The integrated stress response remodels the microtubule-organizing center to clear unfolded proteins following proteotoxic stress

Cells encountering stressful situations activate the integrated stress response (ISR) pathway to limit protein synthesis and redirect translation to better cope. The ISR has also been implicated in cancers, but redundancies in the stress-sensing kinases that trigger the ISR have posed hurdles to dissecting physiological relevance. To overcome this challenge, we targeted the regulatory node of these kinases, namely, the S51 phosphorylation site of eukaryotic translation initiation factor eIF2α and genetically replaced eIF2α with eIF2α-S51A in mouse squamous cell carcinoma (SCC) stem cells of skin. While inconsequential under normal growth conditions, the vulnerability of this ISR-null state was unveiled when SCC stem cells experienced proteotoxic stress. Seeking mechanistic insights into the protective roles of the ISR, we combined ribosome profiling and functional approaches to identify and probe the functional importance of translational differences between ISR-competent and ISR-null SCC stem cells when exposed to proteotoxic stress. In doing so, we learned that the ISR redirects translation to centrosomal proteins that orchestrate the microtubule dynamics needed to efficiently concentrate unfolded proteins at the microtubule-organizing center so that they can be cleared by the perinuclear degradation machinery. Thus, rather than merely maintaining survival during proteotoxic stress, the ISR also functions in promoting cellular recovery once the stress has subsided. Remarkably, this molecular program is unique to transformed skin stem cells, hence exposing a vulnerability in cancer that could be exploited therapeutically

IBEX:A versatile multiplex optical imaging approach for deep phenotyping and spatial analysis of cells in complex tissues

The diverse composition of mammalian tissues poses challenges for understanding the cell–cell interactions required for organ homeostasis and how spatial relationships are perturbed during disease. Existing methods such as single-cell genomics, lacking a spatial context, and traditional immunofluorescence, capturing only two to six molecular features, cannot resolve these issues. Imaging technologies have been developed to address these problems, but each possesses limitations that constrain widespread use. Here we report a method that overcomes major impediments to highly multiplex tissue imaging. “Iterative bleaching extends multiplexity” (IBEX) uses an iterative staining and chemical bleaching method to enable high-resolution imaging of >65 parameters in the same tissue section without physical degradation. IBEX can be employed with various types of conventional microscopes and permits use of both commercially available and user-generated antibodies in an “open” system to allow easy adjustment of staining panels based on ongoing marker discovery efforts. We show how IBEX can also be used with amplified staining methods for imaging strongly fixed tissues with limited epitope retention and with oligonucleotide-based staining, allowing potential cross-referencing between flow cytometry, cellular indexing of transcriptomes and epitopes by sequencing, and IBEX analysis of the same tissue. To facilitate data processing, we provide an open-source platform for automated registration of iterative images. IBEX thus represents a technology that can be rapidly integrated into most current laboratory workflows to achieve high-content imaging to reveal the complex cellular landscape of diverse organs and tissues

The Novel Candida albicans Transporter Dur31 Is a Multi-Stage Pathogenicity Factor

Candida albicans is the most frequent cause of oral fungal infections. However, the exact pathogenicity mechanisms that this fungus employs are largely unknown and many of the genes expressed during oral infection are uncharacterized. In this study we sought to functionally characterize 12 previously unknown function genes associated with oral candidiasis. We generated homozygous knockout mutants for all 12 genes and analyzed their interaction with human oral epithelium in vitro. Eleven mutants caused significantly less epithelial damage and, of these, deletion of orf19.6656 (DUR31) elicited the strongest reduction in pathogenicity. Interestingly, DUR31 was not only involved in oral epithelial damage, but in multiple stages of candidiasis, including surviving attack by human neutrophils, endothelial damage and virulence in vivo. In silico analysis indicated that DUR31 encodes a sodium/substrate symporter with 13 transmembrane domains and no human homologue. We provide evidence that Dur31 transports histatin 5. This is one of the very first examples of microbial driven import of this highly cytotoxic antimicrobial peptide. Also, in contrast to wild type C. albicans, dur31Δ/Δ was unable to actively increase local environmental pH, suggesting that Dur31 lies in the extracellular alkalinization hyphal auto-induction pathway; and, indeed, DUR31 was required for morphogenesis. In agreement with this observation, dur31Δ/Δ was unable to assimilate the polyamine spermidine

