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Congenital stationary night blindness (CSNB), in the complete form, is caused by dysfunctions in ON-bipolar cells (ON-BCs) which are secondary neurons of the retina. We describe the first disease causative variant associated with CSNB in the dog. A genome-wide association study using 12 cases and 11 controls from a research colony determined a 4.6 Mb locus on canine chromosome 32. Subsequent whole-genome sequencing identified a 1 bp deletion in LRIT3 segregating with CSNB. The canine mutant LRIT3 gives rise to a truncated protein with unaltered subcellular expression in vitro. Genetic variants in LRIT3 have been associated with CSNB in patients although there is limited evidence regarding its apparently critical function in the mGluR6 pathway in ON-BCs. We determine that in the canine CSNB retina, the mutant LRIT3 is correctly localized to the region correlating with the ON-BC dendritic tips, albeit with reduced immunolabelling. The LRIT3-CSNB canine model has direct translational potential enabling studies to help understand the CSNB pathogenesis as well as to develop new therapies targeting the secondary neurons of the retina.

Sterile tissue injury recruits myeloid cells (neutrophils and monocytes) from the bone marrow by stimulating a process known as emergency myelopoiesis. It is commonly assumed that myeloid cells activate proinflammatory signaling pathways within the injured tissue after sensing innate immune-activating molecules in the local microenvironment. Here, we show that pathogenic innate immune signaling in myeloid cells begins far from the site of injury, at their origins in the bone marrow. Using myocardial infarction (MI) as a model of sterile injury, we profiled single cell transcriptomes of >50,000 myeloid cells and found evidence of type I interferon signaling characterized by expression of interferon stimulated genes (ISGs) in a subset of neutrophils and monocytes in the heart, blood, and bone marrow after MI. We validated the findings in humans by quantifying ISG-expression in myeloid cells of patients presenting with acute MI, which revealed ISG levels far exceed that of healthy subjects and are comparable to patients with the interferon-associated autoimmune disease, systemic lupus erythematous. The identification of remote innate immune education raises important questions about how distant tissue injury activates interferon signaling in the hematopoietic compartment. Clinically, our data provide a strategy for quantifying pathogenic subsets of emergency myelopoietic cells in the blood as a biomarker that may identify patients who have disproportionately worse outcomes or who may benefit from immune modulatory therapy after acute MI.

Introduction: Ischemic Heart Disease (IHD), the leading cause of death in the world, is often initiated by myocardial infarction (MI). In mice, death typically occurs on day 4 after acute MI is induced by coronary artery ligation. At this time the interferon regulatory factor 3 (IRF3)/type I interferon (IFN) pathway is activated almost exclusively in mononuclear cells in the infarct. We have shown that the genetic or pharmacologic disruption of critical components of this innate immune pathway reduces inflammation, limits ventricular dilation and contractile dysfunction, and improves survival. Hypothesis: Treatment with the IFNAR neutralizing antibody will bias the differentiation of cardiac macrophages and in doing so establish a cardioprotective environment in the infarct. Methods: We conducted single cell RNA-sequencing analysis of mononuclear cells isolated from infarct tissue collected on day 4 after MI from mice that had been treated with an IFNAR neutralizing antibody (Ab) as well as untreated mice. Results: Analyses demonstrate the presence of 3 core cell populations in the untreated sample: monocytes expressing Plac8 and Ms4a4c , phagocytic macrophages expressing Mrc1 and Ccl8 , and reparative macrophages expressing Arg1 and Fn1 . In addition, the dramatic co-expression of related genes in response to environmental stimuli, such as secreted type I IFNs or reactive oxygen species (ROS), resulted in the formation of two more clusters of cells. One cluster was marked by the expression of interferon stimulated genes. The other was marked by the expression of an established anti-oxidant transcriptional program. Anti-interferon therapy with IFNAR Ab inhibited both the interferon stimulated genes and the anti-oxidative transcriptional program. Conclusion: We have defined the differentiation patterns of monocytes and macrophages within the infarcted heart on day 4 after MI. Treatment with the IFNAR Ab after MI alters the composition of macrophage subsets.

Myocardial infarction (MI) is the initiating event in ischemic heart disease, the most common cause of death in the world. Although MI-induced injury is sterile, it nevertheless elicits a vigorous emergency hematopoietic response in the bone marrow that supplies abundant myeloid cells to the heart, which remove dying cell debris and orchestrate healing, repair, and fibrosis. Ensemble methods such as flow sorting of immunostained cells and qPCR have provided some insights into infarct leukocyte phenotypes using candidate markers/genes, but the full diversity remains unknown. Here, we used single cell RNA-Seq to perform genome-wide transcriptomic profiling of >50,000 single cells from the hearts, blood, and bone marrow of infarcted and non-infarcted mice on days 1-4 after MI. We defined the diversification of myeloid cells, from their granulocytic and monocytic origins in the bone marrow, through the blood, and into the infarcted heart. This allowed construction of an atlas of MI-induced myeloid diversification and trafficking. Among the many observations enabled by this data were the origins of the type I interferon response. We recently discovered that MI induces a type I interferon response that can be targeted genetically or pharmacologically for therapeutic benefit. Our single cell data now reveal that interferon induced cells (IFNICs) derive from both neutrophilic and monocytic origins as early as 24 hours after MI. These cells are not only found within the heart, but can also be identified in the post-MI blood and bone marrow. Ongoing studies are investigating whether IFNICs can be detected in human blood after MI. Our results have clinical importance for understanding and modulating the immune response to myocardial injury. We show, using single cell transcriptomics, that post-MI treatment with anti-interferon alpha receptor antibody (IFNAR Ab) (the murine equivalent of a therapeutic antibody currently in Phase 3 clinical trials for lupus), completely abolishes MI-induced interferon signaling and shifts the intracardiac macrophage program towards a reparative posture. Our comprehensive atlas of the emergency hematopoietic response to MI can serve as a resource for others studying the inflammation in ischemic heart disease.

The objectives of the present work were to assess by spectral domain optical coherence tomography (OCT) the changes in thickness of the outer nuclear layer (ONL), the ONL + photoreceptor inner segment (IS), and the retinal thickness, as a function of age in the normal canine retina. OCT retinal scans extending from the edge of the optic nerve head (ONH) along the superior and inferior meridians were captured in both eyes of 17 normal dogs at age ranging from 4 to 119 weeks. The different parameters along the superior and the inferior regions were determined following manual segmentation using the Heidelberg Eye Explorer software. Changes in thickness with age were modeled using one-phase exponential decay models. In vivo OCT imaging results showed no interocular statistically significant differences in ONL, ONL + IS, and retinal thickness at any age. All three parameters were however found to be statistically significantly thicker in the superior vs inferior retina. A rapid thinning of the three layers occurs in both the superior and inferior retina between 4 and 12 weeks of age, before reaching a plateau at around 20 weeks of age. In conclusion, the ONL, ONL + IS, and retinal thickness of the normal canine retina decrease significantly during the first three postnatal months, and is likely attributed to an overall increase in the eye volume and tangential dispersion of the photoreceptor since early photoreceptor developmental cell death is very limited at that age. Establishment of the natural history of ONL, ONL + IS, and retinal thinning will allow a more accurate assessment of the progression of a retinal degenerative condition as well as facilitate the detection of positive rescue effect of novel retinal therapies evaluated in this large animal model.

