Tholey Abbey, located in the town of Tholey in Saarland, is widely recognized as Germany’s oldest monastery, with roots tracing back to the 7th century. Over its long history, the abbey has been rebuilt multiple times, with its current Gothic structure dating to the 13th century The abbey’s most notable recent addition is a set of stained-glass windows designed by the renowned German artist Gerhard Richter in 2020 abstract windows create a striking contrast with the historic surroundings making Tholey Abbey a unique blend of ancient religious heritage and contemporary art welcoming both visitors and those seeking a place of worship These crumbling monastery ruins inspired a master of German Romanticism This 11th-century Cistercian abbey was once seized by Henry VIII The remains of one of Wales’ great medieval buildings The last two abbots of this monastery were both executed by Henry VIII The remains of a 13th-century Franciscan friary have survived multiple attempts at destruction The ruins of a 12th-century Cistercian monastery can be found alongside World War II tank traps and pillboxes An abandoned monastery sits on a far-off island in the Adriatic Sea image: Gel-based sample pre-fractionation using PEPPI-MS allows for deep proteoform analysis using LC-MS protein molecules with different chemical structures called proteoforms are produced from a single gene to perform a variety of physiological protein functions It has been known for quite some time that humans have approximately 22,000 genes but the total number of human proteoforms is still unknown liquid chromatography-mass spectrometry (LC-MS) a highly sensitive method for measuring biomolecules is the main method used to analyze proteoforms Whereas the traditional approach of analyzing proteoforms requires breaking them down into small peptides a newer approach of analyzing intact proteoforms by LC-MS is called top-down proteomics Along the lines of the Human Genome Project that mapped all of our genes there have been attempts to now build an atlas of human proteoforms using top-down proteomics the proteoform components extracted from biological samples are complex and LC-MS alone cannot comprehensively detect all proteoforms the Takemori group at Ehime University originally developed PEPPI-MS an innovative method for high-resolution fractionation of proteoforms using the inexpensive and simple SDS- PAGE system [Ref Sample fractionation using PEPPI-MS has been successful in significantly increasing the number of proteoforms detectable by LC-MS and its performance has led to the adoption of PEPPI in sample preparations Andreas Tholey’s group at the University of Kiel (Christian-Albrechts-Universität zu Kiel) an ultra-sensitive proteoform measurement system was built that combines PEPPI-MS fractionation with FAIMS ion mobility mass spectrometry which can separate proteoforms in the gas phase they achieved detailed analysis of human cultured cells via top-down proteomics and established a methodology for a new middle-down proteomics in which the Glu-C digestion products of proteoforms are analyzed [Refs PEPPI-MS fractionation does not require any special equipment and can be performed with standard biochemical laboratory equipment Its ease and accessibility has led to its becoming a standard method for sample fractionation in deep top-down proteomics the Takemori group established experimental protocols for high-resolution proteoform fractionation applying PEPPI-MS and top-down/middle-down proteomics using the FAIMS-LC-MS system and recently published a streamlined protocol combining their work from 2020 to 2024 in Nature Protocols [Ref The complete set of PEPPI-MS protocols enables high-resolution fractionation of trace biological samples and large-scale proteoform analysis by LC-MS and is expected to contribute to the construction of proteoform atlases for various living species and the development of disease diagnostic methods based on precise proteoform information 10.1038/s41596-024-01100-0 Disclaimer: AAAS and EurekAlert! are not responsible for the accuracy of news releases posted to EurekAlert! by contributing institutions or for the use of any information through the EurekAlert system. Copyright © 2025 by the American Association for the Advancement of Science (AAAS) Please enable JS and disable any ad blocker Volume 11 - 2020 | https://doi.org/10.3389/fpsyg.2020.01885 Lucid dreaming refers to a dream state characterized by the dreamers’ awareness of being in a dream and being able to volitionally control its content The aim of this study was to describe and model neurophysiological evidence for the seven awareness criteria of lucid dreaming based on those proposed by Paul Tholey Each of the awareness criteria was analyzed separately with regard to its underlying neurocircuits but several regions are involved in the state of lucid dreaming Our results have shown a satisfactory overlap of the awareness criteria and the brain regions activated that is something other than the sum of its parts Further research is needed to understand the psychoneurological underpinnings of lucid dreams Lucid dreaming (LD) is a fascinating research topic and has attracted many enthusiasts the scientific field is still lacking a comprehensive definition of LD (3) physical laws need not apply in the dream (4) the dreamer has a clear memory of the waking world He declared criteria 1–4 as essential for a “Klartraum,” while criteria 5–7 are optional and do not make a “Klartraum” by themselves 2. Clarity about the freedom of choice (for experiments on the topic see: Libet et al., 1983; Fried et al., 1991; Haggard and Eimer, 1999; Soon et al., 2008; Liljenström, 2015; Liljenström and Nazir, 2016; for an overview see: Baumeister et al., 2010; Caruso, 2012) The seven criteria used in this article are based on Tholey’s, however, we used an adapted version (Holzinger, 2014) that fits the Gestalt theory terminology better (Yontef, 1993) We suggest these criteria are more closely related to newer neurophysiological findings and reportings of lucid dreaming experiences Awareness being a lasting state seems to describe the process of a lucid dream better compared to a moment of clarity which tends to be momentary the following criteria are in its core the same as those proposed by Tholey 3. Awareness of (intense) concentration – (awareness of “flow” Csikszentmihalyi et al., 2014) Awareness of identity (the “I”) describing this extraordinary state and its potential The definition of LD is still a work in progress and we hope that the discussion about the definition of a pre-lucid a lucid dream and Klartraum will gain momentum in the scientific community and it cannot be reduced to their sum or their difference” (p It therefore occurs when an entity is observed to have properties its parts do not have on their own and in this case we assume a model of brain activation on the basis of the seven awareness criteria first described by Paul Tholey and call it the “lucid brain model,” trying to integrate the varying results of research projects within the last decades former findings regarding the general difference in brain activity during REM sleep and LD will be discussed the matter of consciousness in LD will be introduced and finally neuroscientific evidence for each of our seven proposed awareness criteria will be presented the conscious experience itself relies on a temporo-parietal-occipital cortical “hot zone.” Therefore increased activation of the frontal brain regions and temporo-parietal-occipital regions during LD compared to non-LD seem to have numerous effects on conscious awareness At this point, we would also like to emphasize the notion of consciousness in sleep regarding the understanding and the consequent definition of LD as Harry Hunt did in 1995 (Hunt, 1995) and Jennifer Windt in 2011 (Windt and Noreika, 2011) We do suspect different stages of consciousness and a lucid dreamer does show higher cognitive abilities and reflective awareness than a non-lucid dreamer overall. Empirical data supports the assumption that LD may be defined as a hybrid state, which is still partially ruled by lower level consciousness (Voss et al., 2009; Dresler et al., 2012; Voss et al., 2018) This might be the reason that lucid dreams are “happening” as a result of the subconscious instead of being “created” in the first place they are a reflection of ourselves and our lives Both