Royal jelly is a nutritious substance produced by the young nurse bees and contains significant amounts of proteins which are important for cell growth and proliferation. The aim of this study was to evaluate the effect of royal jelly as an alternative to fetal bovine serum (FBS) in cell culture using cell proliferation assays and live cell imaging.
Epithelial-mesenchymal transition (EMT) is one of the mechanisms that contribute to bronchial remodelling which underlie chronic inflammatory airway diseases such as chronic obstructive pulmonary disorder (COPD) and asthma. Bronchial EMT can be triggered by many factors including transforming growth factor β1 (TGFβ1). The majority of studies on TGFβ1-mediated bronchial EMT used BEGM as the culture medium. LHC-9 medium is another alternative available which is more economical but a less common option. Using normal human bronchial epithelial cells (BEAS-2B) cultured in BEGM as a reference, this study aims to validate the induction of EMT by TGFβ1 in cells cultured in LHC-9. Briefly, the cells were maintained in either LHC-9 or BEGM, and induced with TGFβ1 (5, 10 and 20 ng/ml) for 48 h. EMT induction was confirmed by morphological analysis and EMT markers expression by immunoblotting. In both media, cells induced with TGFβ1 displayed spindle-like morphology with a significantly higher radius ratio compared to non-induced cells which displayed a cobblestone morphology. Correspondingly, the expression of the epithelial marker E-cadherin was significantly lower, whereas the mesenchymal marker vimentin expression was significantly higher in induced cells, compared to non-induced cells. By contrast, a slower cell growth rate was observed in LHC-9 compared to that of BEGM. This study demonstrates that neither LHC-9 nor BEGM significantly influence TGFβ1-induced bronchial EMT. However, LHC-9 is less optimal for bronchial epithelial cell growth compared to BEGM. Thus, LHC-9 may be a more cost-effective substitute for BEGM, provided that time is not a factor.
An optimized cultivation condition is needed to maximize the functional green fluorescent protein (GFP) production. Six process variables (agitation rate, temperature, initial medium pH, concentration of inducer, time of induction, and inoculum density) were screened using the fractional factorial design. Three variables (agitation rate, temperature, and time of induction) exerted significant effects on functional GFP production in E. coli shake flask cultivation and were optimized subsequently using the Box-Behnken design. An agitation rate of 206 rpm at 31°C and induction of the protein expression when the cell density (OD(600nm)) reaches 1.04 could enhance the yield of functional GFP production from 0.025 g/L to 0.241 g/L, which is about ninefold higher than the unoptimized conditions. Unoptimized cultivation conditions resulted in protein aggregation and hence reduced the quantity of functional GFP. The model and regression equation based on the shake flask cultivation could be applied to a 2-L bioreactor for maximum functional GFP production.
Serum deprivation inhibits cell growth and initiates apoptosis cell death in mammalian cell cultures. Since apoptosis is a genetically controlled cell death pathway, over-expression of anti-apoptotic proteins may provide a way to delay apoptosis. This study investigated the ability of the X-linked inhibitor of apoptosis protein (XIAP) to inhibit apoptosis induced by serum deprivation. Study includes evaluation of the ability of XIAP to prolong culture period and its effect on cell proliferation in serum-deprived media. The full length human XIAP was introduced into CHO-K1 cell lines and the effects of XIAP over-expression on the inhibition of apoptosis induced by serum-deprived conditions were examined. In batch cultures, cells over-expressing XIAP showed decreased levels of apoptosis and a higher number of viable cell under serum-deprived conditions compared to the control cell lines. The viability of control cells dropped to 40% after 2days of serum deprivation, the XIAP expressing cells still maintained at a viability higher than 90%. Further investigation revealed that the caspase-3 activity of the CHO-K1 cell line was inhibited as a result of XIAP expression.
