Nutrient removal by Chlorella vulgaris F1068 under cetyltrimethyl ammonium bromide induced hormesis.

Toxicants are generally harmful to biotechnology in wastewater treatment. However, trace toxicant can induce microbial hormesis, but to date, it is still unknown how this phenomenon affects nutrient removal during municipal wastewater treatment process. Therefore, this study focused on the effects of hormesis induced by cetyltrimethyl ammonium bromide (CTAB), a representative quaternary ammonium cationic surfactant, on nutrient removal by Chlorella vulgaris F1068. Results showed that when the concentration of CTAB was less than 10 ng/L, the cellular components chlorophyll a, proteins, polysaccharides, and total lipids increased by 10.11, 58.17, 38.78, and 11.87 %, respectively, and some enzymes in nutrient metabolism of algal cells, such as glutamine synthetase (GS), acid phosphatase (ACP), H(+)-ATPase, and esterase, were also enhanced. As a result, the removal efficiencies of ammonia nitrogen (NH4 (+)) and total phosphorus (TP) increased by 14.66 and 8.51 %, respectively, compared to the control during a 7-day test period. The underlying mechanism was mainly due to an enhanced photosynthetic activity of C. vulgaris F1068 indicated by the increase in chlorophyll fluorescence parameters (the value of Fv/Fm, ΦII, Fv/Fo, and rETR increased by 12.99, 7.56, 25.59, and 8.11 %, respectively) and adenylate energy charge (AEC) (from 0.68 to 0.72). These results suggest that hormesis induced by trace toxicants could enhance the nutrient removal, which would be further considered in the design of municipal wastewater treatment processes. Graphical abstract The schematic mechanism of C. vulgaris F1068 under CTAB induced hormesis. Green arrows ( ) represent the increase and the red arrow ( ) represents the decrease.

Internal charge transfer based ratiometric interaction of anionic surfactant with calf thymus DNA bound cationic surfactant: Study I.

Cetyl trimethyl ammonium bromide (CTAB) binds calf thymus (ct-) DNA like anionic biopolymers electrostatically and established equilibrium both in the ground as well as in excited state in aqueous medium at pH 7. Anionic sodium dodecyl sulfate (SDS) does not show even hydrophobic interaction with ct-DNA at low concentration. On contrary, SDS can establish well defined equilibrium with DNA bound CTAB in ground state where the same CTAB-DNA isosbestic point reappears. First report of internal charge transfer (ICT) based binding of CTAB with ct-DNA as well as ICT based interaction of anionic SDS with DNA bound CTAB that shows dynamic quenching contribution also. The reappearance of anodic peak and slight increase in cathodic peak current with increasing concentration (at lower range) of anionic SDS, possibly reflect the release of CTAB from DNA bound CTAB by SDS.

Impact of efflux in the development of multidrug resistance phenotypes in Staphylococcus aureus.

BACKGROUND: Efflux has been recognized as a resistance mechanism to antimicrobials in Staphylococcus aureus; however its role on the development of clinically relevant resistance is still poorly characterized. This study aimed to examine the impact of efflux on development of resistance to fluoroquinolones and other antimicrobials in S. aureus strains representing relevant phenotypes in terms of antibiotic susceptibility and efflux activity. METHODS: Two closely related methicillin- and ciprofloxacin-resistant Staphylococcus aureus clinical strains, with different efflux capacity and the pan-susceptible strain ATCC25923 were exposed to constant concentrations of the efflux pump (EP) substrates ciprofloxacin, ethidium bromide and cetrimide. Parental and exposed strains were tested regarding their susceptibility towards antibiotics, biocides and ethidium bromide, efflux capacity and levels of EP gene expression. Occurrence of resistance-associated mutations was screened by sequencing. RESULTS: Multidrug resistance phenotypes emerged upon exposure, independently of the substrate or its concentration, which were correlated with increased efflux capacity of the exposed strains. The temporal pattern of EP gene expression disclosed an early-response with high expression of several genes, followed by a late-response, characterized by overexpression of specific genes. The overall cell response was more pronounced for strains with an initial basal efflux activity. Remarkably, detection of the IS256 element in the promoter regions of mgrA and norA, in some cases associated with increased gene expression, suggests that these genes may be hot spots for IS256 insertion events. The results obtained with exposure of ATCC25923 to ciprofloxacin were particularly striking, revealing a step-wise development of fluoroquinolone resistance, with a first efflux-mediated response, followed by the occurrence of a mutation in grlA that resulted in phenotypic resistance. Additionally, challenge by non-fluoroquinolone agents, particularly cetrimide, promoted cross resistance to fluoroquinolones, revealing the potential role of biocides as selective pressure for the emergence of resistance to these antibiotics. CONCLUSIONS: This study reveals efflux as a significant component of S. aureus resistance to fluoroquinolones and biocides and as a primary mechanism to withstand stress imposed by antimicrobials. This efflux-mediated response can result in the emergence of multidrug resistance in healthcare environments and should be taken into account in the management of this major pathogen.

