Monica Gala

Arnaud Lenoble
email: arnaud.lenoble@u-bordeaux.fr

Evidence of the former existence of an endemic macaw in Guadeloupe, Lesser Antilles

Keywords: Ara guadeloupensis, Extinct birds, Guadeloupe, Marie-Galante, Macaw, Pleistocene, fossil-bearing deposit

The discovery of a bone referred to the genus Ara Lacépède, 1799 from a Pleistocene fossil-bearing deposit on Marie-Galante demonstrates macaws to have been present in Guadeloupe before any Amerindian settlement. This directly contradicts the hypothesis that macaws described in historical records concerning the Lesser Antilles were introduced by native peoples from South America. The fossil bone is a terminal phalanx similar in size to a large macaw. Based on its size and geographic arguments, the fossil bone found on Marie-Galante can be attributed to an endemic large macaw (Lesser Antillean Macaw, Ara guadeloupensis Clark in Auk 22: [266][267][268][269][270][271][272][273] 1905) presumed to have inhabited the Guadeloupe Islands. This discovery currently provides the strongest evidence supporting the former existence of this now-extinct macaw.

Introduction

Only a limited number of macaw bones are currently known from the Lesser Antilles. All documented instances of macaw bones have been recovered from archaeological contexts and, consequently, it is impossible to exclude these examples representing birds introduced to the islands as pets or for commerce by Amerindians [START_REF] Olson | New evidence of Ara autochthones from an archeological site in Puerto Rico: a valid species of West Indian macaw of unknown geographical origin (Aves: Psittacidae)[END_REF][START_REF] Wiley | The extinct Macaws of the West Indies with a special reference to Cuban Macaw, Ara tricolor[END_REF]. Moreover, only two Psittacidae are known from the Guadeloupe islands: an Amazon parrot bone (Amazona sp.) from the Amerindian site of Petite-Rivière, La Désirade [START_REF] Grouard | Subsistance, systèmes techniques et gestion territoriale en milieu insulaire antillais précolombien-Exploitation des Vertébrés et des Crustacés aux époques Saladoïdes et Troumassoïdes de Guadeloupe[END_REF]) and an ulna found by Barbotin during his excavations at Folle-Anse, Marie-Galante. This latter specimen, initially thought to belong to the Lesser Antilles Macaw Ara guadeloupensis [START_REF] Williams | The Historic and Prehistoric Distribution of Parrots (Psittacidae) in the West Indies[END_REF], was recently reassigned to the extinct Amazona violacea by [START_REF] Olson | New evidence of Ara autochthones from an archeological site in Puerto Rico: a valid species of West Indian macaw of unknown geographical origin (Aves: Psittacidae)[END_REF].

As it stands, the sparse archaeological record does not support the existence of an endemic macaw formerly inhabiting Guadeloupe. Establishing such a presence would require new material providing evidence for a distinct species, as exemplified by the Ara autochtones described on Hispaniola and Puerto Rico [START_REF] Wetmore | Ancient records of birds from the island of St. Croix with observations on extinct and living birds of Puerto Rico[END_REF][START_REF] Olson | New evidence of Ara autochthones from an archeological site in Puerto Rico: a valid species of West Indian macaw of unknown geographical origin (Aves: Psittacidae)[END_REF], or the documentation of a specimen from a pre-colonization context.

Here, we report a terminal Psittacidae phalanx found in a late Pleistocene fossil deposit from Marie-Galante, whose size and morphology support it representing a member of the genus Ara. This find provides credible evidence for the existence of a now-extinct endemic macaw in Guadeloupe.

Materials and methods

The Psittacidae bone from Blanchard Cave was compared to specimens from the comparative zoology collection held by the PACEA laboratory (PACEA), the Bivaag research project (BIVAAG), both housed in the Bordeaux University, and the Natural History Museum of Paris (MNHN). Samples for radiocarbon dating were pretreated by the Radiocarbon Dating Center in Lyon, France (lab code: Lyon), while isotopic ratio were measured by the mass accelerator Artemis housed at Saclay, France (lab code: SacA).

