Antibodies against Lysophosphatidic Acid Protect against Blast-Induced Ocular Injuries

Exposure to blast overpressure waves is implicated as the major cause of ocular injuries and resultant visual dysfunction in veterans involved in recent combat operations. No effective therapeutic strategies have been developed so far for blast-induced ocular dysfunction. Lysophosphatidic acid (LPA) is a bioactive phospholipid generated by activated platelets, astrocytes, choroidal plexus cells, and microglia and is reported to play major roles in stimulating inflammatory processes. The levels of LPA in the cerebrospinal fluid have been reported to increase acutely in patients with traumatic brain injury (TBI) as well as in a controlled cortical impact (CCI) TBI model in mice. In the present study, we have evaluated the efficacy of a single intravenous administration of a monoclonal LPA antibody (25 mg/kg) given at 1 h post-blast for protection against injuries to the retina and associated ocular dysfunctions. Our results show that a single 19 psi blast exposure significantly increased the levels of several species of LPA in blood plasma at 1 and 4 h post-blast. The anti-LPA antibody treatment significantly decreased glial cell activation and preserved neuronal cell morphology in the retina on day 8 after blast exposure. Optokinetic measurements indicated that anti-LPA antibody treatment significantly improved visual acuity in both eyes on days 2 and 6 post-blast exposure. Anti-LPA antibody treatment significantly increased rod photoreceptor and bipolar neuronal cell signaling in both eyes on day 7 post-blast exposure. These results suggest that blast exposure triggers release of LPAs, which play a major role promoting blast-induced ocular injuries, and that a single early administration of anti-LPA antibodies provides significant protection

One probe, two-channel imaging of nuclear and cytosolic compartments with orange and red emissive dyes

Several new DNA-targeting probes that exhibit binding-induced 'turn on' fluorescence are presented. Two of the dyes, orange emissive 1, (E)-4-(4(-4-methylpiperazin-1-yl)phenyl)6-(4-(4-methylpi-perazin-1-yl)styryl)pyrimidin-2-ol), and red emissive 2, (E)-4-(4(-4-methyl-piperazin-1-yl)-phenyl)6-(4-(4-methylpiperazin-1-yl)styryl)-1,3-propanedionato-κO,κO']difluoroborane), are brightly fluorescent when bound to DNA, but are virtually non-fluorescent in aqueous solutions. Confocal fluorescence microscopy of live BT474, MCF7 and HEK293 cells demonstrates that both probes are cell permeable and rapidly accumulated intracellularly into cell nuclei and the cytosol. Taking advantage of their environmental sensitivity, these two pools of fluorophores are readily resolved into separate channels, and thus, a single dye allows two-color imaging of the nuclear and cytosolic compartments

Two-Photon Spectroscopy as a New Sensitive Method for Determining the DNA Binding Mode of Fluorescent Nuclear Dyes

A new optical strategy to determine the binding modes (intercalation vs groove binding) of small fluorescent organic molecules with calf thymus DNA was developed using two-photon absorption (TPA) spectroscopy. Two-photon excited emission was utilized to investigate a series of fluorescent nuclear dyes. The results show that TPA cross-sections are able to differentiate the fine details between the DNA binding modes. Groove binding molecules exhibit an enhanced TPA cross-section due to the DNA electric field induced enhancement of the transition dipole moment, while intercalative binding molecules exhibit a decrease in the TPA cross-section. Remarkably, the TPA cross-section of 4,6-bis(4-(4-methylpiperazin-1-yl)phenyl) pyrimidine is significantly enhanced (13.6-fold) upon binding with DNA. The sensitivity of our TPA methodology is compared to circular dichroism spectroscopy. TPA demonstrates superior sensitivity by more than an order of magnitude at low DNA concentrations. This methodology can be utilized to probe DNA interactions with other external molecules such as proteins, enzymes, and drugs

A New Design Strategy and Diagnostic to Tailor the DNA-Binding Mechanism of Small Organic Molecules and Drugs

The classical model for DNA groove binding states that groove binding molecules should adopt a crescent shape that closely matches the helical groove of DNA. Here, we present a new design strategy that does not obey this classical model. The DNA-binding mechanism of small organic molecules was investigated by synthesizing and examining a series of novel compounds that bind with DNA. This study has led to the emergence of structure–property relationships for DNA-binding molecules and/or drugs, which reveals that the structure can be designed to either intercalate or groove bind with calf thymus dsDNA by modifying the electron acceptor properties of the central heterocyclic core. This suggests that the electron accepting abilities of the central core play a key role in the DNA-binding mechanism. These small molecules were characterized by steady-state and ultrafast nonlinear spectroscopies. Bioimaging experiments were performed in live cells to evaluate cellular uptake and localization of the novel small molecules. This report paves a new route for the design and development of small organic molecules, such as therapeutics, targeted at DNA as their performance and specificity is dependent on the DNA-binding mechanism

