Enteroendocrine cells are the largest population of hormone-producing cells in the body and play important roles in many aspects of body functions. The enteroendocrine cell population is divided into different subpopulations that secrete different hormones and peptides. Characterization of each subpopulation is particularly useful for analyzing the cellular mechanisms responsible for specific cell types. Therefore, the necessity of a pure cell line for a specific study purpose was the important motivation for the separation of cell lines for each subpopulation of enteroendocrine cells. The present research introduces a method for the isolation of L-cells, one of the important subpopulations of enteroendocrine cells. The antibiotic selection method was conducted in order to isolate the L-cells from a heterogonous population of intestinal cell line. In this method, a neomycin resistance gene (as selected marker) was expressed under the control of a specific promoter of L-cells. After transfection of manipulated plasmid, only the cells which determine the specific promoter and express neomycin resistance protein would be able to survive under Geneticin antibiotic treatment condition. In order to confirm that the isolated cells were L-cells, reverse transcriptase polymerase chain reaction (PCR) and quantitative PCR assays were performed. Based on the results, the isolated cells were pure L-cells that could be able to express specific mRNA of L-cells efficiently. This technique provides a unique method for the isolation and purification of any cell line. The purified isolated L-cells by this method can be used for future studies and for analyzing cellular mechanisms that involve L-cells' functions.
Keratinous wastes have increasingly become a problem and accumulate in the environment mainly in the form of feathers, generated mainly from a large number of poultry industries. As keratins are very difficult to degrade by general proteases, they pose a major environmental problem. Therefore, microorganisms which would effectively degrade keratins are needed for recycling such wastes. A geophilic dermatophyte, Microsporum fulvum IBRL SD3 which was isolated from a soil sample collected from a chicken feather dumping site using a baiting technique, was capable to produce keratinase significantly. The crude keratinase was able to degrade whole chicken feathers effectively. The end product of the degradation was protein that contained essential amino acids and may have potential application in animal feed production. Thus, M. fulvum could be a novel organism to produce keratinase for chicken feathers degradation.
Dendrobium sonia-28 is an important ornamental orchid in the Malaysian flower industry. However, the genus faces both low germination rates and the risk of producing heterozygous progenies. Cryopreservation is currently the favoured long-term storage method for orchids with propagation problems. Vitrification, a frequently used cryopreservation technique, involves the application of pretreatments and cryoprotectants to protect and recover explants during and after storage in liquid nitrogen. However, cryopreservation may cause osmotic injuries and toxicity to cryopreserved explants from the use of highly concentrated additives, and cellular injuries from thawing, devitrification and ice formation. Reactive oxygen species (ROS), occurring during dehydration and cryopreservation, may also cause membrane damage. Plants possess efficient antioxidant systems such as the superoxide dismutase (SOD) and catalase (CAT) enzymes to scavenge ROS during low temperature stress. In this study, protocorm-like bodies (PLBs) of Dendrobium sonia-28 were assayed for the total protein content, and both SOD and CAT activities, at each stage of a vitrification exercise to observe for deleterious stages in the protocol. The results indicated that cryopreserved PLBs of Dendrobium sonia-28 underwent excessive post-thawing oxidative stress due to decreased levels of the CAT enzyme at the post-thawing recovery stage, which contributed to the poor survival rates of the cryopreserved PLBs.
The aeration strategy ranging from intermittent to continuous aeration in the REACT period of moving bed sequencing batch reactor (MBSBR) was evaluated for simultaneous removal of 4-chlorophenol (4-CP) and nitrogen. The results show that the removal rates of 4-CP and ammonium nitrogen (NH(4)(+)-N) increased with the increase of continuous aeration period. In the presence of 4-CP, NH(4)(+)-N removal was mainly by the assimilation process. The removal of NH(4)(+)-N to oxidized nitrogen via oxidation was only observed after 4-CP was completely degraded with sufficient aeration period provided indicating the inhibitory effect of 4-CP on nitrification. As the intermittent aeration strategy would lead to slower 4-CP degradation resulting in the delay of nitrification process, continuous aeration would be the preferred strategy in the simultaneous removal of 4-CP and nitrogen in the MBSBR system.
