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There is an urgent need to develop novel treatments to counter Botulinum neurotoxin (BoNT) poisoning. Currently, the majority of BoNT drug development efforts focus on directly inhibiting the proteolytic components of BoNT, i.e. light chains (LC). Although this is a rational approach, previous research has shown that LCs are extremely difficult drug targets and that inhibiting multi-serotype BoNTs with a single LC inhibitor may not be feasible. An alternative approach would target neuronal pathways involved in intoxication/recovery, rather than the LC itself. Phosphorylation-related mechanisms have been implicated in the intoxication pathway(s) of BoNTs. However, the effects of phosphatase inhibitors upon BoNT activity in the physiological target of BoNTs, i.e. motor neurons, have not been investigated. In this study, a small library of phosphatase inhibitors was screened for BoNT antagonism in the context of mouse embryonic stem cell-derived motor neurons (ES-MNs). Four inhibitors were found to function as BoNT/A antagonists. Subsequently, we confirmed that these inhibitors protect against BoNT/A in a dose-dependent manner in human ES-MNs. Additionally, these compounds provide protection when administered in post-intoxication scenario. Importantly, the inhibitors were also effective against BoNT serotypes B and E. To the best of our knowledge, this is the first study showing phosphatase inhibitors as broad-spectrum BoNT antagonists.

Objective: The fibroblast-like synoviocytes (FLS) in the synovial intimal lining of the joint are key mediators of inflammation and joint destruction in rheumatoid arthritis (RA). In RA, these cells aggressively invade the extracellular matrix, producing cartilage-degrading proteases and inflammatory cytokines. The behavior of FLS is controlled by multiple interconnected signal transduction pathways involving reversible phosphorylation of proteins on tyrosine residues. However, little is known about the role of the protein tyrosine phosphatases (PTPs) in FLS function. This study was undertaken to explore the expression of all of the PTP genes (the PTPome) in FLS., Methods: A comparative screening of the expression of the PTPome in FLS from patients with RA and patients with osteoarthritis (OA) was conducted. The functional effect on RA FLS of SH2 domain-containing phosphatase 2 (SHP-2), a PTP that was up-regulated in RA, was then analyzed by knockdown using cell-permeable antisense oligonucleotides., Results: PTPN11 was overexpressed in RA FLS compared to OA FLS. Knockdown of PTPN11, which encodes SHP-2, reduced the invasion, migration, adhesion, spreading, and survival of RA FLS. Additionally, signaling in response to growth factors and inflammatory cytokines was impaired by SHP-2 knockdown. RA FLS that were deficient in SHP-2 exhibited decreased activation of focal adhesion kinase and mitogen-activated protein kinases., Conclusion: These findings indicate that SHP-2 has a novel role in mediating human FLS function and suggest that it promotes the invasiveness and survival of RA FLS. Further investigation may reveal SHP-2 to be a candidate therapeutic target for RA., (Copyright © 2013 by the American College of Rheumatology.)

We have used differential polarization imaging microscopy to measure the amount and orientation of aligned sickle hemoglobin polymer in quickly deoxygenated sickle red blood cells. Images of the angular orientation of the aligned polymer at each point in the cell allowed for determination of the inclination of individual domains, providing detailed information regarding the polymerization and elongation of sickle hemoglobin polymers ex vivo. We found that the number of aligned polymer domains increased with increasing mean cell hemoglobin concentration. Sickle and holly leaf-shaped cells contained single or few domains of aligned polymer, while more compact cells such as irreversibly sickled cells contained many domains. A new class of cells was discovered by examination of images of the angular orientation of aligned polymer, which contained a single central nucleation site, with growth of polymer occurring outward in all directions in a spherulite-like domain.

The coupled-dipole approximation has been used to model polarized light-scattering data obtained from the sperm of the octopus Eledone cirrhosa. Mueller scattering-matrix elements (which describe how a sample alters the intensity and degree of polarization of scattered light) were measured as a function of angle. The sample was modeled as a helical fiber believed to correspond to a DNA protein complex. It was necessary to propose an inherent anisotropy in the polarizability of the fiber in order to fit the data. The direction of the principle axes of the polarizability were determined by comparing the model with experimental data. The results suggest that the 2-nm DNA fibers are perpendicular to the thick fiber that defines the helical geometry of the octopus sperm head.

