Evaluation of the Single-Use Fixed-Bed Bioreactors in Scalable Virus Production

The accelerating development of gene therapy from research towards clinical trials and beyond has elevated the demand for practical viral vector-manufacturing solutions. The use of disposable upstream technology is gaining traction in clinical manufacturing. Packed-bed or fixed-bed reactors, where column is packed with immobilized biocatalyst particles providing surface to constrain the cells in a particular region of the reactor, have been widely used in bioprocessing applications since mid-1900s. However, the world's first single-use, fully integrated, high cell density, fixed-bed bioreactor was launched only approximately a decade ago. By now, several single-use, fixed-bed technology solutions have been developed in a small scale. Scaling-up the manufacturing can be challenging and for commercial-scale manufacturing, there is practically only one single-use, good manufacturing practice-compliant option available. This study reviews the latest, fully disposable, fixed-bed bioreactors; compares the virus production in the different systems; and discusses important manufacturing cost-related topics. It is predicted that single-use, fixed-bed bioreactors will receive even more attention in the field of viral vector manufacturing and commercialization, especially with the need for higher virus titers and virus yields.     

Kinetic Parameters of Denitrification in a River Continuum

Kinetic parameters for nitrate reduction in intact sediment cores were investigated by using the acetylene blockage method at five sites along the Swale-Ouse river system in northeastern England, including a highly polluted tributary, R. Wiske. The denitrification rate in sediment containing added nitrate exhibited a Michaelis-Menten-type curve. The concentration of nitrate for half-maximal activity (K_map) by denitrifying bacteria increased on passing downstream from 13.1 to 90.4 μM in the main river, but it was highest (640 μM) in the Wiske. The apparent maximal rate (V_maxap) ranged between 35.8 and 324 μmol of N m⁻² h⁻¹ in the Swale-Ouse (increasing upstream to downstream), but it was highest in the Wiske (1,194 μmol N m⁻² h⁻¹). A study of nitrous oxide (N₂O) production at the same time showed that rates ranged from below the detection limit (0.05 μmol of N₂O-N m⁻² h⁻¹) at the headwater site to 27 μmol of N₂O-N m⁻² h⁻¹ at the downstream site. In the Wiske the rate was up to 570 μmol of N₂O-N m⁻² h⁻¹, accounting for up to 80% of total N gas production.     

Usefulness of Kinetic Enzyme Parameters in Biotechnological Practice

The k_cat /K_M ratio, where k_cat is the catalytic constant for the conversion of substrate into product, and K_M is the Michaelis constant, has been widely used as a measure of enzyme performance, but recent analyses have underscored the inadequacy of this ratio to describe the efficiency of a biocatalyst, particularly when employed as a criterion for selecting between enzyme variants for industrial purposes. The main problem with this kinetic relationship is that it neglects the contribution of important factors operating in actual bioprocess conditions, such as substrate concentrations and product inhibition, leading to unreal expectations on enzyme performance and erroneous selection of the most adequate biocatalyst. Two complementary formalisms, the efficiency function and the catalytic effectiveness, have been introduced to incorporate important features of any bio-industrial system. We review herein the rationales underlying each derivation, and the strengths and fields of application of both strategies. Examples of different situations, including continuous and batch-type reactors, as well as reversible and irreversible processes, are provided, together with recommendations on the use of both approaches.     

Study of the Vapor Phase Over Fusarium Fungi Cultured on Various Substrates

The compositions of volatile organic compounds (VOCs) emitted by Fusarium fungi (F. langsethiae, F. sibiricum, F. poae, and F. sporotrichioides) grown on two nutritive substrates: potato sucrose agar (PSA) and autoclaved wheat kernels (WK) were investigated. The culturing of fungi and study of their VOC emissions were performed in chromatographic vials at room temperature (23 – 24 °C) and the VOCs were sampled by a solid‐phase microextraction on a 85 μm carboxen/polydimethylsiloxane fiber. GC/MS was performed using a 60‐m HP‐5 capillary column. Components of the VOC mixture were identified by electron impact mass spectra and chromatographic retention indices (RIs). The most abundant components of the VOC mixture emitted by Fusarium fungi are EtOH, AcOH, ⁱBuOH, 3‐methylbutan‐1‐ol, 2‐methylbutan‐1‐ol, ethyl 3‐methylbutanoate, terpenes with M 136, sesquiterpenes with M 204 (a total of about 25), and trichodiene. It was found that the strains grown on PSA emit a wider spectrum and larger amount of VOCs compared with those grown on wheat kernels. F. langsethiae strain is the most active VOC producer on both substrates. The use of SPME and GC/MS also offers the potential for differentiation of fungal species and strains.     

