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Pellet production represents a critical step for several processes requiring fungal biomass, nevertheless, its optimization is seldom reported. The use of finely ground rice husk as a microcarrier and co-substrate permitted a marked increase (≈ 2.7×) in the productivity of fungal pellet production using Trametes versicolor compared to traditional production methods. The pellets show similar structure and smaller size compared to typical sole-mycelium pellets, as well as comparable laccase activity. The efficiency of the pellets for biodegradation was confirmed by the removal of the crystal violet dye, achieving significantly faster decolorization rates compared to the traditionally produced pellets. The use of these pellets during the continuous treatment of the dye in a stirred tank bioreactor resulted in 97% decolorization operating at a hydraulic residence time of 4.5 d., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Uropathogenic Escherichia coli, the most common cause for urinary tract infections, forms biofilm enhancing its antibiotic resistance. To assess the effects of compounds on biofilm formation of uropathogenic Escherichia coli UMN026 strain, a high-throughput combination assay using resazurin followed by crystal violet staining was optimized for 384-well microplate. Optimized assay parameters included, for example, resazurin and crystal violet concentrations, and incubation time for readouts. For the assay validation, quality parameters Z' factor, coefficient of variation, signal-to-noise, and signal-to-background were calculated. Microplate uniformity, signal variability, edge well effects, and fold shift were also assessed. Finally, a screening with known antibacterial compounds was conducted to evaluate the assay performance. The best conditions found were achieved by using 12 µg/mL resazurin for 150 min and 0.023% crystal violet. This assay was able to detect compounds displaying antibiofilm activity against UMN026 strain at sub-inhibitory concentrations, in terms of metabolic activity and/or biomass., (© 2024. The Author(s).)

Confronting the environmental threat posed by textile dyes, this study highlights bioremediation as a pivotal solution to mitigate the impacts of Crystal Violet, a widely-utilized triphenylmethane dye known for its mutagenic and mitotic toxicity. We isolated and identified several bacterial strains capable of degrading Crystal Violet under various environmental conditions. Newly identified strains, including Mycolicibacterium nivoides , Chryseobacterium sp., Agrobacterium rhizogenes , Pseudomonas crudilactis , and Pseudomonas koreensis demonstrated significant decolorization activity of Crystal Violet, complementing the already known capabilities of Stenotrophomonas maltophilia . Initial experiments using crude extracts confirmed their degradation potential, followed by detailed studies that investigated the impact of different pH levels and temperatures on some strains' degradation efficiency. Depending on the bacteria, the degree of activity change according to pH and temperature was different. At 37 °C, Chryseobacterium sp. and Stenotrophomonas maltophilia exhibited higher degradation activity compared to 25 °C, while Pseudomonas crudilactis and Mycolicibacterium nivoides did not exhibit a statistically significant difference between the two temperatures. Mycolicibacterium nivoides performed optimally at pH 8, while Pseudomonas crudilactis showed high activity at pH 5. Stenotrophomonas maltophilia' s activity remained consistent across the pH range. These findings not only underscore the effectiveness of these bacteria as agents for Crystal Violet degradation but also pave the way for their application in large-scale bioremediation processes for the treatment of textile effluents, marking them as vital to environmental sustainability efforts., Competing Interests: The authors declare that they have no competing interests., (© 2024 Kwak et al.)

Background: Escherichia coli is a common cause of biofilm-associated urinary tract infections (UTIs). Biofilm formation in E. coli is responsible for various indwelling medical device-associated infections, including catheter-associated urinary tract infections (CAUTIs). This study aimed to reduce biofilm formation of E. coli ATCC 25922 by knocking out genes involved in quorum sensing (QS) (luxS) and adhesion (fimH and bolA) using the CRISPR/Cas9-HDR approach., Method: Single-guide RNAs (sgRNAs) were designed to target luxS, fimH and bolA genes. Donor DNA for homologous recombination was constructed to provide accurate repairs of double-strand breaks (DSBs). A biofilm quantification assay (crystal violet assay) was performed to quantify the biofilm formation of mutant and wild-type strains. Morphological changes in biofilm architecture were confirmed by scanning electron microscopy (SEM). Further application of the biofilm formation of mutant and wild-type strains on urinary catheter was tested., Results: Crystal violet assay showed that the biofilm formation of ΔfimH, ΔluxS, and ΔbolA strains was significantly reduced compared to the wild-type strain (P value<0.001). The percentage of biofilm reduction of mutant strains was as follows: ΔluxS1 77.51 %, ΔfimH1 78.37 %, ΔfimH2 84.17 %, ΔbolA1 78.24 %, and ΔbolA2 75.39 %. Microscopic analysis showed that all mutant strains lack extracellular polymeric substances (EPS) production compared to the wild-type strain, which was embedded in its EPS matrix. The adherence, cell aggregation, and biofilm formation of wild-type strain on urinary catheters were significantly higher compared to ΔfimH, ΔluxS and ΔbolA strains., Conclusion: Altogether, our results demonstrated that the knockout of luxS, fimH, and bolA genes reduced EPS matrix production, which is considered the main factor in the development, maturation, and maintenance of the integrity of biofilm. This pathway could be a potential strategy to disrupt E. coli biofilm-associated UTIs. This study suggests that CRISPR/Cas9-HDR system may provide an efficient and site-specific gene editing approach that exhibits a possible antibiofilm strategy through intervention with the QS mechanism and adhesion property to suppress biofilm formation associated with UTI catheter infections., Competing Interests: Conflict of interests The authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