Differential scattering of incident left and right circularly polarized light can be an important contribution to the circular dichroism of macromolecules. In principle both differential absorption and differential scattering of circularly polarized light contribute to circular dichroism, but differential scattering is increasingly important for particles whose dimensions are greater than 1/20th the wavelength of light. The scattering contribution is probably not important for unaggregated proteins and nucleic acids in solution. It can be very important for viruses, membranes, and other protein-nucleic acid complexes. Outside the absorption bands of the scattering, chiral particle, only differential scattering contributes to the circular dichroism. The sign and magnitude of the differential scattering is quantitatively related to the relative orientations and the distances between the scattering units of the particle. The interpretation of the circular differential scattering depends on a simple, classical method. Thus, in understanding a measured circular dichroism, it often will be easier to relate the differential scattering to the structure of a particle (such as a virus) than it is to relate the differential absorption to the structure.

The phenomenon of Duysens flattening of the absorption spectra resulting from the inhomogeneous distribution of the chromophores in the solution is analyzed. These inhomogeneities are treated as localized statistical fluctuations in the concentration of the absorbing species, by using the Gaussian distribution. A law of absorbance is obtained, and the effect of light scattering on the flattening is also characterized. The flattening in the circular dichroism spectra of particulate suspensions is then analyzed. It is shown that the degree of flattening of the circular dichroism of a suspension is, in general, different from the corresponding flattening of its absorption spectrum. A quantitative relationship between the two effects is established.

A fluorescence microscopy technique was used to image the dynamics of individual DNA molecules. Lambda, calf thymus, cosmid (circular), and T4 DNA were studied with the fluorescent dye acridine orange. Experiments with DNAase I were conducted, and the results indicate that these observations correspond to DNA molecules. The results of experiments with circular DNA provide strong evidence that these were single DNA molecules. Molecules were observed free in solution or attached to a glass or copper surface at one or several points. The Brownian motion of these molecules was observed, indicating that DNA in solution exists in a partially supercoiled state. Some molecules appeared stretched and were attached to the surface by their termini; the lengths of these molecules were measured. Such molecules also exhibited elastic behavior upon breaking. The power of this technique is demonstrated in images of cosmid DNA molecules, catenanes, and DNA extending from T4 phage particles. These results suggest immediate applications to molecular biology, such as examining the dynamics of protein-DNA interactions. Areas of ongoing research are discussed.

Ultraviolet circular dichroism spectroscopy in the interval from 190 to 240 nm and infrared spectroscopy in the region of the amide I band (1,600 cm-1 to 1,700 cm-1) has been used to estimate the alpha-helix content and the beta-sheet content of bacteriorhodopsin. Circular dichroism spectroscopy strongly suggests that the alpha-helix content is sufficient for only five helices, if each helix is composed of 20 or more residues. It also suggests that there is substantial beta-sheet conformation in bacteriorhodopsin. The presence of beta-sheet secondary structure is further suggested by the presence of a 1,639 cm-1 shoulder on the amide I band in the infrared spectrum. Although a structural model consisting of seven alpha-helical rods has been generally accepted up to this point, the spectroscopic data are more consistent with a model consisting of five alpha-helices and four strands of beta-sheet. We note that the primary amino acid sequence can be assigned to segments of alpha-helix and beta-sheet in a way that does not require burying more than two charged groups in the hydrophobic membrane interior, contrary to the situation for any seven-helix model.

Phase-dependent forms of microscopy (such as phase contrast, interference, or polarization microscopy) have been used for many years; the amount of phase retardation produces images based mainly on index of refraction. We have developed a microscope that forms images that depend on small differences in extinction for different forms of incident polarized light. By modulating the polarization of light incident on the sample and digitally recording the difference in intensities of transmitted light, we obtain images which specifically reveal either ordered linear structures or chiral (right- or left-handed) structures. Linearly polarized light, incident alternately with two perpendicular directions of polarization, forms images of structures which have linear order or linear orientation. Right-handed and left-handed circularly polarized light incident alternately on a sample forms images of chiral structures. Structures with neither linear order nor chirality are essentially invisible. Thus, images based on linear dichroism, circular dichroism, and linear and circular differential scattering can be used to detect specific types of structures which may be difficult to observe by conventional methods. We have used such 'linear and circular differential imaging' to study the structure of the nucleolus (the site of RNA synthesis) in live primary spermatocytes of Drosophila when they are transcriptionally active or inactive. Some inactive nucleoli are bipartite, with two distinct structures visible by differential scattering of both linearly and circularly polarized light. The active nucleolus is a single domain; it is clearly distinguished from part of the Y chromosome, and it shows different internal structure with linearly and circularly polarized light. Thus, polarization-dependent images reveal structures which can be associated with the transcriptional activity of cells.

