Separation of recombinant antibodies from DNA using divalent cations

New downstream methods for the purification of antibodies are required to meet the demands of current and future antibody applications, e.g. for mass production as cancer therapeutic. The standard chromatographic methods suffer from high material costs and mass transfer limitations. In this study, we established and characterized a method for DNA precipitation for antibody purification using divalent cations, particularly CaCl₂, using four different antibodies. By implementing high‐throughput screening using a factorial design plan, we determined that CaCl₂ concentration and PO₄^3? concentration were significant factors, while temperature and pH were not significant. We detected DNA precipitation as well as host‐cell protein (HCP) reduction. Two‐dimensional difference gel electrophoresis (2D‐DIGE) revealed that improved HCP removal does not occur via an unspecific random mechanism such as the enclosure of proteins in the precipitate. CaCl₂ precipitation of DNA and HCP can be combined with nonchromatographic methods such as precipitation and protein A affinity chromatography. This additional purification method not only enhances DNA removal, but also the removal of HCP and antibody multimers, which will reduce immunogenicity and increase homogeneity of the resulting drug.     

Phagocytosis of the protozoan parasite, Marteilia sydneyi, by Sydney rock oyster (Saccostrea glomerata) hemocytes

QX disease is a fatal disease in Sydney rock oysters caused by the protozoan parasite Marteilia sydneyi. The current study investigates the phagocytosis of M. sydneyi by Sydney rock oyster hemocytes. It also compares the in vitro phagocytic activities of hemocytes from oysters bred for QX disease resistance (QXR) with those of wild-type oysters. After ingestion of M. sydneyi, hemocyte granules fused with phagosome membranes and the pH of phagosomes decreased. Significantly (p = <0.05) more phagosomes in QXR hemocytes showed obvious changes in pH within 40 min of phagocytosis, when compared with wild-type hemocytes. Phenoloxidase deposition was also evident in phagosomes after in vitro phagocytosis. Most importantly, ingested and melanised M. sydneyi were detected in vivo among hemocytes from infected oysters. Overall, the data suggest that Sydney rock oyster hemocytes can recognise and phagocytose M. sydneyi, and that resistance against QX disease may be associated with enhanced phagolysosomal activity in QXR oysters.     

Study of calix[4]resorcinarene-dopamine complexation in mixed phospholipid monolayers formed at the air-water interface

We have studied the physical properties of monolayers formed by calix[4]resorcinarene and in mixtures with dipalmitoyl phosphatidylcholine (DPPC) in various molar ratios formed at the air-water interface and at presence of dopamine in water subphase by means of measurements of surface pressure and dipole potential. We showed that both calix[4]resorcinarene as well as its mixture with DPPC form stable monolayers at the water subphase. The presence of dopamine resulted in an increase of the mean molecular area and in a decrease of the compressibility modulus of the monolayers. For mixed monolayers at higher content of calix[4]resorcinarene (> 0.2 molar fraction) a deviation from ideal miscibility took place especially for monolayers in a solid state. This can be connected with formation of aggregates of calix[4] resorcinarene. Lowest miscibility and weakest interaction of dopamine with a monolayer was observed for calix[4]resorcinarene molar fraction of 0.33 in the monolayer.     

Lipid class dynamics during larval ontogeny of sea scallops, Placopecten magellanicus, in relation to metamorphic success and response to antibiotics

