Selection of Diagnostically Significant Regions of the SLC26A4 Gene Involved in Hearing Loss

Screening pathogenic variants in the SLC26A4 gene is an important part of molecular genetic testing for hearing loss (HL) since they are one of the common causes of hereditary HL in many populations. However, a large size of the SLC26A4 gene (20 coding exons) predetermines the difficulties of its complete mutational analysis, especially in large samples of patients. In addition, the regional or ethno-specific prevalence of SLC26A4 pathogenic variants has not yet been fully elucidated, except variants c.919-2A>G and c.2168A>G (p.His723Arg), which have been proven to be most common in Asian populations. We explored the distribution of currently known pathogenic and likely pathogenic (PLP) variants across the SLC26A4 gene sequence presented in the Deafness Variation Database for the selection of potential diagnostically important parts of this gene. As a result of this bioinformatic analysis, we found that molecular testing ten SLC26A4 exons (4, 6, 10, 11, 13–17 and 19) with flanking intronic regions can provide a diagnostic rate of 61.9% for all PLP variants in the SLC26A4 gene. The primary sequencing of these SLC26A4 regions may be applied as an initial effective diagnostic testing in samples of patients of unknown ethnicity or as a subsequent step after the targeted testing of already-known ethno- or region-specific pathogenic SLC26A4 variants.     

Novel Hydroxypyridine Compound Protects Brain Cells against Ischemic Damage In Vitro and In Vivo

A non-surgical pharmacological approach to control cellular vitality and functionality during ischemic and/or reperfusion-induced phases of strokes remains extremely important. The synthesis of 2-ethyl-6-methyl-3-hydroxypyridinium gammalactone-2,3-dehydro-L-gulonate (3-EA) was performed using a topochemical reaction. The cell-protective effects of 3-EA were studied on a model of glutamate excitotoxicity (GluTox) and glucose-oxygen deprivation (OGD) in a culture of NMRI mice cortical cells. Ca²⁺ dynamics was studied using fluorescent bioimaging and a Fura-2 probe, cell viability was assessed using cytochemical staining with propidium iodide, and gene expression was assessed by a real-time polymerase chain reaction. The compound anti-ischemic efficacy in vivo was evaluated on a model of irreversible middle cerebral artery (MCA) occlusion in Sprague-Dawley male rats. Brain morphological changes and antioxidant capacity were assessed one week after the pathology onset. The severity of neurological disorder was evaluated dynamically. 3-EA suppressed cortical cell death in a dose-dependent manner under the excitotoxic effect of glutamate and ischemia/reoxygenation. Pre-incubation of cerebral cortex cells with 10–100 µM 3-EA led to significant stagnation in Ca²⁺ concentration in a cytosol ([Ca2+]i) of neurons and astrocytes suffering GluTox and OGD. Decreasing intracellular Ca²⁺ and establishing a lower [Ca2+]i baseline inhibited necrotic cell death in an acute experiment. The mechanism of 3-EA cytoprotective action involved changes in the baseline and ischemia/reoxygenation-induced expression of genes encoding anti-apoptotic proteins and proteins of the oxidative status; this led to inhibition of the late irreversible stages of apoptosis. Incubation of brain cortex cells with 3-EA induced an overexpression of the anti-apoptotic genes BCL-2, STAT3, and SOCS3, whereas the expression of genes regulating necrosis and inflammation (TRAIL, MLKL, Cas-1, Cas-3, IL-1β and TNFa) were suppressed. 3-EA 18.0 mg/kg intravenous daily administration for 7 days following MCA occlusion preserved rats’ cortex neuron population, decreased the severity of neurological deficit, and spared antioxidant capacity of damaged tissues. 3-EA demonstrated proven short-term anti-ischemic activity in vivo and in vitro, which can be associated with antioxidant activity and the ability to target necrotic and apoptotic death. The compound may be considered a potential neuroprotective molecule for further pre-clinical investigation.     

Switchable Nanozyme Activity of Porphyrins Intercalated in Layered Gadolinium Hydroxide

