Neutral Red Uptake Research Articles

Ferumoxytol-β-glucan Inhibits Melanoma Growth via Interacting with Dectin-1 to Polarize Macrophages into M1 Phenotype.

Background: Regulating the polarization of macrophages to antitumor M1 macrophages is a promising strategy for overcoming the immunosuppression of the tumor microenvironment for cancer therapy. Ferumoxytol (FMT) can not only serve as a drug deliver agent but also exerts anti-tumor activity. β-glucan has immuno-modulating properties to prevent tumor growth. Thus, a nanocomposite of FMT surface-coated with β-glucan (FMT-β-glucan) was prepared to explore its effect on tumor suppression.Methods: Male B16F10 melanoma mouse model was established to explore the antitumor effect of FMT-β-glucan. The viability and apoptotic rates of B16F10 cells were detected by cell counting kit-8 and Annexin-V/PI experiments. The levels of M1 markers were quantified by quantitative reverse transcription-polymerase chain reaction and enzyme linked immunosorbent assay. Phagocytic activity and intracellular reactive oxygen species (ROS) in macrophages were evaluated by the neutral red uptake assay and flow cytometry, respectively. Small interfering RNA (siRNA) transfection was applied to knock down the Dectin-1 gene in RAW 264.7 cells.Results: FMT-β-glucan suppressed tumor growth to a greater extent and induced higher infiltration of M1 macrophages than the combination of FMT and β-glucan (FMT+β-glucan) in vivo. In vitro, supernatant from FMT-β-glucan-treated RAW 264.7 cells led to lower cell viability and induced more apoptosis of B16F10 cells than that from the FMT+β-glucan group. Moreover, FMT-β-glucan boosted the expression of M1 type markers, and increased phagocytic activity and ROS in RAW 264.7 cells. Further research indicated that FMT-β-glucan treatment promoted the level of Dectin-1 on the surface of RAW 264.7 cells and that knockdown of Dectin-1 abrogated the phosphorylation levels of several components in MAPK and NF-κB signaling.Conclusion: The nanocomposite FMT-β-glucan suppressed melanoma growth by inducing the M1 macrophage-activated tumor microenvironment.

1187. CFTR Function Impacts Proinflammatory Cytokine Expression of Bronchial Epithelial Cells During Pseudomonas aeruginosa and Staphylococcus aureus Infections

BackgroundCystic fibrosis (CF) is a genetic disease in which opportunistic respiratory infections are common, particularly with P. aeruginosa and S. aureus. It has been suggested that dysfunction in the cystic fibrosis transmembrane conductance regulator (CFTR) could impact host immune response to these infections. The aim of this study was to determine if dysfunction in the CFTR gene could impact host cell death and the expression of proinflammatory cytokines.MethodsWe used two human bronchial epithelial (HBE) cell lines NuLi-1 and CuFi-1, which were recovered from individuals without and with CF, respectively. THP-1 cells were also differentiated into a macrophage phenotype with 50ng/mL PMA, and 10 µM CFTRinh-172 was used to impair CFTR function in these cells. Laboratory reference strains PAO1 and S. aureus 6538 were used to conduct in vitro infections. Host cell death at various MOIs was evaluated using a neutral red uptake assay, and cytokine expression was measured using ELISAs.ResultsNo differences in THP-1 cell death or cytokine expression were observed for infections with S. aureus 6538 or PAO1 with and without CFTRinh-172. No differences in cell death for infections with the HBE cells and either bacteria were noted. S. aureus 6538 induced higher levels of IL-1β when infecting CuFi-1 cells in comparison to NuLi-1 cells across all MOIs (P < 0.001) and induced higher levels of IL-6 at an MOI of 100 (P < 0.01). S. aureus 6538 also induced higher levels of IL-8 when infecting CuFi-1 cells in comparison to NuLi-1 at MOIs of 1 and 10 (P < 0.05 and P < 0.01, respectively). Meanwhile, PAO1 induced less IL-6 expression when infecting CuFI-1 cells at an MOI of 1 and 10 (P < 0.01 and P < 0.01, respectively). CuFi-1 cells infected with PAO1 had less IL-8 expression in comparison to infected NuLi-1 cells for all MOIs (P < 0.01), but no differences in IL-1β expression were observed when infecting either cell line with PAO1.ConclusionBronchial epithelial cells with and without functional CFTR appear to respond differently to infections with either P. aeruginosa or S. aureus. Further elucidating how various immune response pathways are impacted by CFTR dysfunction may lead to alternative approaches to therapy to reduce morbidity observed among CF patients.Disclosures All Authors: No reported disclosures

