VIB-UGent Center for Medical Biotechnology

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.

Current yeast interactome network maps contain several hundred molecular complexes with limited and somewhat controversial representation of direct binary interactions. We carried out a comparative quality assessment of current yeast interactome data sets, demonstrating that high-throughput yeast two-hybrid (Y2H) screening provides high-quality binary interaction information. Because a large fraction of the yeast binary interactome remains to be mapped, we developed an empirically controlled mapping framework to produce a "second-generation" high-quality, high-throughput Y2H data set covering approximately 20% of all yeast binary interactions. Both Y2H and affinity purification followed by mass spectrometry (AP/MS) data are of equally high quality but of a fundamentally different and complementary nature, resulting in networks with different topological and biological properties. Compared to co-complex interactome models, this binary map is enriched for transient signaling interactions and intercomplex connections with a highly significant clustering between essential proteins. Rather than correlating with essentiality, protein connectivity correlates with genetic pleiotropy.

Inflammasomes are a set of intracellular protein complexes that enable autocatalytic activation of inflammatory caspases, which drive host and immune responses by releasing cytokines and alarmins into circulation and by inducing pyroptosis, a proinflammatory cell death mode. The inflammasome type mediating these responses varies with the microbial pathogen or stress factor that poses a threat to the organism. Since the discovery that polymorphisms in inflammasome genes are linked to common autoimmune diseases and less frequent periodic fever syndromes, inflammasome signaling has been dissected at the molecular level. In this review, we present recently gained insight on the distinct inflammasome types, their activation and effector mechanisms, and their modulation by microbial virulence factors. In addition, we discuss recently gained knowledge on the role of deregulated inflammasome activity in human autoinflammatory, autoimmune, and infectious diseases.

Peroxisome proliferator-activated receptor α (PPARα) is a nuclear receptor of clinical interest as a drug target in various metabolic disorders. PPARα also exhibits marked anti-inflammatory capacities. The first-generation PPARα agonists, the fibrates, have however been hampered by drug-drug interaction issues, statin drop-in, and ill-designed cardiovascular intervention trials. Notwithstanding, understanding the molecular mechanisms by which PPARα works will enable control of its activities as a drug target for metabolic diseases with an underlying inflammatory component. Given its role in reshaping the immune system, the full potential of this nuclear receptor subtype as a versatile drug target with high plasticity becomes increasingly clear, and a novel generation of agonists may pave the way for novel fields of applications.

