Resazurin Research Articles

Synthesis, Biological Evaluation, Molecular Docking Studies and ADMET Prediction of Oxindole-Based Hybrids for the Treatment of Tuberculosis.

With a projected mortality toll of 1.4 million in 2019, tuberculosis (TB) continues to be a significant public health concern around the world. Studies of novel treatments are required due to decreased bioavailability, increased toxicity, increased side effects, and resistance of several first- and second-line TB therapies, including isoniazid and ethionamide. This study reports the synthesis of oxindole-based hybrids as potent InhA inhibitors targeting Mycobacterium tuberculosis. The synthesized compounds (5a-5e and 8a-8c) were evaluated for their anti-mycobacterial activity against Mycobacterium tuberculosis and nontuberculous mycobacteria (NTMs), viz. M. abscessus (ATCC 19977), M. fortuitum (ATCC 6841), and M. chelonae (ATCC 35752) using the Microplate Alamar Blue Assay (MABA). Molecular docking studies were performed using AutoDock Vina to explore the binding interactions of these compounds with the InhA enzyme (PDB: 2NSD). Additionally, biochemical and histopathological studies were conducted to assess the hepatotoxicity of the lead compounds. Insilico molecular properties and ADMET properties of the synthesized compounds were predicted using SwissADME and Deep-PK online tools to assess their drug-likeness. Among the tested compounds, 8b exhibited significant anti-mycobacterial activity with a minimum inhibitory concentration (MIC = 1 μg/mL) comparable to the reference drug ethambutol. Further, the compound demonstrated a binding affinity and orientation similar to the reference inhibitor 4PI, indicating its potential as a potent InhA inhibitor, and was found to be stabilized within the binding pocket of InhA through H-bonding, hydrophobic and van der Waal's interactions. Besides, the compounds hepatotoxicity assessment studies depicted that 8b showed no significant liver dysfunction or damage to liver tissues. Additionally, 8b adhered to Lipinski's rule of five and Veber's rule, displaying favourable pharmacokinetic and drug-like properties, including high human intestinal absorption, distribution, and acceptable metabolic stability and excretion. Compound 8b emerged as a promising candidate for further optimization and development as a therapeutic agent for tuberculosis, offering a new avenue for tackling tuberculosis.

Evaluation of Selected Plant Phenolics via Beta-Secretase-1 Inhibition, Molecular Docking, and Gene Expression Related to Alzheimer’s Disease

Background: The goal of the current study was to investigate the inhibitory activity of six phenolic compounds, i.e., rosmarinic acid, gallic acid, oleuropein, epigallocatechin gallate (EGCG), 3-hydroxytyrosol, and quercetin, against β-site amyloid precursor protein cleaving enzyme-1 (BACE1), also known as β-secretase or memapsin 2, which is implicated in the pathogenesis of Alzheimer’s disease (AD). Methods and Results: The inhibitory potential against BACE1, molecular docking simulations, as well as neurotoxicity and the effect on the AD-related gene expression of the selected phenolics were tested. BACE1 inhibitory activity was carried out using the ELISA microplate assay via fluorescence resonance energy transfer (FRET) technology. Molecular docking experiments were performed in the human BACE1 active site (PDB code: 2WJO). Neurotoxicity of the compounds was carried out in SH-SY5Y, a human neuroblastoma cell line, by the Alamar Blue method. A gene expression analysis of the compounds on fourteen genes linked to AD was conducted using the real-time polymerase chain reaction (RT-PCR) method. Rosmarinic acid, EGCG, oleuropein, and quercetin (also used as the reference) were able to inhibit BACE1 with their respective IC50 values 4.06 ± 0.68, 1.62 ± 0.12, 9.87 ± 1.01, and 3.16 ± 0.30 mM. The inhibitory compounds were observed to occupy the non-catalytic site of the BACE1. However, hydrogen bonds were found to be present between rosmarinic acid and EGCG and aspartic amino acid D228 in the catalytic site. Oleuropein and quercetin effectively suppressed the expression of PSEN, APOE, and CLU, which are recognized to be linked to the pathogenesis of AD. Conclusions: The outcomes of the work bring quercetin, EGCG, and rosmarinic acid to the forefront as promising BACE1 inhibitors.