lucid and non-lucid dreams may involve a “thinking” dimension as well as a “perceiving” or “experiencing” dimension together with the higher activation of meta-cognitive brain areas possibly supply evidence for the awareness of spatial orientation This includes the awareness of being in a dream – which is Tholey’s first criteria but is also inherent to our first awareness criteria The possible activation of all these cognitive processes during LD might explain the awareness of the option to make sound choices based on thoughts emotions and memories and individual preferences the concentration of oxygenated hemoglobin (oxy-Hb) was significantly increased in the right and left ventrolateral PFC They also found a significant increase in oxy-Hb concentration in the right and left dorsolateral PFC and left ventrolateral PFC while participants were filling out the flow state scale after performing a task in the flow condition These areas have been found to show increased activation during LD flow is associated with activity of the PFC and may therefore be associated with functions such as cognition the flow experience shares many characteristics with the LD state We suggest that this area of the brain might also play a role in LD this is only speculative and requires further exploring The awareness of the “I” is of course closely related to the awareness of memory explained in section “Awareness of Memory,” which determines to a great part what the dreamer might decide wish for or act upon when able to take control of the dream Together with those findings discussed in section “Awareness of (Spatial) Orientation,” the awareness of the dreaming environment during LD might be explained General frontal activation might be the reason for the ability to add meaning to a dream by integrating memory identity and the dreaming environment into a whole Based on empirical and theoretical findings we suggest that a dream becomes meaningful by an integration of emotional content (limbic system) memory (hippothalamus and related structures) and brain structures involved in identity (see section “Awareness of Identity—the “I” Without Which There Would Be No Dialogue”) This might be possible due to an activation of the DMN and executive functions returning when accessing the state of LD compared to non-LD together with the anterior cingulate cortex (ACC) anterior insula and basal ganglia during time perception as well as time perception might play a role when adding meaning to a dream Brain regions showing increased activity during lucid REM sleep contrasted with non-lucid REM sleep Assignment of awareness criteria to brain regions: (1) Awareness of (spatial) orientation: 4 and 8; (2) Awareness of the capacity of choice: 1 and 3; (3) Awareness of (intense) concentration – awareness of “flow”: 1 and 3; (4) Awareness of identity (the “I”) : 4 and 6; (5) Awareness of the dreaming environment : 3 and 8; (6) Awareness of the meaning of the dream : 1 there is still substantial disagreement with regard to the brain regions and frequency bands most activated during lucid dreaming and how they correlate with the theoretical base of lucid dreams Both authors confirm being the only contributors of this work and approved it for publication The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest We would like to express our sincere thanks to the editor and the reviewers for their careful work and thoughtful suggestions The cognitive neuroscience of lucid dreaming CrossRef Full Text | Google Scholar Free Will and Consciousness: How Might They Work? 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) distribution or reproduction in other forums is permitted provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited in accordance with accepted academic practice distribution or reproduction is permitted which does not comply with these terms *Correspondence: Brigitte Holzinger, b2ZmaWNlQHRyYXVtLmFjLmF0; aW5mb0BzY2hsYWZjb2FjaGluZy5vcmc= †ORCID: Brigitte Holzinger, orcid.org/0000-0001-5385-4091 Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher 94% of researchers rate our articles as excellent or goodLearn more about the work of our research integrity team to safeguard the quality of each article we publish Metrics details Top-down proteomics using mass spectrometry facilitates the identification of intact proteoforms Multiple past advances have lead to the development of numerous sample preparation workflows Here we systematically investigated the influence of different sample preparation steps on proteoform and protein identifications We found that all steps in sample preparation influence the subset of proteoforms identified (for example physicochemical properties and artificially generated modifications) The various sample preparation strategies resulted in complementary identifications substantially increasing the proteome coverage we identified 13,975 proteoforms from 2,720 proteins of human Caco-2 cells The results presented can serve as suggestions for designing and adapting top-down proteomics sample preparation strategies to particular research questions we expect that the sampling bias and modifications identified at the intact protein level will also be useful in improving bottom-up proteomics approaches the enrichment of smaller proteoforms or the depletion of larger proteoforms is a common step in TDP sample preparation before liquid chromatography with MS/MS (LC–MS/MS) analysis Upper panel: a suitable LC–MS/MS workflow was established Middle panel: the established LC–MS/MS workflow was used to investigate the influence of various sample preparation steps on proteoform identification various prefractionation strategies to enrich proteoforms smaller than 30 kDa and multidimensional separation schemes Lower panel: data analysis with ProSightPD regarding the proteoform count physicochemical properties and artificially introduced modifications two-dimensional reversed-phase low/low pH proteoform fractionation approach to ensure a fair comparison of different sample preparation strategies To investigate the influence of various lysis solutions on proteoform and protein identifications by TDP Caco-2 cells were lysed in (1) phosphate-buffered saline (PBS) (2) ammonium bicarbonate-buffered urea (Urea–ABC) (4) Tris-buffered sodium dodecyl sulfate (SDS–Tris) (5) acidic acetonitrile-water solution containing sodium chloride (ACN–NaCl) and (6) triethylammonium bicarbonate-buffered ACN-water solution (ACN–TEAB) Number of identified proteoforms (a) and protein accessions (b) (n = 3 replicates of independently performed sample preparations Full-length proteoforms are those deposited in the proteoform database including start methionine excision and previously described proteolytic-processed proteoforms truncated proteoforms refer to all subsequence proteoforms not defined in the database Overlap coefficients of the proteoforms and proteins identified in the replicates from the various cell lysis conditions Source data The lysis with the chaotropic salts urea and GndHCl showed a bias toward more hydrophobic proteins compared to PBS and SDS–Tris lysis The most similar identifications regarding proteoform and protein overlap coefficient (on average, ~56 and 73%, respectively) were obtained between the Urea–ABC, SDS–Tris and PBS lysis buffers (Fig. 2f) maintaining a neutral or alkalic pH during the cell lysis the overlap coefficients with the neutral or alkalic lysis conditions were much lower (45% on proteoform and 67% on protein level) the lysis with ACN–TEAB and ACN–NaCl resulted in very low overlap coefficients compared to the other lysis conditions (23% on proteoform and 45% on protein level) Number of identified proteoforms with or without cysteine residues Fragment maps of selected proteoforms from Glutaredoxin-1 (P35754) identified in the (1) control (2) reduced and (3) reduced/alkylated samples The blue brackets represent b-/y-ions identified after CID fragmentation The cysteine residues highlighted in gray indicate disulfide bridges and those in blue are carbamidomethylated cysteines Analysis of truncated proteoforms in the control (c) and reduced sample (d) The two-dimensional histograms display the potential truncation sites of truncated proteoforms The amino acids N and C terminal of the potential truncation sites are denoted as X and X′ Source data which potentially reduces the formation of artificially truncated proteoforms