Capn5 Expression in the Healthy and Regenerating Zebrafish Retina

PURPOSE. Autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV) is a devastating inherited autoimmune disease of the eye that displays features commonly seen in other eye diseases, such as retinitis pigmentosa and diabetic retinopathy. ADNIV is caused by a gain-of-function mutation in Calpain-5 (CAPN5), a calcium-dependent cysteine protease. Very little is known about the normal function of CAPN5 in the adult retina, and there are conflicting results regarding its role during mammalian embryonic development. The zebrafish (Danio rerio) is an excellent animal model for studying vertebrate development and tissue regeneration, and represents a novel model to explore the function of Capn5 in the eye. METHODS. We characterized the expression of Capn5 in the developing zebrafish central nervous system (CNS) and retina, in the adult zebrafish retina, and in response to photoreceptor degeneration and regeneration using whole-mount in situ hybridization, FISH, and immunohistochemistry. RESULTS. In zebrafish, capn5 is strongly expressed in the developing embryonic brain, early optic vesicles, and in newly differentiated retinal photoreceptors. We found that expression of capn5 colocalized with cone-specific markers in the adult zebrafish retina. We observed an increase in expression of Capn5 in a zebrafish model of chronic rod photoreceptor degeneration and regeneration. Acute light damage to the zebrafish retina was accompanied by an increase in expression of Capn5 in the surviving cones and in a subset of Müller glia. CONCLUSIONS. These studies suggest that Capn5 may play a role in CNS development, photoreceptor maintenance, and photoreceptor regeneration

Proteasome-Mediated Regulation of Cdhr1a by Siah1 Modulates Photoreceptor Development and Survival in Zebrafish

Congenital retinal dystrophies are a major cause of unpreventable and incurable blindness worldwide. Mutations in CDHR1, a retina specific cadherin, are associated with cone-rod dystrophy. The ubiquitin proteasome system (UPS) is responsible for mediating orderly and precise targeting of protein degradation to maintain biological homeostasis and coordinate proper development, including retinal development. Recently, our lab uncovered that the seven in absentia (Siah) family of E3 ubiquitin ligases play a role in optic fissure fusion and identified Cdhr1a as a potential target of Siah. Using two-color whole mount in situ hybridization and immunohistochemistry, we detected siah1 and cdhr1a co-expression as well as protein localization in the retinal outer nuclear layer (ONL), and more precisely in the connecting cilium of rods and cones between 3–5 days post fertilization (dpf). We confirmed that Siah1 targets Cdhr1a for proteasomal degradation by co-transfection and co-immunoprecipitation in cell culture. To analyze the functional importance of this interaction, we created two transgenic zebrafish lines that express siah1 or an inactive siah1 (siah1ΔRING) under the control of the heat shock promoter to modulate Siah activity during photoreceptor development. Overexpression of siah1, but not siah1ΔRING, resulted in a decrease in the number of rods and cones at 72 h post fertilization (hpf). The number of retinal ganglion cells, amacrine and bipolar cells was not affected by Siah1 overexpression, and there was no significant reduction of proliferating cells in the Siah1 overexpressing retina. We did, however, detect increased cell death, confirmed by an increase in the number of TUNEL + cells in the ONL, which was proteasome-dependent, as proteasome inhibition rescued the cell death phenotype. Furthermore, reduction in rods and cones resulting from increased Siah1 expression was rescued by injection of cdhr1a mRNA, and to an even greater extent by injection of a Siah1-insensitive cdhr1a variant mRNA. Lastly, CRISPR induced loss of Cdhr1a function phenocopied Siah1 overexpression resulting in a significant reduction of rods and cones. Taken together, our work provides the first evidence that Cdhr1a plays a role during early photoreceptor development and that Cdhr1a is regulated by Siah1 via the UPS

A VLA Polarimetric Study of the Galactic Center Radio Arc: Characterizing Polarization, Rotation Measure, and Magnetic Field Properties

