Expression levels of selected genes as measured by RNAseq and qPCR.

<p>The expression values generated by RNA-seq (red) or qPCR (blu) for eleven selected genes using the same corneal samples (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133173#pone.0133173.t001" target="_blank">Table 1</a>) are compared. Values detected for each gene were normalized to GAPDH expression and reported as a ratio between APCP-exposed samples and unexposed controls.</p

qPCR analysis of selected genes at different time points.

<p>Two human corneas were used to evaluate the expression of specific genes by qPCR at selected time points from the exposure to APCP. To minimize the variability of response, both corneas, one used as negative control and the other exposed to 2 min APCP, were divided into three pieces, then collected at 3, 6 and 24 h post-treatment. Expression levels of the target genes, detected in the treated corneal sample relative to the untreated sample were normalized to GAPDH levels.</p

Differentially expressed corneal genes (DEGs) at 6 h after exposure to APCP.

<p>(a) over- and under-expressed DEGs are shown as common (overlapping area) or exclusive to the APCP (left) or APCP+NAC (right) treatments; (b) number of total (Baggerly’s test FDR p-value <0.01) and DEG, over- and under-expressed genes detected in HC1-HC6 samples, paired per condition.</p

Gene ID, Ensemble ID and related Forward and Reverse primers used in qPCR.

<p>GAPDH was used as housekeeping gene, other genes were selected for RNA-seq validation (underlined) or time-related expression analysis (*)</p><p>Gene ID, Ensemble ID and related Forward and Reverse primers used in qPCR.</p

Detection of OGG1 in human corneas treated ex-vivo with APCP.

<p>Corneal tissues exposed for 2 min to APCP were analyzed by immunohistochemistry (a-d) and Western Blot (e-f). Frozen sections (5 μm) of corneas treated in the absence (a) or presence (b) of 10 mM NAC were incubated with polyclonal rabbit anti-OGG1 at 6 h post-treatment. Protein immunostaining (in red) was compared to that of untreated controls (c). Negative controls were prepared by omitting the primary antibody (d). For the Western Blot analysis, proteins were extracted at 6 and 24 h post-treatment: the OGG1 protein signal increased at 6 h, and was reduced in the presence of NAC, and returned to values comparable to that of controls within 24 h. Densitometric values of OGG1 autoradiographic bands were normalized to corresponding β-actin and expressed as percentage ± SE of the mean control value.</p

A selection of corneal genes to this study.

<p>A selection of DEGs for at least one treatment or genes selected for RNA-seq validation (^) or time-related expression analysis (*). Where genes are DEG, fold change value and absolute ranks in the transcriptome was reported. FC, Fold Change.</p><p>A selection of corneal genes to this study.</p

Gene Ontologies most represented in the corneal genes up-regulated by APCP.

<p>Gene Ontologies most represented in the corneal genes up-regulated by APCP in the absence (a) or presence (b) of NAC. Size and gray scale color of the circles reflect the importance of cell pathways, represented as functionally connected nodes.</p

Hierarchical clustering performed on the whole normalized dataset.

<p>52,447 genes, Manhattan correlation, complete linkage.</p

Corneal samples used and RNA sequencing data report.

<p>Individual (HC2, HC4, HC6) or pooled (HC1, HC3, HC5) human corneas untreated or treated for 2 min with APCP in the absence or presence of N-acetyl cysteine (NAC) were subjected to RNA sequencing. HC1A-HC1B, HC3A-HC5A, HC3B-HC5B, HC4A-HC6A are cornea pair from the same donor Percentage of obtained Illumina reads on total and mapped reads per sample are shown.</p><p>Corneal samples used and RNA sequencing data report.</p

DNA Damage and Transcriptional Changes in the Gills of <em>Mytilus galloprovincialis</em> Exposed to Nanomolar Doses of Combined Metal Salts (Cd, Cu, Hg)

<div><p>Aiming at an integrated and mechanistic view of the early biological effects of selected metals in the marine sentinel organism <em>Mytilus galloprovincialis</em>, we exposed mussels for 48 hours to 50, 100 and 200 nM solutions of equimolar Cd, Cu and Hg salts and measured cytological and molecular biomarkers in parallel. Focusing on the mussel gills, first target of toxic water contaminants and actively proliferating tissue, we detected significant dose-related increases of cells with micronuclei and other nuclear abnormalities in the treated mussels, with differences in the bioconcentration of the three metals determined in the mussel flesh by atomic absorption spectrometry. Gene expression profiles, determined in the same individual gills in parallel, revealed some transcriptional changes at the 50 nM dose, and substantial increases of differentially expressed genes at the 100 and 200 nM doses, with roughly similar amounts of up- and down-regulated genes. The functional annotation of gill transcripts with consistent expression trends and significantly altered at least in one dose point disclosed the complexity of the induced cell response. The most evident transcriptional changes concerned protein synthesis and turnover, ion homeostasis, cell cycle regulation and apoptosis, and intracellular trafficking (transcript sequences denoting heat shock proteins, metal binding thioneins, sequestosome 1 and proteasome subunits, and GADD45 exemplify up-regulated genes while transcript sequences denoting actin, tubulins and the apoptosis inhibitor 1 exemplify down-regulated genes). Overall, nanomolar doses of co-occurring free metal ions have induced significant structural and functional changes in the mussel gills: the intensity of response to the stimulus measured in laboratory supports the additional validation of molecular markers of metal exposure to be used in Mussel Watch programs.</p> </div