Myeloperoxidase (MPO) is a critical proinflammatory enzyme implicated in cardiovascular, neurological, and rheumatological diseases. Emerging therapies targeting inflammation have raised interest in tracking MPO activity in patients. We describe 18 F-MAPP, an activatable MPO activity radioprobe for positron emission tomography (PET) imaging. The activated radioprobe binds to proteins and accumulates at sites of MPO activity. The radioprobe 18 F-MAPP has a short blood half-life, remains stable in plasma, does not demonstrate cytotoxicity, and crosses the intact blood–brain barrier. The 18 F-MAPP imaging detected sites of elevated MPO activity in living mice embedded with human MPO and in mice induced with chemical inflammation or myocardial infarction. The 18 F-MAPP PET imaging noninvasively differentiated varying amounts of MPO activity, competitive inhibition, and MPO deficiency in living animals, confirming specificity and showing that the radioprobe can quantify changes in in vivo MPO activity. The radiosynthesis has been optimized and automated, an important step in translation. These data indicate that 18 F-MAPP is a promising translational candidate to noninvasively monitor MPO activity and inflammation in patients.

Objective To identify the frequency of vitreous degeneration and its association with ocular comorbidities including cataracts, lens luxation, glaucoma, and retinal detachment. Methods This is a retrospective study of 4217 dogs from the Companion Animal Eye Registry (CAER) that underwent breed screening ophthalmic examinations between 2013 and 2016. The breeds analyzed included the Italian Greyhound, Shih Tzu, Affenpinscher, Bichon Frise, Brussels Griffon, Whippets, and Greyhound. Data collected from CAER included age, gender, number of examinations, and whether vitreous degeneration, along with cataracts, lens luxation, glaucoma and/or retinal detachment were present in either or both eyes. Results The study found that breed and age are significant drivers for developing VD. Italian Greyhounds, Brussels Griffons, and Shih Tzus have a significantly higher likelihood of VD compared to the negative control breed, the Greyhound. Additionally, with every 1-year age increase, there is a 24% higher likelihood of developing VD. However, no association was identified between vitreous degeneration and cataracts, lens luxation, glaucoma, or retinal detachment. Conclusions While the study found that breed and age were significant drivers for developing VD, no association was found between VD and the other ocular comorbidities examined.

The form of hereditary childhood blindness Leber congenital amaurosis (LCA) caused by biallelic RPE65 mutations is considered treatable with a gene therapy product approved in the US and Europe. The resulting vision improvement is well accepted, but long-term outcomes on the natural history of retinal degeneration are controversial. We treated four RPE65-mutant dogs in mid-life (age = 5–6 years) and followed them long-term (4–5 years). At the time of the intervention at mid-life, there were intra-ocular and inter-animal differences in local photoreceptor layer health ranging from near normal to complete degeneration. Treated locations having more than 63% of normal photoreceptors showed robust treatment-related retention of photoreceptors in the long term. Treated regions with less retained photoreceptors at the time of the intervention showed progressive degeneration similar to untreated regions with matched initial stage of disease. Unexpectedly, both treated and untreated regions in study eyes tended to show less degeneration compared to matched locations in untreated control eyes. These results support the hypothesis that successful long-term arrest of progression with RPE65 gene therapy may only occur in retinal regions with relatively retained photoreceptors at the time of the intervention, and there may be heretofore unknown mechanisms causing long-distance partial treatment effects beyond the region of subretinal injection.

PURPOSE. To examine the in vivo microanatomy of retinal folds and geographic lesions in dogs with acquired or inherited retinal dysplasia. MATERIAL AND METHODS. Thirteen dogs had retinal microanatomy evaluation under general anesthesia using cSLO/sdOCT; two eyes had non-inherited multifocal retinal folds, five had inherited multifocal retinal folds (drd1 or drd2), and 10 geographic retinal dysplasia. Retinas from two drd2 carrier dogs were examined by histology and immunohistochemistry (IHC) after in vivo imaging. RESULTS. Retinal folds are the common feature of acquired focal/multifocal or geographic retinal dysplasia, are indistinguishable structurally from those associated with syndromic oculo-skeletal dysplasia, and represent outer nuclear layer invaginations and rosettes visible by sdOCT. In dogs heterozygous for oculo-skeletal dysplasia, the folds form clusters in a perivascular distribution along superior central vessels. IHC confirmed photoreceptor identity in the retinal folds. The geographic dysplasia plaques are not focally detached, but have inner retinal disorganization and intense autofluorescence in cSLO autofluorescence mode that is mainly limited to the geographic lesion, but is not uniform and in some extends beyond the plaques. CONCLUSION. We propose that the autofluorescent characteristic of the geographic lesions is associated with an inner retinal disruption associated with perivascular or infiltrating macrophages and phagocytosis of cellular debris. As well, we suggest restructuring the examination forms to distinguish the folds that are sporadically distributed from those that have a perivascular distribution as the latter likely represent carriers for drd. In this latter group, DNA testing would be a helpful tool to provide specific breeding advice.

The STRs are a widely used tool in forensic genetics and biological anthropology. This study established allele frequencies and some parameters of forensic interest for the autosomal markers included in the PowerPlex ® 16 BIO System kit (Promega). The sample population is from 151 unrelated afro-descendant individuals of San Basilio de Palenque (SBP), Colombia, a population that was founded for free Africans in the colony time and is considered the first free town in America. This population lacks published databases for these markers. DNA was isolated by salting-out. The products were amplified with the PowerPlex ® 16 BIO System kit (Promega) and separated by electrophoresis on 6% polyacrylamide gels and visualized with a FMBIO IIe Genetic Analyzer (HITACHI). Allelic diversity and Hardy-Weinberg equilibrium were performed using the ARLEQUIN software. Bonferroni correction assumes 0.05 significance level used for 15 tests yields an actual significance of 0.0033. The forensic parameters were calculated with PowerStats software (Promega CO). All markers analyzed showed heterozygosity from 66, 89% (TH01) until 87, 42% (Penta E and Penta D). All systems were in Hardy-Weinberg equilibrium after Bonferroni correction. The probabilities of paternity (W), exclusion (PE) and discrimination (PD) accumulated for loci analyzed were 0.99999899, 0.999999 and >0.99999999, respectively. The parameters of forensic interest had values suitable for routine use in forensic genetics.

