Measurement of radiation attenuation parameters of modified defatted soy flour–soy protein isolate-based mangrove wood particleboards to be used for CT phantom production

Radiation and Environmental Biophysics - For the first time, Rhizophora spp. (Rh. spp.) particleboard phantoms were developed using defatted soy flour (DSF) and soy protein isolate (SPI) modified...     

Host cell protein analysis in therapeutic protein bioprocessing – methods and applications

The analysis of host cell proteins (HCPs) is one of the most important analytical requirements during bioprocess development of therapeutic moieties. In this review, we focus on the comparison of different methods for the analysis of HCPs and how cell lines, fermentation conditions, and unit operations influence HCP distribution during the process chain. Current guidelines typically require reduction of HCPs to the ppm level, depending on the intended use, the route of administration of the product, and the production system. A range of immunospecific and non-specific methods are available that have been globally accepted by regulatory bodies. Immunospecific methods, such as ELISA, are simple to use in routine analysis and can quantify low levels of HCPs when specific antibodies are available. Non-specific methods are more complex; however, they provide a holistic view of the HCP profile and qualitative information of the composition of HCP in the sample. Different methods for the comparison of bioprocessing strategies during scale-up and purification development are compared herein. The methods include immunospecific methods, such as ELISA, western blot, and threshold, and non-specific methods, such as 2D-DIGE and 2D-HPLC combined with MS.     

Plant cell culture technology–harnessing a biological approach for competitive cyclotides production

Cyclotides are naturally occurring mini-proteins that have a cyclic backbone and a knotted arrangement of three disulfide bonds. They are remarkably stable and have a diverse range of therapeutically useful biological activities, including antimicrobial and anti-HIV activity, although their natural function appears to be plant defence agents. Cyclotides are amenable to chemical synthesis; however currently most bioactivity studies have involved the use of peptides extracted from plants. Plant cell culture technology shows promise towards the goal of producing therapeutically active cyclotides in qualities and quantities required for drug development.     

Perfusion mammalian cell culture for recombinant protein manufacturing – A critical review

The manufacturing of recombinant protein is traditionally divided in two main steps: upstream (cell culture and synthesis of the target protein) and downstream (purification and formulation of the protein into a drug substance or drug product). Today, cost pressure, market uncertainty and market growth, challenge the existing manufacturing technologies. Leaders in the field are active in designing the process of the future and continuous manufacturing is recurrently mentioned as a potential solution to address some of the current limitations. This review focuses on the application of continuous processing to the first step of the manufacturing process. Enabling technologies and operation modes are described in the first part. In the second part, recent advances in the field that have the potential to support its successful future development are critically discussed.     

Use of cell cycle analysis to characterise growth and interferon-γ production in perfusion culture of CHO cells

The importance of cell cycle analysis in cell culture development has been widely recognised. Whether such analysis is useful in indicating future performance of high cell density culture is uncertain. Using flow cytometric approach to address this question, we utilised the fraction of cells in the S phase to control specific growth rate and productivity in spin filter perfusion cultures and found a significant increase in the accumulated interferon-γ over that obtained from the nutrient-based controlled fed culture. While a general decrease with time exists in both percentage of S phase cells and specific growth rate, a clear oscillatory behaviour of both parameters is found in perfusion cultures. This revised version was published online in July 2006 with corrections to the Cover Date.     

Validation of a dual LC–HRMS platform for clinical metabolic diagnosis in serum,bridging quantitative analysis and untargeted metabolomics

Mass spectrometry-based metabolomics is a rapidly growing field in both research and diagnosis. Generally, the methodologies and types of instruments used for clinical and other absolute quantification experiments are different from those used for biomarkers discovery and untargeted analysis, as the former requires optimal sensitivity and dynamic range, while the latter requires high resolution and high mass accuracy. We used a Q-TOF mass spectrometer with two different types of pentafluorophenyl (PFP) stationary phases, employing both positive and negative ionization, to develop and validate a hybrid quantification and discovery platform using LC–HRMS. This dual-PFP LC–MS platform quantifies over 50 clinically relevant metabolites in serum (using both MS and MS/MS acquisitions) while simultaneously collecting high resolution and high mass accuracy full scans to monitor all other co-eluting non-targeted analytes. We demonstrate that the linearity, accuracy, and precision results for the quantification of a number of metabolites, including amino acids, organic acids, acylcarnitines and purines/pyrimidines, meets or exceeds normal bioanalytical standards over their respective physiological ranges. The chromatography resolved highly polar as well as hydrophobic analytes under reverse-phase conditions, enabling analysis of a wide range of chemicals, necessary for untargeted metabolomics experiments. Though previous LC–HRMS methods have demonstrated quantification capabilities for various drug and small molecule compounds, the present study provides an HRMS quant/qual platform tailored to metabolic disease; and covers a multitude of different metabolites including compounds normally quantified by a combination of separate instrumentation.     