An increase of reactive oxygen species in the placenta and oxidative disbalance has been recognized as a significant factor contributing to pregnancy complications. Dietary intake of food rich in antioxidants during pregnancy could exert a protective role in the prevention of adverse outcomes such as preeclampsia, miscarriage, and others. Flavonoid taxifolin has shown numerous health-promoting effects in a large number of studies conducted on animals, as well as various human cell types in vitro. However, its effects on human placental cells-trophoblasts-have yet to be determined. Therefore, cytoprotective and genoprotective effects of taxifolin on trophoblast cell line HTR-8/SVneo under induced oxidative stress were explored in this study. Cytotoxicity of a range of taxifolin concentrations (1-150 µM) was evaluated using the MTT and crystal violet assays. A model of oxidative stress was achieved by exposing HTR-8/SVneo cells to H2O2. To determine cytoprotective and antigenotoxic effects, the cells were pre-incubated with three concentrations of taxifolin (10, 50, and 100 µM) and then exposed to H2O2. Taxifolin in concentrations of 1, 5, 10, 25, 50, and 100 µM showed no cytotoxic effects on HTR-8/SVneo cells, but 150 µM of taxifolin caused a significant decrease in adherent cell number, as detected by crystal violet assay. Pretreatment with the chosen concentrations of taxifolin showed a significant cytoprotective effect on H2O2-induced cytotoxicity, as determined by the MTT assay. Furthermore, taxifolin showed a significant reduction in H2O2-induced DNA damage, measured by comet assay. This study showed protective effects of taxifolin on human trophoblast cells exposed to oxidative damage. Further studies are needed to explore the underlying mechanisms., (© The Author(s) 2022. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Microscopic filamentous fungi are ubiquitous microorganisms that adapt very easily to a variety of environmental conditions. Due to this adaptability, they can colonize a number of various surfaces where they are able to start forming biofilms. Life in the form of biofilms provides them with many benefits (increased resistance to desiccation, UV radiation, antimicrobial compounds, and host immune response). The aim of this study is to find a reliable and reproducible methodology to determine biofilm growth of selected microscopic filamentous fungi strains. Several methods (crystal violet staining, MTT assay, XTT assay, resazurin assay) for the determination of total biofilm biomass and its metabolic activity were tested on four fungi - Alternaria alternata, Aspergillus niger, Fusarium culmorum and Fusarium graminearum, and their biofilm was also imaged by spinning disc confocal microscopy using fluorescent dyes. A reproducible biofilm quantification method is essential for the subsequent testing of the biofilm growth suppression using antifungal agents or physical methods. Crystal violet staining was found to be a suitable method for the determination of total biofilm biomass of selected strains, and the MTT assay for the determination of metabolic activity of the biofilms. Calcofluor white and Nile red fluorescent stains successfully dyed the hyphae of microscopic fungi., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Psoriasis and atopic dermatitis (AD) are characterized by enhanced skin inflammation, which results in hyperproliferation and the recruitment of immune cells into the skin. For that reason, it is needed a chemical capable to reduce cell proliferation and the recruitment of cells. The search for new molecules for therapeutic skin treatment mainly focuses on the antioxidant and anti-inflammatory properties, highlighting the rheological properties of polymeric polypeptides. We studied L-arginine (L-Arg) grafted (-g-) to enzymatic poly(gallic acid) (PGAL). The latter is a multiradical antioxidant with greater properties and thermal stability. The derivative was enzymatically polymerized in an innocuous procedure. The poly(gallic acid)-g-L-Arg molecule (PGAL-g-L-Arg) inhibits bacterial strains which also have been involved in the progression of psoriasis and AD. However, it is important to analyze their biological effect on skin cells. The cell viability was analyzed by calcein/ethidium homodimer assays and crystal violet. The proliferation and cell attachment were determined by a curve of time and quantitation of the optical density of crystal violet. To analyze the cell migration a wound-healing assay was performed. This synthesis demonstrates that it is not cytotoxic at high concentrations (250 μg/mL). We observed a decrease in the proliferation, migration, and adhesion of dermal fibroblasts in vitro but the compound could not avoid the increase of reactive oxygen species in the cell. Based on our findings, PGAL-g-L-Arg is a promising candidate for treating skin diseases such as psoriasis and AD where decreasing the proliferation and cell migration could help to avoid inflammation.

The cultural parameters of Streptomyces sp. for pectinase production were optimized using the Box-Behnken design. The maximum pectinase production was obtained after 58 h at 35°C and pH 7 upon submerged fermentation in yeast extract-containing media. The enzymes were partially purified with acetone precipitation, and the analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram revealed that Streptomyces sp. produced two pectinases protein with molecular weights of about 25 and 75 kDa. The pectinase activity was detected in a wide range of temperatures (30°C-80°C) and pH (3-9) with maximum pectinase activities observed at 70°C and pH 5 and 9. The enzymes retained about 30-40% of their activities even after incubating the enzyme at different temperatures for 120 min. The pectinase activities of Streptomyces sp. were enhanced in the media containing 1.5% pectin, 1% casein as a nitrogen source, 0.5 mM MgSO4, and 5 mM NaCl. Further, the addition of Tween-20, amino acids, and vitamins to the media also enhanced the pectinase activity. Moreover, the bacterium illustrated the ability to decolorize crystal violet dye efficiently. The decolorization rate ranged from 39.29 to 53.75%, showing the highest bacterial decolorization in the media containing 2 mg/mL crystal violet at 144 h. Therefore, the bacterium has the potential in treating wastewater produced by industries like textile industries., (© 2022 The Author(s). Published by S. Karger AG, Basel.)

Nicotinic acetylcholine receptors (nAChRs) are involved in a great range of physiological and pathological conditions. Since they are transmembrane proteins, they interact strongly with the lipids surrounding them. Thus, the plasma membrane composition and heterogeneity play an essential role for the correct nAChR function, on the one hand, and the nAChR influences its immediate lipid environment, on the other hand. The aim of this work was to investigate in more detail the role of the biophysical properties of the membrane in nAChR function and vice versa, focusing on the relationship between Chol and nAChRs. To this end, we worked with different model systems which were treated either with (i) more Chol, (ii) cholesteryl hemisuccinate, or (iii) the enzyme cholesterol oxidase to generate different membrane sterol conditions and in the absence and presence of γTM4 peptide as a representative model of the nAChR. Fluorescence measurements with crystal violet and patch-clamp recordings were used to study nAChR conformation and function, respectively. Using confocal microscopy of giant unilamellar vesicles we probed the membrane phase state/order and organization (coexistence of lipid domains) and lipid-nAChR interaction. Our results show a feedback relationship between membrane organization and nAChR function, i.e. whereas the presence of a model of nAChRs conditions membrane organization, changing its lipid microenvironment, membrane organization and composition perturb nAChRs function. We postulate that nAChRs have a gain of function in disordered membrane environments but a loss of function in ordered ones, and that Chol molecules at the outer leaflet in annular sites and at the inner leaflet in non-annular sites are related to nAChR gating and desensitization, respectively. Thus, depending on the membrane composition, organization, and/or order, the nAChR adopts different conformations and locates in distinct lipid domains and this has a direct effect on its function., (Copyright © 2022. Published by Elsevier Inc.)

Shewanella putrefaciens (S. putrefaciens), which is a common specific spoilage organism (SSO) of marine fish, has strong spoilage ability even under low-temperature conditions. Carbon dioxide (CO 2 ) was widely applied to control microorganisms in aquatic products package. To explore the regulation mechanism of CO 2 on biofilm formation and cell properties of S. putrefaciens, the dynamic formation process of biofilms, cellular surface properties, and cellular metabolic characteristics of S. putrefaciens at both 30 °C and 4 °C in pure CO 2 gas were evaluated. As evidenced by the crystal violet staining method, confocal laser scanning microscopy (CLSM) analysis, and field emission scanning electron microscopy (FESEM) observation, dynamic formation process of S. putrefaciens biofilms was apparently delayed by CO 2 with integral cellular morphology. The number and viability of sessile cells in S. putrefaciens biofilms was significantly lower than those in normal air composition. The changes in cellular surface properties, such as decreased auto-aggregation and hydrophobicity, might be one of the reasons why biofilms were inhibited by CO 2 . Inhibition of swimming and swarming motility ability by CO 2 could also be observed with significantly shorter bacterial halo diameter. What's more, cellular metabolism was significantly decreased by CO 2 according to the results of ATP content, ATPase activity and extracellular proteolytic activity. The influence of CO 2 could be both observed whether combined with 30 °C or 4 °C. However, the inhibition produced by CO 2 was more pronounced at the incubation temperature of 4 °C. In summary, it could be concluded that the dynamic formation process of S. putrefaciens biofilms and cellular metabolic properties could be inhibited by CO 2 . This research provided a theoretical basis for better application of CO 2 to regulate spoilage microorganisms., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

Aims: In the current study the anti-virulence and anti-biofilm activities of the cinnamic acid derivative, 3-methoxycinnamic acid, was investigated against Agrobacterium tumefaciens., Methods and Results: Based on the disc diffusion test and β-galactosidase activity assay, 3-methoxycinnamic acid was shown to interfere with the quorum sensing (QS) system of A. tumefaciens. Crystal violet staining assay, phenol-sulfuric acid method, Bradford protein assay and confocal laser scanning microscopy (CLSM) revealed that the biofilm formation of A. tumefaciens was inhibited after the treatment of 3-methoxycinnamic acid. Employing high-performance liquid chromatography (HPLC) analysis of culture supernatant revealed that the production of 3-oxo-octanoylhomoserine lactone (3-oxo-C8-HSL) decreased concentration-dependently after treatment with 3-methoxycinnamic acid. Swimming and chemotaxis assays also indicated that 3-methoxycinnamic acid had a good effect on reducing the motility and chemotaxis of A. tumefaciens. In addition, the RT-qPCR, molecular docking and simulations further demonstrated that 3-methoxycinnamic acid could competitively inhibit the binding of 3-oxo-C8-HSL to TraR and down-regulate virulence-related genes., Conclusions: 3-Methoxycinnamic acid is proved to have good anti-virulence and anti-biofilm activities against A. tumefaciens., Significance and Impact of the Study: This is the first study that investigates the anti-virulence and anti-biofilm activities of 3-methoxycinnamic acid against A. tumefaciens. With its potential QS-related virulence and biofilm inhibitory activities, 3-methoxycinnamic acid is expected to be developed as a potent pesticide or adjuvant for the prevention and treatment of crown gall caused by A. tumefaciens., (© 2022 Society for Applied Microbiology.)