Rhizomatoflavonoid D and Other Flavonoids from the Twigs of Ochna Rhizomatosa as a Potential Inhibitor of HIV-1

AbstractCurrently, HIV morbidity and mortality in sub-Saharan Africa remain a huge concern and awaiting interventions. Even though the combination antiretroviral therapy (cART) has recorded significant success, drug resistance and limited access to available therapeutics are major factors responsible for the low impact of cART in several African communities. Herein, as part of our continuous effort on the investigation of bioactive metabolites of Ochna rhizomatosa, we report the isolation of a new flavonoid; Rhizomatoflavonoid D (1), alongside with four known ones (2–5). The structures of these compounds were elucidated by using spectroscopic techniques (1H NMR, 13C NMR, HSQC, HMBC, 1H-1H COSY, and ROESY) and mass spectrometry. The antiviral activity of the resulting compounds was assessed using deCIPhR assay run in parallel with the Alamar Blue based cytotoxicity assay. This assay revealed a moderate activity for compound 4 (72% inhibition at 2.5 µg/mL) while compound 1 had minimal activity (36% inhibition at 2.5 µg/mL). The prominent inhibitory effect on HIV-1 was showed by compound 4 (IC50 = 3.1 µM). Unfortunately, compound 4 proved to be non-selective as it demonstrated also a CC50 = 5.2 µg/mL (Selectivity index of 1.7). The prominent inhibitory effect on HIV-1 showed by compound 4 (IC50 = 3.1 µM) could be due the presence of a methoxy group at C-7, since this group enhances the lipophilicity of biflavonoids, thereby improving its incorporation into cells.

Fabrication of drug loaded polylactic acid/keratin/polyethylene glycol GTR membrane for periodontal application

This study proposes the fabrication of, amoxicillin-loaded composite GTR membranes from polylactic acid (PLA), wool keratin (WK), and polyethylene glycol (PEG) for periodontal therapy. Deep eutectic solvents were employed for the extraction of keratin from wool fibers. For the preparation of the control (C), PLA and PEG were dissolved in a ratio of 60:40 in chloroform. While the experimental groups K2 and K3 were prepared by adding, 2% and 3% WK powder, respectively, to the control mixture. To prepare the experimental groups K2-D and K3-D, 1% AMOX-treated WK powder was mixed with the previously mentioned groups. The prepared GTR membrane was characterized using various analytical techniques. The treatment of sheep wool and the presence of AMOX in the experimental groups were confirmed using FTIR analysis. The Scanning electron microscope (SEM) images of the prepared GTR membranes showed voids and presented a conglomerated appearance. The K2 had the highest tensile strength, whereas K2-D and K3-D which contained drugs had lower tensile strengths but significant antibacterial activity. K2 experienced the greatest mass change after 1 h, while K2-D and K3-D experienced the least. Each membrane was found to be hydrophilic. The Alamar Blue Assay revealed good biocompatibility.

Phytochemical Profile and Antioxidant and Protective Activities of Various Types of Extracts from Hyssopus officinalis L. and Grindelia robusta Nutt. Herb Grown in Poland.

The available literature indicates that Hyssopus officinalis and Grindelia robusta are raw materials with great potential for use in prevention and therapy. Therefore, the aims of this study were to assess the phytochemical profile and antioxidant and cytoprotective properties of extracts prepared using various solvents, additionally taking into account different methods of drying the plant material. Hydrodistilled oil was analysed by GC-MS. The chemical composition of the extracts was estimated by spectrophotometry and the HPLC-DAD method. Antioxidant activity was evaluated using DPPH and FRAP and measuring the intracellular level of ROS. Alamar Blue and Neutral Red tests were used to assess the cytotoxicity of the extracts on skin cells - keratinocytes and fibroblasts. The major components of hyssop essential oil were cis- (44.9%) and trans- (18.2%) pinocamphone, while borneol (16.1%), and α-pinene (12.0%) were predominant in grindelia essential oil. Flavonoids were dominant in the extracts (water:ethanol, water:methanol, and water: glycerol) from hot-air dried hyssop herb, while phenolic acids were the predominant compounds in the grindelia herb extracts. The water:ethanol hyssop extract had the highest total content of flavonoids (42.26 mg CE/mL), among which isoquercitrin and rutin were present in the highest quantities (32.61 mg/mL and 21.47 mg/mL, respectively). In the case of grindelia, the highest total phenolic acid content (26.24 mg CAE/mL) was recorded in the water:ethanol extract, and the dominant compounds among them were 1,5-dicaffeoylquinic and chlorogenic acid (10.85 and 6.39 mg/mL, respectively). The water:ethanol extract from both plants also exhibited the highest antioxidant activity in the DPPH and FRAP tests (79.19% and 1.39 mmol/L, respectively, for grindelia and 67.61% and 1.04 mmol/L for hyssop) and was most effective at reducing the level of ROS in cells. In addition, water:ethanol extracts may have a positive impact on the viability of skin cells in vitro. Water:ethanol extracts from H. officinalis and G. robusta herb are promising sources of active compounds and may find application as natural materials with valuable biological properties, which require further in vitro and in vivo testing.

