A new approach for the quantification of tamoxifen and its metabolites 4-hydroxytamoxifen, N-desmethyltamoxifen, and 4-hydroxy-N-desmethyltamoxifen (endoxifen) in human plasma samples using NACE coupled with contactless conductivity detection (C(4) D) is presented. The buffer system employed consisted of 7.5 mM deoxycholic acid sodium salt, 15 mM acetic acid, and 1 mM 18-crown-6 in 100% methanol. The complete separation of all targeted compounds (including endoxifen racemate) could be achieved within 6 min under optimized conditions. The proposed method was validated and showed good linearity in the range from 100 to 5000 ng/mL with correlation coefficients between 0.9922 and 0.9973, LODs in the range of 25-40 ng/mL, and acceptable reproducibility of the peak area (intraday RSD 2.2-3.1%, n = 4; interday (3 days) RSD 6.0-8.8%, n = 4). The developed method was successfully demonstrated for the quantification of tamoxifen and its metabolites in human plasma samples collected from breast cancer patients undertaking tamoxifen treatment.
The gene coding for the oxygenase component, sadA, of 4-aminobenzenesulfonate (4-ABS) 3,4-dioxygenase in Hydrogenophaga sp. PBC was previously identified via transposon mutagenesis. Expression of wild-type sadA in trans restored the ability of the sadA mutant to grow on 4-ABS. The inclusion of sadB and sadD, coding for a putative glutamine-synthetase-like protein and a plant-type ferredoxin, respectively, further improved the efficiency of 4-ABS degradation. Transcription analysis using the gfp promoter probe plasmid showed that sadABD was expressed during growth on 4-ABS and 4-sulfocatechol. Heterologous expression of sadABD in Escherichia coli led to the biotransformation of 4-ABS to a metabolite which shared a similar retention time and UV/vis profile with 4-sulfocatechol. The putative reductase gene sadC was isolated via degenerate PCR and expression of sadC and sadABD in E. coli led to maximal 4-ABS biotransformation. In E. coli, the deletion of sadB completely eliminated dioxygenase activity while the deletion of sadC or sadD led to a decrease in dioxygenase activity. Phylogenetic analysis of SadB showed that it is closely related to the glutamine-synthetase-like proteins involved in the aniline degradation pathway. This is the first discovery, to our knowledge, of the functional genetic components for 4-ABS aromatic ring hydroxylation in the bacterial domain.
Microbial arsenite oxidation is an essential biogeochemical process whereby more toxic arsenite is oxidized to the less toxic arsenate. Thiomonas strains represent an important arsenite oxidizer found ubiquitous in acid mine drainage. In the present study, the arsenite oxidase gene (aioBA) was cloned from Thiomonas delicata DSM 16361, expressed heterologously in E. coli and purified to homogeneity. The purified recombinant Aio consisted of two subunits with the respective molecular weights of 91 and 21 kDa according to SDS-PAGE. Aio catalysis was optimum at pH 5.5 and 50-55 °C. Aio exhibited stability under acidic conditions (pH 2.5-6). The V max and K m values of the enzyme were found to be 4 µmol min(-1) mg(-1) and 14.2 µM, respectively. SDS and Triton X-100 were found to inhibit the enzyme activity. The homology model of Aio showed correlation with the acidophilic adaptation of the enzyme. This is the first characterization studies of Aio from a species belonging to the Thiomonas genus. The arsenite oxidase was found to be among the acid-tolerant Aio reported to date and has the potential to be used for biosensor and bioremediation applications in acidic environments.
