Gene delivery to respiratory epithelial cells by magnetofection

BACKGROUND: For the topical application of DNA vector complexes to the airways, specific extracellular barriers play a major role. In particular, short contact time of complexes with the cell surface caused by the mucociliary clearance hinders cellular uptake of complexes. The aim of this study was to evaluate the ability of magnetofection, a technique based on the principle of magnetic drug targeting, to overcome these barriers in comparison with conventional nonviral gene transfer methods such as lipofection and polyfection. METHODS: Experiments were carried out on permanent (16HBE14o-) and primary airway epithelial cells (porcine and human), and native porcine airway epithelium ex vivo. Transfection efficiency and dose-response relationship of magnetofection were examined by luciferase reporter gene expression. Sedimentation patterns and uptake of gene transfer complexes were characterized by fluorescence and electron microscopy, respectively. RESULTS: We show that (i) application of a magnetic field allows the magnetofectins to sediment and to enrich at the cell surface within a few minutes, (ii) magnetofection bears an improved dose-response relationship, (iii) magnetofection enhances transfection efficiency in both, permanent and primary airway epithelial cells, and (iv) magnetofection leads to significant transgene expression at very short incubation times in an ex vivo airway epithelium organ model. CONCLUSIONS: Magnetofection provides a potential novel method, which may overcome fundamental limitations of nonviral gene transfer to the airways. Due to the accelerated enrichment at the cell surface it may be of major interest for in vivo applications, where long-term incubation times at the target tissue are hardly achievable.     

Comparative Structure-Function Analysis of Mannose-Specific FimH Adhesins from Klebsiella pneumoniae and Escherichia coli

FimH, the adhesive subunit of type 1 fimbriae expressed by many enterobacteria, mediates mannose-sensitive binding to target host cells. At the same time, fine receptor-structural specificities of FimH from different species can be substantially different, affecting bacterial tissue tropism and, as a result, the role of the particular fimbriae in pathogenesis. In this study, we compared functional properties of the FimH proteins from Escherichia coli and Klebsiella pneumoniae, which are both 279 amino acids in length but differ by some ∼15% of residues. We show that K. pneumoniae FimH is unable to mediate adhesion in a monomannose-specific manner via terminally exposed Manα(1-2) residues in N-linked oligosaccharides, which are the structural basis of the tropism of E. coli FimH for uroepithelial cells. However, K. pneumoniae FimH can bind to the terminally exposed Manα(1-3)Manβ(1-4)GlcNAcβ1 trisaccharide, though only in a shear-dependent manner, wherein the binding is marginal at low shear force but enhanced sevenfold under increased shear. A single mutation in the K. pneumoniae FimH, S62A, converts the mode of binding from shear dependent to shear independent. This mutation has occurred naturally in the course of endemic circulation of a nosocomial uropathogenic clone and is identical to a pathogenicity-adaptive mutation found in highly virulent uropathogenic strains of E. coli, in which it also eliminates the dependence of E. coli binding on shear. The shear-dependent binding properties of the K. pneumoniae and E. coli FimH proteins are mediated via an allosteric catch bond mechanism. Thus, despite differences in FimH structure and fine receptor specificity, the shear-dependent nature of FimH-mediated adhesion is highly conserved between bacterial species, supporting its remarkable