Introduction. Staphylococcus aureus is a major cause of chronic diseases and biofilm formation is a contributing factor. 20S-ginsenoside Rg3 (Rg3) is a natural product extracted from the traditional Chinese medicine red ginseng. Gap statement. The effects of Rg3 on biofilm formation and haemolytic activity as well as its antibacterial mechanism against S. aureus have not been reported. Aim. This study aimed to investigate the effects of Rg3 on biofilm formation and haemolytic activity as well as its antibacterial action against clinical S. aureus isolates. Methodology. The effect of Rg3 on biofilm formation of clinical S. aureus isolates was studied by crystal violet staining. Haemolytic activity analysis was carried out. Furthermore, the influence of Rg3 on the proteome profile of S. aureus was studied by quantitative proteomics to clarify the mechanism underlying its antibacterial action and further verified by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Results. Rg3 significantly inhibited biofilm formation and haemolytic activity in clinical S. aureus isolates. A total of 63 with >1.5-fold changes in expression were identified, including 34 upregulated proteins and 29 downregulated proteins. Based on bioinformatics analysis, the expression of several virulence factors and biofilm-related proteins, containing CopZ, CspA, SasG, SaeR/SaeS two-component system and SaeR/SaeS-regulated proteins, including leukocidin-like protein 2, immunoglobulin-binding protein G (Sbi) and fibrinogen-binding protein, in the S. aureu s of the Rg3-treated group was downregulated. RT-qPCR confirmed that Rg3 inhibited the regulation of SaeR/SaeS and decreased the transcriptional levels of the biofilm-related genes C opZ , CspA and SasG . Conclusions. Rg3 reduces the formation of biofilm by reducing cell adhesion and aggregation. Further, Rg3 can inhibit the SaeR/SaeS two-component system, which acts as a crucial signal transduction system for the anti-virulence activity of Rg3 against clinical S. aureus isolates.

Human adipose stem cells (ASCs) hold great potential for regenerative medicine approaches, including osteogenic regeneration of bone defects, that fail to heal autonomously. Osteogenic differentiation of stem cells is dependent on the stimulation of biophysical factors. In the present study, the effects of hypergravity, hypoxia, and hyperbaric treatment were investigated on adipose stem cell (ASC) metabolic activity, quantified by PrestoBlue conversion, and cell numbers, evaluated by crystal violet staining. Osteogenic differentiation was assessed by alkaline phosphatase (ALP) activity and cresolphthalein staining of calcium deposition. Differentiation was performed for 12 days, which was accompanied by periodical stimulation. Increasing gravity forces up to 50x g did not affect ASC viability, but it enhanced osteogenic markers with a strongest effect between 20 and 30x g. Hyperbaric stimulation at 3 bar decreased ASC cell numbers but increased ALP activity and calcium deposition. Hypoxia at 8 % atmospheric oxygen did not affect ASC proliferation, while cell numbers were reduced at 3 % oxygen. Furthermore, hypoxic conditions produced opposing results on osteogenic markers, as ALP activity increased whereas cresolphthalein staining decreased upon stimulation. These data demonstrated that intermittent short duration of basal physical or chemical impulses interfere with the osteogenic differentiation of ASCs. Our findings could be of specific relevance in ASC based therapies for regenerative medicine and bone tissue engineering approaches., Competing Interests: Declarations of interest No potential conflicts of interest, financial or otherwise are declared by the authors., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Aims: Staphylococcus aureus has emerged as a serious threat to food safety owing to biofilm formation. The study aimed to examine the antibiofilm mechanism of a novel milk-derived antimicrobial peptide BCp12 against it., Methods and Results: Antibiofilm activity of BCp12 was studied by crystal violet staining, MTT assay, motility, SEM and CLSM. TMT proteome, real-time PCR and molecular docking in silico were conducted to evaluate the mechanism of BCp12 against S. aureus biofilm. The results showed that BCp12 had significant antibiofilm activity at 1 × MIC and sub-MIC. BCp12 induced the dispersion of structure of S. aureus biofilm BCp12 inhibited the movement of S. aureus. A total of 703 proteins were downregulated and 334 proteins were upregulated after BCp12 treatment. The proteins (agrA, agrB, agrC and psmβ) of the QS systems were downregulated. Additionally, the expression of the agr-related genes, agrA, agrB, agrC and psmβ, was downregulated. BCp12 was bound to the receptor proteins agrA and agrC through hydrogen bonds and π-π bonds., Conclusions: The results showed the antibiofilm activity of BCp12 and it inhibits the biofilm formation by interfering agr QS system., Significance and Impact of Study: BCp12 has the potential to be a novel antibiofilm agent against S. aureus biofilm and used in the food industry., (© 2022 The Society for Applied Microbiology.)

The cytoprotective effects of a novel hydroalcoholic extract (0.01-5 mg/mL) from Lens culinaria (Terre di Altamura Srl) were investigated within murine native skeletal muscle fibers, bone marrow cells, and osteoblasts, and in cell lines treated with the apoptotic agent staurosporine (2.14 × 10 -6 M), the alkylating drug cisplatin (10 -4 M), the topoisomerase I inhibitor irinotecan (10 -4 M), the antimitotic pro-oxidant doxorubicin (10 -6 M), and the immunosuppressant dexamethasone (2 × 10 -6 M). An amount of 10g of plant material was used to obtain a 70% ethanol/water product, following two-step extraction, evaporation, lyophilization, and storage at -20 °C. For the murine osteoblasts, doxorubicin reduced survival by -65%, dexamethasone by -32% and -60% after 24 and 48 h of incubation time, respectively. The extract was effective in preventing the osteoblast count-reduction induced by dexamethasone; it was also effective at preventing the inhibition of mineralization induced by dexamethasone. Doxorubicin and cisplatin caused a significant reduction in cell growth by -77% for bone marrow cells, -43% for irinotecan, and -60% for dexamethasone, but there was no evidence for the cytoprotective effects of the extract in these cells. Staurosporine and doxorubicin caused a fiber death rate of >-40% after 18 and 24 h of incubation, yet the extract was not effective at preventing these effects. The extract was effective in preventing the staurosporine-induced reduction of HEK293 proliferation and colony formation in the crystal violet DNA staining and the clonogenic assays. It was also effective for the cisplatin-induced reduction in HEK293 cell proliferation. The extract, however, failed to protect the SHSY5Y neurons against cisplatin and irinotecan-induced cytotoxicity. A UV/VIS spectroscopy analysis showed three peaks at the wavelengths of 350, 260, and 190 nm, which correspond to flavonoids, proanthocyanins, salicylates, and AA, constituting the extract. These data suggest the possible development of this extract for use against dexamethasone-induced bone loss and renal chemotherapy-induced damage.

