Visualization of choroidal vasculature in pigmented mouse eyes from experimental models of AMD.

A variety of techniques exist to investigate retinal and choroidal vascular changes in experimental mouse models of human ocular diseases. While all have specific advantages, a method for evaluating the choroidal vasculature in pigmented mouse eyes has been more challenging especially for whole mount visualization and morphometric analysis. Here we report a simple, reliable technique involving bleaching pigment prior to immunostaining the vasculature in whole mounts of pigmented mouse choroids. Eyes from healthy adult pigmented C57BL/6J mice were used to establish the methodology. The retina and anterior segment were separated from the choroid. The choroid with retinal pigment epithelial cells (RPE) and sclera was soaked in 1% ethylenediaminetetraacetic acid (EDTA) to remove the RPE. Tissues were fixed in 2% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). Choroids were subjected to melanin bleaching with 10% hydrogen peroxide (H2O2) at 55 °C for 90 min, washed in PBS and then immunostained with anti-podocalyxin antibody to label vascular endothelium followed by Cy3-AffiniPure donkey anti-goat IgG at 4 °C overnight. Images of immunostained bleached choroids were captured using a Zeiss 710 confocal microscope. In addition to control eyes, this method was used to analyze the choroids from subretinal sodium iodate (NaIO3) RPE atrophy and laser-induced choroidal neovascularization (CNV) mouse models. The H2O2 pretreatment effectively bleached the melanin, resulting in a transparent choroid. Immunolabeling with podocalyxin antibody following bleaching provided excellent visualization of choroidal vasculature in the flat perspective. In control choroids, the choriocapillaris (CC) displayed different anatomical patterns in peripapillary (PP), mid peripheral (MP) and far peripheral (FP) choroid. Morphometric analysis of the vascular area (VA) revealed that the CC was most dense in the PP region (87.4 ± 4.3% VA) and least dense in FP (79.9 ± 6.7% VA). CC diameters also varied depending on location from 11.4 ± 1.97 mm in PP to 15.1 ± 3.15 mm in FP. In the NaIO3-injected eyes, CC density was significantly reduced in the RPE atrophic regions (50.7 ± 5.8% VA in PP and 45.8 ± 6.17% VA in MP) compared to the far peripheral non-atrophic regions (82.8 ± 3.8% VA). CC diameters were significantly reduced in atrophic regions (6.35 ± 1.02 mm in PP and 6.5 ± 1.2 mm in MP) compared to non-atrophic regions (14.16 ± 2.12 mm). In the laser-induced CNV model, CNV area was 0.26 ± 0.09 mm2 and luminal diameters of CNV vessels were 4.7 ± 0.9 mm. Immunostaining on bleached choroids with anti-podocalyxin antibody provides a simple and reliable tool for visualizing normal and pathologic choroidal vasculature in pigmented mouse eyes for quantitative morphometric analysis. This method will be beneficial for examining and evaluating the effects of various treatment modalities on the choroidal vasculature in mouse models of ocular diseases such as age-related macular degeneration, and degenerative genetic diseases.

An online Sexual Health and Rehabilitation eClinic (TrueNTH SHAReClinic) for prostate cancer patients: a feasibility study.

PURPOSE: The primary objective was to determine the feasibility of implementing the TrueNTH SHAReClinic as a pan-Canadian sexual health and rehabilitation intervention for patients treated for localized prostate cancer. METHODS: The feasibility study was designed to evaluate the accessibility and acceptability of the intervention. Participants from five institutions across Canada were enrolled to attend one pre-treatment and five follow-up online clinic visits over 1 year following their prostate cancer (PC) treatment. RESULTS: Sixty-five patients were enrolled in the intervention. Website analytics revealed that 71% completed the intervention in its entirety, including the educational modules, with an additional 10% completing more than half of the intervention. Five thousand eighty-three views of the educational modules were made along with 654 views of the health library items. Over 1500 messages were exchanged between participants and their sexual health coaches. At 12 months, the intervention received an overall average participant rating of 4.1 out of 5 on a single item satisfaction measure. CONCLUSION: Results support the TrueNTH SHAReClinic as highly acceptable to participants as defined by intervention adherence and engagement. The TrueNTH SHAReClinic demonstrated promise for being a feasible and potentially resource-efficient approach to effectively improving the sexual well-being of patients after PC treatment.

