Understanding the mechanism of virus removal by Q sepharose fast flow chromatography during the purification of CHO‐cell derived biotherapeutics

During production of therapeutic monoclonal antibodies (mAbs) in mammalian cell culture, it is important to ensure that viral impurities and potential viral contaminants will be removed during downstream purification. Anion exchange chromatography provides a high degree of virus removal from mAb feedstocks, but the mechanism by which this is achieved has not been characterized. In this work, we have investigated the binding of three viruses to Q sepharose fast flow (QSFF) resin to determine the degree to which electrostatic interactions are responsible for viral clearance by this process. We first used a chromatofocusing technique to determine the isoelectric points of the viruses and established that they are negatively charged under standard QSFF conditions. We then determined that virus removal by this chromatography resin is strongly disrupted by the presence of high salt concentrations or by the absence of the positively charged Q ligand, indicating that binding of the virus to the resin is primarily due to electrostatic forces, and that any non‐electrostatic interactions which may be present are not sufficient to provide virus removal. Finally, we determined the binding profile of a virus in a QSFF column after a viral clearance process. These data indicate that virus particles generally behave similarly to proteins, but they also illustrate the high degree of performance necessary to achieve several logs of virus reduction. Overall, this mechanistic understanding of an important viral clearance process provides the foundation for the development of science‐based process validation strategies to ensure viral safety of biotechnology products. Biotechnol. Bioeng. 2009; 104: 371–380 © 2009 Wiley Periodicals, Inc.     

Mobile larynx in Mongolian gazelle: Retraction of the larynx during rutting barks in male Mongolian gazelle (Procapra gutturosa Pallas, 1777)

This study provides the first evidence of pronounced temporary laryngeal descent in a bovid species. An elaborate acoustic display is prominent in male courtship behavior of polygynous Mongolian gazelle. During rut, rounding up of females is accompanied by continuous head‐up barking by dominant males. Throughout the rut their evolutionarily enlarged larynx descends to a low mid‐neck resting position. In the course of each bark the larynx is additionally retracted toward the sternum by 30% of the resting vocal tract length. A geometric model of active larynx movements was constructed by combining results of video documentation, dissection, skeletonization, and behavioral observation. The considerable distance between resting position and maximal laryngeal descent suggests a backward tilting of the hyoid apparatus and an extension of the thyrohyoid connection during the retraction phase. Return to the resting position is effected by strap muscles and by the elastic recoil of the pharynx and the thyrohyoid connection. An intrapharyngeal inflation of the peculiar palatinal pharyngeal pouch of adult males is inferred from a short‐time expansion of the ventral neck region rostral to the laryngeal prominence. The neck of adult dominant males is accentuated by long gray guard hairs during the rut. The passive swinging of the heavy larynx of adult males during locomotion gives the impression of a handicap imposed on rutting males. Apparently, this disadvantage becomes outweighed by the profits for reproductive success. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

Non-Selective Evolution of Growing Populations

Non-selective effects, like genetic drift, are an important factor in modern conceptions of evolution, and have been extensively studied for constant population sizes (Kimura, 1955; Otto and Whitlock, 1997). Here, we consider non-selective evolution in the case of growing populations that are of small size and have varying trait compositions (e.g. after a population bottleneck). We find that, in these conditions, populations never fixate to a trait, but tend to a random limit composition, and that the distribution of compositions “freezes” to a steady state. This final state is crucially influenced by the initial conditions. We obtain these findings from a combined theoretical and experimental approach, using multiple mixed subpopulations of two Pseudomonas putida strains in non-selective growth conditions (Matthijs et al, 2009) as model system. The experimental results for the population dynamics match the theoretical predictions based on the Pólya urn model (Eggenberger and Pólya, 1923) for all analyzed parameter regimes. In summary, we show that exponential growth stops genetic drift. This result contrasts with previous theoretical analyses of non-selective evolution (e.g. genetic drift), which investigated how traits spread and eventually take over populations (fixate) (Kimura, 1955; Otto and Whitlock, 1997). Moreover, our work highlights how deeply growth influences non-selective evolution, and how it plays a key role in maintaining genetic variability. Consequently, it is of particular importance in life-cycles models (Melbinger et al, 2010; Cremer et al, 2011; Cremer et al, 2012) of periodically shrinking and expanding populations.     

