Hippocampal tandem mass tag (TMT) proteomics analysis during kindling epileptogenesis in rat.

Epilepsy is a neurological disorder that remains difficult to treat due to the lack of a clear molecular mechanism and incomplete understanding of involved proteins. To identify potential therapeutic targets, it is important to gain insight into changes in protein expression patterns related to epileptogenesis. One promising approach is to analyze proteomic data, which can provide valuable information about these changes. In this study, to evaluate the changes in gene expression during epileptogenesis, LC-MC2 analysis was carried out on hippocampus during stages of electrical kindling in rat models. Subsequently, progressive changes in the expression of proteins were detected as a result of epileptogenesis development. In line with behavioral kindled seizure stages and according to the proteomics data, we described epileptogenesis phases by comparing Stage3 versus Control (S3/C0), Stage5 versus Stage3 (S5/S3), and Stage5 versus Control group (S5/C0). Gene ontology analysis on differentially expressed proteins (DEPs) showed significant changes of proteins involved in immune responses like Csf1R, Aif1 and Stat1 during S3/C0, regulation of synaptic plasticity like Bdnf, Rac1, CaMK, Cdc42 and P38 during S5/S3, and nervous system development throughout S5/C0 like Bdnd, Kcc2 and Slc1a3.There were also proteins like Cox2, which were altered commonly among all three phases. The pathway enrichment analysis of DEPs was also done to discover molecular connections between phases and we have found that the targets like Csf1R, Bdnf and Cox2 were analyzed throughout all three phases were highly involved in the PPI network analysis as hub nodes. Additionally, these same targets underwent changes which were confirmed through Western blotting. Our results have identified proteomic patterns that could shed light on the molecular mechanisms underlying epileptogenesis which may allow for novel targeted therapeutic strategies.

CD38 and MGluR1 as possible signaling molecules involved in epileptogenesis: A potential role for NAD+ homeostasis.

In spite of long-term intensive scientific research efforts, there are still many issues concerning the mechanisms of epileptogenesis and epilepsy to be resolved. Temporal lobe, in particular hippocampus, is vulnerable to epileptogenic process. Herein, electrical kindling model of temporal lobe were analyzed using proteomic approach. A dramatic decrease in nicotinamide adenine dinucleotide (NAD+) level was exhibited during the kindling procedure in hippocampus. After stage 3, high CD38 expression was detected by qPCR, nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) and western blot analysis. An increase in expression of CD38/NADase activity was observed during the kindling procedure in hippocampus that suggest it as one of the most important NAD+ degrading enzymes during epileptogenesis. Subsequently, gene expression of CD38 metabolite related proteins (Ryr2, FKBP-12.6, Chrm1, mGluR1 and Cnx43) were examined. Among them, changes in the expression level of mGluR1 was more than other genes, which was also confirmed by LC MS/MS and western blotting analysis. These findings provided valuable information about changes in the expression of CD38/cADPR signaling pathway and suggest its crucial role during epileptogenesis.

Activity, stability and structure of laccase in betaine based natural deep eutectic solvents.

Natural deep eutectic solvents (NADES) play a role as alternative media to water in living organisms. They are formed by mixing two or more natural compounds in certain ratios producing a liquid having a lower melting point than those of the individual NADES components. Although, the eutectics medium bring several advantages as enhanced solubility of non-polar substrates and/or products, however, these advantages would often be limited by a lower stability of biocatalysts in these systems. To examine this matter, biochemical characterization, thermal stability and tertiary structure of laccase from Bacillus HR03 was investigated as a model in betaine and choline based NADES. In eutectics containing choline, a sudden drop in enzyme activity and stability was observed. Betaine based eutectics exhibited a better media for the laccase stability in comparison with the aqueous buffer and choline chloride eutectics. The enzyme highest activity was observed in 20% (v/v) glycerol:betaine (2:1). Among betaine based eutectics, the enzyme exhibited its highest stability in sorbitol:betaine:water (1:1:1) and glycerol:betaine (2:1) compared to the aqueous buffer at 80 and 90°C. Associated conformational changes caused by solvents were monitored using fluorescence technique. Finally, the effects of NADES on the enzyme activity and stability were discussed.