Evolution of oxidative stress, inflammation and neovascularization in the choroid and retina in a subretinal lipid induced age-related macular degeneration model.

Oxidative stress, inflammation and neovascularization are the key pathological events that are implicated in human age-related macular degeneration (AMD). There are a limited number of animal models available for evaluating and developing new therapies. Most models represent late exudative or neovascular AMD (nAMD) but there is a relative paucity of models that mimic early events in AMD. The purpose of this study is to characterize the evolution of oxidative stress, inflammation, retinal degeneration and neovascularization in a rat model of AMD, created by subretinal injection of human lipid hydroperoxide (HpODE) that found in the sub-macular region in aged and AMD patients. Subretinal HpODE induced retinal pigment epithelium (RPE) and retinal degeneration resulting in loss of RPE cells, photoreceptors and retinal thinning. RPE degeneration and atrophy were detected by day 5, followed by neural tissue degeneration at day 12 with robust TUNEL positive cells. Western blot analysis confirmed an increase in pro-apoptotic Bak protein at day 12 in retinal tissues. Oxidative damage biomarkers (4-hydroxynonenal, malondialdehyde, 8-hydroxy-2'-deoxyguanosine, and nitrotyrosine) increased in retinal tissue from days 5-12. Müller glial activation was observed in the HpODE injected area at day 5 followed by its remodeling and migration in the outer retina by day 20. RT-qPCR analysis further indicated upregulation of pro-inflammatory genes (TNF-α, IL-1ß and IL-6) both in retinal and RPE/choroidal tissue as early as day 2 and persisted until day 12. Upregulation of oxidative stress markers such as NADPH oxidase (NOX and DOUX family) was detected early in retinal tissue by day 2 followed by its upregulation in choroidal tissue at day 5. Neovascularization was demonstrated from day 12 to day 20 post HpODE injection in choroidal tissue. The results from this study indicate that subretinal HpODE induces advanced AMD phenotypes comprising many aspects of both dry/early and late) and neovascular/late AMD as observed in humans. Within 3 weeks via oxidative damage, upregulation of reactive oxygen species and pro-inflammatory genes, pro-apoptotic Bak and pro-angiogenic VEGF upregulation occurs leading to CNV formation. This experimental model of subretinal HpODE is an appropriate model for the study of AMD and provides an important platform for translational and basic research in developing new therapies particularly for early/dry AMD where currently no viable therapies are available.

Choriocapillaris dropout in early age-related macular degeneration.

Loss of choriocapillaris (CC) in advanced age-related macular degeneration (AMD) is well documented but changes in early AMD have not been quantified. Postmortem eyes from donors with clinically documented early AMD were examined in choroidal whole mounts to determine the area, pattern, and severity of CC loss. Choroids from postmortem human eyes without AMD (n = 7; mean age = 86.1) and from eyes with a Grade 2 clinical classification of early AMD (n = 7; mean age = 87) were immunolabeled with Ulex europaeus agglutinin (UEA) lectin-FITC to stain blood vessels. Whole mounts were imaged using confocal microscopy and image analysis was performed to determine the area of vascular changes and density of vasculature (percent vascular area, %VA). All areas evaluated had a complete RPE monolayer upon gross examination. In age-matched control eyes, the CC had broad lumens and a homogenous pattern of freely interconnecting capillaries. The mean %VA ± standard deviation in submacula of control subjects was 78.1 ± 3.25%. In eyes with early AMD, there was a significant decrease in mean %VA to 60.1 ± 10.4% (p < 0.0001). The paramacular %VA was not significantly different in eyes with or without AMD. The area of submacular choroid affected by CC dropout was 0.04 ± 0.09 mm2 in control eyes. In eyes with early AMD, the mean area affected by CC dropout was significantly increased (10.4 ± 6.1 mm2; p < 0.001). In some cases, incipient neovascular buds were observed at the border of regions with CC dropout in early AMD choroids. In conclusion, UEA lectin-labeled choroidal whole mounts from donors with clinically documented early AMD has provided a unique opportunity to examine regional changes in vascular pathology associated with choriocapillaris. The study demonstrated attenuation of submacular CC in early AMD subjects but no vascular pathology was observed outside the submacular region. While the affected area in some eyes was quite extensive histologically, these changes may not be detectable clinically using standard in vivo imaging.

