Bioderived dyes-mediated vat photopolymerization 3D printing of chitosan hydrogels for tissue engineering

The importance of 3D printable hydrogels is constantly increasing in the field of tissue engineering, due to their characteristic structure and similarity with the human body extracellular matrix (ECM). Herein, Quinizarin-based bioderived photoactive dyes were employed for the 3D printing of methacrylated chitosan by vat pho-topolymerization (VPP). Dyes are commonly needed in VPP to obtain good resolution avoiding the light scattering in the vat, here the bioderived molecules had further utility granting the printability with good resolution while acting both as photosensitizers and fillers/crosslinkers. Defined and detailed architectures in the range of millimetres were obtained with low printing times showing for the first time the possibility to obtain all-chitosan 3D structures with suspended features by Digital Light Processing (DLP), which is a form of vat photopolymerization; such a result is not achievable without the quinizarin-derived dyes. The same structures were achieved with an opti-mized concentration of poly(ethylene glycol) diacrylate, as a comparison, enhancing the resolution. For all the formulations, the important feature to obtain the gel printability (considering reactiveness, viscosity, shear thinning behaviour, mechanical properties and stability) were evaluated. The hydrogels mechanical character-ization and swelling properties were also tested and reported. Lastly, the possible application in tissue engi-neering was also evaluated through cell proliferation analysis over samples, including metabolic activity and DNA contempt assays. The use of such bioderived photoactive molecules that simultaneously act as photosensitizer enhancing the ink reactivity, as dye improving the definition of the final structures and as filler/crosslinker increasing the me-chanical stability, could represent a valuable route for the DLP printing of soft hydrogels

Late-Onset Stargardt Disease Due to Mild, Deep-Intronic ABCA4 Alleles

PURPOSE. To investigate the role of two deep-intronic ABCA4 variants, that showed a mild splice defect in vitro and can occur on the same allele as the low penetrant c.5603A>T, in Stargardt disease (STGD1). METHODS. Ophthalmic data were assessed of 18 STGD1 patients who harbored c.769-784C>T or c.4253+43G>A in combination with a severe ABCA4 variant. Subjects carrying c.[769784C>T; 5603A>T] were clinically compared with a STGD1 cohort previously published carrying c.5603A>T noncomplex. We calculated the penetrances of the intronic variants using ABCA4 allele frequency data of the general population and investigated the effect of c.769-784C>T on splicing in photoreceptor progenitor cells (PPCs). RESULTS. Mostly, late-onset, foveal-sparing STGD1 was observed among subjects harboring c.769-784C>T or c.4253+43G>A (median age of onset, 54.5 and 52.0 years, respectively). However, ages of onset, phenotypes in fundo, and visual acuity courses varied widely. No significant clinical differences were observed between the c.[769-784C>T; 5603A>T] cohort and the c.4253+43G>A or the c.5603A>T cohort. The penetrances of c.769-784C>T (20.5%-39.6%) and c.4253+43G>A (35.8%-43.1%) were reduced, when not considering the effect of yet unidentified or known factors in cis, such as c.5603A>T (identified in 7/7 probands with c.769-784C>T; 1/8 probands with c.4253+43G>A). Variant c.769-784C>T resulted in a pseudo-exon insertion in 15% of the total mRNA (i.e., similar to 30% of the c.769-784C>T allele alone). CONCLUSIONS. Two mild intronic ABCA4 variants could further explain missing heritability in late-onset STGD1, distinguishing it from AMD. The observed clinical variability and calculated reduced penetrance urge research into modifiers within and outside of the ABCA4 gene

The common ABCA4 variant p.Asn1868ile shows nonpenetrance and variable expression of stargardt disease when present in trans with severe variants

PURPOSE. To assess the occurrence and the disease expression of the common p.Asn1868Ile variant in patients with Stargardt disease (STGD1) harboring known, monoallelic causal ABCA4 variants. METHODS. The coding and noncoding regions of ABCA4 were sequenced in 67 and 63 STGD1 probands respectively, harboring monoallelic ABCA4 variants. In case p.Asn1868Ile was detected, segregation analysis was performed whenever possible. Probands and affected siblings harboring p.Asn1868Ile without additional variants in cis were clinically evaluated retrospe

ABCA4-associated disease as a model for missing heritability in autosomal recessive disorders: novel noncoding splice, cis-regulatory, structural, and recurrent hypomorphic variants

