Non-canonical Wnt pathway expression in the developing mouse and human retina.

The non-canonical Wnt pathway is an evolutionarily conserved pathway essential for tissue patterning and development across species and tissues. In mammals, this pathway plays a role in neuronal migration, dendritogenesis, axon growth, and synapse formation. However, its role in development and synaptogenesis of the human retina remains less established. In order to address this knowledge gap, we analyzed publicly available single-cell RNA sequencing (scRNAseq) datasets for mouse retina, human retina, and human retinal organoids over multiple developmental time points during outer retinal maturation. We identified ligands, receptors, and mediator genes with a putative role in retinal development, including those with novel or species-specific expression, and validated this expression using fluorescence in situ hybridization (FISH). By quantifying outer nuclear layer (ONL) versus inner nuclear layer (INL) expression, we provide evidence for the differential expression of certain non-canonical Wnt signaling components in the developing mouse and human retina during outer plexiform layer (OPL) development. Importantly, we identified distinct expression patterns of mouse and human FZD3 and WNT10A, as well as previously undescribed expression, such as for mouse Wnt2b in Chat+ starburst amacrine cells. Human retinal organoids largely recapitulated the human non-canonical Wnt pathway expression. Together, this work provides the basis for further study of non-canonical Wnt signaling in mouse and human retinal development and synaptogenesis.

HUMAN CELLULAR MODELS FOR RETINAL DISEASE: From Induced Pluripotent Stem Cells to Organoids.

PURPOSE: To provide a concise review of induced pluripotent stem cells (iPSCs) and retinal organoids as models for human retinal diseases and their role in gene discovery and treatment of inherited retinal diseases (IRDs). METHODS: A PubMed literature review was performed for models of human retinal disease, including animal models and human pluripotent stem cell-derived models. RESULTS: There is a growing body of research on retinal disease using human pluripotent stem cells. This is a significant change from just a decade ago when most research was performed on animal models. The advent of induced pluripotent stem cells has permitted not only the generation of two-dimensional human cell cultures such as RPE but also more recently the generation of three-dimensional retinal organoids that better reflect the multicellular laminar architecture of the human retina. CONCLUSION: Modern stem cell techniques are improving our ability to model human retinal disease in vitro, especially with the use of patient-derived induced pluripotent stem cells. In the future, a personalized approach may be used in which the individual's unique genotype can be modeled in two-dimensional culture or three-dimensional organoids and then rescued with an optimized therapy before treating the patient.

MECP2 Mutations in the Rett Syndrome Patients from South India.

Background: Rett syndrome (RTT) is a rare neurological disorder that primarily affects the females. Most cases of RTT are caused by a de novo mutation in the MECP2 gene located on the X chromosome. About 1000 MECP2 mutations have been found to be associated with RTT. Objective: The present study is aimed at the mutation screening of MECP2 gene in the RTT patients belonging to the south Indian state of Kerala. Materials and Methods: In total 22 girls with a clinical suspicion of RTT were recruited for the study. Exons 2, 3, and 4 of MECP2 were amplified and sequenced. Results: MECP2 mutations were observed in 12 patients. While 7 mutations were pathogenic, 4 were benign. All of the mutations were located in exons 3 and 4 of MECP2, spanning the methyl-CpG DNA binding domain (MBD), transcription repression domain (TRD), and C-terminal domain (CTD) domains of the MECP2 protein. Four novel mutations were identified. There were no mutations in the MECP2 gene of 10 patients with a clinical suspicion of RTT. Conclusions: A recommended screening strategy for RTT is to first look for mutations in exons 3 and 4 of MECP2, followed by exons 1 and 2, testing for large deletions in MECP2, and screening for mutations in genes, such as CDKL5 and FOXG1 that are reported to cause a Rett-like phenotype.

Effects of sampling rate on the interpretation of cellular transport measurements.

Electrochemical sensing techniques are increasingly used to study biological processes by monitoring concentration changes of the molecule of interest close to cells. The measured concentration is the result of cellular transport across the cell membrane and diffusion of the released molecules from the cells to the sensing electrode. The objective of such experiments is to understand the cellular processes underlying the observed changes in concentration. Thus, the influence of mass transport on the measured concentration trace has to be removed. This is done by deconvolution of the impulse response function of diffusion from the concentration data. We have recently observed that measuring concentration at a sampling rate that satisfies the Nyquist criterion for the observed concentration dynamics may not be sufficient to correctly reconstruct cellular flux. This is because the impulse response function of diffusion also has to be represented with sufficient temporal resolution. We discuss this problem here using the example of monitoring drug efflux from a monolayer of cancer cells with microvoltammetry, and chloride secretion from an epithelial cell monolayer monitored with an ion-selective electrode.