Blanchard Cave is a fossil-bearing site in south-western Marie-Galante [START_REF] Lenoble | Formes et remplissages du karst littoral guadeloupéen[END_REF], one of the islands forming the Guadeloupe archipelago (Fig. 1). Lying 200 m from the current shoreline, the site comprises a single 30-m-long chamber opening onto the coastal plain. Excavations conducted in the spring of 2013 and 2014 revealed a 4-m-deep deposit overlain by a pre-Columbian layer [START_REF] Goetz | Des fouilles dans la grotte Blanchard, près Capesterre-de-Marie-Galante[END_REF]. The avian assemblage recovered from the non-anthropogenic deposits consists of 341 bones, primarily Passeriformes (54 %) and Procellariiformes (6 %), with a single bone (GB2013-I32c-d23) referred to a Psittacidae.

Twelve layers, containing the remains of frogs, lizards, snakes, birds and bats in variable proportions, have been identified, with the Psittacidae bone recovered from a depth of 1 m in the 15cm-thick layer 5. This layer is preserved at the base of the rearwall of the cave and overlain by a thick rubble layer, which excludes any potential disturbance by crab burrows or Amerindian postholes. This layer, as well as the main portion of the deposit, is a soft organic silt composed of microscopic fragments of vegetal tissues indicative of a fossil guano produced by frugivorous bats [START_REF] Lenoble | Formes et remplissages du karst littoral guadeloupéen[END_REF]. Authigenic phosphates minerals are also abundant, suggesting that others animals, such as insectivorous bats or birds of prey, contributed to the layer's formation [START_REF] Bos | Dynamiques d'accumulations des phosphates en milieu karstique[END_REF]. A radiocarbon date obtained on a sample of the organic fraction from the layer 5 sediment produced an age of 10,690 ± 70 years uncal BP (Lyon-6976_SacA 19445). Good agreement with the dating of the overlying rubble layer (layer 3, 9740 ± 50 years uncal BP, Lyon-5726_SacA-14261) and underlying layer 6 (14,010 ± 80 years, Lyon-6977_SacA 19446) indicates that the formation of layer 5 can be securely placed to the latest Pleistocene. The layer 5 faunal assemblage differs from those recovered from the other layers given its richness and substantial diversity of species, which includes an important proportion of variously-sized birds.

Fossil owl pellets in the form of 2-4 cm long agglomerates of imbricated bones indicate Barn Owls (Tyto) to be the dominant accumulation agent responsible for the bones collected from this layer.

The following specimens were used for osteological comparison: Ara macao (BIVAAG-200913 Bfemale; PACEA-O-609; MNHN 1995-262-adult male); Ara ararauna (MNHN 1995-233-young male); Ara chloroptera (PACEA-O-667, young); Ara rubrogenys (PACEA-O-670-adult female); Amazona vinacea (PACEA-O-666-young); Amazona oratrix (BIVAAG-200913 A, adult male). Anatomical description follows the terminology of [START_REF] Livezey | Higher order phylogeny of modern birds (Theropoda, Aves: Neornithes) based on comparative anatomy. I. Methods and caracters[END_REF] with metric analyses based on the method described by [START_REF] Boev | Early Pliocene avifauna of Muselievo (C Northern Bulgaria)[END_REF], both shown in Fig. 2. The curvature, robustness, conical shape, the presence of a developed, but not prominent tuberculum extensorium, and a distinct sulcus neurovascularis indicate the fossil to be a terminal phalanx from a zygodactyle bird [START_REF] Ostrom | Archaeopteryx and the origin of flight[END_REF][START_REF] Feduccia | Evidence from claw geometry indicating arboreal habits of archaeopteryx[END_REF]. The curvature of the Blanchard specimen is similar to ungual bones of the second or third digit of an Ara (Fig. 3). As large macaws from both the Ara and Amazona genera display no size dimorphism [START_REF] Guedes | Order Psittaciformes (Parrots, Macaws, Conures)[END_REF], size represents a reliable diagnostic criteria. Comparison of the Blanchard specimen to modern comparative material shows the closest analog to be A. macao (Table 1). The fossil phalanx is also similar to modern Ara specimens as the sulcus neurovascularis originates from the margin of cotyla articularis (Fig. 4), even if the sulcus in fossil specimens extends almost across the entire corpus phalangis compared with the partially extending sulcus neurovascularis of the examined modern Ara.