A co-culture consisting of Hydrogenophaga sp. PBC and Ralstonia sp. PBA, isolated from textile wastewater treatment plant could tolerate up to 100 mM 4-aminobenzenesulfonate (4-ABS) and utilize it as sole carbon, nitrogen and sulfur source under aerobic condition. The biodegradation of 4-ABS resulted in the release of nitrogen and sulfur in the form of ammonium and sulfate respectively. Ninety-eight percent removal of chemical oxygen demand attributed to 20 mM of 4-ABS in cell-free supernatant could be achieved after 118 h. Effective biodegradation of 4-ABS occurred at pH ranging from 6 to 8. During batch culture with 4-ABS as sole carbon and nitrogen source, the ratio of strain PBA to PBC was dynamic and a critical concentration of strain PBA has to be reached in order to enable effective biodegradation of 4-ABS. Haldane inhibition model was used to fit the degradation rate at different initial concentrations and the parameters μ(max), K(s) and K(i) were determined to be 0.13 h⁻¹, 1.3 mM and 42 mM respectively. HPLC analyses revealed traced accumulation of 4-sulfocatechol and at least four unidentified metabolites during biodegradation. This is the first study to report on the characterization of 4-ABS-degrading bacterial consortium that was isolated from textile wastewater treatment plant.
Genes involved in the 4-aminobenzenesulfonate (4-ABS) degradation pathway of Hydrogenophaga sp. PBC were identified using transposon mutagenesis. The screening of 10,000 mutants for incomplete 4-ABS biotransformation identified four mutants with single transposon insertion. Genes with insertions that impaired the ability to utilize 4-ABS for growth included (1) 4-sulfocatechol 1,2-dioxygenase β-subunit (pcaH2) and 3-sulfomuconate cycloisomerase involved in the modified β-ketoadipate pathway; (2) 4-aminobenzenesulfonate 3,4-dioxygenase component (sadA) involved in aromatic ring hydroxylation; and (3) transposase gene homolog with a putative cis-diol dehydrogenase gene located downstream. The pcaH2 mutant strain accumulated brown metabolite during growth on 4-ABS which was identified as 4-sulfocatechol through thin layer chromatography and HPLC analyses. Supplementation of wild-type sadA gene in trans restored the 4-ABS degradation ability of the sadA mutant, thus supporting the annotation of its disrupted gene.
Arsenic is a common contaminant in gold mine soil and tailings. Microbes present an opportunity for bio-treatment of arsenic, since it is a sustainable and cost-effective approach to remove arsenic from water. However, the development of existing bio-treatment approaches depends on isolation of arsenic-resistant microbes from arsenic contaminated samples. Microbial cultures are commonly used in bio-treatment; however, it is not established whether the structure of the cultured isolates resembles the native microbial community from arsenic-contaminated soil. In this milieu, a culture-independent approach using Illumina sequencing technology was used to profile the microbial community in situ. This was coupled with a culture-dependent technique, that is, isolation using two different growth media, to analyse the microbial population in arsenic laden tailing dam sludge based on the culture-independent sequencing approach, 4 phyla and 8 genera were identified in a sample from the arsenic-rich gold mine. Firmicutes (92.23%) was the dominant phylum, followed by Proteobacteria (3.21%), Actinobacteria (2.41%), and Bacteroidetes (1.49%). The identified genera included Staphylococcus (89.8%), Pseudomonas (1.25), Corynebacterium (0.82), Prevotella (0.54%), Megamonas (0.38%) and Sphingomonas (0.36%). The Shannon index value (3.05) and Simpson index value (0.1661) indicated low diversity in arsenic laden tailing. The culture dependent method exposed significant similarities with culture independent methods at the phylum level with Firmicutes, Proteobacteria and Actinobacteria, being common, and Firmicutes was the dominant phylum whereas, at the genus level, only Pseudomonas was presented by both methods. It showed high similarities between culture independent and dependent methods at the phylum level and large differences at the genus level, highlighting the complementarity between the two methods for identification of the native population bacteria in arsenic-rich mine. As a result, the present study can be a resource on microbes for bio-treatment of arsenic in mining waste.
