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This White Paper presents the science case of an Electron-Ion Collider (EIC), focused on the structure and interactions of gluon-dominated matter, with the intent to articulate it to the broader nuclear science community. It was commissioned by the managements of Brookhaven National Laboratory (BNL) and Thomas Jefferson National Accelerator Facility (JLab) with the objective of presenting a summary of scientific opportunities and goals of the EIC as a follow-up to the 2007 NSAC Long Range plan. This document is a culmination of a community-wide effort in nuclear science following a series of workshops on EIC physics over the past decades and, in particular, the focused ten-week program on “Gluons and quark sea at high energies” at the Institute for Nuclear Theory in Fall 2010. It contains a brief description of a few golden physics measurements along with accelerator and detector concepts required to achieve them. It has been benefited profoundly from inputs by the users’ communities of BNL and JLab. This White Paper offers the promise to propel the QCD science program in the US, established with the CEBAF accelerator at JLab and the RHIC collider at BNL, to the next QCD frontier.

Predictions made in Albacete et al. [Int. J. Mod. Phys. E 22 (2013) 1330007] prior to the LHC p+Pb run at sNN = 5 TeV are compared to currently available data. Some predictions shown here have been updated by including the same experimental cuts as the data. Some additional predictions are also presented, especially for quarkonia, that were provided to the experiments before the data were made public but were too late for the original publication.

Multiple scattering and induced parton splitting lead to a medium modification of the QCD evolution for jet fragmentation functions and final hadron spectra. Medium-induced parton splittings not only lead to energy loss of leading partons and suppression of leading hadron spectra, but also modify the flavor composition of a jet due to induced flavor conversion via gluon emission and quark pair production and annihilation. Through a numerical study of the medium-modified QCD evolution, leading K- strange meson spectra are found to be particularly sensitive to the medium-induced flavor conversion in semi-inclusive deeply inelastic scatterings (SIDIS) off a large nucleus. The induced flavor conversion can lead to an increased number of gluons and sea quarks in a jet shower and, as a consequence, enhance the leading K- spectrum to counter the effect of parton energy loss in SIDIS with large momentum fractions xB, where the struck quarks are mostly valence quarks of the nucleus.

Rod photoreceptor neurons in the retina detect scotopic light by packaging large quantities of the visual pigment protein rhodopsin (Rho) into stacked membrane discs within their outer segments (OS). Efficient Rho trafficking to the OS through the inner rod compartments is critical for long-term rod health since diseases like retinitis pigmentosa (RP) induce Rho mislocalization in these inner compartments, including in the rod presynaptic terminals (i.e., "spherules"). Given the importance of protein trafficking to the OS, less is known about the trafficking of rod synaptic proteins that maintain critical synapses between rods and inner retina neurons. Furthermore, the subcellular impact of Rho mislocalization on rod spherules has not been investigated. In this study we used super-resolution and electron microscopies, along with proteomic measurements of rod synaptic proteins, to perform an intensive subcellular analysis of Rho synaptic mislocalization in P23H-Rho-RFP RP mutant mice of both sexes. We discovered mutant P23H-Rho-RFP protein mislocalized in distinct ER aggregations within the spherule cytoplasm which we confirmed in wild type (WT) rods overexpressing P23H-Rho-RFP. Additionally, we found significant protein abundance differences in Dystrophin, BASSOON, ELFN1 and other synaptic proteins in P23H-Rho-RFP mice. By comparison, Rho mislocalized along the spherule plasma membrane in WT rods and in rd10 RP mutant rods, in which there was no synaptic protein disruption. Throughout the study, we also identified a network of ER membranes within WT rod presynaptic spherules. Together, our findings establish a previously uncharacterized ER-based secretory system that mediates normal trafficking and turnover at mouse rod synapses., Significance Statement: In the retina, protein trafficking to the outer segments in rod photoreceptor neurons is essential for vision; however, less is known about protein trafficking to the synapses that rods form with downstream retinal neurons. Stressors like retinitis pigmentosa (RP) and other inherited retinal diseases cause widespread rhodopsin (Rho) protein mislocalization in rods, including at the presynaptic terminals. This study examines the subcellular impact of Rho mislocalization on this presynaptic region caused by the P23H-Rho RP mutation and in other contexts. Mutant P23H-Rho-RFP fusion endoplasmic reticulum (ER) aggregation disrupted rod-specific synaptic protein levels, and combined with the detection of an endogenous presynaptic ER network in rods, this study supports a disease-relevant ER-based protein trafficking and turnover mechanism at rod synapses.

Neurodegenerative diseases are characterized by the presence of misfolded and aggregated proteins which are thought to contribute to the development of the disease. In one form of inherited blinding disease, retinitis pigmentosa, a P23H mutation in the light-sensing receptor, rhodopsin causes rhodopsin misfolding resulting in complete vision loss. We investigated whether a xenogeneic protein-unfolding ATPase (unfoldase) from thermophilic Archaea, termed PANet, could counteract the proteotoxicity of P23H rhodopsin. We found that PANet increased the number of surviving photoreceptors in P23H rhodopsin mice and recognized rhodopsin as a substate in vitro. This data supports the feasibility and efficacy of using a xenogeneic unfoldase as a therapeutic approach in mouse models of human neurodegenerative diseases. We also showed that an archaeal proteasome, called the T20S can degrade rhodopsin in vitro and demonstrated that it is feasible and safe to express gateless T20S proteasomes in vivo in mouse rod photoreceptors. Expression of archaeal proteasomes may be an effective therapeutic approach to stimulate protein degradation in retinopathies and neurodegenerative diseases with protein-misfolding etiology., Competing Interests: No competing interests is declared., (Copyright: © 2024 Brooks et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Long-and middle-wavelength cone photoreceptors, which are responsible for our visual acuity and color vision, comprise ~95% of our total cone population and are concentrated in the fovea of our retina. Previously, we characterized the disease mechanisms of the L/M-cone opsin missense mutations N94K, W177R, P307L, R330Q and G338E, all of which are associated with congenital blue cone monochromacy (BCM) or color-vision deficiency. Here, we used a similar viral vector-based gene delivery approach in M-opsin knockout mice to investigate the pathogenic consequences of the BCM or color-vision deficient associated L-cone opsin (OPN1LW) mutants K82E, P187S, and M273K. We investigated their subcellular localization, the pathogenic effects on cone structure, function, and cone viability. K82E mutants were detected predominately in cone outer segments, and its expression partially restored expression and correct localization of cone PDE6α' and cone transducin γ. As a result, K82E also demonstrated the ability to mediate cone light responses. In contrast, expression of P187S was minimally detected by either western blot or by immunohistochemistry, probably due to efficient degradation of the mutant protein. M273K cone opsin appeared to be misfolded as it was primarily localized to the cone inner segment and endoplasmic reticulum. Additionally, M273K did not restore the expression of cone PDE6α' and cone transducin γ in dorsal cone OS, presumably by its inability to bind 11- cis retinal. Consistent with the observed expression pattern, P187S and M273K cone opsin mutants were unable to mediate light responses. Moreover, expression of K82E, P187S, and M273K mutants reduced cone viability. Due to the distinct expression patterns and phenotypic differences of these mutants observed in vivo , we suggest that the pathobiological mechanisms of these mutants are distinct., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Sechrest, Barbera, Ma, Dyka, Ahn, Brothers, Cahill, Hall, Baehr and Deng.)