Results

Class

The oval cotyla articularis of the Blanchard specimen is slightly larger that of the second digit of A. macao, but thinner and less circular that of the third. Similarly, the tuberculum extensorium is slightly larger that of the second digit of A. macao in the comparative sample. In proximal view, the tuberculum flexorium is oval-shaped and flattened, making the Blanchard specimen similar to the ungula bone of the third digit of the A. macao, while, at the same time, differing from the second digit of the distal phalanx, which is more circular. The Blanchard specimen is larger than the examined Amazona unguals. More importantly, the Amazona distal phalanxes have a circular turberculum flexorium separated from the processus neurovascularis by a more or less pronounced concavity depending on the digit. Both of these characters are absent in the Blanchard specimen. Overall, the size and morphology combined with osteological characteristics show the Blanchard specimen to be directly comparable with the distal phalanx of the second or third digit of a member of the genus Ara.

The fossil bone is longer than specimens assigned to A. vinacea and A. oratrix from our reference collection, although both taxa are smaller species of the Amazona genus. The largest extant Amazon parrots are the Lesser Antilles species Amazona guildingui, A. versicolor and A. imperialis. Quantitative comparison with these taxa is precluded by the fact that no specimens were identified in the zoological collections we consulted nor are they available in any publication concerning phalanx. However, while the largest species of their genus, these birds are nevertheless significantly smaller than large macaws such as A. macao [START_REF] Collar | Family Psittacidae (parrots)[END_REF]. Moreover, osteological differences with our comparison material are significant enough to exclude the fossil bone representing a member of the Amazona genus. Several minor details do differ, such as the prominent sulcus neurovascularis in the fossil specimen or the ratio between bone length and a more developed facies articularis compared to modern A.

macao. However, the limited number of modern specimens included in this study does not obviate these differences reflecting intra-species variability. Similarly, the modern specimen shows the development of the sulcus neurovascularis to present significant inter-if not intra-specimen variability.

These small osteological differences are therefore insufficient for referring the Blanchard specimen to a species other than A. macao. A detailed osteological examination of the fossil shows its closest analog to be digit III of A. macao, while its size makes it most comparable to the ungual bone of digit II from the same species. This could suggest that the fossil bone corresponds to a slightly smaller bird than the A. macao specimens we examined.

Discussion

The likelihood that the Blanchard specimen represents a slightly smaller macaw than A. macao fits with the hypothesis of an extinct macaw species having once inhabited Guadeloupe. While two endemic macaws, the Lesser Antilles Macaw (A. guadeloupensis Clark 1905) and the Violet Macaw (Anodorynchus purpurascens [START_REF] Rothschild | Notes on extinct parrots from the West Indies[END_REF], are mentioned in historical documents, it has recently been demonstrated that [START_REF] Rothschild | Notes on extinct parrots from the West Indies[END_REF] incorrectly based his description of the second on the Guadeloupe Amazon A. violacea [START_REF] Lenoble | The Violet Macaw (Anodorhynchus purpurascens, Rothschild 1905) did not exist[END_REF]. Therefore, Guadeloupe would have been home to only a single species of large macaw, the A. guadeloupensis. Size information available in Father Du

Tertre writings (Du Tertre 1667) led [START_REF] Clark | The Lesser Antillean Macaws[END_REF] to conclude that the Lesser Antilles Macaw would have been slightly smaller than A. macao.