Residual CTAB Ligands as Mass Spectrometry Labels to Monitor Cellular Uptake of Au Nanorods.

Gold nanorods have numerous applications in biomedical research, including diagnostics, bioimaging, and photothermal therapy. Even though surfactant removal and surface conjugation with antifouling molecules such as polyethylene glycol (PEG) are required to minimize nonspecific protein binding and cell uptake, the reliable characterization of these processes remains challenging. We propose here the use of laser desorption/ionization mass spectrometry (LDI-MS) to study the ligand exchange efficiency of cetyltrimethylammonium bromide (CTAB)-coated nanorods with different PEG grafting densities and to characterize nanorod internalization in cells. Application of LDI-MS analysis shows that residual CTAB consistently remains adsorbed on PEG-capped Au nanorods. Interestingly, such residual CTAB can be exploited as a mass barcode to discern the presence of nanorods in complex fluids and in vitro cellular systems, even at very low concentrations.

Spectroscopic study on interaction between three cationic surfactants with different alkyl chain lengths and DNA.

In this study, the interaction between cationic surfactants with different alkyl chain lengths, such as hexyltrimethyl ammonium bromide (HTAB), dodecyltrimethyl ammonium bromide (DTAB) and cetyltrimethyl ammonium bromide (CTAB), and DNA was investigated by UV-vis spectroscopy, fluorescence spectroscopy and viscosity techniques. The results showed that these three cationic surfactants with different hydrocarbon chain lengths could all interact with DNA. Their binding modes were estimated and their interaction strength was compared. In addition, the effects of the surfactant, NaCl and phosphate ion concentrations on the interaction were reviewed. It is wished that this work would provide some valuable references to investigate the influence of cationic surfactants with different alkyl chain lengths on DNA.

Thermodynamic and spectroscopic investigation of interactions between reactive red 223 and reactive orange 122 anionic dyes and cetyltrimethyl ammonium bromide (CTAB) cationic surfactant in aqueous solution.

The present study describes the conductometric and spectroscopic study of the interaction of reactive anionic dyes, namely, reactive red 223 and reactive orange 122 with the cationic surfactant cetyltrimethyl ammonium bromide (CTAB). In a systematic investigation, the electrical conductivity data was used to calculate various thermodynamic parameters such as free energy (ΔG), enthalpy (ΔH), and the entropy (ΔS) of solubilization. The trend of change in these thermodynamic quantities indicates toward the entropy driven solubilization process. Moreover, the results from spectroscopic data reveal high degree of solubilization, with strong interactions observed in the cases of both dyes and the CTAB. The spontaneous nature of solubilization and binding was evident from the observed negative values of free energies (ΔG p and ΔG b).

Spectroscopic studies of micelle-enhanced ligand exchange of gallium (III)/4-(2-pyridylazo) resorcinol complex by calf thymus DNA.

