University of Regensburg

Objectives To describe new WHO 2020 guidelines on physical activity and sedentary behaviour. Methods The guidelines were developed in accordance with WHO protocols. An expert Guideline Development Group reviewed evidence to assess associations between physical activity and sedentary behaviour for an agreed set of health outcomes and population groups. The assessment used and systematically updated recent relevant systematic reviews; new primary reviews addressed additional health outcomes or subpopulations. Results The new guidelines address children, adolescents, adults, older adults and include new specific recommendations for pregnant and postpartum women and people living with chronic conditions or disability. All adults should undertake 150–300 min of moderate-intensity, or 75–150 min of vigorous-intensity physical activity, or some equivalent combination of moderate-intensity and vigorous-intensity aerobic physical activity, per week. Among children and adolescents, an average of 60 min/day of moderate-to-vigorous intensity aerobic physical activity across the week provides health benefits. The guidelines recommend regular muscle-strengthening activity for all age groups. Additionally, reducing sedentary behaviours is recommended across all age groups and abilities, although evidence was insufficient to quantify a sedentary behaviour threshold. Conclusion These 2020 WHO guidelines update previous WHO recommendations released in 2010. They reaffirm messages that some physical activity is better than none, that more physical activity is better for optimal health outcomes and provide a new recommendation on reducing sedentary behaviours. These guidelines highlight the importance of regularly undertaking both aerobic and muscle strengthening activities and for the first time, there are specific recommendations for specific populations including for pregnant and postpartum women and people living with chronic conditions or disability. These guidelines should be used to inform national health policies aligned with the WHO Global Action Plan on Physical Activity 2018–2030 and to strengthen surveillance systems that track progress towards national and global targets.

BACKGROUND: Epigenetic silencing of the MGMT (O6-methylguanine-DNA methyltransferase) DNA-repair gene by promoter methylation compromises DNA repair and has been associated with longer survival in patients with glioblastoma who receive alkylating agents. METHODS: We tested the relationship between MGMT silencing in the tumor and the survival of patients who were enrolled in a randomized trial comparing radiotherapy alone with radiotherapy combined with concomitant and adjuvant treatment with temozolomide. The methylation status of the MGMT promoter was determined by methylation-specific polymerase-chain-reaction analysis. RESULTS: The MGMT promoter was methylated in 45 percent of 206 assessable cases. Irrespective of treatment, MGMT promoter methylation was an independent favorable prognostic factor (P<0.001 by the log-rank test; hazard ratio, 0.45; 95 percent confidence interval, 0.32 to 0.61). Among patients whose tumor contained a methylated MGMT promoter, a survival benefit was observed in patients treated with temozolomide and radiotherapy; their median survival was 21.7 months (95 percent confidence interval, 17.4 to 30.4), as compared with 15.3 months (95 percent confidence interval, 13.0 to 20.9) among those who were assigned to only radiotherapy (P=0.007 by the log-rank test). In the absence of methylation of the MGMT promoter, there was a smaller and statistically insignificant difference in survival between the treatment groups. CONCLUSIONS: Patients with glioblastoma containing a methylated MGMT promoter benefited from temozolomide, whereas those who did not have a methylated MGMT promoter did not have such a benefit.

The 2005 National Institutes of Health (NIH) Consensus Conference proposed new criteria for diagnosing and scoring the severity of chronic graft-versus-host disease (GVHD). The 2014 NIH consensus maintains the framework of the prior consensus with further refinement based on new evidence. Revisions have been made to address areas of controversy or confusion, such as the overlap chronic GVHD subcategory and the distinction between active disease and past tissue damage. Diagnostic criteria for involvement of mouth, eyes, genitalia, and lungs have been revised. Categories of chronic GVHD should be defined in ways that indicate prognosis, guide treatment, and define eligibility for clinical trials. Revisions have been made to focus attention on the causes of organ-specific abnormalities. Attribution of organ-specific abnormalities to chronic GVHD has been addressed. This paradigm shift provides greater specificity and more accurately measures the global burden of disease attributed to GVHD, and it will facilitate biomarker association studies.

