Covalent protein modification with ISG15 via a conserved cysteine in the hinge region

The ubiquitin-like protein ISG15 (interferon-stimulated gene of 15 kDa) is strongly induced by type I interferons and displays antiviral activity. As other ubiquitin-like proteins (Ubls), ISG15 is post-translationally conjugated to substrate proteins by an isopeptide bond between the C-terminal glycine of ISG15 and the side chains of lysine residues in the substrates (ISGylation). ISG15 consists of two ubiquitin-like domains that are separated by a hinge region. In many orthologs, this region contains a single highly reactive cysteine residue. Several hundred potential substrates for ISGylation have been identified but only a few of them have been rigorously verified. In order to investigate the modification of several ISG15 substrates, we have purified ISG15 conjugates from cell extracts by metal-chelate affinity purification and immunoprecipitations. We found that the levels of proteins modified by human ISG15 can be decreased by the addition of reducing agents. With the help of thiol blocking reagents, a mutational analysis and miRNA mediated knock-down of ISG15 expression, we revealed that this modification occurs in living cells via a disulphide bridge between the substrates and Cys78 in the hinge region of ISG15. While the ISG15 activating enzyme UBE1L is conjugated by ISG15 in the classical way, we show that the ubiquitin conjugating enzyme Ubc13 can either be classically conjugated by ISG15 or can form a disulphide bridge with ISG15 at the active site cysteine 87. The latter modification would interfere with its function as ubiquitin conjugating enzyme. However, we found no evidence for an ISG15 modification of the dynamin-like GTPases MxA and hGBP1. These findings indicate that the analysis of potential substrates for ISG15 conjugation must be performed with great care to distinguish between the two types of modification since many assays such as immunoprecipitation or metal-chelate affinity purification are performed with little or no reducing agent present.     

Selection of Microsatellite Markers for Bladder Cancer Diagnosis without the Need for Corresponding Blood

Microsatellite markers are used for loss-of-heterozygosity, allelic imbalance and clonality analyses in cancers. Usually, tumor DNA is compared to corresponding normal DNA. However, normal DNA is not always available and can display aberrant allele ratios due to copy number variations in the genome. Moreover, stutter peaks may complicate the analysis. To use microsatellite markers for diagnosis of recurrent bladder cancer, we aimed to select markers without stutter peaks and a constant ratio between alleles, thereby avoiding the need for a control DNA sample. We investigated 49 microsatellite markers with tri- and tetranucleotide repeats in regions commonly lost in bladder cancer. Based on analysis of 50 blood DNAs the 12 best performing markers were selected with few stutter peaks and a constant ratio between peaks heights. Per marker upper and lower cut off values for allele ratios were determined. LOH of the markers was observed in 59/104 tumor DNAs. We then determined the sensitivity of the marker panel for detection of recurrent bladder cancer by assaying 102 urine samples of these patients. Sensitivity was 63% when patients were stratified for LOH in their primary tumors. We demonstrate that up-front selection of microsatellite markers obliterates the need for a corresponding blood sample. For diagnosis of bladder cancer recurrences in urine this significantly reduces costs. Moreover, this approach facilitates retrospective analysis of archival tumor samples for allelic imbalance.     

N-glycosylation as novel strategy to improve pharmacokinetic properties of bispecific single-chain diabodies

The therapeutic efficacy of recombinant antibodies such as single-chain Fv fragments and small bispecific or bifunctional molecules is often limited by rapid elimination from the circulation because of their small size. Here, we have investigated the effects of N-glycosylation on the activity and pharmacokinetics of a small bispecific single-chain diabody (scDb CEACD3) developed for the retargeting of cytotoxic T cells to CEA-expressing tumor cells. We could show that the introduction of N-glycosylation sequons into the flanking linker and a C-terminal extension results in the production of N-glycosylated molecules after expression in transfected HEK293 cells. N-Glycosylated scDb variants possessing 3, 6, or 9 N-glycosylation sites, respectively, retained antigen binding activity and bispecificity for target and effector cells as shown in a target cell-dependent IL-2 release assay, although activity was reduced approximately 3-5-fold compared with the unmodified scDb. All N-glycosylated scDb variants exhibited a prolonged circulation time compared with scDb, leading to a 2-3-fold increase of the area under curve (AUC). In comparison, conjugation of a branched 40-kDa PEG chain increased AUC by a factor of 10.6, while a chimeric anti-CEA IgG1 molecule had the longest circulation time with a 17-fold increase in AUC. Thus, N-glycosylation complements the repertoire of strategies to modulate pharmacokinetics of small recombinant antibody molecules by an approach that moderately prolongs circulation time.     

