Experimental studies and mathematical modeling of the viscoelastic rheology of tracheobronchial mucus from respiratory healthy patients.

Background: Tracheobronchial mucus plays a crucial role in pulmonary function by providing protection against inhaled pathogens. Due to its composition of water, mucins, and other biomolecules, it has a complex viscoelastic rheological behavior. This interplay of both viscous and elastic properties has not been fully described yet. In this study, we characterize the rheology of human mucus using oscillatory and transient tests. Based on the transient tests, we describe the material behavior of mucus under stress and strain loading by mathematical models. Methods: Mucus samples were collected from clinically used endotracheal tubes. For rheological characterization, oscillatory amplitude-sweep and frequency-sweep tests, and transient creep-recovery and stress-relaxation tests were performed. The results of the transient test were approximated using the Burgers model, the Weibull distribution, and the six-element Maxwell model. The three-dimensional microstructure of the tracheobronchial mucus was visualized using scanning electron microscope imaging. Results: Amplitude-sweep tests showed storage moduli ranging from 0.1 Pa to 10,000 Pa and a median critical strain of 4%. In frequency-sweep tests, storage and loss moduli increased with frequency, with the median of the storage modulus ranging from 10 Pa to 30 Pa, and the median of the loss modulus from 5 Pa to 14 Pa. The Burgers model approximates the viscoelastic behavior of tracheobronchial mucus during a constant load of stress appropriately (R2 of 0.99), and the Weibull distribution is suitable to predict the recovery of the sample after the removal of this stress (R2 of 0.99). The approximation of the stress-relaxation test data by a six-element Maxwell model shows a larger fit error (R2 of 0.91). Conclusions: This study provides a detailed description of all process steps of characterizing the rheology of tracheobronchial mucus, including sample collection, microstructure visualization, and rheological investigation. Based on this characterization, we provide mathematical models of the rheological behavior of tracheobronchial mucus. These can now be used to simulate mucus flow in the respiratory system through numerical approaches.

Numerical Investigation of Roughness Effects on Transition on Spherical Capsules.

To address the hitherto unknown mechanism of boundary-layer transition on blunt reentry capsules, the role of roughness-induced disturbance growth on a spherical-section forebody is assessed via optimal transient growth theory and direct numerical simulations (DNS). Optimal transient-growth studies have been performed for the blunt capsule experiments at Mach 5.9 in the Hypersonic Ludwieg tube Braunschweig (HLB) of the Technische Universität Braunschweig, which included measurements behind a patch of controlled, distributed micron-sized surface roughness. Transient-growth results for the HLB capsule indicate similar trends as the corresponding numerical data for a Mach 6 experiment in the Actively Controlled Expansion (ACE) facility of the Texas A&M University (TAMU) at a lower Reynolds number. Both configurations indicate a similar dependence on surface temperature ratio, and more important, rather low values of maximum energy gain. DNS are performed for the conditions of the HLB experiment to understand the generation of stationary disturbances by the roughness patch and the accompanying evolution of unsteady perturbations. However, no evidence of either modal or nonmodal disturbance growth in the wake of the roughness patch is found in the DNS data; thus, the physical mechanism underlying the observed onset of transition still remains unknown.

Moss-based production of asialo-erythropoietin devoid of Lewis A and other plant-typical carbohydrate determinants.

Protein therapeutics represent one of the most increasing areas in the pharmaceutical industry. Plants gain acceptance as attractive alternatives for high-quality and economical protein production. However, as the majority of biopharmaceuticals are glycoproteins, plant-specific N-glycosylation has to be taken into consideration. In Physcomitrella patens (moss), glyco-engineering is an applicable tool, and the removal of immunogenic core xylose and fucose residues was realized before. Here, we present the identification of the enzymes that are responsible for terminal glycosylation (α1,4 fucosylation and ß1,3 galactosylation) on complex-type N-glycans in moss. The terminal trisaccharide consisting of α1,4 fucose and ß1,3 galactose linked to N-acetylglucosamine forms the so-called Lewis A epitope. This epitope is rare on moss wild-type proteins, but was shown to be enriched on complex-type N-glycans of moss-produced recombinant human erythropoietin, while unknown from the native human protein. Via gene targeting of moss galactosyltransferase and fucosyltransferase genes, we identified the gene responsible for terminal glycosylation and were able to completely abolish the formation of Lewis A residues on the recombinant biopharmaceutical.

