Scale‐up of hydrophobin‐assisted recombinant protein production in tobacco BY‐2 suspension cells

Plant suspension cell cultures are emerging as an alternative to mammalian cells for production of complex recombinant proteins. Plant cell cultures provide low production cost, intrinsic safety and adherence to current regulations, but low yields and costly purification technology hinder their commercialization. Fungal hydrophobins have been utilized as fusion tags to improve yields and facilitate efficient low‐cost purification by surfactant‐based aqueous two‐phase separation (ATPS) in plant, fungal and insect cells. In this work, we report the utilization of hydrophobin fusion technology in tobacco bright yellow 2 (BY‐2) suspension cell platform and the establishment of pilot‐scale propagation and downstream processing including first‐step purification by ATPS. Green fluorescent protein‐hydrophobin fusion (GFP‐HFBI) induced the formation of protein bodies in tobacco suspension cells, thus encapsulating the fusion protein into discrete compartments. Cultivation of the BY‐2 suspension cells was scaled up in standard stirred tank bioreactors up to 600 L production volume, with no apparent change in growth kinetics. Subsequently, ATPS was applied to selectively capture the GFP‐HFBI product from crude cell lysate, resulting in threefold concentration, good purity and up to 60% recovery. The ATPS was scaled up to 20 L volume, without loss off efficiency. This study provides the first proof of concept for large‐scale hydrophobin‐assisted production of recombinant proteins in tobacco BY‐2 cell suspensions.     

Different Requirements for GFRα2-Signaling in Three Populations of Cutaneous Sensory Neurons

Many primary sensory neurons in mouse dorsal root ganglia (DRG) express one or several GFRα’s, the ligand-binding receptors of the GDNF family, and their common signaling receptor Ret. GFRα2, the principal receptor for neurturin, is expressed in most of the small nonpeptidergic DRG neurons, but also in some large DRG neurons that start to express Ret earlier. Previously, GFRα2 has been shown to be crucial for the soma size of small nonpeptidergic nociceptors and for their target innervation of glabrous epidermis. However, little is known about this receptor in other Ret-expressing DRG neuron populations. Here we have investigated two populations of Ret-positive low-threshold mechanoreceptors that innervate different types of hair follicles on mouse back skin: the small C-LTMRs and the large Aβ-LTMRs. Using GFRα2-KO mice and immunohistochemistry we found that, similar to the nonpeptidergic nociceptors, GFRα2 controls the cell size but not the survival of both C-LTMRs and Aβ-LTMRs. In contrast to the nonpeptidergic neurons, GFRα2 is not required for the target innervation of C-LTMRs and Aβ-LTMRs in the back skin. These results suggest that different factors drive target innervation in these three populations of neurons. In addition, the observation that the large Ret-positive DRG neurons lack GFRα2 immunoreactivity in mature animals suggests that these neurons switch their GFRα signaling pathways during postnatal development.     

Behavioural responses of naive Arctic charr young to chemical cues from salmonid and non-salmonid fish

The ability to distinguish among chemical cues from multiple predators is of key adaptive value for many prey fish. We examined the attractiveness and repulsiveness of chemical stimuli from different coexisting fish species fed on different diets on the behaviour of hatchery reared Arctic charr young in a Y-maze fluviarum, where the charr could choose between two sides either with control water or stimulus water with fish odour. We used stimuli from (1) matching sized conspecifics, large (2) Arctic charr, (3) salmon, (4) brown trout and (5) brown trout fed on Arctic charr fry. Other salmonids were given pellet food. Additional fish odour treatments included piscivorous (6) pike and (7) burbot. In the control trials both sides received control water. Arctic charr young were expected to respond adaptively to the stimuli from coexisting piscivorous fish. The charr most strongly preferred water with the odour of their matching sized conspecifics, which was the only fish odour they were familiar with before the experiments. They also showed significant preference for other salmonid odours, even though these fish are potential predators on small charr. Chemical stimuli from pike and burbot, on the contrary, were strongly avoided, and burbot odour even prevented the charr to swim and enter the lateral halves of the fluviarum. Moreover, odour from brown trout fed on Arctic charr fry was avoided when compared to stimuli from trout fed on pellets. Although the Arctic charr young were completely naive regarding piscivores, the fact that they could distinguish between different predator taxa and diets on the basis of chemical cues only reflects the long coevolutionary history of these fish populations.     

