Increase In Cellular Viability Research Articles

Electrophoretic Deposition of 58S Bioactive Glass- Polymer Composite Coatings on 316L Stainless Steel: An Optimization for Corrosion, Bioactivity, and Cytocompatibility.

This study presents a facile fabrication of 58S bioactive glass (BG)-polymer composite coatings on a 316L stainless steel (SS) substrate using the electrophoretic deposition technique. The suspension characteristics and deposition kinetics of BG, along with three different polymers, namely ethylcellulose (EC), poly(acrylic acid) (PAA), and polyvinylpyrrolidone (PVP), have been utilized to fabricate the coatings. Among all coatings, 58S BG and EC polymers are selected as the final composite coating (EC6) owing to their homogeneity and good adhesion. EC6 coating exhibits a thickness of ∼18 μm and an average roughness of ∼2.5 μm. Herein, EC6 demonstrates better hydroxyapatite formation compared to PAA and PVP coatings in simulated body fluid-based mineralization studies for a period of 28 days. Corrosion studies of EC6 in phosphate-buffered saline further confirm the higher corrosion resistance properties after 14 days. In vitro cytocompatibility studies using human placental mesenchymal stem cells demonstrate an increase in cellular viability, attachment, and higher proliferation compared to the bare SS substrate. EC6 coatings promote osteogenic differentiation, which is confirmed via the upregulation of the OPN and OCN genes. Moreover, the EC6 sample exhibits improved antibacterial properties against Escherichia coli and Staphylococcus aureus compared to the uncoated ones. The findings of this work emphasize the potential of electrophoretically fabricated BG-EC composite coatings on SS substrates for orthopedic applications.

Direct inkjet writing of polylactic acid/β-tricalcium phosphate composites for bone tissue regeneration: A proof-of-concept study.

There is an ever-evolving need of customized, anatomic-specific grafting materials for bone regeneration. More specifically, biocompatible and osteoconductive materials, that may be configured dynamically to fit and fill defects, through the application of an external stimulus. The objective of this study was to establish a basis for the development of direct inkjet writing (DIW)-based shape memory polymer-ceramic composites for bone tissue regeneration applications and to establish material behavior under thermomechanical loading. Polymer-ceramic (polylactic acid [PLA]/β-tricalcium phosphate [β-TCP]) colloidal gels were prepared of different w/w ratios (90/10, 80/20, 70/30, 60/40, and 50/50) through polymer dissolution in acetone (15% w/v). Cytocompatibility was analyzed through Presto Blue assays. Rheological properties of the colloidal gels were measured to determine shear-thinning capabilities. Gels were then extruded through a custom-built DIW printer. Space filling constructs of the gels were printed and subjected to thermomechanical characterization to measure shape fixity (Rf) and shape recovery (Rr) ratios through five successive shape memory cycles. The polymer-ceramic composite gels exhibited shear-thinning capabilities for extrusion through a nozzle for DIW. A significant increase in cellular viability was observed with the addition of β-TCP particles within the polymer matrix relative to pure PLA. Shape memory effect in the printed constructs was repeatable up to 4 cycles followed by permanent deformation. While further research on scaffold macro-/micro-geometries, and engineered porosities are warranted, this proof-of-concept study suggested suitability of this polymer-ceramic material and the DIW 3D printing workflow for the production of customized, patient specific constructs for bone tissue engineering.

Cellular responses to 3D printed dental resins produced using a manufacturer recommended printer versus a third party printer.

The aim of this study was to evaluate the influence of different 3D dental resins, using a manufacturer recommended printer and a third-party printer, on cellular responses of human gingival cells. Three NextDent resins (Denture 3D+, C&B MFH and Crowntec) were used to produce specimens on printers NextDent 5100 (groups ND, NC and NT, respectively) and Phrozen Sonic Mini 4K (groups PD, PC and PT, respectively). Human gingival fibroblasts were cultured and biocompatibility was evaluated on days 1, 3 and 7. IL-6 and IL-8 concentrations were evaluated at 3 days using ELISA. Surface roughness was evaluated by a contact profilometer. SEM and fluorescence micrographs were analyzed at days 1 and 7. Statistical analyses were performed using SPSS and mean differences were tested using ANOVA and post-hoc Tukey tests (P < .05). There was an increase in cellular viability after 7 days in groups PC and PT, when compared to group PD. ND group resulted in higher concentration of IL-6 when compared to PT group. SEM and fluorescence micrographs showed less adhesion and thinner morphology of fibroblasts from group PD. No significant differences were found regarding surface roughness. The use of different printers or resins did not seem to influence surface roughness. NextDent 5100 and Phrozen Sonic Mini 4K produced resins with similar cellular responses in human gingival fibroblasts. However, Denture 3D+ resin resulted in significantly lower biocompatibility, when compared to C&B MFH and Crowntec resins. Further testing is required to support its long-term use, required for complete dentures.

