One-Tip enables comprehensive proteome coverage in minimal cells and single zygotes.

Mass spectrometry (MS)-based proteomics workflows typically involve complex, multi-step processes, presenting challenges with sample losses, reproducibility, requiring substantial time and financial investments, and specialized skills. Here we introduce One-Tip, a proteomics methodology that seamlessly integrates efficient, one-pot sample preparation with precise, narrow-window data-independent acquisition (nDIA) analysis. One-Tip substantially simplifies sample processing, enabling the reproducible identification of >9000 proteins from ~1000 HeLa cells. The versatility of One-Tip is highlighted by nDIA identification of ~6000 proteins in single cells from early mouse embryos. Additionally, the study incorporates the Uno Single Cell Dispenser™, demonstrating the capability of One-Tip in single-cell proteomics with >3000 proteins identified per HeLa cell. We also extend One-Tip workflow to analysis of extracellular vesicles (EVs) extracted from blood plasma, demonstrating its high sensitivity by identifying >3000 proteins from 16 ng EV preparation. One-Tip expands capabilities of proteomics, offering greater depth and throughput across a range of sample types.

Human-triggered magnification of erosion rates in European Alps since the Bronze Age.

A major feature of the Anthropocene is the drastic increase in global soil erosion. Soil erosion is threatening Earth habitability not only as soils are an essential component of the Earth system but also because societies depend on soils. However, proper quantification of the impact of human activities on erosion over thousands of years is still lacking. This is particularly crucial in mountainous areas, where the highest erosion rates are recorded. Here we use the Lake Bourget catchment, one of the largest in the European Alps, to estimate quantitatively the impact of human activities on erosion. Based on a multi-proxy, source-to-sink approach relying on isotopic geochemistry, we discriminate the effects of climate fluctuations from those of human activities on erosion over the last 10,000 years. We demonstrate that until 3800 years ago, climate is the only driver of erosion. From that time on, climate alone cannot explain the measured rates of erosion. Thanks to an unprecedented regional paleoenvironmental reconstruction, we highlight that the development of pastoralism at high altitudes from the Bronze Age onwards and the extension of agriculture starting in the Middle Ages were key factors in the drastic increase in erosion observed in the Alps.

Wnt/ß-catenin and NFκB signaling synergize to trigger growth factor-free regeneration of adult primary human hepatocytes.

BACKGROUND AND AIMS: The liver has a remarkable capacity to regenerate, which is sustained by the ability of hepatocytes to act as facultative stem cells that, while normally quiescent, re-enter the cell cycle after injury. Growth factor signaling is indispensable in rodents, whereas Wnt/ß-catenin is not required for effective tissue repair. However, the molecular networks that control human liver regeneration remain unclear. METHODS: Organotypic 3D spheroid cultures of primary human or murine hepatocytes were used to identify the signaling network underlying cell cycle re-entry. Furthermore, we performed chemogenomic screening of a library enriched for epigenetic regulators and modulators of immune function to determine the importance of epigenomic control for human hepatocyte regeneration. RESULTS: Our results showed that, unlike in rodents, activation of Wnt/ß-catenin signaling is the major mitogenic cue for adult primary human hepatocytes. Furthermore, we identified TGFß inhibition and inflammatory signaling through NF-κB as essential steps for the quiescent-to-regenerative switch that allows Wnt/ß-catenin-induced proliferation of human cells. In contrast, growth factors, but not Wnt/ß-catenin signaling, triggered hyperplasia in murine hepatocytes. High-throughput screening in a human model confirmed the relevance of NFκB and revealed the critical roles of polycomb repressive complex 2, as well as of the bromodomain families I, II, and IV. CONCLUSIONS: This study revealed a network of NFκB, TGFß, and Wnt/ß-catenin that controls human hepatocyte regeneration in the absence of exogenous growth factors, identified novel regulators of hepatocyte proliferation, and highlighted the potential of organotypic culture systems for chemogenomic interrogation of complex physiological processes.

GLP-1R signaling neighborhoods associate with the susceptibility to adverse drug reactions of incretin mimetics.