Autosomal-Recessive Intellectual Disability with Cerebellar Atrophy Syndrome Caused by Mutation of the Manganese and Zinc Transporter Gene SLC39A8.

Manganese (Mn) and zinc (Zn) are essential divalent cations used by cells as protein cofactors; various human studies and animal models have demonstrated the importance of Mn and Zn for development. Here we describe an autosomal-recessive disorder in six individuals from the Hutterite community and in an unrelated Egyptian sibpair; the disorder is characterized by intellectual disability, developmental delay, hypotonia, strabismus, cerebellar atrophy, and variable short stature. Exome sequencing in one affected Hutterite individual and the Egyptian family identified the same homozygous variant, c.112G>C (p.Gly38Arg), affecting a conserved residue of SLC39A8. The affected Hutterite and Egyptian individuals did not share an extended common haplotype, suggesting that the mutation arose independently. SLC39A8 is a member of the solute carrier gene family known to import Mn, Zn, and other divalent cations across the plasma membrane. Evaluation of these two metal ions in the affected individuals revealed variably low levels of Mn and Zn in blood and elevated levels in urine, indicating renal wasting. Our findings identify a human Mn and Zn transporter deficiency syndrome linked to SLC39A8, providing insight into the roles of Mn and Zn homeostasis in human health and development.

Recessive TRAPPC11 mutations cause a disease spectrum of limb girdle muscular dystrophy and myopathy with movement disorder and intellectual disability.

Myopathies are a clinically and etiologically heterogeneous group of disorders that can range from limb girdle muscular dystrophy (LGMD) to syndromic forms with associated features including intellectual disability. Here, we report the identification of mutations in transport protein particle complex 11 (TRAPPC11) in three individuals of a consanguineous Syrian family presenting with LGMD and in five individuals of Hutterite descent presenting with myopathy, infantile hyperkinetic movements, ataxia, and intellectual disability. By using a combination of whole-exome or genome sequencing with homozygosity mapping, we identified the homozygous c.2938G>A (p.Gly980Arg) missense mutation within the gryzun domain of TRAPPC11 in the Syrian LGMD family and the homozygous c.1287+5G>A splice-site mutation resulting in a 58 amino acid in-frame deletion (p.Ala372_Ser429del) in the foie gras domain of TRAPPC11 in the Hutterite families. TRAPPC11 encodes a component of the multiprotein TRAPP complex involved in membrane trafficking. We demonstrate that both mutations impair the binding ability of TRAPPC11 to other TRAPP complex components and disrupt the Golgi apparatus architecture. Marker trafficking experiments for the p.Ala372_Ser429del deletion indicated normal ER-to-Golgi trafficking but dramatically delayed exit from the Golgi to the cell surface. Moreover, we observed alterations of the lysosomal membrane glycoproteins lysosome-associated membrane protein 1 (LAMP1) and LAMP2 as a consequence of TRAPPC11 dysfunction supporting a defect in the transport of secretory proteins as the underlying pathomechanism.

Increasing thyroid cancer incidence in Queensland, Australia 1982-2008 - true increase or overdiagnosis?