Microporous/Macroporous Polycaprolactone Scaffolds for Dental Applications.

This study leverages the advantages of two fabrication techniques, namely, melt-extrusion-based 3D printing and porogen leaching, to develop multiphasic scaffolds with controllable properties essential for scaffold-guided dental tissue regeneration. Polycaprolactone-salt composites are 3D-printed and salt microparticles within the scaffold struts are leached out, revealing a network of microporosity. Extensive characterization confirms that multiscale scaffolds are highly tuneable in terms of their mechanical properties, degradation kinetics, and surface morphology. It can be seen that the surface roughness of the polycaprolactone scaffolds (9.41 ± 3.01 µm) increases with porogen leaching and the use of larger porogens lead to higher roughness values, reaching 28.75 ± 7.48 µm. Multiscale scaffolds exhibit improved attachment and proliferation of 3T3 fibroblast cells as well as extracellular matrix production, compared with their single-scale counterparts (an approximate 1.5- to 2-fold increase in cellular viability and metabolic activity), suggesting that these structures could potentially lead to improved tissue regeneration due to their favourable and reproducible surface morphology. Finally, various scaffolds designed as a drug delivery device were explored by loading them with the antibiotic drug cefazolin. These studies show that by using a multiphasic scaffold design, a sustained drug release profile can be achieved. The combined results strongly support the further development of these scaffolds for dental tissue regeneration applications.

Abstract 3082: Altered levels of corticosteroid-binding globulin: SerpinA6 and progression of prostate cancer

Abstract SerpinA6 is a main transport protein for cortisol binding, which helps in cortisol tissue delivery particularly at inflammatory sites. Cortisol plays a vital role in adaptation to stress, modulating fuel metabolism, and inflammation. SerpinA6 releases cortisol specifically at inflammatory sites upon cleavage by human neutrophil elastase between residues 344 and 345. Castration induce inflammation in the prostate increases glucocorticoids (GCs) level and elevates pro-inflammatory cytokine IL-6 levels. European and Asian populations represented association between single nucleotide polymorphisms (SNPs) in CYP17A1 and SERPINA6/A1 and circulating glucocorticoid concentrations. We propose increasing IL-6 levels after castration alters SerpinA6 levels in prostate may results in prostate cancer disease progression. Deficient SerpinA6 may not be sufficient to keep steroids inactive, due to the increasing GCs. Our data suggests IL-6 treatment of androgen independent prostate cancer (PC-3) cells decreases SerpinA6 level and conversely increases glucocorticoid receptor (GR) expression in a dose (1-10ng/ml) and time (30-180 minutes) dependent fashion. Moreover, IL-6 dose dependent treatment in PC-3 cells decreases FOXO3a expression, a major apoptotic protein dysregulated in prostate cancer. Next, we determine the role of SerpinA6 (30µg/ml) and IL-6 (5ng/ml) independently and in combination to PC-3 cells. We observed IL-6 and SerpinA6 co-treatment to PC-3 cells (charcoal stripped FBS media) for 6 hours reduced Neutrophil elastase levels significantly. Silencing IL-6 in PC-3 cells increased SerpinA6 expression. Moreover, silencing SerpinA6 in metastatic variant of PC-3 cells increased phospho-Akt-(ser-473), glucocorticoid receptor and increased apoptotic protein FOXO3a expressions. Using dexamethasone (15µM), which is known to suppress PC-3 cell migration, we found increased expression of SerpinA6. Conversely, Mifepristone (15µM), which inhibits PC-3 cell growth, deceases SerpinA6 expression after 24 hours of treatment. In humans the normal range of circulatory SerpinA6 level are 19-45µg/ml. We treated normal prostate epithelial cells (RWPE1) and prostate cancer (PC-3) cells with human recombinant protein 18 and 36µg/ml of doses for 24 hours; RWPE1 cells represented a SerpinA6 dose dependent increase in cellular viability, whereas PC-3 cells demonstrated no change in the viability after a dose response treatment with SerpinA6 for 24 hours. In-vivo data represented altered SerpinA6 levels in castrated TRansgenic Adenocarcinoma Mouse Prostate model after dexamethasone (GR agonist) and Mifepristone (GR antagonist) treatment. We suggest decreasing levels of SerpinA6 in prostate cancer patients may have potential role in disease progression. We need to have an effective agent, to prevent maintain SerpinA6 level to inhibit the progression of prostate cancer. Citation Format: Mohammed Al-Toubat, Jatinder Kumar, Shiva Gautam, Kethandapatti Balaji, Sanjeev Shukla. Altered levels of corticosteroid-binding globulin: SerpinA6 and progression of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3082.

