The SP/NK1R System-Mediated ROS Generation in GBM Cells through Inhibiting Glutaredoxin Protein

Altered redox balance is among the main contributing factors developing glioblastoma multiforme (GBM), a highly aggressive grade IV brain tumor. Neuropeptide substance P (SP) plays a key role in modifying the cellular redox environment by activating the neurokinin-1 receptor (NK1R). In this study, we aimed to investigate the redox-modulating properties of both SP and a commercially available NK1R antagonist, aprepitant in GBM cells. To detect the effect of aprepitant on the viability of U87 glioblastoma cells, resazurin assay was applied. The level of intracellular ROS was assessed using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) assay. The expression of glutaredoxin, a well-known redox-active protein, was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Concurrently, the activity of glutaredoxin was also analyzed by a commercial kit (ZellBio GmbH). We found that SP increased the intracellular levels of reactive oxygen species (ROS) in U87 GBM cells, and aprepitant remarkably decreased this effect. We also explored the effects of SP/NK1R signaling on the glutaredoxin system as a major cellular redox buffer in GBM cells. SP reduced both expression and enzymatic activity of glutaredoxin, and these effects were significantly decreased by aprepitant. In conclusion, our results suggest a possible involvement of SP/NK1R signaling in GBM pathogenesis through oxidative stress and offering new insight for the application of aprepitant as a redox-modulating strategy in GBM patients.

STRUCTURAL AND BIOLOGICAL CHARACTERISTICS OF SELF-ORGANISING CHITOSAN HYDROGELS

Creating innovative methods of treatment and regeneration of damaged tissues or organs is a key challenge of the twenty-first century. The aim of this study was to determine the possibility of producing and characterising the properties of self-organising chitosan hydrogels prepared with the use of chitosan lactate/chloride and disodium hydrogen phosphate dodecahydrate as a cross-linking agent. The structure and supramolecular architecture of the biomaterials were evaluated by Fourier-transform infrared spectroscopy and polarised optical microscopy. Biological studies assessed cytotoxicity by contact with a human colon adenocarcinoma cell line. The colourimetric resazurin assay showed that the obtained chitosan hydrogels are non-cytotoxic materials. Thus, self-organising biomaterialshold great promise for application in tissue engineering.

Antifungal Activity of Extracts, Fractions, and Constituents from Coccoloba cowellii Leaves

Coccoloba cowellii Britton (Polygonaceae, order Caryophyllales) is an endemic and critically endangered plant species that only grows in the municipality of Camagüey, a province of Cuba. A preliminary investigation of its total methanolic extract led to the discovery of promising antifungal activity. In this study, a bioassay-guided fractionation allowed the isolation of quercetin and four methoxyflavonoids: 3-O-methylquercetin, myricetin 3,3′,4′-trimethyl ether, 6-methoxymyricetin 3,4′-dimethyl ether, and 6-methoxymyricetin 3,3′,4′-trimethyl ether. The leaf extract, fractions, and compounds were tested against various fungi and showed strong in vitro antifungal activity against Cryptococcus neoformans and various Candida spp. with no cytotoxicity (CC50 > 64.0 µg/mL) on MRC-5 SV2 cells, determined by a resazurin assay. A Candida albicans SC5314 antibiofilm assay indicated that the antifungal activity of C. cowellii extracts and constituents is mainly targeted to planktonic cells. The total methanolic extract showed higher and broader activity compared with the fractions and mixture of compounds.

Established Gardnerella biofilms can survive metronidazole treatment by reducing metabolic activity

Gardnerella spp., a hallmark of bacterial vaginosis, can form biofilm and it has been suggested that failure of antibiotic treatment of bacterial vaginosis and recurrent vaginosis are linked to its ability to form biofilm. Here, we tested the hypothesis that biofilm formation provides protection from the effects of metronidazole. We performed a broth microdilution assay to measure the minimum inhibitory concentration (MIC) of metronidazole for thirty-five Gardnerella isolates in two different growth media: one medium in which Gardnerella spp. grow primarily as biofilm and the other medium in which Gardnerella spp. grow primarily in planktonic form. The MIC of Gardnerella isolates observed in the two conditions were highly correlated (R2= 0.69, p <0.001) and 27/35 isolates had no difference in MIC between the two growth modes. When established biofilms were treated with metronidazole, live Gardnerella could be recovered following treatment in most cases (7/9 isolates tested). Metabolic activity of established biofilms of thirty-one isolates with and without metronidazole treatment was measured using a resazurin assay. Most (27/31) isolates showed reduced metabolic activity following treatment with μ128 g/ml of metronidazole relative to untreated controls. The amount of biofilm produced by Gardnerella isolates was not enhanced by sub-inhibitory concentrations of metronidazole and scanning electron microscopy revealed no architectural differences between treated and untreated biofilms. Our results suggest that Gardnerella spp. growing in established biofilms reduce metabolic activity as a mechanism of protection from the bactericidal effects of metronidazole.