Platelet lysate: a replacement for fetal bovine serum in animal cell culture?

A new cell culture supplement, platelet lysate, was evaluated with reference to fetal bovine serum (FBS), an established industrial medium for animal cell culture. Chemical and bacteriological profiles were conducted including the presence of platelet-derived growth factor (PDGF). PDGF was detected in the platelet lysate but it was not present in FBS. The platelet lysate medium demonstrated lack of microorganisms, mycoplasma and endotoxins. The platelet lysate was investigated in culture studies (cell growth, viability and product formation) towards a number of target cells including myelomas, hybridomas, hepatocytes, fibroblasts and epithelial cells. In general the platelet lysate medium supported cell growth and maintained viabilities comparable or superior to fetal bovine serum. Productivity studies of antibodies (hybridomas) and transferrin (hepatocytes) showed similar or enhanced production in platelet-derived medium in comparison with FBS. This revised version was published online in July 2006 with corrections to the Cover Date.     

A quantitative approach for understanding small-scale human mesenchymal stem cell culture – implications for large-scale bioprocess development

Human mesenchymal stem cell (hMSC) therapies have the potential to revolutionise the healthcare industry and replicate the success of the therapeutic protein industry; however, for this to be achieved there is a need to apply key bioprocessing engineering principles and adopt a quantitative approach for large-scale reproducible hMSC bioprocess development. Here we provide a quantitative analysis of the changes in concentration of glucose, lactate and ammonium with time during hMSC monolayer culture over 4 passages, under 100% and 20% dissolved oxgen (dO₂), where either a 100%, 50% or 0% growth medium exchange was performed after 72h in culture. Yield coefficients, specific growth rates (h^-1) and doubling times (h) were calculated for all cases. The 100% dO₂ flasks outperformed the 20% dO₂ flasks with respect to cumulative cell number, with the latter consuming more glucose and producing more lactate and ammonium. Furthermore, the 100% and 50% medium exchange conditions resulted in similar cumulative cell numbers, whilst the 0% conditions were significantly lower. Cell immunophenotype and multipotency were not affected by the experimental culture conditions. This study demonstrates the importance of determining optimal culture conditions for hMSC expansion and highlights a potential cost savings from only making a 50% medium exchange, which may prove significant for large-scale bioprocessing.     

Induction of a photomixotrophic plant cell culture of Helianthus annuus and optimization of culture conditions for improved α-tocopherol production

Tocopherols, collectively known as vitamin E, are lipophilic antioxidants, which are synthesized only by photosynthetic organisms. Due to their enormous potential to protect cells from oxidative damage, tocopherols are used, e.g., as nutraceuticals and additives in pharmaceuticals. The most biologically active form of vitamin E is α-tocopherol. Most tocopherols are currently produced via chemical synthesis. Nevertheless, this always results in a racemic mixture of different and less effective stereoisomers because the natural isomer has the highest biological activity. Therefore, tocopherols synthesized in natural sources are preferred for medical purposes. The annual sunflower (Helianthus annuus L.) is a well-known source for α-tocopherol. Within the presented work, sunflower callus and suspension cultures were established growing under photomixotrophic conditions to enhance α-tocopherol yield. The most efficient callus induction was achieved with sunflower stems cultivated on solid Murashige and Skoog medium supplemented with 30 g l^?1 sucrose, 0.5 mg l^?1 of the auxin 1-naphthalene acetic acid, and 0.5 mg l^?1 of the cytokinin 6-benzylaminopurine. Photomixotrophic sunflower suspension cultures were induced by transferring previously established callus into liquid medium. The effects of light intensity, sugar concentration, and culture age on growth rate and α-tocopherol synthesis rate were characterized. A considerable increase (max. 230 %) of α-tocopherol production in the cells was obtained within the photomixotrophic cell culture compared to a heterotrophic cell culture. These results will be useful for improving α-tocopherol yields of plant in vitro cultures.     

