Additional file 1: Table S1. of Molecular evolution of HIV-1 integrase during the 20Â years prior to the first approval of integrase inhibitors

Statistics assessing whether time-trending substitution could have resulted from inheritance. (DOCX 11 kb

Additional file 3: Figure S2. of Molecular evolution of HIV-1 integrase during the 20Â years prior to the first approval of integrase inhibitors

Relation between significant coupling and frequency of single/pairwise substitutions. a) Relation between the relative frequencies of substitution pairs within the MSA and the strength of their coupling values (only significant couplings are considered). b) Relation between the relative frequencies of single substitutions within the MSA and their absolute frequency in significant couplings. (ZIP 133 kb

Additional file 2: Figure S1. of Molecular evolution of HIV-1 integrase during the 20Â years prior to the first approval of integrase inhibitors

dN/dS ratio for different regions of the HIV-1 integrase. The red line depicts the median and the grey shaded area the 25 to 75 percentile range. Black dots mark time-trending positions, i.e. codons 6, 11, 72, 101, 119, 122, 124, 135, 154, 165, 201, 256, 265. A sliding window with window size 5 was applied. (EPS 542 kb

Role of Rac1 in the production of IL-1β in <i>C. pneumoniae</i>-infected cells.

<p>(A) PBMCs were incubated with different concentrations of the Rac1 inhibitor NSC23766 for 30 min and subsequently infected with <i>C. pneumoniae</i> (MOI 3), or (C) cells were first infected with <i>C. pneumoniae</i> (MOI 3) and NSC23766 was added 2.5 h post-infection. After incubating 16 hrs production of IL-1β was quantified by ELISA. Total RNA was harvested for quantification of chlamydial 16s rRNA production (B, D) using real-time PCR as indicated in the Materials and Methods section. (E) THP-1 cells were transfected with control siRNA or siRNA specific for Rac1. After 48 h, cells were infected with <i>C. pneumoniae</i> (MOI 3) for 16 hrs and knock down of Rac1 was assessed by reverse transcription PCR. (F) HEp-2 reinfection assay in which siRNA-transfected THP-1 cells infected with <i>C. pneumoniae</i> (MOI 0.5; 72 h) were harvested and inoculated onto monolayers of HEp-2 cells. Infected cells were then stained for Chlamydia 48 h p.i. and clamydial inclusions were counted. Data shown are representative for at least three (A–D) or two (E, F) experiments performed in duplicates.</p

Caspase-1, ASC and NLRP3 mediate IL-1β production in <i>C. pneumoniae</i>-infected cells.

<p>(A) THP-1 monocytes were infected with <i>C. pneumoniae</i> (MOI 3) for different time intervalls. Cell lysates were harvested and assayed for procaspase-1 and caspase-1 p20. (B, C) PBMCs were transfected with control siRNA or siRNA specific for caspase-1. After 48 h, cells were infected with <i>C. pneumoniae</i> (MOI 3) for 16 hrs. Expression of caspase-1 was examined by reverse transcription PCR, and supernatants were subjected to IL-1β ELISA. (D–G) PBMCs were transfected with control siRNA or siRNA specific for ASC (D, E) or NLRP3 (F, G). After 48 h, cells were infected with <i>C. pneumoniae</i> (MOI 3), expression of ASC (D) and NLRP3 (F) was examined by reverse transcription PCR, and supernatants were subjected to IL-1β ELISA (E, G). (H) Cells were transfected with siRNA as indicated and, after 48 h, infected with <i>C. pneumoniae</i> (MOI 3). Cell lysates were assayed for procaspase-1 and caspase-1 p20. (I) Mouse BMMs obtained from wildtype and Nlrp3−/− mice were infected with <i>C. pneumoniae</i> (MOI 3) for 16 hrs. Production of mIL-1β was quantified by ELISA. Western Blots are representative for at least three independent experiments. Results obtained from ELISAs represent mean ± SD of three independent experiments.</p

Rac1 controls IL-1β production at a posttranscriptional level in <i>C. pneumoniae</i>-infected cells.

<p>PBMCs were transfected with control siRNA or siRNA specific for Rac1. After 48 h, cells were infected with <i>C. pneumoniae</i> (MOI 3) for 16 hrs and knock down of Rac1 was assessed by reverse transcription PCR (A). Cell supernatants were subjected to IL-1β ELISA (B), and levels of pro-IL-1β mRNA were analyzed by Q-PCR (C). (D) THP-1 cells were incubated with the indicated concentrations of NSC23766 for 30 min and afterwards infected with <i>C. pneumoniae</i> (MOI 3) for 8 h. Cell lysates were assayed for pro-caspase-1 and caspase-1 p20 by Western blot. The western Blot is representative of three independent experiments. (E, F) THP-1 cells seeded on coverslips were treated or not treated with NSC23766, and infected with <i>C. pneumoniae</i> for 20 h. Bacteria (red) and ASC (green) were visualized by confocal laser scanning microscopy using specific antibodies. The arrowheads point to ASC foci. Images are representative of three independent experiments (original magnification 63×).</p

Flow chart of patient groups.

<p>Flow chart of patient groups.</p

Characteristics of patients included in treatment success analysis<b>.</b>

<p>^a sc: seroconversion.</p><p>¹ chi-square test.</p><p>² Kruskal-wallis test.</p><p>³ HR: hazard ratio.</p><p>° MSM: men who have sex with men.</p><p>∧ TDR: transmitted drug resistance according to Stanford algorithm.</p><p>* IQR: interquartile range.</p><p>“ CI: 95% confidence interval.</p><p>∼ cc: cell count.</p>×<p>VL: Viral load.</p> = <p>FL-ART: first-line AR.</p

Prescribing practices of NRTIs, NNRTIs and PIs in first-line treatment*.

<p>∧TDR: transmitted drug resistance according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095956#pone.0095956-Bennett1" target="_blank">[42]</a>.</p><p>*Drugs listed also if administered in combined pills.</p

Characteristics of study population.

<p>^asc: seroconversion.</p><p>¹Fisher exact test.</p><p>²Mann-Whitney-U test.</p><p>³Log rank test.</p><p>°MSM: men who have sex with men].</p><p>*IQR: Interquartile range.</p><p>"CI: 95% confidence interval.</p>∼<p>cc: cell count.</p>×<p>FL-ART: first-line cART.</p