BACKGROUND: Thyroid cancer incidence has been increasing worldwide. Some suggest greater ascertainment of indolent tumours is the only driver, but others suggest there has been a true increase. Increases in Australia appear to have been among the largest in the world, so we investigated incidence trends in the Australian state of Queensland to help understand reasons for the rise. METHODS: Thyroid cancers diagnoses in Queensland 1982-2008 were ascertained from the Queensland Cancer Registry. We calculated age-standardized incidence rates (ASR) and used Poisson regression to estimate annual percentage change (APC) in thyroid cancer incidence by socio-demographic and tumour-related factors. RESULTS: Thyroid cancer ASR in Queensland increased from 2·2 to 10·6/100 000 between 1982 and 2008 equating to an APC of 5·5% [95% confidence interval (CI) 4·7-6·4] in men and 6·1% (95% CI 5·5-6·6) in women. The rise was evident, and did not significantly differ, across socio-economic and remoteness-of-residence categories. The largest increase seen was in the papillary subtype in women (APC 7·9%, 95% CI 7·3-8·5). Incidence of localized and more advanced-stage cancers rose over time although the increase was greater for early-stage cancers. CONCLUSION: There has been a marked increase in thyroid cancer incidence in Queensland. The increase is evident in men and women across all adult age groups, socio-economic strata and remoteness-of-residence categories as well as in localized and more advanced-stage cancers. Our results suggest 'overdiagnosis' may not entirely explain rising incidence. Contemporary aetiological data and individual-level information about diagnostic circumstances are required to further understand reasons for rising thyroid cancer incidence.

Idiopathic preretinal glia in aging and age-related macular degeneration.

During analysis of glia in wholemount aged human retinas, frequent projections onto the vitreal surface of the inner limiting membrane (ILM) were noted. The present study characterized these preretinal glial structures. The amount of glial cells on the vitreal side of the ILM was compared between eyes with age-related macular degeneration (AMD) and age-matched control eyes. Retinal wholemounts were stained for markers of retinal astrocytes and activated Müller cells (glial fibrillary acidic protein, GFAP), Müller cells (vimentin, glutamine synthetase) and microglia/hyalocytes (IBA-1). Retinal vessels were labeled with UEA lectin. Images were collected using a Zeiss LSM 710 confocal microscope. Retinas were then cryopreserved. Laminin labeling of cryosections determined the location of glial structures in relation to the ILM. All retinas investigated herein had varied amounts of preretinal glia. These glial structures were classified into three groups based on size: sprouts, blooms, and membranes. The simplest of the glial structures observed were focal sprouts of singular GFAP-positive cells or processes on the vitreal surface of the ILM. The intermediate structures observed, glial blooms, were created by multiple cells/processes exiting from a single point and extending along the vitreoretinal surface. The most extensive structures, glial membranes, consisted of compact networks of cells and processes. Preretinal glia were observed in all areas of the retina but they were most prominent over large vessels. While all glial blooms and membranes contained vimentin and GFAP-positive cells, these proteins did not always co-localize. Many areas had no preretinal GFAP but had numerous vimentin only glial sprouts. In double labeled glial sprouts, vimentin staining extended beyond that of GFAP. Hyalocytes and microglia were detected along with glial sprouts, blooms, and membranes. They did not, however, concentrate in the retina below these structures. Cross sectional analysis identified small breaks in the ILM above large retinal vessels through which glial cells exited the retina. Preretinal glial structures of varied sizes are a common occurrence in aged retinas and, in most cases, are subclinical. While all retinal glia are found in blooms, vimentin labeling suggests that Müller cells form the leading edge. All retinas investigated from eyes with active choroidal neovascularization (CNV) had extensive glial membranes on the vitreal surface of the ILM. Although these structures may be benign, they may exert traction on the retina as they spread along the vitreoretinal interface. In cases with CNV, glial cells in the vitreous could bind intravitreally injected anti-vascular endothelial growth factor. These preretinal glial structures indicate the remodeling of both astrocytes and Müller cells in aged retinas, in particular those with advanced AMD.

A randomised controlled trial of perineural vs intravenous dexamethasone for foot surgery.

