Large-scale rotating filter perfusion system for high-density growth of mammalian suspension cultures

Summary A system has been developed for growth and maintenance of mammalian cells in suspension culture at high density. In principle, the maintenance of constant levels of required nutrients coupled with the removal of toxic cell byproducts can support much higher suspension cell densities than may be obtained in conventional spinners. The system consisted of 4- or 40-liter reaction vessels equipped with a vertically supported rotating cylindrical filter. Agitation was provided by the magnetically driven, rotating filter. Fresh medium was supplied at a rate of 10 to 20 ml/h per 10⁹ cells and the expended medium free of cells was withdrawn through the rotating filter. Both pH and dissolved O₂ and CO₂ were monitored and regulated. Walker 256 carcinosarcoma cells have been grown in these reactors to densities 10-to 30-fold greater than that obtained in Bellco spinners. In addition to high cell densities, the yield of cells per liter of medium used was 2- to 3-fold that obtained in the conventional systems. Both 4-and 40-liter versions of this reactor have been operated without the use of antibiotics. The 40-liter reactor also has been modified for chemostat operation. In a single run, for example, the Walker cell density was maintained between 6 and 10×10⁶ cells/ml with a total yield of 8.7×10¹¹ cells from 360 liters of medium.     

Metal Concentrations in Cerebrospinal Fluid and Blood Plasma from Patients with Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal degenerative disorder of motor neurons. The cause of this degeneration is unknown, and different causal hypotheses include genetic, viral, traumatic and environmental mechanisms. In this study, we have analyzed metal concentrations in cerebrospinal fluid (CSF) and blood plasma in a well-defined cohort (n?=?17) of ALS patients diagnosed with quantitative electromyography. Metal analyses were performed with high-resolution inductively coupled plasma mass spectrometry. Statistically significant higher concentrations of manganese, aluminium, cadmium, cobalt, copper, zinc, lead, vanadium and uranium were found in ALS CSF compared to control CSF. We also report higher concentrations of these metals in ALS CSF than in ALS blood plasma, which indicate mechanisms of accumulation, e.g. inward directed transport. A pattern of multiple toxic metals is seen in ALS CSF. The results support the hypothesis that metals with neurotoxic effects are involved in the pathogenesis of ALS.     

SorLA Controls Neurotrophic Activity by Sorting of GDNF and Its Receptors GFRα1 and RET

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Highlights? SorLA is a sorting receptor for GDNF and its signaling receptors GFRa1 and RET ? The SorLA/GFRa1 complex targets GDNF for lysosomal degradation, while GFRa1 is recycled ? SorLA/GFRa1 targets RET for endocytosis and influences GDNF-induced neurotrophic effects ? SorLA knockout mice display altered dopaminergic function and an ADHD-like phenotype     

Associations between dru Types and SCCmec Cassettes

Molecular typing is an important tool in the investigation of methicillin resistant Staphylococcus aureus (MRSA) outbreaks and in following the evolution of MRSA. The staphylococcal cassette chromosome mec (SCCmec) contains a hypervariable region with a variable number of 40 bp repeats named direct repeat units (dru). The dru region has been suggested as a supplementary typing method for MRSA and an international nomenclature exists. The purpose of this study was to investigate the diversity and variability of the dru region in a diverse collection of MRSA. We studied 302 MRSA isolates harbouring SCCmec types I to VI. The isolates represented a broad genetic background based on Staphylococcal protein A (spa) typing and multilocus sequence typing (MLST) and included 68 isolates (68 patients) from an outbreak with t024-ST8-IVa and 26 isolates from the same patient. Sequencing identified 53 dru types (dt) in 283 isolates, while eighteen isolates contained no dru repeats and one isolate resisted sequencing. The most common dru type, dt10a, was present in 53% of the sequenced isolates and was found in all SCCmec types, except type II. Seven (10%) of the 68 epidemiologically related patients had isolates with dru type variants indicating that dru typing is not useful as a first line epidemiological typing tool. However, MRSA isolates cultured from a single patient over a three year period exhibited a single dru type. The finding of dt10a in most SCCmec types suggests that dru and mecA originate from the same Staphylococcus species.     

