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Background: The most common cause of preventable death after injury is haemorrhage. Resuscitative endovascular balloon occlusion of the aorta is intended to provide earlier, temporary haemorrhage control, to facilitate transfer to an operating theatre or interventional radiology suite for definitive haemostasis., Objective: To compare standard care plus resuscitative endovascular balloon occlusion of the aorta versus standard care in patients with exsanguinating haemorrhage in the emergency department., Design: Pragmatic, multicentre, Bayesian, group-sequential, registry-enabled, open-label, parallel-group randomised controlled trial to determine the clinical and cost-effectiveness of standard care plus resuscitative endovascular balloon occlusion of the aorta, compared to standard care alone., Setting: United Kingdom Major Trauma Centres., Participants: Trauma patients aged 16 years or older with confirmed or suspected life-threatening torso haemorrhage deemed amenable to adjunctive treatment with resuscitative endovascular balloon occlusion of the aorta., Interventions: Participants were randomly assigned 1 : 1 to: standard care, as expected in a major trauma centre standard care plus resuscitative endovascular balloon occlusion of the aorta., Main Outcome Measures: Primary: Mortality at 90 days. Secondary: Mortality at 6 months, while in hospital, and within 24, 6 and 3 hours; need for haemorrhage control procedures, time to commencement of haemorrhage procedure, complications, length of stay (hospital and intensive care unit-free days), blood product use. Health economic: Expected United Kingdom National Health Service perspective costs, life-years and quality-adjusted life-years, modelled over a lifetime horizon., Data Sources: Case report forms, Trauma Audit and Research Network registry, NHS Digital (Hospital Episode Statistics and Office of National Statistics data)., Results: Ninety patients were enrolled: 46 were randomised to standard care plus resuscitative endovascular balloon occlusion of the aorta and 44 to standard care. Mortality at 90 days was higher in the standard care plus resuscitative endovascular balloon occlusion of the aorta group (54%) compared to the standard care group (42%). The odds ratio was 1.58 (95% credible interval 0.72 to 3.52). The posterior probability of an odds ratio > 1 (indicating increased odds of death with resuscitative endovascular balloon occlusion of the aorta) was 86.9%. The overall effect did not change when an enthusiastic prior was used or when the estimate was adjusted for baseline characteristics. For the secondary outcomes (3, 6 and 24 hours mortality), the posterior probability that standard care plus resuscitative endovascular balloon occlusion of the aorta was harmful was higher than for the primary outcome. Additional analyses to account for intercurrent events did not change the direction of the estimate for mortality at any time point. Death due to haemorrhage was more common in the standard care plus resuscitative endovascular balloon occlusion of the aorta group than in the standard care group. There were no serious adverse device effects. Resuscitative endovascular balloon occlusion of the aorta is less costly (probability 99%), due to the competing mortality risk but also substantially less effective in terms of lifetime quality-adjusted life-years (probability 91%)., Limitations: The size of the study reflects the relative infrequency of exsanguinating traumatic haemorrhage in the United Kingdom. There were some baseline imbalances between groups, but adjusted analyses had little effect on the estimates., Conclusions: This is the first randomised trial of the addition of resuscitative endovascular balloon occlusion of the aorta to standard care in the management of exsanguinating haemorrhage. All the analyses suggest that a strategy of standard care plus resuscitative endovascular balloon occlusion of the aorta is potentially harmful., Future Work: The role (if any) of resuscitative endovascular balloon occlusion of the aorta in the pre-hospital setting remains unclear. Further research to clarify its potential (or not) may be required., Trial Registration: This trial is registered as ISRCTN16184981., Funding: This award was funded by the National Institute for Health and Care Research (NIHR) Health Technology Assessment programme (NIHR award ref: 14/199/09) and is published in full in Health Technology Assessment ; Vol. 28, No. 54. See the NIHR Funding and Awards website for further award information.

