Continuous capture of recombinant antibodies by ZnCl2 precipitation without polyethylene glycol

The capture of recombinant antibodies from cell culture broth is the first critical step of downstream processing. We were able to develop a precipitation‐based method for the capture and purification of monoclonal antibodies based on divalent cations, namely ZnCl₂. Traditional precipitation processes have to deal with high dilution factors especially for resolubilization and higher viscosity due to the use of PEG as precipitation or co‐precipitation agent. By the use of the crosslinking nature of divalent cations without the use of PEG, we kept viscosity from the supernatant and resolubilization dilution factors very low. This is especially beneficial for the solid–liquid separation for the harvest and wash of the precipitate in continuous mode. For this harvest and wash, we used tangential flow filtration that benefits a lot from low viscosity solutions, which minimizes the membrane fouling. With this precipitation based on ZnCl_2, we were able to implement a very lean and efficient process. We demonstrated precipitation studies with three different antibodies, Adalimumab, Trastuzumab, and Denosumab, and a continuous capture case study using tangential flow filtration for precipitate recovery. In this study, we achieved yields of 70%.     

New Insights on Cytological and Metabolic Features of Ostreopsis cf. ovata Fukuyo (Dinophyceae): A Multidisciplinary Approach

The harmful dinoflagellate Ostreopsis cf. ovata has been causing toxic events along the Mediterranean coasts and other temperate and tropical areas, with increasing frequency during the last decade. Despite many studies, important biological features of this species are still poorly known. An integrated study, using different microscopy and molecular techniques, Raman microspectroscopy and high resolution liquid chromatography-mass spectrometry (HR LC-MS), was undertaken to elucidate cytological aspects, and identify main metabolites including toxins. The species was genetically identified as O. cf. ovata, Atlantic-Mediterranean clade. The ultrastructural results show unique features of the mucilage network abundantly produced by this species to colonize benthic substrates, with a new role of trichocysts, never described before. The amorphous polysaccharidic component of mucilage appears to derive from pusule fibrous material and mucocysts. In all stages of growth, the cells show an abundant production of lipids. Different developmental stages of chloroplasts are found in the peripheral cytoplasm and in the centre of cell. In vivo Raman microspectroscopy confirms the presence of the carotenoid peridinin in O. cf. ovata, and detects in several specimen the abundant presence of unsaturated lipids structurally related to docosahexaenoic acid. The HR LC-MS analysis reveals that ovatoxin-a is the predominant toxin, together with decreasing amounts of ovatoxin-b, -d/e, -c and putative palytoxin. Toxins concentration on a per cell basis increases from exponential to senescent phase. The results suggest that benthic blooms of this species are probably related to features such as the ability to create a unique mucilaginous sheath covering the sea bottom, associated with the production of potent toxins as palytoxin-like compounds. In this way, O. cf. ovata may be able to rapidly colonize benthic substrates outcompeting other species.     

Anything but Conventional Chromatography Approaches in Bioseparation

While packed bed chromatography, known as conventional chromatography, has been serving the biopharmaceutical industry for decades as the bioseparation method of choice, alternative approaches are likely to take an increasing leading role in the next few years. The high number of new biological drugs under development, and the need to make biopharmaceuticals widely accessible, has been driving the academia and industry in the quest of anything but conventional chromatography approaches. In this perspective paper, these alternative approaches are discussed in view of current and future challenges in the downstream processing field.     

Sharpening of directional selectivity from neural output of rabbit retina

The estimation of motion direction from time varying retinal images is a fundamental task of visual systems. Neurons that selectively respond to directional visual motion are found in almost all species. In many of them already in the retina direction selective neurons signal their preferred direction of movement. Scientific evidences suggest that direction selectivity is carried from the retina to higher brain areas. Here we adopt a simple integrate-and-fire neuron model, inspired by recent work of Casti et al. (2008), to investigate how directional selectivity changes in cells postsynaptic to directional selective retinal ganglion cells (DSRGC). Our model analysis shows that directional selectivity in the postsynaptic cells increases over a wide parameter range. The degree of directional selectivity positively correlates with the probability of burst-like firing of presynaptic DSRGCs. Postsynaptic potentials summation and spike threshold act together as a temporal filter upon the input spike train. Prior to the intricacy of neural circuitry between retina and higher brain areas, we suggest that sharpening is a straightforward result of the intrinsic spiking pattern of the DSRGCs combined with the summation of excitatory postsynaptic potentials and the spike threshold in postsynaptic neurons.     

