Alamar Blue Dye Research Articles

Multiorgan-on-a-chip for cancer drug pharmacokinetics-pharmacodynamics (PK-PD) modeling and simulations.

Cancer is one of the most common and fatal diseases worldwide and kills millions of people every year. Cancer drug resistance, lack of efficacy, and safety are significant problems in cancer patients. A multiorgan-on-a-chip (MOC) device consisting of breast and liver compartments was designed with AutoCAD software. The MOC molds were printed by a Formlabs Form 2 3D printer. MDA-MB-231, HepG2, and MCF-10A cells were used for the MOC experiments. The cell lines were cultured at 37°C with 5% CO2, and cell viability was assessed via Alamar blue dye to generate pharmacodynamics (PD) data. Drug concentrations from the cell culture media were analyzed via Agilent 1260 Infinity II HPLC with a Waters Symmetry C18 column and used to generate pharmacokinetics (PK) data. The PK and PD data were modeled and simulated by Monolix and Simulix software, respectively. The safety and efficacy of drug dosing regimens were compared, and the best dosing regimens were selected. This research designed and fabricated a unique MOC consisting of liver and breast compartments that overcomes the need for sealing or assembling. It was used for PK-PD modeling and simulations, and its functionality was proven experimentally. The new MOC will be helpful in preclinical trials to evaluate the efficacy and safety of drugs.

Green synthesis and in-vitro screening of 3-((4-(arylideneamino)phenyl) imino)indolin-2-one derivatives antimycobacterial agents

In this research article, we attempted to synthesize some new isatin derivatives, 3-((4-(arylideneamino)phenyl)imino)indolin-2-ones (2a-2g) by Schiff\'s base reaction under microwave technique. The structural characterizations of synthesized compounds were done by using different analytical techniques such as IR, 1HNMR, mass spectrometry, and elemental analysis. We aim to develop new compounds with improved antimycobacterial activities. These title compounds were screened for their antimycobacterial activity against the Mycobacterium tuberculosis H37Rv strain by using the microplate Alamar blue dye assay (MABA) method. All title compounds showed significant antimycobacterial activity. Compound 2g was found most potent compound with a MIC value of 6.25 µg/mL among all tested compounds when compared with standard drugs streptomycin (6.25µg/mL) and isoniazid (6.25 µg/mL).

Microplate Alamar Blue assay for detecting anti-tubercular action of Albizia amara leaves

Albizia amara (Roxb.) B. Boivin has been used historically for the treatment of septicaemia, malignancy, delirium, and convulsions. A decoction made from the bark is used to treat rheumatism, hemorrhage, and some bleeding disorders during pregnancy, as well as stomach pain and nasal issues. Albizia has active ingredients like flavonoids, alkaloids, steroids, phenolic compounds, and tannins, according to early phytochemical screening. Flavonoids and phenolic compounds have been shown to play an important role in anti-tubercular activity in previous research. This led us to investigate whether or not this herb possessed anti-tubercular properties. Mycobacterium tuberculosis was found to be sensitive to concentrations of Alamar Blue dye as low as 25 μg/ml when the anti-tubercular activities of petroleum ether, methanol, and an ethanolic extract were tested. The anti-tubercular activity of the extract and fractions is comparable to that of streptomycin, pyrazinamide, and ciprofloxacin. HPTLC has shown that flavonoids and phenolic substances are present, which may explain the activity that can be seen. Taking this research on Albizia amara leaves one step further could lead to the development of a natural medicine that fights TB that is safe and works well.

107 Assessment of in Vitro Toxicity of Inhalable Incidental Nanoparticles Originating in the Ceramic Industry

Abstract Assessing exposure to nanomaterials in industrial workplaces is a widespread challenge. Nanoparticle release was evaluated, under real-world operating conditions, in one thermal spraying and one tile firing facility. Nanoparticles were characterised in terms of mass and number concentrations (µg/m3; /cm3), particle size distribution, chemical composition and morphology, and in vitro toxicity. The present work targets especially the toxicological characterisation of the aerosols sampled. Particles <2 microns in size were sampled in cell-culture medium using an SKC Biosampler connected to a pump operating at 12 lpm. Samples were conserved frozen at -20ºC until analysis. Submerged-cell cultures using the commercially-available A549 cell line were used for the in-vitro assays, where cell viability and generation of reactive-oxygen species (ROS) were assessed. Preliminary results evidenced a relatively large variability across samples in terms of cell viability, with reductions ranging between <5% and up to 60% after 2-hour exposures. Results suggested that the initial INP concentration in the samples may play a relevant role in the results, as in highly concentrated samples nanoparticles may interfere with the cellular uptake of the Alamar Blue dye used and, subsequently, on fluorescence results. ROS generation was detected increasingly over time during the test period (1-4 hours). Readings obtained after 3-4 hour became mostly stable. Finally, these results provide the baseline for the subsequent assessment of the expected reduction in INP concentrations and toxicity resulting from the implementation of risk mitigation measures, which will focus on a custom-designed air filtration system developed in the framework of this project.