Leukemic cells are hard-to-transfect cell lines. Many transfection reagents which can provide high gene transfer efficiency in common adherent cell lines are not effective to transfect established blood cell lines or primary leukemic cells. This study aims to examine a new class of cationic polymer non-viral vector, PEGylated-dextran-spermine (PEG-D-SPM), to determine its ability to transfect the leukemic cells. Here, the optimal conditions of the complex preparation (PEG-D-SPM/plasmid DNA (pDNA)) were examined. Different weight-mixing (w/w) ratios of PEG-D-SPM/pDNA complex were prepared to obtain an ideal mixing ratio to protect encapsulated pDNA from DNase degradation and to determine the optimal transfection efficiency of the complex. Strong complexation between polymer and pDNA in agarose gel electrophoresis and protection of pDNA from DNase were detected at ratios from 25 to 15. Highest gene expression was detected at w/w ratio of 18 in HL60 and K562 cells. However, gene expression from both leukemic cell lines was lower than the control MCF-7 cells. The cytotoxicity of PEG-D-SPM/pDNA complex at the most optimal mixing ratios was tested in HL60 and K562 cells using MTS assay and the results showed that the PEG-D-SPM/pDNA complex had no cytotoxic effect on these cell lines. Spherical shape and nano-nature of PEG-D-SPM/pDNA complex at ratio 18 was observed using transmission electron microscopy. As PEG-D-SPM showed modest transfection efficiency in the leukemic cell lines, we conclude that further work is needed to improve the delivery efficiency of the PEG-D-SPM.
Polyhydroxyalkanoates (PHAs) are hydrophobic biodegradable thermoplastics that have received considerable attention in biomedical applications due to their biocompatibility, mechanical properties, and biodegradability. In this study, the degradation rate was regulated by optimizing the interaction of parameters that influence the enzymatic degradation of P(3HB) film using response surface methodology (RSM). The RSM model was experimentally validated yielding a maximum 21 % weight loss, which represents onefold increment in percentage weight loss in comparison with the conventional method. By using the optimized condition, the enzymatic degradation by an extracellular PHA depolymerase from Acidovorax sp. DP5 was studied at 37 °C and pH 9.0 on different types of PHA films with various monomer compositions. Surface modification of scaffold was employed using enzymatic technique to create highly porous scaffold with a large surface to volume ratio, which makes them attractive as potential tissue scaffold in biomedical field. Scanning electron microscopy revealed that the surface of salt-leached films was more porous compared with the solvent-cast films, and hence, increased the degradation rate of salt-leached films. Apparently, enzymatic degradation behaviors of PHA films were determined by several factors such as monomer composition, crystallinity, molecular weight, porosity, and roughness of the surface. The hydrophilicity and water uptake of degraded salt-leached film of P(3HB-co-70%4HB) were enhanced by incorporating chitosan or alginate. Salt-leached technique followed by partial enzymatic degradation would enhance the cell attachment and suitable for biomedical as a scaffold.
Oil palm empty fruit bunch (EFB) is abundantly available in Malaysia and it is a potential source of xylose for the production of high-value added products. This study aimed to optimize the hydrolysis of EFB using dilute sulfuric acid (H2SO4) and phosphoric acid (H3PO4) via response surface methodology for maximum xylose recovery. Hydrolysis was carried out in an autoclave. An optimum xylose yield of 91.2 % was obtained at 116 °C using 2.0 % (v/v) H2SO4, a solid/liquid ratio of 1:5 and a hydrolysis time of 20 min. A lower optimum xylose yield of 24.0 % was observed for dilute H3PO4 hydrolysis at 116 °C using 2.4 % (v/v) H3PO4, a solid/liquid ratio of 1:5 and a hydrolysis time of 20 min. The optimized hydrolysis conditions suggested that EFB hydrolysis by H2SO4 resulted in a higher xylose yield at a lower acid concentration as compared to H3PO4.
Cellulase is an enzyme that converts the polymer structure of polysaccharides into fermentable sugars. The high market demand for this enzyme together with the variety of applications in the industry has brought the research on cellulase into focus. In this study, crude cellulase was produced from oil palm empty fruit bunch (OPEFB) pretreated with 2% NaOH with autoclave, which was composed of 59.7% cellulose, 21.6% hemicellulose, and 12.3% lignin using Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2. Approximately 0.8 U/ml of FPase, 24.7 U/ml of CMCase and 5.0 U/ml of β-glucosidase were produced by T. asperellum UPM1 at a temperature of 35 °C and at an initial pH of 7.0. A 1.7 U/ml of FPase, 24.2 U/ml of CMCase, and 1.1 U/ml of β-glucosidase were produced by A. fumigatus UPM2 at a temperature of 45 °C and at initial pH of 6.0. The crude cellulase was best produced at 1% of substrate concentration for both T. asperellum UPM1 and A. fumigatus UPM2. The hydrolysis percentage of pretreated OPEFB using 5% of crude cellulase concentration from T. asperellum UPM1 and A. fumigatus UPM2 were 3.33% and 19.11%, with the reducing sugars concentration of 1.47 and 8.63 g/l, respectively.