Direct recovery of hepatitis B core antigen (HBcAg) from unclarified Escherichia coli homogenates via expanded bed adsorption chromatography (EBA) has been explored in this study. Streamline DEAE was selected as the anion exchanger to recover HBcAg from heat-treated and non-heat-treated unclarified feedstocks. The use of anion-exchanger for direct extraction of proteins from unclarified feedstock is not preferred due to lack of specificity of its ligand. In this study, thermal treatment of the unclarified feedstock at 60 degrees C has resulted in 1.2- and 1.8-fold increases in yield and purity of HBcAg, respectively, compared with that purified from non-heat-treated feedstock. Heating the crude feedstock has resulted in denaturation and precipitation of contaminants in the feedstock, hence reducing non-specific interactions between the cell debris and adsorbent. The selectivity of the anion-exchanger has also been increased as shown in the breakthrough curve obtained. Enzyme-linked immunosorbent assay showed that the antigenicity of the HBcAg from heat-treated unclarified feedstock is still preserved.
Over the past few decades, Escherichia coli (E. coli) remains the most favorable host among the microbial cell factories for the production of soluble recombinant proteins. Recombinant protein production (RPP) via E. coli is optimized at the level of gene expression (expression level) and the process condition of fermentation (process level). Presently, the reported studies do not give a clear view on the selection of methods employed in the optimization of RPP. Here, we have reviewed various optimization methods and their preferences with respect to the factors at expression and process levels to achieve the optimal levels of soluble RPP. With a greater understanding of these optimization methods, we proposed a stepwise methodology linking the factors from both levels for optimizing the production of soluble recombinant protein in E. coli. The proposed methodology is further explained through five sets of examples demonstrating the optimization of RPP at both expression and process levels.Key Points• Stepwise methodology of optimizing recombinant protein production is proposed.• In silico tools can facilitate the optimization of gene- and protein-based factors.• Optimization of gene- and protein-based factors aids host-vector selection.• Statistical optimization is preferred for achieving optimal levels of process factors.
Collagen has been widely shown to promote osteogenesis of bone marrow mesenchymal stromal cells (BM-MSCs). Due to the invasive procedure of obtaining BM-MSCs, MSCs from other tissues have emerged as a promising alternative for regenerative therapy. MSCs originated from different sources, exhibiting different differentiation potentials. Therefore, the applicability of collagen type I (COL), combining with amniotic membrane (AM)-MSCs was examined through proliferation and differentiation assays together with the expression of surface markers and genes associated with stemness and differentiation under basal or induction conditions. No increase in cell growth was observed because AM-MSCs might be directed toward spontaneous osteogenesis. This was evidenced by the calcium deposition and elevated expression of osteogenic genes when AM-MSCs were cultured in collagen plate with basal media. Under the osteogenic condition, reciprocal expression of OCN and CEBPA suggested a shift toward adipogenesis. Surprisingly, adipogenic genes were not elevated upon adipogenic induction, although oil droplets deposition was observed. In conclusion, our findings demonstrated that collagen causes spontaneous osteogenesis in AM-MSCs. However, the presence of exogenous inductors could shift the direction of adipo-osteogenic gene regulatory network modulated by collagen.
Chlorella sorokiniana CY-1 was cultivated using palm oil mill effluent (POME) in a novel-designed photobioreactor (NPBR) and glass-made vessel photobioreactor (PBR). The comparison was made on biomass and lipid productions, as well as its pollutants removal efficiencies. NPBR is transparent and is developed in thin flat panels with a high surface area per volume ratio. It is equipped with microbubbling and baffles retention, ensuring effective light and CO2 utilization. The triangular shape of this reactor at the bottom serves to ease microalgae cell harvesting by sedimentation. Both biomass and lipid yields attained in NPBR were 2.3-2.9 folds higher than cultivated in PBR. The pollutants removal efficiencies achieved were 93.7% of chemical oxygen demand, 98.6% of total nitrogen and 96.0% of total phosphorus. Mathematical model revealed that effective light received and initial mass contributes toward successful microalgae cultivation. Overall, the results revealed the potential of NPBR integration in Chlorella sorokiniana CY-1 cultivation, with an aim to achieve greater feasibility in microalgal-based biofuel real application and for environmental sustainability.