Mass spectrometry is a fundamental tool for discovery and analysis in the life sciences. With the rapid advances in mass spectrometry technology and methods, it has become imperative to provide a standard output format for mass spectrometry data that will facilitate data sharing and analysis. Initially, the efforts to develop a standard format for mass spectrometry data resulted in multiple formats, each designed with a different underlying philosophy. To resolve the issues associated with having multiple formats, vendors, researchers, and software developers convened under the banner of the HUPO PSI to develop a single standard. The new data format incorporated many of the desirable technical attributes from the previous data formats, while adding a number of improvements, including features such as a controlled vocabulary with validation tools to ensure consistent usage of the format, improved support for selected reaction monitoring data, and immediately available implementations to facilitate rapid adoption by the community. The resulting standard data format, mzML, is a well tested open-source format for mass spectrometer output files that can be readily utilized by the community and easily adapted for incremental advances in mass spectrometry technology. Mass spectrometry is a fundamental tool for discovery and analysis in the life sciences. With the rapid advances in mass spectrometry technology and methods, it has become imperative to provide a standard output format for mass spectrometry data that will facilitate data sharing and analysis. Initially, the efforts to develop a standard format for mass spectrometry data resulted in multiple formats, each designed with a different underlying philosophy. To resolve the issues associated with having multiple formats, vendors, researchers, and software developers convened under the banner of the HUPO PSI to develop a single standard. The new data format incorporated many of the desirable technical attributes from the previous data formats, while adding a number of improvements, including features such as a controlled vocabulary with validation tools to ensure consistent usage of the format, improved support for selected reaction monitoring data, and immediately available implementations to facilitate rapid adoption by the community. The resulting standard data format, mzML, is a well tested open-source format for mass spectrometer output files that can be readily utilized by the community and easily adapted for incremental advances in mass spectrometry technology. Mass spectrometry (MS) 1The abbreviations used are:MSmass spectrometryHUPOHuman Proteome OrganizationPSI-MSProteomics Standards Initiative working group for mass spectrometry standardsLC-MS/MSliquid chromatography-tandem mass spectrometryCVcontrolled vocabulary.Author contributions: E.W.D. is the chair, P.A.B. is the co-chair, and L.M. is the secretary of PSI-MS WG. All authors actively contributed to the creation and implementation of the standard format. All authors have agreed to all the content in the manuscript, including the data as presented. has recently emerged as a major discovery tool in the life sciences (1.Mind the technology gap.Nat. Methods. 2007; 4 (Editors): 765Crossref PubMed Scopus (5) Google Scholar). This analytical technique is used to analyze the molecular composition of a biological sample by ionizing the sample or analyte molecules and then measuring the mass-to-charge ratios of the resulting ions. The data from an MS experiment consist of mass spectra that are used to identify, characterize, and quantify the abundance of the molecules of interest. The resulting MS spectra, along with their associated metadata (e.g. experimental protocol, MS instrumentation, operational parameters, etc.), are then semi-automatically processed by specialized software packages to identify or quantify the sampled ions. The inherent variability introduced by using different instruments, instrument software, and experimental conditions, however, affects the downstream ability to analyze, integrate, and compare data sets originating from different MS experiments. mass spectrometry Human Proteome Organization Proteomics Standards Initiative working group for mass spectrometry standards liquid chromatography-tandem mass spectrometry controlled vocabulary. Indeed, with the ever-increasing use of mass spectrometry, two issues have arisen in terms of handling MS data: (i) the necessity to share data throughout the scientific community in order to facilitate integration and comparison (2.Prince J.T. Carlson M.W. Wang R. Lu P. Marcotte E.M. The need for a public proteomics repository.Nature Biotechnology. 2004; 22: 471-472Crossref PubMed Scopus (133) Google Scholar), and (ii) the importance of utilizing open and readily accessible standard formats that verifiably capture a consistent amount of crucial information. The importance of addressing these issues has been further emphasized in prominent journal editorials (3.Thou shalt share your data.Nat. Methods. 2008; 5 (Editors): 209Crossref Scopus (23) Google Scholar, 4.Democratizing proteomics data.Nat Biotechnol. 2007; 25 (Editors): 262Crossref Scopus (37) Google Scholar). Data repositories have since been created to allow data to be shared, including Tranche (5.Falkner J.A. Andrews P.C. Tranche: Secure Decentralized Data Storage for the proteomics community.Journal of Biomolecular Techniques. 2007; 18: 3Google Scholar), GPMDB (6.Craig R. Cortens J.P. Beavis R.C. Open source system for analyzing, validating, and storing protein identification data.J. Proteome Res. 2004; 3: 1234-1242Crossref PubMed Scopus (562) Google Scholar), PRIDE (7.Martens L. Hermjakob H. Jones P. Adamski M. Taylor C. States D. Gevaert K. Vandekerckhove J. Apweiler R. PRIDE: the proteomics identifications database.Proteomics. 2005; 5: 3537-3545Crossref PubMed Scopus (422) Google Scholar), and PeptideAtlas (8.Desiere F. Deutsch E.W. King N.L. Nesvizhskii A.I. Mallick P. Eng J. Chen S. Eddes J. Loevenich S.N. Aebersold R. The PeptideAtlas project.Nucleic Acids Res. 2006; 34: D655-658Crossref PubMed Scopus (566) Google Scholar), among others (9.Mead J.A. Bianco L. Bessant C. Recent developments in public proteomic MS repositories and pipelines.Proteomics. 2009; 9: 861-881Crossref PubMed Scopus (35) Google Scholar), and various proposed standard formats for MS data (10.Taylor C.F. Binz P.A. Aebersold R. Affolter M. Barkovich R. Deutsch E.W. Horn D.M. Huhmer A. Kussmann M. Lilley K. Macht M. Mann M. Muller D. Neubert T.A. Nickson J. Patterson S.D. Raso R. Resing K. Seymour S.L. Tsugita A. Xenarios I. Zeng R. Julian Jr., R.K. Guidelines for reporting the use of mass spectrometry in proteomics.Nat Biotechnol. 2008; 26: 860-861Crossref PubMed Scopus (67) Google Scholar, 11.McDonald W.H. Tabb D.L. Sadygov R.G. MacCoss M.J. Venable J. Graumann J. Johnson J.R. Cociorva D. Yates 3rd, J.R. MS1, MS2, and SQT-three unified, compact, and easily parsed file formats for the storage of shotgun proteomic spectra and identifications.Rapid Commun Mass Spectrom. 2004; 18: 2162-2168Crossref PubMed Scopus (288) Google Scholar, 12.Orchard S. Montechi-Palazzi L. Deutsch E.W. Binz P.A. Jones A.R. Paton N. Pizarro A. Creasy D.M. Wojcik J. Hermjakob H. Five years of progress in the Standardization of Proteomics Data 4(th) Annual Spring Workshop of the HUPO-Proteomics Standards Initiative April 23–25, 2007 Ecole Nationale Superieure (ENS), Lyon, France.Proteomics. 2007; 7: 3436-3440Crossref PubMed Scopus (44) Google Scholar, 13.Pedrioli P.G. Eng J.K. Hubley R. Vogelzang M. Deutsch E.W. Raught B. Pratt B. Nilsson E. Angeletti R.H. Apweiler R. Cheung K. Costello C.E. Hermjakob H. Huang S. Julian R.K. Kapp E. McComb M.E. Oliver S.G. Omenn G. Paton N.W. Simpson R. Smith R. Taylor C.F. Zhu W. Aebersold R. A common open representation of mass spectrometry data and its application to proteomics research.Nat Biotechnol. 