Among the properties melatonin is claimed to possess, are the immuno-inflammation inductive capacities that would be responsible of some of the paramount of activities melatonin is reported to have in most of the human pathological conditions. In the present paper, we measured the effect of melatonin on established cellular models of immuno-inflammation, and found none. The discrepancies are discussed, especially because those properties are reported at pharmacological concentration (1 μM and beyond) at which the melatonin receptors are desensitized by internalization, leading to putative non-receptor-dependent mechanism of action., (Copyright © 2024 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Melatonin is a small natural compound, so called a neuro-hormone that is synthesized mainly in pineal gland in animals. Its main role is to master the clock of the body, under the surveillance of light. In other words, it transfers the information concerning night and day to the peripheral organs which, without it, could not "know" which part of the circadian rhythm the body is in. Besides its main circadian and circannual rhythms mastering, melatonin is reported to be a radical scavenger and/or an antioxidant. Because radical scavengers are chemical species able to neutralize highly reactive and toxic species such as reactive oxygen species, one would like to transfer this property to living system, despite impossibilities already largely reported in the literature. In the present commentary, we refresh the memory of the readers with this notion of radical scavenger, and review the possible evidence that melatonin could be an in vivo radical scavenger, while we only marginally discuss here the fact that melatonin is a molecular antioxidant, a feature that merits a review on its own. We conclude four things: (i) the evidence that melatonin is a scavenger in acellular systems is overwhelming and could not be doubted; (ii) the transposition of this property in living (animal) systems is (a) theoretically impossible and (b) not proven in any system reported in the literature where most of the time, the delay of the action of melatonin is over several hours, thus signing a probable induction of cellular enzymatic antioxidant defenses; (iii) this last fact needs a confirmation through the discovery of a nuclear factor-a key relay in induction processes-that binds melatonin and is activated by it and (iv) we also gather the very important description of the radical scavenging capacity of melatonin in acellular systems that is now proven and shared by many other double bond-bearing molecules. We finally discussed briefly on the reason-scientific or else-that led this description, and the consequences of this claim, in research, in physiology, in pathology, but most disturbingly in therapeutics where a vast amount of money, hope, and patient bien-être are at stake., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Over the years, evidence has accumulated on a possible contributive role of the cytosolic quinone reductase NQO2 in models of dopamine neuron degeneration induced by parkinsonian toxin, but most of the data have been obtained in vitro. For this reason, we asked the question whether NQO2 is involved in the in vivo toxicity of MPTP, a neurotoxin classically used to model Parkinson disease-induced neurodegeneration. First, we show that NQO2 is expressed in mouse substantia nigra dopaminergic cell bodies and in human dopaminergic SH-SY5Y cells as well. A highly specific NQO2 inhibitor, S29434, was able to reduce MPTP-induced cell death in a co-culture system of SH-SY5Y cells with astrocytoma U373 cells but was inactive in SH-SY5Y monocultures. We found that S29434 only marginally prevents substantia nigra tyrosine hydroxylase + cell loss after MPTP intoxication in vivo. The compound produced a slight increase of dopaminergic cell survival at day 7 and 21 following MPTP treatment, especially with 1.5 and 3 mg/kg dosage regimen. The rescue effect did not reach statistical significance (except for one experiment at day 7) and tended to decrease with the 4.5 mg/kg dose, at the latest time point. Despite the lack of robust protective activity of the inhibitor of NQO2 in the mouse MPTP model, we cannot rule out a possible role of the enzyme in parkinsonian degeneration, particularly because it is substantially expressed in dopaminergic neurons., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Importance: Bleeding is the most common cause of preventable death after trauma., Objective: To determine the effectiveness of resuscitative endovascular balloon occlusion of the aorta (REBOA) when used in the emergency department along with standard care vs standard care alone on mortality in trauma patients with exsanguinating hemorrhage., Design, Setting, and Participants: Pragmatic, bayesian, randomized clinical trial conducted at 16 major trauma centers in the UK. Patients aged 16 years or older with exsanguinating hemorrhage were enrolled between October 2017 and March 2022 and followed up for 90 days., Intervention: Patients were randomly assigned (1:1 allocation) to a strategy that included REBOA and standard care (n = 46) or standard care alone (n = 44)., Main Outcomes and Measures: The primary outcome was all-cause mortality at 90 days. Ten secondary outcomes included mortality at 6 months, while in the hospital, and within 24 hours, 6 hours, or 3 hours; the need for definitive hemorrhage control procedures; time to commencement of definitive hemorrhage control procedures; complications; length of stay; blood product use; and cause of death., Results: Of the 90 patients (median age, 41 years [IQR, 31-59 years]; 62 [69%] were male; and the median Injury Severity Score was 41 [IQR, 29-50]) randomized, 89 were included in the primary outcome analysis because 1 patient in the standard care alone group declined to provide consent for continued participation and data collection 4 days after enrollment. At 90 days, 25 of 46 patients (54%) had experienced all-cause mortality in the REBOA and standard care group vs 18 of 43 patients (42%) in the standard care alone group (odds ratio [OR], 1.58 [95% credible interval, 0.72-3.52]; posterior probability of an OR >1 [indicating increased odds of death with REBOA], 86.9%). Among the 10 secondary outcomes, the ORs for mortality and the posterior probabilities of an OR greater than 1 for 6-month, in-hospital, and 24-, 6-, or 3-hour mortality were all increased in the REBOA and standard care group, and the ORs were increased with earlier mortality end points. There were more deaths due to bleeding in the REBOA and standard care group (8 of 25 patients [32%]) than in standard care alone group (3 of 18 patients [17%]), and most occurred within 24 hours., Conclusions and Relevance: In trauma patients with exsanguinating hemorrhage, a strategy of REBOA and standard care in the emergency department does not reduce, and may increase, mortality compared with standard care alone., Trial Registration: isrctn.org Identifier: ISRCTN16184981.