PURPOSE: ABCA4-associated disease, a recessive retinal dystrophy, is hallmarked by a large proportion of patients with only one pathogenic ABCA4 variant, suggestive for missing heritability. METHODS: By locus-specific analysis of ABCA4, combined with extensive functional studies, we aimed to unravel the missing alleles in a cohort of 67 patients (p), with one (p = 64) or no (p = 3) identified coding pathogenic variants of ABCA4. RESULTS: We identified eight pathogenic (deep-)intronic ABCA4 splice variants, of which five are novel and six structural variants, four of which are novel, including two duplications. Together, these variants account for the missing alleles in 40.3% of patients. Furthermore, two novel variants with a putative cis-regulatory effect were identified. The common hypomorphic variant c.5603A>T p.(Asn1868Ile) was found as a candidate second allele in 43.3% of patients. Overall, we have elucidated the missing heritability in 83.6% of our cohort. In addition, we successfully rescued three deep-intronic variants using antisense oligonucleotide (AON)-mediated treatment in HEK 293-T cells and in patient-derived fibroblast cells. CONCLUSION: Noncoding pathogenic variants, novel structural variants, and a common hypomorphic allele of the ABCA4 gene explain the majority of unsolved cases with ABCA4-associated disease, rendering this retinopathy a model for missing heritability in autosomal recessive disorders

Comprehensive Rare Variant Analysis via Whole-Genome Sequencing to Determine the Molecular Pathology of Inherited Retinal Disease

Inherited retinal disease is a common cause of visual impairment and represents a highly heterogeneous group of conditions. Here, we present findings from a cohort of 722 individuals with inherited retinal disease, who have had whole-genome sequencing (n = 605), whole-exome sequencing (n = 72), or both (n = 45) performed, as part of the NIHR-BioResource Rare Diseases research study. We identified pathogenic variants (single-nucleotide variants, indels, or structural variants) for 404/722 (56%) individuals. Whole-genome sequencing gives unprecedented power to detect three categories of pathogenic variants in particular: structural variants, variants in GC-rich regions, which have significantly improved coverage compared to whole-exome sequencing, and variants in non-coding regulatory regions. In addition to previously reported pathogenic regulatory variants, we have identified a previously unreported pathogenic intronic variant in $\textit{CHM}$ in two males with choroideremia. We have also identified 19 genes not previously known to be associated with inherited retinal disease, which harbor biallelic predicted protein-truncating variants in unsolved cases. Whole-genome sequencing is an increasingly important comprehensive method with which to investigate the genetic causes of inherited retinal disease.This work was supported by The National Institute for Health Research England (NIHR) for the NIHR BioResource – Rare Diseases project (grant number RG65966). The Moorfields Eye Hospital cohort of patients and clinical and imaging data were ascertained and collected with the support of grants from the National Institute for Health Research Biomedical Research Centre at Moorfields Eye Hospital, National Health Service Foundation Trust, and UCL Institute of Ophthalmology, Moorfields Eye Hospital Special Trustees, Moorfields Eye Charity, the Foundation Fighting Blindness (USA), and Retinitis Pigmentosa Fighting Blindness. M.M. is a recipient of an FFB Career Development Award. E.M. is supported by UCLH/UCL NIHR Biomedical Research Centre. F.L.R. and D.G. are supported by Cambridge NIHR Biomedical Research Centre

Evaluation of nitric oxide (indirect method) in dogs and cats affected with chronic kidney diseases

Human patients affected with chronic kidney disease (CKD) show a decrease in endogenous production of nitric oxide (NO). Similar observations were detected in experimental models in laboratory animals. The aim of this study is to evaluate the metabolism of endogenous NO in dogs and cats affected with CKD by an indirect method: the determination of its stable oxidation products nitrite and nitrate (NOx). Serum and urine samples were analysed from privately owned 12 dogs and 15 cats affected by CKD during the monitoring of the diseases and from 5 healthy dogs and 5 healthy cats during their routine sanitary check-up. Serum urea and creatinine, proteinuria, creatininuria and serum and urinary NOx were assessed. All the parameters were determined by an automated spectrophotometr (Cobas Mira\uae, Roche). Statistic analysis (analysis of variance and linear regression) was performed by JMP7 (SAS Inc). Analysis of data revealed a significant difference between serum NOx levels determined in CKD and healthy groups (p=0.0465) in both species, with a tendency to lower values in the pathological animals. Furthermore serum NOx wasn\u2019t significantly related with the other parameters evaluated. In cats, a significant variance was shown between urinary NOx levels in healthy and CKD groups (p=0.049), with a tendency to higher values in healthy cats. Because only one cat was proteinuric, the relation of urinary NOx vs proteinuria, creatininuria and urine protein to creatinine ratio (UPC) was not evaluated. In dogs, a statistically significant variance between urinary NOx levels in healthy and pathological groups was not detected. The linear regression showed that urinary NOx tends to increase with increasing of proteinuria (p=0.0019) and UPC (p=0.0009). This preliminary study supports the hypothesis that the decrease in serum NO in animals affected by CKD may be due to its lower production or to a greater degradation in the course of this disease, as it has been demonstrated in human medicine. Moreover, this study indicates the need to assess the urinary NOx levels in relation to the degree of proteinuria. In fact, the increase of urinary NOx, in the proteinuric subjects, could arise from its increased local synthesis in course of inflammatory or degenerative glomerular diseases. It has been already demonstrated that in these pathological conditions the inflammatory cytokines stimulate a higher production of NO through the activity of NO sintetase - inducible form