The Radio Arc is one of the brightest systems of non-thermal filaments (NTFs) in the Galactic Center, located near several prominent HII regions (Sickle and Pistol) and the Quintuplet stellar cluster. We present observations of the Arc NTFs using the S-, C-, and X-bands of the Very Large Array interferometer. Our images of total intensity reveal large-scale helical features that surround the Arc NTFs, very narrow sub-filamentation, and compact sources along the NTFs. The distribution of polarized intensity is confined to a relatively small area along the NTFs. There are elongated polarized structures that appear to lack total intensity counterparts. We detect a range of rotation measure values from -1000 to -5800 rad m$\rm^{-2}$, likely caused by external Faraday rotation along the line of sight. After correcting for Faraday rotation, the intrinsic magnetic field orientation is found to generally trace the extent of the NTFs. However, the intrinsic magnetic field in several regions of the Arc NTFs shows an ordered pattern that is rotated with respect to the extent of the NTFs. We suggest this changing pattern may be caused by an additional magnetized source along the line of sight, so that we observe two field systems superposed in our observations. We suggest that the large scale helical segments near the Radio Arc could be components of such a source causing these changes in intrinsic magnetic field, and some variations in the polarization and rotation measure values along the NTFs.Comment: PDF should be 24 pages with 13 figure

Zebrafish Blunt-Force TBI Induces Heterogenous Injury Pathologies That Mimic Human TBI and Responds with Sonic Hedgehog-Dependent Cell Proliferation across the Neuroaxis

Blunt-force traumatic brain injury (TBI) affects an increasing number of people worldwide as the range of injury severity and heterogeneity of injury pathologies have been recognized. Most current damage models utilize non-regenerative organisms, less common TBI mechanisms (penetrating, chemical, blast), and are limited in scalability of injury severity. We describe a scalable blunt-force TBI model that exhibits a wide range of human clinical pathologies and allows for the study of both injury pathology/progression and mechanisms of regenerative recovery. We modified the Marmarou weight drop model for adult zebrafish, which delivers a scalable injury spanning mild, moderate, and severe phenotypes. Following injury, zebrafish display a wide range of severity-dependent, injury-induced pathologies, including seizures, blood–brain barrier disruption, neuroinflammation, edema, vascular injury, decreased recovery rate, neuronal cell death, sensorimotor difficulties, and cognitive deficits. Injury-induced pathologies rapidly dissipate 4–7 days post-injury as robust cell proliferation is observed across the neuroaxis. In the cerebellum, proliferating nestin:GFP-positive cells originated from the cerebellar crest by 60 h post-injury, which then infiltrated into the granule cell layer and differentiated into neurons. Shh pathway genes increased in expression shortly following injury. Injection of the Shh agonist purmorphamine in undamaged fish induced a significant proliferative response, while the proliferative response was inhibited in injured fish treated with cyclopamine, a Shh antagonist. Collectively, these data demonstrate that a scalable blunt-force TBI to adult zebrafish results in many pathologies similar to human TBI, followed by recovery, and neuronal regeneration in a Shh-dependent manner

Using the 7-point checklist as a diagnostic aid for pigmented skin lesions in general practice:a diagnostic validation study

