Sex dependence of opioid-mediated responses to subanesthetic ketamine in rats.

Subanesthetic ketamine is increasingly used for the treatment of varied psychiatric conditions, both on- and off-label. While it is commonly classified as an N-methyl D-aspartate receptor (NMDAR) antagonist, our picture of ketamine's mechanistic underpinnings is incomplete. Recent clinical evidence has indicated, controversially, that a component of the efficacy of subanesthetic ketamine may be opioid dependent. Using pharmacological functional ultrasound imaging in rats, we found that blocking opioid receptors suppressed neurophysiologic changes evoked by ketamine, but not by a more selective NMDAR antagonist, in limbic regions implicated in the pathophysiology of depression and in reward processing. Importantly, this opioid-dependent response was strongly sex-dependent, as it was not evident in female subjects and was fully reversed by surgical removal of the male gonads. We observed similar sex-dependent effects of opioid blockade affecting ketamine-evoked postsynaptic density and behavioral sensitization, as well as in opioid blockade-induced changes in opioid receptor density. Together, these results underscore the potential for ketamine to induce its affective responses via opioid signaling, and indicate that this opioid dependence may be strongly influenced by subject sex. These factors should be more directly assessed in future clinical trials.

Unique pharmacodynamic properties and low abuse liability of the µ-opioid receptor ligand (S)-methadone.

(R,S)-methadone ((R,S)-MTD) is a µ-opioid receptor (MOR) agonist comprised of (R)-MTD and (S)-MTD enantiomers. (S)-MTD is being developed as an antidepressant and is considered an N-methyl-D-aspartate receptor (NMDAR) antagonist. We compared the pharmacology of (R)-MTD and (S)-MTD and found they bind to MORs, but not NMDARs, and induce full analgesia. Unlike (R)-MTD, (S)-MTD was a weak reinforcer that failed to affect extracellular dopamine or induce locomotor stimulation. Furthermore, (S)-MTD antagonized motor and dopamine releasing effects of (R)-MTD. (S)-MTD acted as a partial agonist at MOR, with complete loss of efficacy at the MOR-galanin Gal1 receptor (Gal1R) heteromer, a key mediator of the dopaminergic effects of opioids. In sum, we report novel and unique pharmacodynamic properties of (S)-MTD that are relevant to its potential mechanism of action and therapeutic use. One-sentence summary: (S)-MTD, like (R)-MTD, binds to and activates MORs in vitro, but (S)-MTD antagonizes the MOR-Gal1R heteromer, decreasing its abuse liability.

Mu Opioid Receptor Activation Mediates (S)-ketamine Reinforcement in Rats: Implications for Abuse Liability.

BACKGROUND: (S)-ketamine is an NMDA receptor antagonist, but it also binds to and activates mu opioid receptors (MORs) and kappa opioid receptors in vitro. However, the extent to which these receptors contribute to (S)-ketamine's in vivo pharmacology is unknown. METHODS: We investigated the extent to which (S)-ketamine interacts with opioid receptors in rats by combining in vitro and in vivo pharmacological approaches, in vivo molecular and functional imaging, and behavioral procedures relevant to human abuse liability. RESULTS: We found that the preferential opioid receptor antagonist naltrexone decreased (S)-ketamine self-administration and (S)-ketamine-induced activation of the nucleus accumbens, a key brain reward region. A single reinforcing dose of (S)-ketamine occupied brain MORs in vivo, and repeated doses decreased MOR density and activity and decreased heroin reinforcement without producing changes in NMDA receptor or kappa opioid receptor density. CONCLUSIONS: These results suggest that (S)-ketamine's abuse liability in humans is mediated in part by brain MORs.

6-O-(2-[18F]Fluoroethyl)-6-O-Desmethyl-Diprenorphine ([18F]FE-DPN) Preferentially Binds to Mu Opioid Receptors In Vivo.