Photocatalytic activity of biosynthesized silver nanoparticle fosters oxidative stress at nanoparticle interface resulting in antimicrobial and cytotoxic activities.

Inside the biological milieu, nanoparticles with photocatalytic activity have potential to trigger cell death non-specifically due to production of reactive oxygen species (ROS) upon reacting with biological entities. Silver nanoparticle (AgNP) possessing narrow band gap energy can exhibit high light absorption property and significant photocatalytic activity. This study intends to explore the effects of ROS generated due to photocatalytic activity of AgNP on antimicrobial and cytotoxic propensities. To this end, AgNP was synthesized using the principle of green chemistry from the peel extract of Punica granatum L., and was characterized using UV-Vis spectroscope, transmission electron microscope and x-ray diffraction, and so forth. The antimicrobial activity of AgNP against studied bacteria indicated that, ROS generated at AgNP interface develop stress on bacterial membrane leading to bacterial cell death, whereas Alamar Blue dye reduction assay indicated that increased cytotoxic activity with increasing concentrations of AgNP. The γH2AX activity assay revealed that increasing the concentrations of AgNP increased DNA damaging activity. The results altogether demonstrated that both antimicrobial and cytotoxic propensities are triggered primarily due interfacial ROS generation by photocatalytic AgNP, which caused membrane deformation in bacteria and DNA damage in HT1080 cells resulting in cell death.

Antitubercular study on stem bark of Albizia procera (ROXB.) BENTH using Microplate Alamar Blue assay (MABA)

Albizia procera commonly known as white siris is a large deciduous tree of the family Mimosideae, widely distributed throughout India. Traditionally the plant is used in convulsions, pain, delirium, cancer and septicemia. The decoction of bark is given for rheumatism, hemorrhage and is considered useful in treating pregnancy problems, for stomach ache and sinus.The preliminary phytochemical screening of ethanol extract of Albizia procera shows the presence of active constituents like flavonoids, phenolic compounds and tannins. Earlier studies show the flavonoids and phenolic compounds play major role in antitubercular activity. Based on this, we made an attempt to evaluate the antitubercular potential of this plant. The ethanolic extract and its fractions (Hexane and Ethyl acetate) were investigated for antitubercular activity using Alamar Blue dye method (MABA), in which Mycobacterium tuberculosis was sensitive upto 3.12μg/ml.The extract and fractions show better anti tubercular activity than streptomycin and potentially equal to pyrazinamide and ciprofloxacin. The observed activity may be due to the presence of flavonoids and phenolic compound which is confirmed by HPTLC. This investigation on stem bark of Albizia procera has the potential to be developed further into a natural Anti-TB drug.

Novel method for quantifying cells on carriers and its demonstration during SARS-2 vaccine development.

The most effective way to prevent and control infectious disease outbreak is through vaccines. The increasing use of vaccines has elevated the need to establish new manufacturing strategies. One of the major approaches is cell-based production, which creates a need for high cell density to enable higher cell production levels. This has led to development of the technology of cell carriers, including micro and macro cell carriers. To follow the production process, quantifying the number of cells on these carriers is required, as well as the tracking of their viability and proliferation. However, owing to various carriers' unique structures, tracking the cell's is challenging using current traditional assays that were originally developed for monolayers of adherent cells. The current "gold standard" method is counting cell nuclei, separating cells from the carrier, staining with crystal violet, and visually counting under a microscope. This method is tedious and counts both live and dead cells. A few other techniques were developed but were specific to the carrier type and involved specialized equipment. In this study, we describe a broadly ranging method for counting cells on carriers that was developed and employed as part of the development of severe acute respiratory syndrome coronavirus 2 vaccine. The method is based on the Alamar blue dye, a well-known, common marker for cell activity, and was found to be successful in tracking cell adsorption, cell growth, and viability on carriers. No separation of the cells from the carriers is needed, nor is any specialized equipment; the method is simple and rapid and provides comprehensive details necessary for process control of viral vaccine production in cells. This method can be easily implemented in any of a number of cell-based processes and other unique platforms for measuring the growth of encapsulated cells.