Background: Asian sand dust (ASD) and Aspergillus fumigatus are known risk factors for airway mucosal inflammatory diseases. Bacterial and fungal biofilms commonly coexist in chronic rhinosinusitis and fungus balls. We evaluated the effects of ASD on the development of A. fumigatus biofilm formation on nasal epithelial cells., Methods: Primary nasal epithelial cells were cultured with A. fumigatus conidia with or without ASD for 72 h. The production of interleukin (IL)-6, IL-8, and transforming growth factor (TGF)-β1 from nasal epithelial cells was determined by the enzyme-linked immunosorbent assay. The effects of ASD on A. fumigatus biofilm formation were determined using crystal violet, concanavalin A, safranin staining, and confocal scanning laser microscopy., Results: ASD and A. fumigatus significantly enhanced the production of IL-6 and IL-8 from nasal epithelial cells. By coculturing A. fumigatus with ASD, the dry weight and safranin staining of the fungal biofilms significantly increased in a time-dependent manner. However, the increased level of crystal violet and concanavalin A stain decreased after 72 h of incubation., Conclusions: ASD and A. fumigatus induced the production of inflammatory chemical mediators from nasal epithelial cells. The exposure of A. fumigatus to ASD enhanced the formation of biofilms. The coexistence of ASD and A. fumigatus may increase the development of fungal biofilms and fungal inflammatory diseases in the sinonasal mucosa.

The fungal cell wall and membrane are the principal targets of antifungals. Herein, we report that myricetin exerts antifungal activity against Candida albicans by damaging the cell wall integrity and notably enhancing the membrane permeability. In the presence of sorbitol, an osmotic protectant, the minimum inhibitory concentration (MIC) of myricetin against C. albicans increased from 20 to 40 and 80 μg/ml in 24 and 72 h, respectively, demonstrating that myricetin disturbs the cell wall integrity of C. albicans . Fluorescence microscopic images showed the presence of propidium iodidestained C. albicans cells, indicating the myricetin-induced initial damage of the cell membrane. The effects of myricetin on the membrane permeability of C. albicans cells were assessed using crystal violet-uptake and intracellular material-leakage assays. The percentage uptakes of crystal violet for myricetin-treated C. albicans cells at 1×, 2×, and 4× the MIC of myricetin were 36.5, 60.6, and 79.4%, respectively, while those for DMSO-treated C. albicans cells were 28.2, 28.9, and 29.7%, respectively. Additionally, myricetin-treated C. albicans cells showed notable DNA and protein leakage, compared with the DMSO-treated controls. Furthermore, treatment of C. albicans cells with 1× the MIC of myricetin showed a 17.2 and 28.0% reduction in the binding of the lipophilic probes diphenylhexatriene and Nile red, respectively, indicating that myricetin alters the lipid components or order in the C. albicans cell membrane, leading to increased membrane permeability. Therefore, these data will provide insights into the pharmacological worth of myricetin as a prospective antifungal for treating C. albicans infections.

The control over biodistribution and pharmacokinetics is critical to enhance the efficacy and minimize the side effects of therapeutic agents. To address the need for an on-demand drug delivery system for precise control over the release time and the quantity of drugs, we exploited the mechano-responsiveness of piezoelectric poly(vinylidene fluoride-trifluroethylene) (P(VDF-TrFE)) nanofibers for drug delivery applications. The large surface area-to-volume ratio inherent to nanomaterials, together with the transformative piezoelectric properties, allowed us to use the material as an ultrasensitive and mechano-responsive drug delivery platform driven by the direct piezoelectric effect. The intrinsic negative zeta potential of the nanofibers was utilized to electrostatically load cationic drug molecules, where surface potential changes by exogenous mechanical actuation trigger the release of drug molecules. We show that the drug release kinetics of the P(VDF-TrFE) nanofibers depends on the fiber diameter, thus piezoelectric properties. We further demonstrated that the drug release quantity can be tuned by the applied pressure or dose of physiologically safe corporeal shockwaves as a mechanical stimulus in in vitro and ex vivo models. Overall, we demonstrated the utility of piezoelectric electrospun nanofibers for mechano-responsive controlled drug release.

Synthetic dyes are widely used in various industries and their wastage causes severe environmental problems while being hazardous to human health, leading to the need for eco-friendly degradation techniques. The split-gill fungus Schizophyllum commune , which is found worldwide, has the potential to degrade all components of the lignocellulosic biomass and is a candidate for the treatment of synthetic dyes. A systematic molecular analysis of 75 Korean and 6 foreign S. commune strains has revealed the high genetic diversity of this population and its important contribution to the total diversity of S. commune . We examined the dye decolorization ability of this population and revealed 5 excellent strains that strongly decolorized 3 dyes: Crystal Violet, Congo Red and Methylene Blue. Finally, comparison of dye decolorization ability and the phylogenetic identification of these strains generalized their genetic and physiological diversity. This study provides an initial resource for physiological and genetic research projects as well as the bioremediation of textile dyes.

Triphenylmethane dyes are commonly used in dyeing and printing, but such dyes are recalcitrant to degradation and thus biodegradation of dye compounds has received increasing attention. Here, a recombinant bacterial laccase, CotA, from Bacillus amyloliquefaciens MN-13 was expressed in Escherichia coli BL21(DE3) and used as a biocatalyst to degrade crystal violet (CV). The recombinant CotA remained stable at temperatures in the range 30-40 °C and retained 44-100% enzyme activity at pH 4.5-8.0. The CotA exhibited high activities for decolorization of CV and, after 72 h of incubation, CotA decolorized 70.98% of CV at pH 5.0 and 30 °C. In the UV-visible spectra of CV solution treated by CotA, the full wavelength scan indicated that the chromophore of the triphenylmethane structure of CV was destroyed and CV was degraded into small-molecule aromatic compounds. The main degradation compounds of CV were identified as bis[4-(dimethylamino) phenyl] methanone and its N-demethylation derivative by HPLC/MS/MS. Based on these data, a hypothetical degradation pathway of CV by CotA, including N-demethylation and cleavage of the chromophore structure initiated by radicals, is proposed., (Copyright © 2020 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

