A catalog of human cDNA expression clones and its application to structural genomics

We describe here a systematic approach to the identification of human proteins and protein fragments that can be expressed as soluble proteins in Escherichia coli. A cDNA expression library of 10,825 clones was screened by small-scale expression and purification and 2,746 clones were identified. Sequence and protein-expression data were entered into a public database. A set of 163 clones was selected for structural analysis and 17 proteins were prepared for crystallization, leading to three new structures.     

Patch Connectivity and Genetic Variation in Two Congeneric Grasshopper Species with Different Habitat Preferences

Two congeneric species of grasshopper, Stenobothrus lineatus and S. stigmaticus, are compared in an analysis of genetic structure relative to their observed mobility, and to the spatial structure of their habitat networks. The species differ in their habitat requirements, the latter being rarer and more restricted to isolated patches. We tested for different patch connectivity between the two species in an analysis of genetic variance (based on allozymes) under the assumption that, besides isolation, rarity influences the genetic parameters. Between the species we found no differences in genetic structure as estimated by F_ST; i.e., no isolation effects and no apparent differences between the species in the potential to move between habitat fragments on either a local or regional scale were found. However, the amount of genetic variation in the more widely distributed and less xerothermic S. lineatus was significantly higher than in S. stigmaticus. Some consistency with observed philopatry within patches was found (F_IS > 0), but we consider regular dispersal events of medium and especially long distance to cause the habitat linking. We conclude that the connectivity between occupied patches inferred by genetic analyses can seldom be derived from low observed life-time movements recorded by conventional marking studies. Consequences of applying observed relative to indirect dispersal estimates for the examination of grasshopper metapopulations are discussed.     

Human Rad51 protein displays enhanced homologous pairing of DNA sequences resembling those at genetically unstable loci

DNA strand exchange, the central step of homologous recombination, is considered to occur approximately independently of DNA sequence content. However, certain prokaryotic and eukaryotic genomic loci display either an enhanced or reduced frequency of genetic exchange. Here we show that the Homo sapiens DNA strand exchange protein, HsRad51, shows a preference for binding to single-stranded DNA sequences primarily rich in G-residues and poor in A- and C-residues, and that these DNA sequences manifest enhanced HsRad51 protein-dependent homologous pairing. Both of these properties are common to all DNA strand exchange proteins examined thus far. These preferred DNA pairing sequences resemble those found at genetic loci in human cells that cause genomic instability and lead to genetic diseases.     

A rationalized definition of general tumor suppressor microRNAs excludes miR-34a

While several microRNAs (miRNAs) have been proposed to act as tumor suppressors, a consensual definition of tumor suppressing miRNAs is still missing. Similarly to coding genes, we propose that tumor suppressor miRNAs must show evidence of genetic or epigenetic inactivation in cancers, and exhibit an anti-tumorigenic (e.g., anti-proliferative) activity under endogenous expression levels. Here we observe that this definition excludes the most extensively studied tumor suppressor candidate miRNA, miR-34a. In analyzable cancer types, miR-34a does not appear to be down-regulated in primary tumors relatively to normal adjacent tissues. Deletion of miR-34a is occasionally found in human cancers, but it does not seem to be driven by an anti-tumorigenic activity of the miRNA, since it is not observed upon smaller, miR-34a-specific alterations. Its anti-proliferative action was observed upon large, supra-physiological transfection of synthetic miR-34a in cultured cells, and our data indicates that endogenous miR-34a levels do not have such an effect. Our results therefore argue against a general tumor suppressive function for miR-34a, providing an explanation to the lack of efficiency of synthetic miR-34a administration against solid tumors.     

Forest classification in an Amazonian rainforest landscape using pteridophytes as indicator species

Habitat classification systems are poorly developed for tropical rainforests, where extremely high plant species richness causes numerous methodological difficulties. We used an indicator species approach to classify primary rainforest vegetation for purposes of comparative wildlife habitat studies. We documented species composition of pteridophytes (ferns and fern allies) in 635 plots (2×100 m) along 8 transects within a continuous rainforest landscape in northeastern Peruvian Amazonia. Considerable floristic variation was found when the data were analyzed using multivariate methods. The obtained forest classification was interpreted with the help of indicator value analysis and known soil preferences of the pteridophyte species. The final classification included four forest types: 1) inundated forests, 2) terrace forests, 3) intermediate tierra firme forests and 4) Pebas Formation forests. This rapid and relatively simple vegetation classification technique offers a practical, quantitative method for large-scale vegetation inventory in complex rainforest landscapes.     

Dynamic remodeling of the endosomal system during formation of Salmonella-induced filaments by intracellular Salmonella enterica

The infection by Salmonella enterica results in the massive remodeling of the endosomal system of eukaryotic host cells. One unique consequence is the formation of long tubular endosomal compartments, so-called Salmonella-induced filaments (SIF). Formation of SIF requires the function of type III secretion system and is a requirement of efficient intracellular proliferation of Salmonella. Using high-resolution live cell imaging approaches and electron microscopy, we report for the first time the highly dynamic characteristics of SIF and their ultrastructural properties. In the early phase of infection (4-5 h), SIF display highly dynamic properties in various types of host cells. SIF extend, branch and contract rapidly, and a stabilized network of SIF is formed later (>or=8 h after infection). The velocities of SIF extension and contraction in the different phases of infection were quantified. Our observations lead to novel models for the modification of host cell transport processes by virulence factors of intracellular Salmonella.     