Referring the Blanchard specimen to A. guadeloupensis is further supported by the Pleistocene geography of the Guadeloupe archipelago. The emersion of Les Saintes and Colombie Bank due to lower Pleistocene sea-levels significantly reduced the distance between Marie-Galante and Guadeloupe, which today stands at 26 km [START_REF] Münch | Pliocene to Pleistocene carbonate systems of the Guadeloupe Archipelago, French Lesser Antilles: a land and sea study (the KaShallow Project)[END_REF]. Moreover, the geography of the archipelago during the Last Glacial lowstand (~20 ky, [START_REF] Jouzel | Atmospheric oxygen 18 and sea-level changes[END_REF] reveals Marie-Galante to have been separated from Guadeloupe by a succession of three channels, the largest being less than 6 km wide (Fig. 1). Such short distances would not have presented a significant obstacle for flying vertebrates, most likely rendering the Guadeloupe Islands a single geographical unit during the Pleistocene.

No comparative sample is available for the fossil ungula bone from Blanchard as specimens attributable to the Lesser Antilles Macaw are currently unknown from museum collections. As such, the Blanchard specimen can only tentatively be assigned to the extinct macaw of Guadeloupe (Ara cf.

guadeloupensis). The short distances separating Marie-Galante from Guadeloupe during the Pleistocene as well as the good size agreement between the Blanchard specimen and [START_REF] Clark | The Lesser Antillean Macaws[END_REF] description of the Lesser Antilles Macaw nevertheless makes this hypothesis the most parsimonious.

Current archaeological evidence for the earliest Amerindian occupation of the Lesser Antilles comes from the Pre-ceramic occupation dated to 5.3 ky cal BP at Etang Rouge on Saint-Martin [START_REF] Martias | SAINT-MARTIN, Étang Rouge Lot 411[END_REF][START_REF] Bonnissent | Archéologie précolombienne de l'île de Saint-Martin, Petites Antilles (3300 BC-1600 AD)[END_REF]) and a pollen core drilled in the marshes of the Vieux Fort River on Marie-Galante, which produced evidence for the human-induced modification of the landscape and accumulation of micro-charcoals at around 5 kyr cal BP [START_REF] Siegel | Marie-Galante, analyse préliminaire de prélèvements sédimentaires en provenance de Marie-Galante[END_REF]. The date of 10.7 ky BP (12 ky cal BP) for layer 5 of Blanchard Cave containing the terminal phalanx referred to Ara cf.

guadeloupensis securely predates the initial Amerindian colonization of the Lesser Antilles.

In their review of West Indies parrots, [START_REF] Williams | The Historic and Prehistoric Distribution of Parrots (Psittacidae) in the West Indies[END_REF] assume that 50-60 species of Psittacidae, including 10-12 species of large macaws, would have inhabited the Lesser and Greater

Antilles in the absence of prehistoric and historic human impact, instead of the 12 extant species of Amazon parrots and parakeets. However, this conclusion has been challenged by [START_REF] Olson | New evidence of Ara autochthones from an archeological site in Puerto Rico: a valid species of West Indian macaw of unknown geographical origin (Aves: Psittacidae)[END_REF], who consider the Lesser Antilles land mass insufficient for sustaining indigenous or even endemic species of Ara. Moreover, the variety of West Indies parrots and macaws deduced from historical accounts could also result from misinterpretations of sketchy descriptions [START_REF] Wiley | The extinct Macaws of the West Indies with a special reference to Cuban Macaw, Ara tricolor[END_REF], as was demonstrated for a macaw presumed to have once occupied Hispaniola [START_REF] Olson | Refutation of the historical evidence for a Hispaniolan Macaw (Aves: Psittacidae)[END_REF].

Moreover, because Amerindians kept and transported animals between islands, it cannot be excluded that macaws described in historical sources were in fact introduced from South America [START_REF] Greenway | Extinct and vanishing birds of the world[END_REF][START_REF] Snyder | The parrots of Luquillo: natural history and conservation of the Puerto Rican parrot[END_REF]Olson and Lo´pez 2008). The question remains whether the Lesser Antilles

Figure 1 .

 1 Figure 1. Site location. a Guadeloupe Islands with isobaths (-100 m) indicating lowered sea levels during the Last Glacial (bathymetric data from the IGN-SHOM). b Marie-Galante Island with location of the site (black star).