Species of the genus Ganoderma are a cosmopolitan wood decaying white rot fungi, which has been used by the Asians for therapeutic purposes for centuries. In the present study, solid-substrate fermentation (SSF) of wheat grains (Triticum aestivum L.) was carried out with indigenous Ganoderma australe (KUM60813) and G. neo-japonicum (KUM61076) selected based on ethnomycological knowledge. G. lucidum (VITA GL) (a commercial strain) was also included in the study. Antioxidant activities of the crude ethanol and aqueous extracts of the fermented and unfermented wheat grains were investigated by ferric reducing antioxidant power (FRAP), Trolox equivalent antioxidant capacity (TEAC), diphenyl-1-picryl-hydrazyl (DPPH) free radical scavenging ability, and lipid peroxidation assay. Among the six mycelia extracts tested, the ethanol extract from wheat fermented with KUM61076 mycelia showed the most potent antioxidant activities, whereas the ethanol extract of wheat grains fermented with KUM60813 mycelia has a good potential in protecting frying oils against oxidation. Total phenolic content (TPC) in the ethanol extracts were higher than that in the aqueous extract. The wheat grains fermented with G. australe (KUM60813) and G. neo-japonicum KUM61076 have greater antioxidant potential compared to the commercially available G. lucidum (VITA GL). The antioxidant activities of the mycelia extracts had a positive correlation with their phenolic contents. Thus phenolic compounds may play a vital role in the antioxidant activities of the selected Ganoderma spp.
The PreS domain of hepatitis B virus (HBV) is believed to be involved in virion assembly and attachment to a hepatocyte receptor during infection. In order to study the functions of this region, we fused it to the g3p protein of bacteriophage M13 that allows the fusion protein to be displayed at the tip of the filament. The fusion protein was detected by the anti-E tag antibody on a Western blot. The polypeptide in a soluble form was produced by transfecting a non-suppressor E. coli host cell with the recombinant phagemid. The soluble protein was detected in cytoplasm, in the periplasmic space and also in the medium. The functional display of the PreS domain would provide an alternative means to study its interactions with the nuleocapsid and hepatocytes.
Blastocystis cysts were detected in 38% (47/123) (37 Scottish, 17 Malaysian) of sewage treatment works. Fifty percent of influents (29% Scottish, 76% Malaysian) and 28% of effluents (9% Scottish, 60% Malaysian) contained viable cysts. Viable cysts, discharged in effluent, provide further evidence for the potential for waterborne transmission of Blastocystis.
A potentiometric whole cell biosensor based on immobilized marine bacterium, Pseudomonas carrageenovora producing κ-carrageenase and glycosulfatase enzymes for specific and direct determination of κ-carrageenan, is described. The bacterial cells were immobilized on the self-plasticized hydrogen ion (H+)-selective acrylic membrane electrode surface to form a catalytic layer. Hydrogen ionophore I was incorporated in the poly(n-butyl acrylate) [poly(nBA)] as a pH ionophore. Catalytic decomposition of κ-carrageenan by the bienzymatic cascade reaction produced neoagarobiose, an inorganic sulfate ion and a proton. The latter was detectable by H+ ion transducer for indirect potentiometric quantification of κ-carrageenan concentration. The use of a disposable screen-printed Ag/AgCl electrode (SPE) provided no cleaning requirement and enabled κ-carrageenan detection to be carried out conveniently without cross contamination in a complex food sample. The SPE-based microbial biosensor response was found to be reproducible with high reproducibility and relative standard deviation (RSD) at 2.6% (n = 3). The whole cell biosensor demonstrated a broad dynamic linear response range to κ-carrageenan from 0.2-100 ppm in 20 mM phosphate buffer saline (PBS) at pH 7.5 with a detection limit at 0.05 ppm and a Nernstian sensitivity of 58.78±0.87 mV/decade (R2 = 0.995). The biosensor showed excellent selectivity towards κ-carrageenan compared to other types of carrageenans tested e.g. ι-carrageenan and λ-carrageenan. No pretreatment to the food sample was necessary when the developed whole cell biosensor was employed for direct assay of κ-carrageenan in dairy product.
