Andrew Quinn
email: andrew.quinn@unil.ch

Saurav Subedi

Roshni M Pasanna

Dev Kapil

Singh

Mélina Richard
email: melinadenise.richard@unibas.ch

Dilan Sezer
email: dilan.sezer@unibas.ch

Sarah Bürgler
email: sarah.buergler@unibas.ch

Luana Palermo
email: luana.palermo@stud.unibas.ch

Yannick Schulz
email: yannick.schulz@unibas.ch

Zhifeng Tang

Xin Luo

Xue Li

Jens Gaab
email: jens.gaab@unibas.ch

Pablo Sinues
email: pablo.sinues@unibas.ch

Jessica Medina

Nicolas Goss

Alessia Perino

Kristina Schoonjans

Hector Gallart- Ayala

Julijana Ivanisevic

Tingting Fu
email: tingting.fu@unige.ch

Nicolas Elie

David Touboul

Olivier Geffard

Arnaud Chaumot

Davide Degli-Esposti

Sophie Ayciriex

S Girel

I Meister

N Zamboni
email: zamboni@imsb.biol.ethz.ch

S Rudaz

S Tsouka
email: sofia.tsouka@insel.ch

M Masoodi
email: mojgan.masoodi@insel.ch

Emma Tysinger

Marco Pagni

Olivier Kirchhoffer

Florence Mehl

Fabien Gandon

Jean-Luc Wolfender

Louis-Félix Nothias

Zhihong Yin
email: zhihong.yin@ukbb.ch

Wei Xiong
email: xiongw37@mail.sysu.edu.cn

Ming Wei

Yi Liu

Xia Feng

Philipp Engel
email: philipp.engel@unil.ch

A M Fouassier

P A Andreux

P Aebischer

J Auwerx

C Rinsch

A Singh

D D'amico

Simona Rambousek

Nicholas Paul Hays

Shaillay Kumar Dogra

Jean-Philippe Godin

Jean-Charles Picaud

Viola Jeck
email: viola.jeck@bruker.com

Edward Rudt
email: r_rudt@uni-münster.de

Ansgar Korf
email: ansgar.korf@bruker.com

Sven Meyer
email: sven.meyer@bruker.com

Stefanie Wernisch
email: stefanie.wernisch@bruker.com

Heiko Hayen
email: heiko.hayen@uni-münster.de

Breath pharmacometabolomics of stellate ganglion block intervention 2

Keywords: Breath Metabolomics, Pain, Cold Pressor Test, Validation, Breath Research Lipidomics, Mass spectrometry imaging, MALDI MS, TOF-SIMS, Gammarus fossarum steroidomics, structure elucidation, electron-activated dissociation, prostate cancer pathway analysis, computational, method, functional interpretation Knowledge graph, mass spectrometry, artificial intelligence, natural products stellate ganglion block, real-time and non-invasive breath analysis, secondary electrospray ionization high resolution mass spectrometry Microbiome, Tryptophan, Gut-brain axis, Social insects Urolithin A, muscle function, aging, mitophagy, acylcarnitines NMR metabolomics, infant nutrition, metabolic maturation Lipidomics, Trapped Ion Mobility Spectrometry (TIMS), Mass Spectrometry (MS), Hydrophilic Interaction Liquid Chromatography (HILIC)

A native gut symbiont alters tryptophan catabolism in the gut of the honey bee (Apis mellifera) 3 A.M. Fouassier, Amazentis Urolithin A improves mitochondrial and muscle health from cells to humans -evidence from biochemical and metabolomics-based assays 4 Simona Rambousek, Nestlé Research Impact of infant formulas compared to breast milk on metabolism of infants during the first 3 months of life studied by NMR metabolomics 5 Viola Jeck, Bruker Daltonics Novel Modes of Parallel Accumulation Serial Fragmentation (PASEF) for Mass Spectrometry-Based Lipidomics 6 Simon Isfort, University Hospital Bern Novel pre-treatment using chemical derivatization to allow spatial localization of Cholesterol using MSI in the liver 7 Joachim Kloehn, University of Geneva GNA1-mediated acetylation of glucosamine-6-phosphate is essential for Toxoplasma gondii and cannot be bypassed by GlcNAc salvage 8 Mathieu Galmiche, University of Geneva Towards extended steroid profiling in biological samples with the development of a multitargeted UHPLC-MS/MS workflow 9

Location

The annual meeting takes place in the main building of ETH at Rämistrasse 101 (google maps), floor D, Room HG D1.1. If you enter from the main entrance at the East, in front of the University Hospital, you have to take the stairs down to you right (to the North).

The closest tram stop is ETH/Universitätsspital, right in front of the building. From the main station, it can be reached with tram (#10) or with a 10 minute walk. To avoid the steep climb, take the Polybahn from Central.

A public parking lot exists underneath the building and is accessible from the Karl-Schmid-Strasse.

The Cold Pressor Test (CPT) is a widely used experimental technique for human pain induction, involving immersion of the hand in cold water. Understanding the underlying molecular mechanisms associated with pain responses is crucial for the development of effective pain management strategies. In recent years, metabolomics has emerged as a powerful tool for investigating the comprehensive metabolic changes occurring in response to pain stimuli and metabolomics studies have revealed distinct metabolic profiles associated with pain response. This study is for the first time addressing the question of whether pain induces any measurable changes at the metabolic level in non-invasive realtime measurements of exhaled breath.

We conducted the study at two sites: Switzerland (n=19) and China (n=21). Exhaled breath metabolome was analyzed using Secondary Electrospray Ionization-High Resolution Mass Spectrometry (SESI-HRMS). Each participant performed two sets of exhalations before and after the CPT, with six exhalations in positive and negative modes respectively. A total of 1920 exhalations were examined using standard univariate and multivariate data analysis strategies.

Both for the participants in Switzerland and China, significant changes in peaks before and after the CPT were observed. We found an overlap of 149 significant features [FDR < 0.01, log2(fold change) > 2] between the two study sites with a consistent up-/down-regulation behavior.

Altered metabolite abundances suggest a metabolic response to CPT induced pain. The overlapping features in both study sites indicate consistent metabolic changes associated with pain across populations. We are currently conducting enrichment analysis to investigate the correlation networks of involved metabolic pathways. and bile acid levels. Bile acids have been recognized as enterohepatic-derived hormones which control lipid metabolism and global metabolic health. In the present study, we first evaluated the relative distribution and diversity of lipid species across four organs playing a central role in lipid energy metabolism (including lipid synthesis, utilisation, and storage): subcutaneous white adipose tissue (scWAT), brown adipose tissue (BAT), skeletal muscle and liver. Furthermore, we aimed to determine the effect of diet on lipid composition of specified organs obtained from C57BL/6J mice fed with chow diet (CD) and high-fat diet (HFD). The mice (all males) were sacrificed at 29 weeks of age, following a period of 21 weeks on CD or HFD. The sacrifice was performed in the postprandial state (4h after physiological refeeding). Tissue lipidome was characterized using specific targeted SRM-based HILIC-MS/MS method screening for more than 2400 lipid species in the initial qualitative analysis followed by quantification of robustly detected species. Lipid quantification was achieved by a single point calibration with 75 isotopically labelled standards representative of different lipid classes, covering lipid species with diverse alkyl chain lengths and unsaturation degrees. Correction of isotopic overlap was performed using LICAR (https://slinghub.shinyapps.io/LICAR/). As a result, a wide panel of 24 lipid classes comprising 533 to 838 lipid species were quantified (CV<20%) depending on tissue type. Additionally, bile acids (15 species) were quantified in mice liver and plasma using stable isotope dilution approach. The highest diversity of lipids was detected in quadriceps (n=838 species) while white adipose tissue contained the lowest (n=533 species) number of species. Main differences were found for triacylglycerols (TG) and glycerophospholipids such as glycerophosphocholines (PC) and glycerophosphoethanolamines (PE). In mice fed with HFD, we observed the accumulation of TG as a general trend for all tissues. Besides, a decreasing trend of some hexosylceramides (HexCer), and sphingomyelins (SM) was reported in a tissue-specific manner. The dataset is currently under investigation to identify the lipid signatures associated with measured physiological parameters including body and organ weight, blood glucose and bile acid levels.

Lipids are important component in living organisms and play pivotal roles in various biological functions. In aquatic biomonitoring, lipidomic profiling of sentinel organisms is essential to understanding the molecular mechanism of the toxicity effect of endocrine disrupting chemicals. Currently, the most popular mass spectrometry (MS)-based lipidomic techniques include hyphenated chromatographic MS and direct infusion MS. Both methods require a destructive sample preparation step to isolate the analytes from the biosystems, during which the spatial information of the molecules is inevitably lost. Mass spectrometry imaging (MSI) is a well recognized technique for in situ mapping of a variety of molecular species. Compared to the above mentioned MS workflows, MSI has the unique advantage of preserving the spatial localization of the molecules, thus providing crucial information on biochemical processes within the biofunctional compartments. We herein propose a spatially resolved lipidomics workflow combining shotgun lipidomics and MSI to decipher the lipidome of a freshwater sentinel species Gammarus fossarum. Shotgun lipidomics was performed on whole body gammarids to generate a global reference lipidome of this species. Matrix assisted laser desorption ionization (MALDI) MS and high-resolution time of flightsecondary ion mass spectrometry (TOF-SIMS) imaging were employed to spatially map the lipids in whole-body gammarid tissue sections and specific organs, respectively. This spatial lipidomics workflow enabled a comprehensive examination of lipid composition and spatial distribution in G. fossarum. Particularly, MSI guided the discovery of unknown sulfate-based lipids in this organism and revealed a dynamic lipid composition in gammarids in the course of reproduction.

Due to continuous advancements in the field of metabolomics, the importance of having robust computational tools that facilitate the functional interpretation of large-scale datasets has become essential. One of these tools is pathway analysis, a method that helps link changes in metabolic compounds to biological pathways. Although it is widely used, it is not well standardized and the impact of different considerations on the functional outcome is not well understood. The workflow consists of the selection of a suitable pathway collection, the conversion of these metabolic pathways to graphs, and the calculation of scores for each participating metabolite in the network. Subsequently, pathways are scored and ranked according to the metabolomics dataset. Multiple factors need to be taken into consideration when using pathway analysis in order to produce reliable results. We investigate two main aspects of pathway analysis, namely the consideration of non-human-native enzymatic reactions and the interconnectivity of individual pathways. The exclusion of non-humannative reactions leads to poorly represented reaction networks and to loss of information, particularly when it comes to microbiome related datasets. The connectivity between pathways illustrates better the importance of certain central metabolites in the network, such as aminoacids. However, it might result in hub compound effects, which are mitigated through a penalization scheme. Our findings aim to raise awareness on both the capabilities and limitations of current pathway analysis practices in metabolomics and provide insights on their standardization.

An experimental knowledge graph (KG) driven framework (10.26434/chemrxiv-2023sljbt) was recently introduced to facilitate the integration of heterogenous data types, encompassing both experimental data (mass spectrometry annotation, results from biological screening and fractionation) as well as web-wide meta-information (such as taxonomy and metabolite databases). Although this KG efficiently encapsulates the different data structures and semantic relationships, retrieving specific information visually or programmatically is not trivial.