Quinic acid contributes to neurogenesis: Targeting Notch pathway a key player in hippocampus

Coordinated proliferation and differentiation of neural stem cells (NSCs) results in continuous neurogenesis. The present study provides novel insights into the Notch intracellular signaling in neuronal cell proliferation, maintenance, migration, and differentiation regulated by naturally based Quinic acid (QA) in primary hippocampal cell culture. Further, this study might help in the discovery and development of lead molecules that can overcome the challenges in the treatment of neurodegenerative diseases. The growth supporting effect of QA was studied using Alamar Blue assay. The migratory potential of QA was evaluated using scratch assay. The in vitro H2O2-induced oxidative stress model was used to upregulate neuronal survival after QA treatment. The RT-qPCR and immunocytochemical analysis were performed for selected markers of Notch signaling to determine the proliferation, differentiation, and maintenance of NSCs at gene and molecular levels. The Mash1 and Ngn2 are the upstream proneural genes of the Notch pathway which were included to evaluate the differentiation of NSCs into mature neurons after treatment with QA.Furthermore, regarding the role of QA in maintaining the pool of NPCs, we used Notch1 and Hes1 markers for proliferation analysis. Also, secondary neuronal markers i.e. Pax6, PCNA, and Mcm2 were included in this study and their gene expression analysis was analyzed following treatment with QA. Based on the study’s results, we suggest that naturally based QA can promote the growth and differentiation of neonatal NSCs residing in hippocampal regions into neuronal lineage. Therefore, we propose that the neurogenic potential of QA can be employed to prevent and treat neurodegenerative diseases.

Abstract C035: MAGEA6 Oncogene Upregulates MAPK and AMPK Signaling Pathways in Pancreatic Cancer Cells to Promote Survival under Nutrient Stress: Resistance to Glycolysis Inhibition and Increased Susceptibility to Glutamine Depletion

Abstract Introduction: Melanoma-associated antigens (MAGEs) are linked to aggressive disease progression and treatment resistance in many cancers, but their role in pancreatic cancer is not well understood. Our preliminary TCGA data analysis suggested that 10% of pancreatic tumors express MAGEA3/6 and potentially help pancreatic cancer cells survive and grow under nutrient stress. This study aims to uncover the molecular mechanisms of MAGEA3/6 function in pancreatic cancer cell metabolism and disease progression under nutrient-poor conditions. Methods: To understand the role of highly homologous genes MAGEA3 and MAGEA6 in pancreatic cancer, we expressed them in highly glycolytic MIA PaCa-2 pancreatic cancer cells. Cells were then exposed to cancer-related nutritional stressors, including glycolysis inhibition by 2-deoxy-D-glucose (2DG) treatment and glutamine depletion. We analyzed the effect of MAGEA6 expression on cell viability and cell metabolism by Alamar Blue viability assay (BioRad), Seahorse (Agilent), Phenotype MicroArray Mammalian analysis (Biolog), transmission electron microscopy (TEM), and RNA sequencing. To confirm our results, we performed loss-of- function studies in MAGEA3/6-positive cells, including PANC1. Results: We found that, compared to control GFP-expressing cells, MAGEA6-expressing MIA PaCa-2 cells were resistant to glycolysis inhibition with 2-DG. In contrast, they were more susceptible to glutamine depletion, suggesting the important role of MAGEA3/6 in metabolic phenotype and adaptations of pancreatic cancer cells. Further, we showed that MAGEA6-expressing cells exhibited increased ATP production and mitochondrial oxidative phosphorylation, especially under 2DG stress. After 48 hours of 2DG treatment, TEM images revealed significant differences in nuclear, mitochondrial, and ribosomal structures in MAGEA6-cells, further suggesting MAGEA6's role in metabolic adaptation. The Phenotype MicroArray assay demonstrated that α-D-Glucose is the primary nutrient source consumed by both GFP and MAGEA6-expressing cells among the 93 nutrients tested. Additionally, A6 cells utilize Pyruvic Acid as an alternative nutrient source more than GFP cells. To uncover molecular underpinning, we performed RNA sequencing and found that MAGEA6 expression affects transcriptome under glutamine depletion and 2DG treatment. Intriguingly, one of the major differentially regulated biological processes was associated with the upregulation of several signaling pathways, including MAPK and AMPK. Conclusion: Our findings suggest that in pancreatic cancer, MAGEA6 recruits stress and growth factor- dependent MAPK and AMPK pathways to overcome glycolysis inhibition, even in glutamine- depleted conditions, providing a growth advantage. This implies that patients with MAGEA6- positive tumors may benefit from combination therapy targeting metabolism and signaling pathways. Citation Format: Sima Tozandehjani, Barbara Breznik, Klementina Fon Tacer, Miloš Vittori, Juan Sebastian Solano, Sara Uhan. MAGEA6 Oncogene Upregulates MAPK and AMPK Signaling Pathways in Pancreatic Cancer Cells to Promote Survival under Nutrient Stress: Resistance to Glycolysis Inhibition and Increased Susceptibility to Glutamine Depletion [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C035.

