Methylene blue protects astrocytes against glucose oxygen deprivation by improving cellular respiration.

Astrocytes outnumber neurons and serve many metabolic and trophic functions in the mammalian brain. Preserving astrocytes is critical for normal brain function as well as for protecting the brain against various insults. Our previous studies have indicated that methylene blue (MB) functions as an alternative electron carrier and enhances brain metabolism. In addition, MB has been shown to be protective against neurodegeneration and brain injury. In the current study, we investigated the protective role of MB in astrocytes. Cell viability assays showed that MB treatment significantly protected primary astrocytes from oxygen-glucose deprivation (OGD) & reoxygenation induced cell death. We also studied the effect of MB on cellular oxygen and glucose metabolism in primary astrocytes following OGD-reoxygenation injury. MB treatment significantly increased cellular oxygen consumption, glucose uptake and ATP production in primary astrocytes. In conclusion our study demonstrated that MB protects astrocytes against OGD-reoxygenation injury by improving astrocyte cellular respiration.

BSA Au clusters as a probe for enhanced fluorescence detection using multipulse excitation scheme.

Although BSA Au clusters fluoresce in red region (λmax: 650 nm), they are of limited use due to low fluorescence quantum yield (~6%). Here we report an enhanced fluorescence imaging application of fluorescent bio-nano probe BSA Au clusters using multipulse excitation scheme. Multipulse excitation takes advantage of long fluorescence lifetime (> 1 µs) of BSA Au clusters and enhances its fluorescence intensity 15 times over short lived cellular auto-fluorescence. Moreover we have also shown that by using time gated detection strategy signal (fluorescence of BSA Au clusters) to noise (auto-fluorescence) ratio can be increased by 30 fold. Thereby with multipulse excitation long lifetime probes can be used to develop biochemical assays and perform optical imaging with zero background.

Membrane topology of human presenilin-1 in SK-N-SH cells determined by fluorescence correlation spectroscopy and fluorescent energy transfer.

Presenilin-1 (PS1) protein acts as passive ER Ca(2+) leak channels that facilitate passive Ca(2+) leak across ER membrane. Mutations in the gene encoding PS1 protein cause neurodegeneration in the brains of patients with familial Alzheimer's disease (FAD). FADPS1 mutations abrogate the function of ER Ca(2+) leak channel activity in human neuroblastoma SK-N-SH cells in vitro (Das et al., J Neurochem 122(3):487-500, 2012) and in mouse embryonic fibroblasts. Consequently, genetic deletion or mutations of the PS1 gene cause calcium (Ca(2+)) signaling abnormalities leading to neurodegeneration in FAD patients. By analogy with other known ion channels it has been proposed that the functional PS1 channels in ER may be multimers of several PS1 subunits. To test this hypothesis, we conjugated the human PS1 protein with an NH2-terminal YFP-tag and a COOH-terminal CFP-tag. As expected YFP-PS1, and PS1-CFP were found to be expressed on the plasma membranes by TIRF microscopy, and both these fusion proteins increased ER Ca(2+) leak channel activity similar to PS1 (WT) in SK-N-SH cells, as determined by functional calcium imaging. PS1-CFP was either expressed alone or together with YFP-PS1 into SK-N-SH cell line and the interaction between YFP-PS1 and PS1-CFP was determined by Förster resonance energy transfer analysis. Our results suggest interaction between YFP-PS1 and PS1-CFP confirming the presence of a dimeric or multimeric form of PS1 in SK-N-SH cells. Lateral diffusion of PS1-CFP and YFP-PS1 in the plasma membrane of SK-N-SH cells was measured in the absence or in the presence of glycerol by fluorescence correlation spectroscopy to show that both COOH-terminal and NH2-terminal of human PS1 are located on the cytoplasmic side of the plasma membrane. Therefore, we conclude that both COOH-terminal and NH2-terminal of human PS1 may also be oriented on the cytosolic side of ER membrane.

Fluorescence detection of MMP-9. II. Ratiometric FRET-based sensing with dually labeled specific peptide.