A Gas Lift Bioreactor for Removal of Contaminants from the Vapor Phase

The cometabolic degradation of trichloroethylene (TCE) as a vapor by two aromatic-metabolizing pseudomonads was evaluated in an airlift reactor. These microorganisms were able to degrade 90 to 95% of TCE in air at concentrations at the reactor inlet of 300 to 4,000 μg/liter. Although exposure of the cells to high inlet concentrations of TCE (4 mg/liter) caused a decline in enzyme-specific activity and TCE removal efficiency, this loss in activity could be prevented or delayed by increasing the rate of cosubstrate addition. Under the appropriate operating conditions, the microorganisms were able to degrade even high concentrations of TCE and activity of the cells in the reactor could be maintained for periods of at least 2 weeks.     

Dynamics of Cell Kinetic Parameters During 9l Spheroid Growth

Abstract. Cell population kinetics were followed in 9L tumour spheroids as they grew from aggregates of about 80 μm in diameter to over 800 μm. the kinetic parameters measured were cell cycle time, spheroid-doubling time, and growth fraction; from these the cell loss factor ø was calculated. the rate of cell shedding from the surface was also measured, so that the contribution of shedding to the overall cell loss could be evaluated. the major findings include significant elongation of the cell cycle, a low rate of cell death in spheroids below 500 μ in diameter, and a relatively high GF in large spheroids. the results also indicated that 9L spheroid kinetic parameters may be strongly influenced by the culture methodology.     

Packed-Bed Bioreactors Under the Influence of Chemical Modulators

This report considers the behaviour of packed-bed immobilized enzyme reactors operating in the presence of substrate and/or product sequestrators. In some cases, enzyme inhibition by the reaction product and presence of chemical modulators are also considered. For each case, an appropriate analytical model is developed. Using numerical simulations, it is shown that reactor performance is impaired by substrate sequestration. This effect can be partially reversed when competitive sequestration by product or modulator is operational.

In addition, a comparison is made between some of the predicted characteristics of the reactor and experimental data. It reveals the capabilities and limitations of the models employed.     

Packed-Bed Bioreactors Under the Influence of Chemical Modulators

This report considers the behaviour of packed-bed immobilized enzyme reactors operating in the presence of substrate and/or product sequestrators. In some cases, enzyme inhibition by the reaction product and presence of chemical modulators are also considered. For each case, an appropriate analytical model is developed. Using numerical simulations, it is shown that reactor performance is impaired by substrate sequestration. This effect can be partially reversed when competitive sequestration by product or modulator is operational.

In addition, a comparison is made between some of the predicted characteristics of the reactor and experimental data. It reveals the capabilities and limitations of the models employed.     

Adsorption of the Vapor Phase Components in Cigarette Smoke by Activated Carbon in Vented Filters

Using vented charcoal filters, the adsorption efficiencies of acetaldehyde, isoprene and acetone, the major components in the vapor phase of cigarette smoke, were studied. Filter ventilation was found to raise the adsorption efficiency of the adsorbent. The effect of increasing the ventilation rate through the filter was greatest for the adsorption of acetaldehyde. In order to clarify the effects of decreases of the flow rate and the concentration caused by ventilation, the adsorption by unvented charcoal filters under varied conditions was also measured. Although both raised the adsorptions of the three components, the lowered concentration was contributed to mainly by an increase of adsorption by the vented charcoal filters. Regardless of whether the filter was perforated or not, the adsorptions of the three components depended on the volume of the air drawn in at the top of the lighted end of the cigarette.     

Mathematical Model of Acute Myeloblastic Leukaemia: an Investigation of the Relevant Kinetic Parameters

A simple model of acute myeloblastic leukaemia (AML) development is introduced, explicitly including cell growth, cell differentiation and cell-cell interaction. Each of these processes is described by a single model parameter. It is hypothesized that the leukaemic cell is characterized by an alteration of only one of these processes. the kinetic behaviour of the AML system is examined separately for possible alterations of each of the three parameters describing the three processes involved. It is shown that, on the basis of the existing data on AML kinetics, the alteration of the growth and cell-cell interaction parameters can be eliminated as a possible source of AML. Thus kinetic data support the modification of the differentiation process as the origin of the AML state. Further, the growth characteristics of normal and leukaemic cells in the presence of each other are analysed. It is shown how the initial growth of leukaemic cells depends on the difference in the differentiation of normal and leukaemic cells and how the same difference determines the decay of normal cells in the presence of the predominantly leukaemic population. Correlations between the kinetic parameters of the normal and leukaemic populations are suggested to characterize the leukaemic state.     