To unlock the full potential of KGs for scientists of all fields, we have engineered a KG Artificial Intelligence (AI) agent that can convert natural language questions into programmatic data-mining tasks and generate adapted visualization. By leveraging the potential of emerging Large Language Models (LLMs) to understand semantic relationships encapsulated in KGs, the KGAI-agent autonomously iterates to construct a SPARQL requestthe widely-used query languageof inputted natural question. After retrieving the necessary information from the KG, the KGAI-agent provides a preliminary interpretation of the results in natural language, along with relevant visualizations and statistics. In addition, follow-up interactions between the LLM and the user the AI-agent can be expert-guided to refine the initial results and interpretation. Released as an open-source tool, the KGAI-agent acts as a powerful assistant capable of exploring any KG, democratizes access to such semantic databases without requiring bioinformatics expertise, and is accessible in over 50 human languages.

Here we will describe the core principles and components of the KGAI-agent and illustrate how we can interrogate metabolomics information from a KG to discover bioactive metabolites from a collection of 16,000 plant extracts. Urolithin A (UA) is a natural compound produced by the gut microbiome from dietary precursors found in foods such as pomegranate, berries, and nuts. Our studies showed that UA positively impacts on mitophagy and mitochondrial metabolism. The decline of these processes is considered a hallmark of aging and health. The positive effects by UA was observed across various cell lines, preclinical models, and clinical studies. We first showed that UA induces mitophagy markers and enhances mitochondrial metabolic function in C2C12 muscle cells. Consistent results were confirmed in vivo, where UA increased protein markers of mitophagy and mitochondrial respiration in gastrocnemius muscles of both old mice and dystrophic animals, compared to controls. We also showed improved mitophagy and mitochondrial function by UA in human chondrocytes from both healthy donors and osteoarthritis (OA) patients. Human muscle proteomics data from healthy middle-aged subjects administered with UA for four months described elevated levels of proteins involved in mitophagy and oxidative phosphorylation compared to placebo. Plasma metabolomics analysis of the same subjects, as well as in additional clinical studies in healthy elderly, revealed a significant reduction of several acylcarnitines following UA administration. Such decrease is indicating higher systemic mitochondrial efficiency by UA. Finally, the enhanced mitochondrial health induced by UA was linked to a significant improvement in muscle strength and endurance in both preclinical models and multiple clinical studies. These findings provide evidence for the use of UA as a nutritional intervention targeting mitochondria to support the health of multiple tissues and promote healthy aging.

Urinary metabolomics was performed using nuclear magnetic resonance (NMR) spectroscopy in 0, 1 and 3 months old infants randomized to receive either Control (CTRL; standard starter formula with 1.85g protein/100kcal from enrollment [age ≤7d] until 3 months) or Experimental (EXPL; new formula with 2.5g protein/100kcal from enrollment until age 1 month followed by CTRL formula until 3 months) interventions. The formula regimens were compared to a reference group of breastfed infants using univariate or multivariate analysis and based on their metabolic maturation age derived from changes in urinary metabolites related to infant age.

Metabolomics of infants at age 1 month was influenced by differences in formula protein concentration, inducing changes of microbiota-related metabolites (phenylacetylglutamine, 4hydroxyphenylacetate, indoxyl sulfate, and hippurate) and essential amino acids (isoleucine, leucine, threonine and lysine) as detected in urine, predominantly in EXPL group. The metabolic maturation age trajectories for both infant formula regimens were similar but significantly different from the breastfed trajectory.

In conclusion, urinary metabolomics doesn't indicate superiority of the higher protein content formula (2.5 g protein/100 kcal) from birth to 1 month of age over the standard starter formula (1.85 g protein/100 kcal). The effect of the metabolite differences on health requires further evaluation.

Our contribution outlines the benefits of TIMS-enabled LC-IMS-MS/MS plasma lipidomics workflows based on Parallel Accumulation Serial Fragmentation (PASEF). In PASEF experiments, electrospray generated ions are subjected to cycles of trapping/elution in the TIMS device followed by sequential fragmentation of ions, before entering a time-of-flight (TOF) analyzer. Variation of the TIMS conditions and quadrupole filtering windows enables unique MS/MS experiments with distinct benefits for lipidomics studies.

Data dependent acquisition (dda-)PASEF, suitable for non-targeted approaches, produces highquality CCS and MS/MS data for the identification of phospholipid species based on accurate precursor mass and fragment ion spectra. Compared to dda-PASEF, data independent acquisition (dia-)PASEF and parallel reaction monitoring (prm-)PASEF do not require an MS1 scan for precursor picking. In dia-PASEF, broad-band fragmentation occurs via predefined mass/mobility windows. It leads to a particularly high coverage in the generation of MS/MS spectra. prm-PASEF is based on a target list with user defined retention time and mobility sections, leading to better quality of MS/MS spectra for low abundant lipids. Therefore, prm-PASEF is the method of choice for targeted lipidomics in unknown samples.

Experiments were carried out on a timsTOF flex (Bruker, Germany) coupled to an Ultimate 3000 LC system (Thermo Scientific, Germany) using negative-mode electrospray ionization. Experimental details can be found in Rudt et al., Analytical Chemistry 2023, 95, 9488-9496. Cholesterol plays a crucial role in diverse physiological processes such as regulating cell membrane fluidity and facilitating cellular transport [START_REF] Simons | How Cells Handle Cholesterol[END_REF]. Its perturbation is associated with MASLD [START_REF] Duan | Regulation of cholesterol homeostasis in health and diseases: from mechanisms to targeted therapeutics[END_REF] although its zone-specific distribution is not known.

Swiss Metabolomics Society Annual Meeting

Precise detection and localization are therefore essential to understand its involvement in MASLD.

Desorption electrospray ionization (DESI) mass spectrometry has been employed to detect and identify multiple molecular species and simultaneously map their distribution in tissue sections [START_REF] Ifa | Development of capabilities for imaging mass spectrometry under ambient conditions with desorption electrospray ionization (DESI)[END_REF], [START_REF] Seubnooch | Characterisation of hepatic lipid signature distributed across the liver zonation using mass spectrometry imaging[END_REF]. Cholesterol among other sterols, tends to be poorly ionized by conventional DESI and is often disregarded [START_REF] Liebisch | High throughput quantification of cholesterol and cholesteryl ester by electrospray ionization tandem mass spectrometry (ESI-MS/MS)[END_REF]. To solve this problem, on-tissue derivatization has been developed by adding reagents to the electrospray solvents. Betaine aldehyde, which contains a pre-charged group, reacts selectively and rapidly with alcohols, including cholesterol, and significantly decreases the limits of detection [START_REF] Wu | Rapid, direct analysis of cholesterol by charge labeling in reactive desorption electrospray ionization[END_REF]. This however, imposes limitations to the range of suitable solvents for DESI analysis.

This poster explores the detection of cholesterol throughout the liver, integrating a novel and rapid pre-treatment technique to allow high-resolution spatial mapping of cholesterol in conjunction with other lipids classes. Meanwhile, the integrity of cellular structures remains intact, allowing subsequent pathophysiological assessment of the same tissue slide using conventional staining methods. Given the limited tissue amounts required, this method allows the detection of cholesterol with other lipid classes simultaneously.

Abstract

Like in other eukaryotic cells, many proteins of the apicomplexan parasite Toxoplasma gondii entering the secretory pathway are N-or O-glycosylated or linked to membranes via glycosylphosphatidylinositol (GPI) anchors. Formation of these post-translational modifications relies on activated sugar nucleotides, including uridine diphosphate Nacetylglucosamine (UDP-GlcNAc). A critical enzyme in UDP-GlcNAc synthesis is the glucosamine-phosphate-N-acetyltransferase (GNA1). To decipher the importance of GNA1 for the parasite, conditional depletion of the gene was achieved using the auxin-degron system. While GNA1 was assigned a high positive fitness score in a genome-wide fitness screen, indicating dispensability, we demonstrate here that GNA1 is essential for T. gondii. This discrepancy is likely due to a functional shorter splice variant which is significantly shorter than the annotated protein. Functional characterization of parasites downregulated in GNA1 revealed that GNA1 depletion impairs GPI synthesis via two distinct modes: Depletion of its crucial substrate UDP-GlcNAc as well as a toxic accumulation of glucosamine-6-phosphate (GlcN6P). GNA1-depleted parasites show no or only a modest recovery in fitness following GlcNAc supplementation, despite stable isotope labelling experiments demonstrating T. gondii's ability to take up and phosphorylate GlcNAc. Metabolomic analyses reveal that in glucose-replete conditions, GlcNAc appears not to be efficiently utilized and fails to restore UDP-GlcNAc levels. In glucose-deplete conditions, on the other hand, GlcNAc is efficiently used and fully restores UDP-GlcNAc levels. However, the absence of glucose does not alleviate GlcN6P accumulation, which continues to inhibit GPI synthesis. This vulnerability highlights the evolutionary divergent apicomplexan GNA1 as a promising drug target in T. gondii.

Abstract

Dysregulation of steroidogenesis and steroid metabolism can be responsible for a wide range of diseases such as diabetes, prostate cancer or male infertility.

For extended steroid profiling, untargeted analytical workflows can be implemented. However, they lack absolute quantification and require high-end mass spectrometers for meaningful annotation.

The sensitivity and wide availability of low-resolution mass spectrometers makes the emergence of targeted analysis a promising alternative for extended steroid profiling at very low concentrations.

The main challenges associated with the analysis of steroids in biological samples are the complexity of the matrix causing high background noise and interferences, the wide dynamic range of analyte concentrations (from < 5 ppt to > 5 ppm), the weak ionization efficiencies of some steroids, and the coexistence of multiple isobars with similar MS/MS spectra that can be difficult to separate.

In this work we explore this multi-targeted approach and address these challenges. Sample clean-up was optimized, including phospholipid removal and solid phase extraction of steroids, with comparison of different sorbents. The separation of isobaric pairs was finetuned using a biphenyl stationary phase. Attention was also paid to the optimization of steroid ionization conditions with post-column addition of ammonium fluoride combined with different formic acid concentrations, and to the optimization of MS/MS transitions for improved selectivity and sensitivity.

The method performance was evaluated on pooled plasma samples in which 41% of the target steroids were successfully detected.

Abstract

Lipid metabolism has a proven role in the pathogenesis and progression of many cancers, and growing evidence support a role for dysregulated lipid metabolism and prostate cancer (PCa). Several studies have shown that PCa metabolome could be used as non-invasive diagnostic and prognostic tool. In this study, we analyzed the plasma of 73 prostate cancer patients to identify metabolites with diagnostic or prognostic value. Patient plasma was collected during fasting shortly before radical prostatectomy. Targeted metabolomics was run on the pre-operative plasma. Using machine learning algorithms, we identified a signature of 41 metabolites either positively or negatively associated with the development of bone metastatic PCa. Furthermore, we studied the effect of high-and low-fat diet on the progression and on the morbidity of prostate cancer using inducible genetically engineered mouse models (GEMMs) of prostate cancer of different grades. Mice were switched to either low-or high-fat diet since week 6 and the Cre recombinase was activated during week 8 to induce the phenotype in the GEMMs. Mice were kept on low-or high-fat diet until the time of harvest (1, 3 and 5 months). Faeces were collected longitudinally to perform a metagenomics study on the effect of diet on microbiota, while prostate, plasma and liver tissues were collected at harvest point. In conclusion, our studies associated a metabolic signature to a risk of metastasis development and will provide more insights on the role of diet on prostate cancer metabolism.

Anthropometric measurements were significantly different (p<0.038) at baseline and after 3-months (p<0.0077). Preliminary urinary metabolomic profiles showed a difference between groups in lysine-related metabolites at baseline and alterations with lysine intervention. Metabolites of tryptophan degradation and utilization, threonine, methionine, cysteine, and branched chain amino acid biosynthesis pathways were altered at baseline between groups and with lysine intervention.

