A simple method for quantification of interferon- &alfa;2b through surface plasmon resonance technique

A rapid and efficient immunoassay method for quantification of interferon-2b using surface plasmon resonance was developed with BIAcore 3000 as a sensor. Two different levels of anti-interferon monoclonal antibody were immobilized onto a CM5 chip using an amine coupling method. Similarbinding ratio was observed for both the ligand densities. There was no steric hindrance and loss of antibody activity even at higher ligand density (> 22,000 RU). The sensitivity of the assay was increased up to 45% with the increment in ligand density from 15,400 to 22,360 RU. The binding betweeninterferon-2b and anti-interferon monoclonal antibody was predominantly controlled by mass transfer rate and the relationship was found linear, ranged from 5 to 400 ng/mL. Total cycle time per analysis was less than 8 min and required only 5 L of sample injection

Application of a high density adsorbent in expanded bed adsorption of lipase from Burkholderia pseudomallei

The application of STREAMLINE Direct HST adsorbent in expanded bed adsorption of lipase from Burkholderia pseudomallei was explored in this study. Scouting of optimum binding and elution condition was performed in batch binding mode. The addition of 0.2 M salt in acetate buffer (pH 5)during adsorption has increased the specificity and quantity of lipase binding onto the adsorbent. The addition of 0.4 M salt in phosphate buffer (pH 7) achieved the highest purification fold (2.5) in elution. The high density of the adsorbent allowed the EBA to be operated at linear velocity as high as 657 cm/h with feedstock containing 4.5% (w/v) wet biomass. The Richardson-Zaki correlation obtained for this EBA system at the presence of 4.5% (w/v) wet biomass is 5.14, a value closed to the laminar flow regime of 4.8, demonstrated that a stable bed is achieved under this operating condition. Meanwhile, a flow velocity of 343 cm/h with bed expansion of 3.2 gave highest dynamic binding capacity (4979.28 U/ml)and productivity (61.52 U/ml.min) for this EBA operation. It also demonstrated that biomass concentration up to 4.5% (w/v) wet weight showed slightly drop of sorption efficiency (0.82) compared to lower biomass concentration (0.94). Further increase of biomass concentration above 4.5% (w/v) wet weight has greatly decreased the equilibrium and dynamic capacity. Application of high density adsorbent tolerated to high density and biomass has reduced the processing time and increased theproductivity

Effect of promoter strength and signal sequence on the periplasmic expression of human interferon- &#9452b in Escherichia coli

Two plasmids, pFLAG-ATS and pET 26b(+), were studied for the periplasmic expression of recombinant human interferon-2b (IFN-2b) in Escherichia coli. The pFLAG-ATS contains ompA signal sequence and tac promoter while pET 26b(+) contains pelB signal sequence and T7lac promoter. It was observedthat periplasmic expression of IFN-2b from pET 26b(+) was around 3000 times higher than pFLAGATS. Difference in the expression level was attributed to the difference in the promoters and the signal sequences. In silico analysis of mRNA secondary structures were analyzed using Vienna RNA packageand MFOLD. The results suggested that the increase of expression would mainly due to the difference in the translation initiation associated with secondary structure of mRNA transcribed by both plasmids

The influence of external factors on bacteriophages—review

The ability of bacteriophages to survive under unfavorable conditions is highly diversified. We summarize the influence of different external physical and chemical factors, such as temperature, acidity, and ions, on phage persistence. The relationships between a phage’s morphology and its survival abilities suggested by some authors are also discussed. A better understanding of the complex problem of phage sensitivity to external factors may be useful not only for those interested in pharmaceutical and agricultural applications of bacteriophages, but also for others working with phages

Human cytomegalovirus latency-associated proteins elicit immune-suppressive IL-10 producing CD4⁺ T cells.

