Reduction of biofilm formation of Escherichia coli by targeting quorum sensing and adhesion genes using the CRISPR/Cas9-HDR approach, and its clinical application on urinary catheter.

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.

Inhibition of proliferation, migration, and adhesion of skin fibroblasts by enzymatic poly(gallic acid) grafted with L-Arginine, migration, and adhesion of skin fibroblasts by enzymatic poly(gallic acid) grafted with L-Arginine.

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.

Detection of microscopic filamentous fungal biofilms - Choosing the suitable methodology.

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.

Cytoprotective and genoprotective effects of taxifolin against oxidative damage in HTR-8/SVneo human trophoblast cells.

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.

Counteractions of a Novel Hydroalcoholic Extract from Lens Culinaria against the Dexamethasone-Induced Osteoblast Loss of Native Murine Cells.

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.

Carbon dioxide can inhibit biofilms formation and cellular properties of Shewanella putrefaciens at both 30 °C and 4 °C.

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 (CO2) was widely applied to control microorganisms in aquatic products package. To explore the regulation mechanism of CO2 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 CO2 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 CO2 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 CO2. Inhibition of swimming and swarming motility ability by CO2 could also be observed with significantly shorter bacterial halo diameter. What's more, cellular metabolism was significantly decreased by CO2 according to the results of ATP content, ATPase activity and extracellular proteolytic activity. The influence of CO2 could be both observed whether combined with 30 °C or 4 °C. However, the inhibition produced by CO2 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 CO2. This research provided a theoretical basis for better application of CO2 to regulate spoilage microorganisms.

20S-ginsenoside Rg3 inhibits the biofilm formation and haemolytic activity of Staphylococcus aureus by inhibiting the SaeR/SaeS two-component system.

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. aureus 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 CopZ, 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.

Membrane lipid organization and nicotinic acetylcholine receptor function: A two-way physiological relationship.

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.

The effect of hypergravity, hyperbaric pressure, and hypoxia on osteogenic differentiation of adipose stem cells.

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.

Virulence and biofilm inhibition of 3-methoxycinnamic acid against Agrobacterium tumefaciens.

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 ß-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.

Antibiofilm mechanism of a novel milk-derived antimicrobial peptide against Staphylococcus aureus by downregulating agr quorum sensing system.

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 psmß) of the QS systems were downregulated. Additionally, the expression of the agr-related genes, agrA, agrB, agrC and psmß, 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.

Asian Sand Dust Particles Enhance the Development of Aspergillus fumigatus Biofilm on Nasal Epithelial Cells.

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)-ß1 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.

Myricetin Disturbs the Cell Wall Integrity and Increases the Membrane Permeability of Candida albicans.

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.

Mechano-Responsive Piezoelectric Nanofiber as an On-Demand Drug Delivery Vehicle.

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.

Genetic Diversity and Dye-Decolorizing Spectrum of Schizophyllum commune Population.

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.

Biodegradation of crystal violet mediated by CotA from Bacillus amyloliquefaciens.

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.

Analysis of trace malachite green, crystal violet, and their metabolites in zebrafish by surface-coated probe nanoelectrospray ionization mass spectrometry.

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 (r2) 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.

Detection and differentiation of bacteria by electrical bioimpedance spectroscopy.

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.

Exposure of Lemna minor L. to gentian violet or Congo red is associated with changes in the biosynthesis pathway of biogenic amines.

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.

Characteristics of White-rot Fungus Phlebia brevispora TMIC33929 and Its Growth-Promoting Bacterium Enterobacter sp. TN3W-14 in the Decolorization of Dye-Contaminated Water.

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.