The urine metabolomic profile with EAAD is different between stunted and nonstunted children at baseline and in response to lysine supplementation. A validation of urine metabolomic profiles using the blood metabolomic profiles could provide more insights towards designing targeted nutritional therapies.

Enhancing the mobility resolution for co-eluting compound classes during plasma characterisation using multi-sequence IMS n acquisitions.

Human plasma comprises of many small molecules including amino acids, organic acids, and larger lipid classes. To accurately characterize this biological fluid requires multiple extraction and complimentary LC-MS acquisition methodologies to deconvolve certain compounds not efficiently resolved across methods. When analyzing small polar analytes by HILIC based methods, many moderately polar and non-polar compounds e.g. lipids are also present but co-elute in class specific regions or at the solvent front, meaning specific analytical methods must be employed to separate out these features. Coupling ion mobility separation with mass spectrometry has improved the separation of many of these co-eluting compounds, increase the peak capacity and improving spectral quality. But different compounds require different levels of separation to achieve the desired resolution.

The new multi-sequence IMS n analysis assisted in improving the depth of characterization for plasma metabolomics. Areas of chromatographically resolved small molecules had sufficient separation using a single pass of the cyclic device, whilst larger lipid classes required 5 additional passes to enhance the isolation of co-eluting lipid phosphatidylcholine features. The additional passed for these lipid precursor ions has enabled improved confidence in annotation using drift separated elevated collision energy spectra, reducing the impact of chimeric spectra that result from fragmentation of co-eluting molecules.

Exhaled breath from patients (n = 7) undergoing surgery was collected in Nalophan bags at baseline (5 min prior propofol administration) and every 30 minutes post-propofol. Collected breath samples were immediately transferred within the hospital for mass spectrometric analysis using secondary electrospray ionization combined with high-resolution mass spectrometry. Paired blood samples were collected during surgery to subsequently determine correlations between exhaled metabolites and systemic propofol levels.

A total of 34 breath samples and 27 blood samples from n = 7 patients were collected. Propofol, along with propofol metabolites 4-Hydroxypropofol, 2,6-Diisopropyl-1,4-quinone and 2-propanol-6-isopropyl-1,4-quinol were consistently detected in exhaled breath of all patients. Large inter-patient variabilities were observed for propofol metabolization. A correlation with r = 0.54 of breath propofol and serum propofol concentration was found.

In conclusion, we showed that breath analysis can be deployed in a real clinical setting to monitor propofol and its metabolites during surgical interventions. Integration of this experimental procedure in clinical practice may provide a more personalized approach towards propofol dosage.

Abstract

Lifestyle changes toward a healthy diet and increased physical activity are the cornerstone interventions in the treatment of non-alcoholic fatty liver disease (NAFLD), the most common liver disease worldwide. However, due to its increased prevalence, new therapeutic approaches targeting the gut-liver-axis such as the use of microbial therapeutics and guthormonal interventions have been suggested. The present study introduces a seven-week double-targeted intervention using genetically engineered probiotic Escherichia coli Nissle 1917 to continuously express aldafermin (a non-tumorigenic analog of a human intestinal peptide hormone, fibroblast growth factor 19) along with dietary change (EcNA). The safety, efficacy, and mechanisms of action of the EcNA intervention were demonstrated using a high-fat-diet-induced NAFLD mouse model. The beneficial effects of the EcNA intervention were evidenced by the decrease in body weight, liver steatosis, and plasma concentrations of aspartate aminotransferase and cholesterol. Comprehensive integrated transcriptomics and non-targeted metabolomic analyses further revealed alterations in NAFLD-related genes and metabolites from the host and gut-microbial origin; along with a switch in amino acid, lipid, and their associated receptor signaling pathways. These results suggest the potential efficacy of EcNA in ameliorating NAFLD by decreasing insulin resistance, steatosis, oxidative stress, and maintaining gut-liver axis homeostasis; and highlight the potential of exploring multi-targeted interventions combining microbial therapeutics with the diet for NAFLD.

In natural products (NP) research, efficiently prioritizing samples in natural plant extract (NEs) libraries is crucial for discovering novel bioactive molecules. Here we sought to investigate the UHPLC-HRMS 2 metabolite profiling, dereplication outcomes, and Wntpathway inhibition biological screening results for a set of 1,600 NEs. The results of the biological screening initially drove the selection of NEs. Only non-toxic NEs with an inhibitory IC50 ≤ 5 μg/mL were considered, resulting in a subset of only 30 NEs. To increase the chances of selecting NEs containing novel NPs, Inventa was used to calculate priority scores for structural novelty. Inventa is a computational tool we have developed to automate the prioritization of extracts to assess the potential of NEs to contain novel NPs. It automatically integrates spectral fingerprint specificity, metabolite profiling dereplication results, and previous literature on the profiled organism. The integration of these scores in the selection revealed 9 active NEs with a high potential of containing structurally novel NPs. The most promising candidate was Hymenocardia punctata. Further phytochemical investigations of the H. punctata's leaves ethyl acetate extract resulted in the isolation of 10 novel bicyclo[3.3.1]non-3-ene-2,9-diones and 3 prenylated flavones. Assessment of the Wnt inhibitory activity of these compounds revealed that three novel bicyclic compounds and two prenylated flavones showed interesting activity without apparent cytotoxicity. This study highlights the potential of combining Inventa's structural novelty scores with biological screening to discover potentially novel bioactive compounds in large natural extract collections.

  September 15 th 2023, ETH Zurich Novel pre-treatment using chemical derivatization to allow spatial localization of Cholesterol using MSI in the liver S. Isfort 1 M. Masoodi 1 1 Institute of Clinical Chemistry, University Hospital Bern, Inselspital, Bern, Switzerland

patient outcomes and increased rates of professional errors. In this context, strategies such as mindfulness-based intervention (MBIs) are a promising approach to stress management. Benefits related to MBIs have already been reported in several studies, however, the biological and physiological effects of these interventions on stress reactivity are not well understood. We therefore aimed to characterize the biochemical responses to MBI in a cohort of 28 health care students in Geneva, Switzerland. The intervention was organized over 8 weekly sessions and students were sampled longitudinally before and after the program as well as 6 months later, with the control group was undergoing the MBI after the third sampling. Blood serum was collected and analyzed using untargeted metabolomics and lipidomics platforms and stress was evaluated using several well-established protocols, such as the perceived stress scale and the prosocialness scale in adults. A total of 124 metabolites and 774 lipids were annotated. Metabolic profiles were varied with MBI impacting amino acid and nucleoside in females, while males displayed increased acetylneuraminic acid after the intervention. In terms of lipids, lower triglycerides and phosphatidylethanolamines (PE) and higher etherPE were associated to MBI in male volunteers.

Monitoring of propofol metabolism during surgical interventions via exhaled breath analysis

Abstract

Untargeted mass spectrometry metabolomics is an increasingly popular approach for characterizing complex mixtures. Recent studies have highlighted the impact of data preprocessing for determining the quality of metabolomics data analysis. The first step in data processing with untargeted metabolomics requires that signal thresholds be selected for which features (detected ions) are included in the dataset. Analysts face the challenge of knowing where to set these thresholds; setting them too high could mean missing relevant features, but setting them too low could result in a complex and unwieldy dataset. Our study compared data interpretation for an example metabolomics dataset when intensity thresholds were set at a range of feature heights. The main observations were that low signal thresholds (1) improved the limit of detection, (2) increased the number of features detected with an associated isotope pattern and/or an MS-MS fragmentation spectrum, and

(3) increased the number of in-source clusters and fragments detected for known analytes of interest. When the settings of parameters differing in intensities were applied on a set of 39 samples to discriminate the samples through principal component analyses (PCA), similar results were obtained with both low-and high-intensity thresholds. We conclude that the most information-rich datasets can be obtained by setting low-intensity thresholds. However, in the cases where only a qualitative comparison of samples with PCA is to be performed, it may be sufficient to set high thresholds and thereby reduce the complexity of the data processing and amount of computational time required. reported the successful implementation of real-time breath analysis using secondary electrospray ionization-high resolution mass spectrometry (SESI-HRMS) to provide reliable estimations of systemic drug concentrations along with risk estimates for drug response and side effects in epileptic patients. In this presentation, we intend to show our latest and ongoing efforts to further improve/supplement the abovementioned method. Firstly, we developed a new method (tested on 40 patients) that involves transitory collection of exhaled breath in Nalophan bags prior to SESI-HRMS analysis. We conclude that the clinically relevant free fraction of valproic acid (VPA, an ASM) can be predicted reasonably well by this combination of off-line breath collection with rapid SESI-HRMS analysis (Lin's CCC between actual and predicted free VPA was 0.6). Furthermore, we would also like to show the results from our still ongoing follow-up of previously mentioned study. Finally, we will present another study, where we successfully used SESI-HRMS to characterize the volatolome (and by extension metabolome) of patients undergoing treatment with Methotrexate (MTX, a chemotherapy agent). In conclusion, various studies presented here make an important contribution to the implementation of breath analysis by SESI-HRMS in precision medicine.

Swiss Metabolomics Society Annual Meeting

September 15 th 2023, ETH Zurich Integration of bioactivity and metabolomics structural novelty scores for efficiently targeted isolation of novel bioactive compounds from biodiverse natural extract collections


domain: spi sde

text:
Jordy Charly

Isidore Rabetanetiarimanana

Dominique Morau

Hery Tiana Rakotondramiarana

Mamy Harimisa Radanielina

Physico-chemical characterization of typical Residual Household Waste (RHW) from Reunion Island

Context and Objectives

In Reunion Island, 65%, 13.7% and 21.4% of household wastes are landfilled, recycled and composted, respectively. Significant environmental burdens are caused by current waste management (WM). Indeed, a new WM strategy ensuring energy recovery by gasification of residual household wastes (RHW) will be implemented. Before waste treatment, its characterisation is a primordial action [START_REF] Götze | Physicochemical characterisation of material fractions in residual and sourcesegregated household waste in Denmark[END_REF][START_REF] Elize | The importance of characterizing residual household waste at the local level : A case study of Saguenay, Quebec (Canada)[END_REF]. The present work aims at analyzing the physico-chemical properties of different waste compositions.

Materials and Methods

In this work, direct analysis method is used. Before sampling, 3 mixtures of waste have been established (Fig. 1) based on local context and data from the last waste characterisation campaign in Reunion conducted by ADEME in 2019. RHW-1 and RHW-2 represent respectively the typical RHW without fine particles and the typical RHW. The waste sampling steps began with the collection of mother samples which represent a RHW produced in one week and are from waste storage site and selected dumpsters on the campus of the University of la Réunion. The mother samples are manually sorted into 10 categories such as: putrescible, paper, cardboard, composite, textile, sanitary textile, plastic, combustible, non-combustible and fine particles. Then, a 1 kg sample of each proposed mixture is sent to the laboratory. For the physicochemical characterization of RHW, we have drawn inspiration from the French and European standards that have been put in place as far as possible.