Isolation and Characterization of a Hantavirus from Lemmus sibiricus: Evidence for Host Switch during Hantavirus Evolution

A novel hantavirus, first detected in Siberian lemmings (Lemmus sibiricus) collected near the Topografov River in the Taymyr Peninsula, Siberia (A. Plyusnin et al., Lancet 347:1835-1836, 1996), was isolated in Vero E6 cells and in laboratory-bred Norwegian lemmings (Lemmus lemmus). The virus, named Topografov virus (TOP), was most closely related to Khabarovsk virus (KBR) and Puumala viruses (PUU). In a cross focus reduction neutralization test, anti-TOP Lemmus antisera showed titers at least fourfold higher with TOP than with other hantaviruses; however, a rabbit anti-KBR antiserum neutralized TOP and KBR at the same titer. The TOP M segment showed 77% nucleotide and 88% amino acid identity with KBR and 76% nucleotide and 82% amino acid identity with PUU. However, the homology between TOP and the KBR S segment was disproportionately higher: 88% at the nucleotide level and 96% at the amino acid level. The 3' noncoding regions of KBR and the TOP S and M segments were alignable except for 113- and 58-nucleotide deletions in KBR. The phylogenetic relationships of TOP, KBR, and PUU and their respective rodent carriers suggest that an exceptional host switch took place during the evolution of these viruses; while TOP and KBR are monophyletic, the respective rodent host species are only distantly related.     

Acute Phase Response in Dairy Cows with Experimentally Induced Escherichia coli Mastitis

Six Finnish Ayrshire cows were challenged intramammarily with 1500 CFU of Escherichia coli (E. coli) into single udder quarters, and the challenge was repeated into contralateral quarters 3 weeks later. All cows received flunixine meglumine once, and 3 of them were also treated with enrofloxacin. At the 2nd challenge, treatments were changed vice versa. The development of mastitis was followed by monitoring of systemic and local clinical signs, and with serial milk and serum samples. Intramarnmary challenge with E. coli produced clinical mastitis in all cows, the severity of the disease varying greatly between the animals. No significant changes between the 2 treatment regimens or sequent challenges were found for any of the clinical parameters. The response of each cow followed the same pattern after both challenges; three of the cows became mildly and the other 3 either moderately or severely affected. Two severely affected cows had to be euthanized because of severe mastitis. Serum haptoglobin and amyloid-A concentrations peaked 2–3 days after bacterial challenge. Serum haptoglobin did not correlate with the severity of the disease. Serum amyloid-A rose gradually in the severely affected cows, and significant differences were found between severely versus moderately or mildly affected cows at day 4. Serum tumor necrosis factor alpha concentrations increased only in the severely affected cows. Serum Cortisol response was prolonged in the severely diseased animals, and was significantly lower after the second challenge. Serum nitrite/nitrate concentration increased in the severely affected cows. This indicated excess nitric oxide production during acute E. coli mastitis. Strongly decreased milk production, and high bacterial growth in the infected quarters were best predictors for the outcome from acute E coli mastitis.     