In this study, the ability of Cupriavidus sp. USMAA2-4 to synthesize polyhydroxyalkanoates (PHA) containing 4-hydroxyvalerate monomer (4HV) was studied through one-stage cultivation using γ-valerolactone as the carbon precursor. The presence of 4HV monomer unit in the polymer was detected through gas chromatography analysis, proving the capability of this wild strain bacterium to produce poly(3-hydrxybutyrate-co-3-hydroxyvalerate-co-4-hydroxyvalerate) [P(3HB-co-3HV-co-4HV)] terpolymer. Existence of a 4HV monomer unit in the PHA produced was further confirmed through (13)C and (1)H NMR analysis. P(3HB-co-88 % 3HV-co-1 % 4HV) terpolymer with the highest PHA content of 63 wt% was obtained through combination of 0.14 wt% C of γ-valerolactone with 0.42 wt% C of oleic acid. Various compositions of P(3HB-co-3HV-co-4HV) terpolymer with 3HV and 4HV compositions ranging from 11 to 94 mol% and from 1 to 4 mol%, respectively, were acquired by manipulating γ-valerolactone and oleic acid concentrations. The molecular weight and the thermal and mechanical properties of four different compositions of terpolymers-P(3HB-co-91 % 3HV-co-1 % 4HV), P(3HB-co-55 % 3HV-co-2 % 4HV), P(3HB-co-27 % 3HV-co-2 % 4HV), and P(3HB-co-9 % 3HV-co-1 % 4HV)-were characterized. Among these terpolymers, P(3HB-co-27 % 3HV-co-2 % 4HV) terpolymer with a molecular weight of 5.7 (10(5) Da) exhibited the highest elongation to break (264 %). The monomer unit compositional distributions of these terpolymers were investigated through acetone-water fractionation analysis. The results suggested that these produced terpolymers had broad 3HV compositional distribution and narrow 4HV compositional distribution.
A simple, efficient and economical method for the recovery of P(3HB-co-3HHx) was developed using various chemicals and parameters. The initial content of P(3HB-co-3HHx) in bacterial cells was 50-60 wt%, whereas the monomer composition of 3HHx used in this experiments was 3-5 mol%. It was found that sodium hydroxide (NaOH) was the most effective chemical for the recovery of biodegradable polymer. High polyhydroxyalkanoate purity and recovery yield both in the range of 80-90 wt% were obtained when 10-30 mg/ml of cells were incubated in NaOH at the concentration of 0.1 M for 60-180 min at 30 °C and polished using 20 % (v/v) of ethanol.
Aerobic granulation was developed in overcoming the problem of biomass washout often encountered in activated sludge processes. The novel approach to developing fluffy biosolids into dense and compact granules offers a new dimension for wastewater treatment. Compared with conventional biological flocs, aerobic granules are characterized by well-defined shape and compact buildup, superior biomass retention, enhanced microbial functions, and resilient to toxicity and shock loading. This review provides an up-to-date account on development in aerobic granulation and its applications. Granule characterization, factors affecting granulation, and response of granules to various environmental and operating conditions are discussed. Maintaining granule of adequate structural stability is one of the main challenges for practical applications of aerobic granulation. This paper also reviews recent advances in addressing granule stability and storage for use as inoculums, and as biomass supplement to enhance treatment efficiency. Challenges and future work of aerobic granulation are also outlined.