Studies showed that co-transplantation of mesenchymal stem cells (MSCs) and cord blood-derived CD34+hematopoietic stem cells (HSCs) offered greater therapeutic effects but little is known regarding the effects of human Wharton's jelly derived MSCs on HSC expansion and red blood cell (RBC) generation in vitro. This study aimed to investigate the effects of MSCs on HSC expansion and differentiation. HSCs were co-cultured with MSCs or with 10% MSCs-derived conditioned medium, with HSCs cultured under standard medium served as a control. Cell expansion rates, number of mononuclear cell post-expansion and number of enucleated cells post-differentiation were evaluated. HSCs showed superior proliferation in the presence of MSC with mean expansion rate of 3.5 × 108 ± 1.8 × 107after day 7 compared to the conditioned medium and the control group (8.9 × 107 ± 1.1 × 108and 7.0 × 107 ± 3.3 × 106respectively, P
Mesenchymal stem cells (MSCs) are known to secrete cardioprotective paracrine factors that can potentially activate endogenous cardiac c-kit cells (CCs). This study aims to optimise MSC growth conditions and medium formulation for generating the conditioned medium (CdM) to facilitate CC growth and expansion in vitro. The quality of MSC-CdM after optimisation of seeding density during MSC stabilisation and medium formulation used during MSC stimulation including glucose, ascorbic acid, serum and oxygen levels and the effects of treatment concentration and repeated CdM harvesting were assessed based on CC viability in vitro under growth factor- and serum-deprived condition. Our data showed that functional CdM can be produced from MSCs with a density of 20,000 cells/cm2, which were stimulated using high glucose (25 mM), ascorbic acid supplemented, serum-free medium under normoxic condition. The generated CdM, when applied to growth factor- and serum-deprived medium at 1:1 ratio, improved CC viability, migration and proliferation in vitro. Such an effect could further be augmented by generating CdM concentrates without compromising CC gene and protein expressions, while retaining its capability to undergo differentiation to form endothelial, smooth muscle and cardiomyocytes. Nevertheless, CdM could not be repeatedly harvested from the same MSC culture, as the protein content and its effect on CC viability deteriorated after the first harvest. In conclusion, this study provides a proof-of-concept strategy to standardise the production of CdM from MSCs based on rapid, stepwise assessment of CC viability, thus enabling production of CdM favourable to CC growth for in vitro or clinical applications.
Tioman Island is one of many sources for underexplored actinobacterial diversity in Malaysia. Selective isolation, molecular profiling, 16S rRNA gene sequencing and phylogenetic analyses were carried out to highlight the diversity of the marine actinobacterial community in a sediment collected off Tioman Island. A high number of diverse actinobacteria were recovered using skim milk/HEPES pre-treatment on a mannitol-based medium. A total of 123 actinobacterial strains were isolated, including thirty obligate marine actinobacteria putatively identified as Salinispora spp. Molecular fingerprinting profiles obtained with a double digestion approach grouped the remaining non-Salinispora-like strains into 24 different clusters, with Streptomyces and Blastococcus as the major clusters. A total of 17 strains were identified as novel actinobacterial species within the genera Streptomyces (n = 6), Blastococcus (n = 5), Marinactinospora (n = 3), Nocardiopsis (n = 1), Agromyces (n = 1) and Nonomuraea (n = 1) based on 16S rRNA gene sequence analyses. Polyphasic data from three putative Marinactinospora spp. showed that the strains represent a new genus in the Nocardiopsaceae family. Crude extracts from the strains were also found to inhibit the growth of Gram-positive (Staphylococcus aureus, Bacillus subtilis) and Gram-negative (Providencia alcalifaciens) pathogens. Hierarchical clustering of the bioactivities of an active fraction revealed a unique profile, which is closely related that of fosfomycin.