Introduction: The leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) is a tissue resident stem cell marker, which it is expressed in supporting cells (SCs) in the organ of Corti in the mammalian inner ear. These LGR5+ SCs can be used as an endogenous source of progenitor cells for regeneration of hair cells (HCs) to treat hearing loss and deafness. We have recently reported that LGR5+ SCs survive 1 week after ototoxic trauma. Here, we evaluated Lgr5 expression in the adult cochlea and long-term survival of LGR5+ SCs following severe hearing loss., Methods: Lgr5GFP transgenic mice and wild type mice aged postnatal day 30 (P30) and P200 were used. P30 animals were deafened with a single dose of furosemide and kanamycin. Seven and 28 days after deafening, auditory brainstem responses (ABRs) were recorded. Cochleas were harvested to characterize mature HCs and LGR5+ SCs by immunofluorescence microscopy and quantitative reverse transcription PCR (q-RT-PCR)., Results: There were no significant age-related changes in Lgr5 expression when comparing normal-hearing (NH) mice aged P200 with P30. Seven and 28 days after ototoxic trauma, there was severe outer HC loss and LGR5 was expressed in the third row of Deiters' cells and in inner pillar cells. Seven days after induction of ototoxic trauma there was an up-regulation of the mRNA expression of Lgr5 compared to the NH condition; 28 days after ototoxic trauma Lgr5 expression was similar to NH levels., Discussion: The presence of LGR5+ SCs in the adult mouse cochlea, which persists after severe HC loss, suggests potential regenerative capacity of endogenous cochlear progenitor cells in adulthood. To our knowledge, this is the first study showing not only long-term survival of LGR5+ SCs in the normal and ototoxically damaged cochlea, but also increased Lgr5 expression in the adult mouse cochlea after deafening, suggesting long-term availability of potential target cells for future regenerative therapies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Smith-Cortinez, Hendriksen, Ramekers, Stokroos, Versnel and Straatman.)

D3/D2 sub-specificity is a complex problem to solve. Indeed, in the absence of easy structural biology of the G-protein coupled receptors, and despite key progresses in this area, the systematic knowledge of the ligand/receptor relationship is difficult to obtain. Due to these structural biology limitations concerning membrane proteins, we favored the use of directed mutagenesis to document a rational towards the discovery of markedly specific D3 ligands over D2 ligands together with basic binding experiments. Using our methodology of stable expression of receptors in HEK cells, we constructed the gene encoding for 24 mutants and 4 chimeras of either D2 or D3 receptors and expressed them stably. Those cell lines, expressing a single copy of one receptor mutant each, were stably constructed, selected, amplified and the membranes from them were prepared. Binding data at those receptors were obtained using standard binding conditions for D2 and D3 dopamine receptors. We generated 26 new molecules derived from D2 or D3 ligands. Using 8 reference compounds and those 26 molecules, we characterized their binding at those mutants and chimeras, exemplifying an approach to better understand the difference at the molecular level of the D2 and D3 receptors. Although all the individual results are presented and could be used for minute analyses, the present report does not discuss the differences between D2 and D3 data. It simply shows the feasibility of the approach and its potential., (© 2022 The Protein Society.)

B7-H3 (CD276) has emerged as a target for cancer immunotherapy by virtue of consistent expression in many malignancies, relative absence from healthy tissues, and an emerging role as a driver of tumor immune inhibition. Recent studies have reported B7-H3 to be a suitable target for chimeric antigen receptor-modified T cell (CAR-T) therapy using CARs constructed from established anti-B7-H3 antibodies converted into single-chain Fv format (scFv). We constructed and screened binders in an scFv library to generate a new anti-B7-H3 CAR-T with favorable properties. This allowed access to numerous specificities ready formatted for CAR evaluation. Selected anti-human B7-H3 scFvs were readily cloned into CAR-T and evaluated for anti-tumor reactivity in cytotoxicity, cytokine, and proliferation assays. Two binders with divergent complementarity determining regions were found to show optimal antigen-specific cytotoxicity and cytokine secretion. One binder in second-generation CD28-CD3ζ CAR format induced sustained in vitro proliferation on repeat antigen challenge. The lead candidate CAR-T also demonstrated in vivo activity in a resistant neuroblastoma model. An empirical approach to B7-H3 CAR-T discovery through screening of novel scFv sequences in CAR-T format has led to the identification of a new construct with sustained proliferative capacity warranting further evaluation., Competing Interests: John Anderson declares founder shares in Autolus Ltd and collaborations with Roche and ALX-Oncology., (© 2022 The Author(s).)