Figure

  Figure 2. Psittacidae ungual bone with indication of measurements taken (following Boev 2001); a length of facies articularis, b distance between the base and ventral edge of the facies articularis, c overall length, d maximum width of facies articularis.

Figure 3 .

 3 Figure 3. Right Psittacidea ungula bones (lateral view). a Blanchard specimen. b A. macao (BIVAAG-200913B), digit II. c A. macao (PACEA-O-609), digit III. d-g A. vinacea (PACEA-O-666), digits I, II, III and IV.

Figure 4 .

 4 Figure 4. Proximal extremities (lateral view). a Blanchard specimen. b A. macao (BIVAAG-200913B), digit II. c A. macao (PACEA-O-609), digit III. d A. vinacea (PACEA-O-666), digit III.

  a

  species identified in historical sources, the Martinique Macaw (Ara martinica[START_REF] Rothschild | Notes on extinct parrots from the West Indies[END_REF]), Dominican Green-and-yellow Macaw (A. atwoodi Clark 1908) and the Lesser Antilles Macaw (A. guadeloupensis), were endemic species[START_REF] Hume | Extinct birds[END_REF].[START_REF] Wiley | The extinct Macaws of the West Indies with a special reference to Cuban Macaw, Ara tricolor[END_REF] review of available historical evidence led them to conclude that the Lesser Antilles Macaw is the most credible of all the Lesser Antilles hypothetical species of indigenous Ara. The discovery of an ungual bone in a pre-Amerindian deposit from Blanchard Cave indicates that a large macaw similar in size to the hypothetical Lesser Antilles Macaw was present in the Guadeloupe archipelago before any human settlement. This discovery therefore lends robust support to the hypothesis of the Lesser Antilles Macaw's former existence.

Acknowledgements

This study was conducted as a part of the BIVAAG Program established by the Centre National de la Recherche Scientifique (CNRS), with support from a European POFEDER grant 2007-2013 n°2/2.4/-33456, the Guadeloupe Regional Council, the Direction de l'Environnement et de l'Aménagement du Territoire (DEAL) of Guadeloupe, and the Direction des Affaires Culturelles (DAC) of Guadeloupe. We are very grateful to Veronique Laroulandie for her valuable help and suggestions. We would like to thank Christine Lefevre for access to the bird bone collections of the Laboratoire d'Anatomie Comparée of the Muséum Nationale d'Histoire Naturelle of Paris, Cécile Mourer-Chauviré for helpful comments, Frédéric Fronteneau and Laurent Delavis for donating modern specimens that augmented the Université de Bordeaux osteological collection, and also Maxime Pelletier and Émilie Campmas for preparing the anatomical reference material. Monica Gala thanks Mathieu Langlais, Éric Pubert, Alain Queffelec, Ivana Fiore and Antonio Tagliacozzo for their help.


domain: info info

text:
Mustapha Derras
email: mustapha.derras@berger-levrault.com

Laurent Deruelle
email: laurent.deruelle@berger-levrault.com

Jean Michel Douin

Nicole Levy
email: nicole.levy@cnam.fr

Francisca Losavio
email: francislosavio@gmail.com

Yann Pollet
email: yann.pollet@cnam.fr

Valérie Reiner
email: valerie.reiner@berger-levrault.com

Reference Architecture Design: a Practical Approach

Keywords: Software Product Lines (SPL), Software Architecture, Reference Architecture Design, Software Product Quality, Bottom-up Strategy

Reference Architectures (RA) in a Software Product Line (SPL) context are generic schemas that can be instantiated to configure concrete architectures for particular software systems or products of the SPL family. SPLs claim to be reusable industrial solutions to reduce development cost and time to market; however their development requires a huge effort, since the RA must be evolutionary. The goal of this work in progress is to present a practical RA domain engineering method for the Human Resources domain based on a bottomup strategy applied to the early Scope phase of SPL Engineering. A usual industrial practice in this development is to start from a single existing product built by the enterprise and incrementally derive the RA by adding new elements. Four architectural configurations are developed and quality properties are considered early as major responsible of the SPL variability. Our approach is applied to a real industrial case study.