PURPOSE: 6-O-(2-[18F]Fluoroethyl)-6-O-desmethyl-diprenorphine ([18F]FE-DPN) is regarded as a non-selective opioid receptor radiotracer. PROCEDURE: Here, we report the first characterization of [18F]FE-DPN synthesized from the novel precursor, 6-O-(2-tosyloxyethoxy)-6-O-desmethyl-3-O-trityl-diprenorphine (TE-TDDPN), using a one-pot, two-step nucleophilic radiosynthesis to image opioid receptors in rats and mice using positron emission tomography. RESULTS: We also show that [18F]FE-DPN and [3H]DPN exhibit negligible brain uptake in mu opioid receptor (MOR) knockout mice. CONCLUSIONS: Taken together with prior findings, our results suggest that [18F]FE-DPN and [3H]DPN preferentially bind to MOR in rodents in vivo.

Identifying key risks to the performance of privately protected areas (PPAs) through theory of change (ToC).

Privately protected areas (PPAs) are internationally considered to be important policy implementation instruments to augment and strengthen protected area networks. However, there has been limited reflection on the performance of PPAs over time. This paper aims to identify key risks to the performance of PPAs as policy implementation instruments through the application of Theory of Change (ToC). Identifying and understanding these risks are important to allow for the evaluation and monitoring of PPA performance. The ToC method was applied to a specific PPA policy instrument namely, private nature reserves (PNRs) in the South African context. The research results produced 29 key assumptions translated into 29 key risks. These risk are critically discussed against existing South African and international literature. To test and refine the risks further it is recommended that they be applied to PPA case studies in different contexts.

Nb3Sn multicell cavity coating system at Jefferson Lab.

Superconducting radio frequency niobium cavities are the building blocks of modern accelerators for scientific applications. Lower surface resistance, higher fields, and high operating temperatures advance the reach of the future accelerators for scientific discovery as well as potentially enabling cost-effective industrial solutions. We describe the design and performance of an Nb3Sn coating system that converts the inner surface of niobium cavities to an Nb3Sn film. The niobium surface, heated by radiation from the niobium retort, is exposed to Sn and SnCl2 vapor during the heat cycle, which results in about 2 µm Nb3Sn film on the niobium surface. Film composition and structure as well as radio frequency properties with 1-cell R&D cavities and 5-cell practical accelerator cavities are presented.

Multiple Sclerosis Performance Test: validation of self-administered neuroperformance modules.

BACKGROUND AND PURPOSE: The purpose was to determine the test-retest reliability, practice effects, convergent validity and sensitivity to multiple sclerosis (MS) disability of neuroperformance subtests from the patient self-administered Multiple Sclerosis Performance Test (MSPT) designed to assess low contrast vision (Contrast Sensitivity Test, CST), upper extremity motor function (Manual Dexterity Test, MDT) and lower extremity motor function (Walking Speed Test, WST) and to introduce the concept of regression-based norms to aid clinical interpretation of performance scores using the MSPT cognition test (Processing Speed Test, PST) as an example. METHODS: Substudy 1 assessed test-retest reliability, practice effects and convergent validity of the CST, MDT and WST in 30 MS patients and 30 healthy controls. Substudy 2 examined sensitivity to MS disability in over 600 MS patients as part of their routine clinic assessment. Substudy 3 compared performance on the PST in research volunteers and clinical samples. RESULTS: The CST, MDT and WST were shown to be reliable, valid and sensitive to MS outcomes. Performance was comparable to technician-administered testing. PST performance was poorer in the clinical sample compared with the research volunteer sample. CONCLUSIONS: The self-administered MSPT neuroperformance modules produce reliable, objective metrics that can be used in clinical practice and support outcomes research. Published studies which require patient voluntary consent may underestimate the rate of cognitive dysfunction observed in a clinical setting.

Comparative analysis of two femtosecond LASIK platforms using iTRAQ quantitative proteomics.

