Analysis of the toxicological and pharmacokinetic profile of Kaempferol-3-O-β-D-(6”-E-p-coumaryl) glucopyranoside - Tiliroside: in silico, in vitro and ex vivo assay

Tiliroside is a glycosidic flavonoid present in many plants species including Helicteres velutina K. Schum (Malvaceae sensu lato), commonly known in Brazil as “pitó”. This molecule has been shown to have many biological activities, however no study has been carried out to investigate the toxicity of this substance. The present work aimed to evaluate the possible cellular toxicity in silico, in vitro and ex-vivo of the kaempferol-3-O-β-D-(6”-E-p-coumaroyl) glucopyranoside (tiliroside), through chemical structure analysis, toxicity assessment and predictive bioactive properties, using human samples for in vitro and ex-vivo tests. The in silico analysis suggests that tiliroside exhibited great absorption index when penetrating biological membranes. In addition, it also displayed considerable potential for cellular protection against free radicals, and anticarcinogenic, antioxidant, antineoplastic, anti-inflammatory, anti-hemorrhagic and antithrombotic activities. The assessment of the hemolytic and genotoxic effects of tiliroside showed low hemolysis rates in red blood cells and absence of cellular toxicity in the oral mucosa cells. The data obtained indicate that this molecule could be a promising therapeutic approach as a possible new drug with biotechnological potential.

Hypoxia Marker Carbonic Anhydrase IX Is Present in Abdominal Aortic Aneurysm Tissue and Plasma

Abdominal aortic aneurysms (AAA) are a significant cause of premature deaths worldwide. Since there is no specific treatment for reducing AAA progression, it is crucial to understand the pathogenesis leading to aneurysm wall weakening/remodeling and identify new proteins involved in this process which could subsequently serve as novel therapeutic targets. In this study, we analyzed the presence of the hypoxia-related proteins carbonic anhydrase IX (CA IX), hypoxia-inducible factor 1α (HIF-1α), and AKT as the key molecule in the phosphoinositide-3-kinase pathway in the AAA wall. Additionally, we used a blood-based assay to examine soluble CA IX (s-CA IX) levels in the plasma of AAA patients. Using western blotting, we detected CA IX protein in 12 out of 15 AAA tissue samples. Immunohistochemistry staining proved CA IX expression in the media of the aneurysmal wall. Evaluation of phosphorylated (p-AKT) and total AKT showed elevated levels of both forms in AAA compared to normal aorta. Using ELISA, we determined the concentration of s-CA IX >20 pg/mL in 13 out of 15 AAA patients. Results obtained from in silico analysis of CA9 and aneurysm-associated genes suggest a role for CA IX in aneurysmal wall remodeling. Our results prove the presence of hypoxia-related CA IX in AAA tissues and indicate a possible role of CA IX in hypoxia-associated cardiovascular diseases.

A GP1BA Variant in a Czech Family with Monoallelic Bernard-Soulier Syndrome

Bernard-Soulier syndrome (BSS) is a rare inherited disorder characterized by unusually large platelets, low platelet count, and prolonged bleeding time. BSS is usually inherited in an autosomal recessive (AR) mode of inheritance due to a deficiency of the GPIb-IX-V complex also known as the von Willebrand factor (VWF) receptor. We investigated a family with macrothrombocytopenia, a mild bleeding tendency, slightly lowered platelet aggregation tests, and suspected autosomal dominant (AD) inheritance. We have detected a heterozygous GP1BA likely pathogenic variant, causing monoallelic BSS. A germline GP1BA gene variant (NM_000173:c.98G > A:p.C33Y), segregating with the macrothrombocytopenia, was detected by whole-exome sequencing. In silico analysis of the protein structure of the novel GPIbα variant revealed a potential structural defect, which could impact proper protein folding and subsequent binding to VWF. Flow cytometry, immunoblot, and electron microscopy demonstrated further differences between p.C33Y GP1BA carriers and healthy controls. Here, we provide a detailed insight into its clinical presentation and phenotype. Moreover, the here described case first presents an mBSS patient with two previous ischemic strokes.