In our previous paper we showed that the MMP-9 enzyme recognizes a specific peptide sequence, Lys-Gly- Pro-Arg-Ser-Leu-Ser-Gly-Lys, and cleaves the peptide into two parts [1]. In this study, the peptide is labeled with two dyes, carboxyfluorescein (5-FAM) and Cy5. A highly efficient energy transfer of over 80% results in a dominant emission of Cy5 at ~670 nm with an excitation of 470 nm. Severance of the peptide by the MMP-9 enzyme eliminates Förster Resonance Energy Transfer (FRET) and strongly increases the fluorescence of the 5-FAM dye. In this manuscript we describe the strategy for a FRET-based method for MMP-9 enzyme detection. The basic aim is to apply a ratio-metric sensing technique in which a ratio of green/red fluorescence intensity is measured as a function of enzyme concentration. The ratio-metric method eliminates many experimental variables and enables accurate MMP-9 detection.

Phosphorylation of myosin regulatory light chain has minimal effect on kinetics and distribution of orientations of cross bridges of rabbit skeletal muscle.

Force production in muscle results from ATP-driven cyclic interactions of myosin with actin. A myosin cross bridge consists of a globular head domain, containing actin and ATP-binding sites, and a neck domain with the associated light chain 1 (LC1) and the regulatory light chain (RLC). The actin polymer serves as a "rail" over which myosin translates. Phosphorylation of the RLC is thought to play a significant role in the regulation of muscle relaxation by increasing the degree of skeletal cross-bridge disorder and increasing muscle ATPase activity. The effect of phosphorylation on skeletal cross-bridge kinetics and the distribution of orientations during steady-state contraction of rabbit muscle is investigated here. Because the kinetics and orientation of an assembly of cross bridges (XBs) can only be studied when an individual XB makes a significant contribution to the overall signal, the number of observed XBs was minimized to ∼20 by limiting the detection volume and concentration of fluorescent XBs. The autofluorescence and photobleaching from an ex vivo sample was reduced by choosing a dye that was excited in the red and observed in the far red. The interference from scattering was eliminated by gating the signal. These techniques decrease large uncertainties associated with determination of the effect of phosphorylation on a few molecules ex vivo with millisecond time resolution. In spite of the remaining uncertainties, we conclude that the state of phosphorylation of RLC had no effect on the rate of dissociation of cross bridges from thin filaments, on the rate of myosin head binding to thin filaments, and on the rate of power stroke. On the other hand, phosphorylation slightly increased the degree of disorder of active cross bridges.

Elimination of autofluorescence in fluorescence correlation spectroscopy using the AzaDiOxaTriAngulenium (ADOTA) fluorophore in combination with time-correlated single-photon counting (TCSPC).

Fluorescence correlation spectroscopy (FCS) is a frequently applied technique that allows for the precise and sensitive analysis of molecular diffusion and interactions. However, the potential of FCS for in vitro or ex vivo studies has not been fully realized due in part to artifacts originating from autofluorescence (fluorescence of inherent components and fixative-induced fluorescence). Here, we propose the azadioxatriangulenium (ADOTA) dye as a solution to this problem. The lifetime of the ADOTA probe, about 19.4 ns, is much longer than most components of autofluorescence. Thus, it can be easily separated by time-correlated single-photon counting methods. Here, we demonstrate the suppression of autofluorescence in FCS using ADOTA-labeled hyaluronan macromolecules (HAs) with Rhodamine 123 added to simulate diffusing fluorescent background components. The emission spectrum and decay rate of Rhodamine 123 overlap with the usual sources of autofluorescence, and its diffusion behavior is well known. We show that the contributions from Rhodamine 123 can be eliminated by time gating or by fluorescence lifetime correlation spectroscopy (FLCS). While the pairing of ADOTA and time gating is an effective strategy for the removal of autofluorescence from fluorescence imaging, the loss of photons leads to erroneous concentration values with FCS. On the other hand, FLCS eliminates autofluorescence without such errors. We then show that both time gating and FLCS may be used successfully with ADOTA-labeled HA to detect the presence of hyaluronidase, the overexpression of which has been observed in many types of cancer.

