Synthesis and quantification of oligoesters migrating from starch-based food packaging materials.

The term oligomer refers to structurally diverse compounds coming from incomplete polymerisation or polymer degradation. Their ability to migrate into foodstuffs along with recent studies about their bioavailability and toxicity have risen concerns about the scarcity of standards needed to perform thorough analytical and toxicological studies. In this work, migration extracts of three starch-based biopolymers films for the packaging of fruits and vegetables were analysed according to European legislation 10/2011. Oligoesters analysed by UPLC-MS(QTOF) were the main non-intentionally added substances (NIAS) identified in the food simulants. A stepwise synthesis approach was used to synthesise and isolate eleven cyclic and linear oligoester standards ranging from 2 to 8 monomers based on adipic acid, 1,4-butanediol, isophtalic acid and propylene glycol monomers. These standards were characterised by 1H and 13C NMR as well as high resolution mass spectrometry. An overall high purity of > 98 % was achieved as detected by UPLC-MS(Orbitrap). The standards were then used to unequivocally identify the oligoesters in the migration assay samples by comparing their UPLC-MS/MS spectra, and to semi-quantify or fully quantify these migrant oligoesters. The oligoester quantification results deemed safe only one out of the three biopolymer films according to their threshold of toxicological concern concept. The work herein described aims to contribute towards the oligomers knowledge gaps, opening the door for comprehensive toxicological risk and absorption, distribution, metabolism, excretion and toxicity (ADMET) studies.

O-GlcNAcylation levels remain stable regardless of the anaesthesia in healthy rats.

Anaesthetics are used daily in human and veterinary medicine as well as in scientific research. Anaesthetics have an impact on cell homeostasis especially through modulation of protein post-translational modifications. O-GlcNAcylation, a ubiquitous post-translational modification, plays a role in many biological processes. The aims of this study were to evaluate whether (1) anaesthesia influences O-GlcNAcylation and (2) its stimulation affects physiological parameters. Male Wistar rats (n = 38) were anaesthetized with ketamine-xylazine or isoflurane. They randomly received either an intravenous injection of Ringer's lactate or NButGT (10mg/kg) in order to increase O-GlcNAcylation levels. One hour after induction of anaesthesia, haemodynamic parameters and plasmatic markers were evaluated. Heart, brain and lungs were harvested and O-GlcNAcylation levels and O-GlcNAc-related enzymes were evaluated by western blot. Cardiac and pulmonary O-GlcNAcylation levels and cardiac, cerebral and pulmonary O-GlcNAc associated enzyme expression were not impacted with anaesthesia. Compared with ketamine-xylazine, isoflurane had a lower impact on blood pressure, heart rate and glycaemia. Pharmacological stimulation of O-GlcNAcylation by NButGT did not affect the physiological parameters. This study offers unprecedented insights into the regulation of O-GlcNAcylation and O-GlcNAc related enzymes during anaesthesia. Pharmacological stimulation of O-GlcNAcylation over a 1-h period did not disrupt the physiological balance in healthy anaesthetized rats.

Beneficial Effects of O-GlcNAc Stimulation in a Young Rat Model of Sepsis: Beyond Modulation of Gene Expression.

The young population, which is particularly at risk of sepsis, is, paradoxically, rarely studied. Acute stimulation of O-GlcNAcylation, a post-translational modification involved in metabolic regulation, cell survival and stress response, is beneficial in young rats with sepsis. Considering that sepsis impacts the gene expression profile and that O-GlcNAcylation is a regulator of transcription, the aims of this study are to (i) unveil beneficial mechanisms of O-GlcNAcylation and (ii) decipher the relationship between O-GlcNAcylation and transcription during sepsis. Endotoxemic challenge was induced in 28-day-old male rats using a lipopolysaccharide injection (E. coli O111:B4, 20 mg·kg−1) and compared to control rats (NaCl 0.9%). One hour after, rats were assigned to no therapy or fluidotherapy (NaCl 0.9%, 10 mL.kg−1) ± NButGT (10 mg·kg−1) to stimulate O-GlcNAc levels. Cardiac O-GlcNAcylation levels were evaluated via Western blot and gene transcription using 3' SRP analysis. Lipopolysaccharide injection favorizes inflammatory state with the overexpression of genes involved in the NF-κB, JAK/STAT and MAPK pathways. NButGT treatment increased cardiac O-GlcNAcylation levels (p < 0.05). Yet, the mRNA expression was not impacted two hours after fluidotherapy or NButGT treatment. In conclusion, O-GlcNAc stimulation-induced beneficial effects are not dependent on the gene expression profile at the early phase of sepsis.