Bispecific antibodies (BsAbs) represent an important advance in innovative therapeutic strategies. Among the countless formats of BsAbs, fusion with molecules such as anticalins linked to a monoclonal antibody (mAb), represents an easy and low-cost way to obtain innovative molecules. We fused an anticalin against human fibronectin to a molecule biosimilar to trastuzumab (H0) or rituximab (R0), in four different positions, two on the N terminal region of heavy or light chains and two on the C terminal region. The eight BsAbs (H family (HF) 1 to 4 and R family (RF) 1 to 4) were produced and their affinity parameters and functional properties evaluated. The presence of anticalin did not change the glycosylation of the BsAb, shape or yield. The antigenic recognition of each BsAb family, Her2 for HF1 to 4 and CD20 for RF1 to 4, was slightly decreased (HF) or absent (RF) for the anticalin N-terminal in the light chain position. The anticalin recognition of FN was slightly decreased for the HF family, but a dramatic decrease was observed for RF members with lowest affinity for RF1. Moreover, functional properties of Abs, such as CD16 activation of NK, CD32-dependent phagocytosis and FcRn transcytosis, confirmed that this anticalin position leads to less efficient BsAbs, more so for RF than HF molecules. Nevertheless, all BsAbs demonstrated affinities for CD16, CD32 and FcRn, which suggests that more than affinity for FcRs is needed for a functioning antibody. Our strategy using anticalin and Abs allows for rapid generation of BsAbs, but as suggested by our results, some positions of anticalins on Abs result in less functionality.

