Continuous counter‐current chromatography for capture and polishing steps in biopharmaceutical production

The economic advantages of continuous processing of biopharmaceuticals, which include smaller equipment and faster, efficient processes, have increased interest in this technology over the past decade. Continuous processes can also improve quality assurance and enable greater controllability, consistent with the quality initiatives of the FDA. Here, we discuss different continuous multi‐column chromatography processes. Differences in the capture and polishing steps result in two different types of continuous processes that employ counter‐current column movement. Continuous‐capture processes are associated with increased productivity per cycle and decreased buffer consumption, whereas the typical purity‐yield trade‐off of classical batch chromatography can be surmounted by continuous processes for polishing applications. In the context of continuous manufacturing, different but complementary chromatographic columns or devices are typically combined to improve overall process performance and avoid unnecessary product storage. In the following, these various processes, their performances compared with batch processing and resulting product quality are discussed based on a review of the literature. Based on various examples of applications, primarily monoclonal antibody production processes, conclusions are drawn about the future of these continuous‐manufacturing technologies.     

Isolation of neural crest-derived stem cells from rat embryonic mandibular processes

BACKGROUND INFORMATION: Substantial evidence indicates the existence of NCSCs (neural crest-derived stem cells) in embryonic mandibular processes; however, they have not been fully investigated or isolated. The aim of the present study was to isolate stem cells from mandibular process during embryonic development by MACS (magnetic-activated cell sorting). The findings show that the cells are multipotent and self-renewing. RESULTS: LNGFR (low-affinity nerve-growth-factor receptor)+ cells were isolated from rat embryonic mandibular processes by MACS. The cells were grown in clonal culture by limiting dilution to assess their developmental potential. Clone analysis indicated that, first, LNGFR+ cells are multipotent, being able to generate at least neurons and Schwann cells, similar to peripheral neural crest stem cells. Secondly, multipotent LNGFR+ cells generate multipotent progenies, indicating that they are capable of self-renewal and therefore are stem cells. Thirdly, manipulation of the medium supplementation alters the fate of the isolated LNGFR+ cells. CONCLUSIONS: These results indicate that LNGFR antibodies label NCSCs with high specificity and purity, and suggest that positive selection using these antibodies may become the method of choice for obtaining multipotent cells from rat embryonic mandibular processes for tissue engineering or regenerative therapeutic use.     

Functional analysis of Toll-related genes in Drosophila

The Drosophila genome encodes a total of nine Toll and related proteins. The immune and developmental functions of Toll and 18Wheeler (18W) have been analyzed extensively, while the in vivo functions of the other Toll-related proteins require further investigation. We performed transgenic experiments and found that overexpression of Toll-related genes caused different extents of lethality and developmental defects. Moreover, 18w, Toll-6, Toll-7 and Toll-8 often caused related phenotypic changes, consistent with the idea that these four genes have more conserved molecular structure and thus may regulate similar processes in vivo. Deletion alleles of Toll-6, Toll-7 and Toll-8 were generated by targeted homologous recombination or P element excision. These mutant alleles were viable, fertile, and had no detectable defect in the inducible expression of antimicrobial peptide genes except for the Toll-8 mutant had some defects in leg development. The expression of 18w, Toll-7 and Toll-8 mRNA showed wide and overlapping patterns in imaginal discs and the 18w, Toll-8 double and Toll-7, Toll-8 double mutants showed substantially increased lethality. Overall our results suggest that some of the Toll-related proteins, such as 18W, Toll-7 and Toll-8, may have redundant functions in regulating developmental processes.     

Recent advances in understanding the mechanisms of osteoclast precursor fusion

Bone marrow macrophages fuse on the bone surface to form multinucleated osteoclasts that then organize to efficiently resorb bone. Many, if not all, of the stages of macrophage fusion involve cytoskeletal components that reorganize the cells. Recruitment may involve chemotactic responses to bone matrix protein and calcium ion gradients and/or chemokine production by bone forming osteoblasts. The roles of integrins vary, depending on the particular subunits with some interfering with fusion and others having a participatory role. RANKL is essential for fusion and many identified modulators of fusion influence RANKL signaling pathways. Tetraspanins have been implicated in fusion of macrophages and myoblasts, but differences in impacts exist between these two cell types. Macrophage recruitment to apoptotic cells prior to their engulfment is driven by the exposed phospholipids on the external surface of the apoptotic cells and there is evidence that this same identification mechanism is employed in macrophage fusion. Because loss of cadherin or ADAM family members suppresses macrophage fusion, a crucial role for these membrane glycoproteins is evident. The Ig membrane glycoprotein superfamily members CD200 and MFR/SIRPα are involved in macrophage fusion, although their influences are unresolved. Differential screenings have identified the structurally related membrane proteins DC‐STAMP and OC‐STAMP as required components for fusion and the contributions to fusion remain active areas of investigation. While many of the key components involved in these processes have been identified, a great deal of work remains in resolving the precise processes involved and the interactions between key contributors to multinucleated osteoclast formation. J. Cell. Biochem. 110: 1058–1062, 2010. Published 2010 Wiley‐Liss, Inc.     