Physcomitrella HMGA-type proteins display structural differences compared to their higher plant counterparts.

High mobility group (HMG) proteins of the HMGA family are chromatin-associated proteins that act as architectural factors in nucleoprotein structures involved in gene transcription. To date, HMGA-type proteins have been studied in various higher plant species, but not in lower plants. We have identified two HMGA-type proteins, HMGA1 and HMGA2, encoded in the genome of the moss model Physcomitrella patens. Compared to higher plant HMGA proteins, the two Physcomitrella proteins display some structural differences. Thus, the moss HMGA proteins have six (rather than four) AT-hook DNA-binding motifs and their N-terminal domain lacks similarity to linker histone H1. HMGA2 is expressed in moss protonema and it localises to the cell nucleus. Typical of HMGA proteins, HMGA2 interacts preferentially with A/T-rich DNA, when compared with G/C-rich DNA. In cotransformation assays in Physcomitrella protoplasts, HMGA2 stimulated reporter gene expression. In summary, our data show that functional HMGA-type proteins occur in Physcomitrella.

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

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 HMGB1 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 HMGB1box 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.

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.

Functionality of the beta/six site-specific recombination system in tobacco and Arabidopsis: a novel tool for genetic engineering of plant genomes.

The beta recombinase is a member of the prokaryotic site-specific serine recombinases (invertase/resolvase family), which in the presence of a DNA bending cofactor can catalyse DNA deletions between two directly oriented 90-bp six recombination sites. We have examined here whether the beta recombinase can be expressed in plants and whether it displays in planta its specific catalytic activity excising DNA sequences that are flanked by six sites. In plant protoplasts, the enzyme could be expressed as a GFP-beta recombinase fusion which can localise to the cell nucleus. Beta recombinase stably expressed in tobacco plants can catalyse deletion of a spacer region that is flanked by directly oriented six sites and has been placed between promoter and a GUS reporter gene (preventing GUS expression). In transient transformation experiments, beta recombinase-mediated elimination of the spacer results in transcriptional induction of the GUS gene. Similarly, beta recombinase in stably double-transformed Arabidopsis plants deletes specifically the spacer region of a reporter construct that has been incorporated into the genome. In the segregating T1 generation, plants were identified that contain exclusively the recombined reporter construct. In summary, our results demonstrate that functional / recombinase can be expressed in plants and that the enzyme is suitable to precisely eliminate undesired sequences from plant genomes. Therefore, the beta/six recombination system (and presumably related recombinases) may become an attractive tool for plant genetic engineering.

Targeted knockouts of Physcomitrella lacking plant-specific immunogenic N-glycans.

Using plants as production factories for therapeutic proteins requires modification of their N-glycosylation pattern because of the immunogenicity of plant-specific sugar residues. In an attempt towards such humanization, we disrupted the genes for alpha1,3-fucosyltransferase and beta1,2-xylosyltransferase in Physcomitrella patens by homologous recombination. The single Deltafuc-t and Deltaxyl-t plants, as well as the double knockout, lacked transcripts of the corresponding genes, but did not differ from the wild-type moss in morphology, growth, development, and ability to secrete a recombinant protein, the human vascular endothelial growth factor VEGF(121), into the culture medium. N-Glycan analysis, however, revealed the absence of 1,3-fucosyl and/or 1,2-xylosyl residues, respectively. Therefore, the modifications described here represent the key step towards the generation of moss lines suitable for the production of plant-made glycosylated biopharmaceuticals with nonallergenic N-glycans.