Results and Discussion

Proximate and ultimate analysis

The result of proximate analysis show that, with 11.9 and 10.5 MJ/kg respectively, the LHV of RHW-1 and RHW-2 are indeed low compared to other types of solid wastes, which generally have higher LHV such as RDF and MSW (12.1-22.5 MJ/kg) [START_REF] Milena | Refuse Derived Fuel (RDF) production and gasification in a pilot plant integrated with an Otto cycle ICE through Aspen plus™ modelling: Thermodynamic and economic viability[END_REF]. Moreover, RHW-3 have acceptable LHV (16.77 MJ/kg) compared to LHV of RDF found in the literature [START_REF] Milena | Refuse Derived Fuel (RDF) production and gasification in a pilot plant integrated with an Otto cycle ICE through Aspen plus™ modelling: Thermodynamic and economic viability[END_REF]. According to Fig. 2, RHW-3 has the highest LHV due to the high proportion of plastic in the mixture which leads to a high VM content. The considerable proportion (< 50%) of putrescible, paper and cardboard in RHW-1 and RHW-2 is the origin of their low calorific value. Furthermore, the presence of fine particles in RHW-2 leads to an increase in oxygen (Fig. 3) and moisture content (Fig. 2), which results in a decrease in LHV. According to the ultimate analysis (Fig. 3), RHW-1 and RHW-2 have higher oxygen content due to high proportion of putrescible, paper, cardboard and fine particles.

Van Krevelen diagram

From Fig. 5, the H/C and O/C atomic ratios of the residual wastes show that RSHW-2 is among the biomasses with low calorific values while RHW-1 is outside the biomass zone. Moreover, due to its composition rich in plastics and baby nappyies, RHW-3 is closer to lignin than to biomass.

Conclusion and perspectives

To conclude, typical RHW (RHW-2) in Reunion has a low LHV and a high oxygen and moisture content. Its gasification will probably produces more CO and CO 2 , lower H 2 and chlorinated products concentrations compared to syngas produced by typical RHW without fine particles (RHW-1). The removal of fine particles leads to an increase in the calorific value and decrease in oxygen and moisture content. Furthermore, RHW-3 has the highest LHV and lowest oxygen content, its gasification is likely to yield more hydrocarbons and more dioxin and furans due to its high Cl content. However, the mixture is interesting for heat production. Future surveys should focus on:

• the use of the collected physico-chemical characterization data to model gasification; • pretreatment of RHW in order to improve the fuel quality such as: density, higher heating value, carbon and ash content and fixed carbon

Fig. 1 :

 1 Fig. 1: The different composition of the samples

Fig. 2 :Fig. 3 :

 23 Fig. 2: Proximate analysis of RHW

Fig. 4

 4 Fig.4shows the N, S and Cl content of the analysed samples. In gasification process, the high content of chlorine (N aCl and PVC), N and S in the waste is the cause of the formation of: N H 3 , H 2 S and certain chlorinated products (HCl and Cl 2 ) and a source of emission of dioxins and furans[START_REF] Mukherjee | A review on municipal solid waste-to-energy trends in the USA[END_REF]. As illustrated in Fig.4, the 3 samples have low S content, RHW-1 has the highest N content and RHW-3 has high Cl content because of plastics present in the mixture.

Fig. 4 :

 4 Fig. 4:Inorganic element content of RHW

Fig. 5 :

 5 Fig. 5: Representation of the H/C and O/C atomic ratios in the Van Krevelen

Contact Information * Address: Piment Laboratory, University of La Reunion, 120 Rue Raymond Barre, 97430 Le Tampon, France Email:

jordy-charly-isidore.rabetanetiarimanana@univ-reunion.fr Phone:

(262) 692 628 691 Partnership


domain: phys phys

text:
S Cherukulappurath

J L Godet

G Boudebs

Higher order coefficient measurements in nonlinear absorption process

Keywords: 78.20.C; 42.70.M Chalcogenide glasses, Nonlinear absorption, Multiphoton absorption, Optical limiting

Higher order coefficient measurements in nonlinear absorption process

Introduction

In recent years chalcogenide glasses have been receiving considerable interest due to their large nonlinearities. Highly nonlinear absorbing materials can find applications in the domain of optical limiting. Ideal optical limiters have large transmittance at low intensities, high pulse energy suppression, a good resistance to laser-induced damage, stability over time and ultrafast responses. Glasses that contain chalcogens (S, Se, Te), are considered as good candidates for optical limiting. Thus, it is of great importance to fully characterize nonlinear absorption properties. In references [START_REF] Lawrence | Measurement of the complex nonlinear refractive index of single crystal p-toluene sulfonate at 1064 nm[END_REF] [START_REF] Shim | Four photon absorption in the singlecrystal polymer bis (paratoluene) sulfonate[END_REF], variation of the two (as well as three)-photon absorption coefficients with the intensity have been reported which suggested the inclusion of higher order effects to better describe the nonlinear absorption.

We have previously investigated nonlinear absorption in some chalcogenide glasses [START_REF] Troles | Chalcogenide glasses as solid state optical limiters at 1.064µm[END_REF][START_REF] Troles | Third-order nonlinear optical characterization of new chalcohalogenide glasses containing lead iodine[END_REF]. However, in these references, higher order nonlinear absorption effects were not considered. In reference [START_REF] Bindra | Nonlinear optical properties of chalcogenide glasses: Observation of multiphoton absorption[END_REF], three-, four-and five-photon absorption coefficients are deduced in some chalcogenide glasses from open-aperture z-scan experiments. The coefficients are obtained from a fit of the experimental scan taking into account only one nonlinear absorption process. Recently, in reference [START_REF] Boudebs | Experimental observation of higher order nonlinear absorption in telluride based chalcogenide glasses[END_REF] we presented a study of nonlinear absorption occurring in telluride based chalcogenide glasses. We observed experimentally an effective two-photon absorption (TPA) process as a function of the incident intensity in four chalcogenide glasses containing different amount of tellurium (Ge 10 As 10 Se 80-x Te x where x = 0, 10, 15, 20). We have estimated the values of the respective nonlinear coefficients related to this effective nonlinear absorption. These values were extracted from the experimental data with the help of an approximate theoretical solution of the basic differential equation governing the evolution of the intensity while propagating inside the sample. This solution takes into account the local intensity inside the medium in presence of two-photon absorption. But we have found problem of divergence with the solutions given by this approach.

In this paper, we focus on the exact solution of the basic differential equation relating the intensity inside the medium to the higher order nonlinear absorption coefficients (numerical as well as analytical one). We give the analytical condition necessary to avoid divergence problems. Experimental data are obtained using Z-scan technique [START_REF] Sheik-Bahae | Sensitive measurement of optical nonlinearities using a single beam[END_REF].

Measurement of the nonlinear coefficient is done by fitting these experimental data with the exact solution given by Runge-Kutta numerical integration method. Comparison of the results is done with those obtained with different simplified solutions. We show that, even though the order of magnitude is respected, the error of the estimated values can be large.

Theory

Consider a sample exhibiting the following i) α, the linear absorption coefficient (cm -1 ) ii) β, the two-photon absorption coefficient (cm/GW) and iii) γ, the third order nonlinear coefficient (cm 3 /GW 2 ). The optical intensity I (in GW/cm 2 ) satisfies the equation [START_REF] Shen | Nonlinear Optics[END_REF]:

3 2 I I I dz dI γ - β - α - = ( 1
)
where z is the axis of propagation of light denoting in this section the distance inside the sample. We denote by γ the nonlinear coefficient giving account for the dependency of the TPA coefficient with regard to the intensity I. This coefficient γ denotes here simply a term in the expansion of the absorption in a power series of the intensity that better describes the experimental evolution of the intensity at the output of the specimen (see Refs. [START_REF] Lawrence | Measurement of the complex nonlinear refractive index of single crystal p-toluene sulfonate at 1064 nm[END_REF][START_REF] Shim | Four photon absorption in the singlecrystal polymer bis (paratoluene) sulfonate[END_REF][START_REF] Boudebs | Experimental observation of higher order nonlinear absorption in telluride based chalcogenide glasses[END_REF][START_REF] Smektala | Non-linear optical properties of chalcogenide glasses measured by Z-scan[END_REF]).

In the case of a pure TPA phenomenon, the transmission of the intensity through a sample of thickness L (considered as 'thin') can be written [START_REF] Hermann | Beam propagation and optical power limiting with nonlinear media[END_REF]:

eff 0 L 0 L I 1 e I ) L ( I T β + = = α - (2)
where L eff = (1-e -αL )/α, and I 0 is the peak incident intensity at the focus. When γ cannot be neglected anymore, an effective TPA coefficient β eff has to be introduced. Indeed, it is this value that is usually measured in experiment. An approximate solution of Eq. (1) for the intensity has been provided in reference [START_REF] Boudebs | Experimental observation of higher order nonlinear absorption in telluride based chalcogenide glasses[END_REF]:

( ) [ ] z 0 0 0 1 0 2 I e I(z) I 1 I L (z) Log 1 I L (z) 1 -α = γ α +β γα   + β- + +β   β   β , (3)
where L 1 (z) = (1-e -αz )/α. Comparing relations (2) and ( 3) for z = L, we can easily find:

( ) [ eff 0 eff 0 2 0 eff L I 1 Log L I I β + β ] β + α γ + β γα - β = β (4)
In references [START_REF] Lawrence | Measurement of the complex nonlinear refractive index of single crystal p-toluene sulfonate at 1064 nm[END_REF] and [START_REF] Shim | Four photon absorption in the singlecrystal polymer bis (paratoluene) sulfonate[END_REF], the authors have a more simplified approach to deduce the nonlinear coefficients β and γ. In the case of very thin samples, they write:

0 eff I γ + β = β (5)
We can easily check that when 1 L I eff 0 << β , Eq. ( 4) reduces to relation [START_REF] Bindra | Nonlinear optical properties of chalcogenide glasses: Observation of multiphoton absorption[END_REF]. Thus, considering the sample as 'physically very thin' is not always sufficient to allow the use the simplified expression [START_REF] Bindra | Nonlinear optical properties of chalcogenide glasses: Observation of multiphoton absorption[END_REF]. It depends also on the incident intensity as well as on β, the TPA coefficient. In fact the exact solution for β eff is given by the following (see Eq. (A6) in the appendix):

( ) ∫ α - γ + β = β L 0 z eff eff dz e z I L (6)
We can see from Eq. ( 6) that β eff varies as the intensity propagates inside the sample especially in presence of a large TPA coefficient as it can be noticed from the exact solution of Eq. (1) detailed in the appendix:

z 0 ( ) 2 0 1 0 2 I e I(z) 1 I L I L -α ∞ = + β + γ . ( 7
)
where can be easily computed (cf. Eq. (A7)). In Fig. 1 is shown a numerical simulation of the intensity evolution while propagating inside the sample for large value of β.

The considered parameters are the same as those found in the experiment done with Ge ( ) 2 L ( , , , I , z) ∞ α β γ 0 10 As 10 Se 60 Te 20 : α = 3.18 cm -1 ; β = 13.7 cm/GW, γ = -11.0 cm 3 /GW 2 ; L = 0.134 cm; I 0 = 1.3 GW/cm 2 . The quasi-analytical intensity given by Eq. ( 7) provides the same results when compared to those obtained by using the Runge-Kutta numerical method. These results are significantly different from those deduced from the rough approximations given in Eqs. ( 3), (4), and (5). In section 3 here below, by comparing the measured values of β and γ using Eq. ( 3), Eq. ( 5) and the exact solution, we will illustrate the error induced by these approximations.

Experimental results

The glass preparation is described in reference [START_REF] Boudebs | Experimental observation of higher order nonlinear absorption in telluride based chalcogenide glasses[END_REF]. For optical characterizations, one sample of 16 mm diameter is cut around 1 mm thickness. The sample is polished up to a powder size of 0.5 µm. The optical transparency extends from around 1 µm to 18 µm.