Patient-specific in vitro models for hemodynamic analysis of congenital heart disease – Additive manufacturing approach

Non-invasive hemodynamic assessment of total cavopulmonary connection (TCPC) is challenging due to the complex anatomy. Additive manufacturing (AM) is a suitable alternative for creating patient-specific in vitro models for flow measurements using four-dimensional (4D) Flow MRI. These in vitro systems have the potential to serve as validation for computational fluid dynamics (CFD), simulating different physiological conditions. This study investigated three different AM technologies, stereolithography (SLA), selective laser sintering (SLS) and fused deposition modeling (FDM), to determine differences in hemodynamics when measuring flow using 4D Flow MRI. The models were created using patient-specific MRI data from an extracardiac TCPC. These models were connected to a perfusion pump circulating water at three different flow rates. Data was processed for visualization and quantification of velocity, flow distribution, vorticity and kinetic energy. These results were compared between each model. In addition, the flow distribution obtained in vitro was compared to in vivo. The results showed significant difference in velocities measured at the outlets of the models that required internal support material when printing. Furthermore, an ultrasound flow sensor was used to validate flow measurements at the inlets and outlets of the in vitro models. These results were highly correlated to those measured with 4D Flow MRI. This study showed that commercially available AM technologies can be used to create patient-specific vascular models for in vitro hemodynamic studies at reasonable costs. However, technologies that do not require internal supports during manufacturing allow smoother internal surfaces, which makes them better suited for flow analyses.     

Characterization of haemoglobin from Actinorhizal plants – An in silico approach

Plant haemoglobins (Hbs), found in both symbiotic and non-symbiotic plants, are heme proteins and members of the globin superfamily. Hb genes of actinorhizal Fagales mostly belong to the non-symbiotic type of haemoglobin; however, along with the non-symbiotic Hb, Casuarina sp. posses a symbiotic one (symCgHb), which is expressed specifically in infected cells of nodules. A thorough sequence analysis of 26 plant Hb proteins, currently available in public domain, revealed a consensus motif of 29 amino acids. This motif is present in all the members of symbiotic class II Hbs including symCgHb and non-symbiotic Class II Hbs, but is totally absent in Class I symbiotic and non-symbiotic Hbs. Further, we constructed 3D structures of Hb proteins from Alnus and Casuarina through homology modelling and peeped into their structural properties. Structure-based studies revealed that the Casuarina symbiotic haemoglobin protein shows distinct stereochemical properties from that of the other Casuarina and Alnus Hb proteins. It also showed considerable structural similarities with leghemoglobin structure from yellow lupin (pdb id 1GDI). Therefore, sequence and structure analyses point to the fact that symCgHb protein shows significant resemblance to symbiotic haemoglobin found in legumes and may thus eventually play a similar role in shielding the nitrogenase from oxygen as seen in the case of leghemoglobin.     

An untargeted metabolomic approach in the chemotaxonomic assessment of two Salvia species as a potential source of α-bisabolol

α-Bisabolol is a commercially important aroma chemical currently obtained from the Candeia tree (Vanillosmopsis erythropappa). Continuous unsustainable harvesting of the Candeia tree has prompted the urgent need to identify alternative crops as a source of this commercially important sesquiterpene alcohol. A chemotaxonomic assessment of two Salvia species indigenous to South Africa is presented and recommended as a potential source of α-bisabolol. The essential oil obtained by hydrodistillation of the aerial parts was analysed by gas chromatography coupled to mass spectrometry (GC–MS) and mid-infrared spectroscopy (MIRS). Orthogonal projections to latent structures–discriminant analysis (OPLS–DA) were used for multivariate classification of the oils based on GC–MS and MIRS data. Partial least squares (PLS) calibration models were developed on the MIRS data for the quantification of α-bisabolol using GC–MS as the reference method. A clear distinction between Salvia stenophylla and Salvia runcinata oils was observed using OPLS–DA on both GC–MS and MIRS data. The MIR calibration model showed high coefficient of determination (R² = 0.999) and low error of prediction (RMSEP = 0.540%) for α-bisabolol content.     

