Primer sequences and concentrations used in RT-PCR assays.

<p>Primer sequences and concentrations used in RT-PCR assays.</p

KLF6 protein expression throughout trophoblast cell differentiation.

<p><b>A-</b> Isolated mononuclear villous CTB cells cultured during the indicated hours and stained for KLF6 immunofluorescence detection (middle panels) with the polyclonal R-173 (green) anti-KLF6 antibody. Nuclei were counterstained with Hoechst 33342 dye (blue) and the overlay is shown (right panels). <b>B-</b> Confocal microscopy imaging of KLF6 at the indicated time points of the differentiation process. KLF6 was labelled with the polyclonal R-173 antibody (left panels) and DNA was stained with propidium iodide (IP) (middle panel). Overlay is shown in the right panels. <b>C-</b> Fluorescence intensity profile of KLF6 (green) and IP (red) along the yellow line shown in the confocal microscopy images. <b>D-</b> Morphological and biochemical differentiation of isolated mononuclear CTB cells were confirmed by the disappearance of desmoplakin intercellular staining (red), the appearance of multinucleated structures and the expression of PSG proteins (green). Original magnification, x1000. Scale bar, 10 µm. Immunofluorescence assays were performed with at least three different CTB purifications and representative figures are shown.</p

KLF6 regulates mRNA and protein expression of placental genes.

<p><b>A-</b> JEG-3 cells were cultured in the presence of 1 µM methotrexate to induce cell differentiation during the indicted times and KLF6 mRNA expression was quantified by qRT-PCR (ABI 7500, Applied Biosystems). <b>B-</b> JEG-3 cells transfected with the empty (white bars) or the KLF6 expression vector (black bars) were harvested 24 h after transfection and <i>PSG</i>, <i>βhCG</i> and <i>GCM1</i> gene expression was quantified by qRT- PCR (ABI 7500, Applied Biosystems). For A and B, results were normalized to cyclophilin A and expressed according to the 2^−ΔΔCt method using as calibrator the mRNA level obtained from the control condition. Data are presented as mean ±SEM of three independent experiments performed in triplicates and a one-sample t-test was used to determine whether experimental values were significantly different from the control value set as 1 (*p<0.05). <b>C-</b> Western blot detection of KLF6, PSG, βhCG and GCM1 in protein extracts of JEG-3 cells transfected with the empty (left lane) or KLF6 expression vector (right lane). α-tubulin was used as a loading control in each assay. Representative western blots are shown and the bar graph shows the densitometric analysis of three different experiments. (*p<0.05) <b>D</b>- JEG-3 cells were transiently transfected with the KLF6 expression vector and 24 h later they were immunostained for the detection of KLF6 (red) and βhCG (green) with a monoclonal anti-KLF6 and a polyclonal anti-βhCG antibodies, respectively. Nuclei were counterstained with Hoechst 33342 dye (blue), and the merge of the three channels is shown on the right side. Bar = 10 µm. Original magnification: ×1000. Representative images from three independent transfections are shown. Arrowheads, JEG-3 cells overexpressing KLF6; arrows, cells positive for βhCG.</p

KLF6 transcript and protein levels increase during trophoblast cell differentiation.

<p><b>A-</b> KLF6 mRNA expression was quantified by qRT-PCR (ABI 7500, Applied Biosystems) in CTB cells isolated from three to eight normal term placentas, and cultured in differentiating medium during the indicated times. Results were normalized to cyclophilin A and expressed according to the 2^−ΔΔCt method using as calibrator the expression level at 0 h. Results are depicted as boxplot graphs representing the medians (horizontal bars), the 25–75th percentile interquartile range (box limits), and the lowest and highest values (whiskers) of three to eight experiments performed in triplicates. Inter-group comparisons were made using the Kruskal-Wallis one-way Analysis of variance (ANOVA) with the Dunn's multiple comparisons post-hoc test of statistical significance. *p<0.05 <i>vs</i> 0 h, #p<0.05 <i>vs</i> 2 h. <b>B-</b> Protein extracts (60 µg) prepared from CTB cells cultured for the indicated hours were subjected to western blot analysis using anti-KLF6 and α-tubulin antibodies as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022438#s4" target="_blank"><i>Materials and Methods</i></a>. A representative blot is shown. The bar graph represents the densitometric quantification of KLF6/α-tubulin ratio of three independent experiments expressed as mean ±SEM. *p<0.05 <i>vs</i> 0 h, ‡p<0.05 <i>vs</i> 16 h. (Kruskal-Wallis, Dunn's).</p