Plasma membrane calcium ATPases (PMCAs) are a family of transmembrane proteins responsible for the extrusion of cytosolic Ca 2+ to the extracellular milieu. They are important players of the calcium homeostasis possibly implicated in some important diseases. The reference inhibitors of PMCA extruding activity are on one hand ortho-vanadate (IC 50 in the 30 mM range), and on the other a series of 12- to 20-mer peptides named caloxins (IC 50 in the 100 µM scale). As for all integral membrane proteins, biochemistry and pharmacology are difficult to study on isolated and/or purified proteins. Using a series of reference blockers, we assessed a pharmacological window with which we could study the functionality of PMCAs in living cells. Using this system, we screened for alternative versions of caloxins, aiming at shortening the peptide backbone, introducing non-natural amino acids, and overall trying to get a glimpse at the structure-activity relationship between those new peptides and the protein in a cellular context. We describe a short series of equipotent 5-residue long analogues with IC 50 in the low µM range., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Neuroblastoma is a commonly lethal solid tumor of childhood and intensive chemoradiotherapy treatment cures ~50% of children with high-risk disease. The addition of immunotherapy using dinutuximab, a monoclonal antibody directed against the GD2 disialoganglioside expressed on neuroblasts, improves survival when incorporated into front-line therapy and shows robust activity in regressing relapsed disease when combined with chemotherapy. Still, many children succumb to neuroblastoma progression despite receiving dinutuximab-based immunotherapy, and efforts to counteract the immune suppressive signals responsible are warranted. Animal models of human cancers provide useful platforms to study immunotherapies. TH-MYCN transgenic mice are immunocompetent and develop neuroblastomas at autochthonous sites due to enforced MYCN expression in developing neural crest tissues. However, GD2-directed immunotherapy in this model has been underutilized due to the prevailing notion that TH-MYCN neuroblasts express insufficient GD2 to be targeted. We demonstrate that neuroblasts in TH-MYCN -driven tumors express GD2 at levels comparable to human neuroblastomas but rapidly lose GD2 expression when explanted ex vivo to establish tumor cell lines. This occurs in association with a transition from an adrenergic to mesenchymal differentiation state. Importantly, not only is GD2 expression retained on tumors in situ, treatment with a murine anti-GD2 antibody, 14G2a, markedly extends survival in such mice, including durable complete responses. Tumors in 14G2a-treated mice have fewer macrophage and myeloid-derived suppressor cells in their tumor microenvironment. Our findings support the utility of this model to inform immunotherapy approaches for neuroblastoma and potential opportunities to investigate drivers of adrenergic to mesenchymal fate decisions., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2022 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Melatonin exerts its effects through a series of target proteins/receptors and enzymes. Its antioxidant capacity might be due to its capacity to inhibit a quinone reductase (NQO2) at high concentration (50 μM). Demonstrating the existence of a complex between a compound and a protein is often not easy. It requires either that the compound is an inhibitor-and the complex translates by an inhibition of the catalytic activity-or the compound is radiolabeled-and the complex translates in standard binding approaches, such as in receptology. Outside these two cases, the detection of the protein:small molecule complexes by mass spectrometry has recently been made possible, thanks to the development of so-called native mass spectrometry. Using this approach, one can measure masses corresponding to an intact noncovalent complex between a compound and its target, usually after titration or competition experiments. In the present chapter, we detail the characterization of NQO2:melatonin interaction using native mass spectrometry., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cloning may seem to be a view from the past. The time before software, computers and AI were invented. It seems to us worth discussing these points in view of our favorite target: the melatoninergic system. In a few stances, it might be important to point out that even in the new era of dry science, there is still a need to experiment and to prove at the bench that our in silico assertions are right. Most of the living animals express to some extend the melatonin receptors. Some of these animal genomes were completely or partially sequenced, and it is tempting to extract from this huge information the sequence(s) of our favorite genes (MLT receptors). Then, why bother cloning, as opposed to simply built the gene and express it in a host cell? Because the genetic boundaries of the expressed sequence(s) are not 100% sure. Because the melatonin receptor gene(s) comprise a first exon 25,000 base pair far from the second one and the limits between this Ex1 and In1-as between In1 and Ex2-are subject to changes that might have a huge impact on the biochemical properties of the receptor, once expressed. Because a receptor is a biochemical entity with characteristics that are important for the functioning of this particular pathway, and more generally, for the functioning of life., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The third melatonin binding site MT 3 turned out to be an enzyme, NQO2 (E.C. 1.6.99.2). Its catalytic activity is inhibited by melatonin with an IC 50 in the 50-100 μM range. Some of the functions of melatonin at pharmacological concentrations (1 μM and above) might be explained by this inhibition capacity of melatonin at NQO2. In order to determine precisely these parameters, it is required to comprehend the basic enzymology of this enzyme. In the following chapter, we present the basic conditions of measuring NQO2 catalytic activities and inhibition., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Posttranslational modifications of proteins are catalyzed by a large family of enzymes catalyzing many chemical modifications. One can hijack the natural use of those enzymes to modify targeted proteins with synthetic chemical moieties. The lipoic acid ligase LplA mutants can be used to introduce onto the lysine sidechain lipoic acid moiety synthetic analogues. Substrate protein candidates of the ligase must obey a few a priori rules., Methods and Results: In the present report, we technically detailed the use of a cell line stably expressing both the ligase and a model protein (thioredoxin). Although the goal can be reach, and the protein visualized in situ, many experimental difficulties must be fixed. The sequence of events comprises (i) in cellulo labeling of the target protein with a N 3 -lipoic acid derivative catalyzed by the mutant ligase, (ii) the further introduction by click chemistry onto this lysine sidechain of a fluorophore and (iii) the following of the labeled protein in living cells. One of the main difficulties was to assess the click chemistry step onto the living cells, because images from both control and experimental cells were similar. Alternatively, we describe at that stage, the preferred use of another technique: the Halo-Tag one that led to the obtention of clear images of the targeted protein in its cellular context. Although the ligase-mediated labeling of protein in situ is a rich domain for which many cellular tools must be developed, many difficulties must be considered before entering a systematic use of this approach., Conclusions: In the present contribution, we added several steps of analytical characterization, both in vitro and in cellulo that were previously lacking. Furthermore, we show that the use of the click chemistry should be manipulated with care, as the claimed specificity might be not complete whenever living cells are used. Finally, we added another approach-the Halo Tag-to complete the previously suggested approaches for labelling proteins in cells, as we found difficult to strictly apply the previously reported methodology., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone which possesses a wide range of biological effects. The effects mediated by melatonin are in part attributed to the antioxidant properties of the molecule, which may act as scavenger of free radicals, and also to the binding of melatonin to its protein targets. For a long time, melatonin had been described as a ligand of a putative "receptor" present in the mammalian brain. Several studies were thus carried out with the goal of clarifying the nature of this melatonin "receptor," which led to the discovery of MT3 as the third melatonin binding site. This binding site was confirmed independently by several groups, and it was eventually demonstrated that MT3 was the enzyme quinone reductase 2 (NQO2). Among the different approaches used to validate that MT3 was indeed NQO2, the co-crystallization of NQO2 with melatonin was key in demonstrating the exact binding site and mode of melatonin to the enzyme and led to a clear understanding of the residues important for protein binding and inhibition. In this chapter, we described the details for the cloning, expression, and purification of the human enzyme NQO2. We also describe a detailed protocol for the crystallization of melatonin with this protein., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

A way to study G protein-coupled receptors in a minimal system is to reconstruct artificial membrane mimics, made of detergent and/or of lipids in which the purified receptor is maintained. In particular, it is now possible to generate lipid nanoparticles, such as nanodiscs, in which a single receptor molecule is included. Such objects offer the invaluable potential of studying an isolated receptor stabilized in a finely controlled membrane-like environment to evaluate its pharmacology, its function, and its structure at the molecular level. In this chapter, we detail the different steps from the extraction and isolation of a recombinant MT1 melatonin receptor in detergent, down to its reconstitution into nanodiscs. A G protein activation test is further described in order to exemplify how the functionality of such particles may be investigated., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