An arginine to cysteine substitution at amino acid position 203 (C203R) is the most common missense mutation in human cone opsin. Linked to color blindness and blue cone monochromacy (BCM), C203 is involved in a crucial disulfide bond required for proper folding. It has previously been postulated that expression of mutant C203R cone opsin exerts a toxic effect on cone photoreceptors, similar to some well-characterized missense mutations in rhodopsin that lead to protein misfolding. In this study, we generated and characterized a BCM mouse model carrying the equivalent C203R mutation (Opn1mwC198R Opn1sw-/-) to investigate the disease mechanism and develop a gene therapy approach for this disorder. Untreated Opn1mwC198R Opn1sw-/- cones phenocopied affected cones in human patients with the equivalent mutation, exhibiting shortened or absent cone outer segments and loss of function. We determined that gene augmentation targeting cones specifically yielded robust rescue of cone function and structure when Opn1mwC198R Opn1sw-/- mice were treated at early ages. Importantly, treated cones displayed elaborated outer segments and replenished expression of crucial cone phototransduction proteins. Interestingly, we were unable to detect OPN1MWC198R mutant opsin at any age. We believe this is the first proof-of-concept study exploring the efficacy of gene therapy in BCM associated with a C203R mutation.

An eco-friendly and metal-free method for the synthesis of tetrahydrodibenzo[ b , g ][1,8]naphthyridin-1(2 H )-ones was established. Quinoline-derived dipolarophiles and cyclic enaminones as starting materials undergo a 1,4-Michael addition/S N Ar tandem annulation reaction affording the target products. This approach features transition metal-free conditions, good functional group tolerance and operational simplicity.

Blue cone monochromacy (BCM) is a congenital vision disorder characterized by complete loss or severely reduced long- and middle-wavelength cone function, caused by mutations in the OPN1LW/OPN1MW gene cluster on the X-chromosome. BCM patients typically suffer from poor visual acuity, severely impaired color discrimination, myopia, and nystagmus. In this review, we cover the genetic causes of BCM, clinical features of BCM patients, genetic testing, and clinical outcome measurements for future BCM clinical trials. However, our emphasis is on detailing the animal models for BCM and gene therapy using adeno-associated vectors (AAV). We describe two mouse models resembling the two most common causes of BCM, current progress in proof-of-concept studies to treat BCM with deletion mutations, the challenges we face, and future directions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Optogenetic gene therapies offer a promising strategy for restoring vision to patients with retinal degenerative diseases, such as retinitis pigmentosa (RP). Several clinical trials have begun in this area using different vectors and optogenetic proteins (Clinical Identifiers: NCT02556736, NCT03326336, NCT04945772, and NCT04278131). Here we present preclinical efficacy and safety data for the NCT04278131 trial, which uses an AAV2 vector and Chronos as the optogenetic protein. Efficacy was assessed in mice in a dose-dependent manner using electroretinograms (ERGs). Safety was assessed in rats, nonhuman primates, and mice, using several tests, including immunohistochemical analyses and cell counts (rats), electroretinograms (nonhuman primates), and ocular toxicology assays (mice). The results showed that Chronos-expressing vectors were efficacious over a broad range of vector doses and stimulating light intensities, and were well tolerated: no test article-related findings were observed in the anatomical and electrophysiological assays performed., Competing Interests: B.Y., W.W.H., and S.N. have financial interests in Bionic Sight, Inc. B.Y. is a consultant for Bionic Sight, Inc. S.N. is the principal and founder of Bionic Sight, Inc. W.W.H. is a cofounder of Bionic Sight, Inc., (© 2023 The Author(s).)

Blue cone monochromacy (BCM) is a congenital vision disorder affecting both middle-wavelength (M) and long-wavelength (L) cone photoreceptors of the human retina. BCM results from abolished expression of green and red light-sensitive visual pigments expressed in M- and L-cones, respectively. Previously, we showed that gene augmentation therapy to deliver either human L- or M-opsin rescues dorsal M-opsin dominant cone photoreceptors structurally and functionally in treated M-opsin knockout ( Opn1mw -/- ) mice. Although Opn1mw -/- mice represent a disease model for BCM patients with deletion mutations, at the cellular level, dorsal cones of Opn1mw -/- mice still express low levels of S-opsin, which are different from L- and M-cones of BCM patients carrying a congenital opsin deletion. To determine whether BCM cones lacking complete opsin expression from birth would benefit from AAV-mediated gene therapy, we evaluated the outcome of gene therapy, and determined the therapeutic window and longevity of rescue in a mouse model lacking both M- and S-opsin ( Opn1mw -/- /Opn1sw -/- ). Our data show that cones of Opn1mw -/- /Opn1sw -/- mice are viable at younger ages but undergo rapid degeneration. AAV-mediated expression of human L-opsin promoted cone outer segment regeneration and rescued cone-mediated function when mice were injected subretinally at 2 months of age or younger. Cone-mediated function and visually guided behavior were maintained for at least 8 months post-treatment. However, when mice were treated at 5 and 7 months of age, the chance and effectiveness of rescue was significantly reduced, although cones were still present in the retina. Crossing Opn1mw -/- /Opn1sw -/- mice with proteasomal activity reporter mice (Ub G76V -GFP) did not reveal GFP accumulation in Opn1mw -/- /Opn1sw -/- cones eliminating impaired degradation of ubiquitinated proteins as stress factor contributing to cone loss. Our results demonstrate that AAV-mediated gene augmentation therapy can rescue cone structure and function in a mouse model with a congenital opsin deletion, but also emphasize the importance that early intervention is crucial for successful therapy.

Cone photoreceptors are responsible for the visual acuity and color vision of the human eye. Red/green cone opsin missense mutations N94K, W177R, P307L, R330Q, and G338E have been identified in subjects with congenital blue cone monochromacy or color-vision deficiency. Studies on disease mechanisms due to these cone opsin mutations have been previously carried out exclusively in vitro, and the reported impairments were not always consistent. Here we expressed these mutants via AAV specifically in vivo in M-opsin knockout mouse cones to investigate their subcellular localization, the pathogenic effects on cone structure, function, and cone viability. We show that these mutations alter the M-opsin structure, function, and localization. N94K and W177R mutants appeared to be misfolded since they localized exclusively in cone inner segments and endoplasmic reticulum. In contrast, P307L, R330Q, and G338E mutants were detected predominately in cone outer segments. Expression of R330Q and G338E, but not P307L opsins, also partially restored expression and correct localization of cone PDE6α' and cone transducin γ and resulted in partial rescue of M-cone-mediated light responses. Expression of W177R and P307L mutants significantly reduced cone viability, whereas N94K, R330Q, and G338E were only modestly toxic. We propose that although the underlying biochemical and cellular defects caused by these mutants are distinct, they all seem to exhibit a dominant phenotype, resembling autosomal dominant retinitis pigmentosa associated with the majority of rhodopsin missense mutations. The understanding of the molecular mechanisms associated with these cone opsin mutants is fundamental to developing targeted therapies for cone dystrophy/dysfunction., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Purpose: Postoperative ileus (POI) after abdominal surgery is associated with prolonged hospital stay and increased costs. The aim of this study is to investigate the incidence of, risk factors for, and outcomes associated with POI in patients undergoing hysterectomy for benign indications., Methods: A retrospective review of 1017 consecutive patients undergoing benign hysterectomy over the period 2012-2017 in a single center was performed. POI was predefined as absence of flatus and defecation for more than 2 days with the presence of one or more of the following symptoms: nausea, vomiting, and abdominal distention. The association between perioperative variables and the risk of POI was evaluated by univariate analysis. Independent risk factors were identified by multivariate logistic regression analysis., Results: Overall incidence of POI was 9.2%. Incidence of POI did not differ significantly among three different surgical approaches (abdominal hysterectomy, 10.6%; laparoscopic hysterectomy, 7.8%; vaginal hysterectomy, 11.3%; P = 0.279). Independent risk factors of POI identified by multivariate analysis included anesthesia technique (odds ratio [OR] 2.662, 95% interval [CI] 1.533-4.622, P = 0.001), adhesiolysis (odds ratio [OR] 1.818, 95% interval [CI] 1.533-4.622, P = 0.011), duration of operation (odds ratio [OR] 1.005, 95% interval [CI] 0.942-6.190, P = 0.029), previous cancer (odds ratio [OR] 4.789, 95% interval [CI] 1.232-18.626, P = 0.024), and dysmenorrhea (odds ratio [OR] 1.859, 95% interval [CI] 1.182-2.925, P = 0.007)., Conclusion: POI is a common complication after hysterectomy. This study identified risk factors of POI specifically for gynecologic patients. Patients exposed to these factors should be monitored closely for the development POI.