Non-Cholesterol Sterol Levels Predict Hyperglycemia and Conversion to Type 2 Diabetes in Finnish Men

We investigated the levels of non-cholesterol sterols as predictors for the development of hyperglycemia (an increase in the glucose area under the curve in an oral glucose tolerance test) and incident type 2 diabetes in a 5-year follow-up study of a population-based cohort of Finnish men (METSIM Study, N = 1,050) having non-cholesterol sterols measured at baseline. Additionally we determined the association of 538,265 single nucleotide polymorphisms (SNP) with non-cholesterol sterol levels in a cross-sectional cohort of non-diabetic offspring of type 2 diabetes (the Kuopio cohort of the EUGENE2 Study, N = 273). We found that in a cross-sectional METSIM Study the levels of sterols indicating cholesterol absorption were reduced as a function of increasing fasting glucose levels, whereas the levels of sterols indicating cholesterol synthesis were increased as a function of increasing 2-hour glucose levels. A cholesterol synthesis marker desmosterol significantly predicted an increase, and two absorption markers (campesterol and avenasterol) a decrease in the risk of hyperglycemia and incident type 2 diabetes in a 5-year follow-up of the METSIM cohort, mainly attributable to insulin sensitivity. A SNP of ABCG8 was associated with fasting plasma glucose levels in a cross-sectional study but did not predict hyperglycemia or incident type 2 diabetes. In conclusion, the levels of some, but not all non-cholesterol sterols are markers of the worsening of hyperglycemia and type 2 diabetes.     

Factors affecting the fruiting of bilberries: an analysis of categorical data set

The present study is an application of categorical data analysis in ecological research. The approach is based on logistic regression following an exploratory graphical analysis. The material was collected in an extensive forest inventory in which a set of observations was made by eye in a stratified random sample of 262 mature upland forest stands in South Finland. The problem was to interpret the variation in the fertility of the tillers of bilberry (Vaccinium myrtillus). In the field, the fertility was recorded as a three-class ordinal variable. The information available for the interpretation included the visually estimated density of the tree crowns, the soil fertility class determined using Cajander's forest site types and the percent cover of V. myrtillus.The GLM framework was employed in successive stages of the data analysis in order to find a model to fit the data. For this, the three-class ordinal response variable was reduced to two classes: stands characterized by (a) sterile and (b) fertile bilberry tillers. Successful prediction of the distribution of these two types of forest stands was achieved with a logistic-regression model by using canopy coverage and soil fertility classes as predictor variables. The generalized linear modelling framework is suitable for studying many ecological problems even when only rough categorical estimates of environmental scalars are available.     

Chronic hepatitis B carriers with null genotypes of glutathione S-transferase M1 and T1 polymorphisms who are exposed to aflatoxin are at increased risk of hepatocellular carcinoma.

This study was carried out to elucidate the effect of glutathione S-transferase (GST) Ml and Tl polymorphisms on the aflatoxin-related hepatocarcinogenesis among chronic carriers of hepatitis B surface antigen (HBsAg). A total of 32 newly diagnosed hepatocellular carcinoma (HCC) cases and 73 age-matched controls selected from a cohort of 4,841 chronic HBsAg carriers who had been followed for 5 years were studied. The level of aflatoxin B1 (AFB1)-albumin adducts in their serum samples collected at the recruitment was examined by competitive enzyme-linked immunosorbance assay, and genotypes of GST M1 and T1 were determined by PCR. There was a dose-response relationship between serum level of AFB1-albumin adducts and risk of HCC. The biological gradients between serum AFB1-albumin adducts level and HCC risk were observed among chronic HBsAg carriers who had null genotypes of GST M1 and/or T1 but not among those who had non-null genotypes. The multivariate-adjusted odds ratios of developing HCC for those who had low and high serum levels of AFB1-albumin adducts compared with those who had a undetectable adduct level as the referent (odds ratio = 1.0) were 4.1 and 12.4, respectively, for HBsAg carriers with null GST M1 genotype (P < .01, on the basis of the significance test for trend); 0.7 and 1.4 for those with non-null GST Ml genotype (P = .98); 1.8 and 10.2 for those with null GST T1 genotype (P < .05); and 1.3 and 0.8 for those with non-null GST T1 genotype (P = .93). The interaction between serum AFB1-albumin adduct level and polymorphisms of GST M1 and T1 was at marginal statistical significance levels (.05 < P < .10).