Depending on the composition of tellurium in these glasses, we will denote them hereafter, as Te0, Te10, Te15 and Te20. The Z-scan setup is shown in Fig. 2. Excitation is provided by a Nd:YAG laser (Continuum) delivering 15 ps single pulses at λ = 1.064 µm with 10 Hz repetition rate. The input intensity I 0 can be varied by means of a half-wave plate and a Glan prism (not shown in this figure), in order to maintain linear polarization. In this work I 0 will be fixed at approximately 1.3 GW/cm 2 , lower than the intensity limit value (I lim in the worst case corresponding to Te20 is equal to 1.35 GW/cm 2 ), given by the analytical condition (Eq. A3) in order to avoid divergence problem when resolving numerically Eq. ( 1).

Physically, this condition traduces the fact that there is no light emission or amplification inside the sample for large negative value of γ. A beam splitter at the input of the setup permits to monitor fluctuations occurring in the incident laser beam. Here we assume that the temporal pulse duration is constant and is always equal to 15 ps. Other experimental parameters are: f = 20 cm, (focal length of lens L); d = 26 cm (distance from the focus to the camera). The beam waist at the focal plane of lens L is ω 0 = 35 µm giving a Rayleigh range

6 . 3 z 2 0 0 ≈ λ ω π =
mm. This value is larger than the sample thickness (typically 1 mm). The sample is mounted on a translation stage and moves around the focus region parallel to the beam propagation z-axis. Note that, for Zscan experiment, the origin of this axis is located at the focus of lens L while it was located at the entry of the specimen when considering Eq. ( 1)

in section 2 and in the appendix. The photoreceptor is a 1000 ×1018 pixels cooled camera (Hamamatsu C4880) with a fixed linear gain. The camera pixels have 4095 gray levels and each pixel is 12 × 12 µm 2 . Neutral filters 'nf' are used to keep the camera within its linear response range. Two sets of acquisitions are carried out for the measurement in order to correct for inhomogenities and surface imperfections in the sample: (i) the low irradiance linear scan is performed by placing the high density neutral filters (nf) before the nonlinear medium (NL). (ii) the high irradiance acquisitions are carried out by placing the same (nf)

after the nonlinear material. The latter was subtracted from the first one after normalization as it is recommended in Ref. [START_REF] Sheik-Bahae | Sensitive measurement of optical nonlinearities using a single beam[END_REF]. No damage or photo-induced phenomena occurred in the tested samples because several experiments have been done showing a good reproducibility of data on the same impact point.

In order to take into account only the contributions of the absorption processes the measurement should be realized after spatially integrating the intensity profiles. We perform the measurement of the nonlinear absorption characterized by the coefficient β eff using the relation for normalized power transmittance [START_REF] Sheik-Bahae | Sensitive measurement of optical nonlinearities using a single beam[END_REF]:

( ) ( ) ( ) ( ) z q z q 1 ln e z T 0 0 L p + = α - , (8) where, ( )
[ ]

2 0 2 0 eff eff 0 z z 1 I L z q + β =
, and I 0 within the sample just after the Fresnel reflection. Eq. ( 8) gives the ratio of the total transmitted power to that of the incident one obtained by integrating over the radial and azimuthal coordinates for Gaussian incident intensity.

The experiment uses pulses in which the intensity (and hence nonlinear absorption)

varies during the pulse. We did not take into account the temporal dependence in this study. A first order correction could be given for a temporally Gaussian pulse. Eq. ( 8) can be timeintegrated to give the normalized energy transmittance. This way of correction is valid only if the response time of detector is much lower compared to the pulse duration. In such a case, we can perform the time-integration because the 'detection-system' is considered as linear. In our case, using 15 ps laser pulse, this integration is no more valid. Anyway, a numerical study taking into account a time integration on Gaussian temporal pulses gives a correction factor that shifted our absolute values (given hereafter) without affecting the qualitative evolution of the curves.

The inset of Fig. 3 shows the normalized open aperture Z-scan transmittance for one composition of ChG glass (Ge 10 As 10 Se 60 Te 20 ). In Fig. 3, the transmittance is plotted as a function of I(z, r = 0), the on-axis (peak) intensity. This is calculated using

( ) ( ) z w w I z I 2 2 0 0 = , where
( ) ( ) 2 0 2 2 0 2 z z 1 w z w + =
is the beam radius. The dotted line is the theoretical fit using relation [START_REF] Shen | Nonlinear Optics[END_REF]. This allows to find β eff , the mean value of the effective TPA coefficient over all the incident intensity range (for I(z) varying from approximately 0 up to 1.4 GW/cm 2 ). Note that for low intensities the experimental data lie below the best theoretical fit using TPA. On the contrary, for higher intensities the data are above. Similar results are obtained with the other samples with different tellurium concentrations. The corresponding values of β eff are reported in column 6 of Table 1. One can note an increase of this value with tellurium concentration. The solid line in Fig. 3 fits the experimental data more appropriately because it takes into account the higher order nonlinearity γ. This fitting was done using the function 'fminsearch' in Matlab which is based on the Nelder-Mead simplex (direct search) method. For each couple of trial values (β,γ) we used the function 'ode45' (based on Runge-Kutta integration method) of the same software in order to resolve numerically the differential equation ( 1) taking into account Gaussian intensity profiles at the entry and the output of the specimen. At each step we calculated the normalized power transmittance by integrating over the radial coordinate. In columns 7 and 8 of Table 1, we report the measured values of (β, γ)

for the different compositions. Table 1 gives also the linear parameters for the four different concentrations of tellurium based chalcogenide glasses. The linear characterization were done using the method described in reference [START_REF] Boudebs | Linear optical characterization of chalcogenide glasses[END_REF]. It can be clearly noted that there is an increase in α, the linear absorption and in n, the linear index, with increasing the tellurium concentration. The increase of α is related to the fact that the band-gap energy of the glass shifts towards lower values when the concentration of tellurium in the glass increases,

showing an increase of the semi-conducting character of the compositions. In column 5 we report the normalized photon energy hν/Eg with Eg the band-gap energy defined here from λgap, the band gap wavelength for which α = 10 cm -1 .

Discussion

In references [START_REF] Lawrence | Measurement of the complex nonlinear refractive index of single crystal p-toluene sulfonate at 1064 nm[END_REF] and [START_REF] Shim | Four photon absorption in the singlecrystal polymer bis (paratoluene) sulfonate[END_REF] negative values for gamma have been reported for PTS at 1.06 µm in the picosecond range using the linear relation [START_REF] Bindra | Nonlinear optical properties of chalcogenide glasses: Observation of multiphoton absorption[END_REF]. In reference [START_REF] Boudebs | Experimental observation of higher order nonlinear absorption in telluride based chalcogenide glasses[END_REF], we have reported β 1 and γ 1 values for ChG glasses of the same composition, using an imaging technique, with higher intensities (I 0 varied from 1 to 4 GW/cm 2 ). We have obtained negative value of γ using relations (3) and ( 4). Even though we obtain approximately the same values for β eff (taking into account the difference of the intensity used in both experiments), the inhomogeneity in these glasses, the use of different measurement method combined with the experimental error do not permit us to compare precisely the values of β and γ. Another problem comes from the fact that the differential equation ( 1) diverges for I 0 > I lim (see the Appendix) and this condition was not satisfied experimentally in reference [START_REF] Boudebs | Experimental observation of higher order nonlinear absorption in telluride based chalcogenide glasses[END_REF]. However, the order of magnitude of the γ values are the same as those obtained here. In order to study more precisely the influence of the simplified relations on the measurement, a comparison is made using the same acquisitions at the same I 0 (below I lim ) and the same technique (Z-scan). The obtained values are reported in table 2. In the second column of this table we can find the measured value of β eff . The coefficients β and γ obtained by the exact solution of Eq. ( 1) are given in columns 3 and 4. Using Eq. ( 3), the approximate solution (β 1 , γ 1 ) described in reference ( 6) are given in columns 5 and 6. We also give the solutions β 2 and γ 2 obtained from the simplified approach using the linear relation [START_REF] Bindra | Nonlinear optical properties of chalcogenide glasses: Observation of multiphoton absorption[END_REF]. Note that whatever the simplification is, the measured TPA coefficient (β 1 or β 2 ) is approximately the same (within the experimental error) as the one given from the exact solution (β). For Te0 where the nonlinear absorption is relatively low compared to the other compositions, there is no need to expand the series in Eq. ( 1) to the third order. The corresponding values are not given because the uncertainty of the measurement was too high and the results does not have any physical significance. On the contrary, for Te10, Te15 and Te20, the obtained γ values are dependent on the used model.

Note that the values of γ 1 obtained using relations (3) and ( 4) are close to those obtained with the exact solution, while γ 2 values obtained using the linear relation ( 5) always underestimate γ, the third order nonlinear coefficient. This is due to the fact that in presence of TPA the incident intensity at the output of the specimen is overestimated (see Fig. 1).

In Fig. 4 we can see the evolution of β eff , β and γ with the normalized photon energy.

We note a quasi-linear dependence of β eff with hν/Eg due to an opposite variation of β, the two-photon absorption coefficient and γ, the third-order nonlinear coefficient. Our quasi-linear behavior is observed above the half band-gap. In the case of a pure TPA, Sheik-Bahae et al. [START_REF] Sheik-Bahae | Dispersion of bound electronic nonlinear refraction in solids[END_REF] using a two band model along with Kramers-Krönig transformation have shown that semi-conductors obeys to an universal dispersion curve where a change in the sign of n 2 (nonlinear index coefficient) accompanied with a decrease in β values is predicted for incident pump wavelength close to the band gap (i.e. 0.7 < hν/Eg < 1). However, our results does not follow this trend. In these tellerium ChG glasses, where the normalized photon energy is higher than 0.7, the β values increase with hν/Eg and the sign of n 2 remain positive (shown unambiguously by the peak and valley signature of the Z-scan transmittance giving values of about +20x10 -18 m 2 /W depending on the composition [START_REF] Cherukulappurath | Linear and nonlinear optical characterization of tellurium based chalcogenide glasses[END_REF]). We do not think that this behaviour is in contradiction with the theoretical and the experimental results of reference [START_REF] Sheik-Bahae | Dispersion of bound electronic nonlinear refraction in solids[END_REF]. Indeed, the higher order nonlinear absorption phenomena shown in this work can no longer be ignored. Further theoretical investigations are needed to fully understand the response of these glasses when the fifth-order nonlinear susceptibility can not be neglected any more.

Conclusion

We have investigated the intensity dependent two-photon absorption in chalcogenide glasses both theoretically and experimentally. We have presented a quasi-analytical solution for the basic differential equation giving the intensity at the exit plane of the specimen under study. The analytical condition that should be satisfied by the incident intensity to avoid divergence problem is provided. Second and third-order nonlinear coefficients are deduced from experimental data using nonlinear fitting combined to Runge-Kutta numerical integration method. Comparison of the results of the measured nonlinear coefficients with those obtained by other approximate solutions of the differential equation is made. We have shown that the exact solution of the basic differential equation describing the propagation of the intensity inside the medium would give more convincing results than the simplified relations adopted up to now.

Appendix: quasi-analytical solution for intensity I(z)

The differential equation ( 1) defining the intensity I as a function of the distance z inside the sample can be analytically solved if: i) we are looking for a solution giving z versus I (this kind of solution cannot be used for comparison with the experimental data), ii) either β or γ are equal to zero. In the latter case (γ = 0 or β = 0), we get two Bernoulli's differential equations. The solutions are, respectively:

z 0 0 1 I e I(z) 1 I L -α = + β , (A1) z 0 2 0 2 I e I(z) 1 2 I L -α = + γ , ( A2
)
where the functions L k (α,z) are defined by L k = [1-exp(-kαz)]/(kα) and I 0 stands for the intensity at z=0. Note that L 1 (α,L) = L eff .