2004; 22: 1459-1466Crossref PubMed Scopus (638) Google Scholar, 14..mzData, http://psidev.info/index.php?q=node/80#mzdata, .Google Scholar) were developed. Other formats such as JCAMP-DX (http://www.acornnmr.com/JCAMP.htm; www.jcamp.org), which was designed for IR spectrometry and adapted to NMR and mass spectrometry, and NetCDF are quite variably implemented, difficult to validate, and cannot encode extensive metadata in a standard fashion and therefore have not gained much use for proteomics applications and other complex MS analyses. Analytical Information Markup Language (AnIML; http://animl.sourceforge.net/), which aims to encompass several analytical platforms, including eventually mass spectrometry, is still being designed. For mass spectrometry-based proteomics workflows, mzXML (13.Pedrioli P.G. Eng J.K. Hubley R. Vogelzang M. Deutsch E.W. Raught B. Pratt B. Nilsson E. Angeletti R.H. Apweiler R. Cheung K. Costello C.E. Hermjakob H. Huang S. Julian R.K. Kapp E. McComb M.E. Oliver S.G. Omenn G. Paton N.W. Simpson R. Smith R. Taylor C.F. Zhu W. Aebersold R. A common open representation of mass spectrometry data and its application to proteomics research.Nat Biotechnol. 2004; 22: 1459-1466Crossref PubMed Scopus (638) Google Scholar) and mzData (14..mzData, http://psidev.info/index.php?q=node/80#mzdata, .Google Scholar) have been the most widely used open formats for several years. However, each of these initial efforts to develop an open, vendor-neutral XML data format to store MS information was undertaken with a different underlying purpose. One format, mzData, was developed by HUPO-PSI as a data exchange and archive standard (14..mzData, http://psidev.info/index.php?q=node/80#mzdata, .Google Scholar, 15.Orchard S. Zhu W. Julian Jr., R.K. Hermjakob H. Apweiler R. Further advances in the development of a data interchange standard for proteomics data.Proteomics. 2003; 3: 2065-2066Crossref PubMed Scopus (20) Google Scholar), and was implemented as such in PRIDE (16.Jones P. Cote R.G. Martens L. Quinn A.F. Taylor C.F. Derache W. Hermjakob H. Apweiler R. PRIDE: a public repository of protein and peptide identifications for the proteomics community.Nucleic Acids Res. 2006; 34: D659-663Crossref PubMed Scopus (236) Google Scholar). The other format, mzXML, was developed at the Institute for Systems Biology in an effort to streamline their data processing software (17.Keller A. Eng J. Zhang N. Li X.J. Aebersold R. A uniform proteomics MS/MS analysis platform utilizing open XML file formats.Mol. Syst. Biol. 2005; 1 (2005.0017)Crossref PubMed Google Scholar), and became a popular de-facto standard format. These two formats also differed in their underlying philosophies regarding flexibility. mzData utilized a controlled vocabulary that could be frequently updated as the technology advanced. In contrast, mzXML had a strict schema that used enumerated attributes to describe the auxiliary information, such that support for new annotations required revisions to the schema and software updates. Although each of the proposed formats satisfied the requirements of openness and accessibility, the multiplicity of the formats proved to be confusing and distracting to scientists and computer programmers alike. In order to resolve this situation, the teams that developed mzData and mzXML, along with many other researchers and developers from academia, industry, and vendors joined forces in the Human Proteome Organization (HUPO) Proteomics Standards Initiative working group for mass spectrometry standards (PSI-MS), and set out to create a single MS data standard that would build on the strengths of the previous efforts. The challenge in creating the new unified output format, called mzML, was therefore the resolution of the opposing philosophies of mzXML and mzData, while retaining the best technical attributes of these two formats. In 2006, the unification process was initiated at a PSI workshop based on the guiding design principles determined by members representing instrument and software vendors, data repositories, end users, and the teams that built the mzXML and mzData standards. The designers of mzML focused on four key objectives: (i) creation of a simple format, (ii) elimination of alternate to encode the information, support for all the features of mzXML and mzData, and validation implementation to these would to a single unified format that could support the of mzXML and mzData and that could be easily by vendors and software, with further to be in In order to facilitate adoption and uniform implementation of the new standard format, the of PSI-MS also created open source tool sets that developers as well as end to immediately the format having to their on the format was at PSI as well as to In the mzML standard format was E. a data format for mass spectrometer 2008; PubMed Scopus Google Scholar, E.W. Mass spectrometer output file format Biol. PubMed Scopus Google Scholar). However, the process J.A. Martens L. Hermjakob H. Julian R.K. Paton N.W. The PSI process and its implementation on the PSI 2007; 7: PubMed Scopus Google Scholar), several became as vendors to the new format, most support for and and a file for of which features that had been from the These along with several other were by the PSI-MS working group in with the that had the a mzML was in with the that this new will for quite In to the best technical attributes of the formats, several key were introduced in in order to support new such as the and mzML can multiple operational for an and spectra to a new is the ability to capture data the introduced are also in mzML, such as the ability to encode data to be and the of multiple a liquid R. B. R. K. S. S. Mallick P. mass peptide identification on Proteome Res. 2008; 7: PubMed Scopus Google Scholar). with mzML a controlled vocabulary that and of In mzML with a set of validation These are in a XML to the PSI L. S. F. B. Jones A.R. Martens L. Hermjakob H. The PSI a to of proteomics data.Proteomics. 2009; 9: PubMed Scopus Google and have been implemented in two mzML applications The technical of the mzML standard are available with of its and various files at will the technical of the mzML standard and All of the information from a single MS including the spectra and associated is the mzML its mzML is in XML schema the format and many tools are readily available to an XML to its XML schema The mzML file is as information the controlled in the of the mzML information on the of spectra in the is an that of of controlled vocabulary terms that can be as a throughout a the can information that are in the information the instrument that the and provide a of data processing that the is an that for the mass such as These are by the spectra and and data are by format data for and with This design not has been agreed by the of In order to to the mzML was designed with a for in the as This to a the file having to the file Although is to a of the of years of with mzXML have that these are and are by the of having an To for the of an in the software can easily be to the and the is an To the mzML was designed that the not have an the can still be in a schema that has an an mzML file a or mzML and software is designed to the open and XML it also a that the of the data files by as much as a of for spectra with the However, the files by a of the is of the of XML standard and used tools can be used to this for storage and of mzML for mzML files can by and available on many provide of of the in spectra by the to mzML and therefore to files the A file with in the file with mzML or using and as as the using and file with the applications are to with mzML an in the files are associated with using standard formats are much the associated with to with multiple formats. In an effort to the information in different and to provide support for new with mzML, have designed the format to encode most of the metadata in which provide a to a the PSI MS controlled vocabulary These terms have and including the data and of The controlled vocabulary is and new terms can be of the mzML an a new to describe a new the proposed and can be to the PSI-MS vocabulary the can be and then to the or other can also be used to the for can for be used to the To the use of a was with the data format. validation a simple to the and of the metadata in an mzML such as the of a required the of an source with a or the use of two terms can be at a have the available as a with file or as a tool for the mzML format is designed to support (10.Taylor C.F. Binz P.A. Aebersold R. Affolter M. Barkovich R. Deutsch E.W. Horn D.M. Huhmer A. Kussmann M. Lilley K. Macht M. Mann M. Muller D. Neubert T.A. Nickson J. Patterson S.D. Raso R. Resing K. Seymour S.L. Tsugita A. Xenarios I. Zeng R. Julian Jr., R.K. Guidelines for reporting the use of mass spectrometry in proteomics.Nat Biotechnol. 