Branched pegylated linker-auristatin to control hydrophobicity for the production of homogeneous minibody-drug conjugate against HER2-positive breast cancer.

Trastuzumab emtansine (Kadcyla®) was the first antibody-drug conjugate (ADC) approved by the Food and Drug Administration in 2013 against a solid tumor, and the first ADC to treat human epidermal growth factor receptor 2 positive (HER2+) breast cancer. However, this second generation ADC is burden by several limitations included heterogeneity, limited activity against heterogeneous tumor (regarding antigen expression) and suboptimal tumor penetration. To address this, different development strategies are oriented towards homogeneous conjugation, new drugs, optimized linkers and/or smaller antibody formats. To reach better developed next generation ADCs, a key parameter to consider is the management of the hydrophobicity associated with the linker-drug, increasing with and limiting the drug-to-antibody ratio (DAR) of the ADC. Here, an innovative branched pegylated linker was developed, to control the hydrophobicity of the monomethyl auristatin E (MMAE) and its cathepsin B-sensitive trigger. This branched pegylated linker-MMAE was then used for the efficient generation of internalizing homogeneous ADC of DAR 8 and minibody-drug conjugate of DAR 4, targeting HER2. Both immunoconjugates were then evaluated in vitro and in vivo on breast cancer models. Interestingly, this study highlighted that the minibody-MMAE conjugate of DAR 4 was the best immunoconjugate regarding in vitro cellular internalization and cytotoxicity, gamma imaging, ex vivo biodistribution profile in mice and efficient reduction of tumor size in vivo. These results are very promising and encourage us to explore further fragment-drug conjugate development.

Intra-Domain Cysteines (IDC), a New Strategy for the Development of Original Antibody Fragment-Drug Conjugates (FDCs).

Antibody-drug conjugates (ADCs) derived from a full immunoglobulin-G (IgG) are associated with suboptimal solid-tumor penetration and Fc-mediated toxicities. Antibody fragment-drug conjugates (FDCs) could be an alternative. Nevertheless, innovative solutions are needed to implant cysteines as conjugation sites in the single-chain fragment variable (scFv) format, which is the backbone from which many other antibody formats are built. In addition, the bioconjugation site has the utmost importance to optimize the safety and efficacy of bioconjugates. Our previous intra-tag cysteine (ITC) strategy consisted of introducing a bioconjugation motif at the C-terminal position of the 4D5.2 scFv, but this motif was subjected to proteolysis when the scFv was produced in CHO cells. Considering these data, using three intra-domain cysteine (IDC) strategies, several parameters were studied to assess the impact of different locations of a site-specific bioconjugation motif in the variable domains of an anti-HER2 scFv. In comparison to the ITC strategy, our new IDC strategy allowed us to identify new fragment-drug conjugates (FDCs) devoid of proteolysis and exhibiting enhanced stability profiles, better affinity, and better ability to kill selectively HER2-positive SK-BR-3 cells in vitro at picomolar concentrations. Thus, this work represents an important optimization step in the design of more complex and effective conjugates.

Serodolin, a ß-arrestin-biased ligand of 5-HT7 receptor, attenuates pain-related behaviors.

G protein­coupled receptors (GPCRs) are involved in regulation of manifold physiological processes through coupling to heterotrimeric G proteins upon ligand stimulation. Classical therapeutically active drugs simultaneously initiate several downstream signaling pathways, whereas biased ligands, which stabilize subsets of receptor conformations, elicit more selective signaling. This concept of functional selectivity of a ligand has emerged as an interesting property for the development of new therapeutic molecules. Biased ligands are expected to have superior efficacy and/or reduced side effects by regulating biological functions of GPCRs in a more precise way. In the last decade, 5-HT7 receptor (5-HT7R) has become a promising target for the treatment of neuropsychiatric disorders, sleep and circadian rhythm disorders, and pathological pain. In this study, we showed that Serodolin is unique among a number of agonists and antagonists tested: it behaves as an antagonist/inverse agonist on Gs signaling while inducing ERK activation through a ß-arrestin­dependent signaling mechanism that requires c-SRC activation. Moreover, we showed that Serodolin clearly decreases hyperalgesia and pain sensation in response to inflammatory, thermal, and mechanical stimulation. This antinociceptive effect could not be observed in 5-HT7R knockout (KO) mice and was fully blocked by administration of SB269-970, a specific 5-HT7R antagonist, demonstrating the specificity of action of Serodolin. Physiological effects of 5-HT7R stimulation have been classically shown to result from Gs-dependent adenylyl cyclase activation. In this study, using a ß-arrestin­biased agonist, we provided insight into the molecular mechanism triggered by 5-HT7R and revealed its therapeutic potential in the modulation of pain response.

