Anionic Detergent Research Articles

Preparation of human decellularized peripheral nerve allograft using amphoteric detergent and nuclease.

Animal studies have shown that amphoteric detergent and nuclease (DNase I and ribonuclease A) is the most reliable decellularization method of the peripheral nerve. However, the optimal combination of chemical reagents for decellularization of human nerve allograft needs further investigation. To find the optimal protocol to remove the immunogenic cellular components of the nerve tissue and preserve the basal lamina and extracellular matrix and whether the optimal protocol can be applied to larger-diameter human peripheral nerves, in this study, we decellularized the median and sural nerves from the cadavers with two different methods: nonionic and anionic detergents (Triton X-100 and sodium deoxycholate) and amphoteric detergent and nuclease (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), deoxyribonuclease I, and ribonuclease A). All cellular components were successfully removed from the median and sural nerves by amphoteric detergent and nuclease. Not all cellular components were removed from the median nerve by nonionic and anionic detergent. Both median and sural nerves treated with amphoteric detergent and nuclease maintained a completely intact extracellular matrix. Treatment with nonionic and anionic detergent decreased collagen content in both median and sural nerves, while the amphoteric detergent and nuclease treatment did not reduce collagen content. In addition, a contact cytotoxicity assay revealed that the nerves decellularized by amphoteric detergent and nuclease was biocompatible. Strength failure testing demonstrated that the biomechanical properties of nerves decellularized with amphoteric detergent and nuclease were comparable to those of fresh controls. Decellularization with amphoteric detergent and nuclease better remove cellular components and better preserve extracellular matrix than decellularization with nonionic and anionic detergents, even in large-diameter human peripheral nerves. In Korea, cadaveric studies are not yet legally subject to Institutional Review Board review.

Effect of biomass pre-treatment on supercritical CO2 extraction of lipids from marine diatom Amphora sp. and its biomass evaluation as bioethanol feedstock

Marine diatoms are a potential source for lipids and carbohydrates, which can have several applications ranging from biofuels to nutraceuticals. Due to their siliceous cellular structure and the complex nature of different lipid classes, it is important to understand the effect of biomass pre-treatment on the extractability of marine diatom lipids. In the present study, we tested the effect of four biomass pre-treatments (acid, base, anionic detergent, and non-ionic detergent) conditions on the extractability of lipids from Amphora sp. biomass. Lipids were extracted under identical supercritical fluid extraction (SFE) conditions from each of the above mentioned pre-treated biomass of Amphora sp. grown with or without silica. The fatty acids profile of saponified lipids was analysed by LC-MS. Results obtained in this study suggest each pre-treatment has a specific effect on the fatty acids profile. Therefore, depending on the downstream application of lipids (biodiesel or nutritional), both types of biomass and their pre-treatment conditions need to be considered. From the fermentation study for biomass evaluation as bioethanol feedstock, it was found that the complex carbohydrates of Amphora sp. biomass were easily convertible by autoclaving to monomer sugars, which were suitable for bioethanol production by yeast fermentation.

Disaggregation of amyloid-like protein aggregates isolated from human cataract lens

Crystallins, which represents the major lens protein, play a significant role in ensuring the lens transparency and maintenance of appropriate refractive index of the lens that help in accurate focusing of incident visible light precisely on retina to create clear image perception. Aggregation of lens proteins is known to form the basis of cataract formation. The present study is an attempt to examine the stability of the lens protein aggregates, isolated from human cataract eye lens, against an anionic detergent Sodium dodecyl sulphate (SDS), which is known to disrupt the hydrophobic interaction of protein aggregates. Data that emerged from Congo red (CR), thioflavin T (ThT) and 8-anilino-1-naphthalene sulfonic acid (ANS) binding assaysindicated their amyloidogenic nature. A significant reduction in the bathochromic shift of CR λmax and ThT fluorescence emission intensity were observed after treatment of the aggregated proteins with SDS. In the presence of SDS, a significant change in the number and size of the protein aggregates were observed during their morphological analyses under transmission electron microscopy (TEM). Based on the above data it became evident that the hydrophobic interaction plays a crucial role in formation and stabilizing the protein aggregates during cataract formation.