The effect of cationic micelles of cetyltrimethylammonium bromide (CTAB) on the interaction of gallium (III) with 4-(2-pyridylazo) resorcinol (PAR) under varying conditions has been studied spectrophotometrically. At pH 6.0, CTAB (0.05% w/v) markedly enhanced the absorption intensity of gallium (III)-PAR complex. Furthermore, the introduction of CTAB provided unique selectivity for the ligand exchange of Ga(III)-PAR by calf thymus dsDNA over calf thymus ssDNA. This phenomenon offers a novel spectrophotometric sensing strategy for direct detection of dsDNA.

High throughput quantification of capsular polysaccharides for multivalent vaccines using precipitation with a cationic surfactant.

The increasing requirement for multivalent vaccines containing diverse capsular polysaccharides has created an unmet need for a fast and straightforward assay for polysaccharide titer. We describe a novel and robust assay for the quantitation of anionic capsular polysaccharides. The binding of hexadecyltrimethyammonium bromide (Hb) to anionic capsular polysaccharides results in a precipitation reaction wherein the suspension turbidity is proportional to polysaccharide titer. The turbidity can be quickly measured as absorbance across a range of wavelengths that resolve scattering light. Carbohydrates comprised of repeating units of one to seven monosaccharides with phosphodiester groups, uronic acids, and sialic acids all reacted strongly and there does not appear to be specificity with respect to the particular anionic moiety. The assay is compatible with an array of common buffers across a pH range of 3.0-8.75 and with NaCl concentration exceeding 400 mM. Interference from DNA can be eliminated with a short incubation step with DNase. With these treatments, the assay has been employed in samples as complex as fermentation broth. A two-log dynamic range has been established with a mean relative standard deviation less than 10% across this range although inferior performance has been observed in fermentation broth. The precipitation assay enables the rapid quantitation of anionic polysaccharides. The resulting procedure can robustly measure the titer of myriad anionic capsular polysaccharides (CPS) in 96 samples in less than 30 min using low toxicity reagents and routine laboratory equipment. This development will greatly reduce the effort required to measure polysaccharide titer and yield during process development of polysaccharide vaccines.

Role of charge screening and delocalization for lipophilic cation permeability of model and mitochondrial membranes.

The effects of the mitochondria-targeted lipophilic cation dodecyltriphenylphosphonium (C12TPP, the charge is delocalized and screened by bulky hydrophobic residues) and those of lipophilic cations decyltriethylammonium bromide and cetyltrimethylammonium bromide (C10TEA and C16TMA, the charges are localized and screened by less bulky residues) on bilayer planar phospholipid membranes and tightly-coupled mitochondria from the yeast Yarrowia lipolytica have been compared. In planar membranes, C12TPP was found to generate a diffusion potential as if it easily penetrates these membranes. In the presence of palmitate, C12TPP induced H(+) permeability like plastoquinonyl decyltriphenilphosphonium that facilitates transfer of fatty acid anions (Severin et al., PNAS, 2010, 107, 663-668). C12TPP was shown to stimulate State 4 respiration of mitochondria and caused a mitochondrial membrane depolarization with a half-maximal effect at 6µM. Besides, C12TPP profoundly potentiated the uncoupling effect of endogenous or added fatty acids. C10TEA and C16TMA inhibited State 4 respiration and decreased the membrane potential, though at much higher concentrations than C12TPP, and they did not promote the uncoupling action of fatty acids. These relationships were modeled by molecular dynamics. They can be explained by different membrane permeabilities for studied cations, which in turn are due to different availabilities of the positive charge in these cations to water dipoles.

Interactions of microbicide nanoparticles with a simulated vaginal fluid.