Near-Infrared Emitting Poly(amidoamine) Dendrimers with an Anthraquinone Core toward Versatile Non-Invasive Biological Imaging.

Today, there is a very strong demand for versatile near-infrared (NIR) imaging agents suitable for non-invasive optical imaging in living organisms (in vivo imaging). Here, we created a family of NIR-emitting macromolecules that take advantage of the unique structure of dendrimers. In contrast to existing fluorescent dendrimers bearing fluorophores at their periphery or in their cavities, a NIR fluorescent structure is incorporated into the core of the dendrimer. Using the poly(amidoamine) dendrimer structure, we want to promote the biocompatibility of the NIR-emissive system and to have functional groups available at the periphery to obtain specific biological functionalities such as the ability to deliver drugs or for targeting a biological location. We report here the divergent synthesis and characterization by NMR and mass spectrometries of poly(amidoamine) dendrimers derived from the fluorescent NIR-emitting anthraquinone core (AQ-PAMAF). AQ-PAMAFs ranging from the generation -0.5 up to 3 were synthesized with a good level of control resulting in homogeneous and complete dendrimers. Absorption, excitation, and emission spectra, as well as quantum yields, of AQ-PAMAFs have been determined in aqueous solutions and compared with the corresponding properties of the AQ-core. It has been demonstrated that the absorption bands of AQ-PAMAFs range from UV to 750 nm while emission is observed in the range of 650-950 nm. Fluorescence macroscopy experiments confirmed that the NIR signal of AQ-PAMAFs can be detected with a satisfactory signal-to-noise ratio in aqueous solution, in blood, and through 1 mm thick tissue-mimicking phantom. The results show that our approach is highly promising for the design of an unprecedented generation of versatile NIR-emitting agents.

Screening and Evaluation of Dermo-Cosmetic Activities of the Invasive Plant Species Polygonum cuspidatum.

Polygonum cuspidatum (P. cuspidatum) is among the world's most problematic invasive plant species with negative ecological, socio-economic and security consequences. Management operations in areas invaded systematically generate a large quantity of plant waste, most often without outlets. Using this plant material could constitute a new alternative treatment for sustainable management. P. cuspidatum is well known to have numerous biological properties, containing notably stilbenes, quinones, flavonoids and phenolic acids. The present work proposes a reliable strategy using powerful techniques for the screening and the evaluation of the dermo-cosmetic potential of its aerial parts (AP) and root parts (RP). To the best of our knowledge, only antioxidant and anti-tyrosinase activities were previously evaluated on P. cuspidatum among the targets studied (superoxide dismutase, hyaluronidase, elastase, collagenase and tyrosinase). The results revealed strong antioxidant and anti-collagenase activities, moderate anti-hyaluronidase activity, while weak anti-elastase and anti-tyrosinase activities were observed for ethanolic extracts. Different standards selected and screened on the same targets made it possible to correlate the observed residual activities of produced extracts of P. cuspidatum from Savoie Mont Blanc and their chemical compositions. A structure-activity study was thus conducted on main molecular families, widely represented in the genus Polygonum.

Oak Species Quercus robur L. and Quercus petraea Liebl. Identification Based on UHPLC-HRMS/MS Molecular Networks.

