Optimizing microbial electrolysis cell performance: strategies to mitigate electron mediator degradation on membranes.

This investigation probes the role of the electron mediator, neutral red (NR), in the electrosynthesis process, specifically examining its effect on the production of succinic acid by Actinobacillus succinogenes. Our findings reveal that NR, when integrated into the cell membrane, is pivotal for sustaining MEC efficiency. Nevertheless, it is susceptible to both intrinsic and MECs-induced degradation. Notably, during the exponential growth phase of the bacteria, NR is readily incorporated into the cell membrane. However, the supplemental addition of NR fails to significantly enhance the MEC's capacity for succinic acid synthesis, no matter what stage of bacterial growth. And significant depletion of membrane-associated NR is not adequately compensated by the NR present in the fermentation liquid. The ORP feedback-regulated MECs adeptly conserve the NR on the cell membrane, which is essential for maintaining the efficiency of long-term electrosynthesis. The presence of NR on the cell membrane is essential for the functionality of MECs, yet its external replenishment hard. Implementing precise electro-potential regulation strategies can effectively diminish the degradation of NR, thus maintaining the system's efficiency.

Development of Optical Sensors Based on Neutral Red Absorbance for Real-Time pH Measurements.

Measuring pH with an optical sensor requires the immobilization of a chemical recognition phase on a solid surface. Neutral red (NR), an acid base indicator was used to develop two different optical probe configurations. The chemistry of aryl diazonium salts was chosen for the elaboration of this chemical phase, as it enables strong covalent bonds to be established on the surface of metallized glass or metallic surfaces. It also allows the formation of a thick film required to obtain an exploitable spectral response. The surfaces of interest (metallized optical fiber and 316 L stainless-steel mirror) are modelized by flat surfaces (metallized glass plates and 316 L stainless-steel plates). The analytical characterizations carried out (IR, XPS, UV-Visible, and profilometry) show that NR was covalently grafted onto the model surfaces as well as on the surfaces of interest. The supports grafted with NR to develop optical pH probes exhibit spectral changes, particularly the values of pKa, the pH range, and the isosbestic point wavelength. The experimental results show that the optical probe can be used for pH measurements between 4 and 8.

The Influence of Bacteriophages on the Metabolic Condition of Human Fibroblasts in Light of the Safety of Phage Therapy in Staphylococcal Skin Infections.

Phage therapy has been successfully used as an experimental therapy in the treatment of multidrug-resistant strains of Staphylococcus aureus (MDRSA)-caused skin infections and is seen as the most promising alternative to antibiotics. However, in recent years a number of reports indicating that phages can interact with eukaryotic cells emerged. Therefore, there is a need to re-evaluate phage therapy in light of safety. It is important to analyze not only the cytotoxicity of phages alone but also the impact their lytic activity against bacteria may have on human cells. As progeny virions rupture the cell wall, lipoteichoic acids are released in high quantities. It has been shown that they act as inflammatory agents and their presence could lead to the worsening of the patient's condition and influence their recovery. In our work, we have tested if the treatment of normal human fibroblasts with staphylococcal phages will influence the metabolic state of the cell and the integrity of cell membranes. We have also analyzed the effectiveness of bacteriophages in reducing the number of MDRSA attached to human fibroblasts and the influence of the lytic activity of phages on cell viability. We observed that, out of three tested anti-Staphylococcal phages-vB_SauM-A, vB_SauM-C and vB_SauM-D-high concentrations (109 PFU/mL) of two, vB_SauM-A and vB_SauM-D, showed a negative impact on the viability of human fibroblasts. However, a dose of 107 PFU/mL had no effect on the metabolic activity or membrane integrity of the cells. We also observed that the addition of phages alleviated the negative effect of the MDRSA infection on fibroblasts' viability, as phages were able to effectively reduce the number of bacteria in the co-culture. We believe that these results will contribute to a better understanding of the influence of phage therapy on human cells and encourage even more studies on this topic.

Metal-Assisted Complexation of Fluorogenic Dyes by Cucurbit[7]uril and Cucurbit[8]uril: A DFT Evaluation of the Key Factors Governing the Host-Guest Recognition.