Rationale and Objective: The presence of three conspecifics prevents stress-induced decreases in newly proliferated cells and neuroblasts in mouse dentate gyrus (DG). In this study, we sought to determine how many conspecifics are required to exert these protective effects against stress. In addition, we manipulated the physiological status of those conspecifics in the context of their stress-buffering effects and used airborne oxytocin exposure as a substitute for the presence of conspecifics., Materials and Methods: Bromodeoxyuridine staining was used to indicate the newly proliferated cells and co-staining with doublecortin to reveal the proliferative neuroblasts., Results: Presentation of three intact and lipopolysaccharide-treated conspecifics prevented the stress-induced decreases in the number of newly proliferated cells and neuroblasts in DG. Presentation of one saline- or oxytocin (OT)-treated conspecific did not exert observable stress-buffering effects. In contrast, airborne oxytocin prevented the stress-induced decreases in DG cell proliferation and early neurogenesis, while pretreatment with L-371,257, a selective OT receptor antagonist, abolished the buffering effects of OT., Conclusions: Physical interaction with the conspecifics and conspecifics' sickness, at best, play a minor role in mediating the buffering effects against stress-induced decreases in DG cell proliferation or early neurogenesis. Moreover, stress-buffering effects are negligible with the presence of only one conspecific. Finally, airborne OT produced stress-buffering effects possibly via its stimulation of OT receptors. Oxytocin merits further study as a substitute for the stress-buffering effects of companions.

Purpose: Previously we showed that AAV5-mediated expression of either human M- or L-opsin promoted regrowth of cone outer segments and rescued M-cone function in the treated M-opsin knockout (Opn1mw-/-) dorsal retina. In this study, we determined cone viability and window of treatability in aged Opn1mw-/- mice., Methods: Cone viability was assessed with antibody against cone arrestin and peanut agglutinin (PNA) staining. The rate of cone degeneration in Opn1mw-/- mice was quantified by PNA staining. AAV5 vector expressing human L-opsin was injected subretinally into one eye of Opn1mw-/- mice at 1, 7, and 15 months old, while the contralateral eyes served as controls. M-cone-mediated retinal function was analyzed 2 and 13 months postinjection by full-field ERG. L-opsin transgene expression and cone outer segment structure were examined by immunohistochemistry., Results: We showed that dorsal M-opsin dominant cones exhibit outer segment degeneration at an early age in Opn1mw-/- mice, whereas ventral S-opsin dominant cones were normal. The remaining M-opsin dominant cones remained viable for at least 15 months, albeit having shortened or no outer segments. We also showed that AAV5-mediated expression of human L-opsin was still able to rescue function and outer segment structure in the remaining M-opsin dominant cones when treatment was initiated at 15 months of age., Conclusions: Our results showing that the remaining M-opsin dominant cones in aged Opn1mw-/- mice can still be rescued by gene therapy is helpful for establishing the window of treatability in future blue cone monochromacy clinical trials.

A pair of novel chiral one-dimensional compounds with the general formula [Cd(NO3)2L2]·THF (where L is R- or S-2,2'-bis(methoxymethoxy)-6,6'-bis(4-pyridyl)-1,1'-binaphthyl) for the first time show circularly polarized luminescence, two-photon excited fluorescence, and second harmonic generation activity.

Applied Genetic Technologies Corporation (AGTC) is developing a recombinant adeno-associated virus (rAAV) vector AGTC-501, also designated AAV2tYF-GRK1-RPGRco, to treat retinitis pigmentosa (RP) in patients with mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. The vector contains a codon-optimized human RPGR cDNA (RPGRco) driven by a photoreceptor-specific promoter (G protein-coupled receptor kinase 1, GRK1) and is packaged in an AAV2 capsid with three surface tyrosine residues changed to phenylalanine (AAV2tYF). We conducted a safety and potency study of this vector administered by subretinal a injection in the naturally occurring RPGR-deficient Rd9 mouse model. Sixty Rd9 mice (20 per group) received a subretinal injection in the right eye of vehicle (control) or AAV2tYF-GRK1-RPGRco at one of two dose levels (4 × 10 8 or 4 × 10 9 vg/eye) and were followed for 12 weeks after injection. Vector injections were well tolerated, with no systemic toxicity. There was a trend towards reduced electroretinography b-wave amplitudes in the high vector dose group that was not statistically significant. There were no clinically important changes in hematology or clinical chemistry parameters and no vector-related ocular changes in life or by histological examination. Dose-dependent RPGR protein expression, mainly in the inner segment of photoreceptors and the adjacent connecting cilium region, was observed in all vector-treated eyes examined. Sequence integrity of the codon-optimized RPGR was confirmed by sequencing of PCR-amplified DNA, or cDNA reverse transcribed from total RNA extracted from vector-treated retinal tissues, and by sequencing of RPGR protein obtained from transfected HEK 293 cells. These results support the use of rAAV2tYF-GRK1-RPGRco in clinical studies in patients with XLRP caused by RPGR mutations.

Repeated, recreational ketamine (KE) or methamphetamine (MA) administration seldom produce neurotoxicity, while combining MA and KE administration have been thought to render changes in neural plasticity and motivational behavior. In this study, we sought to assess whether pre-exposure to multiple MA injections and withdrawal may affect low-dose KE-produced rewarding effects, social interaction behavior and its neurochemical underpinnings. A 10-day MA injections (2 mg/kg/day) and 10-day withdrawal regimen was found to cause reliable behavioral sensitization. While KE (1 mg/kg) induced weak conditioned place preference (CPP), pre-exposure to this MA-withdrawal regimen enhanced such KE CPP magnitude. This MA-withdrawal regimen also caused impairments in the social interaction behavior in the sociability, social novelty test. Compared with the mice undergoing the 10-day saline-withdrawal or MA regimen, mice receiving the 10-day MA-withdrawal regimen exhibited lower dopamine-releasing probability in the nucleus accumbens, inferring the MA-withdrawal regimen-primed preference for KE rewarding effects. Likewise, mice receiving the MA-withdrawal regimen had high expression in mGluR5 protein but unaltered EAAT3, Homer2 expression in hippocampal tissues. Pretreatment with MPEP, an mGluR5 antagonist, prevented the MA-withdrawal regimen-induced increment in the KE CPP magnitude and impairments in social interaction behavior. We, thus, conclude that repeated MA administration and abstinence may enhance KE rewarding effects and produce eminent deficits in social recognition and interest. And these effects correlate with the mGluR5 over-expression and modulation of the KE-stimulating effect on dopamine release., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objective: To explore the role of HOTAIR in the pathogenesis of adrenocortical carcinoma (ACC) and its underlying mechanism., Patients and Methods: Differentially expressed lncRNA (HOTAIR) in ACC was screened out from the GEO database. The survival analysis and ROC curve were performed according to HOTAIR expressions in ACC patients. The correlation between HOTAIR expression and clinical information of ACC patients was analyzed by chi-square test. The univariate and multivariate COX regression analysis was carried out to analyze the relationship between HOTAIR expression, disease-free survival (DFS) and overall survival (OS) of ACC patients. We then detected HOTAIR expression in 77 ACC tissues and 30 normal tissues by qRT-PCR (quantitative Real-time polymerase chain reaction). ACC cell lines were further screened out for the following in vitro experiments. After altering HOTAIR expression in ACC cells by plasmid transfection, proliferation and cell cycle were detected by Cell Counting Kit-8 (CCK-8) and colony formation assay, respectively. Finally, Western blot was utilized to detect expressions of cell cycle-related genes in ACC cells., Results: HOTAIR was overexpressed in ACC tissues than that of normal tissues. HOTAIR expression was remarkably increased in ACC with T3 and T4 stage than that of T1 and T2 stage. Moreover, HOTAIR expression was remarkably increased in ACC with stage III and IV than that of stage I and II. HOTAIR was an independent prognostic factor for DFS and OS of ACC patients. For in vitro experiments, inhibited proliferation and arrested cell cycle were observed in H295R cells transfected with si-HOTAIR. Opposite results were obtained after SW-13 cells were transfected with HOTAIR overexpression plasmid. Furthermore, expressions of cell cycle-related genes, including Cyclin D1, p-Rb and p-GSK3β were remarkably decreased after HOTAIR knockdown., Conclusions: We demonstrated for the first time that HOTAIR is overexpressed in ACC and is a prognostic risk factor in ACC patients. HOTAIR participates in the development and progression of ACC via shortening cell cycle and promoting proliferation of ACC cells.

