Differential effects on natural killer cell production by membrane‐bound cytokine stimulations

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A computational study of the interaction of organic surfactants with goethite α-FeO(OH) surfaces

We have studied the adsorption of three organic molecules onto different surfaces of goethite α−FeO(OH) using atomistic simulation techniques. New interatomic potentials for the interaction between goethite and the organic molecules were developed. In the majority of cases the organic molecules were found capable of forming a coordinate bond via their carbonyl oxygen atom with a surface iron ion. In addition, weaker hydrogen-bonds were formed between the organic molecules and the surfaces. The largest adsorption energies were obtained for the modes of adsorption where the organic molecules bridged or spanned the periodic grooves or dips present on the goethite surfaces, thus forming several interactions between the molecule and the surface. Among all adsorbates studied, the hydroxamic acid molecule in the eclipsed conformation releases the largest adsorption energy when it interacts with goethite surfaces, followed by the staggered conformations of hydroxyethanal and methanoic acid molecules. The adsorption energies are in the range of −60.0 to −186.4 kJ∙mol−1. Due to the surface structure, as well as the flexibility and size of hydroxamic acid and hydroxyethanal, in most cases these adsorbate molecules lose their planarity with respect to the structure of the isolated molecules. We found that the replacement of pre-adsorbed water by the organic adsorbates is an exothermic process on all the goethite surfaces studied. The removal by sorption onto iron particles of humic and fulvic acids, the major substituents of natural organic matter (NOM) that pollutes aquifers and soils, is corroborated by our calculations of the adsorption of surfactants with the same functional groups to the surfaces of oxidised iron particles

Product quality control strategy development for non-mAb complex modalities by using combinatorial cell engineering and OMICS screening tools

Product quality control without compromising productivity has been a major goal in biotherapeutics process development. The challenge is further increased for new modalities using complex and hybrid protein structures, such as nanobodies and bispecific antibodies. New product-related impurities and unique product quality attribute (PQA) species have been found to accompany these new protein scaffolds, which usually don’t exist in standard mAb production. Undesired attributes include unique patterns of glycosylation, conformation heterogeneity, mis-pairing, and partial molecules. Many of these PQAs are related to protein folding and assembly efficiency inside the cell, which impact post-translational modifications such as disulfide bond formation and glycosylation processes, directly or indirectly. We have identified multiple intracellular causal factors that link some PQAs directly to host cell lineage. To improve understanding and increase options in developing a successful production cell line with desired product quality profile, we have used this information to develop diversified CHO host lineages using both conditioned-culture adaptation and CRISPR genome editing approaches. The resulting CHO hosts showed significant differences in cell growth and recombinant protein production, including productivity and quality attribute profiles. Furthermore, the hosts respond differently to changes in medium components and process conditions. These differences were more significant for complex/ hybrid proteins such as nanobodies and bispecific antibodies. OMICS tools were systematically utilized to identify the evolutionarily significance of genetic and epigenetic variability of individual host cell lineages, which determine the specific PQA profile of the expressed recombinant protein. Overall, our presentation will illustrate the importance of selecting the appropriate host cell line through screening and/or engineering, as part of quality control strategy to obtain the desired recombinant protein PQA profile.

Associations between one-carbon metabolism and valproic acid-induced liver dysfunction in epileptic patients

Valproic acid (VPA) has been widely used as an antiepileptic drug for decades. Although VPA is effective and well-tolerated, long-term VPA treatment is usually associated with hepatotoxicity. However, the underlying mechanisms of VPA-caused hepatotoxicity remain unclear. In this study, a total of 157 pediatric patients with epilepsy were recruited and divided into normal liver function (NLF, 112 subjects) group and abnormal liver function (ABLF, 45 subjects) group. We observed that MTHFR A1298C and MTHFR C677T variants may be linked to VPA-induced liver dysfunction (p = 0.001; p = 0.023, respectively). We also found that the MTHFR A1298C polymorphism was associated with a higher serum Hcy level (p = 0.001) and a lower FA level (p = 0.001). Moreover, the serum Hcy levels was strongly correlated with the GSH and TBARS concentrations (r = −0.6065, P < 0.001; r = 0.6564, P < 0.001, respectively). Furthermore, logistic analysis indicated that MTHFR A1298C/C677T polymorphisms and increased Hcy concentrations may be risk factors for VPA-induced liver dysfunction. These results suggested that individual susceptibility to VPA-induced liver dysfunction may result from MTHFR A1298C/C677T polymorphisms and increased Hcy levels. This study may be helpful for the prevention and guidance of VPA-induced liver dysfunction

