Additional file 1: Figure S1. of Target antigens for Hs-14 monoclonal antibody and their various expression in normozoospermic and asthenozoospermic men

MALDI/FT-ICR MS analysis of CBB-R250staining spot – peptide map of TERA. (PPTX 224 kb

Chemical Cross-Linking and H/D Exchange for Fast Refinement of Protein Crystal Structure

A combination of chemical cross-linking and hydrogen–deuterium exchange coupled to high resolution mass spectrometry was used to describe structural differences of NKR-P1A receptor. The loop region extended from the compact core in the crystal structure was found to be closely attached to the protein core in solution. Our approach has potential to refine protein structures in solution within a few days and has very low sample consumption

Comparison of methylation status in ctDMRs in HMF (Fb), HMEC and cancer cell lines (CCL)

<p>Methylation status of CpG sites 87 ctDMRs was analyzed by MassARRAY. Individual CpG and sample types were ordered by hierarchical clustering. Within heatmap, red and green correspond to hypermethylation and hypomethylation respectively.</p

Distribution of ctDMRs.

<p>A) Position of ctDMR relative to TSS. Negative and positive distances correspond to sequences upstream and downstream to TSS, respectively. <b>B)</b> Position of ctDMR relative to the nearest CpG island. For both charts, pink lines show the expected distribution if DMRs have random localization and uneven microarray coverage is taken into consideration. 99% confidence interval based on the simulation is shown.</p

Comparison of ctDMRs with breast cancer specific DMRs.

<p>Number of unique and common DMRs is shown in Venn diagrams. Data for breast cancer specific DMR were reported previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052299#pone.0052299-Novak2" target="_blank">[23]</a>. Numbers in Venn diagrams show the number of regions; number of probes on the microarrays which are covering these region are show as numbers in parenthesis. Histograms below Venn diagrams show expected number of overlapping regions if positions of DMRs are random. Histogram is based on 5000 iterations of simulation. Red line shows the observed number of overlapping DMR.</p

Number of DMR.

<p>Number of DMR.</p

Validation of microarray results using MALDI-TOF mass spectrometry (MassARRAY).

<p>CtDMRs are labeled by the gene symbol of the closest gene. Each ctDMR is shown as a heatmap; methylation level for each informative CpG fragment is shown in columns and analyzed samples are organized in rows. Each colored rectangle corresponds to 1 CpG unit. All three genotypes were analyzed. White spaces are shown when the quality of data was not sufficient to assess methylation. <b>A</b>) ctDMRs which are methylated in HMF according to microarray. <b>B)</b> ctDMRs which are methylated in HMEC according to microarray. A more detailed view for each analyzed region is included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052299#pone.0052299.s003" target="_blank">Information S1</a>.</p

Dryad_Repo_for_Submission.tar

A compressed archive of nexus alignments, Beast XML files and MCC trees from the analyses performed in this study. The subfolders are clearly labeled to indicate file and analysis type associated with each file

Lincosamide Synthetase—A Unique Condensation System Combining Elements of Nonribosomal Peptide Synthetase and Mycothiol Metabolism

<div><p>In the biosynthesis of lincosamide antibiotics lincomycin and celesticetin, the amino acid and amino sugar units are linked by an amide bond. The respective condensing enzyme lincosamide synthetase (LS) is expected to be an unusual system combining nonribosomal peptide synthetase (NRPS) components with so far unknown amino sugar related activities. The biosynthetic gene cluster of celesticetin was sequenced and compared to the lincomycin one revealing putative LS coding ORFs shared in both clusters. Based on a bioassay and production profiles of <i>S. lincolnensis</i> strains with individually deleted putative LS coding genes, the proteins LmbC, D, E, F and V were assigned to LS function. Moreover, the newly recognized N-terminal domain of LmbN (LmbN-CP) was also assigned to LS as a NRPS carrier protein (CP). Surprisingly, the homologous CP coding sequence in celesticetin cluster is part of <i>ccbZ</i> gene adjacent to <i>ccbN</i>, the counterpart of <i>lmbN</i>, suggesting the gene rearrangement, evident also from still active internal translation start in <i>lmbN</i>, and indicating the direction of lincosamide biosynthesis evolution. The <i>in vitro</i> test with LmbN-CP, LmbC and the newly identified <i>S. lincolnensis</i> phosphopantetheinyl transferase Slp, confirmed the cooperation of the previously characterized NRPS A-domain LmbC with a <i>holo</i>-LmbN-CP in activation of a 4-propyl-L-proline precursor of lincomycin. This result completed the functional characterization of LS subunits resembling NRPS initiation module. Two of the four remaining putative LS subunits, LmbE/CcbE and LmbV/CcbV, exhibit low but significant homology to enzymes from the metabolism of mycothiol, the NRPS-independent system processing the amino sugar and amino acid units. The functions of particular LS subunits as well as cooperation of both NRPS-based and NRPS-independent LS blocks are discussed. The described condensing enzyme represents a unique hybrid system with overall composition quite dissimilar to any other known enzyme system.</p></div

Lincomycin Biosynthesis Involves a Tyrosine Hydroxylating Heme Protein of an Unusual Enzyme Family

<div><p>The gene lmbB2 of the lincomycin biosynthetic gene cluster of <i>Streptomyces lincolnensis</i> ATCC 25466 was shown to code for an unusual tyrosine hydroxylating enzyme involved in the biosynthetic pathway of this clinically important antibiotic. LmbB2 was expressed in <i>Escherichia coli</i>, purified near to homogeneity and shown to convert tyrosine to 3,4-dihydroxyphenylalanine (DOPA). In contrast to the well-known tyrosine hydroxylases (EC 1.14.16.2) and tyrosinases (EC 1.14.18.1), LmbB2 was identified as a heme protein. Mass spectrometry and Soret band-excited Raman spectroscopy of LmbB2 showed that LmbB2 contains heme b as prosthetic group. The CO-reduced differential absorption spectra of LmbB2 showed that the coordination of Fe was different from that of cytochrome P450 enzymes. LmbB2 exhibits sequence similarity to Orf13 of the anthramycin biosynthetic gene cluster, which has recently been classified as a heme peroxidase. Tyrosine hydroxylating activity of LmbB2 yielding DOPA in the presence of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH₄) was also observed. Reaction mechanism of this unique heme peroxidases family is discussed. Also, tyrosine hydroxylation was confirmed as the first step of the amino acid branch of the lincomycin biosynthesis.</p></div