Use of endogenous signal sequences for transient production and efficient secretion by moss (Physcomitrella patens) cells

BACKGROUND: Efficient targeting to appropriate cell organelles is one of the bottlenecks for the production of recombinant proteins in plant systems. A common practice is to use the native secretory signal peptide of the heterologous protein to be produced. Though general features of secretion signals are conserved between plants and animals, the broad sequence variability among signal peptides suggests differing efficiency of signal peptide recognition. RESULTS: Aiming to improve secretion in moss bioreactors, we quantitatively compared the efficiency of two human signal peptides and six signals from recently isolated moss (Physcomitrella patens) proteins. We therefore used fusions of the different signals to heterologous reporter sequences for transient transfection of moss cells and measured the extra- and intracellular accumulation of the recombinant proteins rhVEGF and GST, respectively. Our data demonstrates an up to fivefold higher secretion efficiency with endogenous moss signals compared to the two utilised human signal peptides. CONCLUSION: From the distribution of extra- and intracellular recombinant proteins, we suggest translational inhibition during the signal recognition particle-cycle (SRP-cycle) as the most probable of several possible explanations for the decreased extracellular accumulation with the human signals. In this work, we report on the supremacy of moss secretion signals over the utilised heterologous ones within the moss-bioreactor system. Though the molecular details of this effect remain to be elucidated, our results will contribute to the improvement of molecular farming systems

Distributional Patterns of Pseudacteon Associated with the Solenopsis saevissima Complex in South America

Classical biological control efforts against imported fire ants have largely involved the use of Pseudacteon parasitoids. To facilitate further exploration for species and population biotypes a database of collection records for Pseudacteon species was organized, including those from the literature and other sources. These data were then used to map the geographical ranges of species associated with the imported fire ants in their native range in South America. In addition, we found geographical range metrics for all species in the genus and related these metrics to latitude and host use. Approximately equal numbers of Pseudacteon species were found in temperate and tropical regions, though the majority of taxa found only in temperate areas were found in the Northern Hemisphere. No significant differences in sizes of geographical ranges were found between Pseudacteon associated with the different host complexes of fire ants despite the much larger and systemic collection effort associated with the S. saevissima host group. The geographical range of the flies was loosely associated with both the number of hosts and the geographical range of their hosts. Pseudacteon with the most extensive ranges had either multiple hosts or hosts with broad distributions. Mean species richnesses of Pseudacteon in locality species assemblages associated with S. saevissima complex ants was 2.8 species, but intensively sampled locations were usually much higher. Possible factors are discussed related to variation in the size of geographical range, and areas in southern South America are outlined that are likely to have been under-explored for Pseudacteon associated with imported fire ants

Pathogens and host immunity in the ancient human oral cavity.

Calcified dental plaque (dental calculus) preserves for millennia and entraps biomolecules from all domains of life and viruses. We report the first, to our knowledge, high-resolution taxonomic and protein functional characterization of the ancient oral microbiome and demonstrate that the oral cavity has long served as a reservoir for bacteria implicated in both local and systemic disease. We characterize (i) the ancient oral microbiome in a diseased state, (ii) 40 opportunistic pathogens, (iii) ancient human-associated putative antibiotic resistance genes, (iv) a genome reconstruction of the periodontal pathogen Tannerella forsythia, (v) 239 bacterial and 43 human proteins, allowing confirmation of a long-term association between host immune factors, 'red complex' pathogens and periodontal disease, and (vi) DNA sequences matching dietary sources. Directly datable and nearly ubiquitous, dental calculus permits the simultaneous investigation of pathogen activity, host immunity and diet, thereby extending direct investigation of common diseases into the human evolutionary past

Likelihood Based Inference and Prediction in Spatio-Temporal Panel Count Models for Urban Crimes

PRELIMINARY DRAFT We discuss maximum likelihood (ML) analysis for panel count data models, in which the observed counts are linked via a measurement density to a latent Gaussian process with spatial as well as temporal dynamics and random effects. For likelihood evaluation requiring high-dimensional integration we rely upon Efficient Importance Sampling (EIS). The algorithm we develop extends existing EIS implementations by constructing importance sampling densities, which closely approximate the nontrivial spatio-temporal correlation structure under dynamic spatial panel models. In order to make this high-dimensional approximation computationally feasible, our EIS implementation exploits the typical sparsity of spatial precision matrices in such a way that all the high-dimensional matrix operations it requires can be performed using computationally fast sparse matrix functions. We use the proposed sparse EIS-ML approach for an extensive empirical study analyzing the socio-demographic determinants and the space-time dynamics of urban crime in Pittsburgh, USA, between 2008 and 2013 for a panel of monthly crime rates at census-tract level

Chromosomal high mobility group (HMG) proteins of the HMGB-type occurring in the moss Physcomitrella patens