Rod and cone phosphodiesterase 6 (PDE6) are key effector enzymes of the vertebrate phototransduction pathway. Rod PDE6 consists of two catalytic subunits PDE6α and PDE6β and two identical inhibitory PDE6γ subunits, while cone PDE6 is composed of two identical PDE6α' catalytic subunits and two identical cone-specific PDE6γ' inhibitory subunits. Despite their prominent function in regulating cGMP levels and therefore rod and cone light response properties, it is not known how each subunit contributes to the functional differences between rods and cones. In this study, we generated an rd10/cpfl1 mouse model lacking rod PDE6β and cone PDE6α' subunits. Both rod and cone photoreceptor cells are degenerated with age and all PDE6 subunits degrade in rd10/cpfl1 mice. We expressed cone PDE6α' in both rods and cones of rd10/cpfl1 mice by adeno-associated virus (AAV)-mediated delivery driven by the ubiquitous, constitutive small chicken β-actin promoter. We show that expression of PDE6α' rescues rod function in rd10/cpfl1 mice, and the restoration of rod light sensitivity is attained through restoration of endogenous rod PDE6γ and formation of a functional PDE6α'γ complex. However, improved photopic cone responses were achieved only after supplementation of both cone PDE6α' and PDE6γ' subunits but not by PDE6α' treatment alone. We observed a two fold increase of PDE6α' levels in the eyes injected with both PDE6α' plus PDE6γ' relative to eyes receiving PDE6α' alone. Despite the presence of both PDE6γ' and PDE6γ, the majority of PDE6α' formed functional complexes with PDE6γ', suggesting that PDE6α' has a higher association affinity for PDE6γ' than for PDE6γ. These results suggest that the presence of PDE6γ' augments cone PDE6 assembly and enhances its stability. Our finding has important implication for gene therapy of PDE6α'-associated achromatopsia.

Objective: Rosai-Dorfman disease (RDD) is a rare histiocytic disorder that has nodal and extranodal manifestations. Currently no guidelines are available for the management of this disease. We report a case of laryngeal RDD that did not present with classical symptoms such as fever or lymphadenopathy and could be easily misdiagnosed as malignant tumor. The patient received minimally invasive surgeries combined with steroid therapy to preserve the laryngeal function as much as possible, and a favorable clinical outcome was obtained. Reports of similar cases in literatures were reviewed to obtain a better understanding of the disease course, diagnosis and treatment of this uncommon condition.

Purpose: Blue cone monochromacy (BCM) is an X-linked congenital vision disorder characterized by complete loss or severely reduced L- and M-cone function. Patients with BCM display poor visual acuity, severely impaired color discrimination, myopia, nystagmus, and minimally detectable cone-mediated electroretinogram. Recent studies of patients with BCM with adaptive optics scanning laser ophthalmoscopy (AOSLO) showed that they have a disrupted cone mosaic with reduced numbers of cones in the fovea that is normally dominated by L- and M-cones. The remaining cones in the fovea have significantly shortened outer segments but retain sufficient structural integrity to serve as potential gene therapy targets. In this study, we tested whether exogenously expressed human L- and M-opsins can rescue M-cone function in an M-opsin knockout ( Opn1mw -/- ) mouse model for BCM., Methods: Adeno-associated virus type 5 (AAV5) vectors expressing OPN1LW, OPN1MW, or C-terminal tagged OPN1LW-Myc, or OPN1MW-HA driven by a cone-specific promoter were injected subretinally into one eye of Opn1mw -/- mice, while the contralateral eye served as the uninjected control. Expression of cone pigments was determined with western blotting and their cellular localization identified with immunohistochemistry. M-cone function was analyzed with electroretinogram (ERG). Antibodies against cone phototransduction proteins were used to study cone outer segment (OS) morphology in untreated and treated Opn1mw -/- eyes., Results: We showed that cones in the dorsal retina of the Opn1mw -/- mouse do not form outer segments, resembling cones that lack outer segments in the human BCM fovea. We further showed that AAV5-mediated expression of either human M- or L-opsin individually or combined promotes regrowth of cone outer segments and rescues M-cone function in the treated Opn1mw -/- dorsal retina., Conclusions: Exogenously expressed human opsins can regenerate cone outer segments and rescue M-cone function in Opn1mw -/- mice, thus providing a proof-of-concept gene therapy in an animal model of BCM.

A new protocol for radical transformations of thioamides promoted by Lewis base-boryl radicals is reported. The desulfurizative reduction to access organic amines was enabled utilizing 4-dimethylaminopyridine-BH 3 as the boryl radical precursor and PhSH as the polarity reversal catalyst. Alternatively, the chain process for unsaturated thioamides was switched to an annulation reaction using N-heterocyclic carbene-BH 3 as the boryl radical precursor and sterically bulky Ph 3 CSH as the catalyst, allowing for the construction of N-heterocyclic and carbocyclic skeletons.

Purpose: We aimed to seek risk factors for AMI among adult patients undergoing cardiac surgery., Materials and Methods: We searched electronic bibliographic databases for studies reporting risk factors for AMI among adult patients undergoing cardiac surgery. Pooled odds ratios (OR) and standard mean differences (SMD or MD) for risk factors between AMI and control group were estimated., Results: 11 studies with 67,195 patients met the inclusion criteria. 14 risk factors were found to be statistically significant: preoperative factors including age (MD 4.62years, 95% CI (1.97,7.27)), cardiac shock (OR 5.17, (1.17,22.81)), peripheral vascular disease (OR 3.53, (2.05,6.09)), need for intra-aortic balloon pump (IABP) (OR 5.89, (3.26,10.65)), emergency surgery (OR 3.75, (1.69,8.33)), and postoperative factors including atrial fibrillation (OR 2.41, (1.79,3.24)), CK-MB level (SMD 1.06, (0.62 to 1.50)), serum creatinine >200μmol/L (OR 23.39, (11.61,47.12)), blood loss (MD 358.32mL, (53.56,663.07)), prolonged ventilation (OR 9.04, (5.24,15.62)), need for IABP (OR 6.32, (3.19,12.54)), inotropic treatment (OR 8.40, (3.19,22.14)), blood transfusion (OR 9.15, (4.79,17.48)), reoperation (OR 3.30, (1.55,7.04))., Conclusions: 14 risk factors were associated with an increased risk of AMI, which indicated that AMI might occur via stenosis or occlusion of mesenteric vessels, reduced blood volume or maldistribution of blood flow., (Copyright © 2017. Published by Elsevier Inc.)