Halogenated chalcones against Mycobacterium tuberculosis targeting InhA: Rational design, in silico and in vitro evaluation

A library of 25-series compounds was designed against Mycobacterium Tuberculosis (M.tb) to identify novel antitubercular drugs. In silico inhibition of InhA, an essential component of FAS-II, was successfully achieved. The drug ability, lead-likeness, and toxicity of the compounds were assessed using Swiss ADME, pkCSM, and Osiris Property Explorer, which revealed the potential for drug development of chalcone compounds. Through in silico research, it was confirmed that toxic-free compounds could bind to InhA. It was found that all of the compounds bind to InhA with binding affinities ranging from −7.78 to −10.29 kcal/mol−1 which is higher than the reference standard Isoniazid and Pyrazinamide. The top five compounds were synthesized from 15 toxic-free compounds. The structural characteristics of the compounds were determined using IR, NMR, and mass spectrometry techniques. These findings indicate that these substances are competitive, reversible, and specific InhA inhibitors of InhA. using the Alamar Blue assay method (H37RV, ATCC No. 27294), the in vitro anti-mycobacterial activity of each of the synthesized compounds against M.tb was evaluated. The two most powerful compounds were (2E)-3-[4-(benzyloxy)-3,5-dimethylphenyl] and (2E)-1-(3,5-dibromophenyl)-3-(3-phenoxyphenyl) prop-2-en-1-one. In the MABA Assay, the MIC for 1-(3,5-dibromophenyl) prop-2-en-1-one was 6.25 μg/ml.

Targeted Gene Delivery to Muscle Cells In Vitro and In Vivo Using Electrostatically Stabilized DNA—Peptide Complexes

Genetic constructs must be delivered selectively to target tissues and intracellular compartments at the necessary concentrations in order to achieve the maximum therapeutic effect in gene therapy. Development of targeted carriers for non-viral delivery of nucleic acids into cells, including those in muscle, which is one of the most challenging tissues to transfect in vivo, remains a topical issue. We have studied ternary complexes of plasmid DNA and an arginine–histidine-rich peptide-based carrier coated with a glutamate–histidine-rich polymer bearing skeletal muscle targeting peptide (SMTP) for the gene delivery to muscle tissue. The relaxation of the ternary complexes after polyanion treatment was assessed using the ethidium bromide displacement assay. The developed polyplexes were used to transfect C2C12 myoblasts in full-media conditions, followed by analysis of their toxic properties using the Alamar Blue assay and expression analysis of lacZ and GFP reporter genes. After delivering plasmids containing the GFP and lacZ genes into the femoral muscles of mdx mice, which are model of Duchenne muscular dystrophy, GFP fluorescence and β-galactosidase activity were detected. We observed that the modification of ternary polyplexes with 10 mol% of SMTP ligand resulted in a 2.3-fold increase in lacZ gene expression when compared to unmodified control polyplexes in vivo. Thus, we have demonstrated that the developed DNA/carrier complexes and SMTP-modified coating are nontoxic, are stable against polyanion-induced relaxation, and can provide targeted gene delivery to muscle cells and tissues. The results of this study are useful for a range of therapeutic applications, from immunization to amelioration of inherited neuromuscular diseases.