Globally, the contamination of water with arsenic is a serious health issue. Recently, several researches have endorsed the efficiency of biomass to remove As (III) via adsorption process, which is distinguished by its low cost and easy technique in comparison with conventional solutions. In the present work, biomass was prepared from indigenous Bacillus thuringiensis strain WS3 and was evaluated to remove As (III) from aqueous solution under different contact time, temperature, pH, As (III) concentrations and adsorbent dosages, both experimentally and theoretically. Subsequently, optimal conditions for As (III) removal were found; 6 (ppm) As (III) concentration at 37 °C, pH 7, six hours of contact time and 0.50 mg/ml of biomass dosage. The maximal As (III) loading capacity was determined as 10.94 mg/g. The equilibrium adsorption was simulated via the Langmuir isotherm model, which provided a better fitting than the Freundlich model. In addition, FESEM-EDX showed a significant change in the morphological characteristic of the biomass following As (III) adsorption. 128 batch experimental data were taken into account to create an artificial neural network (ANN) model that mimicked the human brain function. 5-7-1 neurons were in the input, hidden and output layers respectively. The batch data was reserved for training (75%), testing (10%) and validation process (15%). The relationship between the predicted output vector and experimental data offered a high degree of correlation (R2 = 0.9959) and mean squared error (MSE; 0.3462). The predicted output of the proposed model showed a good agreement with the batch work with reasonable accuracy.
Microbacterium sp. strain SZ1 isolated from gold ores of a Malaysia gold mine was found to be highly resistant to arsenic. Here, we report the draft genome sequence of SZ1, which may provide further insights into understanding its arsenic resistance mechanism. In this draft genome, a complete set of ars operons and two additional scattered ars genes were encoded.
A Gram-negative, arsenite-resistant psychrotolerant bacterial strain, Yersinia sp. strain SOM-12D3, was isolated from a biofilm sample collected from a lake at Svalbard in the Arctic area. To our knowledge, this is the first study on the ability of acid-treated and untreated, non-living biomass of strain SOM-12D3 to absorb arsenic. We conducted batch experiments at pH 7, with an initial As(III) concentration of 6.5 ppm, at 30 °C with 80 min of contact time. The Langmuir isotherm model fitted the equilibrium data better than Freundlich, and the sorption kinetics of As(III) biosorption followed the pseudo-second-order rate equation well for both types of non-living biomass. The highest biosorption capacity of the acid-treated biomass obtained by the Langmuir model was 159 mg/g. Further, a high recovery efficiency of 96% for As(III) was achieved using 0.1 M HCl within four cycles, which indicated high adsorption/desorption. Fourier transformed infrared (FTIR) demonstrated the involvement of hydroxyl, amide, and amine groups in As(III) biosorption. Field emission scanning electron microscopy-energy dispersive analysis (FESEM-EDAX) indicated the different morphological changes occurring in the cell after acid treatment and arsenic biosorption. Our results highlight the potential of using acid-treated non-living biomass of the psychrotolerant bacterium, Yersinia sp. Strain SOM-12D3 as a new biosorbent to remove As(III) from contaminated waters.
The α-amylases from Anoxybacillus species (ASKA and ADTA), Bacillus aquimaris (BaqA) and Geobacillus thermoleovorans (GTA, Pizzo and GtamyII) were proposed as a novel group of the α-amylase family GH13. An ASKA yielding a high percentage of maltose upon its reaction on starch was chosen as a model to study the residues responsible for the biochemical properties. Four residues from conserved sequence regions (CSRs) were thus selected, and the mutants F113V (CSR-I), Y187F and L189I (CSR-II) and A161D (CSR-V) were characterised. Few changes in the optimum reaction temperature and pH were observed for all mutants. Whereas the Y187F (t1/2 43 h) and L189I (t1/2 36 h) mutants had a lower thermostability at 65°C than the native ASKA (t1/2 48 h), the mutants F113V and A161D exhibited an improved t1/2 of 51 h and 53 h, respectively. Among the mutants, only the A161D had a specific activity, k(cat) and k(cat)/K(m) higher (1.23-, 1.17- and 2.88-times, respectively) than the values determined for the ASKA. The replacement of the Ala-161 in the CSR-V with an aspartic acid also caused a significant reduction in the ratio of maltose formed. This finding suggests the Ala-161 may contribute to the high maltose production of the ASKA.