Thorough investigation of non-volatile substances extractible from inner coatings of metallic cans and their occurrence in the canned vegetables.

Since the decline of the use of bisphenol A, the chemistry of the varnishes and coatings which are applied to the inner surfaces of metallic food contact materials is poorly documented. We hypothesised that can coatings are now diverse and bring forth various non-intentionally added substances (NIAS) to be described. Investigating complex components such as NIAS requires demanding non-targeted approaches. We investigated the coatings of 12 vegetable cans from the French market. More than 125 substances were pinpointed, among them 84 oligoester combinations from 8 diols and 4 diacids. Thus, oligoesters were the dominant family. Additives such as epoxidised soybean oil, bisphenol A diglycidyl ether and benzoguanamine derivatives and phenol-formaldehyde oligomers were also identified. A software for exploring databases of theoretical combinations of polyester and phenol-formaldehyde resin components (NIAS-db 1.0) was made available. The stepwise organic synthesis of native and deuterated combinations of neopentyl glycol and isophthalic acid (4 and 8 units, linear and cyclic) enabled a higher confidence level and monitoring in vegetable extracts. Migration of oligoesters averaged 330 µg/kg in the drained vegetables (43-1600 µg/kg). This study sheds light on the need to fulfil a proper risk assessment on this NIAS family (exposure and hazard characterisation).

The PD-L1/4-1BB Bispecific Antibody-Anticalin Fusion Protein PRS-344/S095012 Elicits Strong T-Cell Stimulation in a Tumor-Localized Manner.

PURPOSE: While patients responding to checkpoint blockade often achieve remarkable clinical responses, there is still significant unmet need due to resistant or refractory tumors. A combination of checkpoint blockade with further T-cell stimulation mediated by 4-1BB agonism may increase response rates and durability of response. A bispecific molecule that blocks the programmed cell death 1 (PD-1)/programmed cell death 1 ligand 1 (PD-L1) axis and localizes 4-1BB costimulation to a PD-L1-positive (PD-L1+) tumor microenvironment (TME) or tumor draining lymph nodes could maximize antitumor immunity and increase the therapeutic window beyond what has been reported for anti-4-1BB mAbs. EXPERIMENTAL DESIGN: We generated and characterized the PD-L1/4-1BB bispecific molecule PRS-344/S095012 for target binding and functional activity in multiple relevant in vitro assays. Transgenic mice expressing human 4-1BB were transplanted with human PD-L1-expressing murine MC38 cells to assess in vivo antitumoral activity. RESULTS: PRS-344/S095012 bound to its targets with high affinity and efficiently blocked the PD-1/PD-L1 pathway, and PRS-344/S095012-mediated 4-1BB costimulation was strictly PD-L1 dependent. We demonstrated a synergistic effect of both pathways on T-cell stimulation with the bispecific PRS-344/S095012 being more potent than the combination of mAbs. PRS-344/S095012 augmented CD4-positive (CD4+) and CD8-positive (CD8+) T-cell effector functions and enhanced antigen-specific T-cell stimulation. Finally, PRS-344/S095012 demonstrated strong antitumoral efficacy in an anti-PD-L1-resistant mouse model in which soluble 4-1BB was detected as an early marker for 4-1BB agonist activity. CONCLUSIONS: The PD-L1/4-1BB bispecific PRS-344/S095012 efficiently combines checkpoint blockade with a tumor-localized 4-1BB-mediated stimulation burst to antigen-specific T cells, more potent than the combination of mAbs, supporting the advancement of PRS-344/S095012 toward clinical development. See related commentary by Shu et al., p. 3182.

An O-GlcNAcylomic Approach Reveals ACLY as a Potential Target in Sepsis in the Young Rat.