Background: In recent years, a substantial amount of experimental studies have demonstrated that exogenous administration of corticosterone causes anxiety and depressive-like behaviour in rodents which involves hypothalamic-pituitary-adrenal axis dysregulation. Our present study aimed to explore the neuroprotective potential of mangiferin against corticosterone-induced anxiety and depressive-like behaviour., Methods: Corticosterone (40 mg/kg; subcutaneously) was administered once daily in swiss albino mice for 21 days. Mice were treated simultaneously with mangiferin (40 mg/kg; p.o.), 30 min prior to the corticosterone injection., Results: Chronic administration of corticosterone caused anxiety and depressive-like behaviour in mice which was significantly alleviated by mangiferin treatment. Biochemical analysis revealed that mangiferin treatment significantly attenuated corticosterone-induced oxido-nitrosative stress and neuroinflammation in the hippocampus region. Furthermore, concomitant treatment with mangiferin significantly enhanced the hippocampal brain-derived neurotrophic factor (BDNF) level and decreased the serum corticosterone level in the corticosterone-treated animals. Western blotting analysis revealed that corticosterone administration significantly up-regulated the indoleamine 2,3-dioxygenase (IDO) protein expression level in the hippocampus which was significantly reduced by mangiferin treatment., Conclusion: Taken together, our results suggest that mangiferin exerts anti-anxiety and antidepressant effect in corticosterone-treated rats, which is probably mediated through up-regulation of BDNF level along with inhibition of oxido-nitrosative stress, neuroinflammation and IDO up-regulation in the hippocampus region.

Cones are responsible for daylight, central, high acuity and color vision. Three proteins found in human cones, i.e. long-wavelength (L)-, middle-wavelength (M)-, and short-wavelength sensitive (S)-opsins, are responsible for red, green and blue color recognition, respectively. Human blue cone monochromacy (BCM) is characterized by functional loss of both L- and M-cone opsins due to mutations in the OPN1LW/OPN1MW gene cluster on the X chromosome. BCM patients, who rely on their vision from only S-cones and rods, suffer severely reduced visual acuity and impaired color vision. Recent studies show that there is sufficient cone structure remaining in the central fovea of BCM patients to consider AAV-mediated gene augmentation therapy. In contrast, mouse retina has only two opsins, S-opsin and M-opsin, but no L-opsin. We generated an M-opsin knockout mouse (Opn1mw -/- ) expressing only S-opsin as a model for human BCM. We show that recombinant M-opsin delivered by AAV5 vectors rescues M-cone function in Opn1mw -/- mice. We also show that AAV delivered M-opsin localizes in the dorsal cone outer segments, and co-localizes with S-opsin in the ventral retina. Our study demonstrates that cones without M-opsin remain viable and respond to gene augmentation therapy, thereby providing proof-of-concept for cone function restoration in BCM patients.

Background: Postoperative atrial fibrillation/flutter (POAF) is associated with significant morbidity and mortality after general thoracic surgery, but the need for and the best agent for prophylaxis remains obscure., Methods: A systematic literature search was performed to identify randomized controlled trials that compared regimens for POAF prophylaxis after general thoracic surgery. Random-effects meta-analyses with trial sequential analyses were performed to compare the effects of medical prophylaxis vs placebo/usual care. The risk of POAF among patients receiving various prophylactic regimens was subjected to Bayesian network meta-analysis., Results: Twenty-two trials (2,891 patients and 11 regimens) were included. Overall, medical prophylaxis reduced the incidence of POAF (OR, 0.33; 95% CI, 0.22-0.49) but not short-term mortality (OR, 0.85; 95% CI, 0.41-1.73). There was no significant difference in patient withdrawal due to adverse events (OR, 1.67; 95% CI, 0.67-4.16). Trial sequential analysis showed that as of 2012, sufficient evidence had accrued in support of the effectiveness of medical prophylaxis in reducing POAF after general thoracic surgery. In network meta-analysis, β-blockers, angiotensin-converting enzyme inhibitors, amiodarone, magnesium, and calcium channel blockers significantly reduced the risk of POAF compared with placebo/usual care. β-Blockers had the highest probability of being the most effective agents (OR, 0.12; 95% credible interval [CrI], 0.05-0.27; probability of being best, 77.7%; number needed to treat, 5.2)., Conclusions: The current literature supports the effectiveness and tolerability of medical prophylaxis and the superiority of β-blockers in preventing POAF after general thoracic surgery. β-Blockers are recommended, taking into consideration the status of the bronchopulmonary system., (Copyright © 2016 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.)

Major ozonated autohemotherapy has been shown to promote recovery of upper limb motor function in patients with acute cerebral infarction, but whether major ozonated autohemotherapy affects remote injury remains poorly understood. Here, we assumed that major ozonated autohemotherapy contributes to recovery of clinical function, possibly by reducing remote injury after acute cerebral infarction. Sixty acute cerebral infarction patients aged 30-80 years were equally and randomly allocated to ozone treatment and control groups. Patients in the ozone treatment group received medical treatment and major ozonated autohemotherapy (47 mg/L, 100 mL ozone) for 10 ± 2 days. Patients in the control group received medical treatment only. National Institutes of Health Stroke Scale score, modified Rankin scale score, and reduced degree of fractional anisotropy values of brain magnetic resonance diffusion tensor imaging were remarkably decreased, brain function improved, clinical efficiency significantly increased, and no obvious adverse reactions detected in the ozone treatment group compared with the control group. These findings suggest that major ozonated autohemotherapy promotes recovery of neurological function in acute cerebral infarction patients by reducing remote injury, and additionally, exhibits high safety., Competing Interests: Conflicts of Interest: None declared.

Objective: To observe the difference of the human telomeres RNA component (hTERC) genes' amplification in the cervical tissue by applying the environment-friendly fixative poly hydroxy acrylic acid and the transparent dewaxing solution Van-clear separately or jointly to replace the traditional fixative 4% (volume fraction) neutral buffered formalin and the conventional transparent dewaxing solution xylene in the use of fluorescence in situ hybridization (FISH) for detection., Methods: In the study, 255 cases of cervical tissue specimens submitted by the Department of Gynecology in Zhongshan Boai Hosipital were collected from Mar. 2013 to Apr. 2015. Four samples were taken from the same lesion site. All the cases were divided into 4 groups and named group A, B, C, and D. Group A used 4% neutral buffered formalin fixed and xylene dewaxing to make slices. Group B used poly hydroxy acrylic fixed and xylene dewaxing to make slices. Group C used 4% neutral buffered formalin fixed and Van-clear transparent to make slices. Group D used poly hydroxy acrylic fixed and Van-clear transparent dewaxing to make slices. The amplification of hTERC genes in the four groups of cervical specimens was also detected by FISH technique., Results: When the hTERC genes were detected by FISH method under the fluorescence microscope, it was obvious that the tissue profile and the background of group A, B, C and D were all clear. The probe was fixed in the accurate position so that the bright red or green fluorescence signals were easily found in these four groups. Compared with the positive rate of group A, there was no statistical significance in that of group B, C and D (P>0.05). At the same time, the coincidence rate of the FISH results was high, which showed that the new environment-friendly reagent had no significant difference in the detection of cervical hTERC genes by FISH technique., Conclusion: It is possible for the environment-friendly reagent poly hydroxy acrylic acid and Van-clear to replace 4% neutral buffered formalin and xylene separately or jointly to detect the cervical hTERC genes by adopting FISH technique.

