Early Life Relict Feature in Peptide Mass Distribution

Molecular mass of a biomolecule is characterized in mass spectroscopy by the monoisitopic mass M~mono~ and the average isotopic mass M~av~. We found that peptide masses mapped on a plane made by two parameters derived from M~mono~ and M~av~ form a peculiar global feature in form of a band-gap 5-7 ppm wide stretching across the whole peptide galaxy, with a narrow (FWHM 0.2 ppm) line in the centre. The a priori probability of such a feature to emerge by chance is less than 1:100. Peptides contributing to the central line have elemental compositions following the rules S=0; Z = (2C - N - H)/2 =0, which nine out of 20 amino acid residues satisfy. The relative abundances of amino acids in the peptides contributing to the central line correlate with the consensus order of emergence of these amino acids, with ancient amino acids being overrepresented in on-line peptides. Thus the central line is a relic of ancient life, and likely a signature of its emergence in abiotic synthesis. The linear correlation between M~av~ and M~mono~ reduces the complexity of polypeptide molecules, which may have increased the rate of their abiotic production. This, in turn may have influenced the selection of these amino acid residues for terrestrial life. Assuming the line feature is not spurious, life has emerged from elements with isotopic abundances very close to terrestrial levels, which rules out most of the Galaxy

Early life relict feature in peptide mass distribution

The molecular mass of a biomolecule is characterized by the monoisitopic mass Mmono and the average isotopic mass Mav. We found that tryptic peptide masses mapped on a plane made by two parameters derived from Mmono and Mav form a peculiar feature in the form of a 'band gap' stretching across the whole 'peptide galaxy', with a narrow line in the centre. The purpose of this study was to investigate possible reasons for the emergence of such a feature, provided it is not a random occurrence. The a priori probability of such a feature to emerge by chance was found to be less than 1:100. Peptides contributing to the central line have elemental compositions following the rules S = 0; Z = C - (N + H)/2 = 0, which nine out of 20 amino acid residues satisfy. The relative abundances of amino acids in the peptides contributing to the central line correlate with the consensus order of emergence of these amino acids, with ancient amino acids being overrepresented in on-Line peptides. Since linear correlation between Mav and Mmono reduces the complexity of polypeptide molecules, and the turnover rate of less complex molecules should be faster in non-equilibrium abiotic synthesis, we hypothesize that the line could be a signature of abiotic production of primordial biopolymers. The linear dependence between the average isotopic masses and monoisotopic masses may have influenced the selection of amino acid residues for terrestrial life. © 2010 Versita Warsaw and Springer-Verlag Berlin Heidelberg.link_to_subscribed_fulltex

Multiscale simulation approach to predict the penetration depth of oil between chip and tool during orthogonal cutting of AISI 4140

Cooling lubricants in machining perform important tasks, from cooling and lubrication of the friction partners in contact to the removal of the separated chips. An essential, determining and largely unresolved question in relation to cooling lubricants in ma-chining is to what extent the coolant can get into the cutting zone. The aim of this paper is to address this question by using a multiscale approach to determine the penetration of the cooling lubricant gap. This is achieved by multiscale simulations by means of coupling the results of flow, structural and continuum mechanical simulations. Comparatively, the results of the simulated machining operation are compared with experimental orthogonal cutting tests of AISI 4140

Large-Scale Purification of r28M: A Bispecific scFv Antibody Targeting Human Melanoma Produced in Transgenic Cattle.