Network genomics – A novel approach for the analysis of biological systems in the post-genomic era

Network Genomics studies genomics and proteomics foundations of cellular networks in biological systems. It complements systems biology in providing information on elements, their interaction and their functional interplay in cellular networks. The relationship between genomic and proteomic high-throughput technologies and computational methods are described, as well as several examples of specific network genomic application are presented.     

Trends in greenhouse gas emissions from consumption and production of animal food products – implications for long-term climate targets

To analyse trends in greenhouse gas (GHG) emissions from production and consumption of animal products in Sweden, life cycle emissions were calculated for the average production of pork, chicken meat, beef, dairy and eggs in 1990 and 2005. The calculated average emissions were used together with food consumption statistics and literature data on imported products to estimate trends in per capita emissions from animal food consumption. Total life cycle emissions from the Swedish livestock production were around 8.5 Mt carbon dioxide equivalents (CO₂e) in 1990 and emissions decreased to 7.3 Mt CO₂e in 2005 (14% reduction). Around two-thirds of the emission cut was explained by more efficient production (less GHG emission per product unit) and one-third was due to a reduced animal production. The average GHG emissions per product unit until the farm-gate were reduced by 20% for dairy, 15% for pork and 23% for chicken meat, unchanged for eggs and increased by 10% for beef. A larger share of the average beef was produced from suckler cows in cow–calf systems in 2005 due to the decreasing dairy cow herd, which explains the increased emissions for the average beef in 2005. The overall emission cuts from the livestock sector were a result of several measures taken in farm production, for example increased milk yield per cow, lowered use of synthetic nitrogen fertilisers in grasslands, reduced losses of ammonia from manure and a switch to biofuels for heating in chicken houses. In contrast to production, total GHG emissions from the Swedish consumption of animal products increased by around 22% between 1990 and 2005. This was explained by strong growth in meat consumption based mainly on imports, where growth in beef consumption especially was responsible for most emission increase over the 15-year period. Swedish GHG emissions caused by consumption of animal products reached around 1.1 t CO₂e per capita in 2005. The emission cuts necessary for meeting a global temperature-increase target of 2° might imply a severe constraint on the long-term global consumption of animal food. Due to the relatively limited potential for reducing food-related emissions by higher productivity and technological means, structural changes in food consumption towards less emission-intensive food might be required for meeting the 2° target.     

An information-theoretic classification of amino acids for the assessment of interfaces in protein–protein docking

Docking represents a versatile and powerful method to predict the geometry of protein–protein complexes. However, despite significant methodical advances, the identification of good docking solutions among a large number of false solutions still remains a difficult task. We have previously demonstrated that the formalism of mutual information (MI) from information theory can be adapted to protein docking, and we have now extended this approach to enhance its robustness and applicability. A large dataset consisting of 22,934 docking decoys derived from 203 different protein–protein complexes was used for an MI-based optimization of reduced amino acid alphabets representing the protein–protein interfaces. This optimization relied on a clustering analysis that allows one to estimate the mutual information of whole amino acid alphabets by considering all structural features simultaneously, rather than by treating them individually. This clustering approach is fast and can be applied in a similar fashion to the generation of reduced alphabets for other biological problems like fold recognition, sequence data mining, or secondary structure prediction. The reduced alphabets derived from the present work were converted into a scoring function for the evaluation of docking solutions, which is available for public use via the web service score-MI: http://score-MI.biochem.uni-erlangen.de     

Selective growth of a mutant in continuous culture of Bacillus caldolyticus for production of α-amylase

Summary In a continuous culture of Bacillus caldolyticus strain SP, which requires maltose as an inducer for production of -amylase in batch culture, a predominant mutant strain M1 which produced high amounts of -amylase in the absence of maltose in batch culture, developed. The change of cell population from strain SP to strain M1 in maltose-casitone medium was linear with time in the transient state after the change from batch to continuous culture at a dilution rate of 0.17 h^-1, and was completed in about 11 generations of bacterial growth. The dilution rate effect of continuous culture on -amylase activity was almost the same with both strains SP and M1. The maximum -amylase activity of 380 units/ml was observed at an intermediate dilution rate that was 11.5 times higher than -amylase activity at the end of a batch culture using the same medium. It was deduced that the enhancement of -amylase production in continuous culture was attributed partly to the predominant growth of a mutant strain with higher -amylase productivity.     