Sepsis in the young population, which is particularly at risk, is rarely studied. O-GlcNAcylation is a post-translational modification involved in cell survival, stress response and metabolic regulation. O-GlcNAc stimulation is beneficial in adult septic rats. This modification is physiologically higher in the young rat, potentially limiting the therapeutic potential of O-GlcNAc stimulation in young septic rats. The aim is to evaluate whether O-GlcNAc stimulation can improve sepsis outcome in young rats. Endotoxemic challenge was induced in 28-day-old rats by lipopolysaccharide injection (E. Coli O111:B4, 20 mg·kg-1) and compared to control rats (NaCl 0.9%). One hour after lipopolysaccharide injection, rats were randomly assigned to no therapy, fluidotherapy (NaCl 0.9%, 10 mL·kg-1) ± NButGT (10 mg·kg-1) to increase O-GlcNAcylation levels. Physiological parameters and plasmatic markers were evaluated 2h later. Finally, untargeted mass spectrometry was performed to map cardiac O-GlcNAcylated proteins. Lipopolysaccharide injection induced shock with a decrease in mean arterial pressure and alteration of biological parameters (p < 0.05). NButGT, contrary to fluidotherapy, was associated with an improvement of arterial pressure (p < 0.05). ATP citrate lyase was identified among the O-GlcNAcylated proteins. In conclusion, O-GlcNAc stimulation improves outcomes in young septic rats. Interestingly, identified O-GlcNAcylated proteins are mainly involved in cellular metabolism.

Protein O-GlcNAcylation levels are regulated independently of dietary intake in a tissue and time-specific manner during rat postnatal development.

AIM: Metabolic sources switch from carbohydrates in utero, to fatty acids after birth and then a mix once adults. O-GlcNAcylation (O-GlcNAc) is a post-translational modification considered as a nutrient sensor. The purpose of this work was to assess changes in protein O-GlcNAc levels, regulatory enzymes and metabolites during the first periods of life and decipher the impact of O-GlcNAcylation on cardiac proteins. METHODS: Heart, brain and liver were harvested from rats before and after birth (D-1 and D0), in suckling animals (D12), after weaning with a standard (D28) or a low-carbohydrate diet (D28F), and adults (D84). O-GlcNAc levels and regulatory enzymes were evaluated by western blots. Mass spectrometry (MS) approaches were performed to quantify levels of metabolites regulating O-GlcNAc and identify putative cardiac O-GlcNAcylated proteins. RESULTS: Protein O-GlcNAc levels decrease drastically and progressively from D-1 to D84 (13-fold, P < .05) in the heart, whereas the changes were opposite in liver and brain. O-GlcNAc levels were unaffected by weaning diet in any tissues. Changes in expression of enzymes and levels of metabolites regulating O-GlcNAc were tissue-dependent. MS analyses identified changes in putative cardiac O-GlcNAcylated proteins, namely those involved in the stress response and energy metabolism, such as ACAT1, which is only O-GlcNAcylated at D0. CONCLUSION: Our results demonstrate that protein O-GlcNAc levels are not linked to dietary intake and regulated in a time and tissue-specific manner during postnatal development. We have identified by untargeted MS putative proteins with a particular O-GlcNAc signature across the development process suggesting specific role of these proteins.

Hemisynthesis of 2,3,4-13C3-1,4-Androstadien-3,17-dione: A Key Precursor for the Synthesis of 13C3-Androstanes and 13C3-Estranes.

In this contribution, we describe two simple and efficient routes for the preparation of keto-aldehyde 1, a key intermediate for the synthesis of 13C3-androstanes and 13C3-estranes. In the first route, the targeted aldehyde 1 was obtained in 40% overall yield from 1,4-androstadien-3,17-dione (3 mmol scale) via a two-step sequence involving a one-pot, abnormal ozonolysis/sulfur oxidation/retro-Michael/ozonolysis process. Alternatively, a second route from 4-androsten-3,17-dione, using a six-step sequence, was optimized to produce 40 mmol batches of the key intermediate 1 in 42% overall yield. At the final stage, the A-ring was reconstructed through a Wittig reaction with the 1-triphenylphosphoranylidene-13C3-2-propanone 2, followed by an intramolecular condensation assisted by thioacetic acid via a Michael addition/retro-Michael reaction sequence to provide 2,3,4-13C3-1,4-androstadien-3,17-dione.

Profiling the lymphoid-resident T cell pool reveals modulation by age and microbiota.

Despite being implicated in non-lymphoid tissues, non-recirculating T cells may also exist in secondary lymphoid organs (SLO). However, a detailed characterization of this lymphoid-resident T cell pool has not yet been done. Here we show that a substantial proportion of CD4 regulatory (Treg) and memory (Tmem) cells establish long-term residence in the SLOs of specific pathogen-free mice. Of these SLOs, only T cell residence within Peyer's patches is affected by microbiota. Resident CD4 Treg and CD4 Tmem cells from lymph nodes and non-lymphoid tissues share many phenotypic and functional characteristics. The percentage of resident T cells in SLOs increases considerably with age, with S1PR1 downregulation possibly contributing to this altered homeostasis. Our results thus show that T cell residence is not only a hallmark of non-lymphoid tissues, but can be extended to secondary lymphoid organs.

