N‐linked glycosylation is an important parameter for optimal selection of cell lines producing biopharmaceutical human IgG

We studied the variations in N‐linked glycosylation of human IgG molecules derived from 105 different stable cell lines each expressing one of the six different antibodies. Antibody expression was based on glutamine synthetase selection technology in suspension growing CHO‐K1SV cells. The glycans detected on the Fc fragment were mainly of the core‐fucosylated complex type containing zero or one galactose and little to no sialic acid. The glycosylation was highly consistent for the same cell line when grown multiple times, indicating the robustness of the production and glycan analysis procedure. However, a twofold to threefold difference was observed in the level of galactosylation and/or non‐core‐fucosylation between the 105 different cell lines, suggesting clone‐to‐clone variation. These differences may change the Fc‐mediated effector functions by such antibodies. Large variation was also observed in the oligomannose‐5 glycan content, which, when present, may lead to undesired rapid clearance of the antibody in vivo. Statistically significant differences were noticed between the various glycan parameters for the six different antibodies, indicating that the variable domains and/or light chain isotype influence Fc glycosylation. The glycosylation altered when batch production in shaker was changed to fed‐batch production in bioreactor, but was consistent again when the process was scaled from 400 to 5,000 L. Taken together, the observed clone‐to‐clone glycosylation variation but batch‐to‐batch consistency provides a rationale for selection of optimal production cell lines for large‐scale manufacturing of biopharmaceutical human IgG. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Phasor analysis of multiphoton spectral images distinguishes autofluorescence components of in vivo human skin

Skin contains many autofluorescent components that can be studied using spectral imaging. We employed a spectral phasor method to analyse two photon excited autofluorescence and second harmonic generation images of in vivo human skin. This method allows segmentation of images based on spectral features. Various structures in the skin could be distinguished, including Stratum Corneum, epidermal cells and dermis. The spectral phasor analysis allowed investigation of their fluorescence composition and identification of signals from NADH, keratin, FAD, melanin, collagen and elastin. Interestingly, two populations of epidermal cells could be distinguished with different melanin content. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

A stressor specific multimetric approach for monitoring running waters in Austria using benthic macro-invertebrates

We investigated four stream types in four different bioregions, classified by catchment area and altitude, and stressed by different degrees of organic pollution and habitat alteration. We examined a macro-invertebrate based multimetric approach for Austrian rivers as a potential assessment method within the European Water Framework Directive. Benthic macro-invertebrate data (100 samples including reference sites) were used to develop a multimetric index for each stream type and targeted stressors. Sites were pre-classified based on physical, chemical, and land use criteria into five ecological quality classes. More than 200 biological metrics were tested for their sensitivity to the targeted stressors, their spatial and temporal variability and their ability to discriminate between different types and degrees of stress. Metrics for index development were selected to reflect different levels of information including ecosystem, community, and individual levels (Karr, 1991; Barbour et al., 1995; Gerritsen, 1995). Combinations of metrics were selected to distinguish best between non or slightly impaired and stressed sites (evaluated by calculating discrimination efficiency values and power analysis). The resulting four indices comprised seven to nine metrics from five to seven metric categories, and distinguished reference/slightly disturbed sites from stressed sites with close to 100% efficiency. The indices can form the basis for stressor-specific assessment of stream condition.     

Association of leptin gene polymorphisms with serum leptin concentration in dairy cows

Leptin is a hormone produced by adipocytes, and its expression is regulated by body fatness and energy balance. This study describes the association of four leptin gene polymorphisms in dairy cows (R4C, A59V, RFLP1, and BM1500) with circulating leptin concentrations during the periparturient period. A59V is located at a between-species conserved region of leptin, and R4C might have effect on the tertiary structure of the leptin protein because of the presence of an extra cystein. RFLP1 is an intronic SNP and BM1500 is a microsatellite located 3.6 kb downstream of the leptin locus. The four polymorphisms were genotyped in 323 HF heifers with known pedigree. Leptin concentrations were determined biweekly from 30 days before until 80 days after parturition. The effect of genotype on leptin concentrations was modeled by fitting a spline in ASREML describing leptin concentrations as a function of days relative to parturition for each genotype/allele. Surprisingly, associations were found during pregnancy, but not during lactation. This indicates that the polymorphism could be more effective during pregnancy. If further studies demonstrate that more leptin-binding protein (Ob-Re) is present in this stage, it is hypothesized that a structural difference in the leptin protein could cause a sub-optimal binding stringency to Ob-Re. Free leptin could be cleared faster than bound leptin, and this could result in lower leptin concentrations during pregnancy for the polymorphism. The effects found might be ascribed to R4C. However, more study on the Ob-Re receptor, like binding stringencies between R4C and wild-type leptin and glycosylation during pregnancy, would provide more insight in the results found.     