Overcoming the metabolic burden of protein secretion in Schizosaccharomyces pombe – A quantitative approach using 13C-based metabolic flux analysis

Protein secretion in yeast is generally associated with a burden to cellular metabolism. To investigate this metabolic burden in Schizosaccharomyces pombe, we constructed a set of strains secreting the model protein maltase in different amounts. We quantified the influence of protein secretion on the metabolism applying ¹³C-based metabolic flux analysis in chemostat cultures. Analysis of the macromolecular biomass composition revealed an increase in cellular lipid content at elevated levels of protein secretion and we observed altered metabolic fluxes in the pentose phosphate pathway, the TCA cycle, and around the pyruvate node including mitochondrial NADPH supply. Supplementing acetate to glucose or glycerol minimal media was found to improve protein secretion, accompanied by an increased cellular lipid content and carbon flux through the TCA cycle as well as increased mitochondrial NADPH production. Thus, systematic metabolic analyses can assist in identifying factors limiting protein secretion and in deriving strategies to overcome these limitations.     

Shear-scaling-based approach for irreversible energy loss estimation in stenotic aortic flow – An in vitro study

Today, the functional and risk assessment of stenosed arteries is mostly based on ultrasound Doppler blood flow velocity measurements or catheter pressure measurements, which rely on several assumptions. Alternatively, blood velocity including turbulent kinetic energy (TKE) may be measured using MRI. The aim of the present study is to validate a TKE-based approach that relies on the fact that turbulence production is dominated by the flow’s shear to determine the total irreversible energy loss from MRI scans. Three-dimensional particle tracking velocimetry (3D-PTV) and phase-contrast magnetic resonance imaging (PC-MRI) simulations were performed in an anatomically accurate, compliant, silicon aortic phantom. We found that measuring only the laminar viscous losses does not reflect the true losses of stenotic flows since the contribution of the turbulent losses to the total loss become more dominant for more severe stenosis types (for example, the laminar loss is 0.0094 ± 0.0015 W and the turbulent loss is 0.0361 ± 0.0015 W for the Re_max = 13,800 case, where Re_max is the Reynolds number based on the velocity in the vena-contracta). We show that the commonly used simplified and modified Bernoulli’s approaches overestimate the total loss, while the new TKE-based method proposed here, referred to as “shear scaling” approach, results in a good agreement between 3D-PTV and simulated PC-MRI (mean error is around 10%). In addition, we validated the shear scaling approach on a geometry with post-stenotic dilatation using numerical data by Casas et al. (2016). The shear scaling-based method may hence be an interesting alternative for irreversible energy loss estimation to replace traditional approaches for clinical use. We expect that our results will evoke further research, in particular patient studies for clinical implementation of the new method.     

Comparison of cell disruption methods for determining β-galactosidase activity expressed in animal cells

To determine -galactosidase activity expressed in animal cells, the cells are often disrupted. In this study, four different cell disruption methods (freezing and thawing, sonication, homogenization, and lysis buffer) were compared with regard to their efficiency in the determination of -galactosidase activity. Further, the cell lines' susceptibility to cell disruption methods was investigated by employing three different animal cell lines (HeLa HH, Vero, and HeLa S3 cells) infected by recombinant vaccinia virus (vSC8) expressing -galactosidase gene. Regardless of cell lines used, three different cell disruption methods except homogenization did not show any significant difference in the final -galactosidase activity recovered from the cells. Homogenization was inefficient, and required up to 300 strokes to recover -galactosidase activity fully from HeLa S3 cells. The use of lysis buffer was recommendable because of its convenience. However, if there was no need for an immediate assay, it was convenient to keep cell samples collected during the culture frozen at –20°C until the assay. Since thawing of the cells was enough to recover the -galactosidase, further treatment on the frozen cells after thawing was unnecessary.     

Submerged culture production of chitinase by Trichoderma harzianum in stirred tank bioreactors – the influence of agitator speed

The chitinase fermentation process utilizing chitin as the sole carbon source was investigated in a stirred tank bioreactor. Agitator speed of 224 rpm was found to be most suitable for cell growth as well as for chitinase production. Chitinase yield decreased rapidly at higher agitator speed, while decrease in cell yield at higher agitator speed was not rapid. Probably, mass transfer limitation was predominant in the fermentation process at lower agitator speed. Higher agitator speed appears to reduce chitinase production.