The use of nontoxic biological compounds in the synthesis of nanomaterials is an economic and eco-friendly approach. The present work was undertaken to develop zinc oxide nanoparticles (ZnO-NPs) by a green method using simple precursor from the solution consisting of zinc acetate and the flower extract of Anchusa italica (A. italica). Effect of annealing temperature on structural and antimicrobial properties was investigated. The crystalline structure of ZnO-NPs was shown using X-ray diffraction (XRD) analysis. Transmission electron microscopy (TEM) results showed that ZnO-NPs are hexagonal in shapes with mean particle size of ~8 and ~14nm at 100°C and 200°C annealing temperatures respectively. The optical band gap was increased from 3.27eV to 3.30eV with the decreasing of the particle size. The antimicrobial activity of ZnO-NPs towards Gram positive (Bacillus megaterium and Stapphylococcus aureus) and Gram negative (Escherichia coli and Salmonella typhimurium) pathogens decreased with the increasing of the heat treating temperature. In vitro cytotoxicity studies on Vero cells, a dose dependent toxicity with non-toxic effect of concentration below 142μg/mL was shown. The results indicated that A. italica is an appropriate reaction media to prepare ZnO-NPs for cosmetic and bio-medical productions.
Gamma-aminobutyric acid (GABA) is an important bioactive compound biosynthesized by microorganisms through decarboxylation of glutamate by glutamate decarboxylase (GAD). In this study, a full-length GAD gene was obtained by cloning the template deoxyribonucleic acid to pTZ57R/T vector. The open reading frame of the GAD gene showed the cloned gene was composed of 1410 nucleotides and encoded a 469 amino acids protein. To improve the GABA-production, the GAD gene was cloned into pMG36e-LbGAD, and then expressed in Lactobacillus plantarum Taj-Apis362 cells. The overexpression was confirmed by SDS-PAGE and GAD activity, showing a 53 KDa protein with the enzyme activity increased by sevenfold compared with the original GAD activity. The optimal fermentation conditions for GABA production established using response surface methodology were at glutamic acid concentration of 497.973 mM, temperature 36°C, pH 5.31 and time 60 h. Under the conditions, maximum GABA concentration obtained (11.09 mM) was comparable with the predicted value by the model at 11.23 mM. To our knowledge, this is the first report of successful cloning (clone-back) and overexpression of the LbGAD gene from L. plantarum to L. plantarum cells. The recombinant Lactobacillus could be used as a starter culture for direct incorporation into a food system during fermentation for production of GABA-rich products.
Isoprenoids are a large, diverse group of secondary metabolites which has recently raised a renewed research interest due to genetic engineering advances, allowing specific isoprenoids to be produced and characterized in heterologous hosts. Many researches on metabolic engineering of heterologous hosts for increased isoprenoid production are focussed on Escherichia coli and yeasts. E. coli, as most prokaryotes, use the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway for isoprenoid production. Yeasts on the other hand, use the mevalonate pathway which is commonly found in eukaryotes. However, Lactococcus lactis is an attractive alternative host for heterologous isoprenoid production. Apart from being food-grade, this Gram-positive prokaryote uses the mevalonate pathway for isoprenoid production instead of the MEP pathway. Previous studies have shown that L. lactis is able to produce sesquiterpenes through heterologous expression of plant sesquiterpene synthases. In this work, we analysed the gene expression of the lactococcal mevalonate pathway through RT-qPCR to successfully engineer L. lactis as an efficient host for isoprenoid production. We then overexpressed the mvk gene singly or co-expressed with the mvaA gene as an attempt to increase β-sesquiphellandrene production in L. lactis. It was observed that co-expression of mvk with mvaA doubled the amount of β-sesquiphellandrene produced.
The periplasmic proteome of recombinant E. coli cells expressing human interferon-α2b (INF-α2b) was analysed by 2D-gel electrophoresis to find the most altered proteins. Of some unique up- and down-regulated proteins in the proteome, ten were identified by MS. The majority of the proteins belonged to the ABC transporter protein family. Other affected proteins were ones involved in the regulation of transcription such as DNA-binding response regulator, stress-related proteins and ecotin. Thus, the production of INF-α2b acts as a stress on the cells and results in the induction of various transporters and stress related proteins.
To isolate a bacterial strain capable of biotransforming ferulic acid, a major component of lignin, into vanillin and vanillic acid by a rapid colorimetric screening method.