Combination of gastrin-releasing peptide antagonist with cytotoxic agents produces synergistic inhibition of growth of human experimental colon cancers

We investigated the efficacy of a powerful antagonist of bombesin/gastrin-releasing peptide (BN/GRP) RC-3940-II administered as a single agent or in combination with cytotoxic agents on the growth of HT-29, HCT-116 and HCT-15 human colon cancer in vitro and in vivo. GRP-receptor mRNA and protein were found in all three cell lines tested. Exposure of HT-29 cells to 10 μM RC-3940-II led to an increase in the number of cells blocked in S phase and G₂/M and cells with lower G₀/G₁ DNA content. Similar changes on the cell cycle traverse of HT-29 cells could also be seen at lower concentrations of RC-3940-II (1 μM) after pretreatment with 100 nM GRP (14–27), indicating a dose-dependent mechanism of action based on the blockage of BN/GRP induced proliferation of tumor cells at lower concentrations. Daily in vivo treatment with BN/GRP antagonist RC-3940-II decreased the volume of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic nude mice by 25 to 67% (p < 0.005). Combined treatment with RC-3940-II and chemotherapeutic agents 5-FU and irinotecan resulted in a synergistic tumor growth suppression of HT-29, HCT-116 and HCT-15 xenografts by 43% to 78%. In HT-29 and HCT-116 xenografts the inhibition for the combinations of RC-3940-II and irinotecan vs. single substances (p < 0.05) was significantly greater. These findings support the use of RC-3940-II as an anticancer agent and may help to design clinical trials using RC-3940-II in combinations with cytotoxic agents.     

Objective working hour characteristics and work–life conflict among hospital employees in the Finnish public sector study

This epidemiological cohort study, based on Finnish public sector data, investigated the associations between objective working hour characteristics and work–life conflict in day and shift work. The comprehensive data of hospital workers (n = 8 931, 92% women, average age 45 years), consisted of survey responses from 2012, linked with the payroll data of working hour characteristics from 91 days preceding the survey. Logistic regression analysis was used to investigate the associations between working hour characteristics and experiencing work–life conflict often/very often. The analyses were adjusted for age (< 39, 40–49 and >50 years), sex, level of education, marital status, number of small (0–6 years) and school-aged (7–18 years) children, and the overall stressfulness of the life situation. We also conducted stratified analyses of age and sex on the basis of significant interactions. Difficulties in combining work and life were more often associated with shift work without night shifts and shift work with night shifts than with day work (41% and 34 versus 27%; OR for shift work with night shifts 1.78, 95% CI 1.59–2.00, OR for shift work without night shifts 1.42, 95% CI 1.26–1.60). A high proportion (> 25%) of long (> 40h, (OR 1.26, 95% 1.14–1.39) and very long (> 48h, OR 1.31, 95% CI 1.15–1.49) weekly working hours were associated with work–life conflict, and in the stratified analysis, the latter was also true among women (OR 1.54, 95% CI 1.25–1.89). Of the unsocial working hour characteristics, a relatively large amount (> 10% of all shifts) of evening (OR 1.56, 95% CI 1.41–1.72) and night shifts (OR 1.46, 95%CI 1.32–1.61), a high proportion (> 25% of all shifts) of quick returns (< 11h) (OR 1.46, 95% CI 1.31–1.63), and weekend work (OR 1.44, 95% CI 1.31–1.58) were associated with work–life conflict. A large amount of single days off (> 25% of all days off) was associated with work–life conflict among men (OR 1.90, 95% CI 1.11–3.25), but not in the whole sample. When the two types of shift work were analyzed separately, shift work without night shifts and very long work weeks had higher odds (OR 1.47, 95% CI 1.20–1.80) of work–life conflict than shift work with night shifts. Conversely, weekend work and evening shifts had higher odds of work–life conflict among shift workers with night shifts (OR 1.74, 95% 1.55–1.96; (OR 1.57, 95% CI 1.40–1.77) than among those without night shifts. To conclude, this study shows that shift workers with and without night shifts more often have difficulties combining work and life than day workers. Several unsocial working hour characteristics, including long work weeks, evening and night shifts, weekend work, and quick returns, are associated with work–life conflict.     

Evolution of a core gene network for skeletogenesis in chordates

The skeleton is one of the most important features for the reconstruction of vertebrate phylogeny but few data are available to understand its molecular origin. In mammals the Runt genes are central regulators of skeletogenesis. Runx2 was shown to be essential for osteoblast differentiation, tooth development, and bone formation. Both Runx2 and Runx3 are essential for chondrocyte maturation. Furthermore, Runx2 directly regulates Indian hedgehog expression, a master coordinator of skeletal development. To clarify the correlation of Runt gene evolution and the emergence of cartilage and bone in vertebrates, we cloned the Runt genes from hagfish as representative of jawless fish (MgRunxA, MgRunxB) and from dogfish as representative of jawed cartilaginous fish (ScRunx1–3). According to our phylogenetic reconstruction the stem species of chordates harboured a single Runt gene and thereafter Runt locus duplications occurred during early vertebrate evolution. All newly isolated Runt genes were expressed in cartilage according to quantitative PCR. In situ hybridisation confirmed high MgRunxA expression in hard cartilage of hagfish. In dogfish ScRunx2 and ScRunx3 were expressed in embryonal cartilage whereas all three Runt genes were detected in teeth and placoid scales. In cephalochordates (lancelets) Runt, Hedgehog and SoxE were strongly expressed in the gill bars and expression of Runt and Hedgehog was found in endo- as well as ectodermal cells. Furthermore we demonstrate that the lancelet Runt protein binds to Runt binding sites in the lancelet Hedgehog promoter and regulates its activity. Together, these results suggest that Runt and Hedgehog were part of a core gene network for cartilage formation, which was already active in the gill bars of the common ancestor of cephalochordates and vertebrates and diversified after Runt duplications had occurred during vertebrate evolution. The similarities in expression patterns of Runt genes support the view that teeth and placoid scales evolved from a homologous developmental module.