In this study, organo-inorganic nanohybrids LHGd-MTSPP with enzyme-like activity were prepared by in situ intercalation of anionic 5,10,15,20-tetrakis-(4-sulfonatophenyl)porphyrin and its complexes with Zn(II) and Pd(II) (MTSPP, M = 2H, Zn(II) and Pd(II)) into gadolinium layered hydroxide (LHGd). The combination of powder XRD, CHNS analysis, FT-IR, EDX, and TG confirmed the layered structure of the reaction products. The basal interplanar distances in LHGd-MTSPP samples were 22.3–22.6 Å, corresponding to the size of an intercalated tetrapyrrole molecule. According to SEM data, LHGd-MTSPP hybrids consisted of individual lamellar nanoparticles 20–50 nm in thickness. The enzyme-like activity of individual constituents, LHGd-Cl and sulfoporphyrins TSPP, ZnTSPP and PdTSPP, and hybrid LHGd-MTSPP materials, was studied by chemiluminescence analysis using the ABAP/luminol system in phosphate buffer solution. All the individual porphyrins exhibited dose-dependent antioxidant properties with respect to alkylperoxyl radicals at pH 7.4. The intercalation of free base TSPP porphyrin into the LHGd preserved the radical scavenging properties of the product. Conversely, in LHGd-MTSPP samples containing Zn(II) and Pd(II) complexes, the antioxidant properties of the porphyrins changed to dose-dependent prooxidant activity. Thus, an efficient approach to the design and synthesis of advanced LHGd-MTSPP materials with switchable enzyme-like activity was developed.     

Development of the Periventricular Nucleus as a Brain Center,Containing Dopaminergic Neurons and Neurons Expressing Individual Enzymes of Dopamine Synthesis

We have recently shown that the periventricular nucleus (PeVN) of adult rats is a “mixed dopaminergic (DAergic) center” containing three thousand neurons: DAergic neurons and those expressing one of the dopamine (DA)-synthesizing enzymes. This study aims to evaluate the development of the PeVN as a mixed DAergic center in rats in the perinatal period, critical for brain morphogenesis. During this period, the PeVN contains DAergic neurons and monoenzymatic neurons expressing individual enzymes of DA synthesis: tyrosine hydroxylase (TH) or aromatic L-amino acid decarboxylase (AADC). In the perinatal period, the total number of such neurons triples, mainly due to monoenzymatic neurons; the content of L-DOPA, the end product of monoenzymatic TH neurons, doubles; and the content of DA, the end product of monoenzymatic AADC neurons and DAergic neurons, increases sixfold. Confocal microscopy has shown that, in the PeVN, all types of neurons and their processes are in close relationships, which suggests their mutual regulation by L-DOPA and DA. In addition, monoenzymatic and DAergic fibers are close to the third cerebral ventricle, located in the subependymal zone, between ependymal cells and in the supraependymal zone. These observations suggest that these fibers deliver L-DOPA and DA to the cerebrospinal fluid, participating in the neuroendocrine regulation of the brain.     

Nucleoside Analogs and Perylene Derivatives Modulate Phase Separation of SARS-CoV-2 N Protein and Genomic RNA In Vitro

The life cycle of severe acute respiratory syndrome coronavirus 2 includes several steps that are supposedly mediated by liquid–liquid phase separation (LLPS) of the viral nucleocapsid protein (N) and genomic RNA. To facilitate the rational design of LLPS-targeting therapeutics, we modeled N-RNA biomolecular condensates in vitro and analyzed their sensitivity to several small-molecule antivirals. The model condensates were obtained and visualized under physiological conditions using an optimized RNA sequence enriched with N-binding motifs. The antivirals were selected based on their presumed ability to compete with RNA for specific N sites or interfere with non-specific pi–pi/cation–pi interactions. The set of antivirals included fleximers, 5′-norcarbocyclic nucleoside analogs, and perylene-harboring nucleoside analogs as well as non-nucleoside amphiphilic and hydrophobic perylene derivatives. Most of these antivirals enhanced the formation of N-RNA condensates. Hydrophobic perylene derivatives and 5′-norcarbocyclic derivatives caused up to 50-fold and 15-fold enhancement, respectively. Molecular modeling data argue that hydrophobic compounds do not hamper specific N-RNA interactions and may promote non-specific ones. These findings shed light on the determinants of potent small-molecule modulators of viral LLPS.     

Knowledge mining sensory evaluation data: genetic programming, statistical techniques, and swarm optimization

Knowledge mining sensory evaluation data is a challenging process due to extreme sparsity of the data, and a large variation in responses from different members (called assessors) of the panel. The main goals of knowledge mining in sensory sciences are understanding the dependency of the perceived liking score on the concentration levels of flavors’ ingredients, identifying ingredients that drive liking, segmenting the panel into groups with similar liking preferences and optimizing flavors to maximize liking per group. Our approach employs (1) Genetic programming (symbolic regression) and ensemble methods to generate multiple diverse explanations of assessor liking preferences with confidence information; (2) statistical techniques to extrapolate using the produced ensembles to unobserved regions of the flavor space, and segment the assessors into groups which either have the same propensity to like flavors, or are driven by the same ingredients; and (3) two-objective swarm optimization to identify flavors which are well and consistently liked by a selected segment of assessors.