Vanadium-dependent haloperoxidases belong to a class of vanadium enzymes that may have potential industrial and pharmaceutical applications due to their high stability. In this study, the 5'-flanking genomic sequence and complete reading frame encoding vanadium-dependent bromoperoxidase (GcVBPO1) was cloned from the red seaweed, Fracilaria changii, and the recombinant protein was biochemically characterized. The deduced amino acid sequence of GcVBPO1 is 1818 nucleotides in length, sharing 49% identity with the vanadium-dependent bromoperoxidases from Corralina officinalis and Cor. pilulifera, respectively. The amino acid residues associated with the binding site of vanadate cofactor were found to be conserved. The Km value of recombinant GcVBPO1 for Br(-) was 4.69 mM, while its Vmax was 10.61 μkat mg(-1) at pH 7. Substitution of Arg(379) with His(379) in the recombinant protein caused a lower affinity for Br(-), while substitution of Arg(379) with Phe(379) not only increased its affinity for Br(-) but also enabled the mutant enzyme to oxidize Cl(-). The mutant Arg(379)Phe was also found to have a lower affinity for I(-), as compared to the wild-type GcVBPO1 and mutant Arg(379)His. In addition, the Arg(379)Phe mutant has a slightly higher affinity for H2O2 compared to the wild-type GcVBPO1. Multiple cis-acting regulatory elements associated with light response, hormone signaling, and meristem expression were detected at the 5'-flanking genomic sequence of GcVBPO1. The transcript abundance of GcVBPO1 was relatively higher in seaweed samples treated with 50 parts per thousand (ppt) artificial seawater (ASW) compared to those treated in 10 and 30 ppt ASW, in support of its role in the abiotic stress response of seaweed.
Fifty signal peptides of Pediococcus pentosaceus were characterized by in silico analysis and, based on the physicochemical analysis, (two potential signal peptides Spk1 and Spk3 were identified). The coding sequences of SP were amplified and fused to the gene coding for green fluorescent protein (GFP) and cloned into Lactococcus lactis pNZ8048 and pMG36e vectors, respectively. Western blot analysis indicated that the GFP proteins were secreted using both heterologous SPs. ELISA showed that the secretion efficiency of GFP using Spk1 (0.64 μg/ml) was similar to using Usp45 (0.62 μg/ml) and Spk3 (0.58 μg/ml).
In comparison to monolayer cells, MCTS has been claimed as more suitable candidate for studying drug penetration due to the high resemblance to solid tumors. However, the cultivation of MCTS is cumbersome, time consuming, and most technique fail to generate spheroids with uniform sizes. Therefore, the application of spheroid cultures in high throughput screening has been rather limiting. Besides, the lack of a well established screening protocol method that is applicable to spheroid could also be attributed to this limitation. Here we report a simple way of cultivating homogenous MCTS cultures with compact and rigid structure from the MCF-7 cells. Besides, we had also made some modifications to the standard MTT assay to realize high throughput screening of these spheroids. Using the modified protocol, tamoxifen showed cytotoxicity effect towards MCTS cultures from MCF-7 with high consistency. The results correlated well with the cultures' response assessed by LDH release assay but the latter assay was not ideal for detecting a wide range of cytotoxicity due to high basal background reading. The MTT assay emerged as a better indicator to apoptosis event in comparison to the LDH release assay. Therefore, the method for spheroid generation and the modified MTT assay we reported here could be potentially applied to high throughput screening for response of spheroid cultures generated from MCF-7 as well as other cancer cell lines towards cytotoxic stimuli.
Vanda Mimi Palmer (VMP), an orchid hybrid of Vanda tesselata and Vanda Tan Chay Yan is a highly scented tropical orchid which blooms all year round. Previous studies revealed that VMP produces a variety of isoprenoid volatiles during daylight. Isoprenoids are well known to contribute significantly to the scent of most fragrant plants. They are a large group of secondary metabolites which may possess valuable characteristics such as flavor, fragrance and toxicity and are produced via two pathways, the mevalonate (MVA) pathway or/and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. In this study, a sesquiterpene synthase gene denoted VMPSTS, previously isolated from a floral cDNA library of VMP was cloned and expressed in Lactococcus lactis to characterize the functionality of the protein. L. lactis, a food grade bacterium which utilizes the mevalonate pathway for isoprenoid production was found to be a suitable host for the characterization of plant terpene synthases. Through recombinant expression of VMPSTS, it was revealed that VMPSTS produced multiple sesquiterpenes and germacrene D dominates its profile.
Induction strategies for the periplasmic production of recombinant human IFN-alpha2b (interferon-alpha2b) by recombinant Escherichia coli Rosetta-gami 2(DE3) were optimized in shake-flask cultures using response surface methodology based on the central composite design. The factors included in the present study were induction point, which related to the attenuance of the cell culture, IPTG (isopropyl beta-D-thiogalactoside) concentration and induction temperature. Second-order polynomial models were used to correlate the abovementioned factors to soluble periplasmic IFN-alpha2b formation and percentage of soluble IFN-alpha2b translocated to the periplasmic space of E. coli. The models were found to be significant and subsequently validated. The proposed induction strategies consisted of induction at an attenuance of 4 (measured as D600), IPTG concentration of 0.05 mM and temperature of 25 degrees C. The optimized induction strategy reduced inclusion-body formation as evidenced by electron microscopy and yielded 323.8 ng/ml of IFN-alpha2b in the periplasmic space with translocation of 74% of the total soluble product. In comparison with the non-optimized condition, soluble periplasmic production and the percentage of soluble IFN-alpha2b translocated to the periplasmic space obtained in optimized induction strategies were increased by approx. 20-fold and 1.4-fold respectively.
