Additional file 1: Table S1. of IgG Fc galactosylation predicts response to methotrexate in early rheumatoid arthritis

Demographic characteristics of the nationwide EIRA cohort. Table S2. Characterized complement pathway proteins and IgG-isotype proteins. Table S3. Individual IgG-Fc glycan distribution values in IgG1 and in IgG2 in control subjects, as well as in patients with RA prior to and following MTX treatment. Table S4. Significant differences for the 19 characterized glycan species when comparing healthy versus all, good, moderate, and nonresponding patients prior to and following MTX treatment. Table S5. IgG1 and IgG2 Fc glycan distributions grouped according to structural features, comparing healthy control subjects and patients with RA prior to and following MTX treatment. Table S6. Intra- and interindividual differences in the patients with RA, comparing individual glycan species prior to and following MTX treatment. Table S7. Complete list, ranking, and correlation (with response versus no response to MTX) of the features used in the OPLS-DA model shown in Fig. 3b. Figure S1. Extracted ion chromatograms of IgG1 and IgG2 Fc glycans quantified in a control subject and a patient with RA. Figure S2. Intraindividual changes in galactosylation status on IgG1 and on IgG2 for good and moderate responders and for nonresponders. Figure S3. Intraindividual correlation between the aGal/Gal status of glycans with different types of structural features and of IgG1 and IgG2 substituted glycans. Figure S4. Significant differences between control subjects and patients with early RA at baseline in the classical pathway initiating complements C1 and C9. Figure S5. Intraindividual correlation between the classical and lectin pathway inhibitor C4bBPα versus FA2/(FA2G1 + FA2G2). (DOCX 1180 kb

Normalized thermal melting profiles of <i>PDYN</i>-derived oligonucleotides recorded with CD spectroscopy (275 nm) between 5°C and 60°C.

<p>Normalization was performed using the formula, (S_t−S_60°C)/(S_5°C−S_60°C), where S_t, S_5°C and S_60°C are the signal intensities at 275 nm at a given temperature, 5°C, and 60°C, respectively. A. Black – Dyn A; Blue – Dyn B; Green – Dyn B^5mC₁; Red – α-NE; Turquoise – α-NE^5mC₁. B. Black – Dyn A; Blue – Dyn A^5mC₁; Green – Dyn A^5mC₂; Red – Dyn A^5mC_1,2; Turquoise – Dyn A^5mC_1,3. C. Black – Dyn A; Blue – Dyn A M₁; Green – Dyn A M₂; Red – Dyn A M₃; Turquoise – Dyn A M₄; Orange - Dyn A M₅.</p

Analysis of [γ-³²P]-labeled <i>PDYN</i>-derived oligonucleotides using PAGE (see

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039605#pone-0039605-t001" target="_blank"><b>Table 1</b></a><b>for sequences).</b> Reference oligonucleotides (RO) included 26-, 37- and 54-mer oligomers. Samples were preheated for 10 min at 95°C, incubated in loading buffer (20 mM Tris/HCl pH 7.5, 37.5% glycerol, 15 mM MgCl₂ and 50 mM NaCl) at 4°C (A, B, E,) or at 37°C (C, D, F, G) for 30 minutes before loading on native (A–F) or denaturing 7.5 M urea (G) 15% polyacrylamide gel, and resolved at 4°C (A, B, E,) or at 37°C (C, D, F, G). Images shown were taken from the same gel; equal amounts of radioactive oligonucleotides were loaded.</p

The <i>PDYN</i>-coding sequence, which gives rise to the opioid peptides α-NE, Dyn A, Dyn B.

<p>(<b>A</b>) The oligonucleotides analyzed in this study correspond to <i>PDYN</i> fragments with α-NE-, Dyn A- and Dyn B-coding sequences. Three pathogenic mutations causing the human neurodegenerative disorder SCA23 are shown in red, and two “non-natural” point mutations in blue. (<b>B</b>) <b>DNase hypersensitive site in the human </b><b><i>PDYN</i></b><b>-coding region.</b> Image was taken from the UCSC Genome Browser on Human 2006 (NCBI36/hg18) Assembly with the Dyn A-coding sequence used for the search; the region of DNase hypersensitivity overlaps with the Dyn A-coding sequence in HepG2 cells.</p

1D ¹H NMR spectra showing the imino proton region of the PDYN-derived oligonucleotides.