PURPOSE: New femtosecond laser platforms may reduce ocular surface interference and LASIK-associated dry eye. This study investigated tear protein profiles in subjects who underwent LASIK using two femtosecond lasers to assess differences in protein expression. METHODS: This was a randomized interventional clinical trial involving 22 patients who underwent femtosecond laser refractive surgery with a contralateral paired eye design. Corneal flaps of 22 subjects were created by either Visumax or Intralase laser. Tear samples were collected preoperatively, and at 1 week and 3 months postoperatively using Schirmer's strips. Tear protein ratios were calculated relative to preoperative protein levels at baseline. The main outcome measures were the levels of a panel of dry eye protein markers analyzed using isobaric tagging for relative and absolute quantitation (iTRAQ) mass spectrometry. RESULTS: A total of 824 unique proteins were quantifiable. Tear protein ratios were differentially regulated between the eyes treated with different lasers. The secretoglobulins Lipophilin A (1.80-fold) and Lipophilin C (1.77) were significantly upregulated (P < 0.05) at 1 week postoperatively in Visumax but not in Intralase-treated eyes. At 1 week, orosomucoid1 was upregulated (1.78) in Intralase but not Visumax-treated eyes. In the same eyes, lysozyme, cathepsin B, and lipo-oxygenase were downregulated at 0.44-, 0.64-, and 0.64-folds, respectively. Transglutaminase-2 was downregulated in both groups of eyes. CONCLUSIONS: Different laser platforms induce distinct biological responses in the cornea and ocular surface, which manifests as different levels of tear proteins. This study has implications for surgical technology and modulation of wound healing responses. (ClinicalTrials.gov number, NCT01252654.).

Molecular stress-inducing compounds increase osteoclast formation in a heat shock factor 1 protein-dependent manner.

Many anticancer therapeutic agents cause bone loss, which increases the risk of fractures that severely reduce quality of life. Thus, in drug development, it is critical to identify and understand such effects. Anticancer therapeutic and HSP90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) causes bone loss by increasing osteoclast formation, but the mechanism underlying this is not understood. 17-AAG activates heat shock factor 1 (Hsf1), the master transcriptional regulator of heat shock/cell stress responses, which may be involved in this negative action of 17-AAG upon bone. Using mouse bone marrow and RAW264.7 osteoclast differentiation models we found that HSP90 inhibitors that induced a heat shock response also enhanced osteoclast formation, whereas HSP90 inhibitors that did not (including coumermycin A1 and novobiocin) did not affect osteoclast formation. Pharmacological inhibition or shRNAmir knockdown of Hsf1 in RAW264.7 cells as well as the use of Hsf1 null mouse bone marrow cells demonstrated that 17-AAG-enhanced osteoclast formation was Hsf1-dependent. Moreover, ectopic overexpression of Hsf1 enhanced 17-AAG effects upon osteoclast formation. Consistent with these findings, protein levels of the essential osteoclast transcription factor microphthalmia-associated transcription factor were increased by 17-AAG in an Hsf1-dependent manner. In addition to HSP90 inhibitors, we also identified that other agents that induced cellular stress, such as ethanol, doxorubicin, and methotrexate, also directly increased osteoclast formation, potentially in an Hsf1-dependent manner. These results, therefore, indicate that cellular stress can enhance osteoclast differentiation via Hsf1-dependent mechanisms and may significantly contribute to pathological and therapeutic related bone loss.

Context-dependent role of Grb7 in HER2+ve and triple-negative breast cancer cell lines.

Grb7 is an adapter protein, aberrantly co-overexpressed with HER2 and identified as an independent prognostic marker in breast cancer. It has been established that Grb7 exacerbates the cellular growth and migratory behaviour of HER2+ve breast cancer cells. Less is known about Grb7's role in the context of HER2-ve cells. Here we directly compare the effect of stable Grb7 knockdown in oestrogen sensitive (T47D), HER2+ve (SKBR3) and triple-negative (MDA-MB-468 and MDA-MB-231) breast cancer cell lines on anchorage dependent and independent cell growth, wound healing and chemotaxis. All cell lines showed reduced ability to migrate upon Grb7 knockdown, despite their greatly varied endogenous levels of Grb7. Decreased cell proliferation was not observed in any of the cell lines upon Grb7 knockdown; however, decreased ability to form colonies was observed for all but the oestrogen sensitive cell line, depending upon the stringency of the growth conditions. The data reveal that Grb7 plays an important role in breast cancer progression, beyond the context of HER2+ve cell types.

Patient and surgeon experience during laser in situ keratomileusis using 2 femtosecond laser systems.