The E3 ligase parkin ubiquitinates outer mitochondrial membrane\ud proteins during oxidative stress and is linked to early-onset\ud Parkinson’s disease. Parkin is autoinhibited but is activated by the\ud kinase PINK1 that phosphorylates ubiquitin leading to parkin\ud recruitment, and stimulates phosphorylation of parkin’s N-terminal\ud ubiquitin-like (pUbl) domain. How these events alter the\ud structure of parkin to allow recruitment of an E2~Ub conjugate\ud and enhanced ubiquitination is an unresolved question. We\ud present a model of an E2~Ub conjugate bound to the phosphoubiquitin-loaded\ud C-terminus of parkin, derived from NMR chemical\ud shift perturbation experiments. We show the UbcH7~Ub conjugate\ud binds in the open state whereby conjugated ubiquitin binds to the\ud RING1/IBR interface. Further, NMR and mass spectrometry experiments\ud indicate the RING0/RING2 interface is re-modelled,\ud remote from the E2 binding site, and this alters the reactivity of\ud the RING2(Rcat) catalytic cysteine, needed for ubiquitin transfer.\ud Our experiments provide evidence that parkin phosphorylation\ud and E2~Ub recruitment act synergistically to enhance a weak\ud interaction of the pUbl domain with the RING0 domain and rearrange\ud the location of the RING2(Rcat) domain to drive parkin\ud activity.

Retinitis pigmentosa (RP) is a group of inherited retinal degenerations that lead to progressive vision loss. Over 200 mutations in 60 different genes have been shown to cause RP. Given the diversity of genes and mutations that cause RP, corrective gene therapy approaches currently in development may prove both time-consuming and cost-prohibitive for treatment of all forms of RP. An alternative approach is to find common biological pathways that cause retinal degeneration in various forms of RP, and identify new molecular targets. With this goal, we analyzed the retinal transcriptome of two non-allelic forms of RP in dogs, rcd1 and xlpra2, at clinically relevant advanced stages of the two diseases. Both diseases showed very similar trends in changes in gene expression compared to control normal dogs. Pathway analysis revealed upregulation of various components of the innate immune system in both diseases, including inflammasome and complement pathways. Our results show that the retinal transcriptome at advanced stages of RP is very similar to that of other retinal degenerative diseases such as age-related macular degeneration and diabetic retinopathy. Thus, drugs and therapeutics already in development for targeting these retinopathies may also prove useful for the treatment of many forms of RP.

Purpose To define genetic variants associated with variable severity of X-linked progressive retinal atrophy 1 (XLPRA1) caused by a five-nucleotide deletion in canine RPGR exon ORF15. Methods A genome-wide association study (GWAS) was performed in XLPRA1 phenotype informative pedigree. Whole genome sequencing (WGS) was used for mutational analysis of genes within the candidate genomic region. Retinas of normal and mutant dogs were used for gene expression, gene structure, and RNA duplex analyses. Results GWAS followed by haplotype phasing identified an approximately 4.6 Mb candidate genomic interval on CFA31 containing seven protein-coding genes expressed in retina (ROBO1, ROBO2, RBM11, NRIP1, HSPA13, SAMSN1, and USP25). Furthermore, we identified and characterized two novel lncRNAs, ROBO1-AS and ROBO2-AS, that display overlapping gene organization with axon guidance pathway genes ROBO1 and ROBO2, respectively, producing sense-antisense gene pairs. Notably, ROBO1-AS and ROBO2-AS act in cis to form lncRNA/mRNA duplexes with ROBO1 and ROBO2, respectively, suggesting important roles for these lncRNAs in the ROBO regulatory network. A subsequent WGS identified candidate genes within the genomic region on CFA31 that might be implicated in modifying severity of XLPRA1. This approach led to discovery of genetic variants in ROBO1, ROBO1-AS, ROBO2-AS, and USP25 that are strongly associated with the XLPRA1 moderate phenotype. Conclusions The study provides new insights into the genetic basis of phenotypic variation in severity of RPGRorf15-associated retinal degeneration. Our findings suggest an important role for ROBO pathways in disease progression further expanding on our previously reported changes of ROBO1 expression in XLPRA1 retinas.

Objective Mercaptopurine (MP) and pro-drug azathioprine are ‘first-line’ oral therapies for maintaining remission in IBD. It is believed that their pharmacodynamic action is due to a slow cumulative decrease in activated lymphocytes homing to inflamed gut. We examined the role of host metabolism, lymphocytes and microbiome for the amelioration of colitis by the related thioguanine (TG). Design C57Bl/6 mice with or without specific genes altered to elucidate mechanisms responsible for TG's actions were treated daily with oral or intrarectal TG, MP or water. Disease activity was scored daily. At sacrifice, colonic histology, cytokine message, caecal luminal and mucosal microbiomes were analysed. Results Oral and intrarectal TG but not MP rapidly ameliorated spontaneous chronic colitis in Winnie mice (point mutation in Muc2 secretory mucin). TG ameliorated dextran sodium sulfate-induced chronic colitis in wild-type (WT) mice and in mice lacking T and B lymphocytes. Remarkably, colitis improved without immunosuppressive effects in the absence of host hypoxanthine (guanine) phosphoribosyltransferase (Hprt)-mediated conversion of TG to active drug, the thioguanine nucleotides (TGN). Colonic bacteria converted TG and less so MP to TGN, consistent with intestinal bacterial conversion of TG to so reduce inflammation in the mice lacking host Hprt. TG rapidly induced autophagic flux in epithelial, macrophage and WT but not Hprt−/− fibroblast cell lines and augmented epithelial intracellular bacterial killing. Conclusions Treatment by TG is not necessarily dependent on the adaptive immune system. TG is a more efficacious treatment than MP in Winnie spontaneous colitis. Rapid local bacterial conversion of TG correlated with decreased intestinal inflammation and immune activation.