FRET based ratio-metric sensing of hyaluronidase in synthetic urine as a biomarker for bladder and prostate cancer.

Elevated hyaluronidase levels are found in the urine of bladder and prostate cancer patients. Therefore, HA-ase is regarded as an important biomarker for the detection of these cancers. In this report, we use a FRET based ratiometric sensing approach to detect the level of HA-ase in synthetic urine. For this, we have used a HA-FRET probe (hyaluronan) labeled with fluorescein as a donor and rhodamine as an acceptor. We monitor the digestion of our HA-FRET probe with different concentrations of HA-ase in synthetic urine via fluorescence emission. The extent to which FRET is released depends on the concentration of HA-ase. Our fluorescence intensity results are also supported with time resolved fluorescence decay data. This assay can be used to develop a non-invasive technique for the detection of bladder and/or prostate cancer progression.

Comparison of orientation and rotational motion of skeletal muscle cross-bridges containing phosphorylated and dephosphorylated myosin regulatory light chain.

Calcium binding to thin filaments is a major element controlling active force generation in striated muscles. Recent evidence suggests that processes other than Ca(2+) binding, such as phosphorylation of myosin regulatory light chain (RLC) also controls contraction of vertebrate striated muscle (Cooke, R. (2011) Biophys. Rev. 3, 33-45). Electron paramagnetic resonance (EPR) studies using nucleotide analog spin label probes showed that dephosphorylated myosin heads are highly ordered in the relaxed fibers and have very low ATPase activity. This ordered structure of myosin cross-bridges disappears with the phosphorylation of RLC (Stewart, M. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 430-435). The slower ATPase activity in the dephosporylated moiety has been defined as a new super-relaxed state (SRX). It can be observed in both skeletal and cardiac muscle fibers (Hooijman, P., Stewart, M. A., and Cooke, R. (2011) Biophys. J. 100, 1969-1976). Given the importance of the finding that suggests a novel pathway of regulation of skeletal muscle, we aim to examine the effects of phosphorylation on cross-bridge orientation and rotational motion. We find that: (i) relaxed cross-bridges, but not active ones, are statistically better ordered in muscle where the RLC is dephosporylated compared with phosphorylated RLC; (ii) relaxed phosphorylated and dephosphorylated cross-bridges rotate equally slowly; and (iii) active phosphorylated cross-bridges rotate considerably faster than dephosphorylated ones during isometric contraction but the duty cycle remained the same, suggesting that both phosphorylated and dephosphorylated muscles develop the same isometric tension at full Ca(2+) saturation. A simple theory was developed to account for this fact.

Detection of hyaluronidase activity using fluorescein labeled hyaluronic acid and Fluorescence Correlation Spectroscopy.

The over-expression of hyaluronidase has been observed in many types of cancer, suggesting that it may have utility for diagnosis. Here we present a technique for the detection of hyaluronidase using Fluorescence Correlation Spectroscopy (FCS). Hyaluronan macromolecules (HAs) have been heavily labeled with fluorescein amine resulting in strong self-quenching. In the presence of hyaluronidase, HA is cleaved into smaller, fluorescein-labeled fragments and the self-quenching is released. Such cleavage is manifested by the increased average diffusion rate of the HA fragments, increased concentration of individual, fluorescent HA fragments, and increased intensity. All three of these properties are monitored simultaneously throughout FCS measurements, both as a function of time and hyaluronidase concentration. The method we present provides a sensitive measure of hyaluronidase activity and requires extremely small amounts of the HA substrate.

The mechanical axes of the wrist are oriented obliquely to the anatomical axes.