With the emergence of host-guest systems, a novel branch of complexation chemistry has found wide application in industries such as food, pharmacy, medicine, environmental protection and cosmetics. Along with the extensively studied cyclodextrins and calixarenes, the innovative cucurbiturils (CB) have enjoyed increased popularity among the scientific community as they possess even better qualities as cavitands as compared to the former molecules. Moreover, their complexation abilities could further be enhanced with the assistance of metal cations, which can interestingly exert a dual effect on the complexation process: either by competitively binding to the host entity or cooperatively associating with the CB@guest structures. In our previous work, two metal species (Mg2+ and Ga3+) have been found to bind to CB molecules in the strongest fashion upon the formation of host-guest complexes. The current study focuses on their role in the complex formation with three dye molecules: thiazole orange, neutral red, and thioflavin T. Various key factors influencing the process have been recognized, such as pH and the dielectric constant of the medium, the cavity size of the host, Mn+ charge, and the presence/absence of hydration shell around the metal cation. A well-calibrated DFT methodology, solidly based and validated and presented in the literature experimental data, is applied. The obtained results shed new light on several aspects of the cucurbituril complexation chemistry.

Neutral Red-carbon nanodots for selective fluorescent DNA sensing.

Carbon nanodots modified with Neutral Red covalently inserted in the nanostructure (NR-CDs) have been prepared by a simple synthesis method based on microwave irradiation under controlled temperature and pressure. The synthetized NR-CDs have been characterized by different techniques, demonstrating the covalent bonding of Neutral Red molecules to the carbon dots nanostructure. Fluorescence activity of the prepare NR-CDs has been explored showing different interaction pathways with singled and doubled stranded DNA. These studies have been successfully applied to develop a new fluorescence DNA hybridization assay to the detection of a specific DNA sequence of Escherichia coli bacteria.

Development and Application of Loop-Mediated Isothermal Amplification (LAMP) Assays for Rapid Diagnosis of the Bat White-Nose Disease Fungus Pseudogymnoascus destructans.

Pseudogymnoascus destructans (= Geomyces destructans) is a psychrophilic filamentous fungus that causes White-Nose Disease (WND; the disease associated with White-Nose Syndrome, WNS) in hibernating bats. The disease has caused considerable reductions in bat populations in the USA and Canada since 2006. Identification and detection of the pathogen in pure cultures and environmental samples is routinely based on qPCR or PCR after DNA isolation and purification. Rapid and specific direct detection of the fungus in the field would strongly improve prompt surveillance, and support control measures. Based on the genes coding for ATP citrate lyase1 (acl1) and the 28S-18S ribosomal RNA intergenic spacer (IGS) in P. destructans, two independent LAMP assays were developed for the rapid and sensitive diagnosis of the fungus. Both assays could discriminate P. destructans from 159 tested species of filamentous fungi and yeasts. Sensitivity of the assays was 2.1 picogram per reaction (pg/rxn) and 21 femtogram per reaction (fg/rxn) for the acl1 and IGS based assays, respectively. Moreover, both assays also work with spores and mycelia of P. destructans that are directly added to the master mix without prior DNA extraction. For field-diagnostics, we developed and tested a field-applicable version of the IGS-based LAMP assay. Lastly, we also developed a protocol for preparation of fungal spores and mycelia from swabs and tape liftings of contaminated surfaces or infected bats. This protocol in combination with the highly sensitive IGS-based LAMP-assay enabled sensitive detection of P. destructans from various sources.

Ultrasound-Assisted Dispersive Liquid-Liquid Microextraction Using Deep Eutectic Solvents (DESs) for Neutral Red Dye Spectrophotometric Determination.

Deep eutectic solvents (DES), which have low toxicity and are low cost, biodegradable, and easily synthesized, were used for the extraction of neutral red (NR) dye before its spectrophotometric analysis. DES, containing choline chloride as a hydrogen bond acceptor and phenol as a hydrogen bond donor with a molar ratio of 1:2, was used for the extraction of NR dye from aqueous media. The possible interaction of different DESs with NR was studied using density functional theory (DFT) calculations. Experimentally, a UV-visible spectrophotometer was used for the quantitative analysis. The most important parameters affecting method performance, such as pH, extraction temperature, DES type, its volume, THF volume, sonication time, and centrifugation time, were optimized. The developed method provides exceptional sensitivity in terms of LOD and LOQ, which were 2.2 and 7.3 µg/L respectively. The relative standard deviation was 1.35−1.5% (n = 10), and the pre-concentration factor was 40. The method was found to be linear in the range of 2−300 µg/L (R2 = 0.9967). The method was successfully used for the determination of NR in wastewater samples. Finally, the DES-based method presents operational simplicity, high sensitivity, and rapid determination (<5 min) compared with other analytical procedures.

