Search

To improve assessment of risks associated with pharmaceutical contamination of the environment, it is crucial to understand effects and mode of action of drugs in non-target species. The evidence is accumulating that species with well-conserved drug targets are prone to be at risk when exposed to pharmaceuticals. An interesting group of pharmaceuticals released into the environment is imidazoles, antifungal agents with inhibition of ergosterol synthesis as a primary mode of action in fungi. However, imidazoles have also been identified as competitive antagonists of calmodulin (CaM), a calcium-binding protein with phylogenetically conserved structure and function. Therefore, imidazoles would act as CaM inhibitors in various organisms, including those with limited capacity to synthesize sterols, such as arthropods. We hypothesized that effects observed in crustaceans exposed to imidazoles are related to the CaM inhibition and CaM-dependent nitric oxide (NO) synthesis. To test this hypothesis, we measured (i) CaM levels and its gene expression, (ii) NO accumulation and (iii) gene expression of NO synthase (NOS1 and NOS2), in the cladoceran Daphnia magna exposed to miconazole, a model imidazole drug. Whereas significantly increased CaM gene expression and its cellular allocation were observed, supporting the hypothesized mode of action, no changes occurred in either NO synthase expression or NO levels in the exposed animals. These findings suggest that CaM inhibition by miconazole leads to protein overexpression that compensates for the loss in the protein activity, with no measurable downstream effects on NO pathways. The inhibition of CaM in D. magna may have implications for effect assessment of exposure to mixtures of imidazoles in aquatic non-target species.

Sirtuin 1 (SIRT1), the NAD-dependent histone deacetylase, has been extensively investigated due to its cognitive protective effect. Studies suggest microRNAs (miRNAs) and histone modifications are key epigenetic regulators of gene expression and play important role in brain development. We previously showed that cognitive impairment by lead (Pb) was associated with downregulation of SIRT1, but the epigenetic role of this is unclear. Thus, we exposed 4-week-old male mice to 0.2% lead acetate solution for three months, and subsequently extracted brain homogenate from mice cortex and hippocampus at the age of 1, 4, and 16 months, respectively. In this study, we found that the protein level of SIRT1 was inhibited in the hippocampus and cortex of 16-month-old aged mice exposed to Pb. Moreover, changes in the levels of miR-138-5p and miR-141-3p, which were considered to the mechanistic target of SIRT1 by bioinformatic analysis, were negative correlations SIRT1 protein expression. We also found miR-34c-3p expression was increased in the cortex of mice at the age of 16 months. Collectively, our results showed the expression of neural SIRT1 and three selected microRNAs at different age nodes of mice for the first time of following Pb exposure. Our results suggest that additional efforts should focus on the consequences of early Pb exposure from an epigenetic perspective.

The canonical mode of action (MOA) of microcystins (MC) is the inhibition of protein phosphatases, but complete characterization of toxicity pathways is lacking. The existence of over 200 MC congeners complicates risk estimates worldwide. This work employed RNA-seq to provide an unbiased and comprehensive characterization of cellular targets and impacted cellular processes of hepatocytes exposed to either MC-LR or MC-RR congeners. The human hepatocyte cell line, HepaRG, was treated with three concentrations of MC-LR or -RR for 2 h. Significant reduction in cell survival was observed in LR1000 and LR100 treatments whereas no acute toxicity was observed in any MR-RR treatment. RNA-seq was performed on all treatments of MC-LR and -RR. Differentially expressed genes and pathways associated with oxidative and endoplasmic reticulum (ER) stress, and the unfolded protein response (UPR) were highly enriched by both congeners as were inflammatory pathways. Genes associated with both apoptotic and inflammatory pathways were enriched in LR1000. We present a model of MC toxicity that immediately causes oxidative stress and leads to ER stress and the activation of the UPR. Differential activation of the three arms of the UPR and the kinetics of JNK activation ultimately determine whether cell survival or apoptosis is favored. Extracellular exosomes were enrichment of by both congeners, suggesting a previously unidentified mechanism for MC-dependent extracellular signaling. The complement system was enriched only in MC-RR treatments, suggesting congener-specific differences in cellular effects. This study provided an unbiased snapshot of the early systemic hepatocyte response to MC-LR and MC-RR congeners and may explain differences in toxicity among MC congeners., Highlights • Microcystin-LR and microcystin-RR have similar transcriptional responses. • Genes associated with oxidative stress and the unfolded protein response were enriched by congeners. • Genes associated with extracellular exosomes were enriched, suggesting a potential new mechanism for cell signaling. • Complement associated genes were strongly enriched only by microcystin-RR. • Identified a potential molecular mechanism underlying the cellular fate of hepatocyte.

