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At least 667 records · Page 37Linked to original sources
PubMed2006

Do we live in a quantum world? Advances in multidimensional coherent spectroscopies refine our understanding of quantum coherences and structural dynamics of biological systems.

The issue of quantum effects in biological functions reduces to determining the relevant length and/or time scales over which phase relationships (coherence) in the wave properties of matter are conserved and lead to observable interference effects. Recent advances in femtosecond laser-based two-dimensional spectroscopy and coherent control have made it possible to directly determine the relevant timescales of quantum coherence in biological systems and even manipulate such effects, respectively, and also provide direct information on the interactions between the different degrees of freedom (electronic and nuclear) with sufficient time resolution to catch the very chemical processes driving biological functions in action. The picture that is emerging is that there are primary events in biological processes that occur on timescales commensurate with quantum coherence effects.

Animals↗
PubMed2025

Site-Specific Profiling of RNA-Binding Proteins Enabled by Isotopic Signature-Enhanced Mass Spectrometry.

RNA-binding proteins (RBPs) ubiquitously regulate RNA throughout their lifespan, being extensively involved in cellular metabolism and genetic evolution. Therefore, comprehensive identification of the RNA-protein interactions, especially their interfaces with site-specific resolution, is significant to elucidate the intricate biological activities governed by RNA. Nevertheless, it remains challenging for data-dependent acquisition (DDA)-based proteomics to identify the RNA-cross-linked peptides in depth due to the low abundance and negative charge of modified peptides. To address such limitations, we developed an innovative method named "isoRIC" for profiling RNA-binding proteomes with site-specific resolution, which combines the metabolic labeling of isotopic nucleotides for photo-cross-linking of RNA-binding proteins and the real-time targeted LC-MS/MS analysis of RNA-cross-linked peptides. This method shows a dramatic improvement of sensitivity in identifying RNA-cross-linked peptides with low abundance as compared to the DDA-based proteomic approaches, enabling the discovery of novel RNA-binding proteins and precise mapping of RNA-protein binding interfaces at single amino acid resolution. We applied isoRIC in the context of pathogenic mutations and post-translational modifications to highlight the critical role of RNA-binding sites in modulating the RNA-binding ability.

RNA-Binding Proteins↗
PubMed2006

Correlative microXRF and optical immunofluorescence microscopy of adherent cells labeled with ultrasmall gold particles.

Synchrotron-based X-ray fluorescence microscopy (microXRF) is a powerful tool to study the two-dimensional distribution of a wide range of biologically relevant elements in tissues and cells. By growing mouse fibroblast cells directly on formvar-carbon coated electron microscopy grids, microXRF elemental maps with well-defined subcellular resolution were obtained. In order to colocalize the elemental distribution with the location of specific cellular structures and organelles, we explored the application of a commercially available secondary antibody conjugated to FluoroNanogold, a dual-label that combines a regular organic fluorophore with a 1.4 nm Au-cluster as xenobiotic label for microXRF imaging. Adherent mouse fibroblast cells were grown on silicon nitride windows serving as biocompatible XRF support substrate, and labeled with FluoroNanogold in combination with primary antibodies specific for mitochondria or the Golgi apparatus, respectively. Raster scanning of the in-air dried cells with an incident X-ray energy of 11.95 keV, sufficient to ensure excitation of the Au Lalpha line, provided two-dimensional maps with submicron resolution for Au as well as for most biologically relevant elements. MicroXRF proved to be sufficiently sensitive to image the location and structural details of the Au-labeled organelles, which correlated well with the subcellular distribution visualized by means of optical fluorescence microscopy.

Animals↗
PubMed2003

Measuring and visualizing single molecular interactions in biology.

In recent years, considerable attention has focused upon the biological applications of the atomic force microscope (AFM), and in particular in its ability to explore biomolecular interaction events at the single molecule level. Such measurements can provide considerable advantages, as they remove the data averaging inherent in other biophysical/biochemical approaches that record measurements over large ensembles of molecules. To this end AFM has been used for both the high-resolution imaging of a range of individual biological molecules and their complexes, and to record interaction forces between single interacting molecules. In a recently initiated project we have begun to utilize these approaches to explore the interactions of a range of biologically important peptides with model and cell membrane surfaces. In this review, the potential value of AFM for the investigation of a range of biomolecular interaction events will be discussed, but highlighting in particular its potential for the study of interactions of peptides/proteins with biological membranes.

Animals↗
PubMed2006

Segmentation methodology for automated classification and differentiation of soft tissues in multiband images of high-resolution ultrasonic transmission tomography.

