Characterization of TAP Ambr 250 disposable bioreactors,as a reliable scale‐down model for biologics process development

Demands for development of biological therapies is rapidly increasing, as is the drive to reduce time to patient. In order to speed up development, the disposable Automated Microscale Bioreactor (Ambr 250) system is increasingly gaining interest due to its advantages, including highly automated control, high throughput capacity, and short turnaround time. Traditional early stage upstream process development conducted in 2 ‐ 5 L bench‐top bioreactors requires high foot‐print, and running cost. The establishment of the Ambr 250 as a scale‐down model leads to many benefits in process development. In this study, a comprehensive characterization of mass transfer coefficient (k_La) in the Ambr 250 was conducted to define optimal operational conditions. Scale‐down approaches, including dimensionless volumetric flow rate (vvm), power per unit volume (P/V) and k_La have been evaluated using different cell lines. This study demonstrates that the Ambr 250 generated comparable profiles of cell growth and protein production, as seen at 5‐L and 1000‐L bioreactor scales, when using k_La as a scale‐down parameter. In addition to mimicking processes at large scales, the suitability of the Ambr 250 as a tool for clone selection, which is traditionally conducted in bench‐top bioreactors, was investigated. Data show that cell growth, productivity, metabolite profiles, and product qualities of material generated using the Ambr 250 were comparable to those from 5‐L bioreactors. Therefore, Ambr 250 can be used for clone selection and process development as a replacement for traditional bench‐top bioreactors minimizing resource utilization during the early stages of development in the biopharmaceutical industry. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:478–489, 2017     

Are inbred offspring less fit? Survival in a natural population of Mexican jays

The importance of inbreeding avoidance for the fitness of individualsis currently undergoing reevahation. Especially when the frequencyof inbreeding is low, it may be difficult to accumulate sufficientinformation. In a long-term study of the Mexican jay (Aphelocomaultamarina) in Arizona, we found inbreeding, as determined bypedigree, to be rare even though this species exhibits strongnatal philopatry and commonly lives in social groups of closerelatives. Brood sizes of inbred pairs were significantly lowerthan those of outbred pairs, suggesting hatching failure; butthere was no difference in die probability that one or moreyoung would fledge from a nest. Survival of inbred nestlingsto die next year was significantly lower than that of outbrednestlings.     

Responses of epidermal cell turgor pressure and photosynthetic activity of leaves of the atmospheric epiphyte Tillandsia usneoides (Bromeliaceae) after exposure to high humidity

It has been well-established that many epiphytic bromeliads of the atmospheric-type morphology, i.e., with leaf surfaces completely covered by large, overlapping, multicellular trichomes, are capable of absorbing water vapor from the atmosphere when air humidity increases. It is much less clear, however, whether this absorption of water vapor can hydrate the living cells of the leaves and, as a consequence, enhance physiological processes in such cells. The goal of this research was to determine if the absorption of atmospheric water vapor by the atmospheric epiphyte Tillandsia usneoides results in an increase in turgor pressure in leaf epidermal cells that subtend the large trichomes, and, by using chlorophyll fluorescence techniques, to determine if the absorption of atmospheric water vapor by leaves of this epiphyte results in increased photosynthetic activity. Results of measurements on living cells of attached leaves of this epiphytic bromeliad, using a pressure probe and of whole-shoot fluorescence imaging analyses clearly illustrated that the turgor pressure of leaf epidermal cells did not increase, and the photosynthetic activity of leaves did not increase, following exposure of the leaves to high humidity air. These results experimentally demonstrate, for the first time, that the absorption of water vapor following increases in atmospheric humidity in atmospheric epiphytic bromeliads is mostly likely a physical phenomenon resulting from hydration of non-living leaf structures, e.g., trichomes, and has no physiological significance for the plant's living tissues.     