INTRODUCTION

The architecture is an abstract representation of a software system with related (connected or communicating) components. [START_REF] Shaw | Software Architecture. Perspectives of an emerging discipline[END_REF]) [START_REF] Taylor | Software. Architecture: Foundations, Theory and Practice[END_REF]. A component describes a part of the system holding an individual behaviour. The whole system behaviour is defined by the composition of the individual behaviours of the components. Every component must be specifically implemented and it holds certain quality properties (maintainability, usability, security, etc.); they must be identified and more or less satisfied in order that the component can maintain a suitable functional behaviour. The architecture is composed by abstract components in the sense that the implementation details are not known already. An abstract component can be implemented in different ways, but all the implementations satisfy the behaviour of the abstract component. A concrete architecture is composed by precise implementations of abstract components.

A Reference Architecture (RA) [START_REF] Bosch | Design and Use of Software Architectures -Adopting and evolving a product-line approach[END_REF], [START_REF] Clements | Variability in Software Systems, the key to software reuse[END_REF], [START_REF] Oquendo | Reference Architectures[END_REF], (ISO/IEC 26550, 2015) represents a family of abstract architectures. It highlights the presence of established common components, and of other components that are not common, which are called variants and that are grouped into variation points [START_REF] Pohl | SPL Engineering: Foundations, Principles, and Techniques[END_REF]. A variation point is a particular component defining the variants' common behaviour and their connections with all other architectural components; it identifies a set of variants (eventually empty at first), from which only one can be chosen. RA must satisfy a set of rules describing the constraints that should be satisfied while selecting a configuration with the chosen variants. The goal of RA is to be configured to obtain a particular concrete architecture of a software system by choosing convenient variants. During the configuration of RA, consistency properties between the selected variants must be satisfied. The RA is the main asset of the Domain Engineering (DE) lifecycle of SPL Engineering (SPLE) [START_REF] Pohl | SPL Engineering: Foundations, Principles, and Techniques[END_REF] (ISO/IEC 26550, 2015).

Our research questions are: "how to develop a RA from existing products or systems" and "how to concretise the RA abstract components into executable modules".

Feature models are frequently associated to RA. A feature model [START_REF] Kang | Feature-Oriented Domain Analysis (FODA) Feasibility Study[END_REF], where every node of the feature model tree is a variation point and the sub-nodes are the variants, can be associated to RA. The advantage of the feature model, which provides a tree-like view of RA, is the visualization of the choices to obtain the architecture of the system. However, as RA contains variations points, feature models do not provide any new information. Let us note that it is difficult in general to include properties that are not directly perceived by the user, such as the quality properties [START_REF] Benavides | FAMA: Tooling a Framework for the Automated Analysis of Feature Models[END_REF]. Then, in the present approach we consider building RA without using a feature model.

The main goal of this work in progress is to propose a method to build a RA, inspired in the work of [START_REF] Losavio | Refactoring. Graph for Reference Architecture Design Process[END_REF], and the standard reference model guidelines for SPLE (ISO/IEC 26550, 2015). The method is applied to an industrial case study, the SEDIT Human Resources (HR) System of the French Berger-Levrault Group, based on the one hand, on the behaviour of the abstract functional components representing SEDIT behaviour and on the other hand, considering nonfunctional requirements. We have started the work on this research topic a couple of years ago with the Healthcare Information Systems Domain [START_REF] Losavio | Quality-based Bottom-up Design of Reference Architecture Applied to Healthcare Integrated Information Systems[END_REF]. The RA is developed following a reactive bottom-up strategy [START_REF] Apel | Feature-Oriented Software Product Lines -Concepts and Implementation[END_REF], meaning that the RA will be constructed from a unique product already built by the industrial partner, thus reducing the whole RA design effort. This work considers the experience with the Vacation Request Subsystem (VRS), which is a major complex functionality defining services offered to municipal communities by the SEDIT system.