Prediction of Bioactive Peptides From Chicken Feather and Pig Hair Keratins Using In Silico Analysis Based on Fragmentomic Approach

Background: Keratin is among the most abundant structural proteins of animal origin, however it remains broadly underutilized. Objective: Bioinformatic investigation was performed to evaluate selected keratins originating from mass-produced waste products, i.e., chicken feathers and pig hair, as potential sources of bioactive peptides. Methods: Pepsin, trypsin, chymotrypsin, papain, and subtilisin were used for in silico keratinolysis with the use of “Enzyme(s) action” and fragmentomic analysis of theoretical products was performed using “Profiles of potential biological activity” in BIOPEP-UWM database of bioactive peptides. Bioactivity probability calculation and toxicity prediction of the peptides obtained were estimated using PeptideRanker and ToxinPred tools, respectively. Results: Our results showed that the keratins are a potential source of a variety of biopeptides, including dipeptidyl peptidase IV, angiotensin converting enzyme, prolyl endopeptidase inhibitory and antioxidative. Papain and subtilisin were found to be the most appropriate enzymes for keratin hydrolysis. This study presents possible structures of keratin-derived bioactive peptides that have not been previously described. Conclusion: Our data suggest additional in vitro and in vivo studies to verify theoretical predictions and further investigate the possibility of using keratin-rich waste as a source of peptide nutraceuticals.

Secretome of Stressed Peripheral Blood Mononuclear Cells Alters Transcriptome Signature in Heart, Liver, and Spleen after an Experimental Acute Myocardial Infarction: An In Silico Analysis

Acute myocardial infarction (AMI) is a result of cardiac non-perfusion and leads to cardiomyocyte necrosis, inflammation, and compromised cardiac performance. Here, we showed that the secretome of γ-irradiated peripheral blood mononuclear cells (PBMCsec) improved heart function in a porcine AMI model and displayed beneficial long- and short-term effects. As an AMI is known to strongly affect gene regulation of the ischemia non-affected heart muscle and distal organs, we employed a transcriptomics approach to further study the immediate molecular events orchestrated using the PBMCsec in myocardium, liver, and spleen 24 h post ischemia. In the infarcted area, the PBMCsec mainly induced genes that were essential for cardiomyocyte function and simultaneously downregulated pro-inflammatory genes. Interestingly, genes associated with pro-inflammatory processes were activated in the transition zone, while being downregulated in the remote zone. In the liver, we observed a pronounced inhibition of immune responses using the PBMCsec, while genes involved in urea and tricarboxylic cycles were induced. The spleen displayed elevated lipid metabolism and reduced immunological processes. Together, our study suggested several types of pharmacodynamics by which the PBMCsec conferred immediate cardioprotection. Furthermore, our data supported the assumption that an AMI significantly affects distal organs, suggesting that a holistic treatment of an AMI, as achieved by PBMCsec, might be highly beneficial.

Comprehensive In Silico Analysis and Transcriptional Profiles Highlight the Importance of Mitochondrial Dicarboxylate Carriers (DICs) on Hypoxia Response in Both Arabidopsis thaliana and Eucalyptus grandis

Plant dicarboxylate carriers (DICs) transport a wide range of dicarboxylates across the mitochondrial inner membrane. The Arabidopsis thalianaDIC family is composed of three genes (AtDIC1, 2 and 3), whereas two genes (EgDIC1 and EgDIC2) have been retrieved in Eucalyptus grandis. Here, by combining in silico and in planta analyses, we provide evidence that DICs are partially redundant, important in plant adaptation to environmental stresses and part of a low-oxygen response in both species. AtDIC1 and AtDIC2 are present in most plant species and have very similar gene structure, developmental expression patterns and absolute expression across natural Arabidopsis accessions. In contrast, AtDIC3 seems to be an early genome acquisition found in Brassicaceae and shows relatively low (or no) expression across these accessions. In silico analysis revealed that both AtDICs and EgDICs are highly responsive to stresses, especially to cold and submergence, while their promoters are enriched for stress-responsive transcription factors binding sites. The expression of AtDIC1 and AtDIC2 is highly correlated across natural accessions and in response to stresses, while no correlation was found for AtDIC3. Gene ontology enrichment analysis suggests a role for AtDIC1 and AtDIC2 in response to hypoxia, and for AtDIC3 in phosphate starvation. Accordingly, the investigated genes are induced by submergence stress in A. thaliana and E. grandis while AtDIC2 overexpression improved seedling survival to submergence. Interestingly, the induction of AtDIC1 and AtDIC2 is abrogated in the erfVII mutant that is devoid of plant oxygen sensing, suggesting that these genes are part of a conserved hypoxia response in Arabidopsis.