the Single-Molecular Level.

    PubMed

    Dopfer, Otto; Fujii, Masaaki

    2016-05-11

    Solvation processes play a crucial role in chemical reactions and biomolecular recognition phenomena. Although solvation dynamics of interfacial or biological water has been studied extensively in aqueous solution, the results are generally averaged over several solvation layers and the motion of individual solvent molecules is difficult to capture. This review describes the development and application of a new experimental approach, namely, picosecond time-resolved pump-probe infrared spectroscopy of size- and isomer-selected aromatic clusters, in which for the first time the dynamics of a single individual solvent molecule can be followed in real time. The intermolecular isomerization reaction is triggered by resonant photoionization (pump), and infrared photodissociation (probe) at variable delay generates the spectroscopic signature of salient properties of the reaction, including rates, yields, pathways, branching ratios of competing reactions, existence of reaction intermediates, occurrence of back reactions, and time scales of energy relaxation processes. It is shown that this relevant information can reliably be decoded from the experimental spectra by sophisticated molecular dynamics simulations. This review covers a description of the experimental strategies and spectroscopic methods along with all applications to date, which range from aromatic clusters with nonpolar solvent molecules to aromatic monohydrated biomolecules. PMID:27054835

  11. Characterization of TCP-1 probes for molecular imaging of colon cancer.

    PubMed

    Liu, Zhonglin; Gray, Brian D; Barber, Christy; Bernas, Michael; Cai, Minying; Furenlid, Lars R; Rouse, Andrew; Patel, Charmi; Banerjee, Bhaskar; Liang, Rongguang; Gmitro, Arthur F; Witte, Marlys H; Pak, Koon Y; Woolfenden, James M

    2016-10-10

    Molecular probes capable of detecting colorectal cancer (CRC) are needed for early CRC diagnosis. The objective of this study was to characterize c[CTPSPFSHC]OH (TCP-1), a small peptide derived from phage display selection, for targeting human CRC xenografts using technetium-99m ((99m)Tc)-labeled TCP-1 and fluorescent cyanine-7 (Cy7)-labeled form of the peptide (Cy7-TCP-1). (99m)Tc-TCP-1 was generated by modifying TCP-1 with succinimidyl-6-hydrazino-nicotinamide (S-HYNIC) followed by radiolabeling. In vitro saturation binding experiments were performed for (99m)Tc-TCP-1 in human HCT116 colon cancer cells. SCID mice with human HCT116 cancer xenografts were imaged with (99m)Tc-TCP-1 or control peptide using a small-animal SPECT imager: Group I (n=5) received no blockade; Group II (n=5) received a blocking dose of non-radiolabeled TCP-1. Group III (n=5) were imaged with (99m)Tc-labeled control peptide (inactive peptide). SCID mice with human PC3 prostate cancer xenografts (Group IV, n=5) were also imaged with (99m)Tc-TCP-1. Eight additional SCID mice bearing HCT116 xenografts in dorsal skinfold window chambers (DSWC) were imaged by direct positron imaging of (18)F-fluorodeoxyglucose ((18)F-FDG) and fluorescence microscopy of Cy7-TCP-1. In vitro(99m)Tc-HYNIC-TCP-1 binding assays on HCT 116 cells indicated a mean Kd of 3.04±0.52nM. In cancer xenografts, (99m)Tc-TCP-1 radioactivity (%ID/g) was 1.01±0.15 in the absence of blockade and was reduced to 0.26±0.04 (P<0.01) with blockade. No radioactive uptake was observed in the PC3 tumors with (99m)Tc-TCP-1 or HCT116 tumors with inactive peptide. Cy7-TCP-1 activity localized not only in metabolically active tumors, as defined by (18)F-FDG imaging, but also in peritumoral microvasculature. In conclusion, TCP-1 probes may have a distinct targeting mechanism with high selectivity for CRC and tumor-associated vasculature. Molecular imaging with TCP-1 probes appears promising to detect malignant colorectal lesions. PMID

  12. Targeted next-generation sequencing for the detection of ciprofloxacin resistance markers using molecular inversion probes

    PubMed Central

    Stefan, Christopher P.; Koehler, Jeffrey W.; Minogue, Timothy D.

    2016-01-01

    Antibiotic resistance (AR) is an epidemic of increasing magnitude requiring rapid identification and profiling for appropriate and timely therapeutic measures and containment strategies. In this context, ciprofloxacin is part of the first-line of countermeasures against numerous high consequence bacteria. Significant resistance can occur via single nucleotide polymorphisms (SNP) and deletions within ciprofloxacin targeted genes. Ideally, use of ciprofloxacin would be prefaced with AR determination to avoid overuse or misuse of the antibiotic. Here, we describe the development and evaluation of a panel of 44 single-stranded molecular inversion probes (MIPs) coupled to next-generation sequencing (NGS) for the detection of genetic variants known to confer ciprofloxacin resistance in Bacillus anthracis, Yersinia pestis, and Francisella tularensis. Sequencing results demonstrate MIPs capture and amplify targeted regions of interest at significant levels of coverage. Depending on the genetic variant, limits of detection (LOD) for high-throughput pooled sequencing ranged from approximately 300–1800 input genome copies. LODs increased 10-fold in the presence of contaminating human genome DNA. In addition, we show that MIPs can be used as an enrichment step with high resolution melt (HRM) real-time PCR which is a sensitive assay with a rapid time-to-answer. Overall, this technology is a multiplexable upfront enrichment applicable with multiple downstream molecular assays for the detection of targeted genetic regions. PMID:27174456

  13. Probing the molecular design of hyper-branched aryl polyesters towards lubricant applications

    DOE PAGESBeta

    Robinson, Joshua W.; Zhou, Yan; Bhattacharya, Priyanka; Erck, Robert; Qu, Jun; Bays, J. Timothy; Cosimbescu, Lelia

    2016-01-05

    We report novel polymeric materials that may be used as viscosity index improvers (VII) for lubricant applications. Our efforts included probing the comb-burst hyper-branched aryl polyester architecture for beneficial viscosity and friction behavior when utilized as an additive in a group I oil. The monomer was designed as to undergo polymerization via polycondensation within the architectural construct (AB2), typical of hyperbranched polymers. The monomer design was comprised of aliphatic arms (12 or 16 methylenes) to provide the necessary lipophilicity to achieve solubility in a non-polar medium. Once polymerized, via catalyst and heat, the surface alcohols were functionalized with fatty acidsmore » (lauric and palmitic). Controlling the aliphatic nature of the internal arms and peripheral end-groups provided four unique flexible polymer designs. Changing the reaction time and concentration provided opportunities to investigate the influence of molecular weight and branching density on oil-solubility, viscosity, and friction. Oil-solubility was found to decrease with fewer internal carbons, but the number of internal carbons appears to have little influence on the bulk solution viscosity. Increased branching and degree of polymerization, and thus molecular weight, were found to reduce the solubility of these systems in the base oil. At concentrations of 2 wt % in a group I base oil, these polymer additives demonstrated improved viscosity index and reduced friction coefficient, validating the basic approach.« less

  14. Molecular probes based on microstructured fibers and surface enhanced Raman scattering

    NASA Astrophysics Data System (ADS)

    Zhang, Yi; Shi, Chao; Gu, Claire; Seballos, Leo; Schwartzberg, Adam; Zhang, Jin Z.; Chen, Bin

    2007-09-01

    In recent years, there has been significant interest in using surface enhanced Raman scattering (SERS) and optical fibers for chemical, biological, and environmental detections. The combination of SERS and optical fibers offers the advantages of the molecular specificity of Raman scattering, huge enhancement factor of SERS, and flexibility of optical fibers. In this paper, we report our work on the development of fiber biosensors based on SERS emphasizing on recent progress in the fabrication of photonic crystal fiber (PCF) SERS sensors for highly sensitive molecular detection. To increase the sensitivity, one needs to increase either the excitation laser power or the amount of analyte molecules in the active region of the sensor. The high excitation intensity is not desirable for biosensors due to the low damage threshold of live tissues or bio-molecules. In our investigation of various fiber configurations, hollow core (HC) PCFs show the greatest advantages over all other types of fiber probes because of the large contact area. The hollow core nature allows the analytes and SERS substrate to fill the inner surface of the air channels. In addition, by sealing the cladding holes of the HCPCF, only the central hole will be open and filled with liquid samples. As both the light and the sample are confined in the fiber core, the sensitivity is significantly improved. The newly developed liquid core PCF sensor was tested in the detection of rhodamine 6G (R6G), human insulin, and tryptophan with good sensitivity due to the enhanced interaction volume.

  15. Fluorescence detection of adenosine triphosphate through an aptamer-molecular beacon multiple probe.

    PubMed

    Zeng, Xiaodan; Zhang, Xiaoling; Yang, Wen; Jia, Hongying; Li, Yamin

    2012-05-01

    An aptamer-molecular beacon (MB) multiple fluorescent probe for adenosine triphosphate (ATP) assay is proposed in this article. The ATP aptamer was used as a molecular recognition part, and an oligonucleotide (short strand, SS) partially complementary with the aptamer and an MB was used as the other part. In the presence of ATP, the aptamer bound with it, accompanied by the hybridization of MB and SS and the fluorescence recovering. Wherever there is only very weak fluorescence can be measured in the absence of ATP. Based on the relationship of recovering fluorescence and the concentration of ATP, a method for quantifying ATP has been developed. The fluorescence intensity was proportional to the concentration of ATP in the range of 10 to 500 nM with a detection limit of 0.1 nM. Moreover, this method was able to detect ATP with high selectivity in the presence of guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP). This method is proved to be simple with high sensitivity, selectivity, and specificity.

  16. Single-cell detection of mRNA expression using nanofountain-probe electroporated molecular beacons.

    PubMed

    Giraldo-Vela, Juan P; Kang, Wonmo; McNaughton, Rebecca L; Zhang, Xuemei; Wile, Brian M; Tsourkas, Andrew; Bao, Gang; Espinosa, Horacio D

    2015-05-01

    New techniques for single-cell analysis enable new discoveries in gene expression and systems biology. Time-dependent measurements on individual cells are necessary, yet the common single-cell analysis techniques used today require lysing the cell, suspending the cell, or long incubation times for transfection, thereby interfering with the ability to track an individual cell over time. Here a method for detecting mRNA expression in live single cells using molecular beacons that are transfected into single cells by means of nanofountain probe electroporation (NFP-E) is presented. Molecular beacons are oligonucleotides that emit fluorescence upon binding to an mRNA target, rendering them useful for spatial and temporal studies of live cells. The NFP-E is used to transfect a DNA-based beacon that detects glyceraldehyde 3-phosphate dehydrogenase and an RNA-based beacon that detects a sequence cloned in the green fluorescence protein mRNA. It is shown that imaging analysis of transfection and mRNA detection can be performed within seconds after electroporation and without disturbing adhered cells. In addition, it is shown that time-dependent detection of mRNA expression is feasible by transfecting the same single cell at different time points. This technique will be particularly useful for studies of cell differentiation, where several measurements of mRNA expression are required over time.

  17. Targeted next-generation sequencing for the detection of ciprofloxacin resistance markers using molecular inversion probes.

    PubMed

    Stefan, Christopher P; Koehler, Jeffrey W; Minogue, Timothy D

    2016-01-01

    Antibiotic resistance (AR) is an epidemic of increasing magnitude requiring rapid identification and profiling for appropriate and timely therapeutic measures and containment strategies. In this context, ciprofloxacin is part of the first-line of countermeasures against numerous high consequence bacteria. Significant resistance can occur via single nucleotide polymorphisms (SNP) and deletions within ciprofloxacin targeted genes. Ideally, use of ciprofloxacin would be prefaced with AR determination to avoid overuse or misuse of the antibiotic. Here, we describe the development and evaluation of a panel of 44 single-stranded molecular inversion probes (MIPs) coupled to next-generation sequencing (NGS) for the detection of genetic variants known to confer ciprofloxacin resistance in Bacillus anthracis, Yersinia pestis, and Francisella tularensis. Sequencing results demonstrate MIPs capture and amplify targeted regions of interest at significant levels of coverage. Depending on the genetic variant, limits of detection (LOD) for high-throughput pooled sequencing ranged from approximately 300-1800 input genome copies. LODs increased 10-fold in the presence of contaminating human genome DNA. In addition, we show that MIPs can be used as an enrichment step with high resolution melt (HRM) real-time PCR which is a sensitive assay with a rapid time-to-answer. Overall, this technology is a multiplexable upfront enrichment applicable with multiple downstream molecular assays for the detection of targeted genetic regions. PMID:27174456

  18. Biomarkers and Molecular Probes for Cell Death Imaging and Targeted Therapeutics

    PubMed Central

    Smith, Bryan A.; Smith, Bradley D.

    2012-01-01

    Cell death is a critically important biological process. Disruption of homeostasis, either by excessive or deficient cell death, is a hallmark of many pathological conditions. Recent research advances have greatly increased our molecular understanding of cell death and its role in a range of diseases and therapeutic treatments. Central to these ongoing research and clinical efforts is the need for imaging technologies that can locate and identify cell death in a wide array of in vitro and in vivo biomedical samples with varied spatiotemporal requirements. This review article summarizes community efforts over the past five years to identify useful biomarkers for dead and dying cells, and to develop molecular probes that target these biomarkers for optical, radionuclear, or magnetic resonance imaging. Apoptosis biomarkers are classified as either intracellular (caspase enzymes, mitochondrial membrane potential, cytosolic proteins) or extracellular (plasma membrane phospholipids, membrane potential, surface exposed histones). Necrosis, autophagy, and senescence biomarkers are described, as well as unexplored cell death biomarkers. The article discusses possible chemotherapeutic and theranostic strategies, and concludes with a summary of current challenges and expected eventual rewards of clinical cell death imaging. PMID:22989049

  19. Rational chemical design of the next generation of molecular imaging probes based on physics and biology: mixing modalities, colors and signals

    PubMed Central

    Longmire, Michelle R.; Ogawa, Mikako; Choyke, Peter L.

    2012-01-01

    In recent years, numerous in vivo molecular imaging probes have been developed. As a consequence, much has been published on the design and synthesis of molecular imaging probes focusing on each modality, each type of material, or each target disease. More recently, second generation molecular imaging probes with unique, multi-functional, or multiplexed characteristics have been designed. This critical review focuses on (i) molecular imaging using combinations of modalities and signals that employ the full range of the electromagnetic spectra, (ii) optimized chemical design of molecular imaging probes for in vivo kinetics based on biology and physiology across a range of physical sizes, (iii) practical examples of second generation molecular imaging probes designed to extract complementary data from targets using multiple modalities, color, and comprehensive signals (277 references). PMID:21607237

  20. Rational chemical design of the next generation of molecular imaging probes based on physics and biology: mixing modalities, colors and signals.

    PubMed

    Kobayashi, Hisataka; Longmire, Michelle R; Ogawa, Mikako; Choyke, Peter L

    2011-09-01

    In recent years, numerous in vivo molecular imaging probes have been developed. As a consequence, much has been published on the design and synthesis of molecular imaging probes focusing on each modality, each type of material, or each target disease. More recently, second generation molecular imaging probes with unique, multi-functional, or multiplexed characteristics have been designed. This critical review focuses on (i) molecular imaging using combinations of modalities and signals that employ the full range of the electromagnetic spectra, (ii) optimized chemical design of molecular imaging probes for in vivo kinetics based on biology and physiology across a range of physical sizes, (iii) practical examples of second generation molecular imaging probes designed to extract complementary data from targets using multiple modalities, color, and comprehensive signals (277 references).

  1. A New Probe of the Molecular Gas Content in Galaxies: Application to M101

    NASA Technical Reports Server (NTRS)

    Smith, Denise A.; Allen, Ronald J.; Bohlin, Ralph C.; Nicholson, Natalya; Stecher, Theodore P.

    1999-01-01

    Recent studies of nearby spiral galaxies suggest that photodissoiation regions (PDRS) are capable of producing much of the observed HI in galaxy disks. In that case, measurements of the observed HI column density and the far-ultraviolet (FUV) photon flux responsible for the photodissociation process provide a new probe of the volume density of the local underlying molecular hydrogen. We develop the method and apply it to the giant Scd spiral M101. The HI column density and amount of FUV emission have been measured for a sample of 35 candidate PDRs located throughout the disk of M101 using the Very Large Array and the Ultraviolet Imaging Telescope. We find that, after correction for the known gradient of metallicity in the Interstellar Medium (ISM) of M101 and for the extinction of the ultraviolet emission, molecular gas with a narrow range of density from 30-1000/ cubic cm is found near star-forming regions at all radii in the disk of M101 out to a distance of 12 seconds approximately equals 26 kpc, close to the photometric limit of R(sub 25) approximately equals 13.5 seconds. In this picture, the ISM is virtually all molecular in the inner parts of M101. The strong decrease of the HI column density in the inner disk of the galaxy at R(sub G) < 10 kpc is a consequence of a strong increase in the dust-to-gas ratio there, resulting in an increase of the H(sub 2) formation rate on grains and a corresponding disappearance of hydrogen in its atomic form.

  2. pH-Induced Modulation of One- and Two-Photon Absorption Properties in a Naphthalene-Based Molecular Probe.

    PubMed

    Murugan, N Arul; Kongsted, Jacob; Ågren, Hans

    2013-08-13

    Presently, there is a great demand for small probe molecules that can be used for two-photon excitation microscopy (TPM)-based monitoring of intracellular and intraorganelle activity and pH. The candidate molecules should ideally possess a large two-photon absorption cross section with optical properties sensitive to pH changes. In the present work, we investigate the potential of a methoxy napthalene (MONAP) derivative for its suitability to serve as a pH sensor using TPM. Using an integrated approach rooted in hybrid quantum mechanics/molecular mechanics, the structures, dynamics, and the one- and two-photon properties of the probe in dimethylformamide solvent are studied. It is found that the protonated form is responsible for the optical property of MONAP at moderately low pH, for which the calculated pH-induced red shift is in good agreement with experiments. A 2-fold increase in the two-photon absorption cross section in the IR region of the spectrum is predicted for the moderately low pH form of the probe, suggesting that this can be a potential probe for pH monitoring of living cells. We also propose some design principles aimed at obtaining control of the absorption spectral range of the probe by structural tuning. Our work indicates that the integrated approach employed is capable of capturing the pH-induced changes in structure and optical properties of organic molecular probes and that such in silico tools can be used to draw structure-property relationships to design novel molecular probes suitable for a specific application.

  3. High quality copy number and genotype data from FFPE samples using Molecular Inversion Probe (MIP) microarrays

    SciTech Connect

    Wang, Yuker; Carlton, Victoria E.H.; Karlin-Neumann, George; Sapolsky, Ronald; Zhang, Li; Moorhead, Martin; Wang, Zhigang C.; Richardson, Andrea L.; Warren, Robert; Walther, Axel; Bondy, Melissa; Sahin, Aysegul; Krahe, Ralf; Tuna, Musaffe; Thompson, Patricia A.; Spellman, Paul T.; Gray, Joe W.; Mills, Gordon B.; Faham, Malek

    2009-02-24

    A major challenge facing DNA copy number (CN) studies of tumors is that most banked samples with extensive clinical follow-up information are Formalin-Fixed Paraffin Embedded (FFPE). DNA from FFPE samples generally underperforms or suffers high failure rates compared to fresh frozen samples because of DNA degradation and cross-linking during FFPE fixation and processing. As FFPE protocols may vary widely between labs and samples may be stored for decades at room temperature, an ideal FFPE CN technology should work on diverse sample sets. Molecular Inversion Probe (MIP) technology has been applied successfully to obtain high quality CN and genotype data from cell line and frozen tumor DNA. Since the MIP probes require only a small ({approx}40 bp) target binding site, we reasoned they may be well suited to assess degraded FFPE DNA. We assessed CN with a MIP panel of 50,000 markers in 93 FFPE tumor samples from 7 diverse collections. For 38 FFPE samples from three collections we were also able to asses CN in matched fresh frozen tumor tissue. Using an input of 37 ng genomic DNA, we generated high quality CN data with MIP technology in 88% of FFPE samples from seven diverse collections. When matched fresh frozen tissue was available, the performance of FFPE DNA was comparable to that of DNA obtained from matched frozen tumor (genotype concordance averaged 99.9%), with only a modest loss in performance in FFPE. MIP technology can be used to generate high quality CN and genotype data in FFPE as well as fresh frozen samples.

  4. Continuous sensing of tumor-targeted molecular probes with a vertical cavity surface emitting laser-based biosensor

    PubMed Central

    Parashurama, Natesh; O’Sullivan, Thomas D.; De La Zerda, Adam; El Kalassi, Pascale; Cho, Seongjae; Liu, Hongguang; Teed, Robert; Levy, Hart; Rosenberg, Jarrett; Cheng, Zhen; Levi, Ofer; Harris, James S.

    2012-01-01

    Abstract. Molecular optical imaging is a widespread technique for interrogating molecular events in living subjects. However, current approaches preclude long-term, continuous measurements in awake, mobile subjects, a strategy crucial in several medical conditions. Consequently, we designed a novel, lightweight miniature biosensor for in vivo continuous optical sensing. The biosensor contains an enclosed vertical-cavity surface-emitting semiconductor laser and an adjacent pair of near-infrared optically filtered detectors. We employed two sensors (dual sensing) to simultaneously interrogate normal and diseased tumor sites. Having established the sensors are precise with phantom and in vivo studies, we performed dual, continuous sensing in tumor (human glioblastoma cells) bearing mice using the targeted molecular probe cRGD-Cy5.5, which targets αVβ3 cell surface integrins in both tumor neovasculature and tumor. The sensors capture the dynamic time-activity curve of the targeted molecular probe. The average tumor to background ratio after signal calibration for cRGD-Cy5.5 injection is approximately 2.43±0.95 at 1 h and 3.64±1.38 at 2 h (N=5 mice), consistent with data obtained with a cooled charge coupled device camera. We conclude that our novel, portable, precise biosensor can be used to evaluate both kinetics and steady state levels of molecular probes in various disease applications. PMID:23123976

  5. Molecular probes and microarrays for the detection of toxic algae in the genera Dinophysis and Phalacroma (Dinophyta).

    PubMed

    Edvardsen, Bente; Dittami, Simon M; Groben, René; Brubak, Sissel; Escalera, Laura; Rodríguez, Francisco; Reguera, Beatriz; Chen, Jixin; Medlin, Linda K

    2013-10-01

    Dinophysis and Phalacroma species containing diarrheic shellfish toxins and pectenotoxins occur in coastal temperate waters all year round and prevent the harvesting of mussels during several months each year in regions in Europe, Chile, Japan, and New Zealand. Toxicity varies among morphologically similar species, and a precise identification is needed for early warning systems. Molecular techniques using ribosomal DNA sequences offer a means to identify and detect precisely the potentially toxic species. We designed molecular probes targeting the 18S rDNA at the family and genus levels for Dinophysis and Phalacroma and at the species level for Dinophysis acuminata, Dinophysis acuta, and Dinophysis norvegica, the most commonly occurring, potentially toxic species of these genera in Western European waters. Dot blot hybridizations with polymerase chain reaction (PCR)-amplified rDNA from 17 microalgae were used to demonstrate probe specificity. The probes were modified along with other published fluorescence in situ hybridization and PCR probes and tested for a microarray platform within the MIDTAL project ( http://www.midtal.com ). The microarray was applied to field samples from Norway and Spain and compared to microscopic cell counts. These probes may be useful for early warning systems and monitoring and can also be used in population dynamic studies to distinguish species and life cycle stages, such as cysts, and their distribution in time and space. PMID:23263760

  6. Library synthesis, screening, and discovery of modified Zinc(II)-Bis(dipicolylamine) probe for enhanced molecular imaging of cell death.

    PubMed

    Plaunt, Adam J; Harmatys, Kara M; Wolter, William R; Suckow, Mark A; Smith, Bradley D

    2014-04-16

    Zinc(II)-bis(dipicolylamine) (Zn-BDPA) coordination complexes selectively target the surfaces of dead and dying mammalian cells, and they have promise as molecular probes for imaging cell death. A necessary step toward eventual clinical imaging applications is the development of next-generation Zn-BDPA complexes with enhanced affinity for the cell death membrane biomarker, phosphatidylserine (PS). This study employed an iterative cycle of library synthesis and screening, using a novel rapid equilibrium dialysis assay, to discover a modified Zn-BDPA structure with high and selective affinity for vesicles containing PS. The lead structure was converted into a deep-red fluorescent probe and its targeting and imaging performance was compared with an unmodified control Zn-BDPA probe. The evaluation process included a series of FRET-based vesicle titration studies, cell microscopy experiments, and rat tumor biodistribution measurements. In all cases, the modified probe exhibited comparatively higher affinity and selectivity for the target membranes of dead and dying cells. The results show that this next-generation deep-red fluorescent Zn-BDPA probe is well suited for preclinical molecular imaging of cell death in cell cultures and animal models. Furthermore, it should be possible to substitute the deep-red fluorophore with alternative reporter groups that enable clinically useful, deep-tissue imaging modalities, such as MRI and nuclear imaging.

  7. Molecular Inversion Probes for targeted resequencing in non-model organisms.

    PubMed

    Niedzicka, M; Fijarczyk, A; Dudek, K; Stuglik, M; Babik, W

    2016-01-01

    Applications that require resequencing of hundreds or thousands of predefined genomic regions in numerous samples are common in studies of non-model organisms. However few approaches at the scale intermediate between multiplex PCR and sequence capture methods are available. Here we explored the utility of Molecular Inversion Probes (MIPs) for the medium-scale targeted resequencing in a non-model system. Markers targeting 112 bp of exonic sequence were designed from transcriptome of Lissotriton newts. We assessed performance of 248 MIP markers in a sample of 85 individuals. Among the 234 (94.4%) successfully amplified markers 80% had median coverage within one order of magnitude, indicating relatively uniform performance; coverage uniformity across individuals was also high. In the analysis of polymorphism and segregation within family, 77% of 248 tested MIPs were confirmed as single copy Mendelian markers. Genotyping concordance assessed using replicate samples exceeded 99%. MIP markers for targeted resequencing have a number of advantages: high specificity, high multiplexing level, low sample requirement, straightforward laboratory protocol, no need for preparation of genomic libraries and no ascertainment bias. We conclude that MIP markers provide an effective solution for resequencing targets of tens or hundreds of kb in any organism and in a large number of samples. PMID:27046329

  8. Probing the molecular design of hyper-branched aryl polyesters towards lubricant applications

    NASA Astrophysics Data System (ADS)

    Robinson, Joshua W.; Zhou, Yan; Bhattacharya, Priyanka; Erck, Robert; Qu, Jun; Bays, J. Timothy; Cosimbescu, Lelia

    2016-01-01

    We report novel polymeric materials that may be used as viscosity index improvers (VII) for lubricant applications. Our efforts included probing the comb-burst hyper-branched aryl polyester architecture for beneficial viscosity and friction behavior when utilized as an additive in a group I oil. The monomer was designed as to undergo polymerization via polycondensation within the architectural construct (AB2), typical of hyperbranched polymers. The monomer design was comprised of aliphatic arms (12 or 16 methylenes) to provide the necessary lipophilicity to achieve solubility in a non-polar medium. Once polymerized, via catalyst and heat, the surface alcohols were functionalized with fatty acids (lauric and palmitic). Controlling the aliphatic nature of the internal arms and peripheral end-groups provided four unique flexible polymer designs. Changing the reaction time and concentration provided opportunities to investigate the influence of molecular weight and branching density on oil-solubility, viscosity, and friction. Oil-solubility was found to decrease with fewer internal carbons, but the number of internal carbons appears to have little influence on the bulk solution viscosity. At concentrations of 2 wt % in a group I base oil, these polymer additives demonstrated an improved viscosity index and reduced friction coefficient, validating the basic approach.

  9. Molecular Inversion Probes for targeted resequencing in non-model organisms

    PubMed Central

    Niedzicka, M.; Fijarczyk, A.; Dudek, K.; Stuglik, M.; Babik, W.

    2016-01-01

    Applications that require resequencing of hundreds or thousands of predefined genomic regions in numerous samples are common in studies of non-model organisms. However few approaches at the scale intermediate between multiplex PCR and sequence capture methods are available. Here we explored the utility of Molecular Inversion Probes (MIPs) for the medium-scale targeted resequencing in a non-model system. Markers targeting 112 bp of exonic sequence were designed from transcriptome of Lissotriton newts. We assessed performance of 248 MIP markers in a sample of 85 individuals. Among the 234 (94.4%) successfully amplified markers 80% had median coverage within one order of magnitude, indicating relatively uniform performance; coverage uniformity across individuals was also high. In the analysis of polymorphism and segregation within family, 77% of 248 tested MIPs were confirmed as single copy Mendelian markers. Genotyping concordance assessed using replicate samples exceeded 99%. MIP markers for targeted resequencing have a number of advantages: high specificity, high multiplexing level, low sample requirement, straightforward laboratory protocol, no need for preparation of genomic libraries and no ascertainment bias. We conclude that MIP markers provide an effective solution for resequencing targets of tens or hundreds of kb in any organism and in a large number of samples. PMID:27046329

  10. Determination of viscoelastic properties by analysis of probe-particle motion in molecular simulations

    NASA Astrophysics Data System (ADS)

    Karim, Mir; Kohale, Swapnil C.; Indei, Tsutomu; Schieber, Jay D.; Khare, Rajesh

    2012-11-01

    We present a technique for the determination of viscoelastic properties of a medium by tracking the motion of an embedded probe particle by using molecular dynamics simulations. The approach involves the analysis of the simulated particle motion by continuum theory; it is shown to work in both passive and active modes. We demonstrate that, for passive rheology, an analysis based on the generalized Stokes-Einstein relationship is not adequate to obtain the values of the viscoelastic moduli over the frequency range studied. For both passive and active modes, it is necessary to account for the medium and particle inertia when analyzing the particle motion. For a polymer melt system consisting of short chains, the values calculated from the proposed approach are in good quantitative agreement with previous literature results that were obtained using completely different simulation approaches. The proposed particle rheology simulation technique is general and could provide insight into the characterization of the mechanical properties in biological systems, such as cellular environments and polymeric systems, such as thin films and nanocomposites that exhibit spatial variation in properties over the nanoscale.

  11. Probing the molecular design of hyper-branched aryl polyesters towards lubricant applications

    PubMed Central

    Robinson, Joshua W.; Zhou, Yan; Bhattacharya, Priyanka; Erck, Robert; Qu, Jun; Bays, J. Timothy; Cosimbescu, Lelia

    2016-01-01

    We report novel polymeric materials that may be used as viscosity index improvers (VII) for lubricant applications. Our efforts included probing the comb-burst hyper-branched aryl polyester architecture for beneficial viscosity and friction behavior when utilized as an additive in a group I oil. The monomer was designed as to undergo polymerization via polycondensation within the architectural construct (AB2), typical of hyperbranched polymers. The monomer design was comprised of aliphatic arms (12 or 16 methylenes) to provide the necessary lipophilicity to achieve solubility in a non-polar medium. Once polymerized, via catalyst and heat, the surface alcohols were functionalized with fatty acids (lauric and palmitic). Controlling the aliphatic nature of the internal arms and peripheral end-groups provided four unique flexible polymer designs. Changing the reaction time and concentration provided opportunities to investigate the influence of molecular weight and branching density on oil-solubility, viscosity, and friction. Oil-solubility was found to decrease with fewer internal carbons, but the number of internal carbons appears to have little influence on the bulk solution viscosity. At concentrations of 2 wt % in a group I base oil, these polymer additives demonstrated an improved viscosity index and reduced friction coefficient, validating the basic approach. PMID:26727881

  12. Molecular ions and protonated molecules observed in the atmospheric solids analysis probe analysis of steroids.

    PubMed

    Ray, Andrew D; Hammond, Janet; Major, Hilary

    2010-01-01

    Atmospheric pressure chemical ionisation (APCI) has often been used to ionise steroids in mass spectrometry, usually when interfaced to high-performance liquid chromatography (HPLC). However, in positive ion mode, a dehydrated protonated molecule is often observed with a loss of structural information. The recently introduced technique of atmospheric solids analysis probe (ASAP) has the advantage that the sample can be analysed directly and does not need to be interfaced to HPLC. Existing ionisation sources such as direct analysis in real time (DART) and desorption electrospray ionisation (DESI) have shown the advantage of direct analysis techniques in a variety of applications. ASAP can be performed on commercial atmospheric pressure ionisation (API) mass spectrometers with only simple modifications to API sources. The samples are vaporised by hot nitrogen gas from the electrospray desolvation heater and ionised by a corona discharge. A range of commercially available steroids were analysed by ASAP to investigate the mechanism of ionisation. ASAP analysis of steroids generally results in the formation of the parent molecular ion as either the radical cation M+* or the protonated molecule MH+. The formation of the protonated molecule is a result of proton transfer from ionised water clusters in the source. However, if the source is dry, then formation of the radical cation is the primary ionisation mechanism.

  13. Detection and Quantitation of Heavy Metal Ions on Bona Fide DVDs Using DNA Molecular Beacon Probes.

    PubMed

    Zhang, Lingling; Wong, Jessica X H; Li, Xiaochun; Li, Yunchao; Yu, Hua-Zhong

    2015-01-01

    A sensitive and cost-effective method for the simultaneous quantitation of trace amounts of Hg(2+) and Pb(2+) in real-world samples has been developed using DNA molecular beacon probes bound to bona fide digital video discs (DVDs). With specially designed T-rich or G-rich loop sequences, the detection is based on the strong T-Hg(2+)-T coordination chemistry of Hg(2+) and the formation of G-quadruplexes induced by Pb(2+), respectively. In particular, the presence of metal cations leads to hairpin opening and exposure of the terminal biotin moiety for binding nanogold-streptavidin conjugates. The recognition signal was subsequently enhanced by gold nanoparticle-promoted silver deposition, which leads to quantifiable digital signals upon reading with a standard computer drive. This method exhibits a wide response range and low detection limits for both Hg(2+) and Pb(2+). In addition, the quantitative determination of heavy metals in food products (e.g., rice samples) has been demonstrated and the method compares favorably with other optical sensors developed recently.

  14. Probing the molecular design of hyper-branched aryl polyesters towards lubricant applications.

    PubMed

    Robinson, Joshua W; Zhou, Yan; Bhattacharya, Priyanka; Erck, Robert; Qu, Jun; Bays, J Timothy; Cosimbescu, Lelia

    2016-01-01

    We report novel polymeric materials that may be used as viscosity index improvers (VII) for lubricant applications. Our efforts included probing the comb-burst hyper-branched aryl polyester architecture for beneficial viscosity and friction behavior when utilized as an additive in a group I oil. The monomer was designed as to undergo polymerization via polycondensation within the architectural construct (AB2), typical of hyperbranched polymers. The monomer design was comprised of aliphatic arms (12 or 16 methylenes) to provide the necessary lipophilicity to achieve solubility in a non-polar medium. Once polymerized, via catalyst and heat, the surface alcohols were functionalized with fatty acids (lauric and palmitic). Controlling the aliphatic nature of the internal arms and peripheral end-groups provided four unique flexible polymer designs. Changing the reaction time and concentration provided opportunities to investigate the influence of molecular weight and branching density on oil-solubility, viscosity, and friction. Oil-solubility was found to decrease with fewer internal carbons, but the number of internal carbons appears to have little influence on the bulk solution viscosity. At concentrations of 2 wt % in a group I base oil, these polymer additives demonstrated an improved viscosity index and reduced friction coefficient, validating the basic approach.

  15. Low-dose laulimalide represents a novel molecular probe for investigating microtubule organization

    PubMed Central

    Bennett, Melissa J.; Chan, Gordon K.; Rattner, J.B.; Schriemer, David C.

    2012-01-01

    Laulimalide is a natural product that has strong taxoid-like properties but binds to a distinct site on β-tubulin in the microtubule (MT) lattice. At elevated concentrations, it generates MTs that are resistant to depolymerization, and it induces a conformational state indistinguishable from taxoid-treated MTs. In this study, we describe the effect of low-dose laulimalide on various stages of the cell cycle and compare these effects to docetaxel as a representative of taxoid stabilizers. No evidence of MT bundling in interphase was observed with laulimalide, in spite of the fact that MTs are stabilized at low dose. Cells treated with laulimalide enter mitosis but arrest at prometaphase by generating multiple asters that coalesce into supernumerary poles and interfere with the integrity of the metaphase plate. Cells with a preformed bipolar spindle exist under heightened tension under laulimalide treatment, and chromosomes rapidly shear from the plate, even though the bipolar spindle is well-preserved. Docetaxel generates a similar phenotype for HeLa cells entering mitosis, but when treated at metaphase, cells undergo chromosomal fragmentation and demonstrate reduced centromere dynamics, as expected for a taxoid. Our results suggest that laulimalide represents a new class of molecular probe for investigating MT-mediated events, such as kinetochore-MT interactions, which may reflect the location of the ligand binding site within the interprotofilament groove. PMID:22871740

  16. Low-dose laulimalide represents a novel molecular probe for investigating microtubule organization.

    PubMed

    Bennett, Melissa J; Chan, Gordon K; Rattner, J B; Schriemer, David C

    2012-08-15

    Laulimalide is a natural product that has strong taxoid-like properties but binds to a distinct site on β-tubulin in the microtubule (MT) lattice. At elevated concentrations, it generates MTs that are resistant to depolymerization, and it induces a conformational state indistinguishable from taxoid-treated MTs. In this study, we describe the effect of low-dose laulimalide on various stages of the cell cycle and compare these effects to docetaxel as a representative of taxoid stabilizers. No evidence of MT bundling in interphase was observed with laulimalide, in spite of the fact that MTs are stabilized at low dose. Cells treated with laulimalide enter mitosis but arrest at prometaphase by generating multiple asters that coalesce into supernumerary poles and interfere with the integrity of the metaphase plate. Cells with a preformed bipolar spindle exist under heightened tension under laulimalide treatment, and chromosomes rapidly shear from the plate, even though the bipolar spindle is well-preserved. Docetaxel generates a similar phenotype for HeLa cells entering mitosis, but when treated at metaphase, cells undergo chromosomal fragmentation and demonstrate reduced centromere dynamics, as expected for a taxoid. Our results suggest that laulimalide represents a new class of molecular probe for investigating MT-mediated events, such as kinetochore-MT interactions, which may reflect the location of the ligand binding site within the interprotofilament groove. PMID:22871740

  17. Probing the molecular design of hyper-branched aryl polyesters towards lubricant applications.

    PubMed

    Robinson, Joshua W; Zhou, Yan; Bhattacharya, Priyanka; Erck, Robert; Qu, Jun; Bays, J Timothy; Cosimbescu, Lelia

    2016-01-01

    We report novel polymeric materials that may be used as viscosity index improvers (VII) for lubricant applications. Our efforts included probing the comb-burst hyper-branched aryl polyester architecture for beneficial viscosity and friction behavior when utilized as an additive in a group I oil. The monomer was designed as to undergo polymerization via polycondensation within the architectural construct (AB2), typical of hyperbranched polymers. The monomer design was comprised of aliphatic arms (12 or 16 methylenes) to provide the necessary lipophilicity to achieve solubility in a non-polar medium. Once polymerized, via catalyst and heat, the surface alcohols were functionalized with fatty acids (lauric and palmitic). Controlling the aliphatic nature of the internal arms and peripheral end-groups provided four unique flexible polymer designs. Changing the reaction time and concentration provided opportunities to investigate the influence of molecular weight and branching density on oil-solubility, viscosity, and friction. Oil-solubility was found to decrease with fewer internal carbons, but the number of internal carbons appears to have little influence on the bulk solution viscosity. At concentrations of 2 wt % in a group I base oil, these polymer additives demonstrated an improved viscosity index and reduced friction coefficient, validating the basic approach. PMID:26727881

  18. Molecular engineering of a TBET-based two-photon fluorescent probe for ratiometric imaging of living cells and tissues.

    PubMed

    Zhou, Liyi; Zhang, Xiaobing; Wang, Qianqian; Lv, Yifan; Mao, Guojiang; Luo, Aili; Wu, Yongxiang; Wu, Yuan; Zhang, Jing; Tan, Weihong

    2014-07-16

    In contrast to one-photon microscopy, two-photon probe-based fluorescent imaging can provide improved three-dimensional spatial localization and increased imaging depth. Consequently, it has become one of the most attractive techniques for studying biological events in living cells and tissues. However, the quantitation of these probes is primarily based on single-emission intensity change, which tends to be affected by a variety of environmental factors. Ratiometric probes, on the other hand, can eliminate these interferences by the built-in correction of the dual emission bands, resulting in a more favorable system for imaging living cells and tissues. Herein, for the first time, we adopted a through-bond energy transfer (TBET) strategy to design and synthesize a small molecular ratiometric two-photon fluorescent probe for imaging living cells and tissues in real time. Specifically, a two-photon fluorophore (D-π-A-structured naphthalene derivative) and a rhodamine B fluorophore are directly connected by electronically conjugated bond to form a TBET probe, or Np-Rh, which shows a target-modulated ratiometric two-photon fluorescence response with highly efficient energy transfer (93.7%) and two well-resolved emission peaks separated by 100 nm. This novel probe was then applied for two-photon imaging of living cells and tissues and showed high ratiometric imaging resolution and deep-tissue imaging depth of 180 μm, thus demonstrating its practical application in biological systems.

  19. Complementary optical and nuclear imaging of caspase-3 activity using combined activatable and radio-labeled multimodality molecular probe

    NASA Astrophysics Data System (ADS)

    Lee, Hyeran; Akers, Walter J.; Cheney, Philip P.; Edwards, W. Barry; Liang, Kexian; Culver, Joseph P.; Achilefu, Samuel

    2009-07-01

    Based on the capability of modulating fluorescence intensity by specific molecular events, we report a new multimodal optical-nuclear molecular probe with complementary reporting strategies. The molecular probe (LS498) consists of tetraazacyclododecanetetraacetic acid (DOTA) for chelating a radionuclide, a near-infrared fluorescent dye, and an efficient quencher dye. The two dyes are separated by a cleavable peptide substrate for caspase-3, a diagnostic enzyme that is upregulated in dying cells. LS498 is radiolabeled with 64Cu, a radionuclide used in positron emission tomography. In the native form, LS498 fluorescence is quenched until caspase-3 cleavage of the peptide substrate. Enzyme kinetics assay shows that LS498 is readily cleaved by caspase-3, with excellent enzyme kinetic parameters kcat and KM of 0.55+/-0.01 s-1 and 1.12+/-0.06 μM, respectively. In mice, the initial fluorescence of LS498 is ten-fold less than control. Using radiolabeled 64Cu-LS498 in a controlled and localized in-vivo model of caspase-3 activation, a time-dependent five-fold NIR fluorescence enhancement is observed, but radioactivity remains identical in caspase-3 positive and negative controls. These results demonstrate the feasibility of using radionuclide imaging for localizing and quantifying the distribution of molecular probes and optical imaging for reporting the functional status of diagnostic enzymes.

  20. Molecular imaging probes spy on the body's inner workings: miniaturized microscopes, microbubbles, 7- and 15-T scanners, diffusion-tensor MRI, and other molecular-imaging technologies are pushing molecular imaging into the future.

    PubMed

    Mertz, Leslie

    2013-01-01

    Molecular imaging is one of the hot-button areas within medical imaging. This technology employs imaging techniques in concert with molecular probes, or biomarkers, that together noninvasively spy on cellular function and molecular processes. In some cases, this technology may be able to detect the very earliest stages of diseases and eliminate them on the spot. This paper discusses how miniaturized microscopes, microbubbles, 7T and 15T scanners, diffusion-tensor MRI and other molecular imaging technologies are pushing molecular imaging into the future.

  1. Real-space imaging of molecular structure and chemical bonding by single-molecule inelastic tunneling probe.

    PubMed

    Chiang, Chi-lun; Xu, Chen; Han, Zhumin; Ho, W

    2014-05-23

    The arrangement of atoms and bonds in a molecule influences its physical and chemical properties. The scanning tunneling microscope can provide electronic and vibrational signatures of single molecules. However, these signatures do not relate simply to the molecular structure and bonding. We constructed an inelastic tunneling probe based on the scanning tunneling microscope to sense the local potential energy landscape of an adsorbed molecule with a carbon monoxide (CO)-terminated tip. The skeletal structure and bonding of the molecule are revealed from imaging the spatial variations of a CO vibration as the CO-terminated tip probes the core of the interactions between adjacent atoms. An application of the inelastic tunneling probe reveals the sharing of hydrogen atoms among multiple centers in intramolecular and extramolecular bonding.

  2. Immobilization of ɛ-polylysine onto the probe surface for molecular adsorption type endotoxin detection system

    NASA Astrophysics Data System (ADS)

    Ooe, Katsutoshi; Tsuji, Akihito; Nishishita, Naoki; Hirano, Yoshiaki

    2007-04-01

    adsorption reaction between ɛ-polylysine and endotoxin. ɛ-polylysine has the structure of straight chain molecule composed by 25-30 residues made by lysine, and it is used as an antimicrobial agent, moreover, cellulose beads with immobilized ɛ-polylysine is used as the barrier filter for endotoxin removal. Therefore, it is expected that the endotoxin be adsorbed to the immobilized ɛ-polylysine onto the probe. As the result of this reaction, the mass of the probe is increased, and endotoxin can be detected by using of Quartz Crystal Microbalance (QCM). In our previous research, we have already acquired the proteins immobilization technique onto Au and Si surface. In this report, the proposal of molecular adsorption type endotoxin detection system, and the immobilization of ɛ-polylysine onto the probe are described. We use X-ray Photoelectron Spectroscopy (XPS) to confirm the ɛ-polylysine immobilization, and the adsorptive activity of immobilized ɛ-polylysine is measured by XPS and AFM. The purpose of this study is to bring about the realization of "Real-time endotoxin detection system".

  3. Application of a miniature biochip using the molecular beacon probe in breast cancer gene BRCA1 detection.

    PubMed

    Culha, Mustafa; Stokes, David L; Griffin, Guy D; Vo-Dinh, Tuan

    2004-04-15

    We report for the first time the application of a biochip using the molecular beacon (MB) detection scheme. The usability of this biochip novel detection system for the analysis of the breast cancer gene BRCA1 is demonstrated using molecular beacon probes. The MB is designed for the BRCA1 gene and a miniature biochip system is used for detection. The performance of the biochip-MB detection system is evaluated. The optimum conditions for the MB system for highest fluorescence detection sensitivity are investigated for the detection system. The detection of BRCA1 gene is successfully demonstrated in solution and the limit of detection (LOD) is estimated as 70 nM.

  4. Probing the conformation of FhaC with small-angle neutron scattering and molecular modeling.

    PubMed

    Gabel, Frank; Lensink, Marc F; Clantin, Bernard; Jacob-Dubuisson, Françoise; Villeret, Vincent; Ebel, Christine

    2014-07-01

    Probing the solution structure of membrane proteins represents a formidable challenge, particularly when using small-angle scattering. Detergent molecules often present residual scattering contributions even at their match point in small-angle neutron scattering (SANS) measurements. Here, we studied the conformation of FhaC, the outer-membrane, β-barrel transporter of the Bordetella pertussis filamentous hemagglutinin adhesin. SANS measurements were performed on homogeneous solutions of FhaC solubilized in n-octyl-d17-βD-glucoside and on a variant devoid of the α helix H1, which critically obstructs the FhaC pore, in two solvent conditions corresponding to the match points of the protein and the detergent, respectively. Protein-bound detergent amounted to 142 ± 10 mol/mol as determined by analytical ultracentrifugation. By using molecular modeling and starting from three distinct conformations of FhaC and its variant embedded in lipid bilayers, we generated ensembles of protein-detergent arrangement models with 120-160 detergent molecules. The scattered curves were back-calculated for each model and compared with experimental data. Good fits were obtained for relatively compact, connected detergent belts, which occasionally displayed small detergent-free patches on the outer surface of the β barrel. The combination of SANS and modeling clearly enabled us to infer the solution structure of FhaC, with H1 inside the pore as in the crystal structure. We believe that our strategy of combining explicit atomic detergent modeling with SANS measurements has significant potential for structural studies of other detergent-solubilized membrane proteins. PMID:24988353

  5. Probing the Conformation of FhaC with Small-Angle Neutron Scattering and Molecular Modeling

    PubMed Central

    Gabel, Frank; Lensink, Marc F.; Clantin, Bernard; Jacob-Dubuisson, Françoise; Villeret, Vincent; Ebel, Christine

    2014-01-01

    Probing the solution structure of membrane proteins represents a formidable challenge, particularly when using small-angle scattering. Detergent molecules often present residual scattering contributions even at their match point in small-angle neutron scattering (SANS) measurements. Here, we studied the conformation of FhaC, the outer-membrane, β-barrel transporter of the Bordetella pertussis filamentous hemagglutinin adhesin. SANS measurements were performed on homogeneous solutions of FhaC solubilized in n-octyl-d17-βD-glucoside and on a variant devoid of the α helix H1, which critically obstructs the FhaC pore, in two solvent conditions corresponding to the match points of the protein and the detergent, respectively. Protein-bound detergent amounted to 142 ± 10 mol/mol as determined by analytical ultracentrifugation. By using molecular modeling and starting from three distinct conformations of FhaC and its variant embedded in lipid bilayers, we generated ensembles of protein-detergent arrangement models with 120–160 detergent molecules. The scattered curves were back-calculated for each model and compared with experimental data. Good fits were obtained for relatively compact, connected detergent belts, which occasionally displayed small detergent-free patches on the outer surface of the β barrel. The combination of SANS and modeling clearly enabled us to infer the solution structure of FhaC, with H1 inside the pore as in the crystal structure. We believe that our strategy of combining explicit atomic detergent modeling with SANS measurements has significant potential for structural studies of other detergent-solubilized membrane proteins. PMID:24988353

  6. A Molecular Probe for the Detection of Polar Lipids in Live Cells

    PubMed Central

    Bader, Christie A.; Shandala, Tetyana; Carter, Elizabeth A.; Ivask, Angela; Guinan, Taryn; Hickey, Shane M.; Werrett, Melissa V.; Wright, Phillip J.; Simpson, Peter V.; Stagni, Stefano; Voelcker, Nicolas H.; Lay, Peter A.; Massi, Massimiliano; Brooks, Douglas A.

    2016-01-01

    Lipids have an important role in many aspects of cell biology, including membrane architecture/compartment formation, intracellular traffic, signalling, hormone regulation, inflammation, energy storage and metabolism. Lipid biology is therefore integrally involved in major human diseases, including metabolic disorders, neurodegenerative diseases, obesity, heart disease, immune disorders and cancers, which commonly display altered lipid transport and metabolism. However, the investigation of these important cellular processes has been limited by the availability of specific tools to visualise lipids in live cells. Here we describe the potential for ReZolve-L1™ to localise to intracellular compartments containing polar lipids, such as for example sphingomyelin and phosphatidylethanolamine. In live Drosophila fat body tissue from third instar larvae, ReZolve-L1™ interacted mainly with lipid droplets, including the core region of these organelles. The presence of polar lipids in the core of these lipid droplets was confirmed by Raman mapping and while this was consistent with the distribution of ReZolve-L1™ it did not exclude that the molecular probe might be detecting other lipid species. In response to complete starvation conditions, ReZolve-L1™ was detected mainly in Atg8-GFP autophagic compartments, and showed reduced staining in the lipid droplets of fat body cells. The induction of autophagy by Tor inhibition also increased ReZolve-L1™ detection in autophagic compartments, whereas Atg9 knock down impaired autophagosome formation and altered the distribution of ReZolve-L1™. Finally, during Drosophila metamorphosis fat body tissues showed increased ReZolve-L1™ staining in autophagic compartments at two hours post puparium formation, when compared to earlier developmental time points. We concluded that ReZolve-L1™ is a new live cell imaging tool, which can be used as an imaging reagent for the detection of polar lipids in different intracellular

  7. A Molecular Probe for the Detection of Polar Lipids in Live Cells.

    PubMed

    Bader, Christie A; Shandala, Tetyana; Carter, Elizabeth A; Ivask, Angela; Guinan, Taryn; Hickey, Shane M; Werrett, Melissa V; Wright, Phillip J; Simpson, Peter V; Stagni, Stefano; Voelcker, Nicolas H; Lay, Peter A; Massi, Massimiliano; Plush, Sally E; Brooks, Douglas A

    2016-01-01

    Lipids have an important role in many aspects of cell biology, including membrane architecture/compartment formation, intracellular traffic, signalling, hormone regulation, inflammation, energy storage and metabolism. Lipid biology is therefore integrally involved in major human diseases, including metabolic disorders, neurodegenerative diseases, obesity, heart disease, immune disorders and cancers, which commonly display altered lipid transport and metabolism. However, the investigation of these important cellular processes has been limited by the availability of specific tools to visualise lipids in live cells. Here we describe the potential for ReZolve-L1™ to localise to intracellular compartments containing polar lipids, such as for example sphingomyelin and phosphatidylethanolamine. In live Drosophila fat body tissue from third instar larvae, ReZolve-L1™ interacted mainly with lipid droplets, including the core region of these organelles. The presence of polar lipids in the core of these lipid droplets was confirmed by Raman mapping and while this was consistent with the distribution of ReZolve-L1™ it did not exclude that the molecular probe might be detecting other lipid species. In response to complete starvation conditions, ReZolve-L1™ was detected mainly in Atg8-GFP autophagic compartments, and showed reduced staining in the lipid droplets of fat body cells. The induction of autophagy by Tor inhibition also increased ReZolve-L1™ detection in autophagic compartments, whereas Atg9 knock down impaired autophagosome formation and altered the distribution of ReZolve-L1™. Finally, during Drosophila metamorphosis fat body tissues showed increased ReZolve-L1™ staining in autophagic compartments at two hours post puparium formation, when compared to earlier developmental time points. We concluded that ReZolve-L1™ is a new live cell imaging tool, which can be used as an imaging reagent for the detection of polar lipids in different intracellular

  8. Perspectives of Deuteron Field-Cycling NMR Relaxometry for Probing Molecular Dynamics in Soft Matter.

    PubMed

    Flämig, M; Becher, M; Hofmann, M; Körber, T; Kresse, B; Privalov, A F; Willner, L; Kruk, D; Fujara, F; Rössler, E A

    2016-08-11

    Due to the single-particle character of the quadrupolar interaction in molecular systems, (2)H NMR poses a unique method for probing reorientational dynamics. Spin-lattice relaxation gives access to the spectral density, and its frequency dependency can be monitored by field-cycling (FC) techniques. However, most FC NMR studies employ (1)H; the use of (2)H is still rare. We report on the application of (2)H FC NMR for investigating the dynamics in molecular liquids and polymers. Commercial as well as home-built relaxometers are employed accessing a frequency range from 30 Hz to 6 MHz. Due to low gyromagnetic ratio, high coupling constants, and finite FC switching times, current (2)H FC NMR does not reach the dispersion region in liquids (toluene and glycerol), yet good agreement with the results from conventional high-field (HF) relaxation studies is demonstrated. The pronounced difference at low frequencies between (2)H and (1)H FC NMR data shows the relevance of intermolecular relaxation in the case of (1)H NMR. In the case of the polymers polybutadiene and poly(ethylene-alt-propylene), very similar relaxation dispersion is observed and attributed to Rouse and entanglement dynamics. Combination with HF (2)H relaxation data via applying frequency-temperature superposition allows the reconstruction of the full spectral density reflecting both polymer as well as glassy dynamics. Transformation into the time domain yields the reorientational correlation function C2(t) extending over nine decades in time with a long-time power law, C2(t) ∝ t(-0.45±0.05), which does not conform to the prediction of the tube-reptation model, for which ∝ t(-0.25) is expected. Entanglement sets in below C2(t = τe) ≅ S(2) = 0.001, where τe is the entanglement time and S the corresponding order parameter. Finally, we discuss the future prospects of the (2)H FC NMR technique. PMID:27420118

  9. Isolate-Specific Detection of Grapevine fanleaf virus from Xiphinema index Through DNA-Based Molecular Probes.

    PubMed

    Finetti-Sialer, M M; Ciancio, A

    2005-03-01

    ABSTRACT Tests with a real-time reverse transcription-polymerase chain reaction (RT-PCR) were performed on specimens of Xiphinema index collected from the rhizosphere of Grapevine fanleaf virus (GFLV)-infected grapevines at Palagiano, Italy. A 1,157-bp fragment of the GFLV RNA-2 coat protein (CP) gene was amplified and sequenced. A fluorescent Scorpion probe was designed to detect a highly conserved CP region. A second region with isolate-specific multiple nucleotide polymorphisms was used to detect GFLV isolates using molecular beacons (MB). The Scorpion probe allowed quantitative estimation of GFLV RNA-2 in single nematodes, using a dilution series of a 692-nucleotide transcript of the CP gene. The assay allowed detection of GFLV RNA-2 in individual X. index, with a minimum template threshold of 800 fg or 2.8 x 10(6) RNA-2 molecules per nematode. The CP fragment used for GFLV detection with the Scorpion probe appeared highly conserved among isolates. The probes were tested against other GFLV isolates, which were recognized by the species-specific Scorpion probe and by the corresponding MB specific to the particular isolate. Both tests appeared useful as diagnostic tools or for studies on GFLV in acquisition, retention, and transmission experiments.