BACKGROUND: GPs need to recognise significant pigmented skin lesions, given rising UK incidence rates for malignant melanoma. The 7-point checklist (7PCL) has been recommended by NICE (2005) for routine use in UK general practice to identify clinically significant lesions which require urgent referral. AIM: To validate the Original and Weighted versions of the 7PCL in the primary care setting. DESIGN AND SETTING: Diagnostic validation study, using data from a SIAscopic diagnostic aid randomised controlled trial in eastern England. METHOD: Adults presenting in general practice with a pigmented skin lesion that could not be immediately diagnosed as benign were recruited into the trial. Reference standard diagnoses were histology or dermatology expert opinion; 7PCL scores were calculated blinded to the reference diagnosis. A case was defined as a clinically significant lesion for primary care referral to secondary care (total 1436 lesions: 225 cases, 1211 controls); or melanoma (36). RESULTS: For diagnosing clinically significant lesions there was a difference between the performance of the Original and Weighted 7PCLs (respectively, area under curve: 0.66, 0.69, difference = 0.03, P<0.001). For the identification of melanoma, similar differences were found. Increasing the Weighted 7PCL’s cut-off score from recommended 3 to 4 improved detection of clinically significant lesions in primary care: sensitivity 73.3%, specificity 57.1%, positive predictive value 24.1%, negative predictive value 92.0%, while maintaining high sensitivity of 91.7% and moderate specificity of 53.4% for melanoma. CONCLUSION: The Original and Weighted 7PCLs both performed well in a primary care setting to identify clinically significant lesions as well as melanoma. The Weighted 7PCL, with a revised cut-off score of 4 from 3, performs slightly better and could be applied in general practice to support the recognition of clinically significant lesions and therefore the early identification of melanoma

Ageing well with diabetes: A workshop to co‐design research recommendations for improving the diabetes care of older people

Aims:To identify key research questions where answers could improve care for older people living with diabetes (PLWD), and provide detailed recommendations for researchers and research funders on how best to address them.Methods:A series of online research workshops were conducted, bringing together a range of PLWD and an acknowledged group of academic and clinical experts in their diabetes care to identify areas for future research. Throughout the pre-workshop phase, during each workshop, and in manuscript preparation and editing, PLWD played an active and dynamic role in discussions as part of both an iterative and narrative process.Results:The following key questions in this field were identified, and research recommendations for each were developed:How can we improve our understanding of the characteristics of older people living with diabetes (PLWD) and their outcomes, and can this deliver better person-centred care?How are services to care for older PLWD currently delivered, both for their diabetes and other conditions? How can we optimise and streamline the process and ensure everyone gets the best care, tailored to their individual needs?What tools might be used to evaluate the level of understanding of diabetes in the older population amongst non-specialist Healthcare Professionals (HCPs)?How can virtual experts or centres most effectively provide access to specialist multi-disciplinary team (MDT) expertise for older PLWD and the HCPs caring for them?Is a combination of exercise and a nutrition-dense, high protein diet effective in the prevention of the adverse effects of type 2 diabetes and deterioration in frailty, and how might this be delivered in a way which is acceptable to people with type 2 diabetes?How might we best use continuous glucose monitoring (CGM) in older people and, for those who require support, how should the data be shared?How can older PLWD be better empowered to manage their diabetes in their own home, particularly when living with additional long-term conditions?What are the benefits of models of peer support for older PLWD, both when living independently and when in care?Conclusions:This paper outlines recommendations supported by PLWD through which new research could improve their diabetes care and calls on the research community and funders to address them in future research programmes and strategies

The effect of self-sorting and co-assembly on the mechanical properties of low molecular weight hydrogels

Self-sorting in low molecular weight hydrogels can be achieved using a pH triggered approach. We show here that this method can be used to prepare gels with different types of mechanical properties. Cooperative, disruptive or orthogonal assembled systems can be produced. Gels with interesting behaviour can be also prepared, for example self-sorted gels where delayed switch-on of gelation occurs. By careful choice of gelator, co-assembled structures can also be generated, which leads to synergistic strengthening of the mechanical properties

