The effects of moulting on muscle fibre characteristics of the yabby, Cherax Albidus

In order to grow, crustaceans must shed their exoskeleton in a process known as moulting\u27. Although this process is short and intermittent, it remains dominant over the life of a crustacean. Physiological changes in the period between moults (intermoult) are comparatively quiescent as opposed to the periods directly before and after the actual moult, known as premoult, and postmoult respectively (West, 1997). Moulting is associated with distinct physiological changes including the breakdown of muscle (Musgrove and Geddes, 1985). This muscle breakdown, known as atrophy is the diminution in size of the actual muscle mass and is very specific, occurring to facilitate the withdrawal of the large pinnate claw muscle mass from the narrow basiischial joint (Mykles and Skinner, 1985). It is likely that atrophy does not occur in the abdomen as it is withdrawn through an opening similar in size to the actual muscle mass. This study aimed to determine and compare the effects of moulting on the characteristics of atrophic claw and non-atrophic abdominal muscles in the yabby, Cherax albidus. Although the moult cycle is a continuum, it may be sub-divided into several stages and sub-stages by reference to morphological changes in the integument state, and in development of the uropod setae using light microscopy. Three individuals of the yabby species Cherax albidus, were sacrificed at each of the following four stages, including intermoult (C1 - C2) early premoult (D1 to D1.3), late premoult (D2 – D4) and postmoult A. Claw and abdominal muscle were sectioned in a croystat at -20° C, and stained using three histochemical techniques; mATPase indicative of contractile speed; NADH-TR indicative of fatigue resistance and H&E, which visualizes structural characteristics. In this study, the mATPase and NADH-TR stains were developed for the yabby, Cherax albidus. Subsequently, several important cellular characteristics were determined for both claw and abdominal muscle over the moult cycle, including gross morphology, cellular morphology, fibre diameter, and fibre type distribution, at the four aforementioned strategic stages. In terms of these characteristics, the results suggest that abdominal muscles of the yabby Cherax albidus do not undergo moult-induced atrophy as opposed to the claw, consistent with the hypothesis, however both do exhibit characteristics of growth at postmoult. The main mechanisms of atrophy appeared be associated with mitochondrial aggregates, the proliferation of phagocytcs in fibres, and enlarged intermyofibrillar spaces. These indicators and mechanisms of atrophy were most evident at late premoult. Further studies are required to establish the exact association between these characteristics in claw muscle atrophy, and the underlying mechanisms. At postmoult, the muscle was restored firstly in the proximal region as it resembled the intermoult condition, whilst the distal region remained moderately affected by atrophy, and/or growth. In contrast to claw muscle, the abdominal muscle mass did not exhibit reductions in fibre diameter, or fibre type distributions, however did exhibit slight changes in cellular morphology. The extent of these changes suggest they were associated with growth rather than atrophy. Growth was exhibited as fibre splitting and was observed in both claw and abdominal muscle predominantly at the late premoult and postmoult stages. Five types of fibre splitting were observed, most of which were common to both muscles. The differing extents of particular types of fibre splitting between the claw and abdominal muscles may be related to recovery from atrophy. Hence, great changes associated with moulting were observed in the claw muscle of the yabby, Cherax albidus, whilst abdominal muscle remained relatively unaffected

Driving with retinitis pigmentosa

Background: To establish the proportion of patients with retinitis pigmentosa (RP) meeting the Australian fitness to drive (FTD) visual standards. Methodology: A prospective consecutive case series of patients with a clinical or genetic diagnosis of RP. Data on age at symptom onset, current driving status, inheritance pattern, better eye visual acuity (BEVA), binocular Esterman visual field (BEVF) parameters, genotype and ability to meet the driving standards based on BEVA and BEVF were collected. Outcome measures included the proportion of RP patients overall meeting the standards and clinical predictors for passing. A sub-analysis was performed on those RP patients who reported to drive. Change in BEVA and BEVF parameters across age in specific genotype groups was assessed. Results: Overall, 228 patients with RP had a BEVF assessment. Only 39% (89/228) met the driving standards. Younger age at the time of testing was the only significant predictor (p \u3c 0.01) for passing. Of the 55% of RP patients who reported to drive, 52% (65/125) met the standards, decreasing to 14% in the 56- to 65-year-old age group. RP patients harbouring mutations in HK1 or RHO genes may have slower rates of decline in their VF parameters. Conclusion: Nearly 40% of RP patients met the driving standards. However, almost 50% of RP drivers were unaware of their failure to meet the current standards. BEVF testing is essential in the assessment of RP patients who are still driving. Phenotype and genotype predictors for passing the standards warrant further investigation. Abbreviation: FTD, fitness to drive; IRD, inherited retinal disease; RP, retinitis pigmentosa; RHO, rhodopsin; HK1, hexokinase 1; PRPF31 pre-mRNA processing factor 31; RPGR, retinitis pigmentosa GTPase regulator; VF, visual field; BEVA, better eye visual acuity; BEVF, binocular Esterman visual field

