Max Planck Institute of Molecular Cell Biology and Genetics

The many functional partnerships and interactions that occur between proteins are at the core of cellular processing and their systematic characterization helps to provide context in molecular systems biology. However, known and predicted interactions are scattered over multiple resources, and the available data exhibit notable differences in terms of quality and completeness. The STRING database (http://string-db.org) aims to provide a critical assessment and integration of protein-protein interactions, including direct (physical) as well as indirect (functional) associations. The new version 10.0 of STRING covers more than 2000 organisms, which has necessitated novel, scalable algorithms for transferring interaction information between organisms. For this purpose, we have introduced hierarchical and self-consistent orthology annotations for all interacting proteins, grouping the proteins into families at various levels of phylogenetic resolution. Further improvements in version 10.0 include a completely redesigned prediction pipeline for inferring protein-protein associations from co-expression data, an API interface for the R computing environment and improved statistical analysis for enrichment tests in user-provided networks.

Cell membranes display a tremendous complexity of lipids and proteins designed to perform the functions cells require. To coordinate these functions, the membrane is able to laterally segregate its constituents. This capability is based on dynamic liquid-liquid immiscibility and underlies the raft concept of membrane subcompartmentalization. Lipid rafts are fluctuating nanoscale assemblies of sphingolipid, cholesterol, and proteins that can be stabilized to coalesce, forming platforms that function in membrane signaling and trafficking. Here we review the evidence for how this principle combines the potential for sphingolipid-cholesterol self-assembly with protein specificity to selectively focus membrane bioactivity.

In sexually reproducing organisms, embryos specify germ cells, which ultimately generate sperm and eggs. In Caenorhabditis elegans, the first germ cell is established when RNA and protein-rich P granules localize to the posterior of the one-cell embryo. Localization of P granules and their physical nature remain poorly understood. Here we show that P granules exhibit liquid-like behaviors, including fusion, dripping, and wetting, which we used to estimate their viscosity and surface tension. As with other liquids, P granules rapidly dissolved and condensed. Localization occurred by a biased increase in P granule condensation at the posterior. This process reflects a classic phase transition, in which polarity proteins vary the condensation point across the cell. Such phase transitions may represent a fundamental physicochemical mechanism for structuring the cytoplasm.

Since its initial release in 2000, the human reference genome has covered only the euchromatic fraction of the genome, leaving important heterochromatic regions unfinished. Addressing the remaining 8% of the genome, the Telomere-to-Telomere (T2T) Consortium presents a complete 3.055 billion-base pair sequence of a human genome, T2T-CHM13, that includes gapless assemblies for all chromosomes except Y, corrects errors in the prior references, and introduces nearly 200 million base pairs of sequence containing 1956 gene predictions, 99 of which are predicted to be protein coding. The completed regions include all centromeric satellite arrays, recent segmental duplications, and the short arms of all five acrocentric chromosomes, unlocking these complex regions of the genome to variational and functional studies.

Cells organize many of their biochemical reactions in non-membrane compartments. Recent evidence has shown that many of these compartments are liquids that form by phase separation from the cytoplasm. Here we discuss the basic physical concepts necessary to understand the consequences of liquid-like states for biological functions.

Abstract High-quality and complete reference genome assemblies are fundamental for the application of genomics to biology, disease, and biodiversity conservation. However, such assemblies are available for only a few non-microbial species 1–4 . To address this issue, the international Genome 10K (G10K) consortium 5,6 has worked over a five-year period to evaluate and develop cost-effective methods for assembling highly accurate and nearly complete reference genomes. Here we present lessons learned from generating assemblies for 16 species that represent six major vertebrate lineages. We confirm that long-read sequencing technologies are essential for maximizing genome quality, and that unresolved complex repeats and haplotype heterozygosity are major sources of assembly error when not handled correctly. Our assemblies correct substantial errors, add missing sequence in some of the best historical reference genomes, and reveal biological discoveries. These include the identification of many false gene duplications, increases in gene sizes, chromosome rearrangements that are specific to lineages, a repeated independent chromosome breakpoint in bat genomes, and a canonical GC-rich pattern in protein-coding genes and their regulatory regions. Adopting these lessons, we have embarked on the Vertebrate Genomes Project (VGP), an international effort to generate high-quality, complete reference genomes for all of the roughly 70,000 extant vertebrate species and to help to enable a new era of discovery across the life sciences.