Green Synthesis of Silver Nanoparticles Using Extract of Jasminum officinal L. Leaves and Evaluation of Cytotoxic Activity Towards Bladder (5637) and Breast Cancer (MCF-7) Cell Lines.

IntroductionJasminum officinale L. is a very important medicinal and industrial flowering aromatic plant.MethodsThe present study deals with Jasminum officinale L. leaves extract (JOLE) as a reducing and capping agent for the synthesis of silver nanoparticles (AgNPs) by the green pathway. Phenolic profile of the extract was evaluated using HPLC-PDA/MS/MS technique. Jasminum officinale L. leaves extract silver nanoparticles (JOLE-AgNPs) were characterized by ultraviolet light (UV), Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), zeta potential and X-ray diffraction (XRD). JOLE-AgNPs were examined for their cytotoxic activities by neutral red uptake assay (NRU) against bladder (5637) and breast cancer (MCF-7) cell lines.ResultsHPLC-PDA/MS/MS tentatively identified 51 compounds of different chemical classes. UV spectra showed absorption peak at λmax = 363 nm. The biosynthesized AgNPs were predominantly spherical in shape with an average size of 9.22 nm by TEM. The face cubic center (fcc) nature of silver nanoparticles was proved by XRD diffractogram. JOLE-AgNPs exhibited high cytotoxic activity against 5637 and MCF-7 cell lines compared to the cytotoxic activities of JOLE with IC50 of 13.09 µg/mL and 9.3 µg/mL, respectively.DiscussionThe silver nanoparticles formed by Jasminum officinale L. showed high cytotoxic activities against MCF-7 and 5637 cell lines and can be introduced as a new alternative cytotoxic medication.

Cytotoxicity and Genotoxicity of E-Cigarette Generated Aerosols Containing Diverse Flavoring Products and Nicotine in Oral Epithelial Cell Lines.

Electronic cigarettes are the most commonly used nicotine containing product among teenagers. The oral epithelium is the first site of exposure and our recent work revealed considerable diversity among e-liquids for composition and level of chemical constituents that impact nicotine deposition in a human oral-trachea cast and affect the formation of reactive carbonyls. Here, we evaluate the dose response for cytotoxicity and genotoxicity of e-cigarette-generated aerosols from 10 diverse flavored e-liquid products with and without nicotine compared with unflavored in 3 immortalized oral epithelial cell lines. Three e-liquids, Blue Pucker, Love Potion, and Jamestown caused ≥20% cell toxicity assessed by the neutral red uptake assay. Nine products induced significant levels of oxidative stress up to 2.4-fold quantified by the ROS-Glo assay in at least 1 cell line, with dose response seen for Love Potion with and without nicotine across all cell lines. Lipid peroxidation detected by the thiobarbituric acid reactive substances assay was less common among products; however, dose response increases up to 12-fold were seen for individual cell lines. Micronuclei formation indicative of genotoxicity was increased up to 5-fold for some products. Blue Pucker was the most genotoxic e-liquid, inducing micronuclei across all cell lines irrespective of nicotine status. A potency score derived from all assays identified Blue Pucker and Love Potion as the most hazardous e-liquids. These in vitro acute exposure studies provide new insight about the potential for some flavored vaping products to induce significant levels of oxidative stress and genotoxicity.

An in vitro study of Ocimum sanctum as a chemotherapeutic agent on oral cancer cell-line.

