Tandem affinity purification of functional TAP-tagged proteins from human cells

Tandem affinity purification (TAP) is a generic two-step affinity purification protocol for isolation of TAP-tagged proteins together with associated proteins. We used bacterial artificial chromosome to heterologously express TAP-tagged murine Sgo1 protein in human HeLa cells. This allowed us to test the functionality of the Sgo1-TAP protein by RNA interference-mediated depletion of the endogenous human Sgo1. Here, we present an optimized protocol for purification of TAP-tagged Sgo1 protein as well as KIAA1387 from HeLa cells with detailed instructions. The purification protocol can be completed in 1 day and it should be applicable to other proteins.     

In vivo glyco-engineered antibody with improved lytic potential produced by an innovative non-mammalian expression system

Recent studies have demonstrated that the reduction of the core fucosylation on N-glycans of human IgGs is responsible for a clearly enhanced antibody-dependent cellular cytotoxicity (ADCC). This finding might give access to improved active therapeutic antibodies. Here, the expression of the tumor antigen-specific antibody IGN311 was performed in a glyco-optimized strain of the moss Physcomitrella patens. Removal of plant specific N-glycan structures in this plant expression host was achieved by targeted knockout of corresponding genes and included quantitative elimination of core fucosylation. Antibodies transiently expressed and secreted by such genetically modified moss protoplasts assembled correctly, showed an unaltered antigen-binding affinity and, in extensive tests, revealed an up to 40-fold enhanced ADCC. Thus, the glyco-engineered moss-based transient expression platform combines a rapid technology with the subsequent analysis of glycooptimized therapeutics with regard to advanced properties.     

A genomic background based method for association analysis in related individuals

Background

Feasibility of genotyping of hundreds and thousands of single nucleotide polymorphisms (SNPs) in thousands of study subjects have triggered the need for fast, powerful, and reliable methods for genome-wide association analysis. Here we consider a situation when study participants are genetically related (e.g. due to systematic sampling of families or because a study was performed in a genetically isolated population). Of the available methods that account for relatedness, the Measured Genotype (MG) approach is considered the ‘gold standard’. However, MG is not efficient with respect to time taken for the analysis of genome-wide data. In this context we proposed a fast two-step method called Genome-wide Association using Mixed Model and Regression (GRAMMAR) for the analysis of pedigree-based quantitative traits. This method certainly overcomes the drawback of time limitation of the measured genotype (MG) approach, but pays in power. One of the major drawbacks of both MG and GRAMMAR, is that they crucially depend on the availability of complete and correct pedigree data, which is rarely available.

Methodology

In this study we first explore type 1 error and relative power of MG, GRAMMAR, and Genomic Control (GC) approaches for genetic association analysis. Secondly, we propose an extension to GRAMMAR i.e. GRAMMAR-GC. Finally, we propose application of GRAMMAR-GC using the kinship matrix estimated through genomic marker data, instead of (possibly missing and/or incorrect) genealogy.

Conclusion

Through simulations we show that MG approach maintains high power across a range of heritabilities and possible pedigree structures, and always outperforms other contemporary methods. We also show that the power of our proposed GRAMMAR-GC approaches to that of the ‘gold standard’ MG for all models and pedigrees studied. We show that this method is both feasible and powerful and has correct type 1 error in the context of genome-wide association analysis in related individuals.     

Syk-mediated translocation of PI3Kdelta to the leading edge controls lamellipodium formation and migration of leukocytes

The non-receptor tyrosine kinase Syk is mainly expressed in the hematopoietic system and plays an essential role in beta(2) integrin-mediated leukocyte activation. To elucidate the signaling pathway downstream of Syk during beta2 integrin (CD11/CD18)-mediated migration and extravasation of polymorphonuclear neutrophils (PMN), we generated neutrophil-like differentiated HL-60 (dHL-60) cells expressing a fluorescently tagged Syk mutant lacking the tyrosine residue at the position 323 (Syk-Tyr323) that is known to be required for the binding of the regulatory subunit p85 of the phosphatidylinositol 3-kinase (PI3K) class I(A). Syk-Tyr323 was found to be critical for the enrichment of the catalytic subunit p110delta of PI3K class I(A) as well as for the generation of PI3K products at the leading edge of the majority of polarized cells. In accordance, the translocation of PI3K p110delta to the leading edge was diminished in Syk deficient murine PMN. Moreover, the expression of EGFP-Syk Y323F interfered with proper cell polarization and it impaired efficient migration of dHL-60 cells. In agreement with a major role of beta2 integrins in the recruitment of phagocytic cells to sites of lesion, mice with a Syk-deficient hematopoietic system demonstrated impaired PMN infiltration into the wounded tissue that was associated with prolonged cutaneous wound healing. These data imply a novel role of Syk via PI3K p110delta signaling for beta2 integrin-mediated migration which is a prerequisite for efficient PMN recruitment in vivo.     