2008; 26: 860-861Crossref PubMed Scopus (67) Google Scholar, C.F. Paton N.W. Lilley Binz P.A. Julian Jr., Jones A.R. Zhu W. Apweiler R. Aebersold R. Deutsch E.W. M.J. A. Macht M. Mann M. Martens L. Neubert T.A. Patterson S.D. P. Seymour S.L. P. Tsugita A. Vandekerckhove J. J.P. Xenarios I. Yates 3rd, J.R. Hermjakob H. The information a proteomics experiment Biotechnol. 2007; PubMed Scopus (562) Google Scholar) can be out a by of an mzML data file to the of the required information. is that the metadata can be for different of data, that different of spectra can be using the with different of the mzML format has the HUPO PSI community standard development which in is based on the open source software development A group of of the efforts of the many community members that their and at different and a of the process is an that is accessible to This development has been to be as with and Indeed, the development of mzML has the this the development of the standard The mzML standard is quite as it has been developed with in The required of the format from its of the data are in the XML format such as the necessity to an instrument the that this is quite open, and not by the XML an the for an The different of are controlled vocabulary parameters, and a new source is a simple to the will mzML files to the use of this new terms are this new source will be immediately to software as a it will have an to the This is in in mzML, the format to XML schema or software need be to the which is a simple file that is available in a system and that can be updated and Indeed, the public of mzML, have been introduced to the controlled vocabulary downstream on the XML schema or the of the community in are several implementations of the mzML format in software data and for a of for a In the of software that mzML to and is of the strengths of The software D. M. R. D. Mallick P. open source software for rapid proteomics tools 2008; PubMed Scopus Google Scholar, .Google Scholar) has the for and implementation of mzML in its of of a set of tools and in for proteomic data analyses. The provide a that data file and standard and an to in software that to mzML or is available under a which the to be used in software the terms of that The is used by several software to provide mzML The tool can many different formats to mzML, as well as mzXML files M. A. C. A. R. E. N. A. K. open-source software for mass 2008; 9: PubMed Scopus Google Scholar), an open-source for mass spectrometry, also for and mzML which can be easily in other software it validation and validation of mzML This of was used to an tool for validation of mzML files which is of The Proteomics K. C. E. N. M. proteomics 2007; PubMed Scopus Google Scholar). the and the R.G. F. Martens L. an open-source for mzML, the PSI standard for MS data.Proteomics. PubMed Scopus (44) Google Scholar) provide for and these are available to of mzML several software applications are being with mzML These and software such as C. M. Cheung K. an improved for in on of Proteome Res. 2008; 7: PubMed Scopus Google Scholar), D.L. mass peptide identification by Proteome Res. 2007; PubMed Scopus Google Scholar), the A. Eng J. Zhang N. Li X.J. Aebersold R. A uniform proteomics MS/MS analysis platform utilizing open XML file Biol. 2005; 1 (2005.0017)Crossref PubMed Scopus Google Scholar, P.G. a for proteomic Biol. PubMed Scopus Google Scholar, E.W. L. D. H. N. Nilsson E. Pratt B. B. Eng J.K. Nesvizhskii A.I. Aebersold R. A of the PubMed Scopus Google Scholar), and the J. G. K. F. The software an platform for and analysis of proteomics Proteome Res. 2009; PubMed Scopus Google Scholar). vendors have to provide mzML support in the of their The support for mzML in along with the of several open source software packages and in a of that data in the mzML format is readily accessible to end or software the of open data standards formats are and have two use in the of mass spectrometry-based proteomics to of the of the mzML data standard. many multiple from different vendors for their analyses. Although this in the of strengths of the different it also a at the of data The various data formats by each instrument to its data, are to these different from the can output a the development of software that can on data from such as the tools in the quite difficult This in was of the the mzXML format was developed as of the to the various formats in a open data that of data to support various of downstream including identification and of a from mzXML, mzML these data from many to be the available or the common mzML format, which is in and by all downstream data processing software A use of standard data formats the of data to the scientific an that is in the life sciences J.A. M. Hermjakob H. L. a for 2009; 3: PubMed Scopus Google Scholar). data were in formats, would in in L. Nesvizhskii A.I. Hermjakob H. Adamski M. Omenn Vandekerckhove J. Gevaert K. data mass spectrometry data in public proteomics data 2005; 5: PubMed Scopus Google (i) to data would have and the data and to the and software with the format, (ii) of the data, would in and processing the data, and a all data would become as the required software will be or an open, XML therefore format such as mzML, these key issues are of these of on the of software the format, as can be from the previous many actively and open source implementations in a of and for a of are available for mzML and many other implementations are or will be available with their software it be that the two use are in by to mzML as the format for data processing and the to in mzML The data by mzML will most not in a by sample and sample mass spectrometry data is then further processed to identify or quantify the it is to that HUPO PSI has also standards for protein including based and based for identification of molecules from mass spectra and for the of on the integration of data and metadata in the life sciences is being actively undertaken by the for working group of the which has in the format P. M. A. D. J. J. F. N. Jones P. A. M. N. N. Taylor C. W. G. S. The a simple format for complex 2008; PubMed Scopus Google Scholar). information in all the formats on the other is the C.F. D. J. Apweiler R. M. Binz P.A. M. A. A. Deutsch E.W. J. P. F. G. N.W. Hermjakob H. Julian Jr., R.K. M. C. C. E. M. N.L. J. P. N. H. R. A. N. S. J. P. H. H. J. R.H. D. Smith B. J. Jr., K. P. A. J. S. reporting for biological and the Biotechnol. 2008; 26: PubMed Scopus Google Scholar). In years its mzML was and has to be a format that can easily incremental advances in mass spectrometry while a for to of data from new set of software that support mzML will adoption of the format. However, the formats are also the to is and the adoption of mzML in will be initial of implementations a of to since the of have not been is therefore that will for quite The of instrument vendors in PSI-MS further that mzML will become available on instrument software by