Cryptococcus albidus and C. laurentii were the predominant non-neoformans cryptococci isolated during an environmental sampling study for C. gattii at Klang Valley, Malaysia. Cryptococcus gattii was not isolated from any of the environmental samples. Cryptococcus albidus and C. laurentii were isolated mainly from vegetative samples of Eucalyptus trees and bird droppings. Upon testing on canavanine-glycine-bromothymol blue (CGB) agar, all the C. albidus isolates remained unchanged. Interestingly, a total of 29 (76.3%) C. laurentii isolates formed blue colours on the CGB agar. Sequence analysis of ITS1-5.8rDNA-ITS2 gene sequences (468 bp) of four CGB-blue C. laurentii isolates demonstrated the closest match (99%) with that of C. laurentii CBS 7140. This study demonstrated the diverse environmental niche of C. albidus and C. laurentii in Malaysia.
A successful protocol was established for micropropagation in two selected varieties of exotic ornamental plants, Calathea crotalifera. The effects of different sterilization techniques, explant type, and the combination and concentration of plant growth regulators on shoots induction were studied. The axillary shoot buds explants sprouted from rhizomes in soil free conditions showed high induction rate of shoots with lowest contamination percentage when treated with combination of 30% (v/v) NaOCl, 70% (v/v) ethanol, and 0.3% (w/v) HgCl2. In the present study, the highest number of multiple shoots was obtained in MS basal medium supplemented with 3.5 mg/L 6-Benzylaminopurine (BAP), 1.0 mg/L 1-Naphthaleneacetic acid (NAA), 3% sucrose, and 6 g/L plant agar for both varieties and was used as multiplication medium. Microshoots were highly induced when the young shoot bud explants were incised longitudinally prior subculture. Chlorophyll analysis was studied to test the effects of activated charcoal and L-glutamine on reduction of necrosis problem. The maximum roots induction was recorded on MS medium supplemented with 1.0 mg/L 1-Naphthaleneacetic acid (NAA) compared to indolebutyric acid (IBA). The complete regenerated plantlets were successfully acclimatized in the soilless medium under greenhouse condition. This is the first report of rapid mass propagation for C. crotalifera.
Intact immature flower buds of African violet (Saintpaulia ionantha H. Wendl.) were used as explant sources for in vitro studies. The effect of exogenous hormones, NAA and BAP on the indirect organogenesis of this species was observed. Callus was formed on the cut end (base) of pedicels of floral buds where they were in contact with the medium. When maintained on the same medium, callus was differentiated into adventitious shoots after 10 weeks in culture. MS media supplemented with 2.0 mg L(-1) NAA and 1.0 mg L(-1) BAP gave the highest number of sterile or vegetative floral buds from the surface of callus of the explants, but these buds failed to develop further. The floral buds were expanded as abnormal flowers. The floral structures were smaller in size compared to intact flowers. Petals (corolla) were white to purple in colour but did not form any reproductive organs, i.e., stamens or pistils. All sterile or vegetative floral buds and abnormal flowers survived for 3 months in culture but failed to reach anthesis.
Gymnema sylvestre (R.Br.) is an important diabetic medicinal plant which yields pharmaceutically active compounds called gymnemic acid (GA). The present study describes callus induction and the subsequent batch culture optimization and GA quantification determined by linearity, precision, accuracy, and recovery. Best callus induction of GA was noticed in MS medium combined with 2,4-D (1.5 mg/L) and KN (0.5 mg/L). Evaluation and isolation of GA from the calluses derived from different plant parts, namely, leaf, stem and petioles have been done in the present case for the first time. Factors such as light, temperature, sucrose, and photoperiod were studied to observe their effect on GA production. Temperature conditions completely inhibited GA production. Out of the different sucrose concentrations tested, the highest yield (35.4 mg/g d.w) was found at 5% sucrose followed by 12 h photoperiod (26.86 mg/g d.w). Maximum GA production (58.28 mg/g d.w) was observed in blue light. The results showed that physical and chemical factors greatly influence the production of GA in callus cultures of G. sylvestre. The factors optimized for in vitro production of GA during the present study can successfully be employed for their large-scale production in bioreactors.