Genotypes of methicillin resistant <i>S. aureus</i> (MRSA) isolates recovered from children with Community-onset infections (invasive and non-invasive) in central, northen and eastern regions of Argentina, by epidemiologic case classification.

<p><b>INVI</b>: Invasive infections.</p>a<p>CBAH1-H3: Prospective surveillance of CO-<i>S. aureus</i> infections in children from three children's hospitals of Córdoba (CBAH1, CBAH2 and CBAH3) 2007 and 2008.</p>b<p>CSACHARG and: Prospective surveillance of CO-<i>S. aureus</i> infections in children from Argentina, 2007 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030487#pone.0030487-Paganini1" target="_blank">[16]</a>.</p>c<p>Genotypes are denoted as: type (by PFGE)-Sequence Type (ST by MLST)-SCC<i>mec</i> type.</p

Characteristics, incidence of CA-MRSA infections and location of children's hospitals of northern, eastern and central of Argentina, 2007.

a<p>Number of all CA-MRSA isolates detected in the three Cordoba children's hospitals during 2007 and those recovered in each hospital from the surveillance study for community onset <i>S. aureus</i> infections in children from Argentina-(CSACHARG) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030487#pone.0030487-Paganini1" target="_blank">[16]</a>.</p>b<p>annual visits: include outpatient facility and emergency service.</p>c<p>Incidence: Number of cases/100,000 annual visits.</p

Evolution of community-associated <i>Staphylococcus aureus</i> infections from children in Cordoba-Argentina, 2003–2008.

<p>Evolution of the rates of all community-associated <i>Staphylococcus aureus</i> infections: <i>i)</i> methicillin-resistant <i>S. aureus</i> (CA-MRSA) infections [total (squares) and invasive-INVI (circles)], and <i>ii)</i> total methicillin-susceptible <i>S. aureus</i> (CA-MSSA) infections (triangles) in Córdoba children's hospitals, [H1 (CBAH1): 2003–2008: filled figures, and in H1, H2 and H3 (CBAH1-3): 2005 vs 2007–2008: empty figures] <i>iii)</i> methicillin-resistant community-associated <i>Staphylococcus aureus</i> infections caused by the ST5-IV-PVL⁺clone [total (gray squares) and invasive (INVI) (gray circles)] in H1 (CBAH1): 2003–2008.</p

Characteristics of MRSA clones isolated from children with community onset MRSA infections (CO-MRSA), Argentina.

