Clarification of animal cell cultures on a large scale by continuous centrifugation

Summary Animal cells from 80-L and 2000-L fed batch fermentations were removed by a prototype disc stack centrifuge in order to achieve a fast and reliable separation of solids from large quantities of cell culture fluids. The clarification capacity was excellent for animal cells but particles remained in the liquid phase and affected further downstream processing of the cell-free harvest fluid. No significant loss of product was observed. A number of parameters were monitored to optimize process conditions for use with animal cells.     

Particle sources and trace element reactivity in the Humber plume

Samples of suspended particulate matter (SPM) collected from the Humber Estuary had higher concentrations of particulate metals than SPM from Holderness coastal waters (U.K.). Characterised SPM from both sources was used in laboratory experiments involving the uptake of radiotracer¹⁰⁹Cd,¹³⁷Cs,⁵⁴Mn and⁶⁵Zn. Kinetic experiments, over five days, showed that the rate and extent of uptake was highly dependent on particle type, with¹⁰⁹Cd,⁵⁴Mn and⁶⁵Zn being more reactive with Humber Estuary particles than those from Holderness and¹³⁷Cs having the opposite trend. Adsorption experiments were also carried out on suspensions in which SPM from the Humber Estuary and Holderness coastal water were mixed in various proportions. These experiments revealed that K_d for⁶⁵Zn increased linearly with the proportion of Humber SPM, K_d for¹³⁷Cs decreased linearly with increase in Humber SPM and K_d for⁵⁴Mn and¹⁰⁹Cd displayed non-linear behaviour. The results of the study were used to develop an algorithm for predicting the partition coefficients in the Humber Plume based on the extent of particle mixing from the two source regions. The use of^206/207Pb ratios in determining the extent of particle mixing is discussed, along with the application of the algorithm to the modelling of particulate trace metal behaviour in the Humber-Wash coastal zone.     

Modeling the effects of particles,interstitials, vacancies and tip geometry on indentation-induced plasticity

In this work, which is a follow-up of an earlier work by the authors in this journal, we discuss the effects of particles, interstitials, vacancies and indenter-tip geometry on indentation-induced plasticity. A nano-indentation model based on a close-packed array of straws was used. Upon indentation with a cylindrical indenter, the results observed in situ, match qualitatively computer atomistic and finite-element simulation models. This model helps explain the phenomenon and physical reasons for annihilation of vacancies. It also shows the strong effect of interstitials on the inception of plasticity at their sites. It shows that a rigid particle acts as a stress concentrator even away from the indenter. On indentation with a flat indenter, curling of atoms at the indenter edges was observed, along with slip lines reported in the literature.     

Classification and characterization of therapeutic antibody aggregates

A host of diverse stress techniques was applied to a monoclonal antibody (IgG(2)) to yield protein particles with varying attributes and morphologies. Aggregated solutions were evaluated for percent aggregation, particle counts, size distribution, morphology, changes in secondary and tertiary structure, surface hydrophobicity, metal content, and reversibility. Chemical modifications were also identified in a separate report (Luo, Q., Joubert, M. K., Stevenson, R., Narhi, L. O., and Wypych, J. (2011) J. Biol. Chem. 286, 25134-25144). Aggregates were categorized into seven discrete classes, based on the traits described. Several additional molecules (from the IgG(1) and IgG(2) subtypes as well as intravenous IgG) were stressed and found to be defined with the same classification system. The mechanism of protein aggregation and the type of aggregate formed depends on the nature of the stress applied. Different IgG molecules appear to aggregate by a similar mechanism under the same applied stress. Aggregates created by harsh mechanical stress showed the largest number of subvisible particles, and the class generated by thermal stress displayed the largest number of visible particles. Most classes showed a disruption of the higher order structure, with the degree of disorder depending on the stress process. Particles in all classes (except thermal stress) were at least partially reversible upon dilution in pH 5 buffer. High copper content was detected in isolated metal-catalyzed aggregates, a stress previously shown to produce immunogenic aggregates. In conclusion, protein aggregates can be a very heterogeneous population, whose qualities are the result of the type of stress that was experienced.     

Regulation of Sealing Ring Formation by L-plastin and Cortactin in Osteoclasts

The aim of this study is to identify the exact mechanism(s) by which cytoskeletal structures are modulated during bone resorption. In this study, we have shown the possible role of different actin-binding and signaling proteins in the regulation of sealing ring formation. Our analyses have demonstrated a significant increase in cortactin and a corresponding decrease in L-plastin protein levels in osteoclasts subjected to bone resorption for 18 h in the presence of RANKL, M-CSF, and native bone particles. Time-dependent changes in the localization of L-plastin (in actin aggregates) and cortactin (in the sealing ring) suggest that these proteins may be involved in the initial and maturation phases of sealing ring formation, respectively. siRNA to cortactin inhibits this maturation process but not the formation of actin aggregates. Osteoclasts treated as above but with TNF-α demonstrated very similar effects as observed with RANKL. Osteoclasts treated with a neutralizing antibody to TNF-α displayed podosome-like structures in the entire subsurface and at the periphery of osteoclast. It is possible that TNF-α and RANKL-mediated signaling may play a role in the early phase of sealing ring configuration (i.e. either in the disassembly of podosomes or formation of actin aggregates). Furthermore, osteoclasts treated with alendronate or αv reduced the formation of the sealing ring but not actin aggregates. The present study demonstrates a novel mechanistic link between L-plastin and cortactin in sealing ring formation. These results suggest that actin aggregates formed by L-plastin independent of integrin signaling function as a core in assembling signaling molecules (integrin αvβ3, Src, cortactin, etc.) involved in the maturation process.     