G protein-coupled receptors are important drug targets that engage and activate signaling transducers in multiple cellular compartments. Delineating therapeutic signaling from signaling associated with adverse events is an important step towards rational drug design. The glucagon-like peptide-1 receptor (GLP-1R) is a validated target for the treatment of diabetes and obesity, but drugs that target this receptor are a frequent cause of adverse events. Using recently developed biosensors, we explored the ability of GLP-1R to activate 15 pathways in 4 cellular compartments and demonstrate that modifications aimed at improving the therapeutic potential of GLP-1R agonists greatly influence compound efficacy, potency, and safety in a pathway- and compartment-selective manner. These findings, together with comparative structure analysis, time-lapse microscopy, and phosphoproteomics, reveal unique signaling signatures for GLP-1R agonists at the level of receptor conformation, functional selectivity, and location bias, thus associating signaling neighborhoods with functionally distinct cellular outcomes and clinical consequences.

Pharmacovigilance studies without a priori hypothesis: systematic review highlights inappropriate multiple testing correction procedures.

OBJECTIVES: The purpose of this study was to systematically review the statistical methods used in pharmacovigilance studies without a priori hypotheses. STUDY DESIGN AND SETTING: A systematic review was performed on studies published in the MEDLINE database between 2012 and 2021. The included studies were analyzed for database name and type, statistical methods, anatomical therapeutic chemical class for the studied drug(s), and SOC MedDRA classification for the studied adverse drug reaction. RESULTS: Ninety-two studies were included, with pharmacovigilance databases being the most used type. Disproportionality analysis using frequentist or Bayesian methods was the most common statistical method employed. The most studied drug classes were anti-infectives, nervous system drugs, and antineoplastics and immunomodulators. However, no common procedure was implemented to correct for multiple testing. CONCLUSION: This review highlights the limited number of statistical methods employed for pharmacovigilance studies without a priori hypotheses, with no established consensus-based method and a lack of interest in multiple testing correction. The establishment of guidelines is recommended to improve the performance of such studies.

Antigen-presenting autoreactive B cells activate regulatory T cells and suppress autoimmune arthritis in mice.

B cells undergo several rounds of selection to eliminate potentially pathogenic autoreactive clones, but in contrast to T cells, evidence of positive selection of autoreactive B cells remains moot. Using unique tetramers, we traced natural autoreactive B cells (C1-B) specific for a defined triple-helical epitope on collagen type-II (COL2), constituting a sizeable fraction of the physiological B cell repertoire in mice, rats, and humans. Adoptive transfer of C1-B suppressed arthritis independently of IL10, separating them from IL10-secreting regulatory B cells. Single-cell sequencing revealed an antigen processing and presentation signature, including induced expression of CD72 and CCR7 as surface markers. C1-B presented COL2 to T cells and induced the expansion of regulatory T cells in a contact-dependent manner. CD72 blockade impeded this effect suggesting a new downstream suppressor mechanism that regulates antigen-specific T cell tolerization. Thus, our results indicate that autoreactive antigen-specific naïve B cells tolerize infiltrating T cells against self-antigens to impede the development of tissue-specific autoimmune inflammation.

Analysis of local extracellular matrix identifies different aetiologies behind bicuspid and tricuspid aortic valve degeneration and suggests therapies.

Aortic valve degeneration (AVD) is a life-threatening condition that has no medical treatment and lacks individual therapies. Although extensively studied with standard approaches, aetiologies behind AVD are unclear. We compared abundances of extracellular matrix (ECM) proteins from excised valve tissues of 88 patients with isolated AVD of normal tricuspid (TAV) and congenital bicuspid aortic valves (BAV), quantified more than 1400 proteins per ECM sample by mass spectrometry, and demonstrated that local ECM preserves molecular cues of the pathophysiological processes. The BAV ECM showed enrichment with fibrosis markers, namely Tenascin C, Osteoprotegerin, and Thrombospondin-2. The abnormal physical stress on BAV may cause a mechanical injury leading to a continuous Tenascin C-driven presence of myofibroblasts and persistent fibrosis. The TAV ECM exhibited enrichment with Annexin A3 (p = 1.1 × 10-16 and the fold change 6.5) and a significant deficit in proteins involved in high-density lipid metabolism. These results were validated by orthogonal methods. The difference in the ECM landscape suggests distinct aetiologies between AVD of BAV and TAV; warrants different treatments of the patients with BAV and TAV; elucidates the molecular basis of AVD; and implies possible new therapeutic approaches. Our publicly available database (human_avd_ecm.surgsci.uu.se) is a rich source for medical doctors and researchers who are interested in AVD or heart ECM in general. Systematic proteomic analysis of local ECM using the methods described here may facilitate future studies of various tissues and organs in development and disease.