Human respiratory syncytial virus (HRSV) is a leading pathogen causing lower respiratory tract infections in infants and young children worldwide. In line with the development of an effective vaccine against HRSV, a domain of the fusion (F) glycoprotein of HRSV was produced and its immunogenicity and antigenic properties, namely the effect of deficient glycosylation was examined. A His-tagged recombinant F (rF) protein was expressed in Escherichia coli, solubilized with 8 mol/l urea, purified by the Ni-NTA affinity chromatography and used for the raising of a polyclonal antibody in rabbits. The non-glycosylated rF protein proved to be a strong immunogen that induced a polyclonal antibody that was able to recognize also the glycosylated F1 subunit of native HRSV. The other way around, a polyclonal antibody prepared against the native HRSV was able to react with the rF protein. These results indicated that glycosylation was not necessary for the F domain aa 212-574 in order to be recognized by the specific polyclonal antibody.
This research investigates the use of charge coupled device (abbreviated as CCD) linear image sensors in an optical tomographic instrumentation system used for sizing particles. The measurement system, consisting of four CCD linear image sensors are configured around an octagonal shaped flow pipe for a four projections system is explained. The four linear image sensors provide 2,048 pixel imaging with a pixel size of 14 micron × 14 micron, hence constituting a high-resolution system. Image reconstruction for a four-projection optical tomography system is also discussed, where a simple optical model is used to relate attenuation due to variations in optical density, [R], within the measurement section. Expressed in matrix form this represents the forward problem in tomography [S] [R] = [M]. In practice, measurements [M] are used to estimate the optical density distribution by solving the inverse problem [R] = [S](-1)[M]. Direct inversion of the sensitivity matrix, [S], is not possible and two approximations are considered and compared-the transpose and the pseudo inverse sensitivity matrices.
Osmotic stress is one of the most significant natural abiotic stresses that occur in the intertidal zones. Seaweeds may physiologically acclimate to changing osmolarity by altering their transcriptome. Here, we investigated the transcriptomic changes of Gracilaria changii (B. M. Xia et I. A. Abbott) I. A. Abbott, J. Zhang et B. M. Xia in response to hyper- and hypoosmotic stresses using a cDNA microarray approach. Microarray analysis revealed that 199 and 200 genes from ∼3,300 genes examined were up- and down-regulated by >2-fold in seaweed samples treated at 50 parts per thousand (ppt) artificial seawater (ASW) compared with those at 30 ppt ASW, respectively. The number of genes that were up- and down-regulated by >2-fold in seaweed samples treated at 10 ppt ASW compared with those at 30 ppt ASW were 154 and 187, respectively. A majority of these genes were only differentially expressed under hyper- or hypoosmotic conditions, whereas 67 transcripts were affected by both stresses. The findings of this study have shed light on the expression profiles of many transcripts during the acclimation of G. changii to hyperosmotic and hypoosmotic conditions. This information may assist in the prioritization of genes to be examined in future studies.
This paper explains in detail the solution to the forward and inverse problem faced in this research. In the forward problem section, the projection geometry and the sensor modelling are discussed. The dimensions, distributions and arrangements of the optical fibre sensors are determined based on the real hardware constructed and these are explained in the projection geometry section. The general idea in sensor modelling is to simulate an artificial environment, but with similar system properties, to predict the actual sensor values for various flow models in the hardware system. The sensitivity maps produced from the solution of the forward problems are important in reconstructing the tomographic image.
In this study, the growth kinetics of Lactobacillus rhamnosus and lactic acid production in continuous culture were assessed at a range of dilution rates (0.05 h(-1) to 0.40 h(-1)) using a 2 L stirred tank fermenter with a working volume of 600 ml. Unstructured models, predicated on the Monod and Luedeking-Piret equations, were employed to simulate the growth of the bacterium, glucose consumption, and lactic acid production at different dilution rates in continuous cultures. The maximum specific growth rate of L. rhamnosus, mu-max, was estimated at 0.40 h(-1), and the Monod cell growth saturation constant, Ks, at approximately 0.25 g/L. Maximum cell viability (1.3 x 10(10) CFU/ml) was achieved in the dilution rate range of D = 0.28 h(-1) to 0.35 h(-1). Both maximum viable cell yield and productivity were achieved at D = 0.35 h(-1). The continuous cultivation of L. rhamnosus at D = 0.35 h(-1) resulted in substantial improvements in cell productivity, of 267% (viable cell count) that achieved via batch cultivation.