Nevertheless, it is not so easy to find an analytical solution if both β and γ are different from zero. First, in order to describe an absorption process, the derivative dI/dz must be negative. This is true when the coefficients α, β and γ are positive but can be wrong if γ<0. In the latter case, dI/dz is negative only if the incident intensity I 0 satisfies the condition:

0 l i m 2 2 0 I I 1 1 2   α γ β  ≤ < = + +  γ β     (A3)
Considering that the condition (A3) is checked or that γ is positive, we can build a trial function compatible with Eqs. (A1-A2) at the β=0 or γ=0 limits:

z 0 2 ( ) 0 1 0 2 I e I(z) 1 I L I L -α ∞ = + β + γ , ( A4

L

L ∞ ≈ can be easily found for each z at any order n.

Considering at the zeroth-order approximation that L (0) 2 L 2 = , we can define this nth-order approximation as:

z 2 (n ) 2 2 (n 1) 0 1 0 2 0 e d L (z) 1 I L ( ) I L ( -αζ - ζ = ) + β ζ +γ ζ ∫ (A7)
Except in the close neighbourhood of the I 0 limit defined by (A3) in the γ<0 case, converges rather quickly (at n ≈ 2 to 5) towards a stable function and therefore towards . Putting the latter function in Eq. (A4), we thus get the exact solution I(z) of Eq. ( 1).

(

Figure captions

Fig. 1 Numerical simulations of the intensity evolution while propagating inside a sample for Ge 10 As 10 Se 60 Te 20 with the parameters: α = 3.18 cm -1 ; β = 13.7 cm/GW, γ = -11.0 cm 3 /GW 2 ; L = 0.134 cm; I 0 = 1.3 GW/cm 2 . (+): approximation defined by Eq. ( 5); (ο) approximation given by Eqs. ( 3) and ( 4); (•) exact solution of Eq.(1).

Fig. 2 Experimental setup (Z-scan). M: mirror; BS: beam splitter; L, Lr: lenses, nf: neutral filters, NL: nonlinear material. 'd' is the distance of the CCD camera from the focus. Linear and nonlinear optical coefficients at 1.06 µm for different chalcogenide glasses in the picosecond range.

Table 2.

Comparison of higher order nonlinear absorption coefficients obtained from various approximations for different compositions. In the column 2 is given β eff , β and γ are the values given by the Runge-Kutta numerical integration (exact solution). β 1, γ 1 are those obtained from Eq. (3). β 2 , γ 2 are deduced from the more simplified linear relation [START_REF] Bindra | Nonlinear optical properties of chalcogenide glasses: Observation of multiphoton absorption[END_REF]. The β values are given in cm/GW and γ in cm 3 /GW 2 .

  Indeed, the latter condition and the differential equation (1) are satisfied if: intensity I(z) may be numerically computed step by step by using the integral expression:

  Fig.1

Fig. 4 β

 4 Fig.4

Fig. 3 :

 3 Fig. 3:

Fig. 4 Evolution

 4 Fig.4 Evolution of β eff , β, and γ with the normalized photon energy hν/Eg.

Table 1

 1

	compositions	n	α (m -1 )	λgap (nm) (α = 10 cm -1 )	hν/Eg	<β eff > (cm/GW)	β (cm/GW)	γ (cm 3 /GW 2 )
	Ge 10 As 10 Se 80 2.56 14	755	0.71	2.1	1.4	1.2
	Ge 10 As 10 Se 70 Te 10 2.62 32	900	0.85	6.8	10.8	-8
	Ge 10 As 10 Se 65 Te 15 2.75 72	950	0.89	8.2	13	-10
	Ge 10 As 10 Se 60 Te 20 2.80 318	1010	0.95	9.3	13.7	-11

Table Captions Table 1 .

 Captions1

Acknowledgements

This work is supported by an interregional research program Bretagne-Pays de Loire: "Verres infrarouges pour fonctions optiques télécom". We would like to thank also Prof. F. Smektala and M. Guignard for providing us the tellurium based chalcogenide glasses.


Ke Chen

Hai Liu

Todd Blevins

Jie Hao

Léon Otten
email: leon.otten@ibmp-cnrs.unistra.fr

Extensive natural Agrobacterium-induced transformation in the genus Camellia

Keywords: Camellia taxonomy, cT-DNA, Multiple natural transformation, nGMO, Plant evolution, T-DNA 71 proteins

published or not. The documents may come   L'archive ouverte pluridisciplinaire

Introduction

84

Agrobacterium tumefaciens and Rhizobium rhizogenes (also called A. rhizogenes) induce tumors and 85 hairy roots by genetic transformation. The mechanism for this process is well-known and has been were manually trimmed in MEGA v11.0.13 (Tamura et al. 2021) by deleting gaps with fewer than 217 three sequences. The trimmed sequences were then used to construct a phylogenetic tree using IQtree2 218 (Minh et al. 2020), with the parameter "-m MFP" to automatically detect the optimal model and build 219 a maximum likelihood tree with 1000 bootstrap replicates. The resulting tree was visualized and

Results

224

Investigation of the available Camellia short and long read WGS datasets, as well as chromosome-

225

scale genome assemblies (up to March 1, 2023) (Materials and methods, Suppl. Table S1) yielded 12 Due to incomplete coverage, only about 80% of the two repeats (each about 11.5 kb) could be 304 separated, they are 3% diverged. CaTC carries sequences with homology to acs, orf2, orf3n, c, orf8, 305 rolB, gene plast (truncated at its 5' end, the predicted protein has 87% identity to NTF91499.1 from 306 the R. rhizogenes T155/95 TL-region), and orf13. The rolB, plast, and orf13 genes are part of the 307 unique central region and, according to typical R. rhizogenes T-DNA structures [START_REF] Otten | T-DNA regions from 350 Agrobacterium genomes: maps and phylogeny[END_REF], belong 308 to the left arm (Suppl. Fig. S2). Several ORFs are intact: two copies each of acs, orf2, c, orf8, and the 309 unique rolB copy. CaTC was found in eight species of sect. Paracamellia (Suppl. Table S1). As in the 310 case of CaTB, the CaTC insert could be either present or absent in some species.

317

order to simplify the coordinate numbers, we will transpose 6610000 to 1. This cT-DNA (Fig. 1, Table 318 1, Suppl. Fig. S2) was designated aza1CaTD. aza1CaTD is a partial, inverted repeat, separated by a 319 10.6 kb plant sequence (central plant region, CPR). The repeats are 91% identical. The CPR region

333

The overall structure of aza1CaTD was confirmed by the WGS sequence JAKJMZ (compressed 334 haplotype) from C. oleifera cv. Nanyongensis (CON1). ole1CaTD (Fig. 3) is 13687 nt long and has 335 the same overall structure as aza1CaTA, but its CPR carries a plant sequence not found in aza1CaTD,

354

additional plast gene sequence of about 0.6 kb (Fig. 4), the predicted Plast protein has 31% similarity 355 to the unusual T-DNA Plast protein NTF91499.1 from R. rhizogenes strain T155/9 (Otten 2021). We 356 assume that this plast gene was part of the left arm of the original CaTD insert. In addition, the 357 amp1CaTD region shows a direct repeat in susL.

358

In order to study the distribution of the various CaTD types among Camellia species, reads from 32 359 accessions with sufficient coverage were assembled into 39 contigs (Fig. 4) covering the LA and RA 360 regions. The CPR regions of these accessions could not be assembled because they are composed of 361 highly repeated sequences. Altogether, eight different plant inserts (PL1 to PL8) were found in 362 different CaTD contigs, PL3-PL8 could not be fully reconstructed from the short reads, because they 1, Suppl. Fig. S2). The che1CaTE map (Fig. 1) starts at coordinate 384 1458180, which we redefine as 1. che1CaTE consists of an inverted repeat with 98.6% identity 385 between the two arms. The general structure of the repeat is RB-LB-LB-RB and the gene order of the 386 original T-DNA is acs, orf2, orf3n, c, orf8, rolB, orf13, orf13a, orf14, mas2', and

397

The acs and orf3n genes, one copy of rolB and mas1', and two copies of orf13 and orf13a are intact. S1). In addition, CaTF-like sequences were found in one out of two C.

454

Five C. japonica hybrids also have CaTG-like sequences (Suppl. Table S1). They are over 99% 1, Suppl. Table S1). Only few reads were found per species, but they were over

606

One example is Orf358. Its gene was found close to the left border of tra6CaTH. The predicted protein 607 has 64% similarity to protein MCZ7448279.1 from the recently sequenced R. rhizogenes strain 608 ST15.13.057 (accession JAPYZZ010000006.1, 358 AA), annotated as "hypothetical protein" (HP).

609

We propose to call it Orf358. The orf358 gene of ST15.13.057 is followed by the classical T-DNA 610 sequence orf2-orf3n-orf8-rolB-rolC-orf13-orf14-susL (the corresponding proteins were all annotated 611 as HP). On tra6CaTH, orf358 is also followed by orf2-orf3n-orf8-rolB-rolC-orf13. Thus, both on 612 tra6CaTH and the ST15.13.057 T-DNA, orf358 replaces the usual acs gene. No functional domains 613 were found in Orf358. Similar genes are present in four other members of the Rhizobiaceae (Fig. 6).

614

The corresponding proteins were annotated as HP. In these four strains, the genes are not accompanied 615 by other T-DNA genes, suggesting a bacterial function. Remarkably, Orf358-like proteins are also 616 found in eight different fungi (Fig. 6). PSI-BLAST analysis using MCZ7448279.1 as query, revealed

665

However, in sect. Thea, this is not due to the absence of transformation, but to a CaTD deletion event 666 (Chen et al. 2022 and this work). More data are needed to establish whether the same is true for the 667 other sections. In some accessions of sect. Archecamellia, Theopsis, Eriandria, and Heterogenea,

668

CaTA or CaTD are missing, but present in others, again suggesting deletion events.

669

Compared to CaTA and CaTD, the other cT-DNAs show less divergence between their internal 670 repeats, indicating the following ages: 4.6 Mio yrs for CaTB and CaTC (about 3% divergence), 2.0 671 Mio yrs for CaTE (1.4%) and 1.1 Mio yrs for CaTG (0.7%). CaTH is about 75.000 yrs old (0.05% 672 divergence), and 14 of its 16 genes are intact. Its sequence is still very similar to the T-DNA from the 673 R. rhizogenes strain that introduced this cT-DNA in a C. transarisanensis plant, and the original 674 CaTH-a sequence could be entirely reconstructed from the consensus sequence of its repeats. C.

675

transarisanenesis is endemic to Taiwan (Internal Camellia Register), and it might be possible to study 676 the geographic origin and distribution of plants containing CaTH.

677

Our data provide new insight in the origin and evolution of nGMOs. We have argued (Chen and Otten given species is found to contain a cT-DNA, this species should (strictly speaking) not be designated 696 as an nGMO, but as a species "with nGMOs among its members".

697

Our data show the dynamic nature of the transformed state, as the genus Camellia has both acquired patterns and sequences, including their plant inserts, could be useful to improve the grouping of

  CaTA sequences in different Camellia accessions, we detected a new type of susL 315 sequence, suggesting an additional cT-DNA. The corresponding cT-DNA (17.3 kb) was found in the 316 genome assembly of C. azalea cv. Z01, at coordinates GWHAOSQ00000004:6615474-6632762. In

  320 was most likely linked to the T-DNA sequences during the T-DNA insertion process, as it is unrelated 321 to the CaTD flanking sequences. aza1CaTD 5894-7039 is 72% identical to the susL gene from nGMO 322 Jasminum sambac. The CaTD region shows several unusual features. 8046-8926 has 65% identity to 323 the orf511 cT-DNA gene from nGMO N. tomentosiformis (Chen et al. 2014). 5498-5596 is 80% 324 identical to part of a bacterial insertion sequence (IS)-like sequence of the IS110 family, found in 325 several Rhizobium and Agrobacterium strains, like CP049220.1. LA also contains a 190 nt plant sequence (7361-7551, called PL1) between susL and orf511 with 91% identity to C. sinensis cv. 327 Shuchazao XM_028212253.1:144-333. RA is a shorter version of LA, it contains susL, the bacterial IS fragment was inserted in a T-DNA fragment, which was then duplicated and integrated into the plant 332 genome. However, the LA homolog LA* from che1CaTD and other CaTDs (see below) lacks PL1.

