Avian Feathers as Bioindicators of the Exposure to Heavy Metal Contamination of Food

The aim of this study was to determine the possibility of using feathers of blue tit nestlings to assess the level of endogenous accumulation of lead. For this purpose we conducted an experiment with lead application to randomly chosen nestlings from eight randomly drawn broods. Five days after the exposure, feathers of lead-treated nestlings had significantly higher lead concentrations than control nestlings. This result suggests that feathers can be used as reliable non-destructive bioindicators to assess the level of heavy metals originating from contaminated food, which is of great significance for comparative studies on ecological consequences of pollution

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Hypoxia-activated pro-drugs of the KDAC inhibitor vorinostat (SAHA)

Hypoxia (lower than normal oxygen) is a characteristic of most solid tumours that results in poor cancer patient prognosis. The difference in cellular environment between normoxia (21% oxygen) or physoxia (4–7.5% oxygen) and hypoxia (<2.0% oxygen) causes increased resistance to radio- and chemotherapy, but also provides the opportunity to selectively release hypoxia-activated pro-drugs. This approach potentially allows targeting of chemotherapies, including lysine deacetylase (KDAC) inhibitors, to the hypoxic fraction of cells. Here, we report initial work on the development of KDAC inhibitors that are selectively released in hypoxic conditions. We have shown that the addition of a 4-nitrobenzyl (NB) or 1-methyl-2-nitroimidazole (NI) bioreductive group onto the hydroxamic acid moiety of SAHA, giving NB-SAHA and NI-SAHA, abolishes KDAC inhibition activity. Both NB-SAHA and NI-SAHA undergo enzyme-mediated bioreduction, in a hypoxia-dependent manner, to release SAHA selectively in <0.1% oxygen. This work provides an important foundation for further investigations into the targeted release of KDAC inhibitors in hypoxic tumours

Influence of importin α/β and exportin 1 on equine herpesvirus type 1 (EHV-1) replication in primary murine neurons

Viruses replicating in the nucleus need to cross the nuclear membrane barrier during infection, therefore disruption of specific nuclear transport pathways is crucial for their replication cycle. In the present study we have investigated the influence of nucleo-cytoplasmic transport inhibitors – ivermectin and leptomycin B, on EHV-1 replication in primary murine neurons. Obtained results suggest that the examined proteins – exportin 1 and importin α/β may participate, but are not required, during EHV-1 infection. Based on these results, it can be assumed that EHV-1 is able to use other receptors for nucleo-cytoplasmic transport

The imidazoacridinone C-1311 induces p53-dependent senescence or p53-independent apoptosis and sensitizes cancer cells to radiation

C-1311 is a small molecule, which has shown promise in a number of pre-clinical and clinical studies. However, the biological response to C-1311 exposure is complicated and has been reported to involve a number of cell fates. Here, we investigated the molecular signaling which determines the response to C-1311 in both cancer and non-cancer cell lines. For the first time we demonstrate that the tumor suppressor, p53 plays a key role in cell fate determination after C-1311 treatment. In the presence of wild-type p53, cells exposed to C-1311 entered senescence. In contrast, cells lines without functional p53 underwent mitotic catastrophe and apoptosis. C-1311 also induced autophagy in a non-p53-dependent manner. Cells in hypoxic conditions also responded to C-1311 in a p53-dependent manner, suggesting that our observations are physiologically relevant. Most importantly, we show that C-1311 can be effectively combined with radiation to improve the radiosensitivity of a panel of cancer cell lines. Together, our data suggest that C-1311 warrants further clinical testing in combination with radiotherapy for the treatment of solid tumors

The imidazoacridinone C-1311 induces p53-dependent senescence or p53-independent apoptosis and sensitizes cancer cells to radiation

C-1311 is a small molecule, which has shown promise in a number of pre-clinical and clinical studies. However, the biological response to C-1311 exposure is complicated and has been reported to involve a number of cell fates. Here, we investigated the molecular signaling which determines the response to C-1311 in both cancer and non-cancer cell lines. For the first time we demonstrate that the tumor suppressor, p53 plays a key role in cell fate determination after C-1311 treatment. In the presence of wild-type p53, cells exposed to C-1311 entered senescence. In contrast, cells lines without functional p53 underwent mitotic catastrophe and apoptosis. C-1311 also induced autophagy in a non-p53-dependent manner. Cells in hypoxic conditions also responded to C-1311 in a p53-dependent manner, suggesting that our observations are physiologically relevant. Most importantly, we show that C-1311 can be effectively combined with radiation to improve the radiosensitivity of a panel of cancer cell lines. Together, our data suggest that C-1311 warrants further clinical testing in combination with radiotherapy for the treatment of solid tumors

Zap-Pano: a photocaged prodrug of the KDAC inhibitor panobinostat

We report the synthesis and biological evaluation of a light-activated (caged) prodrug of the KDAC inhibitor panobinostat (Zap-Pano). We demonstrate that addition of the 4,5-dimethoxy-2-nitrobenzyl group to the hydroxamic acid oxygen results in an inactive prodrug. In two cancer cell lines we show that photolysis of this compound releases panobinostat and an unexpected carboxamide analogue of panobinostat. Photolysis of Zap-Pano causes an increase in H3K9Ac and H3K18Ac, consistent with KDAC inhibition, in an oesophageal cancer cell line (OE21). Irradiation of OE21 cells in the presence of Zap-Pano results in apoptotic cell death. This compound is a useful research tool, allowing spatial and temporal control over release of panobinostat