Genotype-specific lesion growth rates in stargardt disease

Reported growth rates (GR) of atrophic lesions in Stargardt disease (STGD1) vary widely. In the present study, we report the longitudinal natural history of patients with confirmed bial-lelic ABCA4 mutations from five genotype groups: c.6079C \u3e T, c.[2588G \u3e C;5603A \u3e T], c.3113C \u3e T, c.5882G \u3e A and c.5603A \u3e T. Fundus autofluorescence (AF) 30◦ × 30◦ images were manually seg-mented for boundaries of definitely decreased autofluorescence (DDAF). The primary outcome was the effective radius GR across five genotype groups. The age of DDAF formation in each eye was calculated using the x-intercept of the DDAF effective radius against age. Discordance between age at DDAF formation and symptom onset was compared. A total of 75 eyes from 39 STGD1 patients (17 male [44%]; mean ± SD age 45 ± 19 years; range 21–86) were recruited. Patients with c.3113C \u3e T or c.6079C \u3e T had a significantly faster effective radius GR at 0.17 mm/year (95% CI 0.12 to 0.22; p \u3c 0.001 and 0.14 to 0.21; p \u3c 0.001) respectively, as compared to those patients harbouring c.5882G \u3e A at 0.06 mm/year (95% CI 0.03–0.09), respectively. Future clinical trial design should consider the effect of genotype on the effective radius GR and the timing of DDAF formation relative to symptom onset

Analysis of the outer retinal bands in ABCA4 and PRPH2-associated retinopathy using OCT

Purpose: To evaluate the outer retinal bands using OCT in ABCA4- and PRPH2-associated retinopathy and develop a novel imaging biomarker to differentiate between these 2 genotypes. Design: Multicenter case-control study. Participants: Patients with a clinical and genetic diagnosis of ABCA4- or PRPH2-associated retinopathy and an age-matched control group. Methods: Macular OCT was used to measure the thickness of the outer retinal bands 2 and 4 by 2 independent examiners at 4 retinal loci. Main Outcome Measures: Outcome measures included the thicknesses of band 2, band 4, and the band 2/band 4 ratio. Linear mixed modeling was used to make comparisons across the 3 groups. Receiver operating characteristic (ROC) analysis determined the optimal cutoff for the band 2/band 4 ratio to distinguish PRPH2- from ABCA4-associated retinopathy. Results: We included 45 patients with ABCA4 variants, 45 patients with PRPH2 variants, and 45 healthy controls. Band 2 was significantly thicker in patients with PRPH2 compared with ABCA4 (21.4 vs. 15.9 m, P \u3c 0.001) variants, whereas band 4 was thicker in patients with ABCA4 variants than those with PRPH2 variants (27.5 vs. 21.7 m, P \u3c 0.001). Similarly, the band 2/band 4 ratio was significantly different (1.0 vs. 0.6 for PRPH2 vs. ABCA4, P \u3c 0.001). The area under the ROC curve was 0.87 for either band 2 ( \u3e 18.58 m) or band 4 ( \u3c 26.17 μm) alone and 0.99 (95% confidence interval: 0.97–0.99) for the band 2/band 4 ratio with a cutoff threshold of 0.79, providing 100% specificity. Conclusions: We report an altered outer retinal band profile whereby the band 2/band 4 ratio was able to discriminate between PRPH2- and ABCA4-associated retinopathy. This may have future clinic utility in predicting the genotype and provide further insight into the anatomic correlate of band 2. Financial Disclosure(s): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article