PURPOSE: To describe the subjective experience of patients and surgeons during laser in situ keratomileusis (LASIK) using the Intralase 60 kHz or the Visumax 500 kHz femtosecond laser. SETTING: Singapore National Eye Centre, Singapore. DESIGN: Prospective randomized clinical study. METHODS: In myopic patients, LASIK was performed with the corneal flap created using the 60 kHz laser in 1 eye and the 500 kHz laser in the contralateral eye. Postoperatively, patients completed a standardized validated questionnaire about their subjective intraoperative experiences (eg, light perception, pain, fear). Surgeons reported their intraoperative experiences and preferences. RESULTS: Loss of light perception occurred in 50.0% of 60 kHz laser cases and 0% of 500 kHz laser cases during docking and in 63.0% and 0% of cases, respectively, during laser flap creation (P < .0001). The mean pain score with the 60 kHz laser was significantly higher during docking (P < .0001) but not during laser flap cutting (P = .006). Subconjunctival hemorrhage occurred in 67.4% of eyes with the 60 kHz laser and in 2.2% of eyes with the 500 kHz laser (P < .0001). The 500 kHz laser was preferred by 78.3% of patients, while 21.7% preferred the 60 kHz laser (P < .0001). The surgeons preferred the 60 kHz laser in 50.0% of cases and the 500 kHz laser in 8.7% (P < .0001); 41.3% had no preference. CONCLUSIONS: Patients preferred surgery with the 500 kHz laser with no loss of light perception, less pain, less fear, and less subconjunctival hemorrhage. Surgeons preferred the 60 kHz laser.

Comparison of efficacy and safety of laser in situ keratomileusis using 2 femtosecond laser platforms in contralateral eyes.

PURPOSE: To compare the efficacy, predictability, and refractive outcomes of laser in situ keratomileusis (LASIK) using 2 femtosecond platforms for flap creation. SETTING: Multisurgeon single center. DESIGN: Clinical trial. METHODS: Bilateral femtosecond LASIK was performed using the Wavelight Allegretto Eye-Q 400 Hz excimer laser system. The Visumax femtosecond platform (Group 1) was used to create the LASIK flap in 1 eye, while the Intralase femtosecond platform (Group 2) was used to create the LASIK flap in the contralateral eye. The preoperative, 1-month, and 3-month postoperative visual acuities, refraction, and contrast sensitivity in the 2 groups were compared. RESULTS: The study enrolled 45 patients. Three months after femtosecond LASIK, 79.5% of eyes in Group 1 and 82.1% in Group 2 achieved an uncorrected distance visual acuity of 20/20 (P=.808). The mean efficacy index was 0.97 in Group 1 and 0.98 in Group 2 at 3 months (P=.735); 89.7% of eyes in Group 1 and 84.6% of eyes in Group 2 were within ± 0.50 diopter of emmetropia at 3 months (P=.498). No eye in either group lost more than 2 lines of corrected distance visual acuity. The mean safety index at 3 months was 1.11 in Group 1 and 1.10 in Group 2 (P=.570). CONCLUSION: The results of LASIK with both femtosecond lasers were similar, and both platforms produced efficacious and predictable LASIK outcomes. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Comparison of corneal sensitivity, tear function and corneal staining following laser in situ keratomileusis with two femtosecond laser platforms.

PURPOSE: To evaluate longitudinal changes in corneal sensitivity, tear function, and corneal staining in patients who underwent laser in situ keratomileusis (LASIK) using two different femtosecond lasers. METHODS: In a prospective, randomized clinical trial, contralateral eyes of 45 patients underwent flap creation by either VisuMax or IntraLase™ femtosecond laser. Corneal sensitivity, tear break up time (TBUT), Schirmer's test, and corneal fluorescein staining were assessed preoperatively and at 1 week, 1 month, and 3 months postoperatively. RESULTS: There were no statistical differences in any clinical outcome measure between the two femtosecond lasers (P > 0.05), although there was a trend towards slightly lower reductions for corneal sensitivity and TBUT in VisuMax-operated eyes. Overall, corneal sensitivity was significantly reduced at 1 week (P < 0.05), 1 month (P < 0 .001), and 3 months (P < 0.001) postoperatively. A significantly greater reduction of corneal sensitivity was noted in eyes with a myopic spherical equivalent of -6.00 diopters (D) to -11.25 D as compared with eyes that had a relatively lower level of myopia of less than -6.00 D (P < 0.001). TBUT and Schirmer's test values were significantly diminished at 1 week postoperatively (P < 0.04). Overall, corneal staining was significantly increased at 1 week postoperatively (P < 0.001). The level of myopia did not significantly affect postoperative changes in TBUT, Schirmer's test values, or corneal staining (P > 0.05). CONCLUSION: This study showed that changes in corneal sensitivity, tear function, and corneal staining were statistically similar in LASIK using VisuMax and IntraLase femtosecond lasers for flap creation. However, the trend towards faster recovery of corneal sensitivity and TBUT observed in VisuMax-operated eyes may be attributable to improved technical specifications.