Functional expression of human prostaglandin E2 receptor 4 (EP4) in E. coli and characterization of the binding property of EP4 with Gα proteins

Human prostaglandin E2 receptor 4 (EP4) is one of the four subtypes of prostaglandin E2 (PGE2) receptors and belongs to the rhodopsin-type G protein-coupled receptor (GPCR) family. Particularly, EP4 is expressed in various cancer cells and is involved in cancer-cell proliferation by a G protein signaling cascade. To prepare an active form of EP4 for biochemical characterization and pharmaceutical application, this study designed a recombinant protein comprising human EP4 fused to the P9 protein (a major envelope protein of phi6 phage) and overexpressed the P9-EP4 fusion protein in the membrane fraction of E. coli. The solubilized P9-EP4 with sarkosyl (a strong anionic detergent) was purified by affinity chromatography. The purified protein was stabilized with amphiphilic polymers derived from poly-γ-glutamate. The polymer-stabilized P9-EP4 showed specific interaction with the alpha subunits of Gs or Gi proteins, and a high content of α-helical structure by a circular dichroism spectroscopy. Furthermore, the polymer-stabilized P9-EP4 showed strong heat resistance compared with P9-EP4 in detergents. The functional preparation of EP4 and its stabilization with amphiphilic polymers could facilitate both the biochemical characterization and pharmacological applications targeting EP4.

Evaluation of side effects of mechanical cleaning with an anionic detergent on granite cladding tiles.

One of the aims of Goal 11 of the UN's 2030 Agenda for Sustainable Development is to make human settlements sustainable. Some effort should therefore focus on developing maintenance strategies for buildings. Polished granite cladding tiles are very popular for use as flooring material in private and public buildings. These tiles are often treated by mechanical cleaning with detergents and scrubbers, and anionic detergents without chelating agents are usually used to prevent damaging the material. In this study, the anionic detergent Teepol® was applied to granite tiles in order to determine the effect of the treatment on the surface properties of this stone, i.e. appearance, colour, gloss, roughness and hydrophobicity. The results obtained were explained by scanning electron microscopy observations, which enabled characterisation of the microtexture of the surfaces, chemical contamination and any damage to the different granite-forming minerals. The results were compared with those obtained for tiles cleaned by the same mechanical procedure but with tap water in place of detergent and for tiles treated by immersion in water without any mechanical action. In summary, the properties evaluated were affected by the extraction of the typical oxyhydroxylated iron forms that fill cracks in the granite and by the formation of new cracks due to the mechanical action. Moreover, colour changes and the chemical contamination (C-rich deposits with Na, S, Cl, Ca and Mg) hindered the effectiveness of the cleaning procedure.

Immobilization of trypsin enzyme on silver nanoparticles

Introduction and Aim:Immobilization of enzyme on silver nanoparticles (AgNPs) is one way to improve their stability and activity and can be reused for large-scaleapplications. The present study was aimed to evaluateand characterizethetrypsin enzyme immobilized on biogenicallysynthesized silver nanoparticles.Materials and
 Methods: Immobilization of trypsin enzyme was optimized with time and varying concentration of silver nanoparticles, which were greensynthesized using avocado seedextracts. The residual activity of trypsin enzyme after immobilization was estimated. The reusabilityand temperature stability of the immobilized enzyme were studied.
 Results:The immobilized enzyme showed maximum activity of 1006.0 U during the time duration of 18 hr of incubation with AgNPs. Reusability of immobilized enzyme in avocado AgNPs was assessed under optimal conditions, the activity of immobilized enzyme was loss after 5 repeated cycles. The enzyme captured on AgNPswas released in the presence of anionic detergent. Temperaturestability was assessed for immobilized enzyme on AgNPs and the immobilized enzyme was stable up to55ºC and later it loss their activity.
 Conclusion: The study reports a feasible method of enzyme immobilization on biogenically synthesized silver nanoparticles. The enzyme immobilized on silver nanoparticles is mainly through non-covalent forces. Further studies are required to improve the reusability of enzyme.
 Keywords: Silver nanoparticles;enzyme immobilization;trypsin;temperature stability;reusability;detergent.