BACKGROUND: the complex motions of the wrist are described in terms of four anatomical directions that are accomplished through the multiple articulations of the carpus. With minimal tendinous insertions, the carpus is primarily a passive structure. This emphasizes the importance of its mechanical properties, which few studies have examined to date. The purpose of the present study was to determine the mechanical properties of the wrist in twenty-four different directions of wrist motion. METHODS: the moment-rotation mechanical behavior of six fresh-frozen cadaver wrists was determined in four directions: flexion, extension, ulnar deviation, and radial deviation. Twenty other directions that were a combination of these anatomical directions were also studied. A custom-designed jig was interfaced with a standard materials testing system to apply unconstrained moments. Moments of ± 2 Nm were applied, and the moment-rotation data were recorded and analyzed to determine the neutral zone, range of motion, and stiffness values as well as the orientation of the envelope of these values. RESULTS: the envelope of wrist range-of-motion values was ellipsoidal in shape and was oriented obliquely (p < 0.001) to the direction of pure flexion-extension by a mean (and standard deviation) of 26.6° ± 4.4°. The largest wrist range of motion was a mean of 111.5° ± 10.2°, in the direction of ulnar flexion, 30° from pure flexion. The largest stiffness (mean, 0.4 Nm/deg) was in the direction of radial flexion, while the smallest stiffness (mean, 0.15 Nm/deg) was in the direction of ulnar flexion. CONCLUSIONS: the mechanical axes of the wrist are oriented obliquely to the anatomical axes. The primary mechanical direction is one of radial extension and ulnar flexion, a direction along a path of the dart thrower's wrist motion. CLINICAL RELEVANCE: understanding the mechanical function of the wrist can aid clinical treatment decisions, arthroplasty, and implant designs. The findings of this study provide new evidence that the mechanical axes of the wrist are not collinear with the anatomical axes.

Retinal detachment despite aggressive management of aggressive posterior retinopathy of prematurity.

Posterior retinopathy of prematurity (ROP) is unusual in its atypical features and its aggressive, rapidly progressive course. It is more difficult to recognize and to treat, with many of these eyes progressing to retinal detachment despite multiple treatments with laser or cryotherapy. The authors present a case of aggressive posterior ROP refractory to multiple laser treatment. This patient was successfully treated with intravitreal bevacizumab, but required repeat treatment 4 months later. The second injection with bevacizumab was followed by progression to retinal detachment requiring surgery. The patient remains stable after surgery.

Treatment of choroidal neovascularization associated with Best's disease in children.

The development of secondary choroidal neovascularization in Best's disease is rare in the pediatric population. A retrospective review of pediatric patients with choroidal neovascularization secondary to Best's disease was performed. The patients' courses and treatments were recorded. Three patients with choroidal neovascularization were identified. All had decreased vision and were treated in an individual manner. Vision improved after treatment in all patients. Treatment may hasten resolution of choroidal neovascularization and improve vision.

Imaging serpiginous choroidopathy with spectral domain optical coherence tomography.

The use of spectral domain optical coherence tomography (SD-OCT) in the study of chronic serpiginous choroiditis was evaluated. Two patients with chronic serpiginous choroiditis were imaged using two prototype SD-OCT systems (6-microm axial resolution). Raster scans covering 6 x 6 X 2-mm regions of the retina were obtained, enabling the study of different retinal cross-sectional images. Thickness maps were obtained after segmentation of retinal layers, which could be compared with those on follow-up. SD-OCT allowed the visualization of the cross-sectional retinal architecture at different horizontal positions. Superimposition of SD-OCT generated reconstructed fundus images with fundus photographs provided accurate images registration. Segmentation of retinal layers provided thickness maps and higher-density improved visualization of photoreceptor layer, cysts, and atrophy, which was useful in following change in disease activity over time. The researchers concluded that SD-OCT is a useful tool to study disease morphology and follow-up of chronic serpiginous choroiditis.

Infectious scleritis after retinal surgery.

PURPOSE: To report a series of patients in whom infectious scleritis developed after vitreoretinal surgery. DESIGN: Interventional case series of four patients. METHODS: Medical records of patients at a single institution in whom infectious scleritis developed after vitreoretinal surgery were reviewed. RESULTS: In three patients, infectious scleritis developed after 20-gauge pars plana vitrectomy, and in one patient, infectious scleritis developed after a scleral buckling procedure. Three cases were had positive culture results; the identified organisms were Pseudomonas aeruginosa in two cases and methicillin-resistant Staphylococcus aureus in one. The fourth patient did not have culture results but responded rapidly to empiric treatment with moxifloxacin. In one patient, surgically induced necrotizing scleritis subsequently developed. CONCLUSIONS: Although infectious scleritis is an uncommon complication after vitreoretinal surgery, it should be a considered cause in patients with persistent postoperative pain and inflammation.