A Simple Method for in situ Quantification of Cells on Carriers

The technology of cell carriers was developed as a response to the need for high cell density to enable higher production levels in cell-based production processes. To follow the production process, quantifying the number of cells on these carriers is required, as well as tracking their viability and proliferation. However, owing to various carriers' unique structures, tracking the cells is challenging using current traditional assays that were originally developed for monolayers of adherent cells. The current "gold standard" method is counting cell nuclei, which is tedious and counts both live and dead cells. A few other techniques have been developed, but they are all specific to a carrier type and involve specialized equipment. Here, we describe a broad ranging method for counting cells on carriers. The method is based on the Alamar blue dye, a well-known, common marker for cell activity. No separation of the cells from the carriers is needed, nor is any specialized equipment. The method is simple and rapid, and provides comprehensive details necessary for control of production processes in cells. This method can be easily implemented in any cell-based process and other unique platforms for measuring growth of cells. Graphic abstract: Schematic of the in situ quantification method.

Anti-VCAM-1 and Anti-IL4Rα Aptamer-Conjugated Super Paramagnetic Iron Oxide Nanoparticles for Enhanced Breast Cancer Diagnosis and Therapy.

The surface protein overexpressed on cancer cells can be used as biomarkers for early detection of specific diseases. Anti-VCAM-1 and anti-IL4Rα DNA aptamers specific to VCAM-1 and IL4Rα receptors that are overexpressed in 4T1 tumor-bearing mice could be used as potential biomarker for both diagnostic and therapeutic applications in cancer biology. Cell Viability and luciferase assay of 4T1-Luc2 cancer cells in the presence of anti-VCAM-1 ssDNA or anti-IL4Rα RNA aptamers was assessed by monitoring the changes in the absorbance and the fluorescence of Alamar blue dye. The aptamer-conjugated SPIO magnetic beads, used for the selective targeting to tumor sites, were monitored using noninvasive MRI and Bioluminescence imaging (BLI). Cell viability and luciferase assays showed that both anti-VCAM-1 and anti-IL4Rα aptamers favor the depletion of cancer cells and limit tumor progression. Microscopic analyses confirmed that the target specific aptamers significantly trigger tumor cell apoptosis and limit cancer cell growth in vitro. The intravenous injection of SPIO nanoparticle-conjugated aptamers were further confirmed using noninvasive MRI and Bioluminescence imaging. Anti-VCAM1 and anti-IL4Rα aptamers, specific to VCAM-1 and IL4Rα receptors overexpressed in 4T1-Luc2 tumor-bearing mice, were used as diagnostic and therapeutic tools.

Inhibitory effect of novel Eugenol Tosylate Congeners on pathogenicity of Candida albicans

BackgroundThe global prevalence of fungal diseases is increasing rapidly, which affects more than a billion people every year with significant mortality rate. On the other hand, the development of new drugs to treat these fungal infections is slow, while the current antifungal therapy is insufficient and associated with adverse side effects and emerging multidrug resistance. Therefore, development of novel antifungal drugs with least or no toxicity and multi-target mechanisms of action is an immediate priority. Natural products have long been known to possess antimicrobial activities and are source of new drugs. Currently, modifying natural products to synthesize derivatives/analogues are of great scientific focus for discovering novel drugs with improved potency and safety. Modifications in eugenol to synthesize eugenol derivatives with enhanced antifungal activity have already been reported.MethodsIn this study, three most active novel eugenol tosylate congeners (ETC-5, ETC-6 and ETC-7) were selected from our previous study to investigate their effect on major virulence factors of Candida albicans which include adherence, morphogenesis, hydrolytic enzymes secretion, biofilm formation and on expression of genes related to these virulence factors. Adherence and biofilm formation were studied by alamarBlue dye and XTT reduction assays respectively, hydrolytic enzyme secretion was evaluated by plate assays. Further, morphological transition was monitored microscopically and RT-qPCR was used to assess the gene expression levels.ResultsETCs significantly inhibited adherence in C. albicans with an inhibition range of 16–66%, and completely inhibited the morphogenesis at MIC values. Inhibition of proteinase and phospholipase activity was in the range of 2–48% and 8–34% respectively. Test compounds also significantly inhibit biofilm formation in C. albicans in the range of 7–77%. Furthermore, RT-qPCR results indicated a significant down regulation in expression levels of genes (ALS1, ALS2, ALS3, ALS9, CPH1, HWP1, SAP1, SAP2, SAP3 and PLB1) in C. albicans cells after treated with ETCs.ConclusionThe results indicated that these novel ETCs target major virulence factors of C. albicans and avert this commensal microbe to turn into pathogenic. However, further in-depth studies may facilitate the mechanisms involved by ETCs in targeting these virulence factors.