Oral squamous cell carcinoma (OSCC) is the most commom cancer in the world. If remain untreated for several years, it may be fatal. Hence, it is important to prevent and treat OSCC at an early stage. In this study the effect of aqueous and dry leaves extract of Ocimum sanctum was observed on Ca9-22 cell line, which is an OSCC cell line. For this, Ca9-22 cell line was cultured and maintained. After 24 h, the cells were treated with aqueous and dry leaves extract of Ocimum sanctum plant. Viability of the cancerous cells were studied by 3-(4, 5-dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and neutral red uptake (NRU) assay. Minimum inhibitory concentrations (MIC), lethal concentration25 (LC25), lethal concentration50 (LC50) and highest permissive concentration (HPC) was calculated by probit computational method. Experimentally, the MIC value was 5 mg/L, whereas the HPC was 30 mg/L of the plant extract in aqueous state. For the dry extract the MIC was 5 mg/L whereas the HPC was 35 mg/L for both MTT and NRU assays. For MTT assay LC values: 7.41 (LC25), 14.79 (LC50) and 26.91 mg/L (LC75) for aqueous extract and 12.58 (LC25), 20.89 (LC50), 29.51 mg/L (LC75) for dry extract. For NRU assay LC values were 10.23 (LC25), 14.79 (LC50) and 20.89 mg/L (LC75) aqueous extract, and 16.59 (LC25), 23.44 (LC50), 30.19 mg/L (LC75) dry extract of the plant. From the above study it was concluded that, Ocimum sanctum have anti-cancerous activity. It can further be used for therapeutic purposes.

Effects of Incubation of Human Brain Microvascular Endothelial Cells and Astrocytes with Pyridostigmine Bromide, DEET, or Permethrin in the Absence or Presence of Metal Salts.

Gulf War Illness (GWI) is a chronic, multi-symptom illness suffered by over one-third of American military veterans who served in the Persian Gulf War between 1990 and 1991. No current single-exposure scenario accounts for all the symptoms observed in GWI, and instead may be due to a multi-exposure scenario. As a larger effort to understand how one category of multi-exposure scenarios of organic compounds such as nerve gas prophylactic pyridostigmine bromide, or insecticides/pesticides such as N,N-diethyl-m-toluamide (DEET) and permethrin, plus heavy metals found in inhaled dust particles (Al, Fe, Ni, Sr, DU, Co, Cu, Mn, and Zn) might play a role in neural aspects of GWI, we begin this initial study to examine the toxicity and oxidative damage markers of human brain endothelial cell and human astrocyte cell cultures in response to these compounds. A battery of cytotoxicity assessments, including the MTT assay, Neutral Red uptake, and direct microscopic observation, was used to determine a non-toxic dose of the test compounds. After testing a wide range of doses of each compound, we chose a sub-toxic dose of 10 µM for the three organic compounds and 1 µM for the nine metals of interest for co-exposure experiments on cell cultures and examined an array of oxidative stress-response markers including nitric oxide production, formation of protein carbonyls, production of thiobarbituric acid-reactive substances, and expression of proteins involved in oxidative stress and cell damage. Many markers were not significantly altered, but we report a significant increase in nitric oxide after exposure to any of the three compounds in conjunction with depleted uranium.

Toxicological effects and bioaccumulation of fullerene C60 (FC60) in the marine bivalve Ruditapes philippinarum

Fullerene C60 (FC60), with its unique physical properties, has been used in many applications in recent decades. The increased likelihood of direct release into the environment has raised interest in understanding the biological effects of FC60 to aquatic organisms. Nowadays, only few studies have analysed FC60 effects and bioaccumulation in marine organisms following in vivo exposure. To provide new data about FC60 toxicity, Ruditapes philippinarum was selected as target species to assess potential adverse effects of the contaminant. Clams were exposed for 1, 3 and 7 days to predicted environmental concentrations of FC60 (1 and 10 μg/L) and cellular and biochemical responses were evaluated in clams’ gills, digestive gland and haemolymph. The FC60 content in gills and digestive gland was determined in all experimental conditions after 7 days of exposure. Results showed an increase in oxidative stress. In particular, a significant modulation in antioxidant enzyme activities, and changes in glutathione S-transferase activity were observed in gills. Moreover, damage to lipids and proteins was detected in FC60-treated (10 µg/L) clams. In digestive gland, slighter variations in antioxidant enzyme activities and damage to molecules were detected. CAT activity was significantly affected throughout the exposure, whereas damage to lipids was evident only at the end of exposure. FC60 accumulation was revealed in both gills and digestive gland, with values up to twelve-fold higher in the latter. Interestingly, haemolymph parameters were slightly affected by FC60 compared to the other tissues investigated. Indeed, only Single Cell Gel Electrophoresis and Neutral Red uptake assays showed increased values in FC60-exposed clams. Moreover, volume and diameter of haemocytes, haemocyte proliferation, and micronucleus assay highlighted significant variations in treated clams, but only in the first phases of exposure, and no changes were detected after 7 days. Our results suggested clam gills as the target tissue for FC60 toxicity under the exposure conditions tested: the high damage detected to lipids and proteins could contribute to long-term problems for the organism.

