Streamlined process development procedure incorporating the selection of various stationary phase types established in a mAb aggregate reduction study with different mixed mode ligands

In biopharmaceutical process development time, cost and reliability are the relevant keywords. During the development of chromatographic processes these targets are challenged by many possible scaffolds, ligands and process parameters. The common response to this diversity is the establishment of platform processes in the development of chromatographic unit operations. However, while developing a platform library to simplify and accelerate chromatographic processes, the potential combination of scaffold, ligands and process parameters need to be characterized. This challenge is addressed in a case study on novel mixed mode (MM) adsorber for the removal of monoclonal antibody (mAb) aggregates. We propose a rigorous strategy to reduce the various experimental design space resulting from possible combinations in scaffolds, backbones and ligands. This strategy is based on theoretical considerations, identification of adsorber selectivity and capacity for the identification of a suitable membrane system. For this system, each potential MM membrane adsorber candidate is investigated in its high molecular weight species reduction potential for a given mAb feed stream and referenced to the performance of Capto™ Adhere. The introduced strategy can reduce the developmental effort in an early stage from three to two possible stationary phases. Thereafter, initial examinations at different ionic capacities enlighten one favorable stationary phase. Finalizing the development strategy procedure by studying five different MM ligands by HTS and confirming the study with a 2–3 MV higher dynamic breakthrough capacity in benchtop experiments and provides an insight in the benefits of a living process platform library.     

Murein hydrolase (N-acetyl-muramyl-l-alanine amidase) in human serum

An enzyme was identified in human serum which unlike lysozyme cleaved the amide bond between N-acetyl-muramic acid and l-alanine of the peptide side chain of the rigid layer (murein) of Escherichia coli. The N-acetylmuramyl-l-alanine amidase released all of the peptide side chains including those to which the lipoprotein is bound. A portion of the peptide side chains of the Micrococcus lysodeikticus murein was also hydrolysed from the polysaccharide chains. E. coli, M. lysodeikticus, Bacillus subtilis and Staphylococcus aureus were not killed by the amidase. Treatment of E. coli with EDTA or osmotic shock rendered the cells sensitive to the amidase and they were killed. Possible biological functions of the amidase are discussed.The enzyme was separated from lysozyme in human serum. Gel permeation chromatography indicated a molecular weight of the active enzyme of 82,000 while gel electrophoresis in the presence of sodium dodecyl sulfate revealed a molecular weight of 75,000. Thus, the enzyme probably consists of a single polypeptide chain. Incubation with neuraminidase rendered the amidase more basic suggesting the release of sialic acid residues. The modified glycoprotein disclosed an increased activity to murein. Enzyme activity was inhibited by p-chloromercuribenzene sulfonate and ethyleneglycol-bis(2-aminomethyl) tetraacetate (EGTA) at 1 and 0.2 mM concentration, respectively, whereas EDTA up to 5 mM was without effect. The amidase was also inactivated by agents that reduce disulfide bridges.     

A population of serum deprivation-induced bone marrow stem cells (SD-BMSC) expresses marker typical for embryonic and neural stem cells

The bone marrow represents an easy accessible source of adult stem cells suitable for various cell based therapies. Several studies in recent years suggested the existence of pluripotent stem cells within bone marrow stem cells (BMSC) expressing marker proteins of both embryonic and tissue committed stem cells. These subpopulations were referred to as MAPC, MIAMI and VSEL-cells. Here we describe SD-BMSC (serumdeprivation-induced BMSC) which are induced as a distinct subpopulation after complete serumdeprivation. SD-BMSC are generated from small-sized nestin-positive BMSC (S-BMSC) organized as round-shaped cells in the top layer of BMSC-cultures. The generation of SD-BMSC is caused by a selective proliferation of S-BMSC and accompanied by changes in both morphology and gene expression. SD-BMSC up-regulate not only markers typical for neural stem cells like nestin and GFAP, but also proteins characteristic for embryonic cells like Oct4 and SOX2. We hypothesize, that SD-BMSC like MAPC, MIAMI and VSEL-cells represent derivatives from a single pluripotent stem cell fraction within BMSC exhibiting characteristics of embryonic and tissue committed stem cells. The complete removal of serum might offer a simple way to specifically enrich this fraction of pluripotent embryonic like stem cells in BMSC cultures.     