A modified metabolic model for mixed culture fermentation with energy conserving electron bifurcation reaction and metabolite transport energy

A modified metabolic model for mixed culture fermentation (MCF) is proposed with the consideration of an energy conserving electron bifurcation reaction and the transport energy of metabolites. The production of H₂ related to NADH/NAD⁺ and Fdred/Fdox is proposed to be divided in three processes in view of energy conserving electron bifurcation reaction. This assumption could fine‐tune the intracellular redox balance and regulate the distribution of metabolites. With respect to metabolite transport energy, the proton motive force is considered to be constant, while the transport rate coefficient is proposed to be proportional to the octanol–water partition coefficient. The modeling results for a glucose fermentation in a continuous stirred tank reactor show that the metabolite distribution is consistent with the literature: (1) acetate, butyrate, and ethanol are main products at acidic pH, while the production shifts to acetate and propionate at neutral and alkali pH; (2) the main products acetate, ethanol, and butyrate shift to ethanol at higher glucose concentration; (3) the changes for acetate and butyrate are following an increasing hydrogen partial pressure. The findings demonstrate that our modified model is more realistic than previous proposed model concepts. It also indicates that inclusion of an energy conserving electron bifurcation reaction and metabolite transport energy for MCF is sound in the viewpoint of biochemistry and physiology. Biotechnol. Bioeng. 2013; 110: 1884–1894. © 2013 Wiley Periodicals, Inc.     

Influence of the conservative force on transport coefficients in the DPD method

The transport coefficients of a dissipative particle dynamics system are investigated numerically taking into account the conservative force. The influence of the conservative force parameter on kinetic and dissipative viscosity is considered and compared with theoretical predictions. The analytical solution of the potential term that arises from the conservative force is generally very complicated; therefore, this term has been ignored in most previous work and deemed negligible. In the present work, an expression for the effect of the conservative force on potential viscosity is semi-empirically obtained revealing the dependence of the latter on various parameters such as conservative force strength, density number and temperature. This relation offers a proper approximation of conservative force impact on potential viscosity.     

Length-weight relationship of 16 fish species from the Negro River basin (Amazonas state,Brazil)

This study aimed to estimate the parameters of length-weight relationship for 16 freshwater fish species that inhabit the Aracá and Demeni Rivers, which are two tributaries of the Negro River (Amazonas state, Brazil). The fishes were sampled using gillnets (15 × 2 m, mesh size 30–120 mm between opposite knots), during 6 days each year, in April 2016, November 2018 and April 2019. The gillnets were placed in the river twice each day; in the morning (5–9 a.m.) and in the evening (5–9 p.m.), with a standardized time of 4 hr. Measurements were taken for standard length (SL – 0.1 cm precision) and total weight (TW – 0.01 g precision). The parameters a and b of the equation W_T = a.L_T^b were estimated. This study also provides new data on of maximum lengths for six species, namely, Argonectes longiceps, Hemiodus atranalis, Chalceus erythrurus, Astrodoras asterifrons, Trachydoras steindachneri and Acarichthys heckelii.     

Artificial neural network and response surface methodology: a comparative analysis for optimizing rice straw pretreatment and saccharification

Abstract

The present study demonstrates a comparative analysis between the artificial neural network (ANN) and response surface methodology (RSM) as optimization tools for pretreatment and enzymatic hydrolysis of lignocellulosic rice straw. The efficacy for both the processes, that is, pretreatment and enzymatic hydrolysis was evaluated using correlation coefficient (R²) & mean squared error (MSE). The values of R² obtained by ANN after training, validation, and testing were 1, 0.9005, and 0.997 for pretreatment and 0.962, 0.923, and 0.9941 for enzymatic saccharification, respectively. On the other hand, the R² values obtained with RSM were 0.9965 for cellulose recovery and 0.9994 for saccharification efficiency. Thus, ANN and RSM together successfully identify the substantial process conditions for rice straw pretreatment and enzymatic saccharification. The percentage of error for ANN and RSM were 0.009 and 0.01 for cellulose recovery and for 0.004 and 0.005 for saccharification efficiency, respectively, which showed the authority of ANN in exemplifying the non-linear behavior of the system.     