Synthesis, mechanical characterization, and biocompatibility evaluation of Graphene-TiO2 reinforced alumina for biomedical applications

The study addresses the limited fracture toughness of Al2O3 ceramics by developing graphene-TiO2 reinforced alumina nanocomposites through pressing. Mechanical properties, including hardness, fracture toughness, and elasticity, were examined, alongside the structural characterization via XRD analysis and microstructure evaluation using SEM. Cytotoxicity and cell adhesion tests were conducted for in vitro biological evaluation. The inclusion of a graphene additive led to a nearly threefold increase in fracture toughness, reaching up to 8.161 MPa m1/2. Graphene doping induced a discernible shift to lower angles in the XRD pattern, revealing the presence of Al2O3, Al2TiO5, and TiO2 phases. Samples immersed in artificial body fluid for 14 days exhibited minimal ion release, with Titanium ion below 0.001 mg/L and aluminum ion below 0.05 mg/L, indicating a lack of significant ion release. The cytotoxic activity of the samples against NIH/3T3 cells was assessed through Alamar Blue analysis, revealing no significant change in cell viability after 48 h of exposure.

Comparative fatigue performance of as-built vs etched Ti64 in TPMS-gyroid and stochastic structures fabricated via PBF-LB for biomedical applications

PurposeThis study compares the fatigue performance and biocompatibility of as-built and chemically etched Ti-6Al-4V alloys in TPMS-gyroid and stochastic structures fabricated via Powder Bed Fusion Laser Beam (PBF-LB). This study aims to understand how complex lattice structures and post-manufacturing treatment, particularly chemical etching, affect the mechanical properties, surface morphology, fatigue resistance and biocompatibility of these metamaterials for biomedical applications.Design/methodology/approachSelective Laser Melting (SLM) technology was used to fabricate TPMS-gyroid and Voronoi stochastic designs with three different relative densities (0.2, 0.3 and 0.4) in Ti-6Al-4V ELI alloy. The as-built samples underwent a chemical etching process to enhance surface quality. Mechanical characterization included static compression and dynamic fatigue testing, complemented by scanning electron microscopy (SEM) for surface and failure analysis. The biocompatibility of the samples was assessed through in-vitro cell viability assays using the Alamar Blue assay and cell proliferation studies.FindingsChemical etching significantly improves the surface morphology, mechanical properties and fatigue resistance of both TPMS-gyroid and stochastic structures. Gyroid structures demonstrated superior mechanical performance and fatigue resistance compared to stochastic structures, with etching providing more pronounced benefits in these aspects. In-vitro biocompatibility tests showed high cytocompatibility for both as-built and etched samples, with etched samples exhibiting notably improved cell viability. The study also highlights the importance of design and post-processing in optimizing the performance of Ti64 components for biomedical applications.Originality/valueThe comparative analysis between as-built and etched conditions, alongside considering different lattice designs, provides valuable information for developing advanced biomedical implants. The demonstration of enhanced fatigue resistance and biocompatibility through etching adds significant value to the field of additive manufacturing, suggesting new avenues for designing and post-processing implantable devices.

Zinc-doped phosphate coatings for enhanced corrosion resistance, antibacterial properties, and biocompatibility of AZ91D Mg alloy

Magnesium and its alloys have been foreseen as promising bone-implant owing to good biocompatibility, high strength, mechanical properties, and biodegradability to replace the conventional bio-metals (Titanium, stainless-steel, Co-Cr alloys) in orthopedic applications. However, high corrosion rate and high degradation rates adversely effects like abrupt evolution of H2 gas and localized alkalization in a physiological environment limit its medical applications. To overcome these issues, phosphate-based surface coating is developed on Mg-alloy to resist the high biodegradation and corrosion rate utilizing magnesium substrate as source of magnesium ion through a single-step hydrothermal process. Controlling the fast corrosion rate and localized alkalization would reduce the antibacterial character of magnesium-based implants therefore, the simple phosphate-based coatings has been induced by doping it with zinc ions due to its physiological tolerance and excellent antibacterial efficacy. The doping of zinc ions may provide a sustained drug-free antibacterial characteristic for potential application in orthopedics. The synthesized phosphate-based coating was characterized using advanced techniques like Scanning Electron Microscopy, X-ray Diffraction, and wettability test, followed by comprehensive electrochemical testing to assess its corrosion behavior. The in vitro immersion test in simulated body fluid (SBF) indicates that deposition of phosphate and zinc doped phosphate coatings reduces the degradation rate consequently minimized the fast dissolution of Mg2+ ions and OH- in physiological environment. Additionally, cytotoxicity and biocompatibility were evaluated using Alamar Blue and live/dead assays on the MC3T3-E1 mouse osteoblast cell line. These assays demonstrated good cell viability, indicating the coatings possess favorable cytocompatibility and support cellular metabolic activity.