Accurate profiling of lipidomes relies upon the quantitative and unbiased recovery of lipid species from analyzed cells, fluids, or tissues and is usually achieved by two-phase extraction with chloroform. We demonstrated that methyl-tert-butyl ether (MTBE) extraction allows faster and cleaner lipid recovery and is well suited for automated shotgun profiling. Because of MTBE's low density, lipid-containing organic phase forms the upper layer during phase separation, which simplifies its collection and minimizes dripping losses. Nonextractable matrix forms a dense pellet at the bottom of the extraction tube and is easily removed by centrifugation. Rigorous testing demonstrated that the MTBE protocol delivers similar or better recoveries of species of most all major lipid classes compared with the “gold-standard” Folch or Bligh and Dyer recipes. Accurate profiling of lipidomes relies upon the quantitative and unbiased recovery of lipid species from analyzed cells, fluids, or tissues and is usually achieved by two-phase extraction with chloroform. We demonstrated that methyl-tert-butyl ether (MTBE) extraction allows faster and cleaner lipid recovery and is well suited for automated shotgun profiling. Because of MTBE's low density, lipid-containing organic phase forms the upper layer during phase separation, which simplifies its collection and minimizes dripping losses. Nonextractable matrix forms a dense pellet at the bottom of the extraction tube and is easily removed by centrifugation. Rigorous testing demonstrated that the MTBE protocol delivers similar or better recoveries of species of most all major lipid classes compared with the “gold-standard” Folch or Bligh and Dyer recipes. Recent developments in mass spectrometric technology enabled the comprehensive characterization of eukaryotic lipidomes, fostering the molecular biology of lipids and metabolism-related disorders (reviewed in Refs. 1.Han X. Gross R.W. Shotgun lipidomics: multidimensional MS analysis of cellular lipidomes.Expert Rev. Proteomics. 2005; 2: 253-264Crossref PubMed Scopus (215) Google Scholar, 2.Wenk M.R. The emerging field of lipidomics.Nat. Rev. Drug Discov. 2005; 4: 594-610Crossref PubMed Scopus (1001) Google Scholar, 3.Piomelli D. Astarita G. Rapaka R. A neuroscientist's guide to lipidomics.Nat. Rev. Neurosci. 2007; 8: 743-754Crossref PubMed Scopus (274) Google Scholar, 4.van Meer G. Cellular lipidomics.EMBO J. 2005; 24: 3159-3165Crossref PubMed Scopus (411) Google Scholar). Typically, lipidome profiling by mass spectrometry proceeds along LC-MS or shotgun approaches. The former identifies and quantifies lipid species preseparated by normal or reversed-phase chromatography coupled online to a mass spectrometer, which is capable of fast acquisition of MS or MS/MS spectra (5.Yetukuri L. Katajamaa M. Medina-Gomez G. Seppanen-Laakso T. Vidal-Puig A. Oresic M. Bioinformatics strategies for lipidomics analysis: characterization of obesity related hepatic steatosis.BMC Syst Biol. 2007; 1: 12-26Crossref PubMed Scopus (193) Google Scholar, 6.Sommer U. Herscovitz H. Welty F.K. Costello C.E. LC-MS-based method for the qualitative and quantitative analysis of complex lipid mixtures.J. Lipid Res. 2006; 47: 804-814Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 7.Hermansson M. Uphoff A. Kakela R. Somerharju P. Automated quantitative analysis of complex lipidomes by liquid chromatography/mass spectrometry.Anal. Chem. 2005; 77: 2166-2175Crossref PubMed Scopus (169) Google Scholar, 8.Haimi P. Uphoff A. Hermansson M. Somerharju P. Software tools for analysis of mass spectrometric lipidome data.Anal. Chem. 2006; 78: 8324-8331Crossref PubMed Scopus (167) Google Scholar). In contrast, in shotgun lipidomics, total lipid extracts are infused directly into a mass spectrometer, and the molecular characterization of lipid species relies either on the accurately determined m/z of precursor ions (9.Schwudke D. Hannich J.T. Surendranath V. Grimard V. Moehring T. Burton L. Kurzchalia T. Shevchenko A. Top-down lipidomic screens by multivariate analysis of high-resolution survey mass spectra.Anal. Chem. 2007; 79: 4083-4093Crossref PubMed Scopus (151) Google Scholar) or on the detection of specific fragment ions or neutral losses in tandem mass spectrometric experiments (1.Han X. Gross R.W. Shotgun lipidomics: multidimensional MS analysis of cellular lipidomes.Expert Rev. Proteomics. 2005; 2: 253-264Crossref PubMed Scopus (215) Google Scholar, 9.Schwudke D. Hannich J.T. Surendranath V. Grimard V. Moehring T. Burton L. Kurzchalia T. Shevchenko A. Top-down lipidomic screens by multivariate analysis of high-resolution survey mass spectra.Anal. Chem. 2007; 79: 4083-4093Crossref PubMed Scopus (151) Google Scholar, 10.Brugger B. Erben G. Sandhoff R. Wieland F.T. Lehmann W.D. Quantitative analysis of biological membrane lipids at the low picomole level by nanoelectrospray ionization tandem mass spectrometry.Proc. Natl. Acad. Sci. USA. 1997; 94: 2339-2344Crossref PubMed Scopus (736) Google Scholar, 11.Han X. Yang K. Yang J. Fikes K.N. Cheng H. Gross R.W. Factors influencing the electrospray intrasource separation and selective ionization of glycerophospholipids.J. Am. Soc. Mass Spectrom. 2006; 17: 264-274Crossref PubMed Scopus (93) Google Scholar, 12.Ejsing C.S. Duchoslav E. Sampaio J. Simons K. Bonner R. Thiele C. Ekroos K. Shevchenko A. Automated identification and quantification of glycerophospholipid molecular species by multiple precursor ion scanning.Anal. Chem. 2006; 78: 6202-6214Crossref PubMed Scopus (331) Google Scholar). Regardless of the analytical approach used, its success depends on the completeness of the extraction of lipids from corresponding cells, fluids, or tissues. Lipids of all major classes could be recovered via chloroform/methanol extraction, typically according to the Folch, Lees, and Sloane Stanley (13.Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipides from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar) or Bligh and Dyer (14.Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43133) Google Scholar) recipes (15.Watson A.D. Thematic review series: systems biology approaches to metabolic and cardiovascular disorders. Lipidomics: a global approach to lipid analysis in biological systems.J. Lipid Res. 2006; 47: 2101-2111Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar), in which they are mostly enriched in the chloroform phase. Electrospray mass spectrometry, a major tool for analyzing complex lipidomes, is particularly sensitive towards the quality of lipid extracts. Coextracted components of biological matrix and salts (often, without further definition, termed background) affect both the sensitivity and specificity of lipid analysis. Often, abundant background ions obscure lipid precursors, and their MS/MS spectra are densely populated with “ghost” peaks and abundant chemical noise. Adducts with common background cations (sodium, potassium) and anions (chloride) increase the ambiguity of molecular species assignment and affect the accuracy of quantitative determination. Because of the higher density of chloroform compared with a water/methanol mixture, it forms the lower phase of the two-phase partitioning system. While collecting the chloroform fraction, a glass pipette or a needle of the pipetting robot reaches it through a voluminous layer of nonextractable insoluble matrix, usually residing at the interface of the water/methanol and chloroform phases. However, even a minute amount of insoluble precipitate accidentally grabbed together with the chloroform fraction clogs the electrospray ion source or LC system, because of the micrometer size of the spraying orifice and/or connecting tubing. We note that, because of high density and the viscosity of chloroform, centrifugation is usually of little help. Although mass spectrometry enables lipid profiling at the low femtomole level, much higher amounts are usually required to circumvent the difficulties in handling microvolumes of total extracts and ensure the sufficient stability of the analytical pipeline. Additionally, the known carcinogenicity of chloroform involves considerable health risk for laboratory personnel (16.Nagano K. Kano H. Arito H. Yamamoto S. Matsushima T. Enhancement of renal carcinogenicity by combined inhalation and oral exposures to chloroform in male rats.J. Toxicol. Environ. Health A. 2006; 69: 1827-1842Crossref PubMed Scopus (22) Google Scholar). Also, chloroform decomposition yields phosgene and hydrochloric acid, inflicting chemical modification of labile lipid species (17.Schmid P. Hunter E. Calvert J. Extraction and purification of lipids. III. Serious limitations of chloroform and chloroform-methanol in lipid investigations.Physiol. Chem. Phys. Med. NMR. 1973; 5: 151-155Google Scholar). Here, we report an extraction protocol specifically developed for shotgun profiling of complex lipidomes from samples with excessive amounts of biological matrices. Lipid extraction by methyl-tert-butyl ether (MTBE)/methanol (18.Thomann W.R. Hill G.B. Modified extraction procedure for gas-liquid chromatography applied to the identification of anaerobic bacteria.J. Clin. Microbiol. 1986; 23: 392-394Crossref PubMed Google Scholar, 19.Kuyukina M.S. Ivshina I.B. Philp J.C. Christofi N. Dunbar S.A. Ritchkova M.I. Recovery of Rhodococcus biosurfactants using methyl tertiary-butyl ether extraction.J. Microbiol. Methods. 2001; 46: 149-156Crossref PubMed Scopus (135) Google Scholar) greatly simplifies sample handling and enables automated processing of minute amounts of biological samples. Rigorous testing established that the recovery of lipid species of almost all major classes is the same or better than was typically achieved by the Folch recipe (13.Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipides from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar), which is generally regarded the “gold standard” in lipid biochemistry. Synthetic lipid standards were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL); MTBE and 2-propanol were from Sigma-Aldrich Chemie GmbH (Munich, Germany). Chloroform, methanol, and ammonium acetate were LC grade; water with 0.1% ammonium acetate was LC-MS grade and purchased from Fluka (Buchs, Switzerland). LC-MS-grade water was purchased from Fisher Scientific (Loughborough, UK). Escherichia coli (NA-22 strain) were grown on Luria-Bertani medium, collected by centrifugation, washed three times by M9 solution (22 mM KH2PO4, 22 mM Na2HPO4, 85 mM NaCl, and 1 mM MgSO4) followed by rinsing with 0.1% ammonium acetate, and frozen. A sample of mouse brain tissue was dissected from adult mouse of NMRI strain. Brain hemispheres were separated and minced into small pieces in ice-cold 0.1% ammonium acetate followed by homogenization in a Potter homogenizer. The daf-22 strain of Caenorhabditis elegans was grown on NGM agar plates with E. coli (NA-22 strain) as a food source (20.Brenner S. The genetics of Caenorhabditis elegans.Genetics. 1974; 77: 71-94Crossref PubMed Google Scholar). To collect eggs, worms were bleached with basic hypochlorite solution as described (21.Sulston J. Hodgkin J. Methods.in: Wood W.B. The Nematode Caenorhabditis elegans. Cold Spring Harbor Laboratory Press, New York1988: 587-606Google Scholar). To remove worm debris, egg suspension was filtered through 80 μm nylon mesh, rinsed with LC-MS-grade water, and frozen in liquid nitrogen in Mass spectrometric analysis was on a mass with a ion source was to to and the source was by lipid samples were in mM ammonium acetate in and infused at the of A sample of of electrospray acquisition experiments were as described D. J. Burton L. E. Hannich J.T. C.S. Kurzchalia T. Shevchenko A. Lipid profiling by multiple precursor and neutral by the Chem. 2006; 78: PubMed Scopus Google Scholar). The analytical was the and were the m/z MS/MS was for the of Lipid species were and using