5-(4-Bromobenzyl)-4-(4-(5-phenyl-1,3,4-oxadiazole-2-yl)phenyl)-2,4-dihydro-3H-1,2,4-triazole-3-one: Synthesis, characterization, DFT study and antimicrobial activity

Since molecules containing 1,3,4-oxadiazoles and 1,2,4-triazole heterocyclic rings in their structures have important biological activities; 5-(4-Bromobenzyl)-4-(4-(5-phenyl-1,3,4-oxadiazole-2-yl)phenyl)-2,4-dihydro-3H-1,2,4-triazole-3-one (II) was synthesized, the synthesized compound was characterized by using NMR and IR spectroscopic methods. HOMO and LUMO energies, geometrical structure parameters, IR parameters and NMR chemical shift values were calculated by using DFT method. IR and NMR experimental values were compared with DFT results. Due to the differences between experimental IR and NMR spectral values and theoretical ones, N–H⋯O type intermolecular strong hydrogen bond was recommended. Antimicrobial activities of the synthesized compound II was determined against fifteen different bacteria and the Leishmania major species by microdilution broth assay with Alamar Blue Dye. Compound II was found to be low effective on three different bacterial species (Minimum Inhibitory Concentration (MIC):5000 μg/ml) and had the high antileishmanial activity (MIC: 1250 μg/mL). In order to use the synthesized compound as a drug candidate, control studies should be performed in experimental animal models in vivo.

Nanosatellites for Biology in Space: In Situ Measurement of Bacillus subtilis Spore Germination and Growth after 6 Months in Low Earth Orbit on the O/OREOS Mission.

We report here complete 6-month results from the orbiting Space Environment Survivability of Living Organisms (SESLO) experiment. The world’s first and only long-duration live-biology cubesat experiment, SESLO was executed by one of two 10-cm cube-format payloads aboard the 5.5-kg O/OREOS (Organism/Organic Exposure to Orbital Stresses) free-flying nanosatellite, which launched to a 72°-inclination, 650-km Earth orbit in 2010. The SESLO experiment measured the long-term survival, germination, metabolic, and growth responses of Bacillus subtilis spores exposed to microgravity and ionizing radiation including heavy-ion bombardment. A pair of radiation dosimeters (RadFETs, i.e., radiation-sensitive field-effect transistors) within the SESLO payload provided an in-situ dose rate estimate of 6–7.6 mGy/day throughout the mission. Microwells containing samples of dried spores of a wild-type B. subtilis strain and a radiation-sensitive mutant deficient in Non-Homologoous End Joining (NHEJ) were rehydrated after 14, 91, and 181 days in space with nutrient medium containing with the redox dye alamarBlue (aB), which changes color upon reaction with cellular metabolites. Three-color transmitted light intensity measurements of all microwells were telemetered to Earth within days of each 24-hour growth experiment. At 14 and 91 days, spaceflight samples germinated, grew, and metabolized significantly more slowly than matching ground-control samples, as measured both by aB reduction and optical density changes; these rate differences notwithstanding, the final optical density attained was the same in both flight and ground samples. After 181 days in space, spore germination and growth appeared hindered and abnormal. We attribute the differences not to an effect of the space environment per se, as both spaceflight and ground-control samples exhibited the same behavior, but to a pair of ~15-day thermal excursions, after the 91-day measurement and before the 181-day experiment, that peaked above 46 °C in the SESLO payload. Because the payload hardware operated nominally at 181 days, the growth issues point to heat damage, most likely to component(s) of the growth medium (RPMI 1640 containing aB) or to biocompatibility issues caused by heat-accelerated outgassing or leaching of harmful compounds from components of the SESLO hardware and electronics.