The effect of acute exposure to various intensities of white light on visual behavior and retinal structure was evaluated in the T4R RHO dog, a naturally-occurring model of autosomal dominant retinitis pigmentosa due to a mutation in the Rhodopsin gene. A total of 14 dogs (ages: 4-5.5 months) were used in this study: 3 homozygous mutant RHO(T4R/T4R), 8 heterozygous mutant RHO(T4R/+), and 3 normal wild-type (WT) dogs. Following overnight dark adaptation, the left eyes were acutely exposed to bright white light with a monocular Ganzfeld dome, while the contralateral right eye was shielded. Each of the 3 homozygous (RHO(T4R/T4R)) mutant dogs had a single unilateral light exposure (LE) to a different (low, moderate, and high) dose of white light (corneal irradiance/illuminance: 0.1 mW/cm(2), 170 lux; 0.5 mW/cm(2), 820 lux; or 1 mW/cm(2), 1590 lux) for 1 min. All 8 heterozygous (RHO(T4R/+)) mutant dogs were exposed once to the same moderate dose of light. The 3 WT dogs had their left eyes exposed 1, 2, or 3 times to the same highest dose of light. Visual function prior to LE and at 2 weeks and 33 weeks after exposure was objectively assessed in the RHO(T4R/T4R) and WT dogs by using an obstacle-avoidance course. Transit time through the obstacle course was measured under different scotopic to photopic ambient illuminations. Morphological retinal changes were evaluated by non-invasive in vivo cSLO/sdOCT imaging and histology before and at several time-points (2-36 weeks) after light exposure. The analysis of the transit time through the obstacle course showed that no differences were observed in any of mutant or WT dogs at 2 weeks and 33 weeks post LE. The RHO(T4R/T4R) retina exposed to the lowest dose of white light showed no obvious changes in ONL thickness at 2 weeks, but mild decrease was noted 36 weeks after LE. The RHO(T4R/T4R) retina that received a moderate dose (showed an obvious decrease in ONL thickness along the superior and temporal meridians at 2 weeks post LE with more severe damage at 36 weeks post LE in all four meridians. The RHO(T4R/T4R) retina exposed to the high dose showed at 2 weeks after LE extensive ONL damage in all four meridians. This light intensity did not cause any retinal damage in WT dogs even after repeated (up to 3) LE. Analysis of ONL thickness in heterozygous mutant dogs exposed to the moderate dose of light confirmed the increased sensitivity to light damage of the superior/tapetal retina, and the occurrence of an ongoing cell death process several weeks after the acute LE. In conclusion, a short single exposure to a dose of white light that is not retinotoxic in WT dogs causes in the T4R RHO retina an acute loss of ONL in the central to mid peripheral region that keeps progressing over the course of several weeks. However, this severe retinal damage does not affect visual behavior presumably because of islands of surviving photoreceptors found in the area centralis including the newly discovered canine fovea-like area, and the lack of damage to peripheral photoreceptors.

Mucopolysaccharidosis VI (MPS VI) is a lysosomal storage disease characterized by an absence or marked reduction of lysosomal N-acetylgalactosamine-4-sulfatase activity. Affected individuals have widespread accumulation of unmetabolized glycosaminoglycan substrates leading to detrimental effects. Recombinant human N-acetylgalactosamine 4-sulfatase (rhASB) is an approved enzyme replacement therapy for patients with MPS VI. Despite the known efficacy of weekly 4-h rhASB infusions, some clinicians wish to treat patients using reduced infusion times. This study compared the pharmacodynamics, pharmacokinetics, and tissue biodistribution of rhASB when administered as 2- and 4-h intravenous infusions using a feline model of MPS VI.Study animals were MPS VI-affected cats that demonstrate clinical signs and biochemical derangements similar to human MPS VI patients. Beginning at age 4weeks, animals received weekly 2-h (N=6) or 4-h (N=6) IV infusions of rhASB for 26weeks (Naglazyme® [galsulfase] Solution for Intravenous Infusion; BioMarin Pharmaceutical, Inc.). The control group consisted of untreated MPS VI-affected cats (N=6). The pharmacokinetic parameters of plasma rhASB and urinary glycosaminoglycan were determined at weeks 13 and 26. Animals were euthanized 48h after the last infusion and tissue concentration of ASB, GAG and β-glucuronidase were measured in the liver, spleen, aorta, and kidney. Skeletal and ophthalmological evaluations were performed within 2weeks of euthanasia.At week 13, the mean AUC0-t in animals treated with 4-h infusions was similar to 2-h infusions while the Cmax of the 4-h infusion was 50% of the 2-h infusion. By week 26, the mean AUC0-t of the 4-h infusion was 1.3-fold higher than the 2-h infusion (p0.05) while Cmax of the 4-h infusion was 70% of the 2-h infusion (p0.05). Among animals treated with 2- and 4-h infusions, there was no difference in urinary GAG excretion, tissue GAG storage, tissue galsulfase activity, and β-glucuronidase but all were significantly different than control animals (for each, p0.001). Radiographic skeletal abnormality scores for animals were also similar for both treatment groups and significantly higher than control animals (p0.001). There was no significant difference in corneal clouding scores among treated and untreated animals.There was no significant difference in clinical outcomes when rhASB was administered to MPS VI affected cats as 2- and 4-h infusions over 26weeks. Additional studies may determine if shorter infusion times are appropriate for MPS VI patients without significant infusion-associated reactions.

Degradation of photoreceptor outer segments (POS) by retinal pigment epithelium (RPE) is essential for vision, and studies have implicated altered POS processing in the pathogenesis of some retinal degenerative diseases. Consistent with this concept, a recently established hiPSC-RPE model of inherited macular degeneration, Best disease (BD), displayed reduced rates of POS breakdown. Herein we utilized this model to determine (i) if disturbances in protein degradation pathways are associated with delayed POS digestion and (ii) whether such defect(s) can be pharmacologically targeted. We found that BD hiPSC-RPE cultures possessed increased protein oxidation, decreased free-ubiquitin levels, and altered rates of exosome secretion, consistent with altered POS processing. Application of valproic acid (VPA) with or without rapamycin increased rates of POS degradation in our model, whereas application of bafilomycin-A1 decreased such rates. Importantly, the negative effect of bafilomycin-A1 could be fully reversed by VPA. The utility of hiPSC-RPE for VPA testing was further evident following examination of its efficacy and metabolism in a complementary canine disease model. Our findings suggest that disturbances in protein degradation pathways contribute to the POS processing defect observed in BD hiPSC-RPE, which can be manipulated pharmacologically. These results have therapeutic implications for BD and perhaps other maculopathies.

Due to improved phenotyping and genetic characterization, the field of ‘incurable’ and ‘blinding’ inherited retinal diseases (IRDs) has moved substantially forward. Decades of ascertainment of IRD patient data from Philadelphia and Toronto centers illustrate the progress from Mendelian genetic types to molecular diagnoses. Molecular genetics have been used not only to clarify diagnoses and to direct counseling but also to enable the first clinical trials of gene-based treatment in these diseases. An overview of the recent reports of gene augmentation clinical trials by subretinal injections is used to reflect on the reasons why there has been limited success in this early venture into therapy. These first-in human experiences have taught that there is a need for advancing the techniques of delivery of the gene products - not only for refining further subretinal trials, but also for evaluating intravitreal delivery. Candidate IRDs for intravitreal gene delivery are then suggested to illustrate some of the disorders that may be amenable to improvement of remaining central vision with the least photoreceptor trauma. A more detailed understanding of the human IRDs to be considered for therapy and the calculated potential for efficacy should be among the routine prerequisites for initiating a clinical trial.

We report the application of cyclic voltammetry and absorption spectroscopy to the characterization and study of the stability of silver colloids in water. The samples are prepared via chemical reduction and the reactions are catalyzed by irradiation with white light. The electrochemical response is related to the characteristic sample surface plasmon resonance (SPR) in the UV-visible absorption spectra. Cyclic voltammetry shows a characteristic reduction peak whose position is specific to each analyzed sample. Optical analysis of a colloid precursor during a 12 h time span, under low-power white-light irradiation, shows that nanoparticles undergo change in size and surface state (absorption bands splitting and inversion) to attain the “stable” colloidal form. While the absorption spectrum bands of the precursor return almost periodically to similar positions, the cyclic voltammogram characteristic reduction peak is displaced as a function of time. Finally, we follow the SPR changes of one “stable” colloid being subjected to electrolysis, heating, and sunlight irradiation, for environmental remediation purposes. Sunlight exposure produces the most significant SPR intensity drop, but the electrochemical technique shows itself promising as well.