Human cytomegalovirus (HCMV) is a widely prevalent human herpesvirus, which, after primary infection, persists in the host for life. In healthy individuals, the virus is well controlled by the HCMV-specific T cell response. A key feature of this persistence, in the face of a normally robust host immune response, is the establishment of viral latency. In contrast to lytic infection, which is characterised by extensive viral gene expression and virus production, long-term latency in cells of the myeloid lineage is characterised by highly restricted expression of viral genes, including UL138 and LUNA. Here we report that both UL138 and LUNA-specific T cells were detectable directly ex vivo in healthy HCMV seropositive subjects and that this response is principally CD4⁺ T cell mediated. These UL138-specific CD4⁺ T cells are able to mediate MHC class II restricted cytotoxicity and, importantly, show IFNγ effector function in the context of both lytic and latent infection. Furthermore, in contrast to CDCD4⁺ T cells specific to antigens expressed solely during lytic infection, both the UL138 and LUNA-specific CD4⁺ T cell responses included CD4⁺ T cells that secreted the immunosuppressive cytokine cIL-10. We also show that cIL-10 expressing CD4⁺ T-cells are directed against latently expressed US28 and UL111A. Taken together, our data show that latency-associated gene products of HCMV generate CD4⁺ T cell responses in vivo, which are able to elicit effector function in response to both lytic and latently infected cells. Importantly and in contrast to CD4⁺ T cell populations, which recognise antigens solely expressed during lytic infection, include a subset of cells that secrete the immunosuppressive cytokine cIL-10. This suggests that HCMV skews the T cell responses to latency-associated antigens to one that is overall suppressive in order to sustain latent carriage in vivo

Semi-continuous anaerobic treatment of fresh leachate from municipal solid waste transfer station

A semi-continuous leachate treatment process was developed in the present study. The fresh leachate was obtained from a municipal solid waste transfer station and palm oil mill effluent (POME) sludge wasused as sources of anaerobic microbial complex. The semi-continuous treatment of leachate was operated in two phases; in Phase 1, the pH of the bioreactor was not adjusted, and in Phase 2, the pH ofthe bioreactor was adjusted by the addition of sodium hydroxide (NaOH). The initial values for both chemical oxygen demand (COD) and biochemical oxygen demand (BOD5) of fresh leachate wereextremely high compared with the stabilized landfill leachate. COD reduction rate for the semicontinuous process for Phase 1 and 2 were 37 and 52.7%, respectively. These results clearly showedthat pH adjustment is important to enhance the COD removal in leachate treatment. In addition, we have analysed the evolution of volatile fatty acid (VFA) in the entire treatment process. The results indicated that the VFA concentration was a rapid indicator of the reactor’s stability

Bcl-2 mediated suppression of apoptosis in myeloma NS0 cultures

The influence of Bcl-2 expression on the suppression of apoptosis during the cultivation of an NS0 cell line expressing a chimeric antibody was investigated. Following selection of transfectants in medium containing G418, Western analysis revealed evidence of some up-regulation of endogenous Bcl-2 expression even in the control vector transfectants. Cultivation of the two cell lines in suspension batch cultures clearly demonstrated the enhanced robustness of the bcl-2 vector transfected cells. Suppression of apoptosis resulted in an approximately 20% increase in maximum viable cell number, and a doubling in culture duration compared to the control transfected cells. However, despite the significant affect on viability, Bcl-2 expression did not result in an increase in final antibody titre in comparison with the control cell line. Exposure of cells to various nutrient limited conditions further emphasised the influence of Bcl-2 on cell survival. After 3 days of exposure to serum, glucose, glutamate and asparagine deprivation, the viable cell number and viability were significantly higher in the bcl-2 transfected cell line. When control cells were deprived of all amino acids, there was a complete loss of viability and viable cell number within 3 days. By contrast, the bcl-2 transfected cell line retained greater than 75% of the initial viable cell number and about 70% viability. In response to exposure to 8 mM thymidine (a cytostatic agent) the control cell line underwent complete loss of viability and viable cell number after 6 days. This compared with 18 days for complete loss of viability in the bcl-2 transfected cell line. As under batch culture conditions, there was no difference between the two cell lines in final antibody titre, which indicated that MAb synthesis is limited by nutrient availability during the latter stages of culture in both cases. When fed batch cultures were carried out using a concentrated essential amino acid feed, the bcl-2 cell line exhibited a 60% increase in maximum viable cell number and a 50% increase in culture duration, when compared to the control cell line. Moreover, the bcl-2 cell line exhibited a greater than 40% increase in maximum antibody titre. (C) 2000 Elsevier Science B.V. All rights reserved