Particulate material and midge larvae (Chironomidae: Diptera) in an upland river

The density of midge larvae (Chironomidae), which dominate the stone-surface fauna (81.3 ± 4.9% of the community by number) in an upland stream in northeast England is regulated by the amount of particulate material available. The amount of material depends on the deposition of suspended particles and on epilithic algal growth. Spates, characteristic of this type of stream, result in scouring and denudation of rock surfaces, preventing the build-up of epilithic material and limiting larval densities by regulating the availability of food and tube-building material. Detritus is the main type of particle in suspension throughout the year (66 ± 6%); detritus and diatoms the chief epilithic kind (65 ± 4 and 29 ± 4% respectively). The probable effects of flow amelioration, resulting from the formation of the proposed Kielder Reservoir are discussed.     

Effect of hot trub and particle addition on fermentation performance of Saccharomyces cerevisiae

In this investigation, the effect of hot trub (a precipitation product of the wort boiling process in beer manufacturing) addition on fermentation performance was observed under variation of yeast vitality, and origin and the amount of hot trub. Its addition improved suspended cell concentrations for all yeast vitalities tested, and the more trub was added, the greater the effect. Further, pilot-scale fermentations showed significantly lower pH values and an accelerated extract degradation, thus, advancing fermentation by roughly 1 day for hot trub addition versus the fermentation of extremely bright wort. Since the positive effect of trub has often been associated with its particulate characteristics, fermentations with fractionated model particles, such as poly(vinylpyrrolidones) and kieselguhr, of different particle sizes were carried out under variation of yeast vitality and particle amounts. The addition of both particle types also improved fermentation performance, however, the effect was not as great as that of hot trub. Particulate material may improve the development of CO₂ from the fermenting medium, thus reducing its concentration and inhibitory effect on yeast metabolism. The most effective fraction of kieselguhr had a 40 μm peak which also occurred in particle size distributions of all hot trubs investigated. This could be of particular interest when discussing particle effects.     

Effects of vegetation status in urban green spaces on particle removal in a street canyon atmosphere

Green spaces contribute to the removal of significant amounts of air pollutants. This quantitative study, using seasonally monitoring data of Total Suspended Particles (TSP) from green spaces along one street of Pudong, Shanghai, China, demonstrates that vegetation can remove large amounts of TSP. Furthermore, correlation analysis was performed between the vegetation status, measured by canopy density and shelterbelt porosity, and the TSP removal percentage. The results show that (1) vegetation in green spaces greatly contributes to reduce TSP pollution. The width of green spaces along the road should not be smaller than 5 m, with the best width being 10 m, and more evergreen trees should be chosen in order to get a noticeable TSP purification effect; (2) The TSP removal percentage is correlated to the canopy density and inversely correlated to the shelterbelt porosity. The optimum intervals of the canopy density and shelterbelt porosity are 0.70–0.85 and 0.25–0.33, respectively. The proposed optimum intervals can help to estimate the vegetation structure of green spaces and provide technical support for the programming and construction of urban green spaces.     

Identification of a Point Mutation Impairing the Binding between Aquaporin-4 and Neuromyelitis Optica Autoantibodies

Neuromyelitis optica (NMO) is characterized by the presence of pathogenic autoantibodies (NMO-IgGs) against supra-molecular assemblies of aquaporin-4 (AQP4), known as orthogonal array of particles (OAPs). NMO-IgGs have a polyclonal origin and recognize different conformational epitopes involving extracellular AQP4 loops A, C, and E. Here we hypothesize a pivotal role for AQP4 transmembrane regions (TMs) in epitope assembly. On the basis of multialignment analysis, mutagenesis, NMO-IgG binding, and cytotoxicity assay, we have disclosed the key role of aspartate 69 (Asp⁶⁹) of TM2 for NMO-IgG epitope assembly. Mutation of Asp⁶⁹ to histidine severely impairs NMO-IgG binding for 85.7% of the NMO patient sera analyzed here. Although Blue Native-PAGE, total internal reflection fluorescence microscopy, and water transport assays indicate that the OAP Asp⁶⁹ mutant is similar in structure and function to the wild type, molecular dynamic simulations have revealed that the D⁶⁹H mutation has the effect of altering the structural rearrangements of extracellular loop A. In conclusion, Asp⁶⁹ is crucial for the spatial control of loop A, the particular molecular conformation of which enables the assembly of NMO-IgG epitopes. These findings provide additional clues for new strategies for NMO treatment and a wealth of information to better approach NMO pathogenesis.     

Membrane specializations in the paired spermatozoa of dytiscid water beetles

The paired spermatozoa of the dytiscid beetles Dytiscus marginalis and Hydaticus seminiger were studied by electron microscopy with the aim of examining whether the regions of the cell membrane in the zones of sperm conjugation might differ from other regions and to explore whether these cells had any other specialized domains of the cell membrane that could be recognized by the freeze-fracturing technique. The spermatozoa are conjugated along one side of the sperm head and proximal tail portion, called the ventral side. The cell membrane was seen to contain tightly packed intramembranous particles (IMPs) that were predominantly located in the external membrane face (the E-face). In thin sections the cell membrane had a ladder-like appearance at these regions and a specialized type of glycocalyx seen as a fluffy material containing granules. Other specialized membrane domains could also be recorded: a ribbon of particles in the protoplasmic face (P-face) of the dorsal side of the spermatozoon at the proximal tail portion and regularly arranged particle rows in the P-face of the distal tail portion. These domains corresponded to regions where the glycocalyx is prominent. Both the E-face and the P-face of the cell membrane were seen to contain numerous intramembranous particles, which suggests an active function for both membrane leaflets; this is in contrast to the situation in most cells where the particles are mainly in the P-face. The functions of the intramembranous particles in the specialized domains of the cell membrane remains unknown. Some particles may represent receptors or ion gates, others proteins with a mechanical function.