Number of identified full-length and truncated proteoforms (a) and protein accessions (n = 3 replicates of independently performed sample preparations showing the percentage of proteoforms identified in one Overlap coefficients between the replicates of all approaches regarding the identified proteins and proteoforms UpSet plot of proteoforms identified with the various approaches (minimum 50 proteoforms) Number of identified proteoforms when combining multiple database search results from the different approaches All raw files were analyzed together in a multiconsensus analysis and the number of proteoforms identified in a combination of various sample preparations was calculated C18 material; PEPPI with subsequent MCW precipitation Source data only 198 proteoforms (4%) and 163 proteins (12%) were identified in all of the seven investigated approaches isoelectric point (pI) (b) and GRAVY score (c) Detection of modifications using MSTopDiff after deconvolution of a randomly selected raw file from each approach with FLASHDeconv Shown are the intensity × count histograms Source data a decent amount of oxidized mass features was detected which may be attributed to the relatively long sample preparation time originating from the sample incubation in Laemmli buffer before SDS–PAGE separation The SPE sample preparation led to the detection of a mass shift assigned to formylation which is due to the use of formic acid at room temperature Furthermore, we examined several variations of the investigated prefractionation approaches presented in the Supplementary Results and Supplementary Fig. 17 Both approaches were used to separate proteoforms from human Caco-2 cells in eight fractions, resulting in the identification of a similar number of proteoforms (~2,150) and proteins (~770) (Supplementary Fig. 18a,b) the two-dimensional separation schemes resulted in more proteoform (~+145%) and protein (~+62%) identifications but required a substantially longer instrument time due to the analysis of eight fractions compared to one sample providing a comprehensive TDP dataset of human Caco-2 cells We systematically investigated the influence of different sample preparation procedures on proteoform and protein identifications in TDP number of replicates and injection amounts ensured a fair comparison of the different sample preparation steps and can be applied to any TDP study We showed that each step in the sample preparation influences the number and the physicochemical properties of identifications, that is, biases toward specific proteoform subgroups, such as small, hydrophobic or acidic proteoforms, were introduced depending on the applied approaches (Supplementary Table 7) we examined the proteoform quality regarding potential artificially introduced modification (for example due to covalent and noncovalent adducts or peptide bond hydrolysis) While many sample preparation steps are similar in BUP and TDP the impact of these steps is often overseen in BUP due to the need for protein inference we suggest that the results of the intact protein level-centric TDP approach presented here can be used for optimizing BUP workflows The application of SPE or MWCO allows direct sample cleanup which may be particularly useful in cases where only limited starting amounts of biological material are available The findings described here allow us to recommend several guidelines for designing a qualitative TDP study Three main questions set the frame for the experimental design: (1) what are the objectives of the study targeting a specific subgroup of proteoforms or reproducible identification of proteoforms with high throughput (2) How much biological material is available and what is the time needed for the analysis (3) How to avoid sample loss and artifacts Specific examples to explain the use of the decision tree are provided in the Supplementary Information the selection of the number and combination of parallel approaches depends on the sample amount and instrument time available should be avoided as this may lead to hydrolysis of peptide bonds C terminal of aspartate residues; (2) the pH value of the cell lysis solution should be neutral or guided by the specific research question with solution with acidic pH can effectively extract alkaline proteoforms and vice versa; (3) reduction and alkylation of proteoforms should be performed if there is no particular interest in assigning disulfides or other reversible cysteine modifications and (4) possible artificial modifications may be introduced by the cell lysis technique applied the components of the protease inhibitor or other chemicals used in sample preparation; if possible reagents that can lead to artificial modifications of proteoforms should be replaced The data presented can help users make an informed decision on sample preparation based on the specific research objectives The cOmplete protease inhibitor was from Roche and Acrylamide/Bis solution was from BioRad Human Caucasian colon adenocarcinoma (Caco-2) cells were from Sigma-Aldrich (cat fetal bovine serum and TrypLE Express Enzyme were from Thermo Fisher Scientific all other chemicals were from Sigma-Aldrich Deionized water (18.2 MΩ cm−1) was prepared by an arium611 VF system (Sartorius) Human Caco-2 cells were maintained according to the recommendations of the European Collection of Cell Cultures The cells were cultured at 37 °C and 5% CO2 in RPMI-1640 medium (25 mM HEPES 0.013 mM phenol red) supplemented with 10% (v/v) fetal bovine serum When a confluence between 90 and 100% was reached the cells were passaged by detaching with TrypLE Express Enzyme the cells were washed three times with PBS (200g The cells were stored at −70 °C until cell lysis and further use Caco-2 cells were lysed in 1× PBS (pH 7.4) supplemented with 1× cOmplete protease inhibitor by ultrasonication (5 × 20 s on ice) the protein concentration was determined by Pierce BCA assays (Thermo Fisher Scientific) following the manufacturer’s instructions protein determination was performed by BCA assay (Thermo Fisher and proteoforms were purified by MCW precipitation and injected twice using the LMW and HMW methods For protein purification, MCW precipitation was used9 Cell lysates containing 100 or 500 µg of protein were made up to 125 µl with MilliQ and 400 µl of methanol 100 µl of chloroform and 275 µl of MilliQ were added The samples were vortexed and centrifuged for 10 min at 14,000g The upper phase was removed and 600 µl of methanol was added and the protein pellet was washed twice with 600 µl of methanol The protein pellet was dried in a fume hood and stored at −20 °C until further processing To investigate the influence of cysteine reduction or alkylation on proteoform identifications Caco-2 cells were lysed and the proteoforms were aliquoted and subjected in triplicates to reduction with or without subsequent alkylation Caco-2 cells were lysed in 8 M GndHCl and 200 mM triethylammonium bicarbonate (pH 8.5) supplemented with 1× cOmplete protease inhibitor The proteoforms (100 µg in 50 µl of lysis buffer) were reduced by adding 1.6 µl of 200 mM tris(2-carboxyethyl)phosphine and incubation at 50 °C for 1 h alkylation was performed by adding 3.3 µl of 375 mM iodoacetamide and incubating for 30 min at room temperature in the dark the proteoforms were purified by MCW precipitation 100 µg of Caco-2 proteins were purified by MCW precipitation The proteoforms were resuspended in 200 µl of LC–MS loading buffer (3% ACN Note that a considerable proportion of the pellet could not be dissolved and was visible as an insoluble precipitate the samples were centrifuged for 20 min at 4 °C For the enrichment of proteoforms below approximately 30 or 50 kDa appropriate MWCO filters were used (Amicon Ultra-0.5 500 µg of Caco-2 proteins were precipitated by MCW precipitation and resuspended in 500 µl of 8 M GndHCl The MWCO filters were equilibrated by adding 500 µl of 8 M GndHCl and centrifugation for 15 min at room temperature The filters were then transferred to a new reaction vial The filters were centrifuged for approximately 15 min at 14,000g until 50 µl of the samples remained on the filter The filter was centrifuged again after adding 8 M GndHCl to a total volume of 500 µl was purified with an equilibrated 3 kDa MWCO filter the sample was centrifuged at 14,000g for 30 min and the flowthrough was discarded (residue in the filter was approximately 50 µl) the