  10. Simultaneous detection of dual single-base mutations by capillary electrophoresis using quantum dot-molecular beacon probe.

    PubMed

    Li, Yong-Qiang; Guan, Li-Yun; Wang, Jian-Hao; Zhang, Hai-Li; Chen, Jun; Lin, Song; Chen, Wei; Zhao, Yuan-Di

    2011-01-15

    Here a novel capillary electrophoresis (CE) for simultaneous detection of dual single-base mutations using quantum dot-molecular beacon (QD-MB) probe is described. Two QD-MB probes were designed using 585 and 650-nm emitting CdTe QDs which were covalently conjugated to MBs with different DNA oligonucleotide sequences by amide linkage and streptavidin-biotin binding, respectively. The hybridizations of QD-MB probes with different DNA targets were then monitored by CE, and results indicated that the two QD-MB probes specifically hybridized with their complementary DNA sequences, respectively. Target DNA identification was observed to have a high sensitivity of 16.2 pg in CE. Furthermore, the simultaneous detection of dual single-base mutations in a given DNA oligonucleotide was successfully achieved in CE using above two QD-MB probes. This novel CE-assisted QD-MB biosensor offers a promising approach for simultaneous detection of multiple single-base mutations, and exhibits potential capability in the single nucleotide polymorphism (SNP) analysis and high-sensitivity DNA detection.

  11. In search of the cochlear amplifier: New mechanical and molecular tools to probe transduction channel function

    NASA Astrophysics Data System (ADS)

    Karavitaki, K. Domenica; Indzhykulian, Artur A.; Zhang, Duan-Sun; Corey, David P.

    2015-12-01

    The study of mechanotransduction in cochlear hair cells requires stimulus methods that mimic the in-vivo stimulation. We have developed a new mechanical probe to better mimic the physiological stimulus delivered to cochlear hair cells through the overlying tectorial membrane. We combine these new probes with electroporation to study the contribution of different components of the transduction apparatus.

  12. Molecular Basis of Glucagon-like Peptide 1 Docking to Its Intact Receptor Studied with Carboxyl-terminal Photolabile Probes*

    PubMed Central

    Chen, Quan; Pinon, Delia I.; Miller, Laurence J.; Dong, Maoqing

    2009-01-01

    The glucagon-like peptide 1 (GLP1) receptor is a member of Family B G protein-coupled receptors and represents an important drug target for type 2 diabetes. Despite recent solution of the structure of the amino-terminal domain of this receptor and that of several close family members, understanding of the molecular basis of natural ligand GLP1 binding to its intact receptor remains limited. The goal of this study was to explore spatial approximations between specific receptor residues within the carboxyl terminus of GLP1 and its receptor as normally docked. Therefore, we developed and characterized two high affinity, full-agonist photolabile GLP1 probes having sites for covalent attachment in positions 24 and 35. Both probes labeled the receptor specifically and saturably. Subsequent peptide mapping using chemical and proteinase cleavages of purified wild-type and mutant GLP1 receptor identified that the Arg131–Lys136 segment at the juxtamembrane region of the receptor amino terminus contained the site of labeling for the position 24 probe, and the specific receptor residue labeled by this probe was identified as Glu133 by radiochemical sequencing. Similarly, nearby residue Glu125 within the same region of the receptor amino-terminal domain was identified as the site of labeling by the position 35 probe. These data represent the first direct demonstration of spatial approximation between GLP1 and its intact receptor as docked, providing two important constraints for the modeling of this interaction. This should expand our understanding of the molecular basis of natural agonist ligand binding to the GLP1 receptor and may be relevant to other family members. PMID:19815559

  13. Molecular Probes for Diagnosis of Clinically Relevant Bacterial Infections in Blood Cultures▿

    PubMed Central

    Hansen, Wendy L. J.; Beuving, Judith; Bruggeman, Cathrien A.; Wolffs, Petra F. G.

    2010-01-01

    Broad-range real-time PCR and sequencing of the 16S rRNA gene region is a widely known method for the detection and identification of bacteria in clinical samples. However, because of the need for sequencing, such identification of bacteria is time-consuming. The aim of our study was to develop a more rapid 16S real-time PCR-based identification assay using species- or genus-specific probes. The Gram-negative bacteria were divided into Pseudomonas species, Pseudomonas aeruginosa, Escherichia coli, and other Gram-negative species. Within the Gram-positive species, probes were designed for Staphylococcus species, Staphylococcus aureus, Enterococcus species, Streptococcus species, and Streptococcus pneumoniae. The assay also included a universal probe within the 16S rRNA gene region for the detection of all bacterial DNA. The assay was evaluated with a collection of 248 blood cultures. In this study, the universal probe and the probes targeting Pseudomonas spp., P. aeruginosa, E. coli, Streptococcus spp., S. pneumoniae, Enterococcus spp., and Staphylococcus spp. all had a sensitivity and specificity of 100%. The probe specific for S. aureus showed eight discrepancies, resulting in a sensitivity of 100% and a specificity of 93%. These data showed high agreement between conventional testing and our novel real-time PCR assay. Furthermore, this assay significantly reduced the time needed for identification. In conclusion, using pathogen-specific probes offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections. PMID:20962139

  14. Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA

    SciTech Connect

    Greulich-Bode, Karin; Wang, Mei; Rhein, Andreas; Weier, Jingly; Weier, Heinz-Ulli

    2008-12-16

    Fluorescence in situ hybridization (FISH) is a sensitive and rapid procedure to detect gene rearrangements in tumor cells using non-isotopically labeled DNA probes. Large insert recombinant DNA clones such as bacterial artificial chromosome (BAC) or P1/PAC clones have established themselves in recent years as preferred starting material for probe preparations due to their low rates of chimerism and ease of use. However, when developing probes for the quantitative analysis of rearrangements involving genomic intervals of less than 100kb, careful probe selection and characterization are of paramount importance. We describe a sensitive approach to quality control probe clones suspected of carrying deletions or for measuring clone overlap with near kilobase resolution. The method takes advantage of the fact that P1/PAC/BAC's can be isolated as circular DNA molecules, stretched out on glass slides and fine-mapped by multicolor hybridization with smaller probe molecules. Two examples demonstrate the application of this technique: mapping of a gene-specific {approx}6kb plasmid onto an unusually small, {approx}55kb circular P1 molecule and the determination of the extent of overlap between P1 molecules homologous to the human NF-?B2 locus. The relatively simple method presented here does not require specialized equipment and may thus find widespread applications in DNA probe preparation and characterization, the assembly of physical maps for model organisms or in studies on gene rearrangements.

  15. Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA

    SciTech Connect

    Greulich-Bode, Karin M.; Wang, Mei; Rhein, Andreas P.; Weier, Jingly F.; Weier, Heinz-Ulli G.

    2008-12-04

    Fluorescence in situ hybridization (FISH) is a sensitive and rapid procedure to detect gene rearrangements in tumor cells using non-isotopically labeled DNA probes. Large insert recombinant DNA clones such as bacterial artificial chromosome (BAC) or P1/PAC clones have established themselves in recent years as preferred starting material for probe preparations due to their low rates of chimerism and ease of use. However, when developing probes for the quantitative analysis of rearrangements involving genomic intervals of less than 100kb, careful probe selection and characterization are of paramount importance. We describe a sensitive approach to quality control probe clones suspected of carrying deletions or for measuring clone overlap with near kilobase resolution. The method takes advantage of the fact that P1/PAC/BAC's can be isolated as circular DNA molecules, stretched out on glass slides and fine-mapped by multicolor hybridization with smaller probe molecules. Two examples demonstrate the application of this technique: mapping of a gene-specific {approx}6kb plasmid onto an unusually small, {approx}55kb circular P1 molecule and the determination of the extent of overlap between P1 molecules homologous to the human NF-{kappa}B2 locus. The relatively simple method presented here does not require specialized equipment and may thus find widespread applications in DNA probe preparation and characterization, the assembly of physical maps for model organisms or in studies on gene rearrangements.

  16. Chemical probes for molecular imaging and detection of hydrogen sulfide and reactive sulfur species in biological systems.

    PubMed

    Lin, Vivian S; Chen, Wei; Xian, Ming; Chang, Christopher J

    2015-07-21

    Hydrogen sulfide (H2S), a gaseous species produced by both bacteria and higher eukaryotic organisms, including mammalian vertebrates, has attracted attention in recent years for its contributions to human health and disease. H2S has been proposed as a cytoprotectant and gasotransmitter in many tissue types, including mediating vascular tone in blood vessels as well as neuromodulation in the brain. The molecular mechanisms dictating how H2S affects cellular signaling and other physiological events remain insufficiently understood. Furthermore, the involvement of H2S in metal-binding interactions and formation of related RSS such as sulfane sulfur may contribute to other distinct signaling pathways. Owing to its widespread biological roles and unique chemical properties, H2S is an appealing target for chemical biology approaches to elucidate its production, trafficking, and downstream function. In this context, reaction-based fluorescent probes offer a versatile set of screening tools to visualize H2S pools in living systems. Three main strategies used in molecular probe development for H2S detection include azide and nitro group reduction, nucleophilic attack, and CuS precipitation. Each of these approaches exploits the strong nucleophilicity and reducing potency of H2S to achieve selectivity over other biothiols. In addition, a variety of methods have been developed for the detection of other reactive sulfur species (RSS), including sulfite and bisulfite, as well as sulfane sulfur species and related modifications such as S-nitrosothiols. Access to this growing chemical toolbox of new molecular probes for H2S and related RSS sets the stage for applying these developing technologies to probe reactive sulfur biology in living systems.

  17. Chemical Probes for Molecular Imaging and Detection of Hydrogen Sulfide and Reactive Sulfur Species in Biological Systems

    PubMed Central

    2014-01-01

    Hydrogen sulfide (H2S), a gaseous species produced by both bacteria and higher eukaryotic organisms, including mammalian vertebrates, has attracted attention in recent years for its contributions to human health and disease. H2S has been proposed as a cytoprotectant and gasotransmitter in many tissue types, including mediating vascular tone in blood vessels as well as neuromodulation in the brain. The molecular mechanisms dictating how H2S affects cellular signaling and other physiological events remain insufficiently understood. Furthermore, the involvement of H2S in metal-binding interactions and formation of related RSS such as sulfane sulfur may contribute to other distinct signaling pathways. Owing to its widespread biological roles and unique chemical properties, H2S is an appealing target for chemical biology approaches to elucidate its production, trafficking, and downstream function. In this context, reaction-based fluorescent probes offer a versatile set of screening tools to visualize H2S pools in living systems. Three main strategies used in molecular probe development for H2S detection include azide and nitro group reduction, nucleophilic attack, and CuS precipitation. Each of these approaches exploit the strong nucleophilicity and reducing potency of H2S to achieve selectivity over other biothiols. In addition, a variety of methods have been developed for the detection of other reactive sulfur species (RSS), including sulfite and bisulfite, as well as sulfane sulfur species and related modifications such as S-nitrosothiols. Access to this growing chemical toolbox of new molecular probes for H2S and related RSS sets the stage for applying these developing technologies to probe reactive sulfur biology in living systems. PMID:25474627

  18. Genome wide DNA copy number analysis in cholangiocarcinoma using high resolution molecular inversion probe single nucleotide polymorphism assay.

    PubMed

    Arnold, Alexander; Bahra, Marcus; Lenze, Dido; Bradtmöller, Maren; Guse, Katrin; Gehlhaar, Claire; Bläker, Hendrik; Heppner, Frank L; Koch, Arend

    2015-10-01

    In order to study molecular similarities and differences of intrahepatic (IH-CCA) and extrahepatic (EH-CCA) cholangiocarcinoma, 24 FFPE tumor samples (13 IH-CCA, 11 EH-CCA) were analyzed for whole genome copy number variations (CNVs) using a new high-density Molecular Inversion Probe Single Nucleotide Polymorphism (MIP SNP) assay. Common in both tumor subtypes the most frequent losses were detected on chromosome 1p, 3p, 6q and 9 while gains were mostly seen in 1q, 8q as well as complete chromosome 17 and 20. Applying the statistical GISTIC (Genomic Identification of Significant Targets in Cancer) tool we identified potential novel candidate tumor suppressor- (DBC1, FHIT, PPP2R2A) and oncogenes (LYN, FGF19, GRB7, PTPN1) within these regions of chromosomal instability. Next to common aberrations in IH-CCA and EH-CCA, we additionally found significant differences in copy number variations on chromosome 3 and 14. Moreover, due to the fact that mutations in the Isocitrate dehydrogenase (IDH-1 and IDH-2) genes are more frequent in our IH-CCA than in our EH-CCA samples, we suggest that the tumor subtypes have a different molecular profile. In conclusion, new possible target genes within regions of high significant copy number aberrations were detected using a high-density Molecular Inversion Probe Single Nucleotide Polymorphism (MIP SNP) assay, which opens a future perspective of fast routine copy number and marker gene identification for gene targeted therapy.

  19. Use of molecular beacon probes for real-time PCR detection of Plasmodium falciparum and other plasmodium species in peripheral blood specimens.

    PubMed

    Elsayed, Sameer; Plewes, Katherine; Church, Deirdre; Chow, Barbara; Zhang, Kunyan

    2006-02-01

    We describe the development and evaluation of a novel pair of real-time, fluorescence-based PCR assays using molecular beacon probes for rapid, sensitive, and specific detection and quantification of Plasmodium falciparum and other Plasmodium species organisms.

  20. A versatile molecular beacon-like probe for multiplexed detection based on fluorescence polarization and its application for a resettable logic gate.

    PubMed

    Zhang, Min; Le, Huynh-Nhu; Wang, Ping; Ye, Bang-Ce

    2012-10-14

    A versatile molecular beacon (MB)-like probe was developed for multiplexed detection based on fluorescence polarization by target-induced allosteric effect and furthermore for resettable logic gate operation.

  1. Oligonucleotide primers, probes and molecular methods for the environmental monitoring of methanogenic archaea

    PubMed Central

    Narihiro, Takashi; Sekiguchi, Yuji

    2011-01-01

    Summary For the identification and quantification of methanogenic archaea (methanogens) in environmental samples, various oligonucleotide probes/primers targeting phylogenetic markers of methanogens, such as 16S rRNA, 16S rRNA gene and the gene for the α‐subunit of methyl coenzyme M reductase (mcrA), have been extensively developed and characterized experimentally. These oligonucleotides were designed to resolve different groups of methanogens at different taxonomic levels, and have been widely used as hybridization probes or polymerase chain reaction primers for membrane hybridization, fluorescence in situ hybridization, rRNA cleavage method, gene cloning, DNA microarray and quantitative polymerase chain reaction for studies in environmental and determinative microbiology. In this review, we present a comprehensive list of such oligonucleotide probes/primers, which enable us to determine methanogen populations in an environment quantitatively and hierarchically, with examples of the practical applications of the probes and primers. PMID:21375721

  2. Specific estrogen sulfotransferase (SULT1E1) substrates and molecular imaging probe candidates

    PubMed Central

    Cole, Graham B.; Keum, Gyochang; Liu, Jie; Small, Gary W.; Satyamurthy, Nagichettiar; Kepe, Vladimir; Barrio, Jorge R.

    2010-01-01

    This work focuses on the development of specific substrates for estrogen sulfotransferase (SULT1E1) to produce molecular imaging probes for this enzyme. SULT1E1 is a key enzyme in estrogen homeostasis, playing a central role in the prevention and development of human disease. In vitro sulfation assays showed alkyl and aryl substitutions to a fused heterocyclic system modeled after β-naphthol (βN), based on compounds that interact with the estrogen receptor, rendered several molecules with enhanced specificity for SULT1E1 over SULT1A1*1, SULT1A1*2, SULT1A3, and SULT2A1. Several 6-hydroxy-2-arylbenzothiazoles tested demonstrated excellent affinity—Vmax/Km ratios—and specificity for SULT1E1. Km values ranged from 0.12–2.36 μM. A strong correlation was observed between polarity of the 4′-sustituent on the 2-aryl moiety (Hammett σp) and the log(Vmax/Km) (r = 0.964). Substrate sensitivity is influenced by the acidity of the 6-phenolic group demonstrated by correlating its 1H NMR chemical shift (δOH) with the log(Vmax/Km) (r = 0.963). Acidity is mediated by the electron withdrawing capacity of the 4′-substituent outlined by the correlation of the C-2 13C NMR chemical shift (δC2) with the log(Vmax/Km) (r = 0.987). 2-[4-(Methylamino)phenyl]-6-hydroxybenzothiazole (2b) was radiolabeled with carbon-11 (11C-(2b)) and used in vivo for microPET scanning and tissue metabolite identification. High PET signal was paralleled with the presence of radiolabeled 11C-(2b)-6-O-sulfate and the SULT1E1 protein detected by western blot. Because this and other members of this family presenting specificity for SULT1E1 can be labeled with carbon-11 or fluorine-18, in vivo assays of SULT1E1 functional activity are now feasible in humans. PMID:20304798

  3. A novel adenosine-based molecular beacon probe for room temperature nucleic acid rapid detection in cotton thread device.

    PubMed

    Du, Ting-E; Wang, Yiyun; Zhang, Yi; Zhang, Tian; Mao, Xun

    2015-02-25

    We used cotton thread as substrate to develop a novel room temperature DNA detection device for low-cost, sensitive and rapid detection of a human genetic disease, hereditary tyrosinemia type I related DNA sequences. A novel adenosine based molecular beacon (ABMB) probe modified on gold nanoparticle was used as reporter probe. In the presence of coralyne, a small molecule which can react with adenosines, the ABMB would form a hairpin structure just like traditional molecular beacon used extensively. In the presence of target DNA sequences, the hairpin structure of ABMB modified on gold nanoparticles will be opened and the biotin group modified at one end of the DNA probes will be released and react with the streptavidin immobilized on the test zone of the cotton thread. The response of the thread based DNA test device is linear over the range of 2.5-100 nM complementary DNA. The ability of our developed device for discriminating the single base mismatched DNA related to a human genetic disease, hereditary tyrosinemia type I, was improved comparing with previous report. It is worth mentioning that the whole assay procedure for DNA test is performed under room temperature which simplified the assay procedures greatly.

  4. Design of molecular beacons for AmpliDet RNA assay--characterization of binding stability and probe specificity.

    PubMed

    Szemes, Marianna; Schoen, Cor D

    2003-04-15

    AmpliDet RNA is a real-time diagnostic method, the specificity of which is defined mainly by the molecular beacon (MB). MBs can be characterized according to the stability of their stem-and-loop structures and that of the probe-target duplex via the free energies accompanying their formation. By the application of thermodynamic models, we propose a prediction method for these deltaG(0) parameters, which was compared to experimental analysis. The average absolute discrepancies for deltaG(0)(41) and for the melting temperatures of MB secondary structures were 0.30 +/- 0.26 kcal/mol and 2.15 +/- 1.5 degrees C, respectively. deltaG(0)(41) of probe-target interaction was predicted with a discrepancy of 1.2 +/- 1.0 kcal/mol. To characterize specificity, we formulated a model system with several MBs of highly similar sequence, but different lengths, and template RNAs carrying different types of mutations. We demonstrated the ability to detect a point mutation, or to tolerate one, irrespective of mismatch type. Of the nucleotide analogues tested, universal pyrimidine was found to increase MB tolerance substantially toward polymorphism. In the present study MBs were characterized under AmpliDet RNA conditions, with respect to probe stability, binding strength, and specificity, which led us to propose a design method, useful for probe design for AmpliDet RNA and adaptable to microarrays.

  5. Highly sensitive and selective detection of biothiols using graphene oxide-based "molecular beacon"-like fluorescent probe.

    PubMed

    Gao, Yuan; Li, Yan; Zou, Xin; Huang, Hui; Su, Xingguang

    2012-06-20

    A fluorometric method for quantity analysis of biothiols was developed using a graphene oxide (GO)-based "molecular beacon"-like probe, which consisted of FITC labeled thymine (T)-rich single-stranded DNA (ssDNA), GO and Hg(2+) ions. The labeled ssDNA containing T-T mismatches would self-hybridize to duplex in the presence of Hg(2+), which can avoid its adsorption on GO and the fluorescence of this GO-based probe was recovered. The fluorescence of the probe quenched after the addition of biothiols such as glutathione (GSH) and cysteine (Cys) owing to thiol groups can selectively competitive ligation of Hg(2+) ions with T-T mismatches. In the present work, the GO-based probe was used for the determination of GSH and Cys. Under the optimal conditions, a linear correlation was established between fluorescence intensity ratio I(0)/I and the concentration of GSH in the range of 2.0×10(-9)-5.0×10(-7) mol L(-1) with a detection limit of 1.0×10(-9) mol L(-1). The linear range for Cys is from 5.0×10(-9) to 4.5×10(-7) mol L(-1) with a detection limit of 2.0×10(-9) mol L(-1). The proposed method was applied to the determination of GSH in human serum and cell extract samples with satisfactory results.

  6. Detection of supercoiled hepatitis B virus DNA and related forms by means of molecular hybridization to an oligonucleotide probe

    SciTech Connect

    Lin, H.J.; Chung, H.T.; Lai, C.L.; Leong, S.; Tam, O.S. )

    1989-12-01

    A novel assay for supercoiled and other fully double-stranded forms of hepatitis B virus (HBV) DNA in blood is presented that utilizes molecular hybridisation to a radiophosphorous-labeled oligonucleotide probe. The probe (5'-d(ACGTGCAGAGGTGAAGCGA)) is complementary to the S(+)-strand sequence furthest downstream, at the end of the gap. We examined blood specimens from 137 healthy HBsAg-positive individuals, applying the probe to dots representing 2-3.5 ml serum or plasma. We found that supercoiled HBV is present in many HBV DNA-positive blood specimens albeit in small quantities. Of the 104 specimens that were positive for HBV DNA of any form, 53 annealed to the probe. Serial specimens from the same subject taken over a period of months showed that the proportion of supercoil to other HBV DNA forms was variable. The presence of supercoil HBV DNA was not closely correlated with the level of serum HBV DNA polymerase. The supercoil is an HBV DNA form that can persist in the liver in the presence or absence of other replicative intermediates. This assay may enable further characterization of the status of HBV infection.

  7. Molecular-Level Insights into Photocatalysis from Scanning Probe Microscopy Studies on TiO2(110)

    SciTech Connect

    Henderson, Michael A.; Lyubinetsky, Igor

    2013-06-12

    The field of heterogeneous photocatalysis has grown considerably in the decades since Fujishima and Honda's ground-breaking publications of photoelectrochemistry on TiO2. Numerous review articles continue to point to both progress made in the use of heterogeneous materials (such as TiO2) to perform photoconversion processes, and the many opportunities and challenges in heterogeneous photocatalysis research such as solar energy conversion and environmental remediation. The past decade has also seen an increase in the use of molecular-level approaches applied to model single crystal surfaces in an effort to obtain new insights into photocatalytic phenomena. In particular, scanning probe techniques (SPM) have enabled researchers to take a ‘nanoscale’ approach to photocatalysis that includes interrogation of the reactivities of specific sites and adsorbates on a model photocatalyst surface. The rutile TiO2(110) surface has become the prototypical oxide single crystal surface for fundamental studies of many interfacial phenomena. In particular, TiO2(110) has become an excellent model surface for probing photochemical and photocatalytic reactions at the molecular level. A variety of experimental approaches have emerged as being ideally suited for studying photochemical reactions on TiO2(110), including desorption-oriented approaches and electronic spectroscopies, but perhaps the most promising techniques for evaluating site-specific properties are those of SPM. In this review, we highlight the growing use of SPM techniques in providing molecular-level insights into surface photochemistry on the model photocatalyst surface of rutile TiO2(110). Our objective is to both illustrate the unique knowledge that scanning probe techniques have already provided the field of photocatalysis, and also to motivate a new generation of effort into the use of such approaches to obtain new insights into the molecular level details of photochemical events occurring at interfaces

  8. A modified molecular beacon combining the properties of TaqMan probe.

    PubMed

    Kong, De-Ming; Gu, Long; Shen, Han-Xi; Mi, Huai-Feng

    2002-04-21

    A modified molecular beacon that possesses a stem-hairpin structure as seen in conventional molecular beacons and can be cleaved during PCR in designed, and it can specifically recognize the presence of the target and was obviously more sensitive than conventional molecular beacons.

  9. Molecular probe dynamics and free volume in organic glass-formers and their relationships to structural relaxation: 1-propanol

    NASA Astrophysics Data System (ADS)

    Bartoš, J.; Švajdlenková, H.; Šauša, O.; Lukešová, M.; Ehlers, D.; Michl, M.; Lunkenheimer, P.; Loidl, A.

    2016-01-01

    A joint study of the rotational dynamics and free volume in amorphous 1-propanol (1-PrOH) as a prototypical monohydroxy alcohol by electron spin resonance (ESR) or positron annihilation lifetime spectroscopy (PALS), respectively, is reported. The dynamic parameters of the molecular spin probe 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and the annihilation ones of the atomic ortho-positronium (o-Ps) probe as a function of temperature are compared. A number of coincidences between various effects in the ESR and PALS responses at the corresponding characteristic ESR and PALS temperatures were found suggesting a common origin of the underlying dynamic processes that were identified using viscosity (VISC) in terms of the two-order parameter (TOP) model and broadband dielectric spectroscopy (BDS) data.

  10. Built-in fields in nanodimensional organic ferroelectric-semiconductor heterostructures: The principal role of molecular probes

    NASA Astrophysics Data System (ADS)

    Lazarev, V. V.; Blinov, L. M.; Yudin, S. G.; Palto, S. P.

    2014-10-01

    The external and internal (built-in) macroscopic fields in a nanodimensional heterostructure consisting of organic ferroelectric and organic semiconductor layers between transparent (indium tin oxide) and semitransparent (aluminum) electrodes have been studied. The fields were measured using optical probe molecules in both the semiconductor layer (copper phthalocyanine, CuPc, with intrinsic specific features of the absorption spectrum) and the ferroelectric layer (poly(vinylidene fluoride)-trifluoroethylene copolymer) in which a molecular probe was introduced as a dopant (palladium tetraphenylporphyrin) possessing a characteristic spectrum. Local fields were measured using electroabsorption, followed by a recalculation in the macroscopic fields. It is established that the amplitude and direction of a macroscopic built-in field in the semiconductor can be controlled by changing the polarization of the heterostructure using pulses of external voltage applied to the heterostructure. This effect can be useful for increasing the efficiency of organic converters of solar radiation energy into electricity.

  11. Probing molecular pathways for DNA orientational trapping, unzipping and translocation in nanopores by using a tunable overhang sensor

    NASA Astrophysics Data System (ADS)

    Wang, Yong; Tian, Kai; Hunter, Lehr L.; Ritzo, Brandon; Gu, Li-Qun

    2014-09-01

    Nanopores provide a unique single-molecule platform for genetic and epigenetic detection. The target nucleic acids can be accurately analyzed by characterizing their specific electric fingerprints or signatures in the nanopore. Here we report a series of novel nanopore signatures generated by target nucleic acids that are hybridized with a probe. A length-tunable overhang appended to the probe functions as a sensor to specifically modulate the nanopore current profile. The resulting signatures can reveal multiple mechanisms for the orientational trapping, unzipping, escaping and translocation of nucleic acids in the nanopore. This universal approach can be used to program various molecular movement pathways, elucidate their kinetics, and enhance the sensitivity and specificity of the nanopore sensor for nucleic acid detection.Nanopores provide a unique single-molecule platform for genetic and epigenetic detection. The target nucleic acids can be accurately analyzed by characterizing their specific electric fingerprints or signatures in the nanopore. Here we report a series of novel nanopore signatures generated by target nucleic acids that are hybridized with a probe. A length-tunable overhang appended to the probe functions as a sensor to specifically modulate the nanopore current profile. The resulting signatures can reveal multiple mechanisms for the orientational trapping, unzipping, escaping and translocation of nucleic acids in the nanopore. This universal approach can be used to program various molecular movement pathways, elucidate their kinetics, and enhance the sensitivity and specificity of the nanopore sensor for nucleic acid detection. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr03195d

  12. Molecular Basis of Secretin Docking to Its Intact Receptor Using Multiple Photolabile Probes Distributed throughout the Pharmacophore*

    PubMed Central

    Dong, Maoqing; Lam, Polo C.-H.; Pinon, Delia I.; Hosohata, Keiko; Orry, Andrew; Sexton, Patrick M.; Abagyan, Ruben; Miller, Laurence J.

    2011-01-01

    The molecular basis of ligand binding and activation of family B G protein-coupled receptors is not yet clear due to the lack of insight into the structure of intact receptors. Although NMR and crystal structures of amino-terminal domains of several family members support consistency in general structural motifs that include a peptide-binding cleft, there are variations in the details of docking of the carboxyl terminus of peptide ligands within this cleft, and there is no information about siting of the amino terminus of these peptides. There are also no empirical data to orient the receptor amino terminus relative to the core helical bundle domain. Here, we prepared a series of five new probes, incorporating photolabile moieties into positions 2, 15, 20, 24, and 25 of full agonist secretin analogues. Each bound specifically to the receptor and covalently labeled single distinct receptor residues. Peptide mapping of labeled wild-type and mutant receptors identified that the position 15, 20, and 25 probes labeled residues within the distal amino terminus of the receptor, whereas the position 24 probe labeled the amino terminus adjacent to TM1. Of note, the position 2 probe labeled a residue within the first extracellular loop of the receptor, a region not previously labeled, providing an important new constraint for docking the amino-terminal region of secretin to its receptor core. These additional experimentally derived constraints help to refine our understanding of the structure of the secretin-intact receptor complex and provide new insights into understanding the molecular mechanism for activation of family B G protein-coupled receptors. PMID:21566140

  13. Molecular Determinants of Matrix Metalloproteinase-12 Covalent Modification by a Photoaffinity Probe

    PubMed Central

    Dabert-Gay, Anne-Sophie; Czarny, Bertrand; Devel, Laurent; Beau, Fabrice; Lajeunesse, Evelyne; Bregant, Sarah; Thai, Robert; Yiotakis, Athanasios; Dive, Vincent

    2008-01-01

    Mass spectroscopy, microsequencing, and site-directed mutagenesis studies have been performed to identify in human matrix metalloelastase (hMMP-12) residues covalently modified by a photoaffinity probe, previously shown to be able to covalently label specifically the active site of matrix metalloproteinases (MMPs). Results obtained led us to conclude that photoactivation of this probe in complex with hMMP-12 affects a single residue in human MMP-12, Lys241, through covalent modification of its side chain ε NH2 group. Because x-ray and NMR studies of hMMP-12 indicate that Lys241 side chain is highly flexible, our data reveal the existence of particular Lys241 side-chain conformation in which the ε NH2 group points toward the photolabile group of the probe, an event explaining the high levels of cross-linking yield between hMMP-12 and the probe. Lys241 is not conserved in MMPs, thus differences in cross-linking yields observed with this probe between MMP members may be linked to the residue variability observed at position 241 in this family. PMID:18775985

  14. Homogeneous scoring of single-nucleotide polymorphisms: comparison of the 5'-nuclease TaqMan assay and Molecular Beacon probes.

    PubMed

    Täpp, I; Malmberg, L; Rennel, E; Wik, M; Syvänen, A C

    2000-04-01

    Homogeneous assays based on real-time fluorescence monitoring during PCR are relevant alternatives for large-scale genotyping of single-nucleotide polymorphisms (SNPs). We compared the performance of the homogeneous TaqMan 5'-nuclease assay and the Molecular Beacon assay using three SNPs in the human estrogen receptor gene as targets. When analyzing a panel of 90 DNA samples, both assays yielded a comparable power of discrimination between the genotypes of a C-to-T transition in codon 10 and a G-to-A transition in codon 594 of the estrogen receptor gene. The Molecular Beacon probes distinguished better than the TaqMan probes between homozygous and heterozygous genotypes of a C-to-G transversion in codon 325. The sensitivity of detecting one allele, present as a minority in a mixed sample, varied between the SNPs and was similar for both assays. With the Molecular Beacon assay, the measured signal ratios were proportional to the amount of the minor allele over a wider range than with the TaqMan assay at all three SNPs.

  15. Molecular beacons: a novel DNA probe for nucleic acid and protein studies.

    PubMed

    Tan, W; Fang, X; Li, J; Liu, X

    2000-04-01

    A new concept has been introduced for molecular beacon DNA molecules. Molecular beacons are a new class of oligonucleotides that can report the presence of specific nucleic acids in both homogeneous solutions and at the liquid-solid interface. They emit an intense fluorescent signal only when hybridized to their target DNA or RNA molecules. Biotinylated molecular beacons have been designed and used for the development of ultrasensitive DNA sensors and for DNA molecular interaction studies at a solid-liquid interface. Molecular beacons have also been used to study protein-DNA interactions. They have provided a variety of exciting opportunities in DNA/RNA/protein studies.

  16. A quantum dot probe conjugated with aβ antibody for molecular imaging of Alzheimer's disease in a mouse model.

    PubMed

    Feng, Li; Long, Hong-Yu; Liu, Ren-Kai; Sun, Dan-Ni; Liu, Chao; Long, Li-Li; Li, Yi; Chen, Si; Xiao, Bo

    2013-08-01

    The treatment of Alzheimer's disease (AD) has been hampered by a lack of sensitive and specific non-invasive diagnostic methods. Quantum dots (QD) are nano-crystals with unique photo-physical properties that bypass some of the limitations of conventional dyes and imaging tools. This study is aimed to evaluate the fluorescence properties of a QD probe conjugated with an anti-Aβ antibody (QD-Aβ-Ab). Healthy mice and mice bearing mutated human APP695swe and APP717 V-F transgenes received intracerebroventricular injection of the probe for subsequent imaging. Immunohistochemistry revealed that Aβ1-42 was distributed in the hippocampus CA1 area in the APP transgenic mice. Fluorescence microscopy demonstrated that fluorescence was mainly observed in the hippocampus area, the cerebral cortex, sagittal septum and striatum of APP transgenic mice. In vivo imaging of mice receiving the QD-Aβ-Ab probe showed that healthy mice exhibited a narrow range of fluorescence and lower fluorescence intensity compared with APP transgenic mice. The mean fluorescence intensity of brain tissues of healthy C57BL mice was 12.3784 ± 3.9826, which was significantly lower than that of 10- and 16-month-old APP transgenic mice (45.03 ± 2.66 and 46.69 ± 3.22, respectively; P < 0.05). In this study we present the first direct evidence that QD-Aβ-Ab conjugate probes can track in vivo state of Aβ accumulation in mice and the findings suggest that such probes may be of potential use for early molecular diagnostic imaging of AD.

  17. TiO2 Nanoparticles as a Soft X-ray Molecular Probe

    SciTech Connect

    Larabell, Carolyn; Ashcroft, Jared M.; Gu, Weiwei; Zhang, Tierui; Hughes, Steven M.; Hartman, Keith B.; Hofmann, Cristina; Kanaras, Antonios G.; Kilcoyne, David A.; Le Gros, Mark; Yin, Yadong; Alivisatos, A. Paul; Larabell, Carolyn A.

    2007-06-30

    With the emergence of soft x-ray techniques for imaging cells, there is a pressing need to develop protein localization probes that can be unambiguously identified within the region of x-ray spectrum used for imaging. TiO2 nanocrystal colloids, which have a strong absorption cross-section within the "water-window" region of x-rays, areideally suited as soft x-ray microscopy probes. To demonstrate their efficacy, TiO2-streptavidin nanoconjugates were prepared and subsequently labeled microtubules polymerized from biotinylated tubulin. The microtubules were imaged using scanning transmission x-ray microscopy (STXM), and the TiO2 nanoparticle tags were specifically identified using x-ray absorption near edge spectroscopy (XANES). These experiments demonstrate that TiO2 nanoparticles are potential probes for protein localization analyses using soft x-ray microscopy.

  18. Dual-Modal Colorimetric/Fluorescence Molecular Probe for Ratiometric Sensing of pH and Its Application.

    PubMed

    Wu, Luling; Li, Xiaolin; Huang, Chusen; Jia, Nengqin

    2016-08-16

    As traditional pH meters cannot work well for minute regions (such as subcellular organelles) and in harsh media, molecular pH-sensitive devices for monitoring pH changes in diverse local heterogeneous environments are urgently needed. Here, we report a new dual-modal colorimetric/fluorescence merocyanine-based molecular probe (CPH) for ratiometric sensing of pH. Compared with previously reported pH probes, CPH bearing the benzyl group at the nitrogen position of the indolium group and the phenol, which is used as the acceptor for proton, could respond to pH changes immediately through both the ratiometric fluorescence signal readout and naked-eye colorimetric observation. The sensing process was highly stable and reversible. Most importantly, the suitable pKa value (6.44) allows CPH to presumably accumulate in lysosomes and become a lysosome-target fluorescent probe. By using CPH, the intralysosomal pH fluctuation stimulated by antimalaria drug chloroquine was successfully tracked in live cells through the ratiometric fluorescence images. Additionally, CPH could be immobilized on test papers, which exhibited a rapid and reversible colorimetric response to acid/base vapor through the naked-eye colorimetric analysis. This proof-of-concept study presents the potential application of CPH as a molecular tool for monitoring intralysosomal pH fluctuation in live cells, as well as paves the way for developing the economic, reusable, and fast-response optical pH meters for colorimetric sensing acid/base vapor with direct naked-eye observation. PMID:27431089

  19. Inner-shell photoexcitations as probes of the molecular ions CH+, OH+, and SiH+: Measurements and theory

    NASA Astrophysics Data System (ADS)

    Mosnier, J.-P.; Kennedy, E. T.; van Kampen, P.; Cubaynes, D.; Guilbaud, S.; Sisourat, N.; Puglisi, A.; Carniato, S.; Bizau, J.-M.

    2016-06-01

    Spectral probes for the CH+, OH+, and SiH+ hydride molecular ions that play key roles in astrophysics and plasma processes are presented. The merged-beam technique at the SOLEIL synchrotron was used to record the photoionization (ion yield) spectra of CH+, OH+, and SiH+ and that of their parent atomic ions, in the K -shell and L -shell regions, respectively. Energies and oscillator strengths for the K α (CH+ and OH+) and L α (SiH+) transitions were determined from the spectra. Ab initio calculations interpret the experimental data in terms of contributions from ground and excited valence electronic states.

  20. Synthesis of new molecular probes for investigation of steroid biosynthesis induced by selective interaction with peripheral type benzodiazepine receptors (PBR).

    PubMed

    Campiani, Giuseppe; Ramunno, Anna; Fiorini, Isabella; Nacci, Vito; Morelli, Elena; Novellino, Ettore; Goegan, Mara; Mennini, Tiziana; Sullivan, Stephen; Zisterer, Daniela M; Williams, Clive D

    2002-09-12

    In the present study, we have synthesized and tested novel pyridopyrrolo- and pyrrolobenzoxazepine derivatives, as novel and selective peripheral type benzodiazepine receptor (PBR) ligands, and their ability to modulate steroid biosynthesis has been investigated. A subset of new ligands bind the PBR (rat brain and testis) with picomolar affinity, representing the most potent ligands that have been identified to date, and elicited effects on endogenous rate of steroidogenesis in MA10 Leydig cells, having similar potency and effect as PK11195. Several compounds, differently substituted at C-7, were used as molecular yardsticks to probe the spatial dimension of the lipophilic pocket L4 in the receptor binding site.

  1. Molecular probes and the polymerase chain reaction for detection and typing of Leishmania species in Mexico.

    PubMed

    Monroy-Ostria, Amalia; Sanchez-Tejeda, Gustavo

    2002-04-01

    Leishmaniasis in Mexico is a public health problem because all the clinical forms have been recorded in most Mexican states. We studied patients showing clinical symptoms of any form of leishmaniasis, from several endemic areas. Bone marrow samples, aspirates or skin biopsies were taken and deoxyribonucleic acid (DNA) was extracted and amplified by the polymerase chain reaction (PCR) with universal primers AJS1 and DeB8, specific for the Leishmania subgenus Leishmania. The PCR products were then hybridized by dot- or Southern blotting and probed with probe 9.2, specific for the L. mexicana complex. If hybridization did not occur, the DNA was amplified with primers D1 and D2, specific for members of the L. donovani complex, and PCR products were hybridized with probe B4Rsa, also specific for the L. donovani complex. DNA was also amplified with primers B1 and B2, specific for the subgenus Viannia, and the PCR products were hybridized with probe B18, specific for the L. braziliensis complex. It was found that in Tabasco and Veracruz, Mexico, localized cutaneous leishmaniasis (LCL) is caused by infection with members of the L. mexicana complex, whereas in the states of Nayarit and Campeche it was due to infection with the L. mexicana and/or L. braziliensis complexes. Visceral leishmaniasis was caused by L. (L.) chagasi, mainly in the states of Chiapas and Guerrero, and by L. (L.) mexicana in one immunocompromised patient from Tabasco.

  2. A robust probe for lighting up intracellular telomerase via primer extension to open a nicked molecular beacon.

    PubMed

    Qian, Ruocan; Ding, Lin; Yan, Liwen; Lin, Manfei; Ju, Huangxian

    2014-06-11

    A nicked molecular beacon (MB)-functionalized probe has been designed for in situ imaging and detection of intracellular telomerase activity. The nick separates the MB into two segments: a shorter telomerase primer (TSP) sequence as a part of the 5'-end stem and a longer sequence to form a loop with one thiol-labeled 3'-end stem. The MB can be opened by substitutional hybridization of the telomerase-triggered stem elongation product, which leads to separation of the Cy5 at the 5'-end nick from the gold nanoparticle (AuNP) as the nanocarrier and thus inhibits the energy transfer from Cy5 to AuNP. Upon endocytosis of the probe, the TSP can be extended by intracellular telomerase at its 3' end to produce the telomeric repeated sequence, which leads to the inner chain substitution and thus turns on the fluorescence of Cy5. The probe provides a one-step incubation technique for quantification and monitoring of the telomerase activity in living cells. The practicality of the proposed approach for distinguishing tumor cells from normal cells and monitoring the decrease of telomerase activity during treatment with antitumor drugs demonstrates its potential in clinical diagnostic and therapeutic monitoring.

  3. Detection of DNA damage by using hairpin molecular beacon probes and graphene oxide.

    PubMed

    Zhou, Jie; Lu, Qian; Tong, Ying; Wei, Wei; Liu, Songqin

    2012-09-15

    A hairpin molecular beacon tagged with carboxyfluorescein in combination with graphene oxide as a quencher reagent was used to detect the DNA damage by chemical reagents. The fluorescence of molecular beacon was quenched sharply by graphene oxide; while in the presence of its complementary DNA the quenching efficiency decreased because their hybridization prevented the strong adsorbability of molecular beacon on graphene oxide. If the complementary DNA was damaged by a chemical reagent and could not form intact duplex structure with molecular beacon, more molecular beacon would adsorb on graphene oxide increasing the quenching efficiency. Thus, damaged DNA could be detected based on different quenching efficiencies afforded by damaged and intact complementary DNA. The damage effects of chlorpyrifos-methyl and three metabolites of styrene such as mandelieaeids, phenylglyoxylieaeids and epoxystyrene on DNA were studied as models. The method for detection of DNA damage was reliable, rapid and simple compared to the biological methods.

  4. Probing the Spatial Organization of Molecular Complexes Using Triple-Pair-Correlation

    PubMed Central

    Yin, Yandong; Rothenberg, Eli

    2016-01-01

    Super-resolution microscopy coupled with multiplexing techniques can resolve specific spatial arrangements of different components within molecular complexes. However, reliable quantification and analysis of such specific organization is extremely problematic because it is frequently obstructed by random co-localization incidents between crowded molecular species and the intrinsic heterogeneity of molecular complexes. To address this, we present a Triple-Pair-Correlation (TPC) analysis approach for unbiased interpretation of the spatial organization of molecular assemblies in crowded three-color super-resolution (SR) images. We validate this approach using simulated data, as well as SR images of DNA replication foci in human cells. This demonstrates the applicability of TPC in deciphering the specific spatial organization of molecular complexes hidden in dense multi-color super-resolution images. PMID:27545293

  5. In vivo quantifying molecular specificity of Cy5.5-labeled cyclic 9-mer peptide probe with dynamic fluorescence imaging

    PubMed Central

    Dai, Yunpeng; Yin, Jipeng; Huang, Yu; Chen, Xueli; Wang, Guodong; Liu, Yajun; Zhang, Xianghan; Nie, Yongzhan; Wu, Kaichun; Liang, Jimin

    2016-01-01

    We quantified molecular specificity of Cy5.5-GX1 in vivo with dynamic fluorescence imaging to better understand its kinetic properties. According to whether or not free GX1 was injected and when it was injected, twelve of BGC-823 xenografted mice were randomly divided into three groups and underwent a 60 minute dynamic fluorescence scanning. Combined with a principal-component analysis, the binding potential (Bp) of the probe was determined by both Logan graphical analysis with reference tissue model (GARTM) and Lammertsma simplified reference tissue model (SRTM). The sum of the pharmacokinetic rate constants (SKRC) was quantified by the Gurfinkel exponential model (GEXPM). Cy5.5-GX1 specifically targeted tumor both in vitro and in vivo. We obtained similar quantification results of Bp (GARTM Bp = 0.582 ± 0.2655, SRTM Bp = 0.618 ± 0.2923), and obtained a good linear relation between the Bp value and the SKRC value. Our results indicate that the SKRC value is more suitable for an early-stage kinetic data analysis, and the Bp value depicts kinetic characteristics under the equilibrium state. Dynamic fluorescence imaging in conjunction with various kinetic models are optimal tools to quantify molecular specificity of the Cy5.5-GX1 probe in vivo. PMID:27446643

  6. In vivo quantifying molecular specificity of Cy5.5-labeled cyclic 9-mer peptide probe with dynamic fluorescence imaging.

    PubMed

    Dai, Yunpeng; Yin, Jipeng; Huang, Yu; Chen, Xueli; Wang, Guodong; Liu, Yajun; Zhang, Xianghan; Nie, Yongzhan; Wu, Kaichun; Liang, Jimin

    2016-04-01

    We quantified molecular specificity of Cy5.5-GX1 in vivo with dynamic fluorescence imaging to better understand its kinetic properties. According to whether or not free GX1 was injected and when it was injected, twelve of BGC-823 xenografted mice were randomly divided into three groups and underwent a 60 minute dynamic fluorescence scanning. Combined with a principal-component analysis, the binding potential (Bp) of the probe was determined by both Logan graphical analysis with reference tissue model (GARTM) and Lammertsma simplified reference tissue model (SRTM). The sum of the pharmacokinetic rate constants (SKRC) was quantified by the Gurfinkel exponential model (GEXPM). Cy5.5-GX1 specifically targeted tumor both in vitro and in vivo. We obtained similar quantification results of Bp (GARTM Bp = 0.582 ± 0.2655, SRTM Bp = 0.618 ± 0.2923), and obtained a good linear relation between the Bp value and the SKRC value. Our results indicate that the SKRC value is more suitable for an early-stage kinetic data analysis, and the Bp value depicts kinetic characteristics under the equilibrium state. Dynamic fluorescence imaging in conjunction with various kinetic models are optimal tools to quantify molecular specificity of the Cy5.5-GX1 probe in vivo. PMID:27446643

  7. Alignment dependence of photoelectron momentum distributions of atomic and molecular targets probed by few-cycle circularly polarized laser pulses

    NASA Astrophysics Data System (ADS)

    Abu-samha, M.; Madsen, Lars Bojer

    2016-08-01

    We present theoretical photoelectron momentum distributions (PMDs) for ionization from Ar(3 p ) and H2+ (σg) orbitals by few-cycle, high-intensity, near-infrared laser fields circularly polarized in the x y plane. The three-dimensional time-dependent Schrödinger equation is solved numerically within the single-active-electron approximation for Ar and within the fixed nuclei approximation for H2+ . The PMDs are investigated for alignment of the probed target orbitals relative to the polarization plane of the laser field. In the atomic case, the PMDs in the polarization plane for aligned 3 p Ar orbitals are, up to an overall scaling factor, insensitive to alignment of the probed orbital, while the lateral PMDs show a signature of the orbital node when that node is sufficiently close to the polarization plane. For the molecular case of H2+ (σg), our results show a significant impact of alignment on the PMDs due to the anisotropic molecular potential and the alignment-dependent coupling between the ground state and excited states.

  8. Molecular Probing of the HPV-16 E6 Protein Alpha Helix Binding Groove with Small Molecule Inhibitors

    PubMed Central

    Rietz, Anne; Petrov, Dino P.; Bartolowits, Matthew; DeSmet, Marsha; Davisson, V. Jo; Androphy, Elliot J.

    2016-01-01

    The human papillomavirus (HPV) HPV E6 protein has emerged as a central oncoprotein in HPV-associated cancers in which sustained expression is required for tumor progression. A majority of the E6 protein interactions within the human proteome use an alpha-helix groove interface for binding. The UBE3A/E6AP HECT domain ubiquitin ligase binds E6 at this helix-groove interface. This enables formation of a trimeric complex with p53, resulting in destruction of this tumor suppressor. While recent x-ray crystal structures are useful, examples of small molecule probes that can modulate protein interactions at this interface are limited. To develop insights useful for potential structure-based design of ligands for HPV E6, a series of 2,6-disubstituted benzopyranones were prepared and tested as competitive antagonists of E6-E6AP helix-groove interactions. These small molecule probes were used in both binding and functional assays to evaluate recognition features of the E6 protein. Evidence for an ionic functional group interaction within the helix groove was implicated by the structure-activity among the highest affinity ligands. The molecular topographies of these protein-ligand interactions were evaluated by comparing the binding and activities of single amino acid E6 mutants with the results of molecular dynamic simulations. A group of arginine residues that form a rim-cap over the E6 helix groove offer compensatory roles in binding and recognition of the small molecule probes. The flexibility and impact on the overall helix-groove shape dictated by these residues offer new insights for structure-based targeting of HPV E6. PMID:26915086

  9. Fluorescence Probe Based on Hybrid Mesoporous Silica/Quantum Dot/Molecularly Imprinted Polymer for Detection of Tetracycline.

    PubMed

    Zhang, Liang; Chen, Ligang

    2016-06-29

    A newly designed fluorescence probe made from a hybrid quantum dot/mesoporous silica/molecularly imprinted polymer (QD/MS/MIP) was successfully created, and the probe was used for the detection of tetracycline (TC) in serum sample. QD/MS/MIP was characterized by transmission electron microscope, Fourier transform infrared spectroscopy, UV spectroscopy, X-ray powder diffraction, nitrogen adsorption-desorption experiment and fluorescence spectroscopy. Tetracycline, which is a type of broad-spectrum antibiotic, was selected as the template. The monomer and the template were combined by covalent bonds. After the template was removed to form a binding site, a hydrogen bonding interaction formed between the hole and the target molecule. Moreover, when rebinding TC, a new complex was produced between the amino group of QD/MS/MIP and the hydroxyl group of TC. After that, the energy of the QDs could transfer to the complex, which explains the fluorescence quenching phenomenon. The fluorescent intensity of QD/MS/MIP decreased in 10 min, and an excellent linearity from 50 to 1000 ng mL(-1) was correspondingly obtained. This composite material has a high selectivity with an imprinting factor of 6.71. In addition, the confirmed probe strategy was successfully applied to serum sample analyses, and the recoveries were 90.2%-97.2% with relative standard deviations of 2.2%-5.7%. This current work offers a novel and suitable method to synthesize QD/MS/MIP with a highly selective recognition ability. This composite material will be valuable for use in fluorescence probe applications. PMID:27280785

  10. How does the molecular linker in dynamic force spectroscopy affect probing molecular interactions at the single-molecule level?

    NASA Astrophysics Data System (ADS)

    Taninaka, Atsushi; Aizawa, Kota; Hanyu, Tatsuya; Hirano, Yuuichi; Takeuchi, Osamu; Shigekawa, Hidemi

    2016-08-01

    Dynamic force spectroscopy (DFS) based on atomic force microscopy, which enables us to obtain information on the interaction potential between molecules such as antigen-antibody complexes at the single-molecule level, is a key technique for advancing molecular science and technology. However, to ensure the reliability of DFS measurement, its basic mechanism must be well understood. We examined the effect of the molecular linker used to fix the target molecule to the atomic force microscope cantilever, i.e., the force direction during measurement, for the first time, which has not been discussed until now despite its importance. The effect on the lifetime and barrier position, which can be obtained by DFS, was found to be ˜10 and ˜50%, respectively, confirming the high potential of DFS.

  11. How does the molecular linker in dynamic force spectroscopy affect probing molecular interactions at the single-molecule level?

    NASA Astrophysics Data System (ADS)

    Taninaka, Atsushi; Aizawa, Kota; Hanyu, Tatsuya; Hirano, Yuuichi; Takeuchi, Osamu; Shigekawa, Hidemi

    2016-08-01

    Dynamic force spectroscopy (DFS) based on atomic force microscopy, which enables us to obtain information on the interaction potential between molecules such as antigen–antibody complexes at the single-molecule level, is a key technique for advancing molecular science and technology. However, to ensure the reliability of DFS measurement, its basic mechanism must be well understood. We examined the effect of the molecular linker used to fix the target molecule to the atomic force microscope cantilever, i.e., the force direction during measurement, for the first time, which has not been discussed until now despite its importance. The effect on the lifetime and barrier position, which can be obtained by DFS, was found to be ∼10 and ∼50%, respectively, confirming the high potential of DFS.

  12. Structural Changes of a Doubly Spin-Labeled Chemically Driven Molecular Shuttle Probed by PELDOR Spectroscopy.

    PubMed

    Franchi, Paola; Bleve, Valentina; Mezzina, Elisabetta; Schäfer, Christian; Ragazzon, Giulio; Albertini, Marco; Carbonera, Donatella; Credi, Alberto; Di Valentin, Marilena; Lucarini, Marco

    2016-06-20

    Gaining detailed information on the structural rearrangements associated with stimuli-induced molecular movements is of utmost importance for understanding the operation of molecular machines. Pulsed electron-electron double resonance (PELDOR) was employed to monitor the geometrical changes arising upon chemical switching of a [2]rotaxane that behaves as an acid-base-controlled molecular shuttle. To this aim, the rotaxane was endowed with stable nitroxide radical units in both the ring and axle components. The combination of PELDOR data and molecular dynamic calculations indicates that in the investigated rotaxane, the ring displacement along the axle, caused by the addition of a base, does not alter significantly the distance between the nitroxide labels, but it is accompanied by a profound change in the geometry adopted by the macrocycle. PMID:27123774

  13. Measurement of molecular length of self-assembled monolayer probed by localized surface plasmon resonance

    NASA Astrophysics Data System (ADS)

    Ito, Juri; Kajikawa, Kotaro

    2016-02-01

    We propose a method to measure the variation of the molecular length of self-assembled monolayers (SAMs) when it is exposed to solutions at different pH conditions. The surface immobilized gold nanospheres (SIGNs) shows strong absorption peak at the wavelengths of 600-800 nm when p-polarized light is illuminated. The peak wavelength depends on the length of the gap distance between the SIGNs and the substrate. The gap is supported by the SAM molecules. According to the analytical calculation based on multiple expansion, the relation between the peak wavelength of the SIGN structures and the gap distance is calculated, to evaluate the molecular length of the SAM through the optical absorption spectroscopy for the SIGN structures. The molecular length of the SIGN structure was measured in air, water, acidic, and basic solutions. It was found that the molecular lengths are longer in acidic solutions.