Two species of oak are dominant in French forests: pedunculate oak (Quercus robur L.) and sessile oak (Quercus petraea Liebl.). Their differentiation is not straightforward but is essential to better understand their respective molecular content in order to better valorize them. Thus, to improve oak species identification, an untargeted UHPLC-HRMS/MS method associated with a two-step data treatment was developed to analyze a wide range of specialized metabolites enabling the comparison of both species of oak extracts. Pooled extracts from sessile and pedunculate oaks, composed of extracts from several trees of pure species from various origins, were compared using first the Venn diagram, as a quick way to get an initial idea of how close the extracts are, and then using a molecular network to visualize, on the one hand, the ions shared between the two species and, on the other hand, the compounds specific to one species. The molecular network showed that the two species shared common clusters mainly representative of tannins derivatives and that each species has specific molecules with similar fragmentation patterns, associated in specific clusters. This methodology was then applied to compare these two pooled extracts to unknown individuals in order to determine the species. The Venn diagram allowed for the quick presumption of the species of the individual and then the species could be assigned more precisely with the molecular network, at the level of specific clusters. This method, developed for the first time, has several interests. First, it makes it possible to discriminate the species and to correctly assign the species of unknown samples. Moreover, it gave an overview of the metabolite composition of each sample to better target oak tree utilization and valorization.

Investigation of lipase-ligand interactions in porcine pancreatic extracts by microscale thermophoresis.

The evaluation of binding affinities between large biomolecules and small ligands is challenging and requires highly sensitive techniques. Microscale thermophoresis (MST) is an emerging biophysical technique used to overcome this limitation. This work describes the first MST binding method to evaluate binding affinities of small ligands to lipases from crude porcine pancreatic extracts. The conditions of the MST assay were thoroughly optimized to successfully evaluate the dissociation constant (Kd) between pancreatic lipases (PL) and triterpenoid compounds purified from oakwood. More precisely, the fluorescent labeling of PL (PL*) using RED-NHS dye was achieved via a buffer exchange procedure. The MST buffer was composed of 20 mM NaH2PO4 + 77 mM NaCl (pH 6.6) with 0.05% Triton-X added to efficiently prevent protein aggregation and adsorption, even when using only standard, uncoated MST capillaries. Storage at -20 °C ensured stability of PL* and its fluorescent signal. MST results showed that crude pancreatic extracts were suitable as a source of PL for the evaluation of binding affinities of small ligands. Quercotriterpenoside-I (QTT-I) demonstrated high PL* binding affinity (31 nM) followed by 3-O-galloylbarrinic acid (3-GBA) (500 nM) and bartogenic acid (BA) (1327 nM). To enrich the 50 kDa lipase responsible for the majority of hydrolysis activity in the crude pancreatic extracts, ammonium sulfate precipitation was attempted and its efficiency confirmed using capillary electrophoresis (CE)-based activity assays and HRMS. Moreover, to accurately explain enzyme modulation mechanism, it is imperative to complement binding assays with catalytic activity ones.

Copper-catalyzed transformation of alkyl nitriles to N-arylacetamide using diaryliodonium salts.

This work reports a simple and efficient method for the copper-catalyzed redox-neutral transformation of alkyl nitriles using eco-friendly diaryliodonium salts and leading to N-arylacetamides. The method features high efficiency, broad substrate scope and good functional group tolerance.

Untargeted metabolomic and molecular network approaches to reveal tomato root secondary metabolites.

INTRODUCTION: The tomato plant, Solanum lycopersicum L. (Solanaceae), is one of the most widely consumed vegetables in the world and plays an important role in human diet. Tomato cultivars are hosts for diverse types of pests, implying diverse chemical defence strategies. Glycoalkaloids are the main specialised metabolites produced by tomato leaves and fruits to protect against pests. However, the roots have received little attention, leading to limited knowledge about their phytochemical content. OBJECTIVE: The main goal of the current study was the development of an untargeted ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS) based metabolomic approach to study phytochemical variations in tomato roots at two different development stages (i.e. 34th and 62nd day after sowing). METHODS: UHPLC-HRMS was used to establish the fingerprint of 24 batches of tomato roots. Statistical analyses were performed to highlight the compounds that discriminated between young and mature tomato roots. A dereplication strategy using molecular networking and HRMS/MS data was set up to identify the metabolites regulated during early root development. KEY FINDINGS: The main biomarkers were guanidine and adenosine derivatives associated with tryptophan. Secondary metabolites such as glycoalkaloids and steroidal alkaloids were also characterised. Most of the metabolites were up-regulated in young tomato roots (34 days old) while tryptophan was up-regulated in the older roots (62 days old). CONCLUSION: The metabolic changes observed in this work contribute to a deeper understanding of early-stage root development and may help our understanding of the complex processes involved in the tomato root defence arsenal.

