Comparison of label-free and label-based strategies for proteome analysis of hepatoma cell lines

Within the past decade numerous methods for quantitative proteome analysis have been developed of which all exhibit particular advantages and disadvantages. Here, we present the results of a study aiming for a comprehensive comparison of ion-intensity based label-free proteomics and two label-based approaches using isobaric tags incorporated at the peptide and protein levels, respectively. As model system for our quantitative analysis we used the three hepatoma cell lines HepG2, Hep3B and SK-Hep-1. Four biological replicates of each cell line were quantitatively analyzed using an RPLC–MS/MS setup. Each quantification experiment was performed twice to determine technical variances of the different quantification techniques. We were able to show that the label-free approach by far outperforms both TMT methods regarding proteome coverage, as up to threefold more proteins were reproducibly identified in replicate measurements. Furthermore, we could demonstrate that all three methods show comparable reproducibility concerning protein quantification, but slightly differ in terms of accuracy. Here, label-free was found to be less accurate than both TMT approaches. It was also observed that the introduction of TMT labels at the protein level reduces the effect of underestimation of protein ratios, which is commonly monitored in case of TMT peptide labeling. Previously reported differences in protein expression between the particular cell lines were furthermore reproduced, which confirms the applicability of each investigated quantification method to study proteomic differences in such biological systems. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.     

人类活动影响下的生物多样性保护:中欧的植被演化及其启示

对历史时期欧洲中部地区植被历史的研究表明,人类活动不仅破坏了自然、导致植被的单一化,同时也对植被与景观的丰富度(多样性)产生影响。人类或家畜的干预使得一些半自然、高多样性的生境类型及相应的植被类型得以保持和发展;但过度的干扰势必造成植被的单一化和贫乏化,威胁生物多样性的保存和保护。当今的中欧植被格局总体上是近几个世纪,某些地区甚至是上千年来,人为干扰叠加在气候变化影响之上的综合产物。中欧大部分地区的潜在植被多为森林,如果没有人为活动的强烈影响,除了岩石、水体、海滩、林线以上的高山以及高位沼泽等特定生境的分布区域以外,当今中欧的大部分地区应当仍覆盖着郁闭的阔叶林,而不会表现出今天的多样性景观。因此,自然保护的对象不应当仅仅是纯自然植被,保护手段也不仅仅是清除人为干扰。在保护多样性思想的指导下,各种典型植被类型都是值得保护的,除了高自然度的自然生态系统外,栽培植被也应当被保存和保护,否则,它们会随着特定耕作或管理方式的消失而消失。同时,既应保护当前植被,也要恢复潜在植被,使目前多样的景观类型、植被类型和栖息地类型得以维持和发展。本文结论为解决当前欧洲自然保护中的争议性问题提供了依据,也可以为中国的生物多样性保护战...     

The relation of workspace and installation space of epicyclic kinematics with six degrees of freedom]

INTRODUCTION: The kinematics of a robotic device significantly determines its installation space when it comes to technical realisation. With regard to the deployment of robotic manipulators in surgery, manipulators with a preferably small installation space are needed. MATERIALS AND METHODS: This study describes six versions of novel epicyclic kinematics with six degrees of freedom (DOF). At first, the kinematics functionality was analysed using Gruebler's formula. Subsequently, the quantitative determination of the relation of workspace and installation space was performed using Matlab algorithms. To qualitatively describe the shape of the workspace, the Matlab visualisation features were utilised. For comparison, the well-known Hexapod was used. RESULTS: The assessed kinematics had 6-DOF-functionality. It became apparent that one version of the epicyclic kinematics having two 3-DOF disk systems mounted in a parallel way featured a particularly good relation of workspace and installation space. Compared to the Hexapod, this is approximately four times better. The shape of the workspaces of all epicyclic kinematics assessed was convex and compact. CONCLUSION: It could be shown that a novel epicyclic kinematics has a notably advantageous relation of workspace and installation space. Apparently, it seems to be well suited for the deployment in robotic machines for surgical procedures.     

Age-Dependent TLR3 Expression of the Intestinal Epithelium Contributes to Rotavirus Susceptibility

Rotavirus is a major cause of diarrhea worldwide and exhibits a pronounced small intestinal epithelial cell (IEC) tropism. Both human infants and neonatal mice are highly susceptible, whereas adult individuals remain asymptomatic and shed only low numbers of viral particles. Here we investigated age-dependent mechanisms of the intestinal epithelial innate immune response to rotavirus infection in an oral mouse infection model. Expression of the innate immune receptor for viral dsRNA, Toll-like receptor (Tlr) 3 was low in the epithelium of suckling mice but strongly increased during the postnatal period inversely correlating with rotavirus susceptibility, viral shedding and histological damage. Adult mice deficient in Tlr3 (Tlr3^−/−) or the adaptor molecule Trif (Trif^Lps2/Lps2) exerted significantly higher viral shedding and decreased epithelial expression of proinflammatory and antiviral genes as compared to wild-type animals. In contrast, neonatal mice deficient in Tlr3 or Trif did not display impaired cell stimulation or enhanced rotavirus susceptibility. Using chimeric mice, a major contribution of the non-hematopoietic cell compartment in the Trif-mediated antiviral host response was detected in adult animals. Finally, a significant age-dependent increase of TLR3 expression was also detected in human small intestinal biopsies. Thus, upregulation of epithelial TLR3 expression during infancy might contribute to the age-dependent susceptibility to rotavirus infection.     

