Torsion and Antero-Posterior Bending in the In Vivo Human Tibia Loading Regimes during Walking and Running

Bending, in addition to compression, is recognized to be a common loading pattern in long bones in animals. However, due to the technical difficulty of measuring bone deformation in humans, our current understanding of bone loading patterns in humans is very limited. In the present study, we hypothesized that bending and torsion are important loading regimes in the human tibia. In vivo tibia segment deformation in humans was assessed during walking and running utilizing a novel optical approach. Results suggest that the proximal tibia primarily bends to the posterior (bending angle: 0.15°–1.30°) and medial aspect (bending angle: 0.38°–0.90°) and that it twists externally (torsion angle: 0.67°–1.66°) in relation to the distal tibia during the stance phase of overground walking at a speed between 2.5 and 6.1 km/h. Peak posterior bending and peak torsion occurred during the first and second half of stance phase, respectively. The peak-to-peak antero-posterior (AP) bending angles increased linearly with vertical ground reaction force and speed. Similarly, peak-to-peak torsion angles increased with the vertical free moment in four of the five test subjects and with the speed in three of the test subjects. There was no correlation between peak-to-peak medio-lateral (ML) bending angles and ground reaction force or speed. On the treadmill, peak-to-peak AP bending angles increased with walking and running speed, but peak-to-peak torsion angles and peak-to-peak ML bending angles remained constant during walking. Peak-to-peak AP bending angle during treadmill running was speed-dependent and larger than that observed during walking. In contrast, peak-to-peak tibia torsion angle was smaller during treadmill running than during walking. To conclude, bending and torsion of substantial magnitude were observed in the human tibia during walking and running. A systematic distribution of peak amplitude was found during the first and second parts of the stance phase.     

Expression and physiological relevance of Agrobacterium tumefaciens phosphatidylcholine biosynthesis genes

Phosphatidylcholine (PC), or lecithin, is the major phospholipid in eukaryotic membranes, whereas only 10% of all bacteria are predicted to synthesize PC. In Rhizobiaceae, including the phytopathogenic bacterium Agrobacterium tumefaciens, PC is essential for the establishment of a successful host-microbe interaction. A. tumefaciens produces PC via two alternative pathways, the methylation pathway and the Pcs pathway. The responsible genes, pmtA (coding for a phospholipid N-methyltransferase) and pcs (coding for a PC synthase), are located on the circular chromosome of A. tumefaciens C58. Recombinant expression of pmtA and pcs in Escherichia coli revealed that the individual proteins carry out the annotated enzyme functions. Both genes and a putative ABC transporter operon downstream of PC are constitutively expressed in A. tumefaciens. The amount of PC in A. tumefaciens membranes reaches around 23% of total membrane lipids. We show that PC is distributed in both the inner and outer membranes. Loss of PC results in reduced motility and increased biofilm formation, two processes known to be involved in virulence. Our work documents the critical importance of membrane lipid homeostasis for diverse cellular processes in A. tumefaciens.     

Isolation and automated ribotyping of Mycobacterium lentiflavum from drinking water distribution system and clinical specimens

Automated ribotyping as a tool for identifying of nontuberculous mycobacteria was evaluated. We created a database comprising of riboprints of 60 strains, representing 32 species of nontuberculous mycobacteria. It was shown that combined ribopatterns generated after digestion with EcoRI and PvuII were distinguishable between species of both slow-growing and rapid-growing mycobacteria. The findings were in good agreement with the 16S rRNA gene sequencing results, allowing correct identification of Mycobacterium lentiflavum isolated from clinical specimens and from biofilms growing in public water distribution system. The automated ribotyping was powerful in discriminating between M. lentiflavum and closely related species M. simiae and M. palustre. Mycobacterium lentiflavum strains from drinking water biofilms were resistant to two to four antimycobacterial drugs. The drinking water distribution system may, thus, be a source of nontuberculous mycobacteria resistant to multiple drugs.     

A novel fed-batch based cultivation method provides high cell-density and improves yield of soluble recombinant proteins in shaken cultures

Background  

Cultivations for recombinant protein production in shake flasks should provide high cell densities, high protein productivity per cell and good protein quality. The methods described in laboratory handbooks often fail to reach these goals due to oxygen depletion, lack of pH control and the necessity to use low induction cell densities. In this article we describe the impact of a novel enzymatically controlled fed-batch cultivation technology on recombinant protein production in Escherichia coli in simple shaken cultures.     

Improved production of human type II procollagen in the yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis> in shake flasks by a wireless-controlled fed-batch system

Background  

Here we describe a new technical solution for optimization of Pichia pastoris shake flask cultures with the example of production of stable human type II collagen. Production of recombinant proteins in P. pastoris is usually performed by controlling gene expression with the strong AOX1 promoter, which is induced by addition of methanol. Optimization of processes using the AOX1 promoter in P. pastoris is generally done in bioreactors by fed-batch fermentation with a controlled continuous addition of methanol for avoiding methanol toxification and carbon/energy starvation. The development of feeding protocols and the study of AOX1-controlled recombinant protein production have been largely made in shake flasks, although shake flasks have very limited possibilities for measurement and control.     