Untargeted UHPLC-Q-TOF-HRMS based determination of discrimating compounds for oak species Quercus robur L. and Quercus petraea Liebl. identification.

INTRODUCTION: Two species of oak are dominant in French forests: pedunculate oak (Quercus robur L.) and sessile oak (Quercus petraea Liebl.). Differentiating oak species is difficult, since features such as morphological characters, geographical origin and grain are not always relevant. Even if the former is generally richer in tannin compounds while the latter is often richer in aromatic compounds, the intra-species variability is high. The characterisation of the oak species remains a suitable indicator of the molecular composition and quality of the wood. OBJECTIVES: The aim of this study was to determine differentiating molecules allowing oak species identification in order to assist in a suitable wood selection for a better oak tree valorisation since the selection of the oak wood to be used in the production of barrels plays an essential role in wine ageing. MATERIALS AND METHODS: Oak wood samples were collected both in forests and in cooperage timber yards. An untargeted metabolomic approach using ultra-high-pressure liquid chromatography qualitative time-of-flight high-resolution mass spectrometry (UHPLC-Q-TOF-HRMS) associated to multivariate statistical analyses (hierarchical ascendant clustering and partial least squares discriminant analysis) was implemented to determine molecular markers of oak species. RESULTS: Heartwood was identified as the suitable wood part to distinguish oak species. Discriminating molecules did not depend on the sample set. The pedunculate species showed overexpression of bartogenic derivatives while sessile oak presented a higher content in oak lactone precursors and in quercotriterpenosids. CONCLUSION: The developed method allowed the identification of relevant compounds for oak species identification to a better wood valorisation and selection.

On-line two-dimensional liquid chromatography hyphenated to mass spectrometry and ion mobility-mass spectrometry for the separation of carbohydrates from lignocellulosic biomass.

Lignocellulosic biomass is a promising resource of renewable energy. Its transformation to ethanol requires efficient pretreatment leading to complex liquid mixtures made of hundreds of oxygenated analytes. A large part of the released compounds belong to the carbohydrates family. To overcome the complexity of such samples, a comprehensive on-line two-dimensional reversed-phase liquid chromatography hyphenated to high-resolution mass spectrometry (RPLC × RPLC-HRMS) was dedicated to the separation of carbohydrates and more specifically oligomers coming from pretreated lignocellulosic biomass. The first part of this study consisted in the optimization of such hyphenation (i.e. selection of stationary phases, mobile phases, sampling time, etc.). Then, the analytical method was applied to an industrial aqueous biomass product coming from the sulfuric acid-based pretreatment of a wheat straw. Around 70 well-resolved chromatographic peaks corresponding to oligomers were obtained. Occupation of the separation space between each chromatographic dimension was estimated to 75%. In the last part of this study, the interest of ion mobility-mass spectrometry in addition to RPLC × RPLC was discussed. Some examples highlighted the additional separation that can bring ion mobility to RPLC × RPLC-IMS-HRMS method. Using this four-dimensional hyphenation method, each analyte was described by two retention times, the collisional cross section and the molecular formula allowing to reach a level of detail never seen for biomass sample compositions.

Dimerization of Uracil in a Simulated Mars-like UV Radiation Environment.