sample was washed twice with 100 mM TEAB (pH 8.5) on the filter The residual containing the re-buffered proteoforms was transferred to a new 1.5-ml reaction tube where the filter was inverted and centrifuged at 2,000g for 3 min The sample was lyophilized and solubilized in 50 µl of LC–MS loading buffer one band below approximately 30 kDa of two adjacent lanes was excised based on the prestained protein marker without previous fixation or staining of the gel After incubation for 10 min at 20 °C and 1,500 rpm the gel pieces were removed using a CoStar-X filter (2,000g the solutions were subjected to 3-kDa MWCO filters and washed (13,500g 25 min) twice with 8 M urea and 100 mM ABC the proteoforms were purified by anion-exchange material via the stage-tip protocol The stage tip was conditioned with 40 µl of methanol and equilibrated with 120 µl of 100 mM ABC (7,000g The sample was loaded on the stage tip and washed with 120 µl of 100 mM ABC The proteoforms were eluted with 80 µl of 50% ethanol and 0.5% formic acid The sample was lyophilized and solubilized in 20 µl of LC–MS loading buffer The proteoform fractionation by GELFrEE (Expedeon) was performed according to the manufacturer’s recommendations 500 µg of Caco-2 proteins were purified by MCW precipitation and resuspended in 30 µl GELFrEE loading buffer 112 µl of MilliQ and 8 µl of 1 M dithiothreitol The sample was incubated for 10 min at 50 °C before loading in the sample loading chamber For the enrichment of proteoforms smaller than approximately 30 kDa an 8% Tris-Acetate cartridge was used and the separation was performed according to the manufacturer’s protocol Only the first fraction containing proteoforms smaller than ~30 kDa was purified by MCW precipitation and resuspended in 50 µl of LC–MS loading buffer before LC–MS/MS analysis The sample was lyophilized and resuspended in 20 µl of LC–MS loading buffer before LC–MS/MS analysis 500 µg of Caco-2 proteins were purified by MCW precipitation and resuspended in 150 µl 8 M GndHCl the sample was centrifuged for 20 min at 21,100g and the supernatant was transferred into a new reaction vial 50 mg; PerkinElmer) cartridge was used for SPE The SPE material was activated by 2× 1 ml 100% ACN and equilibrated by 2× 1 ml 5% formic acid before sample loading The sample was washed twice with 5% formic acid and eluted with 300 µl of 70% ACN and 300 µl of 100% ACN The proteins were resuspended in 50 µl of LC–MS loading buffer before LC–MS/MS analysis 500 µg of Caco-2 proteins were purified by MCW precipitation and 76% ACN supplemented with 50 mM sodium chloride The small protein fraction was resuspended by vigorous vortexing ultrasonication and incubation for 1 h at 20 °C and 1,500 rpm 20 °C) to remove the insoluble (large) proteoforms and the supernatant was transferred in a new reaction vial 100 µl of MilliQ was added and the sample was frozen at −80 °C The proteins were resuspended in 20 µl of LC–MS loading buffer before being subjected to LC–MS/MS analysis The fractions were vacuum-dried by lyophilization and resuspended in 40 µl of LC–MS loading buffer The pools were dried by lyophilization and resuspended in 20 µl of LC–MS loading buffer before LC–MS/MS analysis The processing steps were combined in a multiconsensus search for merging multiple datasets The C-score is a metric that defines well-characterized proteoforms and stringent filtering allows the minimization of false-positive proteoform identifications In the Supplementary Notes various observations that notoriously occur during sample preparation and LC–MS/MS analysis are presented which can provide valuable insights into potential issues to consider when designing TDP experiments The GRAVY score is a measure for the hydrophobicity of the proteoforms: that is The term annotated proteoforms refers to full-length proteoforms deposited in the database and previously described truncated proteoforms subsequence proteoforms are all truncated ones not defined in the database The potential truncation sites were determined by matching the truncated proteoform sequence to the full-length sequence deposited in the database Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article Proteoform: a single term describing protein complexity Proteomes are of proteoforms: embracing the complexity Seeing the complete picture: proteins in top-down mass spectrometry Deep top-down proteomics revealed significant proteoform-level differences between metastatic and nonmetastatic colorectal cancer cells The Blood Proteoform Atlas: a reference map of proteoforms in human hematopoietic cells Mapping the proteoform landscape of five human tissues Laser capture microdissection-capillary zone electrophoresis-tandem mass spectrometry (LCM-CZE-MS/MS) for spatially resolved top-down proteomics: a pilot study of zebrafish brain Quantitative top-down proteomics in complex samples using protein-level tandem mass tag labeling Best practices and benchmarks for intact protein analysis for top-down mass spectrometry On-capillary cell lysis enables top-down proteomic analysis of single mammalian cells by CE-MS/MS Novel strategies to address the challenges in top-down proteomics Comparing top-down proteoform identification: deconvolution Multidimensional separations in top–down proteomics On the scalability and requirements of whole protein mass spectrometry Top-down proteomics and the challenges of true proteoform characterization Characterization of large intact protein ions by mass spectrometry: what directions should we follow Enhancing top-down proteomics of brain tissue with FAIMS Mass spectrometry-based top-down and bottom-up approaches for proteomic analysis of the Moroccan Buthus occitanus scorpion venom PEPPI-MS: polyacrylamide-gel-based prefractionation for analysis of intact proteoforms and protein complexes by mass spectrometry Recent advances in top-down proteome sample processing ahead of MS analysis Optimization of a top-down proteomics platform for closely related pathogenic bacterial discrimination Coupling high-field asymmetric waveform ion mobility spectrometry with capillary zone electrophoresis-tandem mass spectrometry for top-down proteomics Sensitive top-down proteomics analysis of a low number of mammalian cells using a nanodroplet sample processing platform Multiplexed size separation of intact proteins in solution phase for mass spectrometry Solid phase extraction as sample pretreatment method for top-down quantitative analysis of low molecular weight proteins from biological samples using liquid chromatography—triple quadrupole mass spectrometry Multidimensional separation schemes enhance the identification and molecular characterization of low molecular weight proteomes and short open reading frame-encoded peptides in top-down proteomics A top-down proteomics platform coupling serial size exclusion chromatography and Fourier transform ion cyclotron resonance mass spectrometry Deep top-down proteomics using capillary zone electrophoresis-tandem mass spectrometry: identification of 5700 proteoforms from the Escherichia coli proteome Identification of proteoforms by top-down proteomics using two-dimensional 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bottom-up-based N-terminomics reveals pitfalls in top-down-based terminomics workflows Nonproteolytic cleavage of aspartyl proline bonds in the cellulosomal scaffoldin subunit from Clostridium thermocellum Depletion of high-molecular-mass proteins for the identification of small proteins and short open reading frame encoded peptides in cellular proteomes high-quality feature deconvolution for top-down proteomics MSTopDiff: a tool for the visualization of mass shifts in deconvoluted top-down proteomics data for the database-independent detection of protein modifications Artifactual isoform profile modification following treatment of human plasma or serum with protease inhibitor monitored by 2-dimensional electrophoresis and mass spectrometry Site-specific pyrolysis-induced cleavage at aspartic acid residue in peptides and proteins Optimization of protein-level tandem mass tag (TMT) labeling conditions in complex samples with top-down proteomics Toward a universal sample preparation method for denaturing top-down proteomics of complex proteomes Accurate estimation of context-dependent false discovery rates in top-down proteomics Size-based proteome fractionation through polyacrylamide gel electrophoresis combined