  14. Probing structure and phase-transitions in molecular crystals by terahertz time-domain spectroscopy

    NASA Astrophysics Data System (ADS)

    Franz, Morten; Fischer, Bernd M.; Walther, Markus

    2011-12-01

    Since the introduction of ultra-fast laser techniques for the generation and detection of broadband terahertz pulses, terahertz time-domain spectroscopy has become a versatile tool for vibrational spectroscopy of molecular systems in the far-infrared. Due to their highly collective and delocalized character vibrational modes in this part of the spectrum are highly sensitive to molecular structure and arrangement within a molecular crystal. Here we utilize this sensitivity to differentiate between the enantiopure amino acid L-cysteine and its racemic crystalline DL-form. Using terahertz time-domain spectroscopy we are able to observe temperature induced solid-state phase transitions in polycrystalline DL-cysteine, as well as in polycrystalline benzoic acid. The dynamics of the transitions is studied by tracing the temperature dependency of spectral features that are assigned to certain conformational phases.

  15. Complexation of tetrandrine with calcium ion probed by various spectroscopic methods and molecular modeling

    NASA Astrophysics Data System (ADS)

    Stanculescu, Ioana; Mandravel, Cristina; Landy, David; Woisel, Patrice; Surpateanu, Gheorghe

    2003-07-01

    The formation of the complex between tetrandrine and the calcium ion, in solution, was studied using FTIR, UV-Vis, 1H NMR, 13C NMR and electrospray mass spectroscopy spectroscopic methods and molecular modeling. The calcium salts used were: Ca(ClO 4) 2·4H 2O and Ca(Picrate) 2 in the solvents: acetonitrile (CH 3CN), deuterated acetonitrile (CD 3CN) and tetrahydrofurane (THF). The determined complex stability constant was: 20277±67 dm 3 mol -1 and corresponding free energy Δ G0=-5.820±0.002 kcal mol -1. The molecular simulation of the complex formation with the MM3 Augmented force field integrated in CAChe provided useful data about its energy. Combining the experimental results and molecular modeling we propose a model for the structure of tetrandrine-Ca complex with an eight coordinated geometry.

  16. Molecular imaging of hepatocellular carcinoma xenografts with epidermal growth factor receptor targeted affibody probes.

    PubMed

    Zhao, Ping; Yang, Xiaoyang; Qi, Shibo; Liu, Hongguang; Jiang, Han; Hoppmann, Susan; Cao, Qizhen; Chua, Mei-Sze; So, Samuel K; Cheng, Zhen

    2013-01-01

    Hepatocellular carcinoma (HCC) is a highly aggressive and lethal cancer. It is typically asymptomatic at the early stage, with only 10%-20% of HCC patients being diagnosed early enough for appropriate surgical treatment. The delayed diagnosis of HCC is associated with limited treatment options and much lower survival rates. Therefore, the early and accurate detection of HCC is crucial to improve its currently dismal prognosis. The epidermal growth factor receptor (EGFR) has been reported to be involved in HCC tumorigenesis and to represent an attractive target for HCC imaging and therapy. In this study, an affibody molecule, Ac-Cys-ZEGFR:1907, targeting the extracellular domain of EGFR, was used for the first time to assess its potential to detect HCC xenografts. By evaluating radio- or fluorescent-labeled Ac-Cys-ZEGFR:1907 as a probe for positron emission tomography (PET) or optical imaging of HCC, subcutaneous EGFR-positive HCC xenografts were found to be successfully imaged by the PET probe. Thus, affibody-based PET imaging of EGFR provides a promising approach for detecting HCC in vivo. PMID:23710458

  17. Carbon-fiber tips for scanning probe microscopes and molecular electronics experiments.

    PubMed

    Rubio-Bollinger, Gabino; Castellanos-Gomez, Andres; Bilan, Stefan; Zotti, Linda A; Arroyo, Carlos R; Agraït, Nicolás; Cuevas, Juan Carlos

    2012-01-01

    We fabricate and characterize carbon-fiber tips for their use in combined scanning tunneling and force microscopy based on piezoelectric quartz tuning fork force sensors. An electrochemical fabrication procedure to etch the tips is used to yield reproducible sub-100-nm apex. We also study electron transport through single-molecule junctions formed by a single octanethiol molecule bonded by the thiol anchoring group to a gold electrode and linked to a carbon tip by the methyl group. We observe the presence of conductance plateaus during the stretching of the molecular bridge, which is the signature of the formation of a molecular junction.

  18. ATOMIC AND MOLECULAR PHYSICS: Analysis of femtosecond laser ionization/dissociation of polyatomic molecule C6H10O from one-colour pump-probe measurement

    NASA Astrophysics Data System (ADS)

    Hu, Fei-Fei; Zhou, Sheng-Peng; Hu, Zhan; Jin, Ming-Xing; Zhang, Dong-Dong; Wu, Di; Cheng, Xi-Hui; Jiang, Dian-Wu; Ding, Da-Jun

    2009-04-01

    This paper reports that a one-colour fs pump-probe measurement has been carried out for studying photoionization/photodissociation of cyclohexanone (C6H10O) in intense laser field. Two of the fragments from cyclohexanone, C2H3+ and C3H3+, are studied under 800 nm laser pump-probe and the results obtained show similar time evolutions. It proposes a feasible model for analysing the experimental observations of the one-colour fs pump-probe measurement. The results demonstrate that as an intermediate product, the excited molecular parent ions play a very important role in photionization/photodissociation processes in intense laser field.

  19. Which Microbial Communities Are Present? Importance of Selecting Appropriate Primers and Probes for Use in Molecular Microbiological Methods (MMM) in Oilfields

    NASA Astrophysics Data System (ADS)

    Sørensen, Ketil Bernt

    Molecular microbiology techniques play an increasing role in the oil industry. Most of the current applications are based on either Fluorescence in situ Hybridisation (FISH) or polymerase chain reaction (PCR) or some variation thereof. These types of approaches require the use of oligonucleotide primers and probes (i.e. short fragments of DNA that are complementary to the target DNA/RNA of the microorganism of interest). In the case of FISH, the probes are fluorescently labelled in order to identify the target cells. Before undertaking either FISH or PCR approaches, it is important to select the most appropriate primers or probes for targeting the microorganisms of interest in a given environment.

  20. Digoxigenin-labelled molecular probe for the simultaneous detection of three potato pathogens: potato spindle tuber viroid (PSTVd), potato virus Y (PVY), and potato leafroll virus (PLRV).

    PubMed

    Wełnicki, M; Zekanowski, C; Zagórski, W

    1994-01-01

    A molecular probe, p3POT, was constructed of PSTVd, PVY, PLRV cDNA fragments introduced into pUC18 vector. Sequencing of the inserts revealed that cloned fragments covered conservative parts of pathogenic genomes. Dot-blot hybridization of digoxigenin-labelled construct to crude extracts from plants infected with different potato viruses proved high sensitivity and specificity of the p3POT probe. This makes p3POT probe an useful tool for the routine testing, and selection of virus-free potatoes. PMID:7732766

  1. Microfluidic technology platforms for synthesizing, labeling and measuring the kinetics of transport and biochemical reactions for developing molecular imaging probes

    SciTech Connect

    Phelps, Michael E.

    2009-09-01

    Radiotracer techniques are used in environmental sciences, geology, biology and medicine. Radiotracers with Positron Emission Tomography (PET) provided biological examinations of ~3 million patients 2008. Despite the success of positron labeled tracers in many sciences, there is limited access in an affordable and convenient manner to develop and use new tracers. Integrated microfluidic chips are a new technology well matched to the concentrations of tracers. Our goal is to develop microfluidic chips and new synthesis approaches to enable wide dissemination of diverse types of tracers at low cost, and to produce new generations of radiochemists for which there are many unfilled jobs. The program objectives are to: 1. Develop an integrated microfluidic platform technology for synthesizing and 18F-labeling diverse arrays of different classes of molecules. 2. Incorporate microfluidic chips into small PC controlled devices (“Synthesizer”) with a platform interfaced to PC for electronic and fluid input/out control. 3. Establish a de-centralized model with Synthesizers for discovering and producing molecular imaging probes, only requiring delivery of inexpensive [18F]fluoride ion from commercial PET radiopharmacies vs the centralized approach of cyclotron facilities synthesizing and shipping a few different types of 18F-probes. 4. Develop a position sensitive avalanche photo diode (PSAPD) camera for beta particles embedded in a microfluidic chip for imaging and measuring transport and biochemical reaction rates to valid new 18F-labeled probes in an array of cell cultures. These objectives are met within a research and educational program integrating radio-chemistry, synthetic chemistry, biochemistry, engineering and biology in the Crump Institute for Molecular Imaging. The Radiochemistry Training Program exposes PhD and post doctoral students to molecular imaging in vitro in cells and microorganisms in microfluidic chips and in vivo with PET, from new technologies

  2. A probe to study the toxic interaction of tartrazine with bovine hemoglobin at the molecular level.

    PubMed

    Li, Yating; Wei, Haoran; Liu, Rutao

    2014-03-01

    Tartrazine is an artificial azo dye commonly used in food products, but tartrazine in the environment is potentially harmful. The toxic interaction between tartrazine and bovine hemoglobin (BHb) was investigated using fluorescence, synchronous fluorescence, UV-vis absorption, circular dichroism (CD) and molecular modeling techniques under simulated physiological conditions. The fluorescence data showed that tartrazine can bind with BHb to form a complex. The binding process was a spontaneous molecular interaction, in which van der Waals' forces and hydrogen bonds played major roles. Molecular docking results showed that the hydrogen bonds exist between the oxygen atoms at position 31 of tartrazine and the nitrogen atom NZ7 on Lys99, and also between the oxygen atoms at position 15 of tartrazine and the nitrogen atom NZ7 on Lys104, Lys105. The results of UV-vis and CD spectra revealed that tartrazine led to conformational changes in BHb, including loosening of the skeleton structure and decreasing α helix in the secondary structure. The synchronous fluorescence experiment revealed that tartrazine binds into the hemoglobin central cavity, and this was verified using a molecular modeling study. PMID:23653408

  3. Genetic engineered molecular imaging probes for applications in cell therapy: emphasis on MRI approach

    PubMed Central

    Cho, In K; Wang, Silun; Mao, Hui; Chan, Anthony WS

    2016-01-01

    Recent advances in stem cell-based regenerative medicine, cell replacement therapy, and genome editing technologies (i.e. CRISPR-Cas 9) have sparked great interest in in vivo cell monitoring. Molecular imaging promises a unique approach to noninvasively monitor cellular and molecular phenomena, including cell survival, migration, proliferation, and even differentiation at the whole organismal level. Several imaging modalities and strategies have been explored for monitoring cell grafts in vivo. We begin this review with an introduction describing the progress in stem cell technology, with a perspective toward cell replacement therapy. The importance of molecular imaging in reporting and assessing the status of cell grafts and their relation to the local microenvironment is highlighted since the current knowledge gap is one of the major obstacles in clinical translation of stem cell therapy. Based on currently available imaging techniques, we provide a brief discussion on the pros and cons of each imaging modality used for monitoring cell grafts with particular emphasis on magnetic resonance imaging (MRI) and the reporter gene approach. Finally, we conclude with a comprehensive discussion of future directions of applying molecular imaging in regenerative medicine to emphasize further the importance of correlating cell graft conditions and clinical outcomes to advance regenerative medicine. PMID:27766183

  4. Molecular imaging of a cancer-targeting theragnostics probe using a nucleolin aptamer- and microRNA-221 molecular beacon-conjugated nanoparticle.

    PubMed

    Kim, Jin Kyeoung; Choi, Kyung-Ju; Lee, Minhyung; Jo, Mi-hee; Kim, Soonhag

    2012-01-01

    MicroRNAs (miRNA, miR) have been reported as cancer biomarkers that regulate tumor suppressor genes. Hence, simultaneous detecting and inhibiting of miRNA function will be useful as a cancer theragnostics probe to minimize side effects and invasiveness. In this study, we developed a cancer-targeting therangostics probe in a single system using an AS1411 aptamer - and miRNA-221 molecular beacon (miR-221 MB)-conjugated magnetic fluorescence (MF) nanoparticle (MFAS miR-221 MB) to simultaneously target to cancer tissue, image intracellularly expressed miRNA-221 and treat miRNA-221-involved carcinogenesis. AS1411 aptamer-conjugated MF (MFAS) nanoparticles displayed a great selectivity and delivery into various cancer cell lines. The miR-221 MB detached from the MFAS miR-221 MB in the cytoplasm of C6 cells clearly imaged miRNA-221 biogenesis and simultaneously resulted in antitumor therapeutic effects by inhibiting miRNA function, indicating a successful astrocytoma-targeting theragnostics. MFAS miRNA MB can be easily applied to other cancers by simply changing a targeted miRNA highly expressed in cancers.

  5. Semiconductor quantum dots as fluorescent probes for in vitro and in vivo bio-molecular and cellular imaging

    PubMed Central

    Rizvi, Sarwat B.; Ghaderi, Shirin; Keshtgar, Mo; Seifalian, Alexander M.

    2010-01-01

    Over the years, biological imaging has seen many advances, allowing scientists to unfold many of the mysteries surrounding biological processes. The ideal imaging resolution would be in nanometres, as most biological processes occur at this scale. Nanotechnology has made this possible with functionalised nanoparticles that can bind to specific targets and trace processes at the cellular and molecular level. Quantum dots (QDs) or semiconductor nanocrystals are luminescent particles that have the potential to be the next generation fluorophores. This paper is an overview of the basics of QDs and their role as fluorescent probes for various biological imaging applications. Their potential clinical applications and the limitations that need to be overcome have also been discussed. PMID:22110865

  6. Thermal desorption characterisation of molecularly imprinted polymers. Part I: A novel study using direct-probe GC-MS analysis.

    PubMed

    Cummins, Wayne; Duggan, Patrick; McLoughlin, Peter

    2008-06-01

    A novel thermal desorption technique using a direct-probe device (Chromatoprobe) attached to a gas chromatograph-mass spectrometer is presented for the thermal pretreatment, characterisation and analysis of molecularly imprinted polymers. The technique is demonstrated as effective for the removal of volatile materials, including template and unreacted monomers, from methacrylic acid-ethylene glycol dimethacrylate copolymers imprinted with 2-aminopyridine. Mass spectrometry is a powerful technique for polymer bleed characterisation. Thermal desorption studies on reloaded template and related compounds are reported as a means of assessing polymer morphology, specific binding by imprinted polymers compared with reference non-imprinted polymers and selective binding by an imprinted polymer for its template. Calibration studies on the thermal desorption technique using an internal standard are presented with R(2) > 0.999. The technique provides a novel method for assessment of polymer thermal stability, composition and morphology.

  7. Probing surface and interfacial molecular structures of a rubbery adhesion promoter using sum frequency generation vibrational spectroscopy

    NASA Astrophysics Data System (ADS)

    Fang, Yong; Li, Bolin; Yu, Jincheng; Zhou, Jie; Xu, Xin; Shao, Wei; Lu, Xiaolin

    2013-09-01

    The molecular structures of an adhesion promoter, polybutadiene-modified epoxy (PBME) rubber at surfaces and buried interfaces with gold (Au) were studied using sum frequency generation (SFG) vibrational spectroscopy. The SFG spectra showed that the soft butadiene part of PBME can segregate to the surfaces and buried interfaces in two base formulations. This is consistent with its application as an adhesion promoter. For the first time, the orientation of the segregated vinyl methylene groups of PBME at the surface and buried interface was evaluated. We found that the vinyl methylene groups at the surface were highly tilted and twisted by quantitative analysis; while the vinyl methylene groups at the buried Au interface were highly tilted by qualitative estimation. Furthermore, this study confirms that the sandwiched-face-down experimental setup can be employed to study the buried interfaces. This could be developed into a standard way to probe the buried interfaces between the commercialized resins and metal substrates.

  8. Probing the Nanosecond Dynamics of a Designed Three-Stranded Beta-Sheet with a Massively Parallel Molecular Dynamics Simulation

    PubMed Central

    Voelz, Vincent A.; Luttmann, Edgar; Bowman, Gregory R.; Pande, Vijay S.

    2009-01-01

    Recently a temperature-jump FTIR study of a designed three-stranded sheet showing a fast relaxation time of ~140 ± 20 ns was published. We performed massively parallel molecular dynamics simulations in explicit solvent to probe the structural events involved in this relaxation. While our simulations produce similar relaxation rates, the structural ensemble is broad. We observe the formation of turn structure, but only very weak interaction in the strand regions, which is consistent with the lack of strong backbone-backbone NOEs in previous structural NMR studies. These results suggest that either DPDP-II folds at time scales longer than 240 ns, or that DPDP-II is not a well-defined three-stranded β-sheet. This work also provides an opportunity to compare the performance of several popular forcefield models against one another. PMID:19399235

  9. Fluorescence molecular probes for sensitive point detection of amyloid fibrils and protofibrils

    NASA Astrophysics Data System (ADS)

    Lindgren, Mikael; Jonsson, Per; Sörgjerd, Karin; Hammarström, Per

    2005-10-01

    Protein based infections such as prion diseases have lately attracted a large amount of interest, primarily due to the Mad Cow Epidemic in Great Britain, and the increase of Alzheimer's disease and related diseases in the ageing Western society. Infective proteins are very stable and almost untraceable prior to infection making them ideal as biological weapons. Particularly if the used agent is of human origin, the immunoresponse can be avoided, leaving no trace of the infectious agent. The transient nature of infectious oligomeric intermediates of misfolded proteins or peptide fragments that later matures into fibrillar aggregates makes them hard to study, and methods to detect and study these species are sparse. There exist a number of fluorescent probes that bind specifically to protein amyloidic structures. Thioflavins (ThT, ThS), Congo and Nile red, 4-(dicyanovinyl)-julolidine (DCVJ), as well as derivatives amino-8-naphtalene sulphonate (ANS, Bis-ANS) which are known to bind to the fibrillar or pre-fibrillar states with dissociation constants of typically 1 - 20 μM. Here, transthyretin (TTR), insulin and lysozyme were used as model proteins to detect different amyloid precursor states for diseases such as senile systemic amyloidosis, familial amyloidotic polyneuropathy (FAP) and iatrogenic amyloidosis. Specifically, the probes were employed in static assays to characterize protofibrillar and mature amyloid fibrillar states using steady state and time-resolved fluorescence techniques. Particularly, we investigate and report on the possibility to detect protofibrillar states at low concentration levels using modern fluorescence array detector systems in conjunction with lasers operating in the blue or ultraviolett wavelengths as excitation source. Results of ANS, ThT and a ThT analogue (abbreviated ThC) are discussed.

  10. Planck intermediate results. XXXV. Probing the role of the magnetic field in the formation of structure in molecular clouds

    NASA Astrophysics Data System (ADS)

    Planck Collaboration; Ade, P. A. R.; Aghanim, N.; Alves, M. I. R.; Arnaud, M.; Arzoumanian, D.; Ashdown, M.; Aumont, J.; Baccigalupi, C.; Banday, A. J.; Barreiro, R. B.; Bartolo, N.; Battaner, E.; Benabed, K.; Benoît, A.; Benoit-Lévy, A.; Bernard, J.-P.; Bersanelli, M.; Bielewicz, P.; Bock, J. J.; Bonavera, L.; Bond, J. R.; Borrill, J.; Bouchet, F. R.; Boulanger, F.; Bracco, A.; Burigana, C.; Calabrese, E.; Cardoso, J.-F.; Catalano, A.; Chiang, H. C.; Christensen, P. R.; Colombo, L. P. L.; Combet, C.; Couchot, F.; Crill, B. P.; Curto, A.; Cuttaia, F.; Danese, L.; Davies, R. D.; Davis, R. J.; de Bernardis, P.; de Rosa, A.; de Zotti, G.; Delabrouille, J.; Dickinson, C.; Diego, J. M.; Dole, H.; Donzelli, S.; Doré, O.; Douspis, M.; Ducout, A.; Dupac, X.; Efstathiou, G.; Elsner, F.; Enßlin, T. A.; Eriksen, H. K.; Falceta-Gonçalves, D.; Falgarone, E.; Ferrière, K.; Finelli, F.; Forni, O.; Frailis, M.; Fraisse, A. A.; Franceschi, E.; Frejsel, A.; Galeotta, S.; Galli, S.; Ganga, K.; Ghosh, T.; Giard, M.; Gjerløw, E.; González-Nuevo, J.; Górski, K. M.; Gregorio, A.; Gruppuso, A.; Gudmundsson, J. E.; Guillet, V.; Harrison, D. L.; Helou, G.; Hennebelle, P.; Henrot-Versillé, S.; Hernández-Monteagudo, C.; Herranz, D.; Hildebrandt, S. R.; Hivon, E.; Holmes, W. A.; Hornstrup, A.; Huffenberger, K. M.; Hurier, G.; Jaffe, A. H.; Jaffe, T. R.; Jones, W. C.; Juvela, M.; Keihänen, E.; Keskitalo, R.; Kisner, T. S.; Knoche, J.; Kunz, M.; Kurki-Suonio, H.; Lagache, G.; Lamarre, J.-M.; Lasenby, A.; Lattanzi, M.; Lawrence, C. R.; Leonardi, R.; Levrier, F.; Liguori, M.; Lilje, P. B.; Linden-Vørnle, M.; López-Caniego, M.; Lubin, P. M.; Macías-Pérez, J. F.; Maino, D.; Mandolesi, N.; Mangilli, A.; Maris, M.; Martin, P. G.; Martínez-González, E.; Masi, S.; Matarrese, S.; Melchiorri, A.; Mendes, L.; Mennella, A.; Migliaccio, M.; Miville-Deschênes, M.-A.; Moneti, A.; Montier, L.; Morgante, G.; Mortlock, D.; Munshi, D.; Murphy, J. A.; Naselsky, P.; Nati, F.; Netterfield, C. B.; Noviello, F.; Novikov, D.; Novikov, I.; Oppermann, N.; Oxborrow, C. A.; Pagano, L.; Pajot, F.; Paladini, R.; Paoletti, D.; Pasian, F.; Perotto, L.; Pettorino, V.; Piacentini, F.; Piat, M.; Pierpaoli, E.; Pietrobon, D.; Plaszczynski, S.; Pointecouteau, E.; Polenta, G.; Ponthieu, N.; Pratt, G. W.; Prunet, S.; Puget, J.-L.; Rachen, J. P.; Reinecke, M.; Remazeilles, M.; Renault, C.; Renzi, A.; Ristorcelli, I.; Rocha, G.; Rossetti, M.; Roudier, G.; Rubiño-Martín, J. A.; Rusholme, B.; Sandri, M.; Santos, D.; Savelainen, M.; Savini, G.; Scott, D.; Soler, J. D.; Stolyarov, V.; Sudiwala, R.; Sutton, D.; Suur-Uski, A.-S.; Sygnet, J.-F.; Tauber, J. A.; Terenzi, L.; Toffolatti, L.; Tomasi, M.; Tristram, M.; Tucci, M.; Umana, G.; Valenziano, L.; Valiviita, J.; Van Tent, B.; Vielva, P.; Villa, F.; Wade, L. A.; Wandelt, B. D.; Wehus, I. K.; Ysard, N.; Yvon, D.; Zonca, A.

    2016-02-01

    Within ten nearby (d < 450 pc) Gould belt molecular clouds we evaluate statistically the relative orientation between the magnetic field projected on the plane of sky, inferred from the polarized thermal emission of Galactic dust observed by Planck at 353 GHz, and the gas column density structures, quantified by the gradient of the column density, NH. The selected regions, covering several degrees in size, are analysed at an effective angular resolution of 10' FWHM, thus sampling physical scales from 0.4 to 40 pc in the nearest cloud. The column densities in the selected regions range from NH≈ 1021 to1023 cm-2, and hence they correspond to the bulk of the molecular clouds. The relative orientation is evaluated pixel by pixel and analysed in bins of column density using the novel statistical tool called "histogram of relative orientations". Throughout this study, we assume that the polarized emission observed by Planck at 353 GHz is representative of the projected morphology of the magnetic field in each region, i.e., we assume a constant dust grain alignment efficiency, independent of the local environment. Within most clouds we find that the relative orientation changes progressively with increasing NH, from mostly parallel or having no preferred orientation to mostly perpendicular. In simulations of magnetohydrodynamic turbulence in molecular clouds this trend in relative orientation is a signature of Alfvénic or sub-Alfvénic turbulence, implying that the magnetic field is significant for the gas dynamics at the scales probed by Planck. We compare the deduced magnetic field strength with estimates we obtain from other methods and discuss the implications of the Planck observations for the general picture of molecular cloud formation and evolution.

  11. Molecular hydrogen physisorption on boron-nitride nanotubes probed by second harmonic generation

    NASA Astrophysics Data System (ADS)

    Salazar-Aparicio, R. V.; Vázquez-Nava, R. A.; Arzate, N.; Mendoza, B. S.

    2014-10-01

    We present ab initio calculations to investigate second harmonic generation (SHG) response of single wall zigzag pristine boron-nitride nanotubes (BNNTs) and BNNTs modified by the molecular hydrogen adsorption. Calculations have been performed using density functional theory (DFT) within the local-density approximation (LDA) together with the GW Green function method to determine the band gap. A length gauge approach has been used to calculate the nonlinear optical response with the scissors correction to obtain the nonlinear susceptibility χzzz(-2ω ;ω,ω) of the zigzag BNNTs. We have found that, contrary to reports in the literature, the (5,0) and (9,0) BNNTs have a nonvanishing SHG response. We have also found that SHG intensity decreases with the increase of the molecular hydrogen coverage.

  12. Molecular mechanisms of ionic liquid cytotoxicity probed by an integrated experimental and computational approach

    DOE PAGESBeta

    Yoo, Brian; Jing, Benxin; Jones, Stuart E.; Lamberti, Gary A.; Zhu, Yingxi; Shah, Jindal K.; Maginn, Edward J.

    2016-02-02

    Ionic liquids (ILs) are salts that remain liquid down to low temperatures, and sometimes well below room temperature. ILs have been called “green solvents” because of their extraordinarily low vapor pressure and excellent solvation power, but ecotoxicology studies have shown that some ILs exhibit greater toxicity than traditional solvents. A fundamental understanding of the molecular mechanisms responsible for IL toxicity remains elusive. Here we show that one mode of IL toxicity on unicellular organisms is driven by swelling of the cell membrane. Cytotoxicity assays, confocal laser scanning microscopy, and molecular simulations reveal that IL cations nucleate morphological defects in themore » microbial cell membrane at concentrations near the half maximal effective concentration (EC50) of several microorganisms. Lastly, cytotoxicity increases with increasing alkyl chain length of the cation due to the ability of the longer alkyl chain to more easily embed in, and ultimately disrupt, the cell membrane.« less

  13. Molecular mechanisms of ionic liquid cytotoxicity probed by an integrated experimental and computational approach

    PubMed Central

    Yoo, Brian; Jing, Benxin; Jones, Stuart E.; Lamberti, Gary A.; Zhu, Yingxi; Shah, Jindal K.; Maginn, Edward J.

    2016-01-01

    Ionic liquids (ILs) are salts that remain liquid down to low temperatures, and sometimes well below room temperature. ILs have been called “green solvents” because of their extraordinarily low vapor pressure and excellent solvation power, but ecotoxicology studies have shown that some ILs exhibit greater toxicity than traditional solvents. A fundamental understanding of the molecular mechanisms responsible for IL toxicity remains elusive. Here we show that one mode of IL toxicity on unicellular organisms is driven by swelling of the cell membrane. Cytotoxicity assays, confocal laser scanning microscopy, and molecular simulations reveal that IL cations nucleate morphological defects in the microbial cell membrane at concentrations near the half maximal effective concentration (EC50) of several microorganisms. Cytotoxicity increases with increasing alkyl chain length of the cation due to the ability of the longer alkyl chain to more easily embed in, and ultimately disrupt, the cell membrane. PMID:26831599

  14. GAS PHASE MOLECULAR DYNAMICS: HIGH-RESOLUTION SPECTROSCOPIC PROBES OF CHEMICAL DYNAMICS.

    SciTech Connect

    HALL, G.E.

    2006-05-30

    This research is carried out as part of the Gas Phase Molecular Dynamics group program in the Chemistry Department at Brookhaven National Laboratory. High-resolution spectroscopic tools are developed and applied to problems in chemical dynamics. Recent topics have included the state-resolved studies of collision-induced electronic energy transfer, dynamics of barrierless unimolecular reactions, and the kinetics and spectroscopy of transient species.

  15. Fluorescent Resonance Energy Transfer: A Tool for Probing Molecular Cell-Biomaterial Interactions in Three Dimensions

    PubMed Central

    Huebsch, Nathaniel D.; Mooney, David J.

    2007-01-01

    The current paradigm in designing biomaterials is to optimize material chemical and physical parameters based on correlations between these parameters and downstream biological responses, whether in vitro or in vivo. Extensive developments in molecular design of biomaterials have facilitated identification of several biophysical and biochemical variables (e.g. adhesion peptide density, substrate elastic modulus) as being critical to cell response. However, these empirical observations do not indicate whether different parameters elicit cell responses by modulating redundant variables of the cell-material interface (e.g. number of cell-material bonds, cell-matrix mechanics). Recently, a molecular fluorescence technique, Fluorescence Resonance Energy Transfer (FRET) has been applied to quantitatively analyze parameters of the cell-material interface for both two and three-dimensional adhesion substrates. Tools based on FRET have been utilized to quantify several parameters of the cell-material interface relevant to cell response, including molecular changes in matrix proteins induced by interactions both with surfaces and cells, the number of bonds between integrins and their adhesion ligands, and changes in the crosslink density of hydrogel synthetic extracellular matrix analogs. As such techniques allow both dynamic and 3D analyses they will be useful to quantitatively relate downstream cellular responses (e.g. gene expression) to the composition of this interface. Such understanding will allow bioengineers to fully exploit the potential of biomaterials engineered on the molecular scale, by optimizing material chemical and physical properties to a measurable set of interfacial parameters known to elicit a predictable response in a specific cell population. This will facilitate the rational design of complex, multi-functional biomaterials used as model systems for studying diseases or for clinical applications. PMID:17270268

  16. A highly discriminating quencher-free molecular beacon for probing DNA.

    PubMed

    Hwang, Gil Tae; Seo, Young Jun; Kim, Byeang Hyean

    2004-06-01

    We inserted a fluorene-labeled deoxyuridine derivative, synthesized using Sonogashira coupling, efficiently into the loop region of a DNA hairpin using phosphoramidite chemistry. This molecular beacon, which features no additional fluorescence quencher, discriminates between perfect and one-base-mismatched base pairing by changes in its fluorescence intensity. The discrimination factor is 14.7 for the recognition of a single (A/C) base mismatch.

  17. The `Carina Flare' supershell: probing the atomic and molecular ISM in a Galactic chimney

    NASA Astrophysics Data System (ADS)

    Dawson, J. R.; Mizuno, N.; Onishi, T.; McClure-Griffiths, N. M.; Fukui, Y.

    2008-06-01

    The `Carina Flare' supershell, GSH 287+04-17, is a molecular supershell originally discovered in 12CO(J = 1-0) with the NANTEN 4m telescope. We present the first study of the shell's atomic ISM, using HI 21-cm line data from the Parkes 64-m telescope Southern Galactic Plane Survey. The data reveal a gently expanding, ~230 × 360 pc HI supershell that shows strong evidence of Galactic Plane blowout, with a break in its main body at z ~ 280 pc and a capped high-latitude extension reaching z ~ 450 pc. The molecular clouds form comoving parts of the atomic shell, and the morphology of the two phases reflects the supershell's influence on the structure of the ISM. We also report the first discovery of an ionized component of the supershell, in the form of delicate, streamer-like filaments aligned with the proposed direction of blowout. The distance estimate to the shell is re-examined, and we find strong evidence to support the original suggestion that it is located in the Carina Arm at a distance of 2.6 +/- 0.4 kpc. Associated HI and H2 masses are estimated as MHI ~ 7 +/- 3 × 105Msolar and , and the kinetic energy of the expanding shell as EK ~ 1 × 1051 erg. We examine the results of analytical and numerical models to estimate a required formation energy of several 1051 to ~1052 erg, and an age of ~107 yr. This age is compatible with molecular cloud formation time-scales, and we briefly consider the viability of a supershell-triggered origin for the molecular component.

  18. Molecular adsorbates as probes of the local properties of doped graphene

    PubMed Central

    Pham, Van Dong; Joucken, Frédéric; Repain, Vincent; Chacon, Cyril; Bellec, Amandine; Girard, Yann; Rousset, Sylvie; Sporken, Robert; Santos, Maria Cristina dos; Lagoute, Jérôme

    2016-01-01

    Graphene-based sensors are among the most promising of graphene’s applications. The ability to signal the presence of molecular species adsorbed on this atomically thin substrate has been explored from electric measurements to light scattering. Here we show that the adsorbed molecules can be used to sense graphene properties. The interaction of porphyrin molecules with nitrogen-doped graphene has been investigated using scanning tunneling microscopy and ab initio calculations. Molecular manipulation was used to reveal the surface below the adsorbed molecules allowing to achieve an atomic-scale measure of the interaction of molecules with doped graphene. The adsorbate’s frontier electronic states are downshifted in energy as the molecule approaches the doping site, with largest effect when the molecule sits over the nitrogen dopant. Theoretical calculations showed that, due to graphene’s high polarizability, the adsorption of porphyrin induces a charge rearrangement on the substrate similar to the image charges on a metal. This charge polarization is enhanced around nitrogen site, leading to an increased interaction of molecules with their image charges on graphene. Consequently, the molecular states are stabilized and shift to lower energies. These findings reveal the local variation of polarizability induced by nitrogen dopant opening new routes towards the electronic tuning of graphene. PMID:27097555

  19. Transferring biomarker into molecular probe: melanin nanoparticle as a naturally active platform for multimodality imaging.

    PubMed

    Fan, Quli; Cheng, Kai; Hu, Xiang; Ma, Xiaowei; Zhang, Ruiping; Yang, Min; Lu, Xiaomei; Xing, Lei; Huang, Wei; Gambhir, Sanjiv Sam; Cheng, Zhen

    2014-10-29

    Developing multifunctional and easily prepared nanoplatforms with integrated different modalities is highly challenging for molecular imaging. Here, we report the successful transfer of an important molecular target, melanin, into a novel multimodality imaging nanoplatform. Melanin is abundantly expressed in melanotic melanomas and thus has been actively studied as a target for melanoma imaging. In our work, the multifunctional biopolymer nanoplatform based on ultrasmall (<10 nm) water-soluble melanin nanoparticle (MNP) was developed and showed unique photoacoustic property and natural binding ability with metal ions (for example, (64)Cu(2+), Fe(3+)). Therefore, MNP can serve not only as a photoacoustic contrast agent, but also as a nanoplatform for positron emission tomography (PET) and magnetic resonance imaging (MRI). Traditional passive nanoplatforms require complicated and time-consuming processes for prebuilding reporting moieties or chemical modifications using active groups to integrate different contrast properties into one entity. In comparison, utilizing functional biomarker melanin can greatly simplify the building process. We further conjugated αvβ3 integrins, cyclic c(RGDfC) peptide, to MNPs to allow for U87MG tumor accumulation due to its targeting property combined with the enhanced permeability and retention (EPR) effect. The multimodal properties of MNPs demonstrate the high potential of endogenous materials with multifunctions as nanoplatforms for molecular theranostics and clinical translation.

  20. Probing Molecular Recognition at the Solid-Gas Interface by Sum-Frequency Vibrational Spectroscopy.

    PubMed

    Aprile, Arianna; Ciuchi, Federica; Pinalli, Roberta; Dalcanale, Enrico; Pagliusi, Pasquale

    2016-08-01

    Molecular recognition is among the most important chemical events in living systems and has been emulated in supramolecular chemistry, driven by chemical and biochemical sensing potential. Identifying host-guest association in situ at the interface, between the substrate-bound receptors and the analyte-containing media, is essential to predict complexation performances in term of the receptor conformation, orientation and organization. Herein, we report the first sum-frequency vibrational spectroscopy study of molecular recognition at the solid-gas interface. The binding capability of tetraquinoxaline cavitands toward volatile aromatic and aliphatic compounds, namely benzonitrile and acetonitrile, is investigated as test system. We prove the selective complexation of the receptors, organized in a solid-supported hybrid bilayer, toward aromatic compounds. Quantitative analysis allows to correlate the average orientations of the guest molecules and the host binding pockets, establishing "on-axis" complexation of benzonitrile within the cavitand cavity. The study is readily applicable to other receptors, molecular architectures, interfaces and analytes. PMID:27438350

  1. The Chemistry of Interstellar Argonium and Other Probes of the Molecular Fraction in Diffuse Clouds

    NASA Astrophysics Data System (ADS)

    Neufeld, David A.; Wolfire, Mark G.

    2016-08-01

    We present a general parameter study in which the abundance of interstellar argonium (ArH+) is predicted using a model for the physics and chemistry of diffuse interstellar gas clouds. Results have been obtained as a function of UV radiation field, cosmic-ray ionization rate, and cloud extinction. No single set of cloud parameters provides an acceptable fit to the typical ArH+, OH+, and {{{H}}}2{{{O}}}+ abundances observed in diffuse clouds within the Galactic disk. Instead, the observed abundances suggest that ArH+ resides primarily in a separate population of small clouds of total visual extinction of at most 0.02 mag per cloud, within which the column-averaged molecular fraction is in the range {10}-5{--}{10}-2, while OH+ and {{{H}}}2{{{O}}}+ reside primarily in somewhat larger clouds with a column-averaged molecular fraction ˜0.2. This analysis confirms our previous suggestion that the argonium molecular ion is a unique tracer of almost purely atomic gas.

  2. Ultrafast NMR T1 Relaxation Measurements: Probing Molecular Properties in Real Time

    PubMed Central

    Smith, Pieter E. S.; Donovan, Kevin J.; Szekely, Or; Baias, Maria; Frydman, Lucio

    2016-01-01

    The longitudinal relaxation properties of NMR active nuclei carry useful information about the site-specific chemical environments and about the mobility of molecular fragments. Molecular mobility is in turn a key parameter reporting both on stable properties like size, as well as on dynamic ones such as transient interactions and irreversible aggregation. In order to fully investigate the latter, a fast sampling of the relaxation parameters of transiently formed molecular species may be needed. Nevertheless, the acquisition of longitudinal relaxation data is typically slow, being limited by the requirement that the time for which the nucleus relaxes be varied incrementally until a complete build-up curve is generated. Recently a number of single-shot inversion recovery methods have been developed capable of alleviating this need; still, these may be challenged by either spectral resolution restrictions or when coping with very fast relaxing nuclei. Here we present a new experiment to measure the T1s of multiple nuclear spins that experience fast longitudinal relaxation, while retaining full high-resolution chemical shift information. Good agreement is observed between T1s measured with conventional means and T1s measured using the new technique. The method is applied to the real time investigation of the reaction between D-xylose and sodium borate, which is in turn elucidated with the aid of ancillary ultrafast and conventional 2D TOCSY measurements. PMID:23878001

  3. Solution Phase Molecular Dynamics Probed with Synchrotron Hard X-rays

    NASA Astrophysics Data System (ADS)

    March, Anne; Doumy, Gilles; Kanter, Elliot; Southworth, Stephen; Young, Linda; Nemeth, Zoltan; Vankó, Gyorgy; Assefa, Tadesse; Gawelda, Wojciech

    2013-05-01

    The ability to measure short-lived transient states during a chemical reaction is key to understanding many important processes such as oxygen binding in hemeproteins and electron transport in photosynthesis. Time resolved hard x-ray spectroscopies, which are based on laser-pump/x-ray-probe methods, are a unique tool because unlike UV-VIS techniques they are element specific and can provide electronic and structural information with atomic resolution in the vicinity of a particular atom or ion. These characteristics make them particularly powerful for studying molecules in complex environments such as solutions. Using a MHz, picosecond, high average power laser system implemented at Sector 7ID-D of the Advanced Photon Source we have been developing time resolved x-ray emission techniques to track the evolution of photoexcited molecules in solution. We will present recent studies which include the ligand substitution reaction and hydrated electron formation in the coordination complex ferrocyanide Fe(CN)64-. This work was supported by the Chemical Sciences, Geosciences, and Biosciences Division and the Advanced Photon Source by the Office of Basic Energy Sciences, Office of Science, US Dept of Energy, Contract DE-AC02-06CH11357.

  4. Lineage-specific molecular probing reveals novel diversity and ecological partitioning of haplosporidians

    PubMed Central

    Hartikainen, Hanna; Ashford, Oliver S; Berney, Cédric; Okamura, Beth; Feist, Stephen W; Baker-Austin, Craig; Stentiford, Grant D; Bass, David

    2014-01-01

    Haplosporidians are rhizarian parasites of mostly marine invertebrates. They include the causative agents of diseases of commercially important molluscs, including MSX disease in oysters. Despite their importance for food security, their diversity and distributions are poorly known. We used a combination of group-specific PCR primers to probe environmental DNA samples from planktonic and benthic environments in Europe, South Africa and Panama. This revealed several highly distinct novel clades, novel lineages within known clades and seasonal (spring vs autumn) and habitat-related (brackish vs littoral) variation in assemblage composition. High frequencies of haplosporidian lineages in the water column provide the first evidence for life cycles involving planktonic hosts, host-free stages or both. The general absence of haplosporidian lineages from all large online sequence data sets emphasises the importance of lineage-specific approaches for studying these highly divergent and diverse lineages. Combined with host-based field surveys, environmental sampling for pathogens will enhance future detection of known and novel pathogens and the assessment of disease risk. PMID:23966100

  5. Simultaneous detection of kinase and phosphatase activities of polynucleotide kinase using molecular beacon probes.

    PubMed

    Ma, Changbei; Fang, Hefei; Wang, Kemin; Xia, Kun; Chen, Hanchun; He, Hailun; Zeng, Weimin

    2013-12-15

    Phosphorylation and dephosphorylation of DNA by polynucleotide kinase (PNK) has an important role in DNA damage repair, replication, and recombination. Traditionally, it is assayed by denaturing gel electrophoresis and autoradiography, which are tedious and not sensitive. We report on the development of a sensitive and simple method for PNK assay based on DNA ligation using a molecular beacon. Enzyme activity of PNK is measured down to a limit of 0.002 unit/ml. The method not only provides a universal platform for simultaneous monitoring of kinase and phosphatase activities, but also shows great potential in biological research, drug discovery, and clinical diagnostics.

  6. Conformational Sampling and Nucleotide-Dependent Transitions of the GroEL Subunit Probed by Unbiased Molecular Dynamics Simulations

    PubMed Central

    Skjaerven, Lars; Grant, Barry; Muga, Arturo; Teigen, Knut; McCammon, J. Andrew; Reuter, Nathalie; Martinez, Aurora

    2011-01-01

    GroEL is an ATP dependent molecular chaperone that promotes the folding of a large number of substrate proteins in E. coli. Large-scale conformational transitions occurring during the reaction cycle have been characterized from extensive crystallographic studies. However, the link between the observed conformations and the mechanisms involved in the allosteric response to ATP and the nucleotide-driven reaction cycle are not completely established. Here we describe extensive (in total long) unbiased molecular dynamics (MD) simulations that probe the response of GroEL subunits to ATP binding. We observe nucleotide dependent conformational transitions, and show with multiple 100 ns long simulations that the ligand-induced shift in the conformational populations are intrinsically coded in the structure-dynamics relationship of the protein subunit. Thus, these simulations reveal a stabilization of the equatorial domain upon nucleotide binding and a concomitant “opening” of the subunit, which reaches a conformation close to that observed in the crystal structure of the subunits within the ADP-bound oligomer. Moreover, we identify changes in a set of unique intrasubunit interactions potentially important for the conformational transition. PMID:21423709

  7. Probing irradiation induced DNA damage mechanisms using excited state Car-Parrinello molecular dynamics

    NASA Astrophysics Data System (ADS)

    Markwick, Phineus R. L.; Doltsinis, Nikos L.; Schlitter, Jürgen

    2007-01-01

    Photoinduced proton transfer in the Watson-Crick guanine (G)-cytosine (C) base pair has been studied using Car-Parrinello molecular dynamics (CP-MD). A flexible mechanical constraint acting on all three hydrogen bonds in an unbiased fashion has been devised to explore the free energy profile along the proton transfer coordinate. The lowest barrier has been found for proton transfer from G to C along the central hydrogen bond. The resulting charge transfer excited state lies energetically close to the electronic ground state suggesting the possibility of efficient radiationless decay. It is found that dynamic, finite temperature fluctuations significantly reduce the energy gap between the ground and excited states for this charge transfer product, promoting the internal conversion process. A detailed analysis of the internal degrees of freedom reveals that the energy gap is considerably reduced by out-of-plane molecular vibrations, in particular. Consequently, it appears that considering only the minimum energy path provides an upper-bound estimate of the associated energy gap compared to the full-dimension dynamical reaction coordinate. Furthermore, the first CP-MD simulations of the G-C base pair in liquid water are presented, and the effects of solvation on its electronic structure are analyzed.

  8. Building KCNQ1/KCNE1 channel models and probing their interactions by molecular-dynamics simulations.

    PubMed

    Xu, Yu; Wang, Yuhong; Meng, Xuan-Yu; Zhang, Mei; Jiang, Min; Cui, Meng; Tseng, Gea-Ny

    2013-12-01

    The slow delayed rectifier (I(KS)) channel is composed of KCNQ1 (pore-forming) and KCNE1 (auxiliary) subunits, and functions as a repolarization reserve in the human heart. Design of I(KS)-targeting anti-arrhythmic drugs requires detailed three-dimensional structures of the KCNQ1/KCNE1 complex, a task made possible by Kv channel crystal structures (templates for KCNQ1 homology-modeling) and KCNE1 NMR structures. Our goal was to build KCNQ1/KCNE1 models and extract mechanistic information about their interactions by molecular-dynamics simulations in an explicit lipid/solvent environment. We validated our models by confirming two sets of model-generated predictions that were independent from the spatial restraints used in model-building. Detailed analysis of the molecular-dynamics trajectories revealed previously unrecognized KCNQ1/KCNE1 interactions, whose relevance in I(KS) channel function was confirmed by voltage-clamp experiments. Our models and analyses suggest three mechanisms by which KCNE1 slows KCNQ1 activation: by promoting S6 bending at the Pro hinge that closes the activation gate; by promoting a downward movement of gating charge on S4; and by establishing a network of electrostatic interactions with KCNQ1 on the extracellular surface that stabilizes the channel in a pre-open activated state. Our data also suggest how KCNE1 may affect the KCNQ1 pore conductance.

  9. Probing the flexibility of tropomyosin and its binding to filamentous actin using molecular dynamics simulations.

    PubMed

    Zheng, Wenjun; Barua, Bipasha; Hitchcock-DeGregori, Sarah E

    2013-10-15

    Tropomyosin (Tm) is a coiled-coil protein that binds to filamentous actin (F-actin) and regulates its interactions with actin-binding proteins like myosin by moving between three positions on F-actin (the blocked, closed, and open positions). To elucidate the molecular details of Tm flexibility in relation to its binding to F-actin, we conducted extensive molecular dynamics simulations for both Tm alone and Tm-F-actin complex in the presence of explicit solvent (total simulation time >400 ns). Based on the simulations, we systematically analyzed the local flexibility of the Tm coiled coil using multiple parameters. We found a good correlation between the regions with high local flexibility and a number of destabilizing regions in Tm, including six clusters of core alanines. Despite the stabilization by F-actin binding, the distribution of local flexibility in Tm is largely unchanged in the absence and presence of F-actin. Our simulations showed variable fluctuations of individual Tm periods from the closed position toward the open position. In addition, we performed Tm-F-actin binding calculations based on the simulation trajectories, which support the importance of Tm flexibility to Tm-F-actin binding. We identified key residues of Tm involved in its dynamic interactions with F-actin, many of which have been found in recent mutational studies to be functionally important, and the rest of which will make promising targets for future mutational experiments.

  10. Ultrafast probing of ejection dynamics of Rydberg atoms and molecular fragments from electronically excited helium nanodroplets.

    PubMed

    Bünermann, Oliver; Kornilov, Oleg; Haxton, Daniel J; Leone, Stephen R; Neumark, Daniel M; Gessner, Oliver

    2012-12-01

    The ejection dynamics of Rydberg atoms and molecular fragments from electronically excited helium nanodroplets are studied with time-resolved extreme ultraviolet ion imaging spectroscopy. At excitation energies of 23.6 ± 0.2 eV, Rydberg atoms in n = 3 and n = 4 states are ejected on different time scales and with significantly different kinetic energy distributions. Specifically, n = 3 Rydberg atoms are ejected with kinetic energies as high as 0.85 eV, but their appearance is delayed by approximately 200 fs. In contrast, n = 4 Rydberg atoms appear within the time resolution of the experiment with considerably lower kinetic energies. Major features in the Rydberg atom kinetic energy distributions for both principal quantum numbers can be described within a simple elastic scattering model of localized perturbed atomic Rydberg atoms that are expelled from the droplet due to their repulsive interaction with the surrounding helium bath. Time-dependent kinetic energy distributions of He(2) (+) and He(3) (+) ions are presented that support the formation of molecular ions in an indirect droplet ionization process and the ejection of neutral Rydberg dimers on a similar time scale as the n = 3 Rydberg atoms.

  11. Using dSTORM to probe the molecular architecture of filopodia

    NASA Astrophysics Data System (ADS)

    Ahmed, Sohail; Chou, Amy; Sem, K. P.; Thankiah, Sudaharan; Wright, Graham; Lim, John; Hariharan, Srivats

    2014-03-01

    IRSp53 is a Cdc42 effector and a member of the Inverse-Bin-Amphiphysins-Rvs (I-BAR) domain family which can induce negative membrane curvature. IRSp53 generates filopodia by coupling membrane protrusion (I-BAR domain) with actin dynamics through its SH3 domain binding partners. Dynamin 1 (Dyn1), a large GTPase associated with endocytosis, is a novel interacting partner of IRSp53 that localises to filopodia. Using rapid time-lapse TIRF microscopy we have shown that Dyn1 localized to a subcellular region just behind Mena at the leading edge, or in filopodial tip complexes when co-expressed with IRSp53. Dyn1-GFP was strongly localized in the filopodial shaft during the early phase of elongation, after which it moved rearward, suggestive of a role in early filopodia assembly. Mena and Eps8, accumulate at the tip complex in sequence and are involved in filopodial extension and retraction, respectively (Chou at al, 2014 submitted). Here we describe the use of dSTORM to investigate the molecular architecture of filopodia and in particular the size of the F-actin bundle in these structures. The data suggest that direct Stochastic Optical Reconstruction Microscopy (dSTORM) in combination with other techniques will allow the molecular architecture of

  12. Microgravity: Molecular Dynamics Simulations at the NCCS Probe the Behavior of Liquids in Low Gravity

    NASA Technical Reports Server (NTRS)

    2002-01-01

    The life of the very small, whether in something as complicated as a human cell or as simple as a drop of water, is of fundamental scientific interest: By knowing how a tiny amount of material reacts to changes in its environment, scientists maybe able to answer questions about how a bulk of material would react to comparable changes. NASA is in the forefront of computational research into a broad range of basic scientific questions about fluid dynamics and the nature of liquid boundary instability. For example, one important issue for the space program is how drops of water and other materials will behave in the low-gravity environment of space and how the low gravity will affect the transport and containment of these materials. Accurate prediction of this behavior is among the aims of a set of molecular dynamics experiments carried out on the NCCSs Cray supercomputers. In conventional computational studies of materials, matter is treated as continuous - a macroscopic whole without regard to its molecular parts - and the behavior patterns of the matter in various physical environments are studied using well-established differential equations and mathematical parameters based on physical properties such as compressibility density, heat capacity, and vapor pressure of the bulk material.

  13. DNAzyme molecular beacon probes for target-induced signal-amplifying colorimetric detection of nucleic acids.

    PubMed

    Fu, Rongzhan; Li, Taihua; Lee, Soo Suk; Park, Hyun Gyu

    2011-01-15

    A novel DNAzyme molecular beacon (DNAzymeMB) strategy was developed for target-induced signal-amplifying colorimetric detection of target nucleic acids. The DNAzymeMB, which exhibits peroxidase activity in its free hairpin structure, was engineered to form a catalytically inactive hybrid through hybridization with a blocker DNA. The presence of target DNA leads to dissociation of the DNAzymeMB from the inactive hybrid through hybridization with the blocker DNA. This process results in recovery of the catalytically active DNAzymeMB, which can catalyze a colorimetric reaction that signals the presence of the target DNA. In addition, a primer was rationally designed to anneal to the blocker DNA of the blocker/target DNA duplex and displace the bound target DNA during the extension reaction. The released target DNA triggers the next cycle involving hybridization with blocker DNA, DNAzymeMB dissociation, primer extension, and target displacement. This unique amplifying strategy leads to the generation of multiple numbers of active DNAzymeMB molecules from a single target molecule and gives a detection limit down to 1 pM, a value that is nearly 3 or 5 orders of magnitude lower than those of previously reported DNAzyme molecular beacon-based DNA detection methods.

  14. Carotenoid radical cations as a probe for the molecular mechanism of nonphotochemical quenching in oxygenic photosynthesis.

    PubMed

    Amarie, Sergiu; Standfuss, Jörg; Barros, Tiago; Kühlbrandt, Werner; Dreuw, Andreas; Wachtveitl, Josef

    2007-04-01

    Nonphotochemical quenching (NPQ) is a fundamental mechanism in photosynthesis which protects plants against excess excitation energy and is of crucial importance for their survival and fitness. Recently, carotenoid radical cation (Car*+) formation has been discovered to be a key step for the feedback deexcitation quenching mechanism (qE), a component of NPQ, of which the molecular mechanism and location is still unknown. We have generated and characterized carotenoid radical cations by means of resonant two color, two photon ionization (R2C2PI) spectroscopy. The Car*+ bands have maxima located at 830 nm (violaxanthin), 880 nm (lutein), 900 nm (zeaxanthin), and 920 nm (beta-carotene). The positions of these maxima depend strongly on solution conditions, the number of conjugated C=C bonds, and molecular structure. Furthermore, R2C2PI measurements on the light-harvesting complex of photosystem II (LHC II) samples with or without zeaxanthin (Zea) reveal the violaxanthin (Vio) radical cation (Vio*+) band at 909 nm and the Zea*+ band at 983 nm. The replacement of Vio by Zea in the light-harvesting complex II (LHC II) has no influence on the Chl excitation lifetime, and by exciting the Chls lowest excited state, no additional rise and decay corresponding to the Car*+ signal observed previously during qE was detected in the spectral range investigated (800-1050 nm). On the basis of our findings, the mechanism of qE involving the simple replacement of Vio with Zea in LHC II needs to be reconsidered.