We used 20 ml ropivacaine 0.75% for ankle blocks before foot surgery in 90 participants who we allocated in equal numbers to: perineural dexamethasone 8 mg and intravenous saline 0.9%; perineural saline 0.9% and intravenous dexamethasone 8 mg; or perineural and intravenous saline 0.9%. Dexamethasone increased the median (IQR [range]) time for the return of some sensation or movement, from 14.6 (10.8-18.8 [5.5-38.0]) h with saline to 24.1 (19.3-29.3 [5.0-44.0]) h when given perineurally, p = 0.00098, and to 20.9 (18.3-27.8 [8.8-31.3]) h when given intravenously, p = 0.0067. Dexamethasone increased the median (IQR [range]) time for the return of normal neurology, from 17.6 (14.0-21.0 [9.5-40.5]) h with saline to 27.5 (22.0-36.3 [7.0-53.0]) h when given perineurally, p = 0.00016, and to 24.0 (20.5-32.3 [13.0-42.5]) h when given intravenously, p = 0.0022. Dexamethasone did not affect the rates of block success, postoperative pain scores, analgesic use, or nausea and vomiting. The route of dexamethasone administration did not alter its effects.

Matching two independent cohorts validates DPH1 as a gene responsible for autosomal recessive intellectual disability with short stature, craniofacial, and ectodermal anomalies.

Recently, Alazami et al. (2015) identified 33 putative candidate disease genes for neurogenetic disorders. One such gene was DPH1, in which a homozygous missense mutation was associated with a 3C syndrome-like phenotype in four patients from a single extended family. Here, we report a second homozygous missense variant in DPH1, seen in four members of a founder population, and associated with a phenotype initially reminiscent of Sensenbrenner syndrome. This postpublication "match" validates DPH1 as a gene underlying syndromic intellectual disability with short stature and craniofacial and ectodermal anomalies, reminiscent of, but distinct from, 3C and Sensenbrenner syndromes. This validation took several years after the independent discoveries due to the absence of effective methods for sharing both candidate phenotype and genotype data between investigators. Sharing of data via Web-based anonymous data exchange servers will play an increasingly important role toward more efficient identification of the molecular basis for rare Mendelian disorders.

Mutations in SRCAP, encoding SNF2-related CREBBP activator protein, cause Floating-Harbor syndrome.

Floating-Harbor syndrome (FHS) is a rare condition characterized by short stature, delayed osseous maturation, expressive-language deficits, and a distinctive facial appearance. Occurrence is generally sporadic, although parent-to-child transmission has been reported on occasion. Employing whole-exome sequencing, we identified heterozygous truncating mutations in SRCAP in five unrelated individuals with sporadic FHS. Sanger sequencing identified mutations in SRCAP in eight more affected persons. Mutations were de novo in all six instances in which parental DNA was available. SRCAP is an SNF2-related chromatin-remodeling factor that serves as a coactivator for CREB-binding protein (CREBBP, better known as CBP, the major cause of Rubinstein-Taybi syndrome [RTS]). Five SRCAP mutations, two of which are recurrent, were identified; all are tightly clustered within a small (111 codon) region of the final exon. These mutations are predicted to abolish three C-terminal AT-hook DNA-binding motifs while leaving the CBP-binding and ATPase domains intact. Our findings show that SRCAP mutations are the major cause of FHS and offer an explanation for the clinical overlap between FHS and RTS.

TMEM237 is mutated in individuals with a Joubert syndrome related disorder and expands the role of the TMEM family at the ciliary transition zone.