Unveiling the antitumor mechanism of 7α-acetoxy-6β-hydroxyroyleanone from Plectranthus hadiensis in glioblastoma

Ethnopharmacological relevanceGlioblastoma (GB) is the most aggressive and prevalent glioma within the central nervous system. Despite considerable efforts, GB continues to exhibit a dismal 5-year survival rate (∼6%). This is largely attributed to unfavorable prognosis and lack of viable treatment options. Therefore, novel therapies centered around plant-derived compounds emerge as a compelling avenue to enhance patient survival and well-being. The South African species, Plectranthus hadiensis Schweinf. (P. hadiensis), a member of the Lamiaceae family, has a history of use in traditional medicine for treating a range of diseases, including respiratory, digestive, and liver disorders. This species exhibits diverse biological activities, such as anti-inflammatory and antitumoral properties, likely attributed to its rich composition of naturally occurring diterpenes, like the abietane diterpene, 7α-acetoxy-6β-hydroxyroyleanone (Roy). Roy has demonstrated promising antitumor effects in various cancer cell lines, making it a compelling candidate for further investigation into its mechanisms against GB. Aim of the studyThis study aims to investigate the antitumor activity and potential mechanism of Roy, a natural lead compound, in GB cells. Material and methodsRoy was isolated from the acetonic extract of P. hadiensis and its antitumor mechanism was assessed in a panel of human GB cell lines (U87, A172, H4, U373, and U118) to mimic tumor heterogeneity. Briefly, the impact of Roy treatment on the metabolic activity of cells was evaluated by Alamar Blue® assay, while cell death, cell cycle regulation, mitochondrial membrane potential, and activated caspase-3 activity were evaluated by flow cytometry. Measurement of mRNA levels of target genes was performed by qPCR, while protein expression was assessed by Western blotting. Cell uptake and impact on mitochondrial morphology were evaluated by confocal microscopy. ResultsRoy induced G2/M cell cycle arrest, mitochondrial fragmentation, and apoptosis by inhibiting the expression of anti-apoptotic proteins and increasing the levels of activated caspase-3. The concentrations of Roy needed to achieve significant inhibitory outcomes were notably lower (6–9 fold) than those of temozolomide (TMZ), the standard first-line treatment, for achieving comparable effects. In addition, at low concentrations (16 μM), Roy affected the metabolic activity of tumor cells while having no significant impact on non-tumoral cells (microglia and astrocytes). ConclusionOverall, Roy demonstrated a robust antitumor activity against GB cells offering a promising avenue for the development of novel chemotherapeutic approaches.

The Proangiogenic Effects of Melanoma-Derived Ectosomes Are Mediated by αvβ5 Integrin Rather than αvβ3 Integrin.

Ectosomes are carriers of proangiogenic factors during cancer progression. This study investigated whether the proangiogenic effect exerted by melanoma-derived ectosomes on recipient endothelial cells is mediated by ectosomal αvβ3 and αvβ5 integrins. Ectosomes were isolated from the conditioned culture media of four melanoma cell lines and melanocytes. Changes in gene and protein expression of αvβ3 and αvβ5 integrins, as well as VEGF and TNF-α were assessed in ectosome-treated endothelial cells. To confirm the functional involvement of ectosomal integrins in functional tests (Alamar Blue, wound healing and tube formation assays), ectosomes were also pretreated with anti-integrin antibodies and integrin-blocking peptides echistatin and cilengitide. Melanoma-derived ectosomes induced changes in the expression of αvβ3 and αvβ5 integrins in recipient endothelial cells, leading to increased viability, migratory properties, and tube formation potential. The extent of proangiogenic stimulation varied depending on the types of cells releasing ectosomes and the recipient cells. The use of anti-integrin antibodies and integrin-blocking peptides revealed a more significant role for the αvβ5 integrin/VEGF than the αvβ3 integrin/TNF-α pathway in the interactions between ectosomes and endothelial cells. The study demonstrated the functional role of ectosomal αvβ3 and αvβ5 integrins. It also provided a baseline understanding of ectosome-mediated αvβ3 integrin/TNF-α and αvβ5 integrin/VEGF signaling in angiogenesis.