Shockwaves Increase In Vitro Resilience of Rhizopus oryzae Biofilm under Amphotericin B Treatment.

Acoustical biophysical therapies, including ultrasound, radial pressure waves, and shockwaves, have been shown to harbor both a destructive and regenerative potential depending on physical treatment parameters. Despite the clinical relevance of fungal biofilms, little work exits comparing the efficacy of these modalities on the destruction of fungal biofilms. This study evaluates the impact of acoustical low-frequency ultrasound, radial pressure waves, and shockwaves on the viability and proliferation of in vitro Rhizopus oryzae biofilm under Amphotericin B induced apoptosis. In addition, the impact of a fibrin substrate in comparison with a traditional polystyrene well-plate one is explored. We found consistent, mechanically promoted increased Amphotericin B efficacy when treating the biofilm in conjunction with low frequency ultrasound and radial pressure waves. In contrast, shockwave induced effects of mechanotransduction results in a stronger resilience of the biofilm, which was evident by a marked increase in cellular viability, and was not observed in the other types of acoustical pressure waves. Our findings suggest that fungal biofilms not only provide another model for mechanistical investigations of the regenerative properties of shockwave therapies, but warrant future investigations into the clinical viability of the therapy.

Abstract 3983: Dysregulated SerpinA6 levels and progression of prostate cancer

Abstract SerpinA6 is a major cortisol binding transport protein, which helps in cortisol tissue delivery particularly at inflammatory sites. Moreover, SerpinA6 has been reported to participate in stress response by releasing cortisol specifically at inflammatory sites upon cleavage by human neutrophil elastase between residues 344 and 345. In castrate resistant prostate cancer (CRPC) patients, reports suggest castration induces inflammation in the prostate, which increases glucocorticoids (GCs) levels and elevates pro-inflammatory cytokine IL-6 levels. We propose declining SerpinA6 levels in the prostate may play a role in prostate cancer disease progression. This could be due to increasing IL-6 levels after castration, which may inhibit or degrade SerpinA6. Deficient SerpinA6 may not be sufficient to keep steroids inactive, due to the increasing GCs. Our data suggests IL-6 treatment of androgen independent prostate cancer (PC-3) cells decreases SerpinA6 level and conversely increases glucocorticoid receptor (GR) expression in a dose (1-10ng/ml) and time (30-180 minutes) dependent fashion. Moreover, IL-6 dose dependent treatment in PC-3 cells decreases FOXO3a expression, a major apoptotic protein dysregulated in prostate cancer. Next, we determine the role of SerpinA6 (30µg/ml) and IL-6(5ng/ml) independently and in combination to PC-3 cells. We observed IL-6 and SerpinA6 co-treatment to PC-3 cells (charcoal stripped FBS media) for 6 hours reduced Neutrophil elastase levels significantly. Additionally we observed increased FOXO3a and SerpinA6 expression, whereas decreased GR and HNF3α levels. However, silencing IL-6 in PC-3 cells increased SerpinA6 expression. To further support our findings, silencing SerpinA6 in metastatic variant of PC-3 cells increased phosphor-Akt-(ser-473), glucocorticoid receptor and increased apoptotic protein FOXO3a expressions. Using dexamethasone (15µM), which is known to suppress PC-3 cell migration, we found increased expression of SerpinA6. Conversely, Mifepristone (15µM), which inhibits PC-3 cell growth, deceases SerpinA6 expression after 24 hours of treatment. In humans the normal range of circulatory SerpinA6 level are 19-45ug/ml. We treated normal prostate epithelial cells (RWPE1) and prostate cancer (PC-3) cells with human recombinant protein 18 and 36ug/ml of doses for 24 hours; RWPE1 cells represented a SerpinA6 dose dependent increase in cellular viability, whereas PC-3 cells demonstrated no change in the viability after a dose response treatment with SerpinA6 for 24 hours. These findings suggest the SerpinA6 context dependent role. We suggest that decreasing levels of SerpinA6 and FOXO3a expressions in the prostate cancer patients may have potential role in disease progression. Moreover, we need to have an effective agent, which could prevent the decreasing levels of SerpinA6 and FOXO3a in prostate to inhibit the progression of prostate cancer. Citation Format: Carlos Riveros, Victor Chalfant, Jatinder Kumar, Sanjeev Shukla. Dysregulated SerpinA6 levels and progression of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3983.