Joubert syndrome related disorders (JSRDs) have broad but variable phenotypic overlap with other ciliopathies. The molecular etiology of this overlap is unclear but probably arises from disrupting common functional module components within primary cilia. To identify additional module elements associated with JSRDs, we performed homozygosity mapping followed by next-generation sequencing (NGS) and uncovered mutations in TMEM237 (previously known as ALS2CR4). We show that loss of the mammalian TMEM237, which localizes to the ciliary transition zone (TZ), results in defective ciliogenesis and deregulation of Wnt signaling. Furthermore, disruption of Danio rerio (zebrafish) tmem237 expression produces gastrulation defects consistent with ciliary dysfunction, and Caenorhabditis elegans jbts-14 genetically interacts with nphp-4, encoding another TZ protein, to control basal body-TZ anchoring to the membrane and ciliogenesis. Both mammalian and C. elegans TMEM237/JBTS-14 require RPGRIP1L/MKS5 for proper TZ localization, and we demonstrate additional functional interactions between C. elegans JBTS-14 and MKS-2/TMEM216, MKSR-1/B9D1, and MKSR-2/B9D2. Collectively, our findings integrate TMEM237/JBTS-14 in a complex interaction network of TZ-associated proteins and reveal a growing contribution of a TZ functional module to the spectrum of ciliopathy phenotypes.

Development and pilot testing of an online intervention to support young couples' coping and adjustment to breast cancer.

Couplelinks is an original, professionally facilitated online intervention tailored to the unique challenges facing young women with breast cancer and their male partners. The purpose of this evaluation was to determine the feasibility and acceptability of the intervention and areas for improvement. Sixteen couples were sequentially enrolled over an 18-month period. Couples provided feedback via a treatment satisfaction survey, and post-treatment interviews with a sub-group of participants. Qualitative information was analysed for themes relevant to the program's acceptability, perceived benefits and limitations, and directions for improvement. Of the 16 couples who enrolled, six completed four modules or less, and 10 completed the entire program. Completers reported satisfaction with the program overall, as well as with the website useability and professional facilitation. Reported benefits were: enhanced communication and self-other knowledge; creation of opportunities for meaningful, cancer-related discussion; affirmation of relationship strengths; and a greater sense of closeness between partners. The main reported limitation was how program participation disrupted the couple's usual routine. Themes related to non-completion suggest that partners with particularly elevated relational or illness-related distress, or with differential levels of motivation, are less likely to finish. These findings have led to targeted improvements to the website and intervention protocol.

Histologic and Immunohistochemical Characterization of GA-Like Pathology in the Rat Subretinal Sodium Iodate Model.

Purpose: Geographic atrophy (GA) is an advanced form of dry age-related macular degeneration with multifactorial etiology and no well-established treatment. A model recapitulating the hallmarks would serve as a key to understanding the underlying pathologic mechanisms better. In this report, we further characterized our previously reported subretinal sodium iodate model of GA. Methods: Retinal degeneration was induced in rats (6-8 weeks old) by subretinal injections of NaIO3 as described previously. Animals were sacrificed at 3, 8 and 12 weeks after injection and eyes were fixed or cryopreserved. Some choroids were processed as flatmounts while other eyes were cryopreserved, sectioned, and immunolabeled with a panel of antibodies. Finally, some eyes were prepared for transmission electron microscopic (TEM) analysis. Results: NaIO3 subretinal injection resulted in a well-defined focal area of retinal pigment epithelium (RPE) degeneration surrounded by viable RPE. These atrophic lesions expanded over time. RPE morphologic changes at the border consisted of hypertrophy, multilayering, and the possible development of a migrating phenotype. Immunostaining of retinal sections demonstrated external limiting membrane descent, outer retinal tubulation (ORT), and extension of Müller cells toward RPE forming a glial membrane in the subretinal space of the atrophic area. TEM findings demonstrated RPE autophagy, cellular constituents of ORT, glial membranes, basal laminar deposits, and defects in Bruch's membrane. Conclusions: In this study, we showed pathologic features of a rodent model resembling human GA in a temporal order through histology, immunofluorescence, and TEM analysis and gained insights into the cellular and subcellular levels of the GA-like phenotypes. Translational Relevance: Despite its acute nature, the expansion of atrophy and the GA-like border in this rat model makes it ideal for studying disease progression and provides a treatment window to test potential therapeutics for GA.