Generation of an integration-free iPSC line (CSCRi005-A) from erythroid progenitor cells of a healthy Indian male individual.

Reprogramming of somatic cells with higher genome integrity, and use of non-integrating gene delivery methods and xeno-free cell culture conditions aid in the generation of iPSCs which are more suitable for disease modelling and clinical applications. We describe here an iPSC line generated using such conditions, which expressed all the pluripotency markers, retained normal karyotype and exhibited the potential for tri-lineage differentiation, both in-vitro and in-vivo. This is the first iPSC line available from a healthy Indian individual for researchers.

Understanding the Molecular Basis of Heterogeneity in Induced Pluripotent Stem Cells.

Reprogramming of somatic cells to generate induced pluripotent stem cells (iPSCs) has considerable latency and generates epigenetically distinct partially and fully reprogrammed clones. To understand the molecular basis of reprogramming and to distinguish the partially reprogrammed iPSC clones (pre-iPSCs), we analyzed several of these clones for their molecular signatures. Using a combination of markers that are expressed at different stages of reprogramming, we found that the partially reprogrammed stable clones have significant morphological and molecular heterogeneity in their response to transition to the fully pluripotent state. The pre-iPSCs had significant levels of OCT4 expression but exhibited variable levels of mesenchymal-to-epithelial transition. These novel molecular signatures that we identified would help in using these cells to understand the molecular mechanisms in the late of stages of reprogramming. Although morphologically similar mouse iPSC clones showed significant heterogeneity, the human iPSC clones isolated initially on the basis of morphology were highly homogeneous with respect to the levels of pluripotency.

Protection of human erythrocyte using Crinum asiaticum extract and lycorine from oxidative damage induced by 2-amidinopropane.

The intention of this investigation was to evaluate the free radical scavenging activity and erythrocyte protective activity of ethanolic extract of Crinum asiaticum (L) and lycorine. The ethanolic extract of C. asiaticum (L) and lycorine were found to have different levels of antioxidant properties in the test models. Both ethanolic extract of C. asiaticum (L) (0.5-2.5 mg/ml) and lycorine (0.010 mg-0.050 mg/ml) increases the percentage of lipid peroxidation inhibition (26.25 ± 0.23% and 19.25 ± 0.23%) and enhances the free radical scavenging activity (20.92 ± 0.22% and 20.52 ± 0.22%), scavenging of hydrogen peroxide (25.67 ± 0.17% and 23.07 ± 0.3%) superoxide anion scavenging activity (27.69 ± 0.16% and 16.09 ± 0.7%) at concentration of 2.5 and 0.050 mg of C. asiaticum (L) and lycorine, respectively. But compared with tocopherol (P < 0.05) less activity was observed by C. asiaticum (L) and lycorine. The ethanolic extract of C. asiaticum (L) and lycorine were normalized to reduce the level of glutathione and also to sustain the status of protein in erythrocytes during the peroxyl radical [2,2-azobis (2-amidinopropane) dihydrochloride (AAPH)] induced oxidative damage in ex vivo model. The present results of the investigations demonstrated that protective nature of the C. asiaticum (L) and lycorine will be considered as a significant natural antioxidant source.

Multiple soft tissue aneurysmal cysts: An occurrence after resection of primary aneurysmal bone cyst of fibula.

We report a case of multiple extraosseous aneurysmal cysts occurring in the muscle and subcutaneous plane of postero-lateral aspects of the upper right leg. They appeared about 15 months after resection of aneurysmal bone cyst of the upper end of the fibula. They varied in size from 2 cm to 5 cm. Radiologically they were well-defined lesions with central septate areas surrounded by a rim of calcification. Histologically they showed central cystic spaces separated by septa consisting of fibroblasts, osteoclast type of giant cells and reactive woven bone. Thus they showed histological similarity with aneurysmal bone cysts, but did not show any connection with the bone. Only very few examples of aneurysmal cysts of soft tissue had been described in the past one decade and they were reported in various locations including rare sites such as arterial wall and larynx. Recent cytogenetic analyses have shown abnormalities involving 17p11-13 and/or 16q22 in both osseous and extraosseous aneurysmal cysts indicating its probable neoplastic nature. Our case had unique features like multiplicity and occurrence after resection of primary aneurysmal bone cyst of the underlying bone.