MERTK is an essential component of the signaling network that controls phagocytosis in retinal pigment epithelium (RPE), the loss of which results in photoreceptor degeneration. Previous proof-of-concept studies have demonstrated the efficacy of gene therapy using human MERTK (hMERTK) packaged into adeno-associated virus (AAV2) in treating RCS rats and mice with MERTK deficiency. The purpose of this study was to assess the safety of gene transfer via subretinal administration of rAAV2-VMD2-hMERTK in subjects with MERTK-associated retinitis pigmentosa (RP). After a preclinical phase confirming the safety of the study vector in monkeys, six patients (aged 14 to 54, mean 33.3 years) with MERTK-related RP and baseline visual acuity (VA) ranging from 20/50 to <20/6400 were entered in a phase I open-label, dose-escalation trial. One eye of each patient (the worse-seeing eye in five subjects) received a submacular injection of the viral vector, first at a dose of 150 µl (5.96 × 10(10)vg; 2 patients) and then 450 µl (17.88 × 10(10)vg; 4 patients). Patients were followed daily for 10 days at 30, 60, 90, 180, 270, 365, 540, and 730 days post-injection. Collected data included (1) full ophthalmologic examination including best-corrected VA, intraocular pressure, color fundus photographs, macular spectral domain optical coherence tomography and full-field stimulus threshold test (FST) in both the study and fellow eyes; (2) systemic safety data including CBC, liver and kidney function tests, coagulation profiles, urine analysis, AAV antibody titers, peripheral blood PCR and ASR measurement; and (3) listing of ophthalmological or systemic adverse effects. All patients completed the 2-year follow-up. Subretinal injection of rAAV2-VMD2-hMERTK was associated with acceptable ocular and systemic safety profiles based on 2-year follow-up. None of the patients developed complications that could be attributed to the gene vector with certainty. Postoperatively, one patient developed filamentary keratitis, and two patients developed progressive cataract. Of these two patients, one also developed transient subfoveal fluid after the injection as well as monocular oscillopsia. Two patients developed a rise in AAV antibodies, but neither patient was positive for rAAV vector genomes via PCR. Three patients also displayed measurable improved visual acuity in the treated eye following surgery, although the improvement was lost by 2 years in two of these patients. Gene therapy for MERTK-related RP using careful subretinal injection of rAAV2-VMD2-hMERTK is not associated with major side effects and may result in clinical improvement in a subset of patients.

Usher syndrome type III (USH3A) is an autosomal recessive disorder caused by mutations in clarin-1 (CLRN1) gene, leading to progressive retinal degeneration and sensorineural deafness. Efforts to develop therapies for preventing photoreceptor cell loss are hampered by the lack of a retinal phenotype in the existing USH3 mouse models and by conflicting reports regarding the endogenous retinal localization of clarin-1, a transmembrane protein of unknown function. In this study, we used an AAV-based approach to express CLRN1 in the mouse retina in order to determine the pattern of its subcellular localization in different cell types. We found that all major classes of retinal cells express AAV-delivered CLRN1 driven by the ubiquitous, constitutive small chicken β-actin promoter, which has important implications for the design of future USH3 gene therapy studies. Within photoreceptor cells, AAV-expressed CLRN1 is mainly localized at the inner segment region and outer plexiform layer, similar to the endogenous expression of other usher proteins. Subretinal delivery using a full strength viral titer led to significant loss of retinal function as evidenced by ERG analysis, suggesting that there is a critical limit for CLRN1 expression in photoreceptor cells. Taken together, these results suggest that CLRN1 expression is potentially supported by a variety of retinal cells, and the right combination of AAV vector dose, promoter, and delivery method needs to be selected to develop safe therapies for USH3 disorder.

MERTK-associated retinal degenerations are thought to have defects in phagocytosis of shed outer segment membranes by the retinal pigment epithelium (RPE), as do the rodent models of these diseases. We have subretinally injected an RPE-specific AAV2 vector, AAV2-VMD2-hMERTK, to determine whether this would provide long-term photoreceptor rescue in the RCS rat, which it did for up to 6.5 months, the longest time point examined. Moreover, we found phagosomes in the RPE in the rescued regions of RCS retinas soon after the onset of light. The same vector also had a major protective effect in Mertk-null mice, with a concomitant increase in ERG response amplitudes in the vector-injected eyes. These findings suggest that planned clinical trials with this vector will have a favorable outcome.

Objective: The aim of this study was to construct a eukaryotic expression plasmid with a short hairpin RNA (shRNA) targeting Livin in order to obtain a stably transfected Hep-2 cell line with a reduced expression of Livin., Methods: The shRNA targeting Livin mRNA was designed, and a shRNA plasmid and a negative control plasmid were constructed. After amplification in E. coli, restriction endonuclease digestion and sequence confirmation, the plasmids were transfected into Hep-2 cells using Lipofectamine 2000. The stably transfected cell line was screened using G418, and inhibition of Livin mRNA and protein levels were detected using real-time PCR and western blotting, respectively., Results: pGenesil-Livin-shRNA eukaryotic expression plasmid was successfully constructed and identified by sequencing. Green fluorescent protein (GFP) expression was observed in Hep-2 cells transfected with shRNA plasmids by fluorescence microscopy. The expression levels of Livin mRNA and protein decreased significantly in Hep-2 cells transfected with the shRNA recombinant plasmid. The mRNA level was reduced by 47.17 %, and the protein level was reduced by 34.25 %., Conclusion: The shRNA eukaryotic expression plasmid targeting Livin was successfully constructed, which could significantly inhibit the expression of Livin in laryngeal cancer Hep-2 cells. This provides a basis for future research on the function of Livin in Hep-2 cells, and gene therapy for laryngeal cancer.

Inherited retinal degenerations cause progressive loss of photoreceptor neurons with eventual blindness. Corrective or neuroprotective gene therapies under development could be delivered at a predegeneration stage to prevent the onset of disease, as well as at intermediate-degeneration stages to slow the rate of progression. Most preclinical gene therapy successes to date have been as predegeneration interventions. In many animal models, as well as in human studies, to date, retinal gene therapy administered well after the onset of degeneration was not able to modify the rate of progression even when successfully reversing dysfunction. We evaluated consequences of gene therapy delivered at intermediate stages of disease in a canine model of X-linked retinitis pigmentosa (XLRP) caused by a mutation in the Retinitis Pigmentosa GTPase Regulator (RPGR) gene. Spatiotemporal natural history of disease was defined and therapeutic dose selected based on predegeneration results. Then interventions were timed at earlier and later phases of intermediate-stage disease, and photoreceptor degeneration monitored with noninvasive imaging, electrophysiological function, and visual behavior for more than 2 y. All parameters showed substantial and significant arrest of the progressive time course of disease with treatment, which resulted in long-term improved retinal function and visual behavior compared with control eyes. Histology confirmed that the human RPGR transgene was stably expressed in photoreceptors and associated with improved structural preservation of rods, cones, and ON bipolar cells together with correction of opsin mislocalization. These findings in a clinically relevant large animal model demonstrate the long-term efficacy of RPGR gene augmentation and substantially broaden the therapeutic window for intervention in patients with RPGR-XLRP.

Purpose: The mutation S163R in complement C1q tumor necrosis factor-related protein-5 (C1QTNF5) causes an autosomal dominant disorder known as late-onset retinal degeneration (L-ORD). In this study, our goal is to evaluate the consequences of mutant S163R C1QTNF5 expression in mouse RPE following its delivery using an adeno-associated viral (AAV) vector., Methods: We generated AAV vectors containing either human wild-type C1QTNF5 or mutant S163R C1QTNF5 driven by an RPE-specific BEST1 promoter, and delivered them subretinally into one eye of adult C57BL/6 mice. Transgene expression was detected by immunohistochemistry. Retinal function was assessed by full-field ERG. Pathological changes were further examined by digital fundus imaging and spectral-domain optical coherence tomography (SD-OCT)., Results: We show that the AAV-expressed mutant S163R leads to pathological effects similar to some of those found in patients with advanced L-ORD, including RPE thinning, RPE cell loss, and retinal degeneration. In addition, we provide in vivo evidence that mutant S163R C1QTNF5 can form large, transparent, spherical intracellular aggregates throughout the RPE, which are detectable by light microscopy. In contrast to AAV-expressed wild-type C1QTNF5, which is secreted apically from the RPE toward the photoreceptor cells and the outer limiting membrane, the S163R mutant is primarily routed toward the basal side of RPE, where it forms thick, extracellular deposits over time., Conclusions: Adeno-associated viral-targeted expression of mutant S163R in the RPE represents a useful approach for quickly generating animal models that mimic pathological features of L-ORD and offers the potential to understand disease mechanisms and develop therapeutic strategies.