Phosphorylation of maize and Arabidopsis HMGB proteins by protein kinase CK2alpha.

In plants, a variety of chromatin-associated high mobility group (HMG) proteins belonging to the HMGB family have been identified. We have examined the phosphorylation of the HMGB proteins from the monocotyledonous plant maize and the dicotyledonous plant Arabidopsis by protein kinase CK2alpha. Maize CK2alpha phosphorylates the maize HMGB1 and HMGB2/3 proteins and the Arabidopsis HMGB1, HMGB2/3, and HMGB4 proteins. Maize HMGB4 and HMGB5 and Arabidopsis HMGB5 are not phosphorylated by CK2alpha. Depending on the HMGB protein up to five amino acid residues are phosphorylated in the course of the phosphorylation reaction. The HMGB1 proteins from both plants are markedly more slowly phosphorylated by CK2alpha than the other HMGB substrate proteins, indicating that certain HMGB proteins are clearly preferred substrates for CK2alpha. The rate of the phosphorylation reaction appears to be related to the ease of interaction between CK2alpha and the HMGB proteins, as indicated by chemical cross-linking experiments. MALDI/TOF mass spectrometry analyses demonstrate that the HMGB1 and HMGB2/3 proteins occur in various phosphorylation states in immature maize kernels. Thus, HMGB1 exists as monophosphorylated, double-phosphorylated, triple-phosphorylated, and tetraphosphorylated protein in kernel tissue, and the tetraphosphorylated form is the most abundant version. The observed in vivo phosphorylation states indicate that protein kinase(s) other than CK2alpha contribute(s) to the modification of the plant HMGB proteins. The fact that the HMGB proteins are phosphorylated to various extents reveals that the existence of differentially modified forms increases the number of distinct HMGB protein variants in plant chromatin that may be adapted to certain functions.

Protein kinase CK2 phosphorylates the high mobility group domain protein SSRP1, inducing the recognition of UV-damaged DNA.

The structure-specific recognition protein SSRP1 plays a role in transcription and replication in the chromatin context. Mediated by its C-terminal high mobility group (HMG) box domain, SSRP1 binds DNA non-sequence specifically but recognizes certain DNA structures. Using acetic acid urea polyacrylamide gel electrophoresis and mass spectrometry, we have examined the phosphorylation of maize SSRP1 by protein kinase CK2 alpha. The kinase phosphorylated several amino acid residues in the C-terminal part of the SSRP1 protein. Two phosphorylation sites were mapped in the very C-terminal region next to the HMG box domain, and about seven sites are localized within the acidic domain. Circular dichroism showed that the phosphorylation of the two C-terminal sites by CK2 alpha resulted in a structural change in the region of HMG box domain, because the negative peak of the CD spectrum at 222 nm was decreased by approximately 10%. In parallel, the phosphorylation induced the recognition of UV-damaged DNA, whereas the non-phosphorylated protein does not discriminate between UV-damaged DNA and control DNA. The affinity of CK2 alpha-phosphorylated SSRP1 for the DNA correlates with the degree of UV-induced DNA damage. Moreover, maize SSRP1 can restore the increased UV-sensitivity of a yeast strain lacking the NHP6A/B HMG domain proteins to levels of the control strain. Collectively, these findings indicate a role for SSRP1 in the UV response of eukaryotic cells.

The "drought-inducible" histone H1s of tobacco play no role in male sterility linked to alterations in H1 variants.