Spatial proximity of proteins surrounding zyxin under force-bearing conditions

Sensing physical forces is a critical first step in mechano-transduction of cells. Zyxin, a LIM domain-containing protein, is recruited to force-bearing actin filaments and is thought to repair and strengthen them. Yet, the precise force-induced protein interactions surrounding zyxin remain unclear. Using BioID analysis, we identified proximal proteins surrounding zyxin under normal and force-bearing conditions by label-free mass spectrometry analysis. Under force-bearing conditions, increased biotinylation of α-actinin 1, α-actinin 4, and AFAP1 were detected, and these proteins accumulated along force-bearing actin fibers independently from zyxin, albeit at a lower intensity than zyxin. VASP also accumulated along force-bearing actin fibers in a zyxin-dependent manner, but the biotinylation of VASP remained constant regardless of force, supporting the model of a free zyxin–VASP complex in the cytoplasm being corecruited to tensed actin fibers. In addition, ARHGAP42, a RhoA GAP, was also identified as a proximal protein of zyxin and colocalized with zyxin along contractile actin bundles. The overexpression of ARHGAP42 reduced the rate of small wound closure, a zyxin-dependent process. These results demonstrate that the application of proximal biotinylation can resolve the proximity and composition of protein complexes as a function of force, which had not been possible with traditional biochemical analysis.     

Nazca Booby (Sula granti) inputs maintain the terrestrial food web of Malpelo Island

Most of the known food webs are based on organic compounds provided by photoautotrophic organisms. The terrestrial ecosystem of Malpelo Island (Colombia) seems to be an exception, however, since it supports several trophic guilds without hosting an adequate amount of primary producers. It has been suggested that this apparent paradox might be explained by external inputs provided by seabirds, namely Nazca Boobies (Sula granti), forming a huge colony on Malpelo. This hypothesis has never been tested. Here, we present a first approach to quantify the significance of Nazca Booby inputs into the Malpelo ecosystem via excrement, second eggs/chicks (which are prone to die), and carcasses, respectively, during the major breeding season. The total input was calculated to amount to 171.6 t per breeding season, with excrements accounting for almost 99% (170 t) of this input. Second eggs/chicks contributed approximately 1.1 t (0.64%) and carcasses around 0.1 t (0.06%). These finding support the idea of the Nazca Booby facilitating a food chain that pairs the pelagic primary producers of the open ocean with the terrestrial consumers of an island. Species most strongly profiting from this process include three endemic lizard species (Anolis agassizi, Diploglossus millepunctatus, Phyllodactylus transversalis) and the land crab (Johngarthia malpilensis).     

How do soil fauna and soil microbiota respond to beech forest growth?

The dynamics and performance of soil biota during forest rotation were studied in monoculture beech stands forming a chronosequence of four different age-classes(30,62,111,153 yr).Biomass was monitored in major groups of microflora,microfauna,mesofauna,and macrofauna.Resource availability(litter layer,soil organic mater),biomass of the two dominant decomposer groups(microflora,earthworms)as well as the biomass of mesofauna and microfauna were found to remain quite stable during forest succession.Nevertheles...     

Oligomeric structure of ExbB and ExbB-ExbD isolated from Escherichia coli as revealed by LILBID mass spectrometry