Therapy targeting antigen-specific T cells by a peptide-based tolerizing vaccine against autoimmune arthritis.

A longstanding goal has been to find an antigen-specific preventive therapy, i.e., a vaccine, for autoimmune diseases. It has been difficult to find safe ways to steer the targeting of natural regulatory antigen. Here, we show that the administration of exogenous mouse major histocompatibility complex class II protein bounding a unique galactosylated collagen type II (COL2) peptide (Aq-galCOL2) directly interacts with the antigen-specific TCR through a positively charged tag. This leads to expanding a VISTA-positive nonconventional regulatory T cells, resulting in a potent dominant suppressive effect and protection against arthritis in mice. The therapeutic effect is dominant and tissue specific as the suppression can be transferred with regulatory T cells, which downregulate various autoimmune arthritis models including antibody-induced arthritis. Thus, the tolerogenic approach described here may be a promising dominant antigen-specific therapy for rheumatoid arthritis, and in principle, for autoimmune diseases in general.

The legacy of metallurgical atmospheric contamination in a mountainous catchment: A delayed response of Pb contamination.

Metal-rich fumes emitted during ore smelting contribute to widespread anthropogenic contamination. Environmental archives (such as lake sediments) record fallouts deposited on lake and terrestrial surfaces during ancient mining and smelting activities. However, very few is known about the potential buffering effect of soils upon which metal falls out, prior to be released through runoff and or/erosion, hence leading to pervasive contamination fluxes long after the ceasing of metallurgical activities. Here we aim at assessing this long-term remobilisation in a mountainous catchment area. Lake sediments and soils were collected 7 km upward a 200-year-old historic mine. The PbAg mine of Peisey-Nancroix was operated between the 17th and the 19th centuries with a documented smelting period of 80 years. In lake sediments, the total Pb content varies from 29 mg.kg-1 prior smelting to 148 mg.kg-1 during ore smelting. Pb isotopes in lake sediments and soils provide evidence of anthropogenic Pb from the local ore (206Pb/207Pb = 1.173; 208Pb/206Pb = 2.094) during and after smelting, suggesting anthropogenic Pb remobilisation for 200 years. The accumulation rates of anthropogenic Pb calculated in lake sediments after the smelting period confirm such a remobilisation. Despite a decrease in this accumulation rate through time, soils still contain significant stocks of anthropogenic Pb (54-89 % of PbANTH). The distribution of present-day anthropogenic Pb in the catchment area depends mainly on topographic characteristics. Coupling lake sediments and soils investigations is thus necessary to constrain the long-term persistence and remobilisation of a diffuse contamination related to mining activities.

Bariatric Surgery Should Be Proposed in Certain Septuagenarian Patients with Obesity.

Bariatric surgery (BS) is rarely performed on patients aged 70 and over, due to the fear of adverse effects, particularly related to sarcopenia. We examined the outcome of obese patients who underwent BS after the age of 69 in the French population. Operated subjects were matched with non-operated obese patients (n = 1307 in each group after matching). We showed that BS was associated with a reduction in mortality and no increase in the risks of rehospitalization or fracture events.

Intense touristic activities exceed climate change to shape aquatic communities in a mountain lake.

Mountain lakes are especially vulnerable to climate change, but are also increasingly exposed to local anthropogenic development through winter and summer tourism. In this study, we aimed to tease apart the influence of tourism from that of climate in a mountain lake located within one of the largest French ski resorts, by combining paleolimnological and present ecological data. The reconstructed long-term ecological dynamics highlighted an increase in lake biological production from the end of the Little Ice Age up to the 1950s, suggesting a historical dominance of climate control. Afterward, a major drop in pelagic production occurred at the same time as the watershed erosion increased and peaked in the 1990s, concomitant with massive digging for the ski resort expansion. The benthic invertebrates collapsed in the 1980s, concomitantly with the onset of massive salmonid stocking and recent warming. Stable isotope analyses identified benthic invertebrates as the major salmonid diet resource and suggested a possible direct impact of salmonid stocking on benthic invertebrates. However, habitat use may differ among salmonid species as suggested by the way fish DNA was preserved in surficial sediment. The high abundances of macrozooplankton further confirmed the limited reliance of salmonids on pelagic resources. The variable thermal tolerance of benthic invertebrates suggested that the recent warming may mostly affect littoral habitats. Our results indicate that winter and summer tourism may differently affect the biodiversity of mountain lakes and could collectively interfere with the ecological impacts of recent warming, making local management of primary importance to preserve their ecological integrity. Supplementary Information: The online version contains supplementary material available at 10.1007/s00027-023-00968-6.