D. J. Burton L. E. Hannich J.T. C.S. Kurzchalia T. Shevchenko A. Lipid profiling by multiple precursor and neutral by the Chem. 2006; 78: PubMed Scopus Google Scholar). lipid extracts were analyzed by on a mass with an electrospray ion source as described G. B. J. G. quantification of and by electrospray ionization tandem mass spectrometry coupled with PubMed Scopus Google Scholar, G. M. R. T. B. G. quantification of and by electrospray ionization tandem mass spectrometry 2006; PubMed Scopus Google Scholar). of total lipid extracts was for lipid analysis using a and an Germany). A of mM ammonium acetate was through the at the of for followed by for and to for were for of and upon lipid and were analyzed by D. G. R. G. Shevchenko A. Shotgun lipidomics by tandem mass spectrometry acquisition 2007; PubMed Scopus Google Scholar). and was as described G. B. J. G. quantification of and by electrospray ionization tandem mass spectrometry coupled with PubMed Scopus Google Scholar, G. M. B. R. B. A. G. Quantitative of species from cellular extracts by electrospray ionization tandem mass spectrometry Lipid Res. Full Text Full Text PDF PubMed Google mass spectrometric or for species in the acquisition lipidomics approach (22) by of fragment ions in MS/MS for species in the acquisition lipidomics approach D. J. Burton L. E. Hannich J.T. C.S. Kurzchalia T. Shevchenko A. Lipid profiling by multiple precursor and neutral by the Chem. 2006; 78: PubMed Scopus Google Scholar) by of fragment ions Electrospray ionization tandem mass spectrometry of Am. Soc. Mass Spectrom. PubMed Scopus Google Scholar) in MS/MS in a analysis of lipid extracts was on plates were developed with an system, followed by Lipid were by spraying the plates with in and at was to a sample which was into a glass tube with a and the tube was of MTBE was and the was for 1 at in a separation was by of of at the sample was at for The upper phase was and the lower phase was with of the mixture, was to the of the upper phase by and collecting the upper organic were in a To sample of was to the organic phase of centrifugation. lipids were in of for was to of the sample and of was the was for 1 at in a and phase separation was by of The was for at and at for The lower phase was and the upper phase was washed with of the mixture, was to the of the lower phase organic were in a and in of for of of lipid in were into and in a A total of of water was to and lipid extraction was according to the Folch or MTBE protocol as described organic were and in 1 of of lipid of the same that as an and were samples were by pipetting the same of lipid into and in the same amount of the extraction was in and was analyzed three MS spectra were for with an of 1 The lipid recovery was as the of the of peaks of the and the of the same lipid In a of the lipid was as described of water of E. coli suspension was to the The recovery was by multiple in ion as the of the of the fragment with m/z from the and the from grown on were collected by centrifugation, washed with 0.1% ammonium acetate in water, and in 0.1% ammonium in were into glass three were according to Folch and the three were with MTBE as described Lipid extracts were with and analyzed Lipid were by the of by which specific neutral precursor ion and D. G. R. G. Shevchenko A. Shotgun lipidomics by tandem mass spectrometry acquisition 2007; PubMed Scopus Google Scholar). E. coli of and lipid classes were by neutral of with m/z and m/z The was determined in ion by precursor ion for the fragment with m/z of lipid species were to the of of all lipid species of the lipid Brain tissue from adult mouse NMRI strain was rinsed in 0.1% ammonium acetate in water, into small and in 0.1% ammonium acetate in water in a Potter on of of were in and according to the Folch and MTBE The lipid extracts were with and analyzed for and were as described of the suspension in MS water of C. elegans of or were to three of and three were according to the Folch protocol and three were according to the MTBE To lipid was with and analyzed in and lipid were determined as described Lipid extracts were from of into glass lipid extraction, standards solution and in chloroform were into the same and Additionally, samples were by the of known of lipid Bligh and Dyer extraction, of was to the of with of the was and for 1 at (14.Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43133) Google Scholar). separation was achieved by 1 of and 1 of MTBE extraction, 80 of water was to the samples and as described extraction of the lower phase was of organic phase collected according to both was further by a pipetting robot with To the needle was washed with both lipid extraction the organic phase was recovered at a from the lower chloroform and upper MTBE were to by the phase. of the was with for the chloroform phase to sample were to glass In of the organic phase was recovered from of which was for analysis and the was for analysis. The was removed by centrifugation, and lipids were in and of mM ammonium acetate for and To the recoveries of and three samples with medium, and high lipid by a determined total as medium, were in of lipid species were determined by of a sample times as described that MTBE could chloroform in systems for lipid The established extraction in similar to the Folch or Bligh and Dyer In the samples were a extraction and biological Lipids were recovered into the MTBE because of its lower density, was the upper phase of the two-phase system. In to the Folch nonextractable matrix in the phase at the bottom of the extraction the organic phase enriched with lipids was easily by the from the was for lipids from C. which voluminous nonextractable that the chloroform fraction collected with the Folch an was required to it analysis. The MTBE extraction procedure was in three we determined the recoveries of lipid standards of classes and compared with the recoveries achieved by the Folch we established that