SnAvi – a new tandem tag for high-affinity protein-complex purification

Systematic tandem-affinity-purification (TAP) of protein complexes was tremendously successful in yeast and has changed the general concept of how we understand protein function in eukaryotic cells. The transfer of this method to other model organisms has been difficult and may require specific adaptations. We were especially interested to establish a cell-type-specific TAP system for Caenorhabditis elegans, a model animal well suited to high-throughput analysis, proteomics and systems biology. By combining the high-affinity interaction between in vivo biotinylated target-proteins and streptavidin with the usage of a newly identified epitope of the publicly shared SB1 monoclonal antibody we created a novel in vivo fluorescent tag, the SnAvi-Tag. We show the versatile application of the SnAvi-Tag in Escherichia coli, vertebrate cells and in C. elegans for tandem affinity purification of protein complexes, western blotting and also for the in vivo sub-cellular localization of labelled proteins.     

Gender comparisons between unilateral and bilateral landings

The increased number of women participating in sports has led to a higher knee injury rate in women compared with men. Among these injuries, those occurring to the ACL are commonly observed during landing maneuvers. The purpose of this study was to determine gender differences in landing strategies during unilateral and bilateral landings. Sixteen male and 17 female recreational athletes were recruited to perform unilateral and bilateral landings from a raised platform, scaled to match their individual jumping abilities. Three-dimensional kinematics and kinetics of the dominant leg were calculated during the landing phase and reported as initial ground contact angle, ranges of motion (ROM) and peak moments. Lower extremity energy absorption was also calculated for the duration of the landing phase. Results showed that gender differences were only observed in sagittal plane hip and knee ROM, potentially due to the use of a relative drop height versus the commonly used absolute drop height. Unilateral landings were characterized by significant differences in hip and knee kinematics that have been linked to increased injury risk and would best be classified as "stiff" landings. The ankle musculature was used more for impact absorption during unilateral landing, which required increased joint extension at touchdown and may increase injury risk during an unbalanced landing. In addition, there was only an 11% increase in total energy absorption during unilateral landings, suggesting that there was a substantial amount of passive energy transfer during unilateral landings.     

Histone methyltransferases in cancer

Cancer is perceived as a heterogeneous group of diseases that is characterized by aberrant patterns of gene expression. In the last decade, an increasing amount of data has pointed to a key role for epigenetic alterations in human cancer. In this review, we focus on a subclass of epigenetic regulators, namely histone methyltransferases (HMTs). Several HMTs have been linked to different types of cancer; however, in most cases we only have limited knowledge regarding the molecular mechanisms by which the HMTs contribute to disease development. We summarize the current knowledge regarding some of the best-validated examples of HMTs contributing to tumorigenesis and discuss their potential mechanisms of action.     

Translational Selection Is Ubiquitous in Prokaryotes

Codon usage bias in prokaryotic genomes is largely a consequence of background substitution patterns in DNA, but highly expressed genes may show a preference towards codons that enable more efficient and/or accurate translation. We introduce a novel approach based on supervised machine learning that detects effects of translational selection on genes, while controlling for local variation in nucleotide substitution patterns represented as sequence composition of intergenic DNA. A cornerstone of our method is a Random Forest classifier that outperformed previous distance measure-based approaches, such as the codon adaptation index, in the task of discerning the (highly expressed) ribosomal protein genes by their codon frequencies. Unlike previous reports, we show evidence that translational selection in prokaryotes is practically universal: in 460 of 461 examined microbial genomes, we find that a subset of genes shows a higher codon usage similarity to the ribosomal proteins than would be expected from the local sequence composition. These genes constitute a substantial part of the genome—between 5% and 33%, depending on genome size—while also exhibiting higher experimentally measured mRNA abundances and tending toward codons that match tRNA anticodons by canonical base pairing. Certain gene functional categories are generally enriched with, or depleted of codon-optimized genes, the trends of enrichment/depletion being conserved between Archaea and Bacteria. Prominent exceptions from these trends might indicate genes with alternative physiological roles; we speculate on specific examples related to detoxication of oxygen radicals and ammonia and to possible misannotations of asparaginyl–tRNA synthetases. Since the presence of codon optimizations on genes is a valid proxy for expression levels in fully sequenced genomes, we provide an example of an “adaptome” by highlighting gene functions with expression levels elevated specifically in thermophilic Bacteria and Archaea.     

