Identification of glycosylated nucleosides in small synthetic glyco-RNAs.

Recently, the post-transcriptional modification of RNA with N-glycans was reported, changing the paradigm that RNAs are not commonly N-glycosylated. Moreover, glycan modifications of RNA are investigated for therapeutic targeting purposes. But the glyco-RNA field is in its infancy with many challenges to overcome. One question is how to accurately characterize glycosylated RNA constructs. Thus, we generated glycosylated forms of Y5 RNA mimics, a short non-coding RNA. The simple glycans lactose and sialyllactose were attached to the RNA backbone using azide-alkyne cycloadditions. Using nuclease digestion followed by LC-MS, we confirmed the presence of the glycosylated nucleosides, and characterized the chemical linkage. Next, we probed if glycosylation would affect the cellular response to Y5 RNA. We treated human foreskin fibroblasts in culture with the generated compounds. Key transcripts in the innate immune response were quantified by RT-qPCR. We found that under our experimental conditions, exposure of cells to the Y5 RNA did not trigger an interferon response, and glycosylation of this RNA did not have an impact. Thus, we have identified a successful approach to chemically characterize synthetic glyco-RNAs, which will be critical for further studies to elucidate how the presence of complex glycans on RNA affects the cellular response.

Improvement of camel milk-clotting: Usefulness of crude extract from green pods of carob (Ceratonia siliqua. L) as a substitute for commercial rennet.

Camel milk transformation into cheese remains an objective to be improved today. This study aimed to improve camel milk clotting using a crude extract from green pods of carob as a substitute for commercial rennet. The composition of the crude carob extract was determined for dry matter and protein content. Milk clotting conditions were studied at different temperatures, pH and CaCl2 concentrations. Milk clotting properties were assessed by milk clotting activity, specific activity and proteolytic activity. Enzymatic hydrolysis of camel milk caseins by crude carob extract and its inhibition were demonstrated by SDS-polyacrylamide gel electrophoresis. Crude carob extract analysis showed a protein and dry matter content of 23.26±0.5 mg/ml and 30.66±0.5 g/l, respectively. Optimal milk clotting activity was observed at 53.6 °C, pH 4.5, and 0.09 M CaCl2. The crude carob extract showed a high milk clotting activity (4.97 U/ml) and a low proteolytic activity (0.04U/ml) with camel milk. The cheese yield of curd produced from camel milk using crude carob extract was the highest (23.95%) compared with that of Camel chymosin (20.5%). The high ratio of milk-clotting to proteolytic activity shows the potential of this extract as a substitute for commercial rennet in the dairy industry.

Effect of heat treatment on the antioxidant activities of camel milk alpha, beta and total caseins.

This study aimed to evaluate the effect of various heating temperatures on the antioxidant activities of camel milk caseins. The samples were processed with three different heat treatments: Pasteurization at low and high temperatures and boiling. Fresh camel milk (unheated) was used as a control. Camel milk caseins were separated by fast ion exchange liquid chromatography (FPLC) and identified by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS page). The antioxidant activities of caseins were measu- red by three different in vitro methods: 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, 2, 2'-azino-bis (3-ethylbenzthiazoline-6-sulfonate) (ABTS) radical scavenging activity and ferric reducing power assay (FRAP). The antioxidant activity evaluated by the DPPH assay decreased significantly (p<0.05) with the increase in heat treatment of caseins. However, there was no significant difference in ABTS radical scavenging activity and Ferric Reducing Antioxidant Power assay (FRAP) of heat-treated camel caseins compared to unheated onesStill, a decrease was observed in those activities by the increase of temperature in the different casein concentrations. Besides, whatever the concentration tested and the methods applied, the antioxidant activity of beta-casein (ß-CN) was more pronounced than the alpha-casein (α-CN). Therefore, camel milk casein could be used as a natural source of antioxidants which may have a potential application in the food and nutraceutical industries. Throughout the different heat treatments applied, pasteurization at low temperature could be the most suitable alternative to preserve the antioxidant properties of camel milk.