the recovery of both was almost we the Folch recipe as a to lipid yields were by biological and they on the of lipid MTBE extraction was automated and applied for of we determined lipid recoveries were using the Folch and MTBE recipes. To of lipid standards of lipid classes was and their recoveries were determined by mass spectrometry using standards of the same and similar mass and were known on the lipid the recovery achieved by both was The was the which was by The same was in the into E. coli total lipid was recovered by Folch and by we that, in further we could MTBE extraction to the Folch upon the of peaks of of lipid standards by MTBE and Folch extraction are of of lipid standards Folch or MTBE extraction were determined by with the of peaks of the corresponding in a are of of lipid standards Folch or MTBE extraction were determined by with the of peaks of the corresponding The MTBE and Folch were applied to lipids from as E. mouse and C. an of and animal the same samples were in by both recovered lipids were analyzed by and lipid species were by shotgun profiling on a mass D. J. Burton L. E. Hannich J.T. C.S. Kurzchalia T. Shevchenko A. Lipid profiling by multiple precursor and neutral by the Chem. 2006; 78: PubMed Scopus Google Scholar). The E. coli lipidome mostly of and in lipid of Escherichia coli from with organic and with food Environ. Microbiol. PubMed Google Scholar). to species and survey mass spectra were that the extraction yields were similar for both and were of lipid and the of the was by analysis the m/z of background peaks were lipid profiling in ion the same total and of and species In we species of and species of which with D. C. N. S. V. Lipid of of Escherichia coli by liquid mass spectrometry using electrospray Mass Spectrom. 2007; PubMed Scopus Google Scholar). the Folch and MTBE were applied to lipids from adult mouse brain MS analysis of the extracts almost spectra and was further by analysis The profiling lipid species from lipid classes A total of of species lipids from the most abundant and classes the were to species from and the of major and species recovered by both was almost that corresponding organic and were with lipids. MTBE and Folch extraction of C. elegans of lipid in lipid species from major lipid classes of in ion species with of were in both extracts The MTBE protocol was further for automated extraction of and in lipidomics screens G. B. J. G. quantification of and by electrospray ionization tandem mass spectrometry coupled with PubMed Scopus Google Scholar, G. M. R. T. B. G. quantification of and by electrospray ionization tandem mass spectrometry 2006; PubMed Scopus Google Scholar). In the same samples were by the Bligh and Dyer method using the same The extraction recovery was by processing three samples with medium, and high lipid Bligh and Dyer and MTBE recovered the same amount of lipids of major classes with similar of which was MTBE extraction recovered from samples compared with the Bligh and Dyer of lipids extraction of samples according to the MTBE or Bligh and Dyer of lipid recovered automated MTBE or Bligh and Dyer The are of experiments in to the total of the samples medium, determined by Lipid to the total of the samples medium, determined by of lipid recovered automated MTBE or Bligh and Dyer The are of experiments in in a To the accuracy of automated lipid of the same sample were according to either the MTBE or the Bligh and Dyer Mass spectrometric analysis of the extracts lipid species of major lipid classes and which were the of quantification of with of the total of the corresponding lipid are in in the lipid of the most abundant lipid and or for the and were we that the MTBE recipe was well suited for automated lipid extraction of biological and the same or better recoveries as the established Bligh and Dyer Recent developments in mass spectrometry enabled comprehensive quantitative profiling of eukaryotic Although lipid extraction from cells, fluids, or tissues is a in the automated lipidomics it little and the is typically to the Folch or Bligh and Dyer recipes. The MTBE extraction procedure faster and cleaner recovery of most of the major lipid classes and was well suited for shotgun in which total extracts were infused directly into a mass with The of MTBE extraction two-phase systems from the low density of the lipid-containing organic phase that forms the upper layer during phase greatly its collection and dripping losses. compared with chloroform, MTBE is and water review and approach to Health PubMed Scopus Google Scholar, H. U. of in the by the for the 2001; PubMed Scopus Google Scholar), which the as well as the health for is and forms during and of labile lipids H. M. of MTBE and Press, New Google Scholar). Rigorous testing that in biological species from major lipid classes demonstrated that the MTBE protocol similar or better compared with the Folch or Bligh and Dyer and specific limitations of the enabled processing of cells, biological fluids, and tissues and was to using a the to shotgun profiling of complex lipidomes in a automated (9.Schwudke D. Hannich J.T. Surendranath V. Grimard V. Moehring T. Burton L. Kurzchalia T. Shevchenko A. Top-down lipidomic screens by multivariate analysis of high-resolution survey mass spectra.Anal. Chem. 2007; 79: 4083-4093Crossref PubMed Scopus (151) Google Scholar, 12.Ejsing C.S. Duchoslav E. Sampaio J. Simons K. Bonner R. Thiele C. Ekroos K. Shevchenko A. Automated identification and quantification of glycerophospholipid molecular species by multiple precursor ion scanning.Anal. Chem. 2006; 78: 6202-6214Crossref PubMed Scopus (331) Google Scholar, D. J. Burton L. E. Hannich J.T. C.S. Kurzchalia T. Shevchenko A. Lipid profiling by multiple precursor and neutral by the Chem. 2006; 78: PubMed Scopus Google Scholar). The are to of and and developed the for mass and to of the Kurzchalia and Shevchenko for and The Sampaio of and for of the with

