Engineering a mammalian super producer

Mammalian cells are the preferred host for the manufacture of a wide range of biopharmaceuticals, but production costs are high owing to low productivity. A range of rational engineering strategies have been pursued in order to increase volumetric product titres from mammalian cells, such as delaying apoptosis, manipulation of the cell cycle, and improving metabolism and protein processing. Unfortunately, outcomes from these strategies have been mixed, with few instances where significant improvements in product yield have been achieved. This article reviews and contrasts many of the engineering strategies attempted to date, highlighting the variability and context specificity in outcome. The paper argues that this is a reflection of the complexity of mammalian cells, and that a deeper understanding of the biology underpinning protein production for biotechnological purposes is required. Copyright © 2011 Society of Chemical Industry     

Lipids and endothelium-dependent vasodilation—A review

Studies using both in vitro and in vivo techniques have repeatedly shown that endothelium-dependent vasodilation (EDV) is impaired in different forms of experimental as well as human hypercholesterolemia. Clearly this impaired EDV can be reversed by lowering cholesterol levels by diet or medical therapy. Competitive blocking of l-arginine, changes in nitric oxide synthase acitivity, increased release of endothelin-1, and inactivation of nitric oxide due to superoxide ions all contribute to the impairment in EDV during dyslipidemia. The oxidation of low density lipoprotein, with its compound lysophosphatidylcholine, plays a critical role in these events. However, data on the role of triglycerides and fat-rich meals regarding EDV are not so consistent as data for cholesterol, although a view that the compositions of individual fatty acids and antioxidants are of major importance is emerging. Thus, this review shows that while impaired FDV is a general feature of hypercholesterolemia, the mechanisms involved and the therapeutic opportunities available still have to be investigated. Furthermore, discrepancies regarding the role of triglycerides and fat content in food may be explained by divergent effects of different fatty acids on the endothelium.     

Combined TPS, XPS, EXAFS, and NO-TPD study of the sulfiding of Mo/Al2O3

The sulfiding of Mo/Al₂O₃ in H₂S/Ar versus in H₂S/H₂ has been studied by temperature-programmed sulfiding (TPS), X-ray photon electron spectroscopy (XPS), extended X-ray absorption fine structure (EXAFS), and temperature-programmed desorption of NO (NO-TPD). All the applied techniques agree on the sulfur content in the sulfided catalysts and the findings are in accord with a model for the H₂S production reaction. The nucleation and growth of well-ordered MoS₂ clusters are probed by XPS during sulfiding with and without the presence of hydrogen. The resulting dispersion of the MoS₂ phase is evaluated on the basis of XPS, EXAFS, and NO-TPD, and is found to be highest when the sulfiding occurs in the presence of hydrogen.     

Synthesis and Reactivity of Iron(II) Complexes with a New Tripodal Imine Ligand

A new tripodal imine ligand tris(2-(propan-2-ylideneamino)ethyl)amine (imine₃tren) was prepared in order to stabilize high valent iron-oxido complexes. Iron complexes were synthesized in template reactions from iron(II) salts, tris(2-aminoethyl)amine (tren) and acetone. Due to the reversibility of the imine formation, complexes with different ligands were obtained depending on the reaction conditions. Three complexes, [Fe(imine₃tren)(OAc)₂] ( 1 ), [Fe(imine₃tren)(OAc)]OTf ( 2 ) and [(imine₃tren)₂Fe₂(F)₂](SbF₆)₂ ( 3 ), could be synthesized and structurally characterized. However, reactions with hydrogen peroxide, iodosobenzene or ozone did not lead to any kind of “oxygen adduct” complex that could be spectroscopically observed.     

Competitive effects of free volume,rigidity, and self-adaptivity on indentation response of silicoaluminoborate glasses

Lithium aluminoborate glasses have recently been found to feature high resistance to crack initiation during indentation, but suffer from relatively low hardness and chemical durability. To further understand the mechanical properties of this glass family and their correlation with the network structure, we here study the effect of adding SiO₂ to a 25Li₂O–20Al₂O₃–55B₂O₃ glass on the structure and mechanical properties. Addition of silica increases the average network rigidity, but meanwhile its open tetrahedral structure decreases the atomic packing density. Consequently, we only observe a minor increase in hardness and glass transition temperature, and a decrease in Poisson's ratio. The addition of SiO₂, and thus removal of Al₂O₃ and/or B₂O₃, also makes the network less structurally adaptive to applied stress, since Al and B easily increase their coordination number under pressure, while this is not the case for Si under modest pressures. As such, although the silica-containing networks have more free volume, they cannot densify more during indentation, which in turn leads to an overall decrease in crack resistance upon SiO₂ addition. Our work shows that, although pure silica glass has very high glass transition temperature and relatively high hardness, its addition in oxide glasses does not necessarily lead to significant increase in these properties due to the complex structural interactions in mixed network former glasses and the competitive effects of free volume and network rigidity.     

Aggregation‐Prone Amyloid‐β⋅CuII Species Formed on the Millisecond Timescale under Mildly Acidic Conditions

Metal ions and their interaction with the amyloid beta (Aβ) peptide might be key elements in the development of Alzheimer's disease. In this work the effect of Cu^II on the aggregation of Aβ is explored on a timescale from milliseconds to days, both at physiological pH and under mildly acidic conditions, by using stopped‐flow kinetic measurements (fluorescence and light‐scattering), ¹H NMR relaxation and ThT fluorescence. A minimal reaction model that relates the initial Cu^II binding and Aβ folding with downstream aggregation is presented. We demonstrate that a highly aggregation prone Aβ ? Cu^II species is formed on the sub‐second timescale at mildly acidic pH. This observation might be central to the molecular origin of the known detrimental effect of acidosis in Alzheimer's disease.     

One Question,Multiple Answers: Biochemical and Biophysical Screening Methods Retrieve Deviating Fragment Hit Lists

Fragment‐based lead discovery is gaining momentum in drug development. Typically, a hierarchical cascade of several screening techniques is consulted to identify fragment hits which are then analyzed by crystallography. Because crystal structures with bound fragments are essential for the subsequent hit‐to‐lead‐to‐drug optimization, the screening process should distinguish reliably between binders and non‐binders. We therefore investigated whether different screening methods would reveal similar collections of putative binders. First we used a biochemical assay to identify fragments that bind to endothiapepsin, a surrogate for disease‐relevant aspartic proteases. In a comprehensive screening approach, we then evaluated our 361‐entry library by using a reporter‐displacement assay, saturation‐transfer difference NMR, native mass spectrometry, thermophoresis, and a thermal shift assay. While the combined results of these screening methods retrieve 10 of the 11 crystal structures originally predicted by the biochemical assay, the mutual overlap of individual hit lists is surprisingly low, highlighting that each technique operates on different biophysical principles and conditions.