Copepod carcasses in the western Bay of Bengal and associated ecology.

Although a portion of all copepods in the natural environment are physiologically inactive or dead, there is little data on this aspect in Indian seas. Using the classical neutral red live staining technique, the dead percentage of copepods in the western Bay of Bengal (BoB) is determined for the first time in this study, which takes into account hydrography and zooplankton samples collected on April 2015 (Spring Intermonsoon [SIM]) from 6 inshore and 8 offshore locations in the western BoB. The offshore parts of the research area were infested with a persistent and extensive warm-core eddy/gyre during the sampling time, as demonstrated by satellite data of sea surface temperature, mean sea level anomaly, and surface currents. As a result, the mixed layer depth in the offshore zone was higher, whereas nutrients, chlorophyll a, and turbidity were significantly lower than inshore. Copepods dominated the zooplankton community (> 80%), with abundances ranging from 112 to 2580 No m-3 in the study area. Calanoids (particularly Paracalanidae) dominated nearshore waters (35-65%), whereas cyclopoids (notably Corycaeidae and Oncaeidae) dominated offshore (49-71%). There were 48 species of copepods identified in total, and all of them had dead individuals (carcasses). Offshore waters had a larger percentage of copepod carcasses (27-39%) than inshore seas (18-28%). The larger percentage of carcasses is possibly related to a higher top-down feeding pressure on cyclopoids in offshore waters due to extreme oligotrophy and the dominance of the microbial food web.

Seasonal effect on biomarker responses in sentinel species: innovation in mangrove conservation status assessment.

We evaluated the environmental quality in mangrove areas of the Western Atlantic with different levels and history of contamination, considering biomarkers for the crab Ucides cordatus. For this purpose, specimens were collected in two climatic seasons (rainy and dry seasons) and assays of genotoxicity (MN, micronucleus), cytotoxicity (NRRT, neutral red retention time) and biochemical (MT, metallothionein; and LPO, lipoperoxidation) were conducted. In the most impacted mangroves, there was an increase in the mean of micronucleus (frequency of MN/1000), which was associated with a shorter retention time (minutes of NRRT). In contrast, the most pristine areas showed MN < 3 and NRRT < 100 min, with no seasonal effect, indicating a lower effect of degenerative processes by xenobiotics. The rainy season was more harmful, especially regarding cytogenotoxicity. The use of bioindicator species for environmental monitoring should be guided by an analysis of biomarkers considering the season, because during the period of highest rainfall, biomarkers values can change.

Electrophoresis on a polyester thread coupled with an end-channel pencil electrode detector.

We present the design and characterization of a low cost, thread-based electrophoretic device with integrated electrochemical detection. The device has an end-channel pencil graphite electrode placement system for performing electrochemical detection on the thread electrophoresis platform with direct sample pipetting onto the thread. We also established the use of methylene blue and neutral red as a pair of reference migration markers for separation techniques coupled with electrochemical detection, as they have different colors for visual analysis and are both electroactive. Importantly, neutral red was also found to migrate at a similar rate to the EOF, indicating that it can be used as a visual identifier of EOF. The utility of our system was demonstrated by electrophoretic separation and electrochemical detection of physiologically relevant concentrations of pyocyanin in a solution containing multiple electroactive compounds. Pyocyanin is a biomarker for the detection of pathogenic Pseudomonas aeruginosa and has a redox potential that is similar to that of methylene blue. The system was able to effectively resolve methylene blue, neutral red, and pyocyanin in less than 7 min of electrophoretic separation. The theoretical limit of detection for pyocyanin was determined to be 559 nM. The electrophoretic mobilities of methylene blue (0.0236 ± 0.0007 mm2 /V·s), neutral red (0.0149 ± 0.0007 mm2 /V·s), and pyocyanin (0.0107 ± 0.0003 mm2 /V·s) were also determined.

Screening of different cytotoxicity methods for the assessment of ENDS toxicity relative to tobacco cigarettes.