Short-term safety and efficacy of intravitreal bevacizumab (Avastin) for neovascular age-related macular degeneration.

PURPOSE: To evaluate the safety and efficacy of intravitreal bevacizumab (Avastin, Genentech Inc.) for the treatment of neovascular age-related macular degeneration (ARMD). METHODS: A retrospective review was performed on consented patients with neovascular ARMD receiving intravitreal bevacizumab therapy. All patients received intravitreal bevacizumab at baseline with additional monthly injections given at the discretion of the treating physician. At each visit, a routine Snellen visual acuity assessment was performed followed by an ophthalmic examination and optical coherence tomography (OCT) imaging. RESULTS: Fifty-three eyes of 50 patients received an intravitreal bevacizumab injection between May and August 2005. Including the month 3 visit, the average number of injections was 2.3 out of a maximum of 4 injections. No serious drug-related ocular or systemic adverse events were identified. Improvements in visual acuity and central retinal thickness measurements were evident by week 1 and continued through month 3. At month 3, the mean visual acuity improved from 20/160 to 20/125 (P < 0.001) and the mean central retinal thickness decreased by 99.6 microm (P < 0.001). CONCLUSION: Off-label intravitreal bevacizumab therapy for neovascular ARMD was well tolerated over 3 months with improvements in visual acuity and OCT central retinal thickness measurements. While the long-term safety and efficacy of intravitreal bevacizumab remain unknown, these short-term results suggest that intravitreal bevacizumab may be the most cost effective therapy for the treatment of neovascular ARMD.

Use of autologous platelet concentrate in blepharoplasty surgery.

PURPOSE: To evaluate postoperative edema and ecchymosis after blepharoplasty surgery with or without autologous platelet gel. METHODS: In this prospective, randomized, controlled trial, patients received autologous platelet concentrate in the eyelid incisions of one side during bilateral blepharoplasty surgery. The opposing eye was not treated and was used as a control. Autologous platelet concentrate was prepared by the Harvest system (Harvest Technologies, Plymouth, MA, U.S.A.). The blood was centrifuged and platelet-rich plasma isolated. Platelet-rich plasma was mixed with thrombin and instilled in the wound on a randomly selected side before wound closure. Patients were examined and completed a questionnaire at postoperative days 1, 3, 7, and 30. Photographs were taken at each visit and were graded by masked, trained observers for edema and ecchymosis. RESULTS: Significantly less edema (p<0.05) was noted by the photograders at day 1 and by the patients at day 30. There were non-statistically significant trends toward decreased ecchymosis and edema in the treated group. Questionnaire data showed no significant difference in postoperative pain between the treated and untreated sides. Photographic and questionnaire data showed no clinically meaningful difference between the treated and control sides. CONCLUSIONS: Although there were statistically significant differences in edema using autologous platelet gel in blepharoplasty surgery, trends toward improvement in postoperative edema and ecchymosis did not achieve clinical significance.

HER2 expression in thyroid tumors.

Similarities exist in hormone receptors of breast, prostate, and thyroid tumors. HER2 oncogene expression is known to be present in breast and prostate tumors, but conflicting data have been published about its presence in thyroid tumors. This uncertainty prompted us to examine the incidence of HER2 overexpression in normal and malignant thyroid tissue. Normal and neoplastic thyroid tissue samples from 46 female and 9 male patients were assayed for HER2 expression by immunohistochemical assay. Of the 55 total samples, 36 were from neoplasms and 19 were from benign tissues. Significant HER2 overexpression was not found in any benign or malignant thyroid tissue. Two of 6 thyroid carcinomas from male patients showed 1+ reactivity for HER2 expression on immunohistochemistry assay, but remained negative on fluorescene in sito hybridization confirmatory testing. No significant expression of HER2 was noted in benign or malignant thyroid tissue. These results cast doubt on the value of HER2 as a prognostic factor or possible target for specific antitumor therapy for thyroid cancer.