with LC–FAIMS–MS for in-depth top-down proteomics Orbitrap mass spectrometry and high-field asymmetric waveform ion mobility spectrometry (FAIMS) enable the in-depth analysis of human serum proteoforms Revealing Corynebacterium glutamicum proteoforms through top-down proteomics Advancing top-down analysis of the human proteome using a benchtop quadrupole-orbitrap mass spectrometer UniProt: the Universal Protein Knowledgebase in 2023 PyOpenMS: a Python-based interface to the OpenMS mass-spectrometry algorithm library Pyteomics 4.0: five years of development of a Python proteomics framework ProteomeXchange provides globally coordinated proteomics data submission and dissemination Download references Steinbach for cultivating the human Caco-2 cells This work was supported by the DFG Cluster of Excellence ‘Precision Medicine in Inflammation’ decision to publish or preparation of the manuscript Open access funding provided by Christian-Albrechts-Universität zu Kiel Systematic Proteome Research and Bioanalytics Institute for Bioinformatics and Medical Informatics performed the experiments and LC–MS/MS measurements wrote the original draft of the manuscript review and editing of the final manuscript The authors declare no competing interests Nature Methods thanks Claire Boos, Alexander Leitner and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available in collaboration with the Nature Methods team Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Supplementary Table 5 Proteoforms identified Supplementary Table 6 Proteins with associates proteoforms identified Download citation DOI: https://doi.org/10.1038/s41592-024-02481-6 Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. World Journal of Microbiology and Biotechnology (2025) Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma. The small village of Tholey in southwestern Germany is home to the oldest monastery on German soil. Founded in the seventh century, the Abbey of St Maurice was plundered and badly damaged 1000 years later during the wars of the French Revolution, and only re-established in 1949. But local philanthropists have breathed new life into the old walls. The highlight of the extensive works are the stained glass windows for the north and south aisles of the abbey church by Afghanistan-born artist Mahbuba Maqsoodi and the three choir windows designed by Germany’s most famous living artist The art establishment are calling this the most significant project of its kind on the continent of Europe The dates displayed for an article provide information on when various publication milestones were reached at the journal that has published the article activities on preceding journals at which the article was previously under consideration are not shown (for instance submission All content on this site: Copyright © 2025 Elsevier B.V. This article contains an account of an attempted suicide If you or a loved one is thinking about suicide call or text the National Suicide Prevention Lifeline at 988 Crystal Tetu had a deep relationship with her grandmother "I was raised by my grandparents," said Tetu Very structured — we always had dinner together As Tholey aged and moved into an independent living facility Tetu said Tholey was known for showing new residents around and helping them feel comfortable "She just took it upon herself to help out new people." She was hospitalized several times for heart failure "This happened in May and June and the end of July," Tetu said and I missed that — she didn't tell me because she didn't want to ruin my honeymoon." Tholey ultimately told Tetu that she wasn't improving A problem arose: A doctor explained to Tetu that Tholey was no longer getting nutrients from food Her body was no longer digesting food because it needed that energy to keep her heart beating "She's not going to get better," Tetu said "and she was wondering why she had to suffer like that She just had to stick it out until she starved to death." because I strongly believe that my grandma shouldn't have had to suffer that way," Tetu said "There's suffering that is required as part of being alive Tetu said she supports medical aid in dying — also known as physician-assisted suicide and assisted death — a practice that is legal in 10 U.S a bill is in play at the Minnesota Legislature this year The potential passage of a MAID law in Minnesota has inspired passionate testimony from those who support — and those who oppose — the practice "My grandma should have had the option," Tetu said "It's modeled after the Oregon Death with Dignity law that's been in effect since 1997," said Dr a Minneapolis physician and Compassion & Choices' Doctors for Dignity program director "They must be able to self-ingest the medication through the GI tract Medical aid in dying is currently legal in 10 states and the District of Columbia Oregon was the first state to enact MAID policy in 1997 have based their MAID laws off of Oregon's the national campaign strategist for policy reform organization Death with Dignity are among the co-authors for Minnesota's End-of-Life Option Act in their respective chambers said he first heard about medical aid in dying from a constituent on the campaign trail in 2022 just through discussions about this being something that was important to them," Smith said how tragic some of these end-of-life moments are this great law in place that allowed some of these more horrific situations to be alleviated." she said her support for the bill comes through the lens of bodily autonomy have experienced patients' deaths," Boldon said "and I know what that can look like and how awful that can be for some people for people wanting to make their own decisions about what that looks like for them." has introduced MAID legislation several times since 2016 the End-of-Life Option Act has gained more traction compared to previous years is the longest bill hearing I've had so far as I've been in the Legislature and tragic hearing about people's loss of close family members and very difficult and it has since passed the House's public safety and judiciary committees and will be considered by the Commerce Finance and Policy Committee next "We've had it through the health committee which is a big barrier because the health committee is very limited on time," Smith said says a lot about what she thinks about the issue and what can be done with that bill this session." Boldon said she would be surprised if the Senate's End-of-Life Option Act (SF 1813) received a committee hearing due to time constraints and not having enough support to pass "It's something that I very much hope we can continue talking about because it's important but there is a diversity of thought around this within my caucus in the Senate," Boldon said "And so I don't see it having the votes to pass this session." Thoman said whether this bill makes it to Gov Tim Walz's desk depends on "whether Minnesotans get out there and start talking about how important this is to them." "Lawmakers have said they're dedicated to respect for bodily autonomy privacy in medical decision-makings," Thoman continued "And so this fits right along with their priorities and their values." Not all Minnesotans who have publicly commented on the End-of-Life Option Act have been in favor of the proposal there were three testifiers who said they live in Rochester the healthy and the sick," said Karin Charron of Rochester "The catechism of the Catholic Church says we are stewards Many testifiers — from a group of 40 Muslim doctors to the National Association of Catholic Nurses — shared Charron's opposition to MAID on religious and moral grounds "From our standpoint (at Death with Dignity)," Sugerman said "forcing all people in the country to adhere to a particular religious belief is just not the way we should be operating." at least three Rochester physicians submitted written testimony for the MAID bill "I believe this would be a significant detriment to the way medicine is practiced in Minnesota," wrote Dr "The supporters of this bill are undoubtedly well-intentioned in their desire to relieve suffering for terminally ill patients the means that they would legalize are highly unethical and should not be condoned by the state." That impact on practicing medicine is something the Minnesota Alliance for Ethical Healthcare is concerned about — in a statement alliance president Nancy Utoft said the proposal "introduces risks