The following review summarizes the state of the art in representative aspects of gene therapy/translational medicine and evolves from a symposium held at the School of Veterinary Medicine, University of Pennsylvania on November 16, 2017 honoring Dr. Gustavo Aguirre, recipient of ARVO's 2017 Proctor Medal. Focusing on the retina, speakers highlighted current work on moving therapies for inherited retinal degenerative diseases from the laboratory bench to the clinic.

Inherited retinal degenerations are caused by mutations in >250 genes that affect photoreceptor cells or the retinal pigment epithelium and result in vision loss. For autosomal recessive and X-linked retinal degenerations, significant progress has been achieved in the field of gene therapy as evidenced by the growing number of clinical trials and the recent commercialization of the first gene therapy for a form of congenital blindness. However, despite significant efforts to develop a treatment for the most common form of autosomal dominant retinitis pigmentosa (adRP) caused by >150 mutations in the rhodopsin (RHO) gene, translation to the clinic has stalled. Here, we identified a highly efficient shRNA that targets human (and canine) RHO in a mutation-independent manner. In a single adeno-associated viral (AAV) vector we combined this shRNA with a human RHO replacement cDNA made resistant to RNA interference and tested this construct in a naturally occurring canine model of RHO-adRP. Subretinal vector injections led to nearly complete suppression of endogenous canine RHO RNA, while the human RHO replacement cDNA resulted in up to 30% of normal RHO protein levels. Noninvasive retinal imaging showed photoreceptors in treated areas were completely protected from retinal degeneration. Histopathology confirmed retention of normal photoreceptor structure and RHO expression in rod outer segments. Long-term (>8 mo) follow-up by retinal imaging and electroretinography indicated stable structural and functional preservation. The efficacy of this gene therapy in a clinically relevant large-animal model paves the way for treating patients with RHO-adRP.

Ischemic heart disease (IHD), the leading cause of death in the world, begins with myocardial infarction (MI). Overly exuberant innate immune responses are detrimental to post-MI healing and repair. Efforts to achieve post-MI cardioprotection focused on blocking NFκB-mediated inflammation have been largely unsuccessful. We recently discovered that MI leads to cytosolic DNA sensing in leukocytes and triggers the interferon regulatory factor 3 (IRF3)-dependent expression of interferon stimulated genes (ISGs). We found that genetic inhibition of IRF3 after MI reduces inflammation, limits ventricular dilation and contractile dysfunction, and improves survival. In this work, we test two hypotheses. 1) Type I interferons (IFNs), secreted by a few DNA-sensing/IRF3-activated leukocytes, signal via type I interferon alpha/beta receptors (IFNARs), to amplify ISG expression after MI. 2) Pharmacologic inhibition of type I IFN/IFNAR signaling blocks response amplification and significantly reduces ISG expression. We established DNA-stimulated bone marrow derived macrophages (BMDMs) as an in vitro model of the post-MI type I IFN response and measured the expression of ISGs ( Cxcl10 , Irf7 , Ifit1 , and Ifit3 ) by qPCR. We quantified and compared changes in ISG expression caused by direct DNA stimulation, incubation with conditioned media containing secreted factors from DNA-stimulated cells, and treatment with an IFNAR neutralizing antibody. Compared to unstimulated BMDMs, ISG expression was amplified by >35-fold in BMDMs stimulated by either DNA or conditioned media. However, there was no significant amplification when the experiments were performed in the presence of an IFNAR neutralizing antibody. In a mouse model of MI, single cell RNA-Seq revealed that IFNAR-deficient mice have reduced numbers of ISG-expressing leukocytes compared to wild type controls, which indicates that IFNAR-dependent amplification of innate immune responses occurs after MI in vivo . Collectively, these results demonstrate that IFNAR ligands are important amplifiers of DNA-induced innate immune responses after MI and suggest that anti-interferon therapy should be further investigated in vivo to improve clinical outcomes after MI.

Practice guidelines for preoperative fasting have not clearly established the fasting time needed after oral administration of water-soluble contrast media. The aim of this study was to determine the time required for the gastric emptying during the water-soluble contrast media in patients with acute abdominal pain.This prospective longitudinal study included sixty-eight patients older than 18 years of age with acute abdominal pain, who required a water-soluble contrast media enhanced abdominal computed tomography study. Plain radiographs were obtained hourly until complete the gastric emptying. Patients with probable bowel obstruction were not included in the study.A total of 31 (45,6%), 54 (79,4%), and 64 (94,1%) patients achieved a complete gastric clearance of barium in 1, 2 and 3 hours, respectively. All patients achieved complete emptying of water-soluble contrast media within 6 hours. Gastric emptying time was not associated with gender (P=0,44), body mass index (P=.35), fasting time prior to water-soluble contrast media intake (P=0,12), administration of opioids in the emergency room (P=0,7), and the presence of comorbidities (P=0,36).Ninety-four percent of the patients with acute abdominal pain achieved complete gastric emptying within 3hours after the administration of water-soluble contrast media. All of them achieved complete gastric emptying within 6hours. The results suggested 6hours after oral intake of the contrast media is enough to complete transit of water-soluble contrast media through the stomach and avoid unnecessary risks.

Ndr2/Stk38l encodes a protein kinase associated with the Hippo tumor suppressor pathway and is mutated in a naturally-occurring canine early retinal degeneration (erd). To elucidate the retinal functions of Ndr2 and its paralog Ndr1/Stk38, we generated Ndr1 and Ndr2 single knockout mice. Although retinal lamination appeared normal in these mice, Ndr deletion caused a subset of Pax6-positive amacrine cells to proliferate in differentiated retinas, while concurrently decreasing the number of GABAergic, HuD and Pax6-positive amacrine cells. Retinal transcriptome analyses revealed that Ndr2 deletion increased expression of neuronal stress genes and decreased expression of synaptic organization genes. Consistent with the latter, Ndr deletion dramatically reduced levels of Aak1, an Ndr substrate that regulates vesicle trafficking. Our findings indicate that Ndr kinases are important regulators of amacrine and photoreceptor cells and suggest that Ndr kinases inhibit the proliferation of a subset of terminally differentiated cells and modulate interneuron synapse function via Aak1.

Canine bestrophinopathy (cBest) is an important translational model for BEST1-associated maculopathies in man that recapitulates the broad spectrum of clinical and molecular disease aspects observed in patients. Both human and canine bestrophinopathies are characterized by focal to multifocal separations of the retina from the RPE. The lesions can be macular or extramacular, and the specific pathomechanism leading to formation of these lesions remains unclear. We used the naturally occurring canine BEST1 model to examine factors that underlie formation of vitelliform lesions and addressed the susceptibility of the macula to its primary detachment in BEST1-linked maculopathies.