Autophagy and apoptosis are two important and interconnected stress-response mechanisms. However, the molecular interplay between these two pathways is not fully understood. To study the fate and function of autophagic proteins at the onset of apoptosis, we used a cellular model system in which autophagy precedes apoptosis. IL-3 depletion of Ba/F3 cells caused caspase (casp)-mediated cleavage of Beclin-1 and PI3KC3, two crucial components of the autophagy-inducing complex. We identified two casp cleavage sites in Beclin-1, TDVD(133) and DQLD(149), cleavage at which yields fragments lacking the autophagy-inducing capacity. Noteworthy, the C-terminal fragment, Beclin-1-C, localized predominantly at the mitochondria and sensitized the cells to apoptosis. Moreover, on isolated mitochondria, recombinant Beclin-1-C was able to induce the release of proapoptotic factors. These findings point to a mechanism by which casp-dependent generation of Beclin-1-C creates an amplifying loop enhancing apoptosis upon growth factor withdrawal.

Plants exhibit a unique developmental flexibility to ever-changing environmental conditions. To achieve their profound adaptability, plants are able to maintain permanent stem cell populations and form new organs during the entire plant life cycle. Signaling substances, called plant hormones, such as auxin, cytokinin, abscisic acid, brassinosteroid, ethylene, gibberellin, jasmonic acid, and strigolactone, govern and coordinate these developmental processes. Physiological and genetic studies have dissected the molecular components of signal perception and transduction of the individual hormonal pathways. However, over recent years it has become evident that hormones do not act only in a linear pathway. Hormonal pathways are interconnected by a complex network of interactions and feedback circuits that determines the final outcome of the individual hormone actions. This raises questions about the molecular mechanisms underlying hormonal cross talk and about how these hormonal networks are established, maintained, and modulated throughout plant development.