Biochemical and structural characterization of benzenoid carboxyl methyltransferases involved in floral scent production in Stephanotis floribunda and Nicotiana suaveolens

Flower-specific benzenoid carboxyl methyltransferases from Stephanotis floribunda and Nicotiana suaveolens were biochemically and structurally characterized. The floral scents of both these species contain higher levels of methyl benzoate and lower levels of methyl salicylate. The S. floribunda enzyme has a 12-fold lower K(m) value for salicylic acid (SA) than for benzoic acid (BA), and results of in silico modeling of the active site of the S. floribunda enzyme, based on the crystal structure of Clarkia breweri salicylic acid methyltransferase (SAMT), are consistent with this functional observation. The enzyme was therefore designated SAMT. The internal concentration of BA in S. floribunda flowers is three orders of magnitude higher than the SA concentration, providing a rationale for the observation that these flowers synthesize and emit more methyl benzoate than methyl salicylate. The N. suaveolens enzyme has similar K(m) values for BA and SA, and the in silico modeling results are again consistent with this in vitro observation. This enzyme was therefore designated BSMT. However, the internal concentration of BA in N. suaveolens petals was also three orders of magnitude higher than the concentration of SA. Both S. floribunda SAMT and N. suaveolens BSMT are able to methylate a range of other benzenoid-related compounds and, in the case of S. floribunda SAMT, also several cinnamic acid derivatives, an observation that is consistent with the larger active site cavity of each of these two enzymes compared to the SAMT from C. breweri, as shown by the models. Broad substrate specificity may indicate recent evolution or an adaptation to changing substrate availability.     

Effects of pH and cholesterol on DMPA membranes: a solid state 2H- and 31P-NMR study.

The effect of pH and cholesterol on the dimyristoylphosphatidic acid (DMPA) model membrane system has been investigated by solid state 2H- and 31P-NMR. It has been shown that each of the three protonation states of the DMPA molecule corresponds to a 31P-NMR powder pattern with characteristic delta sigma values; this implies additionally that the proton exchange on the membrane surface is slow on the NMR time scale (millisecond range). Under these conditions, the 2H-labeled lipid chains sense only one magnetic environment, indicating that the three spectra detected by 31P-NMR are related to charge-dependent local dynamics or orientations of the phosphate headgroup or both. Chain ordering in the fluid phase is also found to depend weakly on the charge at the interface. In addition, it has also been found that the first pK of the DMPA membrane is modified by changes in the lipid lateral packing (gel or fluid phases or in the presence of cholesterol) in contrast to the second pK. The incorporation of 30 mol% cholesterol affects the phosphatidic acid bilayer in a way similar to what has been reported for phosphatidylcholine/cholesterol membranes, but to an extent comparable to 10-20 mol % sterol in phosphatidylcholines. However, the orientation and molecular order parameter of cholesterol in DMPA are similar to those found in dimyristoylphosphatidylcholine.     

Epithelial autophagy controls chronic colitis by reducing TNF-induced apoptosis

Genome-wide association studies (GWAS) linking polymorphisms in ATG16L1 with susceptibility to inflammatory bowel disease (IBD) have prompted mucosal immunologists to investigate the functional roles of macroautophagy/autophagy in different cell types in the gut. Here we present a recent study that addressed 2 key questions: in which cell type is autophagy deficiency most detrimental during chronic colitis and what is the functional role of autophagy in those cells? We report that autophagy in intestinal epithelial cells (IECs) acts to limit intestinal inflammation by protecting them from TNF-induced apoptosis and we discuss the potential implications for IBD treatment.     

Observations on parasitic gastroenteritis and bronchitis in grazing calves: Untreated calves

Pott J. M., Jpones R. M. and Cornwell R. L. 1978. Observations on parasitic gastroenteritis and bronchitis in grazing calves: untreated calves. International Journal for Parasitology8: 331–339. The epidemiology of parasitic gastroenteritis (p.g.e.) and incidental observations on lungworm infection were studied on two equal-sized paddocks of permanent pasture on a Kent farm in 1970. Groups of 10 Friesian calves were exposed for the first time to each paddock in May and grazed until the end of October. Faeces were monitored, body weights recorded and clinical signs observed. Herbage samples were examined for larvae throughout the year. Five ‘cumulative’ tracer calves exposed at the commencement were removed at monthly intervals and 5 ‘short term’ tracer calves exposed at successive monthly intervals for 1 month were killed for worm counts. A similar pattern of events occurred on both paddocks. Moderate herbage larval levels of Ostertagia and Cooperia fell to almost zero during April/May but rose quickly in July to high levels in August/September. Lower levels continued until the end of the year. Faecal egg excretion began in the test calves at the end of May, showed peaks in July and September and fell to low levels in October. D. viviparus larval excretion occurred in most calves with greater counts on Paddock 2. Clinical signs of p.g.e. complicated by lungworm infection occurred in August/September. Body weight gain which was initially rapid levelled off in June due partly to drought in addition to the parasitic infection. Weight loss occurred in August and there were 2 deaths on Paddock 1 and one on Paddock 2. Survivors gained weight again in September/October.Worm burdens in tracer calves showed quite heavy infections with Ostertagia and Cooperia as early as June/July. Massive infections complicated by lungworm disease accompanied clinical signs and death. There was evidence that aquisition of resistance to Cooperia and Dictyocaulus was acquired more readily than to Ostertagia. Inhibition of development of Ostertagia and Cooperia also became evident at the end of the trial period.