  336 and a 5802 nt deletion removes part of the CPR and the adjacent right arm. This shorter right arm was 337 called shRA. An assembled CaTD sequence from C. fluviatilis var. megalantha (C. lanceoleosa, 338 JANUSC) is fully colinear with ole1CaTD and 99.6% identical. 339 Another modified CaTD sequence was found in accession C. chekiangoleosa Hu (che1, Shen et al. 340 2022). A 18799 nt deletion has removed most of the CPR and the entire right arm (Fig. 3). The 341 che1CaTD LA sequence (called LA*) lacks PL1, but carries another plant sequence, PL2.

  342C. handelii, C. euryoides, and C. cuspidata (all sect. Theopsis), carry a modified CaTD (Fig.3) with a 343 16426 nt deletion with respect to aza1CaTD (6879-23305), removing RA and part of LA (leaving a 344 shorter LA, shLA). 345 Some accessions from C. nitidissima var. nitidissima (sect. Archecamellia) show a different type of 346 CaTD deletion (Fig. 3, Fig. 4, nit2, nit5), removing half of the LA (the rest being called sh*LA) and 347 the complete RA. Other C. nitidissima accessions (Fig. 4, nit1, nit3, nit4) do not (Suppl.

363

  are highly repeated in the Camellia genome. The gigCaTD insert shares PL4 with han1CaTD, aza1CaTD:5420 and 28617. further cT-DNA in C. chekiangoleosa Hu (CCH) in GWHBGBN00000017, called 383 che1CaTE (41.2 kb, Fig. 1, Table

  392 found immediately adjacent to the left and right border (che1CaTE:987-1277 and che1CaTE:41495-393 41785). IS1 and IS2 flank the mas1'-mas2' region and were probably part of the original T-DNA. The 394 central part of che1CaTE is most similar to JAAMCX01000012 from R. rhizogenes T155-95, whereas 395 acs and mas are more similar to sequences from R. rhizogenes A4. This reflects the well-known 396 chimeric nature of the T-DNAs (Otten 2021).

398

  che1CaTE was used as a query to investigate its presence in other accessions. Significant parts of the 399 CaTE insert were found in C. sasanqua sas1. C. japonica cv. 'Huaheling' jap4CaTE is very similar to 400 sas1CaTE. The C. sasanqua reads are not sufficient for complete reconstruction, but the simplest 401 model shows three deletions in sas1CaTE compared to che1CaTE (Fig. 1, del1-3). The left part of LA 402 is missing (del1), with a shift of the left border to the left (now corresponding to japonica 'Naidong' (SRR17085750.18983087) suggests a 5193 nt internal deletion (a region which 405 contains acs, orf2 and orf3n), probably due to homologous recombination between the inverted repeats, 406 which removes the unique region and 700 nt each of the adjacent inverted repeat (del2). Another 407 deletion in sas1CaTE occurs in the RA mas2' gene (del3). 408 CaTE traces were found in C. sasanqua cv. 'Xiaomeigui' and 'Zhaohe Zhi Rong', and in C. japonica 409 cv. 'HXZ', 'Naidong 768', 'red,' 'Chidan', 'Fendan' and 'Yudan'. Ten C. chekiangoleosa accessions 410 (Suppl.Table S1) contain large numbers of CaTD reads, but no CaTE reads. Thus, the presence of 411 CaTE in C. chekiangoleosa Hu (che1CaTE) is an exception in this species. It may be derived from 412 another Camellia species by hybridization. several C. oleifera (sect. Paracamellia) accessions showed homology to CaTD, but differed 417 from CaTD. Those from C. oleifera cv. XL210 (ole4) were assembled and extended into an 8 kb cT-418 DNA sequence, ole4CaTF (Fig. 1, Table 1, Suppl.Fig. S2). ole4CaTF contains two plant DNA inserts, 419 called pl1 and pl2. The overall CaTF structure is: orf8 interrupted by pl1, orf511, susL-1, interrupted 420 by pl2, and a second susL-like gene, susL-2 (72% identity to susL-1). The R. rhizogenes LMG152 TL-421 DNA (Otten 2021) also carries two dissimilar susL genes in the same order, but their sequences are 422 different. The ole4CaTF orf8 and orf511 genes are truncated at both ends. A search for CaTF in a 423 large collection of cultivated and wild C. oleifera accessions showed that CaTF was present in 14 out 424 of 68 accessions (Suppl. Table

  425kissii accessions, but not in other Camellia species. C. kissii has been considered as a variant of C.

  426oleifera by Sealy(1958).

  search for cT-DNAs in sect. Theopsis, we sequenced different accessions of C. 431 transarisanensis (Materials and methods). In one of these (tra3, SRR22071522), we found a new cT-432 DNA, designated tra3CaTG (41.5 kb, Fig. 1, Table 1, Suppl. Fig. S2). It consists of a partial, inverted 433 repeat of the LB-RB-RB-LB type. Part of LA and RA could be aligned with R. rhizogenes T155/95 T-434 DNA. Another region is similar to the nGMO N. tomentosiformis TD region (KJ599829.1). The 435 remaining regions showed no detectable DNA homology to either plant or Agrobacterium sequences, 436 but BLASTX analysis showed homology to various T-DNA proteins.

  437tra3CaTG carries the following T-DNA genes: acs, orf8, rolBTR , orf14-1, orf2, orf3n, orf13, orf13a, 438 orf14-2, and cus. This represents a new type of T-DNA with two different orf14 genes (30% identity 439 at the protein level), and an orf2-orf3n fragment in an unusual position. For a discussion on the origin eur1CaTG is similar to tra3CaTG. Its repeats are 99.3% identical. The high similarity between 449 tra3CaTG and eur1CaTG on the one hand, and between LA and RA from each of the two species on 450 the other hand, show that CaTG is of recent origin. CaTG is also found in sect. Eriandria, but not in 451 other sections. Thus, CaTG must have been introduced in the immediate ancestor of these two sections.

452

  This corresponds well to the fact that sect. Theopsis and Eriandria are considered to be closely related 453(Viyajan et al. 2009; Zhang et al. 2021a).

  455identical to each other and 98.3% identical to tra1CaTG. Each hybrid contains DNA from C. synaptica assume that these CaTG sequences originated from the sect. Theopsis partner. The CaTG-like reads 458 from the C. japonica hybrids were assembled into 16 contigs. They aligned with the rolB TR -orf8-acs 459 part of the right arm of tra1CaTG (Fig.1). The remaining sequence is missing. Inspection of the right-460 most contig showed that the C. synaptica sequence diverges from tra1CaTG at tra1CaTG:41764, 461 which represents the right border. The C. synaptica CaTG-like sequence is thus inserted in another 462 plant sequence (see below). We therefore called it synCaTG* (Fig.1). cT-DNA was found in C. transarisanensis tra4 and tra6. Two different units were found:467CaTH-a (24.5 kb, several copies) and CaTH-b (9 kb, one copy), assembled together in one long 468 fragment (Fig.1, Table1, Suppl. Fig. S2). The gene order of CaTH-a is orf358 (a new type of T-DNA 469 gene, see below), orf2, orf3n, orf8-1, rolA, rolB, rolC, orf13, orf13a, orf8-2, rolBTR, and susL. All, 470 except orf8-1 and rolA, are intact ORFs. The orf8-1 gene shows a stop codon at 6327 in all copies of 471 the CaTH-a unit, which was therefore present in the original T-DNA. The truncated orf8-1 gene would 472 still allow the synthesis of a 281AA N-Orf8 protein with potential biological activity (see below). The 473 predicted Orf8-1 and Orf8-2 proteins show only 45% similarity. Long read SRR22198564.1153256 474 from tra4 carries the single CaTH-b unit, linked on the left to the RB of a CaTH-a unit, and on the 475 right to the LB of the next CaTH-a unit. As the long reads are imprecise, the sequence of CaTH-b was 476 assembled using the tra6 short reads (Fig. 1, Suppl. Fig. S5). CaTH-b shows a 1.5 kb fragment on the complete insert is shown in Suppl. Fig. S5a. To the right of the e-d-c'-c region, a 2 kb junction region 483 (intercal 1, Suppl. Fig. S5b), composed of two short CaTH-a fragments, links this unique fragment to 484 the LB of the neighboring CaTH-a unit. A second rearranged sequence composed of three short 485 CaTH-a fragments (intercal 2, Suppl. Fig. S5b) is found more to the right. The CaTH-a unit from tra6 486 shows only 14 SNPs, showing the very recent origin of this insert. A short, partial CaTH fragment 487 from the susL-RB-LB region was found in tra5. No other Camellia accessions contained CaTH of cT-DNA, CaTL, was found in C. impressinervis, C. huana, C. pingguoensis, C. 493 fascicularis, and C. aurea (all from sect. Archecamellia) and C. insularis (not yet attributed to a 494 section) (Fig. 1, Table

495

  99% similar. Two separate fragments could be assembled (called CaTI-a and CaTI-b, Fig.1, Suppl.

  496

Fig

  Fig.S2). CaTI-a (2010 nt) contains an acs-like region (129-1367) with 77% identity to the acs region

617

  several phosphodiester glycosidases in the second iteration, for example WP_150671122.1 from the 618 bacterium Pandoraea anhela (20% similarity). Thus, orf358 from ST15.13.057 and CaTH may encode 619 a new opine synthase. The T-DNA protein Acs (agrocinopine phosphodiesterase) is also a 620 phosphodiester glycosidase, but Orf358 and Acs show no similarity. 621 Another enigmatic T-DNA protein is protein C, the gene is found on CaTB, CaTC, CaTE, CaTH, and 622 CaTL. We noted previously that gene c also occurs in two fungi, Pestalothiopsis fici and Melampsora 623 larici-populina, possibly introduced through natural transformation by Agrobacterium (Chen et al.

  624 2014). A recent homology search found that gene c sequences not only occur on T-DNAs or cT-DNAs, are available for sect. Corallinae and Piquetia. Fig. 7 shows the relationship between the divergence 650 of the internal repeats in the cT-DNAs (indicative for their order of introduction and age) and the cT-651 DNA distribution over different sections. the genus Camellia carry 12 different cT-DNAs, CaTA to CaTL, which result from 656 multiple transformation events. All 72 investigated Camellia species are natural transformants, 657 showing the prevalence of genetic transformation in this genus. Although we do not have a complete 658 overview because of insufficient coverage in different Camellia species, the overall pattern is fairly 659 clear. CaTA and CaTD were inserted early, as shown by the divergence of their repeats (about 10%) 660 and wide distribution. However, CaTA is lacking in sect. Camellia and Paracamellia, and the 661 corresponding insertion sites are intact. Previously, we estimated that the CaTA insert is about 15 Mio seems different. It is absent from sect. Thea, Longipedicellatae, Calpandria, and Tuberculatae.