Protocols for photoreceptor outer segment (POS) isolation that can be used in phagocytosis assays of retinal pigment epithelium (RPE) cells have routinely used a large number of cow or pig eyes. However, when working with large animal models (e.g., dog, cats, transgenic pigs) of inherited retinal degenerative diseases, access to retinal tissues may be limited. An optimized protocol is presented in this paper to isolate sufficient POS from a single canine retina for use in RPE phagocytosis assays.

Retinitis pigmentosa (RP) is a group of inherited retinal degenerations that lead to progressive vision loss. Many mutations in 60 different genes have been shown to cause RP. Given the diversity of genes and mutations that cause RP, corrective gene therapy approaches currently in development may prove both time-consuming and cost-prohibitive for treatment of all forms of RP. An alternative approach is to find common biological pathways that cause retinal degeneration in various forms of RP, and identify new molecular targets. With this goal, we analyzed the retinal transcriptome of two non-allelic forms of RP in dogs, rcd1 and xlpra2, at clinically relevant advanced stages of the two diseases. Both diseases showed very similar trends in changes in gene expression compared to control normal dogs. Pathway analysis revealed upregulation of various components of the innate immune system in both diseases, including inflammasome and complement pathways. Our results show that the retinal transcriptome at advanced stages of RP is very similar to that of other retinal degenerative diseases such as age-related macular degeneration and diabetic retinopathy. Thus, drugs and therapeutics already in development for targeting these retinopathies may also prove useful for the treatment of many forms of RP.

In this paper, two LED emitting circuits are implemented in a VLC positioning system, a regular Bias-Tee and a circuit proposed by Ghassemlooy, aiming to provide the system with portability and functionality. For this, an Arduino Nano is used in the emitting circuit together with an AD9850 signal generator circuit. For the receiver circuit a Thorlabs PDA36-EC photodetector, an XMOS for data acquisition and a Raspberry Pi for data processing are used. Subsequently a brief comparison is made between the two emitting circuits, comparing the power received by the photodetector as a function of the emitter-receiver distance.

Background Previous studies have demonstrated that statin therapy improves cardiac outcomes, probably by stabilizing thin‐cap fibroatheroma in patients with coronary artery disease. However, major adverse cardiac events still occur in some patients, despite statin therapy. The aim of this study is to identify clinical predictors for the lack of a favorable vascular response to statin therapy in patients with coronary artery disease. Methods and Results A total of 140 nonculprit plaques from 84 patients with coronary artery disease who were treated with a statin and had serial optical coherence tomography imaging (median interval, 6.3 months) were included. Thin‐cap area (fibrous cap thickness, 2 ; 25 th –75 th percentile, 1.023–6.157 mm 2 ) to follow‐up (median, 1.210 mm 2 ; 25 th –75 th percentile, 0.250–3.192 mm 2 ; P P 2 ; 95% confidence interval, 0.350–3.033 mm 2 ; P =0.013) and lower in patients with acute coronary syndrome (regression coefficient b, −1.535 mm 2 ; 95% confidence interval, −2.561 to −0.509 mm 2 ; P =0.003). Conclusions Chronic kidney disease is an independent predictor for the lack of a favorable vascular response to statin therapy, whereas acute coronary syndrome is an independent predictor for favorable vascular response to statin therapy. These findings should be further warranted in future prospective studies. Clinical Trial Registration URL: http://www.clinicaltrials.gov . Unique identifier: NCT01110538.

Real-time microscopic imaging of moving organs at single-cell resolution represents a major challenge in studying complex biology in living systems. Motion of the tissue from the cardiac and respiratory cycles severely limits intravital microscopy by compromising ultimate spatial and temporal imaging resolution. However, significant recent advances have enabled single-cell resolution imaging to be achieved in vivo. In this protocol, we describe experimental procedures for intravital microscopy based on a combination of thoracic surgery, tissue stabilizers and acquisition gating methods, which enable imaging at the single-cell level in the beating heart in the mouse. Setup of the model is typically completed in 1 h, which allows 2 h or more of continuous cardiac imaging. This protocol can be readily adapted for the imaging of other moving organs, and it will therefore broadly facilitate in vivo high-resolution microscopy studies.

Achromatopsia is an inherited retinal disorder of cone photoreceptors characterized by markedly reduced visual acuity, extreme light sensitivity, and absence of color discrimination. Approximately 50% of cases are caused by mutations in the cone photoreceptor-specific cyclic nucleotide gated channel beta subunit (CNGB3) gene. Studies in CNGB3-mutant dogs showed that subretinal injection of an AAV vector expressing human CNGB3, which has 76% amino acid identity with canine CNGB3, driven by a 2.1 kb human red cone opsin promoter (PR2.1) and packaged in AAV5 capsids (AAV5-PR2.1-hCNGB3) rescued cone photoreceptor function, but at high doses was associated with an inflammatory response (focal chorioretinitis) consistent with immune-mediated toxicity. AAV vectors containing the PR2.1 promoter packaged in AAV5 capsids and expressing either the native canine CNGB3 (AAV5-PR2.1-cCNGB3) or the human CNGB3 (AAV5-PR2.1-hCNGB3) were evaluated at different dose levels in CNGB3-mutant dogs. The vector expressing canine CNGB3 achieved somewhat better rescue of cone function but unexpectedly was associated with a greater degree of retinal toxicity than the vector expressing human CNGB3. Very low-level T-cell immune responses to some AAV or CNGB3 peptides were observed in animals that received the higher vector dose. There was a more than twofold increase in serum neutralizing antibodies to AAV in one of three animals in the low-dose group and in two of three animals in the high-dose group. No serum anti-hCNGB3 antibodies were detected in any animal. The results of this study do not support the hypothesis that the focal chorioretinitis seen with high doses of AAV5-PR2.1-hCNGB3 in the initial studies was due to an immune response to human CNGB3.