Interaction of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> non-O1/non-O139 with Copepods,Cladocerans and Competing Bacteria in the Large Alkaline Lake Neusiedler See,Austria

Vibrio cholerae is a human pathogen and natural inhabitant of aquatic environments. Serogroups O1/O139 have been associated with epidemic cholera, while non-O1/non-O139 serogroups usually cause human disease other than classical cholera. V. cholerae non-O1/non-O139 from the Neusiedler See, a large Central European lake, have caused ear and wound infections, including one case of fatal septicaemia. Recent investigations demonstrated rapid planktonic growth of V. cholerae non-O1/non-O139 and correlation with zooplankton biomass. The aim of this study was to elucidate the interaction of autochthonous V. cholerae with two dominant crustacean zooplankton species in the lake and investigate the influence of the natural bacterial community on this interaction. An existing data set was evaluated for statistical relationships between zooplankton species and V. cholerae and co-culture experiments were performed in the laboratory. A new fluorescence in situ hybridisation protocol was applied for quantification of V. cholerae non-O1/non-O139 cells, which significantly reduced analysis time. The experiments clearly demonstrated a significant relationship of autochthonous V. cholerae non-O1/non-O139 with cladocerans by promoting growth of V. cholerae non-O1/non-O139 in the water and on the surfaces of the cladocerans. In contrast, copepods had a negative effect on the growth of V. cholerae non-O1/non-O139 via competing bacteria from their surfaces. Thus, beside other known factors, biofilm formation by V. cholerae on crustacean zooplankton appears to be zooplankton taxon specific and may be controlled by the natural bacterial community.     

The aryl hydrocarbon receptor-dependent deregulation of cell cycle control induced by polycyclic aromatic hydrocarbons in rat liver epithelial cells

Disruption of cell proliferation control by polycyclic aromatic hydrocarbons (PAHs) may contribute to their carcinogenicity. We investigated role of the aryl hydrocarbon receptor (AhR) in disruption of contact inhibition in rat liver epithelial WB-F344 'stem-like' cells, induced by the weakly mutagenic benz[a]anthracene (BaA), benzo[b]fluoranthene (BbF) and by the strongly mutagenic benzo[a]pyrene (BaP). There were significant differences between the effects of BaA and BbF, and those of the strongly genotoxic BaP. Both BaA and BbF increased percentage of cells entering S-phase and cell numbers, associated with an increased expression of Cyclin A and Cyclin A/cdk2 complex activity. Their effects were significantly reduced in cells expressing a dominant-negative AhR mutant (dnAhR). Roscovitine, a chemical inhibitor of cdk2, abolished the induction of cell proliferation by BbF. However, neither BaA nor BbF modulated expression of the principal cdk inhibitor involved in maintenance of contact inhibition, p27(Kip1), or pRb phosphorylation. The strongly mutagenic BaP induced apoptosis, a decrease in total cell numbers and significantly higher percentage of cells entering S-phase than either BaA or BbF. Given that BaP induced high levels of Cyclin A/cdk2 activity, downregulation of p27(Kip1) and hyperphosphorylation of pRb, the accumulation of cells in S-phase was probably due to cell proliferation, although S-phase arrest due to blocked replication forks can not be excluded. Both types of effects of BaP were significantly attenuated in dnAhR cells. Transfection of WB-F344 cells with siRNA targeted against AhR decreased induction of Cyclin A induced by BbF or BaP, further supporting the role of AhR in proliferative effects of PAHs. This suggest that activation of AhR plays a significant role both in disruption of contact inhibition by weakly mutagenic PAHs and in genotoxic effects of BaP possibly leading to enhanced cell proliferation. Thus, PAHs may increase proliferative rate and the likelihood of fixation of mutations.     

Screening of ACE-inhibitory peptides from a random peptide-displayed phage library using ACE-coupled liposomes

Angiotensin I converting enzyme (ACE)-inhibitory peptides were screened from a random peptide-displayed phage library using ACE-coupled liposomes. Among four kinds of inhibitory peptides selected by biopanning with two different elution strategies, a peptide (LSTLRSFCA) showed the highest inhibitory activity with an IC(50) value of 3microM. By measuring inhibitory activities of fragments of the peptide, it was found that the RSFCA region was a functional site to inhibit strongly the ACE catalytic activity, and particularly both Arg and Cys residues were essential for the strong inhibitory activity. The inhibitory activity of RRFCA was slightly increased, while that of the RSFRA, in which the Cys residue was replaced by Arg, was decreased to greater extent in comparison with the inhibitory activity of RSFCA. Taking into account the results obtained from the SPOT analysis, it was suggested that the Arg and Phe residues in RSFCA were important for a specific interaction with ACE, and the Cys residue inhibited the ACE activity. The cystein-based ACE-inhibitory peptides have not been isolated from processed food materials. These findings suggested that the biopanning method utilizing protein-coupled liposomes and random peptide libraries might have a possibility to screen new functional peptides that are not found in processed food materials.