This paper presents a novel segmentation methodology for automated classification and differentiation of soft tissues using multiband data obtained with the newly developed system of high-resolution ultrasonic transmission tomography (HUTT) for imaging biological organs. This methodology extends and combines two existing approaches: the L-level set active contour (AC) segmentation approach and the agglomerative hierarchical kappa-means approach for unsupervised clustering (UC). To prevent the trapping of the current iterative minimization AC algorithm in a local minimum, we introduce a multiresolution approach that applies the level set functions at successively increasing resolutions of the image data. The resulting AC clusters are subsequently rearranged by the UC algorithm that seeks the optimal set of clusters yielding the minimum within-cluster distances in the feature space. The presented results from Monte Carlo simulations and experimental animal-tissue data demonstrate that the proposed methodology outperforms other existing methods without depending on heuristic parameters and provides a reliable means for soft tissue differentiation in HUTT images.

Algorithms↗
PubMed2006

Independent component analysis reveals new and biologically significant structures in micro array data.

BACKGROUND: An alternative to standard approaches to uncover biologically meaningful structures in micro array data is to treat the data as a blind source separation (BSS) problem. BSS attempts to separate a mixture of signals into their different sources and refers to the problem of recovering signals from several observed linear mixtures. In the context of micro array data, "sources" may correspond to specific cellular responses or to co-regulated genes. RESULTS: We applied independent component analysis (ICA) to three different microarray data sets; two tumor data sets and one time series experiment. To obtain reliable components we used iterated ICA to estimate component centrotypes. We found that many of the low ranking components indeed may show a strong biological coherence and hence be of biological significance. Generally ICA achieved a higher resolution when compared with results based on correlated expression and a larger number of gene clusters with significantly enriched for gene ontology (GO) categories. In addition, components characteristic for molecular subtypes and for tumors with specific chromosomal translocations were identified. ICA also identified more than one gene clusters significant for the same GO categories and hence disclosed a higher level of biological heterogeneity, even within coherent groups of genes. CONCLUSION: Although the ICA approach primarily detects hidden variables, these surfaced as highly correlated genes in time series data and in one instance in the tumor data. This further strengthens the biological relevance of latent variables detected by ICA.

Algorithms↗
PubMedDate not supplied

Non-invasive image acquisition and advanced processing in optical bioimaging.

Light is a most versatile tool for investigating biological systems and phenomena; the range, non-destructiveness, spatial discrimination and speed of optical imaging are all important for investigating structure and function at the cellular, tissue or even whole organism level. In live biological imaging, where the technological requirements are heightened, other features of light, such as coherence and wavelength, are used to generate the additional contrast and resolution needed. We report here recent improvements in our ability to image biological specimens optically, focusing on (a) spectral resolution and the related image processing issues, and (b) tomographic three-dimensional fluorescence imaging in vivo.

Fluorescence↗
PubMed1988

Direct enantiomeric separation of betaxolol with applications to analysis of bulk drug and biological samples.

A direct method is described for the resolution of the enantiomers of betaxolol, a novel cardioselective beta-adrenergic blocking agent, using a tris(3,5-dimethylphenylcarbamate)cellulose chiral column. An excellent resolution of the two antipodes is obtained (Rs greater than 2) with high peak symmetries. The method is simple and ideally suited to the routine control of the enantiomeric excess in the bulk drug and the analysis of the enantiomers of betaxolol in hepatocyte suspensions. With modification of the polar modifier in a hexane-based mobile phase, most commercially available beta-blockers can be baseline resolved.

Adrenergic beta-Antagonists↗
PubMed2003

Noninvasive in situ observation of the crystallization kinetics of biological macromolecules by confocal laser scanning microscopy.

High-resolution confocal laser scanning microscopy (CLSM) is a powerful tool for in situ observation and analysis of protein crystal growth kinetics. Because the resolution of CLSM is not diffraction-limited by the object, it is possible to visualize, under certain conditions, objects in molecular dimensions. A modified batch technique is applied which allows the growth kinetics of sufficiently small crystallites fixed at the lower side of a cover glass, within a hanging drop, to be studied in reflected light near the total reflection angle. A gap, or cavity, filled with solution is formed between the cover glass and the upper crystal face, which acts to fix small crystallites by hydrodynamic friction forces. The cavity height enables the propagation of molecular steps across the upper crystal face without constraint, so that the propagation velocity and geometrical parameters can be measured by CLSM. The layer growth kinetics of monoclinic crystallites of a long-acting insulin derivative (Insulin Glargine) is investigated. For a twofold supersaturation of the solution, the growth is governed by 2D nucleation at the edges of the crystallites followed by a spreading of molecular steps. The layer growth kinetics are well fitted by the simple cubic kinetic lattice model. We find that only about one of a thousand solute (protein) molecules which push a kink place due to their Brownian motion becomes really incorporated into the growing crystal.