Flow and Diffusion in Channel-Guided Cell Migration

Collective migration of mechanically coupled cell layers is a notable feature of wound healing, embryonic development, and cancer progression. In confluent epithelial sheets, the dynamics have been found to be highly heterogeneous, exhibiting spontaneous formation of swirls, long-range correlations, and glass-like dynamic arrest as a function of cell density. In contrast, the flow-like properties of one-sided cell-sheet expansion in confining geometries are not well understood. Here, we studied the short- and long-term flow of Madin-Darby canine kidney (MDCK) cells as they moved through microchannels. Using single-cell tracking and particle image velocimetry (PIV), we found that a defined averaged stationary cell current emerged that exhibited a velocity gradient in the direction of migration and a plug-flow-like profile across the advancing sheet. The observed flow velocity can be decomposed into a constant term of directed cell migration and a diffusion-like contribution that increases with density gradient. The diffusive component is consistent with the cell-density profile and front propagation speed predicted by the Fisher-Kolmogorov equation. To connect diffusion-mediated transport to underlying cellular motility, we studied single-cell trajectories and occurrence of vorticity. We discovered that the directed large-scale cell flow altered fluctuations in cellular motion at short length scales: vorticity maps showed a reduced frequency of swirl formation in channel flow compared with resting sheets of equal cell density. Furthermore, under flow, single-cell trajectories showed persistent long-range, random-walk behavior superimposed on drift, whereas cells in resting tissue did not show significant displacements with respect to neighboring cells. Our work thus suggests that active cell migration manifests itself in an underlying, spatially uniform drift as well as in randomized bursts of short-range correlated motion that lead to a diffusion-mediated transport.     

Weathering-Associated Bacteria from the Damma Glacier Forefield: Physiological Capabilities and Impact on Granite Dissolution

Several bacterial strains isolated from granitic rock material in front of the Damma glacier (Central Swiss Alps) were shown (i) to grow in the presence of granite powder and a glucose-NH₄Cl minimal medium without additional macro- or micronutrients and (ii) to produce weathering-associated agents. In particular, four bacterial isolates (one isolate each of Arthrobacter sp., Janthinobacterium sp., Leifsonia sp., and Polaromonas sp.) were weathering associated. In comparison to what was observed in abiotic experiments, the presence of these strains caused a significant increase of granite dissolution (as measured by the release of Fe, Ca, K, Mg, and Mn). These most promising weathering-associated bacterial species exhibited four main features rendering them more efficient in mineral dissolution than the other investigated isolates: (i) a major part of their bacterial cells was attached to the granite surfaces and not suspended in solution, (ii) they secreted the largest amounts of oxalic acid, (iii) they lowered the pH of the solution, and (iv) they formed significant amounts of HCN. As far as we know, this is the first report showing that the combined action of oxalic acid and HCN appears to be associated with enhanced elemental release from granite, in particular of Fe. This suggests that extensive microbial colonization of the granite surfaces could play a crucial role in the initial soil formation in previously glaciated mountain areas.Glaciers in alpine regions are highly sensitive to changes in climatic conditions (29). Increasing global atmospheric temperatures over the last decades have resulted in the recession of alpine glaciers (18). Forefields of temperate alpine glaciers provide unique opportunities to study initial soil formation as well as microbial and plant succession along the chronosequences (12, 26, 34, 36). The forefields close to the glacier terminus are initially vegetation free and consist mainly of rock material with high fractions of silt-sized grains with low C and N content and small amounts of available nutrients (14). Mineral weathering is a key process in the formation of soils (1, 26), and the crucial importance of microbially promoted mineral weathering for nutrient acquisition is increasingly recognized (2, 4, 39, 46). Recently exposed rock surfaces can be considered primary ecosystems where only a few microbes that are adapted due to their mineral-weathering abilities can grow (17). Some cations of rock-forming minerals are essential for proper cell functions. However, our understanding of geochemically significant microbes in forefields of temperate alpine glacier is still very limited but is crucial for increasing our knowledge of nutrient mobilization and the buildup of organic matter that is essential for the development of macroorganisms.The area of the Damma glacier in Central Switzerland is characterized by a relatively homogenous granitic rock basement and is used as field site of the interdisciplinary research project “Biosphere-Geosphere interactions: Linking climate change, weathering, soil formation and ecosystem evolution (BigLink)” (5). In the frame of this research project, we studied the functional roles of granite-colonizing microbes as biotic weathering agents in previously glaciated areas. So far, relatively little is known about microbe-granite interactions, especially regarding the release of trace elements. Several studies have examined the dissolution of specific granite-forming minerals in the presence of actively metabolizing bacteria or compounds that simulate metabolic activity (24, 30, 31, 37, 38, 44). There is a general agreement that microbially produced organic acids, siderophores, and extracellular polysaccharides can all promote dissolution of minerals. Previous dissolution experiments have mainly been performed with (i) commercially obtained minerals (23, 45), (ii) model microorganisms that were commercially obtained from culture collections (3, 35, 45), or (iii) laboratory strains, such as those of Bacillus subtilis (23) and Burkholderia fungorum (47). Most studies have focused on individual mineral specimens rather than on the mixture of minerals that are present in granite rock (47). Few studies observed mineral weathering of collected rock and bacteria isolated from volcanic areas covered with vegetation (30, 31). Moreover, there are no studies on microbial weathering for such immediately deglaciated environments combining functional and taxonomic investigations, probably due to the difficulties in obtaining heterotrophic bacterial isolates from granitic glacier forefields. In spite of this, a comprehensive culture collection containing approximately 500 bacterial strains, which were isolated from the glacier tongue of the Damma glacier, was established. Full-length 16S rRNA gene sequences of 120 isolates revealed that many isolates obtained from oligotrophic media were closely related to readily cultivable heterotrophic bacteria (e.g., Arthrobacter sp., Collimonas sp., Paenibacillus sp., and Pseudomonas sp.). These bacteria have been found to enhance mineral dissolution (39).Our aim was to characterize the impact of microorganisms on granite weathering. We performed laboratory dissolution experiments with sterile crushed granite rock material, 12 bacterial strains, and 1 algal strain. To investigate the potential weathering abilities of these isolates, granite dissolution experiments were performed abiotically with model agents, such as HCl for proton-promoted weathering or oxalate and citrate and KCN for ligand-promoted weathering.     

