Combining M-FISH and Quantum Dot technology for fast chromosomal assignment of transgenic insertions

<p>Abstract</p> <p>Background</p> <p>Physical mapping of transgenic insertions by Fluorescence in situ Hybridization (FISH) is a reliable and cost-effective technique. Chromosomal assignment is commonly achieved either by concurrent G-banding or by a multi-color FISH approach consisting of iteratively co-hybridizing the transgenic sequence of interest with one or more chromosome-specific probes at a time, until the location of the transgenic insertion is identified.</p> <p>Results</p> <p>Here we report a technical development for fast chromosomal assignment of transgenic insertions at the single cell level in mouse and rat models. This comprises a simplified 'single denaturation mixed hybridization' procedure that combines multi-color karyotyping by Multiplex FISH (M-FISH), for simultaneous and unambiguous identification of all chromosomes at once, and the use of a Quantum Dot (QD) conjugate for the transgene detection.</p> <p>Conclusions</p> <p>Although the exploitation of the unique optical properties of QD nanocrystals, such as photo-stability and brightness, to improve FISH performance generally has been previously investigated, to our knowledge this is the first report of a purpose-designed molecular cytogenetic protocol in which the combined use of QDs and standard organic fluorophores is specifically tailored to assist gene transfer technology.</p

Induced four fold anisotropy and bias in compensated NiFe/FeMn double layers

A vector spin model is used to show how frustrations within a multisublattice antiferromagnet such as FeMn can lead to four-fold magnetic anisotropies acting on an exchange coupled ferromagnetic film. Possibilities for the existence of exchange bias are examined and shown to exist for the case of weak chemical disorder at the interface in an otherwise perfect structure. A sensitive dependence on interlayer exchange is found for anisotropies acting on the ferromagnet through the exchange coupling, and we show that a wide range of anisotropies can appear even for a perfect crystalline structure with an ideally flat interface.Comment: 7 pages, 7 figure

Long-term follow-up of chronic central serous chorioretinopathy after successful treatment with photodynamic therapy or micropulse laser

Purpose To describe the treatment outcomes and recurrence risk of chronic central serous chorioretinopathy (cCSC) in patients who had complete resolution of subretinal fluid (SRF) after either primary half-dose photodynamic therapy (PDT) or high-density subthreshold micropulse laser (HSML) in the PLACE trial.Methods This multicentre prospective follow-up study evaluated cCSC patients at 1 year after completion of the PLACE trial. Outcomes included: complete resolution of SRF on OCT, best-corrected visual acuity (BCVA) in Early Treatment of Diabetic Retinopathy Study (ETDRS) letters, retinal sensitivity on microperimetry and a visual function questionnaire (NEI-VFQ25).Results Twenty-nine out of 37 patients who received half-dose PDT and 15 out of 17 patients who received HSML could be evaluated at final visit. At final visit, 93% of the patients treated with half-dose PDT had complete resolution of SRF, compared with 53% of HSML-treated patients (p = 0.006). At final visit, the mean estimate increase in the PDT group compared with the HSML group was + 2.1 ETDRS letters, +0.15 dB for the retinal sensitivity and + 5.1 NEI-VFQ25 points (p = 0.103, p = 0.784 and p = 0.071, respectively). The mean estimated central retinal thickness in the half-dose PDT group was -7.0 mu m compared with the HSML group (p = 0.566). The mean estimated subfoveal choroidal thickness in the half-dose PDT group was -16.6 mu m compared with the HSML group (p = 0.359).Conclusion At 20 months after treatment, cCSC patients successfully treated with half-dose PDT are less likely to have recurrences of SRF compared with those successfully treated with HSML. However, functional outcomes did not differ.Development and application of statistical models for medical scientific researc

The T309G MDM2 gene polymorphism is a novel risk factor for proliferative vitreoretinopathy

Proliferative vitreoretinopathy (PVR) is still the major cause of failure in retinal detachment (RD) surgery. It is believed that down-regulation in the p53 pathway could be an important key in PVR pathogenesis. The purpose was to evaluate the impact of T309G MDM2 polymorphism (rs2279744) in PVR. Distribution of T309G MDM2 genotypes among European subjects undergoing RD surgery was evaluated. Proportions of genotypes between subsamples from different countries were analyzed. Also, a genetic interaction between rs2279744 in MDM2 and rs1042522 in p53 gene was analyzed. Significant differences were observed comparing MDM2 genotype frequencies at position 309 of intron 1 between cases (GG: 21.6%, TG: 54.5%, TT: 23.8%) and controls (GG: 7.3%, TG: 43.9%, TT: 48.7%). The proportions of genotypes between sub-samples from different countries showed a significant difference. Distribution of GG genotype revealed differences in Spain (35.1-53.0)/(22.6-32.9), Portugal (39.0-74.4)/(21.4-38.9), Netherlands (40.6-66.3)/(25.3-38.8) and UK (37.5-62.4)/(23.3-34.2). The OR of G carriers in the global sample was 5.9 (95% CI: 3.2 to 11.2). The OR of G carriers from Spain and Portugal was 5.4 (95% CI: 2.2-12.7), whereas in the UK and the Netherlands was 7.3 (95% CI: 2.8-19.1). Results indicate that the G allele of rs2279744 is associated with a higher risk of developing PVR in patients undergoing a RD surgery. Further studies are necessary to understand the role of this SNP in the development of PVR. Copyright

Optic nerve regeneration

SIGLEAvailable from British Library Document Supply Centre-DSC:D192342 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

