Santucci, A. | Search bioRxiv

bioRxiv2022

Evaluation of targeted oxidative stress induced by oxygen-ozone gas mixtures in vitro after ischemic induction

Encephalic vascular accident, or stroke, is the most common pathology of the central nervous system in humans. It is the second leading cause of death, as well as physical and cognitive disabilities, in developing countries. It is a vascular disorder that may present in an ischemic (more common) or hemorrhagic form. Ozone is a gas capable of oxidizing double bonds of organic molecules, thereby producing lipoperoxides and aldehydes. Stimulation of the immune and antioxidant system, and improvement in tissue vascularization and oxygenation, are few among several effects exerted by ozone. Ozone therapy has previously been shown to be effective in neuromodulation, neuroprotection, and nerve regeneration. The present study aimed to evaluate the effect of targeted mild ozone after inducing cerebral ischemia in vitro. The experiment was divided into two steps; in the first step, neuroblastoma lineage cells (SH-SY5Y) were subjected to 24 hours of hypoxia in an incubator culture chamber. Treatment with different concentrations of ozone (2-10 {micro}g/mL), followed by an 2,5-diphenyl-2H-tetrazolium bromide assay, indicated a possible neuroregenerative effect at low concentrations. The same protocol was applied to canine amniotic membrane stem cells that were evaluated via colorimetric assay spectrophotometry, fluorescence microscopy, and flow cytometry. The metabolic conditions and cellular regeneration in cells at low ozone concentrations (3-8 {micro}g/mL) correlated with lower levels of apoptosis and oxidative stress compared to cells not subjected to hypoxia. High concentrations of ozone (18-30 {micro}g/mL) promoted an increase in rate of apoptosis and cell death. Therefore, we developed a novel protocol that mimics ozone therapy for ischemic stroke, using ozonized culture medium after hypoxia induction. Although more studies are needed to open new avenues for translational medicine, we conclude that ozone has a dose-dependent hormetic effect and can reverse the effect of ischemia in vitro at low concentrations.

molecular biology↗

bioRxiv2021

HGDiscovery: an online tool providing functional and phenotypic information on novel variants of homogentisate 1,2- dioxigenase

Alkaptonuria (AKU), a rare genetic disorder, is characterized by the accumulation of homogentisic acid (HGA) in the body. Affected individuals lack enough functional levels of an enzyme required to breakdown HGA. Mutations in the HGD gene cause AKU and they are responsible for deficient levels of functional homogentisate 1,2-dioxygenase (HGD), which, in turn, leads to excess levels of HGA. Although HGA is rapidly cleared from the body by the kidneys, in the long term it starts accumulating in various tissues, especially cartilage. Over time (rarely before adulthood), it eventually changes the color of affected tissue to slate blue or black. Here we report a comprehensive mutation analysis of 111 pathogenic and 190 non-pathogenic HGD missense mutations using protein structural information. Using our comprehensive suite of graph-based signature methods, mCSM complemented with sequence-based tools, we studied the functional and molecular consequences of each mutation on protein stability, interaction and evolutionary conservation. The scores generated from the structure and sequence-based tools were used to train a supervised machine learning algorithm with 84% accuracy. The empirical classifier was used to generate the variant phenotype for novel HGD missense mutations. All this information is deployed as a user friendly freely available web server called HGDiscovery (http://biosig.unimelb.edu.au/hgdiscovery/).

bioinformatics↗