Some endocytic proteins have recently been shown to play a role in tumorigenesis. In this study, we demonstrate that APPL2, an adapter protein with known endocytic functions, is upregulated in 40% cases of glioblastoma multiforme, the most common and aggressive cancer of the central nervous system. The silencing of APPL2 expression by small interfering RNAs (siRNAs) in glioma cells markedly reduces cell survival under conditions of low growth factor availability and enhances apoptosis (measured by executor caspase activity). Long‐term depletion of APPL2 by short hairpin RNAs (shRNAs), under regular growth factor availability, suppresses the cell transformation abilities, assessed by inhibited colony formation in soft agar and by reduced xenograft tumor growth in vivo. At the molecular level, the negative effect of APPL2 knockdown on cell survival is not due to the alterations in AKT or GSK3β activities which were reported to be modulated by APPL proteins. Instead, we attribute the reduced cell survival upon APPL2 depletion to the changes in gene expression, in particular to the upregulation of apoptosis‐related genes, such as UNC5B (a proapoptotic dependence receptor) and HRK (harakiri, an activator of apoptosis, which antagonizes anti‐apoptotic function of Bcl2). In support of this notion, the loss of glioma cell survival upon APPL2 knockdown can be rescued either by an excess of netrin‐1, the prosurvival ligand of UNC5B or by simultaneous silencing of HRK. Consistently, APPL2 overexpression reduces expression of HRK and caspase activation in cells treated with apoptosis inducers, resulting in the enhancement of cell viability. This prosurvival activity of APPL2 is independent of its endosomal localization. Cumulatively, our data indicate that a high level of APPL2 protein might enhance glioblastoma growth by maintaining low expression level of genes responsible for cell death induction., Highlights ► APPL2 protein levels are elevated in 40% cases of glioblastoma multiforme.► Overexpression of APPL2 exhibits cytoprotective effects in glioma cells.► APPL2 depletion reduces survival and transformation abilities of glioma cells.► Silencing of APPL2 promotes expression of proapoptotic genes HRK and UNC5B.

MicroRNAs (miRNAs) are small 21–25 nucleotide-long non-coding RNAs that have emerged as key negative post-transcriptional regulators of gene expression [1, 2]. Currently there are more than 700 mammalian miRNAs that can potentially target up to one-third of protein-coding human genes [1] involved in diverse physiological and pathological processes, including cancer [3, 4]. Indeed, aberrant levels of miRNAs have been reported in all major human malignancies [5, 6]. In tumors, altered expression of miRNAs has been demonstrated to inhibit tumor suppressor genes or inappropriately activate oncogenes and has been associated with every aspect of tumor biology, including tumor progression, invasiveness, metastasis, and acquisition of resistance by malignant cells to chemotherapeutic agents [3, 4, 7, 8]. These observations lead to the suggestion that aberrant expression of miRNAs may contribute to tumorigenesis [9]. However, most of the tumor-miRNA-related studies are based on expression analysis of miRNAs in tumors in comparison with corresponding adjacent normal tissues [4, 5, 6]. The altered expression of any given miRNA in neoplastic cells is not sufficient to address conclusively the role of these changes in tumorigenesis [10]. Additionally, despite the established biological significance of miRNA dysregulation in neoplastic cells, there is a lack of knowledge on the role of miRNAs during early stages of tumor development, especially if variations in the expression of specific miRNAs are associated with differences in the susceptibility to tumorigenesis.