Thermodynamic Parameters and Influence of Kinetic Factors on the Self-Assembly of Acid-Soluble Collagen Nanofibrils

In this study, the acid-soluble collagen (ASC), extracted from the fish scales of the Caspian white fish (Rutilus Firisikutum) was studied. The thermo-gravimetric analysis (TGA) showed the maximum demineralization accomplished after 48 h of EDTA treatment. SDS-PAGE and FT-IR spectroscopy confirmed that extracted ASC was mainly type I collagen. FE-SEM images confirmed the porous and filamentary structure. The denaturation temperature (T_d) of ASC was 19 °C, and the transition heat achieved 9.6 J/g. Collagen self-assembly exhibit important potential because for biomedical applications and green technologies. Various inter- and intra-molecular no-covalent interactions such as hydrogen bonding, hydrophobic, electrostatic and Van der Waals interactions influence the formation of self-assembled collagen. Therefore, critical factors as concentration of ASC, temperature, pH, and ionic strength play crucial role in function integration and structural modulation. The impacts of those external triggers on the kinetic self-assembly of ASC demonstrated a two-phase kinetic process, a sigmoidal plot. ACS showed pronounced self-assembly behavior when temperature and concentration reach above 14 °C and 0.125 mg/ml, higher concentration and/or temperature could stimulate the ASC self-assembly. The optimum pH value for ASC self-assembly was pH = 7. The effect of ionic strength on ASC self-assembly showed the turbidity increases significantly in 131.2 mM salt concentration. The process of self-assembly is mainly driven by thermodynamics. The thermodynamic study of collagen self-assembly illustrated that the activation energy, E_a = 44.3 kJ/mol, the frequency factor, A = 117 × 10⁵ s^?1, the enthalpy transition, ΔH^? = 42.98 kJ/mol, and the entropy transition, ΔS^? = ?0.12 kJ/mol.K, respectively. These findings show that kinetics factors not only influence the self-assembly structure of ASC but also regulate the activation complex structure in the transition state.     

Influence of Process Parameters on Tablet Bed Microenvironmental Factors During Pan Coating

Recent studies have shown the importance of monitoring microenvironmental conditions (temperature, relative humidity) experienced by the tablet bed during a pan coating process, thereby necessitating the need to understand how various process parameters influence these microenvironmental conditions. The process parameters studied in this work include exhaust air temperature, spray rate, inlet airflow rate, gun-to-bed distance, coating suspension percent solids, and atomization and pattern air pressure. Each of these process parameters was found to have an impact on the tablet bed relative humidity (RH), as measured using PyroButton data logging devices. A higher tablet bed RH was obtained with an increase in spray rate and atomization air pressure and with a decrease in exhaust air temperature, inlet airflow rate, gun-to-bed distance, suspension percent solids, and pattern air pressure. Based on this work, it can be concluded that the tablet bed thermodynamic conditions are a cumulative effect of the various process conditions. A strong correlation between the tablet bed RH and the frequency of tablet coating defect (logo bridging) was established, with increasing RH resulting in a higher percent of logo bridging events.     

Kinetic Parameters of the Conversion of Methane Precursors to Methane in a Hypereutrophic Lake Sediment

The kinetic parameters K_m, V_max, T_t (turnover time), and v (natural velocity) were determined for H₂ and acetate conversion to methane by Wintergreen Lake sediment, using short-term (a few hours) methods and incubation temperatures of 10 to 14°C. Estimates of the Michaelis-Menten constant, K_m, for both the consumption of hydrogen and the conversion of hydrogen to methane by sediment microflora averaged about 0.024 μmol g⁻¹ of dry sediment. The maximal velocity, V_max, averaged 4.8 μmol of H₂ g⁻¹ h⁻¹ for hydrogen consumption and 0.64 μmol of CH₄ g⁻¹ h⁻¹ for the conversion of hydrogen to methane during the winter. Estimated natural rates of hydrogen consumption and hydrogen conversion to methane could be calculated from the Michaelis-Menten equation and estimates of K_m, V_max, and the in situ dissolved-hydrogen concentration. These results indicate that methane may not be the only fate of hydrogen in the sediment. Among several potential hydrogen donors tested, only formate stimulated the rate of sediment methanogenesis. Formate conversion to methane was so rapid that an accurate estimate of kinetic parameters was not possible. Kinetic experiments using [2-¹⁴C]acetate and sediments collected in the summer indicated that acetate was being converted to methane at or near the maximal rate. A minimum natural rate of acetate conversion to methane was estimated to be about 110 nmol of CH₄ g⁻¹ h⁻¹, which was 66% of the V_max (163 nmol of CH₄ g⁻¹ h⁻¹). A 15-min preincubation of sediment with 5.0 × 10⁻³ atm of hydrogen had a pronounced effect on the kinetic parameters for the conversion of acetate to methane. The acetate pool size, expressed as the term K_m + S_n (S_n is in situ substrate concentration), decreased by 37% and T_t decreased by 43%. The V_max remained relatively constant. A preincubation with hydrogen also caused a 37% decrease in the amount of labeled carbon dioxide produced from the metabolism of [U-¹⁴C]valine by sediment heterotrophs.     