The HEK293 human cell lineage is widely used in cell biology and biotechnology. Here we use whole-genome resequencing of six 293 cell lines to study the dynamics of this aneuploid genome in response to the manipulations used to generate common 293 cell derivatives, such as transformation and stable clone generation (293T); suspension growth adaptation (293S); and cytotoxic lectin selection (293SG). Remarkably, we observe that copy number alteration detection could identify the genomic region that enabled cell survival under selective conditions (i.c. ricin selection). Furthermore, we present methods to detect human/vector genome breakpoints and a user-friendly visualization tool for the 293 genome data. We also establish that the genome structure composition is in steady state for most of these cell lines when standard cell culturing conditions are used. This resource enables novel and more informed studies with 293 cells, and we will distribute the sequenced cell lines to this effect. The human embryonic kidney 293 (HEK293) cell lineage is widely used in cell biology and biotechnology. Here, the authors apply whole genome resequencing methods to characterise genomic variation in six HEK293 cell lines and suggest that this variation could affect experiments using these cell lines.

While long non-coding RNA (lncRNA) research in the past has primarily focused on the discovery of novel genes, today it has shifted towards functional annotation of this large class of genes. With thousands of lncRNA studies published every year, the current challenge lies in keeping track of which lncRNAs are functionally described. This is further complicated by the fact that lncRNA nomenclature is not straightforward and lncRNA annotation is scattered across different resources with their own quality metrics and definition of a lncRNA. To overcome this issue, large scale curation and annotation is needed. Here, we present the fifth release of the human lncRNA database LNCipedia (https://lncipedia.org). The most notable improvements include manual literature curation of 2482 lncRNA articles and the use of official gene symbols when available. In addition, an improved filtering pipeline results in a higher quality reference lncRNA gene set.

Ongoing debates surrounding Open Access to the scholarly literature are multifaceted and complicated by disparate and often polarised viewpoints from engaged stakeholders. At the current stage, Open Access has become such a global issue that it is critical for all involved in scholarly publishing, including policymakers, publishers, research funders, governments, learned societies, librarians, and academic communities, to be well-informed on the history, benefits, and pitfalls of Open Access. In spite of this, there is a general lack of consensus regarding the potential pros and cons of Open Access at multiple levels. This review aims to be a resource for current knowledge on the impacts of Open Access by synthesizing important research in three major areas: academic, economic and societal. While there is clearly much scope for additional research, several key trends are identified, including a broad citation advantage for researchers who publish openly, as well as additional benefits to the non-academic dissemination of their work. The economic impact of Open Access is less well-understood, although it is clear that access to the research literature is key for innovative enterprises, and a range of governmental and non-governmental services. Furthermore, Open Access has the potential to save both publishers and research funders considerable amounts of financial resources, and can provide some economic benefits to traditionally subscription-based journals. The societal impact of Open Access is strong, in particular for advancing citizen science initiatives, and leveling the playing field for researchers in developing countries. Open Access supersedes all potential alternative modes of access to the scholarly literature through enabling unrestricted re-use, and long-term stability independent of financial constraints of traditional publishers that impede knowledge sharing. However, Open Access has the potential to become unsustainable for research communities if high-cost options are allowed to continue to prevail in a widely unregulated scholarly publishing market. Open Access remains only one of the multiple challenges that the scholarly publishing system is currently facing. Yet, it provides one foundation for increasing engagement with researchers regarding ethical standards of publishing and the broader implications of 'Open Research'.