Macrophages Induce Long-Term Trapping of γδ T Cells with Innate-like Properties within Secondary Lymphoid Organs in the Steady State.

So far, peripheral T cells have mostly been described to circulate between blood, secondary lymphoid organs (SLOs), and lymph in the steady state. This nomadic existence would allow them to accomplish their surveying task for both foreign Ags and survival signals. Although it is now well established that γδ T cells can be rapidly recruited to inflammatory sites or in certain tumor microenvironments, the trafficking properties of peripheral γδ T cells have been poorly studied in the steady state. In the present study, we highlight the existence of resident γδ T cells in the SLOs of specific pathogen-free mice. Indeed, using several experimental approaches such as the injection of integrin-neutralizing Abs that inhibit the entry of circulating lymphocytes into lymph nodes and long-term parabiosis experiments, we have found that, contrary to Ly-6C-/+CD44lo and Ly-6C+CD44hi γδ T cells, a significant proportion of Ly-6C-CD44hi γδ T cells are trapped for long periods of time within lymph nodes and the spleen in the steady state. Specific in vivo cell depletion strategies have allowed us to demonstrate that macrophages are the main actors involved in this long-term retention of Ly-6C-CD44hi γδ T cells in SLOs.

Molecular basis of fatty acid selectivity in the zDHHC family of S-acyltransferases revealed by click chemistry.

S-acylation is a major posttranslational modification, catalyzed by the zinc finger DHHC domain containing (zDHHC) enzyme family. S-acylated proteins can be modified by different fatty acids; however, very little is known about how zDHHC enzymes contribute to acyl chain heterogeneity. Here, we used fatty acid-azide/alkyne labeling of mammalian cells, showing their transformation into acyl-CoAs and subsequent click chemistry-based detection, to demonstrate that zDHHC enzymes have marked differences in their fatty acid selectivity. This difference in selectivity was apparent even for highly related enzymes, such as zDHHC3 and zDHHC7, which displayed a marked difference in their ability to use C18:0 acyl-CoA as a substrate. Furthermore, we identified isoleucine-182 in transmembrane domain 3 of zDHHC3 as a key determinant in limiting the use of longer chain acyl-CoAs by this enzyme. This study uncovered differences in the fatty acid selectivity profiles of cellular zDHHC enzymes and mapped molecular determinants governing this selectivity.

Adaptive Immune-like γ/δ T Lymphocytes Share Many Common Features with Their α/ß T Cell Counterparts.

To better apprehend γ/δ T cell biological functions in the periphery, it appears crucial to identify markers highlighting the existence of distinct phenotypic and functional γ/δ T cell subsets. Interestingly, the expression of CD44 and Ly-6C subdivides murine peripheral γ/δ T cells into several subsets, with Ly-6C(-) CD44(hi) γ/δ T cells corresponding to the IL-17-producing CD27(-) γ/δ T cell subset exhibiting innate-like features. By comparing the other subsets to naive and memory CD8(+) α/ß T cells, in this study, we show that Ly-6C(- or +) CD44(lo) and Ly-6C(+)CD44(hi) γ/δ T cells greatly resemble, and behave like, their CD8(+) α/ß T cell counterparts. First, like memory CD8(+) α/ß T cells, Ly-6C(+)CD44(hi) γ/δ T cells are sparse in the thymus but largely increased in proportion in tissues. Second, similarly to naive CD8 α/ß T cells, CD44(lo) γ/δ T cells are poorly cycling in vivo in the steady state, and their proportion declines with age in secondary lymphoid organs. Third, CD44(lo) γ/δ T cells undergo spontaneous proliferation and convert to a memory-like Ly-6C(+)CD44(hi) phenotype in response to lymphopenia. Finally, CD44(lo) γ/δ T cells have an intrinsic high plasticity as, upon appropriate stimulation, they are capable of differentiating nonetheless into Th17-like and Th1-like cells but also into fully functional Foxp3(+) induced regulatory T cell-like γ/δ T cells. Thus, peripheral CD27(+) γ/δ T cells, commonly considered as a functionally related T cell compartment, actually share many common features with adaptive α/ß T cells, as both lineages include naive-like and memory-like lymphocytes with distinct phenotypic, functional, and homeostatic characteristics.