The interaction with cervicovaginal mucus presents the potential to impact the performance of drug nanocarriers. These systems must migrate through this biological fluid in order to deliver their drug payload to the underlying mucosal surface. We studied the ability of dapivirine-loaded polycaprolactone (PCL)-based nanoparticles (NPs) to interact with a simulated vaginal fluid (SVF) incorporating mucin. Different surface modifiers were used to produce NPs with either negative (poloxamer 338 NF and sodium lauryl sulfate) or positive (cetyltrimethylammonium bromide) surface charge. Studies were performed using the mucin particle method, rheological measurements, and real-time multiple particle tracking. Results showed that SVF presented rheological properties similar to those of human cervicovaginal mucus. Analysis of NP transport indicated mild interactions with mucin and low adhesive potential. In general, negatively charged NPs underwent subdiffusive transport in SVF, i.e., hindered as compared to their diffusion in water, but faster than for positively charged NPs. These differences were increased when the pH of SVF was changed from 4.2 to 7.0. Diffusivity was 50- and 172-fold lower in SVF at pH 4.2 than in water for negatively charged and positively charged NPs, respectively. At pH 7.0, this decrease was around 20- and 385-fold, respectively. The estimated times required to cross a layer of SVF were equal to or lower than 1.7 h for negatively charged NPs, while for positively charged NPs these values were equal to or higher than 7 h. Overall, our results suggest that negatively charged PCL NPs may be suitable to be used as carriers in order to deliver dapivirine and potentially other antiretroviral drugs to the cervicovaginal mucosal lining. Also, they further reinforce the importance in characterizing the interactions of nanosystems with mucus fluids or surrogates when considering mucosal drug delivery.

A novel dodine-free selective medium based on the use of cetyl trimethyl ammonium bromide (CTAB) to isolate Beauveria bassiana, Metarhizium anisopliae sensu lato and Paecilomyces lilacinus from soil.

This study evaluated the quaternary ammonium compound cetyl trimethyl ammonium bromide (CTAB) as an alternative to the chemically related dodecylguanidine (dodine) for the selective isolation of entomopathogenic fungi. Oatmeal agar (OA) with chloramphenicol was used as basal medium, and three concentrations of CTAB (0.5, 0.6, 0.7 g/L) were evaluated and compared against OA + 0.46 g/L dodine. Selective isolation and growth studies were performed with the entomopathogens Beauveria bassiana, Metarhizium anisopliae s.l. and Paecilomyces lilacinus and five common non-entomopathogenic non-target species. The three entomopathogenic fungi sporulated earlier on OA + 0.6 g/L CTAB than on OA + 0.46 g/L dodine, while none of the non-target fungi sporulated on OA + 0.6 g/L CTAB. All entomopathogenic fungal isolates grew on OA + 0.6 g/L CTAB, despite some intra-species variation, whereas non-target fungi showed no growth or sporulation. OA + 0.6 g/L CTAB resulted in an efficient medium to isolate B. bassiana, M. anisopliae s. l. and P. lilacinus from soil samples. Results of our study suggest that OA + 0.6 g/L CTAB is a suitable, simple and inexpensive to prepare medium to replace OA + 0.46 g/L dodine for the selective isolation of these fungi.

Degradation of transgene DNA in genetically modified herbicide-tolerant rice during food processing.

In order to assess the effect of food processing on the degradation of exogenous DNA components in sweet rice wine and rice crackers made from genetically modified (GM) rice (Oryza sativa L.), we developed genomic DNA extraction methods and compared the effect of different food processing procedures on DNA degradation. It was found that the purity, quantity and quality of DNA by alkaline lysis method were higher than by CTAB (cetyltrimethylammonium bromide) method. For sweet rice wine, CAMV35S (cauliflower mosaic virus 35S) promoter and NOS (nopaline synthase) terminator were degraded by the third day, whereas the exogenous gene Bar (bialaphos resistance) remained unaffected. For rice crackers, boiling, drying and microwaving contributed to the initial degradations of DNA. Baking resulted in further degradations, and frying led to the most severe changes. These results indicated that the stability of DNA in GM rice was different under different processing conditions. For sweet rice wine, Bar was most stable, followed by NOS, CAMV35S, and SPS. For rice crackers, CAMV35S was most stable, followed by SPS, NOS, and Bar.