Background Differentiation of Systemic Lupus Erythematosus (SLE) activity and infection in a febrile SLE patient become difficult, since initial clinical presentation may be similar. Several biological markers (including procalcitonin and CRP) have been evaluated, with discordant results. Soluble CD14 (sCD14), also called presepsin, is the receptor for lipopolysaccharide - lipopolysaccharide binding protein (LPS-LBP) complexes. CD14 could activate a series of signal transduction pathways and inflammatory cascades, and lead to systemic inflammatory responses.(1) Objectives The aim of this study is to evaluate the utility of sCD14 as a biomarker to differentiate infection vs. activity in SLE patients admitted with systemic inflammatory response (SIRS). Methods We included 11 patients with SLE (ACR criteria 1997) and SIRS (International conference 2001) admitted to the ER and/or ICU. The measurement of sCD14 in plasma by enzymatic immunoassay of chemiluminescence in vitro was performed to differentiate active SLE vs. infection. Infection was considered if a positive culture/PCR was obtained. Mann-Whitney test was used to evaluate the association of variables with infection. Results All patients were female; mean age 37.9 years. An infectious disease was confirmed in 5 cases (3 bacterial including urinary tract infection, pneumonia and bacteremia; 1 viral infection by Chikungunya virus and 1 fungal by histoplasma capsulatum). sCD14 was elevated in the infected SLE patients (median: 1005 pg/mL- RIC: 533–1415-) vs patients with lupus flare (median: 431.5 pg/mL - RIC: 369–579-) (p=0,04). Conclusions High values of sCD14 levels seem to be useful to differentiate infections from activity in SLE patients with SIRS. More patients and further analysis are necessary to define the clinical use of this biomarker. References Zou Q, Wen W, Zhang X. Presepsin as a novel sepsis biomarker. World J Emerg Med [Internet]. Second Affiliated Hospital of Zhejiang University School of Medicine; 2014 Aug 22;5(1):16–9. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4129857. Disclosure of Interest None declared

X-linked retinitis pigmentosa (XLRP) caused by mutations in the RPGR gene is an early onset and severe cause of blindness. Successful proof-of-concept studies in a canine model have recently shown that development of a corrective gene therapy for RPGR-XLRP may now be an attainable goal. In preparation for a future clinical trial, we have here optimized the therapeutic AAV vector construct by showing that GRK1 (rather than IRBP) is a more efficient promoter for targeting gene expression to both rods and cones in non-human primates. Two transgenes were used in RPGR mutant (XLPRA2) dogs under the control of the GRK1 promoter. First was the previously developed stabilized human RPGR (hRPGRstb). Second was a new full-length stabilized and codon-optimized human RPGR (hRPGRco). Long-term (>2 years) studies with an AAV2/5 vector carrying hRPGRstb under control of the GRK1 promoter showed rescue of rods and cones from degeneration and retention of vision. Shorter term (3 months) studies demonstrated comparable preservation of photoreceptors in canine eyes treated with an AAV2/5 vector carrying either transgene under the control of the GRK1 promoter. These results provide the critical molecular components (GRK1 promoter, hRPGRco transgene) to now construct a therapeutic viral vector optimized for RPGR-XLRP patients.

Background Heart murmurs are commonly detected at the Newborn Infant Physical Examination (NIPE). Routine use of antenatal and pulse oximetry screening means isolated murmurs are unlikely to be due to missed critical Congenital Heart Disease (CHD). We have developed a local guideline for assessment and follow up of these babies and share our experience of this service. Aim To assess the outcomes of neonatal heart murmurs detected on routine NIPE and review utilisation of neonatal and PEC (Paediatrician with Expertise in Cardiology) clinics. Methods All babies with murmurs on NIPE over one year (July 2015–June 2016) were retrospectively identified from the NIPE Smart system. Data was gathered from electronic and paper hospital records. All babies had follow-up outcomes for minimum 6 months. Babies with antenatal CHD diagnosis or having NICU admission were excluded. Results Out of about 6000 deliveries, 139 patients had murmurs detected (50.4% Male). 96 murmurs were noted at Conclusion Most murmurs in neonates with normal pulse-oximetry are innocent, only 4% diagnosed with underlying CHD. CXR and ECGs have little role in the routine investigation of isolated neonatal murmurs. The current department referral pathway is working well with only 10% of referrals to neonatal clinic requiring PEC clinic referral, thus optimising PEC clinic utilisation.

Organ-specific functions of tissue-resident macrophages in the steady-state heart are unknown. Here, we show that cardiac macrophages facilitate electrical conduction through the distal atrioventricular node, where conducting cells densely intersperse with elongated macrophages expressing connexin 43. When coupled to spontaneously beating cardiomyocytes via connexin-43-containing gap junctions, cardiac macrophages have a negative resting membrane potential and depolarize in synchrony with cardiomyocytes. Conversely, macrophages render the resting membrane potential of cardiomyocytes more positive and, according to computational modeling, accelerate their repolarization. Photostimulation of channelrhodopsin-2-expressing macrophages improves atrioventricular conduction, whereas conditional deletion of connexin 43 in macrophages and congenital lack of macrophages delay atrioventricular conduction. In the Cd11b(DTR) mouse, macrophage ablation induces progressive atrioventricular block. These observations implicate macrophages in normal and aberrant cardiac conduction.

Bestrophinopathies, one of the most common forms of inherited macular degenerations, are caused by mutations in the BEST1 gene expressed in the retinal pigment epithelium (RPE). Both human and canine BEST1-linked maculopathies are characterized by abnormal accumulation of autofluorescent material within RPE cells and bilateral macular or multifocal lesions; however, the specific mechanism leading to the formation of these lesions remains unclear. We now provide an overview of the current state of knowledge on the molecular pathology of bestrophinopathies, and explore factors promoting formation of RPE-neuroretinal separations, using the first spontaneous animal model of BEST1-associated retinopathies, canine Best (cBest). Here, we characterize the nature of the autofluorescent RPE cell inclusions and report matching spectral signatures of RPE-associated fluorophores between human and canine retinae, indicating an analogous composition of endogenous RPE deposits in Best Vitelliform Macular Dystrophy (BVMD) patients and its canine disease model. This study also exposes a range of biochemical and structural abnormalities at the RPE-photoreceptor interface related to the impaired cone-associated microvillar ensheathment and compromised insoluble interphotoreceptor matrix (IPM), the major pathological culprits responsible for weakening of the RPE-neuroretina interactions, and consequently, formation of vitelliform lesions. These salient alterations detected at the RPE apical domain in cBest as well as in BVMD- and ARB-hiPSC-RPE model systems provide novel insights into the pathological mechanism of BEST1-linked disorders that will allow for development of critical outcome measures guiding therapeutic strategies for bestrophinopathies.

OBJECTIVES Certain neointimal patterns including neoatherosclerosis (NA) are known to be associated with poor clinical outcome. The prevalence and time course of different neointimal patterns have not been studied systematically. The aim of this study was to investigate the serial changes in neointimal pattern after drug-eluting stent implantation. PATIENTS AND METHODS A total of 132 patients with 207 drug-eluting stents, who underwent two follow-up optical coherence tomography studies at 6 and 12 months, were included. Neointimal patterns were categorized as homogeneous, heterogeneous, layered, or NA using optical coherence tomography. Quantitative and qualitative analyses of neointima were carried out. RESULTS Both at 6 and at 12 months, the homogenous neointima was the predominant type (>75%), followed by the layered and the heterogeneous pattern. At 12 months, progression to NA was observed in 0.6% of the patients in the homogeneous group, in 5.6% of the patients in the heterogeneous group, and in 3.9% of the patients in the layered group. Regression to the homogeneous pattern was observed in 5.6% of the patients in the heterogeneous group and 11.5% of the patients in the layered group. CONCLUSION The homogenous neointima is the predominant pattern both at 6 and at 12 months. The neointimal pattern changed between 6 and 12 months in 10.6% of stents. Further studies are needed to understand the mechanisms of these neointimal changes and their clinical significance.