Tools and methods for providing assurance of clonality for legacy cell lines

Over the last several years demonstration of cell line clonality has been a topic of many industry and regulatory presentations and papers. Guidance has been provided by the regulatory authorities, especially the FDA, on a path forward for providing evidence of clonality with high probability. It has been recommended that two-rounds of limiting dilution cloning (LDC) at sufficiently low seeding densities (≤0.5 cells/well) provides sufficient evidence that a cell line is clonal. Furthermore, one-round of LDC may also suffice if supplemental data from a characterized FACS or plate-imaging workflow are also included in the package. Cell lines generated by methods that do not demonstrate high probability of clonal derivation, including legacy cell lines, may require additional studies to provide assurance and/or process control strategies to satisfy regulatory expectations. Within the Biologics function of the IQ Consortium the “Clonality” Working Group is focusing on methods and tools which could be utilized to provide a high assurance of clonality for legacy cell lines. The presentation will outline a three tier approach to address legacy cell line clonality assurance: standard practices already used in industry to support limit of in vitro cell age studies, enhanced control strategies to ensure process consistency, and emerging technologies that could be used to further support cell line clonality

A computer modelling study of the uptake, structure and distribution of carbonate defects in hydroxy-apatite

Computer modelling techniques have been employed to qualitatively and quantitatively investigate the uptake and distribution of carbonate groups in the hydroxyapatite lattice. Two substitutional defects are considered: the type-A defect, where the carbonate group is located in the hydroxy channel, and the type-B defect, where the carbonate group is located at the position of a phosphate group. A combined type A–B defect is also considered and different charge compensations have been taken into account. The lowest energy configuration of the A-type carbonate has the O–C–O axis aligned with the channel in the c-direction of the apatite lattice and the third oxygen atom lying in the a/b plane. The orientation of the carbonate of the B-type defect is strongly affected by the composition of the apatite material, varying from a position (almost) flat in the a/b plane to being orientated with its plane in the b/c plane. However, Ca–O interactions are always maximised and charge compensating ions are located near the carbonate ion. When we make a direct comparison of the energies per substitutional carbonate group, the results of the different defect simulations show that the type-A defect where two hydroxy groups are replaced by one carbonate group is energetically preferred View the MathML source, followed by the combined A–B defect, where both a phosphate and a hydroxy group are replaced by two carbonate groups View the MathML source. The type-B defect, where we have replaced a phosphate group by both a carbonate group and another hydroxy group in the same location is energetically neutral View the MathML source, but when the replacement of the phosphate group by a carbonate is charge compensated by the substitution of a sodium or potassium ion for a calcium ion, the resulting type-B defect is energetically favourable View the MathML source and its formation is also promoted by A-type defects present in the lattice. Our simulations suggest that it is energetically possible for all substitutions to occur, which are calculated as ion-exchange reactions from aqueous solution. Carbonate defects are widely found in biological hydroxy-apatite and our simulations, showing that incorporation of carbonate from solution into the hydroxyapatite lattice is thermodynamically feasible, hence agree with experiment

Variations in calcite growth kinetics with surface topography: molecular dynamics simulations and process-based growth kinetics modelling

It is generally accepted that cation dehydration is the rate-limiting step to crystal growth from aqueous solution. Here we employ classical molecular dynamics simulations to show that the water exchange frequency at structurally distinct calcium sites in the calcite surface varies by about two orders of magnitude. The decrease in water exchange frequency with progressive embedding of surface calcium ions is thought to be rate limiting to subsequent attachment of carbonate ions during calcite growth. Therefore, a process-based calcite growth kinetics model, reparameterized using the water exchange frequencies computed from molecular dynamics simulations, is used to illustrate the impact of these variations on kink-formation rate, step velocities and bulk growth rate. The calculated frequencies of kink formation show a strong variation with surface structures, which can be amplified depending on the saturation state and calcium to carbonate ratio of the solution. Modelled and measured step velocities and bulk growth rates are generally in agreement, showing that variations in calcite growth rates and step velocities observed experimentally might be at least partially induced by surface topography

A computational study of the interaction of organic surfactants with goethite α-FeO(OH) surfaces

We have studied the adsorption of three organic molecules onto different surfaces of goethite α-FeO(OH) using atomistic simulation techniques. New interatomic potentials for the interaction between goethite and the organic molecules were developed. In the majority of cases the organic molecules were found to be capable of forming a coordinate bond via their carbonyl oxygen atom with a surface iron ion. In addition, weaker hydrogen bonds were formed between the organic molecules and the surfaces. The largest adsorption energies were obtained for the modes of adsorption where the organic molecules bridged or spanned the periodic grooves or dips present on the goethite surfaces, thus forming several interactions between the molecule and the surface. Among all adsorbates studied, the hydroxamic acid molecule in the eclipsed conformation releases the largest adsorption energy when it interacts with goethite surfaces, followed by the staggered conformations of hydroxyethanal and methanoic acid molecules. The adsorption energies are in the range of -60.0 to -186.4 kJ mol-1. Due to the surface structure, as well as the flexibility and size of hydroxamic acid and hydroxyethanal, in most cases these adsorbate molecules lose their planarity with respect to the structure of the isolated molecules. We found that the replacement of pre-adsorbed water by the organic adsorbates is an exothermic process on all the goethite surfaces studied. The removal by sorption onto iron particles of humic and fulvic acids, the major substituents of natural organic matter (NOM) that pollutes aquifers and soils, is corroborated by our calculations of the adsorption of surfactants with the same functional groups as the surfaces of oxidised iron particles