The ability of non-living biomass of an arsenic-hypertolerant Bacillus cereus strain SZ2 isolated from a gold mining environment to adsorb As (III) from aqueous solution in batch experiments was investigated as a function of contact time, initial As (III) concentration, pH, temperature and biomass dosage. Langmuir model fitted the equilibrium data better in comparison to Freundlich isotherm. The maximum biosorption capacity of the sorbent, as obtained from the Langmuir isotherm, was 153.41 mg/g. The sorption kinetic of As (III) biosorption followed well the pseudo-second-order rate equation. The Fourier transform infrared spectroscopy analysis indicated the involvement of hydroxyl, amide and amine groups in As (III) biosorption process. Field emission scanning electron microscopy-energy dispersive X-ray analysis of the non-living B. cereus SZ2 biomass demonstrated distinct cell morphological changes with significant amounts of As adsorbed onto the cells compared to non-treated cells. Desorption of 94 % As (III) was achieved at acidic pH 1 showing the capability of non-living biomass B. cereus SZ2 as potential biosorbent in removal of As (III) from arsenic-contaminated mining effluent.
To date, the genus Parvularcula consists of 6 species and no potential application of this genus was reported. Current study presents the genome sequence of Parvularcula flava strain NH6-79 T and its cellulolytic enzyme analysis. The assembled draft genome of strain NH6-79 T consists of 9 contigs and 7 scaffolds with 3.68 Mbp in size and GC content of 59.87%. From a total of 3,465 genes predicted, 96 of them are annotated as glycoside hydrolases (GHs). Within these GHs, 20 encoded genes are related to cellulosic biomass degradation, including 12 endoglucanases (5 GH10, 4 GH5, and 3 GH51), 2 exoglucanases (GH9) and 6 β-glucosidases (GH3). In addition, highest relative enzyme activities (endoglucanase, exoglucanase, and β-glucosidase) were observed at 27th hour when the strain was cultured in the carboxymethyl cellulose/Avicel®-containing medium for 45 h. The combination of genome analysis with experimental studies indicated the ability of strain NH6-79 T to produce extracellular endoglucanase, exoglucanase, and β-glucosidase. These findings suggest the potential of Parvularcula flava strain NH6-79 T in cellulose-containing biomass degradation and that the strain could be used in cellulosic biorefining process.
Cellulose acetate (CA) is a remarkable biomaterial most extensively used in biomedical applications due to their properties. This review highlighted the synthesis and chemical structure of CA polymer as well as focused on the mechanical, chemical, thermal, biocompatible, and biodegradable properties of electrospun CA nanofibers. These properties are essential in the evaluation of the CA nanofibers and provide information as a reference for the further utilization and improvement of CA nanofibers. Moreover, we have summarized the use of electrospun CA nanofibers in the drug delivery system as a carrier for drugs and classify them according to the drug class, including anti-inflammatory, anticancer, antioxidant, antimicrobial agents, vitamins and amino acids. Our review has been concluded that CA nanofibers cannot wholly be biodegraded within the human body due to the absence of cellulase enzyme but degraded by microorganisms. Hence, the biodegradation of CA nanofibers in vivo has addressed as a critical challenge.
Distinctive bioactivities possessed by luteolin (3', 4', 5, 7-tetrahydroxy-flavone) are advantageous for sundry practical applications. This paper reports the in vitro selection and characterization of single stranded-DNA (ssDNA) aptamers, specific for luteolin (LUT). 76-mer library containing 1015 randomized ssDNA were screened via systematic evolution of ligands by exponential enrichment (SELEX). The recovered ssDNA pool from the 8th round was amplified with unlabeled primers and cloned into PSTBlue-1 vector prior to sequencing. 22 of LUT-binding aptamer variants were further classified into one of the seven groups based on their N40 random sequence regions, wherein one representative from each group was characterized. The dissociation constant of aptamers designated as LUT#28, LUT#20 and LUT#3 was discerned to be 107, 214 and 109 nM, respectively with high binding affinity towards LUT. Prediction analysis of the secondary structure suggested discrete features with typical loop and stem motifs. Furthermore, LUT#3 displayed higher specificity with insignificant binding toward kaempferol and quercetin despite its structural and functional similarity compared to LUT#28 and LUT#20. Further LUT#3 can detect free luteolin within 0.2-1 mM in solution. It was suggested that LUT#3 aptamer were the most suitable for LUT recognition tool at laboratory scale based on the condition tested.