In the literature, there is a lack of data on the effect of gentian violet (GV) and congo red (CR) dyes on the biosynthesis pathway of biogenic amines (BAs) in Lemna minor L. (common duckweed). This plant species is an important link in the food chain. Both dyes inhibited growth, biomass yield and the biosynthesis of chlorophyll a in common duckweed. The predicted toxic units demonstrated that GV had a more toxic effect on the growth rate and biomass yield of common duckweed than CR. Decarboxylase activity in the biosynthesis of BAs in common duckweed is also a useful indicator for evaluating the toxicity of both dyes. Gentian violet also exerted more phytotoxic effects on the analyzed biochemical features of common duckweed because it changed the putrescine (Put) biosynthesis pathway, increased tyramine content 1.6 fold, inhibited the activity of S-adenosylmethionine decarboxylase by 40% and the activity of ornithine decarboxylase (ODC) by 80%. Tyrosine decarboxylase (TDC) was most active in plants exposed to the highest concentration of GV. Similarly to control plants, in common duckweed exposed to CR, Put was synthesized from ornithine; however, spermidine content was 86% higher, Put content was 51% lower, and ODC activity was 86% lower., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Malachite green (MG) and crystal violet (CV) are the typical triphenylmethane dyes, which are recalcitrant molecules exerting mutagenic and carcinogenic effects on living organisms. Characterization of the residues of MG, CV, and their metabolites in biological organisms is of importance, especially for in vivo and in situ characterization. In this study, a method for determination of trace MG, CV, and their leuco metabolites in zebrafish by surface-coated probe nanoelectrospray ionization mass spectrometry (SCP-nanoESI-MS) was developed. A microscale solid-phase microextraction (SPME) probe was developed and used for extraction and enrichment of trace MG, CV, and their leuco metabolites in zebrafish after exposure. After that, the loaded SPME probe was directly employed for nanoESI-MS analysis under ambient and open-air conditions. Under the optimum conditions, the method demonstrated good linearity, with correlation coefficient values (r 2 ) no less than 0.9925. The limits of detection and quantification were 0.014-0.023 ng mL -1 and 0.046-0.077 ng mL -1 , respectively. By using the proposed method, the bioaccumulation of MG and CV in zebrafish was investigated, and the distribution of MG, CV, and their leuco metabolites in different organs of zebrafish was studied. MG, CV, and their leuco metabolites were all found in zebrafish tissues including brain, muscle, heart, and kidney after exposure, with highest concentration in intestine followed in ovary., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Detecting bacteria in samples and differentiating between Gram-negative and Gram-positive species is an important challenge, and the most common method, Gram staining, is very time consuming. The aim of this study was to evaluate the electrical bioimpedance spectroscopy (EBIS) technique as an inexpensive and practical tool for real-time detection of bacteria and differentiation between Gram-positive and Gram-negative species. The relevant sensitivity for differentiating between species was found in the magnitude and phase at frequencies of 158,489 and 5248 Hz, respectively, at a bacterial concentration of 1 μg/μl. Subsequently, the sensitivity was estimated as a function of bacterial concentration. Our results demonstrated that EBIS can potentially distinguish between presence and absence of bacteria as well as between different types of bacteria.

The dye decolorization potential of the white-rot fungus Phlebia brevispora TMIC33929 when grown alone or in coculture with its growth-promoting bacterium Enterobacter sp. TN3W-14 was evaluated in low nitrogen liquid medium at different pHs. Axenic fungus removed a similar amount of Congo red and crystal violet at pH 4.5 and 7.0, respectively. The bacterium alone achieved only slightly better decolorization of crystal violet than the fungus at pH 9.0. Compared with axenic fungus, cocultures provided no increased crystal violet removal but achieved higher removal of crystal violet in mixed dye at all pHs, and the best-mixed dye decolorization at pH 9.0. Unlike bacterial growth on dyes, growth of fungal mycelia was not inhibited by the dyes at all pH but the cocultures gave comparably higher mycelial growth.

Purpose: Diagnosis of the depth of invasion is crucial in the endoscopic management of early colorectal cancer. Image-enhanced endoscopy (IEE) is a method for easily evaluating the depth of invasion. Linked colour imaging (LCI) is an IEE method that enables clearer identification of neoplastic lesions and mucosal inflammation. The aim of this experimental study was to explore the efficacy of LCI in vessel and pit pattern recognition when used in magnifying chromoendoscopy with crystal violet staining for superficial colorectal neoplasms., Methods: This was an experimental study. Colour difference (CD) values between the surrounding mucosa and vessels and pits were measured on white light (WLI), blue laser (BLI), and LCI images. The CD values of 10 neoplastic lesions were calculated and compared between WLI and the other techniques., Results: The CD value was 9.8 (interquartile range, 7.3-12.4) for WLI, 9.7 (6.7-13.4) for LCI, and 6.8 (5.1-9.3) for BLI. The CD value was statistically different between WLI and BLI but not between WLI and LCI. With regard to vessel description, the CD value was 7.5 (4.0-11.0) for WLI, 15.6 (11.6-23.9) for LCI, and 23.3 (15.8-30.4) for BLI., Conclusions: LCI provides more diagnostic information than other light modes. Further, it is superior to the other techniques in terms of vessel visibility and is comparable to them in terms of pit recognition. These unique features of LCI may lead to its use as an alternative to WLI and BLI for pit and vessel pattern evaluation in the future.

The feasibility of producing biochar and its steam-activated counterpart in a large scale (1000 kg) from spent mushroom substrate (SMS) and their usage as effective environmental remediation tools to augment current SMS management strategies were explored. Steam-activated SMS biochar exhibited enhanced surface area (332 m 2 /g), pore volume (0.29 cm 3 /g), and porosity (77.1%). The effectiveness of activation was higher on the cationic dye, crystal violet (CV) by 4.1 times increase from 255 mg/g to 1057 mg/g. The biochar and its steam-activated counterpart, respectively exhibited high COD and color removal efficiencies of 49.6% and 40.1%, and 67.7 and 99.6% for CV-spiked real wastewater. Reusability studies confirmed the dominant role of chemisorption in the adsorption process. The lower production cost coupled with the superior physicochemical properties and adsorption performances rendered the biochar with/without steam activation, as a promising alternative adsorbent to serve as a green, viable and effective environmental remediation tool., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The present study focuses on the biodegradation of triphenylmethane dye crystal violet (CV) by Cedecea davisae. The degradation of CV was evaluated via ultraviolet absorbance at 254 nm (UV 254 ) and chemical oxygen demand (COD) removal, and the kinetics was used to evaluate the degradation efficiency. Intermediate products were analyzed via UV-vis spectroscopy (UV), Fourier transform infrared spectroscopy (FTIR), and high-performance liquid chromatography (HPLC). Results showed that C. davisae was able to decolorize the CV, and the maximum decolorization ratio reached 97%. COD reduction was observed after decolorization, with average removal rates of >90% after 48 h. Moreover, 50% of UV 254 can be removed after 14 h. The removal efficiency of CV by C. davisae followed first- and second-order reaction kinetics at temperature ranged from 20 °C to 40 °C and pH 4.0 to 6.0, respectively. By using UV, the peak representing the CV disappeared 14 h after CV decolorization, and the degradation of aromatic and naphthalene rings was attributed to the formation of a new metabolite. The FTIR spectra of metabolites showed that a new functional group of OH, CH, CH 2 , CH 3 , NH, CN, CN, or CO was produced. The chromatograms of HPLC recorded at 589 nm at retention time decreased and were not detected following incubation for 8 h by C. davisae., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Monomeric, dimeric, and trimeric derivatives of the triphenylmethane dye crystal violet (1a-1f) have been synthesized for the purpose of evaluating their affinity and sequence selectivity for duplex DNA. Competitive ethidum displacement assays indicate that 1a-1f have apparent association constants for CT DNA in the range of 1.80-16.2 × 10 7  M -1 and binding site sizes of 10-14 bp. Viscosity experiments performed on ligand 1f confirmed that these dyes associate with duplex DNA by a non-intercalative mode of binding. Circular dichroism and competition binding studies of the tightest binding ligand 1e with known major and minor groove binding molecules suggest that these dye derivatives likely occupy the major groove of DNA. Data from the binding of 1e to polynucleotides indicate close to an order of magnitude preference for associating with AT rich homopolymers over GC rich homopolymers, suggesting a shape-selective match of the sterically bulky ligand with DNA containing a wider major groove., (Copyright © 2018 Elsevier Inc. All rights reserved.)