Kinetic and Morphological Observations on the Yeast Phase of Histoplasma capsulatum During Protoplast Formation

Protoplast formation by Histoplasma capsulatum yeasts using high concentrations of MgSO(4) occurs either by lysis of the bud or lysis of the entire cell wall. Both mechanisms may also occur simultaneously. Neither the protoplast emerging through a hole in the cell wall nor the freshly released protoplast has a recognizable cell wall or the remnant of such. The protoplast contains all the organelles of the normal cell except for mesosomes. During protoplast formation the nucleus increases in size and produces several nuclear masses by the invaginations of the internal layer of the nuclear membrane. All these nuclear masses are surrounded by the external layer of the nuclear membrane. Several nuclei with a normal nuclear membrane are formed later.     

Perception of Elasticity in the Kinetic Illusory Object with Phase Differences in Inducer Motion

Background

It is known that subjective contours are perceived even when a figure involves motion. However, whether this includes the perception of rigidity or deformation of an illusory surface remains unknown. In particular, since most visual stimuli used in previous studies were generated in order to induce illusory rigid objects, the potential perception of material properties such as rigidity or elasticity in these illusory surfaces has not been examined. Here, we elucidate whether the magnitude of phase difference in oscillation influences the visual impressions of an object''s elasticity (Experiment 1) and identify whether such elasticity perceptions are accompanied by the shape of the subjective contours, which can be assumed to be strongly correlated with the perception of rigidity (Experiment 2).

Methodology/Principal Findings

In Experiment 1, the phase differences in the oscillating motion of inducers were controlled to investigate whether they influenced the visual impression of an illusory object''s elasticity. The results demonstrated that the impression of the elasticity of an illusory surface with subjective contours was systematically flipped with the degree of phase difference. In Experiment 2, we examined whether the subjective contours of a perceived object appeared linear or curved using multi-dimensional scaling analysis. The results indicated that the contours of a moving illusory object were perceived as more curved than linear in all phase-difference conditions.

Conclusions/Significance

These findings suggest that the phase difference in an object''s motion is a significant factor in the material perception of motion-related elasticity.     

气垫床在预防压疮中的效果评价

目的：探讨气垫床在预防压疮中的应用,为减少住院患者压疮的发生提供科学依据,提高患者生存质量。方法：选择我院2011年6月-2012年6月住院患者116例为研究对象,对患者进行褥疮风险评估后将116例患者随机分为两组,一组患者使用气垫床,另一组患者普通褥疮预防和护理,对两组患者发生褥疮的情况进行比较。结果：普通组患者发生压疮26例,对照组患者发生压疮5例,经卡方比较,对照组好于普通组,P〈0.05。结论：气垫床的使用不仅可以很好的杜绝压疮的发生,还可以提高患者的生存质量。     

Rate Equations and Kinetic Parameters of the Reactions Involved in Pyrite Oxidation by Thiobacillus ferrooxidans

Rate equations and kinetic parameters were obtained for various reactions involved in the bacterial oxidation of pyrite. The rate constants were 3.5 μM Fe²⁺ per min per FeS₂ percent pulp density for the spontaneous pyrite dissolution, 10 μM Fe²⁺ per min per mM Fe³⁺ for the indirect leaching with Fe³⁺, 90 μM O₂ per min per mg of wet cells per ml for the Thiobacillus ferrooxidans oxidation of washed pyrite, and 250 μM O₂ per min per mg of wet cells per ml for the T. ferrooxidans oxidation of unwashed pyrite. The K_m values for pyrite concentration were similar and were 1.9, 2.5, and 2.75% pulp density for indirect leaching, washed pyrite oxidation by T. ferrooxidans, and unwashed pyrite oxidation by T. ferrooxidans, respectively. The last reaction was competitively inhibited by increasing concentrations of cells, with a K_i value of 0.13 mg of wet cells per ml. T. ferrooxidans cells also increased the rate of Fe²⁺ production from Fe³⁺ plus pyrite.