English

In vitro antiviral activity of three ellagitannins - castalagin, vescalagin and grandinin was investigated against replication of three of the most common herpes viruses infecting animals, namely bovine herpesvirus-1 (BoHV-1), suid alphaherpesvirus-1 (SuHV-1), and caprine herpesvirus-1 (CapHV-1). The cytopathic effect inhibition test via the neutral red uptake assay in Madin-Darby bovine kidney (MDBK) cell culture was applied. The highest activity was demonstrated by castalagin followed by vescalagin against replication of SuHV-1, strain A2, with selectivity index values of 336.8 and 309, respectively, which are close to that of acyclovir (SI = 540). The activity of grandinin against the replication of SuHV-1 A2 strain (SI = 40.8) as well as the activity of all three substances against the replication of BoHV-1 strain K22 was lower but still significant (castalagin SI = 45; vescalagin SI = 42.5; grandinin SI = 32.3). Against the E/CH strain of CapHV-1, the three ellagitannins showed a moderate to weak activity: Castalagin SI = 19.3, vescalagin SI = 18.8, grandinin SI = 11.8. The results obtained characterize compounds tested as perspective antivirals. Key words: bovine herpesvirus-1, suid alphaherpesvirus-1, caprine herpesvirus-1, ellagitannins, alpha acyclovir, antiviral activity.

Acute Effect of Electronic Cigarette-Generated Aerosol From Flavored CBD-Containing Refill Solutions on Human Bronchial Epithelial Cells.

Introduction: Although electronic cigarettes (e-cigarettes) were originally developed to deliver aerosolized nicotine to lungs, recent data have shown that consumers also use them for inhalation of other drugs, including cannabidiol (CBD). The aim of this study was to test the acute inhalation toxicity of flavored CBD-containing aerosols emitted from e-cigarettes. Methods: Bronchial epithelial cells (H292) cells were exposed to aerosol generated from e-cigarettes refilled either with (1) propylene glycol solvent only (PG, control), (2) commercially purchased unflavored solution with CBD, or (3) commercially purchased solutions with and without CBD and with different flavors. The in vitro toxicological effects were assessed using the following methods: (1) trypan blue exclusion assay (cell viability), (2) neutral red uptake assay (metabolic activity), and (3) ELISA (concentrations of inflammatory mediators). Results: Most flavored products with or without CBD were cytotoxic as compared to the air control. Overall, aerosols with CBD were more cytotoxic than aerosols without CBD irrelevant of the flavoring used in the product. Although, unflavored aerosols containing CBD in PG were significantly more cytotoxic than aerosols containing only PG, not all flavored products containing CBD were significantly more toxic than the same flavored products without CBD. Most CBD containing products significantly increase the concentration of cytokines released as compared to the same flavored products without CBD. Conclusion: Different flavors show different cytotoxic effects in CBD-containing e-cigarettes. Aerosols emitted from CBD containing e-cigarettes were more cytotoxic than those emitted from CBD-free e-cigarettes.

A Novel Bioactive Glass Containing Therapeutic Ions with Enhanced Biocompatibility.