Reference phylogenies are crucial for providing a taxonomic framework for interpretation of marker gene and metagenomic surveys, which continue to reveal novel species at a remarkable rate. Greengenes is a dedicated full-length 16S rRNA gene database that provides users with a curated taxonomy based on de novo tree inference. We developed a 'taxonomy to tree' approach for transferring group names from an existing taxonomy to a tree topology, and used it to apply the Greengenes, National Center for Biotechnology Information (NCBI) and cyanoDB (Cyanobacteria only) taxonomies to a de novo tree comprising 408,315 sequences. We also incorporated explicit rank information provided by the NCBI taxonomy to group names (by prefixing rank designations) for better user orientation and classification consistency. The resulting merged taxonomy improved the classification of 75% of the sequences by one or more ranks relative to the original NCBI taxonomy with the most pronounced improvements occurring in under-classified environmental sequences. We also assessed candidate phyla (divisions) currently defined by NCBI and present recommendations for consolidation of 34 redundantly named groups. All intermediate results from the pipeline, which includes tree inference, jackknifing and transfer of a donor taxonomy to a recipient tree (tax2tree) are available for download. The improved Greengenes taxonomy should provide important infrastructure for a wide range of megasequencing projects studying ecosystems on scales ranging from our own bodies (the Human Microbiome Project) to the entire planet (the Earth Microbiome Project). The implementation of the software can be obtained from http://sourceforge.net/projects/tax2tree/.

BACKGROUND: Mesenchymal stem cells (MSC) are promising tools for tissue-engineering and musculoskeletal regeneration. They reside within various tissues, like adipose tissue, periosteum, synovia, muscle, dermis, blood and bone marrow, latter being the most common tissue used for MSC isolation. A promising alternative source for MSC is adipose tissue due to better availability and higher yield of MSC in comparison to bone marrow. A drawback is the yet fragmentary knowledge of adipose-derived stem cell (ASC) physiology in order to make them a safe tool for in vivo application. METHODS/RESULTS: Here, we identified Sox9 as a highly expressed and crucial transcription factor in undifferentiated rat ASC (rASC). In comparison to rat bone marrow-derived stem cells (rBMSC), mRNA and protein levels of Sox9 were significantly higher in rASC. To study the role of Sox9 in detail, we silenced Sox9 with shRNA in rASC and examined proliferation, apoptosis and the expression of osteogenic differentiation markers. Our results clearly point to a difference in the expression profile of osteogenic marker genes between undifferentiated rASC and rBMSC in early passages. Sox9 silencing induced the expression of osteocalcin, Vegfα and Mmp13, and decreased rASC proliferation accompanied with an induction of p21 and cyclin D1 expression and delayed S-phase entry. CONCLUSIONS: We suggest a pro-proliferative role for Sox9 in undifferentiated rASC which may explain the higher proliferation rate of rASC compared to rBMSC. Moreover, we propose an osteogenic differentiation delaying role of Sox9 in rASC which suggests that Sox9 expression is needed to maintain rASC in an undifferentiated, proliferative state.

Plant functional traits are the features (morphological, physiological, phenological) that represent ecological strategies and determine how plants respond to environmental factors, affect other trophic levels and influence ecosystem properties. Variation in plant functional traits, and trait syndromes, has proven useful for tackling many important ecological questions at a range of scales, giving rise to a demand for standardised ways to measure ecologically meaningful plant traits. This line of research has been among the most fruitful avenues for understanding ecological and evolutionary patterns and processes. It also has the potential both to build a predictive set of local, regional and global relationships between plants and environment and to quantify a wide range of natural and human-driven processes, including changes in biodiversity, the impacts of species invasions, alterations in biogeochemical processes and vegetation–atmosphere interactions. The importance of these topics dictates the urgent need for more and better data, and increases the value of standardised protocols for quantifying trait variation of different species, in particular for traits with power to predict plant- and ecosystem-level processes, and for traits that can be measured relatively easily. Updated and expanded from the widely used previous version, this handbook retains the focus on clearly presented, widely applicable, step-by-step recipes, with a minimum of text on theory, and not only includes updated methods for the traits previously covered, but also introduces many new protocols for further traits. This new handbook has a better balance between whole-plant traits, leaf traits, root and stem traits and regenerative traits, and puts particular emphasis on traits important for predicting species’ effects on key ecosystem properties. We hope this new handbook becomes a standard companion in local and global efforts to learn about the responses and impacts of different plant species with respect to environmental changes in the present, past and future.