This study examines ontogenetic changes in lipid class composition in relation to survival and growth of five batches of sea scallops, Placopecten magellanicus, from egg to dissoconch stages. It also investigates the effects of antibiotic addition during metamorphosis to determine whether transient treatment during this critical period is effective in increasing metamorphic success. The top-performing larvae (growth rate: 4.7-5.0 μm day^− 1, dissoconch survival: 32-57%) were characterized by a pronounced increase in absolute (ng larva^− 1) and relative concentrations (wt.% of total lipid) of triacylglycerol (TAG) during the pre-metamorphic period, followed by utilization during metamorphosis (0.5-1.4 μg day^− 1 larva^− 1). In contrast, the low-performing scallops (growth rate: 3.6-4.5 μm day^− 1, dissoconch survival < 1%) exhibited a constant, low level of TAG. These results strongly suggest that the accumulation of TAG during the pre-metamorphic period is a good predictor of scallop performance as measured by survival to dissoconch stage. Antibiotic treatment enhanced absolute and relative TAG concentrations in pre-metamorphic scallops and resulted in higher post-metamorphic survival and TAG concentration than in non-treated controls. However, the proportion of dissoconch to live larvae at the end of the experiment was significantly lower with antibiotic treatment (8% vs. 20% in controls), thus resulting in comparable yields of dissoconch larvae ∼ 8% irrespective of treatment. A possible negative effect of antibiotic treatment on bacterial induction of settlement and metamorphosis of sea scallop larvae is suggested.     

Filtration and respiration of the deep living bivalve Acesta excavata (J.C. Fabricius, 1779) (Bivalvia; Limidae)

Here we report the first study of clearance rate and respiration rate of a deep living bivalve, Acesta excavata (J.C. Fabricius, 1779) (Mollusca: Limidae). We found that A. excavata had extreme values both for clearance and respiration rates compared to other bivalves. It has the second largest clearance rate ever reported, 13.36 l h^− 1 g^− 1, and the second lowest value of respiration rate, 0.12 ml O₂ h^− 1 g^− 1. The gill area of 7063 mm² g^− 1 is one of the largest found in bivalves so far. We suggest that these values indicate a physiological adaptation to the low and irregular food supply in the deep sea rather that a specific adaptation to depth.     

Heterogeneity and tissue specificity of tropomyosin isoforms from four species of bivalves

Heterogeneity and tissue specificity of tropomyosin isoforms obtained from four species of bivalves (Scapharca broughtonii (ark shell), Mytilus galloprovincialis (mussel), Atrina pectinata (surf clam) and Crassostrea gigas (Pacific oyster)), were examined. Tropomyosins were extracted from translucent and opaque portions of posterior adductor muscle, respectively, and cardiac muscle of each bivalve. There were two tropomyosin isoforms in the ark shell, the surf clam and the Pacific oyster. They were designated as TMa and TMb. In the ark shell, TMa was the common isoform and TMb was specific for the opaque portion of the adductor muscle. In the surf clam, TMb was the common isoform present in all tissues. TMa was found only in the translucent portion of muscle. In the Pacific oyster, TMb was the major component in both portions of adductor muscle and TMa was the major component in cardiac muscle, although both tropomyosins were included in all tissues. The mussel had only one tropomyosin.     

Feeding activity of scallops and mussels measured simultaneously in the field: Repeated measures sampling and implications for modelling

Many studies on the biology of bivalves have focused on identifying the importance of various environmental factors in regulating feeding behaviour in order to predict ingestion and growth responses in a variety of habitats. When taking into consideration the diversity of studies undertaken and the variety of methodologies employed, it is perhaps not surprising that there have been inconsistencies in both interspecific and intraspecific comparisons. In order to compare and contrast the way in which feeding activities of a mussel species (Mytilus edulis) and a scallop species (Placopecten magellanicus) respond to environmental factors and seston characteristics, we exposed both species simultaneously to a fluctuating assemblage of natural particles found in their local environment. Similar sizes of mussels (40-73 mm) and scallops (40-88 mm) were held in flow through chambers at two different sites in Atlantic Canada on four separate occasions, during which environmental conditions, including seston characteristics, were monitored and individual clearance rates were measured. Because the same individuals were measured over time, repeated-measures statistical procedures were used to analyze the data. Results indicate that weight standardized clearance rates of mussels were significantly higher than those of scallops only at the highest concentration tested (12.8 mg l^− 1), which translates into almost 3 times more material being filtered. Scallops appeared to respond to fluctuations in environment parameters and suspended food particles more consistently than mussels. These data suggest that the feeding behaviour of these two species is mediated differently by the same environmental parameters. Our study also demonstrates the importance of selecting appropriate statistical tests (e.g., repeated measures) for the analyses of data obtained from repeated sampling of the same individuals over time, and the need to consider species-specific feeding responses when developing models that predict the influence of bivalve feeding on ecosystem function.     