30 years ago, the potential of bispecific antibodies to engage cytotoxic T cells for the lysis of cancer cells was discovered. Today a variety of bispecific antibodies against diverse cell surface structures have been developed, the majority of them produced in mammalian cell culture systems. Beside the r28M, described here, no such bispecific antibody is known to be expressed by transgenic livestock, although various biologicals for medical needs are already harvested-mostly from the milk-of these transgenics. In this study we investigated the large-scale purification and biological activity of the bispecific antibody r28M, expressed in the blood of transgenic cattle. This tandem single-chain variable fragment antibody is designed to target human CD28 and the melanoma/glioblastoma-associated cell surface chondroitin sulfate proteoglycan 4 (CSPG4).With the described optimized purification protocol an average yield of 30 mg enriched r28M fraction out of 2 liters bovine plasma could be obtained. Separation of this enriched fraction by size exclusion chromatography into monomers, dimers and aggregates and further testing regarding the biological activity revealed the monomer fraction as being the most appropriate one to continue working with. The detailed characterization of the antibody's activity confirmed its high specificity to induce the killing of CSPG4 positive cells. In addition, first insights into tumor cell death pathways mediated by r28M-activated peripheral blood mononuclear cells were gained. In consideration of possible applications in vivo we also tested the effect of the addition of different excipients to r28M.Summing up, we managed to purify monomeric r28M from bovine plasma in a large-scale preparation and could prove that its biological activity is unaffected and still highly specific and thus, might be applicable for the treatment of melanoma

Selection of scFv Antibody Fragments Binding to Human Blood versus Lymphatic Endothelial Surface Antigens by Direct Cell Phage Display

<div><p>The identification of marker molecules specific for blood and lymphatic endothelium may provide new diagnostic tools and identify new targets for therapy of immune, microvascular and cancerous diseases. Here, we used a phage display library expressing human randomized single-chain Fv (scFv) antibodies for direct panning against live cultures of blood (BECs) and lymphatic (LECs) endothelial cells in solution. After six panning rounds, out of 944 sequenced antibody clones, we retrieved 166 unique/diverse scFv fragments, as indicated by the V-region sequences. Specificities of these phage clone antibodies for respective compartments were individually tested by direct cell ELISA, indicating that mainly pan-endothelial cell (EC) binders had been selected, but also revealing a subset of BEC-specific scFv antibodies. The specific staining pattern was recapitulated by twelve phage-independently expressed scFv antibodies. Binding capacity to BECs and LECs and differential staining of BEC versus LEC by a subset of eight scFv antibodies was confirmed by immunofluorescence staining. As one antigen, CD146 was identified by immunoprecipitation with phage-independent scFv fragment. This antibody, B6-11, specifically bound to recombinant CD146, and to native CD146 expressed by BECs, melanoma cells and blood vessels. Further, binding capacity of B6-11 to CD146 was fully retained after fusion to a mouse Fc portion, which enabled eukaryotic cell expression. Beyond visualization and diagnosis, this antibody might be used as a functional tool. Overall, our approach provided a method to select antibodies specific for endothelial surface determinants in their native configuration. We successfully selected antibodies that bind to antigens expressed on the human endothelial cell surfaces <i>in situ</i>, showing that BECs and LECs share a majority of surface antigens, which is complemented by cell-type specific, unique markers.</p></div

LC-MS/MS identification of CD146 binding to scFv B6-11, and antigen confirmation by immunoprecipitation and ELISA.

<p>A: Alignment of LC-MS/MS identified peptides (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127169#pone.0127169.s004" target="_blank">S4 Fig</a>) with the sequence of MCAM/CD146MUC18. The eluates from scFv B6-11 immunoprecipitation were subjected to trypsin digestion (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127169#sec002" target="_blank">Materials and Methods</a> section) and subsequently analyzed by LC-MS/MS. B: Immunoprecipitation of CD146 by soluble scFv B6-11 from BEC lysates. Immune complexes were tested by Western blot in reducing conditions using commercial anti-CD146 antibody. Lane 1: input BEC lysate, lanes 2–5: immunoprecipitates with scFv B6-11, lanes 2 and 4: under addition of PNGase F. Treatment of BEC lysates with PNGase prior or after addition of scFv B6-11 had no influence on co-immunoprecipitation capacity of scFv B6-11, showing that scFv B6-11 binding to CD146 is glycosylation-independent. C: scFv B6-11 binds to immobilized extracellular domain of recombinant human CD146 in ELISA. An irrelevant antigen (BSA), a non-binding scFv and uncoated wells served as controls. scFv binding was detected with peroxidase-conjugated anti-His tag antibody (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127169#pone.0127169.s011" target="_blank">S1 Table</a>), and absorbance was measured at 450nm. Mean and standard deviation of triplicate experiments are given. D: CD146 expression of different cell lines as shown by immunoprobing with anti-CD146 antibody. CD146 is expressed in BECs, in A375, CRL1676, HTB71 melanoma cells, but not in primary LECs and HEK293 cells. The same blot was probed with anti-tubulin antibody for control of equal protein loads. E: scFv B6-11 stains cell lines expressing CD146 with similar intensity as commercial anti-CD146 antibody in ELISA. Negative controls were a non-binding scFv and 2^nd antibody only. F: Similar to commercial anti-CD146 antibody, scFv B6-11 stains BECs (upper lane, red) but not LECs (lower lane, green) in immunofluorescence. Size bars: 50μm. G: scFv B6-11 stains blood, but not lymphatic vessels in human skin. Double immunofluorescence staining of skin sample with Cy3-labeled scFv-B6-11 or anti-CD146 (red) and anti-PDPN (green) antibodies. Blood (BV) and lymphatic (LV) vessels are indicated by lines. Nuclei were counterstained with DAPI. Size bars: 50μm.</p