Effects of two different eight-week training programs on military physical performance

Various physical demands are placed on soldiers, whose effectiveness and survivability depend on their combat-specific physical fitness. Because sport training programs involving weight-based training have proven effective, this study examined the value of such a program for short-term military training using combat-relevant tests. A male weight-based training (WBT) group (n = 15; mean +/- SD: 27.0 +/- 4.7 years, 173.8 +/- 5.8 cm, 80.9 +/- 12.7 kg) performed full-body weight-based training workouts, 3.2-km runs, interval training, agility training, and progressively loaded 8-km backpack hikes. A male Army Standardized Physical Training (SPT) group (n = 17; mean +/- SD: 29.0 +/- 4.6 years, 179.7 +/- 8.2 cm, 84.5 +/- 10.4 kg) followed the new Army Standardized Physical Training program of stretching, varied calisthenics, movement drills, sprint intervals, shuttle running, and distance runs. Both groups exercised for 1.5 hours a day, 5 days a week for 8 weeks. The following training-induced changes were statistically significant (P < 0.05) for both training groups: 3.2-km run or walk with 32-kg load (minutes), 24.5 +/- 3.2 to 21.0 +/- 2.8 (SPT) and 24.9 +/- 2.8 to 21.1 +/- 2.2 (WBT); 400-m run with 18-kg load (seconds), 94.5 +/- 14.2 to 84.4 +/- 11.9 (SPT) and 100.1 +/- 16.1 to 84.0 +/- 8.4 (WBT); obstacle course with 18-kg load (seconds), 73.3 +/- 10.1 to 61.6 +/- 7.7 (SPT) and 66.8 +/- 10.0 to 60.1 +/- 8.7 (WBT); 5 30-m sprints to prone (seconds), 63.5 +/- 4.8 to 59.8 +/- 4.1 (SPT) and 60.4 +/- 4.2 to 58.9 +/- 2.7 (WBT); and 80-kg casualty rescue from 50 m (seconds), 65.8 +/- 40.0 to 42.1 +/- 9.9 (SPT) and 57.6 +/- 22.0 to 44.2 +/- 8.8 (WBT). Of these tests, only the obstacle course showed significant difference in improvement between the two training groups. Thus, for short-term (i.e., 8-week) training of relatively untrained men, the Army's new Standardized Physical Training program and a weight-based training experimental program can produce similar, significant, and meaningful improvements in military physical performance. Further research would be needed to determine whether weight-based training provides an advantage over a longer training period.     

Metabolite-based clustering and visualization of mass spectrometry data using one-dimensional self-organizing maps

Background  

One of the goals of global metabolomic analysis is to identify metabolic markers that are hidden within a large background of data originating from high-throughput analytical measurements. Metabolite-based clustering is an unsupervised approach for marker identification based on grouping similar concentration profiles of putative metabolites. A major problem of this approach is that in general there is no prior information about an adequate number of clusters.     

Microbial responses to solvent and alcohol stress

Increasing fuel prices and doubts over the long-term availability of oil are currently major global concerns. Such concerns have led to national policies and objectives to develop microbially produced alcohols as fuel additives or substitutes. However, in South Africa this solution poses the further dilemma of sourcing a suitable fermentative carbohydrate that will not impact negatively on the availability of staple foods. The solution lies in the use of lignocellulosic materials, currently a waste product of the food and agriculture industries, which could be used in conjunction with a catabolically suitable production strain. In the pursuit of lignocellulosic biofuel production, conventional fermentation strains have been shown to have limited catabolic versatility. However, catabolically versatile engineered strains and novel isolates engineered with ethanologenic pathways have subsequently been shown to exhibit limitations in solvent tolerance, hindering their full potential as economically viable production strains. A considerable volume of research has been reported on the general cellular mechanisms and physiological responses to solvent shock as well as adaptive changes responsible for solvent tolerant phenotypes in mutant progeny. Here we review a number of the more common cell responses to solvents with particular focus on alcohol tolerance.     

Native and alien plant species richness in relation to spatial heterogeneity on a regional scale in Germany

Aim The aim of our study was to reveal relationships between richness patterns of native vs. alien plant species and spatial heterogeneity across varying landscape patterns at a regional scale. Location The study was carried out in the administrative district of Dessau (Germany), covering around 4000 km². Methods Data on plant distribution of the German vascular flora available in grid cells covering 5′ longitude and 3′ latitude (c. 32 km²) were divided into three status groups: native plants, archaeophytes (pre 1500 AD aliens) and neophytes (post 1500 AD aliens). Land use and abiotic data layers were intersected with 125 grid cells comprising the selected area. Using novel landscape ecological methods, we calculated 38 indices of landscape composition and configuration for each grid cell. Principal components analysis (PCA) with a set of 29 selected, low correlated landscape indices was followed by multiple linear regression analysis. Results PCA reduced 29 indices to eight principal components (PCs) that explained 80% cumulative variance. Multiple linear regression analysis was highly significant and explained 41% to 60% variance in plant species distribution (adjusted R²) with three significant PCs (tested for spatial autocorrelation) expressing moderate to high disturbance levels and high spatial heterogeneity. Comparing the significance of the PCs for the species groups, native plant species richness is most strongly associated with riverine ecosystems, followed by urban ecosystems, and then small‐scale rural ecosystems. Archaeophyte and neophyte richness are most strongly associated with urban ecosystems, followed by small‐scale rural ecosystems and riverine ecosystems for archaeophytes, and riverine ecosystems and small‐scale rural ecosystems for neophytes. Main conclusions Our overall results suggest that species richness of native and alien plants increases with moderate levels of natural and/or anthropogenic disturbances, coupled with high levels of habitat and structural heterogeneity in urban, riverine, and small‐scale rural ecosystems. Despite differences in the order of relevance of PCs for the three plant groups, we conclude that at the regional scale species richness patterns of native plants as well as alien plants are promoted by similar factors.