Mass spectrometric protein structure characterization reveals cause of migration differences of haptoglobin alpha chains in two-dimensional gel electrophoresis

Haptoglobin belongs to the major constituents of plasma and acts as hemoglobin-binding and acute-phase protein. Due to the occurrence of three major allelic variants and further structural modifications, the alpha chains of haptoglobin form varying spot patterns in two-dimensional gel electrophoresis (2-DE) gels, which is generally observed in differential proteome analyses using plasma or related body fluids of humans. In the present study plasma samples from 10 donors of initially unknown haptoglobin phenotype were separated by 2-DE and tryptic digests of excised haptoglobin alpha chain spots were analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and MALDI-quadrupole ion trap TOF-MS. Haptoglobin alpha1S, alpha1F, as well as alpha2 chains were found to occur each with at least three structurally differing protein species: (i) the unmodified form, which corresponds to the sequence database entries; (ii) derivatives, in which asparagine at position five is deamidated to aspartic acid; and (iii) derivatives with an additional C-terminal arginine residue. These structural variants account for the most commonly observed spot patterns of haptoglobin alpha chains in Coomassie-stained gels. Additionally, a minor derivative of the haptoglobin alpha2 chain carrying both modifications, deamidation at position five and the C-terminal arginine residue, was identified. Theoretical pI values of the characterized structural variants are, consistent with their observed migration in the 2-DE gels.     

Bone marrow as a priming site for T-cell responses to blood-borne antigen

Although bone marrow is known as a primary lymphoid organ, its potential to serve as a secondary immune organ has hardly been explored. Here we demonstrate that naive, antigen-specific T cells home to bone marrow, where they can be primed. Antigen presentation to T cells in bone marrow is mediated via resident CD11c+ dendritic cells. They are highly efficient in taking up exogenous blood-borne antigen and processing it via major histocompatibility complex class I and class II pathways. T-cell activation correlates with dendritic cell-T cell clustering in bone marrow stroma. Primary CD4+ and CD8+ T-cell responses generated in bone marrow occur in the absence of secondary lymphoid organs. The responses are not tolerogenic and result in generation of cytotoxic T cells, protective anti-tumor immunity and immunological memory. These findings highlight the uniqueness of bone marrow as an organ important for hemato- and lymphopoiesis and for systemic T cell-mediated immunity.     

Cryodetector mass spectrometry profiling of plasma samples for HELLP diagnosis: an exploratory study

The cryodetector MS-based screening method enables the rapid and reliable separation of hemolysis, elevated liver enzymes, and low platelets syndrome (HELLP) patients from healthy women. For developing the assay, plasma protein abundances from patients suffering from HELLP were profiled before and after delivery and compared to healthy pregnant and nonpregnant control individuals, using a TOF mass spectrometer equipped with a cryodetector system. The spectra were well reproducible when acquisition conditions were kept constant and the overall appearance of the profiles was well comparable. Peak areas of ten selected ion signals (9.5, 11.8, 14.0, 28.1, 43.4, 50.5, 60.1, 66.6, 74.5, and 79.8 kDa) from each spectrum were subjected to statistical analysis. Significant differences in ion intensities between the groups could be measured even without sample fractionation. The most striking difference between most of the spectra from HELLP patients and nonHELLP samples was the presence/absence of the 11.8 kDa ion signal (p-value: 0.0000365), most likely belonging to serum amyloid A (SAA). Other significant differences in ion intensities between HELLP and control samples were observed in the peak areas of the 14.0, 28.1, 50.5, and 74.5 kDa ion signals. Using the cryodetector MS data for sorting plasma samples into either the HELLP group (n = 8) or the nonHELLP group (n = 22), a sensitivity of 87.5% and a specificity of 100% were achieved. The positive predictive value of our screening assay was 100% and a negative predictive value of 95.6% was obtained with the samples included in this study.     

Manganese peroxidase of Agaricus bisporus: grain bran-promoted production and gene characterization

The main manganese peroxidase (MnP) isoenzyme of Agaricus bisporus ATCC 62459 produced in lignocellulose-containing cultures was isolated, cloned and sequenced. In liquid medium, where MnP was previously detected only in trace amounts, the production of MnP was enhanced by rye and wheat bran supplements. The pI (3.25) and N-terminal amino acid sequence (25 aa) of the enzyme from bran-containing cultures were identical to those reported from compost-isolated MnP1. MnP1 is a 328-aa long polypeptide preceded by a 26-aa leader peptide. The nucleotide sequence and putative amino acid sequence of MnP1 reveal its similarity to Pleurotus ostreatus MnP3 (62.5%), Lepista irina versatile peroxidase (VP) (61.8%) and Pleurotus eryngii VPs VPL2 and VPL1 (61.9% and 61.2%, respectively). The intron-exon structure resembles that of P. ostreatus MnP1 and P. eryngii VPL1. Despite the sequence similarity to VPs, in the A. bisporus MnP1 sequence, alanine (A163) is present instead of tryptophane (W164), distinguishing it from the veratryl alcohol oxidising P. eryngii VPLs. The MnP sequence can be used as a tool to examine the pattern of ligninolytic gene expression during the growth and fruiting of A. bisporus to optimise compost composition, fungal growth and mushroom production.