Nanohydroxyapatite, Nanosilicate-Reinforced Injectable, and Biomimetic Gelatin-Methacryloyl Hydrogel for Bone Tissue Engineering.

PurposeGiven that autologous bone graft for bone defects is limited by insufficient supply and morbidity at the donor site, developing biomimetic graft materials as an alternative has gained consistent attention. However, obstacles in designing bone-mimetic materials that could integrate the biomimetic nature of the bone extracellular matrix, osteogenic cells, and osteoinductive ingredients with a fast and convenient strategy still exist.MethodsThis study designed and fabricated a mesenchymal stem cell (MSC)-laden, nanohydroxyapatite (HAP), and nanosilicate (SN)-loaded bone mimetic and injectable gelatin-methacryloyl hydrogel (GelMA-HAP-SN) system for bone tissue engineering, and systemically investigated the osteogenic capacity of GelMA-HAP-SN in vitro and in vivo.ResultsIntroducing HAP enhanced the compositional similarity to the natural bone extracellular matrix, and SN loading endowed the hydrogel with injectable and osteogenic ability. As a result, the GelMA-HAP-SN hydrogel demonstrated an increase in cellular viability, proliferation, and spreading behavior. The GelMA-HAP-SN hydrogel also amplified the embedded MSCs’ osteogenic biomarkers’ expression and matrix mineralization. Furthermore, the MSC-encapsulated GelMA-HAP-SN hydrogel was injected into rats’ critical-sized calvaria defect, and micro-CT and histomorphometry staining results further confirmed its excellent bone regeneration ability.ConclusionThese MSC-loaded GelMA-HAP-SN hydrogels are potential graft materials for bone defect treatment.

Ameliorating hydroxychloroquine induced retinal toxicity through cerium oxide nanoparticle treatments.

The present study investigated the potential protective effects of cerium oxide nanoparticles (CNP) on human retinal pigment epithelium (ARPE-19) cells damaged by hydroxychloroquine (HCQ). Toxicity of HCQ on the ARPE-19 cells was explored with a dose response trial. CNP rescue both a pre-treatment protocol, where CNP were applied 24 hours prior to HCQ application and a simultaneous treatment protocol where both CNP and HCQ were applied together, were used. In the dose response trial, 250 µM HCQ showed 51.84% cell viability after 24 hours and 32.75% after 48 hours time period. This was selected as model HCQ dose for rescue trials. The simultaneous treatment trials did not show a significant increase in viability compared to model toxic dose. The CNP pre-treatment trials showed a significant increase in cellular viability compared to model toxic dose with 68.03% ± 3.27 viability (p = 4.56E-05) at 24 hours and 51.85% ± 4.96 (p = 1.18E-05) at 48 hours time period. CNP pre-treatment showed significant protection of cells from HCQ induced toxicity. The difference in efficacy of simultaneous and pre-treatment is hypothesized to lie in the cellular localization of CNP. Furthermore, including the reactive oxygen species (ROS) scavenging properties of CNP seems to be responsible for protection, the effect of CNP on autophagosome and lysosome colocalization are also hypothesized to play a significant role.

MiR-744/eNOS/NO axis: A novel target to halt triple negative breast cancer progression.