Two-Photon Spectroscopy as a New Sensitive Method for Determining the DNA Binding Mode of Fluorescent Nuclear Dyes

A new optical strategy to determine the binding modes (intercalation vs groove binding) of small fluorescent organic molecules with calf thymus DNA was developed using two-photon absorption (TPA) spectroscopy. Two-photon excited emission was utilized to investigate a series of fluorescent nuclear dyes. The results show that TPA cross-sections are able to differentiate the fine details between the DNA binding modes. Groove binding molecules exhibit an enhanced TPA cross-section due to the DNA electric field induced enhancement of the transition dipole moment, while intercalative binding molecules exhibit a decrease in the TPA cross-section. Remarkably, the TPA cross-section of 4,6-bis­(4-(4-methylpiperazin-1-yl)­phenyl) pyrimidine is significantly enhanced (13.6-fold) upon binding with DNA. The sensitivity of our TPA methodology is compared to circular dichroism spectroscopy. TPA demonstrates superior sensitivity by more than an order of magnitude at low DNA concentrations. This methodology can be utilized to probe DNA interactions with other external molecules such as proteins, enzymes, and drugs

Turn-On, Fluorescent Nuclear Stains with Live Cell Compatibility

DNA-binding, green and yellow fluorescent probes with excellent brightness and high on/off ratios are reported. The probes are membrane permeable, live-cell compatible, and optimally matched to 405 nm and 514 nm laser lines, making them attractive alternatives to UV-excited and blue emissive Hoechst 33342 and DAPI nuclear stains. Their electronic structure was investigated by optical spectroscopy supported by TD-DFT calculations. DNA binding is accompanied by 27- to 75-fold emission enhancements, and linear dichroism demonstrates that one dye is a groove binder while the other intercalates into DNA

Two-Photon Spectroscopy as a New Sensitive Method for Determining the DNA Binding Mode of Fluorescent Nuclear Dyes

A new optical strategy to determine the binding modes (intercalation vs groove binding) of small fluorescent organic molecules with calf thymus DNA was developed using two-photon absorption (TPA) spectroscopy. Two-photon excited emission was utilized to investigate a series of fluorescent nuclear dyes. The results show that TPA cross-sections are able to differentiate the fine details between the DNA binding modes. Groove binding molecules exhibit an enhanced TPA cross-section due to the DNA electric field induced enhancement of the transition dipole moment, while intercalative binding molecules exhibit a decrease in the TPA cross-section. Remarkably, the TPA cross-section of 4,6-bis­(4-(4-methylpiperazin-1-yl)­phenyl) pyrimidine is significantly enhanced (13.6-fold) upon binding with DNA. The sensitivity of our TPA methodology is compared to circular dichroism spectroscopy. TPA demonstrates superior sensitivity by more than an order of magnitude at low DNA concentrations. This methodology can be utilized to probe DNA interactions with other external molecules such as proteins, enzymes, and drugs

Base Pair Sensitivity and Enhanced ON/OFF Ratios of DNA-Binding: Donor–Acceptor–Donor Fluorophores

The photophysical properties of two recently reported live cell compatible, DNA-binding dyes, 4,6-bis­(4-(4-methylpiperazin-1-yl)­phenyl)­pyrimidin-2-ol, <b>1</b>, and [1,3-bis­[4-(4-methylpiperazin-1-yl)­phenyl]-1,3-propandioato-κ<i>O</i>, κ<i>O</i>′]­difluoroboron, <b>2</b>, are characterized. Both dyes are quenched in aqueous solutions, while binding to sequences containing only AT pairs enhances the emission. Binding of the dyes to sequences containing only GC pairs does not produce a significant emission enhancement, and for sequences containing both AT and GC base pairs, emission is dependent on the length of the AT pair tracts. Through emission lifetime measurements and analysis of the dye redox potentials, photoinduced electron transfer with GC pairs is implicated as a quenching mechanism. Binding of the dyes to AT-rich regions is accompanied by bathochromic shifts of 26 and 30 nm, respectively. Excitation at longer wavelengths thus increases the ON/OFF ratio of the bound probes significantly and provides improved contrast ratios in solution as well as in fluorescence microscopy of living cells