Decentralised water and wastewater treatment technologies to produce functional water for irrigation

International audienceThe EU project SAFIR aimed to help farmers solve problems related to the use of low quality water for irrigation in a context of increasing scarcity of conventional freshwater resources. New decentralised water treatment devices (prototypes) were developed to allow a safe direct or indirect reuse of wastewater produced by small communities/industries or the use of polluted surface water. Water treatment technologies were coupled with irrigation strategies and technologies to obtain a flexible, easy to use, integrated management of the system. The challenge is to apply new strategies and technologies which allow using the lowest irrigation water quality without harming food safety or yield and fruit or derivatives quality. This study presents the results of prototype testing of a small-scale compact pressurized membrane bioreactor and of a modular field treatment system including commercial gravel filters and heavy-metal specific adsorption materials. Decentralised compact pressurised membrane biobooster (MBR), was able to remove up to 99.99% of the inlet Escherichia coli and 98.52% of total coliforms. E. coli was completely removed from irrigation water in 53% of the samples by the last MBR prototype version. In 2008, 100% of samples fulfilled WHO standards (1989) and Global Gap requirement for faecal contamination. MBR removed from inlet flow in the average 82% of arsenic, 82% of cadmium, 97% of chromium, 93% of copper and 99% of lead. Boron and manganese were not removed from permeate. The field treatment system (FT'S) proved to be effective against faecal contamination when applied with its complete set up including UV treatment. The sole gravel filter and heavy metal removal device (HMR) cannot provide sufficient and steadily treatment for microbial contamination. Nevertheless, gravel filter can remove up to 60% of E. coli but the removal process was not stable nor predictable. FTS removed 76% of arsenic, 80% of cadmium and copper, 88% of chromium and lead, and up to 97% of zinc. Like the MBR. boron and manganese were not removed from the irrigation water. Gravel filter directly fed with secondary treated wastewater was found able to remove 41% of arsenic, 36% of cadmium and lead, 48% of chromium and 46% of copper. The residual heavy metals concentration after the gravel filter was further reduced by the HMR: 35% for arsenic, 22% for cadmium, 25% for chromium, 33% for copper and 53% for lead

Decentralised water and wastewater treatment technologies to produce functional water for irrigation

The EU project SAFIR aimed to help farmers solve problems related to the use of low quality water for irrigation in a context of increasing scarcity of conventional freshwater resources. New decentralised water treatment devices (prototypes) were developed to allow a safe direct or indirect reuse of wastewater produced by small communities/industries or the use of polluted surface water. Water treatment technologies were coupled with irrigation strategies and technologies to obtain a flexible, easy to use, integrated management of the system. The challenge is to apply new strategies and technologies which allow using the lowest irrigation water quality without harming food safety or yield and fruit or derivatives quality. This study presents the results of prototype testing of a small-scale compact pressurized membrane bioreactor and of a modular field treatment system including commercial gravel filters and heavy-metal specific adsorption materials. Decentralised compact pressurised membrane biobooster (MBR), was able to remove up to 99.99% of the inlet Escherichia coli and 98.52% of total coliforms. E. coli was completely removed from irrigation water in 53% of the samples by the last MBR prototype version. In 2008, 100% of samples fulfilled WHO standards (1989) and Global Gap requirement for faecal contamination. MBR removed from inlet flow in the average 82% of arsenic, 82% of cadmium, 97% of chromium, 93% of copper and 99% of lead. Boron and manganese were not removed from permeate. The field treatment system (FTS) proved to be effective against faecal contamination when applied with its complete set up including UV treatment. The sole gravel filter and heavy metal removal device (HMR) cannot provide sufficient and steadily treatment for microbial contamination. Nevertheless, gravel filter can remove up to 60% of E. coli but the removal process was not stable nor predictable. FTS removed 76% of arsenic, 80% of cadmium and copper, 88% of chromium and lead, and up to 97% of zinc. Like the MBR, boron and manganese were not removed from the irrigation water. Gravel filter directly fed with secondary treated wastewater was found able to remove 41% of arsenic, 36% of cadmium and lead, 48% of chromium and 46% of copper. The residual heavy metals concentration after the gravel filter was further reduced by the HMR: 35% for arsenic, 22% for cadmium, 25% for chromium, 33% for copper and 53% for lead.Agricultural water reuse Decentralised treatments Faecal contamination Heavy metals