Phyto-fabricated ZnO nanoparticles from Canthium dicoccum (L.) for antimicrobial, anti-tuberculosis and antioxidant activity

Abstract Bioactivity exhibited by nanoparticles has been interesting for researchers globally. The phytogenic synthesis of zinc oxide nanoparticles (ZnO-NPs) has been an eco-friendly approach due to its low toxicity and biological potential. In this pretext, the present study has been conducted. The bacterial inhibition by ZnO-NPs could be attributed to its high specific surface charge and reactive oxygen species generation. The present study states the phyto-fabrication of ZnO-NPs employing aqueous leaf extract of Canthium dicoccum (L.). The synthesized nanoparticles (NPs) displayed characteristic excitation at 293 nm. The dynamic light scattering (DLS) analysis revealed an average 33 nm size of ZnO-NPs. The FT-IR functional groups C H stretch, C H bend, aromatic C N stretch, and C O stretch were observed in phyto-fabricated ZnO-NPs. Results obtained from antibacterial activities inferred that ZnO-NPs were most effective against Bacillus subtilis, least sensitive to Staphylococcus aureus. The minimum inhibitory concentration (MIC) was observed in the range 78.12 to 625 μg mL−1 which was further confirmed by bacterial cell viability test. The Anti-TB activity by Alamar Blue Dye test revealed phyto-fabricated ZnO-NPs inhibited M. tuberculosis at 25 μg mL−1. The result of the antioxidant activity of the DPPH method was dose-dependent. The application of ZnO-NPs as potential antibacterial applications could be envisioned.

Synthesis and characterization of metallophthalocyanine with morpholine containing Schiff base and determination of their antimicrobial and antioxidant activities

In this work, novel metallophthalocyanine compounds (M = Zn, Cu, Co) bearing four 2-methoxy-4-{(Z)-[(4-morpholin-4-ylphenyl)imino]methyl}phenol at the peripheral positions were sythesized starting from the new phthalonitrile derivative (2). These new compounds (2–5) have been characterized by a combination of FT-IR, 1H NMR (for compounds 2 and 3), 13C NMR (for compound 2), UV–Vis (for compounds 3,4 and 5) and mass spectrophotometry techniques. Antioxidant activities of the metallophthalocyanines were investigated by using DPPH free radical scavenging assay and FRAP (ferric ion reducing antioxidant power) method. The antimicrobial activity of the synthesized metallo phthalocyanine compounds (3–5) were determined against the selected different six standart bacteria isolates by microdilution broth assay with Alamar Blue Dye. Most affected bacteria from the compounds were standard E. coli and S. typhimurium (MIC 625 μg/ml). Standart Y. enterocolitica and S. aureus have been less affected by the compounds (MIC 10.000 μg/ml).

Abstract 4605: Exosomes harvested from patient-derived-explants or cells-enhance proliferation and migration in TNBC breast cancer cells and breast epithelial cells

Abstract Triple negative breast cancer (TNBC) is a clinically aggressive breast cancer subtype due in part by high rates of metastasis and poor prognoses. TNBC tumors are molecularly heterogeneous and lack targetable receptors common in other breast cancer subtypes (estrogen receptor, HER2/Neu amplification), hindering the discovery of effective targeted therapies. Our group has designed a series of experiments to gain insight into the role of the tumor microenvironment in TNBC proliferation, migration and invasion. Patient-derived xenografts (PDXs) are translational models that are necessary to use in the laboratory setting when studying the complex tumor microenvironment of TNBC tumors. Recent breast cancer-focused studies show exosomes can be local and systemic cell-to-cell transporters of oncogenic information. Here, we aim to better understand how tumor-derived exosomes harvested from TNBC PDX models affect cell proliferation and migration when cultured with either triple negative breast cancer cell line MDA-MB-231 or non-tumorigenic breast epithelial cell line MCF-10A. PDX tumors or cells were plated in 2D culture conditions, and exosomes were isolated and purified from the cell culture media. Proliferation and migration assays were then performed after co-culture of the exosomes (or RPMI-1640 media as a negative control) with breast cell lines (MDA-MB-231, MCF-10A). The Alamar blue dye exclusion assay was used to measure proliferation, and absorbance was measured to quantify relative proliferation amongst the different treatment groups. To evaluate cell migration, a transwell migration assay was utilized where the breast cancer cell lines were plated with either the exosomal media or standard growth media as the chemoattractant for 48 hours. For invasion experiments, the transwell migration assay was employed. Our data indicate that TNBC PDX-derived exosomes increased cell proliferation in the MDA-MB-231 cell line by an average of approximately 60% compared to the control (p ≤ 0.05). We observed similar patterns of increased cell proliferation in the MCF-10A cell line, with an increase of 34% when cells were exposed to exosomes (p ≤ 0.05) compared to the control. Exosomes from TNBC PDX models increased migration in MDA-MB-231 cells by 4% and in MCF-10A cells by 10 percent. Interestingly, when we screened the exosomes from different TNBC PDX models, we found that one invasive TNBC PDX model contributed to the increased proliferation and migration in both the MDA-MB-231 and MCF-10A cell lines. Exosomes harvested and purified from TNBC patient-derived explants, and 2D culture conditions models increased proliferation and migration, cell line MDA-MB-231, and the non-tumorigenic breast epithelial cell line MCF-10A. Our data suggests that exosomes are key mediators in the progression of cancer. Note: This abstract was not presented at the meeting. Citation Format: Jordon Coward, Margarite D. Matossian, Paige A. Roberts, Jasmine Johnson, Matthew E. Burow, KiTani P. Lemieux. Exosomes harvested from patient-derived-explants or cells-enhance proliferation and migration in TNBC breast cancer cells and breast epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4605.