High-throughput sequencing of full-length transcripts using long reads has paved the way for the discovery of thousands of novel transcripts, even in well-annotated mammalian species. The advances in sequencing technology have created a need for studies and tools that can characterize these novel variants. Here, we present SQANTI, an automated pipeline for the classification of long-read transcripts that can assess the quality of data and the preprocessing pipeline using 47 unique descriptors. We apply SQANTI to a neuronal mouse transcriptome using Pacific Biosciences (PacBio) long reads and illustrate how the tool is effective in characterizing and describing the composition of the full-length transcriptome. We perform extensive evaluation of ToFU PacBio transcripts by PCR to reveal that an important number of the novel transcripts are technical artifacts of the sequencing approach and that SQANTI quality descriptors can be used to engineer a filtering strategy to remove them. Most novel transcripts in this curated transcriptome are novel combinations of existing splice sites, resulting more frequently in novel ORFs than novel UTRs, and are enriched in both general metabolic and neural-specific functions. We show that these new transcripts have a major impact in the correct quantification of transcript levels by state-of-the-art short-read-based quantification algorithms. By comparing our iso-transcriptome with public proteomics databases, we find that alternative isoforms are elusive to proteogenomics detection. SQANTI allows the user to maximize the analytical outcome of long-read technologies by providing the tools to deliver quality-evaluated and curated full-length transcriptomes.

Many computational methods have been developed to infer cell type proportions from bulk transcriptomics data. However, an evaluation of the impact of data transformation, pre-processing, marker selection, cell type composition and choice of methodology on the deconvolution results is still lacking. Using five single-cell RNA-sequencing (scRNA-seq) datasets, we generate pseudo-bulk mixtures to evaluate the combined impact of these factors. Both bulk deconvolution methodologies and those that use scRNA-seq data as reference perform best when applied to data in linear scale and the choice of normalization has a dramatic impact on some, but not all methods. Overall, methods that use scRNA-seq data have comparable performance to the best performing bulk methods whereas semi-supervised approaches show higher error values. Moreover, failure to include cell types in the reference that are present in a mixture leads to substantially worse results, regardless of the previous choices. Altogether, we evaluate the combined impact of factors affecting the deconvolution task across different datasets and propose general guidelines to maximize its performance.

Hydrogen is reviewed as a possible new marine fuel, with emphasis on the challenges concerning sustainable production, on board use and safety and specifically the challenges concerning hydrogen storage.

In the PINK1 Pathogenic mutations in the kinase PINK1 are causally related to Parkinson's disease (PD). One hypothesis proposes that PINK1 regulates mitophagy—the clearance of dysfunctional mitochondria. A second hypothesis suggests that PINK1 has a direct effect on mitochondrial complex I, affecting the maintenance of the electron transport chain (ETC) resulting in decreased mitochondrial membrane potential and dysfunctional mitochondria. In support of the second hypothesis, Morais et al. (p. 203 , published online 20 March) observed a complex I deficit in fibroblasts and neurons derived from induced pluripotent stem cells from PINK1 patients before any mitophagy was induced. The phosphoproteome of complex I in liver and brain from mice deficient for Pink1, compared to wild-type animals, revealed that Ser 250 in complex I subunit NdufA10 was differentially phosphorylated. Ser 250 is critically involved in the reduction of ubiquinone by complex I, explaining why Pink1 knockout mice, flies, and patient cell lines show decreased mitochondrial membrane potential. Synaptic defects in pink1 null mutant Drosophila could be rescued using phosphomimetic NdufA10.

Immunogenic cell death significantly contributes to the success of anti-cancer therapies, but immunogenicity of different cell death modalities widely varies. Ferroptosis, a form of cell death that is characterized by iron accumulation and lipid peroxidation, has not yet been fully evaluated from this perspective. Here we present an inducible model of ferroptosis, distinguishing three phases in the process-'initial' associated with lipid peroxidation, 'intermediate' correlated with ATP release and 'terminal' recognized by HMGB1 release and loss of plasma membrane integrity-that serves as tool to study immune cell responses to ferroptotic cancer cells. Co-culturing ferroptotic cancer cells with dendritic cells (DC), reveals that 'initial' ferroptotic cells decrease maturation of DC, are poorly engulfed, and dampen antigen cross-presentation. DC loaded with ferroptotic, in contrast to necroptotic, cancer cells fail to protect against tumor growth. Adding ferroptotic cancer cells to immunogenic apoptotic cells dramatically reduces their prophylactic vaccination potential. Our study thus shows that ferroptosis negatively impacts antigen presenting cells and hence the adaptive immune response, which might hinder therapeutic applications of ferroptosis induction.