CP2K is an open source electronic structure and molecular dynamics software package to perform atomistic simulations of solid-state, liquid, molecular, and biological systems. It is especially aimed at massively parallel and linear-scaling electronic structure methods and state-of-the-art ab initio molecular dynamics simulations. Excellent performance for electronic structure calculations is achieved using novel algorithms implemented for modern high-performance computing systems. This review revisits the main capabilities of CP2K to perform efficient and accurate electronic structure simulations. The emphasis is put on density functional theory and multiple post-Hartree-Fock methods using the Gaussian and plane wave approach and its augmented all-electron extension.

Abstract Molpro (available at http://www.molpro.net ) is a general‐purpose quantum chemical program. The original focus was on high‐accuracy wave function calculations for small molecules, but using local approximations combined with explicit correlation treatments, highly accurate coupled‐cluster calculations are now possible for molecules with up to approximately 100 atoms. Recently, multireference correlation treatments were also made applicable to larger molecules. Furthermore, an efficient implementation of density functional theory is available. © 2011 John Wiley &amp; Sons, Ltd. This article is categorized under: Software &gt; Quantum Chemistry

These guidelines provide an up-date of previous IFCN report on "Non-invasive electrical and magnetic stimulation of the brain, spinal cord and roots: basic principles and procedures for routine clinical application" (Rossini et al., 1994). A new Committee, composed of international experts, some of whom were in the panel of the 1994 "Report", was selected to produce a current state-of-the-art review of non-invasive stimulation both for clinical application and research in neuroscience. Since 1994, the international scientific community has seen a rapid increase in non-invasive brain stimulation in studying cognition, brain-behavior relationship and pathophysiology of various neurologic and psychiatric disorders. New paradigms of stimulation and new techniques have been developed. Furthermore, a large number of studies and clinical trials have demonstrated potential therapeutic applications of non-invasive brain stimulation, especially for TMS. Recent guidelines can be found in the literature covering specific aspects of non-invasive brain stimulation, such as safety (Rossi et al., 2009), methodology (Groppa et al., 2012) and therapeutic applications (Lefaucheur et al., 2014). This up-dated review covers theoretical, physiological and practical aspects of non-invasive stimulation of brain, spinal cord, nerve roots and peripheral nerves in the light of more updated knowledge, and include some recent extensions and developments.

Microbial-type rhodopsins are found in archaea, prokaryotes, and eukaryotes. Some of them represent membrane ion transport proteins such as bacteriorhodopsin, a light-driven proton pump, or channelrhodopsin-1 (ChR1), a recently identified light-gated proton channel from the green alga Chlamydomonas reinhardtii. ChR1 and ChR2, a related microbial-type rhodopsin from C. reinhardtii, were shown to be involved in generation of photocurrents of this green alga. We demonstrate by functional expression, both in oocytes of Xenopus laevis and mammalian cells, that ChR2 is a directly light-switched cation-selective ion channel. This channel opens rapidly after absorption of a photon to generate a large permeability for monovalent and divalent cations. ChR2 desensitizes in continuous light to a smaller steady-state conductance. Recovery from desensitization is accelerated by extracellular H+ and negative membrane potential, whereas closing of the ChR2 ion channel is decelerated by intracellular H+. ChR2 is expressed mainly in C. reinhardtii under low-light conditions, suggesting involvement in photoreception in dark-adapted cells. The predicted seven-transmembrane alpha helices of ChR2 are characteristic for G protein-coupled receptors but reflect a different motif for a cation-selective ion channel. Finally, we demonstrate that ChR2 may be used to depolarize small or large cells, simply by illumination.

