Quality and applicability of cadaveric donor eyes for molecular biology research: An Indian experience.

PURPOSE: Human ocular tissue banking plays an important part in the advancement of translational research for identifying the molecular processes involved in disease etiology and pathogenesis. Timely obtaining a good-quality ocular tissue from a cadaveric donor is exceedingly difficult, especially in remote areas, with a variable transportation time (within 12-24 h), raising concerns about RNA quality and its subsequent applications. Therefore, we assessed the utility of retinal tissues from cadaver donor and enucleated eyes based on the RNA quality and gene expression by real-time polymerase chain reaction (PCR). SETTINGS AND DESIGN: Prospective study. METHODS: Retina tissues were separated from the donor/enucleated eyes received in the eye bank within 24 h of death (n = 15) and within an hour from OR (n = 3), respectively, and stored immediately at -80 degree. RNA was isolated using trizol, and the quantity and quality were assessed using Qubit and agarose gel electrophoresis, respectively. QPCR was performed for measuring the expression of different retinal-specific genes. The cellular viability of the retina was assessed by establishing explant primary cell cultures. STATISTICAL ANALYSIS: The data were calculated as an average of normalised Ct values ± standard error of the mean. RESULTS: RNA obtained from cadaveric tissues despite being partially degraded showed a uniform strong gene expression of several retinal-specific genes such as PAX6, RHO, TUBB3, CRX , and ALDH1L1 . The primary cultures established from cadaveric tissues showed viable cells. CONCLUSION: The cadaver donor tissues collected within 24 hours of death can be effectively utilized for gene expression profiling.

Exploring the involvement of the alternative complement pathway in non-infectious uveitis pathogenesis.

Purpose: Non-infectious uveitis is a complex disease characterized by intraocular inflammation of the uveal area and the leading cause of vision impairment and blindness in young people globally. However, what triggers inflammation and contributes to its recurrence remains unclear. The complement system has been linked to various immunological and inflammatory conditions. In the present study, we have systematically evaluated the role of the alternative complement pathway in the pathogenesis of non-infectious uveitis. Methodology: Quantitative PCR was done in the peripheral leukocytes to study the expression of genes and regulatory miRNA in both anterior and posterior uveitis (n=28 in each category). Multiplex ELISA was performed to measure alternative pathway complement components, such as C3b, factor B, and CFH, and aqueous humor of infectious and non-infectious uveitis patients and non-inflammatory controls (n=10 each). Western blotting was done to validate the ELISA findings in a subset of patients and controls. Results: Downregulation of C3 and CFH mRNA in the peripheral blood was shown by quantitative PCR in the group of anterior uveiits (AU), while the opposite result was found in the group of posterior uveitis (PU). ELISA levels of C3b and CFH proteins were significantly higher in aqueous humor of infectious and non-infectious uveitis (*p = 0.03 and **p = 0.0007 respectively) as compared to the control group. Western blotting further validated (VitH) the activation of the complement cascade in the aqueous (AH) and vitreous humor of patients with non-infectious uveitis, with an increased level of C3b (n=6) and CFH (n=4) in aqueous humor. C3b level was significantly increased while CFH was reduced relative to controls in the vitreous humor (VitH) of posterior uveitis patients compared to controls (n=27 in each category). A C3b to CFH ratio was computed to assess the regulation of complement activation and this index was several folds higher in both anterior and posterior uveitis (n=10 each). The expression of miRNA-hsa-miR-146a and miRNA-hsa-miR-155-5p that regulates CFH was downregulated and nicely correlated with the increased complement proteins in both anterior and posterior uveitis (n=10 each). Conclusion: Our results demonstrate a clear role of CFH and the activation of the alternative complement pathway in the pathogenesis of non-infectious uveitis; however, its therapeutic potential warrants further investigations.

Chronic intake of 4-Methylimidazole induces Hyperinsulinemia and Hypoglycaemia via Pancreatic Beta Cell Hyperplasia and Glucose Dyshomeostasis.

Caramel colours are the preferential food colouring agent globally, reaches wide age groups through eatables. Colas, a sweetened carbonated drink are most common caramel coloured beverage and its consumption is linked with diabetes, obesity, pancreatic cancer and other endocrine disorders. A major by-product produced during caramelization is 4-methylimidazole (4-MEI) that is detected in noteworthy concentrations in colas and other beverages. Previous studies revealed the neurotoxic and carcinogenic potential of 4-MEI in animals at higher doses but the effect of 4-MEI at theoretical maximum daily intake dose on glucose homeostasis is unexplored. Here, mice treated with 4-MEI (32 µg/kg bodyweight/day) for seven weeks exhibited severe hypoglycaemia and hyperinsulinemia mediated by hyperplasia of pancreatic beta cells and induces metabolic alterations. On combinatorial treatment, 4-MEI suppressed the glucogenic potential of non-artificial sweeteners and promotes lipogenesis. Furthermore, increased levels of C-peptide, LDL-cholesterol and triglycerides were observed in the humans with regular intake of 4-MEI containing beverages. In summary, 4-MEI induced pancreatic beta cell hyperplasia and leads to disruption of glucose and lipid homeostasis. This study suggests the need for further assessment and reconsideration of the wide usage of 4-MEI containing caramels as food additives.