Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Czech Lexico-Semantic Database 0.1

A lexicographical project, whose aim is to digitize and align two Czech onomasiological dictionaries (Haller 1969–77; Klégr 2007) in order to create an integrated digital multi-purpose lexico-semantic database of Czech

Level of invasion and invasibility of European habitats, assessed on the basis of large phytosociological databases

Habitats vary considerably in the level of invasion (LI), i.e. the number or proportion of alien plant species. To determine typical LIs for different habitats, we compiled a database of 52 480 relevés from three contrasting regions: Catalonia, Czech Republic and Great Britain. We classified plants into neophytes, archaeophytes and native, and calculated the proportion of each group in 33 EUNIS habitats. Only 8 of 545 aliens found in the relevés occurred in all three regions. Despite this large difference in alien species composition, habitat LIs were consistent among regions. Few aliens were found in nutrient-poor habitats, e.g. mires, heaths and alpine grasslands. Many aliens were found in frequently disturbed habitats with fluctuating nutrient availability, e.g. in man-made habitats. Neophytes were also frequently found in coastal, littoral and riverine habitats. Besides local habitat properties LI depends on propagule pressure of aliens. To determine real susceptibility of habitats to invasions (invasibility), it is necessary to factor out the effects of propagule pressure on the LI. We did it by statistical modelling based on phytosociological data and digital maps. Using regression trees, the proportion of aliens was related to variables representing habitat properties, propagule pressure and climate. Propagule pressure was expressed through urban, industrial or agricultural land cover and human population density in the region. Urban and industrial land use positively affected the proportion of both archaeophytes and neophytes. Agricultural land use and higher population density positively affected the proportion of archaeophytes. After removing the propagule pressure effect, some habitats with intermediate LI had very low relative proportions of aliens. This indicates that these habitats, e.g., dry, wet and saline grasslands, base-rich fens or deciduous forests are invasion-resistant. Overall, habitats explained much more variation in the LI than propagule pressure. Our findings that (1) habitat LIs are consistent across contrasting parts of Europe, and (2) LI mainly depends on habitat type and less so on propagule pressure, enabled us to extrapolate our data to wider Europe. By plotting the quantitative information on the LIs from Catalonian, Czech and British data on the CORINE land cover map of Europe, we produced the first European LI map

A Potent Autophagy Inhibitor (Lys05) Enhances the Impact of Ionizing Radiation on Human Lung Cancer Cells H1299

Autophagy inhibition through small-molecule inhibitors is one of the approaches to increase the efficiency of radiotherapy in oncological patients. A new inhibitor—Lys05—with the potential to accumulate within lysosomes and to block autophagy was discovered a few years ago. Several studies have addressed its chemosensitizing effects but nothing is known about its impact in the context of ionizing radiation (IR). To describe its role in radiosensitization, we employed radioresistant human non-small cell lung carcinoma cells (H1299, p53-negative). Combined treatment of H1299 cells by Lys05 together with IR decreased cell survival in the clonogenic assay and real-time monitoring of cell growth more than either Lys05 or IR alone. Immunodetection of LC3 and p62/SQSTM1 indicated that autophagy was inhibited, which correlated with increased SQSTM1 and decreased BNIP3 gene expression determined by qRT-PCR. Fluorescence microscopy and flow cytometry uncovered an accumulation of lysosomes. Similarly, transmission electron microscopy demonstrated the accumulation of autophagosomes confirming the ability of Lys05 to potentiate autophagy inhibition in H1299 cells. We report here for the first time that Lys05 could be utilized in combination with IR as a promising future strategy in the eradication of lung cancer cells

Testing of library preparation methods for transcriptome sequencing of real life glioblastoma and brain tissue specimens: A comparative study with special focus on long non-coding RNAs.

Current progress in the field of next-generation transcriptome sequencing have contributed significantly to the study of various malignancies including glioblastoma multiforme (GBM). Differential sequencing of transcriptomes of patients and non-tumor controls has a potential to reveal novel transcripts with significant role in GBM. One such candidate group of molecules are long non-coding RNAs (lncRNAs) which have been proved to be involved in processes such as carcinogenesis, epigenetic modifications and resistance to various therapeutic approaches. To maximize the value of transcriptome sequencing, a proper protocol for library preparation from tissue-derived RNA needs to be found which would produce high quality transcriptome sequencing data and increase the number of detected lncRNAs. It is important to mention that success of library preparation is determined by the quality of input RNA, which is in case of real-life tissue specimens very often altered in comparison to high quality RNA commonly used by manufacturers for development of library preparation chemistry. In the present study, we used GBM and non-tumor brain tissue specimens and compared three different commercial library preparation kits, namely NEXTflex Rapid Directional qRNA-Seq Kit (Bioo Scientific), SENSE Total RNA-Seq Library Prep Kit (Lexogen) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB). Libraries generated using SENSE kit were characterized by the most normal distribution of normalized average GC content, the least amount of over-represented sequences and the percentage of ribosomal RNA reads (0.3-1.5%) and highest numbers of uniquely mapped reads and reads aligning to coding regions. However, NEBNext kit performed better having relatively low duplication rates, even transcript coverage and the highest number of hits in Ensembl database for every biotype of our interest including lncRNAs. Our results indicate that out of three approaches the NEBNext library preparation kit was most suitable for the study of lncRNAs via transcriptome sequencing. This was further confirmed by highly consistent data reached in an independent validation on an expanded cohort

Distribution of neutrons and protons in elongated targets

Analysis of neutron distribution was carried out for two elongated targets. The targets have cylindrical shape and are made of lead and carbon, respectively. The dimensions are approximately one meter in length and 19 cm in diameter. The targets were irradiated with 660 MeV proton beam at Phasotron accelerator at the Joint Institute for Nuclear Research. The total number of protons was 2.35(18)E15 for the experiment with carbon target and the total number of particles at the second experiment was 2.32(19)E15. The produced neutron field was monitored by cobalt threshold activation detectors at various positions. The activation detectors were measured by means of gamma spectroscopy using HPGe detectors. Reaction rates of different radionuclides produced in the activation detectors were determined and the results from both experiments were compared. The ratios were calculated for 7 reactions produced in cobalt detectors. The ratio of the reaction rates shows that the number of residual nuclei with higher threshold energies is higher for experiment with carbon target than for the experiment with the lead target