Kiilerich B, Stemmer C, Merkle T, Launholt D, Gorr G, Grasser KD. Chromosomal high mobility group (HMG) proteins of the HMGB-type occurring in the moss Physcomitrella patens. Gene. 2008;407(1-2):86-97.High mobility group (HMG) proteins of the HMGB family are chromatin-associated proteins that act as architectural factors in nucleoprotein structures, which regulate DNA-dependent processes including transcription. Members of the HMGB family have been characterised from various mono-and dicot plants, but not from lower plant species. Here, we have identified three candidate HMGB proteins encoded in the genome of the moss Physcomitrella patens. The structurally similar HMGB2 and HMGB3 proteins display the typical overall structure of higher plant HMGB proteins consisting of a central HMG-box DNA-binding domain that is flanked by a basic N-terminal and an acidic C-terminal domain. The HMGB1 protein differs from higher plant HMGB proteins by having a very extensive N-terminal domain and by lacking the acidic C-terminal domain. Like higher plant HMGB proteins, HMGB3 localises to the cell nucleus, but HMGB I is targeted to plastids. Analysis of the HMG-box domains of HMGB1 and HMGB3 by CD revealed that HMGB1box and the HMGB3box have an alpha-helical structure. While the HMGB3box interacts with DNA comparable to typical higher plant counterparts, the HMGB I box has only a low affinity for DNA. Cotransformation assays in Physcomitrella protoplasts demonstrated that expression of HMGB3 resulted in repression of reporter gene expression. In summary, our data show that functional HMGB-type proteins occur in Physcomitrella and most likely in other lower plant species. (c) 2007 Elsevier B.V All rights reserved

Increased natural mortality at low abundance can generate a demographic Allee effect

Negative density-dependent regulation of population dynamics promotes population growth at low abundance and is therefore vital for recovery following depletion. Inversely, any process that reduces the compensatory density-dependence of population growth can negatively affect recovery. Here, we show that increased adult mortality at low abundance can reverse compensatory population dynamics into its opposite—a demographic Allee effect. Northwest Atlantic cod (Gadus morhua) stocks collapsed dramatically in the early 1990s and have since shown little sign of recovery. Many experienced dramatic increases in natural mortality, ostensibly attributable in some populations to increased predation by seals. Our findings show that increased natural mortality of a magnitude observed for overfished cod stocks has been more than sufficient to fundamentally alter the dynamics of density-dependent population regulation. The demographic Allee effect generated by these changes can slow down or even impede the recovery of depleted populations even in the absence of fishing

Use of endogenous signal sequences for transient production and efficient secretion by moss () cells-0

<p><b>Copyright information:</b></p><p>Taken from "Use of endogenous signal sequences for transient production and efficient secretion by moss () cells"</p><p>BMC Biotechnology 2005;5():30-30.</p><p>Published online 7 Nov 2005</p><p>PMCID:PMC1291358.</p><p>Copyright © 2005 Schaaf et al; licensee BioMed Central Ltd.</p>rowth factor (VSP) were fused to the coding sequence of vascular endothelial growth factor (hVEGF). Moss protoplasts were transiently transfected with the expression constructs and concentration of extracellular as well as intracellular rhVEGF was determined by ELISA. Mean values were taken from three transfections. Error bars indicate the absolute average deviation (AAD)

Use of endogenous signal sequences for transient production and efficient secretion by moss () cells-5

<p><b>Copyright information:</b></p><p>Taken from "Use of endogenous signal sequences for transient production and efficient secretion by moss () cells"</p><p>BMC Biotechnology 2005;5():30-30.</p><p>Published online 7 Nov 2005</p><p>PMCID:PMC1291358.</p><p>Copyright © 2005 Schaaf et al; licensee BioMed Central Ltd.</p>pFLP, PpLTP, PpPME1, PpXTH1, PpCALP, and VEGF are shown as predicted by SignalP. Letters with yellow background represent hydrophobic amino acid residues (V, L, I, W, F, M), orange letters positive (K, R) and green letters small and neutral residues (A, C, G, N, P, S, T, V). The predicted cleavage sites are indicated as well as the positions adjacent to this site. In the right part of the figure probabilities for each signal peptide and the cleavage site given by the SignalP-HMM prediction as well as the length of the signal peptides are shown

Use of endogenous signal sequences for transient production and efficient secretion by moss () cells-1

<p><b>Copyright information:</b></p><p>Taken from "Use of endogenous signal sequences for transient production and efficient secretion by moss () cells"</p><p>BMC Biotechnology 2005;5():30-30.</p><p>Published online 7 Nov 2005</p><p>PMCID:PMC1291358.</p><p>Copyright © 2005 Schaaf et al; licensee BioMed Central Ltd.</p>s. After 5 days the concentrations of secreted and intracellularly-retained recombinant protein were measured by ELISA. Additionally, GST was affinity-purified from both medium and supernatant and detected by western blot. Culture medium of two transfections was pooled. Mean values of eight transfections are given. Protoplasts of eight transfections were pooled and GST was affinity-purified from the crude extracts. Values reflect the means of the two measurements. Control: mock-transfected protoplasts

Use of endogenous signal sequences for transient production and efficient secretion by moss () cells-2

<p><b>Copyright information:</b></p><p>Taken from "Use of endogenous signal sequences for transient production and efficient secretion by moss () cells"</p><p>BMC Biotechnology 2005;5():30-30.</p><p>Published online 7 Nov 2005</p><p>PMCID:PMC1291358.</p><p>Copyright © 2005 Schaaf et al; licensee BioMed Central Ltd.</p> and seven (pASP-VEGF) independent transfections. After 5 days the concentrations of secreted and intracellularly retained recombinant protein were measured by ELISA. mean values of concentrations of extra- and intracellular rhVEGF measured by ELISA. Western blot with culture medium of transiently rhVEGF-producing cell. Control: untransfected moss protoplasts