MOTIVATION: Modern anatomical and developmental studies often require high-resolution imaging of large specimens in three dimensions (3D). Confocal microscopy produces high-resolution 3D images, but is limited by a relatively small field of view compared with the size of large biological specimens. Therefore, motorized stages that move the sample are used to create a tiled scan of the whole specimen. The physical coordinates provided by the microscope stage are not precise enough to allow direct reconstruction (Stitching) of the whole image from individual image stacks. RESULTS: To optimally stitch a large collection of 3D confocal images, we developed a method that, based on the Fourier Shift Theorem, computes all possible translations between pairs of 3D images, yielding the best overlap in terms of the cross-correlation measure and subsequently finds the globally optimal configuration of the whole group of 3D images. This method avoids the propagation of errors by consecutive registration steps. Additionally, to compensate the brightness differences between tiles, we apply a smooth, non-linear intensity transition between the overlapping images. Our stitching approach is fast, works on 2D and 3D images, and for small image sets does not require prior knowledge about the tile configuration. AVAILABILITY: The implementation of this method is available as an ImageJ plugin distributed as a part of the Fiji project (Fiji is just ImageJ: http://pacific.mpi-cbg.de/).

eggNOG is a public resource that provides Orthologous Groups (OGs) of proteins at different taxonomic levels, each with integrated and summarized functional annotations. Developments since the latest public release include changes to the algorithm for creating OGs across taxonomic levels, making nested groups hierarchically consistent. This allows for a better propagation of functional terms across nested OGs and led to the novel annotation of 95 890 previously uncharacterized OGs, increasing overall annotation coverage from 67% to 72%. The functional annotations of OGs have been expanded to also provide Gene Ontology terms, KEGG pathways and SMART/Pfam domains for each group. Moreover, eggNOG now provides pairwise orthology relationships within OGs based on analysis of phylogenetic trees. We have also incorporated a framework for quickly mapping novel sequences to OGs based on precomputed HMM profiles. Finally, eggNOG version 4.5 incorporates a novel data set spanning 2605 viral OGs, covering 5228 proteins from 352 viral proteomes. All data are accessible for bulk downloading, as a web-service, and through a completely redesigned web interface. The new access points provide faster searches and a number of new browsing and visualization capabilities, facilitating the needs of both experts and less experienced users. eggNOG v4.5 is available at http://eggnog.embl.de.

Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, are too small, or occur too rapidly to see clearly with existing tools. We crafted ultrathin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at subsecond intervals, at the diffraction limit and beyond. We applied this to systems spanning four orders of magnitude in space and time, including the diffusion of single transcription factor molecules in stem cell spheroids, the dynamic instability of mitotic microtubules, the immunological synapse, neutrophil motility in a 3D matrix, and embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. The results provide a visceral reminder of the beauty and the complexity of living systems.

The genomes of prokaryotes and eukaryotic organelles are usually circular as are most plasmids and viral genomes. In contrast, the nuclear genomes of eukaryotes are organized on linear chromosomes, which require mechanisms to protect and replicate DNA ends. Eukaryotes navigate these problems with the advent of telomeres, protective nucleoprotein complexes at the ends of linear chromosomes, and telomerase, the enzyme that maintains the DNA in these structures. Mammalian telomeres contain a specific protein complex, shelterin, that functions to protect chromosome ends from all aspects of the DNA damage response and regulates telomere maintenance by telomerase. Recent experiments, discussed here, have revealed how shelterin represses the ATM and ATR kinase signaling pathways and hides chromosome ends from nonhomologous end joining and homology-directed repair.

BACKGROUND: PacBio high fidelity (HiFi) sequencing reads are both long (15-20 kb) and highly accurate (> Q20). Because of these properties, they have revolutionised genome assembly leading to more accurate and contiguous genomes. In eukaryotes the mitochondrial genome is sequenced alongside the nuclear genome often at very high coverage. A dedicated tool for mitochondrial genome assembly using HiFi reads is still missing. RESULTS: MitoHiFi was developed within the Darwin Tree of Life Project to assemble mitochondrial genomes from the HiFi reads generated for target species. The input for MitoHiFi is either the raw reads or the assembled contigs, and the tool outputs a mitochondrial genome sequence fasta file along with annotation of protein and RNA genes. Variants arising from heteroplasmy are assembled independently, and nuclear insertions of mitochondrial sequences are identified and not used in organellar genome assembly. MitoHiFi has been used to assemble 374 mitochondrial genomes (368 Metazoa and 6 Fungi species) for the Darwin Tree of Life Project, the Vertebrate Genomes Project and the Aquatic Symbiosis Genome Project. Inspection of 60 mitochondrial genomes assembled with MitoHiFi for species that already have reference sequences in public databases showed the widespread presence of previously unreported repeats. CONCLUSIONS: MitoHiFi is able to assemble mitochondrial genomes from a wide phylogenetic range of taxa from Pacbio HiFi data. MitoHiFi is written in python and is freely available on GitHub ( https://github.com/marcelauliano/MitoHiFi ). MitoHiFi is available with its dependencies as a Docker container on GitHub (ghcr.io/marcelauliano/mitohifi:master).

Interactions between proteins and small molecules are an integral part of biological processes in living organisms. Information on these interactions is dispersed over many databases, texts and prediction methods, which makes it difficult to get a comprehensive overview of the available evidence. To address this, we have developed STITCH ('Search Tool for Interacting Chemicals') that integrates these disparate data sources for 430 000 chemicals into a single, easy-to-use resource. In addition to the increased scope of the database, we have implemented a new network view that gives the user the ability to view binding affinities of chemicals in the interaction network. This enables the user to get a quick overview of the potential effects of the chemical on its interaction partners. For each organism, STITCH provides a global network; however, not all proteins have the same pattern of spatial expression. Therefore, only a certain subset of interactions can occur simultaneously. In the new, fifth release of STITCH, we have implemented functionality to filter out the proteins and chemicals not associated with a given tissue. The STITCH database can be downloaded in full, accessed programmatically via an extensive API, or searched via a redesigned web interface at http://stitch.embl.de.

Caveolae are plasma membrane invaginations that may play an important role in numerous cellular processes including transport, signaling, and tumor suppression. By targeted disruption of caveolin-1, the main protein component of caveolae, we generated mice that lacked caveolae. The absence of this organelle impaired nitric oxide and calcium signaling in the cardiovascular system, causing aberrations in endothelium-dependent relaxation, contractility, and maintenance of myogenic tone. In addition, the lungs of knockout animals displayed thickening of alveolar septa caused by uncontrolled endothelial cell proliferation and fibrosis, resulting in severe physical limitations in caveolin-1-disrupted mice. Thus, caveolin-1 and caveolae play a fundamental role in organizing multiple signaling pathways in the cell.

Unwanted side effects of drugs are a burden on patients and a severe impediment in the development of new drugs. At the same time, adverse drug reactions (ADRs) recorded during clinical trials are an important source of human phenotypic data. It is therefore essential to combine data on drugs, targets and side effects into a more complete picture of the therapeutic mechanism of actions of drugs and the ways in which they cause adverse reactions. To this end, we have created the SIDER ('Side Effect Resource', http://sideeffects.embl.de) database of drugs and ADRs. The current release, SIDER 4, contains data on 1430 drugs, 5880 ADRs and 140 064 drug-ADR pairs, which is an increase of 40% compared to the previous version. For more fine-grained analyses, we extracted the frequency with which side effects occur from the package inserts. This information is available for 39% of drug-ADR pairs, 19% of which can be compared to the frequency under placebo treatment. SIDER furthermore contains a data set of drug indications, extracted from the package inserts using Natural Language Processing. These drug indications are used to reduce the rate of false positives by identifying medical terms that do not correspond to ADRs.