Nitric oxide (NO) may have a dual role in cancer. At low concentrations, endogenous NO promotes tumor growth and proliferation. However, at very high concentrations, it mediates cancer cell apoptosis and inhibits cancer growth. High levels of NO have been observed in blood of breast cancer (BC) patients, which increases tumor blood flow and promotes angiogenesis. To date, the regulation of NO-synthesizing enzyme, eNOS, by miRNAs has not been adequately investigated in BC. Therefore, the main aim of this study is to unravel the possible regulation of eNOS by miRNAs in BC and to examine their influence on NO production and BC progression. Expression profile of eNOS in Egyptian BC patients and MDA-MB-231 cell lines was investigated using qRT-PCR. In-silico analysis was performed to predict a putative upstream regulator of eNOS. miR-744-5p was selected and its expression was quantified in BC tissues using qRT-PCR. MDA-MB-231 cells were cultured and transfected with miR-744-5p using lipofection method. NO levels were determined using Griess Reagent. Cellular viability and colony-forming ability were assessed using MTT and colony-forming assays; respectively. eNOS and miR-744-5p were significantly up-regulated in BC tissues compared to paired normal tissues. In-silico analysis revealed that miR-744-5p putatively binds to eNOS transcript with high binding scores. Transfection of MDA-MB-231 cells with miR-744-5p mimics resulted in a significant up-regulation of eNOS and consequently NO levels. In addition, miR-744-5p transfection led to an increase in cellular viability and colony-forming ability of the MDA-MB-231. miR-744-5p acts as an upstream positive regulator of the NO synthesizing enzyme, eNOS which in turn elevates NO levels. Furthermore, miR-744-5p is a novel oncogenic miRNA in BC. Thus, targeting miR-744/eNOS/NO axis may act as a therapeutic tool in TNBC.

Polysaccharides from acerola, cashew apple, pineapple, mango and passion fruit co-products: Structure, cytotoxicity and gastroprotective effects

This study aimed to characterize the chemical structure of water-soluble polysaccharide fractions extracted from co-products of acerola, cashew apple, pineapple, mango and passion fruit, evaluating their gastroprotective effect in vivo associated to their antioxidant activity. The results from the structural analysis revealed that all fractions extracted from the co-products were heteropolysaccharides composed by the monosaccharides glucose, arabinose, galactose and d-galacturonic acid, and presented a pectic nature, which was evidenced by the predominant presence of arabinogalactan in their constitutions. The polysaccharides obtained from acerola, cashew apple and pineapple exhibited similarity in their morphology compared to those from mango and passion fruit. The fractions did not show cytotoxic effects for epithelial culture IEC-6A, and stimulated cell growth as visualized by the increase in cellular viability. Notable antioxidant activities were found for cashew apple (147 μM Trolox.g−1), pineapple (80.0 μM Trolox.g−1) and acerola (79.0 μM Trolox.g−1). These same polysaccharides promoted higher preservation of glutathione levels in mice stomach tissue, resulting in its effective protection from the damage caused by ethanol. On the other hand, high malondialdehyde levels were observed in the stomach tissues of the animals treated with the polysaccharides from mango and passion fruit. The co-products from acerola and cashew were able to protect the gastric mucosa of mice from the lesions induced by ethanol. The results found in this research introduce polysaccharide fractions extracted from acerola, cashew apple and pineapple co-products as safe and natural antioxidants, with a high potential to be applied in different industry fields.

EVALUATION OF THE PROTECTIVE EFFECT OF GALLIC ACID AGAINST ARSENIC-INDUCED GENOTOXICITY IN HEPG2 CELL LINE

Objective: Arsenic has cytotoxic as well as mutagenic effect in human health due to its indirect effect on oxidative stress on the cells. We aimed to find out the effect of gallic acid (GA), a well-known natural antioxidant in ameliorating in heavy metal toxicity.&#x0D; Methods: MTT assay was performed to determine the cytotoxicity of sodium arsenite (NaAsO2) on HepG2 cells with the cytoprotectant GA at varying concentrations for exposure durations of 6 h, 12 h, and 24 h. Similarly, the alkaline version of the comet assay was performed to investigate the genotoxicity and assessment of oxidative stress of the cells using flow cytometry.&#x0D; Results: Cells treated with NaAsO2 at various doses spanning a broad range of concentrations (5–500 μM) showed a dose- and time-dependent decrease in cellular viability as observed. However, the effect of the proposed protectant, GA showed an increase in cellular viability in a concentration-dependent manner.&#x0D; Conclusion: We assessed the cytotoxicity and genotoxicity induced by NaAsO2 to provide insight into the role of GA on arsenic-induced toxicity in liver cells and to shed light on its possible ameliorative effect at low concentrations in a time-dependent manner.