This paper investigates the degradation of polyhydroxyalkanoates and its biofiber composites in both soil and lake environment. Time-dependent changes in the weight loss of films were monitored. The rate of degradation of poly(3-hydroxybutyrate) [P(3HB)], poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-23 mol% 4HB)] and poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) [P(3HB-co-9 mol% 3HV-co-19 mol% 4HB)] were investigated. The rate of degradation in the lake is higher compared to that in the soil. The highest rate of degradation in lake environment (15.6% w/w week(-1)) was observed with P(3HB-co-3HV-co-4HB) terpolymer. Additionally, the rate of degradation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-38 mol% 3HV)] was compared to PHBV biofiber composites containing compatibilizers and empty fruit bunch (EFB). Here, composites with 30% EFB displayed the highest rate of degradation both in the lake (25.6% w/w week(-1)) and soil (15.6% w/w week(-1)) environment.
Human adipose-derived stem cells (ASCs) have generated a great deal of excitement in regenerative medicine. However, their safety and efficacy issue remain a major concern especially after long-term in vitro expansion. The aim of this study was to investigate the fundamental changes of ASCs in long-term culture by studying the morphological feature, growth kinetic, surface marker expressions, expression level of the senescence-associated genes, cell cycle distribution and ß-galactosidase activity. Human ASCs were harvested from lipoaspirate obtained from 6 patients. All the parameters mentioned above were measured at P5, P10, P15 and P20. Data were subjected to one-way analysis of variance with a Tukey post hoc test to determine significance difference (P < 0.05). The data showed that growth of ASCs reduced in long-term culture and the ß-galactosidase activity was significantly increased at later passage (P20). The morphology of ASCs in long-term culture showed the manifestation of senescent feature at P15 and P20. Significant alteration in the senescence-associated genes expression levels was observed in MMP1, p21, Rb and Cyclin D1 at P15 and P20. Significant increase in CD45 and HLA DR DQ DP surface marker was observed at P20. While cell cycle analysis showed significant decrease in percentage of ASCs at S and G2/M phase at later passage (P15). Our data showed ASCs cultured beyond P10 favours the senescence pathway and its clinical usage in cell-based therapy may be limited.
Acetone-butanol-ethanol (ABE) production from renewable resources has been widely reported. In this study, Clostridium butyricum EB6 was employed for ABE fermentation using fermentable sugar derived from treated oil palm empty fruit bunch (OPEFB). A higher amount of ABE (2.61 g/l) was produced in a fermentation using treated OPEFB as the substrate when compared to a glucose based medium that produced 0.24 g/l at pH 5.5. ABE production was increased to 3.47 g/l with a yield of 0.24 g/g at pH 6.0. The fermentation using limited nitrogen concentration of 3 g/l improved the ABE yield by 64%. The study showed that OPEFB has the potential to be applied for renewable ABE production by C. butyricum EB6.
In silico and experimental investigations were conducted to explore the effects of substituting hydrophobic residues, Val, Met, Leu, Ile, Trp, and Phe into Gln 114 of T1 lipase. The in silico investigations accurately predicted the enzymatic characteristics of the mutants in the experimental studies and provided rationalization for some of the experimental observations. Substitution with Leu successfully improved the conformational stability and enzymatic characteristics of T1 lipase. However, replacement of Gln114 with Trp negatively affected T1 lipase and resulted in the largest disruption of protein stability, diminished lipase activity and inferior enzymatic characteristics. These results suggested that the substitution of a larger residue in a densely packed area of the protein core can have considerable effects on the structure and function of an enzyme. This is especially true when the residue is next to the catalytic serine as demonstrated with the Phe and Trp mutation.
Sago pith residue is one of the most abundant lignocellulosic biomass which can serve as an alternative cheap substrate for fermentable sugars production. This residue is the fibrous waste left behind after the starch extraction process and contains significant amounts of starch (58%), cellulose (23%), hemicellulose (9.2%) and lignin (3.9%). The conversion of sago pith residue into fermentable sugars is commonly performed using cellulolytic enzymes or known as cellulases. In this study, crude cellulases were produced by two local isolates, Trichoderma asperellum UPM1 and Aspergillus fumigatus, UPM2 using sago pith residue as substrate. A. fumigatus UPM2 gave the highest FPase, CMCase and β-glucosidase activities of 0.39, 23.99 and 0.78 U/ml, respectively, on day 5. The highest activity of FPase, CMCase and β-glucosidase by T. asperellum UPM1 was 0.27, 12.03 and 0.42 U/ml, respectively, on day 7. The crude enzyme obtained from A. fumigatus UPM2 using β-glucosidase as the rate-limiting enzyme (3.9, 11.7 and 23.4 IU) was used for the saccharification process to convert 5% (w/v) sago pith residue into reducing sugars. Hydrolysis of sago pith residue using crude enzyme containing β-glucosidase with 23.4 IU, produced by A. fumigatus UPM2 gave higher reducing sugars production of 20.77 g/l with overall hydrolysis percentage of 73%.