A model of human muscle energy expenditure

A model of muscle energy expenditure was developed for predicting thermal, as well as mechanical energy liberation during simulated muscle contractions. The model was designed to yield energy (heat and work) rate predictions appropriate for human skeletal muscle contracting at normal body temperature. The basic form of the present model is similar to many previous models of muscle energy expenditure, but parameter values were based almost entirely on mammalian muscle data, with preference given to human data where possible. Nonlinear phenomena associated with submaximal activation were also incorporated. The muscle energy model was evaluated at varying levels of complexity, ranging from simulated contractions of isolated muscle, to simulations of whole body locomotion. In all cases, acceptable agreement was found between simulated and experimental energy liberation. The present model should be useful in future studies of the energetics of human movement using forward dynamic computer simulation.     

Photic entrainment of circadian activity patterns in the tropical labrid fish Halichoeres chrysus

Yellow wrasses (Halichoeres chrysus) show clear daily activity patterns. The fish hide in the substrate at (subjective) night, during the distinct rest phase. Initial entrainment in a 12h:12h light-dark (12:12 LD) cycle (mean period 24.02h, SD 0.27h, n = 16 was followed by a free run (mean period 24.42h, SD 1.33h) after transition into constant dim light conditions. Light pulses of a comparable intensity as used in the light part of the LD cycles did not result in significant phase shifts of the free-running rhythm in constant darkness. Application of much brighter 3h light pulses resulted in a phase-response curve (PRC) for a fish species, with pronounced phase advances during late subjective night. The PRCs differed from those mainly obtained in other vertebrate taxa by the absence of significant phase delays in the early subjective night. At that circadian phase, significant tonic effects of the light pulses caused a shortening of the circadian period length. Entrainment to skeleton photoperiods of 1:11 LD was observed in five of six wrasses exposed, also after a 3h phase advance of this LD cycle. Subsequently, a 1:11.25 LD cycle resulted in entrainment in four of the six fish. It is suggested that the expression of the circadian system in fish can be interpreted as a functional response to a weak natural zeitgeber, as present in the marine environment. This response allows photic entrainment as described here in the yellow wrasse. (Chronobiology International, 17(5), 613-622, 2000)     

Patient-specific initial wall stress in abdominal aortic aneurysms with a backward incremental method

Patient-specific wall stress simulations on abdominal aortic aneurysms may provide a better criterion for surgical intervention than the currently used maximum transverse diameter. In these simulations, it is common practice to compute the peak wall stress by applying the full systolic pressure directly on the aneurysm geometry as it appears in medical images. Since this approach does not account for the fact that the measured geometry is already experiencing a substantial load, it may lead to an incorrect systolic aneurysm shape. We have developed an approach to compute the wall stress on the true diastolic geometry at a given pressure with a backward incremental method. The method has been evaluated with a neo-Hookean material law for several simple test problems. The results show that the method can predict an unloaded configuration if the loaded geometry and the load applied are known. The effect of incorporating the initial diastolic stress has been assessed by using three patient-specific geometries acquired with cardiac triggered MR. The comparison shows that the commonly used approach leads to an unrealistically smooth systolic geometry and therefore provides an underestimation for the peak wall stress. Our backward incremental modelling approach overcomes these issues and provides a more plausible estimate for the systolic aneurysm volume and a significantly different estimate for the peak wall stress. When the approach is applied with a more complex material law which has been proposed specifically for abdominal aortic aneurysm similar effects are observed and the same conclusion can be drawn.     

Quantitative linkage genome scan for atopy in a large collection of Caucasian families

Quantitative phenotypes correlated with a complex disorder offer increased power to detect linkage in comparison to affected-unaffected classifications. Asthma is a complex disorder characterized by periods of bronchial obstruction and increased bronchial hyper reactivity. In childhood and early adulthood, asthma is frequently associated also with quantitative measures of atopy. Genome wide quantitative multipoint linkage analysis was conducted for serum IgE levels and percentage of positive skin prick test (SPT(per)) using three large groups of families originally ascertained for asthma. In this report, 438 and 429 asthma families were informative for linkage using IgE and SPT(per) which represents 690 independent families. Suggestive linkage (LOD > or = 2) was found on chromosomes 1, 3, and 8q with maximum LODs of 2.34 (IgE), 2.03 (SPT(per)), and 2.25 (IgE) near markers D1S1653, D3S2322-D3S1764, and D8S2324, respectively. The results from chromosomes 1 and 3 replicate previous reports of linkage. We also replicate linkage to 5q with peak LODs of 1.96 (SPT(per)) and 1.77 (IgE) at or near marker D5S1480. Our results provide further evidence implicating chromosomes 1, 3, and 5q. The current report represents one of the biggest genome scans so far reported for asthma related phenotypes. This study also demonstrates the utility of increased sample sizes and quantitative phenotypes in linkage analysis of complex disorders.