Fusion antibodies scFv-Fc B6-11, B6-112 and B6-117 stain blood vessels in the dermis.

<p>A: Representative images of double immunofluorescence stainings of frozen human skin sections with scFv-Fc fusion antibodies (red) in combination with anti-CD31 (green) show overlapping staining, but B: not with anti-PDPN antibody (green) as control. Nuclei were counter-stained with DAPI. Size bars: 50μm.</p

Production of scFv-Fc B6-11 fusion antibody and reconfirmation of binding to CD146.

<p>A: Western Blot analysis showing molecular size of scFv-Fc fusion antibodies B6-11, B6-112 and B6-117. The scFv-region of the phagemids was cloned into the pFUSE-mIgG2B-vector (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127169#pone.0127169.s005" target="_blank">S5 Fig</a>). Constructs were transfected into HEK293-cells. Cell culture supernatants were loaded on reducing (R) and non-reducing (NR) 10% SDS-PAGE, and nitrocellulose blots were probed with peroxidase-labeled anti-mouseFc antibodies. scFv-Fc fusion antibodies are secreted as approximate 140 kDa dimers, as seen under non-reducing condition (-DTT). Control: Fc only protein, produced from pFUSE vector without scFv insert. B: Immunoprecipitation with scFv-Fc B6-11 reconfirms CD146-binding. G1S1 lysates were incubated with scFv-Fc B6-11 and Fc only as control. Blots of immunoprecipitates were probed with anti-CD146 and anti-mouseFc antibodies. C: scFv-Fc B6-11 binds to recombinant CD146 in ELISA. scFv-Fc B6-11, commercial anti-CD146 antibody and Fc only were used on respective dilutions of recombinant human CD146 or BSA as control antigen coated on 96-well ELISA plates. Absorbance was measured at 450nm. D: scFv-Fc B6-11 fusion antibody binds to cells expressing CD146 in ELISA. Purified scFv-Fc B6-11 (light grey bars), commercial anti-CD146 antibody (dark grey bars) and Fc only (black bars) were added to monolayers of respective cell lines. Bound antibodies were detected using anti-Fc and HRP-conjugated anti-rabbit antibodies, and absorbance was measured at 450nm. Mean and standard deviation of triplicate experiments are given. E: On HDMECs, scFv-Fc B6-11 fusion antibody shows the same reactivity as a commercial anti-CD146 antibody. F: scFv-Fc fusion antibodies (red) reveal diverse membraneous labling patterns in co-immunofluorescent stainings with anti-CD31 antibody (green). Nuclei were counterstained with DAPI. Size bars: 50μm.</p

Frequency of diverse scFv antibody sequences among 557 intact clones.

<p>Out of 994 sequenced phage clones, 557 intact scFv sequences were derived, among which 166 diverse scFv sequences were identified (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127169#pone.0127169.s012" target="_blank">S2 Table</a>).</p><p>^a Frequency of unique scFv antibody sequences</p><p>^b Number of different scFv sequences</p><p>^c Number of scFv sequences with respective sequence diversity</p><p>^d Sequence occurrence was calculated as percentage of sequence count.</p><p>Frequency of diverse scFv antibody sequences among 557 intact clones.</p