Molecular modelling of the mass density of single proteins

Using molecular dynamics (MD) simulations, the density of single proteins and its temperature dependence was modelled starting from the experimentally determined protein structure and a generic, transferable force field, without the need of prior parameterization. Although all proteins consist of the same 20 amino acids, their density in aqueous solution varies up to 10% and the thermal expansion coefficient up to twofold. To model the protein density, systematic MD simulations were carried out for 10 proteins with a broad range of densities (1.32–1.43?g/cm³) and molecular weights (7–97?kDa). The simulated densities deviated by less than 1.4% from their experimental values that were available for four proteins. Further analyses of protein density showed that it can be essentially described as a consequence of amino acid composition. For five proteins, the density was simulated at different temperatures. The simulated thermal expansion coefficients ranged between 4.3 and 7.1?×?10^?4?K^?1 and were similar to the experimentally determined values of ribonuclease-A and lysozyme (deviations of 2.4 and 14.6%, respectively). Further analyses indicated that the thermal expansion coefficient is linked to the temperature dependence of atomic fluctuations: proteins with a high thermal expansion coefficient show a low increase in flexibility at increasing temperature. A low increase in atomic fluctuations with temperature has been previously described as a possible mechanism of thermostability. Thus, a high thermal expansion coefficient might contribute to protein thermostability.     

Phosphorus nutrition of barley, buckwheat and rape seedlings. II. Influx and efflux of phosphorous by intact roots of different P status

Seedlings of barley (Hordeum vulgare L. cvs Salka and Zita), buckwheat (Fagopyrum esculentum Moench) and rape (Brassica napus L. ssp. napus cv. Line) were raised at 8 or 10 different extenral P concentrations in the range 0–2000 μM. Apart from P, the nutrient solutions were complete. Phosphate influx in roots of different P status was determined by use of a nutrient solution containing 0.1 mM³²P-labelled phosphate. A double labelling technique was used for simultaneous determination of influx (³³P) and efflux (³²P) of phosphorus by roots of barley and rape with three selected P levels. Flux determinations were also done in presence of a metabolic uncoupler (2,4-dinitrophenol) and a protein synthesis inhibitor (cycloheximide). Influx of phosphate was maximal at a certin optimal P level of the roots and decreased at both lower and higher P levels. Maximum phosphate influex [μmol (g root)-^?1 h^?1] were: rape 4,4, buckwheat 2.2, barley cv. Salka 1.6, barley cv. Zita 1.5. Both Hill plots and plots of the untransformed decreasing phosphate influx vs root P concentrations above the optimal were linear and had high correlation coefficients. The Hill coefficient varied between -3.1 and -4.2. The decrease of phosphate influx from the maximum to the lowest value at the highest P concentration of the root was 60–70%. Hence, phosphate influex appeared to be regulated through negative feedback by the internal level of phosphorous in the roots. The regulation mechanism seems bascially similar for the three species and may be of an allosteric type. P efflux from roots of low and optimal (with regard to P influx) P status was 15–20% of the simultaneous P influx. Contary to P influx, P efflux increased at high P status and almost eliminated (barley) or halved (rape) net P uptake. 2,4-Dinitrophenol reduced both P influx and P efflux by low P roots and gave linearly increasing P efflux with increasing root P status. This indicates that P efflux partly occurred by counter transport and ion exchange at the uptake sites, partly by passive P efflux along an electrochemical potential gradient. Phosphate influx was not affected by inhibition of barley root growth with cycloheximide, but P efflux increased considerably.     

The genetic consequences of selection in natural populations

The selection coefficient, s, quantifies the strength of selection acting on a genetic variant. Despite this parameter's central importance to population genetic models, until recently we have known relatively little about the value of s in natural populations. With the development of molecular genetic techniques in the late 20th century and the sequencing technologies that followed, biologists are now able to identify genetic variants and directly relate them to organismal fitness. We reviewed the literature for published estimates of natural selection acting at the genetic level and found over 3000 estimates of selection coefficients from 79 studies. Selection coefficients were roughly exponentially distributed, suggesting that the impact of selection at the genetic level is generally weak but can occasionally be quite strong. We used both nonparametric statistics and formal random‐effects meta‐analysis to determine how selection varies across biological and methodological categories. Selection was stronger when measured over shorter timescales, with the mean magnitude of s greatest for studies that measured selection within a single generation. Our analyses found conflicting trends when considering how selection varies with the genetic scale (e.g., SNPs or haplotypes) at which it is measured, suggesting a need for further research. Besides these quantitative conclusions, we highlight key issues in the calculation, interpretation, and reporting of selection coefficients and provide recommendations for future research.