physiological significance.The most common type of adhesive organelle in the Enterobacteriaceae is the type 1 fimbria, which has been most extensively studied in Escherichia coli. The corresponding structures of Klebsiella pneumoniae are similar to those of E. coli with regard to genetic composition and regulation (15). Type 1 fimbriae are composed primarily of the structural subunit FimA, with minor amounts of three ancillary subunits, FimF, FimG, and the mannose-specific adhesin FimH. The FimH adhesin is an allosteric protein that mediates the catch bond mechanism of adhesion where the binding is increased under increased shear stress (48).It has been demonstrated in E. coli that FimH has two domains, the mannose-binding lectin domain (from amino acid [aa] 1 through 156) and the fimbria-incorporating pilin domain (from aa 160 through 279), connected via a 3-aa-long linker chain (6). A mannose-binding site is located at the top of the lectin domain, at the opposite end from the interdomain linker (17).Several studies have demonstrated that type 1 fimbriae play an important role in E. coli urinary tract infection (UTI) (7, 21, 23, 35). In addition, in urinary E. coli isolates, the FimH adhesin accumulates amino acid replacements which increase tropism for the uroepithelium and various components of basement membranes (21, 30, 35, 37, 49). Most of the replacements increase the monomannose binding capability of FimH under low shear, by altering allosteric catch bond properties of the protein (48). The mutated FimH variants were shown to provide an advantage in colonization of the urinary tract in the mouse model (35) and correlate with the overall extraintestinal virulence of E. coli (16). Thus, FimH mutations are pathoadaptive in nature.Klebsiella pneumoniae is recognized as an important opportunistic pathogen frequently causing UTIs, septicemia, or pneumonia in immunocompromised individuals (29). It is responsible for up to 10% of all nosocomial bacterial infections (18, 41). K. pneumoniae is ubiquitous in nature, and it has been shown that environmental isolates are phenotypically indistinguishable from clinical isolates (22, 26, 27, 29, 33). Furthermore, it has been demonstrated that environmental isolates of K. pneumoniae are as virulent as clinical isolates (28, 45).K. pneumoniae possesses a number of known virulence factors, including a pronounced capsule, type 3 fimbriae, and type 1 fimbriae (29, 44). Type 1 fimbriae produced by K. pneumoniae are described as functionally and structurally similar to type 1 fimbriae from E. coli (25) and have been shown to play a significant role in K. pneumoniae UTI (32, 43).We have previously shown that mature FimH from 54 isolates of K. pneumoniae (isolated from urine, blood, liver, and the environment) is represented by seven protein variants due to point amino acid replacements. (42) When K. pneumoniae FimH was aligned with the FimH of E. coli, they showed ∼85% similarity at the amino acid level. Furthermore, a majority (14 out of 21 isolates) of the K. pneumoniae strains isolated from patients with UTI grouped into a single clonal group based on multilocus sequence typing, but fimH in one isolate in the group differed from the others by a single nucleotide mutation resulting in an amino acid change, serine to alanine, in position 62 (42). The same mutation has been found in FimH of a highly uropathogenic clone of E. coli and significantly increases the adhesin''s ability to adhere to monomannose under low or no shear (19, 39, 50).In this study, we describe the extent and pattern of structural variability of the FimH protein from K. pneumoniae and perform comparative analyses of the functional properties of FimH from both K. pneumonae and E. coli.     