Holocene variations in Lake Titicaca water level and their implications for sociopolitical developments in the central Andes.

Holocene climate in the high tropical Andes was characterized by both gradual and abrupt changes, which disrupted the hydrological cycle and impacted landscapes and societies. High-resolution paleoenvironmental records are essential to contextualize archaeological data and to evaluate the sociopolitical response of ancient societies to environmental variability. Middle-to-Late Holocene water levels in Lake Titicaca were reevaluated through a transfer function model based on measurements of organic carbon stable isotopes, combined with high-resolution profiles of other geochemical variables and paleoshoreline indicators. Our reconstruction indicates that following a prolonged low stand during the Middle Holocene (4000 to 2400 BCE), lake level rose rapidly ~15 m by 1800 BCE, and then increased another 3 to 6 m in a series of steps, attaining the highest values after ~1600 CE. The largest lake-level increases coincided with major sociopolitical changes reported by archaeologists. In particular, at the end of the Formative Period (500 CE), a major lake-level rise inundated large shoreline areas and forced populations to migrate to higher elevation, likely contributing to the emergence of the Tiwanaku culture.

Temporal pesticide dynamics alter specific eukaryotic taxa in a coastal transition zone.

Land use change and anthropogenic forcing can drastically alter the rates and patterns of sediment transport and modify biodiversity and ecosystem functions in coastal transition zones, such as the coastal ecosystems. Molecular studies of sediment extracted DNAs provide information on currently living organisms within the upper layers or buried from various periods of time, but might also provide knowledge on species dynamics, replacement and turnover. In this study, we evaluated the eukaryotic communities of a marine core that present a shift in soil erosion that was linked to glyphosate usage and correlated to chlordecone resurgence since 2000. We show differences in community composition between samples from the second half of the last century and those from the last two decades. Temporal analyses of the relative abundance, alpha diversity, and beta diversity for the two periods demonstrated different temporal dynamics depending on the considered taxonomic group. In particular, Ascomycetes showed a decrease in abundance over the most recent period associated with changes in community membership but not community structure. Two photosynthetic groups, Bacillariophyceae and Prasinophytes clade VII, showed a different pattern with an increase in abundance since the beginning of the 21st century with a decrease in diversity and evenness to form more heterogeneous communities dominated by a few abundant OTUs. Altogether, our data reveal that agricultural usages such as pesticide use can have long-term and species-dependent implications for microeukaryotic coastal communities on a tropical island.

System-Wide Profiling by Proteome Integral Solubility Alteration Assay of Drug Residence Times for Target Characterization.

Most drugs are used in the clinic and drug candidate target multiple proteins, and thus detailed characterization of their efficacy targets is required. While current methods rely on quantitative measurements at thermodynamic equilibrium, kinetic parameters such as the residence time of a drug on its target provide a better proxy for efficacy in vivo. Here, we present a residence time proteome integral solubility alteration (ResT-PISA) assay, which facilitates monitoring temporal protein solubility profiles after drug removal ("off-curve") in cell lysates or intact cells, quantifying the lifetime of drug-target interaction. A compressed version of the assay measures the integral under the off-curve enabling the multiplexing of binding affinity and residence time assessments into a single proteomic analysis. We introduce a combined scoring system for three parametric dimensions to improve prioritization of targets. By providing complementary information to other characteristics of drug-target interaction, the ResT-PISA approach will be useful in drug development and precision medicine.

240Pu/239Pu signatures allow refining the chronology of radionuclide fallout in South America.

Atmospheric nuclear tests (1945-1980) have led to radioactive fallout across the globe. French tests in Polynesia (1966-1974) may influence the signature of fallout in South America in addition to those conducted by USA and former USSR until 1963 in the Northern hemisphere. Here, we compiled the 240Pu/239Pu atom ratios reported for soils of South America and conducted additional measurements to examine their latitudinal distributions across this continent. Significantly lower ratio values were found in the 20-45° latitudinal band (0.04 to 0.13) compared to the rest of the continent (up to 0.20) and attributed to the contribution of the French atmospheric tests to the ultra-trace plutonium levels found in these soils. Based on sediment cores collected in lakes of Chile and Uruguay, we show the added value of measuring 240Pu/239Pu atom ratios to refine the age models of environmental archives in this region of the world.