Rapid Method for Detecting Bactericidal Effects of Mangrove Derived Silver Nanoparticles Using Resazurin Microdilution Assay

Nanoparticles plays a vital role in the field of antimicrobial agents against pathogenic microorganisms. Screening of nanoparticles for antimicrobial activities is a time consuming and cumbersome process. Recently, a simple technique of using the dye resazurin has been used as an indicator of bacterial growth for testing antimicrobial activity on microtitre plate. However, this technique does not quantify the microbial load. Therefore, the present work was attempted to find a new antibacterial method employing the dye resazurin assay and haemocytometric counting of microbes for testing silver nanoparticles synthesised from Xylocarpus mekongensis .The bacterial strains E. coli, S. aureus and P. aeruginosa (multi-drug resistant strain) were used to evaluate the screening of mangrove extracts. Minimum inhibition concentration (MIC) was also calculated for the silver nanoparticles using ciproflaxcin as reference antibiotic. The antibacterial activity Xylocarpus mekongensis was carried out against all the three bacteria by the same method and the values were compared with reference antibiotic. The present study has suggested a rapid, dependable, easy and inexpensive method, suitable for testing the antibacterial activity of silver nanoparticles which are promising to develop as new antibacterials. Keywords: Mangroves, Silver, Nanoparticles, Resazurin, Antibacterials

Establishment of a resazurin-based aortic valve tissue viability assay for dynamic culture in a microphysiological system

BACKGROUND/AIM: Tissue pathogenesis of aortic valve (AV) stenosis is research focus in cardiac surgery. Model limitations of conventional 2D culture of human or porcine valvular interstitial/endothelial cells (VIC/VECs) isolated from aortic valve tissues but also limited ability of (small) animal models to reflect human (patho)physiological situation in AV position raise the need to establish an in vitro setup using AV tissues. Resulting aim is to approximate (patho)physiological conditions in a dynamic pulsatile Microphysiological System (MPS) to culture human and porcine AV tissue with preservation of tissue viability but also defined ECM composition. MATERIALS/METHODS: A tissue incubation chamber (TIC) was designed to implement human or porcine tissues (3×5 mm) in a dynamic pulsatile culture in conventional cell culture ambience in a MPS. Cell viability assays based on lactate dehydrogenase (LDH)-release or resazurin-conversion were tested for applicability in the system and applied for a culture period of 14 days with interval evaluation of tissue viability on every other day. Resazurin-assay setup was compared in static vs. dynamic culture using varying substance saturation settings (50–300μM), incubation times and tissue masses and was consequently adapted. RESULTS: Sterile dynamic culture of human and porcine AV tissue segments was established at a pulsatile flow rate range of 0.9–13.4μl/s. Implementation of tissues was realized by stitching the material in a thermoplastic polyurethane (TPU) –ring and insertion in the TIC-MPS-system. Culture volume of 2 ml caused LDH dilution not detectable in standard membrane integrity assay setup. Therefore, detection of resazurin-conversion of viable tissue was investigated. Optimal incubation time for viability conversion was determined at two hours at a saturated concentration of 300μM resazurin. Measurement in static conditions was shown to offer comparable results as dynamic condition but allowing optimal handling and TIC sterilization protocols for long term culture. Preliminary results revealed favourable porcine AV tissue viability over a 14 day period confirmed via resazurin-assay comparing statically cultured tissue counterparts. CONCLUSIONS: Human and porcine AV tissue can be dynamically cultured in a TIC-MPS with monitoring of tissue viability using an adapted resazurin-assay setup. Preliminary results reveal advantageous viability of porcine AV tissues after dynamic TIC-MPS culture compared to static control.

Adaptation of the Start-Growth-Time Method for High-Throughput Biofilm Quantification

Colony forming unit (CFU) determination by agar plating is still regarded as the gold standard for biofilm quantification despite being time- and resource-consuming. Here, we propose an adaption of the high-throughput Start-Growth-Time (SGT) method from planktonic to biofilm analysis, which indirectly quantifies CFU/mL numbers by evaluating regrowth curves of detached biofilms. For validation, the effect of dalbavancin, rifampicin and gentamicin against mature biofilms of Staphylococcus aureus and Enterococcus faecium was measured by accessing different features of the viability status of the cell, i.e., the cultivability (conventional agar plating), growth behavior (SGT) and metabolic activity (resazurin assay). SGT correlated well with the resazurin assay for all tested antibiotics, but only for gentamicin and rifampicin with conventional agar plating. Dalbavancin treatment-derived growth curves showed a compared to untreated controls significantly slower increase with reduced cell doubling times and reduced metabolic rate, but no change in CFU numbers was observed by conventional agar plating. Here, unspecific binding of dalbavancin to the biofilm interfered with the SGT methodology since the renewed release of dalbavancin during detachment of the biofilms led to an unintended antimicrobial effect. The application of the SGT method for anti-biofilm testing is therefore not suited for antibiotics which stick to the biofilm and/or to the bacterial cell wall. Importantly, the same applies for the well-established resazurin method for anti-biofilm testing. However, for antibiotics which do not bind to the biofilm as seen for gentamicin and rifampicin, the SGT method presents a much less labor-intensive method suited for high-throughput screening of anti-biofilm compounds.