An inactive biomass of a new fungus recently discovered, Diaporthe schini, was evaluated for the biosorption of crystal violet (CV) in simulated textile effluents. The characterization assays were performed using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and N 2 adsorption/desorption isotherms. The influences of pH and biosorbent dosage on the biosorption capacity were evaluated. Kinetics, equilibrium and thermodynamic studies were also carried out. Characterization techniques showed an amorphous biosorbent, with a rough surface containing irregular particles and surface area of 6.5 m 2 g -1 . The most adequate values of pH and biosorbent dosage were 7.5 and 0.4 g L -1 , respectively. The Elovich kinetic model and the Sips equilibrium model were suitable to fit the experimental data. The biosorption capacity increased with temperature, reaching a maximum biosorption capacity of 642.3 mg g -1 at 328 K. The biosorption was a spontaneous and endothermic process. Diaporthe schini inactive biomass was an interesting biosorbent to treat colored effluents, presenting efficiency of 87% in the decolorization of a simulated dye house effluent.

Laccases can oxidize a variety of phenolic and non-phenolic substrates including synthetic dyes. In this research, a laccase gene Lcc9 from Laccaria bicolor was chemically synthesized and optimized to heterogeneous expression in Pichia pastoris and Arabidopsis thaliana . The properties of recombinant laccase expressed by P. pastoris were investigated. The laccase activity was optimal at 3.6 pH and 40°C. It exhibited K m and V max values of 0.565 mmol l⁻¹ and 1.51 μmol l⁻¹ min⁻¹ for ABTS respectively. As compared with untransformed control plants, the laccase activity in crude extracts of transgenic lines exhibited a 5.4 to 12.4-fold increase. Both laccases expressed in transgenic P. pastoris or A. thaliana could decolorize crystal violet. These results indicated that L. bicolor laccase gene may be transgenically exploited in fungi or plants for dye decolorization.

Persistent candidemia refers to the continued isolation of the same Candida species in the blood of a candidemic patient despite appropriate therapy. Despite the clinical importance of persistent candidemia, studies have superficially addressed the biological conditions behind this phenomenon. The aim of this study was to evaluate the correlation between the biofilm-forming ability by Candida bloodstream isolates and the persistence of infection. A total of 55 isolates of Candida were tested and characterized in two groups: (i) group I, which included seven patients with persistent candidemia, and (ii) group II, which included 18 patients with nonpersistent candidemia. Microorganisms were identified at the species level by sequencing the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA). Biofilm quantification was evaluated by the crystal violet staining method and confocal scanning laser microscopy (CSLM). Molecular tests confirmed the identification of Candida albicans (92% group I and 94% group II) and Candida dubliniensis isolates (8% group I and 6% group II). All 55 isolates were able to form biofilms, but a higher biofilm mass was produced by C. albicans/C. dubliniensis strains cultured from the persistent group (P < .05). Our data suggest that Candida sp. biofilm production should be considered a relevant biologic variable in explaining patients who fail to clear a bloodstream infection despite adequate antifungal treatment with triazoles.

Leuco crystal violet (LCV) and leuco malachite green (LMG) are the main metabolites of two dyes that are forbidden for use in food production, but can be present at low background concentration in novel Atlantic salmon feed ingredients such as processed animal proteins (animal by-product [ABP]). In this study, the potential transfer of dietary LCV or LMG to the fillet of farmed Atlantic salmon was investigated. The uptake and elimination rate kinetics were determined in seawater-adapted Atlantic salmon (initial weight 587 ± 148 g) fed two levels of either LCV- or LMG-enriched diets (~500 and 4000 µg kg -1 , respectively) for 40 days, followed by a 90-day depuration period with feeding on control diets (<0.15 μg kg -1 LCV and LMG). A three-compartmental model was developed, based on a fillet fat, fillet muscle and a central body compartment comprising all other tissues. Model calibrations showed a good fit with measured values during overall uptake and elimination period; however, the model poorly predicted the short-term (days) peak measured values at the end of the exposure period. The model was used to simulate the long-term (>16 months) LCV and LMG feed-to-fillet transfer in Atlantic salmon under realistic farming conditions such as the seasonal fluctuations in feed intake, growth and fillet fat deposition. The model predictions gave highest expected LCV and LMG fillet concentrations of approximately 0.12 and 0.45 μg kg -1 , depending on the dietary levels of ABP and background level of LCV and LMG contamination. These levels are under the reference point for action of 2 µg kg -1 for the sum of MG and LMG that EFSA assessed as adequate to protect public health. However, for LCV, the predicted highest levels exceed the analytical decision limit (CCα) of 0.15 µg kg -1 for the method used in this paper.

Guanine-rich sequences may fold into highly ordered structures known as G-quadruplexes. Apart from the monomeric G-quadruplex, these sequences may form multimeric structures that are not usually considered when studying interaction with ligands. This work studies the interaction of a ligand, crystal violet, with three guanine-rich DNA sequences with the capacity to form multimeric structures. These sequences correspond to short stretches found near the promoter regions of c-kit and SMARCA4 genes. Instrumental techniques (circular dichroism, molecular fluorescence, size-exclusion chromatography and electrospray ionization mass spectrometry) and multivariate data analysis were used for this purpose. The polymorphism of G-quadruplexes was characterized prior to the interaction studies. The ligand was shown to interact preferentially with the monomeric G-quadruplex; the binding stoichiometry was 1:1 and the binding constant was in the order of 10 5 M -1 for all three sequences. The results highlight the importance of DNA treatment prior to interaction studies., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cell-to-cell communication mediated by the diffusible signal factor (DSF) is a common form of gene regulation and plays an important role in virulence of many plant pathogenic bacteria including Xanthomonas spp. Here we describe several approaches to study the involvement of DSF-dependent QS system of the plant pathogenic bacteria Xanthomonas campestris pv. pelargonii (Xhp) as an example of the Xanthomonas spp. The methods described include detection and measurement of DSF, movement in planta, colonization, and aggregate formation.

Candida haemulonii species complex have emerged as multidrug-resistant yeasts able to cause fungemia worldwide. However, very little is known regarding their physiology and virulence factors. In this context, planktonic growth and biofilm formation of Brazilian clinical isolates of Candida haemulonii (n = 5), Candida duobushaemulonii (n = 4), and Candida haemulonii var. vulnera (n = 3) were reported. Overall, the fungal planktonic growth curves in Sabouraud dextrose broth reached the exponential phase in 48 h at 37°C. All the clinical isolates formed biofilm on polystyrene in a time-dependent event, as judged by the parameters evaluated: biomass (crystal violet staining), metabolic activity (XTT reduction), and extracellular matrix (safranin incorporation). No statistically significant differences were observed when the average measurements among the three Candida species were compared regarding both planktonic and biofilm lifestyles; however, typical isolate-specific differences were clearly noticed in fungal growth kinetics., (© The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The bacterial diversity associated with biofilm-forming ability was studied. Eighteen bacterial strains were isolated from a microbial film collected from the roof of an old house located in Sfax, Tunisia. The purity of these microorganisms was confirmed by microscopic observation after repeated streaking on a Tryptic Soy agar medium. Biofilm formation was estimated using preliminary tests including a motility test, microbial adhesion to solvents (MATS), and the Congo Red Agar method (CRA). Since these tests showed no significant result, microplate tests, such as crystal violet and resazurin assays, were used. The results obtained showed that strain S61 was able to form a biofilm within 24 h (OD 570 = 4.87). The viability of the S61 biofilm with resazurin assessed with fluorescence measurement was about 1.5 × 10 3 . The S61 strain was identified as Staphylococcus epidermidis. In the biofilm studied here, it was the most biofilm-forming bacterium and will be used as a bacterial model for studying anti-biofilm activity.