Mechanical properties and biocompatibility of multilayer systems based on amorphous SiN:H/SiCN:H layers on Ti6Al7Nb titanium alloy

Surfaces of metallic implants that replace diseased joints are bound to degrade over time under constant mechanical wear and corrosion, caused by the presence of biological fluids. Production of wear particles and toxic metal ions that follows can undermine both implant stability and patient’s health. To counteract this process multiple surface modifications have been proposed in scientific literature, ranging from simple nitriding to protective layers (e.g.: DLC, CN:H) and multilayer systems. This work presents a combined approach to outlined issue, using multi-staged plasma deposition with SiCN:H layer as a key component, realized using the MW CVD (Microwave Chemical Vapour Deposition) system. The study covers chemical characterization by EDS and FTIR analysis and measurements of relevant functional parameters, such as hardness, Young’s modulus, coefficient of friction, specific wear rate and wettability. Additionally, biocompatibility of modified surfaces are tested on MG-63 cells using Alamar Blue assay, flow cytometry and alkaline phosphatase (ALP) assay. Chemical and microscopic studies revealed amorphous cauliflower-like microstructures that have proven to be hydrogenated SiCN/SiN structures. The obtained layers are turned out to be biocompatible (>70 % of cell survival rate), additionally layers based on amorphous SiCN:H boost ALP activity due to combination of their chemistry and mechanical properties.

74 Unraveling the Complexities of Obesity in Clear Cell Renal Cell Carcinoma Carcinogenesis

Abstract Background Obesity has been identified as an established risk factor in the development of clear cell renal cell carcinoma (ccRCC). ccRCC arises from the proximal convoluted tubule (PCT) of the kidney which is also the site of obesity-associated chronic kidney disease. Obesity can lead to chronic cellular insults which may lead to DNA injury and whether these mechanisms are critical to cancer initiation in the kidney is unclear. One potential way that obesity can lead to cancer is by altering lipid metabolism, including increasing circulating saturated fatty acids. In particular, palmitic acid is known to be lipotoxic to proximal tubules of the kidney. Understanding how free fatty acids initiate cancer is critical to understanding the role of obesity in cancer initiation and development. We hypothesize that chronic injury and repair due to excess FFA exposure could lead to ccRCC carcinogenesis and aim to define the metabolic phenotype of obesity associated ccRCC. Methods Using publicly available data from The KIRC Cancer Genome Atlas Program (TCGA) and clinicopathologic data, we compared the RNAseq profiles of obese (BMI >= 30) to normal weight (BMI =< 25) sample origins. Of the 337, a total of 333 had BMI available for analysis. We evaluated differences in Hallmark and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Using DESeq, we adjusted for age and sex. In vitro, HK PCT cells were cultured in DMEM+10%FBS treated with bovine serum albumin (BSA)-palmitic acid complex for 48 hrs. We measured cell viability using Alamar Blue. Results were normalized to controls with BSA only. We extracted protein and performed western blots to identify DNA damage (pATM, H2AX) and endoplasmic reticulum stress (ATF4, eFI2alpha). Results Most of the patients in our clinical data cohort are male (64.4%), while 45.4% of patients are categorized by BMI as obese and 33.2% are categorized as normal. We utilized 333 individuals from the Kyoto Encyclopedia of Genes and Genomes (KEGG) to link genomic information to pathways associated with fatty acid metabolism as well as other cancer-associated hallmark metabolic pathways. We confirmed that excess free fatty acid supplementation leads to decreased cell viability in renal proximal tubule cells and renal embryonal cells and aim to assess differences in gene expression and metabolomic variation with fatty acid supplementation. Conclusions Through the investigation of these questions, we hope to improve understanding of obesity associated ccRCC and ultimately impact oncological outcomes for patients with this cancer. Understanding changes in lipid metabolism around ccRCC initiation will help identify novel pathways for pharmacologic intervention at earlier stages of the disease. DOD CDMRP Funding: yes

The Repurposing of FDA-Approved Drugs as FtsZ Inhibitors against Mycobacterium tuberculosis: An In Silico and In Vitro Study.

Using an in silico pharmacological repositioning strategy, four FDA-based drugs that bind to the catalytic site FtsZ were selected. The Alamar Blue colorimetric assay was used to assess antimicrobial activity and the effect of drugs on Mtb growth through growth curves. Bacterial load was determined with an in vitro infection model using colony-forming units (CFU)/mL, and cytotoxicity on human monocyte-derived macrophages (MDMhs) was assessed by flow cytometry. Paroxetine and nebivolol exhibited antimycobacterial activity against both reference TB and MDR strains at a concentration of 25 µg/mL. Furthermore, both paroxetine and nebivolol demonstrated a significant reduction (p < 0.05) in viable bacteria compared to the untreated group in the in vitro infection model. Collectively, our findings demonstrate that the use of paroxetine and nebivolol is a promising strategy to help in the control of tuberculosis infection.