P granules and other RNA/protein bodies are membrane-less organelles that may assemble by intracellular phase separation, similar to the condensation of water vapor into droplets. However, the molecular driving forces and the nature of the condensed phases remain poorly understood. Here, we show that the Caenorhabditis elegans protein LAF-1, a DDX3 RNA helicase found in P granules, phase separates into P granule-like droplets in vitro. We adapt a microrheology technique to precisely measure the viscoelasticity of micrometer-sized LAF-1 droplets, revealing purely viscous properties highly tunable by salt and RNA concentration. RNA decreases viscosity and increases molecular dynamics within the droplet. Single molecule FRET assays suggest that this RNA fluidization results from highly dynamic RNA-protein interactions that emerge close to the droplet phase boundary. We demonstrate than an N-terminal, arginine/glycine rich, intrinsically disordered protein (IDP) domain of LAF-1 is necessary and sufficient for both phase separation and RNA-protein interactions. In vivo, RNAi knockdown of LAF-1 results in the dissolution of P granules in the early embryo, with an apparent submicromolar phase boundary comparable to that measured in vitro. Together, these findings demonstrate that LAF-1 is important for promoting P granule assembly and provide insight into the mechanism by which IDP-driven molecular interactions give rise to liquid phase organelles with tunable properties.

The field of image denoising is currently dominated by discriminative deep learning methods that are trained on pairs of noisy input and clean target images. Recently it has been shown that such methods can also be trained without clean targets. Instead, independent pairs of noisy images can be used, in an approach known as Noise2Noise (N2N). Here, we introduce Noise2Void (N2V), a training scheme that takes this idea one step further. It does not require noisy image pairs, nor clean target images. Consequently, N2V allows us to train directly on the body of data to be denoised and can therefore be applied when other methods cannot. Especially interesting is the application to biomedical image data, where the acquisition of training targets, clean or noisy, is frequently not possible. We compare the performance of N2V to approaches that have either clean target images and/or noisy image pairs available. Intuitively, N2V cannot be expected to outperform methods that have more information available during training. Still, we observe that the denoising performance of Noise2Void drops in moderation and compares favorably to training-free denoising methods.

Although the exact etiology of Alzheimer's disease (AD) is a topic of debate, the consensus is that the accumulation of beta-amyloid (Abeta) peptides in the senile plaques is one of the hallmarks of the progression of the disease. The Abeta peptide is formed by the amyloidogenic cleavage of the amyloid precursor protein (APP) by beta- and gamma-secretases. The endocytic system has been implicated in the cleavages leading to the formation of Abeta. However, the identity of the intracellular compartment where the amyloidogenic secretases cleave and the mechanism by which the intracellularly generated Abeta is released into the extracellular milieu are not clear. Here, we show that beta-cleavage occurs in early endosomes followed by routing of Abeta to multivesicular bodies (MVBs) in HeLa and N2a cells. Subsequently, a minute fraction of Abeta peptides can be secreted from the cells in association with exosomes, intraluminal vesicles of MVBs that are released into the extracellular space as a result of fusion of MVBs with the plasma membrane. Exosomal proteins were found to accumulate in the plaques of AD patient brains, suggesting a role in the pathogenesis of AD.

For most intracellular structures with larger than molecular dimensions, little is known about the connection between underlying molecular activities and higher order organization such as size and shape. Here, we show that both the size and shape of the amphibian oocyte nucleolus ultimately arise because nucleoli behave as liquid-like droplets of RNA and protein, exhibiting characteristic viscous fluid dynamics even on timescales of < 1 min. We use these dynamics to determine an apparent nucleolar viscosity, and we show that this viscosity is ATP-dependent, suggesting a role for active processes in fluidizing internal contents. Nucleolar surface tension and fluidity cause their restructuring into spherical droplets upon imposed mechanical deformations. Nucleoli exhibit a broad distribution of sizes with a characteristic power law, which we show is a consequence of spontaneous coalescence events. These results have implications for the function of nucleoli in ribosome subunit processing and provide a physical link between activity within a macromolecular assembly and its physical properties on larger length scales.

Cholesterol plays an indispensable role in regulating the properties of cell membranes in mammalian cells. Recent advances suggest that cholesterol exerts many of its actions mainly by maintaining sphingolipid rafts in a functional state. How rafts contribute to cholesterol metabolism and transport in the cell is still an open issue. It has long been known that cellular cholesterol levels are precisely controlled by biosynthesis, efflux from cells, and influx of lipoprotein cholesterol into cells. The regulation of cholesterol homeostasis is now receiving a new focus, and this changed perspective may throw light on diseases caused by cholesterol excess, the prime example being atherosclerosis.