If an optically active (chiral) sample is placed in a microscope and illuminated with circularly polarized light, an image can be formed that is related to the circular dichroism of each feature of the sample. A theoretical investigation has been done for the circular differential image obtained by subtracting the images formed under right- and left-circularly polarized light. Two types of differential images are possible: (i) dark-field images formed from light reflected or scattered by the sample and (ii) bright-field images formed from light transmitted through the sample. The sign and magnitude of each feature in a circular differential image strongly depend on the structure of the sample. The dark-field circular differential images are most sensitive to large features with dimensions similar to the wavelength of illumination whereas the bright-field images are most sensitive to the short-range molecular order. Applications of circular differential imaging may include clinical fingerprinting of normal and transformed cells and structural analysis of individual cellular components.

The distribution of oriented, polymerized sickle cell hemoglobin (hemoglobin S) in erythrocytes is visualized with a microscope that produces an image proportional to linear dichroism. Monochromatic light alternately polarized along two perpendicular directions is incident on the sample. The image is focused on a diode array, and the digital output is used to form two images. One is the usual image proportional to the average transmitted light intensity of the two incident polarizations of light; the other is a linear differential image proportional to the linear dichroism of the sample. This quantitative image can specifically reveal oriented hemoglobin molecules with a sensitivity of about 4000 oriented molecules per picture element of the image.

We have used differential polarization microscopy, which provides images of linear dichroism, to measure the percentage of aligned hemoglobin (Hb) in 1086 deoxygenated red blood cells from subjects with sickle cell anemia. The percentage was found to be only slightly dependent on the speed of deoxygenation, thus showing that the percentage of aligned Hb was thermodynamically controlled (as has been found previously for the percentage of polymerized Hb in vitro). A slight decrease in the percentage of aligned Hb due to increasing speed of deoxygenation is primarily due to the increase in the number of cells containing no detectable aligned Hb. This class of cells was also the most variable between the different subjects studied. We were able to identify two other groups of cells that contain different numbers of domains of aligned Hb and show that these groups contain statistically different percentages of aligned Hb. The differences between these classes of cells was shown to be primarily due to different numbers of initial nucleation sites within each cell. It appears that the presence of preformed nucleation sites within cells at ambient oxygen tensions results in the thermodynamic control of aligned Hb polymer.

Experimental techniques are presented that can be used to assay and correct for differential light scattering effects in circular dichroism spectra of biological macrostructures. The assay is based upon use of variable detector geometries that collect light over large solid angles. Disrupted T2 virus suspensions and purified T2 phage DNA exhibit geometry-independent spectra; the spectrum of intact T2 virus is highly sensitive to detection geometry. On the basis of spectra obtained after light-scattering correction, the structure of T2 DNA in the phage particle is assigned to the C form. We conclude that: (i) The measured circular dichroism of a light-scattering specimen may be highly sensitive to light-detection geometry of the instrument. This effect is indicative of differential scattering intensity for left and right circularly polarized light. (ii) Some optically active particles, although they scatter light intensely, exhibit circular dichroism that is independent of detection geometry and, therefore, apparently uninfluenced by differential light scattering. We infer that whether differential light scattering arises may depend upon the presence or absence of ordered asymmetry in the organization of the scattering particle. (iii) The circular dichroism of any light-scattering specimen should be measured again in apparatus designed for differential light-scattering correction as a prerequisite to meaningful structural conclusions. (iv) Differential scattering effects in circular dichroism may be potentially useful as a probe for large-order organization of the scattering particle.

The intrinsic birefringence of multiple-coiled DNA is computed in terms of an equally dense array of parallel DNA molecules. The birefringence for n times-coiled DNA molecules is given by [Formula: see text] where beta(o) = 0, beta(i) = tan(-1) (p(i)/2pir(i)), p(i) = period of the i(th) helix coil and r(i) = radius of i(th) helix coil. The formula is applied to two cases of helically coiled DNA in biological material and found to agree quantitatively with experimental results.