Resistance of the insect cell line IPLB-LdFB to salsolinol-induced apoptosis.

Apoptosis is a form of cell death that is manifested in Parkinson's disease (PD) and certain other neurodegenerative disorders. Metabolites of salsolinol (SAL), an intraneuronal, dopamine-derived tetrahydroisoquinoline (TIQ), have been shown to induce apoptosis in human dopaminergic neuroblastoma cells, implicating these molecules as causative or contributory factors in the selective killing of nigrostriatal dopaminergic neurons, a cardinal manifestation of Parkinson's disease. Since insects employ dopamine and related catecholamines in a variety of processes including cuticular sclerotization and cellular immune reactions, it was of interest to know how insect cells metabolized exogenous SAL. Propidium iodide staining combined with flow cytometry showed that IPLB-LdFB cells from Lymantria dispar exhibited no significant (P < 0.05) increase in apoptosis when incubated for 48 h with concentrations of SAL ranging from 10 microM to 1 mM. A significant increase in apoptosis (P < 0.05) was observed in cell cultures containing the highest concentration of SAL tested (5 mM), but only 12.4% of the cells manifested this form of cell death. High pressure liquid chromatography with electrochemical detection (HPLC-ED) was used to document the production of two potentially cytotoxic quinonoids generated during the autoxidation of SAL, a reaction that was found to be significantly (P < 0.05) enhanced by peroxidase. The resistance of IPLB-LdFB cells to SAL-induced apoptosis is attributed to the ability of these insect cells to metabolize and/or detoxify such dopamine-derived catecholic TIQs. Thus, the biochemical pathways employed by insect cells in these processes may be of considerable interest to individuals investigating certain neurodegenerative disorders.     

Potential of cell retention techniques for large-scale high-density perfusion culture of suspended mammalian cells

This review focuses on cultivation of mammalian cells in a suspended perfusion mode. The major technological limitation in the scaling-up of these systems is the need for robust retention devices to enable perfusion of medium as needed. For this, cell retention techniques available to date are presented, namely, cross-flow filters, hollow fibers, controlled-shear filters, vortex-flow filters, spin-filters, gravity settlers, centrifuges, acoustic settlers, and hydrocyclones. These retention techniques are compared and evaluated for their respective advantages and potential for large-scale utilization in the context of industrial manufacturing processes. This analysis shows certain techniques have a limited range of perfusion rate where they can be implemented (most microfiltration techniques). On the other hand, techniques were identified that have shown high perfusion capacity (centrifuges and spin-filters), or have a good potential for scale-up (acoustic settlers and inclined settlers). The literature clearly shows that reasonable solutions exist to develop large-scale perfusion processes.     

Bayesian analysis of wildlife age-at-harvest data

SUMMARY: State and federal natural resource management agencies often collect age-structured harvest data. These data represent finite realizations of stochastic demographic and sampling processes and have long been used by biologists to infer population trends. However, different sources of data have been combined in ad hoc ways and these methods usually failed to incorporate sampling error. In this article, we propose a "hidden process" (or state-space) model for estimating abundance, survival, recovery rate, and recruitment from age-at-harvest data that incorporate both demographic and sampling stochasticity. To this end, a likelihood for age-at-harvest data is developed by embedding a population dynamics model within a model for the sampling process. Under this framework, the identification of abundance parameters can be achieved by conducting a joint analysis with an auxiliary data set. We illustrate this approach by conducting a Bayesian analysis of age-at-harvest and mark-recovery data from black bears (Ursus americanus) in Pennsylvania. Using a set of reasonable prior distributions, we demonstrate a substantial increase in precision when posterior summaries of abundance are compared to a bias-corrected Lincoln-Petersen estimator. Because demographic processes link consecutive abundance estimates, we also obtain a more realistic biological picture of annual changes in abundance. Because age-at-harvest data are often readily obtained, we argue that this type of analysis provides a valuable strategy for wildlife population monitoring.     

