Engineering adeno-associated viral vectors to evade innate immune and inflammatory responses.

Nucleic acids are used in many therapeutic modalities, including gene therapy, but their ability to trigger host immune responses in vivo can lead to decreased safety and efficacy. In the case of adeno-associated viral (AAV) vectors, studies have shown that the genome of the vector activates Toll-like receptor 9 (TLR9), a pattern recognition receptor that senses foreign DNA. Here, we engineered AAV vectors to be intrinsically less immunogenic by incorporating short DNA oligonucleotides that antagonize TLR9 activation directly into the vector genome.

Efficacy of fluralaner spot-on solution against induced infestations with Rhipicephalus sanguineus on dogs.

Background: The efficacy of fluralaner spot-on solution administered once topically against induced infestations with Rhipicephalus sanguineus was evaluated in dogs over a 12-week post-treatment period.

Methods: Six negative-controlled studies were conducted, involving a total of 112 adult dogs (57 mixed breed, 47 Beagles, eight Labradors). In each study, dogs were randomized to two groups of eight to ten dogs each.

Phosphatidylinositol synthase is required for lens structural integrity and photoreceptor cell survival in the zebrafish eye.

The zebrafish lens opaque (lop) mutant was previously isolated in a genetic screen and shown to lack rod and cone photoreceptors and exhibit lens opacity, or cataract, at 7 days post-fertilization (dpf). In this manuscript, we provide four different lines of evidence demonstrating that the lop phenotype results from a defect in the cdipt (phosphatidylinositol (PI) synthase; CDP-diacylglycerol-inositol 3-phosphatidyltransferase) gene. First, DNA sequence analysis revealed that the lop mutant contained a missense mutation in the lop open reading frame, which yields a nonconservative amino acid substitution (Ser-111-Cys) within the PI synthase catalytic domain.

The loss of vacuolar protein sorting 11 (vps11) causes retinal pathogenesis in a vertebrate model of syndromic albinism.

Purpose: To establish the zebrafish platinum mutant as a model for studying vision defects caused by syndromic albinism diseases such as Chediak-Higashi syndrome, Griscelli syndrome, and Hermansky-Pudlak syndrome (HPS).

Methods: Bulked segregant analysis and candidate gene sequencing revealed that the zebrafish platinum mutation is a single-nucleotide insertion in the vps11 (vacuolar protein sorting 11) gene. Expression of vps11 was determined by RT-PCR and in situ hybridization.

Cellular expression of midkine-a and midkine-b during retinal development and photoreceptor regeneration in zebrafish.

In the retina of adult teleosts, stem cells are sustained in two specialized niches: the ciliary marginal zone (CMZ) and the microenvironment surrounding adult Müller glia. Recently, Müller glia were identified as the regenerative stem cells in the teleost retina. Secreted signaling molecules that regulate neuronal regeneration in the retina are largely unknown.

Teleost lens development and degeneration.

The transparent properties of the lens and its ability to focus light onto the retina are critical for normal vision. Optical clarity of the lens is achieved and maintained by a unique, highly regulated integration of lens cell proliferation and differentiation that persists throughout life. Zebrafish is a powerful genetic model for studying vertebrate lens differentiation and growth because the structural organization of the lens and gene functions are largely conserved with mammals, including humans.

Immunohistochemical evidence for multiple photosystems in box jellyfish.

Cubomedusae (box jellyfish) possess a remarkable visual system with 24 eyes distributed in four sensory structures termed rhopalia. Each rhopalium is equipped with six eyes: two pairs of pigment cup eyes and two unpaired lens eyes. Each eye type probably captures specific features of the visual environment.

Age-related cone abnormalities in zebrafish with genetic lesions in sonic hedgehog.

Purpose: Sonic hedgehog (Shh) signaling is essential for photoreceptor differentiation and retinal cell survival in embryonic zebrafish. The study was conducted to determine whether adult heterozygous carriers of mutant alleles for the shh gene display retinal abnormalities.

Methods: Retinal cryosections from young, middle-aged, and senescent wild-type and sonic-you(+/-) (syu(+/-)) zebrafish were probed with retinal cell type-specific markers.

Lengsin expression and function during zebrafish lens formation.

A zebrafish ortholog of human lengsin was identified by EST analysis of an adult lens cDNA library. During zebrafish development, lengsin transcription is first detected at 24 h post-fertilization (hpf). Immunolocalization, using polyclonal antiserum generated against a Lengsin bacterial fusion protein, detects lens-specific protein in whole-mount embryos at 30 hpf.