Our collaborative successful gene replacement therapy using AAV vectors expressing a variant of human RPGR-ORF15 in two canine models provided therapeutic proof of concept for translation into human treatment. The ORF15 sequence contained within this AAV vector, however, has ORF15 DNA sequence variations compared to the published sequence that are likely due to its unusual composition of repetitive purine nucleotides. This mutability is a concern for AAV vector production and safety when contemplating a human trial. In this study, we establish the safety profile of AAV-hIRBP-hRPGR and AAV-hGRK1-hRPGR vectors used in the initial canine proof-of-principle experiments by demonstrating hRPGR-ORF15 sequence stability during all phases of manipulation, from plasmid propagation to vector production to its stability in vivo after subretinal administration to animals. We also evaluate potential toxicity in vivo by investigating protein expression, retinal structure and function, and vector biodistribution. Expression of hRPGR is detected in the inner segments and synaptic terminals of photoreceptors and is restricted to the connecting cilium when the vector is further diluted. Treated eyes exhibit no toxicity as assessed by retinal histopathology, immunocytochemistry, optical coherence tomography, fundoscopy, electroretinogram, and vector biodistribution. Therefore, the hRPGR-ORF15 variant in our AAV vectors appears to be a more stable form than the endogenous hRPGR cDNA when propagated in vitro. Its safety profile presented here in combination with its proven efficacy supports future gene therapy clinical trials.

The CNGA3(-/-)/Nrl(-/-) mouse is a cone-dominant model with Cnga3 channel deficiency, which partially mimics the all cone foveal structure of human achromatopsia 2 with CNGA3 mutations. Although subretinal (SR) AAV vector administration can transfect retinal cells efficiently, the injection-induced retinal detachment can cause retinal damage, particularly when SR vector bleb includes the fovea. We therefore explored whether cone function-structure could be rescued in CNGA3(-/-)/Nrl(-/-) mice by intravitreal (IVit) delivery of tyrosine to phenylalanine (Y-F) capsid mutant AAV8. We find that AAV-mediated CNGA3 expression can restore cone function and rescue structure following IVit delivery of AAV8 (Y447, 733F) vector. Rescue was assessed by restoration of the cone-mediated electroretinogram (ERG), optomotor responses, and cone opsin immunohistochemistry. Demonstration of gene therapy in a cone-dominant mouse model by IVit delivery provides a potential alternative vector delivery mode for safely transducing foveal cones in achromatopsia patients and in other human retinal diseases affecting foveal function., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Endomorphin-2 (EM2) and Substance P (SP) exert suppressive and facilitative influences upon nociception, respectively. Although EM2 and SP were often co-expressed in single neurons in dorsal root ganglion (DRG), it is still unknown if and how the nociception-suppressive influences of EM2 might be exerted upon nociception-facilitative effects of SP in the DRG neurons. We examined these issues in the inflammatory pain model rats produced by subcutaneous injection of the complete Freund's adjuvant into the hind paw. The paw withdrawal threshold for mechanical allodynia was measured. Changes of EM2 and SP release were estimated by measuring intrathecal levels of EM2 and SP through in vivo microdialysis analysis of cerebrospinal fluid. The mechanical allodynia was dose-dependently attenuated by intrathecal injection of EM2 or a neurokinin-1 receptor antagonist, and facilitated by intrathecal injection of SP or a mu-opioid receptor (MOR) antagonist. Importantly, intrathecal level of SP was found to be lowered by intrathecal injection of EM2. Morphologically, colocalization of EM2-, MOR- and SP-immunoreactivity in single DRG neurons was observed by immunofluorescent histochemistry, and co-expression of EM2 and SP in large, dense-cored presynaptic vesicles in primary afferents, as well as localization of MOR on pre- and postsynaptic membrane in spinal dorsal horn, was also confirmed electron miscroscopically. Thus, the results indicated that analgesic influences of EM2 upon inflammatory pain might be exerted through suppression of SP release, supporting the assumptions that binding of EM2 to presynaptic MOR might induce such effects., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Purpose: The retinal degeneration 11 (rd11) mouse is a newly discovered, naturally occurring animal model with early photoreceptor dysfunction and rapid rod photoreceptor degeneration followed by cone degeneration. The rd11 mice carry a spontaneous mutation in the lysophosphatidylcholine acyltransferase 1 (Lpcat1) gene. Here, we evaluate whether gene replacement therapy using the fast-acting tyrosine-capsid mutant AAV8 (Y733F) can arrest retinal degeneration and restore retinal function in this model., Methods: The AAV8 (Y733F)-smCBA-Lpcat1 was delivered subretinally to postnatal day 14 (P14) rd11 mice in one eye only. At 10 weeks after injection, treated rd11 mice were examined by visually-guided behavior, electroretinography (ERG) and spectral domain optical coherence tomography (SD-OCT), and then killed for morphologic and biochemical examination., Results: Substantial scotopic and photopic ERG signals were maintained in treated rd11 eyes, whereas untreated eyes in the same animals showed extinguished signals. The SD-OCT (in vivo) and light microscopy (in vitro) showed a substantial preservation of the outer nuclear layer in most parts of the treated retina only. Almost wild-type LPCAT1 expression in photoreceptors with strong rod rhodopsin and M/S cone opsin staining, and normal visually-guided water maze behavioral performances were observed in treated rd11 mice., Conclusions: The results demonstrate that the tyrosine-capsid mutant AAV8 (Y733F) vector is effective for treating rapidly degenerating models of retinal degeneration and, moreover, is more therapeutically effective than AAV2 (Y444, 500, 730F) vector with the same promoter-cDNA payload. To our knowledge, this is the first demonstration of phenotypic rescue by gene therapy in an animal model of retinal degeneration caused by Lpcat1 mutation.

In this work, a metal-mediated assembling strategy has been used to organize a series of new assemblies based on tetrapyridylporphyrin () on nanostructured TiO2 electrode surfaces, wherein the metal ions (, = Zn(2+), Cd(2+), Hg(2+) and Mn(2+)) bridge the pyridyl units of and (E)-4-[(pyridin-4-ylmethylene)-amino]benzoic acid (), resulting in a assembled mode. The assembled structures were characterized by transmission electron microscopy (TEM), computational calculations, energy-dispersive X-ray spectroscopy (EDX), IR, UV-vis absorption and fluorescence spectra. The performances of the assembly-sensitized solar cells were also measured under an irradiance of 100 mW cm(-2) AM 1.5G sunlight. Photoelectrochemical results reveal a relatively large photocurrent of the device. Simultaneously, a large open-circuit photovoltage and a significantly improved conversion efficiency of the device are also observed. These findings may serve as another good testing ground for the fabrication of supramolecular solar cells in future.

