A Novel Strategy for Selective Thyroid Hormone Determination Based on an Electrochemical Biosensor with Graphene Nanocomposite.

This article presents a novel and selective electrochemical bioassay with antibody and laccase for the determination of free thyroid hormone (free triiodothyronine, fT3). The biosensor was based on a glassy carbon electrode modified with a Fe3O4@graphene nanocomposite with semiconducting properties, an antibody (anti-PDIA3) with high affinity for fT3, and laccase, which was responsible for catalyzing the redox reaction of fT3. The electrode modification procedure was investigated using a cyclic voltammetry technique, based on the response of the peak current after modifications. All characteristic working parameters of the developed biosensor were analyzed using differential pulse voltammetry. Obtained experimental results showed that the biosensor revealed a sensitive response to fT3 in a concentration range of 10-200 µM, a detection limit equal to 27 nM, and a limit of quantification equal to 45.9 nM. Additionally, the constructed biosensor was selective towards fT3, even in the presence of interference substances: ascorbic acid, tyrosine, and levothyroxine, and was applied for the analysis of fT3 in synthetic serum samples with excellent recovery results. The designed biosensor also exhibited good stability and can find application in future medical diagnostics.

Inhibition of Protein-tyrosine Phosphatase PTP1B and LMPTP Promotes Palmitate/Oleate-challenged HepG2 Cell Survival by Reducing Lipoapoptosis, Improving Mitochondrial Dynamics and Mitigating Oxidative and Endoplasmic Reticulum Stress.

OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) is considered a well-known pathology that is determined without using alcohol and has emerged as a growing public health problem. Lipotoxicity is known to promote hepatocyte death, which, in the context of NAFLD, is termed lipoapoptosis. The severity of NAFLD correlates with the degree of hepatocyte lipoapoptosis. Protein-tyrosine phosphatases (PTP) including PTP1B and Low molecular weight PTP (LMPTP), are negative regulators of the insulin signaling pathway and are considered a promising therapeutic target in the treatment of diabetes. In this study, we hypothesized that the inhibition of PTP1B and LMPTP may potentially prevent hepatocyte apoptosis, mitochondrial dysfunction and endoplasmic reticulum (ER) stress onset, following lipotoxicity induced using a free fatty acid (FFA) mixture. METHODS: HepG2 cells were cultured in the presence or absence of two PTP inhibitors, namely MSI-1436 and Compound 23, prior to palmitate/oleate overloading. Apoptosis, ER stress, oxidative stress, and mitochondrial dynamics were then evaluated by either MUSE or RT-qPCR analysis. RESULTS: The obtained data demonstrate that the inhibition of PTP1B and LMPTP prevents apoptosis induced by palmitate and oleate in the HepG2 cell line. Moreover, mitochondrial dynamics were positively improved following inhibition of the enzyme, with concomitant oxidative stress reduction and ER stress abrogation. CONCLUSION: In conclusion, PTP's inhibitory properties may be a promising therapeutic strategy for the treatment of FFA-induced lipotoxicity in the liver and ultimately in the management of the NAFLD condition.

Age-dependent impairment of adipose-derived stem cells isolated from horses.

BACKGROUND: Progressive loss of cell functionality caused by an age-related impairment in cell metabolism concerns not only mature specialized cells but also its progenitors, which significantly reduces their regenerative potential. Adipose-derived stem cells (ASCs) are most commonly used in veterinary medicine as an alternative treatment option in ligaments and cartilage injuries, especially in case of high-value sport horses. Therefore, the main aim of this study was to identify the molecular alternations in ASCs derived from three age-matched horse groups: young (< 5), middle-aged (5-15), and old (> 15 years old). METHODS: ASCs were isolated from three age-matched horse groups using an enzymatic method. Molecular changes were assessed using qRT-PCR, ELISA and western blot methods, flow cytometry-based system, and confocal and scanning electron microscopy. RESULTS: Our findings showed that ASCs derived from the middle-aged and old groups exhibited a typical senescence phenotype, such as increased percentage of G1/G0-arrested cells, binucleation, enhanced ß-galactosidase activity, and accumulation of γH2AX foci, as well as a reduction in cell proliferation. Moreover, aged ASCs were characterized by increased gene expression of pro-inflammatory cytokines and miRNAs (interleukin 8 (IL-8), IL-1ß, tumor necrosis factor α (TNF-α), miR-203b-5p, and miR-16-5p), as well as apoptosis markers (p21, p53, caspase-3, caspase-9). In addition, our study revealed that the protein level of mitofusin 1 (MFN1) markedly decreased with increasing age. Aged ASCs also displayed a reduction in mRNA levels of genes involved in stem cell homeostasis and homing, like TET-3, TET-3 (TET family), and C-X-C chemokine receptor type 4 (CXCR4), as well as protein expression of DNA methyltransferase (DNMT1) and octamer transcription factor 3/4 (Oct 3/4). Furthermore, we observed a higher splicing ratio of XBP1 (X-box binding protein 1) mRNA, indicating elevated inositol-requiring enzyme 1 (IRE-1) activity and, consequently, increased endoplasmic reticulum (ER) stress. We also observed reduced levels of glucose transporter 4 (GLUT-4) and insulin receptor (INSR) which indicated impaired insulin sensitivity. CONCLUSIONS: Obtained data suggest that ASCs derived from horses older than 5 years old exhibited several molecular alternations which markedly limit their regenerative capacity. The results provide valuable information that allows for a better understanding of the molecular events occurring in ASCs in the course of aging and may help to identify new potential drug targets to restore their regenerative potential.