Crystallization↗
PubMed2001

Probing the machinery of intracellular trafficking with the atomic force microscope.

Atomic force microscopy has emerged as a powerful tool for characterizing single biological macromolecules, macromolecular assemblies, and whole cells in aqueous buffer, in real time, and at molecular-scale spatial and force resolution. Many of the central elements of intracellular transport are tens to hundreds of nanometers in size and highly dynamic. Thus, atomic force microscopy provides a valuable means of addressing questions of structure and mechanism in intracellular transport. We begin this review of recent efforts to apply atomic force microscopy to problems in intracellular transport by discussing the technical principles behind atomic force microscopy. We then turn to three specific areas in which atomic force microscopy has been applied to problems with direct implications for intracellular trafficking: cytoskeletal structure and dynamics, vesicular transport, and receptor-ligand interactions. In each case, we discuss studies which use both intact cellular elements and reconstituted models. While many technical challenges remain, these studies point to several areas where atomic force microscopy can be used to provide valuable insight into intracellular transport at exquisite spatial and energetic resolution.

Biological Transport↗
PubMed2000

Two-photon excitation fluorescence microscopy.

Two-photon fluorescence microscopy is one of the most important recent inventions in biological imaging. This technology enables noninvasive study of biological specimens in three dimensions with submicrometer resolution. Two-photon excitation of fluorophores results from the simultaneous absorption of two photons. This excitation process has a number of unique advantages, such as reduced specimen photodamage and enhanced penetration depth. It also produces higher-contrast images and is a novel method to trigger localized photochemical reactions. Two-photon microscopy continues to find an increasing number of applications in biology and medicine.

Animals↗
PubMed2001

The papillomavirus E2 proteins: structure, function, and biology.

Nearly twenty years after the first high-resolution crystal structures of specific protein-DNA complexes were determined, the stereo-chemical basis for protein-DNA recognition remains an active area of investigation. One outstanding question is, how are proteins able to detect noncontacted sequences in their binding sites? The papillomavirus E2 proteins represent a particularly suitable group of proteins in which to examine the mechanisms of "indirect readout." Coordinated structural and thermodynamic studies of the E2-DNA interaction conducted over the past five years are summarized in this review. The data support a model in which the electrostatic properties of the individual E2 proteins correlate with their affinities for intrinsically flexible or rigidly prebent DNA targets.

Models, Molecular↗
PubMed2003

Studying large viruses.

In this article we have attempted to describe some structural aspects of large viruses. Although this may seem a straightforward task, it is complicated by the fact that large viruses do not represent a distinctive class of organisms and any grouping under this heading will include a range of unrelated viruses with different structures, replication strategies, and host types. To simplify matters we limited our definition to dsDNA viruses with genomes of 100 kbp or larger. However, even this restricted grouping includes viruses with diverse and seemingly unrelated structures. Furthermore, few if any structural features are exclusive to large viruses and most of what appears distinctive about their structure or assembly can also be found in smaller, and usually better characterized, viruses. Therefore we have not attempted to provide a comprehensive catalog of the properties of large viruses but have tried to illustrate particular structural points with examples from a few of the better known forms, notably herpes simplex virus (HSV) and phage T4. The two techniques used to provide rigorous analyses of virus structures are X-ray crystallography and electron cryomicroscopy with computer-assisted reconstruction. To date, X-ray crystallography has been successful only with smaller viruses, and what is known about the structures of these large viruses has come primarily from electron cryomicroscopy. However, with the notable exception of the HSV capsid, such studies have been limited in extent and of relatively low resolution, and the information obtained has been confined largely to describing the spatial distributions and relationships between the subunits. Nevertheless, these studies have given us our clearest insights into the biology of these complex particles and increases in resolution promise to extend these insights by bridging the gap between gross and atomic structures, as exemplified by the identification and mapping of secondary structural elements in the HSV capsid.

Genome, Viral↗
PubMed1997

An optical submicrometer calcium sensor with conductance sensing capability.

The identification of chemical species and the measurement of their concentrations with high (submicrometer) spatial resolution are of considerable importance in cell biology. In this article we report the first successful development of a > or = 0.1-micron Ca2+ sensor based on a pulled micropipet, filled with a conducting porous sol-gel glass which was doped with the fluorescent calcium green 1 Ca2+ indicator. Such sensors are potentially capable of measuring Ca2+ concentrations as low as 10(-8) M, in confined volumes, with a three-dimensional resolution which exceeds approximately 0.1 micron. A major advantage of the sensor is its capability to be integrated into a multifunctional probe which will measure chemical analyte concentrations and ion conductance.