A novel bioactive glass containing therapeutic ions with enhanced biocompatibility was designed and produced by the classical melt-quenching route. Starting from a very promising composition (Bio_MS), which combined bioactivity and high crystallization temperature, the ratio between some oxides was tailored to obtain a new and more reactive (in terms of dissolution rate) bioactive glass, called BGMSN (composition in mol%: 6.1 Na2O, 31.3 CaO, 5 MgO, 10 SrO, 2.6 P2O5, 45 SiO2). The aim of this work was to produce a bioactive glass with a good biological performance, preserving, at the same time, the high crystallization temperature achieved for Bio_MS; this is strategic in order to avoid undesired crystalline phases during thermal treatments, which can undermine the bioactivity and even the stability of final products. A complete characterization of the novel bioactive glass was performed in terms of thermal, mechanical and biological properties and in vitro bioactivity. The thermal behavior of the bioactive glass was studied by heating microscopy, differential thermal analysis (DTA) and optical dilatometry; BGMSN showed a very high crystallization temperature and a high sinterability parameter, thus being suitable for applications where thermal treatments are required, such as sintered samples, coatings and scaffolds. Mechanical properties were investigated by the micro-indentation technique. The in vitro biological properties were evaluated by means of both direct and indirect cell tests, i.e., neutral red (NR) uptake and MTT assay, using murine long bone osteocyte Y4 (MLO-Y4) cells: the cellular viability of BGMSN was higher compared to cellular viability of 45S5, both in direct and indirect tests. Finally, the in vitro bioactivity test by soaking samples in simulated body fluid (SBF) showed high dissolution rate, with a good rate of formation of hydroxyapatite.

Theoretical and experimental electronic transition behaviour study of 2-((4-(dimethylamino)benzylidene)amino)-4-methylphenol and its cytotoxicity

In this study, a Schiff base (2-((4-(dimethylamino)benzylidene)amino)-4-methylphenol; 7S2) has been synthesized and characterized with 1H, 13C-NMR, IR and elemental analysis methods. An electronic transition behaviour of the Schiff base has been investigated in the different eight solvents by UV-Vis. spectroscopy. The stable geometry of 7S2 has been determined by DFT method with Gaussian09 program (B3LYP/6-311G(2d, p)). 7S2 has been analysed for its target region selection using the SwissTarget program. A docking study has been performed against DHODH protein while its pharmacokinetic properties has been evaluated using SwissADME, Osiris and Molinspiration. 7S2 has important pharmacokinetic and drug-likeness properties against other FDA approved DHODH inhibitors. Further, 7S2 has been tested with the neutral red uptake assay and found cytotoxic for several cancer cell lines, having IC50 values ranged between 18-23 µg/mL. According to the pharmacokinetic properties and cytotoxic activities, we suggest that 7S2 may have a potential as an anticancer drug.

Smoke Chemistry, In Vitro Cytotoxicity, and Genotoxicity Demonstrates Enhanced Toxicity of Cigarillos Compared With Cigarettes

There has been limited toxicity testing of cigarillos, including comparison to cigarettes. This study compared the smoke chemistry and the cytotoxic and genotoxic potential of 10 conventional cigarettes and 10 cigarillos based on the greatest market share. Whole smoke and total particulate matter (TPM) were generated using the Canadian Intense and International Organization for Standardization puffing protocols. Tobacco-specific nitrosamines, carbonyls, and polycyclic aromatic hydrocarbons were measured using gas chromatography-mass spectrometry. TPM smoke extracts were used for the in vitro assays. Cytotoxicity was assessed in human bronchial epithelial continuously cultured cell line cells using the neutral red uptake assay. Genotoxic potential was assessed using the micronucleus (human lung adenocarcinoma continuously cultured cell line cells), Ames, and thymidine kinase assays. TPM from all cigarillos tested was more cytotoxic than cigarettes. Micronucleus formation was significantly greater for cigarillos compared with cigarettes at the highest dose of TPM, with or without rat liver S9 fraction. In the Ames test +S9, both tobacco products exhibited significant dose-dependent increases in mutation frequency, indicating metabolic activation is required for genotoxicity. In the thymidine kinase assay +S9, cigarillos showed a significantly enhanced mutation frequency although both tobacco products were positive. The levels of all measured polycyclic aromatic hydrocarbons, tobacco-specific nitrosamines, and carbonyls (except acrolein) were significantly greater in cigarillos than cigarettes. The Canadian Intense puffing protocol demonstrated increased smoke constituent levels compared with International Organization for Standardization. Even though the gas vapor phase was not tested, the results of this study showed that under the tested conditions the investigated cigarillos showed greater toxicity than comparator cigarettes. This study found that there is significantly greater toxicity in the tested U.S. marketed cigarillos than cigarettes for tobacco constituent levels, cytotoxicity, and genotoxicity. These findings are important for understanding the human health toxicity from the use of cigarillos relative to cigarettes and for building upon knowledge regarding harm from cigarillos to inform risk mitigation strategies.