B Cell Activation by Outer Membrane Vesicles—A Novel Virulence Mechanism

Secretion of outer membrane vesicles (OMV) is an intriguing phenomenon of Gram-negative bacteria and has been suggested to play a role as virulence factors. The respiratory pathogens Moraxella catarrhalis reside in tonsils adjacent to B cells, and we have previously shown that M. catarrhalis induce a T cell independent B cell response by the immunoglobulin (Ig) D-binding superantigen MID. Here we demonstrate that Moraxella are endocytosed and killed by human tonsillar B cells, whereas OMV have the potential to interact and activate B cells leading to bacterial rescue. The B cell response induced by OMV begins with IgD B cell receptor (BCR) clustering and Ca²⁺ mobilization followed by BCR internalization. In addition to IgD BCR, TLR9 and TLR2 were found to colocalize in lipid raft motifs after exposure to OMV. Two components of the OMV, i.e., MID and unmethylated CpG-DNA motifs, were found to be critical for B cell activation. OMV containing MID bound to and activated tonsillar CD19⁺ IgD⁺ lymphocytes resulting in IL-6 and IgM production in addition to increased surface marker density (HLA-DR, CD45, CD64, and CD86), whereas MID-deficient OMV failed to induce B cell activation. DNA associated with OMV induced full B cell activation by signaling through TLR9. Importantly, this concept was verified in vivo, as OMV equipped with MID and DNA were found in a 9-year old patient suffering from Moraxella sinusitis. In conclusion, Moraxella avoid direct interaction with host B cells by redirecting the adaptive humoral immune response using its superantigen-bearing OMV as decoys.     

Inactivation of Vibrio anguillarum by Attached and Planktonic Roseobacter Cells

The purpose of the present study was to investigate the inhibition of Vibrio by Roseobacter in a combined liquid-surface system. Exposure of Vibrio anguillarum to surface-attached roseobacters (10⁷ CFU/cm²) resulted in significant reduction or complete killing of the pathogen inoculated at 10² to 10⁴ CFU/ml. The effect was likely associated with the production of tropodithietic acid (TDA), as a TDA-negative mutant did not affect survival or growth of V. anguillarum.Antagonistic interactions among marine bacteria are well documented, and secretion of antagonistic compounds is common among bacteria that colonize particles or surfaces (8, 13, 16, 21, 31). These marine bacteria may be interesting as sources for new antimicrobial drugs or as probiotic bacteria for aquaculture.Aquaculture is a rapidly growing sector, but outbreaks of bacterial diseases are a limiting factor and pose a threat, especially to young fish and invertebrates that cannot be vaccinated. Because regular or prophylactic administration of antibiotics must be avoided, probiotic bacteria are considered an alternative (9, 18, 34, 38, 39, 40). Several microorganisms have been able to reduce bacterial diseases in challenge trials with fish or fish larvae (14, 24, 25, 27, 33, 37, 39, 40). One example is Phaeobacter strain 27-4 (17), which inhibits Vibrio anguillarum and reduces mortality in turbot larvae (27). The antagonism of Phaeobacter 27-4 and the closely related Phaeobacter inhibens is due mainly to the sulfur-containing tropolone derivative tropodithietic acid (TDA) (2, 5), which is also produced by other Phaeobacter strains and Ruegeria mobilis (28). Phaeobacter and Ruegeria strains or their DNA has been commonly found in marine larva-rearing sites (6, 17, 28).Phaeobacter and Ruegeria (Alphaproteobacteria, Roseobacter clade) are efficient surface colonizers (7, 11, 31, 36). They are abundant in coastal and eutrophic zones and are often associated with algae (3, 7, 41). Surface-attached Phaeobacter bacteria may play an important role in determining the species composition of an emerging biofilm, as even low densities of attached Phaeobacter strain SK2.10 bacteria can prevent other marine organisms from colonizing solid surfaces (30, 32).In continuation of the previous research on roseobacters as aquaculture probiotics, the purpose of this study was to determine the antagonistic potential of Phaeobacter and Ruegeria against Vibrio anguillarum in liquid systems that mimic a larva-rearing environment. Since production of TDA in liquid marine broth appears to be highest when roseobacters form an air-liquid biofilm (5), we addressed whether they could be applied as biofilms on solid surfaces.     