Sibling concordance in symptom onset and atrophy growth rates in Stargardt disease using ultra-widefield fundus autofluorescence

Purpose: To investigate concordance in symptom onset, area of dark autofluorescence (DAF), and growth rate (GR) between Stargardt disease siblings at an age-matched time point. Methods: In this retrospective longitudinal study of sibling pairs with identical biallelic ABCA4 variants, age at symptom onset, best-corrected visual acuity, atrophy area, and effective radius of DAF on ultra-widefield fundus autofluorescence were recorded. Absolute intersibling differences for both eyes were compared with absolute interocular differences using the Mann-Whitney test. Results: Overall 39 patients from 19 families were recruited. In 16 families, age-matched best-corrected visual acuity and DAF were compared between siblings. In 8 families, DAF GR was compared. The median (range) absolute difference in age at symptom onset between siblings was 3 (0-35) years. Absolute intersibling differences in age-matched best-corrected visual acuity were greater than interocular differences (P = 0.01). Similarly, absolute intersibling differences in DAF area and radius were greater than interocular differences (P = 0.04 for area and P = 0.001 for radius). Differences between absolute interocular and intersibling GR were not statistically significant (P = 0.44 for area GR and P = 0.61 for radius GR). Conclusion: There was significant discordance in age-matched best-corrected visual acuity and DAF beyond the expected limits of interocular asymmetry. Lack of significant intersibling differences in GR warrants further investigation

Retinal Dystrophies Associated With Peripherin-2: Genetic Spectrum and Novel Clinical Observations in 241 Patients.

PURPOSE: To describe the clinical, electrophysiological and genetic spectrum of inherited retinal diseases associated with variants in the PRPH2 gene. METHODS: A total of 241 patients from 168 families across 15 sites in 9 countries with pathogenic or likely pathogenic variants in PRPH2 were included. Records were reviewed for age at symptom onset, visual acuity, full-field ERG, fundus colour photography, fundus autofluorescence (FAF), and SD-OCT. Images were graded into six phenotypes. Statistical analyses were performed to determine genotype-phenotype correlations. RESULTS: The median age at symptom onset was 40 years (range, 4-78 years). FAF phenotypes included normal (5%), butterfly pattern dystrophy, or vitelliform macular dystrophy (11%), central areolar choroidal dystrophy (28%), pseudo-Stargardt pattern dystrophy (41%), and retinitis pigmentosa (25%). Symptom onset was earlier in retinitis pigmentosa as compared with pseudo-Stargardt pattern dystrophy (34 vs 44 years; P = 0.004). The median visual acuity was 0.18 logMAR (interquartile range, 0-0.54 logMAR) and 0.18 logMAR (interquartile range 0-0.42 logMAR) in the right and left eyes, respectively. ERG showed a significantly reduced amplitude across all components (P < 0.001) and a peak time delay in the light-adapted 30-Hz flicker and single-flash b-wave (P < 0.001). Twenty-two variants were novel. The central areolar choroidal dystrophy phenotype was associated with 13 missense variants. The remaining variants showed marked phenotypic variability. CONCLUSIONS: We described six distinct FAF phenotypes associated with variants in the PRPH2 gene. One FAF phenotype may have multiple ERG phenotypes, demonstrating a discordance between structure and function. Given the vast spectrum of PRPH2 disease our findings are useful for future clinical trials

Retinal Dystrophies Associated With Peripherin-2: Genetic Spectrum and Novel Clinical Observations in 241 Patients