To ensure the physical interaction between a protein and its ligand, many techniques can be applied. One of them, isothermal titration calorimetry (ITC), measures the heat exchange between a forming molecular complex and its milieu. From this heat exchange, it is possible to acquire the thermodynamic parameters, the binding stoichiometry and the affinity constant (K a ) between the two interacting binding partners, which can then be used to determine the dissociation constant (K d ). We made use of ITC to determine the true K d of melatonin for its putative receptor MT3, also known as the enzyme quinone reductase 2 (NQO2). In this chapter, we describe the step-by-step procedure for performing this experiment and extend it to 2-iodomelatonin, a melatonin derivative that was used in the initial identification and characterization of MT3. The dissociation constants of melatonin and 2-iodomelatonin toward NQO2 derived from these experiments are in line with data reported previously, albeit using alternative techniques., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Melatonin, (N-acetyl-5-methoxytryptamine), is a neurohormone which possesses a wide range of biological effects. The effects mediated by melatonin are in part attributed to the antioxidant properties of the molecule. For a long time, melatonin had been described as a ligand of a putative "receptor" present in mammalian brains named MT 3 . Several studies were thus carried out with the goal of clarifying the nature of this melatonin "receptor." The experimental setup of the binding measurements is unusual. The present chapter aims at describing this technique. This binding site was confirmed independently by several groups, and it was eventually demonstrated that MT3 was the enzyme quinone reductase 2 (NQO2)., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

EPIDemio study is a multicenter, prospective and observational study. The objective is to estimate the prevalence and incidence of fibrosing interstitial lung diseases (ILDs) in the department of Haute Garonne (31) in France. Fifty-five pulmonologists from the Toulouse university hospital and 8 private establishments participated in this study. Two hundred and fifty-six cases of fibrosing ILDs were reported (gross overall prevalence: 22.8/100,000 and estimated 30.1/100,000. Idiopathic ILDs represent 55.8% of fibrosing ILDs ahead of systemic disease-related ILDs (24.6%) and ILDs associated with environmental exposure (13.3%). Idiopathic pulmonary fibrosis (IPF) represents 35.9% of fibrosing ILDs, which corresponds to a minimal prevalence of 8.2/100,000 and an estimated prevalence of 11.2/100,000. This study confirms epidemiological data collected in France and Europe., (Copyright © 2021 SPLF. Published by Elsevier Masson SAS. All rights reserved.)

Sensorineural hearing loss is mainly caused by irreversible damage to sensory hair cells (HCs). A subgroup of supporting cells (SCs) in the cochlea express leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), a marker for tissue-resident stem cells. LGR5+ SCs could be used as an endogenous source of stem cells for regeneration of HCs to treat hearing loss. Here, we report long-term presence of LGR5+ SCs in the mature adult cochlea and survival of LGR5+ SCs after severe ototoxic trauma characterized by partial loss of inner HCs and complete loss of outer HCs. Surviving LGR5+ SCs (confirmed by GFP expression) were located in the third row of Deiters' cells. We observed a change in the intracellular localization of GFP, from the nucleus in normal-hearing to cytoplasm and membrane in deafened mice. These data suggests that the adult mammalian cochlea possesses properties essential for regeneration even after severe ototoxic trauma., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Smith-Cortinez, Yadak, Hendriksen, Sanders, Ramekers, Stokroos, Versnel and Straatman.)

VHH stands for the variable regions of heavy chain only of camelid IgGs. The VHH family forms a set of interesting proteins derived from antibodies that maintain their capacity to recognize the antigen, despite their relatively small molecular weight (in the 12,000 Da range). Continuing our exploration of the possibilities of those molecules, we chose to design alternative molecules with maintained antigen recognition, but enhanced capacity, by fusing four VHH with one Fc, the fragment crystallizable region of antibodies. In doing so, we aimed at having a molecule with superior quantitative antigen recognition (×4) while maintaining its size below the 110 kDa. In the present paper, we described the building of those molecules that we coined VHH 2 -Fc-VHH 2 . The structure of VHH 2 -Fc-VHH 2 in complex with HER2 antigen was determined using electronic microscopy and modeling. The molecule is shown to bind four HER2 proteins at the end of its flexible arms. VHH 2 -Fc-VHH 2 also shows an internalization capacity via HER2 receptor superior to the reference anti-HER2 monoclonal antibody, Herceptin®, and to a simple fusion of two VHH with one Fc (VHH 2 -Fc). This new type of molecules, VHH 2 -Fc-VHH 2 , could be an interesting addition to the therapeutic arsenal with multiple applications, from diagnostic to therapy., (© 2021 The Protein Society.)