Expression and characterization of family 40 Carbohydrate Binding Module (CBM) from Vibrio cholerae Non-O1 sialidase

Carbohydrate Binding Module (CBM) is a non-catalytic protein domain found in carbohydrate-active enzyme (glycoside hydrolase) and its role is to bring carbohydrates in close proximity to the enzyme catalytic site for complete hydrolysis. The removal of this CBM from most protein domains often leads to reduced enzyme activity and efficiency. In this study, a gene encoding for family 40 CBM from Vibrio cholerae Non-O1 sialidase was cloned and successfully expressed in E. coli BL21 (DE3) strain. The CBM40 encoded 195 amino acids with 585 bp of nucleotide sequence. The protein was successfully expressed at 18°C when induced with 1 mM IPTG. Maximum expression was achieved at 20 hours after post-induction time. For purification of the protein, an anionic denaturing detergent method was used containing 1% SDS and 0.1% sarkosyl with gradient affinity elution at 50 mM imidazole concentrations. SDS-PAGE analysis of the purified CBM40 protein displayed a protein band with a molecular mass of 21 kDa. Protein characterization showed optimum stability in 100 mM citrate buffer pH 5.5, with the highest Tm value of 40 °C. The protein was stable between pH 5.5–6.2 and able to retain its activity at 27–56°C. The addition of Mn2+ and Mg2+ increased the protein melting temperature to 56°C. Meanwhile, the addition of reagents, such as 1% SDS and 1 M urea increased the protein melting temperature (Tm) to approximately 55°C. Protein stability can be influenced by many factors, including different buffers, pHs, temperatures, ionic strengths, and chemical reagents used in a study. The optimum characterization conditions established would further lead to the discovery of CBM40 protein true potential in enhancing substrate binding affinity and protein-carbohydrate recognition, which underpins its broad applications in biotechnology and protein engineering fields.

Iron-crosslinked Rososome with robust stability and high drug loading for synergistic cancer therapy

Development of liposomal nanomedicine with robust stability, high drug loading and synergistic efficacy is a promising strategy for effective cancer therapy. Here, we present an iron-crosslinked rosmarinic liposome (Rososome) which can load high contents of drugs (including 25.8% rosmarinic acid and 9.04% doxorubicin), keep stable in a high concentration of anionic detergent and exhibit synergistic anti-cancer efficacy. The Rososomes were constructed by rosmarinic acid-lipid conjugates which not only work synergistically with doxorubicin by producing reactive oxygen species but also provide catechol moieties for the iron cross-linkages. The cross-linkages can lock the payloads tightly, endowing the crosslinked Rososome with better stability and pharmacokinetics than its non-crosslinked counterpart. On the syngeneic mouse model of breast cancer, the iron-crosslinked Rososomes exhibit better anticancer efficacy than free rosmarinic acid, doxorubicin, non-crosslinked Rososome and commercial liposomal formulation of doxorubicin (DOXIL). This study introduces a novel strategy for the development of liposomes with robust stability, high drug loading and synergistic anti-cancer efficacy.

Investigations of Detergents and Metal Components for Supporting the Effective Treatment of Greywater

Abstract The aim of our research was to investigate the detergent content and several micro and macro elements in greywater samples, a necessary step in determining the water quality for the further reuse of the greywater. We determined the anionic detergent content of synthetic greywater with a two-phased titration method prepared in laboratory conditions. The current study leads to a new suggestion for a more accurate and reliable titrimetric method for determination of anionic detergent content of synthetic greywater samples. Following these investigations, we also examined the metal content of synthetic bath water. We wanted to highlight how detergents in greywater influence the micro and macro element content of the given synthetic bath water samples.

Contact/Release Coordinated Antibacterial Cotton Fabrics Coated with N-Halamine and Cationic Antibacterial Agent for Durable Bacteria-Killing Application.