Evaluation of cellular safety and the chemical composition of the peanut (Arachis hypogaea L.) ethanolic extracts

Arachis hypogaea L. (Leguminosae) is distributed in tropical and subtropical areas. Peanut has high nutritional and commercial value. Scientific research showed that peanut has biological properties such as anticancer, antioxidant, antiinflammatory. However, it is necessary to know if consumption of peanut, either as food or as a phytopharmaceutical implies a health risk. The aim was to evaluate cytotoxicity and genotoxicity of ethanolic extracts from A. hypogaea. Also, chemical characterization of these extracts was performed. Cytotoxicity was evaluated by MTT and Neutral Red Uptake (NRU) assays on Vero cells. Genotoxicity was studied by Micronuclei and comet assays on Balb/C mice. Qualitative and quantitative chemical analysis of extracts were performed. Results showed that extracts have low cytotoxicity. Tegument ethanolic extract (TEE) and Seed ethanolic extract (SEE) were not genotoxic. The treatments with TEE at 250 mg/kg and SEE at 2000 mg/kg revealed (highest concentrations evaluated) some toxicity on blood marrow cells of mice. Chemical characterization indicated that TEE had 74.33 ± 1.10 mg GAE/g of dried extract and SEE had 15.05 ± 0.06 mg GAE/g of dried extract of total phenolic content. Also, proanthocyanidins (O.D. at 550 nm 1.39 ± 0.15) and caffeic acid (2.46%) were identified in TEE. While, linoleic acid (58.84%) oleic acid (11.31%) and palmitic acid (8.37%) were major compounds of SEE. In conclusion, peanut consumption is safe at concentrations recommended for healthy uses, such as nutrition, and phytomedicine.

Methylated Xanthones from the Rootlets of Metaxya rostrata Display Cytotoxic Activity in Colorectal Cancer Cells.

The tree fern Metaxya rostrata (Kunth) C. Presl is common in the rainforests of Central and South America, where suspensions of the dried rhizome are traditionally used to treat intestinal diseases. Two compounds from this plant, 2-deprenyl-rheediaxanthone B (XB) and 2-deprenyl-7-hydroxy-rheediaxanthone B (OH-XB), have been shown to be biologically highly active against colorectal cancer (CRC) cells in previous studies. The current investigation resulted in the isolation of the previously undescribed methylated xanthones 2-deprenyl-6-O-methyl-7-hydroxy-rheediaxanthone B, 2-deprenyl-5-O-methyl-7-methoxy-rheediaxanthone B, 2-deprenyl-5-O-methyl- 7-hydroxy-rheediaxanthone B and 2-deprenyl-7-methoxy-rheediaxanthone B. All compounds were isolated by column chromatography, structures were elucidated by one- and two-dimensional NMR-experiments and the identities of the compounds were confirmed by LC-HRMS. In logarithmically growing SW480 CRC cell cultures, cytotoxicity by neutral red uptake and MTT assays as well as caspase activation was analyzed. Cellular targets were examined by Western blot, and topoisomerase I (topo I) inhibition potential was tested. Comparing the structure-activity relationship with XB and OH-XB, the monomethylated derivatives showed qualitatively similar effects/mechanisms to their nonmethylated analogues, while dimethylation almost abolished the activity. Inhibition of topo I was dependent on the presence of an unmethylated 7-OH group.