and unintended consequences for Minnesotans who already face severe health care disparities and marginalization people of color and people with limited income." "This proposed legislation would turn the practice of medicine on its head by creating a new standard of care that views assisted suicide the same as treatments that ease pain and increase quality of life," Utoft continued "It would force providers to inform patients of their option to die or to refer patients to a provider that will prescribe the lethal dosage How are patients — especially those who have previously experienced marginalization in health care — supposed to trust that doctors are always working in their best interests if this is a requirement of our care system?" Physicians and other medical providers are able to opt out of providing MAID to a patient who requests it some doctors have voiced concern over having to present MAID as an option to patients even if they would not offer that option "The only requirement is that if a patient requests that their medical records be transferred that that's done in a timely manner," Thoman said "That's really the only requirement for health professionals or organizations in this bill." No matter how Minnesota's End-of-Life Option Act shakes out this year the issue of medical aid in dying is something that will stay on Julianne Donaldson's mind until 2015 when they moved to Dundas to be closer to their hobby farm Don was diagnosed with esophageal and stomach cancer in which she asked her husband if he — faced with a dire diagnosis — ever thought of committing suicide I would never do something like that,'" Donaldson said we had a grandson who took his own life .. and until you experience something like that I don't think anyone can realize the depth of that action Don went through chemotherapy and radiation it became apparent that "he was going to lose his life to that horrible cancer." Donaldson said she was brushing her teeth when she heard Don I'm going to shoot myself,'" Donaldson said you're not,' and I turned him around and got him back in bed." After a conversation with a social worker and a nurse she learned what had changed for Don: the physical and mental pain was overwhelming "I didn't know it would be this hard," she remembers him saying and the hospice caretakers did so for the next two weeks until he passed away on Jan Donaldson now advocates for MAID legalization in Minnesota they should be allowed that bodily autonomy especially when there is no hope," Donaldson said it would have been so much of an easier passing for him and for us The years leading up to 1861 saw polarised politics One of the US’s foremost historians reflects on America’s Disunion – then and now “I tremble for my country when I reflect that God is just,” Thomas Jefferson wrote in 1781. The American revolution still raged, many of his own slaves had escaped, his beloved Virginia teetered on social and political chaos who had crafted the Declaration of Independence for this fledgling nation at war with the world’s strongest empire felt deeply worried about whether his new country could survive with slavery “daily exercised in tyranny,” with slaveholders practicing “unremitting despotism” He admitted that slaveholding rendered his own class depraved “despots” and destroyed the “amor patriae” of their bondsmen “Can the liberties of a nation be thought secure when we have removed their only firm basis a conviction in the minds of the people that these liberties are of the gift of God?” This advocate of the natural rights tradition ended his rumination with the vague entreaty that his countrymen “be contented to hope” that a “mollifying” of the conditions of slaves and a new “spirit” from the revolution would in the “order of events” save his country Danger cannot come from abroad … If destruction be our lot we must ourselves be its author and finisherAbraham Lincoln in 1838For that republic to survive it took far more than hope and a faith in progress it did not survive; in roughly four score years it tore itself asunder over the issue of racial slavery as well as over fateful contradictions in its constitution and the reimagining of the second republic that resulted form the pivot of American history The civil war sits like the giant sleeping dragon of American history ever ready to rise up when we do not expect it and strike us with unbearable fire It has happened here – existential civil war fought with unspeakable death and suffering for fundamentally different visions of the future as our first and second founders well understood Americans love to believe their history is blessed and exceptional the story of a people with creeds born of the Enlightenment that will govern the worst of human nature and inspire our “better angels” to hold us together But this most diverse nation in the world is still an experiment and we are once again in a political condition that has made us ask if we are on the verge of some kind of new civil conflict Abraham Lincoln worried about politicians’ unbridled ambition and about the “perpetuation of our political institutions” The abolitionist Elijah Lovejoy had just been murdered by a mob the previous year in Illinois Lincoln saw an “ill omen” across the land due to the slavery question He felt a deep sense of responsibility inherited from the “fathers” of the revolution How to preserve and renew “the edifice of liberty and equal rights” “At what point shall we expect the approach of danger?” Lincoln asked “By what means shall we fortify against it?” His worries made him turn inward “Shall we expect some transatlantic military giant to step the ocean Asia and Africa combined … could not by force in a trial of a thousand years.” Lincoln did not fear foreign enemies we must ourselves be its author and finisher Those words were prescient in Lincoln’s own century But they have a frightful clarity even today Are Americans on the verge of some kind of social disintegration as the writer Paul Starobin has recently asked in which he revisits the old thesis that the secession moment represented a “crisis of fear” that led tragically to disunion and war It remains to be seen whether we have a deep enough well of tolerance and faith in free speech to endure this “catharsis” we seem to seek Far-right protesters clash with anti-fascist demonstrators in Charlottesville Photograph: Michael N/Pacific/BarcroftImagesPsychological explanations do not fully explain America’s current political condition We are in conflict about real and divergent ideas Are we engaged in a “cold civil war” as one writer has suggested with so much rhetorical and real violence in the air have many serious people using words like “unprecedented” or phrases like “where in time are we” or “we haven’t been here before” Commentators and ordinary citizens have been asking how or where in the past we can find parallels for our current condition Trump has been the gift that keeps on giving his flouting of political and constitutional traditions his own party’s moral bankruptcy in its inability to confront him have forced the media to endlessly ask historians for help That moral cowardice by Republicans shows some glimmers of hope; Mitt Romney has just called out Trump accusing him of “unraveling … our national fabric” by his coziness with white supremacists and Senator Bob Corker of Tennessee charged Trump with putting the nation “in great peril” by his incompetence and racism Sixteen years ago, in the book, Race and Reunion: The Civil War in American Memory I made a simple claim: “As long as America has a politics of race it will have a politics of civil war memory.” Unfortunately as well as conferences and courses taught on the same subject that prescription seems truer now than ever The line from the killings of Travon Martin and Michael Brown through a myriad of other police shootings and then especially from the mass murder of nine African Americans in Charleston in June to the recent white supremacist demonstration and violence in Charlottesville mark a dizzying America is in the midst of yet another of its racial reckonings which always confront us with a shock of events we are Just now we are engaged in a frenzied wave of Confederate monument removals; it is a manifestation of how well-meaning Americans can demonstrate their anti-racism and full of admirable impulses But this too in all likelihood will not itself prepare