Abstract Plant traits – the morphological, anatomical, physiological, biochemical and phenological characteristics of plants and their organs – determine how primary producers respond to environmental factors, affect other trophic levels, influence ecosystem processes and services and provide a link from species richness to ecosystem functional diversity. Trait data thus represent the raw material for a wide range of research from evolutionary biology, community and functional ecology to biogeography. Here we present the global database initiative named TRY, which has united a wide range of the plant trait research community worldwide and gained an unprecedented buy‐in of trait data: so far 93 trait databases have been contributed. The data repository currently contains almost three million trait entries for 69 000 out of the world's 300 000 plant species, with a focus on 52 groups of traits characterizing the vegetative and regeneration stages of the plant life cycle, including growth, dispersal, establishment and persistence. A first data analysis shows that most plant traits are approximately log‐normally distributed, with widely differing ranges of variation across traits. Most trait variation is between species (interspecific), but significant intraspecific variation is also documented, up to 40% of the overall variation. Plant functional types (PFTs), as commonly used in vegetation models, capture a substantial fraction of the observed variation – but for several traits most variation occurs within PFTs, up to 75% of the overall variation. In the context of vegetation models these traits would better be represented by state variables rather than fixed parameter values. The improved availability of plant trait data in the unified global database is expected to support a paradigm shift from species to trait‐based ecology, offer new opportunities for synthetic plant trait research and enable a more realistic and empirically grounded representation of terrestrial vegetation in Earth system models.

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

Cholangiocarcinoma (CCA) includes a cluster of highly heterogeneous biliary malignant tumours that can arise at any point of the biliary tree. Their incidence is increasing globally, currently accounting for ~15% of all primary liver cancers and ~3% of gastrointestinal malignancies. The silent presentation of these tumours combined with their highly aggressive nature and refractoriness to chemotherapy contribute to their alarming mortality, representing ~2% of all cancer-related deaths worldwide yearly. The current diagnosis of CCA by non-invasive approaches is not accurate enough, and histological confirmation is necessary. Furthermore, the high heterogeneity of CCAs at the genomic, epigenetic and molecular levels severely compromises the efficacy of the available therapies. In the past decade, increasing efforts have been made to understand the complexity of these tumours and to develop new diagnostic tools and therapies that might help to improve patient outcomes. In this expert Consensus Statement, which is endorsed by the European Network for the Study of Cholangiocarcinoma, we aim to summarize and critically discuss the latest advances in CCA, mostly focusing on classification, cells of origin, genetic and epigenetic abnormalities, molecular alterations, biomarker discovery and treatments. Furthermore, the horizon of CCA for the next decade from 2020 onwards is highlighted.

Ecological data often show temporal, spatial, hierarchical (random effects), or phylogenetic structure. Modern statistical approaches are increasingly accounting for such dependencies. However, when performing cross‐validation, these structures are regularly ignored, resulting in serious underestimation of predictive error. One cause for the poor performance of uncorrected (random) cross‐validation, noted often by modellers, are dependence structures in the data that persist as dependence structures in model residuals, violating the assumption of independence. Even more concerning, because often overlooked, is that structured data also provides ample opportunity for overfitting with non‐causal predictors. This problem can persist even if remedies such as autoregressive models, generalized least squares, or mixed models are used. Block cross‐validation, where data are split strategically rather than randomly, can address these issues. However, the blocking strategy must be carefully considered. Blocking in space, time, random effects or phylogenetic distance, while accounting for dependencies in the data, may also unwittingly induce extrapolations by restricting the ranges or combinations of predictor variables available for model training, thus overestimating interpolation errors. On the other hand, deliberate blocking in predictor space may also improve error estimates when extrapolation is the modelling goal. Here, we review the ecological literature on non‐random and blocked cross‐validation approaches. We also provide a series of simulations and case studies, in which we show that, for all instances tested, block cross‐validation is nearly universally more appropriate than random cross‐validation if the goal is predicting to new data or predictor space, or for selecting causal predictors. We recommend that block cross‐validation be used wherever dependence structures exist in a dataset, even if no correlation structure is visible in the fitted model residuals, or if the fitted models account for such correlations.

