Safety and efficacy of CRISPR-mediated genome ablation of VEGFA as a treatment for choroidal neovascularization in nonhuman primate eyes.

CRISPR-based genome editing enables permanent suppression of angiogenic factors like vascular endothelial growth factor (VEGF) as potential treatment for choroidal neovascularization (CNV) - a major cause of blindness in age-related macular degeneration. We previously designed adeno-associated viral (AAV) vectors with S. pyogenes Cas 9 (SpCas9) and guide RNAs (gRNAs) to target conserved sequences in VEGFA across mouse, rhesus macaque, and human, with successful suppression of VEGF and laser-induced CNV in mice. Here, we advanced the platform to nonhuman primates and found that subretinal AAV8-SpCas9 with gRNAs targeting VEGFA may reduce VEGF and CNV severity as compared to SpCas9 without gRNAs. However, all eyes that received AAV8-SpCas9 regardless of gRNA presence developed subfoveal deposits, concentric macular rings, and outer retinal disruption that worsened at higher dose. Immunohistochemistry showed subfoveal accumulation of retinal pigment epithelial cells, collagen, and vimentin, disrupted photoreceptor structure, and retinal glial and microglial activation. Subretinal AAV8-SpCas9 triggered aqueous elevations in CCL2, but minimal systemic humoral or cellular responses against AAV8, SpCas9, or GFP reporter. Our findings suggest that CRISPR-mediated VEGFA ablation in NHP eyes may suppress VEGF and CNV, but can also lead to unexpected subretinal fibrosis, photoreceptor damage, and retinal inflammation despite minimal systemic immune responses.

Stability of R-phycoerythrin from Furcellaria lumbricalis - Dependence on purification strategies and purity.

R-phycoerythrin (R-PE) is the most abundant, naturally occurring phycobiliproteins found in red algae. The spectroscopic and structural properties of phycobiliproteins exhibit unique absorption characteristics with two significant absorption maxima at 498 and 565 nm, indicating two different chromophores of R-PE, phycourobilin and phycoerythrobilin respectively. This study aimed to clarify how the stability of R-PE purified from F. lumbricalis was affected by different purification strategies. Crude extracts were compared to R-PE purified by i) microfiltration, ii) ultrafiltration, and iii) multi-step ammonium sulphate precipitation followed by dialysis. The stability of the different R-PE preparations was evaluated with respect to pH (2, 4, 6, 7, 8, 10 and 12) and temperature (20, 40, 60, 80 and 100 °C). The absorbance spectra indicated higher stability of phycourobilin as compared to phycoerythrobilin for heat and pH stability in the samples. All preparations of R-PE showed heat stability till 40 °C from the findings of color, concentration of R-PE and fluorescence emission. The crude extract showed stability from pH 6 to 8, whereas R-PE purified by ultrafiltration and multi-step ammonium sulphate precipitation were both stable from pH 4 to 8 and R-PE purified by microfiltration exhibited stability from pH 4 to 10 from the results of color, SDS-PAGE, and concentration of R-PE. At pH 2, the color changed to violet whereas a yellow color was observed at pH 12 in the samples along with the precipitation of the protein.

Gelling properties of black soldier fly (Hermetia illucens) larvae protein after ultrasound treatment.

H. illucens, black soldier fly larvae (BSFL) is one of the sustainable sources of protein. However, the research on the functionality of BSFL proteins is limited and need to be explored to increase consumer acceptance. The aim of this study is to create a gel system from BSFL protein and evaluate the impact of ultrasound treatment at different exposure time (5, 15, 30 min) on the physicochemical properties of BSFL protein. The highest values for surface hydrophobicity, size, ζ-potential were obtained after 15 min of ultrasound treatment and the same was found for the elastic modulus. Finally, confocal laser scanning microscopy (CLSM) along with image analysis revealed the lowest pore size after 15 min of treatment. The high protein content of BSFL protein extract and its promising gel system herein created, are important features to be considered for further development of insect-based food.

Resveratrol protects the brain of obese mice from oxidative damage.

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a polyphenolic phytoalexin that exerts cardioprotective, neuroprotective, and antioxidant effects. Recently it has been shown that obesity is associated with an increase in cerebral oxidative stress levels, which may enhance neurodegeneration. The present study evaluates the neuroprotective action of resveratrol in brain of obese (ob/ob) mice. Resveratrol was administered orally at the dose of 25 mg kg(-1) body weight daily for three weeks to lean and obese mice. Resveratrol had no effect on body weight or blood glucose levels in obese mice. Lipid peroxides were significantly increased in brain of obese mice. The enzymatic antioxidants superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase and nonenzymatic antioxidants tocopherol, ascorbic acid, and glutathione were decreased in obese mice brain. Administration of resveratrol decreased lipid peroxide levels and upregulated the antioxidant activities in obese mice brain. Our findings indicate a neuroprotective effect of resveratrol by preventing oxidative damage in brain tissue of obese mice.