We have analyzed the complex pattern of the inflammatory response in early-onset canine models of human retinitis pigmentosa, rcd1, xlpra2 and erd, as well as late-onset xlpra1, in comparative manner. The time course of immune response genes and proteins expression was examined along the timeline of photoreceptors degeneration. Gene expression analysis of the early-onset models prior to and after the peak of photoreceptors death identified the involvement of multiple immune response genes including those encoding constituents of the NLRP3 inflammasome, its substrates, pro-IL1B, pro-IL18, and common components of IL1B, IL18 and TLR4 pathways. Out of two activated caspase-1 cleavage products, IL1B and IL18, only IL1B was detected in rcd1 and xlpra2 while precursor IL18 remained unprocessed in the same protein extract highlighting prominence of IL1B pathway. An overall immune response was most prominent in rcd1 followed by xlpra2 and least prominent in erd. Noticeably, in rcd1 and xlpra2, but not in erd, early induction of the immune response was accompanied by sustained intraretinal migration and activation of retinal microglia. Lastly, delayed activation of the anti-inflammatory factors in all early-onset models was insufficient to counterbalance rapidly progressing inflammation. In contrast to early-onset models, in late-onset xlpra1 retinas a subset of the pro-inflammatory genes was highly upregulated long before any disease-related structural changes occurred, but was counterbalanced by an adequate anti-inflammatory response. Results point out to upregulated immune response accompanying disease progression in animal models of retinal degeneration, and to potential benefits of early anti-inflammatory therapy.

Background— Recent reports show that plaque erosion can be diagnosed in vivo using optical coherence tomography in patients with acute coronary syndrome. However, quantitative optical coherence tomographic image criteria for computer-aided diagnosis of plaque erosion have not been established. Methods and Results— A total of 42 patients with acute coronary syndrome caused by plaque erosion were included. Plaque erosion was identified according to the previously established optical coherence tomography criteria. Both optical properties and morphological features of the focal-eroded region as well as erosion-adjacent region were analyzed using a custom-designed computer algorithm. Noneroded fibrous plaques remote from the erosion site within the same vessel were used as controls. Eroded plaques have significantly lower surface intensity ( P P P P P P P P =0.008), higher attenuation ( P P =0.005). Using a logistic regression model built on the quantitative features, plaque erosion can be accurately classified against intact fibrous plaques. There was low inter- and intraobserver variability associated with the algorithm-assisted quantitative assessment. Conclusions— Plaque erosion has distinctive optical properties and morphological features when compared with noneroded fibrous plaques. Quantitative image analysis may enhance diagnostic accuracy for plaque erosion in vivo.

Significance Despite advances in cardiac imaging technologies such as MRI, computed tomography, and ultrasound it has not been possible to image cardiac function in animal models at the cellular level owing to insufficient resolution and excess motion. As a result, our understanding of cardiac biology and function is derived from either whole-organ imaging techniques or from isolated in vitro heart preparations and cellular model systems. We demonstrate a technique for intravital optical microscopy that compensates for motion of the contracting heart and allows for measurement of contractile function at the single-cell level. These approaches will enable studies of complex and dynamic cellular physiology in the beating heart and should be widely useful for studying heart disease in animal models.

© 2014 John Wiley & Sons Ltd. The use of next-generation sequencing technologies is revolutionizing microbial ecology by allowing a deep phylogenetic coverage of tens to thousands of samples simultaneously. Double Principal Coordinates Analysis (DPCoA) is a multivariate method, developed in community ecology, able to integrate a distance matrix describing differences among species (e.g. phylogenetic distances) in the analysis of a species abundance matrix. This ordination technique has been used recently to describe microbial communities taking into account phylogenetic relatedness. In this work, we extend DPCoA to integrate the information of external variables measured on communities. The constrained Double Principal Coordinates Analysis (cDPCoA) is able to enforce a priori classifications to retrieve subtle differences and (or) remove the effect of confounding factors. We describe the main principles of this new approach and demonstrate its usefulness by providing application examples based on published 16S rRNA gene data sets.

The development path described for JNJ-26489112 provides perspectives on interpretation of retinal effects observed in nonclinical studies and their implications for clinical development. JNJ-26489112 is a CNS-active investigational drug that has potential as a novel treatment for treatment-resistant and bipolar depression, epilepsy, and neuropathic/inflammatory pain. In a 6-month toxicity study in albino rats, retinal atrophy was observed at supratherapeutic exposures to JNJ-26489112. The histopathological changes and topography of the lesions were characteristic of light-induced damage specific to albino rats. The species/strain specificity is supported by an absence of any ocular effects in dogs and in pigmented and albino rats, housed under standard and reduced lighting, respectively. To further evaluate its potential to cause ocular effects, in vivo functional and structural ocular analyses were included in a 9-month monkey toxicity study. Reductions in rod- and cone-mediated electroretinograms were observed at supratherapeutic exposures but without any histopathologic changes. These data suggested that the effects of JNJ-26489112 in monkeys were neuromodulatory and not neurotoxic. Taken together, data related to the light-induced atrophy in albino rats and reversible neuromodulatory effects in monkeys, supported the safe evaluation of JNJ-26489112 in a clinical proof-of-concept study that included comprehensive functional and structural ocular monitoring.

A magnetic ligation method is utilized for the detection of microRNAs amongst a complex biological background without polymerase chain reaction or nucleotide modification. The sandwich probes assay can be adapted to analyze a panel of microRNAs associated with cardiovascular diseases in heart tissue samples.

Mutations in PARK2 and PARK6 genes are responsible for the majority of hereditary Parkinson’s disease cases. These genes encode the E3 ubiquitin ligase parkin and the protein kinase PTEN-induced kinase 1 (PINK1), respectively. Together, parkin and PINK1 regulate the mitophagy pathway, which recycles damaged mitochondria following oxidative stress. Native parkin is inactive and exists in an autoinhibited state mediated by its ubiquitin-like (UBL) domain. PINK1 phosphorylation of serine 65 in parkin’s UBL and serine 65 of ubiquitin fully activate ubiquitin ligase activity; however, a structural rationale for these observations is not clear. Here, we report the structure of the phosphorylated UBL domain from parkin. We find that destabilization of the UBL results from rearrangements to hydrophobic core packing that modify its structure. Altered surface electrostatics from the phosphoserine group disrupt its intramolecular association, resulting in poorer autoinhibition in phosphorylated parkin. Further, we show that phosphorylation of both the UBL domain and ubiquitin are required to activate parkin by releasing the UBL domain, forming an extended structure needed to facilitate E2–ubiquitin binding. Together, the results underscore the importance of parkin activation by the PINK1 phosphorylation signal and provide a structural picture of the unraveling of parkin’s ubiquitin ligase potential.