The balance between detoxification and toxicity is linked to enzymes of the drug metabolism Phase I (cytochrome P450 or oxidoreductases) and phase II conjugating enzymes (such as the UGTs). After the reduction of quinones, the product of the reaction, the quinols-if not conjugated-re-oxidizes spontaneously to form the substrate quinone with the concomitant production of the toxic reactive oxygen species (ROS). Herein, we documented the modulation of the toxicity of the quinone menadione on a genetically modified neuroblastoma model cell line that expresses both the quinone oxidoreductase 2 (NQO2, E.C. 1.10.5.1) alone or together with the conjugation enzyme UDP-glucuronosyltransferase (UGT1A6, E.C. 2.4.1.17), one of the two UGT isoenzymes capable to conjugate menadione. As previously shown, NQO2 enzymatic activity is concomitant to massive ROS production, as previously shown. The quantification of ROS produced by the menadione metabolism was probed by electron-paramagnetic resonance (EPR) on cell homogenates, while the production of superoxide was measured by liquid chromatography coupled to mass spectrometry (LC-MS) on intact cells. In addition, the dysregulation of the redox homeostasis upon the cell exposure to menadione was studied by fluorescence measurements. Both EPR and LCMS studies confirmed a significant increase in the ROS production in the NQO2 overexpressing cells due to the fast reduction of quinone into quinol that can re-oxidize to form superoxide radicals. However, the effect of NQO2 inhibition was drastically different between cells overexpressing only NQO2 vs. both NQO2 and UGT. Whereas NQO2 inhibition decreases the amount of superoxide in the first case by decreasing the amount of quinol formed, it increased the toxicity of menadione in the cells co-expressing both enzymes. Moreover, for the cells co-expressing QR2 and UGT the homeostasis dysregulation was lower in presence of menadione than for the its counterpart expressing only QR2. Those results confirmed that the cooperation of the two enzymes plays a fundamental role during the cells' detoxification process. The fluorescence measurements of the variation of redox homeostasis of each cell line and the detection of a glucuronide form of menadiol in the cells co-expressing NQO2 and UGT1A6 enzymes further confirmed our findings., Competing Interests: Author RG was employed by company Flash Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chhour, Perio, Gayon, Ternet-Fontebasso, Ferry, Nepveu, Boutin, Sudor and Reybier.)

Melanin-concentrating hormone (MCH) is a 19 amino acid long peptide found in the brain of animals, including fishes, batrachians, and mammals. MCH is implicated in appetite and/or energy homeostasis. Antagonists at its receptor (MCH-R1) could be major tools (or ultimately drugs) to understand the mechanism of MCH action and to fight the obesity syndrome that is a worldwide societal health problem. Ever since the deorphanisation of the MCH receptor, we cloned, expressed, and characterized the receptor MCH-R1 and started a vast medicinal chemistry program aiming at the discovery of such usable compounds. In the present final work, we describe GPS18169, a pseudopeptide antagonist at the MCH-R1 receptor with an affinity in the nanomolar range and a Ki for its antagonistic effect in the 20 picomolar range. Its metabolic stability is rather ameliorated compared to its initial parent compound, the antagonist S38151. We tested it in an in vivo experiment using high diet mice. GPS18169 was found to be active in limiting the accumulation of adipose tissues and, correlatively, we observed a normalization of the insulin level in the treated animals, while no change in food or water consumption was observed.

N -ribosyldihydronicotinamide:quinone oxidoreductase 2 (NQO2/QR2, Enzyme Commission number 1.10.99.2) is a cytosolic enzyme, abundant in the liver and variably expressed in mammalian tissues. Cloned 30 years ago, it was characterized as a flavoenzyme catalyzing the reduction of quinones and pseudoquinones. To do so, it uses exclusively N -alkyl nicotinamide derivatives, without being able to recognize NADH, the reference hydrure donor compound, in contrast to its next of a kind, NAD(P)H:quinone oxidoreductase 1 (NQO1). For a long time both enzymes have been considered as key detoxifying enzymes in quinone metabolism, but more recent findings point to a more toxifying function of NQO2, particularly with respect to ortho -quinones. In fact, during the reduction of substrates, NQO2 generates fairly unstable intermediates that reoxidize immediately back to the original quinone, creating a futile cycle, the byproducts of which are deleterious reactive oxygen species. Beside this peculiarity, it is a target for numerous drugs and natural compounds such as melatonin, chloroquine, imiquimod, resveratrol, piceatannol, quercetin, and other flavonoids. Most of these enzyme-ligand interactions have been documented by numerous crystallographic studies, and now NQO2 is one of the best represented proteins in the structural biology database. Despite evidence for a causative role in several important diseases, the functional role of NQO2 remains poorly explored. In the present review, we aimed at detailing the main characteristics of NQO2 from a molecular pharmacology perspective. By drawing a clear border between facts and speculations, we hope to stimulate the future research toward a better understanding of this intriguing drug target. SIGNIFICANCE STATEMENT: Evidence is reviewed on the prevalent toxifying function of N -ribosyldihydronicotinamide:quinone oxidoreductase 2 while catalyzing the reduction of ortho -quinones such as dopamine quinone. The product of this reaction is unstable and generates a futile but harmful cycle (substrate/product/substrate) associated with reactive oxygen species generation., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