Lovastatin alters neurotrophin expression in rat hippocampus-derived neural stem cells in vitro

Neural stem/progenitor cells hold valuable potential for the treatment of neurodegenerative disorders. The modulation of intrinsic growth factor expression, such as neurotrophins and their receptors, is a necessary step in achieving neural stem cells (NSCs) therapy. The statins have recently been reported to provide both anti‑inflammatory and neuroprotective effects. In the developing and mature nervous systems, neurotrophic factors are known to impact neuronal growth and survival. In this study, we investigated for a positive effect of lovastatin on the expression of neurotrophins in the neonatal rat hippocampus‑derived NSCs. NSCs were isolated and cultured up to passage three. To confirm cellular identity, immunocytochemical evaluation and flow cytometry analysis were performed using specific antibodies. To determine the optimum concentration of lovastatin, the MTT assay was used. Neurotrophin expression was evaluated using quantitative real‑time reverse transcription‑polymerase chain reaction (RT‑qPCR). Flow cytometry results demonstrated that NSCs were positive for nestin, a marker for neural progenitor cells. An increase in cellular viability was observed with a 24 h exposure of lovastatin. Moreover, results showed an increase in mRNA expression for all neurotrophins compared to the control group. Taken together, the results of this study add to the growing body of literature on the neuroprotective effects of statins in neurological disorders. Lovastatin is a promising therapeutic agent for the treatment of neurodegenerative disorders.

TMIC-43. ROLE OF EXTRACELLULAR FERRITIN HEAVY CHAIN PROTEIN IN GLIOBLASTOMA MULTIFORME

Abstract Increased expression of Ferritin heavy chain (FHC) protein has been associated with poor prognosis in Glioblastoma Multiforme (GBM) which is one of the most aggressive and common types of brain cancer. GBM patients have also been found to have increased extracellular ferritin levels, in their serum and cerebrospinal fluid (CSF), which are lowered once the source/tumor has been resected. Extracellular FHC can function as an iron delivery protein, and increasing amount of iron has been known to contribute to tumor initiation and proliferation. To study the effect of extracellular FHC in GBM cells we used patient derived GBM, CD133+ cancer stem cells (GSCs) from the pro-neural (T3691) and mesenchymal (T387) subtypes. Using recombinant FHC, conjugated with quantum dots (QD), we observed significant increase in cellular viability and intracellular uptake of FHC by the GSCs in a dose dependent manner. Our lab has previously shown that extracellular FHC interacts with T-Cell Immunoglobulin Mucin Receptor 1 (Tim-1) in the human oligodendrocytes. In order to determine if GSCs express the Tim-1 receptor we first confirmed its expression on GSCs using immunoblotting and immunocytochemistry. To test if FHC interacts with Tim-1, we performed knockdown of Tim-1 using siRNAs. However, the siRNA was not able to downregulate the Tim-1 receptors. Next, we exposed the GSCs to Sema4A, which has been shown in our previous studies to interact with Tim-1 receptor on human oligodendrocytes and is toxic to oligodendrocytes. The GSCs however were not affected by the saturable concentration of Sema4A. Thus, through this study we have shown the expression of potential FHC receptors on GSCs and a robust effect of H-ferritin on GSCs proliferation. Further experiments are warranted in this direction to understand this extracellular FHC uptake pathway and its role in GBM cell proliferation.

Effect of size and silica coating on structural, magnetic as well as cytotoxicity properties of copper ferrite nanoparticles

Copper ferrite nanoparticles, synthesized by conventional sol–gel method were calcined at different temperatures. The magnetic, structural, morphological and cytotoxicity analyses of the uncalcined and calcined nanoparticles (NPs) were investigated and compared. Formation of tetragonal structure of CuFe2O4 NPs was observed in XRD patterns. On increasing the temperature, better crystallinity and increased crystallite size were also observed. In the FTIR spectra, bonds corresponding to CH, OH and carboxylate groups gradually disappeared with increasing temperature, while peak corresponding to FeO existed more prominently. NPs calcined at 300 °C (Cu3) exhibited the highest magnetic saturation and lowest retentivity, thereby indicating its superparamagnetic behaviour. Concentration-dependent cytotoxicity values were obtained by invitro MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, a tetrazole) assay, Cell Titer assay and Cell Flow Cytometry with Propidium Iodide. NPs calcined at 300 °C, 500 °C and 700 °C exhibited non-toxicity at all the concentrations. Based on magnetic and biocompatibility analyses, Cu3 NPs were found to be the most suitable one to investigate the influence of silica coating on its surface. Presence of silica was confirmed by XRD pattern, FTIR spectrum, SEM and HRTEM micrographs as well as SAED pattern. In M-H curve, superparamagnetic behaviour of the CuFe2O4 core was retained but with reduced magnetic saturation due to magnetically dead layer of silica. An increase in cellular viability was witnessed in case of silica coated CuFe2O4 NPs as compared to uncoated NPs, thus reflecting on its enhanced biocompatibility. Nanosized, superparamagnetic and highly biocompatible characteristics make silica coated CuFe2O4 NPs a potential claimant for biomedical applications.