<p>Hydrogen-bonded imino protons originating from A-T base pairs (14–15 ppm), G-C base pairs (12–13 ppm) and G-T base pairs (10–12 ppm) are observed. Spectra were recorded at 4°C (<b>A</b>) or 37°C (<b>B</b>). The oligonucleotide abbreviations used in the figure are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039605#pone-0039605-t001" target="_blank">Table 1</a>.</p

Mobility on native PAGE and number of base-pair hydrogen bonds obtained from NMR experiments for <i>PDYN</i> derived oligonucleotides.

<p>The oligonucleotide sequences and corresponding names are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039605#pone-0039605-t001" target="_blank">Table 1</a>.</p>a<p>, standard deviation for relative mobility values calculated using data of 2–6 experiments did not exceed 0.02.</p><p>-, no imino protons were observed.</p><p>n/a, no measurements were carried out.</p>b<p>, R_f was calculated for dominant bands, except Dyn B^5mC₁, where calculation was carried out for the lower (left value) and upper (right value) bands showing similar intensity (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039605#pone-0039605-g002" target="_blank">Fig. 2</a>).</p

Oligonucleotides used in the study.

<p>α-NE, Dyn A and Dyn B correspond to the α-neoendorphin-, dynorphin A- and dynorphin B-coding sequences of the human prodynorphin (<i>PDYN</i>) gene. AS, antisense oligonucleotide. ^5mC, 5-methylcytosine. RO, reference oligonucleotide. (+), plus strand. (−), minus strand. M, mutations are shown in bold italic underlined letters. CpG dinucleotides are shown in bold letters. Dyn A M₁–₃ are oligonucleotides with human pathogenic SCA 23 mutations <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039605#pone.0039605-Bakalkin1" target="_blank">[35]</a>. Dyn A M_4,5 are oligonucleotides with nonsense and silent mutations.</p

Effects of excess of unlabeled Dyn A and Dyn A^5mC₁ oligonucleotides on migration of [γ-³²P]-labeled Dyn A^5mC₁ oligonucleotide on a native gel.

<p>Samples were preheated for 10 min at 95°C, mixed with loading buffer (20 mM Tris/HCl pH 7.5, 37.5% glycerol, 15 mM MgCl₂ and 50 mM NaCl), incubated for 30 minutes at 4°C, and resolved on native 15% polyacrylamide gel at 4°C. Lane 1, [γ-³²P]-labeled α-NE^5mC₁ oligonucleotide; lane 2, [γ-³²P]-labeled Dyn A^5mC₁ oligonucleotide; lane 3, double-stranded (ds) oligonucleotide produced by preincubation of [γ-³²P]-labeled Dyn A oligonucleotide with the corresponding antisense oligonucleotide; lanes 4 to 6 and 7 to 9, [γ-³²P]-labeled Dyn A^5mC₁ oligonucleotide preincubated with 0.7, 7.0 or 700.0 ng of unlabeled Dyn A^5mC₁ or Dyn A oligonucleotide.</p

Predicted secondary structures of the Dyn A-coding sequence, calculated with the mFold software (shown as B and C on Fig. S3).

<p>Methylated cytosines are shown in red letters, and mutated bases are shown in blue circles.</p

Multiparametric profiling of engineered nanomaterials: unmasking the surface coating effect

Despite considerable efforts, the properties that drive the cytotoxicity of engineered nanomaterials (ENMs) remain poorly understood. Here, the authors inverstigate a panel of 31 ENMs with different core chemistries and a variety of surface modifications using conventional in vitro assays coupled with omics-based approaches. Cytotoxicity screening and multiplex-based cytokine profiling reveals a good concordance between primary human monocyte-derived macrophages and the human monocyte-like cell line THP-1. Proteomics analysis following a low-dose exposure of cells suggests a nonspecific stress response to ENMs, while microarray-based profiling reveals significant changes in gene expression as a function of both surface modification and core chemistry. Pathway analysis highlights that the ENMs with cationic surfaces that are shown to elicit cytotoxicity downregulated DNA replication and cell cycle responses, while inflammatory responses are upregulated. These findings are validated using cell-based assays. Notably, certain small, PEGylated ENMs are found to be noncytotoxic yet they induce transcriptional responses reminiscent of viruses. In sum, using a multiparametric approach, it is shown that surface chemistry is a key determinant of cellular responses to ENMs. The data also reveal that cytotoxicity, determined by conventional in vitro assays, does not necessarily correlate with transcriptional effects of ENMs