Comprehensive Target Screening and Cellular Profiling of the Cancer-Active Compound b-AP15 Indicate Abrogation of Protein Homeostasis and Organelle Dysfunction as the Primary Mechanism of Action.

Dienone compounds have been demonstrated to display tumor-selective anti-cancer activity independently of the mutational status of TP53. Previous studies have shown that cell death elicited by this class of compounds is associated with inhibition of the ubiquitin-proteasome system (UPS). Here we extend previous findings by showing that the dienone compound b-AP15 inhibits proteasomal degradation of long-lived proteins. We show that exposure to b-AP15 results in increased association of the chaperones VCP/p97/Cdc48 and BAG6 with proteasomes. Comparisons between the gene expression profile generated by b-AP15 to those elicited by siRNA showed that knock-down of the proteasome-associated deubiquitinase (DUB) USP14 is the closest related to drug response. USP14 is a validated target for b-AP15 and we show that b-AP15 binds covalently to two cysteines, Cys203 and Cys257, in the ubiquitin-binding pocket of the enzyme. Consistent with this, deletion of USP14 resulted in decreased sensitivity to b-AP15. Targeting of USP14 was, however, found to not fully account for the observed proteasome inhibition. In search for additional targets, we utilized genome-wide CRISPR/Cas9 library screening and Proteome Integral Solubility Alteration (PISA) to identify mechanistically essential genes and b-AP15 interacting proteins respectively. Deletion of genes encoding mitochondrial proteins decreased the sensitivity to b-AP15, suggesting that mitochondrial dysfunction is coupled to cell death induced by b-AP15. Enzymes known to be involved in Phase II detoxification such as aldo-ketoreductases and glutathione-S-transferases were identified as b-AP15-targets using PISA. The finding that different exploratory approaches yielded different results may be explained in terms of a "target" not necessarily connected to the "mechanism of action" thus highlighting the importance of a holistic approach in the identification of drug targets. We conclude that b-AP15, and likely also other dienone compounds of the same class, affect protein degradation and proteasome function at more than one level.

Ion-Based Proteome-Integrated Solubility Alteration Assays for Systemwide Profiling of Protein-Molecule Interactions.

Unbiased drug target engagement deconvolution and mechanism of action elucidation are major challenges in drug development. Modification-free target engagement methods, such as thermal proteome profiling, have gained increasing popularity in the last several years. However, these methods have limitations, and, in any case, new orthogonal approaches are needed. Here, we present a novel isothermal method for comprehensive characterization of protein solubility alterations using the effect on protein solubility of cations and anions in the Hofmeister series. We combine the ion-based protein precipitation approach with Proteome-Integrated Solubility Alteration (PISA) analysis and use this I-PISA assay to delineate the targets of several anticancer drugs both in cell lysates and intact cells. Finally, we demonstrate that I-PISA can detect solubility changes in minute amounts of sample, opening chemical proteomics applications to small and rare biological material.

Accelerating High-Dimensional Temporal Modelling Using Graphics Processing Units for Pharmacovigilance Signal Detection on Real-Life Data.

Adverse drug reaction is a major public health issue. The increasing availability of medico-administrative databases offers major opportunities to detect real-life pharmacovigilance signals. We have recently adapted a pharmacoepidemiological method to the large dimension, the WCE (Weigthed Cumulative Exposure) statistical model, which makes it possible to model the temporal relationship between the prescription of a drug and the appearance of a side effect without any a priori hypothesis. Unfortunately, this method faces a computational time problem. The objective of this paper is to describe the implementation of the WCE statistical model using Graphics Processing Unit (GPU) programming as a tool to obtain the spectrum of adverse drug reactions from medico-administrative databases. The process is divided into three steps: pre-processing of care pathways using the Python library Panda, calculation of temporal co-variables using the Python library "KeOps", estimation of the model parameters using the Python library "PyTorch" - standard in deep learning. Programming the WCE method by distributing the heaviest portions (notably spline calculation) on the GPU makes it possible to accelerate the time required for this method by 1000 times using a computer graphics card and up to 10,000 times with a GPU server. This implementation makes it possible to use WCE on all the drugs on the market to study their spectrum of adverse effects, to highlight new vigilance signals and thus to have a global vigilance tool on medico-administrative database. This is a proof of concept for the use of this technology in epidemiology.