  15. Probing the interaction of troxerutin with transfer RNA by spectroscopic and molecular modeling.

    PubMed

    Subastri, A; Ramamurthy, C H; Suyavaran, A; Lokeswara Rao, P; Preedia Babu, E; Hari Krishna, K; Suresh Kumar, M; Thirunavukkarasu, C

    2015-12-01

    The studies on the interaction between tRNA (transfer RNA) and small molecules are an area of remarkable recent attention. For this notion a fundamental knowledge of the molecular features involving the interaction of small molecules with tRNA is crucial. Hence, in the present study we have investigated the interaction of TXER (troxerutin), natural bioflavonoid rutin derivative with yeast tRNA by using various spectroscopic techniques and molecular docking studies. The UV absorption and fluorescence emission studies demonstrated external binding of TXER on tRNA with low binding constant values as compared to strong binders. Circular dichroism (CD) spectroscopy study revealed that TXER did not show any significant modification on native conformation of tRNA. Furthermore in electrochemical study, the complex of TXER-tRNA did not expose any noticeable positive potential peak shift which indicated an interaction of TXER with tRNA by electrostatic or external binding mode. The docking study showed that the hydrogen and hydrophobic interactions were involved in binding of TXER-tRNA with docking score -7.0 kcal/mol. These findings led us to confirm the interaction of TXER on tRNA through external binding with low binding affinity, indicating its potential bioapplication in the future.

  16. Probing confinement resonances by photoionizing Xe inside a C60+ molecular cage

    NASA Astrophysics Data System (ADS)

    Phaneuf, R. A.; Kilcoyne, A. L. D.; Aryal, N. B.; Baral, K. K.; Thomas, C. M.; Esteves-Macaluso, D. A.; Lomsadze, R.; Gorczyca, T. W.; Ballance, C. P.; Manson, S. T.; Hasoglu, M. F.; Hellhund, J.; Schippers, S.; Müller, A.

    2014-05-01

    Double photoionization accompanied by loss of n C atoms (n = 0 , 2 , 4 , 6) was investigated by merging beams of Xe@C60+ ions and synchrotron radiation and measuring the yields of product ions. The giant 4 d dipole resonance of the caged Xe atom has a prominent signature in the cross section for these product channels, which together account for 6 . 2 +/- 1 . 4 of the total Xe 4 d oscillator strength of 10. Compared to that for a free Xe atom, the oscillator strength is redistributed in photon energy due to multipath interference of outgoing Xe 4 d photoelectron waves that may be transmitted or reflected by the spherical C60+ molecular cage, yielding so-called confinement resonances. The data are compared with an earlier measurement and with theoretical predictions for this single-molecule photoelectron interferometer system. Relativistic R-matrix calculations for the Xe atom in a spherical potential shell representing the fullerene cage show the sensitivity of the interference pattern to the molecular geometry.

  17. Probing confinement resonances by photoionizing Xe inside a C60+ molecular cage

    NASA Astrophysics Data System (ADS)

    Phaneuf, R. A.; Kilcoyne, A. L. D.; Aryal, N. B.; Baral, K. K.; Esteves-Macaluso, D. A.; Thomas, C. M.; Hellhund, J.; Lomsadze, R.; Gorczyca, T. W.; Ballance, C. P.; Manson, S. T.; Hasoglu, M. F.; Schippers, S.; Müller, A.

    2013-11-01

    Double photoionization accompanied by loss of n C atoms (n=0, 2, 4, 6) was investigated by merging beams of Xe@C60+ ions and synchrotron radiation and measuring the yields of product ions. The giant 4d dipole resonance of the caged Xe atom has a prominent signature in the cross section for these product channels, which together account for 6.2 ± 1.4 of the total Xe 4d oscillator strength of 10. Compared to that for a free Xe atom, the oscillator strength is redistributed in photon energy due to multipath interference of outgoing Xe 4d photoelectron waves that may be transmitted or reflected by the spherical C60+ molecular cage, yielding so-called confinement resonances. The data are compared with an earlier measurement and with theoretical predictions for this single-molecule photoelectron interferometer system. Relativistic R-matrix calculations for the Xe atom in a spherical potential shell representing the fullerene cage show the sensitivity of the interference pattern to the molecular geometry.

  18. Artemisinin activity-based probes identify multiple molecular targets within the asexual stage of the malaria parasites Plasmodium falciparum 3D7

    PubMed Central

    Ismail, Hanafy M.; Barton, Victoria; Phanchana, Matthew; Charoensutthivarakul, Sitthivut; Wong, Michael H. L.; Hemingway, Janet; Biagini, Giancarlo A.; O’Neill, Paul M.; Ward, Stephen A.

    2016-01-01

    The artemisinin (ART)-based antimalarials have contributed significantly to reducing global malaria deaths over the past decade, but we still do not know how they kill parasites. To gain greater insight into the potential mechanisms of ART drug action, we developed a suite of ART activity-based protein profiling probes to identify parasite protein drug targets in situ. Probes were designed to retain biological activity and alkylate the molecular target(s) of Plasmodium falciparum 3D7 parasites in situ. Proteins tagged with the ART probe can then be isolated using click chemistry before identification by liquid chromatography–MS/MS. Using these probes, we define an ART proteome that shows alkylated targets in the glycolytic, hemoglobin degradation, antioxidant defense, and protein synthesis pathways, processes essential for parasite survival. This work reveals the pleiotropic nature of the biological functions targeted by this important class of antimalarial drugs. PMID:26858419

  19. Artemisinin activity-based probes identify multiple molecular targets within the asexual stage of the malaria parasites Plasmodium falciparum 3D7.

    PubMed

    Ismail, Hanafy M; Barton, Victoria; Phanchana, Matthew; Charoensutthivarakul, Sitthivut; Wong, Michael H L; Hemingway, Janet; Biagini, Giancarlo A; O'Neill, Paul M; Ward, Stephen A

    2016-02-23

    The artemisinin (ART)-based antimalarials have contributed significantly to reducing global malaria deaths over the past decade, but we still do not know how they kill parasites. To gain greater insight into the potential mechanisms of ART drug action, we developed a suite of ART activity-based protein profiling probes to identify parasite protein drug targets in situ. Probes were designed to retain biological activity and alkylate the molecular target(s) of Plasmodium falciparum 3D7 parasites in situ. Proteins tagged with the ART probe can then be isolated using click chemistry before identification by liquid chromatography-MS/MS. Using these probes, we define an ART proteome that shows alkylated targets in the glycolytic, hemoglobin degradation, antioxidant defense, and protein synthesis pathways, processes essential for parasite survival. This work reveals the pleiotropic nature of the biological functions targeted by this important class of antimalarial drugs. PMID:26858419

  20. Synthesis of fluorescent molecular probes specific for the receptor of blepharismone, a mating-inducing pheromone of the ciliate Blepharisma japonicum.

    PubMed

    Uruma, Yoshiyuki; Sugiura, Mayumi; Harumoto, Terue; Usuki, Yoshinosuke; Iio, Hideo

    2007-02-15

    Blepharismone (gamone 2) is a mating-inducing pheromone of the ciliate Blepharisma japonicum. N-Pyrenylbutyryl-blepharismone and N-biphenylacetyl-blepharismone, which are fluorescent derivatives of blepharismone, were synthesized as molecular probes for the gamone 2 receptor. Further, we proved that they have inhibitory activities against the blepharismone-induced monotypic pairing of B. japonicum.

  1. Probing molecular dynamics at the nanoscale via an individual paramagnetic centre

    PubMed Central

    Staudacher, T.; Raatz, N.; Pezzagna, S.; Meijer, J.; Reinhard, F.; Meriles, C. A.; Wrachtrup, J.

    2015-01-01

    We demonstrate a protocol using individual nitrogen-vacancy centres in diamond to observe the time evolution of proton spins from organic molecules located a few nanometres from the diamond surface. The protocol records temporal correlations among the interacting protons, and thus is sensitive to the local dynamics via its impact on the nuclear spin relaxation and interaction with the nitrogen vacancy. We gather information on the nanoscale rotational and translational diffusion dynamics by analysing the time dependence of the nuclear magnetic resonance signal. Applying this technique to liquid and solid samples, we find evidence that liquid samples form a semi-solid layer of 1.5-nm thickness on the surface of diamond, where translational diffusion is suppressed while rotational diffusion remains present. Extensions of the present technique could be exploited to highlight the chemical composition of molecules tethered to the diamond surface or to investigate thermally or chemically activated dynamical processes such as molecular folding. PMID:26456017

  2. Probing nuclear motion by frequency modulation of molecular high-order harmonic generation.

    PubMed

    Bian, Xue-Bin; Bandrauk, André D

    2014-11-01

    Molecular high-order harmonic generation (MHOHG) in a non-Born-Oppenheimer treatment of H(2)(+), D(2)(+), is investigated by numerical simulations of the corresponding time-dependent Schrödinger equations in full dimensions. As opposed to previous studies on amplitude modulation of intracycle dynamics in MHOHG, we demonstrate redshifts as frequency modulation (FM) of intercycle dynamics in MHOHG. The FM is induced by nuclear motion using intense laser pulses. Compared to fixed-nuclei approximations, the intensity of MHOHG is much higher due to the dependence of enhanced ionization on the internuclear distance. The width and symmetry of the spectrum of each harmonic in MHOHG encode rich information on the dissociation process of molecules at the rising and falling parts of the laser pulses, which can be used to retrieve the nuclear dynamics. Isotope effects are studied to confirm the FM mechanism.

  3. A modified staining technique for arbuscular mycorrhiza compatible with molecular probes.

    PubMed

    Pitet, M; Camprubí, A; Calvet, C; Estaún, V

    2009-02-01

    The effects of the different steps of the root staining on the arbuscular mycorrhizal (AM) fungal rDNA extraction and amplification have been assessed. The results obtained using molecular techniques are compared with those obtained from fresh, non-stained leek roots. A modified staining procedure that eliminates heating, the use of hydrochloric acid and trypan blue, has been proved to be the most adequate to observe the AM colonisation in different plant species with/without lignified roots allowing at the same time the subsequent rDNA extraction and amplification from the stained roots. The staining technique decreased the sensitivity of the process and a higher number of roots had to be used to obtain enough material for a positive amplification. The extraction and amplification process was reliable up to 3 days after staining. A week after staining, the amplification was not dependable and after 2 weeks there was no amplification from stained material.

  4. Probing the Spatial Structure of a Molecular Attosecond Electron Wave Packet Using Shaped Recollision Trajectories

    NASA Astrophysics Data System (ADS)

    Niikura, Hiromichi; Wörner, Hans Jakob; Villeneuve, D. M.; Corkum, P. B.

    2011-08-01

    Using orthogonally polarized 800 nm and 400 nm laser pulses, we have generated high harmonics in ethane (C2H6). We observe that the intensity of each harmonic order modulates with the attosecond delay between the two laser fields. The modulation period of the low even harmonics is twice that of the period of modulation of the other harmonics. By comparing with theoretical calculation, we show that the double periodicity is a result of the electron wave packet motion in the valence shell of C2H6 on the attosecond time-scale. Our method is a general approach to measuring internal electron dynamics which does not require molecular alignment, making it applicable to more complex molecules than previous approaches.

  5. On the dynamic and static manifestation of molecular absorption in thin films probed by a microcantilever

    SciTech Connect

    Finot, Eric; Fabre, Arnaud; Passian, Ali; Thundat, Thomas

    2014-03-01

    Mechanical resonators shaped like microcantilevers have been demonstrated as a platform for very sensitive detection of chemical and biological analytes. However, its use as an analytical tool will require fundamental understanding of the molecular absorption-induced effects in the static and dynamic sensor response. The effect of absorption-induced surface stress on the microcantilever response is here investigated using palladium hydride formation. It is shown that the resonance and deformation states of the cantilever monitored simultaneously exhibit excellent correlation with the phase of the hydride formation. However, the associated frequency shifts and quasistatic bending are observed to be independent during solid solution phase. Importantly, absorption-induced changes in the elastic parameters of the palladium film are found to play a dominant role in the static and dynamic response. The presented results help in discerning the parameters that control the cantilever response as well as the relationships between these parameters.

  6. Operation mechanism of rotary molecular motor F1 probed by single-molecule techniques

    NASA Astrophysics Data System (ADS)

    Iino, Ryota

    2013-03-01

    F1 is a rotary motor protein. Three catalytic β - subunitsinthestator 33ring are torque generators, and rotate the rotor γ-subunit by sequential and cooperative conformational changes coupled with adenosine triphosphate (ATP) hydrolysis reaction. F1 shows remarkable performances such as rotation rate faster than 10,000 rpm, high reversibility and efficiency in chemo-mechanical energy conversion. I will introduce basic characteristics of F1 revealed by single-molecule imaging and manipulation techniques based on optical microscopy and high-speed atomic force microscopy. I will also discuss the possible operation mechanism behind the F1, along with structurally-related hexameric ATPases, also mentioning the possibility of generating hybrid molecular motors.

  7. Engineering Agatoxin, a Cystine-Knot Peptide from Spider Venom, as a Molecular Probe for In Vivo Tumor Imaging

    PubMed Central

    Norton, Heidi K.; Cochran, Jennifer R.

    2013-01-01

    -targeting knottins as probes for in vivo molecular imaging. PMID:23573262

  8. Advancing molecular-guided surgery through probe development and testing in a moderate cost evaluation pipeline

    NASA Astrophysics Data System (ADS)

    Pogue, Brian W.; Paulsen, Keith D.; Hull, Sally M.; Samkoe, Kimberley S.; Gunn, Jason; Hoopes, Jack; Roberts, David W.; Strong, Theresa V.; Draney, Daniel; Feldwisch, Joachim

    2015-03-01

    Molecular guided oncology surgery has the potential to transform the way decisions about resection are done, and can be critically important in areas such as neurosurgery where the margins of tumor relative to critical normal tissues are not readily apparent from visual or palpable guidance. Yet there are major financial barriers to advancing agents into clinical trials with commercial backing. We observe that development of these agents in the standard biological therapeutic paradigm is not viable, due to the high up front financial investment needed and the limitations in the revenue models of contrast agents for imaging. The hypothesized solution to this problem is to develop small molecular biologicals tagged with an established fluorescent reporter, through the chemical agent approval pathway, targeting a phase 0 trials initially, such that the initial startup phase can be completely funded by a single NIH grant. In this way, fast trials can be completed to de-risk the development pipeline, and advance the idea of fluorescence-guided surgery (FGS) reporters into human testing. As with biological therapies the potential successes of each agent are still moderate, but this process will allow the field to advance in a more stable and productive manner, rather than relying upon isolated molecules developed at high cost and risk. The pathway proposed and tested here uses peptide synthesis of an epidermal growth factor receptor (EGFR)-binding Affibody molecules, uniquely conjugated to IRDye 800CW, developed and tested in academic and industrial laboratories with well-established records for GMP production, fill and finish, toxicity testing, and early phase clinical trials with image guidance.

  9. Probing the origins of aromatase inhibitory activity of disubstituted coumarins via QSAR and molecular docking

    PubMed Central

    Worachartcheewan, Apilak; Suvannang, Naravut; Prachayasittikul, Supaluk; Prachayasittikul, Virapong; Nantasenamat, Chanin

    2014-01-01

    This study investigated the quantitative structure-activity relationship (QSAR) of imidazole derivatives of 4,7-disubstituted coumarins as inhibitors of aromatase, a potential therapeutic protein target for the treatment of breast cancer. Herein, a series of 3,7- and 4,7-disubstituted coumarin derivatives (1-34) with R1 and R2 substituents bearing aromatase inhibitory activity were modeled as a function of molecular and quantum chemical descriptors derived from low-energy conformer geometrically optimized at B3LYP/6-31G(d) level of theory. Insights on origins of aromatase inhibitory activity was afforded by the computed set of 7 descriptors comprising of F10[N-O], Inflammat-50, Psychotic-80, H-047, BELe1, B10[C-O] and MAXDP. Such significant descriptors were used for QSAR model construction and results indicated that model 4 afforded the best statistical performance. Good predictive performance were achieved as verified from the internal (comprising the training and the leave-one-out cross-validation (LOO-CV) sets) and external sets affording the following statistical parameters: R2Tr = 0.9576 and RMSETr = 0.0958 for the training set; Q2CV = 0.9239 and RMSECV = 0.1304 for the LOO-CV set as well as Q2Ext = 0.7268 and RMSEExt = 0.2927 for the external set. Significant descriptors showed correlation with functional substituents, particularly, R1 in governing high potency as aromatase inhibitor. Molecular docking calculations suggest that key residues interacting with the coumarins were predominantly lipophilic or non-polar while a few were polar and positively-charged. Findings illuminated herein serve as the impetus that can be used to rationally guide the design of new aromatase inhibitors. PMID:26417339

  10. Advancing Molecular-Guided Surgery through probe development and testing in a moderate cost evaluation pipeline

    PubMed Central

    Pogue, Brian W; Paulsen, Keith D; Hull, Sally M.; Samkoe, Kimberly S.; Gunn, Jason; Hoopes, Jack; Roberts, David W.; Strong, Theresa V.; Draney, Daniel; Feldwisch, Joachim

    2015-01-01

    Molecular guided oncology surgery has the potential to transform the way decisions about resection are done, and can be critically important in areas such as neurosurgery where the margins of tumor relative to critical normal tissues are not readily apparent from visual or palpable guidance. Yet there are major financial barriers to advancing agents into clinical trials with commercial backing. We observe that development of these agents in the standard biological therapeutic paradigm is not viable, due to the high up front financial investment needed and the limitations in the revenue models of contrast agents for imaging. The hypothesized solution to this problem is to develop small molecular biologicals tagged with an established fluorescent reporter, through the chemical agent approval pathway, targeting a phase 0 trials initially, such that the initial startup phase can be completely funded by a single NIH grant. In this way, fast trials can be completed to de-risk the development pipeline, and advance the idea of fluorescence-guided surgery (FGS) reporters into human testing. As with biological therapies the potential successes of each agent are still moderate, but this process will allow the field to advance in a more stable and productive manner, rather than relying upon isolated molecules developed at high cost and risk. The pathway proposed and tested here uses peptide synthesis of an epidermal growth factor receptor (EGFR)-binding Affibody molecules, uniquely conjugated to IRDye 800CW, developed and tested in academic and industrial laboratories with well-established records for GMP production, fill & finish, toxicity testing, and early phase clinical trials with image guidance. PMID:25914500

  11. Probing the organic-mineral interface at the molecular level in model biominerals.

    PubMed

    Metzler, Rebecca A; Kim, Il Won; Delak, Katya; Evans, John Spencer; Zhou, Dong; Beniash, Elia; Wilt, Fred; Abrecht, Mike; Chiou, Jau-Wern; Guo, Jinghua; Coppersmith, Susan N; Gilbert, P U P A

    2008-03-18

    It is widely known that macromolecules, such as proteins, can control the nucleation and growth of inorganic solids in biomineralizing organisms. However, what is not known are the complementary molecular interactions, organization, and rearrangements that occur when proteins interact with inorganic solids during the formation of biominerals. The organic-mineral interface (OMI) is expected to be the site for these phenomena, and is therefore extraordinarily interesting to investigate. In this report, we employ X-ray absorption near edge (XANES) spectromicroscopy to investigate the electronic structure of both calcium carbonate mineral crystals and polypeptides, and detect changing bonds at the OMI during crystal growth in the presence of polypeptides. We acquired XANES spectra from calcium carbonate crystals grown in the presence of three mollusk nacre-associated polypeptides (AP7N, AP24N, n16N) and in the presence of a sea urchin spicule matrix protein, LSM34. All these model biominerals gave similar results, including the disruption of CO bonds in calcite and enhancement of the peaks associated with C-H bonds and C-O bonds in peptides, indicating ordering of the amino acid side chains in the mineral-associated polypeptides and carboxylate binding. This is the first evidence of the mutual effect of calcite on peptide chain and peptide chain on calcite during biomineralization. We also show that these changes do not occur when Asp and Glu are replaced in the n16N sequence with Asn and Gln, respectively, demonstrating that carboxyl groups in Asp and Glu do participate in polypeptide-mineral molecular associations.

  12. Molecular Interactions of Lipopolysaccharide with an Outer Membrane Protein from Pseudomonas aeruginosa Probed by Solution NMR.

    PubMed

    Kucharska, Iga; Liang, Binyong; Ursini, Nicholas; Tamm, Lukas K

    2016-09-13

    Pseudomonas aeruginosa is an opportunistic human pathogen causing pneumonias that are particularly severe in cystic fibrosis and immunocompromised patients. The outer membrane (OM) of P. aeruginosa is much less permeable to nutrients and other chemical compounds than that of Escherichia coli. The low permeability of the OM, which also contributes to Pseudomonas' significant antibiotic resistance, is augmented by the presence of the outer membrane protein H (OprH). OprH directly interacts with lipopolysaccharides (LPS) that constitute the outer leaflet of the OM and thus contributes to the structural stability of the OM. In this study, we used solution NMR spectroscopy to characterize the interactions between LPS and OprH in molecular detail. NMR chemical shift perturbations observed upon the addition of LPS to OprH in DHPC micelles indicate that this interaction is predominantly electrostatic and localized to the extracellular loops 2 and 3 and a number of highly conserved basic residues near the extracellular barrel rim of OprH. Single-site mutations of these residues were not enough to completely abolish binding, but OprH with cumulative mutations of Lys70, Arg72, and Lys103 no longer binds LPS. The dissociation constant (∼200 μM) measured by NMR is sufficient to efficiently bind LPS to OprH in the OM. This work highlights that solution NMR is suitable to study specific interactions of lipids with integral membrane proteins and provides a detailed molecular model for the interaction of LPS with OprH; i.e., an interaction that contributes to the integrity of the OM of P. aeruginosa under low divalent cation and antibiotic stress conditions. These methods should thus be useful for screening antibiotics that might disrupt OprH-LPS interactions and thereby increase the permeability of the OM of P. aeruginosa. PMID:27532487

  13. Probing the importance of lipid diversity in cell membranes via molecular simulation.

    PubMed

    Khakbaz, Pouyan; Klauda, Jeffery B

    2015-11-01

    Lipid membranes in prokaryotes and eukaryotes have a wide array of lipids that are necessary for proper membrane structure and function. In this paper, an introduction to lipid diversity in biology and a mini-review on how molecular simulations have been used to model biological membranes (primarily limited to one to three lipid types in most simulation-based models) is provided, which motivates the use of all-atom molecular dynamics (MD) simulations to study the effect of lipid diversity on properties of realistic membrane models of prokaryotes and eukaryotes. As an example, cytoplasmic membrane models of Escherichia coli were developed at different stages of the colony growth cycle (early-log, mid-log, stationary and overnight). The main difference between lipid compositions at each stage was the concentration of a cyclopropane-containing moiety on the sn-2 lipid acyl chain (cyC17:0). Triplicate MD simulations for each stage were run for 300 ns to study the influence of lipid diversity on the surface area per lipid, area compressibility modulus, deuterium order parameters, and electron density profiles. The overnight stage (also known as the death stage) had the highest average surface area per lipid, highest rigidity, and lowest bilayer thickness compare to other stages of E. coli cytoplasmic membrane. Although bilayer thickness did depend on the growth stage, the changes between these were small suggesting that the hydrophobic core of transmembrane proteins fit well with the membrane in all growth stages. Although it is still common practise in MD simulations of membrane proteins to use simple one- or two-component membranes, it can be important to use diverse lipid model membranes when membrane protein structure and function are influenced by changes in lipid membrane composition.

  14. High sensitivity of diamond resonant microcantilevers for direct detection in liquids as probed by molecular electrostatic surface interactions.

    PubMed

    Bongrain, Alexandre; Agnès, Charles; Rousseau, Lionel; Scorsone, Emmanuel; Arnault, Jean-Charles; Ruffinatto, Sébastien; Omnès, Franck; Mailley, Pascal; Lissorgues, Gaëlle; Bergonzo, Philippe

    2011-10-01

    Resonant microcantilevers have demonstrated that they can play an important role in the detection of chemical and biological agents. Molecular interactions with target species on the mechanical microtransducers surface generally induce a change of the beam's bending stiffness, resulting in a shift of the resonance frequency. In most biochemical sensor applications, cantilevers must operate in liquid, even though damping deteriorates the vibrational performances of the transducers. Here we focus on diamond-based microcantilevers since their transducing properties surpass those of other materials. In fact, among a wide range of remarkable features, diamond possesses exceptional mechanical properties enabling the fabrication of cantilever beams with higher resonant frequencies and Q-factors than when made from other conventional materials. Therefore, they appear as one of the top-ranked materials for designing cantilevers operating in liquid media. In this study, we evaluate the resonator sensitivity performances of our diamond microcantilevers using grafted carboxylated alkyl chains as a tool to investigate the subtle changes of surface stiffness as induced by electrostatic interactions. Here, caproic acid was immobilized on the hydrogen-terminated surface of resonant polycrystalline diamond cantilevers using a novel one-step grafting technique that could be also adapted to several other functionalizations. By varying the pH of the solution one could tune the -COO(-)/-COOH ratio of carboxylic acid moieties immobilized on the surface, thus enabling fine variations of the surface stress. We were able to probe the cantilevers resonance frequency evolution and correlate it with the ratio of -COO(-)/-COOH terminations on the functionalized diamond surface and consequently the evolution of the electrostatic potential over the cantilever surface. The approach successfully enabled one to probe variations in cantilevers bending stiffness from several tens to hundreds of

  15. Probing the origins of human acetylcholinesterase inhibition via QSAR modeling and molecular docking.

    PubMed

    Simeon, Saw; Anuwongcharoen, Nuttapat; Shoombuatong, Watshara; Malik, Aijaz Ahmad; Prachayasittikul, Virapong; Wikberg, Jarl E S; Nantasenamat, Chanin

    2016-01-01

    Alzheimer's disease (AD) is a chronic neurodegenerative disease which leads to the gradual loss of neuronal cells. Several hypotheses for AD exists (e.g., cholinergic, amyloid, tau hypotheses, etc.). As per the cholinergic hypothesis, the deficiency of choline is responsible for AD; therefore, the inhibition of AChE is a lucrative therapeutic strategy for the treatment of AD. Acetylcholinesterase (AChE) is an enzyme that catalyzes the breakdown of the neurotransmitter acetylcholine that is essential for cognition and memory. A large non-redundant data set of 2,570 compounds with reported IC50 values against AChE was obtained from ChEMBL and employed in quantitative structure-activity relationship (QSAR) study so as to gain insights on their origin of bioactivity. AChE inhibitors were described by a set of 12 fingerprint descriptors and predictive models were constructed from 100 different data splits using random forest. Generated models afforded R (2), [Formula: see text] and [Formula: see text] values in ranges of 0.66-0.93, 0.55-0.79 and 0.56-0.81 for the training set, 10-fold cross-validated set and external set, respectively. The best model built using the substructure count was selected according to the OECD guidelines and it afforded R (2), [Formula: see text] and [Formula: see text] values of 0.92 ± 0.01, 0.78 ± 0.06 and 0.78 ± 0.05, respectively. Furthermore, Y-scrambling was applied to evaluate the possibility of chance correlation of the predictive model. Subsequently, a thorough analysis of the substructure fingerprint count was conducted to provide informative insights on the inhibitory activity of AChE inhibitors. Moreover, Kennard-Stone sampling of the actives were applied to select 30 diverse compounds for further molecular docking studies in order to gain structural insights on the origin of AChE inhibition. Site-moiety mapping of compounds from the diversity set revealed three binding anchors encompassing both hydrogen bonding and van der Waals

  16. Probing the origins of human acetylcholinesterase inhibition via QSAR modeling and molecular docking.

    PubMed

    Simeon, Saw; Anuwongcharoen, Nuttapat; Shoombuatong, Watshara; Malik, Aijaz Ahmad; Prachayasittikul, Virapong; Wikberg, Jarl E S; Nantasenamat, Chanin

    2016-01-01

    Alzheimer's disease (AD) is a chronic neurodegenerative disease which leads to the gradual loss of neuronal cells. Several hypotheses for AD exists (e.g., cholinergic, amyloid, tau hypotheses, etc.). As per the cholinergic hypothesis, the deficiency of choline is responsible for AD; therefore, the inhibition of AChE is a lucrative therapeutic strategy for the treatment of AD. Acetylcholinesterase (AChE) is an enzyme that catalyzes the breakdown of the neurotransmitter acetylcholine that is essential for cognition and memory. A large non-redundant data set of 2,570 compounds with reported IC50 values against AChE was obtained from ChEMBL and employed in quantitative structure-activity relationship (QSAR) study so as to gain insights on their origin of bioactivity. AChE inhibitors were described by a set of 12 fingerprint descriptors and predictive models were constructed from 100 different data splits using random forest. Generated models afforded R (2), [Formula: see text] and [Formula: see text] values in ranges of 0.66-0.93, 0.55-0.79 and 0.56-0.81 for the training set, 10-fold cross-validated set and external set, respectively. The best model built using the substructure count was selected according to the OECD guidelines and it afforded R (2), [Formula: see text] and [Formula: see text] values of 0.92 ± 0.01, 0.78 ± 0.06 and 0.78 ± 0.05, respectively. Furthermore, Y-scrambling was applied to evaluate the possibility of chance correlation of the predictive model. Subsequently, a thorough analysis of the substructure fingerprint count was conducted to provide informative insights on the inhibitory activity of AChE inhibitors. Moreover, Kennard-Stone sampling of the actives were applied to select 30 diverse compounds for further molecular docking studies in order to gain structural insights on the origin of AChE inhibition. Site-moiety mapping of compounds from the diversity set revealed three binding anchors encompassing both hydrogen bonding and van der Waals

  17. Theory of quasicrystal surfaces: Probing the chemical reactivity by atomic and molecular adsorption

    NASA Astrophysics Data System (ADS)

    Krajčí, M.; Hafner, J.

    The adsorption of oxygen and carbon atoms and of carbon monoxide molecules on a fivefold surface of icosahedral Al-Pd-Mn quasicrystals has been investigated using ab initio density-functional calculations. The quasicrystalline surface has been modeled by periodically repeated slabs cut from rational approximants to the quasicrystalline structure. Atomic and molecular adsorption have been studied for a large number of possible adsorption sites by performing three-dimensional static relaxations of the adsorbate/substrate complex. Four different scenarios for the dissociative adsorption of the CO molecule have been investigated via nudged-elastic band calculations of the transition states. Al and Mn-metal atoms present at the surface bind C and O atoms rather strongly, while Pd atoms are unstable adsorption sites: during relaxation, the adsorbate drifts to the nearest strong-binding site. The chemical reactivity with respect to a CO molecule varies very strongly across the surface. The adsorption close to Mn sites is promoted by rather strong covalent effects, but CO is only physisorbed at Al sites via weak polarization forces. On the basis of the observed local variations of the adsorption strength, we develop scenarios for dissociation and determine the potential energy barriers for this processes. We find that CO adsorbed close to a transition-metal atom can dissociate via an activated process, but the dissociation rate is expected to be rather low because of a high dissociation barrier and a "late" transition state. CO adsorbed close to Al atoms will desorb before dissociation. Surface vacancies present as a consequence of the irregular coordination of the Mackay cluster in the quasiperiodic structure will act as strongly attractive traps for diffusing molecules. Mn surface atoms are located in the center of truncated Mackay clusters. In scanning tunneling electron microscopy (STM) these truncated clusters are imaged as "white flowers". Surface vacancies are

  18. Probing self assembly in biological mixed colloids by SANS, deuteration and molecular manipulation

    SciTech Connect

    Hjelm, R.P.; Thiyagarajan, P.; Hoffman, A.; Alkan-Onyuksel, H.

    1994-12-31

    Small-angle neutron scattering was used to obtain information on the form and molecular arrangement of particles in mixed colloids of bile salts with phosphatidylcholine, and bile salts with monoolein. Both types of systems showed the same general characteristics. The particle form was highly dependent on total lipid concentration. At the highest concentrations the particles were globular mixed micelles with an overall size of 50{Angstrom}. As the concentration was reduced the mixed micelles elongated, becoming rodlike with diameter about 50{Angstrom}. The rods had a radial core-shell structure in which the phosphatidylcholine or monoolein fatty tails were arranged radially to form the core with the headgroups pointing outward to form the shell. The bile salts were at the interface between the shell and core with the hydrophilic parts facing outward as part of the shell. The lengths of the rods increased and became more polydispersed with dilution. At sufficiently low concentrations the mixed micelles transformed into single bilayer vesicles. These results give insight on the physiological function of bile and on the rules governing the self assembly of bile particles in the hepatic duct and the small intestine.

  19. Probing the interaction of anthraquinone with DNA by spectroscopy, molecular modeling and cancer cell imaging technique.

    PubMed

    Yang, Lei; Fu, Zheng; Niu, Xiaoqing; Zhang, Guisheng; Cui, Fengling; Zhou, Chunwu

    2015-05-25

    A new anthraquinone derivative, (E)-2-(1-(4,5-dihydroxy-6-(hydroxymethyl)-tetrahydro-2H-pyran-2-yloxyimino)ethyl)-1,4-dihydroxyanthracene-9,10-dione (AODGlc), was synthesized and its binding properties towards DNA were explored under physiological conditions by fluorescence spectroscopy, DNA melting as well as docking techniques. The experimental results revealed that AODGlc could bind to calf thymus DNA (ctDNA) through intercalation between DNA base pairs. The values of thermodynamic parameters at different temperatures including ΔG, ΔH, and ΔS and the molecular modeling study implied that hydrophobic interactions and hydrogen bonds were the main interactions in the AODGlc-ctDNA system. Cervical cancer cells (HepG2 cells) were used in cell viability assay and cell imaging experiment. AODGlc could interact with HepG2 cells and kill HepG2 cells under high concentration with nice curative effect, indicating its potential bioapplication in the future.

  20. Probing ice-nucleation processes on the molecular level using second harmonic generation spectroscopy

    NASA Astrophysics Data System (ADS)

    Abdelmonem, A.; Lützenkirchen, J.; Leisner, T.

    2015-08-01

    We present and characterize a novel setup to apply second harmonic generation (SHG) spectroscopy in total internal reflection geometry (TIR) to heterogeneous freezing research. It allows to monitor the evolution of water structuring at solid surfaces at low temperatures prior to heterogeneous ice nucleation. Apart from the possibility of investigating temperature dependence, a major novelty in our setup is the ability of measuring sheet-like samples in TIR geometry in a direct way. As a main experimental result, we find that our method can discriminate between good and poor ice nucleating surfaces. While at the sapphire basal plane, which is known to be a poor ice nucleator, no structural rearrangement of the water molecules is found prior to freezing, the basal plane surface of mica, an analogue to ice active mineral dust surfaces, exhibits a strong change in the nonlinear optical properties at temperatures well above the freezing transition. This is interpreted as a pre-activation, i.e. an increase in the local ordering of the interfacial water which is expected to facilitate the crystallization of ice at the surface. The results are in line with recent predictions by molecular dynamics simulations on a similar system.

  1. Probing of molecular replication and accumulation in shallow heat gradients through numerical simulations.

    PubMed

    Keil, Lorenz; Hartmann, Michael; Lanzmich, Simon; Braun, Dieter

    2016-07-27

    How can living matter arise from dead matter? All known living systems are built around information stored in RNA and DNA. To protect this information against molecular degradation and diffusion, the second law of thermodynamics imposes the need for a non-equilibrium driving force. Following a series of successful experiments using thermal gradients, we have shown that heat gradients across sub-millimetre pores can drive accumulation, replication, and selection of ever longer molecules, implementing all the necessary parts for Darwinian evolution. For these lab experiments to proceed with ample speed, however, the temperature gradients have to be quite steep, reaching up to 30 K per 100 μm. Here we use computer simulations based on experimental data to show that 2000-fold shallower temperature gradients - down to 100 K over one metre - can still drive the accumulation of protobiomolecules. This finding opens the door for various environments to potentially host the origins of life: volcanic, water-vapour, or hydrothermal settings. Following the trajectories of single molecules in simulation, we also find that they are subjected to frequent temperature oscillations inside these pores, facilitating e.g. template-directed replication mechanisms. The tilting of the pore configuration is the central strategy to achieve replication in a shallow temperature gradient. Our results suggest that shallow thermal gradients across porous rocks could have facilitated the formation of evolutionary machines, significantly increasing the number of potential sites for the origin of life on young rocky planets. PMID:27153345

  2. Probing the molecular interstellar medium of M82 with Herschel-SPIRE spectroscopy

    NASA Astrophysics Data System (ADS)

    Panuzzo, P.; Rangwala, N.; Rykala, A.; Isaak, K. G.; Glenn, J.; Wilson, C. D.; Auld, R.; Baes, M.; Barlow, M. J.; Bendo, G. J.; Bock, J. J.; Boselli, A.; Bradford, M.; Buat, V.; Castro-Rodríguez, N.; Chanial, P.; Charlot, S.; Ciesla, L.; Clements, D. L.; Cooray, A.; Cormier, D.; Cortese, L.; Davies, J. I.; Dwek, E.; Eales, S. A.; Elbaz, D.; Fulton, T.; Galametz, M.; Galliano, F.; Gear, W. K.; Gomez, H. L.; Griffin, M.; Hony, S.; Levenson, L. R.; Lu, N.; Madden, S.; O'Halloran, B.; Okumura, K.; Oliver, S.; Page, M. J.; Papageorgiou, A.; Parkin, T. J.; Pérez-Fournon, I.; Pohlen, M.; Polehampton, E. T.; Rigby, E. E.; Roussel, H.; Sacchi, N.; Sauvage, M.; Schulz, B.; Schirm, M. R. P.; Smith, M. W. L.; Spinoglio, L.; Stevens, J. A.; Srinivasan, S.; Symeonidis, M.; Swinyard, B.; Trichas, M.; Vaccari, M.; Vigroux, L.; Wozniak, H.; Wright, G. S.; Zeilinger, W. W.

    2010-07-01

    We present the observations of the starburst galaxy M82 taken with the Herschel SPIRE Fourier-transform spectrometer. The spectrum (194-671 μm) shows a prominent CO rotational ladder from J = 4-3 to 13-12 emitted by the central region of M82. The fundamental properties of the gas are well constrained by the high J lines observed for the first time. Radiative transfer modeling of these high-S/N 12CO and 13CO lines strongly indicates a very warm molecular gas component at ~500 K and pressure of ~3×106 K cm-3, in good agreement with the H2 rotational lines measurements from Spitzer and ISO. We suggest that this warm gas is heated by dissipation of turbulence in the interstellar medium (ISM) rather than X-rays or UV flux from the straburst. This paper illustrates the promise of the SPIRE FTS for the study of the ISM of nearby galaxies. Herschel is an ESA space observatory with science instruments provided by European-led Principal Investigator consortia and with important participation from NASA.

  3. Hydrogen-bonding molecular ruler surfactants as probes of specific solvation at liquid/liquid interfaces.

    PubMed

    Siler, A Renee; Brindza, Michael R; Walker, Robert A

    2009-10-01

    Resonance-enhanced, second harmonic generation (SHG) is used to measure the electronic structure of solutes sensitive to specific solvation adsorbed to liquid/liquid and liquid/solid interfaces. Here, specific solvation refers to solvent-solute interactions that are directional and localized. N-methyl-p-methoxyaniline (NMMA) is a solute whose first allowed electronic transition wavelength remains almost constant (approximately 315 nm) in non-hydrogen-bonding solvents regardless of solvent polarity. However, in hydrogen-bond-accepting solvents such as dimethylsulfoxide, NMMA's absorbance shifts to longer wavelengths (320 nm), whereas in hydrogen-bond-donating solvents (e.g., water), the absorbance shifts to shorter wavelengths (approximately 300 nm). SHG experiments show that at alkane/silica interfaces, surface silanol groups serve as moderately strong hydrogen-bond donors as evidenced by NMMA's absorbance of 307 nm. At the carbon tetrachloride/water interface, NMMA absorbance also shifts to slightly shorter wavelengths (298 nm) implying that water molecules at this liquid/liquid interface are donating strong hydrogen bonds to the adsorbed NMMA solutes. In contrast, experiments using newly developed molecular ruler surfactants with NMMA as a model hydrophobic solute and a hydrophilic, cationic headgroup imply that, as NMMA migrates across an aqueous/alkane interface, it carries with it water that functions as a hydrogen-bond-accepting partner.

  4. Spectroscopic investigation of a FRET molecular beacon containing two fluorophores for probing DNA/RNA sequences.

    PubMed

    Jockusch, Steffen; Martí, Angel A; Turro, Nicholas J; Li, Zengmin; Li, Xiaoxu; Ju, Jingyue; Stevens, Nathan; Akins, Daniel L

    2006-05-01

    We report the design, synthesis, and characterization of a molecular beacon (MB) consisting of two fluorescent dyes (Alexa 488 and RedX) for DNA and RNA analysis. In the absence of the target DNA or RNA the MB is in its stem-closed form and shows efficient energy transfer from the donor (Alexa) to the acceptor (RedX), generating mostly fluorescence from RedX. In the presence of the complementary target DNA the MB opened efficiently, hybridizes with the target DNA, and energy transfer is blocked in the stem-open form. This attachment to the target generates a fluorescence signature, which is clearly distinguishable from the fluorescence signature of the stem-closed form, allowing for ratiometric analysis of the fluorescence signal. In addition to steady-state fluorescence analysis, time resolved fluorescence (ps time range) and fluorescence depolarization studies were performed. We show that fluorescence lifetime and fluorescence depolarization measurements are useful analytical tools to optimize the MB design.

  5. Probing the origins of human acetylcholinesterase inhibition via QSAR modeling and molecular docking

    PubMed Central

    Shoombuatong, Watshara; Malik, Aijaz Ahmad; Prachayasittikul, Virapong; Wikberg, Jarl E.S.

    2016-01-01

    {mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}${Q}_{\\mathrm{CV }}^{2}$\\end{document}QCV2 and \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}${Q}_{\\mathrm{Ext}}^{2}$\\end{document}QExt2 values of 0.92 ± 0.01, 0.78 ± 0.06 and 0.78 ± 0.05, respectively. Furthermore, Y-scrambling was applied to evaluate the possibility of chance correlation of the predictive model. Subsequently, a thorough analysis of the substructure fingerprint count was conducted to provide informative insights on the inhibitory activity of AChE inhibitors. Moreover, Kennard–Stone sampling of the actives were applied to select 30 diverse compounds for further molecular docking studies in order to gain structural insights on the origin of AChE inhibition. Site-moiety mapping of compounds from the diversity set revealed three binding anchors encompassing both hydrogen bonding and van der Waals interaction. Molecular docking revealed that compounds 13, 5 and 28 exhibited the lowest binding energies of −12.2, −12.0 and −12.0 kcal/mol, respectively, against human AChE, which is modulated by hydrogen bonding, π–π stacking and hydrophobic interaction inside the binding pocket. These information may be used as guidelines for the design of novel and robust AChE inhibitors.

  6. Probing the origins of human acetylcholinesterase inhibition via QSAR modeling and molecular docking

    PubMed Central

    Shoombuatong, Watshara; Malik, Aijaz Ahmad; Prachayasittikul, Virapong; Wikberg, Jarl E.S.

    2016-01-01

    {mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}${Q}_{\\mathrm{CV }}^{2}$\\end{document}QCV2 and \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}${Q}_{\\mathrm{Ext}}^{2}$\\end{document}QExt2 values of 0.92 ± 0.01, 0.78 ± 0.06 and 0.78 ± 0.05, respectively. Furthermore, Y-scrambling was applied to evaluate the possibility of chance correlation of the predictive model. Subsequently, a thorough analysis of the substructure fingerprint count was conducted to provide informative insights on the inhibitory activity of AChE inhibitors. Moreover, Kennard–Stone sampling of the actives were applied to select 30 diverse compounds for further molecular docking studies in order to gain structural insights on the origin of AChE inhibition. Site-moiety mapping of compounds from the diversity set revealed three binding anchors encompassing both hydrogen bonding and van der Waals interaction. Molecular docking revealed that compounds 13, 5 and 28 exhibited the lowest binding energies of −12.2, −12.0 and −12.0 kcal/mol, respectively, against human AChE, which is modulated by hydrogen bonding, π–π stacking and hydrophobic interaction inside the binding pocket. These information may be used as guidelines for the design of novel and robust AChE inhibitors. PMID:27602288

  7. Probing molecular interaction between concanavalin A and mannose ligands by means of SFM.

    PubMed

    Lekka, M; Laidler, P; Dulińska, J; Łabedź, M; Pyka, G

    2004-11-01

    Recently, the scanning force microscope (SFM) has been widely used for direct monitoring of specific interactions between biologically active molecules. Such studies have employed the SFM liquid-cell setup, which allows measurements to be made in the native environment with force resolution down to a tenth of a picoNewton. In this study, the ligand-receptor strength of monoclonal anti-human prostatic acid phosphatase and prostatic acid phosphatase, representing an antigen-antibody system with a single type of interaction, was determined. Then, the interaction force occurring between concanavalin A and the carbohydrate component of the glycoproteins arylsulfatase A and carboxypeptidase Y was measured. High mannose-type glycans were sought on the human prostate carcinoma cell surface. Application of an analysis based on the Poisson distribution of the number of bonds formed in all these measured systems allowed the strength of the molecular interaction to be calculated. The values of the force acting between two single molecules were 530+/-25, 790+/-32, and 940+/-39 pN between prostatic acid phosphatase and monoclonal anti-human prostatic acid phosphatase, between concanavalin A and arylsulfatase A, and between concanavalin A and carboxypeptidase Y, respectively. The value calculated from data collected for the force between concanavalin A and mannose-containing ligands present on the surface of human prostate carcinoma cells was smaller, 116+/-17 pN. The different values of the binding force between concanavalin A and mannose-containing ligands were attributed to the structural changes of the carbohydrate components.

  8. Use of molecular modelling to probe the mechanism of the nucleoside transporter NupG

    PubMed Central

    Vaziri, Hamidreza; Baldwin, Stephen A.; Baldwin, Jocelyn M.; Adams, David G.; Young, James D.

    2013-01-01

    Nucleosides play key roles in biology as precursors for salvage pathways of nucleotide synthesis. Prokaryotes import nucleosides across the cytoplasmic membrane by proton- or sodium-driven transporters belonging to the Concentrative Nucleoside Transporter (CNT) family or the Nucleoside:H+ Symporter (NHS) family of the Major Facilitator Superfamily. The high resolution structure of a CNT from Vibrio cholerae has recently been determined, but no similar structural information is available for the NHS family. To gain a better understanding of the molecular mechanism of nucleoside transport, in the present study the structures of two conformations of the archetypical NHS transporter NupG from Escherichia coli were modelled on the inward- and outward-facing conformations of the lactose transporter LacY from E. coli, a member of the Oligosaccharide:H+ Symporter (OHS) family. Sequence alignment of these distantly related proteins (∼ 10% sequence identity), was facilitated by comparison of the patterns of residue conservation within the NHS and OHS families. Despite the low sequence similarity, the accessibilities of endogenous and introduced cysteine residues to thiol reagents were found to be consistent with the predictions of the models, supporting their validity. For example C358, located within the predicted nucleoside binding site, was shown to be responsible for the sensitivity of NupG to inhibition by p-chloromercuribenzene sulphonate. Functional analysis of mutants in residues predicted by the models to be involved in the translocation mechanism, including Q261, E264 and N228, supported the hypothesis that they play important roles, and suggested that the transport mechanisms of NupG and LacY, while different, share common features. PMID:23256604

  9. Probing molecular conformations in momentum space: The case of n-pentane

    NASA Astrophysics Data System (ADS)

    Knippenberg, S.; Huang, Y. R.; Hajgató, B.; François, J.-P.; Deng, J. K.; Deleuze, M. S.

    2007-11-01

    A comprehensive study, throughout the valence region, of the electronic structure and electron momentum density distributions of the four conformational isomers of n-pentane is presented. Theoretical (e,2e) valence ionization spectra at high electron impact energies (1200eV+electron binding energy) and at azimuthal angles ranging from 0° to 10° in a noncoplanar symmetric kinematical setup are generated according to the results of large scale one-particle Green's function calculations of Dyson orbitals and related electron binding energies, using the third-order algebraic-diagrammatic construction [ADC(3)] scheme. The results of a focal point analysis (FPA) of relative conformer energies [A. Salam and M. S. Deleuze, J. Chem. Phys. 116, 1296 (2002)] and improved thermodynamical calculations accounting for hindered rotations are also employed in order to quantitatively evaluate the abundance of each conformer in the gas phase at room temperature and reliably predict the outcome of experiments on n-pentane employing high resolution electron momentum spectroscopy. Comparison with available photoelectron measurements confirms the suggestion that, due to entropy effects, the trans-gauche (tg) conformer strongly dominates the conformational mixture characterizing n-pentane at room temperature. Our simulations demonstrate therefore that experimental measurements of (e,2e) valence ionization spectra and electron momentum distributions would very consistently and straightforwardly image the topological changes and energy variations that molecular orbitals undergo due to torsion of the carbon backbone. The strongest fingerprints for the most stable conformer (tt) are found for the electron momentum distributions associated with ionization channels at the top of the inner-valence region, which sensitively image the development of methylenic hyperconjugation in all-staggered n-alkane chains.

  10. Low molecular weight alkyl-polycation wrapped magnetite nanoparticle clusters as MRI probes for stem cell labeling and in vivo imaging.

    PubMed

    Liu, Gang; Wang, Zhiyong; Lu, Jian; Xia, Chunchao; Gao, Fabao; Gong, Qiyong; Song, Bin; Zhao, Xuna; Shuai, Xintao; Chen, Xiaoyuan; Ai, Hua; Gu, Zhongwei

    2011-01-01

    Superparamagnetic iron oxide (SPIO) nanoparticles are potential probes for noninvasive cell tracking, but the design of safe probes coupled with high labeling efficiency is still an important objective for such application. In this study, an efficient SPIO probe has been developed for mesenchymal stem cells (MSCs) labeling and tracking. Different from many other systems involving high molecular polycations, we chose low molecular weight amphiphilic PEI2k to form stable nanocomplexes with SPIO nanoparticles. The probe can hold multiple SPIO nanoparticles with a controlled clustering structure, leading to much higher T(2) relaxivities compared to single SPIO nanoparticles. Labeled MSCs are unaffected in their viability, proliferation, or differentiation capacity. The iron uptake process in MSCs displays a time- and dose-dependent behavior. Transmission electron microscopy reveals that the nanoprobes are internalized into the cytoplasm of MSCs. Subcutaneous injection of the labeled MSCs dispersed in a collagen type I hydrogel showed strong image contrast against unlabeled cells under a clinical 3T magnetic resonance imaging (MRI) scanner up to 19 days post-transplantation. This study provides an important alternative to label MSCs at optimized low dosages with high efficiency, and the probe may be useful to label other biologically important cells for imaging studies.

  11. Effect of cholesterol on molecular transport of organic cations across liposome bilayers probed by second harmonic generation.

    PubMed Central

    Yan, E C; Eisenthal, K B

    2000-01-01

    The effect of cholesterol on the molecular transport of an organic cation, malachite green (MG), across large unilamellar dioleolyphosphatidylglycerol (DOPG) liposome bilayers with 0-50 mol% cholesterol was studied by second harmonic generation (SHG). Because SHG is a surface-specific technique, it requires no labeled molecule, quencher, or shifting agent to distinguish the location of the solute molecules. An additional important feature of SHG is that it is sensitive only to the probe molecules bound to the liposome, whereas other methods can only differentiate between molecules that are outside and those inside the liposome. The transport kinetics of MG across the liposome bilayers was observed in real time, and the results show that cholesterol retards the rate of transport of MG across liposome bilayers. The rate was found to decrease by six times for 50 mol% cholesterol content compared with cholesterol-free liposomes. This demonstrates the applicability of SHG to investigation of the effect of liposome composition on the transport kinetics across the liposome bilayers. PMID:10920021

  12. Probing the Molecular Complexity of Cometary Volatiles: The Case of C/2012 K1 (PanSTARRS)

    NASA Astrophysics Data System (ADS)

    Milam, Stefanie N.; Cordiner, Martin; Remijan, Anthony; Gicquel, Adeline; Charnley, Steven; Colom, Pierre; Crovisier, Jacques; Mumma, Michael; Boissier, Jeremie; Bockelee-Morvan, Dominique; Biver, Nicolas; Villanueva, Geronimo; Paganini, Lucas; Lis, Dariusz; Kuan, Yi-Jehng; Coulson, Iain

    2014-11-01

    Small bodies are considered to contain the most primitive material remaining from the formation of our solar system. Cometary ices trace the pristine volatile component and provide an important source of information regarding the physical and chemical conditions of the early Solar Nebula. However, not all observed species are native to the comet nucleus and some are likely formed in the coma (at least in part) with previous observations unable to ascertain the precise origin of fundamental species including H2CO, HCN, CO, CS, and HNC. Simultaneous, spatially and spectrally-resolved molecular emission maps of comets at mm and sub-mm wavelengths provide the key information required to probe the origin and nature of these volatiles. After months of monitoring cometary activity through OH observations at the NRAO GBT and the Nançay radio telescope, we observed comet C/2012 K1 (PanSTARRS) using ALMA Bands 6 and 7, sampling emission lines from HCN, CO, CS, HCO+, CH3OH, H2CO and HNC. In order to recover any extended flux resolved out by the interferometer, we simultaneously measured HCN and CH3OH with the APEX observatory. We will present full details of these unique observations, and an analysis of the observed spectra.

  13. Direct Monitoring of γ-Glutamyl Transpeptidase Activity In Vivo Using a Hyperpolarized (13) C-Labeled Molecular Probe.

    PubMed

    Nishihara, Tatsuya; Yoshihara, Hikari A I; Nonaka, Hiroshi; Takakusagi, Yoichi; Hyodo, Fuminori; Ichikawa, Kazuhiro; Can, Emine; Bastiaansen, Jessica A M; Takado, Yuhei; Comment, Arnaud; Sando, Shinsuke

    2016-08-26

    The γ-glutamyl transpeptidase (GGT) enzyme plays a central role in glutathione homeostasis. Direct detection of GGT activity could provide critical information for the diagnosis of several pathologies. We propose a new molecular probe, γ-Glu-[1-(13) C]Gly, for monitoring GGT activity in vivo by hyperpolarized (HP) (13) C magnetic resonance (MR). The properties of γ-Glu-[1-(13) C]Gly are suitable for in vivo HP (13) C metabolic analysis since the chemical shift between γ-Glu-[1-(13) C]Gly and its metabolic product, [1-(13) C]Gly, is large (4.3 ppm) and the T1 of both compounds is relatively long (30 s and 45 s, respectively, in H2 O at 9.4 T). We also demonstrate that γ-Glu-[1-(13) C]Gly is highly sensitive to in vivo modulation of GGT activity induced by the inhibitor acivicin. PMID:27483206

  14. High potency olfactory receptor agonists discovered by virtual high-throughput screening: molecular probes for receptor structure and olfactory function

    PubMed Central

    Triballeau, Nicolas; Van Name, Eric; Laslier, Guillaume; Cai, Diana; Paillard, Guillaume; Sorensen, Peter W.; Hoffmann, Rémy; Bertrand, Hugues-Olivier; Ngai, John; Acher, Francine C.

    2008-01-01

    The detection and discrimination of diverse chemical structures by the vertebrate olfactory system is accomplished by the recognition of odorous ligands by their cognate receptors. In the present study we used a computational high-throughput screening strategy to discover novel high affinity agonists of an olfactory G protein-coupled receptor tuned to recognize amino acid ligands. Functional testing of the top candidates validated several agonists with potencies higher than any of the receptor’s known natural ligands. Computational modeling revealed molecular interactions involved in ligand recognition by this receptor, and further highlighted interactions that have been conserved in evolutionarily divergent amino acid receptors. Significantly, the top compounds display robust activities as odorants in vivo, and include a natural product that may be used to signal the presence of bacteria in the aquatic environment. Our virtual screening approach should be applicable to the identification of new bioactive molecules for probing the structure of chemosensory receptors and the function of chemosensory systems in vivo. PMID:19081373

  15. Unfolding stabilities of two structurally similar proteins as probed by temperature-induced and force-induced molecular dynamics simulations.