Outcome of Full-Thickness Macular Hole Surgery in Choroideremia

Ophthalmic researc

Retinal gene therapy in patients with choroideremia: initial findings from a phase 1/2 clinical trial

Background: Choroideremia is an X-linked recessive disease that leads to blindness due to mutations in the CHM gene, which encodes the Rab escort protein 1 (REP1). We assessed the effects of retinal gene therapy with an adeno-associated viral (AAV) vector encoding REP1 (AAV.REP1) in patients with this disease.Methods: In a multicentre clinical trial, six male patients (aged 35-63 years) with choroideremia were administered AAV.REP1 (0·6-1·0×10(10) genome particles, subfoveal injection). Visual function tests included best corrected visual acuity, microperimetry, and retinal sensitivity tests for comparison of baseline values with 6 months after surgery. This study is registered with ClinicalTrials.gov, number NCT01461213.Findings: Despite undergoing retinal detachment, which normally reduces vision, two patients with advanced choroideremia who had low baseline best corrected visual acuity gained 21 letters and 11 letters (more than two and four lines of vision). Four other patients with near normal best corrected visual acuity at baseline recovered to within one to three letters. Mean gain in visual acuity overall was 3·8 letters (SE 4·1). Maximal sensitivity measured with dark-adapted microperimetry increased in the treated eyes from 23·0 dB (SE 1·1) at baseline to 25·3 dB (1·3) after treatment (increase 2·3 dB [95% CI 0·8-3·8]). In all patients, over the 6 months, the increase in retinal sensitivity in the treated eyes (mean 1·7 [SE 1·0]) was correlated with the vector dose administered per mm(2) of surviving retina (r=0·82, p=0·04). By contrast, small non-significant reductions (p&gt;0·05) were noted in the control eyes in both maximal sensitivity (-0·8 dB [1·5]) and mean sensitivity (-1·6 dB [0·9]). One patient in whom the vector was not administered to the fovea re-established variable eccentric fixation that included the ectopic island of surviving retinal pigment epithelium that had been exposed to vector.Interpretation: The initial results of this retinal gene therapy trial are consistent with improved rod and cone function that overcome any negative effects of retinal detachment. These findings lend support to further assessment of gene therapy in the treatment of choroideremia and other diseases, such as age-related macular degeneration, for which intervention should ideally be applied before the onset of retinal thinning<br/

CNTF gene therapy confers lifelong neuroprotection in a mouse model of human retinitis pigmentosa.

The long-term outcome of neuroprotection as a therapeutic strategy for preventing cell death in neurodegenerative disorders remains unknown, primarily due to slow disease progression and the inherent difficulty of assessing neuronal survival in vivo. Employing a murine model of retinal disease, we demonstrate that ciliary neurotrophic factor (CNTF) confers life-long protection against photoreceptor degeneration. Repetitive retinal imaging allowed the survival of intrinsically fluorescent cone photoreceptors to be quantified in vivo. Imaging of the visual cortex and assessment of visually-evoked behavioural responses demonstrated that surviving cones retain function and signal correctly to the brain. The mechanisms underlying CNTF-mediated neuroprotection were explored through transcriptome analysis, revealing widespread up-regulation of proteolysis inhibitors, which may prevent cellular/extracellular matrix degradation and complement activation in neurodegenerative diseases. These findings provide insights into potential novel therapeutic avenues for diseases such as retinitis pigmentosa and amyotrophic lateral sclerosis, for which CNTF has been evaluated unsuccessfully in clinical trials.Molecular Therapy (2015); doi:10.1038/mt.2015.68

Outcome of half-dose photodynamic therapy in chronic central serous chorioretinopathy with fovea-involving atrophy

Purpose To evaluate the clinical outcomes after half-dose photodynamic therapy (PDT) in chronic central serous chorioretinopathy (cCSC) patients with pre-existent fovea-involving atrophy. Methods In this retrospective study, cCSC patients who had a window defect of the retinal pigment epithelium (RPE) on fluorescein angiography (FA), compatible with RPE atrophy, prior to half-dose PDT were included. Results Thirty-four cCSC eyes with typical findings of cCSC on multimodal imaging, and fovea-involving RPE atrophy on FA, were included. At the first visit after PDT (at a median of 1.8 months after half-dose PDT), 20 eyes (59%) had a complete resolution of SRF (p < 0.001), while this was the case in 19 eyes (56%) at final visit (median of 11.3 months after half-dose PDT; p < 0.001). The mean BCVA in Early Treatment of Diabetic Retinopathy Study letters was 71. 2 +/- 15.9 at last visit before PDT, which increased to 74.1 +/- 14.1 at first visit after PDT (p = 0.093, compared with baseline), and changed to 73.0 +/- 19.1 at final visit (p = 0.392, compared with baseline). Both at first visit after PDT and at final visit, a significant decrease in subfoveal choroidal thickness was observed (p = 0.032 and p = 0.004, respectively). Conclusions Half-dose PDT in cCSC patients with pre-existing fovea-involving atrophy may lead to anatomical changes, but not to functional improvements. Ideally, cCSC should be treated with half-dose PDT before the occurrence of such atrophy

Characterization of a Dominant Cone Degeneration in a Green Fluorescent Protein–Reporter Mouse with Disruption of Loci Associated with Human Dominant Retinal Dystrophy

The authors characterized a dominant cone degeneration in the cone green fluorescent protein (GFP) reporter mouse that is not mediated by GFP toxicity. Transgenic insertion has been mapped to chromosome 10 and disrupts loci that in humans are associated with dominant cone dystrophies