Application of the Resazurin Cell Viability Assay to Monitor Escherichia coli and Salmonella Typhimurium Inactivation Mediated by Phages

Bacterial inactivation using bacteriophages (or phages) has emerged as an effective solution for bacterial infections, but the screening methods used to evaluate the effectiveness of the phages to inactivate bacteria are not fast, reliable or precise enough. The efficiency of bacterial inactivation by phages has been evaluated by monitoring bacterial concentration either by counting colony-forming units (CFU), a laborious and time-consuming method, or by monitoring the optical density (OD), a less sensitive method. In this study, the resazurin cell viability assay was used to monitor the viability of bacteria from different genera during the inactivation by different phages, and the results were compared with the standard methods used to assess bacterial inactivation. The results showed that the resazurin colorimetric cell viability assay produces similar results to the standard method of colony-counting and giving, and also more sensitive results than the OD method. The resazurin assay can be used to quickly obtain the results of the cell viability effect profile using two different bacterial strains and several different phages at the same time, which is extremely valuable in screening studies. Moreover, this methodology is established as an effective, accurate and rapid method when compared to the ones widely used to monitor bacterial inactivation mediated by phages.

Multi-Faceted Role of Silver and Gold Nanoparticles Synthesized from Biowaste and its in vitroAntibacterial, Antifungal and Antidiabetic Activities

Increasing global fish production, increasing the amount of waste generated, unsafe disposal of waste tissues like head, bones, skin, scales, fins etc., into land and open water reservoirs leads to environmental pollution. The role of nanobiotechnology in biowaste management is an innovative strategy to handle environmental issues. This study synthesized silver and gold nanoparticles from prawn heads using one millimolar AgNO3 and HAuCl4. Biosynthesized nanoparticles were characterized by UV -Visible spectroscopy, XRD, FTIR, SEM and EDAX. The maximum absorption spectrum was monitored at 437 nm for silver and 552 for gold nanoparticles. Antimicrobial activity was assessed using the resazurin assay method. MIC values obtained for the tested organisms revealed antimicrobial activities. P aeruginosa, K. pneumoniae, showed MIC at 15.6 µg for silver nanoparticles, and A. niger, A.flavus and C.albicans showed MIC at 125 µg for gold nanoparticles synthesized from the prawn head extract. IC 50 values of α- amylase activity were found to be 296 and 356 µg/ml for the silver and gold nanoparticles, respectively. IC50 values are about 705 and 2475 µg/ml for the silver and gold nanoparticles, respectively, in α-glucosidase activity. Conclusively silver and gold nanoparticles synthesized from prawn head extract (PHE) showed antibacterial, antifungal, and antidiabetic activities.

Antiplasmodial and Cytotoxic Flavonoids from Pappea capensis (Eckl. & Zeyh.) Leaves

Ethnobotanical surveys indicate that the Masai and Kikuyu in Kenya, the Venda in South Africa, and the Gumuz people of Ethiopia use Pappea capensis for the treatment of malaria. The present study aimed to investigate the phytochemical and antiplasmodial properties of the plant leaves. The bioactive compounds were isolated using chromatographic techniques. The structures were established using NMR, HRMS, and UV spectroscopy. Antiplasmodial activity of P. capensis leaf extract and isolated compounds against chloroquine-sensitive 3D7 P. falciparum was evaluated using the parasite lactate dehydrogenase assay. Cytotoxicity against HeLa (human cervix adenocarcinoma) cells was determined using the resazurin assay. The extract inhibited the viability of Plasmodium falciparum by more than 80% at 50 µg/mL, but it was also cytotoxic against HeLa cells at the same concentration. Chromatographic purification of the extract led to the isolation of four flavonoid glycosides and epicatechin. The compounds displayed a similar activity pattern with the extract against P. falciparum and HeLa cells. The results from this study suggest that the widespread use of P. capensis in traditional medicine for the treatment of malaria might have some merits. However, more selectivity studies are needed to determine whether the leaf extract is cytotoxic against noncancerous cells.