Background: MxA gene expression is one of the most appropriate markers of biological activity of exogenous interferon (IFN) beta. Methods: We quantified MxA mRNA for five consecutive days in 62 patients treated with IFN beta (16, Avonex; 10, Betaferon; 24, Rebif 22; 12, Rebif 44), by quantitative-competitive polymerase chain reaction. Every three months, IFN beta induced neutralising antibodies (NAbs) were evaluated in sera using a cytopathic effect assay. Results: Two categories of patients were identified: one group (49/62) had a sharp post-injection increase in MxA expression (defined as "IFN beta biological responder"), whereas the other group (13/62) had no MxA induction after IFN beta administrations (defined as "IFN beta biological non-responder"). In 11/13 biological non-responders, the persistent presence of NAbs correlated with abolished biological activity, independently of treatment regimen. The two remaining IFN beta biological non-responders were NAb–. Among the 49 IFN beta biological responders, biological activity was comparable between the four preparations on day 2 and 3 (+12 and +36 hours post-injection), but it was greater in Betaferon and both Rebif preparations on day 1, 4, and 5. In biological responders treated three times a week, only 82% (59/72) of injections were considered effective, compared with 100% (13/13) of Avonex injections. Conclusion: Our results suggest that an optimal IFN beta regimen is not yet available: Avonex, given once a week, shows lower cumulative biological activity. On the other hand, both Betaferon and Rebif, given three times a week, show 18% biologically ineffective injections and higher risk of developing NAbs, which abolish biological activity.

International audience; The agronomic and pulping performance of transgenic trees with altered lignin has been evaluated in duplicated, long-term field trials. Poplars expressing cinnamyl alcohol dehydrogenase (CAD) or caffeate/5-hydroxy-ferulate O-methyltransferase (COMT) antisense transgenes were grown for four years at two sites, in France and England. The trees remained healthy throughout the trial. Growth indicators and interactions with insects were normal. No changes in soil microbial communities were detected beneath the transgenic trees. The expected modifications to lignin were maintained in the transgenics over four years, at both sites. Kraft pulping of tree trunks showed that the reduced-CAD lines had improved characteristics, allowing easier delignification, using smaller amounts of chemicals, while yielding more high-quality pulp. This work highlights the potential of engineering wood quality for more environmentally benign papermaking without interfering with tree growth or fitness.

Background/Aims—Loss of function of the retinoblastoma (Rb) tumour suppressor gene, located on chromosome 13, is common in many inherited and sporadic forms of cancer. Inactivation of its gene product by oncogenic human papillomaviruses (HPV) plays a key role in the genesis of cervical cancer. It has been shown previously that non-melanoma skin cancers of renal transplant recipients and immunocompetent patients with skin cancer also frequently harbour potentially oncogenic HPV types. This study aimed to examine the integrity of the Rb gene in histologically confirmed squamous cell carcinomas (SCCs) from renal transplant recipients and immunocompetent patients with skin cancer. Methods—Loss of heterozygosity (LOH) at the Rb locus was examined in 13 histologically confirmed SCCs using the D13S153 microsatellite marker, which is located in exon 2 of the Rb gene. Loss of a second marker, D13S118, distal telomerically to the Rb gene at 13q14.3 was also analysed. Results—Of the 13 HPV associated SCCs examined 11 were informative (two SCCs were homozygous for both microsatellite markers). LOH at the D13S153 locus was found in four of the 10 informative SCCs and LOH at the D13S118 locus was found in five of the 11 informative cases. Overall, seven of the 11 informative cases showed LOH at one or other locus. This represents a high degree of chromosomal instability in these tumours. The expression of the Rb gene product in the 11 informative cases was analysed immunohistochemically. Expression of Rb was detected in 10 of the 11 SCCs examined. No correlation between the HPV status of the tumours and the expression of Rb was found. Although the only SCC not to express Rb also demonstrated LOH at the D13S153 locus, the remaining SCCs that had LOH at 13q14 were able to express Rb. Conclusion—Another tumour suppressor gene located at 13q14 might be responsible for the genesis of these tumours.