The protective effect of parthenolide in an in vitro model of Parkinson's disease through its regulation of nuclear factor-kappa B and oxidative stress.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor and non-motor symptoms, and is due to the degeneration of dopaminergic neurons. It is multifactorial, caused by genetic and environmental factors and currently has no definitive cure. We have investigated the protective effects of parthenolide (PTN), a compound with known anti-inflammatory and antioxidant properties, in an in vitro model of PD, that is induced by 6-OHDA, and that causes neurotoxicity in SH-SY5Y human neuroblastoma cells. SH-SY5Y cells were pretreated with PTN to assess its protective effects in 6-OHDA-induced cellular damage. Cell viability was measured using Alamar blue. Apoptosis was evaluated using an Annexin V-FITC/PI kit. Reactive oxygen species (ROS) levels were quantified, and expression levels of apoptotic markers (Bax, Bcl-2, p53) and NF-κB were analyzed via Western blotting and Quantitative real-time- (qRT-) PCR. We found that 6-OHDA reduced cell viability, that was inhibited significantly by pre-treatment with PTN (p < 0.05). Flow cytometry revealed that PTN reduced apoptosis induced by 6-OHDA. PTN also reduced the ROS levels raised by 6-OHDA (p < 0.05). Moreover, PTN decreased the expression of Bax, p53, NF-κB, and p-NF-κB that were increased by treatment with 6-OHDA. These findings indicate the potential beneficial effects of PTN in an in vitro model of PD via mitigating oxidative stress and inflammation, suggested PTN as a promising agent to be used for PD therapy, warranting further investigation in preclinical and clinical studies.

Venturicidin A affects the mitochondrial membrane potential and induces kDNA loss in Trypanosoma brucei.

Neglected tropical diseases caused by trypanosomatid parasites have devastating health and economic consequences, especially in tropical areas. New drugs or new combination therapies to fight these parasites are urgently needed. Venturicidin A, a macrolide extracted from Streptomyces, inhibits the ATP synthase complex of fungi and bacteria. However, its effect on trypanosomatids is not fully understood. In this study, we tested venturicidin A on a panel of trypanosomatid parasites using Alamar Blue assays and found it to be highly active against Trypanosoma brucei and Leishmania donovani, but much less so against Trypanosoma evansi. Using fluorescence microscopy, we observed a rapid loss of the mitochondrial membrane potential in T. brucei bloodstream forms upon venturicidin A treatment. Additionally, we report the loss of mitochondrial DNA in approximately 40%-50% of the treated parasites. We conclude that venturicidin A targets the ATP synthase of T. brucei, and we suggest that this macrolide could be a candidate for anti-trypanosomatid drug repurposing, drug combinations, or medicinal chemistry programs.

Impact of CAD/CAM burs lifetime on surface properties of dental zirconia: Implications for biocompatibility of custom abutments.

To evaluate the effect of the deterioration of computer aided design/computer aided manufacturing (CAD/CAM) burs during zirconia milling, on surface roughness, contact angle, and fibroblast viability. Ceramic blocks were milled and 75 ceramic disks (8 × 1.5 mm) made and allocated into three groups (n = 25): G1-brand new 2L and 1L burs, G2-2L bur at the end of lifetime and brand new 1L bur and G3-both burs at the end of their lifetimes. Roughness (Ra, Rq, and Rz) was evaluated using a 3D optical profilometer, the contact angle by the sessile drop method and the cell viability of the mouse NIH/3T3 fibroblast, using the Alamar Blue assay at intervals of 24, 48, and 72 h (ISO 10993-5). Data were analyzed by one-way ANOVA and Kruskal-Wallis tests (p ≤ 0.05). Roughness increased as the burs deteriorated and G3 (0.27 ± 0.04) presented a higher value for Ra (p < 0.001). The highest contact angle was observed in G3 (86.2 ± 2.66) when compared with G1 (63.7 ± 12.49) and G2 (75.3 ± 6.36) (p < 0.001). Alamar Blue indicated an increase in cell proliferation, with no significant differences among the groups at 24 and 72 h (p > 0.05). The deterioration of the burs increased the surface roughness and decreased the wettability, but did not interfere in cell viability and proliferation. The use of custom zirconia abutments represents an effective strategy for single crowns restorations. Our findings suggest that these abutments can be efficiently milled using CAD/CAM burs within their recommended lifetime.