Energy-coupled transporters in the outer membrane of Escherichia coli and other Gram-negative bacteria allow the entry of scarce substrates, toxic proteins, and bacterial viruses (phages) into the cells. The required energy is derived from the proton-motive force of the cytoplasmic membrane, which is coupled to the outer membrane via the ExbB-ExbD-TonB protein complex. Knowledge of the structure of this complex is required to elucidate the mechanisms of energy harvesting in the cytoplasmic membrane and energy transfer to the outer membrane transporters. Here we solubilized an ExbB oligomer and an ExbB-ExbD subcomplex from the cytoplasmic membrane with the detergent undecyl maltoside. Using laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS), we determined at moderate desorption laser energies the oligomeric structure of ExbB to be mainly hexameric (ExbB(6)), with minor amounts of trimeric (ExbB(3)), dimeric (ExbB(2)), and monomeric (ExbB(1)) oligomers. Under the same conditions ExbB-ExbD formed a subcomplex consisting of ExbB(6)ExbD(1), with a minor amount of ExbB(5)ExbD(1). At higher desorption laser intensities, ExbB(1) and ExbD(1) and traces of ExbB(3)ExbD(1), ExbB(2)ExbD(1), ExbB(1)ExbD(1), ExbB(3), and ExbB(2) were observed. Since the ExbB(6) complex and the ExbB(6)ExbD(1) complex remained stable during solubilization and subsequent chromatographic purification on nickel-nitrilotriacetate agarose, Strep-Tactin, and Superdex 200, and during native blue gel electrophoresis, we concluded that ExbB(6) and ExbB(6)ExbD(1) are subcomplexes on which the final complex including TonB is assembled.     

Effects of low frequency electromagnetic fields on the chondrogenic differentiation of human mesenchymal stem cells

Electromagnetic fields (EMF) have been shown to exert beneficial effects on cartilage tissue. Nowadays, differentiated human mesenchymal stem cells (hMSCs) are discussed as an alternative approach for cartilage repair. Therefore, the aim of this study was to examine the impact of EMF on hMSCs during chondrogenic differentiation. HMSCs at cell passages five and six were differentiated in pellet cultures in vitro under the addition of human fibroblast growth factor 2 (FGF‐2) and human transforming growth factor‐β₃ (TGF‐β₃). Cultures were exposed to homogeneous sinusoidal extremely low‐frequency magnetic fields (5 mT) produced by a solenoid or were kept in a control system. After 3 weeks of culture, chondrogenesis was assessed by toluidine blue and safranin‐O staining, immunohistochemistry, quantitative real‐time polymerase chain reaction (PCR) for cartilage‐specific proteins, and a DMMB dye‐binding assay for glycosaminoglycans. Under EMF, hMSCs showed a significant increase in collagen type II expression at passage 6. Aggrecan and SOX9 expression did not change significantly after EMF exposure. Collagen type X expression decreased under electromagnetic stimulation. Pellet cultures at passage 5 that had been treated with EMF provided a higher glycosaminoglycan (GAG)/DNA content than cultures that had not been exposed to EMF. Chondrogenic differentiation of hMSCs may be improved by EMF regarding collagen type II expression and GAG content of cultures. EMF might be a way to stimulate and maintain chondrogenesis of hMSCs and, therefore, provide a new step in regenerative medicine regarding tissue engineering of cartilage. Bioelectromagnetics 32:283–290, 2011. © 2010 Wiley‐Liss, Inc.     

Scaling properties of multivariate landscape structure

Analyses of landscape context is essential for understanding how ecological patterns and processes relate to space. This requires that we quantify variation patterns of different landscape parameters, which may change relative to one another at different spatial scales. Here, we analyzed how statistical relationships of land-use composition parameters changed as a function of extent in 20 real agricultural landscapes. Furthermore, we tested the generality of these scaling relations in numerical simulations using 300 artificial landscapes. We analyzed proportions of artificial habitat types at different extent and compared these patterns with three dominant habitat types in real landscapes (forest, arable land and grassland) at four spatial scales (quadrates of 1–4 km). Both real and simulated landscapes showed that variance of landscape parameters (data differentiation) decreased and their correlations (data consistency) increased as scale increased, thereby suggesting general scaling laws. The potential statistical impact of these scaling relationships is revealed in simultaneous analyses of variation of (local) site parameters of 20 arable fields and their surrounding landscape context. At small and medium extent (quadrates of 1–3 km), variability of local site parameters (e.g. fertilization, pH-value) was high relative to those of landscape parameters. In contrast, at large extent (quadrates of 4 km) variability of landscape parameters was greater than that of site parameters indicating a fundamental shift in the relationship between these sets of parameters at different scales. Hence, it is clear that there is a high risk of artefactual correlations in hierarchical multi-scale landscape analyses when ecological data are related to the landscape context. Accordingly, there is a necessity for multi-scale analyses in landscape ecology to accurately evaluate the relative importance of landscape context at different spatial scales.