The auditory system of non-calling grasshoppers (Melanoplinae: Podismini) and the evolutionary regression of their tympanal ears

Reduction of tympanal hearing organs is repeatedly found amongst insects and is associated with weakened selection for hearing. There is also an associated wing reduction, since flight is no longer required to evade bats. Wing reduction may also affect sound production. Here, the auditory system in four silent grasshopper species belonging to the Podismini is investigated. In this group, tympanal ears occur but sound signalling does not. The tympanal organs range from fully developed to remarkably reduced tympana. To evaluate the effects of tympanal regression on neuronal organisation and auditory sensitivity, the size of wings and tympana, sensory thresholds and sensory central projections are compared. Reduced tympanal size correlates with a higher auditory threshold. The threshold curves of all four species are tuned to low frequencies with a maximal sensitivity at 3–5 kHz. Central projections of the tympanal nerve show characteristics known from fully tympanate acridid species, so neural elements for tympanal hearing have been strongly conserved across these species. The results also confirm the correlation between reduction in auditory sensitivity and wing reduction. It is concluded that the auditory sensitivity of all four species may be maintained by stabilising selective forces, such as predation.     

Random walk in genome space: A key ingredient of intermittent dynamics of community assembly on evolutionary time scales

Community assembly is studied using individual-based multispecies models. The models have stochastic population dynamics with mutation, migration, and extinction of species. Mutants appear as a result of mutation of the resident species, while migrants have no correlation with the resident species. It is found that the dynamics of community assembly with mutations are quite different from the case with migrations. In contrast to mutation models, which show intermittent dynamics of quasi-steady states interrupted by sudden reorganizations of the community, migration models show smooth and gradual renewal of the community. As a consequence, instead of the 1/f diversity fluctuations found for the mutation models, 1/f², random-walk like fluctuations are observed for the migration models. In addition, a characteristic species-lifetime distribution is found: a power law that is cut off by a “skewed” distribution in the long-lifetime regime. The latter has a longer tail than a simple exponential function, which indicates an age-dependent species-mortality function. Since this characteristic profile has been observed, both in fossil data and in several other mathematical models, we conclude that it is a universal feature of macroevolution.     

Allogeneic Human Mesenchymal Stem Cells Restore Epithelial Protein Permeability in Cultured Human Alveolar Type II Cells by Secretion of Angiopoietin-1

Acute lung injury is characterized by injury to the lung epithelium that leads to impaired resolution of pulmonary edema and also facilitates accumulation of protein-rich edema fluid and inflammatory cells in the distal airspaces of the lung. Recent in vivo and in vitro studies suggest that mesenchymal stem cells (MSC) may have therapeutic value for the treatment of acute lung injury. Here we tested the ability of human allogeneic mesenchymal stem cells to restore epithelial permeability to protein across primary cultures of polarized human alveolar epithelial type II cells after an inflammatory insult. Alveolar epithelial type II cells were grown on a Transwell plate with an air-liquid interface and injured by cytomix, a combination of IL-1β, TNFα, and IFNγ. Protein permeability measured by ¹³¹I-labeled albumin flux was increased by 5-fold over 24 h after cytokine-induced injury. Co-culture of human MSC restored type II cell epithelial permeability to protein to control levels. Using siRNA knockdown of potential paracrine soluble factors, we found that angiopoietin-1 secretion was responsible for this beneficial effect in part by preventing actin stress fiber formation and claudin 18 disorganization through suppression of NFκB activity. This study provides novel evidence for a beneficial effect of MSC on alveolar epithelial permeability to protein.     

Expression of estrogen receptor β increases integrin α1 and integrin β1 levels and enhances adhesion of breast cancer cells

Estrogen effects on mammary gland development and differentiation are mediated by two receptors (ERα and ERβ). Estrogen‐bound ERα induces proliferation of mammary epithelial and cancer cells, while ERβ is important for maintenance of the differentiated epithelium and inhibits proliferation in different cell systems. In addition, the normal breast contains higher ERβ levels compared to the early stage breast cancers, suggesting that loss of ERβ could be important in cancer development. Analysis of ERβ?/? mice has consistently revealed reduced expression of cell adhesion proteins. As such, ERβ is a candidate modulator of epithelial homeostasis and metastasis. Consequently, the aim of this study was to analyze estrogenic effects on adhesion of breast cancer cells expressing ERα and ERβ. As ERβ is widely found in breast cancer but not in cell lines, we used ERα positive T47‐D and MCF‐7 human breast cancer cells to generate cells with inducible ERβ expression. Furthermore, the colon cancer cell lines SW480 and HT‐29 were also used. Integrin α1 mRNA and protein levels increased following ERβ expression. Integrin β1—the unique partner for integrin α1—increased only at the protein level. ERβ expression enhanced the formation of vinculin containing focal complexes and actin filaments, indicating a more adhesive potential. This was confirmed by adhesion assays where ERβ increased adhesion to different extracellular matrix proteins, mostly laminin. In addition, ERβ expression was associated to less cell migration. These results indicate that ERβ affects integrin expression and clustering and consequently modulates adhesion and migration of breast cancer cells. J. Cell. Physiol. 222:156–167, 2010. © 2009 Wiley‐Liss, Inc.     