Coating a cationic antibacterial layer on the surface of cotton fabric is an effective strategy to provide it with excellent antibacterial properties and to protect humans from bacterial cross-infection. However, washing with anionic detergent will inactivate the cationic antibacterial coating. Although this problem can be solved by increasing the amount of cationic antibacterial coating, excessive cationic antibacterial coating reduces the drapability of cotton fabric and affects the comfort of wearing it. In this study, a coordinated antibacterial coating strategy based on quaternary ammonium salt and a halogenated amine compound was designed. The results show that the antibacterial effect of the modified cotton fabric was significantly improved. In addition, after mechanically washing the fabric 50 times in the presence of anionic detergent, the antibacterial effect against Staphylococcus aureus and Escherichia coli was still more than 95%. Furthermore, the softness of the obtained cotton fabric showed little change compared with the untreated cotton fabric. This easy-to-implement and cost-effective approach, combined with the cationic contact and the release effect of antibacterial agents, can endow cotton textiles with durable antibacterial properties and excellent wearability.

Overexpression and refolding of human Cyclin D3. A reliable method or not?

Cyclin D3 is a regulatory protein associated in a variety of human tumors. Despite several studies involving Cyclin D1 and D2, there are few articles that investigate the role of Cyclin D3. Therefore, this study aimed to produce and characterize Cyclin D3 using the Escherichia coli expression system and a protein refolding protocol. The anionic detergent SDS was used to solubilize the protein, then the solution were kept at 4 °C to precipitate SDS. After removing the precipitate by centrifugation, the supernatant was applied to the Ni-NTA column to purify His- tagged Cyclin D3. The recombinant protein shown to be refolded in a denaturation study by fluorescence spectroscopy at increasing concentrations of guanidine hydrochloride. The protein secondary structure, evaluated by circular dichroism, is composed of 39 % α helix and 15 % β-strands. In addition, the study aimed to validate the refolding protocol used to obtain Cyclin D3 from E. coli inclusion bodies.

Membrane interactions of the anuran antimicrobial peptide HSP1-NH2: Different aspects of the association to anionic and zwitterionic biomimetic systems

Studies have suggested that antimicrobial peptides act by different mechanisms, such as micellisation, self-assembly of nanostructures and pore formation on the membrane surface. This work presents an extensive investigation of the membrane interactions of the 14 amino-acid antimicrobial peptide hylaseptin P1-NH2 (HSP1-NH2), derived from the tree-frog Hyla punctata, which has stronger antifungal than antibacterial potential. Biophysical and structural analyses were performed and the correlated results were used to describe in detail the interactions of HSP1-NH2 with zwitterionic and anionic detergent micelles and phospholipid vesicles. HSP1-NH2 presents similar well-defined helical conformations in both zwitterionic and anionic micelles, although NMR spectroscopy revealed important structural differences in the peptide N-terminus. 2H exchange experiments of HSP1-NH2 indicated the insertion of the most N-terminal residues (1–3) in the DPC-d38 micelles. A higher enthalpic contribution was verified for the interaction of the peptide with anionic vesicles in comparison with zwitterionic vesicles. The pore formation ability of HSP1-NH2 (examined by dye release assays) and its effect on the size and surface charge as well as on the lipid acyl chain ordering (evaluated by Fourier-transform infrared spectroscopy) of anionic phospholipid vesicles showed membrane disruption even at low peptide-to-phospholipid ratios, and the effect increases proportionately to the peptide concentration. On the other hand, these biophysical investigations showed that a critical peptide-to-phospholipid ratio around 0.6 is essential for promoting disruption of zwitterionic membranes. In conclusion, this study demonstrates that the binding process of the antimicrobial HSP1-NH2 peptide depends on the membrane composition and peptide concentration.