Isolation, quantification, and analysis of chloroplast DNA.

Many areas of chloroplast research require methods that can assess the quality and quantity of chloroplast DNA (cpDNA). The study of chloroplast functions that depend on the proper maintenance and expression of the chloroplast genome, understanding cpDNA replication and repair, and the development of technologies for chloroplast transformation are just some of the disciplines that require the isolation of high-quality cpDNA. Arabidopsis thaliana offers several advantages for studying these processes because of the sizeable collection of mutants and natural varieties (accessions) available from stock centers and a broad community of researchers that has developed many other genetic resources. Several approaches for the isolation and quantification of cpDNA have been developed, but little consideration has been given to the strengths and weaknesses and the type of information obtained by each method, especially with respect to A. thaliana. Here, we provide protocols for obtaining high-quality cpDNA for PCR and other applications, and we evaluate several different isolation and analytical methods in order to build a robust framework for the study of cpDNA with this model organism.

Isolation of plant DNA for PCR and genotyping using organic extraction and CTAB.

A general difficulty in isolation of DNA from plant cells is the presence of a cell wall. It is necessary to degrade plant cell walls, either physically or enzymatically, in order to effectively isolate plant DNA. Additionally, some tissues (such as endosperm) or some species contain high levels of starches or phenolic compounds that can complicate DNA isolation. A number of plant DNA isolation protocols are designed to overcome species-specific difficulties. This is a relatively simple protocol that uses an extraction buffer containing cetyltrimethylammonium bromide (CTAB); it can be used for many plant species. It provides a substantial amount of high-quality DNA that is suitable for polymerase chain reaction (PCR) procedures and is stable for long periods of time. The cost per sample is very low. In addition, this protocol is relatively robust and can be performed by individuals who have had relatively little training. A typical undergraduate student can perform ~200-300 isolations in a day using this protocol. The disadvantages are that it requires a freeze-dryer and a mill or paint-shaker-like device and that it utilizes an organic extraction step, requiring the use of a fume hood.

DNA extraction from freeze-dried plant tissue with CTAB in a 96-well format.

This protocol is a modified version of DNA isolation using cetyltrimethylammonium bromide (CTAB) and 96-well plates. It is high-throughput, which facilitates the analysis of large mapping populations. The DNA yield is adequate for at least 100-500 polymerase chain reaction (PCR) procedures.

Fluorescence dynamics of double- and single-stranded DNA bound to histone and micellar surfaces.

The study of structure and dynamics of bound DNA has special implications in the context of its biological as well as material functions. It is of fundamental importance to understand how a binding surface affects different positions of DNA with respect to its open ends. Because double-stranded (ds) and single-stranded (ss) DNA are the predominant functional forms, we studied the site-specific dynamics of these DNA forms, bound to the oppositely charged surface of histones, and compared the effects with that of DNA bound to cetyltrimethyl ammonium bromide micelles. We utilized a time-resolved fluorescence technique using fluorescent base analogue 2-aminopurine located at specific positions of synthetic poly-A DNA strands to obtain fluorescence lifetime and anisotropy information. It is observed that the binding leads to overall rigidification of the DNA backbone, and the highly flexible ends show drastic dampening of their internal dynamics as well as the fraying motions. In the case of ds-DNA, we find that the binding not only decreases the flexibility but also leads to significant weakening of base-stacking interactions. An important revelation that strong binding between DNA and the binding agents (histones as well as micelles) does not dampen the internal dynamics of the bases completely suggests that the DNA in its bound form stays in some semiactive state, retaining its full biological activity. Considering that the two binding agents (histones and micelles) are chemically very different, an interesting comparison is made between DNA-histones and DNA-micelle interactions.

Enhancement in thermal stability and resistance to denaturants of lipase encapsulated in mesoporous silica with alkyltrimethylammonium (CTAB).