Direct interaction of actin filaments with F‐BAR protein pacsin2

Two mechanisms have emerged as major regulators of membrane shape: BAR domain‐containing proteins, which induce invaginations and protrusions, and nuclear promoting factors, which cause generation of branched actin filaments that exert mechanical forces on membranes. While a large body of information exists on interactions of BAR proteins with membranes and regulatory proteins of the cytoskeleton, little is known about connections between these two processes. Here, we show that the F‐BAR domain protein pacsin2 is able to associate with actin filaments using the same concave surface employed to bind to membranes, while some other tested N‐BAR and F‐BAR proteins (endophilin, CIP4 and FCHO2) do not associate with actin. This finding reveals a new level of complexity in membrane remodeling processes.     

Historical demography of four gecko species specializing in boulder cave habitat: Implications in the evolutionary dead end hypothesis and conservation

Specialization in narrow ecological niches may not only help species to survive in competitive or unique environments but also contribute to their extermination over evolutionary time. Although the “evolutionary dead end” hypothesis has long been debated, empirical evidence from species with detailed information on niche specialization and evolutionary history remains rare. Here we use a group of four closely related Cnemaspis gecko species that depend highly on granite boulder caves in the Mekong Delta to investigate the potential impact of ecological specialization on their evolution and population dynamics. Isolated by unsuitable floodplain habitats, these boulder‐dwelling geckos are among the most narrowly distributed Squamata in the world. We applied several coalescence‐based approaches combined with the RAD‐seq technique to estimate their divergence times, gene flow and demographic fluctuations during the speciation and population differentiation processes. Our results reveal long‐term population shrinkage in the four geckos and limited gene flow during their divergence. The results suggest that the erosion and fragmentation of the granite boulder hills have greatly impacted population divergence and declines. The habitat specialization of these geckos has led to fine‐scaled speciation in these granite rocky hills; in contrast, specialization might also have pushed these species toward the edge of extinction. Our study also emphasizes the conservation urgency of these vulnerable, cave‐dependent geckos.     

Phylogenomics unravels Quaternary vicariance and allopatric speciation patterns in temperate‐montane plant species: A case study on the Ranunculus auricomus species complex

The time frame and geographical patterns of diversification processes in European temperate‐montane herbs are still not well understood. We used the sexual species of the Ranunculus auricomus complex as a model system to understand how vicariance versus dispersal processes in the context of Pleistocene climatic fluctuations have triggered speciation in temperate‐montane plant species. We used target enrichment sequence data from about 600 nuclear genes and coalescent‐based species tree inference methods to resolve phylogenetic relationships among the sexual taxa of the complex. We estimated absolute divergence times and, using ancestral range reconstruction, we tested if speciation was enhanced by vicariance or by dispersal processes. Phylogenetic relationships among taxa were fully resolved with some incongruence in the position of the tetraploid R. marsicus. Speciation events took place in a very short time at the end of the Mid‐Pleistocene Transition (830–580 thousand years ago [ka]). A second wave of intraspecific geographical differentiation occurred at the end of the Riss glaciation or during the Eemian interglacial between 200 and 100 ka. Ancestral range reconstruction suggests a widespread European ancestor of the R. auricomus complex. Vicariance has triggered allopatric speciation in temperate‐montane plant species during the climatic deterioration that occurred during the last phase of the Mid‐Pleistocene Transition. Vegetation restructuring from forest into tundra could have confined these forest species into isolated glacial macro‐ and microrefugia. During subsequent warming periods, range expansions of these species could have been hampered by apomictic derivatives and by other congeneric competitors in the same habitat.     

New oxa-bridged macrocycles

In creating new aroma molecules, the fragrance chemist can make use of several tools: receptor or combinatorial research as well as lead structure optimization of existing chemicals or substances from the natural pool. Sometimes, it is also possible to discover new structures via another way: the careful analysis of existing products and their production processes. In analyzing the production process of 1-oxacyclohexadecan-2-one (6), we identified at least two new oxa-bridged macrocyclic molecules. In continuation, these results inspired us to synthesize and evaluate more representatives with similar structures. In this contribution, presented at the RSC/SCI conference 'flavours & fragrances 2007' in London, September 24-26, 2007, the synthesis and olfactory properties of several new oxa-bridged macrocycles will be introduced and discussed.     