Marasmius scorodoniu s secretes an extracellular laccase in potato dextrose broth, and this enzyme was purified up to 206-fold using (NH 4 ) 2 SO 4 precipitation and a Hi-trap Q Sepharose column. The molecular mass of the purified laccase was estimated to be ~67 kDa by SDS-PAGE. The UV/vis spectrum of the enzyme was nontypical for laccases, and metal content analysis revealed that the enzyme contains 1 mole of Fe and Zn and 2 moles of Cu per mole of protein. The optimal pH for the enzymatic activity was 3.4, 4.0, and 4.6 with 2,2'-azino- bis (3-ethylbenzothazoline-6-sulfonate) (ABTS), guaiacol, and 2,6-dimethoxy phenol as the substrate, respectively. The optimal temperature of the enzyme was 75°C with ABTS as the substrate. The enzyme was stable in the presence of some metal ions such as Ca 2+ , Cu 2+ , Ni 2+ , Mg 2+ +, Mn 2+ , Ba 2+ , Co 2+ , and Zn 2+ at a low concentration (1 mM), whereas Fe 2+ completely inhibited the enzymatic activity. The enzymatic reaction was strongly inhibited by metal chelators and thiol compounds except for EDTA. This enzyme directly decolorized Congo red, Malachite green, Crystal violet, and Methylene green dyes at various decolorization rates of 63-90%. In the presence of 1-hydroxybenzotriazole as a redox mediator, the decolorization of Reactive orange 16 and Remazol brilliant blue R was also achieved.

A novel nanoscale zero-valent iron-Sargassum swartzii (nZVI-SS) biocomposite was synthesized and evaluated for its ability to adsorb crystal violet (CV) from aqueous solutions. Involvement of various functional groups of the biosorbent in preferential adsorption of cationic dye was observed using Fourier transform infrared (FTIR) spectroscopy. Morphological changes occurring on the biocomposite materials were characterized using scanning electron microscopy (SEM). Significant increase (∼90%) in the biosorption of cationic dye was observed with gradual increase in pH of the medium from 3 to 12. The effect of biosorbent concentration, initial pH, temperature, agitation rate, adsorption time, and initial dye concentration was studied for the biosorption of CV using nZVI biocomposite. During the optimization study, maximum biosorption capacity was observed at pH of 8. At various initial CV concentrations (20-100 mg/L), attainment of batch sorption equilibrium was observed within 120 min of reaction time. The Langmuir isotherm model expressed high coefficient of determination (R 2 = 0.999). The maximum dye uptake of 200 mg/g was reported at pH 8. Kinetics and temperature profiles were evaluated and reported. Desorption study was carried out with 0.1 M HCl. Investigations proved that nZVI-SS is an excellent biosorbent for the sequestration of CV in aqueous media.

Smart antimicrobial materials with on-demand drug release are highly desired for biomedical applications. Herein, we report about temperature-responsive poly(N-isopropylacrylamide) (PNIPAM) nanospheres doped with crystal violet (CV) and incorporated into the poly-l-lactide (PLLA) nanofibers. The nanofibers were prepared by electrospinning, using different initial polymers ratios. The morphology of the nanofibers and polymers distribution in the nanofibers were characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The interaction between PNIPAM and PLLA in the nanofibers was studied by Fourier transform infrared spectroscopy (FTIR) and its effect on the PNIPAM phase transition was also investigated. It was shown that by the changing of the environmental temperature across the lower critical solution temperature (LCST) of PNIPAM, the switchable wettability and controlled CV release can be achieved. The temperature-dependent release kinetics of CV from polymer nanofibers was investigated by ultraviolet-visible spectroscopy (UV-Vis). The temperature-responsive release of antibacterial CV was also tested for triggering of antibacterial activity, which was examined on Staphylococcus epidermidis (S. epidermidis) and Escherichia coli (E. coli). Thus, the proposed material is promising value for controllable drug-release., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Laccases of white-rot fungi provide a promising future as a tool to be used in the field of biodegradation of synthetic dyes with different chemical structures. The aim of this study was production, characterization, and application of laccases from the white-rot fungus Ceriporiopsis subvermispora ATCC 90467 for decolorization of triphenylmethane dyes that could remain persistent in wastewater. Laccase was purified from a C. subvermispora culture by a four-step method resulting high specific activity of 2,571 U g -1 , 88-fold higher than crude laccase. Purified laccase (molecular weight 45 kDa) had the optimum activity at pH 2.0 and the optimum temperature 50 °C using ABTS as chromogenic substrate. Laccases efficiently decolorized triphenylmethane dyes such as Malachite Green (87.8%), Bromocresol Purple (71.6%), and Methyl Violet (68.1%) without redox mediator. However, decolorization percentage of hardly degradable triphenylmethane dyes such as Phenol Red, Bromophenol Blue, and Brilliant Blue R-250 was increased the presence of some low-molecular weight compounds (natural or synthetic redox mediators). Purified laccases were resistant to Mg 2+ , Ca 2+ , Ba 2+ , Mn 2+ , Fe 2+ , Cu 2+ , Zn 2+ , and Sn 2+ (10 mmol L -1 ). These findings suggest that laccases from C. subvermispora are able to decolorize triphenylmethane dyes without the negative influence of metal ions that can be found in wastewater., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Adherent cells detach from cell culture plates during cell death. This characteristic can be used for the indirect quantification of cell death and to determine differences in proliferation upon stimulation with death-inducing agents. One simple method to detect maintained adherence of cells is the staining of attached cells with crystal violet dye, which binds to proteins and DNA. Cells that undergo cell death lose their adherence and are subsequently lost from the population of cells, reducing the amount of crystal violet staining in a culture. This protocol describes a quick and reliable screening method that is suitable for the examination of the impact of chemotherapeutics or other compounds on cell survival and growth inhibition. However, characterization of the cause of reduced crystal violet staining requires additional methods detailed elsewhere., (© 2016 Cold Spring Harbor Laboratory Press.)

In the present study, microwave treated Salvadora oleoides (MW-SO) has been investigated as a potential biosorbent for the removal of toxic methyl violet dye. A batch adsorption method was experimented for biosorptive removal of toxic methyl violet dye from the aqueous solution. The effect of various operating variables, viz., adsorbent dosage, pH, contact time and temperature on the removal of the dye was studied and it was found that nearly 99% removal of the dye was possible under optimum conditions. Kinetic study revealed that a pseudo-second-order mechanism was predominant and the overall process of the dye adsorption involved more than one step. Hence, in order to investigate the rate determining step, intra-particle diffusion model was applied. Adsorption equilibrium study was made by analyzing Langmuir, Freundlich, and Dubinin-Radushkevich (D-R) adsorption isotherm models and the biosorption data was found to be best represented by the Langmuir model. The biosorption efficiency of MW-SO was also compared with unmodified material, Salvadora oleoides (SO). It was found that the sorption capacity (qmax) increased from 58.5 mg/g to 219.7 mg/g on MW treatment. Determination of thermodynamic parameters such as free energy change (ΔG°), enthalpy change (ΔH°) and entropy change (ΔS°) confirmed the spontaneous, endothermic and feasible nature of the adsorption process. The preparation of MW-SO did not require any additional chemical treatment and a high percentage removal of methyl violet dye was obtained in much lesser time. Thus, it is in agreement with the principles of green chemistry. The results of the present research work suggest that MW-SO can be used as an environmentally friendly and economical alternative biosorbent for the removal of methyl violet dye from aqueous solutions.