Haploinsufficiency of HDAC4 causes brachydactyly mental retardation syndrome, with brachydactyly type E, developmental delays, and behavioral problems.

Brachydactyly mental retardation syndrome (BDMR) is associated with a deletion involving chromosome 2q37. BDMR presents with a range of features, including intellectual disabilities, developmental delays, behavioral abnormalities, sleep disturbance, craniofacial and skeletal abnormalities (including brachydactyly type E), and autism spectrum disorder. To date, only large deletions of 2q37 have been reported, making delineation of a critical region and subsequent identification of candidate genes difficult. We present clinical and molecular analysis of six individuals with overlapping deletions involving 2q37.3 that refine the critical region, reducing the candidate genes from >20 to a single gene, histone deacetylase 4 (HDAC4). Driven by the distinct hand and foot anomalies and similar cognitive features, we identified other cases with clinical findings consistent with BDMR but without a 2q37 deletion, and sequencing of HDAC4 identified de novo mutations, including one intragenic deletion probably disrupting normal splicing and one intragenic insertion that results in a frameshift and premature stop codon. HDAC4 is a histone deacetylase that regulates genes important in bone, muscle, neurological, and cardiac development. Reportedly, Hdac4(-/-) mice have severe bone malformations resulting from premature ossification of developing bones. Data presented here show that deletion or mutation of HDAC4 results in reduced expression of RAI1, which causes Smith-Magenis syndrome when haploinsufficient, providing a link to the overlapping findings in these disorders. Considering the known molecular function of HDAC4 and the mouse knockout phenotype, taken together with deletion or mutation of HDAC4 in multiple subjects with BDMR, we conclude that haploinsufficiency of HDAC4 results in brachydactyly mental retardation syndrome.

Mutation in the ßA3/A1-crystallin gene impairs phagosome degradation in the retinal pigmented epithelium of the rat.

Phagocytosis of the shed outer segment discs of photoreceptors is a major function of the retinal pigmented epithelium (RPE). We demonstrate for the first time that ßA3/A1-crystallin, a major structural protein of the ocular lens, is expressed in RPE cells. Further, by utilizing the Nuc1 rat, in which the ßA3/A1-crystallin gene is mutated, we show that this protein is required by RPE cells for proper degradation of outer segment discs that have been internalized in phagosomes. We also demonstrate that in wild-type RPE, ßA3/A1-crystallin is localized to the lysosomes. However, in the Nuc1 RPE, ßA3/A1-crystallin fails to translocate to the lysosomes, perhaps because misfolding of the mutant protein masks sorting signals required for proper trafficking. The digestion of phagocytized outer segments requires a high level of lysosomal enzyme activity, and cathepsin D, the major enzyme responsible for proteolysis of the outer segments, is decreased in mutant RPE cells. Interestingly, our results also indicate a defect in the autophagy process in the Nuc1 RPE, which is probably also linked to impaired lysosomal function, because phagocytosis and autophagy might share common mechanisms in degradation of their targets. ßA3/A1-crystallin is a novel lysosomal protein in RPE, essential for degradation of phagocytosed material.

Mutant chromatin remodeling protein SMARCAL1 causes Schimke immuno-osseous dysplasia.

Schimke immuno-osseous dysplasia (SIOD, MIM 242900) is an autosomal-recessive pleiotropic disorder with the diagnostic features of spondyloepiphyseal dysplasia, renal dysfunction and T-cell immunodeficiency. Using genome-wide linkage mapping and a positional candidate approach, we determined that mutations in SMARCAL1 (SWI/SNF2-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like 1), are responsible for SIOD. Through analysis of data from persons with SIOD in 26 unrelated families, we observed that affected individuals from 13 of 23 families with severe disease had two alleles with nonsense, frameshift or splicing mutations, whereas affected individuals from 3 of 3 families with milder disease had a missense mutation on each allele. These observations indicate that some missense mutations allow retention of partial SMARCAL1 function and thus cause milder disease.