microRNAs are noncoding RNAs which downregulate a large number of target mRNAs and modulate cell activity. Despite continued progress, bioinformatics prediction of microRNA targets remains a challenge since available software still suffer from a lack of accuracy and sensitivity. Moreover, these tools show fairly inconsistent results from one another. Thus, in an attempt to circumvent these difficulties, we aggregated all human results of four important prediction algorithms (miRanda, PITA, SVmicrO, and TargetScan) showing additional characteristics in order to rerank them into a single list. Instead of deciding which prediction tool to use, our method clearly helps biologists getting the best microRNA target predictions from all aggregated databases. The resulting database is freely available through a webtool called miRabel which can take either a list of miRNAs, genes, or signaling pathways as search inputs. Receiver operating characteristic curves and precision-recall curves analysis carried out using experimentally validated data and very large data sets show that miRabel significantly improves the prediction of miRNA targets compared to the four algorithms used separately. Moreover, using the same analytical methods, miRabel shows significantly better predictions than other popular algorithms such as MBSTAR, miRWalk, ExprTarget and miRMap. Interestingly, an F-score analysis revealed that miRabel also significantly improves the relevance of the top results. The aggregation of results from different databases is therefore a powerful and generalizable approach to many other species to improve miRNA target predictions. Thus, miRabel is an efficient tool to guide biologists in their search for miRNA targets and integrate them into a biological context., (Copyright © 2020 Quillet, Saad, Ferry, Anouar, Vergne, Lecroq and Dubessy.)

Among the biological approaches to therapeutics, are the cells, such as CAR-T cells engineered or not, the antibodies armed or not, and the smaller protein scaffolds that can be modified to render them specific of other proteins, à la façon of antibodies. For several years, we explored ways to substitute antibodies by nanobodies (also known as VHHs), the smallest recognizing part of camelids' heavy-chain antibodies: production of those small proteins in host microorganisms, minute analyses, characterization, and qualification of their affinity towards designed targets. Here, we present three standard VHHs described in the literature: anti-albumin, anti-EGF receptor and anti-HER2, a typical cancer cell surface -associated protein. Because they differ slightly in global structure, they are good models to assess our body of analytical methodologies. The VHHs were expressed in several bacteria strains in order to identify and overcome the bottlenecks to obtain homogeneous preparations of this protein. A large panel of biophysical tools, ranging from spectroscopy to mass spectrometry, was here combined to assess VHH structural features and the impact of the disulfide bond. The routes are now ready to move to more complex VHHs raised against specific targets in numerous areas including oncology., (Copyright © 2019. Published by Elsevier Inc.)

Dunnione, a natural product isolated from the leaves of Streptocarpus dunnii (Gesneriaceae), acts as a substrate for quinone-reductases that may be associated with its antimalarial properties. Following our exploration of reactive oxygen species-producing compounds such as indolones, as possible new approaches for the research of new ways to treat this parasitosis, we explored derivatives of this natural product and their possible antiplasmodial and antimalarial properties, in vitro and in vivo, respectively. Apart from one compound, all the products tested had weak to moderate antiplasmodial activities, the best IC 50 value being equal to 0.58 µM. In vivo activities in the murine model were moderate (at a dose of 50 mg/kg/mice, five times higher than the dose of chloroquine). These results encourage further pharmacomodulation steps to improve the targeting of the parasitized red blood cells and antimalarial activities.

In the continuous exploration of the VHH chemistry, biochemistry and therapeutic future use, we investigated two different production strategies of this small antibody-like protein, using an anti-HER2 VHH as a model. The total chemical synthesis of the 125 amino-acid peptide was performed with reasonable yield, even if optimization will be necessary to upgrade this kind of production. In parallel, we expressed the same sequence in two different hosts: Escherichia coli and Pichia pastoris. Both productions were successful and led to a fair amount of VHHs. The integrity and conformation of the VHH were characterized by complementary mass spectrometry approaches, while surface plasmon resonance experiments were used to assess the VHH recognition capacity and affinity toward its "antigen." Using this combination of orthogonal techniques, it was possible to show that the three VHHs-whether synthetic or recombinant ones-were properly and similarly folded and recognized the "antigen" HER2 with similar affinities, in the nanomolar range. This opens a route toward further exploration of modified VHH with unnatural amino acids and subsequently, VHH-drug conjugates., (© 2019 The Protein Society.)