Skin Protective Activity of LactoSporin-the Extracellular Metabolite from Bacillus Coagulans MTCC 5856

Background: Probiotics and their products are increasingly used in skincare in recent years. Postbiotics are defined as any substance derived through the metabolic activity of a probiotic microorganism, which exerts a direct or indirect beneficial effect on the host. The extracellular metabolites of probiotic bacteria have antimicrobial activities, protect against acne, and improve skin condition. We studied skin protective activities of the extracellular metabolite (LactoSporin) of a spore-forming probiotic Bacillus coagulans MTCC 5856 in vitro. Methods: LactoSporin was evaluated for antioxidant activity by free radical scavenging activity and reactive oxygen quenching activity in human dermal fibroblast cells. Protection of fibroblasts from UV-induced apoptosis and cell death was studied by flow cytometry and neutral red uptake assays. Enzyme inhibition assays were carried out for collagenase, Elastase, and Hyaluronidase. Gene expression studies were carried out using polymerase chain reaction. Results: LactoSporin showed antioxidant activity and was found to protect skin cells from UV-induced apoptosis and cell death. LactoSporin inhibited collagenase, elastase, and hyaluronidase activity and upregulated the expression of hyaluronan synthase, transforming growth factor and epidermal growth factor, which are associated with extracellular matrix integrity. Conclusions: These results suggest LactoSporin is a skin protective postbiotic with wide application in cosmetic formulations.

Lens-Free Imaging as a Sensor for Dynamic Cell Viability Detection Using the Neutral Red Uptake Assay.

Neutral red is a low-cost supravital stain for determining cell viability. The standard protocol relies on a destructive extraction process to release the accumulated dye for endpoint spectrophotometric quantification. We report a non-destructive, live-cell quantification of neutral red uptake using a compact lens-free system. Two light sources indentify the cell perimeter and quantify neutral red uptake. The quantification occurs during staining, thus eliminating the destructive extraction process and reducing assay time. Our system enables live quantification for continuous high-throughput screening of cell viability within confined spaces such as incubators.

Isothymusin, a Potential Inhibitor of Cancer Cell Proliferation: An In Silico and In Vitro Investigation.

Since centuries plant-based compounds are known for the treatment of cancer in both traditional and contemporary medicine. The problems like target non-specificity and toxicity are well-known regarding anticancer drugs. Therefore, target specific search of novel entities is constant. Isothymusin is a dimethoxy, trihydroxy flavone present in plants like Ocimum sanctum, and Limnophilla geoffrayi. There are limited reports available on the anticancer potential of isothymusin. The effects of isothymusin on redox status, cell cytotoxicity, and targets involved in the promotion and progression of the cancer cells have been investigated. Antiproliferative efficacy was evaluated by MTT, Neutral Red Uptake, and Sulforhodamine-B assays. The spectrophotometric methods were adopted to study the effect against selected targets. Redox activity was assessed by in vitro antioxidant assays and the interaction study, ADMET profiling, and toxicity assessments were done in silico. Isothymusin scavenges the radicals, i.e., DPPH and nitric oxide with moderate ferric reducing potential. It affected the proliferation of leukemia, colon, skin, and breast cancer cell lines by more than 50% but moderately affected prostate, kidney, lung, hepatic, and breast adenocarcinoma (up to 48%). Isothymusin inhibited the enzymes associated with the promotion stage of cancer, including cycloxygenase- 2 and lipoxygenase-5. Additionally, it also inhibited the activity of proliferation markers like cathepsin- D, dihydrofolate reductase, hyaluronidase, and ornithine-decarboxylase. Besides, in silico studies supported the in vitro enzyme inhibition assays outcome. Toxicity studies showed promising results of chemical descriptors and non-skin-irritant, moderate ocular-irritancy, and in vitro Ames test confirmed non-mutagenic nature. Isothymusin showed radical scavenging and anti-proliferative activities, which may be taken up as a phytochemical lead for the synthesis of analogues possessing enhanced anticancer potential.

Anti-cancer efficacy of Aloe vera capped hematite nanoparticles in human breast cancer (MCF-7) cells