The molecular basis of quantitative variation in foliar secondary metabolites in Eucalyptus globulus

Eucalyptus is characterized by high foliar concentrations of plant secondary metabolites with marked qualitative and quantitative variation within a single species. Secondary metabolites in eucalypts are important mediators of a diverse community of herbivores. We used a candidate gene approach to investigate genetic associations between 195 single nucleotide polymorphisms (SNPs) from 24 candidate genes and 33 traits related to secondary metabolites in the Tasmanian Blue Gum (Eucalyptus globulus). We discovered 37 significant associations (false discovery rate (FDR) Q < 0.05) across 11 candidate genes and 19 traits. The effects of SNPs on phenotypic variation were within the expected range (0.018 < r(2) < 0.061) for forest trees. Whereas most marker effects were nonadditive, two alleles from two consecutive genes in the methylerythritol phosphate pathway (MEP) showed additive effects. This study successfully links allelic variants to ecologically important phenotypes which can have a large impact on the entire community. It is one of very few studies to identify the genetic variants of a foundation tree that influences ecosystem function.     

Helices in peptoids of alpha- and beta-peptides

Peptoids of alpha- and beta-peptides (alpha- and beta-peptoids) can be obtained by shifting the amino acid side chains from the backbone carbon atoms of the monomer constituents to the peptide nitrogen atoms. They are, therefore, N-substituted poly-glycines and poly-beta-alanines, respectively. Due to the substituted nitrogen atoms, the ability for hydrogen bond formation between peptide bonds gets lost. It may be very interesting to see whether such non-natural oligomers could be regarded as foldamers, which fold into definite backbone conformers. In this paper, we provide a complete overview on helix formation in alpha- and beta-peptoids on the basis of systematic theoretical conformational analyses employing the methods of ab initio molecular orbital (MO) theory. It can be shown that the alpha- and beta-peptoid structures form helical structures with both trans and cis peptide bonds despite the missing hydrogen bonds. Obviously, the conformational properties of the backbone are more important for folding than the possibility of hydrogen bonding. There are close relationships between the helices of alpha-peptoids and poly-glycine and poly-proline helices of alpha-peptides, whereas the helices of beta-peptoids correspond to the well-known helical structures of beta-peptides as, for instance, the 3(1)-helix of beta-peptides with 14-membered hydrogen-bonded rings. Thus, alpha- and beta-peptoids enrich the field of foldamers and may be used as useful tools in peptide and protein design.     

Identification of clinically relevant protein targets in prostate cancer with 2D-DIGE coupled mass spectrometry and systems biology network platform

Prostate cancer (PCa) is the most common type of cancer found in men and among the leading causes of cancer death in the western world. In the present study, we compared the individual protein expression patterns from histologically characterized PCa and the surrounding benign tissue obtained by manual micro dissection using highly sensitive two-dimensional differential gel electrophoresis (2D-DIGE) coupled with mass spectrometry. Proteomic data revealed 118 protein spots to be differentially expressed in cancer (n = 24) compared to benign (n = 21) prostate tissue. These spots were analysed by MALDI-TOF-MS/MS and 79 different proteins were identified. Using principal component analysis we could clearly separate tumor and normal tissue and two distinct tumor groups based on the protein expression pattern. By using a systems biology approach, we could map many of these proteins both into major pathways involved in PCa progression as well as into a group of potential diagnostic and/or prognostic markers. Due to complexity of the highly interconnected shortest pathway network, the functional sub networks revealed some of the potential candidate biomarker proteins for further validation. By using a systems biology approach, our study revealed novel proteins and molecular networks with altered expression in PCa. Further functional validation of individual proteins is ongoing and might provide new insights in PCa progression potentially leading to the design of novel diagnostic and therapeutic strategies.     

Deep Proteomic Evaluation of Primary and Cell Line Motoneuron Disease Models Delineates Major Differences in Neuronal Characteristics