Cannabinoid-mediated modulation of neuropathic pain and microglial accumulation in a model of murine type I diabetic peripheral neuropathic pain

Background

Despite the frequency of diabetes mellitus and its relationship to diabetic peripheral neuropathy (DPN) and neuropathic pain (NeP), our understanding of underlying mechanisms leading to chronic pain in diabetes remains poor. Recent evidence has demonstated a prominent role of microglial cells in neuropathic pain states. One potential therapeutic option gaining clinical acceptance is the cannabinoids, for which cannabinoid receptors (CB) are expressed on neurons and microglia. We studied the accumulation and activation of spinal and thalamic microglia in streptozotocin (STZ)-diabetic CD1 mice and the impact of cannabinoid receptor agonism/antagonism during the development of a chronic NeP state. We provided either intranasal or intraperitoneal cannabinoid agonists/antagonists at multiple doses both at the initiation of diabetes as well as after establishment of diabetes and its related NeP state.

Results

Tactile allodynia and thermal hypersensitivity were observed over 8 months in diabetic mice without intervention. Microglial density increases were seen in the dorsal spinal cord and in thalamic nuclei and were accompanied by elevation of phosphorylated p38 MAPK, a marker of microglial activation. When initiated coincidentally with diabetes, moderate-high doses of intranasal cannabidiol (cannaboid receptor 2 agonist) and intraperitoneal cannabidiol attenuated the development of an NeP state, even after their discontinuation and without modification of the diabetic state. Cannabidiol was also associated with restriction in elevation of microglial density in the dorsal spinal cord and elevation in phosphorylated p38 MAPK. When initiated in an established DPN NeP state, both CB1 and CB2 agonists demonstrated an antinociceptive effect until their discontinuation. There were no pronociceptive effects demonstated for either CB1 or CB2 antagonists.

Conclusions

The prevention of microglial accumulation and activation in the dorsal spinal cord was associated with limited development of a neuropathic pain state. Cannabinoids demonstrated antinociceptive effects in this mouse model of DPN. These results suggest that such interventions may also benefit humans with DPN, and their early introduction may also modify the development of the NeP state.     

<Emphasis Type="Italic">Glyphiteuthis rhinophora</Emphasis> n. sp., a trachyteuthidid (Coleoidea,Cephalopoda) from the Cenomanian (Late Cretaceous) of Mexico

A new vampyropod coleoid from the Cenomanian limestones of Coahuila (Mexico) is described. Glyphiteuthis rhinophora n. sp. is classified as a member of the Trachyteuthididae because of its general gladius morphology. Within the genus Glyphiteuthis, Gl. rhinophora n. sp. is unique by its nose-shaped extension of the anterior median field extremity. The ventral gladius surface reflects the dorsal surface and lacks evidence of a phragmocone, so affiliations with sepiids are unlikely. Gl. rhinophora n. sp. represents the first Cenomanian record of a vampyropod coleoid in the New World and the first evidence of the genus outside the Tethyan and Boreal realm. The paleoenvironment indicates a nektonic lifestyle for Gl. rhinophora n. sp.     

Distribution and expression of elicitin genes in the interspecific hybrid oomycete Phytophthora alni

Phytophthora alni subsp. alni, P. alni subsp. multiformis, and P. alni subsp. uniformis are responsible for alder disease in Europe. Class I and II elicitin gene patterns of P. alni subsp. alni, P. alni subsp. multiformis, P. alni subsp. uniformis, and the phylogenetically close species P. cambivora and P. fragariae were studied through mRNA sequencing and 3' untranslated region (3'UTR)-specific PCRs and sequencing. The occurrence of multiple 3'UTR sequences in association with identical elicitin-encoding sequences in P. alni subsp. alni indicated duplication/recombination events. The mRNA pattern displayed by P. alni subsp. alni demonstrated that elicitin genes from all the parental genomes are actually expressed in this allopolyploid taxon. The complementary elicitin patterns resolved confirmed the possible involvement of P. alni subsp. multiformis and P. alni subsp. uniformis in the genesis of the hybrid species P. alni subsp. alni. The occurrence of multiple and common elicitin gene sequences throughout P. cambivora, P. fragariae, and P. alni sensu lato, not observed in other Phytophthora species, suggests that duplication of these genes occurred before the radiation of these species.