    PubMed

    Gorai, Biswajit; Prabhavadhni, Arasu; Sivaraman, Thirunavukkarasu

    2015-09-01

    Unfolding stabilities of two homologous proteins, cardiotoxin III and short-neurotoxin (SNTX) belonging to three-finger toxin (TFT) superfamily, have been probed by means of molecular dynamics (MD) simulations. Combined analysis of data obtained from steered MD and all-atom MD simulations at various temperatures in near physiological conditions on the proteins suggested that overall structural stabilities of the two proteins were different from each other and the MD results are consistent with experimental data of the proteins reported in the literature. Rationalization for the differential structural stabilities of the structurally similar proteins has been chiefly attributed to the differences in the structural contacts between C- and N-termini regions in their three-dimensional structures, and the findings endorse the 'CN network' hypothesis proposed to qualitatively analyse the thermodynamic stabilities of proteins belonging to TFT superfamily of snake venoms. Moreover, the 'CN network' hypothesis has been revisited and the present study suggested that 'CN network' should be accounted in terms of 'structural contacts' and 'structural strengths' in order to precisely describe order of structural stabilities of TFTs.

  16. Cloning, sequencing, and use as a molecular probe of a gene encoding an aminoglycoside 6'-N-acetyltransferase of broad substrate profile.

    PubMed Central

    Terán, F J; Suárez, J E; Mendoza, M C

    1991-01-01

    A gene coding for an aminoglycoside 6'-N-acetyltransferase that was able to modify amikacin was cloned from a plasmid isolated from a clinical strain of Enterobacter cloacae. Sequencing of a 955-bp segment which mediates the modifying activity revealed a single open reading frame of 432 nucleotides that predicted a polypeptide of 144 amino acid residues with a molecular weight of 16,021. Putative ribosomal binding sites and -10 and -35 sequences were located at the 5' end of the gene. The size of the polypeptide was confirmed through minicell analysis of the expression products of plasmids containing the sequence. The use of the gene as a molecular probe revealed its specificity toward strains harboring genes coding for related enzymes. This probe is therefore useful for epidemiological studies. Images PMID:2069376

  17. Molecular rotor-based fluorescent probe for selective recognition of hybrid G-quadruplex and as a K+ sensor.

    PubMed

    Liu, Lingling; Shao, Yong; Peng, Jian; Huang, Chaobiao; Liu, Hua; Zhang, Lihua

    2014-02-01

    This work demonstrates the significant fluorescence enhancement of thioflavin T (ThT) when binding to G-quadruplexes possessing hybrid structures by using UV-vis absorption spectra, fluorescence spectra, and Tm experiments to confirm the binding events. ThT binding does not disturb native G-quadruplex structures preformed in Na(+) and K(+) solutions. The fluorescence enhancement is caused by the rotation restriction of benzothiazole (BZT) and dimethylaminobenzene (DMAB) rings in the ThT excited state upon its G-quadruplex binding. This molecular rotor mechanism as a means of fluorescence enhancement is confirmed using a nonrotor analogue of ThT. Hydroxylation and electrolyte experiments demonstrate that ThT stacks on the tetrad of the hybrid G-quadruplexes, whereas electrostatic forces contribute more to ThT binding for other G-quadruplex structures. By stacking on the tetrad, the ThT binding favors selective identification of DNA hybrid G-quadruplex structures with enhanced fluorescence and can serve as a conformation probe to monitor G-quadruplex structure conversion between hybrid and other structures. Using these properties, we developed a selective and label-free fluorescent K(+) sensor with a detection limit of 1 mM for K(+) in the presence of 100 mM Na(+). The coexistence of other metal ions produces a fluorescence response comparable to K(+) alone. We believe that ThT can potentially provide structure identification of hybrid G-quadruplexes and aid in the construction of G-quadruplex-based sensors.

  18. Characterization of sorbent properties of soil organic matter and carbonaceous geosorbents using n-alkanes and cycloalkanes as molecular probes

    SciTech Connect

    Satoshi Endo; Peter Grathwohl; Stefan B. Haderlein; Torsten C. Schmidt

    2009-01-15

    Nonspecific interactions and modes (i.e., adsorption vs absorption) of sorption by noncondensed, amorphous organic phases (here termed organic matter; OM) in soils and by rigid, aromatic, and condensed phases (termed carbonaceous geosorbents; CGs) were investigated using n-alkanes and cycloalkanes as molecular probes. Sorption isotherms of and cyclooctane from water for seven CGs (charcoal, lignite coke, activated carbon, graphite, partially oxidized graphite, diesel soot, bituminous coal), four sorbents with a predominance of OM (lignite, peat, two sapric soils), and two soils containing OM and high amounts of CGs were measured in batch systems. The peat and the sapric soils showed extensively linear sorption, while the CGs exhibited highly nonlinear and strong (K{sub oc} values being up to 105 times those for the OM-rich materials at low concentrations) sorption for the alkanes studied, showing that enhanced sorption by CGs can occur to completely apolar sorbates that do not undergo any specific interaction. The n-octane-to-cyclooctane sorption coefficient ratios for adsorption to CGs were {ge}1, being distinctly different from those for absorption to the OM-rich materials. The measured sorption isotherms and the CG compositions in the soils determined by quantitative petrography analysis suggest, however, that CGs occurring in soils may be far less effective sorbents than the reference CGs used in the sorption experiments at least for nonspecifically interacting sorbates, probably because of competitive sorption and/or pore blocking by natural OM. The presented approaches and results offer a basis for interpreting sorption data for other organic compounds, as nonspecific interactions and sorption modes are relevant for any compound. 47 refs., 4 figs., 2 tabs.

  19. Nanoscopic electrode molecular probes

    DOEpatents

    Krstic, Predrag S.; Meunier, Vincent

    2012-05-22

    The present invention relates to a method and apparatus for enhancing the electron transport property measurements of a molecule when the molecule is placed between chemically functionalized carbon-based nanoscopic electrodes to which a suitable voltage bias is applied. The invention includes selecting a dopant atom for the nanoscopic electrodes, the dopant atoms being chemically similar to atoms present in the molecule, and functionalizing the outer surface and terminations of the electrodes with the dopant atoms.

  20. Improving the visualization of fluorescently tagged nanoparticles and fluorophore-labeled molecular probes by treatment with CuSO(4) to quench autofluorescence in the rat inner ear.

    PubMed

    Zhang, Ya; Zhang, Weikai; Johnston, Alexander H; Newman, Tracey A; Pyykkö, Ilmari; Zou, Jing

    2010-10-01

    Fluorescent tags and fluorophore-conjugated molecular probes have been extensively employed in histological studies to demonstrate nanoparticle distribution in inner ear cell populations. However, autofluorescence that exists in the rodent cochleae disturbs visualization of the fluorescent tags and fluorophore labeling. In the present work, we aimed to improve the visualization of fluorescently tagged nanoparticles and fluorophore-labeled molecular probes by treatment with CuSO(4) to quench autofluorescence in the rat inner ear. The in vivo study was performed on eight- to nine-month-old rats using confocal laser scanning microscopy, and the in vitro study was carried out with DiI-tagged poly(ethylene glycol) and poly(capro-lactone) polymersomes and different fluorescent-labeling agents using a spectrofluorometer. The nanoparticles were intratympanically administered using either an osmotic pump or transtympanic injection. Abundant autofluorescence was detected in spiral ganglion cells (SGCs), stria marginal cells, spiral ligament fibrocytes (SL) and the subcuticular cytoplasm of inner hair cells (IHCs). Sparsely distributed faint autofluorescence was also visualized in outer hair cells (OHCs). The autofluorescence was eliminated by treatment with 1 mM CuSO(4) (in 0.01 M ammonium acetate buffer) for 70-90 min, while the fluorescent tag in the nanoparticle was absolutely preserved and the labeling fluorescence signals of the molecular probes were mostly retained. PMID:20659540

  1. Cystic Fibrosis Transmembrane Conductance Regulator: Using Differential Reactivity toward Channel-Permeant and Channel-Impermeant Thiol-Reactive Probes To Test a Molecular Model for the Pore†

    PubMed Central

    2009-01-01

    The sixth transmembrane segment (TM6) of the CFTR chloride channel has been intensively investigated. The effects of amino acid substitutions and chemical modification of engineered cysteines (cysteine scanning) on channel properties strongly suggest that TM6 is a key component of the anion-conducting pore, but previous cysteine-scanning studies of TM6 have produced conflicting results. Our aim was to resolve these conflicts by combining a screening strategy based on multiple, thiol-directed probes with molecular modeling of the pore. CFTR constructs were screened for reactivity toward both channel-permeant and channel-impermeant thiol-directed reagents, and patterns of reactivity in TM6 were mapped onto two new, molecular models of the CFTR pore: one based on homology modeling using Sav1866 as the template and a second derived from the first by molecular dynamics simulation. Comparison of the pattern of cysteine reactivity with model predictions suggests that nonreactive sites are those where the TM6 side chains are occluded by other TMs. Reactive sites, in contrast, are generally situated such that the respective amino acid side chains either project into the predicted pore or lie within a predicted extracellular loop. Sites where engineered cysteines react with both channel-permeant and channel-impermeant probes occupy the outermost extent of TM6 or the predicted TM5−6 loop. Sites where cysteine reactivity is limited to channel-permeant probes occupy more cytoplasmic locations. The results provide an initial validation of two, new molecular models for CFTR and suggest that molecular dynamics simulation will be a useful tool for unraveling the structural basis of anion conduction by CFTR. PMID:19754156

  2. Refinement of Glucagon-like Peptide 1 Docking to Its Intact Receptor Using Mid-region Photolabile Probes and Molecular Modeling*

    PubMed Central

    Miller, Laurence J.; Chen, Quan; Lam, Polo C.-H.; Pinon, Delia I.; Sexton, Patrick M.; Abagyan, Ruben; Dong, Maoqing

    2011-01-01

    The glucagon-like peptide 1 (GLP1) receptor is an important drug target within the B family of G protein-coupled receptors. Its natural agonist ligand, GLP1, has incretin-like actions and the receptor is a recognized target for management of type 2 diabetes mellitus. Despite recent solution of the structure of the amino terminus of the GLP1 receptor and several close family members, the molecular basis for GLP1 binding to and activation of the intact receptor remains unclear. We previously demonstrated molecular approximations between amino- and carboxyl-terminal residues of GLP1 and its receptor. In this work, we study spatial approximations with the mid-region of this peptide to gain insights into the orientation of the intact receptor and the ligand-receptor complex. We have prepared two new photolabile probes incorporating a p-benzoyl-l-phenylalanine into positions 16 and 20 of GLP1(7–36). Both probes bound to the GLP1 receptor specifically and with high affinity. These were each fully efficacious agonists, stimulating cAMP accumulation in receptor-bearing CHO cells in a concentration-dependent manner. Each probe specifically labeled a single receptor site. Protease cleavage and radiochemical sequencing identified receptor residue Leu141 above transmembrane segment one as its site of labeling for the position 16 probe, whereas the position 20 probe labeled receptor residue Trp297 within the second extracellular loop. Establishing ligand residue approximation with this loop region is unique among family members and may help to orient the receptor amino-terminal domain relative to its helical bundle region. PMID:21454562

  3. Evaluation of molecular-Beacon, TaqMan, and fluorescence resonance energy transfer probes for detection of antibiotic resistance-conferring single nucleotide polymorphisms in mixed Mycobacterium tuberculosis DNA extracts.

    PubMed

    Yesilkaya, Hasan; Meacci, Francesca; Niemann, Stefan; Hillemann, Doris; Rüsch-Gerdes, Sabine; Barer, Michael R; Andrew, Peter W; Oggioni, Marco R

    2006-10-01

    The ability of fluorescence resonance energy transfer, molecular-beacon, and TaqMan probes to detect single nucleotide polymorphism (SNP) in the presence of a wild-type allele was evaluated using drug resistance-conferring SNPs in mixed Mycobacterium tuberculosis DNA. It was found that both the absolute quantity and the ratio of alleles determine the detection sensitivity of the probe systems.

  4. A novel sandwich assay with molecular beacon as report probe for nucleic acids detection on one-dimensional microfluidic beads array.

    PubMed

    Zuo, Xinbing; Yang, Xiaohai; Wang, Kemin; Tan, Weihong; Wen, Jianhui

    2007-03-21

    A novel sandwich assay with molecular beacons as report probes has been developed and integrated into one-dimensional microfluidic beads array (1-D chip) to pursue a label-free and elution-free detection of DNA/mRNA targets. In contrast with the immobilized molecular beacons, this sandwich assay can offer lower fluorescence background and correspondingly higher sensitivity. Furthermore, this sandwich assay on 1-D chip operating in conjunction with molecular beacon technique allows multiple targets detection without the need of laborious and time-consuming elution, which makes the experiment process simple, easy to handle, and reproducible results. In the experiment, the synthesized DNA targets with different concentrations were detected with a detection limit of approximately 0.05 nM. Moreover, the mRNA expression changes in A549 cells before and after anticancer drug 5-flouorouracil treatments were detected and the results were validated by the conventional RT-PCR method.

  5. Molecular cytogenetic analysis of Inv Dup(15) chromosomes, using probes specific for the Pradar-Willi/Angelman syndrome region: Clinical implications

    SciTech Connect

    Leana-Cox, J. ); Jenkins, L. ); Palmer, C.G.; Plattner, R. ); Sheppard, L. ); Flejter, W.L. ); Zackowski, J. ); Tsien, F. ); Schwartz, S. )

    1994-05-01

    Twenty-seven cases of inverted duplications of chromosome 15 (inv dup[15]) were investigated by FISH with two DNA probes specific for the Prader-Willi syndrome/Angelman syndrome (PWS/AS) region on proximal 15q. Sixteen of the marker chromosomes displayed two copies of each probe, while in the remaining 11 markers no hybridization was observed. A significant association was found between the presence of this region and an abnormal phenotype (P<.01). This is the largest study to date of inv dup(15) chromosomes, that uses molecular cytogenetic methods and is the first to report a significant association between the presence of a specific chromosomal region in such markers and an abnormal phenotype. 30 refs., 1 fig., 4 tabs.

  6. Design, synthesis and biological evaluation of a simplified fluorescently labeled discodermolide as a molecular probe to study the binding of discodermolide to tubulin.

    PubMed

    Qi, Jun; Blanden, Adam R; Bane, Susan; Kingston, David G I

    2011-09-01

    The design, synthesis, and biological evaluation of a simplified fluorescently labeled discodermolide analogue possessing a dimethylaminobenzoyl fluorophore has been achieved. Stereoselective Suzuki coupling and Horner-Wadsworth-Emmons reaction comprised the key tactics for its construction. The analogue exhibited qualitatively similar activity to paclitaxel in a tubulin assembly assay, and it can thus be used as a fluorescent molecular probe to explore the local environment of the discodermolide binding site on tubulin. The results of fluorescence measurements on the tubulin-bound analogue are reported.

  7. Premutation for the Martin-Bell syndrome analyzed in a large Sardinian family: III. Molecular analysis with the StB12.3 probe

    SciTech Connect

    Grasso, M.; Perroni, L.; Dagna-Bricarelli, F.

    1996-08-09

    This report complements a series of clinical, cytogenetical, and psychological studies previously reported on a large Sardinian pedigree segregating for premutations and full mutations associated with the Martin-Bell syndrome (MBS). Using the StB12.3 probe, we report now the molecular classification of all of the critical members of the pedigree. These molecular findings are evaluated against the variable phenotypic manifestations of the disease in the course of a six-generation segregation of an MBS premutation allegedly present in a common female progenitor of 14 MBS male patients and 9 female MBS heterozygotes seen in the last two generations. The nature and stepwise progression of MBS-premutations toward the fully manifested Martin-Bell syndrome and the possibility of reverse mutational events toward the normal allele are discussed with respect to the application of the presently available diagnostic tools in genetic counseling. 12 refs., 1 fig.

  8. Binding hotspots on K-ras: consensus ligand binding sites and other reactive regions from probe-based molecular dynamics analysis.

    PubMed

    Prakash, Priyanka; Hancock, John F; Gorfe, Alemayehu A

    2015-05-01

    We have used probe-based molecular dynamics (pMD) simulations to search for interaction hotspots on the surface of the therapeutically highly relevant oncogenic K-Ras G12D. Combining the probe-based query with an ensemble-based pocket identification scheme and an analysis of existing Ras-ligand complexes, we show that (i) pMD is a robust and cost-effective strategy for binding site identification, (ii) all four of the previously reported ligand binding sites are suitable for structure-based ligand design, and (iii) in some cases probe binding and expanded sampling of configurational space enable pocket expansion and increase the likelihood of site identification. Furthermore, by comparing the distribution of hotspots in nonpocket-like regions with known protein- and membrane-interacting interfaces, we propose that pMD has the potential to predict surface patches responsible for protein-biomolecule interactions. These observations have important implications for future drug design efforts and will facilitate the search for potential interfaces responsible for the proposed transient oligomerization or interaction of Ras with other biomolecules in the cellular milieu.

  9. The study of a curcumin-resembling molecular probe for the pH-responsive fluorometric assay and application in cell imaging.

    PubMed

    Xiang, Decheng; Meng, Qinghua; Liu, Heng; Lan, Minbo; Wei, Gang

    2016-01-01

    A molecular probe of DibOH (2,6-bis(4-hydroxybenzylidene)cyclohexanone) designed as the ameliorant to the curcumin was prepared in which one enol unit was removed to avoid the intramolecular hydrogen bond and thus more rigidity and better coplanarity were achieved. Deprotonation of the phenolic hydroxyl group led to the negative charge and the intramolecular charge transfer (ICT) functioned accordingly. The DibOH probe in basic conditions exhibited the absorption peak at 468 nm, which indicated a red shift of 183 nm relative to that in acid conditions and was visible as a brown color to naked eyes. The ratio of fluorescence intensity at 612 nm to that at 520 nm (I612/I520) was calculated and a clear correlation with the pH value was obtained. The density functional theory (DFT) was performed on theoretical investigation of the acidic and basic forms of DibOH. The DibOH probe was evaluated in fluorescent imaging of human breast cancer cells (MCF-7) wherein the heterogeneous distribution of the intracellular pH microenvironment was observed. PMID:26695339

  10. The study of a curcumin-resembling molecular probe for the pH-responsive fluorometric assay and application in cell imaging.

    PubMed

    Xiang, Decheng; Meng, Qinghua; Liu, Heng; Lan, Minbo; Wei, Gang

    2016-01-01

    A molecular probe of DibOH (2,6-bis(4-hydroxybenzylidene)cyclohexanone) designed as the ameliorant to the curcumin was prepared in which one enol unit was removed to avoid the intramolecular hydrogen bond and thus more rigidity and better coplanarity were achieved. Deprotonation of the phenolic hydroxyl group led to the negative charge and the intramolecular charge transfer (ICT) functioned accordingly. The DibOH probe in basic conditions exhibited the absorption peak at 468 nm, which indicated a red shift of 183 nm relative to that in acid conditions and was visible as a brown color to naked eyes. The ratio of fluorescence intensity at 612 nm to that at 520 nm (I612/I520) was calculated and a clear correlation with the pH value was obtained. The density functional theory (DFT) was performed on theoretical investigation of the acidic and basic forms of DibOH. The DibOH probe was evaluated in fluorescent imaging of human breast cancer cells (MCF-7) wherein the heterogeneous distribution of the intracellular pH microenvironment was observed.

  11. Molecular imaging of human tumor cells that naturally overexpress type 2 cannabinoid receptors using a quinolone-based near-infrared fluorescent probe

    NASA Astrophysics Data System (ADS)

    Wu, Zhiyuan; Shao, Pin; Zhang, Shaojuan; Ling, Xiaoxi; Bai, Mingfeng

    2014-07-01

    Cannabinoid CB2 receptors (CB2R) hold promise as therapeutic targets for treating diverse diseases, such as cancers, neurodegenerative diseases, pain, inflammation, osteoporosis, psychiatric disorders, addiction, and immune disorders. However, the fundamental role of CBR in the regulation of diseases remains unclear, largely due to a lack of reliable imaging tools for the receptors. The goal of this study was to develop a CBR-targeted molecular imaging probe and evaluate the specificity of the probe using human tumor cells that naturally overexpress CBR. To synthesize the CBR-targeted probe (NIR760-Q), a conjugable CBR ligand based on the quinolone structure was first prepared, followed by bioconjugation with a near-infrared (NIR) fluorescent dye, NIR760. In vitro fluorescence imaging and competitive binding studies showed higher uptake of NIR760-Q than free NIR760 dye in Jurkat human acute T-lymphoblastic leukemia cells. In addition, the high uptake of NIR760-Q was significantly inhibited by the blocking agent, 4-quinolone-3-carboxamide, indicating specific binding of NIR760-Q to the target receptors. These results indicate that the NIR760-Q has potential in diagnostic imaging of CBR positive cancers and elucidating the role of CBR in the regulation of disease progression.

  12. Ratiometric Molecular Probes Based on Dual Emission of a Blue Fluorescent Coumarin and a Red Phosphorescent Cationic Iridium(III) Complex for Intracellular Oxygen Sensing.

    PubMed

    Yoshihara, Toshitada; Murayama, Saori; Tobita, Seiji

    2015-06-09

    Ratiometric molecular probes RP1 and RP2 consisting of a blue fluorescent coumarin and a red phosphorescent cationic iridium complex connected by a tetra- or octaproline linker, respectively, were designed and synthesized for sensing oxygen levels in living cells. These probes exhibited dual emission with good spectral separation in acetonitrile. The photorelaxation processes, including intramolecular energy transfer, were revealed by emission quantum yield and lifetime measurements. The ratios (R(I) = (I(p)/I(f))) between the phosphorescence (I(p)) and fluorescence (I(f)) intensities showed excellent oxygen responses; the ratio of R(I) under degassed and aerated conditions ( R(I)(0) was 20.3 and 19.6 for RP1 and RP2. The introduction of the cationic Ir (III) complex improved the cellular uptake efficiency compared to that of a neutral analogue with a tetraproline linker. The emission spectra of the ratiometric probes internalized into living HeLa or MCF-7 cells could be obtained using a conventional microplate reader. The complex RP2 with an octaproline linker provided ratios comparable to the ratiometric measurements obtained using a microplate reader: the ratio of the R(I)) value of RP2 under hypoxia (2.5% O2) to that under normoxia (21% O2) was 1.5 and 1.7 for HeLa and MCF-7 cells, respectively. Thus, the intracellular oxygen levels of MCF-7 cells could be imaged by ratiometric emission measurements using the complex RP2.

  13. SERS Detection of Dopamine Using Label-Free Acridine Red as Molecular Probe in Reduced Graphene Oxide/Silver Nanotriangle Sol Substrate

    NASA Astrophysics Data System (ADS)

    Luo, Yanghe; Ma, Lu; Zhang, Xinghui; Liang, Aihui; Jiang, Zhiliang

    2015-05-01

    The reduced graphene oxide/silver nanotriangle (rGO/AgNT) composite sol was prepared by the reduction of silver ions with sodium borohydride in the presence of H2O2 and sodium citrate. In the nanosol substrate, the molecular probe of acridine red (AR) exhibited a weak surface-enhanced Raman scattering (SERS) peak at 1506 cm-1 due to its interaction with the rGO of rGO/AgNT. Upon addition of dopamine (DA), the competitive adsorption between DA and AR with the rGO took place, and the AR molecules were adsorbed on the AgNT aggregates with a strong SERS peak at 1506 cm-1 that caused the SERS peak increase. The increased SERS intensity is linear to the DA concentration in the range of 2.5-500 μmol/L. This new analytical system was investigated by SERS, fluorescence, absorption, transmission electron microscope (TEM), and scanning electron microscope (SEM) techniques, and a SERS quantitative analysis method for DA was established, using AR as a label-free molecular probe.

  14. Microbead-based ligase detection reaction assay using a molecular beacon probe for the detection of low-abundance point mutations.

    PubMed

    Watanabe, Sho; Hagihara, Kenta; Tsukagoshi, Kazuhiko; Hashimoto, Masahiko

    2014-01-01

    A microbead-based ligase detection reaction (LDR) assay using a molecular beacon probe was developed for the facile and rapid detection of point mutations present in low copy numbers in a mixed population of wild-type DNA. Biotin-tagged ligation products generated in the LDR were captured on the surface of streptavidin-modified magnetic beads for purification and concentration. The resulting product-tethered microbeads were combined with a molecular beacon probe solution, and the suspension was directly flowed into a capillary. The microbeads were accumulated in a confined space within the capillary using a bar magnet. The packed bead sample was then scanned by a fluorescence scanning imager to detect the presence of any mutations. With the developed methodology, we were able to successfully detect one cancer mutation in a mixture of 400 wild-type templates (t test at 95% confidence level). Furthermore, the post-LDR processing, typically the most laborious and time-consuming step in LDR-based mutation detection assays, could be carried out much more rapidly (approximately 20 min). This was enabled by the simple bead and fluid manipulations involved in the present assay.

  15. Molecular cytogenetic analysis of the crucian carp, Carassius carassius (Linnaeus, 1758) (Teleostei, Cyprinidae), using chromosome staining and fluorescence in situ hybridisation with rDNA probes

    PubMed Central

    Spoz, Aneta; Boron, Alicja; Porycka, Katarzyna; Karolewska, Monika; Ito, Daisuke; Abe, Syuiti; Kirtiklis, Lech; Juchno, Dorota

    2014-01-01

    Abstract The crucian carp Carassius carassius (Linnaeus, 1758) is a species with restricted and decreasing distribution in Europe. Six males and six females of the species from the Baltic Sea basin in Poland were examined to show sequentially CMA3/AgNO3 staining pattern, DAPI staining, and, for the first time in literature, molecular cytogenetic analysis using double-colour fluorescence in situ hybridisation (FISH) with 28S and 5S rDNA probes. The karyotype consisted of 20 m, 36 sm and 44 sta chromosomes, NF=156. The AgNO3 stained NORs were most frequently located terminally in the short arms of two sm and two sta elements, and CMA3-positive sites were also observed suggesting abundant GC-rich repetitive DNA in the regions. Other CMA3-positive sites in the short arms of six to ten sm and sta chromosomes were detected. The results based on 28S rDNA FISH confirmed the location of rDNA sites. DAPI-negative staining of NORs suggested the scarcity of AT-rich DNA in the regions. FISH with 5S rDNA probe revealed 8–14 loci (ten and 12 in respectively 49 and 29% of metaphases). They were located in two sm and eight to ten sta chromosomes and six of them were larger than others. Simultaneously, mapping of the two rDNA families on the chromosomes of C. carassius revealed that both 28S and 5S rDNA probes were located in different chromosomes. Molecular cytogenetic data of C. carassius presented here for the first time give an important insight into the structure of chromosomes of this polyploid and declining species and may be useful in its systematics. PMID:25349674

  16. Novel super-resolution capable mitochondrial probe, MitoRed AIE, enables assessment of real-time molecular mitochondrial dynamics

    PubMed Central

    Lo, Camden Yeung-Wah; Chen, Sijie; Creed, Sarah Jayne; Kang, Miaomiao; Zhao, Na; Tang, Ben Zhong; Elgass, Kirstin Diana

    2016-01-01

    Mitochondria and mitochondrial dynamics play vital roles in health and disease. With the intricate nanometer-scale structure and rapid dynamics of mitochondria, super-resolution microscopy techniques possess great un-tapped potential to significantly contribute to understanding mitochondrial biology and kinetics. Here we present a novel mitochondrial probe (MitoRed AIE) suitable for live mitochondrial dynamics imaging and single particle tracking (SPT), together with a multi-dimensional data analysis approach to assess local mitochondrial (membrane) fluidity. The MitoRed AIE probe localizes primarily to mitochondrial membranes, with 95 ms fluorophore on-time delivering 106 photons/ms, characteristics which we exploit to demonstrate live cell 100 fps 3D time-lapse tracking of mitochondria. Combining our experimental and analytical approaches, we uncover mitochondrial dynamics at unprecedented time scales. This approach opens up a new regime into high spatio-temporal resolution dynamics in many areas of mitochondrial biology. PMID:27492961

  17. Development of a highly sensitive semi-quantitative real-time PCR and molecular beacon probe assay for the detection of respiratory syncytial virus.

    PubMed

    O'Shea, Matthew K; Cane, Patricia A

    2004-06-15

    Molecular beacons are a novel class of oligonucleotide probe capable of reporting the accumulation of target amplicon during real-time PCR by the emission of a fluorescent signal. A novel assay for the detection and estimation of respiratory syncytial virus (RSV) nucleic acid in clinical specimens based on real-time PCR utilising such a probe was developed. The probe consisted of two short arm sequences and a central loop sequence complementary to a region of the N gene (the target amplicon). The probe was characterised and a semi-quantitative nested real-time PCR using a LightCycler instrument was optimised. Standard curves were generated using cycle threshold (C(t)) values calculated from several assays over a range of logarithmic RSV titres. Linear coefficient correlations were close to one ( r(2) = 0.998) and the detection limit of the optimised assay was reproducibly shown to be 1 x 10 (-4) pfu/ml. The intra-assay coefficient of variation (CV) of C(t) values of the optimal assay was 0.8% and the CV of quantification data was 6.6%. The interassay CV of C(t) values was 2.0% and the quantification CV was 6.7%. The validity of the assay for the detection of RSV in clinical specimens was assessed by analysing ten specimens previously assayed in a different laboratory. Real-time PCR analysis was completely consistent with the results of prior analysis. The rapidity, sensitivity and specificity of the assay should greatly facilitate epidemiological studies, particularly in adults as existing methods perform better on clinical specimens from children.

  18. A Fluorescent Molecular Probe for the Detection of Hydrogen Based on Oxidative Addition Reactions with Crabtree-Type Hydrogenation Catalysts.

    PubMed

    Kos, Pavlo; Plenio, Herbert

    2015-11-01

    A Crabtree-type Ir(I) complex tagged with a fluorescent dye (bodipy) was synthesized. The oxidative addition of H2 converts the weakly fluorescent Ir(I) complex (Φ=0.038) into a highly fluorescent Ir(III) species (Φ=0.51). This fluorogenic reaction can be utilized for the detection of H2 and to probe the oxidative addition step in the catalytic hydrogenation of olefins.

  19. Molecular imaging of biothiols and in vitro diagnostics based on an organic chromophore bearing a terbium hybrid probe.

    PubMed

    Zhou, Zhan; Wang, Qianming; Zhang, Cheng Cheng; Gao, Jinwei

    2016-04-25

    In this research, a novel terbium-based luminescent hybrid inorganic/organic probe was designed and synthesized. Mesoporous silica nanospheres dispersed in water were used as the appropriate host for the covalently linked lanthanide-containing organic structures. The lanthanide structure was linked to a sulfonate ester unit, which, in the presence of biothiols, was cleaved to result in terbium emission. The hybrid probe exhibited the capabilities of quantitative determination and detection limits for biothiols were presented (36.8 nM for Cys, 32.5 nM for GSH, and 34.7 nM for Hcy). Evaluation of luminescence changes in cell culture demonstrated that this smart probe is cell membrane permeable and selectively lights up in the presence of cysteine and glutathione in human embryonic kidney cells and human lung adenocarcinoma cells. This variation in the presence of biothiols can be controlled by the treatment with N-methylmaleimide. The narrow line-like bands and long-lived excited states of this terbium luminescent sensor allows the discrimination of scattering signals and interfering fluorescence derived from biological tissues.

  20. Molecular imaging of biothiols and in vitro diagnostics based on an organic chromophore bearing a terbium hybrid probe.

    PubMed

    Zhou, Zhan; Wang, Qianming; Zhang, Cheng Cheng; Gao, Jinwei

    2016-04-25

    In this research, a novel terbium-based luminescent hybrid inorganic/organic probe was designed and synthesized. Mesoporous silica nanospheres dispersed in water were used as the appropriate host for the covalently linked lanthanide-containing organic structures. The lanthanide structure was linked to a sulfonate ester unit, which, in the presence of biothiols, was cleaved to result in terbium emission. The hybrid probe exhibited the capabilities of quantitative determination and detection limits for biothiols were presented (36.8 nM for Cys, 32.5 nM for GSH, and 34.7 nM for Hcy). Evaluation of luminescence changes in cell culture demonstrated that this smart probe is cell membrane permeable and selectively lights up in the presence of cysteine and glutathione in human embryonic kidney cells and human lung adenocarcinoma cells. This variation in the presence of biothiols can be controlled by the treatment with N-methylmaleimide. The narrow line-like bands and long-lived excited states of this terbium luminescent sensor allows the discrimination of scattering signals and interfering fluorescence derived from biological tissues. PMID:27041001

  1. Optical probes for molecular-guided surgery: Using photomedicine to prevent recurrence in the surgical bed (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Spring, Bryan Q.; Sears, R. Bryan; Zheng, Lei Z.; Mai, Zhiming; Watanabe, Reika; Villa, Elizabeth; Hasan, Tayyaba

    2016-03-01

    Residual tumor deposits missed by conventional treatments frequently seed local and distal recurrence utilizing a network of molecular signaling mechanisms. Beyond providing contrast for molecular-guided surgery, this talk will highlight new concepts in phototherapy to address residual cancer cells in danger zones of recurrence, including selective treatment of microscopic disease using molecular-targeted, activatable immunoconjugates, and photo-initiated release of multikinase inhibitors that suppress multiple modes of tumor escape using optically active nanoparticles. These new approaches support an expanded role for the use of light in fluorescence-guided surgery—for phototherapy and for focused drug release to maximize tumor debulking with suppression of disease recurrence.

  2. An in vivo molecular imaging probe (18)F-Annexin B1 for apoptosis detection by PET/CT: preparation and preliminary evaluation.

    PubMed

    Wang, Ming-Wei; Wang, Fang; Zheng, Yu-Jia; Zhang, Ying-Jian; Zhang, Yong-Ping; Zhao, Qing; Shen, Clifton Kwang-Fu; Wang, Yue; Sun, Shu-Han

    2013-02-01

    There is an increasing need to develop non-invasive molecular imaging strategies for visualizing and quantifying apoptosis status of diseases (especially for cancer) for diagnosis and monitoring treatment response. Since externalization of phosphatidylserine (PS) is one of the early molecular events during apoptosis, Annexin B1 (AnxB1), a member of Annexins family with high affinity toward the head group of PS, could be a potential positron emission tomography (PET) imaging probe for imaging cell death process after labeled by positron-emitting nuclides, such as (18)F. In the present study, we investigated a novel PET probe, (18)F-labeled Annexin B1 ((18)F-AnxB1), for apoptosis imaging. (18)F-AnxB1 was prepared reliably by conjugating AnxB1 with a (18)F-tag, N-succinimidyl 4-[(18)F]fluorobenzoate ([(18)F]SFB), in a radiolabeling yield of about 20 % within 40 min. The in vitro binding of (18)F-AnxB1 with apoptotic cells induced by anti-Fas antibody showed twofold increase compared to those without treatment, confirmed by flow cytometric analysis with AnxV-FITC/PI staining. Stability tests demonstrated (18)F-AnxB1 was rather stable in vitro and in vivo without degradation. The serial (18)F-AnxB1 PET/CT scans in healthy rats outlined its biodistribution and pharmacokinetics, indicating a rapid renal clearance and predominant accumulation into kidney and bladder at 2 h p.i. (18)F-AnxB1 PET/CT imaging was successfully applied to visualize in vivo apoptosis sites in tumor induced by chemotherapy and in kidney simulated by ischemia-reperfusion injury. The high-contrast images were obtained at 2 h p.i. to delineate apoptotic tumor. Apoptotic region could be still identified by (18)F-AnxB1 PET 4 h p.i., despite the high probe retention in kidneys. In summary, we have developed (18)F-AnxB1 as a PS-specific PET probe for the apoptosis detection and quantification which could have broad applications from disease diagnosis to treatment monitoring, especially in the cases of

  3. Classical Wolf-Hirschhorn Syndrome confirmed molecularly despite normal results using commercially available probes: Redefinition of critical region

    SciTech Connect

    Zackai, E.H.; McDonald-McGinn, D.M.; Spinner, N.

    1994-09-01

    Wolf-Hirschhorn Syndrome, WHS, (4p-) is a clinically recognized entity where the deletion ranges from one half of the short arm of 4p to being subtle and cytogenetically undetectable. Because such variations do not result in significant differences in the WMS phenotype it has been suggested that them is a critical region involved in the distal portion of chromosome 4, within 4p16.3. This has been recently localized to a 2.5 Mb segment 100-300 kb from the telomere. A cosmid probe, pC847.351, that maps to distal 4p16.3 (locus D4F26) is commercially available for diagnostic use. We present a child with classical feature of Wolf-Hirschhorn Syndrome whose chromosome analyses, including high resolution banding, looking specifically at the 4p region, were normal, and in whom FISH using the commercially available cosmid probe for the 4p16.3 region did not demonstrate a deletion. Insistence on the clinician`s part that the child`s features were classic for WHS prompted further investigation. Four additional cosmid clones distal to the HD gene were tested by FISH on metaphase chromosomes from the proband. Cosmids representing the loci D4S95 and D4S43, which are {approximately}3.5 and 2.5 Mb, Respectively, proximal to D4F26 were present. However, cosmids for the loci D4S98 and FGFR3, which are within 100 kb of each other and {approximately}300 kb distal to D4S43 were deleted. This deletion is the smallest reported to date in a patient with typical WHS. Previous studies of patients both lacking the WHS phenotype and deleted using cosmid probe pC847.351 have suggested that the most distal region (150 kb) of 4p16.3 is not part of the WHS critical region. This is confirmed by our study. Estabrooks` report, together with our case, suggests redefinition of the WHS critical region proximal to D4F26 and distal to D4S43. We caution against ruling out WHS with the above probe since there may be other cases where the most distal region is intact.

  4. Phenotyping hepatocellular metabolism using uniformly labeled carbon-13 molecular probes and LC-HRMS stable isotope tracing.

    PubMed

    Meissen, John K; Pirman, David A; Wan, Min; Miller, Emily; Jatkar, Aditi; Miller, Russell; Steenwyk, Rick C; Blatnik, Matthew

    2016-09-01

    Metabolite stable isotope tracing is a powerful bioanalytical strategy that has the potential to unravel phenotypic markers of early pharmaceutical efficacy by monitoring enzymatic incorporation of carbon-13 atoms into targeted pathways over time. The practice of probing biological systems with carbon-13 labeled molecules using broad MS-based screens has been utilized for many years in academic laboratories but has had limited application in the pharmaceutical R&D environment. The goal of this work was to establish a LCMS analytical workflow that was capable of monitoring carbon-13 isotope changes in glycolysis, the TCA and urea cycles, and non-essential amino acid metabolism. This work applies a standardized protein precipitation with 80% cold methanol and two distinct reverse-phase ion-pair liquid chromatography methods coupled to either a positive- or negative-ion mode high-resolution accurate mass spectrometry screening method. The data herein combines thousands of single-point peak integrations into a novel metabolite network map as a visualization aid to probe and monitor stable isotope incorporation in murine hepatocytes using uniformly labeled (13)C6 glucose, (13)C3 lactate, and (13)C5 glutamine. This work also demonstrates that nitrogen metabolism may have a large influence on the TCA cycle and gluconeogenic carbon fluxes in hepatocyte cell culture. PMID:27343766

  5. Development of real-time PCR assays for the quantitative detection of Epstein-Barr virus and cytomegalovirus, comparison of TaqMan probes, and molecular beacons.

    PubMed

    Jebbink, Jiska; Bai, Xin; Rogers, Beverly Barton; Dawson, D Brian; Scheuermann, Richard H; Domiati-Saad, Rana

    2003-02-01

    Human Epstein-Barr virus (EBV) and cytomegalovirus (CMV) can cause serious complications in immunocompromised patients. Rapid diagnosis of EBV and CMV infection is critical in the management of the disease so that anti-viral therapy can be started early. Here we describe the development of real-time PCR assays using TaqMan probes and molecular beacons and compare the performance of both assays with a well-established, validated, gel-based PCR method for the quantification of EBV and CMV in patients' samples. The TaqMan and molecular beacon assays were linear between 10 to 10(7) viral genomes/reaction. Both assays generated calibration curves with strong correlation and low intra-assay and interassay variation. Results of EBV and CMV viral load determination inpatient samples obtained by the gel-based and real-time PCR were very similar. The real-time PCR assays showed increases in viral load before clinical measures of viral disease and decreases in viral load during anti-viral therapy in two of six pediatric patients. The data indicate that these TaqMan and molecular beacon approaches are accurate, rapid, and reliable assays for the diagnosis and monitoring of EBV and CMV infections in patients.

  6. Probing ligand-binding modes and binding mechanisms of benzoxazole-based amide inhibitors with soluble epoxide hydrolase by molecular docking and molecular dynamics simulation.

    PubMed

    Chen, Hang; Zhang, Ying; Li, Liang; Han, Ju-Guang

    2012-08-30

    Soluble epoxide hydrolase (sEH) has become a new therapeutic target for treating a variety of human diseases. The inhibition of human sEH hydrolase activity was studied by molecular docking and molecular dynamics (MD) simulation techniques. A set of six benzoxazole-based amide inhibitors binding to sEH has been studied through molecular docking, MD simulation, free energy calculations, and energy decomposition analysis. On the basis of molecular mechanics-generalized Born/surface area (MM-GB/SA) computation and normal-mode analysis (NMA), the obtained results indicate that the rank of calculated binding free energies (ΔΔGTOT) of these inhibitors is in excellent agreement with that of experimental bioactivity data (IC50). The correlation coefficient (r(2)) between the predicted ΔΔGTOT and IC50 is 0.88. van der Waals energies are the largest component of the total energies, and the entropy changes play an indispensable role in determining the ΔΔGTOT. Rational binding modes were discussed and determined by the docking results and binding free energies. The free energy decomposition of each residue reveals that the residue Trp334 dominates the most binding free energies among all residues and that the activities for these molecules to the sEH are not decided by hydrogen bonds or a certain residue but by the common effect of multiple side chains in the active site.

  7. Molecular beacon-functionalized gold nanoparticles as probes in dry-reagent strip biosensor for DNA analysis.

    PubMed

    Mao, Xun; Xu, Hui; Zeng, Qingxiang; Zeng, Lingwen; Liu, Guodong

    2009-06-01

    The highly specific molecule recognition properties of molecular beacons (MB) are combined with the unique optical properties of gold nanoparticles (Au-NPs) for the development of a dry-reagent strip-type nucleic acid biosensor (DSNAB) that enables sensitive and low-cost detection of nucleic acid samples within 15 min.

  8. Interactions between a surface-active cationic 3H-indole molecular probe and β-cyclodextrin. Design of a novel type of rotaxane

    NASA Astrophysics Data System (ADS)

    Shen, Xinghai; Belletête, Michel; Durocher, Gilles

    1999-02-01

    We report herein the interactions of a cationic surface-active molecular probe having long aliphatic chains, i.e., iodo-methyldioctadecyl 2-( p-hexylaminophenyl)-3,3-dimethyl-5-carboethoxy-3H-indole ammonium, with β-CD investigated by spectral and photophysical characterizations. It is found through lifetime measurements that only two species exist within the whole range of β-CD concentrations. Both the steady-state and the time-resolved fluorescence results further show that the stoichiometry of the inclusion complex is 1:3. It is also suggested that an interaction of the aliphatic chains of the cationic 3H-indole with β-CD takes place. Finally it is shown that a new rotaxane forms spontaneously in solution.

  9. Molecular beacon-based junction probes for efficient detection of nucleic acids via a true target-triggered enzymatic recycling amplification.

    PubMed

    Kong, Rong-Mei; Zhang, Xiao-Bing; Zhang, Liang-Liang; Huang, Yan; Lu, Dan-Qing; Tan, Weihong; Shen, Guo-Li; Yu, Ru-Qin

    2011-01-01

    This work reports the development of a new molecular beacon-based junction sensing system with highly sensitive DNA detection and a strong capability to identify SNPs. The single linear probe typically labels the midsection of the oligonucleotide, but our next-generation junction sensing system uses a hairpin-structured MB with labels on each end of the oligonucleotide to maintain the cleaving activity of our newly designed ssDNA-cleaved endonuclease, Nt.BbvCI, rather than the typical dsDNA-cleaved endonuclease. These design improvements guarantee a true and efficient target-triggered enzymatic recycling amplification process in our sensing system. They also afford a faster and more sensitive response toward target DNA than the first-generation junction sensing system.

  10. Real time PCR for the rapid detection of vanA gene in surface waters and aquatic macrophyte by molecular beacon probe.

    PubMed

    Lata, Pushpa; Ram, Siya; Agrawal, Madhoolika; Shanker, Rishi

    2009-05-01

    Enterococci serve as an "indicator" of fecal contamination for recreational water quality. The vancomycin-resistant-enterococci (VRE) are emerging environmental contaminants in the surface waters. The aim ofthis study wasto develop a rapid and specific molecular beacon probe (MBP)-based real-time PCR assay for detection of vanA gene in surface waters and aquatic macrophyte. The limit of detection (LOD) of the MBP assay was 1 CFU/mL of VRE [r = 0.943; PCR efficiency = 99.7%] in 2-fold dilution format within 2.5 h and demonstrated high specificityfor environmental enterococci isolates exhibiting VanA phenotype (n=25). VRE were detected from downstream surface waters of the rivers impacted by point sources of pollution and recreational activities.The probe detected vanA gene in rootmat associated microbiota of E. crassipes (Mart) Solms. an aquatic nuisance weed, at eutrophic sites of the surface waters (ANOVA p < 0.001). In addition, the assay enabled detection of otherwise nondetectable vanA gene concentration in the upstream sites of two Indian rivers (Student's ttest p < 0.001). The MBP assay developed can be used for sensitive and rapid detection of VRE in surface waters and identification of nonpoint sources of pollution for implementation of preventive measures to protect human health.

  11. The distribution of cosmic-ray ionization rates in diffuse molecular clouds as probed by H3+.

    PubMed

    Indriolo, Nick

    2012-11-13

    Owing to its simple chemistry, H(3)(+) is widely regarded as the most reliable tracer of the cosmic-ray ionization rate in diffuse interstellar clouds. At present, H(3)(+) observations have been made in over 50 sight lines that probe the diffuse interstellar medium (ISM) throughout the Galaxy. This small survey presents the opportunity to investigate the distribution of cosmic-ray ionization rates in the ISM, as well as any correlations between the ionization rate and line-of-sight properties. Some of the highest inferred ionization rates are about 25 times larger than the lowest upper limits, suggesting variations in the underlying low-energy cosmic-ray flux across the Galaxy. Most likely, such variations are caused predominantly by the distance between an observed cloud and the nearest site of particle acceleration.

  12. Water at a hydrophilic solid surface probed by ab-initio molecular dynamics: inhomogeneous thin layers of dense fluid

    SciTech Connect

    Cicero, G; Grossman, J; Galli, G; Catellani, A

    2005-01-28

    We present a microscopic model of the interface between liquid water and a hydrophilic, solid surface, as obtained from ab-initio molecular dynamics simulations. In particular, we focused on the (100)surface of cubic SiC, a leading candidate semiconductor for bio-compatible devices. Our results show that, in the liquid in contact with the clean substrate, molecular dissociation occurs in a manner unexpectedly similar to that observed in the gas phase. After full hydroxylation takes place, the formation of a thin ({approx}3 {angstrom})interfacial layer is observed, which has higher density than bulk water and forms stable hydrogen bonds with the substrate. The liquid does not uniformly wet the surface, rather molecules preferably bind along directions parallel to the Si dimer rows. Our calculations also predict that one dimensional confinement between two hydrophilic surfaces at about 1.3 nm distance does not affect the structural and electronic properties of the whole water sample.

  13. Complete Photo-Induced Breakup of the H2 Molecule as a Probe of Molecular Electron Correlation

    NASA Astrophysics Data System (ADS)

    Vanroose, Wim; Martín, Fernando; Rescigno, Thomas N.; McCurdy, C. William

    2005-12-01

    Despite decades of progress in quantum mechanics, electron correlation effects are still only partially understood. Experiments in which both electrons are ejected from an oriented hydrogen molecule by absorption of a single photon have recently demonstrated a puzzling phenomenon: The ejection pattern of the electrons depends sensitively on the bond distance between the two nuclei as they vibrate in their ground state. Here, we report a complete numerical solution of the Schrödinger equation for the double photoionization of H2. The results suggest that the distribution of photoelectrons emitted from aligned molecules reflects electron correlation effects that are purely molecular in origin.

  14. Complete Photo-Induced Breakup of the H2 Molecule as a Probe ofMolecular Electron Correlation

    SciTech Connect

    Vanroose, Wim; Martin, Fernando; Rescigno, Thomas N.; McCurdy, C.William

    2005-11-17

    Despite decades of progress in quantum mechanics, electron correlation effects are still only partially understood. Experiments in which both electrons are ejected from an oriented hydrogen molecule by absorption of a single photon have recently demonstrated a puzzling phenomenon: The ejection pattern of the electrons depends sensitively on the bond distance between the two nuclei as they vibrate in their ground state. Here we report a complete numerical solution of the Schrodinger equation for the double photoionization of H2. The results suggest that the distribution of photoelectrons emitted from aligned molecules reflects electron correlation effects that are purely molecular in origin.

  15. Probing molecular interaction in ionic liquids by low frequency spectroscopy: Coulomb energy, hydrogen bonding and dispersion forces.

    PubMed

    Fumino, Koichi; Reimann, Sebastian; Ludwig, Ralf

    2014-10-28

    Ionic liquids are defined as salts composed solely of ions with melting points below 100 °C. These remarkable liquids have unique and fascinating properties and offer new opportunities for science and technology. New combinations of ions provide changing physical properties and thus novel potential applications for this class of liquid materials. To a large extent, the structure and properties of ionic liquids are determined by the intermolecular interaction between anions and cations. In this perspective we show that far infrared and terahertz spectroscopy are suitable methods for studying the cation-anion interaction in these Coulomb fluids. The interpretation of the measured low frequency spectra is supported by density functional theory calculations and molecular dynamics simulations. We present results for selected aprotic and protic ionic liquids and their mixtures with molecular solvents. In particular, we focus on the strength and type of intermolecular interaction and how both parameters are influenced by the character of the ions and their combinations. We show that the total interaction between cations and anions is a result of a subtle balance between Coulomb forces, hydrogen bonds and dispersion forces. For protic ionic liquids we could measure distinct vibrational modes in the low frequency spectra indicating clearly the cation-anion interaction characterized by linear and medium to strong hydrogen bonds. Using isotopic substitution we have been able to dissect frequency shifts related to pure interaction strength between cations and anions and to different reduced masses only. In this context we also show how these different types of interaction may influence the physical properties of ionic liquids such as the melting point, viscosity or enthalpy of vaporization. Furthermore we demonstrate that low frequency spectroscopy can also be used for studying ion speciation. Low vibrational features can be assigned to contact ion pairs and solvent separated

  16. Trimodal color-fluorescence-polarization endoscopy aided by a tumor selective molecular probe accurately detects flat lesions in colitis-associated cancer

    PubMed Central

    Charanya, Tauseef; York, Timothy; Bloch, Sharon; Sudlow, Gail; Liang, Kexian; Garcia, Missael; Akers, Walter J.; Rubin, Deborah; Gruev, Viktor; Achilefu, Samuel

    2014-01-01

    Abstract. Colitis-associated cancer (CAC) arises from premalignant flat lesions of the colon, which are difficult to detect with current endoscopic screening approaches. We have developed a complementary fluorescence and polarization reporting strategy that combines the unique biochemical and physical properties of dysplasia and cancer for real-time detection of these lesions. Using azoxymethane-dextran sodium sulfate (AOM-DSS) treated mice, which recapitulates human CAC and dysplasia, we show that an octapeptide labeled with a near-infrared (NIR) fluorescent dye selectively identified all precancerous and cancerous lesions. A new thermoresponsive sol-gel formulation allowed topical application of the molecular probe during endoscopy. This method yielded high contrast-to-noise ratios (CNR) between adenomatous tumors (20.6±1.65) and flat lesions (12.1±1.03) and surrounding uninvolved colon tissue versus CNR of inflamed tissues (1.62±0.41). Incorporation of nanowire-filtered polarization imaging into NIR fluorescence endoscopy shows a high depolarization contrast in both adenomatous tumors and flat lesions in CAC, reflecting compromised structural integrity of these tissues. Together, the real-time polarization imaging provides real-time validation of suspicious colon tissue highlighted by molecular fluorescence endoscopy. PMID:25473883

  17. Trimodal color-fluorescence-polarization endoscopy aided by a tumor selective molecular probe accurately detects flat lesions in colitis-associated cancer

    NASA Astrophysics Data System (ADS)

    Charanya, Tauseef; York, Timothy; Bloch, Sharon; Sudlow, Gail; Liang, Kexian; Garcia, Missael; Akers, Walter J.; Rubin, Deborah; Gruev, Viktor; Achilefu, Samuel

    2014-12-01

    Colitis-associated cancer (CAC) arises from premalignant flat lesions of the colon, which are difficult to detect with current endoscopic screening approaches. We have developed a complementary fluorescence and polarization reporting strategy that combines the unique biochemical and physical properties of dysplasia and cancer for real-time detection of these lesions. Using azoxymethane-dextran sodium sulfate (AOM-DSS) treated mice, which recapitulates human CAC and dysplasia, we show that an octapeptide labeled with a near-infrared (NIR) fluorescent dye selectively identified all precancerous and cancerous lesions. A new thermoresponsive sol-gel formulation allowed topical application of the molecular probe during endoscopy. This method yielded high contrast-to-noise ratios (CNR) between adenomatous tumors (20.6±1.65) and flat lesions (12.1±1.03) and surrounding uninvolved colon tissue versus CNR of inflamed tissues (1.62±0.41). Incorporation of nanowire-filtered polarization imaging into NIR fluorescence endoscopy shows a high depolarization contrast in both adenomatous tumors and flat lesions in CAC, reflecting compromised structural integrity of these tissues. Together, the real-time polarization imaging provides real-time validation of suspicious colon tissue highlighted by molecular fluorescence endoscopy.

  18. Spectroscopic and molecular modeling methods to investigate the interaction between 5-Hydroxymethyl-2-furfural and calf thymus DNA using ethidium bromide as a probe.

    PubMed

    Zhu, Jinhua; Chen, Lanlan; Dong, Yingying; Li, Jiazhong; Liu, Xiuhua

    2014-04-24

    In this work, the interaction of 5-Hydroxymethyl-2-furfural (5-HMF) with calf thymus DNA (ctDNA) under simulated physiological conditions (Tris-HCl buffer of pH 7.40), was explored by UV absorption spectroscopy, fluorescence spectroscopy and molecular modeling method, using ethidium bromide (EB) as a fluorescence probe of DNA. The fluorescence quenching mechanism of EB-ctDNA by 5-HMF was confirmed to be a static quenching, which derived from the formation of a new complex. The binding constants of 5-HMF with DNA in the presence of EB were calculated to be 2.17×10(3), 4.24×10(3) and 6.95×10(3) L mol(-1) at 300, 305 and 310 K, respectively. The calculated thermodynamic parameters, enthalpy change ΔH and entropy change ΔS, suggested that both hydrophobic interactions and hydrogen bonds played a predominant role in the binding of 5-HMF to DNA. According to the UV absorption spectroscopy and melting temperature (Tm) curve results, the binding mode of 5-HMF with DNA was indicative of a non-intercalative binding, which was supposed to be a groove binding. The molecular modeling results showed that 5-HMF could bind into the hydrophobic region of ctDNA and supported the conclusions obtained from the above experiments.

  19. Pd0-mediated rapid cross-coupling reactions, the rapid C-[11C]methylations, revolutionarily advancing the syntheses of short-lived PET molecular probes.