Electronic Nicotine Delivery Systems (ENDS), i.e., electronic-cigarettes (e-cigs) and Tobacco Heating Products (THPs), are rapidly growing in popularity. Nonetheless, comprehensive quality and safety requirements for regulatory purposes are still under development. Cytotoxicity studies are important initial steps in appraising the potential ENDS toxicity. The aim of the present study was to screen different in vitro cytotoxicity methods for the assessment of ENDS toxicity. We evaluated NRU, MTT, Annexin V apoptosis (AN-V), High-Content Screening (HCS) assays and Real-Time Cell Analysis (RTCA), to compare two e-cigs and two THPs with the 1R6F reference tobacco cigarette. Human adenocarcinoma lung epithelial cells (H292) were exposed to tobacco smoke and ENDS vapor at air-liquid interface. All tests showed reduced cell viability following 1R6F smoke exposure and slight or no reduction with ENDS at 24 h. AN-V and RTCA exhibited a further significant reduction in cell viability following 1R6F exposure. AN-V allowed to discriminate viable cells from those in early/late apoptosis. RTCA and HCS being time-resolved analyses elucidate the kinetic dependency parameter for toxicity of smoke/vapor chemicals on cell viability. In conclusion, NRU assay may be considered a suitable test, especially when combined with a time-resolved analysis, for assessing the kinetic of cytotoxicity induced by these products.

The viability of human cells irradiated with 470-nm light at various radiant energies in vitro.

Blue light is known to be antimicrobial, but its effect on normal cutaneous and subcutaneous cells remains unclear. Therefore, we studied the effect of 470-nm light on the viability of adult and neonatal human dermal fibroblasts, Jurkat T-cells, and THP-1 monocytes in vitro. Each culture was irradiated with 0, 3, 55, or 110 J/cm2 of 470-nm light and subjected to trypan blue assay to ascertain viability. Further, MTT, neutral red, and fluorescence assays of fibroblasts were performed, and cell morphology visualized using bright field and fluorescence microscopy. At each dose and in each of the four cell lines, there was no significant difference in cell concentration between irradiated and non-irradiated cultures, even though irradiation with 55 J/cm2 or 110 J/cm2 slightly decreased cell count. Light microscopy showed progressive morphological changes in the fibroblasts as energy fluence increased from 55 to 110 J/cm2. Irradiation at 3 J/cm2 produced a slight but non-significant increase in the viability of Jurkat T-cells and THP-1 monocytes. In contrast, at 110 J/cm2 radiant exposure, irradiation slightly decreased the viability of all four cells. While 3 J/cm2 appears stimulatory, our finding that 110 J/cm2 produces a slight decrease in viability and engenders morphological changes in fibroblasts, suggesting that such high doses should be avoided in blue light treatments.

The cytotoxic and oxidative effects of restorative materials in cultured human gingival fibroblasts.

The aim of this study was to evaluate the cytotoxic and oxidative effects of the most commonly used dental restorative materials on human gingival fibroblast cells (HGFCs). HGFCs were obtained from healthy individuals. The tested restorative materials were a microhybrid resin based composite, a compomer resin, a glass ionomer cement, and an amalgam alloy. One hundred eight cylindirical samples, 10 mm in diameter and 2 mm in height, were prepared according to ISO 10993-12:2002 specifications (n = 9 in the tested subgroups). Freshly prepared and aged samples in artificial saliva at 37 °C (7 and 21 d) were placed into well plates and incubated. Wells without dental materials were constituted as the control group. After 72 h incubation period, cytotoxicity was determined using the neutral red (NR) assay. Oxidative alterations were assessed using total antioxidant capacity (TAC) and total oxidant status (TOS) assay kits. Data were analyzed using the ANOVA and LSD post hoc tests. All tested materials led to significant decreases in the cell viability rates (33-73%) compared to the control group. Glass ionomer and resin composite were found to be more cytotoxic than amalgam alloy and compomer. The highest TAC level was observed in glass ionomer after seven-day aging and these changes prevented an increase in TOS levels. Increases in TAC levels after seven-day aging in all groups exhibited significant differences with freshly prepared samples (p < 0.05). In all material groups, TOS levels of freshly prepared samples differed statistically and significantly from samples aged for 7 and 21 d (p < 0.05). The data obtained suggested that all the tested materials exhibited cytotoxic and pro-oxidant features. Freshly prepared samples caused higher TOS levels. However, oxidant status induced by materials decreased over time.