Tobacco ( Nicotiana tabacum L.) has two major H1 variants (H1A and H1B), which account for over 80% of chromatin linker histones, and four minor variants: H1C, H1D, H1E and H1F. We have shown previously [M. Prymakowska-Bosak et al. (1999) Plant Cell 11:2317-2329] that reversal of the natural proportion of major to minor H1 variants in transgenic tobacco plants results in a characteristic male-sterility phenotype identical to that occurring in many plant species subjected to water deficit at the time of male meiosis. It has been proposed by others that the drought-induced arrest of male gametophyte development is linked to decreased sugar delivery to reproductive tissues. Within the family of angiosperm H1s there is a well-defined class of minor H1 variants named "drought inducible" because some of its members have been shown to be induced by water deficit. We have identified and cloned the tobacco H1C gene, which, based on sequence similarity, represents a "drought-inducible" minor H1 variant. Analysis of the un-translated mRNA and promoter regions of H1C suggests a regulation by sucrose concentration. Antisense silencing of H1C and its close homologue H1D in plants that do not express H1A and H1B does not affect the characteristic H1A(-)/ H1B(-) male-sterility phenotype. Silencing of H1C and H1D also has no effect on growth and development of plants. Our findings demonstrate that H1C and H1D are dispensable for normal growth and development of tobacco, and that the compensatory up-regulation of "drought-inducible" H1s observed in H1A(-)/ H1B(-) plants is not the direct cause of male sterility linked to alterations in H1 variants.

Plant chromosomal HMGB proteins efficiently promote the bacterial site-specific beta-mediated recombination in vitro and in vivo.

In the presence of an accessory DNA bending protein, the bacterial site-specific beta recombinase catalyzes resolution and DNA inversion. Five different maize high mobility group B (HMGB) proteins were examined for their potential to facilitate beta recombination in vitro using DNA substrates with different intervening distances (73-913 bp) between two directly oriented recombination (six) sites. All analyzed HMGB proteins (HMGB1 to HMGB5) could promote beta recombination, but depending on the DNA substrate with different efficiencies. The HMGB1 protein displayed an activity comparable to that of the natural promoting protein Hbsu, whereas the other HMGB proteins were less effective. Phosphorylation of the HMGB1 protein resulted in an increased efficiency of HMGB1 to promote beta recombination. Analyses of DNA substrates with closely spaced six sites demonstrated that in the presence of HMGB1 the recombination rate was correlated to the distance between the six sites, but independent of the helical orientation of the six sites. Using a Bacillus subtilis strain defective in Hbsu, the coexpression of beta recombinase and HMGB1 (or a truncated HMGB1 derivative) revealed that a plant HMG-box domain protein is sufficient for assisting beta to catalyze recombination in vivo. Our results using beta recombination as a model system suggest that the various plant HMGB proteins (and their posttranslationally modified versions) have the potential of forming a repertoire of different DNA structures, which is compatible with the idea that the HMGB proteins can act as architectural factors in a variety of nucleoprotein structures.

Protein kinase CK2 differentially phosphorylates maize chromosomal high mobility group B (HMGB) proteins modulating their stability and DNA interactions.

The high mobility group (HMG) proteins of the HMGB family are architectural factors in eukaryotic chromatin, which are involved in the regulation of various DNA-dependent processes. We have examined the post-translational modifications of five HMGB proteins from maize suspension cultured cells, revealing that HMGB1 and HMGB2/3, but not HMGB4 and HMGB5, are phosphorylated by protein kinase CK2. The phosphorylation sites have been mapped to the acidic C-terminal domains by analysis of tryptic peptides derived from HMGB1 and HMGB2/3 using nanospray ion trap mass spectrometry. In native HMGB1, Ser(149) is constitutively phosphorylated, whereas Ser(133) and Ser(136) are differentially phosphorylated. The functional significance of the CK2-mediated phosphorylation of HMGB proteins was analyzed by circular dichroism measurements showing that the phosphorylation increases the thermal stability of the HMGB proteins. Electrophoretic mobility shift assays demonstrate that the phosphorylation reduces the affinity of the HMGB proteins for linear DNA. The specific recognition of DNA minicircles is not affected by the phosphorylation, but a different pattern of protein-DNA complexes is formed. Collectively, these findings show that phosphorylation of residues within the acidic C-terminal domain of the HMGB proteins can modulate protein stability and the DNA binding properties of the HMGB proteins.