    PubMed

    Suzuki, Masaaki; Doi, Hisashi; Koyama, Hiroko; Zhang, Zhouen; Hosoya, Takamitsu; Onoe, Hirotaka; Watanabe, Yasuyoshi

    2014-06-01

    Positron emission tomography is a noninvasive method for monitoring drug (or diagnostic) behavior and its localization on the target molecules in the living systems, including the human body, using a short-lived positron-emitting radionuclide. New methodologies for introducing representative short-lived radionuclides, (11)C and (18)F, into the carbon frameworks of biologically active organic compounds have been established by developing rapid C-[(11)C]methylations and C-[(18)F]fluoromethylations using rapid Pd(0)-mediated cross-coupling reactions between [(11)C]methyl iodide (sp(3)-hybridized carbon) and an excess amount of organotributylstannane or organoboronic acid ester having sp(2) (phenyl, heteroaromatic, or alkenyl), sp(alkynyl), or sp(3) (benzyl and cinnamyl)-hybridized carbons; and [(18)F]fluoromethyl halide (iodide or bromide) and an organoboronic acid ester, respectively. These rapid reactions provide a firm foundation for an efficient and general synthesis of short-lived (11)C- or (18)F-labeled PET molecular probes to promote in vivo molecular imaging studies.

  20. Combining cryogenic fiber optic probes with commercial spectrofluorimeters for the synchronous fluorescence Shpol'skii spectroscopy of high molecular weight polycyclic aromatic hydrocarbons.

    PubMed

    Moore, Anthony F T; Barbosa, Fernando; Campiglia, Andres D

    2014-01-01

    Cryogenic fiber optic probes are combined for the first time with a commercial spectrofluorometer for Shpol'skii spectroscopy measurements at liquid nitrogen (77 K) and liquid helium (4.2 K) temperatures. Accurate and reproducible acquisition of fluorescence spectra and signal intensities is demonstrated with three well known Shpol'skii systems, namely, anthracene/heptane, pyrene/hexane, and benzo[a]pyrene/octane. The ability to adjust the excitation and emission bandpass of the spectrofluorimeter to reach both site-resolution and analytically valuable signal-to-noise ratios was illustrated with benzo[a]pyrene in n-octane. The analytical potential of 4.2 K synchronous fluorescence Shpol'skii spectroscopy for the analysis of high molecular weight-polycyclic aromatic hydrocarbons was then explored for the first time. The judicious optimization of wavelength offsets permitted the successful determination of dibenzo[a,l]pyrene, dibenzo[a,e]pyrene, dibenzo[a,h]pyrene, dibenzo[a,i]pyrene, and naphtho[2,3-a]pyrene without previous chromatographic separation from a soil extract with complex matrix composition. The simplicity of the experimental procedure, the competitive analytical figures of merit, and the selectivity of analysis turn 4.2 K synchronous fluorescence Shpol'skii spectroscopy into a valuable alternative for screening isomers of high molecular weight polycyclic aromatic hydrocarbons in environmental samples.

  1. Probing the role of chemical enhancers in facilitating drug release from patches: Mechanistic insights based on FT-IR spectroscopy, molecular modeling and thermal analysis.

    PubMed

    Song, Wenting; Quan, Peng; Li, Shanshan; Liu, Chao; Lv, Siji; Zhao, Yongshan; Fang, Liang

    2016-04-10

    In patches, a drug must release from patches prior to its percutaneous absorption. Chemical enhancers have been used for several decades, but their roles in drug release from patches are poorly understood. In this work, the roles of chemical enhancers in bisoprolol tartrate (BSP-T) release from patches were probed in vitro and in vivo. More importantly, an innovative mechanism insight of chemical enhancers in drug release process was provided at molecular level. FT-IR spectroscopy and molecular modeling were employed to investigate the influence of chemical enhancers on drug-adhesive interaction. The results showed chemical enhancers like Span 80, which had a strong ability forming hydrogen bonds, could decrease drug-adhesive interaction leading to the release of drug from adhesive of patches. Thermal analysis was conducted to research the influence of chemical enhancers on the thermodynamic properties of patch system. It showed that chemical enhancers promoted the formation of free volume of adhesive, which facilitated drug release process. By contrast, the influence on the thermodynamic properties of BSP-T was less effective in influencing BSP-T release process. In conclusion, chemical enhancers played an important role in facilitating BSP-T release from the adhesive DURO-TAK® 87-2287 of patches by decreasing drug-adhesive interaction and promoting the formation of free volume of adhesive. This work may be an important step in understanding the important roles of chemical enhancers in drug release process.

  2. A rapid and sensitive method to detect siRNA-mediated mRNA cleavage in vivo using 5' RACE and a molecular beacon probe.

    PubMed

    Lasham, Annette; Herbert, Mike; Coppieters 't Wallant, Natacha; Patel, Rachna; Feng, Sheryl; Eszes, Marika; Cao, Helen; Reid, Glen

    2010-01-01

    Specific detection of mRNA cleavage by 5'RACE is the only method to confirm the knockdown of mRNA by RNA interference, but is rarely reported for in vivo studies. We have combined 5'-RNA-linker-mediated RACE (5'-RLM-RACE) with real-time PCR using a molecular beacon to develop a rapid and specific method termed MBRACE, which we have used to detect small-interfering RNA (siRNA)-induced cleavage of ApoB, RRM1 and YBX1 transcripts in vitro, and ApoB in vivo. When RNA from siRNA-transfected cells was used for 5'-RLM-RACE and a cleavage site-specific molecular beacon probe was included in subsequent real-time PCR analysis, the specific mRNA cleavage product was detected. Detection of siRNA-mediated cleavage was also observed when RNA from mouse liver following administration of ApoB-specific siRNA was analysed, even in cases where ApoB knockdown measured by real-time PCR was <10%. With its sensitivity and specificity, this variation on the 5'RACE method should prove a useful tool to detect mRNA cleavage and corroborate knockdown studies following siRNA use in vivo.

  3. Probing non-covalent interactions with a second generation energy decomposition analysis using absolutely localized molecular orbitals.

    PubMed

    Horn, Paul R; Mao, Yuezhi; Head-Gordon, Martin

    2016-08-17

    An energy decomposition analysis (EDA) separates a calculated interaction energy into as many interpretable contributions as possible; for instance, permanent and induced electrostatics, Pauli repulsions, dispersion and charge transfer. The challenge is to construct satisfactory definitions of all terms in the chemically relevant regime where fragment densities overlap, rendering unique definitions impossible. Towards this goal, we present an improved EDA for Kohn-Sham density functional theory (DFT) with properties that have previously not been simultaneously attained. Building on the absolutely localized molecular orbital (ALMO)-EDA, this second generation ALMO-EDA is variational and employs valid antisymmetric electronic wavefunctions to produce all five contributions listed above. These contributions moreover all have non-trivial complete basis set limits. We apply the EDA to the water dimer, the T-shaped and parallel-displaced benzene dimer, the p-biphthalate dimer "anti-electrostatic" hydrogen bonding complex, the biologically relevant binding of adenine and thymine in stacked and hydrogen-bonded configurations, the triply hydrogen-bonded guanine-cytosine complex, the interaction of Cl(-) with s-triazine and with the 1,3-dimethyl imidazolium cation, which is relevant to the study of ionic liquids, and the water-formaldehyde-vinyl alcohol ter-molecular radical cationic complex formed in the dissociative photoionization of glycerol. PMID:27492057

  4. Molecular beam studies of unimolecular and bimolecular chemical reaction dynamics using VUV synchrotron radiation as a product probe

    SciTech Connect

    Blank, D.A.

    1997-08-01

    This dissertation describes the use of a new molecular beam apparatus designed to use tunable VUV synchrotron radiation for photoionization of the products from scattering experiments. The apparatus was built at the recently constructed Advanced Light Source at Lawrence Berkeley National Laboratory, a third generation 1-2 GeV synchrotron radiation source. The new apparatus is applied to investigations of the dynamics of unimolecular reactions, photodissociation experiments, and bimolecular reactions, crossed molecular beam experiments. The first chapter describes the new apparatus and the VUV radiation used for photoionization. This is followed by a number of examples of the many advantages provided by using VUV photoionization in comparison with the traditional technique of electron bombardment ionization. At the end of the chapter there is a discussion of the data analysis employed in these scattering experiments. The remaining four chapters are complete investigations of the dynamics of four chemical systems using the new apparatus and provide numerous additional examples of the advantages provided by VUV photoionizaiton of the products. Chapters 2-4 are photofragment translational spectroscopy studies of the photodissociation dynamics of dimethyl sulfoxide, acrylonitrile, and vinyl chloride following absorption at 193 mn. All of these systems have multiple dissociation channels and provide good examples of the ability of the new apparatus to unravel the complex UV photodissociation dynamics that can arise in small polyatomic molecules.

  5. Molecular gels in the gas phase? Gelator-gelator and gelator-solvent interactions probed by vibrational spectroscopy.

    PubMed

    Lozada-Garcia, Rolando; Mu, Dan; Plazanet, Marie; Çarçabal, Pierre

    2016-08-10

    Benzylidene glucose (BzGlc) is a member of the benzylidene glycoside family. These molecules have the ability to form molecular physical gels. These materials are formed when gelator molecules create a non-covalently bound frame where solvent molecules are trapped. Since the gel formation process and its properties are determined by the subtle balance between non-covalent forces, it is difficult to anticipate them. Quantitative and qualitative understanding of the gelator-gelator and gelator-solvent interactions is needed to better control these materials for important potential applications. We have used gas phase vibrational spectroscopy and theoretical chemistry to study the conformational choices of BzGlc, its dimer and the complexes it forms with water or toluene. To interpret the vibrational spectra we have used the dispersion corrected functional B97D which we have calibrated for the calculation of OH stretching frequencies. Even at the most basic molecular level, it is possible to interrogate a large range of non-covalent interactions ranging from OH → OH hydrogen bonding, to OH → π, and CH → π, all being at the center of gel properties at the macroscopic level. PMID:27443393

  6. Spatially resolved variations of the IMF mass normalisation in early-type galaxies as probed by molecular gas kinematics

    NASA Astrophysics Data System (ADS)

    Davis, Timothy A.; McDermid, Richard M.

    2016-09-01

    We here present the first spatially-resolved study of the IMF in external galaxies derived using a dynamical tracer of the mass-to-light ratio. We use the kinematics of relaxed molecular gas discs in seven early-type galaxies (ETGs) selected from the ATLAS3D survey to dynamically determine mass-to-light ratio (M/L) gradients. These M/L gradients are not very strong in the inner parts of these objects, and galaxies that do show variations are those with the highest specific star formation rates. Stellar population parameters derived from star formation histories are then used in order to estimate the stellar initial mass function function (IMF) mismatch parameter, and shed light on its variation within ETGs. Some of our target objects require a light IMF, otherwise their stellar population masses would be greater than their dynamical masses. In contrast, other systems seem to require heavier IMFs to explain their gas kinematics. Our analysis again confirms that IMF variation seems to be occurring within massive ETGs. We find good agreement between our IMF normalisations derived using molecular gas kinematics and those derived using other techniques. Despite this, we do not see find any correlation between the IMF normalisation and galaxy dynamical properties or stellar population parameters, either locally or globally. In the future larger studies which use molecules as tracers of galaxy dynamics can be used to help us disentangle the root cause of IMF variation.

  7. Probing environment fluctuations by two-dimensional electronic spectroscopy of molecular systems at temperatures below 5 K.

    PubMed

    Rancova, Olga; Jankowiak, Ryszard; Abramavicius, Darius

    2015-06-01

    Two-dimensional (2D) electronic spectroscopy at cryogenic and room temperatures reveals excitation energy relaxation and transport, as well as vibrational dynamics, in molecular systems. These phenomena are related to the spectral densities of nuclear degrees of freedom, which are directly accessible by means of hole burning and fluorescence line narrowing approaches at low temperatures (few K). The 2D spectroscopy, in principle, should reveal more details about the fluctuating environment than the 1D approaches due to peak extension into extra dimension. By studying the spectral line shapes of a dimeric aggregate at low temperature, we demonstrate that 2D spectra have the potential to reveal the fluctuation spectral densities for different electronic states, the interstate correlation of static disorder and, finally, the time scales of spectral diffusion with high resolution.

  8. Simultaneously probing two ultrafast condensed-phase molecular symmetry breaking events by two-dimensional infrared spectroscopy.

    PubMed

    Yang, Fan; Yu, Pengyun; Zhao, Juan; Wang, Jianping

    2013-08-01

    In condensed phases, a highly symmetric gas-phase molecule lowers its symmetry under perturbation of the solvent, which is vital to a variety of structural chemistry related processes. However, the dynamical aspects of solvent-mediated symmetry-breaking events remain largely unknown. Herein, direct evidence for two types of solvent-mediated symmetry-breaking events that coexist on the picosecond timescale in a highly symmetric anion, namely, hexacyanocobaltate, is presented: 1) an equilibrium symmetry-breaking event in which a solvent-bound species having lowered symmetry undergoes a population exchange reaction with the symmetry-retaining species; 2) a dynamic symmetry-breaking event that is composed of many dynamic population-exchange reactions under fluctuating solvent interactions. Ultrafast two-dimensional infrared spectroscopy is used to simultaneously observe and dynamically characterize these two events. This work opens a new window into molecular symmetry and structural dynamics under equilibrium and non-equilibrium conditions.

  9. Molecular recognition of curcumin (Indian Ayurvedic medicine) by the supramolecular probe, p-t-butyl calix(8)arene

    NASA Astrophysics Data System (ADS)

    Meenakshi, C.; Jayabal, P.; Ramakrishnan, V.

    2014-06-01

    The thermodynamic property of the host-guest complexes formed between the curcumin, component of Indian Ayurvedic medicine turmeric, a drug molecule, with the supra molecule, p-t-butyl calix(8)arene was studied. p-t-Butyl calix(8)arene has been used as a host molecule and curcumin as a guest molecule. Optical absorption spectral studies were carried out to investigate the molecular recognition properties of p-t-butyl calix(8)arene with curcumin. The stochiometry of the host-guest complexes formed and the binding constant were determined. An interesting 1:1 and 4:1 stochiometric host-guest complexes were formed. Job's continuous method of variation and Benesi-Hildebrand expression were used for the determination of binding constant and the stochiometry of the host-guest complex formed.

  10. Molecular recognition of curcumin (Indian Ayurvedic medicine) by the supramolecular probe, p-t-butyl calix(8)arene.

    PubMed

    Meenakshi, C; Jayabal, P; Ramakrishnan, V

    2014-06-01

    The thermodynamic property of the host-guest complexes formed between the curcumin, component of Indian Ayurvedic medicine turmeric, a drug molecule, with the supra molecule, p-t-butyl calix(8)arene was studied. p-t-Butyl calix(8)arene has been used as a host molecule and curcumin as a guest molecule. Optical absorption spectral studies were carried out to investigate the molecular recognition properties of p-t-butyl calix(8)arene with curcumin. The stochiometry of the host-guest complexes formed and the binding constant were determined. An interesting 1:1 and 4:1 stochiometric host-guest complexes were formed. Job's continuous method of variation and Benesi-Hildebrand expression were used for the determination of binding constant and the stochiometry of the host-guest complex formed.

  11. Probing environment fluctuations by two-dimensional electronic spectroscopy of molecular systems at temperatures below 5 K

    SciTech Connect

    Rancova, Olga; Abramavicius, Darius; Jankowiak, Ryszard

    2015-06-07

    Two-dimensional (2D) electronic spectroscopy at cryogenic and room temperatures reveals excitation energy relaxation and transport, as well as vibrational dynamics, in molecular systems. These phenomena are related to the spectral densities of nuclear degrees of freedom, which are directly accessible by means of hole burning and fluorescence line narrowing approaches at low temperatures (few K). The 2D spectroscopy, in principle, should reveal more details about the fluctuating environment than the 1D approaches due to peak extension into extra dimension. By studying the spectral line shapes of a dimeric aggregate at low temperature, we demonstrate that 2D spectra have the potential to reveal the fluctuation spectral densities for different electronic states, the interstate correlation of static disorder and, finally, the time scales of spectral diffusion with high resolution.

  12. Effect of sequence variation on the mechanical response of amyloid fibrils probed by steered molecular dynamics simulation.

    PubMed

    Ndlovu, Hlengisizwe; Ashcroft, Alison E; Radford, Sheena E; Harris, Sarah A

    2012-02-01

    The mechanical failure of mature amyloid fibers produces fragments that act as seeds for the growth of new fibrils. Fragmentation may also be correlated with cytotoxicity. We have used steered atomistic molecular dynamics simulations to study the mechanical failure of fibrils formed by the amyloidogenic fragment of human amylin hIAPP20-29 subjected to force applied in a variety of directions. By introducing systematic variations to this peptide sequence in silico, we have also investigated the role of the amino-acid sequence in determining the mechanical stability of amyloid fibrils. Our calculations show that the force required to induce mechanical failure depends on the direction of the applied stress and upon the degree of structural order present in the β-sheet assemblies, which in turn depends on the peptide sequence. The results have implications for the importance of sequence-dependent mechanical properties on seeding the growth of new fibrils and the role of breakage events in cytotoxicity.

  13. Probing into the binding interaction between medroxyprogesterone acetate and bovine serum albumin (BSA): spectroscopic and molecular docking methods.

    PubMed

    Fang, Fang; Pan, Dong-Qi; Qiu, Min-Jie; Liu, Ting-Ting; Jiang, Min; Wang, Qi; Shi, Jie-Hua

    2016-09-01

    To further understand the mechanism of action and pharmacokinetics of medroxyprogesterone acetate (MPA), the binding interaction of MPA with bovine serum albumin (BSA) under simulated physiological conditions (pH 7.4) was studied using fluorescence emission spectroscopy, synchronous fluorescence spectroscopy, circular dichroism and molecular docking methods. The experimental results reveal that the fluorescence of BSA quenches due to the formation of MPA-BSA complex. The number of binding sites (n) and the binding constant for MPA-BSA complex are ~1 and 4.6 × 10(3)  M(-1) at 310 K, respectively. However, it can be concluded that the binding process of MPA with BSA is spontaneous and the main interaction forces between MPA and BSA are van der Waals force and hydrogen bonding interaction due to the negative values of ΔG(0) , ΔH(0) and ΔS(0) in the binding process of MPA with BSA. MPA prefers binding on the hydrophobic cavity in subdomain IIIA (site II'') of BSA resulting in a slight change in the conformation of BSA, but BSA retaining the α-helix structure. Copyright © 2016 John Wiley & Sons, Ltd.

  14. Using singlet molecular oxygen to probe the solute and temperature dependence of liquid-like regions in/on ice.

    PubMed

    Bower, Jonathan P; Anastasio, Cort

    2013-08-01

    Liquid-like regions (LLRs) are found at the surfaces and grain boundaries of ice and as inclusions within ice. These regions contain most of the solutes in ice and can be (photo)chemically active hotspots in natural snow and ice systems. If we assume all solutes partition into LLRs as a solution freezes, freezing-point depression predicts that the concentration of a solute in LLRs is higher than its concentration in the prefrozen (or melted) solution by the freeze-concentration factor (F). Here we use singlet molecular oxygen production to explore the effects of total solute concentration ([TS]) and temperature on experimentally determined values of F. For ice above its eutectic temperature, measured values of F agree well with freezing-point depression when [TS] is above ∼1 mmol/kg; at lower [TS] values, measurements of F are lower than predicted from freezing-point depression. For ice below its eutectic temperature, the influence of freezing-point depression on F is damped; the extreme case is with Na2SO4 as the solute, where F shows essentially no agreement with freezing-point depression. In contrast, for ice containing 3 mmol/kg NaCl, measured values of F agree well with freezing-point depression over a range of temperatures, including below the eutectic. Our experiments also reveal that the photon flux in LLRs increases in the presence of salts, which has implications for ice photochemistry in the lab and, perhaps, in the environment. PMID:23841666

  15. Probing the molecular forces involved in binding of selected volatile flavour compounds to salt-extracted pea proteins.

    PubMed

    Wang, Kun; Arntfield, Susan D

    2016-11-15

    Molecular interactions between heterologous classes of flavour compounds with salt-extracted pea protein isolates (PPIs) were determined using various bond disrupting agents followed by GC/MS analysis. Flavour bound by proteins decreased in the order: dibutyl disulfide>octanal>hexyl acetate>2-octanone=benzaldehyde. Benzaldehyde, 2-octanone and hexyl acetate interacted non-covalently with PPIs, whereas octanal bound PPIs via covalent and non-covalent forces. Dibutyl disulfide reacted with PPIs covalently, as its retention was not diminished by urea and guanidine hydrochloride. Using propylene glycol, H-bonding and ionic interactions were implicated for hexyl acetate, benzaldehyde, and 2-octanone. A protein-destabilising salt (Cl3CCOONa) reduced bindings for 2-octanone, hexyl acetate, and benzaldehyde; however, retention for octanal and dibutyl disulfide increased. Conversely, a protein-stabilising salt (Na2SO4) enhanced retention for benzaldehyde, 2-octanone, hexyl acetate and octanal. Formation of a volatile flavour by-product, 1-butanethiol, from dibutyl disulfide when PPIs were treated with dithiothreitol indicated occurrence of sulfhydryl-disulfide interchange reactions. PMID:27283627

  16. Assessing the potential of atomistic molecular dynamics simulations to probe reversible protein-protein recognition and binding

    PubMed Central

    Abriata, Luciano A.; Dal Peraro, Matteo

    2015-01-01

    Protein-protein recognition and binding are governed by diffusion, noncovalent forces and conformational flexibility, entangled in a way that only molecular dynamics simulations can dissect at high resolution. Here we exploited ubiquitin’s noncovalent dimerization equilibrium to assess the potential of atomistic simulations to reproduce reversible protein-protein binding, by running submicrosecond simulations of systems with multiple copies of the protein at millimolar concentrations. The simulations essentially fail because they lead to aggregates, yet they reproduce some specificity in the binding interfaces as observed in known covalent and noncovalent ubiquitin dimers. Following similar observations in literature we hint at electrostatics and water descriptions as the main liable force field elements, and propose that their optimization should consider observables relevant to multi-protein systems and unfolded proteins. Within limitations, analysis of binding events suggests salient features of protein-protein recognition and binding, to be retested with improved force fields. Among them, that specific configurations of relative direction and orientation seem to trigger fast binding of two molecules, even over 50 Å distances; that conformational selection can take place within surface-to-surface distances of 10 to 40 Å i.e. well before actual intermolecular contact; and that establishment of contacts between molecules further locks their conformations and relative orientations. PMID:26023027

  17. Probing thermal stability of the β-lactoglobulin-oleic acid complex by fluorescence spectroscopy and molecular modeling

    NASA Astrophysics Data System (ADS)

    Simion (Ciuciu), Ana-Maria; Aprodu, Iuliana; Dumitrașcu, Loredana; Bahrim, Gabriela Elena; Alexe, Petru; Stănciuc, Nicoleta

    2015-09-01

    Bovine β-lactoglobulin is able to interact with different bioactive compounds, thus being an important candidate in the development of delivery systems with improved functionality. The heat induced changes in the β-lactoglobulin-oleic acid complex were examined by means of fluorescence spectroscopy and molecular modeling techniques. Fluorescence spectroscopy results indicated a rigid protein structure in the temperature range 25-70 °C, whereas at temperatures over 75 °C, the rearrangements of the polypeptide chains led to higher exposure of hydrophobic residues. The most significant increase of the accessible surface area with temperature increase was identified in case of Tyr99 and Tyr102. The phase diagram method indicated an all or none transition between two conformations. Due to conformational changes, no contact between Ile56 or Lys60 and the fatty acid could be identified at 85 °C, but new non-bonding interaction were established with Ile12 and Val15. The results obtained in this study provide important details about thermal induced changes in the conformation of β-lactoglobulin-oleic acid complex. Significant conformational changes were registered above 75 °C, suggesting the possibility of obtaining highly functional complexes between whey proteins and natural unsaturated fatty acids.

  18. A disposable evanescent wave fiber optic sensor coated with a molecularly imprinted polymer as a selective fluorescence probe.

    PubMed

    Ton, Xuan-Anh; Acha, Victor; Bonomi, Paolo; Tse Sum Bui, Bernadette; Haupt, Karsten

    2015-02-15

    We have developed a disposable evanescent wave fiber optic sensor by coating a molecularly imprinted polymer (MIP) containing a fluorescent signaling group on a 4-cm long polystyrene optical waveguide. The MIP is composed of a naphthalimide-based fluorescent monomer, which shows fluorescence enhancement upon binding with carboxyl-containing molecules. The herbicide 2,4-dichlorophenoxyacetic acid and the mycotoxin citrinin were used as model analytes. The coating of the MIP was either performed ex-situ, by dip-coating the fiber with MIP particles synthesized beforehand, or in-situ by evanescent-wave photopolymerization on the fiber. The sensing element was interrogated with a fiber-coupled spectrofluorimeter. The fiber optic sensor detects targets in the low nM range and exhibits specific and selective recognition over structural analogs and non-related carboxyl-containing molecules. This technology can be extended to other carboxyl-containing analytes, and to a broader spectrum of targets using different fluorescent monomers.

  19. Molecular interactions in the ionic liquid emim acetate and water binary mixtures probed via NMR spin relaxation and exchange spectroscopy.

    PubMed

    Allen, Jesse J; Bowser, Sage R; Damodaran, Krishnan

    2014-05-01

    Interactions of ionic liquids (ILs) with water are of great interest for many potential IL applications. 1-Ethyl-3-methylimidazolium (emim) acetate, in particular, has shown interesting interactions with water including hydrogen bonding and even chemical exchange. Previous studies have shown the unusual behavior of emim acetate when in the presence of 0.43 mole fraction of water, and a combination of NMR techniques is used herein to investigate the emim acetate-water system and the unusual behavior at 0.43 mole fraction of water. NMR relaxometry techniques are used to describe the effects of water on the molecular motion and interactions of emim acetate with water. A discontinuity is seen in nuclear relaxation behavior at the concentration of 0.43 mole fraction of water, and this is attributed to the formation of a hydrogen bonded network. EXSY measurements are used to determine the exchange rates between the H2 emim proton and water, which show a complex dependence on the concentration of the mixture. The findings support and expand our previous results, which suggested the presence of an extended hydrogen bonding network in the emim acetate-water system at concentrations close to 0.50 mole fraction of H2O. PMID:24654003

  20. A molecular simulation probing of structure and interaction for supramolecular sodium dodecyl sulfate/single-wall carbon nanotube assemblies.

    PubMed

    Xu, Zhijun; Yang, Xiaoning; Yang, Zhen

    2010-03-10

    Here we report a larger-scale atomic-level molecular dynamics (MD) simulation for the self-assembly of sodium dodecyl sulfate (SDS) surfactant on single-walled carbon nanotube (SWNT) surfaces and the interaction between supramolecular SDS/SWNT aggregates. We make an effort to address several important problems in regard to carbon nanotube dispersion/separation. At first, the simulation provides comprehensive direct evidence for SDS self-assembly structures on carbon nanotube surfaces, which can help to clarify the relevant debate over the exact adsorption structure. We also, for the first time, simulated the potential of mean force (PMF) between two SWNTs embedded in SDS surfactant micelles. A novel unified PMF approach has been applied to reveal various cooperative interactions between the SDS/SWNT aggregates, which is different from the previous electrostatic repulsion explanation. The unique role of sodium ions revealed here provides a new microscopic understanding of the recent experiments in the electrolyte tuning of the interfacial forces on the selective fractionation of SDS surrounding SWNTs.

  1. Probing the ATP-induced conformational flexibility of the PcrA helicase protein using molecular dynamics simulation.

    PubMed

    Mhashal, Anil R; Choudhury, Chandan Kumar; Roy, Sudip

    2016-03-01

    Helicases are enzymes that unwind double-stranded DNA (dsDNA) into its single-stranded components. It is important to understand the binding and unbinding of ATP from the active sites of helicases, as this knowledge can be used to elucidate the functionality of helicases during the unwinding of dsDNA. In this work, we investigated the unbinding of ATP and its effect on the active-site residues of the helicase PcrA using molecular dynamic simulations. To mimic the unbinding process of ATP from the active site of the helicase, we simulated the application of an external force that pulls ATP from the active site and computed the free-energy change during this process. We estimated an energy cost of ~85 kJ/mol for the transformation of the helicase from the ATP-bound state (1QHH) to the ATP-free state (1PJR). Unbinding led to conformational changes in the residues of the protein at the active site. Some of the residues at the ATP-binding site were significantly reoriented when the ATP was pulled. We observed a clear competition between reorientation of the residues and energy stabilization by hydrogen bonds between the ATP and active-site residues. We also checked the flexibility of the PcrA protein using a principal component analysis of domain motion. We found that the ATP-free state of the helicase is more flexible than the ATP-bound state.

  2. Probing molecular packing at engineered interfaces in organic field effect transistor and its correlation with charge carrier mobility.

    PubMed

    Maheshwari, Priya; Mukherjee, Saurabh; Bhattacharya, Debarati; Sen, Shashwati; Tokas, Raj Bahadur; Honda, Yoshihide; Basu, Saibal; Padma, Narayanan; Pujari, Pradeep Kumar

    2015-05-20

    Surface engineering of SiO2 dielectric using different self-assembled monolayer (SAM) has been carried out, and its effect on the molecular packing and growth behavior of copper phthalocyanine (CuPc) has been studied. A correlation between the growth behavior and performance of organic field effect transistors is examined. Depth profiling using positron annihilation and X-ray reflectivity techniques has been employed to characterize the interface between CuPc and the modified and/or unmodified dielectric. We observe the presence of structural defects or disorder due to disorientation of CuPc molecules on the unmodified dielectric and ordered arrangement on the modified dielectrics, consistent with the high charge carrier mobility in organic field effect transistors in the latter. The study also highlights the sensitivity of these techniques to the packing of CuPc molecules on SiO2 modified using different SAMs. Our study also signifies the sensitivity and utility of these two techniques in the characterization of buried interfaces in organic devices. PMID:25922969

  3. Substrate specificity of pyrimidine nucleoside phosphorylases of NP-II family probed by X-ray crystallography and molecular modeling

    NASA Astrophysics Data System (ADS)

    Balaev, V. V.; Lashkov, A. A.; Prokofev, I. I.; Gabdulkhakov, A. G.; Seregina, T. A.; Mironov, A. S.; Betzel, C.; Mikhailov, A. M.

    2016-09-01

    Pyrimidine nucleoside phosphorylases, which are widely used in the biotechnological production of nucleosides, have different substrate specificity for pyrimidine nucleosides. An interesting feature of these enzymes is that the three-dimensional structure of thymidine-specific nucleoside phosphorylase is similar to the structure of nonspecific pyrimidine nucleoside phosphorylase. The three-dimensional structures of thymidine phosphorylase from Salmonella typhimurium and nonspecific pyrimidine nucleoside phosphorylase from Bacillus subtilis in complexes with a sulfate anion were determined for the first time by X-ray crystallography. An analysis of the structural differences between these enzymes demonstrated that Lys108, which is involved in the phosphate binding in pyrimidine nucleoside phosphorylase, corresponds to Met111 in thymidine phosphorylases. This difference results in a decrease in the charge on one of the hydroxyl oxygens of the phosphate anion in thymidine phosphorylase and facilitates the catalysis through SN2 nucleophilic substitution. Based on the results of X-ray crystallography, the virtual screening was performed for identifying a potent inhibitor (anticancer agent) of nonspecific pyrimidine nucleoside phosphorylase, which does not bind to thymidine phosphorylase. The molecular dynamics simulation revealed the stable binding of the discovered compound—2-pyrimidin-2-yl-1H-imidazole-4-carboxylic acid—to the active site of pyrimidine nucleoside phosphorylase.

  4. Probing the alpha-helical structural stability of stapled p53 peptides: molecular dynamics simulations and analysis.

    PubMed

    Guo, Zuojun; Mohanty, Udayan; Noehre, Justin; Sawyer, Tomi K; Sherman, Woody; Krilov, Goran

    2010-04-01

    Reactivation of the p53 cell apoptosis pathway through inhibition of the p53-hDM2 interaction is a viable approach to suppress tumor growth in many human cancers and stabilization of the helical structure of synthetic p53 analogs via a hydrocarbon cross-link (staple) has been found to lead to increased potency and inhibition of protein-protein binding (J. Am. Chem. Soc. 129: 5298). However, details of the structure and dynamic stability of the stapled peptides are not well understood. Here, we use extensive all-atom molecular dynamics simulations to study a series of stapled alpha-helical peptides over a range of temperatures in solution. The peptides are found to exhibit substantial variations in predicted alpha-helical propensities that are in good agreement with the experimental observations. In addition, we find significant variation in local structural flexibility of the peptides with the position of the linker, which appears to be more closely related to the observed differences in activity than the absolute alpha-helical stability. These simulations provide new insights into the design of alpha-helical stapled peptides and the development of potent inhibitors of alpha-helical protein-protein interfaces.

  5. Efficient fluorescence turn-on probe for zirconium via a target-triggered DNA molecular beacon strategy.

    PubMed

    Meng, Hong-Min; Fu, Ting; Zhang, Xiao-Bing; Wang, Nan-Nan; Tan, Weihong; Shen, Guo-Li; Yu, Ru-Qin

    2012-03-01

    It is well-known that Zr(4+) could selectively bind with two phosphate-functionalized molecules through a coordinate covalent interaction to form a sandwich-structured complex (-PO(3)(2-)-Zr(4+)-PO(3)(2-)-). In this paper, we for the first time converted such interaction into fluorescence sensing systems for Zr(4+) via a target-triggered DNA molecular beacon strategy. In the new designed sensing system, two phosphorylated and pyrene-labeled oligonucleotides were chosen as both recognition and reporter units, which will be linked by target Zr(4+) to form a hairpin structure and bring the two labeled pyrene molecules into close proximity, resulting in a "turn-on" excimer fluorescence signal. Moreover, γ-cyclodextrin was introduced to afford an amplified fluorescence signal and, therefore, provided an improved sensitivity for the target Zr(4+). This allows detection of Zr(4+) with high sensitivity (limit of detection, LOD = 200 nM) and excellent selectivity. The proposed sensing system has also been used for detection of Zr(4+) in river water samples with satisfactory result.

  6. Probing the interactions between carboxylated multi-walled carbon nanotubes and copper-zinc superoxide dismutase at a molecular level.

    PubMed

    Guan, Jin; Liu, Guiliang; Cai, Kai; Gao, Canzhu; Liu, Rutao

    2015-08-01

    In order to evaluate the toxicity of multi-walled carbon nanotubes (MWCNTs-COOH) at a molecular level, the effect of MWCNTs-COOH on antioxidant enzyme copper-zinc superoxide dismutase (Cu/ZnSOD) was investigated using fluorescence spectroscopy, UV/vis absorption spectroscopy, circular dichroism (CD) spectroscopy and isothermal titration calorimetry (ITC). By deducting the inner filter effect (IFE), the fluorescence emission spectra and synchronous fluorescence spectra indicated that there were interactions between MWCNTs-COOH and Cu/ZnSOD. Moreover, the microenvironment of the amino acid residues in the enzyme was changed slightly. The UV/vis absorption and CD spectroscopic results showed appreciable conformational changes in Cu/ZnSOD. However, the results of a Cu/ZnSOD activity determination did not show any significant difference. In other words, MWCNTs-COOH has no significant effect on enzyme activity. The ITC results showed that the binding of MWCNTs-COOH to Cu/ZnSOD was a weak endothermic process, indicating that the predominant force of the binding was hydrophobic interaction. Moreover, it was essential to consider the IFE in fluorescence assays, which might affect the accuracy and precision of the results. The above results are helpful in evaluating the oxidative stress induced by MWCNTs-COOH in vivo.

  7. Green's function-stochastic methods framework for probing nonlinear evolution problems: Burger's equation, the nonlinear Schroedinger's equation, and hydrodynamic organization of near-molecular-scale vorticity

    SciTech Connect

    Keanini, R.G.

    2011-04-15

    Research Highlights: > Systematic approach for physically probing nonlinear and random evolution problems. > Evolution of vortex sheets corresponds to evolution of an Ornstein-Uhlenbeck process. > Organization of near-molecular scale vorticity mediated by hydrodynamic modes. > Framework allows calculation of vorticity evolution within random strain fields. - Abstract: A framework which combines Green's function (GF) methods and techniques from the theory of stochastic processes is proposed for tackling nonlinear evolution problems. The framework, established by a series of easy-to-derive equivalences between Green's function and stochastic representative solutions of linear drift-diffusion problems, provides a flexible structure within which nonlinear evolution problems can be analyzed and physically probed. As a preliminary test bed, two canonical, nonlinear evolution problems - Burgers' equation and the nonlinear Schroedinger's equation - are first treated. In the first case, the framework provides a rigorous, probabilistic derivation of the well known Cole-Hopf ansatz. Likewise, in the second, the machinery allows systematic recovery of a known soliton solution. The framework is then applied to a fairly extensive exploration of physical features underlying evolution of randomly stretched and advected Burger's vortex sheets. Here, the governing vorticity equation corresponds to the Fokker-Planck equation of an Ornstein-Uhlenbeck process, a correspondence that motivates an investigation of sub-sheet vorticity evolution and organization. Under the assumption that weak hydrodynamic fluctuations organize disordered, near-molecular-scale, sub-sheet vorticity, it is shown that these modes consist of two weakly damped counter-propagating cross-sheet acoustic modes, a diffusive cross-sheet shear mode, and a diffusive cross-sheet entropy mode. Once a consistent picture of in-sheet vorticity evolution is established, a number of analytical results, describing the motion

  8. In Vivo Detection of Oxidation-Specific Epitopes in Atherosclerotic Lesions Using Bio-Compatible Mn(II) Molecular Magnetic Imaging Probes

    PubMed Central

    Briley-Saebo, Karen C.; Hoang, Tuyen; Saeboe, Alexander M.; Cho, Young Seok; Ryu, Sung Kee; Volkava, Eugenia; Dickson, Stephen; Leibundgut, Gregor; Weisner, Philipp; Green, Simone; Casanada, Florence; Miller, Yury I.; Shaw, Walter; Witztum, Joseph L; Fayad, Zahi A.; Tsimikas, Sotirios

    2012-01-01

    Objectives To evaluate the in vivo magnetic resonance (MR) imaging efficacy of manganese (Mn(II)) molecular imaging probes targeted to oxidation-specific epitopes (OSE). Background OSE are critical in the initiation, progression and de-stabilization of atherosclerotic plaques. Gadolinium (Gd(III)) based MR imaging agents can be associated with systemic toxicity. Mn is an endogenous, bio-compatible, paramagnetic metal ion that has poor MR efficacy when chelated, but strong efficacy when released within cells. Methods Multimodal Mn-micelles were generated to contain rhodamine for confocal microscopy and conjugated with either the murine monoclonal IgG antibody MDA2 targeted to malondialdehyde (MDA)-lysine epitopes or the human single-chain Fv antibody fragment IK17 targeted to MDA-like epitopes (‘targeted micelles”). Micelle formulations were characterized in vitro and in vivo and their MR efficacy (9.4 Tesla) evaluated in apoE−/− and LDLR−/− mice (0.05 mmol Mn/Kg dose) (total of 120 mice for all experiments). In vivo competitive inhibition studies were performed to evaluate target specificity. Untargeted, MDA2-Gd and IK17-Gd micelles (0.075 mmol Gd/Kg) were included as controls. Results In vitro studies demonstrated that targeted Mn-micelles accumulate in macrophages when pre-exposed to MDA-LDL with ~10X increase in longitudinal relativity. Following intravenous injection, strong MR signal enhancement was observed 48–72 hours after administration of targeted Mn-micelles, with co-localization within intraplaque macrophages. Co-injection of free MDA2 with the MDA2-Mn micelles resulted in full suppression of MR signal in the arterial wall confirming target specificity. Similar MR efficacy was noted in apoE−/− and LDLR−/− mice with aortic atherosclerosis. No significant differences in MR efficacy were noted between targeted Mn and Gd micelles. Conclusions This study demonstrates that bio-compatible multimodal Mn-based molecular imaging probes

  9. Strong electric fields at a prototypical oxide/water interface probed by ab initio molecular dynamics: MgO(001).

    PubMed

    Laporte, Sara; Finocchi, Fabio; Paulatto, Lorenzo; Blanchard, Marc; Balan, Etienne; Guyot, François; Saitta, Antonino Marco

    2015-08-21

    We report a density-functional theory (DFT)-based study of the interface of bulk water with a prototypical oxide surface, MgO(001), and focus our study on the often-overlooked surface electric field. In particular, we observe that the bare MgO(001) surface, although charge-neutral and defectless, has an intense electric field on the Å scale. The MgO(001) surface covered with 1 water monolayer (1 ML) is investigated via a supercell accounting for the experimentally-observed (2 × 3) reconstruction, stable at ambient temperature, and in which two out of six water molecules are dissociated. This 1 ML-hydrated surface is also found to have a high, albeit short-ranged, normal component of the field. Finally, the oxide/water interface is studied via room-temperature ab initio molecular dynamics (AIMD) using 34 H2O molecules between two MgO(001) surfaces. To our best knowledge this is the first AIMD study of the MgO(001)/liquid water interface in which all atoms are treated using DFT and including several layers above the first adsorbed layer. We observe that the surface electric field, averaged over the AIMD trajectories, is still very strong on the fully-wet surface, peaking at about 3 V Å(-1). Even in the presence of bulk-like water, the structure of the first layer in contact with the surface remains similar to the (2 × 3)-reconstructed ice ad-layer on MgO(001). Moreover, we observe proton exchange within the first layer, and between the first and second layers - indeed, the O-O distances close to the surface are found to be distributed towards shorter distances, a property which has been shown to directly promote proton transfer.

  10. Steered Molecular Dynamics Simulations of a Type IV Pilus Probe Initial Stages of a Force-Induced Conformational Transition

    PubMed Central

    Baker, Joseph L.; Biais, Nicolas; Tama, Florence

    2013-01-01

    Type IV pili are long, protein filaments built from a repeating subunit that protrudes from the surface of a wide variety of infectious bacteria. They are implicated in a vast array of functions, ranging from bacterial motility to microcolony formation to infection. One of the most well-studied type IV filaments is the gonococcal type IV pilus (GC-T4P) from Neisseria gonorrhoeae, the causative agent of gonorrhea. Cryo-electron microscopy has been used to construct a model of this filament, offering insights into the structure of type IV pili. In addition, experiments have demonstrated that GC-T4P can withstand very large tension forces, and transition to a force-induced conformation. However, the details of force-generation, and the atomic-level characteristics of the force-induced conformation, are unknown. Here, steered molecular dynamics (SMD) simulation was used to exert a force in silico on an 18 subunit segment of GC-T4P to address questions regarding the nature of the interactions that lead to the extraordinary strength of bacterial pili. SMD simulations revealed that the buried pilin α1 domains maintain hydrophobic contacts with one another within the core of the filament, leading to GC-T4P's structural stability. At the filament surface, gaps between pilin globular head domains in both the native and pulled states provide water accessible routes between the external environment and the interior of the filament, allowing water to access the pilin α1 domains as reported for VC-T4P in deuterium exchange experiments. Results were also compared to the experimentally observed force-induced conformation. In particular, an exposed amino acid sequence in the experimentally stretched filament was also found to become exposed during the SMD simulations, suggesting that initial stages of the force induced transition are well captured. Furthermore, a second sequence was shown to be initially hidden in the native filament and became exposed upon stretching. PMID

  11. A study of dynamical processes in the Orion KL region using ALMA—probing molecular outflow and inflow

    SciTech Connect

    Wu, Yuefang; Liu, Tie; Qin, Sheng-Li

    2014-08-20

    This work reports high spatial resolution observations toward the Orion KL region with high critical density lines of CH{sub 3}CN (12{sub 4}-11{sub 4}) and CH{sub 3}OH (8{sub –1,8}-7{sub 0,7}), as well as a continuum at ∼1.3 mm band. The observations were made using the Atacama Large Millimeter/Submillimeter Array with a spatial resolution of ∼1.''5 and sensitivity of about 0.07 K and ∼0.18 K for continuum and line, respectively. The observational results showed that the gas in the Orion KL region consists of jet-propelled cores at the ridge and dense cores east and south of the region that are shaped like a wedge ring. The outflow has multiple lobes, which may originate from an explosive ejection, and is not driven by young stellar objects. Four infrared bubbles were found in the Spitzer/IRAC emissions. These bubbles, the distributions of the previously found H{sub 2} jets, the young stellar objects, and molecular gas suggest that BN is the explosive center. The burst time was estimated to be ≤1300 yr. At the same time, signatures of gravitational collapse toward Source I and the hot core were detected with material infall velocities of 1.5 km s{sup –1} and ∼0.6 km s{sup –1}, corresponding to mass accretion rates of 1.2 × 10{sup –3} M {sub ☉}/yr and 8.0 × 10{sup –5} M {sub ☉}/yr, respectively. These observations may support the belief that high-mass stars form via the accretion model, similar to their low-mass counterparts.

  12. The bandmerged Planck Early Release Compact Source Catalogue: probing sub-structure in the molecular gas at high Galactic latitude

    NASA Astrophysics Data System (ADS)

    Chen, X.; Chary, R.; Pearson, T. J.; McGehee, P.; Fowler, J. W.; Helou, G.

    2016-06-01

    The Planck Early Release Compact Source Catalogue (ERCSC) includes nine lists of highly reliable sources, individually extracted at each of the nine Planck frequency channels. To facilitate the study of the Planck sources, especially their spectral behaviour across the radio/infrared frequencies, we provide a `bandmerged' catalogue of the ERCSC sources. This catalogue consists of 15 191 entries, with 79 sources detected in all nine frequency channels of Planck and 6818 sources detected in only one channel. We describe the bandmerging algorithm, including the various steps used to disentangle sources in confused regions. The multifrequency matching allows us to develop spectral energy distributions of sources between 30 and 857 GHz, in particular across the 100 GHz band, where the energetically important CO J = 1→0 line enters the Planck bandpass. We find ˜3σ-5σ evidence for contribution to the 100 GHz intensity from foreground CO along the line of sight to 147 sources with |b|>{30°}. The median excess contribution is 4.5 ± 0.9 per cent of their measured 100 GHz flux density which cannot be explained by calibration or beam uncertainties. This translates to 0.5 ± 0.1 K km s-1 of CO which must be clumped on the scale of the Planck 100 GHz beam, i.e. ˜10 arcmin. If this is due to a population of low-mass (˜15 M⊙) molecular gas clumps, the total mass in these clumps may be more than 2000 M⊙. Further, high-spatial-resolution, ground-based observations of the high-latitude sky will help shed light on the origin of this diffuse, clumpy CO emission.

  13. Novel fluorescent antagonist as a molecular probe in A(3) adenosine receptor binding assays using flow cytometry.

    PubMed

    Kozma, Eszter; Kumar, T Santhosh; Federico, Stephanie; Phan, Khai; Balasubramanian, Ramachandran; Gao, Zhan-Guo; Paoletta, Silvia; Moro, Stefano; Spalluto, Giampiero; Jacobson, Kenneth A

    2012-06-01

    The physiological role of the A(3) adenosine receptor (AR) was explored in cardiac ischaemia, inflammatory diseases and cancer. We report a new fluorophore-conjugated human (h) A(3)AR antagonist for application to cell-based assays in ligand discovery and for receptor imaging. Fluorescent pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-ylamine (pyrazolo-triazolo-pyrimidine, PTP) and triazolo[1,5-c]quinazolin-5-yl)amine (triazolo-quinazoline, TQ) AR antagonists were compared. A chain-extended and click-conjugated Alexa Fluor-488 TQ derivative (MRS5449) displayed a radioligand binding K(i) value of 6.4±2.5nM in hA(3)AR-expressing CHO cell membranes. MRS5449 antagonized hA(3)AR agonist-induced inhibition of cyclic AMP accumulation in a concentration-dependent manner (K(B)=4.8nM). Using flow cytometry (FCM), MRS5449 saturated hA(3)ARs with very high specific-to-nonspecific binding ratio with an equilibrium binding constant 5.15nM, comparable to the K(d) value of 6.65nM calculated from kinetic experiments. K(i) values of known AR antagonists in inhibition of MRS5449 binding in whole cell FCM were consistent with radioligand binding in membranes, but agonist binding was 5-20 fold weaker than obtained with agonist radioligand [(125)I]I-AB-MECA. Further binding analysis of MRS5549 suggested multiple agonist binding states of the A(3)AR. Molecular docking predicted binding modes of these fluorescent antagonists. Thus, MRS5449 is a useful tool for hA(3)AR characterization.

  14. Probing Molecular Composition of Soil Organic Matter with Nanospray Desorption Electrospray Ionization (nano-DESI) High-Resolution Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Laskin, A.; Laskin, J.; Nizkorodov, S.

    2014-12-01

    The standard techniques for chemical analysis of SOM often lack molecular detail necessary for characterization of the key classes of compounds in soil necessary for the development of predictive models. High-resolution mass spectrometry (HR-MS) combined with tandem mass spectrometry (MSn) is the technique of choice for structural characterization of individual molecules in complex environmental mixtures because HR-MS enables chemical characterization of complex samples with a level of detail that is not attainable using other techniques. However, the power of this technique for characterization of SOM has been demonstrated only recently. Nanospray desorption ionization (nano-DESI) bypasses the traditional SOM extraction steps and provides the unique ability to record SOM mass spectra from small whole-soil samples. Nano-DESI benefits from a short sample preparation time (dozens of samples can be analyzed in a matter of hours by simply swapping the samples on the sample holder and running a pre-programmed positioning stage protocol), fast analysis time (useful signal results from only a few seconds of contact between the sample and the liquid bridge), and highly sensitive detection (less than 10 ng of organic mass is required for analysis). The nano-DESI method is minimally destructive. Only a small spot (typically < 1mm) is in contact with the solvent bridge during the analysis, and the rest of the sample is unaffected. Sampling over multiple spots on the same sample in effect replaces the need to extract a large amount of SOM from a soil sample in order to homogenize it, as done in conventional soil analysis. Another critical advantage of nano-DESI is that it makes it possible to observe organic components that cannot be easily detected using traditional ESI method (69). Specifically, by minimizing the residence time of analyte in the spray solvent, it enables detection and structural characterization of chemically labile molecules in environmental samples, which

  15. Electrokinetic characterization of superparamagnetic nanoparticle-aptamer conjugates: design of new highly specific probes for miniaturized molecular diagnostics.

    PubMed

    Girardot, Marie; d'Orlyé, Fanny; Varenne, Anne

    2014-02-01

    With the view of designing new nanoparticle (NP)-aptamer conjugates and proving their suitability as biorecognition tools for miniaturized molecular diagnostics, new maghemite-silica core-shell NP-aptamer conjugates were characterized for the first time in terms of grafting rate and colloidal stability under electrophoretic conditions using capillary electrophoresis. After the grafting rate (on the order of six to 50) of the lysozyme-binding aptamer had been estimated, the electrophoretic stability and peak dispersion of the resulting oligonucleotide-NP conjugates were estimated so as to determine the optimal separation conditions in terms of buffer pH, ionic strength and nature, as well as temperature and electric field strength. The effective surface charge density of the NPs was close to zero for pH lower than 5, which led to some aggregation. The NPs were stable in the pH range from 5 to 9, and an increase in electrophoretic mobility was evidenced with increasing pH. Colloidal stability was preserved at physiological pH for both non-grafted NPs and grafted NPs in the 10-100 mM ionic strength range and in the 15-60 °C temperature range. A strong influence of the nature of the buffer counterion on NP electrophoretic mobility and peak dispersion was evidenced, thus indicating some interactions between buffer components and NP-aptamer conjugates. Whereas an electric field effect (50-900 V cm(-1)) on NP electrophoretic mobility was evidenced, probably linked to counterion dissociation, temperature seems to have an appreciable effect on the zeta potential and aptamer configuration as well. This information is crucial for estimating the potentialities of such biorecognition tools in electrophoretic systems.

  16. Novel Fluorescent Antagonist as a Molecular Probe in A3 Adenosine Receptor Binding Assays Using Flow Cytometry

    PubMed Central

    Kozma, Eszter; Kumar, T. Santhosh; Federico, Stephanie; Phan, Khai; Balasubramanian, Ramachandran; Gao, Zhan-Guo; Paoletta, Silvia; Moro, Stefano; Spalluto, Giampiero; Jacobson, Kenneth A.

    2012-01-01

    The physiological role of the A3 adenosine receptor (AR) was explored in cardiac ischaemia, inflammatory diseases and cancer. We report a new fluorophore-conjugated human (h) A3AR antagonist for application to cell-based assays in ligand discovery and for receptor imaging. Fluorescent pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-ylamine (pyrazolo-triazolo-pyrimidine, PTP) and triazolo[1,5-c]quinazolin-5-yl)amine (triazolo-quinazoline, TQ) AR antagonists were compared. A chain-extended and click-conjugated Alexa Fluor-488 TQ derivative (MRS5449) displayed a radioligand binding Ki value of 6.4 ± 2.5 nM in hA3AR-expressing CHO cell membranes. MRS5449 antagonized hA3AR agonist-induced inhibition of cyclic AMP accumulation in a concentration-dependent manner (KB 4.8 nM). Using flow cytometry (FCM), MRS5449 saturated hA3ARs with very high specific-to-nonspecific binding ratio with an equilibrium binding constant 5.15 nM, comparable to the Kd value of 6.65 nM calculated from kinetic experiments. Ki values of known AR antagonists in inhibition of MRS5449 binding in whole cell FCM were consistent with radioligand binding in membranes, but agonist binding was 5–20 fold weaker than obtained with agonist radioligand [125I]I-AB-MECA. Further binding analysis of MRS5549 suggested multiple agonist binding states of the A3AR. Molecular docking predicted binding modes of these fluorescent antagonists. Thus, MRS5449 is a useful tool for hA3AR characterization. PMID:22402302

  17. Foldable polymers as probes

    DOEpatents

    Li, Alexander D. Q.; Wang, Wei

    2007-07-03

    Disclosed herein are novel probes, which can be used to detect and identify target molecules of interest in a sample. The disclosed probes can be used to monitor conformational changes induced by molecular recognition events in addition to providing signaling the presence and/or identity of a target molecule. Methods, including solid phase synthesis techniques, for making probe molecules that exhibit changes in their optical properties upon target molecule binding are described in the disclosure. Also disclosed herein are novel chromophore moieties, which have tailored fluorescent emission spectra.

  18. Foldable polymers as probes

    DOEpatents

    Li, Alexander D. Q.; Wang, Wei

    2009-07-07

    Disclosed herein are novel probes, which can be used to detect and identify target molecules of interest in a sample. The disclosed probes can be used to monitor conformational changes induced by molecular recognition events in addition to providing signaling the presence and/or identity of a target molecule. Methods, including solid phase synthesis techniques, for making probe molecules that exhibit changes in their optical properties upon target molecule binding are described in the disclosure. Also disclosed herein are novel chromophore moieties, which have tailored fluorescent emission spectra.