Cytotoxicity and antigenotoxicity evaluation of acetylshikonin and shikonin.

Shikonin (SH) is used as a red pigment for food coloring and cosmetics, and has cytotoxic activity towards cancer cells. However, due to strong toxicity SH has limited potential as an anticancer drug. Acetylshikonin (ASH) is one of the SH derivatives with promising anticancer potential. In present study, we attempted to evaluate and compare the cytotoxicity of SH and ASH towards a normal cell line (V79) and in addition to evaluate their antigenotoxic activity. The evaluation was made with the use of the set of cytotoxicity assays with V79 line and the micronucleus test in vitro performed using clinafloxacin (CLFX), ethyl methanesulfonate (EMS) as direct genotoxins and cyclophosphamide (CPA) as indirect genotoxin. For CPA and EMS the simultaneous protocol was used and for CLFX three different variants were performed: pretreatment, simultaneous, and post-treatment. A higher cytotoxic effect was observed for SH. The EC50 values obtained for SH were approximately twofold lower compared to that of ASH. Moreover, ASH exhibited an antigenotoxic potential against CPA-induced genotoxicity, whereas SH has no activity. However, ASH increased the EMS-induced genotoxicity, when SH exhibited no effect. Both compounds decreased the genotoxicity of CLFX in pretreatment and simultaneous protocol. Based on the results of the present study it can be concluded that ASH is less cytotoxic than SH to normal cells and has comparable antigenotoxic potential.

Human-relevant approaches to assess eye corrosion/irritation potential of agrochemical formulations.

There are multiple in vitro and ex vivo eye irritation and corrosion test methods that are available as internationally harmonized test guidelines for regulatory use. Despite their demonstrated usefulness to a broad range of substances through inter-laboratory validation studies, they have not been widely adopted for testing agrochemical formulations due to a lack of concordance with parallel results from the traditional regulatory test method for this endpoint, the rabbit eye test. The inherent variability of the rabbit test, differences in the anatomy of the rabbit and human eyes, and differences in modelling exposures in rabbit eyes relative to human eyes contribute to this lack of concordance. Ultimately, the regulatory purpose for these tests is protection of human health, and, thus, there is a need for a testing approach based on human biology. This paper reviews the available in vivo, in vitro and ex vivo test methods with respect to their relevance to human ocular anatomy, anticipated exposure scenarios, and the mechanisms of eye irritation/corrosion in humans. Each of the in vitro and ex vivo methods described is generally appropriate for identifying non-irritants. To discriminate among eye irritants, the human three-dimensional epithelial and full thickness corneal models provide the most detailed information about the severity of irritation. Consideration of the mechanisms of eye irritation, and the strengths and limitations of the in vivo, in vitro and ex vivo test methods, show that the in vitro/ex vivo methods are as or more reflective of human biology and less variable than the currently used rabbit approach. Suggestions are made for further optimizing the most promising methods to distinguish between severe (corrosive), moderate, mild and non-irritants and provide information about the reversibility of effects. Also considered is the utility of including additional information (e.g. physical chemical properties), consistent with the Organization for Economic Cooperation and Development's guidance document on an integrated approach to testing and assessment of potential eye irritation. Combining structural and functional information about a test substance with test results from human-relevant methods will ensure the best protection of humans following accidental eye exposure to agrochemicals.

In vitro effects of glyphosate-based herbicides and related adjuvants on primary culture of hemocytes from Haliotis tuberculata.

Glyphosate-based herbicides are among the most produced and widely-used herbicides. Studies have shown that commercial formulations and adjuvants may be more toxic to non-target organisms than the active ingredients alone, but the mechanisms of action of these chemicals remain unclear. The aim of this study was to investigate the in vitro effects of glyphosate, a commercial formulation and adjuvant alone using primary culture of hemocytes from the European abalone Haliotis tuberculata, a commonly farmed shellfish. Glyphosate was found to have negligible effects on viability, phagocytic activities and lysosome stability even with very high doses (i.e. 100 mg L-1). By contrast, greater effects on viability were observed for the commercial formulation and adjuvant alone, with EC50 values of 41.42 mg L-1 and 1.85 mg L-1, respectively. These results demonstrate that the toxic sublethal effects (i.e. phagocytic activity and destabilization of lysosomal membranes) of formulated glyphosate came from adjuvants and suggest they may be related to cell and organelle membrane destabilization.