Failure to detect functional transfer of active K-Ras protein from extracellular vesicles into recipient cells in culture.

Exosomes, extracellular nanovesicles that carry nucleic acids, lipids, and proteins, have been the subject of several studies to assess their ability to transfer functional cargoes to cells. We recently characterized extracellular nanovesicles released from glioblastoma cells that carry active Ras in complex with proteins regulating exosome biogenesis. Here, we investigated whether a functional transfer of Ras from exosomes to other cells can initiate intercellular signaling. We observed that treatment of serum-starved, cultured glioblastoma cells with exogenous glioblastoma exosomes caused a significant increase in cellular viability over time. Moreover, we detected fluorescent signal transfer from lipophilic dye-labeled exogenous glioblastoma exosomes into cultured glioblastoma cells. To probe possible signaling from cell-to-cell, we utilized bimolecular luciferase complementation to examine the ability of K-Ras in exosomes to interact with the Raf-Ras Binding domain (Raf-RBD) expressed in a recipient cell line. Although the K-Ras/Raf-RBD interaction was readily detectable upon co-expression in a single cell line, or following lysis of co-cultured cell lines separately expressing K-Ras and RBD, bearing in mind the limitations of our assay, we were unable to detect the interaction in the intact, co-cultured cell lines or upon treatment of the Raf-RBD-expressing cells with exosomes containing K-Ras. Furthermore, HA-Tag-BFP fused to the K-Ras hypervariable region and CAAX sequence failed to be transferred at significant levels from extracellular vesicles into recipient cells, but remained detectable in the cell supernatants even after 96 hours of culture of naïve cells with extracellular vesicles. We conclude that if transfer of functional K-Ras from extracellular vesicles into the cytoplasm of recipient cells occurs, it must do so at an extremely low efficiency and therefore is unlikely to initiate Ras-ERK MAP kinase pathway signaling. These results suggest that studies claiming functional transfer of protein cargoes from exosomes should be interpreted with caution.

ATM Is Required for the Prolactin-Induced HSP90-Mediated Increase in Cellular Viability and Clonogenic Growth After DNA Damage.

Prolactin (PRL) acts as a survival factor for breast cancer cells, but the PRL signaling pathway and the mechanism are unknown. Previously, we identified the master chaperone, heat shock protein 90 (HSP90) α, as a prolactin-Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5) target gene involved in survival, and here we investigated the role of HSP90 in the mechanism of PRL-induced viability in response to DNA damage. The ataxia-telangiectasia mutated kinase (ATM) protein plays a critical role in the cellular response to double-strand DNA damage. We observed that PRL increased viability of breast cancer cells treated with doxorubicin or etoposide. The increase in cellular resistance is specific to the PRL receptor, because the PRL receptor antagonist, Δ1-9-G129R-hPRL, prevented the increase in viability. Two different HSP90 inhibitors, 17-allylamino-17-demethoxygeldanamycin and BIIB021, reduced the PRL-mediated increase in cell viability of doxorubicin-treated cells and led to a decrease in JAK2, ATM, and phosphorylated ATM protein levels. Inhibitors of JAK2 (G6) and ATM (KU55933) abolished the PRL-mediated increase in cell viability of DNA-damaged cells, supporting the involvement of each, as well as the crosstalk of ATM with the PRL pathway in the context of DNA damage. Drug synergism was detected between the ATM inhibitor (KU55933) and doxorubicin and between the HSP90 inhibitor (BIIB021) and doxorubicin. Short interfering RNA directed against ATM prevented the PRL-mediated increase in cell survival in two-dimensional cell culture, three-dimensional collagen gel cultures, and clonogenic cell survival, after doxorubicin treatment. Our results indicate that ATM contributes to the PRL-JAK2-STAT5-HSP90 pathway in mediating cellular resistance to DNA-damaging agents.

Emodin protects against oxidative stress and apoptosis in HK-2 renal tubular epithelial cells after hypoxia/reoxygenation.