Heparin promotes fibrillation of most phenol-soluble modulin virulence peptides from Staphylococcus aureus

Phenol-soluble modulins (PSMs), such as α-PSMs, β-PSMs, and δ-toxin, are virulence peptides secreted by different Staphylococcus aureus strains. PSMs are able to form amyloid fibrils, which may strengthen the biofilm matrix that promotes bacterial colonization of and extended growth on surfaces (e.g., cell tissue) and increases antibiotic resistance. Many components contribute to biofilm formation, including the human-produced highly sulfated glycosaminoglycan heparin. Although heparin promotes S. aureus infection, the molecular basis for this is unclear. Given that heparin is known to induce fibrillation of a wide range of proteins, we hypothesized that heparin aids bacterial colonization by promoting PSM fibrillation. Here, we address this hypothesis using a combination of thioflavin T-fluorescence kinetic studies, CD, FTIR, electron microscopy, and peptide microarrays to investigate the mechanism of aggregation, the structure of the fibrils, and identify possible binding regions. We found that heparin accelerates fibrillation of all α-PSMs (except PSMα2) and δ-toxin but inhibits β-PSM fibrillation by blocking nucleation or reducing fibrillation levels. Given that S. aureus secretes higher levels of α-PSM than β-PSM peptides, heparin is therefore likely to promote fibrillation overall. Heparin binding is driven by multiple positively charged lysine residues in α-PSMs and δ-toxins, the removal of which strongly reduced binding affinity. Binding of heparin did not affect the structure of the resulting fibrils, that is, the outcome of the aggregation process. Rather, heparin provided a scaffold to catalyze or inhibit fibrillation. Based on our findings, we speculate that heparin may strengthen the bacterial biofilm and therefore enhance colonization via increased PSM fibrillation.     

Repression of both isoforms of disproportionating enzyme leads to higher malto-oligosaccharide content and reduced growth in potato

Two glucanotransferases, disproportionating enzyme 1 (StDPE1) and disproportionating enzyme 2 (StDPE2), were repressed using RNA interference technology in potato, leading to plants repressed in either isoform individually, or both simultaneously. This is the first detailed report of their combined repression. Plants lacking StDPE1 accumulated slightly more starch in their leaves than control plants and high levels of maltotriose, while those lacking StDPE2 contained maltose and large amounts of starch. Plants repressed in both isoforms accumulated similar amounts of starch to those lacking StDPE2. In addition, they contained a range of malto-oligosaccharides from maltose to maltoheptaose. Plants repressed in both isoforms had chlorotic leaves and did not grow as well as either the controls or lines where only one of the isoforms was repressed. Examination of photosynthetic parameters suggested that this was most likely due to a decrease in carbon assimilation. The subcellular localisation of StDPE2 was re-addressed in parallel with DPE2 from Arabidopsis thaliana by transient expression of yellow fluorescent protein fusions in tobacco. No translocation to the chloroplasts was observed for any of the fusion proteins, supporting a cytosolic role of the StDPE2 enzyme in leaf starch metabolism, as has been observed for Arabidopsis DPE2. It is concluded that StDPE1 and StDPE2 have individual essential roles in starch metabolism in potato and consequently repression of these disables regulation of leaf malto-oligosaccharides, starch content and photosynthetic activity and thereby plant growth possibly by a negative feedback mechanism.     

Integrin-linked kinase controls microtubule dynamics required for plasma membrane targeting of caveolae

Caveolae are specialized compartments of the plasma membrane that are involved in signaling, endocytosis, and cholesterol transport. Their formation requires the transport of caveolin-1 to the plasma membrane, but the molecular mechanisms regulating the transport are largely unknown. Here, we?identify a critical role for adhesion-mediated signaling through β1 integrins and integrin-linked kinase (ILK) in caveolae formation. Mice lacking β1 integrins or ILK in keratinocytes have dramatically reduced numbers of plasma membrane caveolae in?vivo, which is due to impaired transport of caveolin-1-containing vesicles along microtubules (MT) to the plasma membrane. Mechanistically, ILK promotes the recruitment of the F-actin binding protein IQGAP1 to the cell cortex, which, in turn, cooperates with its?effector mDia1 to locally stabilize MTs and to allow?stable insertion of caveolae into the plasma membrane. Our results assign an important role to the integrin/ILK complex for caveolar trafficking to the cell surface.