Engineering of an Orthogonal Aminoacyl-tRNA Synthetase for Efficient Incorporation of the Non-natural Amino Acid O-Methyl-L-tyrosine using Fluorescence-based Bacterial Cell Sorting

We describe a strategy for the rapid selection of mutant aminoacyl-tRNA synthetases (aaRS) with specificity for a novel amino acid based on fluorescence-activated cell sorting of transformed Escherichia coli using as reporter the enhanced green fluorescent protein (eGFP) whose gene carries an amber stop codon (TAG) at a permissive site upstream of the fluorophore. To this end, a one-plasmid expression system was developed encoding an inducible modified Methanocaldococcus jannaschii (Mj) tyrosyl-tRNA synthetase, the orthogonal cognate suppressor tRNA, and eGFP_UAG in an individually regulatable fashion. Using this system a previously described aaRS with specificity for O-methyl-L-tyrosine (MeTyr) was engineered for 10-fold improved incorporation of the foreign amino acid by selection from a mutant library, prepared by error-prone as well as focused random mutagenesis, for MeTyr-dependent eGFP fluorescence. Applying alternating cycles of positive and negative fluorescence-activated bacterial cell sorting in the presence or in the absence, respectively, of the foreign amino acid was crucial to select for high specificity of MeTyr incorporation. The optimized synthetase was used for the preparative expression of a modified uvGFP carrying MeTyr at position 66 as part of its fluorophore. This biosynthetic protein showed quantitative incorporation of the non-natural amino acid, as determined by mass spectrometry, and it revealed a unique emission spectrum due to the altered chemical structure of its fluorophore. Our combined genetic/selection system offers advantages over earlier approaches that relied wholly or in part on antibiotic selection schemes, and it should be generally useful for the engineering and optimization of orthogonal aaRS/tRNA pairs to incorporate non-natural amino acids into recombinant proteins.     

Recovery after shift work: Relation to coronary risk factors in women

Shift work increases the risk for developing cardiovascular disease. There is, however, little knowledge of what aspects of shift scheduling that are detrimental and what characteristics promote good health. The aim of the present study was to evaluate whether coronary risk factors deteriorate after a hard work period and whether recovery, in the form of a week off, was sufficient to improve them. A total of 19 women worked an extremely rapidly rotating and clockwise shift schedule at a paper and pulp factory. They underwent two health examinations, one at the end of the work period and one after the week off. In addition, the women were divided into a tolerant and a vulnerable group, depending on their satisfaction with their work hours. Most risk factors did not change, but total cholesterol and low-density lipoprotein (LDL)-cholesterol were lower after the working period than after the week-off. In addition, vulnerable women had higher levels of total cholesterol and a higher ratio of total cholesterol/high-density lipoprotein (HDL) than tolerant ones. In conclusion, the finding that a week-off worsens cholesterol levels was against our hypothesis and suggests further studies on how activities/responsibilities outside the workplace affect shift-working women. It was also shown that susceptible shift workers had worse lipid profiles.     

Free energy calculations show that acidic P1 variants undergo large pKa shifts upon binding to trypsin

Serine proteinases and their protein inhibitors belong to one of the most comprehensively studied models of protein-protein interactions. It is well established that the narrow trypsin specificity is caused by the presence of a negatively charged aspartate at the specificity pocket. X-ray crystallography as well as association measurements revealed, surprisingly, that BPTI with glutamatic acid as the primary binding (P1) residue was able to bind to trypsin. Previous free energy calculations showed that there was a substantially unfavorable binding free energy associated with accommodation of ionized P1 Glu at the S1-site of trypsin. In this study, the binding of P1 Glu to trypsin has been systematically investigated in terms of the protonation states of P1 Glu and Asp189, the orientation of Gln192, as well as the possible presence of counterions using the linear interaction energy (LIE) approach and the free energy perturbation (FEP) method. Twenty-four conceivable binding arrangements were evaluated and quantitative agreement with experiments is obtained when the P1 Glu binds in its protonated from. The results suggest that P1 Glu is one of the variants of BPTI that inhibit trypsin strongest at low pH, contrary to the specificity profile of trypsin, suggesting a new regulation mechanism of trypsin-like enzymes.     

Haemophilus influenzae surface fibrils contribute to serum resistance by interacting with vitronectin

Vitronectin inhibits the membrane attack complex of the complement system and is found both in plasma and the extracellular matrix. In this study, we have identified the outer membrane protein Haemophilus surface fibrils (Hsf) as the major vitronectin-binding protein in encapsulated H. influenzae type b. A H. influenzae mutant devoid of Hsf showed a significantly decreased binding to both soluble and immobilized vitronectin as compared with the wild-type counterpart. Moreover, Escherichia coli-expressing Hsf at the surface strongly adhered to immobilized vitronectin. Importantly, the H. influenzae Hsf mutant had a markedly reduced survival as compared with the wild-type bacterium when incubated with normal human serum. A series of truncated Hsf fragments were recombinantly manufactured in E. coli. The vitronectin binding regions were located within two separate binding domains. In conclusion, Hsf interacts with vitronectin and thereby inhibits the complement-mediated bactericidal activity, and thus is a major H. influenzae virulence factor.     

Metabolic regulation of type III secretion gene expression in Pseudomonas aeruginosa

Type III secretion-mediated cytotoxicity is one of the key virulence mechanisms of the opportunistic pathogen Pseudomonas aeruginosa. Prior data from several laboratories have established that metabolism is a key factor in the regulation of type III secretion gene expression in P. aeruginosa. Here we use a fluorescence-activated cell sorter (FACS)-based approach to investigate expression of type III secretion genes at a single-cell level. The data demonstrate that the metabolic state regulates the percentage of cells that are able to induce type III secretion gene expression under inducing conditions. We also present evidence that this regulation is the result of an effect of the growth conditions on the ability of P. aeruginosa to assemble a functional type III secretion apparatus. Preliminary data suggest that the metabolite that controls type III secretion gene expression is derived from acetyl-CoA and that this regulation may, in part, be mediated by changes in the intracellular concentration of cyclic-AMP.