The discovery of phenylbenzamide derivatives as Grb7-based antitumor agents.

Grb7 is a non-catalytic protein, the overexpression of which has been associated with the proliferative and migratory potentials of cancer cells. Virtual screening strategies involving a shape-based similarity search, molecular docking, and 2D-similarity searches complemented by experimental binding studies (Thermofluor and isothermal titration calorimetry) resulted in the identification of nine novel phenylbenzamide-based antagonists of the Grb7 SH2 domain. Moderate binding affinities were observed, ranging from K(d)=32.3 µM for lead phenylbenzamide NSC 104999 (1) to K(d)=1.1 µM for a structurally related compound, NSC 57148 (2). Deconvolution of the affinity data into its components revealed differences in lead binding, from being entropy based (lead 1) to enthalpically driven (NSC 100874 (3), NSC 55158 (4), and compound 2). Finally, the lead compound 1 was found to decrease the growth of MDA-MB-468 breast cancer cells, with an IC(50) value of 39.9 µM. It is expected that these structures will serve as novel leads in the development of Grb7-based anticancer therapeutics.

Calorimeters for precision power dissipation measurements on controlled-temperature superconducting radiofrequency samples.

Two calorimeters, with stainless steel and Cu as the thermal path material for high precision and high power versions, respectively, have been designed and commissioned for the 7.5 GHz surface impedance characterization system at Jefferson Lab to provide low temperature control and measurement for CW power up to 22 W on a 5 cm diameter disk sample which is thermally isolated from the radiofrequency (RF) portion of the system. A power compensation method has been developed to measure the RF induced power on the sample. Simulation and experimental results show that with these two calorimeters, the whole thermal range of interest for superconducting radiofrequency materials has been covered. The power measurement error in the interested power range is within 1.2% and 2.7% for the high precision and high power versions, respectively. Temperature distributions on the sample surface for both versions have been simulated and the accuracy of sample temperature measurements have been analyzed. Both versions have the ability to accept bulk superconductors and thin film superconducting samples with a variety of substrate materials such as Al, Al(2)O(3), Cu, MgO, Nb, and Si.

Note: Radio frequency surface impedance characterization system for superconducting samples at 7.5 GHz.

A radio frequency (RF) surface impedance characterization (SIC) system that uses a novel sapphire-loaded niobium cavity operating at 7.5 GHz has been developed as a tool to measure the RF surface impedance of flat superconducting material samples. The SIC system can presently make direct calorimetric RF surface impedance measurements on the central 0.8 cm(2) area of 5 cm diameter disk samples from 2 to 20 K exposed to RF magnetic fields up to 14 mT. To illustrate system utility, we present first measurement results for a bulk niobium sample.

Laser in situ keratomileusis flap measurements: Comparison between observers and between spectral-domain and time-domain anterior segment optical coherence tomography.