Consortium of Marsilea crenata and Ludwigia adscendens for Linear Alkylbenzene Sulfonate Detergent Phytoremediator

Water clover (Marsilea crenata Presl.) and water primrose (Ludwigia adscendens L.) are plants grow in wetlands, polluted by inorganic or organic materials, including detergent. This study aims to evaluate the capability of M. crenata and L. adscendens individually or as a consortium to remediate li near alkylbenzene sulfonate (LAS) detergent-polluted water, and to measure the growth and chlorophyll content of these plants. M. crenata and L. adscendens were grown in a hydroponic system exposed to LAS at 0, 10, 20, and 30 ppm for 10 days. Concentration of LAS (as anionic detergent) in treated media was analysed by UV-Vis spectrophotometer at 652 nm, wet biomass was measured using analytical balance, and chlorophyll content was analysed using spectrophotometer at 649 and 655 nm. Data were analysed statistically using analysis of variance followed by the Duncan test. The results shows that (1) at the end of experiment the concentration of residual detergent increased with increasing LAS in the media and (2) the plants biomass and the chlorophyll concentration decreased with increasing the LAS concentration in media. However, since both M. crenata and L. ascendens can significantly reduce LAS detergent concentration and can survive in 30 ppm concentration of LAS detergent, both species can be applied as phytoremediator agents.

An eco-friendly dyeing method: bromophenol blue (BPB) applied for dyeing cotton fabrics coated with cationic finishing agents

Poly(hexamethylene biguanide) (PHMB) is a commercially available cationic bactericide which can form stable and water-insoluble blue complex salt with bromophenol blue (BPB) at room temperature. Herein, we reported the feasibility and applicability of BPB for dyeing PHMB-coated fabrics. The colour yield, levelling property, dye uptake, fastness behavior and antimicrobial efficacy of PHMB-coated fabrics were fully investigated by a variety of analytical methods. The controllable blue colour shade depth depended on the concentration of BPB dyebath and the quantities of PHMB coated on the fabrics, and the colour levelness showed good and above rating. The dye uptake was positively proportional to the quantities of PHMB coated on the fabric, but showed negatively correlation to BPB concentration. The colour fastness to rubbing and washing were excellent, excluding anionic detergents washing. Continuous dyeing was found to be feasible at room temperature without any additives simply via replenishing BPB dyebath, hence avoiding dye wastewater discharge, which may provide an eco-friendly and sustainable dyeing method to reduce generation and emission of wastewater in textile industry. Meanwhile, the antibacterial efficacy of PHMB-coated fabrics was slightly compromised after BPB dyeing, but was still satisfactory, particularly against the gram-positive S. aureus.

Sodium oleate, arachidonate, and linoleate enhance fibrinogenolysis by Russell's viper venom proteinases and inhibit FXIIIa; a role for phospholipase A2 in venom induced consumption coagulopathy

Life-threatening symptoms produced by Russell's viper (RV, Daboia russelii) envenomation result largely from venom induced consumption coagulopathy (VICC). VICC is thought to be mediated to a large degree by venom serine and metalloproteinases, as well as by snake venom phospholipase A2 (svPLA2), the most abundant constituent of RV venom (RVV). The observation that the phenolic lipid anacardic acid markedly enhances proteolytic degradation of fibrinogen by RVV proteinases led us to characterize the chemical basis of this phenomenon with results indicating that svPLA2 products may be major contributors to VICC. ResultsOf the chemical analogs tested, the anionic detergents sodium dodecyl sulfate, sodium deoxycholate, N-lauryl sodium sarcosine, and the sodium salts of the fatty acids arachidonic, oleic and to a lesser extend linoleic acid were able to enhance fibrinogenolysis by RVV proteinases. Enhanced Fibrinogenolysis (EF) was observed with various venom size exclusion fractions containing different proteinases, and also with trypsin, indicating that conformational changes of the substrate and increased accessibility of otherwise cryptic cleavage sites are likely to be responsible for EF. In addition to enhancing fibrinogenolysis, sodium arachidonate and oleate were found to partially inhibit thrombin induced, factor XIIIa (FXIIIa) mediated ligation of fibrin chains. In clotting experiments with fresh blood RVV was found to disrupt normal coagulation, leading to small, partial clot formation, whereas RVV pretreated with the PLA2 inhibitor Varespladib induced rapid and complete clot formation (after 5 min) compared to blood alone. ConclusionThe observations that fatty acid anions and anionic detergents induce conformational changes that render fibrin(ogen) more susceptible to proteolysis by RVV proteinases and that RVV-PLA2 activity (which produces FFA) is required to render blood incoagulable in clotting experiments with RVV indicate a mechanism by which the activity of highly abundant RVV-PLA2 promotes degradation and depletion of fibrin(ogen) resulting in incoagulable blood seen following RVV envenomation (VICC).