The search for organic molecules at the surface of Mars is a key objective in astrobiology, given that many organic compounds are possible biosignatures and their presence is of interest with regard to the habitability of Mars. Current environmental conditions at the martian surface are harsh and affect the stability of organic molecules. For this reason, and because current and future Mars rovers collect samples from the upper surface layer, it is important to assess the fate of organic molecules under the conditions at the martian surface. Here, we present an experimental study of the evolution of uracil when exposed to UV radiation, pressure, and temperature conditions representative of the surface of Mars. Uracil was selected because it is a nucleobase that composes RNA, and it has been detected in interplanetary bodies that could be the exogenous source of this molecule by meteoritic delivery to the surface of Mars. Our results show that the experimental quantum efficiency of photodecomposition of uracil is 0.16 ± 0.14 molecule/photon. Although these results suggest that uracil is quickly photodegraded when directly exposed to UV light on Mars, such exposure produces dimers that are more stable over time than the monomer. The identified dimers could be targets of interest for current and future Mars space missions.

Capillary Electrophoresis-Mass Spectrometry at Trial by Metabo-Ring: Effective Electrophoretic Mobility for Reproducible and Robust Compound Annotation.

Capillary zone electrophoresis-mass spectrometry (CE-MS) is a mature analytical tool for the efficient profiling of (highly) polar and ionizable compounds. However, the use of CE-MS in comparison to other separation techniques remains underrepresented in metabolomics, as this analytical approach is still perceived as technically challenging and less reproducible, notably for migration time. The latter is key for a reliable comparison of metabolic profiles and for unknown biomarker identification that is complementary to high resolution MS/MS. In this work, we present the results of a Metabo-ring trial involving 16 CE-MS platforms among 13 different laboratories spanning two continents. The goal was to assess the reproducibility and identification capability of CE-MS by employing effective electrophoretic mobility (µeff) as the key parameter in comparison to the relative migration time (RMT) approach. For this purpose, a representative cationic metabolite mixture in water, pretreated human plasma, and urine samples spiked with the same metabolite mixture were used and distributed for analysis by all laboratories. The µeff was determined for all metabolites spiked into each sample. The background electrolyte (BGE) was prepared and employed by each participating lab following the same protocol. All other parameters (capillary, interface, injection volume, voltage ramp, temperature, capillary conditioning, and rinsing procedure, etc.) were left to the discretion of the contributing laboratories. The results revealed that the reproducibility of the µeff for 20 out of the 21 model compounds was below 3.1% vs 10.9% for RMT, regardless of the huge heterogeneity in experimental conditions and platforms across the 13 laboratories. Overall, this Metabo-ring trial demonstrated that CE-MS is a viable and reproducible approach for metabolomics.

Input of an Off-Line, Comprehensive, Three-Dimensional Method (CPC×SFC/HRMS) to Quantify Polycyclic Aromatic Hydrocarbons in Vacuum Gas Oils.

Heavy polycyclic aromatic hydrocarbons (HPAHs) are known to cause undesirable effects in petroleum hydrocracking processes by deactivating the catalysts and accumulating in the downstream of reactors. Polycyclic aromatic hydrocarbons with less than seven rings (PAHs) naturally contained in vacuum gas oils (VGOs) act as precursors in the HPAHs formation. However, getting a detailed quantitative characterization of such polycyclic hydrocarbons has never been done until now, because of the high chemical complexity of VGOs. Thus, an off-line, comprehensive, three-dimensional methodology was required to achieve a quantitative analysis: centrifugal partition chromatography (CPC) as the first dimension of separation, supercritical fluid chromatography (SFC) as the second dimension hyphenated to Fourier transform ion cyclotron resonance mass spectrometry as the third dimension. In this study, we demonstrated that the developed CPC method fractionated samples according to the hydrocarbons' alkylation degree, whereas our SFC method provided an elution order according to their double bond equivalent. Finally, high-resolution mass spectrometry (HRMS) brought crucial information on the identity of analytes and proved to be essential in the event of unresolved peaks from CPC and SFC chromatograms. To assess the ability of the three-dimensional method for quantification purposes, matrix effects were evaluated by spiking VGO samples with deuterated pyrene. A strong ion suppression phenomenon was highlighted when using only SFC/HRMS, whereas no significant matrix effect was observed with the CPC×SFC/HRMS approach. These experiments revealed the great potential of this innovative methodology to quantify both PAH and HPAH in VGOs for the first time.