Promotion through external magnetic field of osteogenic differentiation potential in adipose-derived mesenchymal stem cells: Design of polyurethane/poly(lactic) acid sponges doped with iron oxide nanoparticles.

Recently, iron oxide nanoparticles (IONPs) have gathered special attention in regenerative medicine. Owing to their magnetic and bioactive properties, IONPs are utilized in the fabrication of novel biomaterials. Yet, there was no report regarding thermoplastic polyurethane (TPU) and poly(lactic acid) (PLA) polymer doped with IONPs on osteogenic differentiation of mesenchymal stem cells. Thus the objectives of presented study was to: (a) fabricate magnetic TPU + PLA sponges doped with iron (III) oxide Fe2 O3 nanoparticles; (b) investigate the effects of biomaterial and its exposition to static magnetic field (MF) on osteogenic differentiation, proliferation, and apoptosis in adipose-derived mesenchymal stem cells (ASCs). TPU + PLA sponges were prepared using solvent casting technique while incorporation of the Fe2 O3 nanoparticles was performed with solution cast method. RT-PCR was applied to evaluate expression of osteogenic-related genes and integrin's in cells cultured on fabricated materials with or without the stimulation of static MF. MF stimulation enhanced the expression of osteopontin and collagen type I while decreased expression of bone morphogenetic protein 2 in tested magnetic materials-TPU + PLA/1% Fe2 O3 and TPU + PLA/5% Fe2 O3 . Therefore, TPU + PLA sponges doped with IONPs and exposure to MF resulted in improved osteogenic differentiation of ASC.

New diphenylphosphane derivatives of ketoconazole are promising antifungal agents.

Four new derivatives of ketoconazole (Ke) were synthesized: diphenylphosphane (KeP), and phosphane chalcogenides: oxide (KeOP), sulphide (KeSP) and selenide (KeSeP). These compounds proved to be promising antifungal compounds towards Saccharomyces cerevisiae and Candida albicans, especially in synergy with fluconazole. Simulations of docking to the cytochrome P450 14α-demethylase (azoles' primary molecular target) proved that the new Ke derivatives are capable of inhibiting this enzyme by binding to the active site. Cytotoxicity towards hACSs (human adipose-derived stromal cells) of the individual compounds was studied and the IC50 values were higher than the MIC50 for C. albicans and S. cerevisiae. KeP and KeOP increased the level of the p21 gene transcript but did not change the level of p53 gene transcript, a major regulator of apoptosis, and decreased the mitochondrial membrane potential. Taken together, the results advocate that the new ketoconazole derivatives have a similar mechanism of action and block the lanosterol 14α-demethylase and thus inhibit the production of ergosterol in C. albicans membranes.

Inhibition of the Low Molecular Weight Protein Tyrosine Phosphatase (LMPTP) as a Potential Therapeutic Strategy for Hepatic Progenitor Cells Lipotoxicity-Short Communication.

Equine metabolic syndrome (EMS) is a cluster of metabolic disorders, such as obesity, hyperinsulinemia, and hyperleptinemia, as well as insulin resistance (IR). In accordance with the theory linking obesity and IR, excessive accumulation of lipids in insulin-sensitive tissues (lipotoxicity), like liver, alters several cellular functions, including insulin signaling. Therefore, the purpose of the study was to isolate equine hepatic progenitor-like cells (HPCs) and assess whether inhibition of low molecular weight protein tyrosine phosphatase (LMPTP) affects the expression of genes involved in macroautophagy, chaperone-mediated autophagy (CMA), endoplasmic reticulum stress, and mitochondrial dynamics in a palmitate-induced IR model. We demonstrated that LMPTP inhibition significantly enhanced expression of heat shock cognate 70 kDa protein (HSC70), lysosome-associated membrane protein 2 (LAMP2), and parkin (PRKN), all master regulators of selective autophagy. We also observed downregulation of C/EBP homologous protein (CHOP), activating transcription factor 6 (ATF6) and binding immunoglobulin protein encoded by the HSPA gene. Moreover, LMPTP inhibition increased alternative splicing of X-box binding protein 1 (XBP1), suggesting high endonuclease activity of inositol-requiring enzyme 1 alpha (IRE1α). Taken together, our data provide convincing evidence that LMPTP inhibition reverses palmitate-induced insulin resistance and lipotoxicity. In conclusion, this study highlights the role of LMPTP in the regulation of CMA, mitophagy, and ER stress, and provides a new in vitro model for studying HPC lipotoxicity in pre-clinical research.