PURPOSE: To evaluate the between-observer (interobserver) and between-instrument (intraobserver) variability in flap thickness measurements after laser in situ keratomileusis (LASIK) using spectral-domain and time-domain anterior segment optical coherence tomography (AS-OCT). SETTING: Singapore National Eye Centre. DESIGN: Evaluation of diagnostic test or technology. METHODS: Two independent masked observers measured flap thickness 1 month after LASIK using spectral-domain (RTVue) or time-domain (Visante) AS-OCT. The measurements were taken at central (0.0 mm), -1.5 mm, and +1.5 mm locations. Measurements were repeated to assess between-instrument variability. RESULTS: There was no statistically significant difference in mean flap thickness between the 2 observers at -1.5 mm, 0.0 mm, and +1.5 mm on spectral-domain AS-OCT and at -1.5 mm and +1.5 mm on time-domain AS-OCT (P < .01). There was a statistically significant difference between the 2 observers in the central (0.0 mm) values on time-domain AS-OCT (P=.0008). There was stronger interobserver correlation for spectral-domain AS-OCT at -1.5 mm (r = 0.82), 0.0 mm (r = 0.88), and +1.5 mm (r = 0.88) than for time-domain AS-OCT (r = 0.73, r = 0.62, and r = 0.79, respectively). There was no statistically significant difference in between-instrument measurements. There was stronger between-instrument correlation with spectral-domain AS-OCT than with time-domain AS-OCT at all locations. The mean standard deviation (measure of instrument repeatability) for spectral-domain AS-OCT was 4.19 µm. CONCLUSION: Spectral-domain AS-OCT had closer agreement in between-observer and between-instrument measurements than time-domain AS-OCT and provided more consistent measurements of post-LASIK flap thickness.

Uptake of a cell permeable G7-18NATE contruct into cells and binding with the Grb-7-SH2 domain.

Grb7 is an adapter protein found to be overexpressed in several breast and other cancer cell types along with ErbB2. Grb7 is normally an interaction partner with focal adhesion kinase and in cancer cells also aberrantly interacts with ErbB2. It is thus implicated in the migratory and proliferative potential of cancer cells. Previous studies have shown that the phage display-derived cyclic nonphosphorylated inhibitor peptide, G7-18NATE, when linked to Penetratin, is able to interfere with the interaction of Grb7 with its upstream binding partners and to impact on both cell migration and proliferation. Here we report the synthesis of a biotinylated G7-18NATE covalently attached to just the last seven residues of Penetratin (G7-18NATE-P-Biotin). We demonstrate that this construct is taken up efficiently into MDA-MB-468 breast cancer cells and colocalizes with Grb7 in the cytoplasm. We also used isothermal titration calorimetry to determine the binding affinity of G7-18NATE-P-Biotin to the Grb7-SH2 domain, and showed that it binds with micromolar affinity (K(d) = 14.4 microM), similar to the affinity of G7-18NATE (K(d) = 35.4 microM). Together this shows that this shorter G7-18NATE-P-Biotin construct is suitable for further studies of the antiproliferative and antimigratory potential of this inhibitor.

Benzopyrazine derivatives: A novel class of growth factor receptor bound protein 7 antagonists.

Growth factor receptor bound protein 7 (Grb7) is an adapter protein that functions as a downstream effector of growth factor mediated signal transduction. Over-expression of Grb7 has been implicated in a variety of cancers such as breast, blood, pancreatic, esophageal, and gastric carcinomas. Inhibition of Grb7 has been shown to reduce the migratory and proliferative potential of these cancers, making it an attractive therapeutic target. Starting with a known peptide antagonist, the present work reports the application of a succession of computational ligand design tools comprising a ligand shape based similarity search, molecular docking and a 2D-similarity search to identify small molecular antagonists of the Grb7-SH2 domain from the NCI chemical database. Binding to the Grb7-SH2 domain was then experimentally tested using melting point shift assays and isothermal titration calorimetry. Overall, a total of 11 benzopyrazine based small molecular antagonists were identified with affinity for the Grb7-SH2 domain. Representative compounds tested using ITC were revealed to possess moderate binding affinity in the low micromolar range. Finally, the lead compound (NSC642056) was found to reduce the growth of a Grb7-expressing breast cancer cell line with an IC(50) of 86µM. It is expected that the identified antagonists will be useful additions to further explore the function of Grb7 and for the development of inhibitors with therapeutic potential.

Photodynamic Therapy with Verteporfin for Corneal Neovascularisation.

Corneal neovascularization can be a difficult problem to treat. The authors describe a patient with lipid keratopathy secondary to corneal neovascularization treated with photodynamic therapy. Six months following treatment the neovascularization has not returned and the lipid keratopathy has not increased in size. No significant side effects from the treatment occurred. Photodynamic therapy with Verteporfin was a useful treatment modality in this case of corneal neovascularization with associated lipid keratopathy.