The rd6 mouse is a natural model of an RPE-based (retinal pigment epithelium) autosomal recessive retinitis pigmentosa (RP) caused by mutations in the Mfrp (membrane-type frizzled related protein) gene. Previously, we showed that subretinal delivery of the wild-type mouse Mfrp mediated by a tyrosine-capsid mutant scAAV8 (Y733F) vector prevented photoreceptor cell death, and rescued retinal function as assessed by electroretinography. In this study, we describe the effect of gene therapy on the retinal structure and function in rd6 mice using a quadruple (Y272, 444, 500, 730F) tyrosine-capsid mutant scAAV2 viral vector delivered subretinally at postnatal day 14 (P14). We show that therapy is effective at slowing the photoreceptor degeneration, and in preventing the characteristic accumulation of abnormal phagocytic cells in the subretinal space. MFRP expression as driven by the ubiquitous chicken β-actin (smCBA) promoter in treated rd6 mice was found predominantly in the RPE apical membrane and the entire length of its microvilli, as well as in the photoreceptor inner segments, suggesting a potential interaction with actin filaments. In spite of preserving retinal morphology, the effects of gene therapy on retinal function were minimal, suggesting that the scAAV8 (Y733F) vector may be more efficient for the treatment of RP caused by Mfrp mutations.

Phosphodiesterase-6 (PDE6) is the key effector enzyme of the vertebrate phototransduction pathway in rods and cones. Rod PDE6 catalytic core is composed of two distinct subunits, PDE6α and PDE6β, whereas two identical PDE6α' subunits form the cone PDE6 catalytic core. It is not known whether this difference in PDE6 catalytic subunit identity contributes to the functional differences between rods and cones. To address this question, we expressed cone PDE6α' in the photoreceptor cells of the retinal degeneration 10 (rd10) mouse that carries a mutation in rod PDEβ subunit. We show that adeno-associated virus-mediated subretinal delivery of PDE6α' rescues rod electroretinogram responses and preserves retinal structure, indicating that cone PDE6α' can couple effectively to the rod phototransduction pathway. We also show that restoration of light sensitivity in rd10 rods is attributable to assembly of PDE6α' with rod PDE6γ. Single-cell recordings revealed that, surprisingly, rods expressing cone PDE6α' are twofold more sensitive to light than wild-type rods, most likely because of the slower shutoff of their light responses. Unlike in wild-type rods, the response kinetics in PDE6α'-treated rd10 rods accelerated with increasing flash intensity, indicating a possible direct feedback modulation of cone PDE6α' activity. Together, these results demonstrate that cone PDE6α' can functionally substitute for rod PDEαβ in vivo, conferring treated rods with distinct physiological properties.

Abstract Proof of concept for MERTK gene replacement therapy has been demonstrated using different viral vectors in the Royal College of Surgeon (RCS) rat, a well characterized model of recessive retinitis pigmentosa that contains a mutation in the Mertk gene. MERTK plays a key role in renewal of photoreceptor outer segments (OS) by phagocytosis of shed OS tips. Mutations in MERTK cause impaired phagocytic activity and accumulation of OS debris in the interphotoreceptor space that ultimately leads to photoreceptor cell death. In the present study, we conducted a series of preclinical potency and GLP-compliant safety evaluations of an adeno-associated virus type 2 (AAV2) vector expressing human MERTK cDNA driven by the retinal pigment epithelium-specific, VMD2 promoter. We demonstrate the potency of the vector in RCS rats by improved electroretinogram (ERG) responses in treated eyes compared with contralateral untreated controls. Toxicology and biodistribution studies were performed in Sprague-Dawley (SD) rats injected with two different doses of AAV vectors and buffer control. Delivery of vector in SD rats did not result in a change in ERG amplitudes of rod and cone responses relative to balanced salt solution control-injected eyes, indicating that administration of AAV vector did not adversely affect normal retinal function. In vivo fundoscopic analysis and postmortem retinal morphology of the vector-injected eyes were normal compared with controls. Evaluation of blood smears showed the lack of transformed cells in the treated eyes. All injected eyes and day 1 blood samples were positive for vector genomes, and all peripheral tissues were negative. Our results demonstrate the potency and safety of the AAV2-VMD2-hMERTK vector in animal models tested. A GMP vector has been manufactured and is presently in clinical trial.

Purpose: The absence of Mertk in RCS rats results in defective RPE phagocytosis, accumulation of outer segment (OS) debris in the subretinal space, and subsequent death of photoreceptors. Previous research utilizing Mertk gene replacement therapy in RCS rats provided proof of concept for treatment of this form of recessive retinitis pigmentosa (RP); however, the beneficial effects on retinal function were transient. In the present study, we evaluated whether delivery of a MERTK transgene using a tyrosine-mutant AAV8 capsid could lead to more robust and longer-term therapeutic outcomes than previously reported., Methods: An AAV8 Y733F vector expressing a human MERTK cDNA driven by a RPE-selective promoter was administrated subretinally at postnatal day 2. Functional and morphological analyses were performed at 4 months and 8 months post-treatment. Retinal vasculature and Müller cell activation were analyzed by quantifying acellular capillaries and glial fibrillary acidic protein immunostaining, respectively., Results: Electroretinographic responses from treated eyes were more than one-third of wild-type levels and OS were well preserved in the injection area even at 8 months. Rescue of RPE phagocytosis, prevention of retinal vasculature degeneration, and inhibition of Müller cell activation were demonstrated in the treated eyes for at least 8 months., Conclusions: This research describes a longer and much more robust functional and morphological rescue than previous studies. We also demonstrate for the first time that an AAV8 mutant capsid serotype vector has a substantial therapeutic potential for RPE-specific gene delivery. These results suggest that tyrosine-mutant AAV8 vectors hold promise for the treatment of individuals with MERTK-associated RP.

Autosomal recessive retinitis pigmentosa (RP), a heterogeneous group of degenerations of the retina, can be due to mutations in the MFRP (membrane-type frizzled-related protein) gene. A patient with RP with MFRP mutations, one of which is novel and the first splice site mutation reported, was characterized by noninvasive retinal and visual studies. The phenotype, albeit complex, suggested that this retinal degeneration may be a candidate for gene-based therapy. Proof-of-concept studies were performed in the rd6 Mfrp mutant mouse model. The fast-acting tyrosine-capsid mutant AAV8 (Y733F) vector containing the small chicken β-actin promoter driving the wild-type mouse Mfrp gene was used. Subretinal vector delivery on postnatal day 14 prevented retinal degeneration. Treatment rescued rod and cone photoreceptors, as assessed by electroretinography and retinal histology at 2 months of age. This AAV-mediated gene delivery also resulted in robust MFRP expression predominantly in its normal location within the retinal pigment epithelium apical membrane and its microvilli. The clinical features of MFRP-RP and our preliminary data indicating a response to gene therapy in the rd6 mouse suggest that this form of RP is a potential target for gene-based therapy.

Hereditary retinal blindness is caused by mutations in genes expressed in photoreceptors or retinal pigment epithelium. Gene therapy in mouse and dog models of a primary retinal pigment epithelium disease has already been translated to human clinical trials with encouraging results. Treatment for common primary photoreceptor blindness, however, has not yet moved from proof of concept to the clinic. We evaluated gene augmentation therapy in two blinding canine photoreceptor diseases that model the common X-linked form of retinitis pigmentosa caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene, which encodes a photoreceptor ciliary protein, and provide evidence that the therapy is effective. After subretinal injections of adeno-associated virus-2/5-vectored human RPGR with human IRBP or GRK1 promoters, in vivo imaging showed preserved photoreceptor nuclei and inner/outer segments that were limited to treated areas. Both rod and cone photoreceptor function were greater in treated (three of four) than in control eyes. Histopathology indicated normal photoreceptor structure and reversal of opsin mislocalization in treated areas expressing human RPGR protein in rods and cones. Postreceptoral remodeling was also corrected: there was reversal of bipolar cell dendrite retraction evident with bipolar cell markers and preservation of outer plexiform layer thickness. Efficacy of gene therapy in these large animal models of X-linked retinitis pigmentosa provides a path for translation to human treatment.