us for the next shock of events nor our next reckoning All parallels are unsteady or untrustworthy But the present is always embedded in the past the fateful decade that led to the civil war After the Great Hunger in Ireland the US experienced an unprecedented immigration wave between 1845 and the mid-1850s prompting a rapid and powerful rise of nativism Irish and German Catholics were unwelcome and worse the nation’s first expansionist foreign conflict stimulated an explosive political struggle over the expansion of slavery The Fugitive Slave Act of 1850 caused a wave of “refugee” former slaves escaping the northern states into Canada as well as a widespread crisis over violent rescues of fugitive slaves the constant flight of slaves from the South to free states was became increasingly politicized as it became more radical Americans had to ask whether their institutions would last either disintegrated or broke into sectional parts Third parties suddenly emerged with success like no other time in our history whose xenophobia and anti-Catholicism got them elected in droves in New England in the early 1850s And the most successful third party in our history were born in direct resistance to the Kansas-Nebraska Act of 1854 and which opened up the western territories to the perpetual expansion of slavery A succession of weak and pro-slavery presidents from 1844 through 1860 either tarnished the institution of the presidency or deepened the sectional and partisan divide the supreme court weighed in by declaring in Dred Scott v Sandford that blacks were not and could never be citizens of the US “for more than a century been regarded as beings of an inferior order … so far inferior that they had no rights which the white man was bound to respect.” This most notorious court decision legally opened up all of the west all of the north to the presence of slavery So discredited was the supreme court among many northerners in the wake of the decision that the Republicans made resistance to the judiciary a rallying cry of their political insurgency That impulse led to the election of Lincoln in 1860 who firmly controlled that region’s politics as the primary impulse to secede from the union They believed they could not co-exist in a nation now led by a political organization devoted to their destruction By the time of the sectionalized and polarized election of 1860 conducted in a climate of violence and danger caused by John Brown’s raid on Harper’s Ferry in 1859 north and south had developed broad-based mutual conspiracy theories of each other They did so through a thriving and highly partisan press Both sides tended to have their own sets of facts and their own conceptions of both history and the constitution White southerners feared and loathed abolitionists and now they faced anti-slavery politicians who could truly affect power and legislation if elected pro-slavery interests had developed a widespread theory about a “black Republican” conspiracy in the north determined on taking hold of all reins of government to put slavery one southern leader after another pronounced against what they perceived as an abolitionist conspiracy against their livelihoods and their lives the secession commissioner for Mississippi 1860 that Republicans “now demand equality between the white and negro races under our constitution; equality in representation equality in the right of suffrage … equality in the social circle equality in the rights of matrimony.” He concluded therefore the deep south faced a stark choice: “Sink or swim she will never submit to the principles and policy of this black Republican administration.” advanced an equally potent idea of a “slave power” conspiracy that had grown into a staple of antislavery politics consisted of the southern slaveholding political class; they were obsessively bent on control of every level of government and every institution – presidency The slave power especially demanded control over future expansion of the United States in order for its system to survive The theory made greater sense with time to many people was increasingly a minority interest in the federal government No one made this case about the slave power better than the black abolitionist Frederick Douglass 1853 Douglass gave the slave power clear definition It was “a purely slavery party” in national affairs and its branches reached “far and wide in church and state” The conspiracy’s “cardinal objects” were suppression of abolitionist speech removal of free blacks from the United States the “nationalization” of slavery in every state of the union and the expansion of slavery to Mexico and South America Douglass saw the slave power as an all-encompassing national plague with “instinctive rapacity” with a “natural craving after human flesh and blood” It was a “murderous onslaught” upon the rights of all Americans to sustain the claims of a few would be “thawing a deadly viper instead of killing it” He had faith in the “monster’s” inherent tendency to over-reach and destroy itself “it is also crushing itself.” It had “made such a frightful noise” with the “Fugitive Slave Act… the Nebraska bill the recent marauding movements of the oligarchy in Kansas,” that it now performed as the abolitionists’ “most potent ally” Douglass detected a great change in northern public opinion Instead of regarding the abolitionists as mere fanatics “crying wolf” the masses now perceived the evil in their midst and themselves cried “kill the wolf” Thus we might see one of the strongest parallels of all between the road to disunion and our current predicament The rhetoric about the slave power and about black Republicans has a familiar ring today Millions of Americans on the right who garner their information from selective websites radio shows and Fox News possess all sorts conspiratorial conceptions of liberals and the alleged radical views of professors on university campuses Many on the left also know precious little about people in rural and suburban America who voted for Trump; coastal elites do sometimes hold contemptuous views bordering on the conspiratorial about the people they “fly over” Americans are more than politically polarized; we are bitterly divided about our expanding diversity about the right to vote and how to protect it over whether we even believe in a social contract between citizens and the polity we are divided over conflicting visions of our future Let us hope that we find ways to fight out our current conflicts within politics and not between each other in our over-armed society the white supremacist far right will become its own worst enemy As Americans consider the survival of their own amor patriae we might reflect on just how old our story is When Moses sent the Israelites across the Jordan he instructed them to put up memory stones to mark their journey and their story Americans have put up more than their share of memory stones and are just now living through a profound process of deciding which ones will remain But as we look deeply into just what our own amor patriae means we might think hard about what inscriptions we want written on the memory stones of our own times We might draw one from Douglass in 1867: “We ought to have our government so shaped that even when in the hands of a bad man we shall be safe.” View upcoming auction estimates and receive personalized email alerts for the artists you follow Gerhard Richter Gerhard Richter has completed his final major artwork On the occasion of the unveiling of three stained-glass windows in the Tholey Abbey in the Saarland in South Germany which were produced at the direction of the world’s foremost painter he spoke to the Deutsche Presse-Agentur (German press agency) and said the windows were surely his last ‘work number.’ Starting with Tisch in 1962 all of Richter’s works are registered in a directory which will now be complete with the windows assigned the number 957 The new Gerhard Richter stained glass windows in the Tholey Abbey The good news is that this does not mean that his creation will cease altogether While his age of 88 doesn’t permit Richter to continue with painting large-scale artworks he will create a new directory for drawings and sketches that he is now dedicating himself to “Not so great,” the coy and humble painter told German press The windows aren’t the first of their kind by Richter the “Richter-Fenster,’ created by use of 72 colors and a random pattern of small squares The execution of the Tholey Abbey windows was carried out by the Gustva van Treeck glass workshop in Munich Both the window patterns stand in close relation to paintings Richter has made in the past his 4096 Colours in the case of the Cologne window and motives from his book Patterns in the newly unveiled one It is not by accident that Richter came to design church windows with their unique effects Many of his abstract paintings in their colorful harmony evoke this spiritual effect like the meeting of a setting sun with the colorful stained glass of a house of prayer The majority of Richter’s work is dedicated to abstraction but also to photorealistic paintings and his own kind of surrealism His oeuvre stands somewhere between Modernist Art and developed into the Contemporary but he stands as living witness to a period that has largely faded The secondary market has long endowed his masterpieces with eight-figure values albeit the figures for such a major persona in the chronicles of art history still come in short compared to some of his contemporaries and successors but the test of time might change that balance # of Lots Offered and Sold: Gerhard Richter The most expensive recorded artwork by the German sold for $46.4 million at Sotheby’s in 2015 establishing a new record for an artwork by a living artist earning Richter’s dismay at the astronomical sums spent on his art The record was broken the same year still by Jeff Koons’ Balloon Dog (Orange) but Richter’s paintings are still the fourth fifth and sixth most expensive works by a living artist The mania in the first half of the previous decade surrounding his works has cooled slightly in the last years evident by the decline of yearly performances in the second half This is not to say that an average yearly sales value north of $150 million is not substantial but the records of 2012 and 2014 with $293.8 million and $289.6 million Not that he would have any ambition to do so the master painter was granted another special honor when Sotheby’s named its summer concluding sale Rembrandt to Richter demonstrating his importance in a sale filled with major names of 500 years of art history The monumentally-sized Wolken (Fenster) only reservedly fulfilled its expectations as it achieved $13.5 million after a $11.6 million - $15.5 million estimate This year’s high is expected to be broken on October 06 when Sotheby’s will offer Abstraktes Bild (649-2) at its Hong Kong Contemporary Sale with an estimate of $15.3 million - $17.9 million is not looking to break records and is and was largely interested in art for its own sake It is surely a large part of why he has achieved such depth and brilliance and is revered by the world The completion of his last major work is therefore a sad development for the art world but a fitting and well-deserved one for an artist who has been creating for almost 60 years.   For more on auctions, exhibitions, and current trends, visit our Magazine Page By Flora Graham (Image: EURAC Institute for Mummies and the Iceman) When peering at these magnified blood cells, you’re not just looking closer than the naked eye can – you’re also looking through the mists of time. The dark areas on the cells show bruising that occurred over 5000 years ago, when Ötzi the iceman met his end on the Schnalstal glacier in the Alps Ötzi seems to have died just hours after sustaining serious injuries in hand-to-hand combat. Now Frank Maixner from the Institute for Mummies and the Iceman in Bolzano and Andreas Tholey from Kiel University in Germany have found evidence that he received a blow to the forehead that caused his brain to knock against the back of his skull the blood cell samples were extracted through tiny holes that already existed By investigating the cells’ proteins the researchers were able to see a snapshot of Ötzi’s last hours regardless of where it originates in the body whereas proteins provide precise information about what is happening in specific regions within the body,” says Tholey “When you think that we have succeeded in identifying actual tissue changes in a human who lived over 5000 years ago you can begin to understand how pleased we are.” Ötzi has already been photographed more than the most paparazzi-loved celebrity – the Iceman photoscan website puts many of these images online Journal reference: Cellular and Molecular Life Sciences, DOI: 10.1007/s00018-013-1360-y Clarification: When this article was first published the Leipzig-based startup behind the vegan chocolate and protein brands Nucao and Numove has just bagged €14 million in a Series A round of financing Proceeds from the round will fuel Nu’s ongoing expansion amid a record-breaking year of sales for the company driven by rising consumer demand for health-conscious and plant-based foods.   German startup The Nu Company has announced the closing of its Series A funding with more than €14 million also saw participation from Paris-headquartered food tech investors Five Seasons Ventures and DX Ventures the VC arm of food delivery giant Delivery Hero Other notable investors include former racing driver and Formula 1 world racing champion Nico Rosberg who has since his retirement backed a number of green tech startups like Tier Mobility as well as BackBone Ventures and the Austrian F&B investors Square One Foods which is the startup’s first financing round will help it grow its distribution across Europe and internationally.  “This Series A will help us bring more products to market that can bring about a real and positive change in the food industry,” said co-founder and CMO Christian Fenner “We want our products to be even easier to access throughout Europe whether at the local supermarket or with a single click online.”  Fenner launched the startup back in 2016 with Thomas Stoffels and Mathias Tholey and it has since rolled out two successful consumer-facing brands plastic-free and low-sugar vegan chocolate and protein bars and snacks and free from refined sugars and packaged in home compostable cellulose-based wrappers.  For every product sold, Nu sets aside a portion of proceeds to support reforestation projects led by nonprofit organisation Eden Nu’s funding round comes amid surging demand for more sustainable and healthy foods, especially climate-friendly snacks, which is quickly becoming a big trend in the food industry Nu itself recorded triple-digit sales growth for 2021 for the fourth consecutive year and the brand is now retailed in more than 10,000 stores Co-founder and CFO Stoffels says that the startup is now on track to plant more than 10 million trees before the end of 2021.  “We don‘t stop at our own products. There needs to be a change in consciousness in the industry as a whole,” said Tholey, who issued with his co-founders an open call for climate action on the part of German politicians and corporations “We’re putting the pressure on and demanding that politicians and large corporations follow suit so we can treat consumers and our planet with respect and operate in a way that’s appropriate for all generations.”  Nu’s purpose-driven business is a major part of why it believes the brand is poised for growth amid changing consumer trends “The goal is always to bring a healthier option to everyone’s table without neglecting the environment,” said principal Clément Helinckx.  “The result is excellent consumer engagement and outstanding growth rates,” Helinckx continued “It’s exciting to see a brand spread its wings so quickly This shows a clear adjustment of the market and a great deal of potential to trigger long-term changes.”  Founded by serial entrepreneur Sonalie Figueiras in 2011 Green Queen is a multi-channel digital news platform and a trusted global impact media brand Our award-winning reporting reaches millions of readers globally Green Queen is the world’s leading food and climate media with a focus on future food innovation and food system decarbonization one of the most important consumer products and investment opportunities of our time Our coverage includes breaking news and product launches and exclusive interviews with entrepreneurs and key ecosystem players from every continent Green Queen is an editorially-driven media publication Over 98% of our content is editorial and independent Paid posts are clearly marked as such: look for 'This is a Green Queen Partner Post' at the bottom of the page.