Evaluation of the Photosonolysis Process Efficacy for the Removal of Anionic Surfactant Linear Alkyl Benzene Sulfonate from Aqueous Solutions

In this study, the experiments on the sulfonate linear alkylbenzene (LAS) solution were performed using the methylene blue active substances (MBAS) method. The effectiveness of the sonochemical and photochemical reactors for LAS degradation was evaluated with an emphasis on the effect of the contact time, pH values, type of process and initial concentrations. The experiments were carried out at the initial concentrations of 0.5 mg/L, 14.00 mg/L and 21.00 mg/L, the contact time of 8, 16, and 24 minutes, pH of 3, 7, and 11, the ultrasonic frequency of 42 kHz, the acoustic power value of 160 W, an ultraviolet lamp with 150W power and the wavelength of 254 nm. This study showed that the LAS degradation was increased along with the contact time. In addition, at alkaline pH, the removal efficiency increased as well. Overall, the results obtained from this research demonstrated that by themselves, both the sonochemical and photochemical reactors may not be useful for completely reducing the complex wastewaters with high surfactant loads, but the succession of the UV/US process has a significant effect on the removal of anionic detergents. These findings demonstrated the removal efficiency under alkaline conditions and the contact time of 24 minutes was equal to 96.97 percent.

Synthesis of FAU-Type Zeolite Membranes with Antimicrobial Activity.

In this study, a layer of a pure and dense phase of FAU-type zeolite was synthesized directly on the surface of α-Al2O3 plane macroporous support. Before hydrothermal synthesis, a step of cleaning of the support by an anionic detergent was performed, a roughness surface is created, allowing the anchoring of the zeolite nuclei and then their growth, favoring in this sense the formation of a homogeneous zeolite layer. The obtained membranes were fully characterized using X-ray diffraction analysis (XRD), nitrogen sorption, scanning electron microscopy (SEM), and mercury porosimetry. After 24 h of thermal treatment at 75 °C, a homogeneous zeolite layer composed of bipyramidal crystals of FAU-type zeolite is obtained with a thickness of about 2.5 µm. No obvious defects or cracks can be observed. It was found that the increase in heating temperature could lead to the appearance of an impurity phase, GIS-type zeolite. Then the ideal zeolite membrane was exchanged with Ag+ or Zn2+ cations to studies their antimicrobial properties. Zeolites membranes exchanged with Ag+ showed an agar-diffusive bactericidal activity against gram negative Escherichia coli (E. coli) bacteria. Zn2+ exchanged zeolite membrane presented a bacteriostatic activity that is less diffusive in agar. As expected, non-exchanged zeolite membrane (in its Na+ form) have no effect on bacterial activity. This process is particularly interesting for the synthesis of a good quality FAU-type zeolite membranes with antimicrobial properties.

Study of Nickel Nanoparticles in Highly Porous Nickel Metal–Organic Framework for Efficient Heterogeneous Catalytic Ozonation of Linear Alkyl Benzene Sulfonate in Water