Adipose-Derived Mesenchymal Stem Cells Isolated from Patients with Type 2 Diabetes Show Reduced "Stemness" through an Altered Secretome Profile, Impaired Anti-Oxidative Protection, and Mitochondrial Dynamics Deterioration.

The widespread epidemic of obesity and type 2 diabetes (T2D), suggests that both disorders are closely linked. Several pre-clinical and clinical studies have showed that adipose-derived mesenchymal stem cells (ASC) transplantation is efficient and safe. Moreover, scientists have already highlighted the therapeutic capacity of their secretomes. In this study, we used quantitative PCR, a flow cytometry-based system, the ELISA method, spectrophotometry, and confocal and scanning electron microscopy, to compare the differences in proliferation activity, viability, morphology, mitochondrial dynamics, mRNA and miRNA expression, as well as the secretory activity of ASCs derived from two donor groups-non-diabetic and T2D patients. We demonstrated that ASCs from T2D patients showed a reduced viability and a proliferative potential. Moreover, they exhibited mitochondrial dysfunction and senescence phenotype, due to excessive oxidative stress. Significant differences were observed in the expressions of miRNA involved in cell proliferations (miR-16-5p, miR-146a-5p, and miR-145-5p), as well as miRNA and genes responsible for glucose homeostasis and insulin sensitivity (miR-24-3p, 140-3p, miR-17-5p, SIRT1, HIF-1α, LIN28, FOXO1, and TGFß). We have observed a similar correlation of miR-16-5p, miR-146a-5p, miR-24-3p, 140-3p, miR-17-5p, and miR-145-5p expression in extracellular vesicles fraction. Furthermore, we have shown that ASCT2D exhibited a lower VEGF, adiponectin, and CXCL-12 secretion, but showed an overproduction of leptin. We have shown that type 2 diabetes attenuated crucial functions of ASC, like proliferation, viability, and secretory activity, which highly reduced their therapeutic efficiency.

Identification of a potent inhibitor of type II secretion system from Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen infecting human population. The pathogen is becoming a serious health problem due to its ability to evade normal immune response of the host and multiple drug resistance to many antibiotics. The pathogen has 2 major virulence systems of which the type III secretion system (T3SS) is of major concern to humans. A third system, type 2 secretion system (T2SS), is common to bacteria and used to secrete exotoxin A (ExoA) responsible for human cell destruction. To help bypass the drug resistance, a strategy to block the T2SS based on a low similarity between human ATPases and the essential ATPases of the T3SS and T2SS of P. aeruginosa, was used. An in silico-optimized inhibitor of T3SS, made directly from the computer-optimized of previously published compounds and their combinatorial libraries, showed IC50 = 1.3 ±â€¯0.2 µM in the T2SS ExoA secretion blocking test. The compound was non-toxic to human lung epithelial cell line A549 and could block cellular destruction of those cells in a cell infection model at 200 µM for at least 24 h. The compound could be a lead candidate for the development of T2SS virulence blockers of Pseudomonas aeruginosa.

The Effect of Chronic Inflammation and Oxidative and Endoplasmic Reticulum Stress in the Course of Metabolic Syndrome and Its Therapy.

Metabolic syndrome (MetS) is highly associated with a modern lifestyle. The prevalence of MetS has reached epidemic proportion and is still rising. The main cause of MetS and finally type 2 diabetes occurrence is excessive nutrient intake, lack of physical activity, and inflammatory cytokines secretion. These factors lead to redistribution of body fat and oxidative and endoplasmic reticulum (ER) stress occurrence, resulting in insulin resistance, increase adipocyte differentiation, and much elevated levels of proinflammatory cytokines. Cellular therapies, especially mesenchymal stem cell (MSC) transplantation, seem to be promising in the MetS and type 2 diabetes treatments, due to their immunomodulatory effect and multipotent capacity; adipose-derived stem cells (ASCs) play a crucial role in MSC-based cellular therapies. In this review, we focused on etiopathology of MetS, especially on the crosstalk between chronic inflammation, oxidative stress, and ER stress and their effect on MetS-related disease occurrence, as well as future perspectives of cellular therapies. We also provide an overview of therapeutic approaches that target endoplasmic reticulum and oxidative stress.