Development,characterization, and application of a 2‐Compartment system to investigate the impact of pH inhomogeneities in large‐scale CHO‐based processes

Large‐scale bioreactors for the production of monoclonal antibodies reach volumes of up to 25 000 L. With increasing bioreactor size, mixing is however affected negatively, resulting in the formation of gradients throughout the reactor. These gradients can adversely affect process performance at large scale. Since mammalian cells are sensitive to changes in pH, this study investigated the effects of pH gradients on process performance. A 2‐Compartment System was established for this purpose to expose only a fraction of the cell population to pH excursions and thereby mimicking a large‐scale bioreactor. Cells were exposed to repeated pH amplitudes of 0.4 units (pH 7.3), which resulted in decreased viable cell counts, as well as the inhibition of the lactate metabolic shift. These effects were furthermore accompanied by increased absolute lactate levels. Continuous assessment of molecular attributes of the expressed target protein revealed that subunit assembly or N‐glycosylation patterns were only slightly influenced by the pH excursions. The exposure of more cells to the same pH amplitudes further impaired process performance, indicating this is an important factor, which influences the impact of pH inhomogeneity. This knowledge can aid in the design of pH control strategies to minimize the effects of pH inhomogeneity in large‐scale bioreactors.     

Metabotropic glutamate receptor subtype 7 controls maternal care,maternal motivation and maternal aggression in mice

The group III metabotropic glutamate receptor subtype 7 (mGlu7) is an important regulator of glutamatergic and GABAergic neurotransmission and known to mediate emotionality and male social behavior. However, a possible regulatory role in maternal behavior remains unknown to date. Adequate expression of maternal behavior is essential for successful rearing and healthy development of the young. By understanding genetic and neural mechanisms underlying this important prosocial behavior, we gain valuable insights into possible dysregulations. Using genetic ablation as well as pharmacological modulation, we studied various parameters of maternal behavior in two different mouse strains under the influence of mGlu7. We can clearly show a regulatory role of mGlu7 in maternal behavior. Naïve virgin female C57BL/6 mGlu7 knockout mice showed more often nursing postures and less spontaneous maternal aggression compared to their heterozygous and wildtype littermates. In lactating C57BL/6 wildtype mice, acute central activation of mGlu7 by the selective agonist AMN082 reduced arched back nursing and accelerated pup retrieval without affecting maternal aggression. In addition, in lactating CD1 wildtype mice the selective mGlu7 antagonist XAP044 increased both pup retrieval and maternal aggression. With respect to receptor expression levels, mGlu7 mRNA expression was higher in lactating vs virgin C57BL/6 mice in the prefrontal cortex, but not hypothalamus or hippocampus. In conclusion, these findings highlight a significant role of the mGlu7 receptor subtype in mediating maternal behavior in mice. Region‐dependent studies are warranted to further extend our knowledge on the specific function of the brain glutamate system in maternal behavior.     

Boosting Escherichia coli’s heterologous production rate of ectoines by exploiting the non-halophilic gene cluster from Acidiphilium cryptum

Extremophiles - The compatible solutes ectoine and hydroxyectoine are synthesized by many microorganisms as potent osmostress and desiccation protectants. Besides their successful implementation...     

Staphylococcus aureus Lpl protein triggers human host cell invasion via activation of Hsp90 receptor

Staphylococcus aureus is a facultative intracellular pathogen. Recently, it has been shown that the protein part of the lipoprotein‐like lipoproteins (Lpls), encoded by the lpl cluster comprising of 10 lpls paralogue genes, increases pathogenicity, delays the G2/M phase transition, and also triggers host cell invasion. Here, we show that a recombinant Lpl1 protein without the lipid moiety binds directly to the isoforms of the human heat shock proteins Hsp90α and Hsp90ß. Synthetic peptides covering the Lpl1 sequence caused a twofold to fivefold increase of S. aureus invasion in HaCaT cells. Antibodies against Hsp90 decrease S. aureus invasion in HaCaT cells and in primary human keratinocytes. Additionally, inhibition of ATPase function of Hsp90 or silencing Hsp90α expression by siRNA also decreased the S. aureus invasion in HaCaT cells. Although the Hsp90ß is constitutively expressed, the Hsp90α isoform is heat‐inducible and appears to play a major role in Lpl1 interaction. Pre‐incubation of HaCaT cells at 39°C increased both the Hsp90α expression and S. aureus invasion. Lpl1‐Hsp90 interaction induces F‐actin formation, thus, triggering an endocytosis‐like internalisation. Here, we uncovered a new host cell invasion principle on the basis of Lpl‐Hsp90 interaction.     

A framework for modelling soil structure dynamics induced by biological activity

Soil degradation is a worsening global phenomenon driven by socio‐economic pressures, poor land management practices and climate change. A deterioration of soil structure at timescales ranging from seconds to centuries is implicated in most forms of soil degradation including the depletion of nutrients and organic matter, erosion and compaction. New soil–crop models that could account for soil structure dynamics at decadal to centennial timescales would provide insights into the relative importance of the various underlying physical (e.g. tillage, traffic compaction, swell/shrink and freeze/thaw) and biological (e.g. plant root growth, soil microbial and faunal activity) mechanisms, their impacts on soil hydrological processes and plant growth, as well as the relevant timescales of soil degradation and recovery. However, the development of such a model remains a challenge due to the enormous complexity of the interactions in the soil–plant system. In this paper, we focus on the impacts of biological processes on soil structure dynamics, especially the growth of plant roots and the activity of soil fauna and microorganisms. We first define what we mean by soil structure and then review current understanding of how these biological agents impact soil structure. We then develop a new framework for modelling soil structure dynamics, which is designed to be compatible with soil–crop models that operate at the soil profile scale and for long temporal scales (i.e. decades, centuries). We illustrate the modelling concept with a case study on the role of root growth and earthworm bioturbation in restoring the structure of a severely compacted soil.     

Stochastic combinations of actin regulatory proteins are sufficient to drive filopodia formation

Assemblies of actin and its regulators underlie the dynamic morphology of all eukaryotic cells. To understand how actin regulatory proteins work together to generate actin-rich structures such as filopodia, we analyzed the localization of diverse actin regulators within filopodia in Drosophila embryos and in a complementary in vitro system of filopodia-like structures (FLSs). We found that the composition of the regulatory protein complex where actin is incorporated (the filopodial tip complex) is remarkably heterogeneous both in vivo and in vitro. Our data reveal that different pairs of proteins correlate with each other and with actin bundle length, suggesting the presence of functional subcomplexes. This is consistent with a theoretical framework where three or more redundant subcomplexes join the tip complex stochastically, with any two being sufficient to drive filopodia formation. We provide an explanation for the observed heterogeneity and suggest that a mechanism based on multiple components allows stereotypical filopodial dynamics to arise from diverse upstream signaling pathways.     

Natural transformation in Gram-negative bacteria thriving in extreme environments: from genes and genomes to proteins,structures and regulation

Extremophiles - Extremophilic prokaryotes live under harsh environmental conditions which require far-reaching cellular adaptations. The acquisition of novel genetic information via natural...