678 2017 ;

 2017 Chen et al. 2022) that the appearance of an nGMO requires different steps: the induction of 679 hairy roots, the spontaneous regeneration of a hairy root into a fertile nGMO plant, and the survival of 680 the descendants over evolutionary time. Transformed plants could rapidly replace non-transformed 681 siblings if they are sufficiently different to cause reproductive isolation. The present study shows that 682 new nGMO plants might not always be able to rapidly establish a population with a genetically fixed 683 cT-DNA insert. Indeed, age and distribution of the CaTB and CaTC inserts indicate that it may take 684 millions of years for a cT-DNA to become fixed. The rapidity of fixation probably depends on the 685 selective value of the cT-DNA. Crosses between closely related accessions containing either a cT-686DNA or not, may yield more information on this. Another conclusion from these findings is that 687 several accessions must be investigated before one can state that a given species never underwent 688 genetic transformation.689From our Camellia results, it appears that the approximate average natural transformation frequency in 690 Camellia is about one event every 1.25 Mio yrs (12 cT-DNAs in 15 Mio yrs), but this figure could 691 change as more Camellia species are sequenced. The Camellia data hint at the possibility that many 692 plant species could be mixtures of transformed and non-transformed individuals. This means that if 693 nGMO detection is based on one or a few accessions (as in Matveeva and Otten 2019), the overall 694 frequency of species with nGMOs could be severely underestimated. Also, if a single accession of a

  695

Sinicae 963 Fig. 2 a 972 Fig. 3 976 Fig. 4 Fig. 5 987 Fig. 6

 96329723976459876 Fig. 2 a Maps of the hypothetical ancestor CaTA, sin1CaTA, fan1aCaTA, cus1bCaTA, tra1bCaTA,

Fig. 7

 7 Fig. 7 Hypothetical order of cT-DNA insertion events in the genus Camellia. The cT-DNA sequences

  TableS1).

	348
	349	Inspection of long reads of sect. Theopsis species C. transarisanensis (tra1) revealed a 23.3 kb
	350	deletion compared to aza1CaTD 5409-28675, removing the entire CaTD. In sect. Theopsis, 15 out of
	351	30 accessions lack the CaTD region, in sect. Eriandria, five out of six.
	352	C. crapnelliana (sect. Heterogenea) has a CaTD sequence with a similar overall structure as
	353	han1CaTD, cus1CaTD, and eur1CaTD, but the sequence is only 89-90% identical (see below). A
		further type of CaTD was found in C. amplexicaulis. amp1CaTD lacks orf511, but contains an

  A. tumefaciens MD_2022a, KJF65760.1), on one T-DNA (MCZ7448279.1, R. 994 rhizogenes ST15.13.057), and in one nGMO (tra6CaTH, C. transarisanensis).

	988
	989	(Amorphotheca resinae, PSS15205.1; Glarea lozoyensis 74030, EHL02794.1; Glarea lozoyensis
	990	ATCC 20868, EPE26289.1; Infundibulicybe gibba, KAF8887076.1; Xylariaceae sp. FL0255,
	991	KAI1325734.1; Xylariaceae sp. FL1272, KAI0153137.1; Xylariaceae sp. FL1019, KAI1258539.1;
	992	Tephrocybe rancida, KAG6916237.1), in free-living bacteria (Pandoraea anhela, WP_150671122.1;
	993	Rhizobium terrae, WP_117196202+203; R. nepotum, NSZ14673.1; A. fabrum ID132,
		WP_265613673.1;

and Otten 2019; Matveeva 2021). Such plants were called natural Agrobacterium transformants or

rhizogenes T-DNA proteins[START_REF] Otten | T-DNA regions from 350 Agrobacterium genomes: maps and phylogeny[END_REF]. In a few cases, cT-DNA sequences without similarity to

Protein or nucleotide sequences were aligned using MUSCLE(Edgar 2004). The aligned sequences

annotated using iTOL (Letunic and Bork 2021).

different cT-DNA inserts, which we will call CaTA to CaTL (see below). Their sequences are

provided in Suppl. Fig.S2, maps are shown in Fig.1. Various properties (size of insert, size of left and

right arms, size of flanking plant sequences, percentage of identity of repeats) of these inserts are listed

in Table1, and their distribution among the various Camellia accessions is given in Suppl. TableS1.

Suppl. Fig.S2). sas1CaTC consists of a partial, inverted repeat of 28.4 kb of the LB-RB-RB-LB type.

fragment and a PL1-like sequence. The latter is 27 nt shorter than PL1 (PL1*, 20530-20685) and a 66

nt sequence adjacent to PL1 in LA (7552-7618) is found on the other side of PL1* in RA (20686-

20756). The presence of PL1 and PL1* in both CaTD arms seems to indicate that this plant DNA

cus1CaTD, eur1CaTD, confirming their close relationship. Although the ancestral insert carries LA

and RA, we found 13 cases (Fig.4, lower part) which only contained LA* with PL2, as in che1CaTD

(Fig.3). C. sasanqua sas1CaTD is an exception, as it contains both LA*, but also LA and RA. This

and LA, but no RA (the extent of the deletion around RA could not be established).

between the LA, RA, shLA, shRA, sh*LA, and LA* sequences. It indicates five independent losses of

RA (red arrows), partial deletions of LA and RA, and insertion of different plant sequences in various

branches.

Compared to aza1, the C. sinensis cv. Shuchazao sequence shows a 23.2 kb gap, between

JAKJMZ010000013.1:68158951). The central part is also modified. An unusual CaTE read in C.

of the orf2-orf3n region, see below. It should be noted that only 10 different R. rhizogenes T-DNAs

Agrobacterium and in nGMOs, clearly suggests that many R. rhizogenes strains remain to be

discovered. Two copies each of orf8, rolB TR , orf14-1 and orf3n, and one copy of orf13, orf13a, orf14-2

and cus are intact and could code for active proteins (see below). The CaTG structure varied in

different C. transarisanensis accessions, these variants are shown in Suppl. Fig.S4.

CaTG was also found in other species from sect. Theopsis (Suppl. TableS1), but is lacking in one

accession of C. cuspidata and the two accessions of C. lutchuensis (sect. Theopsis). C. euryoides

var. parviovata (sect. Theopsis, International Camellia Register; Zhang et al. 2021a). We therefore

left (FR, Fig.1) with 68% identity to the right end of the A. vitis CG412 TB region (Group IIa, Otten

2021). This fragment includes a partial IS66-like sequence, and two vis gene fragments. It is followed

by the intact genes e-d-c'-c. The e-d-c'-c gene combination is found (in the reverse order) on the T-

nopaline strain C58), but has not been reported in R. rhizogenes or nGMOs so far. The structure of the

ole1CaTD. JAKJMZ010000001 refers to chromosome 1, and for simplification, JAKJMZ0100000

monooxygenase, and could lead to indoleacetamide synthesis. The amp1CaTK Orf8 protein is highly

but also in several free-living Rhizobium strains outside a T-DNA context. Remarkably, in those cases,

gene. R. leguminosarum Cermik105A and Sinorhizobium meliloti M270 also carry gene c and an acs

gene. In R. nepotum 39/7, gene c is linked to a vitopine synthase (vis) gene. Thus, gene c could have

yrs old. As the Camellia group arose some 14.3 Mio yrs ago(Wu et al. 2022), this suggests that sect.

and lost cT-DNAs. Species carrying CaTA and CaTD have persisted for millions of years, with

accessions carry three cT-DNAs (in different combinations), C. sasanqua accession CM-1 has four.

interspecific and intersectional hybridization, both natural and man-made, as this will lead to

abnormalities in the phylogenetic tree. We detected three cases of cT-DNA transmission by

intersectional breeding. The first involves transfer of synCaTG* from C. synaptica to five C. japonica

(as expected for the plast genes, including orf8). The functions of several cT-DNA genes remain

unknown. These include orf358, orf8, orf13a, orf14, gene c, gene c', gene d, gene e and orf511, all

found in Camellia. The new T-DNA gene orf358 is found at a position normally occupied by an acs

gene and might code for an enzyme, as the predicted protein is weakly related to phosphodiester

glycosidases. The orf358 gene occurs on the T-DNA of a single Agrobacterium strain, in one nGMO

(C. transarisanensis), and strikingly, in several fungi. Thus, this gene could have been transferred

from fungi to a T-DNA in Agrobacterium, and then to Camellia, or from Agrobacterium to fungi and

Mol Biol 215:403-410. https://doi.org/10.1016/S0022-2836(05)80360-2

Cai Y, Meng J, Cui Y, Tian M, Shi Z, Wang J (2022) Transcriptome and targeted hormone

metabolome reveal the molecular mechanisms of flower abscission in Camellia. Front Plant Sci

13:1076037. https://doi.org/10.3389/fpls.2022.1076037

Chang HT (1998) Genus Camellia. In: Chang HT, Ren SX (eds) Theaceae, Flora Republicae Popularis

(eds) Molecular form and function of the plant genome, Plenum Press, New York, pp 625-636

ole4CaTF: plant insertion sequences. susL-1 and susL-2 in ole4CaTF: two different susL genes. del a

In two fungi (Melampsora larici-populina, EGG11381.1; Pestalotiopsis fici, ETS73997.1). acs, c:

unlinked genes, acs-c: linked genes.

1091 1092 1093 1094 1095 1096 1097

This work was supported by the National Natural Science Foundation of China (82170045 to JH and 27 KC, 31800253 to KC and JH). It was also supported by a special fund for scientific research of 28 Shanghai landscaping and city appearance administrative bureau (G222410), and 29 ����������� (Zhongkeyuanqingnian yingcai peiyu jihua) grant from the Chinese Academy 30 of Sciences to KC. TB was supported by the Interdisciplinary Thematic Institute IMCBio (ITI 2021-31 2028 program), including funds from IdEx Unistra (ANR-10-IDEX-0002), SFRI-STRAT'US (ANR 32 20-SFRI-0012) and EUR IMCBio (ANR-17-EURE-0023) in the framework of the French Investments 33 for the Future Program.

Abbreviations

Although CaTD is widespread, some Camellia accessions lack CaTD. These include all accessions 374 from sect. Thea and Tuberculatae, two out of seven C. indochinensis accessions (sect. Archecamellia),

711

The oldest cT-DNAs, CaTA and CaTD, have been modified by insertion of different plant sequences.

712

These plant sequences likely originate from transposition, with different inserts having been

713

introduced at different times and in different evolutionary branches (Fig. 5). They could not be fully 714 reconstructed from short reads, because of their highly repetitive nature. Altogether, we found 12

727

amplexicaulis. This species carries the unique CaTK sequence with its highly unusual orf8 gene, and a

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unique CaTD sequence with a plast gene, lacking in all other CaTDs. The precise taxonomic status of 729 this species is not clear (Vijayan et al. 2009;Zhang et al. 2019;Zhao et al. 2019; International 730 Camellia register). Further studies on its two unusual cT-DNAs and their occurrence in other Camellia 731 species may help clarify its affinity to other species.

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The Camellia cT-DNAs contain altogether 47 ORFs (Fig. 1). These need to be further investigated for 733 their possible influence on metabolism (as expected for the opine synthesis genes) and development Camellia. The possibility that some fungi were transformed by Agrobacterium (Chen and Otten 2017)

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A large effort will be required to elucidate their biological effects, both on the level of the individual 760 genes, and on the level of the species that carry them. Analysis of rarely studied Camellia species will 761 help us to trace these inserts through evolutionary time, and thereby provide new tools to study 762 Camellia evolution. We believe that the genus Camellia with its numerous cT-DNAs represents an 763 excellent model for the origin, function and evolution of plant nGMOs. Table 3

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