We assembled a highly durable conjugate with both a high-density accumulation and a regular array of lipase, by encapsulating it in mesoporous silica (FSM) with alkyltrimethylammonium (CTAB) chains on the surface. The activity for hydrolyzing esters of the lipase immobilized in mesoporous silica was linearly related to the concentration of lipase, whereas that of non-immobilized lipase showed saturation due to self-aggregation at a high concentration. The lipase conjugate also had increased resistance to heating when stayed in the silica coupling with CTAB. In addition, encapsulating the enzyme with FSM coupled CTAB caused the lipase to remain stable even in the presence of urea and trypsin, suggesting that the encapsulation prevented dissociation and denaturing. This conjugate had much higher activity and much higher stability for hydrolyzing esters when compared to the native lipase. These results show that FSM provides suitable support for the immobilization and dispersion of proteins in mesopores with disintegration of the aggregates.

Beta-cyclodextrin in DNA decompaction: an imaging approach.

In the present work we investigate the CTAB:DNA complexes decompaction process using beta-CD as a decompacting agent. The transition from globules (compacted DNA) to coils (decompacted DNA) was achieved without a coexistence region between coils and globules. This non first-order transition was investigated combining fluorescence microscopy (FM), cryo-transmission electron microscopy (cryo-TEM) and TEM of negatively stained samples. Additionally the presence of multi-globular aggregates in the proximity of the critical transition was elucidated. A possible mechanism for the decompaction process was suggested.

Interaction of bovine (BSA), rabbit (RSA), and porcine (PSA) serum albumins with cationic single-chain/gemini surfactants: a comparative study.

The interactions among bovine, rabbit, and porcine serum albumins and single-chain cationic surfactant cetyltrimethylammonium bromide (CTAB) versus its gemini counterpart (designated as G4) have been studied. The studies were carried out in an aqueous medium at pH 7.0 using UV, intrinsic and extrinsic fluorescence spectroscopy, and far-UV circular dichroism techniques. The results indicate that compared to CTAB, G4 interacts strongly with the serum albumins, resulting in a significantly larger unfolding or decrease in alpha-helical content as reflected by the significantly larger decrease in ellipticity in the far-UV range. Unlike CTAB, a remarkable increase in the alpha-helical content of BSA at 625 microM G4 and at 250 microM G4 for RSA and PSA is observed. The appearance of conformational changes and saturation points in the proteins occurs at considerably lower [G4] compared to [CTAB]. The results obtained from the multi-technique approach are ascribed to the stronger forces in G4 owing to the presence of two charged headgroups and two hydrocarbon tails. Keeping the results in view, it is suggested that the gemini surfactants may be effectively used in the renaturation of proteins produced in genetically engineered cells via the artificial chaperone protocol and may also prove useful in drug delivery as solubilizing agents to recover proteins from insoluble inclusion bodies.

Simple formation of an abiotic porphyrinogen in aqueous solution.

Porphyrins have long been proposed as key ingredients in the emergence of life yet plausible routes for forming their essential pyrrole precursor have heretofore not been identified. Here we show that the anaerobic reaction of δ-aminolevulinic acid (ALA, 1-5 mM) with the ß-ketoester methyl 4-methoxyacetoacetate (2-40 mM) in water (pH5-7) at 70-100°C for >6 h affords the porphyrinogen, which upon chemical oxidation gives the corresponding porphyrin in overall yield of up to 10%. The key intermediate is the α-methoxymethyl-substituted pyrrole, which undergoes tetramerization and macrocycle formation under kinetic control. The resulting type-I porphyrin bears four propionic acid and four carbomethoxy groups, is distinct from porphyrins (e.g., uroporphyrin or coproporphyrin) derivable from ALA alone via the extant universal biosynthetic path to tetrapyrroles, and is photoactive upon assembly into cationic micelles in aqueous solution. The simple self-organization of eight acyclic molecules into a tetrapyrrole macrocycle, from which a porphyrin is derived that is photoactive in lipid assemblies, augurs well for the spontaneous origin of catalysts and pigments essential for prebiotic metabolism and proto-photosynthesis.