PURPOSE: To describe the clinical, electrophysiological and genetic spectrum of inherited retinal diseases associated with variants in the PRPH2 gene. METHODS: A total of 241 patients from 168 families across 15 sites in 9 countries with pathogenic or likely pathogenic variants in PRPH2 were included. Records were reviewed for age at symptom onset, visual acuity, full-field ERG, fundus colour photography, fundus autofluorescence (FAF), and SD-OCT. Images were graded into six phenotypes. Statistical analyses were performed to determine genotype-phenotype correlations. RESULTS: The median age at symptom onset was 40 years (range, 4-78 years). FAF phenotypes included normal (5%), butterfly pattern dystrophy, or vitelliform macular dystrophy (11%), central areolar choroidal dystrophy (28%), pseudo-Stargardt pattern dystrophy (41%), and retinitis pigmentosa (25%). Symptom onset was earlier in retinitis pigmentosa as compared with pseudo-Stargardt pattern dystrophy (34 vs 44 years; P = 0.004). The median visual acuity was 0.18 logMAR (interquartile range, 0-0.54 logMAR) and 0.18 logMAR (interquartile range 0-0.42 logMAR) in the right and left eyes, respectively. ERG showed a significantly reduced amplitude across all components (P \u3c 0.001) and a peak time delay in the light-adapted 30-Hz flicker and single-flash b-wave (P \u3c 0.001). Twenty-two variants were novel. The central areolar choroidal dystrophy phenotype was associated with 13 missense variants. The remaining variants showed marked phenotypic variability. CONCLUSIONS: We described six distinct FAF phenotypes associated with variants in the PRPH2 gene. One FAF phenotype may have multiple ERG phenotypes, demonstrating a discordance between structure and function. Given the vast spectrum of PRPH2 disease our findings are useful for future clinical trials

Investigation of Pax7 and its transcripts in the regulation of foetal and adult mouse myogenesis: A natural history

The development and regeneration of skeletal muscle is fuelled by muscle stem/progenitor cell biogenesis. The ability of these cells to survive and contribute to muscle growth and regeneration is dependent on master regulator genes, Pax3 and Pax7. These genes control key aspects of cell behaviour by their regulation of, and interactions with, other genes and proteins. Exactly how the Pax7 protein recognises and regulates specific genes to effect appropriate muscle progenitor behaviour is not fully understood. However as for other Pax genes, which similarly regulate the development and maintenance of different tissue lineages, this is likely to be linked to the production of multiple splice forms. Surprisingly little is known about 1) the contribution of Pax7 at the later, foetal, stages of muscle development and, 2) Pax7 splice forms to the regulation of muscle stem cell biogenesis in both developing and adult muscles. This gap in knowledge was the impetus for this PhD research. As the roles of many Pax genes are specific to the stage of cell biogenesis as well as the status of the tissue in which the cell is found, an in vivo approach was taken to characterise the natural history of Pax7 transcript expression and investigate the relationship of Pax7 with the powerful myogenic inducer, MyoD. Using mouse models of development and muscle maintenance and regeneration, this work suggests that the functional diversity of Pax7 in normal foetal and adult myogenesis in vivo is due to a factor other than changes in the relative proportions of alternate paired box transcripts. A second key finding of this work was the demonstrated expression of an alternate transcript, Pax7A, in the early mouse embryo as well as foetal muscle and brain, arising from proximal polyadenylation and splicing. Dynamic expression during foetal myogenesis suggests this mechanism may be functionally important during this time. Computational analyses predicted that Pax7A lacks miRNA control elements in the 3’UTR, leading to the hypothesis that changes in the ratios of Pax7A to Pax7B could affect the known reciprocal inhibition between Pax7 and MyoD. Endogenous levels of Pax7 and MyoD proteins were investigated to explore the spatio-temporal contribution of Pax7 to foetal limb myogenesis. This work showed that Pax7 and MyoD are co-expressed in foetal limb muscles and that the Pax7 and MyoD expression profile is highly likely to play a role in myogenic progression of foetal precursors in vivo, as reported for adult satellite cells. Further, temporal expression patterns suggest that MyoD protein can be downregulated in Pax7 precursors in foetal muscles. This could be related to the specification of satellite cells to the sublaminar position that occurs under the guidance of Notch, which is known to inhibit MyoD-mediated differentiation. It could be important that these cells do not differentiate during this time. Lastly, to investigate if Pax3 cells may also regulate muscle progenitor biogenesis during normal foetal myogenesis, spatio-temporal expression of endogenous Pax3 protein in situ was qualitatively analysed. Whilst Pax3 is mostly absent, a novel finding is that endogenous Pax3 protein may be re-expressed in some foetal limb muscles. In conclusion, this thesis reveals new insights into the contributions of Pax7 and its transcripts to adult and foetal myogenesis in vivo, particularly of the foetal limb