Valorization of discarded Marine Eel fish skin for collagen extraction as a 3D printable blue biomaterial for tissue engineering

Discarded marine Eel fish skin has essential properties of biomaterials for potential use in tissue engineering application. Processing and preparation of eel fish for edible purpose requires the removal of skin due to its thick size, which is treated as a waste. A huge amount of Eel skin is dumped as a waste material which leads to marine environmental pollution. To overcome this issue, we have isolated collagen from the discarded marine Eel skin as a potential blue biomaterial. Further, the isolated collagen was incorporated into alginate hydrogel to fabricate scaffolds using extrusion-based 3D printing technology. Swelling, degradation and biocompatibility were evaluated for lyophilized scaffolds. Biocompatibility studies were performed on hUMSCs (Human Umbilical cord Derived Mesenchymal Stem Cells) by live/dead staining using FDA (fluorescein diacetate)/PI (Propidium Iodide). The quantitative evaluation of metabolic activity was performed using Alamar Blue (AB) dye reduction assay. All the hydrogels with collagen show enhanced metabolic activity and cell proliferation compared to alginate hydrogels without collagen. The utilization of Eel skin derived collagen for 3D printing application was not yet reported. Moreover, sustainable utilization of renewable marine Eel skin discard as a novel blue biomaterial is of immense value due to its low cost and has great potential for further tissue engineering applications.

Mechanochemically synthesized phase stable and biocompatible β-tricalcium phosphate from avian eggshell for the development of tissue ingrowth system

Abstract Bioceramics obtained from naturally derived materials are gaining much interest as implants for bone and dental defects. The present study aims to synthesize phase stable β-tricalcium phosphate (β-TCP) from avian eggshell assisted with ball milling process followed by a wet chemical precipitation method (Group CPM). The effect of mechanical stimulation on phase conversion of CaO was also studied. The study was carried alongside the powders synthesized from chemical precursors (Group CPS) as well as eggshell derived powders without ball milling process (Group CPN). The phase behaviour and surface morphology were studied by XRD, FT-IR, and SEM analysis. Scaffolds were fabricated using sponge replication method to simulate a potential bone graft analogue. The cytocompatibility study was performed by human adipose-derived stem cells (ADSCs) over a period of 21 days by live-dead assay and Alamar blue dye reduction assay. The process of mechanically stimulating CaO precursor through extensive milling plays a major role on phase stabilization of β-TCP, as compared to the mixed phases of Hydroxyapatite (HAp) and β-TCP formed from unmilled CaO. Group CPN scaffolds were found to be biologically equivalent to group CPS scaffolds. This novel route, aided with ball-milling process for the synthesis of β-TCP from naturally occurring eggshell waste seems promising enough to replace commercially available β-TCP produced from harmful nitrate precursors and has the capability to develop implantable biomaterial for tissue regeneration.