VEGF 165 b in the developing vasculatures of the fetal human eye.

VEGF(165) b is an anti-angiogenic form of VEGF(165) produced by alternative splicing. The localization of pro-angiogenic VEGF(165) and anti-angiogenic VEGF(165) b was investigated during development of the vasculatures in fetal human eyes from 7 to 21 weeks gestation (WG). The fetal vasculature of vitreous, which includes tunica vasculosa lentis (TVL), had moderate VEGF(165) immunoreactivity at 7WG and very little VEGF(165) b. Both forms were elevated at 12WG. VEGF(165) then decreased around 17WG when the TVL regresses but VEGF(165) b remained elevated. In choroid, VEGF(165) was present in forming choriocapillaris (CC) and retinal pigment epithelium (RPE) at 7WG while VEGF165b was present in CC and mesenchymal precursors within the choroidal stroma. By 21WG, both forms were elevated in RPE and choroidal blood vessels but VEGF(165) b was apical and VEGF(165) basal in RPE. Diffuse VEGF(165) immunoreactivity was prominent in 12WG innermost retina where blood vessels will form while VEGF(165) b was present in most CXCR4(+) progenitors in the inner neuroblastic layer and migrating angioblasts in the putative nerve fiber layer. By 21WG, VEGF(165) was present in nerve fibers and VEGF(165) b in the inner Muller cell process. The localization of VEGF(165) b was distinctly different from VEGF(165) both spatially and temporally and it was often associated with nucleus in progenitors.

Endocrine therapy for postmenopausal women with hormone receptor-positive her2-negative advanced breast cancer after progression or recurrence on nonsteroidal aromatase inhibitor therapy: a Canadian consensus statement.

Approximately 22,700 Canadian women were expected to be diagnosed with breast cancer in 2012. Despite improvements in screening and adjuvant treatment options, a substantial number of postmenopausal women with hormone receptor positive (hr+) breast cancer will continue to develop metastatic disease during or after adjuvant endocrine therapy. Guidance on the selection of endocrine therapy for patients with hr+ disease that is negative for the human epidermal growth factor receptor 2 (her2-) and that has relapsed or progressed on earlier nonsteroidal aromatase inhibitor (nsai) therapy is of increasing clinical importance. Exemestane, fulvestrant, and tamoxifen are approved therapeutic options in this context. Four phase iii trials involving 2876 patients-efect, sofea, confirm, and bolero-2-have assessed the efficacy of various treatment options in this clinical setting. Data from those trials suggest that standard-dose fulvestrant (250 mg monthly) and exemestane are of comparable efficacy, that doubling the dose of fulvestrant from 250 mg to 500 mg monthly results in a 15% reduction in the risk of progression, and that adding everolimus to exemestane (compared with exemestane alone) results in a 57% reduction in the risk of progression, albeit with increased toxicity. Multiple treatment options are now available to women with hr+ her2- advanced breast cancer recurring or progressing on earlier nsai therapy, although current clinical trial data suggest more robust clinical efficacy with everolimus plus exemestane. Consideration should be given to the patient's age, functional status, and comorbidities during selection of an endocrine therapy, and use of a proactive everolimus safety management strategy is encouraged.

When jurisdictional boundaries become barriers to good patient care.

Canada is a pioneer in remote cancer care delivery to underserved populations; however, it is trailing behind on policies that would support clinicians in providing care using distance technologies. The current policy framework is disjointed, and discussions by professional boards about online jurisprudence associated with licensure appear to be regressive. We hope that by addressing the discrepancies in interjurisdictional practice and focusing on the key issue of "where therapy resides," we will be able to nudge dialogue and thinking closer toward the reasoning and recommendations of national telehealth organizations. We present this discussion of jurisdictional issues and e-health practice in the context of a pan-Canadian online support program developed for cancer patients and family members. Although the present paper uses online support groups as a springboard to advocate for e-health, it ultimately addresses a broader audience: that of all Canadian health care stakeholders.