Genetic determinants of hyaloid and retinal vasculature in zebrafish.

Background: The retinal vasculature is a capillary network of blood vessels that nourishes the inner retina of most mammals. Developmental abnormalities or microvascular complications in the retinal vasculature result in severe human eye diseases that lead to blindness. To exploit the advantages of zebrafish for genetic, developmental and pharmacological studies of retinal vasculature, we characterised the intraocular vasculature in zebrafish.

Time course analysis of gene expression during light-induced photoreceptor cell death and regeneration in albino zebrafish.

Constant intense light causes apoptosis of rod and cone photoreceptors in adult albino zebrafish. The photoreceptors subsequently regenerate from proliferating inner nuclear layer (INL) progenitor cells that migrate to the outer nuclear layer (ONL) and differentiate into rods and cones. To identify gene expression changes during this photoreceptor regeneration response, a microarray analysis was performed at five time points during the light treatment.

Mutations in laminin alpha 1 result in complex, lens-independent ocular phenotypes in zebrafish.

We report phenotypic and genetic analyses of a recessive, larval lethal zebrafish mutant, bal(a69), characterized by severe eye defects and shortened body axis. The bal(a69) mutation was mapped to chromosome 24 near the laminin alpha 1 (lama1) gene. We analyzed the lama1 gene sequence within bal(a69) embryos and two allelic mutants, bal(arl) and bal(uw1).

Zebrafish foxe3: roles in ocular lens morphogenesis through interaction with pitx3.

Foxe3 is a winged helix/forkhead domain transcription factor necessary for mammalian and amphibian lens development. Human FOXE3 mutations cause anterior segment dysgenesis and cataracts. The zebrafish foxe3 cDNA was PCR amplified from 24 h post-fertilization (hpf) embryo cDNA.

Gene duplication and separation of functions in alphaB-crystallin from zebrafish (Danio rerio).

We previously reported that zebrafish alphaB-crystallin is not constitutively expressed in nervous or muscular tissue and has reduced chaperone-like activity compared with its human ortholog. Here we characterize the tissue expression pattern and chaperone-like activity of a second zebrafish alphaB-crystallin. Expressed sequence tag analysis of adult zebrafish lens revealed the presence of a novel alpha-crystallin transcript designated cryab2 and the resulting protein alphaB2-crystallin.

Expressed sequence tag analysis of zebrafish eye tissues for NEIBank.

Purpose: To characterize gene expression patterns in various tissues of the zebrafish (Danio rerio) eye and identify zebrafish orthologs of human genes by expressed sequence tag (EST) analysis for NEIBank.

Methods: mRNA was extracted from adult zebrafish eye tissues, including lenses, anterior segments (minus lens), retinas, posterior segments lacking retinas, and whole eyes. Five different cDNA libraries were constructed in the pCMVSport6 vector.

Retinal regional differences in photoreceptor cell death and regeneration in light-lesioned albino zebrafish.

Teleost fish regenerate retinal cells from a population of inner nuclear layer (INL) stem cells. To characterize photoreceptor regeneration in zebrafish (Danio rerio), adult albino fish were subjected to constant intense light to cause photoreceptor cell death. Retinal morphometry was performed on histological sections of control and light-lesioned albino retinas to compare the extent of light damage in the ventral, central and dorsal retinal regions.

Zebrafish pitx3 is necessary for normal lens and retinal development.

The human PITX3 gene encodes a bicoid-like homeodomain transcription factor associated with a variety of congenital ocular conditions, including anterior segment dysgenesis, Peter's anomaly, and cataracts. We identified a zebrafish pitx3 gene encoding a protein (Pitx3) that possesses 63% amino acid identity with human PITX3. The zebrafish pitx3 gene encompasses approximately 16.

Lens opacity and photoreceptor degeneration in the zebrafish lens opaque mutant.

The zebrafish lens opaque (lop) mutant was identified in a chemical mutagenesis screen. The lop mutant, which develops normally through 4 days postfertilization (dpf), exhibits several signs of lens and retinal degeneration at 7 dpf. Histology revealed disrupted lens fibers and increased numbers of nucleated cells within the mutant lens and anterior chamber.

Zebrafish mutagenesis yields eye morphological mutants with retinal and lens defects.

A chemical mutagenesis to identify zebrafish eye morphological mutants was performed by screening F(3) larvae at 5 and 7 days post-fertilization (dpf) for changes in eye or pupil size. Based on histological analysis, four different phenotypic classes were obtained. The two Class I and three Class II mutants are all characterized by small eyes and exhibit defects in early retinal development or unregulated cell death, respectively.