<p><i>agr</i> type, type of accessory gene regulator, ST: Sequence Type, SCC<i>mec</i>: Staphylococcal Cassette Chromosome <i>mec</i>, PFGE, Pulsed Field Gel Electrophoresis; RIDOM <i>spa</i> type: staphylococcal protein A (<i>spa</i>) type assigned through the RIDOM databases (<a href="http://spaserver.ridom.de" target="_blank">http://spaserver.ridom.de</a>).</p>a<p>n (%), total number and % of strains with this molecular characteristic [PFGE subtype (only those more frequent are indicated) or ST or <i>spa</i> type or <i>SCCmec</i> type]. % is not expressed when only one isolate with this characteristic was detected.</p>b<p><i>pvl</i>, Panton-Valentine leukocidin genes (<i>lukS</i>-PV-<i>lukF</i>-PV); indicated as number and % of isolates harboring (PVl⁺) or not (PVL⁻) <i>pvl</i> genes.</p>c<p>virulence genes profile: From all virulence genes analyzed, only those detected are indicated (number and % of positive isolates is expressed when not all isolates harbor this virulence factor).</p>d<p>Drug resistance to non-β-Lactams (%), is indicated as follows: Gentamicin (GEN), Ciprofloxacin (CIP), Erythromycin (ERY), Clindamycin (CLIc and CLIi: constitutive and inducible resistance to macrolides, lincosamide and streptogramine B, respectively), rifampin (RIF), chloramphenicol (CHL), trimethoprim/sulfamethoxazole (SXT) and minocycline (MIN) (%) of strains resistant to these antibiotics within each pulsotype is indicated when more than one isolate was detected. h-VISA (1): means one isolate belonging to this clone with phenotype h-VISA.</p>e<p>IV NT: SCC<i>mec</i> type IV non typable.</p>f<p>Vr: SCC<i>mec</i> related to V.</p

Molecular characteristics and proportion from different regions of Argentina of dominant community methicillin-resistant-<i>Staphylococcus aureus</i> clones.

<p><b>A</b>. PFGE pattern analysis for representative isolates belonging to the most prevalent subtypes of community-onset-MRSA clones (CA-MRSA and HA-MRSA) detected in central, eastern and northern regions of Argentina during 2007–2008. The schematic presentation of <i>SmaI</i> restriction patterns (middle) and dendrogram (left) by the unweighted-pair group method using average linkage clusterings are shown. Genotypes are denoted as subtype (by PFGE)-ST (by MLST)-SCC<i>mec</i> type-<i>spa</i> type (right). CA-MRSA clones appear in gray. The presence (+) or absence (−) of <i>pvl</i> genes (by PCR) is also indicated for each subtype; strains with and other without <i>pvl</i> genes belonging to the same PFGE subtype (I9) are indicated as +/−. The PFGE pattern of USA300-0114 (ST8-IVa-<i>t008</i>-ACME+) is shown for comparison purposes. The first (A1-ST5-I-t149-Cordobes/Chilean), second (B1-ST239-IIIA-t037-Brazilian) and third (C1-ST100-IVNv-t002-Pediatric) more frequent HA-MRSA clones in our country, detected among community-onset MRSA infections, are also shown (dotted gray). <b>B</b>: Proportion of CA-MRSA clones among representative isolates from different regions of Argentina in 2007.</p

Demographic and clinical characteristics of children with Community-onset methicillin resistant <i>S. aureus</i> infections in central, northern and eastern regions of Argentina.

<p>CA-MRSA: Community-associated methicillin resistant <i>Staphylococcus aureus</i>, HACO-MRSA: Healthcare-associated community-onset methicillin resistant <i>Staphylococcus aureus</i> infections.</p>a<p>CBAH1-H3: Prospective surveillance of CO-<i>S. aureus</i> infections in children from three children's hospitals of Córdoba (CBAH1, CBAH2 and CBAH3), 2007 and 2008.</p>b<p>CSACHARG: Prospective surveillance of CO-<i>S. aureus</i> infections in children from Argentina, 2007 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030487#pone.0030487-Paganini1" target="_blank">[16]</a>.</p>c<p><i>P</i> values are based on chi-square test or Fisher's exact test, as appropriate, for CA-MRSA,CBAH1-H3 vs. CA-MRSA, CSACHARG and total CA-MRSA vs. HACO-MRSA comparisons by each of categorical variables (males and infection type); <i>p</i><0.05 was considered statistically significant.</p>d<p>Values are number of patients and the percentages are indicated in parentheses. [n]: number of patients with this secondary infection focus (CA-MRSA: 342 infections in 312 patients).</p>e<p>Abscess and cellulites: include 9 cases of impetigo(1 from CBAH1-H3 and 8 cases from CSACHARG).</p>f<p>Deep abscess included: breast (2 cases), psoas (4 cases), liver and renal (1 case each one) abscesses.</p