Multiple biogeographical barriers identified across the monsoon tropics of northern Australia: phylogeographic analysis of the brachyotis group of rock-wallabies

The monsoon tropics of northern Australia are a globally significant biodiversity hotspot, but its phylogeography is poorly known. A major challenge for this region is to understand the biogeographical processes that have shaped the distribution and diversity of taxa, without detailed knowledge of past climatic and environmental fluctuations. Although molecular data have great potential to address these questions, only a few species have been examined phylogeographically. Here, we use the widely distributed and abundant short-eared rock-wallaby (Petrogale brachyotis; n = 101), together with the sympatric monjon (P. burbidgei; n = 11) and nabarlek (P. concinna; n = 1), to assess historical evolutionary and biogeographical processes in northern Australia. We sequenced ～1000 bp of mitochondrial DNA (control region, ND2) and ～3000 bp of nDNA (BRCA1, ω-globin and two anonymous loci) to investigate phylogeographic structuring and delineate the time-scale of diversification within the region. Our results indicate multiple barriers between the Top End (Northern Territory) and Kimberley (Western Australia), which have caused divergence throughout the Plio-Pleistocene. Eight geographically discrete and genetically distinct lineages within the brachyotis group were identified, five of which are separated by major river valleys (Ord, Victoria, Daly), arid lowlands and discontinuous sandstone ranges. It is likely that these barriers have similarly influenced genetic structure in other monsoonal biota.     

Parallel declines in survival of adult Northern Fulmars Fulmarus glacialis at colonies in Scotland and Ireland

Assessing broad‐scale changes in seabird populations across the North Atlantic requires an integration of available datasets to understand the spatial extent of potential drivers and demographic change. Here, we compared survival of Northern Fulmars Fulmarus glacialis from a Scottish and an Irish colony from 1974 to 2009. Despite lower recapture probabilities of monel‐ringed Irish birds compared with colour‐ringed Scottish birds, survival probability decreased at both colonies. The extent to which the decline in survival is related to density‐dependent processes or other external drivers remains uncertain, but our results suggest that these changes in survival are possibly indicative of larger‐scale processes and are not confined to local colony dynamics.     

Measuring the hierarchy of molecular events during clathrin-mediated endocytosis

A well-orchestrated hierarchy of molecular events is required for successful initiation and maturation of clathrin-coated pits (CCPs). Nevertheless, CCPs display a broad range of lifetimes. This dynamic heterogeneity could either reflect differences in the temporal hierarchy of molecular events, or similar CCP maturation processes with variable kinetics. To address this question, we have used multi-channel image acquisition and automated analysis of CCP dynamics in combination with a new method to quantify the time courses of recruitment of endocytic factors to CCPs of different lifetimes. Using this approach we have extracted the kinetics of recruitment and disassembly of fluorescently labeled clathrin and/or AP-2 throughout the entire lifetime of temporally defined CCP cohorts. On the basis of these analyses, we can (i) directly correlate recruitment profiles of these two proteins; (ii) define five distinct CCP maturation phases, i.e. initiation, growth, maturation, separation and departure; (iii) distinguish events with absolute versus fractional timing and (iv) provide information on the spatial distribution of fluorophores during CCP maturation. Emerging from these analyses is a more clearly defined role for AP-2 in determining the temporal hierarchy for clathrin recruitment and CCP maturation. This method provides a new means to identify other such hierarchies during CCP maturation.     

Community assembly in Lake Tanganyika cichlid fish: quantifying the contributions of both niche‐based and neutral processes

The cichlid family features some of the most spectacular examples of adaptive radiation. Evolutionary studies have highlighted the importance of both trophic adaptation and sexual selection in cichlid speciation. However, it is poorly understood what processes drive the composition and diversity of local cichlid species assemblages on relatively short, ecological timescales. Here, we investigate the relative importance of niche‐based and neutral processes in determining the composition and diversity of cichlid communities inhabiting various environmental conditions in the littoral zone of Lake Tanganyika, Zambia. We collected data on cichlid abundance, morphometrics, and local environments. We analyzed relationships between mean trait values, community composition, and environmental variation, and used a recently developed modeling technique (STEPCAM) to estimate the contributions of niche‐based and neutral processes to community assembly. Contrary to our expectations, our results show that stochastic processes, and not niche‐based processes, were responsible for the majority of cichlid community assembly. We also found that the relative importance of niche‐based and neutral processes was constant across environments. However, we found significant relationships between environmental variation, community trait means, and community composition. These relationships were caused by niche‐based processes, as they disappeared in simulated, purely neutrally assembled communities. Importantly, these results can potentially reconcile seemingly contrasting findings in the literature about the importance of either niche‐based or neutral‐based processes in community assembly, as we show that significant trait relationships can already be found in nearly (but not completely) neutrally assembled communities; that is, even a small deviation from neutrality can have major effects on community patterns.     