The bacterial reduction of Cr(VI) from industrial wastewater was evaluated using a 2.0-m(3) bioreactor. Liquid pineapple waste was used as a nutrient for the biofilm community formed inside the bioreactor. The use of rubber wood sawdust as packing material was able to immobilize more than 10(6) CFU mL(-1) of Acinetobacter haemolyticus cells after 3 days of contact time. Complete reduction of 15-240 mg L(-1) of Cr(VI) was achieved even after 3 months of bioreactor operation. Cr(VI) was not detected in the final effluent fraction indicating complete removal of Cr from solution from the flocculation/coagulation step and the unlikely re-oxidation of Cr(III) into Cr(VI). Impatiens balsamina L. and Gomphrena globosa L. showed better growth in the presence of soil-sludge mixture compared to Coleus scutellarioides (L.) Benth. Significant amounts of Cr accumulated at different sections of the plants indicate its potential application in Cr phytoremediation effort. The bacterial-based system was also determined not to be detrimental to human health based on the low levels of Cr detected in the hair and nail samples of the plant operators. Thus, it can be said that bacterial-based Cr(VI) treatment system is a feasible alternative to the conventional system especially for lower Cr(VI) concentrations, where sludge generated can be used as growth supplement for ornamental plant as well as not detrimental to the health of the workers.
Agitation speed was found to influence the tannase production and fungal growth of Aspergillus niger FETL FT3. The optimal agitation speed was at 200 rpm which produced 1.41 U/ml tannase and 3.75 g/l of fungal growth. Lower or higher agitation speeds than 200 rpm produced lower enzyme production and fungal growth. Based on the SEM and TEM micrograph observation, there was a significant correlation between agitation speed and the morphology of the fungal mycelia. The results revealed an increase of the enzyme production with the change of the fungal growth morphology from filamentous to pelleted growth forms. However, the exposure to higher shear stress with an increasing agitation speed of the shaker also resulted in lower biomass yields as well as enzyme production.
The present work highlighted the production of violacein by the locally isolated Chromobacterium violaceum (GenBank accession no. HM132057) in various agricultural waste materials (sugarcane bagasse, solid pineapple waste, molasses, brown sugar), as an alternative to the conventional rich medium. The highest yield for pigment production (0.82 g L⁻¹) was obtained using free cells when grown in 3 g of sugarcane bagasse supplemented with 10% (v/v) of L-tryptophan. A much lower yield (0.15 g L⁻¹) was obtained when the cells were grown either in rich medium (nutrient broth) or immobilized onto sugarcane bagasse. Violacein showed similar chemical properties as other natural pigments based on the UV-Vis, Fourier transform infrared spectroscopy, thin-layer chromatography, nuclear magnetic resonance, and mass spectrometry analysis. The pigment is highly soluble in acetone and methanol, insoluble in water or non-polar organic solvents, and showed good stability between pH 5-9, 25-100 °C, in the presence of light metal ions and oxidant such as H₂O₂. However, violacein would be slowly degraded upon exposure to light. This is the first report on the use of cheap and easily available agricultural wastes as growth medium for violacein-producing C. violaceum.
Gynura procumbens (Lour.) Merr. is a traditionally used medicinal plant to decrease cholesterol level, reduce high blood pressure, control diabetics, and for treatment of cancer. In our present study, a proteomic approach was applied to study the proteome of the plant that had never analyzed before. We have identified 92 abundantly expressed proteins from the leaves of G. procumbens (Lour.) Merr. Amongst the identified proteins was miraculin, a taste-masking agent with high commercial value. Miraculin made up ∼0.1% of the total protein extracted; the finding of miraculin gave a great commercial value to G. procumbens (Lour.) Merr. as miraculin's natural source is limited while the production of recombinant miraculin faced challenges of not being able to exhibit the taste-masking effect as in the natural miraculin. We believe the discovery of miraculin in G. procumbens (Lour.) Merr., provides commercial feasibility of miraculin in view of the availability of G. procumbens (Lour.) Merr. that grow wildly and easily in tropical climate.