Electroenzymatic Nitrogen Fixation Using a MoFe Protein System Immobilized in an Organic Redox Polymer.

We report an organic redox-polymer-based electroenzymatic nitrogen fixation system using a metal-free redox polymer, namely neutral-red-modified poly(glycidyl methacrylate-co-methylmethacrylate-co-poly(ethyleneglycol)methacrylate) with a low redox potential of -0.58 V vs. SCE. The stable and efficient electric wiring of nitrogenase within the redox polymer matrix enables mediated bioelectrocatalysis of N3- , NO2- and N2 to NH3 catalyzed by the MoFe protein via the polymer-bound redox moieties distributed in the polymer matrix in the absence of the Fe protein. Bulk bioelectrosynthetic experiments produced 209±30 nmol NH3 nmol MoFe-1 h-1 from N2 reduction. 15 N2 labeling experiments and NMR analysis were performed to confirm biosynthetic N2 reduction to NH3 .

Study of biocompatibility of peritoneal dialysis solutions measured as in vitro cells viability.

This paper presents the comparable viability study results of the HepG2 and Vero cells in the presence of traditional peritoneal dialysis (PD) solutions determined by three methods (3-[4,5-dimethylthiazol]-2-yl-2,5-diphenyl tetrazolium bromide (MTT), neutral red (NR) and sulforhodamine B assays) with establishing different correlations between viability and quality indexes of the tested PD solutions. The obtained results confirmed cytotoxicity of the PD solutions even compared with an isotonic solution of sodium chloride. PD solutions action resulted in a similar reduction in the HepG2 and Vero cells. Moreover, this research found that metabolic cellular activity is more vulnerable to the action of PD solutions measured in the MTT-test. One more point is that cytotoxicity is related to pH of a solution and other unknown mechanisms, while glucose degradation products, glucose or lactate did not exert an exceptional negative action on PD solutions cytotoxicity. It is concluded that MTT-test is the best suitable for comparative studies of PD solutions which differ in pH values.

An efficient method for eccrine gland isolation from human scalp.

We describe a simple and efficient method to isolate eccrine sweat glands from the human scalp. This method is inspired by the hair graft harvesting method used in hair transplantation. Based on the recently described anatomical relationship between the scalp hair follicle and the eccrine gland, we have found that scalp follicular unit grafts are an excellent eccrine gland isolation source, especially for the coiled component. In order to make the gland visible for stereoscopic microdissection, the follicular units need to be previously stained with a vital dye like methylene blue or neutral red. The simplicity and efficiency of this isolation method should encourage further research into human eccrine sweat gland function which has always been hindered by the difficulty of gland isolation.

Assessment of tobacco heating product THP1.0. Part 5: In vitro dosimetric and cytotoxic assessment.

Tobacco heating products (THPs) represent a subset of the next-generation nicotine and tobacco product category, in which tobacco is typically heated at temperatures of 250-350 °C, thereby avoiding many of the harmful combustion-related toxicant emissions of conventional cigarettes. In this study, we have assessed aerosol generation and cytotoxicity from two commercially available THPs, THP1.0 and THS, relative to tobacco smoke from 3R4F reference cigarettes, using an adapted Borgwaldt RM20S Smoking Machine. Quantification of nicotine in the exposed cell-culture media showed greater delivery of nicotine from both THPs than from the cigarette. Using Neutral Red Uptake assay, THPs demonstrated reduced in vitro cytotoxicity in H292 human bronchial epithelial cells as compared with 3R4F cigarette exposure at the air-liquid interface (p < 0.0001). Both THPs demonstrated a statistically similar reduction in biological response, with >87% viability relative to 3R4F at a common aerosol dilution (1:40, aerosol:air). A similar response was observed when plotted against nicotine; a statistical difference between 3R4F and THPs (p < 0.0001) and no difference between the THPs (p = 0.0186). This pre-clinical in vitro biological testing forms part of a larger package of data to help assess the safety and risk reduction potential of next-generation tobacco products relative to cigarettes, using a weight of evidence approach.