Surveillance of the Sensitivity towards Antiparasitic Bath-Treatments in the Salmon Louse (Lepeophtheirus salmonis)

The evolution of drug resistant parasitic sea lice is of major concern to the salmon farming industry worldwide and challenges sustainable growth of this enterprise. To assess current status and development of L. salmonis sensitivity towards different pesticides used for parasite control in Norwegian salmon farming, a national surveillance programme was implemented in 2013. The programme aims to summarize data on the use of different pesticides applied to control L. salmonis and to test L. salmonis sensitivity to different pesticides in farms along the Norwegian coast. Here we analyse two years of test-data from biological assays designed to detect sensitivity-levels towards the pesticides azamethiphos and deltamethrin, both among the most common pesticides used in bath-treatments of farmed salmon in Norway in later years. The focus of the analysis is on how different variables predict the binomial outcome of the bioassay tests, being whether L. salmonis are immobilized/die or survive pesticide exposure. We found that local kernel densities of bath treatments, along with a spatial geographic index of test-farm locations, were significant predictors of the binomial outcome of the tests. Furthermore, the probability of L. salmonis being immobilized/dead after test-exposure was reduced by odds-ratios of 0.60 (95% CI: 0.42–0.86) for 2014 compared to 2013 and 0.39 (95% CI: 0.36–0.42) for low concentration compared to high concentration exposure. There were also significant but more marginal effects of parasite gender and developmental stage, and a relatively large random effect of test-farm. We conclude that the present data support an association between local intensities of bath treatments along the coast and the outcome of bioassay tests where salmon lice are exposed to azamethiphos or deltamethrin. Furthermore, there is a predictable structure of L. salmonis phenotypes along the coast in the data, characterized by high susceptibility to pesticides in the far north and far south, but low susceptibility in mid Norway. The study emphasizes the need to address local susceptibility to pesticides and the need for restrictive use of pesticides to preserve treatment efficacy.     

Nitrogen Loss and Denitrification as Studied in Relation to Reductions in Nitrogen Loading in a Shallow,Hypertrophic Lake (Lake Søbygård,Denmark)

A detailed mass balance on nitrogen was carried out in shallow and hypertrophic Lake Søbygård during 4.5 years before through 2.5 years after a 36 % reduction in nitrogen loading. Annual mean loss rate of nitrogen was 159–229 mg N m⁻² d⁻¹ before the loading reduction and 125 mg N m⁻² d⁻¹ after. In spite of a short hydraulic retention time (18–27 days) the proportion of nitrogen loading lost in the lake was high (38–53 %) and not affected by changes in loading. Calculated denitrification accounted for 86–93% of the loss rate, while 7–14% was permanently buried. Marked seasonal variations in the loss percentage were found during the season, ranging from 23 % in first quarter to 65 % in third quarter. The seasonal variation in the loss percentage of nitrogen showed a hysteresis like relationship to temperature, with a high percentage in fourth quarter. This suggests that the amount of available substrate, which mainly consists of sedimentated phytoplankton, accumulated during summer, is an important regulating factor. The ability of various published input-output models to predict the observed changes in in-lake nitrogen concentration in Lake Søbygård was tested. This study has further confirmed that small lakes with short retention and high nitrogen loading may significantly reduce the nitrogen loading of downstream aquatic environments.     

Cyclomorphosis in Daphnia lumholtzi induced by temperature

• 1 Cyclomorphosis is a well known phenomenon in Daphnia that involves a regular, seasonal, or induced change in body allometry. Long helmets and tail spines were induced in laboratory cultures of Daphnia lumholtzi with temperature of 31 °C as the proximal cue (temperature of locally occurring peak abundance in Kentucky Lake). The effect was greater in embryos than juveniles or adults exposed to the temperature cue.
• 2 The temperature cue appears to have a threshold value (animals cultured at 25 or 28 °C did not develop elongated helmets or spines). The helmet and spine length receded both with D. lumholtzi kept at a constant 31 °C temperature and when water temperature was decreased.
• 3 The induced helmet in this experiment (0.66 mm, 1.0 mm animal) was significantly longer than values reported in the literature for induction by planktivorous fish kairomones (0.25 mm, 1.2 mm animal). The strong response to a proximal cue of temperature may require the second weaker chemical cue for maintenance. It is suggested that a synergistic explanation with two cues may be more appropriate for cyclomorphosis induction and maintenance in Daphnia lumholtzi that could be tested with further studies.

     

Implications of an invertebrate predator's ( Bythotrephes cederstroemi) atypical effects on a pelagic zooplankton community

The traditional view of predaceous zooplankton is that they prefer small-bodied prey, are hindered by morphological anti-predator defenses, and have a minor influence on zooplankton communities when fish are present. We performed a series of experiments with the large-bodied onychopod (cladoceran) Bythotrephes cederstroemi, in which we incubated this predator with known prey to determine prey preference and predation rates. We also performed an allozyme analysis of prey tissue in the gut of B. cederstroemi collected from several stations around Lake Michigan to determine what prey types are chosen in the field. We found that B. cederstroemi does not fit the standard invertebrate predator mold: adult B. cederstroemi prefer large (>2.0 mm) Daphnia pulicaria over smaller individuals; the elongated tailspine and helmet of Daphnia galeata mendotae are not effective deterrents to B. cederstroemi predation; and B. cederstroemi is a generalist predator with the potential to consume a significant portion of cladoceran production in Lake Michigan. This revised version was published online in July 2006 with corrections to the Cover Date.