Emergence of SARS-CoV-2 causing COVID-19 has resulted in hundreds of thousands of deaths. In search for key targets of effective therapeutics, robust animal models mimicking COVID-19 in humans are urgently needed. Here, we show that Syrian hamsters, in contrast to mice, are highly permissive to SARS-CoV-2 and develop bronchopneumonia and strong inflammatory responses in the lungs with neutrophil infiltration and edema, further confirmed as consolidations visualized by micro-CT alike in clinical practice. Moreover, we identify an exuberant innate immune response as key player in pathogenesis, in which STAT2 signaling plays a dual role, driving severe lung injury on the one hand, yet restricting systemic virus dissemination on the other. Our results reveal the importance of STAT2-dependent interferon responses in the pathogenesis and virus control during SARS-CoV-2 infection and may help rationalizing new strategies for the treatment of COVID-19 patients.

Rapid response to tissue damage Damaged plants are susceptible to microbial attack. In response to physical damage, plants proactively generate signal peptides to activate their immune systems. Hander et al. examined wound responses in the model plant Arabidopsis thaliana . They identified a metacaspase that releases an immunomodulatory signal peptide from its precursor form within 30 seconds of the damage. The metacaspase itself was activated by a burst of calcium released by tissue damage. Science , this issue p. eaar7486

Identification and validation of extracellular vesicle (EV)-associated biomarkers requires robust isolation and characterization protocols. We assessed the impact of some commonly implemented pre-analytical, analytical and post-analytical variables in EV research. Centrifugal filters with different membrane types and pore sizes are used to reduce large volume biofluids prior to EV isolation or to concentrate EVs. We compared five commonly reported filters for their efficiency when using plasma, urine and EV-spiked PBS. Regenerated cellulose membranes with pore size of 10 kDa recovered EVs the most efficient. Less than 40% recovery was achieved with other filters. Next, we analyzed the effect of the type of protein assays to measure EV protein in colorimetric and fluorometric kits. The fluorometric assay Qubit measured low concentration EV and BSA samples the most accurately with the lowest variation among technical and biological replicates. Lastly, we quantified Optiprep remnants in EV samples from density gradient ultracentrifugation and demonstrate that size-exclusion chromatography efficiently removes Optiprep from EVs. In conclusion, choice of centrifugal filters and protein assays confound EV analysis and should be carefully considered to increase efficiency towards biomarker discovery. SEC-based removal of Optiprep remnants from EVs can be considered for downstream applications.

The field of computational proteomics is approaching the big data age, driven both by a continuous growth in the number of samples analyzed per experiment as well as by the growing amount of data obtained in each analytical run. In order to process these large amounts of data, it is increasingly necessary to use elastic compute resources such as Linux-based cluster environments and cloud infrastructures. Unfortunately, the vast majority of cross-platform proteomics tools are not able to operate directly on the proprietary formats generated by the diverse mass spectrometers. Here, we present ThermoRawFileParser, an open-source, cross-platform tool that converts Thermo RAW files into open file formats such as MGF and the HUPO-PSI standard file format mzML. To ensure the broadest possible availability and to increase integration capabilities with popular workflow systems such as Galaxy or Nextflow, we have also built Conda package and BioContainers container around ThermoRawFileParser. In addition, we implemented a user-friendly interface (ThermoRawFileParserGUI) for those users not familiar with command-line tools. Finally, we performed a benchmark of ThermoRawFileParser and msconvert to verify that the converted mzML files contain reliable quantitative results.

Peer review of research articles is a core part of our scholarly communication system. In spite of its importance, the status and purpose of peer review is often contested. What is its role in our modern digital research and communications infrastructure? Does it perform to the high standards with which it is generally regarded? Studies of peer review have shown that it is prone to bias and abuse in numerous dimensions, frequently unreliable, and can fail to detect even fraudulent research. With the advent of web technologies, we are now witnessing a phase of innovation and experimentation in our approaches to peer review. These developments prompted us to examine emerging models of peer review from a range of disciplines and venues, and to ask how they might address some of the issues with our current systems of peer review. We examine the functionality of a range of social Web platforms, and compare these with the traits underlying a viable peer review system: quality control, quantified performance metrics as engagement incentives, and certification and reputation. Ideally, any new systems will demonstrate that they out-perform and reduce the biases of existing models as much as possible. We conclude that there is considerable scope for new peer review initiatives to be developed, each with their own potential issues and advantages. We also propose a novel hybrid platform model that could, at least partially, resolve many of the socio-technical issues associated with peer review, and potentially disrupt the entire scholarly communication system. Success for any such development relies on reaching a critical threshold of research community engagement with both the process and the platform, and therefore cannot be achieved without a significant change of incentives in research environments.