Amsterdam urban canals contain novel niches for methane-cycling microorganisms

Urbanised environments have been identified as hotspots of anthropogenic methane emissions. Especially urban aquatic ecosystems are increasingly recognised as important sources of methane. However, the microbiology behind these emissions remains unexplored. Here, we applied microcosm incubations and molecular analyses to investigate the methane-cycling community of the Amsterdam canal system in the Netherlands. The sediment methanogenic communities were dominated by Methanoregulaceae and Methanosaetaceae, with co-occurring methanotrophic Methanoperedenaceae and Methylomirabilaceae indicating the potential for anaerobic methane oxidation. Methane was readily produced after substrate amendment, suggesting an active but substrate-limited methanogenic community. Bacterial 16S rRNA gene amplicon sequencing of the sediment revealed a high relative abundance of Thermodesulfovibrionia. Canal wall biofilms showed the highest initial methanotrophic potential under oxic conditions compared to the sediment. During prolonged incubations the maximum methanotrophic rate increased to 8.08 mmol g_DW⁻¹ d⁻¹ that was concomitant with an enrichment of Methylomonadaceae bacteria. Metagenomic analysis of the canal wall biofilm lead to the recovery of a single methanotroph metagenome-assembled genome. Taxonomic analysis showed that this methanotroph belongs to the genus Methyloglobulus. Our results underline the importance of previously unidentified and specialised environmental niches at the nexus of the natural and human-impacted carbon cycle.     

Variations in carotenoid content and acyl chain composition in exponential,stationary and biofilm states of Staphylococcus aureus,and their influence on membrane biophysical properties

Bacteria are often found in close association with surfaces, resulting in the formation of biofilms. In Staphylococcus aureus (S. aureus), biofilms are implicated in the resilience of chronic infections, presenting a serious clinical problem world-wide. Here, S. aureus biofilms are grown under flow within clinical catheters at 37 °C. The lipid composition and biophysical properties of lipid extracts from these biofilms are compared with those from exponential growth and stationary phase cells. Biofilms show a reduction in iso and anteiso branching compensated by an increase in saturated fatty acids compared to stationary phase. A drastic reduction in carotenoid levels is also observed during biofilm formation. Thermotropic measurements of Laurdan GP and DPH polarization, show a reduction of lipid packing at 37 °C for biofilms compared to stationary phase. We studied the effects of carotenoid content on DMPG and DPPG model membranes showing trends in thermotropic behavior consistent with those observed in bacterial isolates, indicating that carotenoids participate in modulating lipid packing. Additionally, bending elastic constant (k_c) measurements using vesicle fluctuation analysis (VFA) show that the presence of carotenoids can increase membrane bending rigidity. The antimicrobial peptide Magainin H2 was less activity on liposomes composed of stationary phase compared to biofilms or exponential growth isolates. This study contributes to an understanding of how Staphylococcus aureus modulates the composition of its membrane lipids, and how those changes affect the biophysical properties of membranes, which in turn may play a role in its virulence and its resistance to different membrane-active antimicrobial agents.     

Generation and characterization of a functional human adipose-derived multipotent mesenchymal stromal cell line

Multipotent mesenchymal stromal cells (MSC) and MSC-derived products have emerged as promising therapeutic tools. To fully exploit their potential, further mechanistic studies are still necessary and bioprocessing needs to be optimized, which requires an abundant supply of functional MSC for basic research. To address this need, here we used a novel technology to establish a human adipose-derived MSC line with functional characteristics representative of primary MSC. Primary MSC were isolated and subjected to lentiviral transduction with a library of expansion genes. Clonal cell lines were generated and evaluated on the basis of their morphology, immunophenotype, and proliferation potential. One clone (K5 iMSC) was then selected for further characterization. This clone had integrated a specific transgene combination including genes involved in stemness and maintenance of adult stem cells. Favorably, the K5 iMSC showed cell characteristics resembling juvenile MSC, as they displayed a shorter cell length and enhanced migration and proliferation compared with the non-immortalized original primary MSC (p < 0.05). Still, their immunophenotype and differentiation potential corresponded to the original primary MSC and the MSC definition criteria, and cytogenetic analyses revealed no clonal aberrations. We conclude that the technology used is applicable to generate functional MSC lines for basic research and possible future bioprocessing applications.     

Cell fate specification and differentiation in the nervous system of Caenorhabditis elegans

Neuronal cell fates are specified by a hierarchy of events mediated by cell-intrinsic determinants and cell-cell interactions. The determination of cell fate can be subdivided into three general steps. First, cell fate is restricted by the cell's position in the animal. For example, neurons are specified along the anterior-posterior body axis through the action of the Hox genes lin-39, mab-5, and egl-5. Second, a decision is made to generate a particular cell type, such as the progenitor of a neurogenic lineage as opposed to that of an epidermal lineage. Among the genes that influence this decision is the proneural gene lin-32. Third, characteristics of a particular cell type are specified. For example, in a neurogenic lineage, a decision may be made to generate a specific neuron type such as a sensory or motor neuron. Genes that affect neuronal fate can act in different ways to influence the development of different types of neurons. © 1996 Wiley-Liss, Inc.