Primary Adenocarcinoma of the Gland of the Nictitating Membrane in a Cat

An 11-year-old, neutered, male domestic shorthair was presented with a five-month history of recurrent, unilateral, seromucoid discharge from the right eye. A verrucous mass extended from the posterior aspect of the nictitating membrane. Adenocarcinoma of the gland of the nictitating membrand (GNM) was diagnosed upon biopsy. The cat subsequently developed metastases to the lungs, pleura, mediastinum, liver, and kidneys and died six months after clinical signs first were observed. Little is known about the biological behavior of adenocarcinoma of the GNM in cats. This is the first report that describes the natural progression of this disease

Targeting Gene Expression to Cones With Human Cone Opsin Promoters in Recombinant AAV

Specific cone-directed therapy is of high priority in the treatment of human hereditary retinal diseases. However, not much information exists about the specific targeting of photoreceptor subclasses. Three versions of the human red cone opsin promoter (PR0.5, 3LCR-PR0.5 and PR2.1), and the human blue cone opsin promoter HB569, were evaluated for their specificity and robustness in targeting green fluorescent protein (GFP) gene expression to subclasses of cones in the canine retina when used in recombinant adeno-associated viral vectors of serotype 5. The vectors were administered by subretinal injection. The promoter PR2.1 led to most effective and specific expression of GFP in the long- and medium-wavelength-absorbing cones (L/M cones) of normal and diseased retinas. The PR0.5 promoter was not effective. Adding three copies of the 35-bp LCR in front of PR0.5 lead to weak GFP expression in L/M cones. The HB569 promoter was not specific, and GFP was expressed in a few L/M cones, some rods and the retinal pigment epithelium. These results suggest that L/M cones, the predominant class of cone photoreceptors in the retinas of dogs and most mammalian species can be successfully targeted using the human red cone opsin promoter

Recombinant AAV-Mediated \u3cem\u3eBEST1\u3c/em\u3e Transfer to the Retinal Pigment Epithelium: Analysis of Serotype-Dependent Retinal Effects

Mutations in the BEST1 gene constitute an underlying cause of juvenile macular dystrophies, a group of retinal disorders commonly referred to as bestrophinopathies and usually diagnosed in early childhood or adolescence. The disease primarily affects macular and paramacular regions of the eye leading to major declines in central vision later in life. Currently, there is no cure or surgical management for BEST1-associated disorders. The recently characterized human disease counterpart, canine multifocal retinopathy (cmr), recapitulates a full spectrum of clinical and molecular features observed in human bestrophinopathies and offers a valuable model system for development and testing of therapeutic strategies. In this study, the specificity, efficiency and safety of rAAV-mediated transgene expression driven by the human VMD2 promoter were assessed in wild-type canine retinae. While the subretinal delivery of rAAV2/1 vector serotype was associated with cone damage in the retina when BEST1 and GFP were co-expressed, the rAAV2/2 vector serotype carrying either GFP reporter or BEST1 transgene under control of human VMD2 promoter was safe, and enabled specific transduction of the RPE cell monolayer that was stable for up to 6 months post injection. These encouraging studies with the rAAV2/2 vector lay the groundwork for development of gene augmentation therapy for human bestrophinopathies

A Novel Genome-Wide Association Study Approach Using Genotyping by Exome Sequencing Leads to the Identification of a Primary Open Angle Glaucoma Associated Inversion Disrupting ADAMTS17

Closed breeding populations in the dog in conjunction with advances in gene mapping and sequencing techniques facilitate mapping of autosomal recessive diseases and identification of novel disease-causing variants, often using unorthodox experimental designs. In our investigation we demonstrate successful mapping of the locus for primary open angle glaucoma in the Petit Basset Griffon Vendéen dog breed with 12 cases and 12 controls, using a novel genotyping by exome sequencing approach. The resulting genome-wide association signal was followed up by genome sequencing of an individual case, leading to the identification of an inversion with a breakpoint disrupting the ADAMTS17 gene. Genotyping of additional controls and expression analysis provide strong evidence that the inversion is disease causing. Evidence of cryptic splicing resulting in novel exon transcription as a consequence of the inversion in ADAMTS17 is identified through RNAseq experiments. This investigation demonstrates how a novel genotyping by exome sequencing approach can be used to map an autosomal recessive disorder in the dog, with the use of genome sequencing to facilitate identification of a disease-associated variant

Human Retinal Gene Therapy for Leber Congential Amaurosis Shows Advancing Retinal Degeneration Despite Enduring Visual Improvement

Leber congenital amaurosis (LCA) associated with retinal pigment epithelium-specific protein 65 kDa (RPE65) mutations is a severe hereditary blindness resulting from both dysfunction and degeneration of photoreceptors. Clinical trials with gene augmentation therapy have shown partial reversal of the dysfunction, but the effects on the degeneration are not known. We evaluated the consequences of gene therapy on retinal degeneration in patients with RPE65-LCA and its canine model. In untreated RPE65-LCA patients, there was dysfunction and degeneration of photoreceptors, even at the earliest ages. Examined serially over years, the outer photoreceptor nuclear layer showed progressive thinning. Treated RPE65-LCA showed substantial visual improvement in the short term and no detectable decline from this new level over the long term. However, retinal degeneration continued to progress unabated. In RPE65-mutant dogs, the first one-quarter of their lifespan showed only dysfunction, and there was normal outer photoreceptor nuclear layer thickness retina-wide. Dogs treated during the earlier dysfunction-only stage showed improved visual function and dramatic protection of treated photoreceptors from degeneration when measured 5–11 y later. Dogs treated later during the combined dysfunction and degeneration stage also showed visual function improvement, but photoreceptor loss continued unabated, the same as in human RPE65-LCA. The results suggest that, in RPE65 disease treatment, protection from visual function deterioration cannot be assumed to imply protection from degeneration. The effects of gene augmentation therapy are complex and suggest a need for a combinatorial strategy in RPE65-LCA to not only improve function in the short term but also slow retinal degeneration in the long term