  19. scVEGF Microbubble Ultrasound Contrast Agents: A Novel Probe for Ultrasound Molecular Imaging of Tumor Angiogenesis

    PubMed Central

    Christopher R., Anderson; Joshua J., Rychak; Marina, Backer; Joseph, Backer; Klaus, Ley; Alexander L., Klibanov

    2012-01-01

    significantly higher ultrasound contrast signal enhancement in tumors (8.46 ± 1.61 dB) compared with nontargeted control MB (1.58 ± 0.83 dB). Conclusions These results demonstrate the functionality of a novel scVEGF-bearing MB contrast agent, which could be useful for molecular imaging of VEGFR-2 in basic science and drug discovery research. PMID:20733505

  20. Comparative Evaluation of Sloppy Molecular Beacon and Dual-Labeled Probe Melting Temperature Assays to Identify Mutations in Mycobacterium tuberculosis Resulting in Rifampin, Fluoroquinolone and Aminoglycoside Resistance

    PubMed Central

    Lee, Jong Seok; Via, Laura E.; Barry, Clifton E.; Alland, David; Chakravorty, Soumitesh

    2015-01-01

    Several molecular assays to detect resistance to Rifampin, the Fluoroquinolones, and Aminoglycosides in Mycobacterium tuberculosis (M. tuberculosis) have been recently described. A systematic approach for comparing these assays in the laboratory is needed in order to determine the relative advantage of each assay and to decide which ones should be advanced to evaluation. We performed an analytic comparison of a Sloppy Molecular Beacon (SMB) melting temperature (Tm) assay and a Dual labeled probe (DLP) Tm assay. Both assays targeted the M. tuberculosis rpoB, gyrA, rrs genes and the eis promoter region. The sensitivity and specificity to detect mutations, analytic limit of detection (LOD) and the detection of heteroresistance were tested using a panel of 56 clinical DNA samples from drug resistant M. tuberculosis strains. Both SMB and DLP assays detected 29/29 (100%) samples with rpoB RRDR mutations and 3/3 (100%) samples with eis promoter mutations correctly. The SMB assay detected all 17/17 gyrA mutants and 22/22 rrs mutants, while the DLP assay detected 16/17 (94%) gyrA mutants and 12/22 (55%) rrs mutants. Both assays showed comparable LODs for detecting rpoB and eis mutations; however, the SMB assay LODs were at least two logs better for detecting wild type and mutants in gyrA and rrs targets. The SMB assay was also moderately better at detecting heteroresistance. In summary, both assays appeared to be promising methods to detect drug resistance associated mutations in M. tuberculosis; however, the relative advantage of each assay varied under each test condition. PMID:25938476

  1. A cost-effective sandwich electrochemiluminescence immunosensor for ultrasensitive detection of HIV-1 antibody using magnetic molecularly imprinted polymers as capture probes.

    PubMed

    Zhou, Jing; Gan, Ning; Li, Tianhua; Hu, Futao; Li, Xing; Wang, Lihong; Zheng, Lei

    2014-04-15

    In this report, a rapid and cost-effective sandwich electrochemiluminescence (ECL) immunosensor was constructed for the ultrasensitive detection of human immunodeficiency virus type 1 antibody (anti-HIV-1) using magnetic molecularly imprinted polymers (MMIPs) as capture probes by combining surface and epitope imprinting techniques and antigen conjugated with horseradish peroxidase (HRP-HIV-1) as labels. First, 3-aminobenzeneboronic acid (APBA) was used as the functional monomer and cross-linking reagent, which was polymerized on the surface of silicate-coated magnetic iron oxide nanoparticles (Fe3O4@SiO2 NPs) in the presence of human immunoglobulin G (HIgG), as the template exhibiting the same Fc region but different Fab region to anti-HIV-1 after the addition of the initiator, ammonium persulfate. This process resulted in grafting a hydrophilic molecularly imprinted polymer (MIP) film on the Fe3O4@SiO2 NPs. Thus, MMIPs, which could be reused after eluting the template, were used to recognize and enrich ultra-trace levels of anti-HIV-1. Subsequently, a novel sandwich ECL immunosensor was formed through the immunoreaction between MMIPs conjugated with varied concentrations of anti-HIV-1 and HRP-HIV-1. By the catalysis of HRP immobilized onto HRP-HIV-1 on the ECL system of Luminol-H2O2, a linear response range of the anti-HIV-1 dilution ratio (standard positive serum) was achieved from 1:20,000 to 1:50, with a detection limit of 1:60,000 (S/N=3). The developed method provides a low-cost, simple, and sensitive way for the early diagnosis of HIV infected patients.

  2. A cost-effective sandwich electrochemiluminescence immunosensor for ultrasensitive detection of HIV-1 antibody using magnetic molecularly imprinted polymers as capture probes.

    PubMed

    Zhou, Jing; Gan, Ning; Li, Tianhua; Hu, Futao; Li, Xing; Wang, Lihong; Zheng, Lei

    2014-04-15

    In this report, a rapid and cost-effective sandwich electrochemiluminescence (ECL) immunosensor was constructed for the ultrasensitive detection of human immunodeficiency virus type 1 antibody (anti-HIV-1) using magnetic molecularly imprinted polymers (MMIPs) as capture probes by combining surface and epitope imprinting techniques and antigen conjugated with horseradish peroxidase (HRP-HIV-1) as labels. First, 3-aminobenzeneboronic acid (APBA) was used as the functional monomer and cross-linking reagent, which was polymerized on the surface of silicate-coated magnetic iron oxide nanoparticles (Fe3O4@SiO2 NPs) in the presence of human immunoglobulin G (HIgG), as the template exhibiting the same Fc region but different Fab region to anti-HIV-1 after the addition of the initiator, ammonium persulfate. This process resulted in grafting a hydrophilic molecularly imprinted polymer (MIP) film on the Fe3O4@SiO2 NPs. Thus, MMIPs, which could be reused after eluting the template, were used to recognize and enrich ultra-trace levels of anti-HIV-1. Subsequently, a novel sandwich ECL immunosensor was formed through the immunoreaction between MMIPs conjugated with varied concentrations of anti-HIV-1 and HRP-HIV-1. By the catalysis of HRP immobilized onto HRP-HIV-1 on the ECL system of Luminol-H2O2, a linear response range of the anti-HIV-1 dilution ratio (standard positive serum) was achieved from 1:20,000 to 1:50, with a detection limit of 1:60,000 (S/N=3). The developed method provides a low-cost, simple, and sensitive way for the early diagnosis of HIV infected patients. PMID:24280050

  3. Comparative Evaluation of Sloppy Molecular Beacon and Dual-Labeled Probe Melting Temperature Assays to Identify Mutations in Mycobacterium tuberculosis Resulting in Rifampin, Fluoroquinolone and Aminoglycoside Resistance.

    PubMed

    Roh, Sandy S; Smith, Laura E; Lee, Jong Seok; Via, Laura E; Barry, Clifton E; Alland, David; Chakravorty, Soumitesh

    2015-01-01

    Several molecular assays to detect resistance to Rifampin, the Fluoroquinolones, and Aminoglycosides in Mycobacterium tuberculosis (M. tuberculosis) have been recently described. A systematic approach for comparing these assays in the laboratory is needed in order to determine the relative advantage of each assay and to decide which ones should be advanced to evaluation. We performed an analytic comparison of a Sloppy Molecular Beacon (SMB) melting temperature (Tm) assay and a Dual labeled probe (DLP) Tm assay. Both assays targeted the M. tuberculosis rpoB, gyrA, rrs genes and the eis promoter region. The sensitivity and specificity to detect mutations, analytic limit of detection (LOD) and the detection of heteroresistance were tested using a panel of 56 clinical DNA samples from drug resistant M. tuberculosis strains. Both SMB and DLP assays detected 29/29 (100%) samples with rpoB RRDR mutations and 3/3 (100%) samples with eis promoter mutations correctly. The SMB assay detected all 17/17 gyrA mutants and 22/22 rrs mutants, while the DLP assay detected 16/17 (94%) gyrA mutants and 12/22 (55%) rrs mutants. Both assays showed comparable LODs for detecting rpoB and eis mutations; however, the SMB assay LODs were at least two logs better for detecting wild type and mutants in gyrA and rrs targets. The SMB assay was also moderately better at detecting heteroresistance. In summary, both assays appeared to be promising methods to detect drug resistance associated mutations in M. tuberculosis; however, the relative advantage of each assay varied under each test condition.

  4. Thermal desorption characterisation of molecularly imprinted polymers. Part II: Use of direct probe GC-MS analysis to study crosslinking effects.

    PubMed

    Holland, Niamh; Duggan, Patrick; Owens, Eleanor; Cummins, Wayne; Frisby, June; Hughes, Helen; McLoughlin, Peter

    2008-06-01

    A powerful method utilising direct probe thermal desorption GC-MS is presented for the study of molecularly imprinted polymers (MIPs). A series of 2-aminopyridine (2-apy)-imprinted methacrylic acid-ethyleneglycol dimethacrylate (MAA-EGDMA) copolymers were prepared under identical conditions but with varying amounts of EGDMA (crosslinking monomer). The use of appropriate temperature programmes permitted template removal, and the subsequent assessment of polymer affinity and specificity, all of which were found to be dependent on polymer composition and morphology. The system was sufficiently sensitive to identify a specific response of imprinted polymers over nonimprinted counterparts. Correlations were found to exist between thermal desorption analysis and solution phase binding, which was assessed by UV spectroscopy, where specificity was found to diminish with decreasing EGDMA concentration. This was attributed to the increased number of free carboxyl groups in those polymers containing a lower percentage of EGDMA. Thermal desorption profiles obtained for the analyte were found to be unaffected by the physical and chemical properties of the solvent used for analyte reloading.

  5. Low molecular weight fluorescent probes with good photostability for imaging RNA-rich nucleolus and RNA in cytoplasm in living cells.

    PubMed

    Song, Guofen; Sun, Yuming; Liu, Yong; Wang, Xiankun; Chen, Meiling; Miao, Fang; Zhang, Weijia; Yu, Xiaoqiang; Jin, Jianling

    2014-02-01

    We have synthesized two low molecular weight organic molecules, PY and IN successfully, which selectively stain nucleolus and cytoplasm of living cells in 30 min, with a much lower uptake in the nucleus. Nucleic acids electrophoresis and digest test of ribonuclease indicate their markedly higher affinity for RNA, especially PY. Moreover their RNA localization in cells is further supported by digest test of ribonuclease, namely, the nucleolar fluorescence signal is distinctly lost upon treatment with RNase. And, the fact that live cells stained by PY and IN still possess physiological function can be confirmed: 1) MTT assay demonstrates that the mitochondria of cells stained remains its electron mediating ability, 2) Double assay of PY/IN and propidium iodide as well as trypan blue testing show that the membrane of cells stained still is intact. Importantly, compared with the only commercial RNA probe, SYTO RNA-Select, PY and IN exhibit much better photostability when continuously illuminated with 488 nm laser and mercury lamp. These results prove that PY and IN are very attractive staining reagents for visualizing RNA in living cells.

  6. Use of molecular beacons to probe for messenger RNA release from ribosomes during 5'-translational blockage by consecutive low-usage codons in Escherichia coli

    NASA Astrophysics Data System (ADS)

    Gao, Wenwu; Tyagi, Sanjay; Kramer, Fred R.; Goldman, Emanuel

    2000-03-01

    In `5'-translational blockage,' significantly reduced yields of proteins are synthesized in Escherichia coli when consecutive low-usage codons are inserted near translation starts of messages (with reduced or no effect when these same codons are inserted downstream). We tested the hypothesis that ribosomes encountering these low-usage codons prematurely release the mRNA. RNA from polysome gradients was fractionated into pools of polysomes, monosomes and ribosomes-free. New hybridization probes, called `molecular beacons,' and standard slot-blots, were used to detect test messages containing either consecutive low-usage AGG (arginine) or synonymous high-usage CGU insertions near the 5' end. The results show an approximately twofold increase in the ratio of free to bound mRNA when the low-usage codons were present compared to high-usage codons. In contrast, there was no difference in the ratio of free to bound mRNA when consecutive low-usage CUA or high-usage CUG (leucine) codons were inserted, or when the arginine codons were inserted near the 3' end. These data indicate that at least some mRNA is released from ribosomes during 5'-translational blockage by arginine but not leucine codons, and they support proposals that premature termination of translation can occur in some conditions in vivo in the absence of a stop codon.

  7. Study on vibrational relaxation dynamics of phenol-water complex by picosecond time-resolved IR-UV pump-probe spectroscopy in a supersonic molecular beam

    NASA Astrophysics Data System (ADS)

    Miyazaki, Yasunori; Inokuchi, Yoshiya; Ebata, Takayuki; Petković, Milena

    2013-06-01

    A comparative study of vibrational energy relaxation (VER) between the monohydrated complexes of phenol-d0 and phenol-d1 is investigated in a supersonic molecular beam. The direct time-resolved measurement of energy redistribution from the phenolic OH/OD stretching mode of the phenol-d0-H2O/phenol-d1-D2O is performed by picosecond IR-UV pump-probe spectroscopy. Two complexes follow the same relaxation process that begins with the intramolecular vibrational energy redistribution (IVR) and the intermolecular vibrational energy redistribution (IVR), which is followed by the vibrational predissociation (VP). The difference in the relaxation lifetimes between them is discussed by anharmonic force field and RRKM calculations. Anharmonic analysis implies that intra- (IVR) and intermolecular (IVR) relaxations occur in parallel in the complexes. The RRKM-predicted dissociation (VP) lifetimes show qualitative agreement with the observed results, suggesting that VP takes place after the statistical energy distribution in the complexes.

  8. Detection of trace anthracene in soil samples with real-time fluorescence quantitative immuno-PCR using a molecular beacon probe.

    PubMed

    Ye, Qi-Yan; Zhuang, Hui-Sheng; Zhou, Chun

    2009-11-01

    We developed a highly sensitive and robust real-time fluorescence quantitative immuno-PCR (RTFQ-IPCR) method which uses molecular beacon (MB) probe to detect trace anthracene in the environment. This method was performed on serial dilutions of known anthracene concentrations equivalent to 10-fold dilutions of 10fg/mL to 100pg/mL. We obtained a linear relationship between 10fg/mL and 100pg/mL, with y=0.684x+13.221. A correlation coefficient of 0.994 was also identified, with a detection limit of 4.5fg/mL. After investigating the presence of anthracene in soil samples via RTFQ-IPCR, the obtained concentrations were confirmed by ELISA to be correct and believable, with the recovery ratio ranging from 82% to 112.5%. Based on its sensitivity and reproducibility, MB-based RTFQ-IPCR was found to be acceptable for use in on-site field tests to provide rapid, quantitative, and reliable test results for making environmental decisions.

  9. Using EPR spectroscopy as a unique probe of molecular-scale reorganization and solvation in self-assembled gel-phase materials.

    PubMed

    Caragheorgheopol, Agneta; Edwards, William; Hardy, John G; Smith, David K; Chechik, Victor

    2014-08-01

    We describe the synthesis of spin-labeled bis-ureas which coassemble with bis-urea gelators and report on self-assembly as detected using electron paramagnetic resonance spectroscopy (EPR). Specifically, EPR detects the gel-sol transition and allows us to quantify how much spin-label is immobilized within the gel fibers and how much is present in mobile solvent pools-as controlled by temperature, gelator structure, and thermal history. EPR is also able to report on the initial self-assembly processes below the gelation threshold which are not macroscopically visible and appears to be more sensitive than NMR to intermediate-sized nongelating oligomeric species. By studying dilute solutions of gelator molecules and using either single or double spin-labels, EPR allows quantification of the initial steps of the hierarchical self-assembly process in terms of cooperativity and association constant. Finally, EPR enables us to estimate the degree of gel-fiber solvation by probing the distances between spin-labels. Comparison of experimental data against the predicted distances assuming the nanofibers are only composed of gelator molecules indicates a significant difference, which can be assigned to the presence of a quantifiable number of explicit solvent molecules. In summary, EPR provides unique data and yields powerful insight into how molecular-scale mobility and solvation impact on assembly of supramolecular gels.

  10. Spectrophotometric probe

    DOEpatents

    Prather, William S.; O'Rourke, Patrick E.

    1994-01-01

    A support structure bearing at least one probe for making spectrophotometric measurements of a fluid using a source of light and a spectrophotometer. The probe includes a housing with two optical fibers and a planoconvex lens. A sleeve bearing a mirror surrounds the housing. The lens is separated from the mirror by a fixed distance, defining an interior space for receiving a volume of the fluid sample. A plurality of throughholes extending through the sleeve communicate between the sample volume and the exterior of the probe, all but one hole bearing a screen. A protective jacket surrounds the probe. A hollow conduit bearing a tube is formed in the wall of the probe for venting any air in the interior space when fluid enters. The probe is held at an acute angle so the optic fibers carrying the light to and from the probe are not bent severely on emergence from the probe.

  11. Spectrophotometric probe

    DOEpatents

    Prather, W.S.; O'Rourke, P.E.

    1994-08-02

    A support structure is described bearing at least one probe for making spectrophotometric measurements of a fluid using a source of light and a spectrophotometer. The probe includes a housing with two optical fibers and a planoconvex lens. A sleeve bearing a mirror surrounds the housing. The lens is separated from the mirror by a fixed distance, defining an interior space for receiving a volume of the fluid sample. A plurality of throughholes extending through the sleeve communicate between the sample volume and the exterior of the probe, all but one hole bearing a screen. A protective jacket surrounds the probe. A hollow conduit bearing a tube is formed in the wall of the probe for venting any air in the interior space when fluid enters. The probe is held at an acute angle so the optic fibers carrying the light to and from the probe are not bent severely on emergence from the probe. 3 figs.

  12. Assessing the acidity of high silica chabazite H-SSZ-13 by FTIR using CO as molecular probe: Comparison with H-SAPO-34.

    PubMed

    Bordiga, Silvia; Regli, Laura; Cocina, Donato; Lamberti, Carlo; Bjørgen, Morten; Lillerud, Karl Petter

    2005-02-24

    Zeolitic materials based on the chabazite topology, such as H-SAPO-34, possess unique shape-selectivity properties for converting methanol into light olefins. In addition to the topology, zeolite acidity is inherently linked to catalyst activity and selectivity. The acidic properties of high silica chabazite (H-SSZ-13) have attracted much attention in the past decade because the material represents an idealized model system having one acidic site per cage. Conclusions drawn so far have essentially been founded on quantum chemical methods. An experimentally based benchmark of the acidity of H-SSZ-13 has hitherto not been available. In this work, transmission FTIR spectroscopy provides a description of the different acidic sites of H-SSZ-13 by using CO as molecular probe at 70 K. The results demonstrate that H-SSZ-13 is a strongly Brønsted acidic material, essentially having two distinct families of acidic sites. In contrast to numerous preceding reports, we find it fundamental to consider proton distributions among all four possible sites, and do not delimit the interpretations to only two sites. The present data consistently suggest the most abundant family of protons to have three members being located on different crystalline positions on the eight-membered-ring window giving access to the chabazite cage. Consequently, these protons are exposed to two neighboring cages. The second, and less abundant family, is constituted by only one site that is situated on the six-membered ring defining the top/bottom of the barrel-shaped chabazite cage. This proton is therefore only exposed to one cage and requires a higher CO pressure to form adducts. Toward CO, both families of sites possess the same acidity. Parallel experiments were also carried out for the isostructural and commercially important H-SAPO-34 having an equal density of acidic sites. This is the first attempt to directly compare, on an experimental basis, the acidity of these two materials.

  13. Assessing the acidity of high silica chabazite H-SSZ-13 by FTIR using CO as molecular probe: Comparison with H-SAPO-34.

    PubMed

    Bordiga, Silvia; Regli, Laura; Cocina, Donato; Lamberti, Carlo; Bjørgen, Morten; Lillerud, Karl Petter

    2005-02-24

    Zeolitic materials based on the chabazite topology, such as H-SAPO-34, possess unique shape-selectivity properties for converting methanol into light olefins. In addition to the topology, zeolite acidity is inherently linked to catalyst activity and selectivity. The acidic properties of high silica chabazite (H-SSZ-13) have attracted much attention in the past decade because the material represents an idealized model system having one acidic site per cage. Conclusions drawn so far have essentially been founded on quantum chemical methods. An experimentally based benchmark of the acidity of H-SSZ-13 has hitherto not been available. In this work, transmission FTIR spectroscopy provides a description of the different acidic sites of H-SSZ-13 by using CO as molecular probe at 70 K. The results demonstrate that H-SSZ-13 is a strongly Brønsted acidic material, essentially having two distinct families of acidic sites. In contrast to numerous preceding reports, we find it fundamental to consider proton distributions among all four possible sites, and do not delimit the interpretations to only two sites. The present data consistently suggest the most abundant family of protons to have three members being located on different crystalline positions on the eight-membered-ring window giving access to the chabazite cage. Consequently, these protons are exposed to two neighboring cages. The second, and less abundant family, is constituted by only one site that is situated on the six-membered ring defining the top/bottom of the barrel-shaped chabazite cage. This proton is therefore only exposed to one cage and requires a higher CO pressure to form adducts. Toward CO, both families of sites possess the same acidity. Parallel experiments were also carried out for the isostructural and commercially important H-SAPO-34 having an equal density of acidic sites. This is the first attempt to directly compare, on an experimental basis, the acidity of these two materials. PMID:16851287

  14. Simultaneous detection of pathogens in clinical samples from patients with community-acquired pneumonia by real-time PCR with pathogen-specific molecular beacon probes.

    PubMed

    Morozumi, Miyuki; Nakayama, Eiichi; Iwata, Satoshi; Aoki, Yasuko; Hasegawa, Keiko; Kobayashi, Reiko; Chiba, Naoko; Tajima, Takeshi; Ubukata, Kimiko

    2006-04-01

    In this study, real-time PCR with pathogen-specific molecular beacons (MB) and primers was evaluated for prediction of community-acquired pneumonia (CAP) causative agents, detecting six main CAP agents, Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, and Streptococcus pyogenes, simultaneously. The PCR assay was evaluated for fresh clinical specimens from infants and children (n = 389) and from adults (n = 40). The MB probes and primers are both pathogen specific, namely, the lytA gene for S. pneumoniae, the mip gene for L. pneumophila, and 16S rRNA genes for the remaining four organisms. DNA extraction of clinical specimens was performed with a commercially available EXTRAGEN II kit, and amplification was performed with Stratagene Mx3000P. The limit of detection for these pathogens ranged from 2 copies to 18 copies. The whole process from DNA extraction to the analysis was finished in less than 2 h. The obtained sensitivity and specificity of this real-time PCR study relative to those of conventional cultures were as follows: 96.2% and 93.2% for S. pneumoniae, 95.8% and 95.4% for H. influenzae, 100% and 100% for S. pyogenes, and 100% and 95.4% for M. pneumoniae, respectively. The sensitivity and specificity for M. pneumoniae relative to those of a serologic assay were 90.2% and 97.9%, respectively. In six clinical samples of C. pneumoniae, the real-time PCR gave positive predictable values, and in those cases, elevation of the titer value was also observed. In conclusion, we demonstrated that a real-time PCR assay with pathogen-specific MB is useful in identifying CAP causative agents rapidly and in examining the clinical course of empirical chemotherapy in a timely manner, supporting conventional culture methods.

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

    PubMed

    Christensen, Geoff A; Wymore, Ann M; King, Andrew J; Podar, Mircea; Hurt, Richard A; Santillan, Eugenio U; Soren, Ally; Brandt, Craig C; Brown, Steven D; Palumbo, Anthony V; Wall, Judy D; Gilmour, Cynthia C; Elias, Dwayne A

    2016-10-01

    discovery of the Hg-methylating gene pair, hgcA and hgcB, has allowed us to design and optimize molecular probes against these genes within the genomic DNA for microorganisms known to methylate Hg. The protocols designed in this study allow for both qualitative and quantitative assessments of pure-culture or environmental samples. With these protocols in hand, we can begin to study the distribution of Hg-methylating organisms in nature via a cultivation-independent strategy. PMID:27422835

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

    PubMed

    Christensen, Geoff A; Wymore, Ann M; King, Andrew J; Podar, Mircea; Hurt, Richard A; Santillan, Eugenio U; Soren, Ally; Brandt, Craig C; Brown, Steven D; Palumbo, Anthony V; Wall, Judy D; Gilmour, Cynthia C; Elias, Dwayne A

    2016-10-01

    discovery of the Hg-methylating gene pair, hgcA and hgcB, has allowed us to design and optimize molecular probes against these genes within the genomic DNA for microorganisms known to methylate Hg. The protocols designed in this study allow for both qualitative and quantitative assessments of pure-culture or environmental samples. With these protocols in hand, we can begin to study the distribution of Hg-methylating organisms in nature via a cultivation-independent strategy.

  17. A conveniently prepared and hypersensitized small molecular fluorescent probe: Rapidly detecting free zinc ion in HepG2 cells and Arabidopsis.

    PubMed

    Gan, Xiaoping; Sun, Ping; Li, Hong; Tian, Xiaohe; Zhang, Baowei; Wu, Jieying; Tian, Yupeng; Zhou, Hongping

    2016-12-15

    In this paper, we reported a conveniently prepared fluorescent probe for zinc ions detection, which constructed by the condensation reaction between p-(benzothiazolyl)aniline with 4, 4- diethylaminesalicylaldehyde. The sensing ability of the probe toward zinc ions in vitro was tested by a series of UV-Vis and fluorescence spectroscopy studies, which showed that the probe possessed high sensitivity with a detection limit of 5.8nM and a rapid response time of 10s. We also carried out fluorescent bio-imaging of the probe for zinc ions in human liver hepatocellular carcinoma cells (HepG2), which showed that the probe could be utilized to detect the intracellular endogenous zinc ions visually without introducing external zinc sources. Meanwhile, co-staining experiment with organelle selective trackers was performed to illustrate that the probe could locate at endoplasmic reticulum. Finally, we successfully used it as a zinc ion developer in plant tissue, which clearly demonstrated the distribution of zinc ions in the growth stage of plant tissue. PMID:27414244

  18. Design, synthesis, and biological evaluation of 4-(5-dimethylamino-naphthalene-1-sulfon-amido)-3-(4-iodophenyl)butanoic acid as a novel molecular probe for apoptosis imaging

    SciTech Connect

    Zeng, Wenbin; Miao, Weimin; Le Puil, Michael; Shi, Guangqing; Biggerstaff, John; Kabalka, George W.; Townsend, David

    2010-07-30

    Research highlights: {yields} Annexin V is the gold standard probe for imaging apoptosis. {yields} Unfavorable profiles of Annexin V make it difficult to apply in the clinic. {yields} A novel small-molecular probe DNSBA was designed as an alternative to Annexin V. {yields} DNSBA specifically and selectively detect apoptotic cancer cells at all stages. {yields} DNSBA is a potential SPECT and PET agent when labeled with radioiodine. -- Abstract: Apoptosis (programmed cell death) plays a crucial role in the pathogenesis of many disorders, thus the detection of apoptotic cells can provide the physician with important information to further therapeutic strategies and would substantially advance patient care. A small molecule, 4-(5-dimethylamino-naphthalene-1-sulfonamido)-3-(4-iodo-phenyl)butanoic acid (DNSBA), was designed as a novel probe for imaging apoptosis and synthesized with good yield. The biological characterization demonstrated that DNSBA can be used to specifically and selectively detect apoptotic cancer cells at all stages. DNSBA is also designed as a potential SPECT and PET probe when labeled with radioiodine (I-123, -124, and -131).

  19. In situ molecular hybridization for detection of Aleutian mink disease parvovirus DNA by using strand-specific probes: identification of target cells for viral replication in cell cultures and in mink kits with virus-induced interstitial pneumonia.

    PubMed

    Alexandersen, S; Bloom, M E; Wolfinbarger, J; Race, R E

    1987-08-01

    Strand-specific hybridization probes were utilized in in situ molecular hybridization specifically to localize replicative form DNA of Aleutian mink disease parvovirus (ADV). Throughout in vitro infection, duplex replicative form DNA of ADV was located in the cell nuclei. Single-stranded virion DNA and capsid proteins were present in the nuclei early in infection, but were later translocated to the cytoplasm. In neonatal mink, ADV causes acute interstitial pneumonia, and replicative forms of viral DNA were found predominantly in alveolar type II cells of the lung. Viral DNA was also found in other organs, but strand-specific probes made it possible to show that most of this DNA represented virus sequestration. In addition, glomerular immune complexes containing intact virions were detected, suggesting that ADV virions may have a role in the genesis of ADV-induced glomerulonephritis.

  20. Optical probe

    DOEpatents

    Hencken, Kenneth; Flower, William L.

    1999-01-01

    A compact optical probe is disclosed particularly useful for analysis of emissions in industrial environments. The instant invention provides a geometry for optically-based measurements that allows all optical components (source, detector, rely optics, etc.) to be located in proximity to one another. The geometry of the probe disclosed herein provides a means for making optical measurements in environments where it is difficult and/or expensive to gain access to the vicinity of a flow stream to be measured. Significantly, the lens geometry of the optical probe allows the analysis location within a flow stream being monitored to be moved while maintaining optical alignment of all components even when the optical probe is focused on a plurality of different analysis points within the flow stream.

  1. Development and application of multiple-probe scanning probe microscopes.

    PubMed

    Nakayama, Tomonobu; Kubo, Osamu; Shingaya, Yoshitaka; Higuchi, Seiji; Hasegawa, Tsuyoshi; Jiang, Chun-Sheng; Okuda, Taichi; Kuwahara, Yuji; Takami, Kazuhiro; Aono, Masakazu

    2012-04-01

    In the research of advanced materials based on nanoscience and nanotechnology, it is often desirable to measure nanoscale local electrical conductivity at a designated position of a given sample. For this purpose, multiple-probe scanning probe microscopes (MP-SPMs), in which two, three or four scanning tunneling microscope (STM) or atomic force microscope (AFM) probes are operated independently, have been developed. Each probe in an MP-SPM is used not only for observing high-resolution STM or AFM images but also for forming an electrical contact enabling nanoscale local electrical conductivity measurement. The world's first double-probe STM (DP-STM) developed by the authors, which was subsequently modified to a triple-probe STM (TP-STM), has been used to measure the conductivities of one-dimensional metal nanowires and carbon nanotubes and also two-dimensional molecular films. A quadruple-probe STM (QP-STM) has also been developed and used to measure the conductivity of two-dimensional molecular films without the ambiguity of contact resistance between the probe and sample. Moreover, a quadruple-probe AFM (QP-AFM) with four conductive tuning-fork-type self-detection force sensing probes has been developed to measure the conductivity of a nanostructure on an insulating substrate. A general-purpose computer software to control four probes at the same time has also been developed and used in the operation of the QP-AFM. These developments and applications of MP-SPMs are reviewed in this paper.

  2. Development and Application of Multiple-Probe Scanning Probe Microscopes

    SciTech Connect

    Nakayama, T.; Kubo, O.; Shingaya, Y.; Higuchi, S.; Hasegawa, T.; Jiang, C. S.; Okuda, T.; Kuwahara, Y.; Takami, K.; Aono, M.

    2012-04-03

    the research of advanced materials based on nanoscience and nanotechnology, it is often desirable to measure nanoscale local electrical conductivity at a designated position of a given sample. For this purpose, multiple-probe scanning probe microscopes (MP-SPMs), in which two, three or four scanning tunneling microscope (STM) or atomic force microscope (AFM) probes are operated independently, have been developed. Each probe in an MP-SPM is used not only for observing high-resolution STM or AFM images but also for forming an electrical contact enabling nanoscale local electrical conductivity measurement. The world's first double-probe STM (DP-STM) developed by the authors, which was subsequently modified to a triple-probe STM (TP-STM), has been used to measure the conductivities of one-dimensional metal nanowires and carbon nanotubes and also two-dimensional molecular films. A quadruple-probe STM (QP-STM) has also been developed and used to measure the conductivity of two-dimensional molecular films without the ambiguity of contact resistance between the probe and sample. Moreover, a quadruple-probe AFM (QP-AFM) with four conductive tuning-fork-type self-detection force sensing probes has been developed to measure the conductivity of a nanostructure on an insulating substrate. A general-purpose computer software to control four probes at the same time has also been developed and used in the operation of the QP-AFM. These developments and applications of MP-SPMs are reviewed in this paper.

  3. Alternative methods for estimating common descriptors for QSAR studies of dyes and fluorescent probes using molecular modeling software. 2. Correlations between log P and the hydrophilic/lipophilic index, and new methods for estimating degrees of amphiphilicity.

    PubMed

    Dapson, Richard W; Horobin, Richard W

    2013-11-01

    The log P descriptor, despite its usefulness, can be difficult to use, especially for researchers lacking skills in physical chemistry. Moreover this classic measure has been determined in numerous ways, which can result in inconsistant estimates of log P values, especially for relatively complex molecules such as fluorescent probes. Novel measures of hydrophilicity/lipophilicity (the Hydrophilic/Lipophilic Index, HLI) and amphiphilicity (hydrophilic/lipophilic indices for the head group and tail, HLIT and HLIHG, respectively) therefore have been devised. We compare these descriptors with measures based on log P, the standard method for quantitative structure activity relationships (QSAR) studies. HLI can be determined using widely available molecular modeling software, coupled with simple arithmetic calculations. It is based on partial atomic charges and is intended to be a stand-alone measure of hydrophilicity/lipophilicity. Given the wide application of log P, however, we investigated the correlation between HLI and log P using a test set of 56 fluorescent probes of widely different physicochemical character. Overall correlation was poor; however, correlation of HLI and log P for probes of narrowly specified charge types, i.e., non-ionic compounds, anions, conjugated cations, or zwitterions, was excellent. Values for probes with additional nonconjugated quaternary cations, however, were less well correlated. The newly devised HLI can be divided into domain-specific descriptors, HLIT and HLIHG in amphiphilic probes. Determinations of amphiphilicity, made independently by the authors using their respective methods, showed excellent agreement. Quantifying amphiphilicity from partial log P values of the head group (head group hydrophilicity; HGH) and tail (amphiphilicity index; AI) has proved useful for understanding fluorescent probe action. The same limitations of log P apply to HGH and AI, however. The novel descriptors, HLIT and HLIHG, offer analogous advantages

  4. Preferential molecular encapsulation of an ICT fluorescence probe in the supramolecular cage of cucurbit[7]uril and β-cyclodextrin: an experimental and theoretical approach.

    PubMed

    Samanta, Anuva; Guchhait, Nikhil; Bhattacharya, Subhash Chandra

    2014-11-20

    Supramolecular interaction between an intramolecular charge transfer (ICT) probe, N,N-dimethylaminonaphthyl-(acrylo)-nitrile (DMANAN), and two well-recognized macrocyclic hosts, cucurbit[7]uril (CB7) and β-cyclodextrin (β-CD), has been studied in aqueous medium by absorption, emission, time-resolved measurements, and (1)H NMR spectroscopic methods. The changes in the profiles of the fluorescence spectra illustrate significant modifications in fluorescence intensity, decay time, and quantum yield upon confinement of probe within the hydrophobic cavity of the hosts. Using the Benesi-Hildebrand relationship, the stoichiometric ratio as well as the binding constant of the host-guest complexation has been estimated. The stable inclusion complexes of the probe with different hosts have been supported by DFT and ONIOM based quantum chemical calculations. These methods of measurement establish that the acceptor group of the probe resides inside the hydrophobic cavity of the macrocycle. The competitive binding of metal ions and cationic surfactants to CB7 has been excellently mapped with this guest fluorosensor.

  5. In vivo imaging of intraperitoneally disseminated tumors in model mice by using activatable fluorescent small-molecular probes for activity of cathepsins.

    PubMed

    Fujii, Tomohiko; Kamiya, Mako; Urano, Yasuteru

    2014-10-15

    It is difficult to completely remove carcinomas in unguided ablative surgery because they cannot be distinguished with the unaided human eye. Therefore, in order to precisely visualize tiny tumors and the borders between cancerous lesions and normal tissues, we have been developing fluorescence probes activatable only in cancer cells. We previously reported the hydroxymethylrhodamine green (HMRG)-based fluorescence probe gGlu-HMRG for γ-glutamyltransferase (GGT), which is overexpressed in a variety of cancer cells, and we showed that it enables in vivo rapid detection of human ovarian cancer SHIN-3 nodules with a high tumor-to-background (T/B) fluorescence ratio in model mice. However, cancer cell lines with low GGT expression could hardly be detected with gGlu-HMRG. Here we developed two new HMRG-based fluorescence probes for the cathepsin family of cysteine proteases, including cathepsin B (CatB) and cathepsin L (CatL), which show increased expression and/or activity, secretion, and altered localization in many kinds of cancer cells. The developed probes, Z-Phe-Arg-HMRG and Z-Arg-Arg-HMRG, are colorless and nonfluorescent at the physiological pH of 7.4, but are hydrolyzed to HMRG upon reaction with purified cathepsins, resulting in a more than 200-fold fluorescence increase. These probes could visualize human ovarian cancer cell lines SHIN-3, SK-OV-3, and OVCAR-3, of which the latter two were hardly detectable with gGlu-HMRG. Z-Phe-Arg-HMRG showed higher applicability than Z-Arg-Arg-HMRG for in vivo imaging, and we confirmed that 0.5-mm-sized SK-OV-3 tumor nodules disseminated on the mesentery in a mouse model could be rapidly visualized by Z-Phe-Arg-HMRG, with a T/B fluorescence ratio of 4.2. Further, intraperitoneally disseminated tumor could be visualized in real time in vivo by fluorescence endoscopy after spraying Z-Phe-Arg-HMRG, with a T/B ratio of 3. In conclusion, our HMRG-based activatable probes targeted to cathepsins have expanded the detectable range

  6. Conductivity Probe

    NASA Technical Reports Server (NTRS)

    2008-01-01

    The Thermal and Electrical Conductivity Probe (TECP) for NASA's Phoenix Mars Lander took measurements in Martian soil and in the air.

    The needles on the end of the instrument were inserted into the Martian soil, allowing TECP to measure the propagation of both thermal and electrical energy. TECP also measured the humidity in the surrounding air.

    The needles on the probe are 15 millimeters (0.6 inch) long.

    The Phoenix Mission is led by the University of Arizona, Tucson, on behalf of NASA. Project management of the mission is by NASA's Jet Propulsion Laboratory, Pasadena, Calif. Spacecraft development is by Lockheed Martin Space Systems, Denver.

  7. A naturally occurring mutation within the probe-binding region compromises a molecular-based West Nile virus surveillance assay for mosquito pools (Diptera: Culicidae).

    PubMed

    Brault, Aaron C; Fang, Ying; Dannen, Maureen; Anishchenko, Michael; Reisen, William K

    2012-07-01

    A naturally occurring mutation was detected within the probe binding region targeting the envelope gene sequence of West Nile virus used in real-time polymerase chain reaction assays to test mosquito pools and other samples. A single C-->T transition 6nt from the 5' end of the 16mer in the envelope gene probe-binding region at genomic position 1,194 reduced assay sensitivity. The mutation first was detected in 2009 and persisted at a low prevalence into 2011. The mutation caused a 0.4% false negative error rate during 2011. These data emphasized the importance of confirmational testing and redundancy in surveillance systems relying on highly specific nucleic acid detection platforms.

  8. Five Years' Evaluation of the BD ProbeTec System for the Direct Molecular Detection of Mycobacterium tuberculosis Complex in Respiratory and Nonrespiratory Clinical Samples.

    PubMed

    Bicmen, Can; Karaman, Onur; Gunduz, Ayriz T; Erer, Onur F; Coskun, Meral; Kaftan, Osman; Demirel, Mahmut M; Senol, Gunes; Akarca, Tulay; Dereli, Sevket; Ozsoz, Ayse

    2015-01-01

    In this study, Mycobacterium tuberculosis complex was detected by BD ProbeTec ET system in 4716 respiratory and 167 nonrespiratory samples [mostly (98%) smear negative). Sensitivity, specificity, positive and negative predictive values were 81.8%, 98.3, 85.1 and 97.9 for respiratory and 100%, 96.2, 64.7 and 100, for nonrespiratory samples, respectively. Among 149 (3.1%) ProbeTec DTB positive and culture negative samples, 72 (65 respiratory and seven nonrespiratory) (48.3%) were recovered from the patients who were evaluated as having TB infection. The system has been found as useful in early diagnosis of tuberculosis infection in association with the clinical, radiological and histopathological findings.

  9. Diagnostic Molecular Mycobacteriology in Regions With Low Tuberculosis Endemicity: Combining Real-time PCR Assays for Detection of Multiple Mycobacterial Pathogens With Line Probe Assays for Identification of Resistance Mutations.

    PubMed

    Deggim-Messmer, Vanessa; Bloemberg, Guido V; Ritter, Claudia; Voit, Antje; Hömke, Rico; Keller, Peter M; Böttger, Erik C

    2016-07-01

    Molecular assays have not yet been able to replace time-consuming culture-based methods in clinical mycobacteriology. Using 6875 clinical samples and a study period of 35months we evaluated the use of PCR-based assays to establish a diagnostic workflow with a fast time-to-result of 1-2days, for 1. detection of Mycobacterium tuberculosis complex (MTB), 2. detection and identification of nontuberculous mycobacteria (NTM), and 3. identification of drug susceptible MTB. MTB molecular-based detection and culture gave concordant results for 97.7% of the specimens. NTM PCR-based detection and culture gave concordant results for 97.0% of the specimens. Defining specimens on the basis of combined laboratory data as true positives or negatives with discrepant results resolved by clinical chart reviews, we calculated sensitivity, specificity, PPV and NPV for PCR-based MTB detection as 84.7%, 100%, 100%, and 98.7%; the corresponding values for culture-based MTB detection were 86.3%, 100%, 100%, and 98.8%. PCR-based detection of NTM had a sensitivity of 84.7% compared to 78.0% of that of culture-based NTM detection. Molecular drug susceptibility testing (DST) by line-probe assay was found to predict phenotypic DST results in MTB with excellent accuracy. Our findings suggest a diagnostic algorithm to largely replace lengthy culture-based techniques by rapid molecular-based methods.

  10. Pollution Probe.

    ERIC Educational Resources Information Center

    Chant, Donald A.

    This book is written as a statement of concern about pollution by members of Pollution Probe, a citizens' anti-pollution group in Canada. Its purpose is to create public awareness and pressure for the eventual solution to pollution problems. The need for effective government policies to control the population explosion, conserve natural resources,…

  11. A disulfide bound-molecular beacon as a fluorescent probe for the detection of reduced glutathione and its application in cells.

    PubMed

    Guo, Yingshu; Wang, Hao; Sun, Yuanshun; Qu, Bin

    2012-03-28

    A disulfide-bound molecular beacon (MB) is reported to respond sensitively to changing levels of glutathione in vitro. Importantly, this successful application of a MB has exciting potential for monitoring cellular thiol.

  12. Comparison of DC and AC Transport in 1.5-7.5 nm Oligophenylene Imine Molecular Wires across Two Junction Platforms: Eutectic Ga-In versus Conducting Probe Atomic Force Microscope Junctions.

    PubMed

    Sangeeth, C S Suchand; Demissie, Abel T; Yuan, Li; Wang, Tao; Frisbie, C Daniel; Nijhuis, Christian A

    2016-06-15

    We have utilized DC and AC transport measurements to measure the resistance and capacitance of thin films of conjugated oligophenyleneimine (OPI) molecules ranging from 1.5 to 7.5 nm in length. These films were synthesized on Au surfaces utilizing the imine condensation chemistry between terephthalaldehyde and 1,4-benzenediamine. Near edge X-ray absorption fine structure (NEXAFS) spectroscopy yielded molecular tilt angles of 33-43°. To probe DC and AC transport, we employed Au-S-OPI//GaOx/EGaIn junctions having contact areas of 9.6 × 10(2) μm(2) (10(9) nm(2)) and compared to previously reported DC results on the same OPI system obtained using Au-S-OPI//Au conducting probe atomic force microscopy (CP-AFM) junctions with 50 nm(2) areas. We found that intensive observables agreed very well across the two junction platforms. Specifically, the EGaIn-based junctions showed: (i) a crossover from tunneling to hopping transport at molecular lengths near 4 nm; (ii) activated transport for wires >4 nm in length with an activation energy of 0.245 ± 0.008 eV for OPI-7; (iii) exponential dependence of conductance with molecular length with a decay constant β = 2.84 ± 0.18 nm(-1) (DC) and 2.92 ± 0.13 nm(-1) (AC) in the tunneling regime, and an apparent β = 1.01 ± 0.08 nm(-1) (DC) and 0.99 ± 0.11 nm(-1) (AC) in the hopping regime; (iv) previously unreported dielectric constant of 4.3 ± 0.2 along the OPI wires. However, the absolute resistances of Au-S-OPI//GaOx/EGaIn junctions were approximately 100 times higher than the corresponding CP-AFM junctions due to differences in metal-molecule contact resistances between the two platforms.

  13. Validation of a simple and fast method to quantify in vitro mineralization with fluorescent probes used in molecular imaging of bone

    SciTech Connect

    Moester, Martiene J.C.; Schoeman, Monique A.E.; Oudshoorn, Ineke B.; Beusekom, Mara M. van; Mol, Isabel M.; Kaijzel, Eric L.; Löwik, Clemens W.G.M.; Rooij, Karien E. de

    2014-01-03

    Highlights: •We validate a simple and fast method of quantification of in vitro mineralization. •Fluorescently labeled agents can detect calcium deposits in the mineralized matrix of cell cultures. •Fluorescent signals of the probes correlated with Alizarin Red S staining. -- Abstract: Alizarin Red S staining is the standard method to indicate and quantify matrix mineralization during differentiation of osteoblast cultures. KS483 cells are multipotent mouse mesenchymal progenitor cells that can differentiate into chondrocytes, adipocytes and osteoblasts and are a well-characterized model for the study of bone formation. Matrix mineralization is the last step of differentiation of bone cells and is therefore a very important outcome measure in bone research. Fluorescently labelled calcium chelating agents, e.g. BoneTag and OsteoSense, are currently used for in vivo imaging of bone. The aim of the present study was to validate these probes for fast and simple detection and quantification of in vitro matrix mineralization by KS483 cells and thus enabling high-throughput screening experiments. KS483 cells were cultured under osteogenic conditions in the presence of compounds that either stimulate or inhibit osteoblast differentiation and thereby matrix mineralization. After 21 days of differentiation, fluorescence of stained cultures was quantified with a near-infrared imager and compared to Alizarin Red S quantification. Fluorescence of both probes closely correlated to Alizarin Red S staining in both inhibiting and stimulating conditions. In addition, both compounds displayed specificity for mineralized nodules. We therefore conclude that this method of quantification of bone mineralization using fluorescent compounds is a good alternative for the Alizarin Red S staining.

  14. Molecular interactions of a model bile salt and porcine bile with (1,3:1,4)-β-glucans and arabinoxylans probed by (13)C NMR and SAXS.

    PubMed

    Gunness, Purnima; Flanagan, Bernadine M; Mata, Jitendra P; Gilbert, Elliot P; Gidley, Michael J

    2016-04-15

    Two main classes of interaction between soluble dietary fibres (SDFs), such as (1,3:1,4)-β-D-glucan (βG) and arabinoxylan (AX) and bile salt (BS) or diluted porcine bile, were identified by (13)C NMR and small angle X-ray scattering (SAXS). Small chemical shift differences of BS NMR resonances were consistent with effective local concentration or dilution of BS micelles mostly by βG, suggesting dynamic interactions; whilst the reduced line widths/intensities observed were mostly caused by wheat AX and the highest molecular size and concentrations of βG. SAXS showed evidence of changes in βG but not AX in the presence of BS micelles, at >13 nm length scale consistent with molecular level interactions. Thus intermolecular interactions between SDF and BS depend on both SDF source and its molecular weight and may occur alone or in combination. PMID:26617003

  15. Length of hydrocarbon chain influences location of curcumin in liposomes: Curcumin as a molecular probe to study ethanol induced interdigitation of liposomes.

    PubMed

    El Khoury, Elsy; Patra, Digambara

    2016-05-01

    Using fluorescence quenching of curcumin in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes by brominated derivatives of fatty acids, the location of curcumin has been studied, which indicates length of hydrocarbon chain has an effect on the location of curcumin in liposomes. Change of fluorescence intensity of curcumin with temperature in the presence of liposomes helps to estimate the phase transition temperature of these liposomes, thus, influence of cholesterol on liposome properties has been studied using curcumin as a molecule probe. The cooperativity due to the interactions between the hydrocarbon chains during melting accelerates the phase transition of DPPC liposomes in the presence of high percentage of cholesterol whereas high percentage of cholesterol generates a rather rigid DMPC liposome over a wide range of temperatures. We used ethanol to induce interdigitation between the hydrophobic chains of the lipids and studied this effect using curcumin as fluorescence probe. As a result of interdigitation, curcumin fluorescence is quenched in liposomes. The compact arrangement of the acyl chains prevents curcumin from penetrating deep near the midplane. In the liquid crystalline phase ethanol introduces a kind of order to the more fluid liposome, and does not leave space for curcumin to be inserted away from water.

  16. Validation of a simple and fast method to quantify in vitro mineralization with fluorescent probes used in molecular imaging of bone.

    PubMed

    Moester, Martiene J C; Schoeman, Monique A E; Oudshoorn, Ineke B; van Beusekom, Mara M; Mol, Isabel M; Kaijzel, Eric L; Löwik, Clemens W G M; de Rooij, Karien E

    2014-01-01

    Alizarin Red S staining is the standard method to indicate and quantify matrix mineralization during differentiation of osteoblast cultures. KS483 cells are multipotent mouse mesenchymal progenitor cells that can differentiate into chondrocytes, adipocytes and osteoblasts and are a well-characterized model for the study of bone formation. Matrix mineralization is the last step of differentiation of bone cells and is therefore a very important outcome measure in bone research. Fluorescently labelled calcium chelating agents, e.g. BoneTag and OsteoSense, are currently used for in vivo imaging of bone. The aim of the present study was to validate these probes for fast and simple detection and quantification of in vitro matrix mineralization by KS483 cells and thus enabling high-throughput screening experiments. KS483 cells were cultured under osteogenic conditions in the presence of compounds that either stimulate or inhibit osteoblast differentiation and thereby matrix mineralization. After 21 days of differentiation, fluorescence of stained cultures was quantified with a near-infrared imager and compared to Alizarin Red S quantification. Fluorescence of both probes closely correlated to Alizarin Red S staining in both inhibiting and stimulating conditions. In addition, both compounds displayed specificity for mineralized nodules. We therefore conclude that this method of quantification of bone mineralization using fluorescent compounds is a good alternative for the Alizarin Red S staining.

  17. Multiplexed color-coded probe-based gene expression assessment for clinical molecular diagnostics in formalin-fixed paraffin-embedded human renal allograft tissue.

    PubMed

    Adam, Benjamin; Afzali, Bahman; Dominy, Katherine M; Chapman, Erin; Gill, Reeda; Hidalgo, Luis G; Roufosse, Candice; Sis, Banu; Mengel, Michael

    2016-03-01

    Histopathologic diagnoses in transplantation can be improved with molecular testing. Preferably, molecular diagnostics should fit into standard-of-care workflows for transplant biopsies, that is, formalin-fixed paraffin-embedded (FFPE) processing. The NanoString(®) gene expression platform has recently been shown to work with FFPE samples. We aimed to evaluate its methodological robustness and feasibility for gene expression studies in human FFPE renal allograft samples. A literature-derived antibody-mediated rejection (ABMR) 34-gene set, comprised of endothelial, NK cell, and inflammation transcripts, was analyzed in different retrospective biopsy cohorts and showed potential to molecularly discriminate ABMR cases, including FFPE samples. NanoString(®) results were reproducible across a range of RNA input quantities (r = 0.998), with different operators (r = 0.998), and between different reagent lots (r = 0.983). There was moderate correlation between NanoString(®) with FFPE tissue and quantitative reverse transcription polymerase chain reaction (qRT-PCR) with corresponding dedicated fresh-stabilized tissue (r = 0.487). Better overall correlation with histology was observed with NanoString(®) (r = 0.354) than with qRT-PCR (r = 0.146). Our results demonstrate the feasibility of multiplexed gene expression quantification from FFPE renal allograft tissue. This represents a method for prospective and retrospective validation of molecular diagnostics and its adoption in clinical transplantation pathology.

  18. Mapping Cannabinoid 1 Receptor Allosteric Site(s): Critical Molecular Determinant and Signaling Profile of GAT100, a Novel, Potent, and Irreversibly Binding Probe.

    PubMed

    Laprairie, Robert B; Kulkarni, Abhijit R; Kulkarni, Pushkar M; Hurst, Dow P; Lynch, Diane; Reggio, Patricia H; Janero, David R; Pertwee, Roger G; Stevenson, Lesley A; Kelly, Melanie E M; Denovan-Wright, Eileen M; Thakur, Ganesh A

    2016-06-15

    One of the most abundant G-protein coupled receptors (GPCRs) in brain, the cannabinoid 1 receptor (CB1R), is a tractable therapeutic target for treating diverse psychobehavioral and somatic disorders. Adverse on-target effects associated with small-molecule CB1R orthosteric agonists and inverse agonists/antagonists have plagued their translational potential. Allosteric CB1R modulators offer a potentially safer modality through which CB1R signaling may be directed for therapeutic benefit. Rational design of candidate, druglike CB1R allosteric modulators requires greater understanding of the architecture of the CB1R allosteric endodomain(s) and the capacity of CB1R allosteric ligands to tune the receptor's information output. We have recently reported the synthesis of a focused library of rationally designed, covalent analogues of Org27569 and PSNCBAM-1, two prototypic CB1R negative allosteric modulators (NAMs). Among the novel, pharmacologically active CB1R NAMs reported, the isothiocyanate GAT100 emerged as the lead by virtue of its exceptional potency in the [(35)S]GTPγS and β-arrestin signaling assays and its ability to label CB1R as a covalent allosteric probe with significantly reduced inverse agonism in the [(35)S]GTPγS assay as compared to Org27569. We report here a comprehensive functional profiling of GAT100 across an array of important downstream cell-signaling pathways and analysis of its potential orthosteric probe-dependence and signaling bias. The results demonstrate that GAT100 is a NAM of the orthosteric CB1R agonist CP55,940 and the endocannabinoids 2-arachidonoylglycerol and anandamide for β-arrestin1 recruitment, PLCβ3 and ERK1/2 phosphorylation, cAMP accumulation, and CB1R internalization in HEK293A cells overexpressing CB1R and in Neuro2a and STHdh(Q7/Q7) cells endogenously expressing CB1R. Distinctively, GAT100 was a more potent and efficacious CB1R NAM than Org27569 and PSNCBAM-1 in all signaling assays and did not exhibit the inverse

  19. Stable Isotope Probing of an Algal Bloom To Identify Uncultivated Members of the Rhodobacteraceae Associated with Low-Molecular-Weight Polycyclic Aromatic Hydrocarbon Degradation▿

    PubMed Central

    Gutierrez, Tony; Singleton, David R.; Aitken, Michael D.; Semple, Kirk T.

    2011-01-01

    Polycyclic aromatic hydrocarbon (PAH)-degrading bacteria associated with an algal bloom in Tampa Bay, FL, were investigated by stable isotope probing (SIP) with uniformly labeled [13C]naphthalene. The dominant sequences in clone libraries constructed from 13C-enriched bacterial DNA (from naphthalene enrichments) were identified as uncharacterized members of the family Rhodobacteraceae. Quantitative PCR primers targeting the 16S rRNA gene of these uncultivated organisms were used to determine their abundance in incubations amended with unlabeled naphthalene and phenanthrene, both of which showed substantial increases in gene copy numbers during the experiments. As demonstrated by this work, the application of uniformly 13C-labeled PAHs in SIP experiments can successfully be used to identify novel PAH-degrading bacteria in marine waters. PMID:21926219

  20. Molecular characterization of Yersinia enterocolitica by pulsed-field gel electrophoresis and hybridization of DNA fragments to ail and pYV probes.

    PubMed Central

    Buchrieser, C; Weagant, S D; Kaspar, C W

    1994-01-01

    Sixty strains of Yersinia enterocolitica from five serogroups (O:3; O:9; O:8; O:5; and O:5,27) and eight non-Y. enterocolitica strains, recovered from diverse sources (humans, animals, food, and the environment) in Europe, Argentina, and the United States, were examined by the pulsed-field gel electrophoresis (PFGE) technique of contour clamped homogeneous electric field electrophoresis (CHEF) by using NotI and XbaI as restriction enzymes. NotI and XbaI generated 36 and 33 restriction endonuclease digestion profiles (REDP), respectively. By combining the results of both enzymes, 42 unique genomic groups were differentiated. DNA fragments were transferred to nylon membranes and hybridized with digoxigenin-labelled oligonucleotide probes to the ail gene and virulence plasmid to determine hybridization patterns and the potential virulence of the strains. The strains were tested for the presence of the plasmid by PFGE-CHEF and phenotypic characteristics encoded for by the virulence plasmid. Thirty of the 60 Y. enterocolitica strains tested harbored the virulence plasmid. The specificity of the ail and pYV probes was 100% when tested with 68 Yersinia strains and 19 different non-Yersinia strains. Sixteen selected Y. enterocolitica strains were tested for their virulence by lethality in iron- and desferrioxamine-sensitized mice. No correlation between REDP and the virulence of the strains was observed. The observed REDP and the hybridization patterns were very homogeneous within a serogroup and independent of the source of isolation. In addition, PFGE-CHEF was shown to be valuable in identifying and confirming serogroups. Principal component analysis of Dice similarity indices from REDP was an excellent tool for determining genetic relatedness among strains. Images PMID:7811077

  1. Array of Synthetic Oligonucleotides to Generate Unique Multi-Target Artificial Positive Controls and Molecular Probe-Based Discrimination of Liposcelis Species

    PubMed Central

    Arif, Mohammad; Opit, George; Mendoza-Yerbafría, Abigail; Dobhal, Shefali; Li, Zhihong; Kučerová, Zuzana; Ochoa-Corona, Francisco M.