ABSTRACT Metal–organic frameworks (MOFs), as a relatively new class of crystalline porous materials with large surface area and ultrahigh porosity, are extensively used in heterogeneous catalysts. In this study, the efficiency of heterogeneous catalytic ozonation process (COP) with a new nickel nanoparticle-based catalyst in highly porous carbon derived from nickel-MOF was investigated for Linear Alkyl benzene Sulfonate (LAS) degradation in aqueous solutions. LAS is the most frequently consumed anionic detergent and is used mainly in the household detergents such as washing powder, dishwashing liquid, and other household cleaners. Furthermore, if LAS gets in the water, it can produce eutrophication in water resources. In recent years, emphasis has been put on heterogeneous COP, which is principally based on producing the hydroxyl-free radicals. The influence of important parameters including solution pH (4–10), catalyst dosage (0.1–0.3 g/L), and ozone contact time (10–30 min) on the efficiency of COP were examined. The experimental results showed that catalytic ozonation with Ni/NHPC was the most effective at pH 10 with ozone reaction time of 30 min and the catalyst dosage of 0.3 g. Compared to the sole ozonation process (SOP), the removal percentage of LAS were 98.1% and 32.3%, respectively. These findings show that Ni/NHPC acts as an appropriate catalyst derived from nickel-MOF in COP to remove organic pollutants from aqueous solution.

Effective production of recombinant Δ60VP1 chicken anemia virus protein in Escherichia coli and its application to a serodiagnostic indirect ELISA

Chicken anemia virus (CAV) causes severe anemia and immunosuppression in chickens. VP1 is the main capsid protein, and is suitable for diagnostic kit development, however, it has 24 arginine residues in the first forty N-terminal amino acids of the protein causing toxicity to bacteria leading to reduced prokaryotic expression. In this study, a 60 amino acid N-terminally truncated VP1 (Δ60VP1) which removes the toxic region was expressed in Escherichia coli and the resultant insoluble recombinant protein was purified by Ni-NTA affinity chromatography with anionic denaturing detergents. The high amounts of purified Δ60VP1 produced (150 mg/L) retained appropriate antigenicity and the antigen was used to develop an indirect enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of CAV. One hundred fifty-two chicken serum samples (n = 152) were evaluated using the newly developed Δ60VP1 indirect ELISA (cutoff value = 7.58 % S/P). The sensitivity and specificity of the Δ60VP1 indirect ELISA were 87.50 % and 95.31 %, respectively, while the agreement between the Δ60VP1 indirect ELISA and the commercial IDEXX CAV ELISA was 90.79 % (kappa = 0.814). In this study, we have developed an alternative VP1 production platform in E. coli by truncating the N-terminal 60 amino acids (Δ60VP1) and using anionic denaturing detergents during the purification to successfully solubilize the insoluble Δ60VP1. The antigen was purified with high yield and good immunoreactivity, and an indirect ELISA was developed. The assay could potentially be applied to large-scale CAV serosurveillance.

Regulatory Role of the Two-Component System BasSR in the Expression of the EmrD Multidrug Efflux in Escherichia coli.

Due to excessive use of antimicrobial agents in the treatment of infectious diseases, bacteria have developed resistance to antibacterial drugs and toxic compounds. The development of multidrug efflux pumps is one of the important mechanisms of bacterial drug resistance. A multidrug efflux pump, EmrD, belonging to the major facilitator superfamily of transporters, confers resistance to many antimicrobial agents. BasSR, a typical two-component signal transduction system (TCS), regulates susceptibility to the cationic antimicrobial peptide, polymyxin B, and the anionic bile detergent, deoxycholic acid, in Escherichia coli. However, whether or not the BasSR TCS affects susceptibility or resistance to other antimicrobial agents and transcription of emrD has not been reported in E. coli. In the present study, we constructed the basSR mutants of wild-type MG1655 and clinical strain APECX40 and performed antimicrobial susceptibility testing, antibacterial activity assays, real-time reverse transcription-PCR experiments and electrophoretic mobility shift assays (EMSA) to investigate the molecular mechanism by which BasSR regulates the EmrD multidrug efflux pump. Results showed that the basSR mutation increased cell susceptibility to eight antimicrobial agents, including ciprofloxacin, norfloxacin, doxycycline, tetracycline, clindamycin, lincomycin, erythromycin, and sodium dodecyl sulfate, by downregulating the transcriptional levels of emrD. Furthermore, EMSA indicated that BasR could directly bind to the emrD promoter. Therefore, this study was the first to demonstrate that BasSR activates transcription of emrD by binding directly to its promoter region, and then decreases susceptibility to various antimicrobial agents in E. coli strains, APECX40 and MG1655.