We have applied a green approach employing the Aloe vera bioactives to encapsulate the hematite nanoparticles (ALE-α-Fe2O3NPs), and invested its potential to kill human breast cancer cells (MCF-7). Cytotoxicity analysis by [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)] (MTT) and neutral red uptake (NRU) assays showed 36% and 33% reduction in cell viability at 100 μg/ml of ALE-α-Fe2O3NPs. Relative to untreated control, flow cytometric (FCM) analysis revealed 68.97% greater internalization of ALE-α-Fe2O3NPs in MCF-7 cells. FCM study further confirmed ALE-α-Fe2O3NPs induced reduction in mitochondrial membrane potential (ΔΨm), and increase in intracellular reactive oxygen species (ROS). MCF-7 cells treated with ALE-α-Fe2O3NPs demonstrated DNA damage quantified by the comet assay. MCF-7 cells explicitly showed an increase in sub-G1 peak (36.3%) at 100 μg/ml of ALE-α-Fe2O3NPs, confirming apoptotic cell death. qPCR data of apoptotic genes (p53, bax, caspase-3 and caspase-9) in MCF-7 cells showed enhanced upregulation upon ALE-α-Fe2O3NPs exposure. Overall, our data give a comprehensive, safe, cost-effective, and scalable biogenic nano-formulation of ALE-α-Fe2O3NPs for its putative clinical applications as anti-cancer drug.

Anti-inflammatory and antinociceptive activity profile of a new lead compound – LQFM219

LQFM219 is a molecule designed from celecoxibe (COX-2 inhibitor) and darbufelone (inhibitor of COX-2 and 5-LOX) lead compounds through a molecular hybridisation strategy. Therefore, this work aimed to investigate the antinociceptive and anti-inflammatory activities of this new hybrid compound. The acute oral systemic toxicity of LQFM219 was evaluated via the neutral red uptake assay. Acetic acid-induced abdominal writhing and CFA-induced mechanical hyperalgesia were performed to evaluate the antinociceptive activity, and the anti-oedematogenic activity was studied by CFA-induced paw oedema and croton oil-induced ear oedema. Moreover, the acute anti-inflammatory activity was determined by carrageenan-induced pleurisy. In addition, cell migration, myeloperoxidase enzyme activity, and TNF-α and IL-1β levels were determined in pleural exudate. Moreover, a redox assay was conducted using electroanalytical and DPPH methods. The results demonstrated that LQFM219 was classified as GHS category 4, and it showed better free radical scavenger activity compared to BHT. Besides, LQFM219 decreased the number of writhings induced by acetic acid and the response to the mechanical stimulus in the CFA-induced mechanical hyperalgesia test. Furthermore, LQFM219 reduced oedema formation, cell migration, and IL-1β and TNF-α levels in the pleural cavity and inhibited myeloperoxidase enzyme activity. Thus, our study provides that the new pyrazole derivative, LQFM219, demonstrated low toxicity, antinociceptive and anti-inflammatory potential in vitro and in vivo.

Guidelines for cell viability assays

AbstractRecently, the interest in the application of cell viability assays has been increasing in various fields. Cell viability assays may be broadly classified as (a) dye exclusion assays, (b) colorimetric assays, (c) fluorometric assays, (d) luminometric assays, and (e) flow cytometric assays. Dye exclusion assays include trypan blue, eosin, congo red, and erythrosine B stain assays, whereas 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5 diphenyl tetrazolium bromide (MTT), 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium (MTS), 2,3‐bis‐(2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐2H‐tetrazolium‐5‐carboxanilide (XTT), 2‐(4‐iodophenyl)‐3‐(4‐nitrophenyl)‐5‐(2,4‐disulfophenyl)‐2H tetrazolium, monosodium salt (WST‐1), 2‐(2‐methoxy‐4‐nitrophenyl)‐3‐(4‐nitrophenyl)‐5‐(2,4‐disulfophenyl)‐2H‐tetrazolium, monosodium salt (WST‐8), lactate dehydrogenase (LDH), sulforhodamine B (SRB), neutral red uptake (NRU), and crystal violet stain (CVS) assays are among the colorimetric assays. Similarly, resazurin and 5‐carboxyfluorescein diacetate acetoxymethyl ester (5‐CFDA‐AM) assays are based on fluorometric measurements, whereas luminometric assays comprise adenosine triphosphate and real‐time viability assays. Major flow cytometric assays include membrane asymmetry, membrane permeability, and mitochondria assays. In this guideline, the mechanisms and the practice of assessment of the most common cell viability assays applied in research labs are discussed in detail. An ideal cell viability assay should be safe, rapid, reliable, efficient, and time‐ and cost‐effective, and should not interfere with the test compound. Overall, it can be concluded that more than one cell viability assay should be applied in order to obtain reliable results.