Ozone-Induced Aryl Hydrocarbon Receptor Activation Controls Lung Inflammation via Interleukin-22 Modulation.

Airborne ozone exposure causes severe lung injury and inflammation. The aryl hydrocarbon Receptor (AhR) (1), activated in pollutant-induced inflammation, is critical for cytokine production, especially IL-22 and IL-17A. The role of AhR in ozone-induced lung inflammation is unknown. We report here that chronic ozone exposure activates AhR with increased tryptophan and lipoxin A4 production in mice. AhR-/- mice show increased lung inflammation, airway hyperresponsiveness, and tissue remodeling with an increased recruitment of IL-17A and IL-22-expressing cells in comparison to control mice. IL-17A- and IL-22-neutralizing antibodies attenuate lung inflammation in AhR-/- and control mice. Enhanced lung inflammation and recruitment of ILC3, ILC2, and T cells were observed after T cell-specific AhR depletion using the AhRCD4cre-deficient mice. Together, the data demonstrate that ozone exposure activates AhR, which controls lung inflammation, airway hyperresponsiveness, and tissue remodeling via the reduction of IL-22 expression.

Development of an LC-MS multivariate nontargeted methodology for differential analysis of the peptide profile of Asian hornet venom (Vespa velutina nigrithorax): application to the investigation of the impact of collection period variation.

Insect venom is a highly complex mixture of bioactive compounds, containing proteins, peptides, and small molecules. Environmental factors can alter the venom composition and lead to intraspecific variation in its bioactivity properties. The investigation of discriminating compounds caused by variation impacts can be a key to manage sampling and explore the bioactive compounds. The present study reports the development of a peptidomic methodology based on UHPLC-ESI-QTOF-HRMS analysis followed by a nontargeted multivariate analysis to reveal the profile variance of Vespa velutina venom collected in different conditions. The reliability of the approach was enhanced by optimizing certain XCMS data processing parameters and determining the sample peak threshold to eliminate the interfering features. This approach demonstrated a good repeatability and a criterion coefficient of variation (CV) > 30% was set for deleting nonrepeatable features from the matrix. The methodology was then applied to investigate the impact of collection period variation. PCA and PLS-DA models were used and validated by cross-validation and permutation tests. A slight discrimination was found between winter and summer hornet venom in two successive years with 10 common discriminating compounds. Graphical abstract.

Asian hornet Vespa velutina nigrithorax venom: Evaluation and identification of the bioactive compound responsible for human keratinocyte protection against oxidative stress.

The present study aimed to explore the potential antioxidant molecules of the Asian hornet venom (Vespa velutina nigrithorax) responsible for radical scavenging activity and human keratinocyte protection against oxidative stress. We developed a first technical platform that combined a DPPH radical scavenging chemical assay and cytotoxicity and ROS (reactive oxygen species) production in HaCaT keratinocyte cells exposed to UVB to evaluate the antioxidant property of V. velutina venom. We further employed Thin Layer Chromatography (TLC) combined with the DPPH assay as a targeted separation approach to isolate the antioxidant compounds responsible for the free radical scavenging property of V. velutina venom. In parallel, the latter was fractionated by a HPLC-DAD non-targeted separation approach. From this experiment, nine fractions were generated which were again evaluated separately for their antioxidant properties using DPPH assays. Results showed that only one fraction exhibited significant antioxidant activity in which serotonin was identified as the major compound by a UHPLC-ESI-QTOF HRMS/MS approach. We finally demonstrated, using purified serotonin molecule that this bioactive structure is mostly responsible for the free radical scavenging property of the crude venom as evidenced by DPPH and ROS assays in HaCaT cells exposed to UVB.

Characterization of liquid-liquid extraction fractions from lignocellulosic biomass by high performance liquid chromatography hyphenated to tandem high-resolution mass spectrometry.