Modulatory effects of hard clam (Mercenaria mercenaria) tissue extracts on the in vitro growth of its pathogen QPX

Quahog parasite unknown (QPX) is a fatal protistan parasite affecting cultured and wild hard clams Mercenaria mercenaria along the northeastern coasts of the USA and maritime Canada. Field investigations and laboratory transmission studies revealed some variations in the susceptibility of different hard clam stocks to QPX infection. In this study, we used in vitro QPX cultures to investigate the effect of plasma and tissue extracts from two different clam stocks on parasite survival and growth. Results demonstrated the presence of factors in clams that significantly modulate QPX growth. Extracts from gills and mantle tissues as well as plasma inhibited in vitro QPX growth, whereas foot and adductor muscle extracts enhanced parasite growth. Investigations of anti-QPX activities in plasma from two clam stocks displaying different susceptibility toward QPX disease in vivo demonstrated higher inhibition of QPX growth by plasma from New York (resistant) clams compared to Florida (susceptible) clams. Some clams appeared to be deficient in inhibitory factors, suggesting that such animals may become more easily infected by the parasite.     

Neural-specific Deletion of FIP200 Leads to Cerebellar Degeneration Caused by Increased Neuronal Death and Axon Degeneration

FIP200 (FAK family-interacting protein of 200 kDa) is a conserved protein recently identified as a potential mammalian counterpart of yeast autophagy protein Atg17. However, it remains unknown whether mammalian FIP200 regulates autophagy in vivo. Here we show that neural-specific deletion of FIP200 resulted in cerebellar degeneration accompanied by progressive neuronal loss, spongiosis, and neurite degeneration in the cerebellum. Furthermore, deletion of FIP200 led to increased apoptosis in cerebellum as well as accumulation of ubiquitinated protein aggregates without any deficiency in proteasome catalytic functions. We also observed an increased p62/SQSTM1 accumulation in the cerebellum and reduced autophagosome formation as well as accumulation of damaged mitochondria in the mutant mice. Lastly, analysis of cerebellar neurons in vitro showed reduced JNK activation and increased susceptibility to serum deprivation-induced apoptosis in cerebellar neurons from the mutant mice. Taken together, these results provide strong genetic evidence for a role of FIP200 in the regulation of neuronal homeostasis through its function in autophagy in vivo.     

Stacking Increments: A New Model and Morphospace for the Analysis of Bivalve Shell Growth

A model employing stacking increments is introduced for the analysis of bivalve shell growth and form. The model is based on the components of shell growth that are potentially independent: the rate of mantle cell proliferation, the rate of precipitation of shell material, and the rate of translation of the pallial line, where the mantle is attached to the shell. This model is defined in terms of the following parameters: (1) the ratio of accretion of shell material at the shell margin to growth of the mantle by cell division, (2) the ratio of shell accretion at the pallial line to mantle growth, and (3) the ratio of the amount of pallial muscle translation, away from the umbo toward the shell margin, to mantle growth. In this model, the shape of a radial section through the shell is simulated by stacking of internal microgrowth increments. The mode of stacking of the increments is determined by the balance among the parameters defining growth. A theoretical morphospace defined on the basis of this model is largely consistent with the range of forms of naturally occurring bivalve shells. Analysis of the distribution of actual shell forms in relation to this morphospace suggests that the absolute rate of shell precipitation and the gradient in precipitation rate away from the shell margin along a radial cross-section are physiologically as well as geometrically constrained.