The fatal neurodegenerative disorders amyotrophic lateral sclerosis and spinal muscular atrophy are, respectively, the most common motoneuron disease and genetic cause of infant death. Various in vitro model systems have been established to investigate motoneuron disease mechanisms, in particular immortalized cell lines and primary neurons. Using quantitative mass-spectrometry-based proteomics, we compared the proteomes of primary motoneurons to motoneuron-like cell lines NSC-34 and N2a, as well as to non-neuronal control cells, at a depth of 10,000 proteins. We used this resource to evaluate the suitability of murine in vitro model systems for cell biological and biochemical analysis of motoneuron disease mechanisms. Individual protein and pathway analysis indicated substantial differences between motoneuron-like cell lines and primary motoneurons, especially for proteins involved in differentiation, cytoskeleton, and receptor signaling, whereas common metabolic pathways were more similar. The proteins associated with amyotrophic lateral sclerosis also showed distinct differences between cell lines and primary motoneurons, providing a molecular basis for understanding fundamental alterations between cell lines and neurons with respect to neuronal pathways with relevance for disease mechanisms. Our study provides a proteomics resource for motoneuron research and presents a paradigm of how mass-spectrometry-based proteomics can be used to evaluate disease model systems.Motoneurons are extremely extended neurons that mediate the control of all muscle types by the central nervous system. Therefore, diseases involving progressive motoneuron degeneration such as amyotrophic lateral sclerosis (ALS)¹ (OMIM: 105400) or spinal muscle atrophy (OMIM: 253300) are particularly devastating and generally fatal disorders. Today, ALS is believed to form a phenotypic continuum with the disease entity frontotemporal lobe degeneration (OMIM: 600274) (1, 2). About 10% of ALS cases are known to be inherited, but the vast majority are considered sporadic. The number of inherited cases might be underestimated because of incomplete family histories, non-paternity, early death of family members, or incomplete penetrance (3).Mutations in several genes have been reported for the familial form, including in Sod1 (4), Als2 (5), Setx (6), Vapb (7), Tardbp (8, 9), Fus/Tls (10, 11), Vcp (12), Pfn1 (13), and several others (reviewed in Ref. 14). The most frequent genetic cause of inherited ALS was recently shown to be a hexanucleotide repeat expansion in an intron of a gene of unknown function called C9orf72 (15–17). Based on the spectrum of known mutations, several disease mechanisms for ALS have been proposed, including dysfunction of protein folding, axonal transport, RNA splicing, and metabolism (reviewed in Refs. 14, 18, and 19). Despite intensive research, it is still unclear whether a main common molecular pathway or mechanism underlies motoneuron degeneration in ALS and frontotemporal lobe degeneration. Spinal muscle atrophy is caused by homozygous mutations or deletions in the survival of motor neuron gene (Smn1) that presumably impair the RNA metabolism through diminished functionality of the Smn1 gene product (20). Over recent decades several model systems have been established to investigate ALS (21). These include transgenic animal models such as mouse (22), drosophila (23), and zebrafish (24). In cell-based studies, primary motoneurons cultured from rodent embryos (25) or motoneuron-like cell lines are employed. Primary cells are considered to more closely mimic the in vivo situation, but they are more challenging to establish and maintain. In contrast, the degree of functional relevance of cell lines can be difficult to establish, but they can be propagated without limitation and are well suited for high-throughput analysis. In particular, the spinal cord neuron–neuroblastoma hybrid cell line NSC-34 (26) and the mouse neuroblastoma cell line N2a (27) are widely used not only to assess motoneuron function, but also to study disease mechanisms in motoneurons (28, 29).As proteins are the functional actors in cells, proteomics should be able to make important contributions to the characterization and evaluation of cellular models. In particular, by identifying and quantifying the expressed proteins and bioinformatically interpreting the results, one can obtain enough information to infer functional differences. Our laboratory has previously shown proof of concept of such an approach by comparing the expression levels of about 4,000 proteins between primary hepatocytes and a hepatoma cell line (30). Very recently, mass-spectrometry-based proteomics has achieved sufficient depth and accuracy to quantify almost the entire proteome of mammalian cell lines (31–33). Furthermore, new instrumentation and algorithms now make it possible to perform label-free quantification between multiple cellular systems and with an accuracy previously associated only with stable isotope labeling techniques (34, 35).To evaluate the suitability of motoneuron-like cell lines as cellular model systems for research on ALS and related disorders, we characterized the proteomes of two widely used cell lines, NSC-34 and N2a, and compared them with the proteomes of mouse primary motoneurons and non-neuronal control cell lines. To generate primary motoneurons, we employed a recently described culturing system that makes it possible to isolate highly enriched motoneuron populations in less than 8 h (25). We identified more than 10,000 proteins and investigated differences in quantitative levels of individual neuron-associated proteins and pathways related to motoneuron function and disease mechanisms.     

Identification of the dynein light chains required for human papillomavirus infection

Human papillomaviruses (HPVs) are a family of small non-enveloped DNA viruses. Some genital HPV types, including HPV type 16 (HPV16), are the causative agent for the development of cancer at the site of infection. HPVs encode two capsid proteins, L1 and L2. After endocytic cell entry and egress from endosomes, L2 accompanies the viral DNA to the nucleus where replication is initiated. For cytoplasmic transport, L2 interacts with the microtubule network via the motor protein complex dynein. We have performed yeast two-hybrid screening and identified the dynein light chain DYNLT1 (previously called Tctex1) as interaction partner of HPV16 L2. Using co-immunoprecipitation and immunofluorescence colocalization studies we confirmed the L2-DYNLT1 interaction in mammalian cells. Further studies revealed that DYNLT3, the second member of the Tctex-light chain family, also interacts with L2 in vitro and in vivo, whereas other constituents of the dynein complex were not found to associate with L2. Depletion of DYNLT1 and DYNLT3 by specific siRNAs or cytosolic delivery of light chain-specific antibodies inhibited infection of HPV16. Therefore, this work identified two host cell proteins involved in HPV16 infection that are most likely required for transport purposes towards the nucleus.