This study aims to investigate the biological processes related to the biodegradable potential of growing microbial cells for contaminated water treatment. Thus, the use of the Streptomyces fulvissimus CKS 7 (CKS7) has been evaluated for decolorizing efficiency of a solution containing a cationic triphenylmethane dye, crystal violet. The color reduction was monitored by UV-Vis spectroscopic analysis, through changes in their absorption spectrum and comparing the results with those of the respective controls. It was found that the CKS7 performed well and reached up to 100% effectiveness. The required process parameters have been apparently mild and include the reaction temperature of 27-30 °C, 10% inoculum size, under shaking conditions, whereas the time course of decolorization had been concentration dependent. A possible mechanism for removing dye from the working medium was accomplished in two steps: the binding of the dye on the bacterial cell surface, in addition to the dye biodegradation by the bacterial intracellular enzymes. After one cycle of the complete dye removal, the adapted culture was successfully reused for the same purpose. The phytotoxicity analysis revealed that non-toxic compounds were present in decolorized medium, indicating that the CKS7 bacteria seem to be a promising application for contaminated water treatment.

Crystal Violet (CV), a triphenylmethane dye, has been extensively used in human and veterinary medicine as a biological stain, as a textile dye in textile processing industries and also used to provide a deep violet color to paints and printing ink. CV is also used as a mutagenic and bacteriostatic agent in medical solutions and antimicrobial agent to prevent the fungal growth in poultry feed. Inspite of its many uses, CV has been reported as a recalcitrant dye molecule that persists in environment for a long period and pose toxic effects in environment. It acts as a mitotic poison, potent carcinogen and a potent clastogene promoting tumor growth in some species of fish. Thus, CV is regarded as a biohazard substance. Although, there are several physico-chemical methods such as adsorption, coagulation and ion-pair extraction reported for the removal of CV, but these methods are insufficient for the complete removal of CV from industrial wastewaters and also produce large quantity of sludge containing secondary pollutants. However, biological methods are regarded as cost-effective and eco-friendly for the treatment of industrial wastewaters, but these methods also have certain limitations. Therefore, there is an urgent need to develop such eco-friendly and cost-effective biological treatment methods, which can effectively remove the dye from industrial wastewaters for the safety of environment, as well as human and animal health.

Dyes are used to stain inks, paints, textile, paper, leather and household products. They are omnipresent, some are toxic and may threaten our environment, especially aquatic ecosystems. The presence of residues of sixteen dyes (triarylmethanes, xanthenes, phenothiazines and phenoxazines) and their metabolites was analyzed in muscle tissue samples of individual yellow-phased European eels (Anguilla anguilla) from 91 locations in Belgian rivers, canals and lakes sampled between 2000 and 2009 using ultra performance liquid chromatography-tandem mass spectrometry. Eel was contaminated by dyes in 77% of the sites. Malachite Green, Crystal Violet and Brilliant Green were present in 25-58% of the samples. Dye occurrence was related to the distribution of textile and dye production industries. This field study is the first large-scale survey to document the occurrence of artificial dyes in wildlife. Considering the annual amounts of dyes produced worldwide and the unintentional spillage during their use, our observations warrant additional research in other parts of the world. The presence of these highly toxic dyes in the European eel may form an additional threat to this critically endangered species. The contaminated eels should be considered as not suitable for consumption., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Lactobacillus plantarum has been associated with food spoilage in a wide range of products and the biofilm growth mode has been implicated as a possible source of contamination. In this study we analysed the biofilm forming capacity of L. plantarum WCFS1 and six food spoilage isolates. Biofilm formation as quantified by crystal violet staining and colony forming units was largely affected by the medium composition, growth temperature and maturation time and by strain specific features. All strains showed highest biofilm formation in Brain Heart Infusion medium supplemented with manganese and glucose. For L. plantarum biofilms the crystal violet (CV) assay, that is routinely used to quantify total biofilm formation, correlates poorly with the number of culturable cells in the biofilm. This can in part be explained by cell death and lysis resulting in CV stainable material, conceivably extracellular DNA (eDNA), contributing to the extracellular matrix. The strain to strain variation may in part be explained by differences in levels of eDNA, likely as result of differences in lysis behaviour. In line with this, biofilms of all strains tested, except for one spoilage isolate, were sensitive to DNase treatment. In addition, biofilms were highly sensitive to treatment with Proteinase K suggesting a role for proteins and/or proteinaceous material in surface colonisation. This study shows the impact of a range of environmental factors and enzyme treatments on biofilm formation capacity for selected L. plantarum isolates associated with food spoilage, and may provide clues for disinfection strategies in food industry., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Sperm morphometry has some value as an indicator of reproductive capacity in males. In laboratory practice a variety of slide-staining methods are used during morphological evaluation of semen to predict male fertility. The aim of this study was to determine the effect of staining of semen using four different techniques on the morphometry of the bull sperm cell. The material for the study consisted of semen collected from test bulls of the Black-and-White variety of Holstein-Friesians. The results obtained in the study indicate differences in the dimensions of bull sperm heads when different slide staining techniques were used. The most similar results for sperm head dimensions were obtained in the case of SpermBlue(®) and eosin+gentian violet complex, although statistically significant differences were found between all the staining techniques. Extreme values were noted for the other staining techniques - lowest for the Papanicolaou and highest for silver nitrate, which may indicate more interference in the cell by the reagents used in the staining process. However, silver nitrate staining was best at identifying the structures of the sperm cell. Hence it is difficult to determine which of the staining methods most faithfully reveals the dimensions and shape of the bull sperm., (Copyright © 2015 Elsevier B.V. All rights reserved.)

For well over a century, Hans Christian Gram's famous staining protocol has been the standard go-to diagnostic for characterizing unknown bacteria. Despite continuous and ubiquitous use, we now demonstrate that the current understanding of the molecular mechanism for this differential stain is largely incorrect. Using the fully complementary time-resolved methods: second-harmonic light-scattering and bright-field transmission microscopy, we present a real-time and membrane specific quantitative characterization of the bacterial uptake of crystal-violet (CV), the dye used in Gram's protocol. Our observations contradict the currently accepted mechanism which depicts that, for both Gram-negative and Gram-positive bacteria, CV readily traverses the peptidoglycan mesh (PM) and cytoplasmic membrane (CM) before equilibrating within the cytosol. We find that not only is CV unable to traverse the CM but, on the time-scale of the Gram-stain procedure, CV is kinetically trapped within the PM. Our results indicate that CV, rather than dyes which rapidly traverse the PM, is uniquely suited as the Gram stain.

Phenotypic smooth cells of the fish pathogenic bacterium Flavobacterium psychrophilum have previously been reported to be more adhesive to polystyrene surfaces than corresponding rough cells. In this study, the adhesion ability of smooth and rough cells of F. psychrophilum to polystyrene surfaces was investigated in detail with a crystal violet staining method. By treating both polystyrene surfaces with fish mucus and carbohydrates and the bacterial cells with carbohydrates, the involvement of lectins in the adhesion process was investigated. Smooth cells showed significantly higher adhesion ability to untreated polystyrene surfaces compared with corresponding rough cells and increasing water hardness had an inhibitory effect on the adhesion. Treatment of polystyrene surfaces with D-glucose, D-galactose and fish mucus increased the adhesion ability of smooth cells to polystyrene. Furthermore, treatment of the smooth cells with D-glucose, D-galactose and sialic acid decreased the adhesion ability of the cells, indicating that the adhesion is likely mediated by complementary lectins on the surface of the cells. Sodium (meta)periodate treatment of smooth cells also decreased the adhesion ability to polystyrene, suggesting that the lectins, such as the dominating sialic acid-binding lectin, are probably localized in the extracellular polysaccharides surrounding the cells., (© 2014 John Wiley & Sons Ltd.)