This study focuses on the biodegradation of recalcitrant, coloured compounds resulting from auto-oxidation of Acid Orange 7 (AO7) in a sequential facultative anaerobic-aerobic treatment system. A novel mixed bacterial culture, BAC-ZS, consisting of Brevibacillus panacihumi strain ZB1, Lysinibacillus fusiformis strain ZB2, and Enterococcus faecalis strain ZL bacteria were isolated from environmental samples. The acclimatisation of the mixed culture was carried out in an AO7 decolourised solution. The acclimatised mixed culture showed 98 % decolourisation within 2 h of facultative anaerobic treatment using yeast extract and glucose as co-substrate. Subsequent aerobic post treatment caused auto-oxidation reaction forming dark coloured compounds that reduced the percentage decolourisation to 73 %. Interestingly, further agitations of the mixed culture in the solution over a period of 48 h significantly decolourise the coloured compounds and increased the decolourisation percentage to 90 %. Analyses of the degradation compounds using UV-visible spectrophotometer, Fourier transform infrared spectroscopy (FTIR) and high performance liquid chromatography (HPLC) showed complete degradation of recalcitrant AO7 by the novel BAC-ZS. Phytotoxicity tests using Cucumis sativus confirmed the dye solution after post aerobic treatment were less toxic compared to the parent dye. The quantitative real-time PCR revealed that E. faecalis strain ZL was the dominant strain in the acclimatised mix culture.
Several parts of the world have been facing the problem of nitrite and nitrate contamination in ground and surface water. The acute toxicity of nitrite has been shown to be 10-fold higher than that of nitrate. In the present study, aminated silica carbon nanotube (ASCNT) was synthesised and tested for nitrite removal. The synergistic effects rendered by both amine and silica in ASCNT have significantly improved the nitrite removal efficiency. The IEP increased from 2.91 for pristine carbon nanotube (CNT) to 8.15 for ASCNT, and the surface area also increased from 178.86 to 548.21 m2 g-1. These properties have promoted ASCNT a novel adsorbent to remove nitrite. At optimum conditions of 700 ppm of nitrite concentration at pH 7 and 5 h of contact with 15 mg of adsorbent, the ASCNT achieved the maximal loading capacity of 396 mg/g (85% nitrite removal). The removal data of nitrite onto ASCNT fitted the Langmuir isotherm model better than the Freundlich isotherm model with the highest regression value of 0.98415, and also, the nonlinear analysis of kinetics data showed that the removal of nitrite followed pseudo-second-order kinetic. The positive values of both ΔS° and ΔH° suggested an endothermic reaction and an increase in randomness at the solid-liquid interface. The negative ΔG° values indicated a spontaneous adsorption process. The ASCNT was characterised using FESEM-EDX and FTIR, and the results obtained confirmed the removal of nitrite. Based on the findings, ASCNT can be considered as a novel and promising candidate for the removal of nitrite ions from wastewater.
This review discusses the application of bacterial nanocellulose (BNC) and modified BNC in treating wastewater containing heavy metals and dye contaminants. It also highlights the challenges and future perspectives of BNC and its composites. Untreated industrial effluents containing toxic heavy metals are systematically discharged into public waters. In particular, lead (Pb), copper (Cu), cadmium (Cd), nickel (Ni), zinc (Zn), and arsenic (As) are very harmful to human health and, in some cases, may lead to death. Several methods such as chemical precipitation, ion exchange, membrane filtration, coagulation, and Fenton oxidation are used to remove these heavy metals from the environment. However, these methods involve the use of numerous chemicals whilst producing high amount of toxic sludge. Meanwhile, the development of the adsorption-based technique has provided an alternative way of treating wastewater using BNC. Bacterial nanocellulose requires less energy for purification and has higher purity than plant cellulose. In general, the optimum growth parameters are crucial in BNC production. Even though native BNC can be used for the removal of heavy metals and dyes, the incorporation of other materials, such as polyethyleneimine, graphene oxide, calcium carbonate and polydopamine can improve sorption efficiencies.