Influence of Bcl-2 on cell death during the cultivation of a Chinese hamster ovary cell line expressing a chimeric antibody

The influence of Bcl-2 expression on the robustness of a CHO cell line (22H11) developed for the industrial production of a chimeric antibody was evaluated. Western blot analysis following transfection with the expression vector unexpectedly revealed upregulation of endogenous Bcl-2 expression in the control (Neo) cell line in response to exposure to the selection drug G418. This indicated that geneticin may function by inducing apoptosis in cells not carrying the control plasmid or expressing very low levels of survival genes. Thus, exposure to the drug enriched the culture for a population of cells which expressed enhanced levels of endogenous Bcl-2. In batch cultures, ectopic bcl-2 expression resulted in a 75% increase in maximum viable cell density over control cultures. Moreover, the rate of decrease in viability in the Bcl-2 cultures was significantly lower than that in the control cultures. After 18 days, the Bcl-2 viability was around 90%, compared to 20% in the control cultures. Evaluation of the mechanism of cell death revealed very few cells with classical apoptotic morphology. Around 10% were clearly necrotic, but the majority of dead cells were seen as chromatin free but otherwise relatively intact structures. Because of the relatively low rate of cell death in both cell lines, few cells were observed in the transitional, easily identifiable early stages of apoptosis. However, DNA gel electrophoresis revealed a clear ladder-pattern, but only in the control cultures, thus confirming high levels of apoptotic death. Antibody concentrations during both sets of cultures were very similar, both during the growth and death phases, with a maximum titer of around 40 mu g/ml. Analysis of Bcl-2 expression by flow cytometry revealed that the cultures contained two populations of cells: a large population which expressed high levels of Bcl-2 and a relatively smaller low-expressing population. During the course of the batch, the smaller, low-expressing population declined in frequency, suggesting that these cells were more sensitive to cell death. In addition, the mean level of Bcl-2 expression in the overexpressing population also declined significantly, presumably reflecting the exhaustion of precursors for protein synthesis following nutrient depletion. Importantly, when cells were taken from day 40 of the significantly extended Bcl-2 batch cultures, they immediately proliferated, confirming that they had retained their replicative potential. Cultivation of the cells in basal medium lacking (individually) serum, all amino acids, glutamate/asparagine, and, finally, glucose, resulted in relatively lower viable cell numbers and viability in the control cell line compared to the Bcl-2 cell line. Exposure of cells to ammonia toxicity also revealed the relative robustness of the bcl-2 transfected cells. When growth was arrested by treatment with 4 mM thymidine, Bcl-2 overexpressing cells exhibit a viability of over 80% after 5 days in culture, compared to only 40% in the control cell line. However, under growth-arrested conditions, there was no major difference in antibody titer between the two cell lines. (C) 2000 John Wiley & Sons, Inc

Physicochemical stability of calcium-alginate beads immobilizing TiO2 nanoparticles for removal of cationic dye under UV irradiation

Recently, there have been considerable interests to immobilize photocatalyst in alginate beads for removing pollutants from water sources. However, the feasibility of using alginate beads in industry largely depends on its long-term stability during operation. This study investigated the physicochemical stability of alginate/titanium dioxide beads (Alg/TiO2) when exposed to UV irradiation in aqueous environment. The degradation of Alg/TiO2 beads was evident because the diameter and mass of the beads was reduced by 12% and 40%, respectively, after 120 h of irradiation. A substantial amount of TiO2 was leached into the external medium. Consequently, the removal efficiency of model cationic dye was found to reduce after every process cycle. Morphological analysis showed the formation of cavities on the surface of the Alg/TiO2 beads. Interestingly, the blank alginate beads degraded more rapidly than the Alg/TiO2 beads, confirming the UV shielding effect of TiO2. Nevertheless, this study reveals the need to improve the UV stability of alginate-based beads before they can be considered for practical application. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017, 133, 45002