    2015-01-01

    Several species of the genus Liposcelis are common insect pests that cause serious qualitative and quantitative losses to various stored grains and processed grain products. They also can contaminate foods, transmit pathogenic microorganisms and cause allergies in humans. The common occurrence of multi-species infestations and the fact that it is difficult to identify and discriminate Liposcelis spp. make accurate, rapid detection and discriminatory tools absolutely necessary for confirmation of their identity. In this study, PCR primers and probes specific to different Liposcelis spp. were designed based on nucleotide sequences of the cytochrome oxidase 1 (CO1) gene. Primer sets ObsCo13F/13R, PeaCo15F/14R, BosCO7F/7R, BruCo5F/5R, and DecCo11F/11R were used to specifically detect Liposcelis obscura Broadhead, Liposcelis pearmani Lienhard, Liposcelis bostrychophila Badonnel, Liposcelis brunnea Motschulsky and Liposcelis decolor (Pearman) in multiplex endpoint PCRs, which amplified products of 438-, 351-, 191-, 140-, and 87-bp, respectively. In multiplex TaqMan qPCR assays, orange, yellow, red, crimson and green channels corresponding to reporter dyes 6-ROXN, HEX, Cy5, Quasar705 and 6-FAM specifically detected L. obscura, L. brunnea, L. bostrychophila, L. pearmani and L. decolor, respectively. All developed primer and probe sets allowed specific amplification of corresponding targeted Liposcelis species. The development of multiplex endpoint PCR and multiplex TaqMan qPCR will greatly facilitate psocid identification and their management. The use of APCs will streamline and standardize PCR assays. APC will also provide the opportunity to have all positive controls in a single tube, which reduces maintenance cost and labor, but increases the accuracy and reliability of the assays. These novel methods from our study will have applications in pest management, biosecurity, quarantine, food safety, and routine diagnostics. PMID:26086728

  2. HOPS + MALT90 + Hi-GAL: Probing star formation on a Galactic scale through mm molecular line and far-IR continuum Galactic plane surveys

    NASA Astrophysics Data System (ADS)

    Longmore, Steven N.; Rathborne, Jill; Bastian, Nate; Alves, Joao; Ascenso, Joana; Bally, John; Testi, Leonardo; Longmore, Andy; Battersby, Cara; Bressert, Eli; Purcell, Cormac; Walsh, Andrew; Jackson, James; Foster, Jonathan; Molinari, Sergio; Meingast, Stefan; Amorim, A.; Lima, J.; Marques, R.; Moitinho, A.; Pinhao, J.; Rebordao, J.; Santos, F. D.

    2012-03-01

    With the HOPS and MALT90 Galactic plane surveys we are mapping a significant fraction of the dense molecular gas in the Galaxy in over 20 dense-gas-tracing transitions (e.g. from H2O, NH3, HC3N, HC5N, N2H+, HCN, HNC, HCO+, CH3CN, SiO, C2H, ...). Combining this with the far-IR continuum emission from Hi-GAL we can derive the physical/chemical/kinematic properties and evolutionary state of much of the molecular gas in the Galaxy destined to form stars. I will present results from three science projects based on this combined dataset, namely: i) looking for variations in the star formation rate across the Galaxy as a function of environment, in particular, comparing the CMZ with the rest of the Galactic disk -- we find the rate of star formation per unit mass of dense gas in the CMZ may be an order of magnitude lower than that in the disk; ii) seeing if Galactic dense molecular clouds follow the empirical relations observed in extragalactic systems (e.g. the Kennicutt-Schmidt and Gao & Solomon relations) and what this implies for interpretating the extragalactic relations; iii) searching for molecular cloud progenitors of the most extreme (massive and dense) stellar clusters. I will present one cloud we have studied as part of project iii) which lies close to the Galactic center and which is clearly extreme compared to the rest of the Galactic molecular cloud population. With a mass of 10^5 Msun, a radius of only ~3pc and almost no signs of star formation it appears to be the progenitor of an Arches-like stellar cluster. As such, we speculate this molecular cloud may be a local-universe-analogue of the initial conditions of a super star cluster or potentially even a small globular cluster. From our Galactic plane survey data this object appears to be unique in the Galaxy, making it extremely important for testing massive cluster formation models. We have been awarded 6 hours of ALMA Cycle 0 observing time to study this object in detail and I hope to show preliminary

  3. One-step, multiplex, real-time PCR assay with molecular beacon probes for simultaneous detection, differentiation, and quantification of human T-cell leukemia virus types 1, 2, and 3.

    PubMed

    Besson, Guillaume; Kazanji, Mirdad

    2009-04-01

    A single-tube, multiplex, real-time PCR assay with molecular beacons was established in which various probes were used for the simultaneous detection, differentiation, and quantification of human T-cell leukemia virus types 1, 2, and 3 (HTLV-1, HTLV-2, and HTLV-3, respectively) and of simian T-cell leukemia virus types 1 and 3 (STLV-1 and STLV-3, respectively). The quantitative amplification of the standards with MT4 (HTLV-1) and C19 (HTLV-2) cell lines and a molecular clone of HTLV-3 was linear, with the simplex and multiplex methods having similar efficiencies. A maximum difference of 0.9 (mean, 0.4; range, 0.0 to 0.9) was found between threshold cycle values in single and multiplex reactions. The efficiency with each probe in the multiplex reaction was close to 100%, indicating strong linear amplification. The albumin gene was used to standardize the copy number. Comparable results for the detection and quantification of HTLV-1 were obtained with our new methods and with other real-time PCR methods described previously. With our new multiplex assay, however, we were able to detect and quantify HTLV-2 and -3 and STLV-1 and -3 in clinical specimens, with an excellent dynamic range of 10(6) to 10(0) copies per assay, which the other assays could not do. Thus, it will be possible to determine a wide range of HTLV types in both standard and clinical samples, with a detection of 1 to 10 HTLV copies in samples containing at least 100 cells. Furthermore, our system can provide evidence for multiple infections with the three HTLV types, with separate proviral load results. Our new method also could be used for epidemiological studies in Africa and in countries where HTLVs and STLVs are endemic.

  4. Visualizing genomes with Oligopaint FISH probes

    PubMed Central

    Beliveau, Brian J.; Apostolopoulos, Nicholas; Wu, Chao-ting

    2014-01-01

    Oligopaint probes are fluorescently-labeled, single-stranded DNA oligonucleotides that can be used to visualize genomic regions ranging in size from tens of kilobases to many megabases. This unit details how Oligopaint probes can be synthesized using basic molecular biological techniques as well as provides protocols for FISH, 3D-FISH, and sample preparation. PMID:24510436

  5. Probing Cellular and Molecular Mechanisms of Cigarette Smoke-Induced Immune Response in the Progression of Chronic Obstructive Pulmonary Disease Using Multiscale Network Modeling

    PubMed Central

    Pan, Zhichao; Yu, Haishan; Liao, Jie-Lou

    2016-01-01

    Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disorder characterized by progressive destruction of lung tissues and airway obstruction. COPD is currently the third leading cause of death worldwide and there is no curative treatment available so far. Cigarette smoke (CS) is the major risk factor for COPD. Yet, only a relatively small percentage of smokers develop the disease, showing that disease susceptibility varies significantly among smokers. As smoking cessation can prevent the disease in some smokers, quitting smoking cannot halt the progression of COPD in others. Despite extensive research efforts, cellular and molecular mechanisms of COPD remain elusive. In particular, the disease susceptibility and smoking cessation effects are poorly understood. To address these issues in this work, we develop a multiscale network model that consists of nodes, which represent molecular mediators, immune cells and lung tissues, and edges describing the interactions between the nodes. Our model study identifies several positive feedback loops and network elements playing a determinant role in the CS-induced immune response and COPD progression. The results are in agreement with clinic and laboratory measurements, offering novel insight into the cellular and molecular mechanisms of COPD. The study in this work also provides a rationale for targeted therapy and personalized medicine for the disease in future. PMID:27669518

  6. Molecular ordering of PAH/MA-co-DR13 azopolymer layer-by-layer films probed by second-harmonic generation.

    PubMed

    Silva, Heurison S; Lopes, Fábio J S; Miranda, Paulo B

    2016-09-14

    Molecular orientation within azopolymer thin films is important for their nonlinear optical properties and photonic applications. We have used optical second-harmonic generation (SHG) to study the molecular orientation of Layer-by-Layer (LbL) films of a cationic polyelectrolyte (poly(allylamine hydrochloride)) and an anionic polyelectrolyte containing azochromophore side groups (MA-co-DR13) on a glass substrate. The SHG measurements indicate that there is a preferential orientation of the azochromophores in the film, leading to a significant optical nonlinearity. However, both the signal strength and its anisotropy are not homogeneous throughout the sample, indicating the presence of large orientational domains. This is corroborated with Brewster angle microscopy. The average SHG signal does not increase with film thickness, in contrast to some reports in the literature, indicating an independent orientational order for successive bilayers. Analyzing the SHG signal as a function of the input and output polarizations, a few parameters of the azochromophore orientational distribution can be deduced. Fitting the SHG signal to a simple model distribution, we have concluded that the chromophores have an angular distribution with a slight in-plane anisotropy and a mean polar angle ranging from 45° to 80° with respect to substrate normal direction, with a relatively large width of about 25°. These results show that SHG is a powerful technique for a detailed investigation of the molecular orientation in azopolymer LbL films, allowing a deeper understanding of their self-assembling mechanism and nonlinear optical properties. The inhomogeneity and anisotropy of these films may have important consequences for their applications in nonlinear optical devices. PMID:27634274

  7. Molecular ordering of PAH/MA-co-DR13 azopolymer layer-by-layer films probed by second-harmonic generation

    NASA Astrophysics Data System (ADS)

    Silva, Heurison S.; Lopes, Fábio J. S.; Miranda, Paulo B.

    2016-09-01

    Molecular orientation within azopolymer thin films is important for their nonlinear optical properties and photonic applications. We have used optical second-harmonic generation (SHG) to study the molecular orientation of Layer-by-Layer (LbL) films of a cationic polyelectrolyte (poly(allylamine hydrochloride)) and an anionic polyelectrolyte containing azochromophore side groups (MA-co-DR13) on a glass substrate. The SHG measurements indicate that there is a preferential orientation of the azochromophores in the film, leading to a significant optical nonlinearity. However, both the signal strength and its anisotropy are not homogeneous throughout the sample, indicating the presence of large orientational domains. This is corroborated with Brewster angle microscopy. The average SHG signal does not increase with film thickness, in contrast to some reports in the literature, indicating an independent orientational order for successive bilayers. Analyzing the SHG signal as a function of the input and output polarizations, a few parameters of the azochromophore orientational distribution can be deduced. Fitting the SHG signal to a simple model distribution, we have concluded that the chromophores have an angular distribution with a slight in-plane anisotropy and a mean polar angle ranging from 45° to 80° with respect to substrate normal direction, with a relatively large width of about 25°. These results show that SHG is a powerful technique for a detailed investigation of the molecular orientation in azopolymer LbL films, allowing a deeper understanding of their self-assembling mechanism and nonlinear optical properties. The inhomogeneity and anisotropy of these films may have important consequences for their applications in nonlinear optical devices.

  8. Molecular cytogenetic analysis of the Appenine endemic cyprinid fish Squalius lucumonis and three other Italian leuciscines using chromosome banding and FISH with rDNA probes.

    PubMed

    Rossi, Anna Rita; Milana, Valentina; Hett, Anne Kathrin; Tancioni, Lorenzo

    2012-12-01

    Karyotype and other chromosomal characteristics of the Appenine endemic cyprinid fish, Toscana stream chub Squalius lucumonis, were analysed using conventional banding and FISH with 45S and 5S rDNA probes. The diploid chromosome number (2n = 50) and karyotype characteristics including pericentromeric heterochromatic blocks and GC-rich CMA(3)-positive sites corresponding to both positive Ag-NORs and 45S rDNA loci on the short arms of a single medium-sized submetacentric chromosome pair were consistent with those found in most European leuciscine cyprinids. On other hand, 5S rDNA FISH in the Toscana stream chub and three other Italian leuciscines, S. squalus, Rutilus rubilio and Telestes muticellus, revealed a species-specific hybridization pattern, i.e. signals on four (S. lucumonis), three (S. squalus and R. rubilio) and two (T. muticellus) chromosome pairs. Whereas all the species shared the 5S rDNA loci on the largest subtelocentric chromosome pair, a "leuciscine" cytotaxonomic marker, S. lucumonis showed both classes of rDNA loci tandem aligned on the short arms of chromosome pair No. 12. The present findings suggest that the observed high variability of 5S rDNA loci provides a powerful tool for investigation of karyotype differentiation in karyologically conservative leuciscine fishes.

  9. Molecular cytogenetic analysis of the Appenine endemic cyprinid fish Squalius lucumonis and three other Italian leuciscines using chromosome banding and FISH with rDNA probes.

    PubMed

    Rossi, Anna Rita; Milana, Valentina; Hett, Anne Kathrin; Tancioni, Lorenzo

    2012-12-01

    Karyotype and other chromosomal characteristics of the Appenine endemic cyprinid fish, Toscana stream chub Squalius lucumonis, were analysed using conventional banding and FISH with 45S and 5S rDNA probes. The diploid chromosome number (2n = 50) and karyotype characteristics including pericentromeric heterochromatic blocks and GC-rich CMA(3)-positive sites corresponding to both positive Ag-NORs and 45S rDNA loci on the short arms of a single medium-sized submetacentric chromosome pair were consistent with those found in most European leuciscine cyprinids. On other hand, 5S rDNA FISH in the Toscana stream chub and three other Italian leuciscines, S. squalus, Rutilus rubilio and Telestes muticellus, revealed a species-specific hybridization pattern, i.e. signals on four (S. lucumonis), three (S. squalus and R. rubilio) and two (T. muticellus) chromosome pairs. Whereas all the species shared the 5S rDNA loci on the largest subtelocentric chromosome pair, a "leuciscine" cytotaxonomic marker, S. lucumonis showed both classes of rDNA loci tandem aligned on the short arms of chromosome pair No. 12. The present findings suggest that the observed high variability of 5S rDNA loci provides a powerful tool for investigation of karyotype differentiation in karyologically conservative leuciscine fishes. PMID:23238894

  10. Manipulating the charge state of Au clusters on rutile TiO2(110) single crystal surfaces through molecular reactions probed by infrared spectroscopy.

    PubMed

    Cao, Yunjun; Hu, Shujun; Yu, Min; Wang, Tingting; Huang, Shiming; Yan, Shishen; Xu, Mingchun

    2016-07-14

    The charge state of Au clusters deposited on rutile TiO2(110) single crystal surfaces was studied by UHV-FTIRS using CO as a probe. The as-deposited Au clusters on oxidized TiO2(110) surfaces are electrically neutral and are identified by the 2105-2112 cm(-1) vibrational frequency of adsorbed CO depending on Au coverage. Annealing Au/TiO2(110) in a moderate O2 atmosphere at 400 K blue shifts the CO vibrational frequency by only 2-3 cm(-1) both on bare TiO2(110) surfaces and on Au clusters. However, NO exposure blue shifts the CO vibrational frequency by 16-26 cm(-1) for CO adsorbed on Au atoms near the interface and by 3-4 cm(-1) for CO adsorbed on top of Au clusters. As the acceptors of the intense charge transfer from Au, the Oa atoms generated through (NO)2→ N2O + Oa reactions on the small fraction of the bare TiO2(110) surface reside around the Au/TiO2(110) interface perimeter, causing the neutral Au(0) to be cationic Au(δ+) states. This is a new approach to manipulate the charge state of Au clusters on oxide surfaces, which may be helpful in regulating the catalytic redox reactions on oxide supported metal systems.

  11. Novel molecular beacon probe-based real-time RT-PCR assay for diagnosis of Crimean-Congo hemorrhagic fever encountered in India.

    PubMed

    Kamboj, Aman; Pateriya, Atul Kumar; Mishra, Anamika; Ranaware, Pradip; Kulkarni, Diwakar D; Raut, Ashwin Ashok

    2014-01-01

    Crimean-Congo hemorrhagic fever (CCHF) is an emerging zoonotic disease in India and requires immediate detection of infection both for preventing further transmission and for controlling the infection. The present study describes development, optimization, and evaluation of a novel molecular beacon-based real-time RT-PCR assay for rapid, sensitive, and specific diagnosis of Crimean-Congo hemorrhagic fever virus (CCHFV). The developed assay was found to be a better alternative to the reported TaqMan assay for routine diagnosis of CCHF.

  12. Probing the binding mechanisms of α-tocopherol to trypsin and pepsin using isothermal titration calorimetry, spectroscopic, and molecular modeling methods.

    PubMed

    Li, Xiangrong; Ni, Tianjun

    2016-06-01

    α-Tocopherol is a required nutrient for a variety of biological functions. In this study, the binding of α-tocopherol to trypsin and pepsin was investigated using isothermal titration calorimetry (ITC), steady-state and time-resolved fluorescence measurements, circular dichroism (CD) spectroscopy, and molecular modeling methods. Thermodynamic investigations reveal that α-tocopherol binds to trypsin/pepsin is synergistically driven by enthalpy and entropy. The fluorescence experimental results indicate that α-tocopherol can quench the fluorescence of trypsin/pepsin through a static quenching mechanism. The binding ability of α-tocopherol with trypsin/pepsin is in the intermediate range, and one molecule of α-tocopherol combines with one molecule of trypsin/pepsin. As shown by circular dichroism (CD) spectroscopy, α-tocopherol may induce conformational changes of trypsin/pepsin. Molecular modeling displays the specific binding site and gives information about binding forces and α-tocopherol-tryptophan (Trp)/tyrosine (Tyr) distances. In addition, the inhibition rate of α-tocopherol on trypsin and pepsin was studied. The study provides a basic data set for clarifying the binding mechanisms of α-tocopherol with trypsin and pepsin and is helpful for understanding its biological activity in vivo.

  13. The Release of Trapped Gases from Amorphous Solid Water Films: I. “Top-Down” Crystallization-Induced Crack Propagation Probed using the Molecular Volcano

    SciTech Connect

    May, Robert A.; Smith, R. Scott; Kay, Bruce D.

    2013-03-14

    In this (Paper I) and the companion paper (Paper II) we investigate the mechanisms for the release of trapped gases from underneath of amorphous solid water (ASW) films. In prior work, we reported the episodic release of trapped gases in concert with the crystallization ASW, a phenomenon that we termed the "molecular volcano". The observed abrupt desorption is due to the formation of cracks that span the film to form a connected pathway for release. In this paper we utilize the "molecular volcano" desorption peak to characterize the formation of crystallization-induced cracks. We find that the crack length and distribution are independent of the trapped gas (Ar, Kr, Xe, CH4, N2, O2 or CO). Selective placement of the inert gas layer is used to show that cracks form near the top of the film and propagate downward into the film. Isothermal experiments reveal that, after some induction time, cracks propagate linearly in time with an Arrhenius dependent velocity corresponding to an activation energy of 54 kJ/mol. This value is consistent with the crystallization growth rate reported by others and establishes a direct connection between crystallization growth rate and the crack propagation rate. A two-step model in which nucleation and crystallization occurs in an induction zone near the top of the film followed by the propagation of a crystallization/crack front into the film is in good agreement with the temperature programmed desorption results.

  14. The release of trapped gases from amorphous solid water films. I. "Top-down" crystallization-induced crack propagation probed using the molecular volcano.

    PubMed

    May, R Alan; Smith, R Scott; Kay, Bruce D

    2013-03-14

    In this (Paper I) and the companion paper (Paper II; R. May, R. Smith, and B. Kay, J. Chem. Phys. 138, 104502 (2013)), we investigate the mechanisms for the release of trapped gases from underneath amorphous solid water (ASW) films. In prior work, we reported the episodic release of trapped gases in concert with the crystallization of ASW, a phenomenon that we termed the "molecular volcano." The observed abrupt desorption is due to the formation of cracks that span the film to form a connected pathway for release. In this paper, we utilize the "molecular volcano" desorption peak to characterize the formation of crystallization-induced cracks. We find that the crack length distribution is independent of the trapped gas (Ar, Kr, Xe, CH4, N2, O2, or CO). Selective placement of the inert gas layer is used to show that cracks form near the top of the film and propagate downward into the film. Isothermal experiments reveal that, after some induction time, cracks propagate linearly in time with an Arrhenius dependent velocity corresponding to an activation energy of 54 kJ∕mol. This value is consistent with the crystallization growth rates reported by others and establishes a direct connection between crystallization growth rate and the crack propagation rate. A two-step model in which nucleation and crystallization occurs in an induction zone near the top of the film followed by the propagation of a crystallization∕crack front into the film is in good agreement with the temperature programmed desorption results.

  15. Probing Origin of Binding Difference of inhibitors to MDM2 and MDMX by Polarizable Molecular Dynamics Simulation and QM/MM-GBSA Calculation

    NASA Astrophysics Data System (ADS)

    Chen, Jianzhong; Wang, Jinan; Zhang, Qinggang; Chen, Kaixian; Zhu, Weiliang

    2015-11-01

    Binding abilities of current inhibitors to MDMX are weaker than to MDM2. Polarizable molecular dynamics simulations (MD) followed by Quantum mechanics/molecular mechanics generalized Born surface area (QM//MM-GBSA) calculations were performed to investigate the binding difference of inhibitors to MDM2 and MDMX. The predicted binding free energies not only agree well with the experimental results, but also show that the decrease in van der Walls interactions of inhibitors with MDMX relative to MDM2 is a main factor of weaker bindings of inhibitors to MDMX. The analyses of dihedral angles based on MD trajectories suggest that the closed conformation formed by the residues M53 and Y99 in MDMX leads to a potential steric clash with inhibitors and prevents inhibitors from arriving in the deep of MDMX binding cleft, which reduces the van der Waals contacts of inhibitors with M53, V92, P95 and L98. The calculated results using the residue-based free energy decomposition method further prove that the interaction strength of inhibitors with M53, V92, P95 and L98 from MDMX are obviously reduced compared to MDM2. We expect that this study can provide significant theoretical guidance for designs of potent dual inhibitors to block the p53-MDM2/MDMX interactions.

  16. Putative membrane lytic sites of P-type and S-type cardiotoxins from snake venoms as probed by all-atom molecular dynamics simulations.

    PubMed

    Gorai, Biswajit; Karthikeyan, Muthusamy; Sivaraman, Thirunavukkarasu

    2016-10-01

    Cardiotoxins (CTXs) belonging to the three-finger toxin superfamily of snake venoms are one of principal toxic components and the protein toxins exhibit membrane lytic activities when the venoms are injected into victims. In the present study, complex formations between CTX VI (a P-type CTX from Naja atra) and CTX1 (an S-type CTX from Naja naja) on zwitterionic POPC bilayers (a major lipid component of cell membranes) have been studied in near physiological conditions for a total dynamic time scale of 1.35 μs using all-atom molecular dynamics (MD) simulations. Comprehensive analyses of the MD data revealed that residues such as Leu1, Lys2, Tyr11, Lys31, Asp57 and Arg58 of CTX VI, and Ala16, Lys30 and Arg58 of CTX1 were crucial for establishing interactions with the POPC bilayer. Moreover, loop I, along with globular head and loop II of CTX VI, and loop II of CTX1 were found to be the structural regions chiefly governing complex formation of the respective proteins with POPC. Rationalizations for the differential binding modes of CTXs and implications of the findings for designing small molecular inhibitors to the toxins are also discussed. Graphical Abstract Binding modes of a P-type CTX and an S-type CTX towards the POPC bilayer. PMID:27628673

  17. Probing Origin of Binding Difference of inhibitors to MDM2 and MDMX by Polarizable Molecular Dynamics Simulation and QM/MM-GBSA Calculation

    PubMed Central

    Chen, Jianzhong; Wang, Jinan; Zhang, Qinggang; Chen, Kaixian; Zhu, Weiliang

    2015-01-01

    Binding abilities of current inhibitors to MDMX are weaker than to MDM2. Polarizable molecular dynamics simulations (MD) followed by Quantum mechanics/molecular mechanics generalized Born surface area (QM//MM-GBSA) calculations were performed to investigate the binding difference of inhibitors to MDM2 and MDMX. The predicted binding free energies not only agree well with the experimental results, but also show that the decrease in van der Walls interactions of inhibitors with MDMX relative to MDM2 is a main factor of weaker bindings of inhibitors to MDMX. The analyses of dihedral angles based on MD trajectories suggest that the closed conformation formed by the residues M53 and Y99 in MDMX leads to a potential steric clash with inhibitors and prevents inhibitors from arriving in the deep of MDMX binding cleft, which reduces the van der Waals contacts of inhibitors with M53, V92, P95 and L98. The calculated results using the residue-based free energy decomposition method further prove that the interaction strength of inhibitors with M53, V92, P95 and L98 from MDMX are obviously reduced compared to MDM2. We expect that this study can provide significant theoretical guidance for designs of potent dual inhibitors to block the p53-MDM2/MDMX interactions. PMID:26616018

  18. Structure-Function Analysis of Mammalian CYP2B Enzymes Using 7-Substituted Coumarin Derivatives as Probes: Utility of Crystal Structures and Molecular Modeling in Understanding Xenobiotic Metabolism.

    PubMed

    Shah, Manish B; Liu, Jingbao; Huo, Lu; Zhang, Qinghai; Dearing, M Denise; Wilderman, P Ross; Szklarz, Grazyna D; Stout, C David; Halpert, James R

    2016-04-01

    Crystal structures of CYP2B35 and CYP2B37 from the desert woodrat were solved in complex with 4-(4-chlorophenyl)imidazole (4-CPI). The closed conformation of CYP2B35 contained two molecules of 4-CPI within the active site, whereas the CYP2B37 structure demonstrated an open conformation with three 4-CPI molecules, one within the active site and the other two in the substrate access channel. To probe structure-function relationships of CYP2B35, CYP2B37, and the related CYP2B36, we tested the O-dealkylation of three series of related substrates-namely, 7-alkoxycoumarins, 7-alkoxy-4-(trifluoromethyl)coumarins, and 7-alkoxy-4-methylcoumarins-with a C1-C7 side chain. CYP2B35 showed the highest catalytic efficiency (kcat/KM) with 7-heptoxycoumarin as a substrate, followed by 7-hexoxycoumarin. In contrast, CYP2B37 showed the highest catalytic efficiency with 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC), followed by 7-methoxy-4-(trifluoromethyl)coumarin (7-MFC). CYP2B35 had no dealkylation activity with 7-MFC or 7-EFC. Furthermore, the new CYP2B-4-CPI-bound structures were used as templates for docking the 7-substituted coumarin derivatives, which revealed orientations consistent with the functional studies. In addition, the observation of multiple -Cl and -NH-π interactions of 4-CPI with the aromatic side chains in the CYP2B35 and CYP2B37 structures provides insight into the influence of such functional groups on CYP2B ligand binding affinity and specificity. To conclude, structural, computational, and functional analysis revealed striking differences between the active sites of CYP2B35 and CYP2B37 that will aid in the elucidation of new structure-activity relationships.

  19. Infrared absorption by molecular gases as a probe of nanoporous silica xerogel and molecule-surface collisions: Low-pressure results

    NASA Astrophysics Data System (ADS)

    Vander Auwera, J.; Ngo, N. H.; El Hamzaoui, H.; Capoen, B.; Bouazaoui, M.; Ausset, P.; Boulet, C.; Hartmann, J.-M.

    2013-10-01

    Transmission spectra of gases confined (but not adsorbed) within the pores of a 1.4-cm-thick silica xerogel sample have been recorded between 2.5 and 5 μm using a high-resolution Fourier transform spectrometer. This was done for pure CO, CO2, N2O, H2O, and CH4 at room temperature and pressures of a few hectopascals. Least-squares fits of measured absorption lines provide the optical-path lengths within the confined (LC) and free (LF) gas inside the absorption cell and the half width at half maximum ΓC of the lines of the confined gases. The values of LC and LF retrieved using numerous transitions of all studied species are very consistent. Furthermore, LC is in satisfactory agreement with values obtained from independent measurements, thus showing that reliable information on the open porosity volume can be retrieved from an optical experiment. The values of ΓC, here resulting from collisions of the molecules with the inner surfaces of the xerogel pores, are practically independent of the line for each gas and inversely proportional to the square root of the probed-molecule molar mass. This is a strong indication that, for the studied transitions, a single collision of a molecule with a pore surface is sufficient to change its rotational state. A previously proposed simple model, used for the prediction of the line shape, leads to satisfactory agreement with the observations. It also enables a determination of the average pore size, bringing information complementary to that obtained from nitrogen adsorption porosimetry.

  20. LAT1 targeted delivery of methionine based imaging probe derived from M(III) metal ions for early diagnosis of proliferating tumours using molecular imaging modalities.

    PubMed

    Hazari, Puja Panwar; Prakash, Surbhi; Meena, Virendra K; Jaswal, Ambika; Khurana, Harleen; Mishra, Surabhi Kirti; Bhonsle, Hemanth Kumar; Singh, Lokendra; Mishra, Anil K

    2015-01-01

    We investigated the potential of DTPA-bis(Methionine), a target specific amino acid based probe for detection of L-type amino acid transporters (LAT1) known to over express in proliferating tumours using multimodality imaging. The ligand, DTPA-bis(Met) was readily converted to lanthanide complexes and was found capable of targeting cancer cells using multimodality imaging. DTPA-bis(Met) complexes were synthesized and characterized by mass spectroscopy. MR longitudinal relaxivity, r₁ = 4.067 ± 0.31 mM⁻¹s⁻¹ and transverse relaxivity, r₂ = 8.61 ± 0.07 mM⁻¹s⁻¹ of Gd(III)-DTPA-bis(Met) were observed at pH 7.4 at 7 T. Bright, localized fluorescence of Eu(III)-DTPA-bis(Met) was observed with standard microscopy and displacement studies indicated ligand functionality. K(D) value determined for Eu(III)-DTPA-bis(Met) on U-87 MG cells was found to be 17.3 pM and showed appreciable fluorescence within the cells. Radio HPLC showed a radiochemical purity more than 95% (specific activity = 400-500 MBq/μmol, labelling efficiency 78 %) for ⁶⁸Ga(III)-DTPA-bis(Met). Pre-treatment of xenografted U-87 MG athymic mice with ⁶⁸Ga(III)-DTPA-bis(Met) following unlabelled L-methionine administration reduced tumour uptake by 10-folds in Micro PET. These data support the specific binding of ⁶⁸Ga(III)-DTPA-bis(Met) to the LAT1 transporter. To summarize, this agent possesses high stability in biological environment and exhibits effective interaction with its LAT1 transporters giving high accumulation in tumour area, excellent tumour/non-tumour ratio and low non-specific retention in vivo. PMID:25329672

  1. LAT1 targeted delivery of methionine based imaging probe derived from M(III) metal ions for early diagnosis of proliferating tumours using molecular imaging modalities.

    PubMed

    Hazari, Puja Panwar; Prakash, Surbhi; Meena, Virendra K; Jaswal, Ambika; Khurana, Harleen; Mishra, Surabhi Kirti; Bhonsle, Hemanth Kumar; Singh, Lokendra; Mishra, Anil K

    2015-01-01

    We investigated the potential of DTPA-bis(Methionine), a target specific amino acid based probe for detection of L-type amino acid transporters (LAT1) known to over express in proliferating tumours using multimodality imaging. The ligand, DTPA-bis(Met) was readily converted to lanthanide complexes and was found capable of targeting cancer cells using multimodality imaging. DTPA-bis(Met) complexes were synthesized and characterized by mass spectroscopy. MR longitudinal relaxivity, r₁ = 4.067 ± 0.31 mM⁻¹s⁻¹ and transverse relaxivity, r₂ = 8.61 ± 0.07 mM⁻¹s⁻¹ of Gd(III)-DTPA-bis(Met) were observed at pH 7.4 at 7 T. Bright, localized fluorescence of Eu(III)-DTPA-bis(Met) was observed with standard microscopy and displacement studies indicated ligand functionality. K(D) value determined for Eu(III)-DTPA-bis(Met) on U-87 MG cells was found to be 17.3 pM and showed appreciable fluorescence within the cells. Radio HPLC showed a radiochemical purity more than 95% (specific activity = 400-500 MBq/μmol, labelling efficiency 78 %) for ⁶⁸Ga(III)-DTPA-bis(Met). Pre-treatment of xenografted U-87 MG athymic mice with ⁶⁸Ga(III)-DTPA-bis(Met) following unlabelled L-methionine administration reduced tumour uptake by 10-folds in Micro PET. These data support the specific binding of ⁶⁸Ga(III)-DTPA-bis(Met) to the LAT1 transporter. To summarize, this agent possesses high stability in biological environment and exhibits effective interaction with its LAT1 transporters giving high accumulation in tumour area, excellent tumour/non-tumour ratio and low non-specific retention in vivo.

  2. Probing the binding interaction of human serum albumin with three bioactive constituents of Eriobotrta japonica leaves: Spectroscopic and molecular modeling approaches.

    PubMed

    Wang, Qing; Sun, Qiaomei; Ma, Xiangling; Rao, Zaisheng; Li, Hui

    2015-07-01

    Corosolic acid (CRA), maslinic acid (MA), and tormentic acid (TA) are three kind of bioactive constituents of Eriobotrta japonica leaves. In this study, plasma protein binding model prediction suggested that the binding ability to HSA was CRA>MA>TA. Furthermore, fluorescence spectroscopy confirmed this prediction. The results from emission and time resolved fluorescence studies revealed that the emission quenching of HSA with CRA, MA, and TA were all initiated by static quenching mechanism. From molecular docking results and site marker competitive experimental results it was possible to make good estimates about CRA, MA, and TA mainly bound to subdomain IIA of HSA. 3D fluorescence, FT-IR and CD spectra indicated that the local conformation of HSA molecules was affected by the presence of CRA, MA, and TA, but at different extents.

  3. Probing the wild-type HRas activation mechanism using steered molecular dynamics, understanding the energy barrier and role of water in the activation.

    PubMed

    Sharma, Neeru; Sonavane, Uddhavesh; Joshi, Rajendra

    2014-03-01

    Ras is one of the most common oncogenes in human cancers. It belongs to a family of GTPases that functions as binary conformational switches by timely switching of their conformations from GDP to GTP and vice versa. It attains the final active state structure via an intermediate GTP-bound state. The transition between these states is a millisecond-time-scale event. This makes studying this mechanism beyond the scope of classical molecular dynamics. In the present study, we describe the activation pathway of the HRas protein complex along the distance-based reaction coordinate using steered molecular dynamics. Approximately ~720 ns of MD simulations using CMD and SMD was performed. We demonstrated the change in orientation and arrangement of the two switch regions and the role of various hydrogen bonds during the activation process. The weighted histogram analysis method was also performed, and the potential of mean force was calculated between the inactive and active via the intermediate state (state 1) of HRas. The study indicates that water seems to play a crucial role in the activation process and to transfer the HRas protein from its intermediate state to the fully active state. The implications of our study hereby suggest that the HRas activation mechanism is a multistep process. It starts from the inactive state to an intermediate state 1 followed by trapping of water molecules and flipping of the Thr35 residue to form a fully active state (state 2). This state 2 also comprises Gly60, Thr35, GTP, Mg(2+) and water-forming stable interactions.

  4. The release of trapped gases from amorphous solid water films. I. ``Top-down'' crystallization-induced crack propagation probed using the molecular volcano

    NASA Astrophysics Data System (ADS)

    May, R. Alan; Smith, R. Scott; Kay, Bruce D.

    2013-03-01

    In this (Paper I) and the companion paper (Paper II; R. May, R. Smith, and B. Kay, J. Chem. Phys. 138, 104502 (2013), 10.1063/1.4793312), we investigate the mechanisms for the release of trapped gases from underneath amorphous solid water (ASW) films. In prior work, we reported the episodic release of trapped gases in concert with the crystallization of ASW, a phenomenon that we termed the "molecular volcano." The observed abrupt desorption is due to the formation of cracks that span the film to form a connected pathway for release. In this paper, we utilize the "molecular volcano" desorption peak to characterize the formation of crystallization-induced cracks. We find that the crack length distribution is independent of the trapped gas (Ar, Kr, Xe, CH4, N2, O2, or CO). Selective placement of the inert gas layer is used to show that cracks form near the top of the film and propagate downward into the film. Isothermal experiments reveal that, after some induction time, cracks propagate linearly in time with an Arrhenius dependent velocity corresponding to an activation energy of 54 kJ/mol. This value is consistent with the crystallization growth rates reported by others and establishes a direct connection between crystallization growth rate and the crack propagation rate. A two-step model in which nucleation and crystallization occurs in an induction zone near the top of the film followed by the propagation of a crystallization/crack front into the film is in good agreement with the temperature programmed desorption results.

  5. Probing Molecular Associations of Secondary Organic Aerosol (SOA) Samples from CalNex 2010 with Nano-DESI High-Resolution Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    O'Brien, R. E.; Nguyen, T. B.; Laskin, A.; Laskin, J.; Hayes, P. L.; Liu, S.; Jimenez, J. L.; Russell, L. M.; Nizkorodov, S.; Goldstein, A. H.

    2012-12-01

    This project focuses on analyzing the identities of molecules that comprise oligomers in size resolved aerosol fractions. Since oligomers are generally too large and polar to be measured by typical GC/MS analysis, soft ionization with high resolution mass spectrometry is used to extend the range of observable compounds. Samples collected during CalNex 2010 in Bakersfield and Los Angeles and secondary organic aerosol (SOA) produced in a photochemical chamber by photooxidation of diesel (DSL) fuel and isoprene (ISO) under humid, high-NOx conditions have been analyzed with nanospray desorption electrospray ionization (nano-DESI) and a high-resolution Orbitrap mass spectrometer. The nano-DESI is a soft ionization technique that allows molecular ions to be observed and the Orbitrap has sufficient resolution to determine the elemental composition of almost all species above the detection limit. A large fraction of SOA is made up of high molecular weight oligomers which are thought to form through acid catalyzed reactions of photo-chemically processed volatile organic compounds (VOC). The formation of oligomers is influenced by the VOCs available, the amount of atmospheric sulfate and nitrate, and the magnitude of photo-chemical processing, among other potential influences. We present the elemental composition of chemical species in size resolved SOA samples with six-hour time resolution, providing the first time resolved data set for the study of these oligomers in atmospheric samples. We present a comparison of the degree of overlap between the ambient and chamber experiments as a novel method to examine sources for this fraction of SOA. Possible formation pathways and sources of observed compounds are analyzed by comparison to other concurrent measurements at the site.

  6. Herschel/HIFI observations of interstellar OH+ and H2O+ towards W49N: a probe of diffuse clouds with a small molecular fraction

    NASA Astrophysics Data System (ADS)

    Neufeld, D. A.; Goicoechea, J. R.; Sonnentrucker, P.; Black, J. H.; Pearson, J.; Yu, S.; Phillips, T. G.; Lis, D. C.; de Luca, M.; Herbst, E.; Rimmer, P.; Gerin, M.; Bell, T. A.; Boulanger, F.; Cernicharo, J.; Coutens, A.; Dartois, E.; Kazmierczak, M.; Encrenaz, P.; Falgarone, E.; Geballe, T. R.; Giesen, T.; Godard, B.; Goldsmith, P. F.; Gry, C.; Gupta, H.; Hennebelle, P.; Hily-Blant, P.; Joblin, C.; Kołos, R.; Krełowski, J.; Martín-Pintado, J.; Menten, K. M.; Monje, R.; Mookerjea, B.; Perault, M.; Persson, C.; Plume, R.; Salez, M.; Schlemmer, S.; Schmidt, M.; Stutzki, J.; Teyssier, D.; Vastel, C.; Cros, A.; Klein, K.; Lorenzani, A.; Philipp, S.; Samoska, L. A.; Shipman, R.; Tielens, A. G. G. M.; Szczerba, R.; Zmuidzinas, J.

    2010-10-01

    We report the detection of absorption by interstellar hydroxyl cations and water cations, along the sight-line to the bright continuum source W49N. We have used Herschel's HIFI instrument, in dual beam switch mode, to observe the 972 GHz N = 1-0 transition of OH+ and the 1115 GHz 111-000 transition of ortho-H2O+. The resultant spectra show absorption by ortho-H2O+, and strong absorption by OH+, in foreground material at velocities in the range 0 to 70 km s-1 with respect to the local standard of rest. The inferred OH+/H2O+ abundance ratio ranges from ~3 to ~15, implying that the observed OH+ arises in clouds of small molecular fraction, in the 2-8% range. This conclusion is confirmed by the distribution of OH+ and H2O+ in Doppler velocity space, which is similar to that of atomic hydrogen, as observed by means of 21 cm absorption measurements, and dissimilar from that typical of other molecular tracers. The observed OH+/H abundance ratio of a few × 10-8 suggests a cosmic ray ionization rate for atomic hydrogen of 0.6-2.4×10-16 s-1, in good agreement with estimates inferred previously for diffuse clouds in the Galactic disk from observations of interstellar H3+ and other species. Herschel is an ESA space observatory with science instruments provided by European-led Principal Investigator consortia and with important participation from NASA.

  7. A Total Molecular Gas Mass Census in Z ˜ 2-3 Star-forming Galaxies: Low-J CO Excitation Probes of Galaxies’ Evolutionary States

    NASA Astrophysics Data System (ADS)

    Sharon, Chelsea E.; Riechers, Dominik A.; Hodge, Jacqueline; Carilli, Chris L.; Walter, Fabian; Weiß, Axel; Knudsen, Kirsten K.; Wagg, Jeff

    2016-08-01

    We present CO(1-0) observations obtained at the Karl G. Jansky Very Large Array for 14 z˜ 2 galaxies with existing CO(3-2) measurements, including 11 galaxies that contain active galactic nuclei (AGNs) and three submillimeter galaxies (SMGs). We combine this sample with an additional 15 z˜ 2 galaxies from the literature that have both CO(1-0) and CO(3-2) measurements in order to evaluate differences in CO excitation between SMGs and AGN host galaxies, to measure the effects of CO excitation on the derived molecular gas properties of these populations, and to look for correlations between the molecular gas excitation and other physical parameters. With our expanded sample of CO(3-2)/CO(1-0) line ratio measurements, we do not find a statistically significant difference in the mean line ratio between SMGs and AGN host galaxies as can be found in the literature; we instead find {r}{3,1}=1.03+/- 0.50 for AGN host galaxies and {r}{3,1}=0.78+/- 0.27 for SMGs (or {r}{3,1}=0.90+/- 0.40 for both populations combined). We also do not measure a statistically significant difference between the distributions of the line ratios for these populations at the p = 0.05 level, although this result is less robust. We find no excitation dependence on the index or offset of the integrated Schmidt-Kennicutt relation for the two CO lines, and we obtain indices consistent with N = 1 for the various subpopulations. However, including low-z “normal” galaxies increases our best-fit Schmidt-Kennicutt index to N˜ 1.2. While we do not reproduce correlations between the CO line width and luminosity, we do reproduce correlations between CO excitation and star-formation efficiency.

  8. Herschel Survey of Galactic OH+, H2O+, and H3O+: Probing the Molecular Hydrogen Fraction and Cosmic-Ray Ionization Rate

    NASA Astrophysics Data System (ADS)

    Indriolo, Nick; Neufeld, D. A.; Gerin, M.; Schilke, P.; Benz, A. O.; Winkel, B.; Menten, K. M.; Chambers, E. T.; Black, John H.; Bruderer, S.; Falgarone, E.; Godard, B.; Goicoechea, J. R.; Gupta, H.; Lis, D. C.; Ossenkopf, V.; Persson, C. M.; Sonnentrucker, P.; van der Tak, F. F. S.; van Dishoeck, E. F.; Wolfire, Mark G.; Wyrowski, F.

    2015-02-01

    In diffuse interstellar clouds the chemistry that leads to the formation of the oxygen-bearing ions OH+, H2O+, and H3O+ begins with the ionization of atomic hydrogen by cosmic rays, and continues through subsequent hydrogen abstraction reactions involving H2. Given these reaction pathways, the observed abundances of these molecules are useful in constraining both the total cosmic-ray ionization rate of atomic hydrogen (ζH) and molecular hydrogen fraction (f_H_2). We present observations targeting transitions of OH+, H2O+, and H3O+ made with the Herschel Space Observatory along 20 Galactic sight lines toward bright submillimeter continuum sources. Both OH+ and H2O+ are detected in absorption in multiple velocity components along every sight line, but H3O+ is only detected along 7 sight lines. From the molecular abundances we compute f_H_2 in multiple distinct components along each line of sight, and find a Gaussian distribution with mean and standard deviation 0.042 ± 0.018. This confirms previous findings that OH+ and H2O+ primarily reside in gas with low H2 fractions. We also infer ζH throughout our sample, and find a lognormal distribution with mean log (ζH) = -15.75 (ζH = 1.78 × 10-16 s-1) and standard deviation 0.29 for gas within the Galactic disk, but outside of the Galactic center. This is in good agreement with the mean and distribution of cosmic-ray ionization rates previously inferred from H_3^+ observations. Ionization rates in the Galactic center tend to be 10-100 times larger than found in the Galactic disk, also in accord with prior studies. Herschel is an ESA space observatory with science instruments provided by European-led Principal Investigator consortia and with important participation from NASA.

  9. The release of trapped gases from amorphous solid water films. I. "Top-down" crystallization-induced crack propagation probed using the molecular volcano.

    PubMed

    May, R Alan; Smith, R Scott; Kay, Bruce D

    2013-03-14

    In this (Paper I) and the companion paper (Paper II; R. May, R. Smith, and B. Kay, J. Chem. Phys. 138, 104502 (2013)), we investigate the mechanisms for the release of trapped gases from underneath amorphous solid water (ASW) films. In prior work, we reported the episodic release of trapped gases in concert with the crystallization of ASW, a phenomenon that we termed the "molecular volcano." The observed abrupt desorption is due to the formation of cracks that span the film to form a connected pathway for release. In this paper, we utilize the "molecular volcano" desorption peak to characterize the formation of crystallization-induced cracks. We find that the crack length distribution is independent of the trapped gas (Ar, Kr, Xe, CH4, N2, O2, or CO). Selective placement of the inert gas layer is used to show that cracks form near the top of the film and propagate downward into the film. Isothermal experiments reveal that, after some induction time, cracks propagate linearly in time with an Arrhenius dependent velocity corresponding to an activation energy of 54 kJ∕mol. This value is consistent with the crystallization growth rates reported by others and establishes a direct connection between crystallization growth rate and the crack propagation rate. A two-step model in which nucleation and crystallization occurs in an induction zone near the top of the film followed by the propagation of a crystallization∕crack front into the film is in good agreement with the temperature programmed desorption results. PMID:23514503

  10. A mixed quantum-classical molecular dynamics study of the hydroxyl stretch in methanol/carbon tetrachloride mixtures III: nonequilibrium hydrogen-bond dynamics and infrared pump-probe spectra.

    PubMed

    Kwac, Kijeong; Geva, Eitan

    2013-06-27

    We present a mixed quantum-classical molecular dynamics study of the nonequilibrium hydrogen-bond dynamics following vibrational energy relaxation of the hydroxyl stretch in a 10 mol % methanol/carbon tetrachloride mixture and pure methanol. The ground and first-excited energy levels and wave functions are identified with the eigenvalues and eigenfunctions of the hydroxyl's adiabatic Hamiltonian and as such depend parametrically on the configuration of the remaining, classically treated, degrees of freedom. The dynamics of the classical degrees of freedom are in turn governed by forces obtained by taking the expectation value of the force with respect to the ground or excited vibrational wave functions. Polarizable force fields and nonlinear mapping relations between the hydroxyl transition frequencies and dipole moments and the electric field along the hydroxyl bond are used, which were previously shown to quantitatively reproduce the experimental infrared steady-state absorption spectra and excited state lifetime [Kwac, K.; Geva, E. J. Phys. Chem. B 2011, 115, 9184; 2012, 116, 2856]. The relaxation from the first-excited state to the ground state is treated as a nonadiabatic transition. Within the mixed quantum-classical treatment, relaxation from the excited state to the ground state is accompanied by a momentum-jump in the classical degrees of freedom, which is in turn dictated by the nonadiabatic coupling vector. We find that the momentum jump leads to breaking of hydrogen bonds involving the relaxing hydroxyl, thereby blue-shifting the transition frequency by more than the Stokes shift between the steady-state emission and absorption spectra. The subsequent nonequilibrium relaxation toward equilibrium on the ground state potential energy surface is thereby accompanied by red shifting of the transition frequency. The signature of this nonequilibrium relaxation process on the pump-probe spectrum is analyzed in detail. The calculated pump-probe spectrum is found

  11. Probing the Probes: Fitness Factors For Small Molecule Tools

    PubMed Central

    Workman, Paul; Collins, Ian

    2010-01-01

    Chemical probes for interrogating biological processes are of considerable current interest. Cell permeable small molecule tools have a major role in facilitating the functional annotation of the human genome, understanding both physiological and pathological processes, and validating new molecular targets. To be valuable, chemical tools must satisfy necessary criteria and recent publications have suggested objective guidelines for what makes a useful chemical probe. Although recognizing that such guidelines may be valuable, we caution against overly restrictive rules that may stifle innovation in favor of a “fit-for-purpose” approach. Reviewing the literature and providing examples from the cancer field, we recommend a series of “fitness factors” to be considered when assessing chemical probes. We hope this will encourage innovative chemical biology research while minimizing the generation of poor quality and misleading biological data, thus increasing understanding of the particular biological area, to the benefit of basic research and drug discovery. PMID:20609406

  12. Probing exchange kinetics and atomic resolution dynamics in high-molecular-weight complexes using dark-state exchange saturation transfer NMR spectroscopy.

    PubMed

    Fawzi, Nicolas L; Ying, Jinfa; Torchia, Dennis A; Clore, G Marius

    2012-07-19

    We present the protocol for the measurement and analysis of dark-state exchange saturation transfer (DEST), a novel solution NMR method for characterizing, at atomic resolution, the interaction between an NMR-'visible' free species and an NMR-'invisible' species transiently bound to a very high-molecular-weight (>1 MDa) macromolecular entity. The reduced rate of reorientational motion in the bound state that precludes characterization by traditional NMR methods permits the observation of DEST. (15)N-DEST profiles are measured on a sample comprising the dark state in exchange with an NMR-visible species; in addition, the difference (ΔR(2)) in (15)N transverse relaxation rates between this sample and a control sample comprising only the NMR-visible species is also obtained. The (15)N-DEST and ΔR(2) data for all residues are then fitted simultaneously to the McConnell equations for various exchange models describing the residue-specific dynamics in the bound state(s) and the interconversion rate constants. Although the length of the experiments depends strongly on sample conditions, approximately 1 week of NMR spectrometer time was sufficient for full characterization of samples of amyloid-β (Aβ) at concentrations of ~100 μM.

  13. Probing the molecular and electronic structure of norhipposudoric and hipposudoric acids from the red sweat of Hippopotamus amphibius: a DFT investigation.

    PubMed

    Galasso, Vinicio; Pichierri, Fabio

    2009-03-19

    Molecular structure and tautomeric/conformational preferences of norhipposudoric and hipposudoric acids, the recently isolated pigments of the Hippopotamus amphibius' sweat, were investigated using the density functional theory (DFT) PBE0 formalism. Among a large variety of possible structures, two similar keto-enol tautomer/conformers are nearly isoenergetic and markedly more stable than the others both in the gas phase and aqueous solution. The bulk solvent effect was accounted for with the polarizable continuum model (PCM). A distinctive structural feature is the strong intramolecular hydrogen bonding in the keto-enol O-H...O bridge, as shown by analysis of the atoms-in-molecules topological properties of the electron density. To elucidate the claimed strong acidity of these pigments, the site-specific microscopic dissociation constants were also calculated using the cluster-continuum model, a hybrid approach based on inclusion of explicit solvent molecules and solvation of the cluster by the dielectric continuum. Notably, the first deprotonation should occur predominantly from the enolic group with a remarkably low pk(i) value. This factor could play an important role in the potent antibiotic activity of the pigments. The absorption spectra of the undissociated and dissociated compounds in aqueous solution were interpreted with time-dependent DFT/PCM calculations. The pi-pi* diquinoid excitations, mainly occurring in the fluorenoid nucleus, are the major contributors to the color and strong absorption bands in the UVA and UVB regions, which are closely related to the efficient sunscreen activity exerted by the pigments.

  14. A molecular dynamics simulation study of the α-relaxation in a 1,4-polybutadiene melt as probed by the coherent dynamic structure factor

    NASA Astrophysics Data System (ADS)

    Smith, Grant D.; Bedrov, Dmitry; Paul, Wolfgang

    2004-09-01

    The dynamic coherent structure factor Scoh(q,t) for a 1,4-polybutadiene (PBD) melt has been investigated using atomistic molecular dynamics simulations. The relaxation of Scoh(q,t) at q=1.44 Å-1 and q=2.72 Å-1, corresponding to the first and second peaks in the static structure factor for PBD, was studied in detail over a wide range of temperature. It was found that time-temperature superposition holds for the α-relaxation for both q values over a wide temperature range and that the α-relaxation can be well described by a stretched (Kohlrauch-William-Watts) exponential with temperature independent but q dependent amplitude and stretching exponent. The α-relaxation times for both q values were found to exhibit the same non-Arrhenius temperature dependence, indicating that the same physical processes are responsible for relaxation on both length scales. The α-relaxation time was found to depend strongly upon the dynamical range of data utilized in determining the relaxation time, accounting for qualitative discrepancies between α-relaxation times reported here and those extracted for PBD from experimentally measured Scoh(q,t).

  15. Probing molecular associations of field-collected and laboratory-generated SOA with nano-DESI high-resolution mass spectrometry

    NASA Astrophysics Data System (ADS)

    O'Brien, Rachel E.; Nguyen, Tran B.; Laskin, Alexander; Laskin, Julia; Hayes, Patrick L.; Liu, Shang; Jimenez, Jose L.; Russell, Lynn M.; Nizkorodov, Sergey A.; Goldstein, Allen H.

    2013-01-01

    Aerosol samples from the 2010 CalNex field study in Bakersfield (BF) and Pasadena (LA) were analyzed using positive mode nanospray-desorption electrospray ionization mass spectrometry. Secondary organic aerosol (SOA) produced in a photochemical chamber by photooxidation of diesel (DSL) fuel and isoprene (ISO) under humid, high-NOx conditions, was analyzed for comparison. Three groups of organic compounds with zero, one, or two nitrogen atoms in their molecular formulas (0N, 1N, 2N) were compared in detail. The composition of ambient SOA exhibited greater overlap with DSL than with ISO. The overlap of the chamber experiments with the BF data was relatively consistent throughout the day while the overlap with LA data increased significantly in the noon to 6 P.M. sample, consistent with the SOA plume arriving from downtown Los Angeles. BF samples were more oxidized, contained more organic nitrogen, and had more overlap with the chamber data compared to LA samples. The addition of gaseous ammonia (NH3) to the DSL experiment was necessary to generate many of the 2N compounds observed in BF. This analysis demonstrates that DSL and ISO were important sources but cannot account for all of the observed ambient compounds indicating that other sources of organics were also likely important.