Medication and road test performance among cognitively healthy older adults

IMPORTANCE: Older adults are increasingly prescribed medications that have adverse effects. Prior studies have found a higher risk of motor vehicle crashes to be associated with certain medication use. OBJECTIVE: To determine whether specific medication classes were associated with performance decline as assessed by a standardized road test in a community sample of cognitively healthy older adults, to evaluate additional associations of poor road test performance with comorbid medical conditions and demographic characteristics, and to test the hypothesis that specific medication classes (ie, antidepressants, benzodiazepines, sedatives or hypnotics, anticholinergics, antihistamines, and nonsteroidal anti-inflammatory drugs or acetaminophen) would be associated with an increase in risk of impaired driving performance over time. DESIGN, SETTING, AND PARTICIPANTS: This was a prospective cohort study of 198 cognitively healthy adults 65 years and older with a valid driver\u27s license who were followed up annually, with rolling enrollment. Data were collected from participants in St Louis, Missouri, and neighboring Illinois who were enrolled in the Knight Alzheimer\u27s Disease Research Center. Data were collected from August 28, 2012, to March 14, 2023, and analyzed from April 1 to 25, 2023. Participants with healthy cognition, defined as a Clinical Dementia Rating score of 0 at baseline and subsequent visits, who had available clinical, neuropsychological, road tests, and self-reported medication data were included. EXPOSURE: Potentially driver-impairing medication use. MAIN OUTCOMES AND MEASURES: The primary outcome measure was performance on the Washington University Road Test (pass or marginal/fail). Multivariable Cox proportional hazards models were used to evaluate associations between potentially driver-impairing medication use and road test performance. RESULTS: Of the 198 included adults (mean [SD] baseline age, 72.6 [4.6] years; 87 female [43.9%]), 70 (35%) received a marginal/fail rating on the road test over a mean (SD) follow-up of 5.70 (2.45) years. Any use of antidepressants (adjusted hazard ratio [aHR], 2.68; 95% CI, 1.69-4.71), serotonin and norepinephrine reuptake inhibitors (aHR, 2.68; 95% CI, 1.54-4.64), sedatives or hypnotics (aHR, 2.70; 95% CI, 1.40-5.19), or nonsteroidal anti-inflammatory drugs (aHR, 2.72; 95% CI, 1.31-5.63) was associated with an increase in risk of receiving a marginal/fail rating on the road test compared with control individuals. Conversely, participants taking lipid-lowering agents had a lower risk of receiving a marginal/fail rating compared to control individuals. There were no statistically significant associations found between anticholinergic or antihistamines and poor performance. CONCLUSIONS AND RELEVANCE: In this prospective cohort study, specific medication classes were associated with an increase in risk of poor road test performance over time. Clinicians should consider this information and counsel patients accordingly when prescribing these medications

Naturalistic driving measures of route selection associate with resting state networks in older adults

Our objective was to identify functional brain changes that associate with driving behaviors in older adults. Within a cohort of 64 cognitively normal adults (age 60+), we compared naturalistic driving behavior with resting state functional connectivity using machine learning. Functional networks associated with the ability to interpret and respond to external sensory stimuli and the ability to multi-task were associated with measures of route selection. Maintenance of these networks may be important for continued preservation of driving abilities

Her9/Hes4 Is Required for Retinal Photoreceptor Development, Maintenance, and Survival

The intrinsic and extrinsic factors that regulate vertebrate photoreceptor specification and differentiation are complex, and our understanding of all the players is far from complete. Her9, the zebrafish ortholog of human HES4, is a basic helix-loop-helix-orange transcriptional repressor that regulates neurogenesis in several developmental contexts. We have previously shown that her9 is upregulated during chronic rod photoreceptor degeneration and regeneration in adult zebrafish, but little is known about the role of her9 during retinal development. To better understand the function of Her9 in the retina, we generated zebrafish her9 CRISPR mutants. Her9 homozygous mutants displayed striking retinal phenotypes, including decreased numbers of rods and red/green cones, whereas blue and UV cones were relatively unaffected. The reduction in rods and red/green cones correlated with defects in photoreceptor subtype lineage specification. The remaining rods and double cones displayed abnormal outer segments, and elevated levels of apoptosis. In addition to the photoreceptor defects, her9 mutants also possessed a reduced proliferative ciliary marginal zone, and decreased and disorganized Müller glia. Mutation of her9 was larval lethal, with no mutants surviving past 13 days post fertilization. Our results reveal a previously undescribed role for Her9/Hes4 in photoreceptor differentiation, maintenance, and survival