Transient Photoreceptor Deconstruction by CNTF Enhances rAAV-Mediated Cone Functional Rescue in Late Stage CNGB3-Achromatopsia

Achromatopsia is a genetic disorder of cones, and one of the most common forms is a channelopathy caused by mutations in the β-subunit, CNGB3, of the cone cyclic nucleotide-gated (CNG) channel. Recombinant adeno-associated virus of serotype 5 (rAAV5)-mediated gene transfer of human CNGB3 cDNA to mutant dog cones results in functional and structural rescue in dogs \u3c0.5 years of age, but treatment is minimally effective in dogs \u3e1 year. We now test a new therapeutic concept by combining gene therapy with the administration of ciliary neurotrophic factor (CNTF). Intravitreal CNTF causes transient dedifferentiation of photoreceptors, a process called deconstruction, whereby visual cells become immature with short outer segments, and decreased retinal function and gene expression that subsequently return to normal. Cone function was successfully rescued in all mutant dogs treated between 14 and 42 months of age with this strategy. CNTF-mediated deconstruction and regeneration of the photoreceptor outer segments prepares the mutant cones optimally for gene augmentation therapy

BEST1 Gene Therapy Corrects a Diffuse Retina-Wide Microdetachment Modulated by Light Exposure

Mutations in the BEST1 gene cause detachment of the retina and degeneration of photoreceptor (PR) cells due to a primary channelopathy in the neighboring retinal pigment epithelium (RPE) cells. The pathophysiology of the interaction between RPE and PR cells preceding the formation of retinal detachment remains not well-understood. Our studies of molecular pathology in the canine BEST1 disease model revealed retina-wide abnormalities at the RPE-PR interface associated with defects in the RPE microvillar ensheathment and a cone PR-associated insoluble interphotoreceptor matrix. In vivo imaging demonstrated a retina-wide RPE-PR microdetachment, which contracted with dark adaptation and expanded upon exposure to a moderate intensity of light. Subretinal BEST1 gene augmentation therapy using adeno-associated virus 2 reversed not only clinically detectable subretinal lesions but also the diffuse microdetachments. Immunohistochemical analyses showed correction of the structural alterations at the RPE-PR interface in areas with BEST1 transgene expression. Successful treatment effects were demonstrated in three different canine BEST1 genotypes with vector titers in the 0.1-to-5E11 vector genomes per mL range. Patients with biallelic BEST1 mutations exhibited large regions of retinal lamination defects, severe PR sensitivity loss, and slowing of the retinoid cycle. Human translation of canine BEST1 gene therapy success in reversal of macro- and microdetachments through restoration of cytoarchitecture at the RPE-PR interface has promise to result in improved visual function and prevent disease progression in patients affected with bestrophinopathies

Applications of yeast flocculation in biotechnological processes

A review on the main aspects associated with yeast flocculation and its application in biotechnological processes is presented. This subject is addressed following three main aspects – the basics of yeast flocculation, the development of “new” flocculating yeast strains and bioreactor development. In what concerns the basics of yeast flocculation, the state of the art on the most relevant aspects of mechanism, physiology and genetics of yeast flocculation is reported. The construction of flocculating yeast strains includes not only the recombinant constitutive flocculent brewer’s yeast, but also recombinant flocculent yeast for lactose metabolisation and ethanol production. Furthermore, recent work on the heterologous β-galactosidase production using a recombinant flocculent Saccharomyces cerevisiae is considered. As bioreactors using flocculating yeast cells have particular properties, mainly associated with a high solid phase hold-up, a section dedicated to its operation is presented. Aspects such as bioreactor productivity and culture stability as well as bioreactor hydrodynamics and mass transfer properties of flocculating cell cultures are considered. Finally, the paper concludes describing some of the applications of high cell density flocculation bioreactors and discussing potential new uses of these systems.Fundação para a Ciência e a Tecnologia (FCT) – PRAXIS XXI - BD11306/97

Risk factors of migraine-related brain white matter hyperintensities: an investigation of 186 patients

Brain white matter hyperintensities are more prevalent in migraine patients than in the general population, but the pathogenesis and the risk factors of these hyperintensities are not fully elucidated. The authors analyzed the routine clinical data of 186 migraine patients who were referred to the Outpatient Headache Department of the Department of Neurology, Medical School, University of Pécs, Hungary between 2007 and 2009: 58 patients with white matter hyperintensities and 128 patients without white matter hyperintensities on 3 T MRI. Significant associations between the presence of white matter hyperintensities and longer disease duration (14.4 vs. 19.9 years, p = 0.004), higher headache frequency (4.1 vs. 5.5 attacks/month, p = 0.017), hyperhomocysteinemia (incidence of hyperintensity is 9/9 = 100%, p = 0.009) and thyroid gland dysfunction (incidence of hyperintensity is 8/14 = 57.1%, p = 0.038) were found. These data support the theory that both the disease duration and the attack frequency have a key role in the formation of migraine-related brain white matter hyperintensities, but the effects of comorbid diseases may also contribute to the development of the hyperintensities