Arsenic (As) is an increasing threat across the globe, widely known as a non-threshold carcinogen, and it is reaching harmful values in several areas of the world. In this study, the effect of plant growth promoting bacteria (Microbacterium foliorum) on inorganic arsenic (Arsenate) phytoremediation by Melastoma malabathricum plants was investigated through histological analysis and proteome profiling of the M. malabathricum plants. Two-dimensional gel electrophoresis and transmission electron microscopy were used to conduct the proteome and histological analysis. When arsenic-treated cells were compared to untreated cells, substantial changes were found (1) severely altered the morphology of the cells, intensely disturbed; (2) the cell wall was thicker; (3) drastically changed the cytoplasm, the cells were polygonal in shape, different in size (scattered), and relatively dense. Compared to the control group, the ultra-structure of the root cells of the control group revealed intact cytoplasm, vacuole, and cell wall under exposure to As + bacteria that had a minor effect on the cell form. To further understand As + bacteria interaction, proteome profiling of the root cell was analyzed. The As-induced oxidative stress enrichment was confirmed by the up-regulation of tubulin, nucleoside diphosphate kinase, and major allergen during As + bacteria exposure It was observed that the profusion of proteins involved in defence, protein biogenesis, signaling, photosynthesis, nucleoside and energy metabolism was greater in As + bacteria as compared to the rooting out of As only. Overall, it can be obviously seen that the current study demonstrates the effectiveness of phytoremediation by M. foliorum on proteins involved and responsive pathways in dealing with As toxicity in M. malabathricum plant.
Vitamin D deficiency is now a global health problem; despite several drug delivery systems for carrying vitamin D due to low bioavailability and loss bioactivity. Developing a new drug delivery system to deliver vitamin D3 is a strong incentive in the current study. Hence, an implantable drug delivery system (IDDS) was developed from the electrospun cellulose acetate (CA) and ε-polycaprolactone (PCL) nanofibrous membrane, in which the core of implants consists of vitamin D3-loaded CA nanofiber (CAVD) and enclosed in a thin layer of the PCL membrane (CAVD/PCL). CA nanofibrous mat loaded with vitamin D3 at the concentrations of 6, 12, and 20% (w/w) of vitamin D3 were produced using electrospinning. The smooth and bead-free fibers with diameters ranged from 324 to 428 nm were obtained. The fiber diameters increased with an increase in vitamin D3 content. The controlled drug release profile was observed over 30-days, which fit with the zero-order model (R2 > 0.96) in the first stage. The mechanical properties of IDDS were improved. Young's modulus and tensile strength of CAVD/PCL (dry) were161 ± 14 and 13.07 ± 2.5 MPa, respectively. CA and PCL nanofibers are non-cytotoxic based on the results of the in-vitro cytotoxicity studies. This study can further broaden in-vivo study and provide a reference for developing a new IDDS to carry vitamin D3 in the future.
Aim: Time-based microfluidic absorption sampling was proposed using cotton fiber-based device made in swab stick. The assay was optimized and compared with conventional pipetted drop sampling using the same device. Materials & methods: Reagents were integrated into cotton fiber device for assessing concentration of analytes by the colorimetric detection method through time-based absorption sampling microfluidic system. All assay parameters were first optimized using conventional pipette-based drop sampling. Results: The color intensity is linear in the relevant concentration range of the analytes. The LOD are 0.189 mM for glucose and 6.56 μM for nitrite, respectively. These values are better than conventional drop sampling. The fiber-containing swab itself functions as sampling, assay and calibration device. Conclusion: Microfluidic cotton fiber-based assay device was fabricated and can determine analyte concentration in artificial salivary samples, colorimetrically, by time-based absorption sampling without the need of complex equipments.