Animals↗
PubMed1981

Use of an encapsulated fluorescent probe to measure intracellular PO2.

The objectives of this investigation were to produce a reliable, sensitive probe to measure intracellular PO2 with a high degree of resolution and to apply this technique to biological systems. A fluorescent molecule, pyrene dissolved in paraffin oil, was encapsulated in polyacrylamide to form a probe of nanometer dimensions. The quantitative and microscopic oxygen values were determined by analyzing the quenching of the fluorescence of the probe by oxygen, as displayed on a television monitor by a silicon-intensified-target camera. The nanocapsules had a sensitivity of approximately 1 mm PO2, a spatial resolution of 0.5 micrometer, and a temporal resolution of milliseconds. Calibrated nanocapsules within nonrespiring Amoeba proteus responded to ambient partial pressures of oxygen. At two different ambient partial pressures, nanocapsules engulfed by respiring amoebas indicated an intracellular PO2 28 mm Hg less than extracellular PO2. The capsules retained their sensitivity to oxygen for at least 8 months.

Amoeba↗
PubMed2005

Toward high-resolution de novo structure prediction for small proteins.

The prediction of protein structure from amino acid sequence is a grand challenge of computational molecular biology. By using a combination of improved low- and high-resolution conformational sampling methods, improved atomically detailed potential functions that capture the jigsaw puzzle-like packing of protein cores, and high-performance computing, high-resolution structure prediction (<1.5 angstroms) can be achieved for small protein domains (<85 residues). The primary bottleneck to consistent high-resolution prediction appears to be conformational sampling.

Amino Acid Sequence↗
PubMed2005

Quantitation of clovamide-type phenylpropenoic acid amides in cells and plasma using high-performance liquid chromatography with a coulometric electrochemical detector.

A high-performance liquid chromatography (HPLC) method was developed for measuring the concentrations of clovamide-type phenylpropenoic acid amides (N-caffeoyldopamine and N-caffeoyltyramine) in cell and plasma samples. The separation was performed on a Nova-Pak C18 column using an isocratic buffer with a coulometric electrochemical detector with four electrode channels. Using the HPLC method, N-caffeoyldopamine and N-caffeoyltyramine could be detected with good peak resolutions at respective retention times (4 and 6.4 min). The calibration curves were linear over the ranges (0.1 and 100 microM), and their lower limit of detection was as little as 100 fmol. For quantifying N-caffeoyldopamine and N-caffeoyltyramine in cell and plasma samples, the samples were extracted by extraction methods with more than 95% recoveries. After extraction, the amides were detected with the same sensitivity, peak resolutions, and retention times. Using this method, plasma concentrations of N-caffeoyltyramine were determined in blood samples collected at 12, 24, 30, 36, 48, 60, and 75 min after the oral administrations of N-caffeoyltyramine (0.5 mg and 2 mg/30 g body weight). This HPLC method with an electrochemical detector is the first reported method able to quantify N-caffeoyldopamine and N-caffeoyltyramine in biological samples with excellent detection limits, peak resolutions, discrete retention times, and consistent reproducibility.

Animals↗
PubMed2002

Modeling a parallel L4 neuron array of the fly (Musca domestica) vision system with a sequential processor.

At RMBS 2001 Olson presented a novel approach to image edge detection based on the vision system of the common house fly, Musca domestica [1]. Biologically based vision systems are inherently parallel and the vision related cells form a self-contained cartridge, ommatidium, which is duplicated across the surface of the fly's eye. Histological evidence provides the interconnection both within the vision cartridge and the connections to adjacent cartridges. Due to the parallel nature of biologically inspired vision systems, they outperform computer based digital vision systems in speed performance and memory requirements. Olson provided a model of the cartridge with its intra- and inter-connections. This model, rendered in MATLAB and Excel, demonstrated the feasibility of edge detection in the first several synaptic cellular connections within the cartridge. His results demonstrated how edge detection and object movements are easily obtained using a biologically based vision model. He demonstrated the model using simple rectangular and circular objects. We term this work Olson's Algorithm. We have extended Olson's Algorithm into a high-resolution model using a standard off-the-shelf frame grabber. Although, the frame grabber is a digitally based instrument, its image planes are used to model the photoreceptor layer (R1-R6), the L1, L2 monopolar cell layer, and also the monopolar L4 cell layer. The connections between these cells are programmed in "C". The high-resolution model demonstrates the feasibility of using a biologically based vision system in a real world application. Furthermore, it allows object segmentation, movement, and tracking to be modeled prior to implementation in parallel analog hardware.

Algorithms↗