Twenty-one Vibrio parahaemolyticus isolates representing 21 samples of coastal seawater from three beaches in peninsular Malaysia were found to be sensitive to streptomycin, norfloxacin and chloramphenicol. Resistance was observed to penicillin (100%), ampicillin (95.2%), carbenicilin (95.2%), erythromycin (95.2%), bacitracin (71.4%), cephalothin (28.6%), moxalactam (28.6%), kanamycin (19.1%), tetracycline (14.3%), nalidixic acid (9.5%) and gentamicin (9.5%). Plasmids of 2.6 to 35.8 mDa were detected among plasmid-containing isolates. All isolates carried the Vp-toxR gene specific to V. parahaemolyticus and were negative for the tdh gene, but only one isolate was positive for the trh gene. DNA fingerprinting of the isolates using ERIC-PCR and PFGE showed that the isolates belong to two major clonal groups, with several isolates from different locations in the same group, indicating the presence of similar strains in the different locations.
The complexity of residual toxic organics from biologically treated effluents of pulp and paper mills is a serious concern. To date, it has been difficult to choose the best treatment technique because each of the available options has advantages and drawbacks. In this study, two different treatment techniques using laboratory-scale aerobic sequencing batch reactors (SBRs) were tested with the same real recycled paper mill effluent to evaluate their treatment efficiencies. Two attached-growth SBRs using granular activated carbon (GAC) with and without additional biomass and a suspended-growth SBR were used in the treatment of real recycled paper mill effluent at a chemical oxygen demand (COD) level in the range of 800-1300 mg/L, a fixed hydraulic retention time of 24 h and a COD:N:P ratio of approximately 100:5:1. The efficiency of this biological treatment process was studied over a 300-day period. The six most important wastewater quality parameters, namely, chemical oxygen demand (COD), turbidity, ammonia (expressed as NH3-N), phosphorus (expressed as PO4(3)-P), colour, and suspended solids (SS), were measured to compare the different treatment techniques. It was determined that these processes were able to almost completely and simultaneously eliminate COD (99%) and turbidity (99%); the removals of NH3-N (90-100%), PO4(3)-P (66-78%), colour (63-91%), and SS (97-99%) were also sufficient. The overall performance results confirmed that an attached-growth SBR system using additional biomass on GAC is a promising configuration for wastewater treatment in terms of performance efficiency and process stability under fluctuations of organic load. Hence, this hybrid system is recommended for the treatment of pulp and paper mill effluents.
An oral lactococcal-based vaccine which haboured the haemagglutinin1 (HA1) antigen fused to nisP anchor protein for the purpose of surface displaying the HA1 antigen was developed against H1N1 virus.
Dominant strains of lactic acid bacteria (LAB) isolated from honey bees were evaluated for their γ-aminobutyric acid (GABA)-producing ability. Out of 24 strains, strain Taj-Apis362 showed the highest GABA-producing ability (1.76 mM) in MRS broth containing 50 mM initial glutamic acid cultured for 60 h. Effects of fermentation parameters, including initial glutamic acid level, culture temperature, initial pH and incubation time on GABA production were investigated via a single parameter optimization strategy. The optimal fermentation condition for GABA production was modeled using response surface methodology (RSM). The results showed that the culture temperature was the most significant factor for GABA production. The optimum conditions for maximum GABA production by Lactobacillus plantarum Taj-Apis362 were an initial glutamic acid concentration of 497.97 mM, culture temperature of 36 °C, initial pH of 5.31 and incubation time of 60 h, which produced 7.15 mM of GABA. The value is comparable with the predicted value of 7.21 mM.
Enterococcus species isolated from poultry sources were characterized for their resistance to antibiotics, plasmid content, presence of van genes and their diversity by randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). The results showed that all isolates were multi-resistance to the antibiotics tested. Ampicillin (15/70) followed by chloramphenicol (37/70) were the most active antibiotics tested against the Enterococcus spp. isolates, while the overall resistant rates against the other antibiotics were between 64.3% to 100%. All vancomycin-resistant E. faecalis, E. durans, E. hirae and E. faecium isolates tested by the disk diffusion assay were positive in PCR detection for presence of vanA gene. All E. casseliflavus isolates were positive for vanC2/C3 gene. However, none of the Enterococcus spp. isolates were positive for vanB and vanC1 genes. Plasmids ranging in sizes between 1.1 to ca. 35.8 MDa were detected in 38/70 of the Enterococcus isolates. When the genetic relationship among all isolates of the individual species were tested by RAPD-PCR, genetic differences detected suggested a high genetic polymorphisms of isolates in each individual species. Our results indicates that further epidemiological studies are necessary to elucidate the role of food animals as reservoir of VRE and the public health significance of infections caused by Enterococcus spp.