The aim of the present study was to determine the effects of emodin, a natural compound with antioxidant properties, on oxidative stress and apoptosis induced by hypoxia/reoxygenation (H/R) in HK-2 human renal tubular cells. In HK-2 cells subjected to H/R, it was observed that pre-treatment with emodin lead to an increase in cellular viability and a reduction in the rate of apoptosis and the B-cell lymphoma 2 (Bcl-2)-associated X protein/Bcl-2 ratio. H/R alone caused a significant increase in the levels of reactive oxygen species and malondialdehyde (P<0.05) and a significant decrease in the activities of superoxide dismutase, catalase and glutathione peroxidase (P<0.05), relative to normoxic cells. In turn, parameters of oxidative stress were improved by emodin pre-treatment. In addition, emodin pre-treatment significantly inhibited the phosphorylation of extracellular signal-regulated protein kinase and c-Jun N-terminal kinase mitogen-activated protein kinases (MAPKs) induced by H/R (P<0.05). These data suggest that emodin may prevent H/R-induced apoptosis in human renal tubular cells through the regulation of cellular oxidative stress, MAPK activation and restoration of the Bax/Bcl-2 ratio.

Morphology and surface chemistry of bicomponent scaffolds in terms of mesenchymal stromal cell viability

Biological interaction between cells and scaffolds is mediated through events at surfaces. Proteins present in the culture medium adsorb on substrates, generating a protein adlayer that triggers further downstream events governing cell adhesion. Polymer blends often combine the properties of the individual components, for example, can provide mechanical as well as surface properties in one fibre. Therefore, mixtures of synthetic polycaprolactone and gelatin as a denatured form of collagen were electrospun at selected conditions and polymer weight ratios. Fibre morphologies and chemical properties of the surfaces were analysed. These scaffolds were seeded with human mesenchymal stromal cells and their viability was studied. Gelatin addition to polycaprolactone leads to a reduction in fibre diameter. A linear increase in gelatin at the fibre surface was observed in function of the weighed polymers, except for polycaprolactone/gelatin fibres incorporating equal weight ratios. Thereby, a depletion of gelatin at the fibre surface is stated for equally mixed polymers. The depletion of gelatin at the fibre surface is most probably due to hydrophobic interactions between hydrophobic segments of polycaprolactone and gelatin, affecting the spinning mechanism and thus fibre structure. Furthermore, polycaprolactone/gelatin blends show enhanced wettability properties compared to pure gelatin, at least partly due to molecular segregation. Results of in vitro studies reveal an increase in cellular viability and proliferation for cells cultivated on nanofibres containing gelatin, caused by the cell-attractive surface composition as well as the hydrophilic nature of the scaffolds. Contact guidance of cells seeded on parallelised fibres is observed, and DNA tests show evidently enhanced cell numbers on nanofibres containing 20 wt% of gelatin.

Cannabinoid receptor ligands prevent dopaminergic neurons death induced by neurotoxic, inflammatory and oxidative stimuli in vitro

Title: Cannabinoid receptor ligands prevent dopaminergic neurons death induced by neurotoxic, inflammatory and oxidative stimuli in vitro. Background: During the last 25 years increasing efforts have been invested for the comprehension of the cannabinoid system in a wide range of healthy and pathological conditions. Previous investigations have indicated the possible protective role of synthetic cannabinoids like arachidonyl-2’-chloroethylamide, ACEA (CB1 agonist), during neurodegenerative diseases such as Parkinson’s disease. Other synthetic CB1 receptor ligands such as N-(Piperidin-1-yl)-5-(4- iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide, AM251, commonly known as an antagonist/inverse agonist, have recently been presented as an allosteric modulator of this receptor. In this study, we show the protective effect of ACEA against oxidative and inflammatory damage in in vitro dopaminergic neurons. Methods and Findings: Neuro2A cells differentiated into dopaminergic-like neurons were challenged with neurotoxic, inflammatory and oxidative treatments, 6-hydroxidopamine (6OHDA), lipopolysaccharide (LPS) and hydrogen peroxide (H2O2). Cannabinoid-dependent cell protection was evaluated by means of cellular viability, reactive oxygen species (ROS) production and the pro apoptotic protein caspase 3 expression after CB1 agonist ACEA and antagonist AM251. The ACEA treatment resulted in an increase of cellular viability after the 6OHDA, LPS and H2O2 challenges. When cells were co-treated with ACEA and AM251, an increase in cell death prevention was observed, with a reduction in reactive oxygen species production and caspase 3 expression. Conclusions: Together, we show an ACEA-mediated neuronal protection by means of ROS expression reduction and pro apoptotic protein caspase 3 expression inhibition.