A group of European experts reappraised the guidelines on the therapeutic efficacy of repetitive transcranial magnetic stimulation (rTMS) previously published in 2014 [Lefaucheur et al., Clin Neurophysiol 2014;125:2150-206]. These updated recommendations take into account all rTMS publications, including data prior to 2014, as well as currently reviewed literature until the end of 2018. Level A evidence (definite efficacy) was reached for: high-frequency (HF) rTMS of the primary motor cortex (M1) contralateral to the painful side for neuropathic pain; HF-rTMS of the left dorsolateral prefrontal cortex (DLPFC) using a figure-of-8 or a H1-coil for depression; low-frequency (LF) rTMS of contralesional M1 for hand motor recovery in the post-acute stage of stroke. Level B evidence (probable efficacy) was reached for: HF-rTMS of the left M1 or DLPFC for improving quality of life or pain, respectively, in fibromyalgia; HF-rTMS of bilateral M1 regions or the left DLPFC for improving motor impairment or depression, respectively, in Parkinson's disease; HF-rTMS of ipsilesional M1 for promoting motor recovery at the post-acute stage of stroke; intermittent theta burst stimulation targeted to the leg motor cortex for lower limb spasticity in multiple sclerosis; HF-rTMS of the right DLPFC in posttraumatic stress disorder; LF-rTMS of the right inferior frontal gyrus in chronic post-stroke non-fluent aphasia; LF-rTMS of the right DLPFC in depression; and bihemispheric stimulation of the DLPFC combining right-sided LF-rTMS (or continuous theta burst stimulation) and left-sided HF-rTMS (or intermittent theta burst stimulation) in depression. Level A/B evidence is not reached concerning efficacy of rTMS in any other condition. The current recommendations are based on the differences reached in therapeutic efficacy of real vs. sham rTMS protocols, replicated in a sufficient number of independent studies. This does not mean that the benefit produced by rTMS inevitably reaches a level of clinical relevance.

Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly ‘housekeeping’, whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research. A study from the FANTOM consortium using single-molecule cDNA sequencing of transcription start sites and their usage in human and mouse primary cells, cell lines and tissues reveals insights into the specificity and diversity of transcription patterns across different mammalian cell types. FANTOM5 (standing for functional annotation of the mammalian genome 5) is the fifth major stage of a major international collaboration that aims to dissect the transcriptional regulatory networks that define every human cell type. Two Articles in this issue of Nature present some of the project's latest results. The first paper uses the FANTOM5 panel of tissue and primary cell samples to define an atlas of active, in vivo bidirectionally transcribed enhancers across the human body. These authors show that bidirectional capped RNAs are a signature feature of active enhancers and identify more than 40,000 enhancer candidates from over 800 human cell and tissue samples. The enhancer atlas is used to compare regulatory programs between different cell types and identify disease-associated regulatory SNPs, and will be a resource for studies on cell-type-specific enhancers. In the second paper, single-molecule sequencing is used to map human and mouse transcription start sites and their usage in a panel of distinct human and mouse primary cells, cell lines and tissues to produce the most comprehensive mammalian gene expression atlas to date. The data provide a plethora of insights into open reading frames and promoters across different cell types in addition to valuable annotation of mammalian cell-type-specific transcriptomes.

Visible-light photocatalysis has evolved over the last decade into a widely used method in organic synthesis. Photocatalytic variants have been reported for many important transformations, such as cross-coupling reactions, α-amino functionalizations, cycloadditions, ATRA reactions, or fluorinations. To help chemists select photocatalytic methods for their synthesis, we compare in this Review classical and photocatalytic procedures for selected classes of reactions and highlight their advantages and limitations. In many cases, the photocatalytic reactions proceed under milder reaction conditions, typically at room temperature, and stoichiometric reagents are replaced by simple oxidants or reductants, such as air, oxygen, or amines. Does visible-light photocatalysis make a difference in organic synthesis? The prospect of shuttling electrons back and forth to substrates and intermediates or to selectively transfer energy through a visible-light-absorbing photocatalyst holds the promise to improve current procedures in radical chemistry and to open up new avenues by accessing reactive species hitherto unknown, especially by merging photocatalysis with organo- or metal catalysis.