HIV-1, ubiquitin and ubiquitin-like proteins: the dialectic interactions of a virus with a sophisticated network of post-translational modifications

The modification of intracellular proteins by ubiquitin (Ub) and ubiquitin-like (UbL) proteins is a central mechanism for regulating and fine-tuning all cellular processes. Indeed, these modifications are widely used to control the stability, activity and localisation of many key proteins and, therefore, they are instrumental in regulating cellular functions as diverse as protein degradation, cell signalling, vesicle trafficking and immune response. It is thus no surprise that pathogens in general, and viruses in particular, have developed multiple strategies to either counteract or exploit the complex mechanisms mediated by the Ub and UbL protein conjugation pathways. The aim of this review is to provide an overview on the intricate and conflicting relationships that intimately link HIV-1 and these sophisticated systems of post-translational modifications.     

Cellular functions and intrinsic attributes of the ATP‐binding Eps15 homology domain‐containing proteins

Several cellular processes rely on a cohort of dedicated proteins that manage tubulation, fission, and fusion of membranes. A notably large number of them belong to the dynamin superfamily of proteins. Among them is the evolutionarily conserved group of ATP‐binding Eps15‐homology domain‐containing proteins (EHDs). In the two decades since their discovery, EHDs have been linked to a range of cellular processes that require remodeling or maintenance of specific membrane shapes such as during endocytic recycling, caveolar biogenesis, ciliogenesis, formation of T‐tubules in skeletal muscles, and membrane resealing after rupture. Recent work has shed light on their structure and the unique attributes they possess in linking ATP hydrolysis to membrane remodeling. This review summarizes some of these recent developments and reconciles intrinsic protein functions to their cellular roles.     

Presentation of Phagocytosed Antigens by MHC Class I and II

Phagocytosis provides innate immune cells with a mechanism to take up and destroy pathogenic bacteria, apoptotic cells and other large particles. In some cases, however, peptide antigens from these particles are preserved for presentation in association with major histocompatibility complex (MHC) class I or class II molecules in order to stimulate antigen‐specific T cells. Processing and presentation of antigens from phagosomes presents a number of distinct challenges relative to antigens internalized by other means; while bacterial antigens were among the first discovered to be presented to T cells, analyses of the cellular mechanisms by which peptides from phagocytosed antigens assemble with MHC molecules and by which these complexes are then expressed at the plasma membrane have lagged behind those of conventional model soluble antigens. In this review, we cover recent advances in our understanding of these processes, including the unique cross‐presentation of phagocytosed antigens by MHC class I molecules, and in their control by signaling modalities in phagocytic cells.     

Recent Progress on Spray Pyrolysis for High Performance Electrode Materials in Lithium and Sodium Rechargeable Batteries

Advanced electrode materials have been intensively explored for next‐generation lithium‐ion batteries (LIBs), and great progresses have been achieved for many potential candidates at the lab‐scale. To realize the commercialization of these materials, industrially‐viable synthetic approaches are urgently needed. Spray pyrolysis (SP), which is highly scalable and compatible with on‐line continuous production processes, offers great fidelity in synthesis of electrode materials with complex architectures and chemistries. In this review, motivated by the rapid advancement of the given technology in the battery area, we have summarized the recent progress on SP for preparing a great variety of anode and cathode materials of LIBs with emphasis on their unique structures generated by SP and how the structures enhanced the electrochemical performance of various electrode materials. Considering the emerging popularity of sodium‐ion batteries (SIBs), recent electrode materials for SIBs produced by SP will also be discussed. Finally, the powerfulness and limitation along with future research efforts of SP on preparing electrode materials are concisely provided. Given current worldwide interests on LIBs and SIBs, we hope this review will greatly stimulate the collaborative efforts among different communities to optimize existing approaches and to develop innovative processes for preparing electrode materials.