The conversion of lignocellulosic biomass is a major challenge in the field of renewable energies and bio-based chemicals. The diversity of biomasses and processes leads to complex products having a wide range of polarities and molecular weights. Nowadays, the molecular description of these oxygenated matrices is still largely incomplete and new analytical strategies are required to have a better understanding of biomass products properties. The present study proposes a reliable protocol based on successive liquid-liquid extractions prior to high performance liquid chromatography hyphenated to high-resolution tandem mass spectrometry (HPLC/MSn) using a linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer (LTQ/FT-ICR). The protocol allowed to fractionate an industrial sample coming from the sulfuric acid-based pretreatment of a wheat straw into four key chemical families: carbohydrates, organic acids, phenols and neutral compounds. Each fraction was separately analyzed, which limited matrix effects during mass spectrometry ionization step. Electrospray and atmospheric pressure chemical ionization sources were used in both positive and negative modes in order to ionize and detect a maximum of compounds. Thanks to HPLC/MSn, structures of heavy lignin-carbohydrate complexes (LCC) were elucidated (up to 600 g/mol) as well as carbohydrate oligomers having acid functionalities. Mono, di, tri and tetra-aromatic compounds coming from lignin were also detected. The results reported in this paper demonstrate the complexity of pretreated biomass samples and propose an analytical approach from sample simplification to data treatment in order to describe the biomass composition.

Combination of molecular network and centrifugal partition chromatography fractionation for targeting and identifying Artemisia annua L. antioxidant compounds.

Artemisia annua L. is an annual weedy herb belonging to the Asteraceae family. As a traditional Chinese herb, Artemisia annua is a major source of artemisinin, an antimalarial drug. In addition to artemisinin, this plant contains several other molecular families presenting a wide range of biological properties. To facilitate the screening and the identification of active compounds, the present study describes their targeting by combining the dereplication information obtained by means of Molecular Networks and a crude extract fractionation by Centrifugal Partition Chromatography to obtain and test simplified fractions. This simple and fast approach was developed focused on the antioxidant activity of Artemisia annua with the aim of screening and identifying the antioxidant molecules for further cosmetic uses. Firstly, the aerial parts of Artemisia annua were extracted and their antioxidant activity was evaluated by DPPH, ABTS, CUPRAC, FRAP and iron (II) chelating assays. Extract with a positive response was subjected to UHPLC-HRMS with autoMS/MS experiments in order to build a Molecular Network using the GNPS (Global Natural Products Social Molecular Networking) platform. Secondly, the crude extract was fractionated using CPC with an adapted Arizona solvent system. The fractions obtained were evaluated for antioxidant activity to focus on active compounds, which were located on the Molecular Network and identified thanks to their MS/MS spectra. Using this approach, the major phenolic compound contributing to the antioxidant activity of Artemisia annua extract was identified.

In Vitro Characterization and Stability Profiles of Antibody-Fluorophore Conjugates Derived from Interchain Cysteine Cross-Linking or Lysine Bioconjugation.

Fluorescent labelling of monoclonal antibodies (mAbs) is classically performed by chemical bioconjugation methods. The most frequent labelling technique to generate antibody-fluorophore conjugates (AFCs) involves the bioconjugation onto the mAb lysines of a dye bearing an N-hydroxysuccinimide ester or an isothiocyanate group. However, discrepancies between labelling experiments or kits can be observed, related to reproducibility issues, alteration of antigen binding, or mAb properties. The lack of information on labelling kits and the incomplete characterization of the obtained labelled mAbs largely contribute to these issues. In this work, we generated eight AFCs through either lysine or interchain cysteine cross-linking bioconjugation of green-emitting fluorophores (fluorescein or BODIPY) onto either trastuzumab or rituximab. This strategy allowed us to study the influence of fluorophore solubility, bioconjugation technology, and antibody nature on two known labelling procedures. The structures of these AFCs were thoroughly analyzed by mass spectroscopy, and their antigen binding properties were studied. We then compared these AFCs in vitro by studying their respective spectral properties and stabilities. The shelf stability profiles and sensibility to pH variation of these AFCs prove to be dye-, antibody- and labelling-technology-dependent. Fluorescence emission in AFCs was higher when lysine labelling was used, but cross-linked AFCs were revealed to be more stable. This must be taken into account for the design of any biological study involving antibody labelling.