Plant traits-the morphological, anatomical, physiological, biochemical and phenological characteristics of plants-determine how plants respond to environmental factors, affect other trophic levels, and influence ecosystem properties and their benefits and detriments to people. Plant trait data thus represent the basis for a vast area of research spanning from evolutionary biology, community and functional ecology, to biodiversity conservation, ecosystem and landscape management, restoration, biogeography and earth system modelling. Since its foundation in 2007, the TRY database of plant traits has grown continuously. It now provides unprecedented data coverage under an open access data policy and is the main plant trait database used by the research community worldwide. Increasingly, the TRY database also supports new frontiers of trait-based plant research, including the identification of data gaps and the subsequent mobilization or measurement of new data. To support this development, in this article we evaluate the extent of the trait data compiled in TRY and analyse emerging patterns of data coverage and representativeness. Best species coverage is achieved for categorical traits-almost complete coverage for 'plant growth form'. However, most traits relevant for ecology and vegetation modelling are characterized by continuous intraspecific variation and trait-environmental relationships. These traits have to be measured on individual plants in their respective environment. Despite unprecedented data coverage, we observe a humbling lack of completeness and representativeness of these continuous traits in many aspects. We, therefore, conclude that reducing data gaps and biases in the TRY database remains a key challenge and requires a coordinated approach to data mobilization and trait measurements. This can only be achieved in collaboration with other initiatives.

BACKGROUND: Modern genotyping platforms permit a systematic search for inherited components of complex diseases. We performed a joint analysis of two genomewide association studies of coronary artery disease. METHODS: We first identified chromosomal loci that were strongly associated with coronary artery disease in the Wellcome Trust Case Control Consortium (WTCCC) study (which involved 1926 case subjects with coronary artery disease and 2938 controls) and looked for replication in the German MI [Myocardial Infarction] Family Study (which involved 875 case subjects with myocardial infarction and 1644 controls). Data on other single-nucleotide polymorphisms (SNPs) that were significantly associated with coronary artery disease in either study (P<0.001) were then combined to identify additional loci with a high probability of true association. Genotyping in both studies was performed with the use of the GeneChip Human Mapping 500K Array Set (Affymetrix). RESULTS: Of thousands of chromosomal loci studied, the same locus had the strongest association with coronary artery disease in both the WTCCC and the German studies: chromosome 9p21.3 (SNP, rs1333049) (P=1.80x10(-14) and P=3.40x10(-6), respectively). Overall, the WTCCC study revealed nine loci that were strongly associated with coronary artery disease (P<1.2x10(-5) and less than a 50% chance of being falsely positive). In addition to chromosome 9p21.3, two of these loci were successfully replicated (adjusted P<0.05) in the German study: chromosome 6q25.1 (rs6922269) and chromosome 2q36.3 (rs2943634). The combined analysis of the two studies identified four additional loci significantly associated with coronary artery disease (P<1.3x10(-6)) and a high probability (>80%) of a true association: chromosomes 1p13.3 (rs599839), 1q41 (rs17465637), 10q11.21 (rs501120), and 15q22.33 (rs17228212). CONCLUSIONS: We identified several genetic loci that, individually and in aggregate, substantially affect the risk of development of coronary artery disease.

Most two-dimensional (2D) semiconductors are interesting materials as quantum confinement enhances the Coulomb interaction between carriers, leading to a strong attraction between conduction electrons and valence holes, forming stable excitons and the optical response of 2D semiconductors can be extraordinary. In this Colloquium the progress and open questions in the study of excitons in 2D semiconductors from both the experimental and theoretical perspectives are reviewed.