5-Phenyl thiophene amino phenol derivatives: Synthesis, spectroscopic characterization, computational study and antimicrobial activity

(((5-phenylthiophen-2-yl) methylene) amino) phenol derivatives were synthezised and characterized by IR and NMR. The density functional theory (DFT) method with 6–311++G (d,p) basis set was used for both calculations of molecular geometries and spectroscopic parameters. Frontier molecular orbitals and molecular electrostatic potential maps for compounds 1a, 1b and 1c were obtained. Due to the differences observed in the theoretical spectral parameters, NH⋯O type intermolecular/intramolecular hydrogen bonds in the molecular structures of compounds were proposed. To determine the nonlinear optical properties of compounds, the mean polarizability (α) and the first hyperpolarizability (β) obtained as 44.2388 × 10−24 esu and 373.1267 × 10−31 esu for compound 1a, 43.8141 × 10−24 esu and 195.2893 × 10−31 esu for compound 1b, 36.9489 × 10−24 esu and 120.73825 × 10−31 esu for compound 1c, respectively were used. These values indicate that compounds can be suggested as a good nonlinear optical material. In addition, the antimicrobial properties of compounds were investigated by considering antileishmanial and antibacterial activities. The compounds were evaluated against Leishmania infantum promastigots for antileishmanial activity and against five standard bacteria (Escherichia coli, Shigella flexneri, Yersinia enterocolitica, Salmonella typhimurium and Salmonella enterica) for antibacterial activity, by microdilution broth assay with Alamar Blue Dye. Results show that only compound 1a has high antileishmanial activity and can be evaluated in leishmaniasis treatment after in vivo studies and also considered in pharmacological studies for antibacterial activity.

Insulin adsorption onto zinc oxide nanoparticle mediates conformational rearrangement into amyloid-prone structure with enhanced cytotoxic propensity

BackgroundInjection localized amyloidosis is one of the most prevalent disorders in type II diabetes mellitus (TIIDM) patients relying on insulin injections. Previous studies have reported that nanoparticles can play a role in the amyloidogenic process of proteins. Hence, the present study deals with the effect of zinc oxide nanoparticles (ZnONP) on the amyloidogenicity and cytotoxicity of insulin. MethodsZnONP is synthesised and characterized using XRD, Zeta Sizer, UV-Visible spectroscope and TEM. The characterization is followed by ZnONP interaction with insulin, which is studied employing fluorescence spectroscopes, isothermal titration calorimetry and molecular dynamics simulations. The interaction leads insulin conformational rearrangement into amyloid-like fibril, which is studied using thioflavin T dye binding assay, circular dichroism spectroscopy and TEM, followed by cytotoxicity propensity using Alamar Blue dye reduction assay. ResultsInsulin has very weak interaction with ZnONP interface. Insulin at studied concentration forms amorphous aggregates at physiological pH, whereas in presence of ZnONP interface amyloid-like fibrils are formed. While the amyloid-like fibrils are cytotoxic to MIN6 and THP-1 cell lines, insulin and ZnONP individual solutions and their fresh mixtures enhance the cells proliferation. ConclusionsThe presence of ZnONP interface enhances insulin fibrillation at physiological pH by providing a favourable template for the nucleation and growth of insulin amyloids. General significanceThe studied protein-nanoparticle system from protein conformational dynamics point of view throws caution over nanoparticle use in biological applications, especially in vivo applications, considering the amyloidosis a very slow but non-curable degenerative disease.

1,2,4-triazole derivatives with morpholine; DFT study and antileishmanial activity

1,2,4-triazole derivatives with morpholine; 4-((3-methylthiophene–2-yl)methylenamino)-1-((4-(3-methylthiophene–2-yl)methylene amino)-1-(morpholinomethyl)-5-thioxo-4,5-dihydro-1H-1,2,4-triazole-3-yl)methyl)-3-(thiophene–2-ylmethyl)-1H-1,2,4-triazole-5(4H)-one (compound I) and 1-((1-(morpholinomethyl)–4-(5-nitrothiophene–2-yl)methyleneamino)-5-thioxo-4,5-dihydro-1H-1,2,4-triazole-3-yl)methyl)-4-((5-nitrothiophene–2-yl)methyleneamino)-3-(thiophene–2-ylmethyl)-1H-1,2,4-triazole-5(4H)-one (compound II), were optimized using a density functional theory (DFT) method with 6-311G(d,p) basis set and structural and spectral parameters were determined. In vitro antileishmanial activities of compounds were performed against Leishmania infantum promastigots by microdilution broth assay with Alamar Blue dye. Amphotericin B was used as standard drug. The results show that both compounds are antiparasitic and especially compound II has considerable antileishmanial activity due to the minimal inhibitory concentration value of 312 μg/mL.