Functional Requirements (FR) are the system basic functionalities accomplishing prescribed functional goals, expressed in our case by the enterprise business processes. However, nonfunctional goals that are required by functionalities to behave properly, i.e., to be functionally suitable (ISO/IEC 25010, 2011), must also be considered. Non-functional Requirements (NFR) must be taken into account while studying existing products. There is a general agreement on the fact that NFR must be considered early in the software development lifecycle to increase software maintainability. The SPLE reference model claims that NFR are greatly responsible of RA variability. However, these aspects are considered only at the late domain design stage when the RA is already built, contradicting this assumption. In usual industrial practice these issues are poorly treated, being often left to the responsibility of the particular development platform used. Notice that SPLE favours a global top-down design to build the SPL from scratch considering a very huge and time consuming domain analysis; however, the first SPL scope phase recommends bottom-up strategies since domain existing products have to be analysed. We start using this strategy that also contributes to the light-weightiness of our methodology, reducing the effort in the subsequent DE phases, namely Domain Requirements Engineering (DRE) and Domain Design (DD).

The structure of this paper, besides this introduction, is the following: section 2 details the method and the underlying process; section 3 presents the application of the process to the VRS case study; section 4 analyses related works, and finally section 5 provides the conclusion and some perspectives.

METHOD TO BUILD RA

SPLE includes two lifecycles: DE where domain knowledge is captured to build RA, and Application Engineering (AE) where the concrete products of the SPL family are configured according to the variability model contained in the RA. The proposed method is framed into the SPLE Domain Engineering lifecycle and follows the systematic process shown in Figure 1; it concerns a four-phases process; the first two steps can be used to develop any quality driven single system architecture; the third step defines the Domain Requirement Engineering Architecture, and the fourth step defines the RA, maintaining quality traceability through functional and non-functional components. Figure 1 presents the process as a UML Activity Diagram.

APPLICATION OF THE PROCESS

The phases of the process shown in Figure 1 will be detailed in the following sections; however, note that Phase 0.1 was performed for only one product and will not be discussed here.

3.1 Phase 0.2, Activities 0.2.1. 0.2.2, 0.

Identification of Domain Style and Quality Properties

VRS belongs to the family of HR Information Systems (HRIS) that combines a number of systems and processes to ensure the easy management of business employees and data. The programming of data processing systems evolved into standardized routines and packages of Enterprise Resource Planning (ERP) software. Another important issue that HRIS should support is the changing of legal requirements, such as laws and country specific regulations, for example the SAP Company has to handle manually these issues.

We consider a business goal as an observable and measurable end-result having one or more objectives to be achieved within a more or less fixed timeframe. The main business goal of the company is HR and financial management on French local authorities, i.e., communities, departments, regions, etc., where people invested in public offices exercise authority over their territory. The hierarchy of assigned authority is relevant to provide the correct signature to authorize different administrative transactions. SEDIT verifies the HRIS domain hybrid architectural style [START_REF] Shaw | Software Architecture. Perspectives of an emerging discipline[END_REF]: event-based, layers and client/server model for communication and main quality properties.

One of its most important qualities, the functional suitability (appropriateness), is to be compliant at any time with laws that are constantly changing; the system must guarantee this compliance. The hierarchy of people in office can also change at any time, impacting signature circuits, and this compliance must be also assured. The VRS system studied is a classic Web-based information system. The availability of services is conditioned also by the network connection.

Phase 0.2, Activities 0.2.4, 0.2.5 -Specify Business Processes and Criteria to Conform Architectural Components

The stakeholders of the company were interviewed to capture the information about the functional behaviour of the system. The workflows relative to the participant (person or system) activities were specified using BPMN; they will not be shown here to abridge the presentation. The idea is to "translate" into architectural components these activities or tasks expressing functional behaviours to accomplish a specific goal. Some automatic tools are available to "translate" business processes into choreography/orchestration of services, requiring in general optimization processes; but there are few automatic tools to translate business processes activities into functional architectural components, such as ArchiMate (The Open Group Architecture Framework) (Open Group, 2012).

The whole VRS representing a service to be accomplished by the SEDIT HRIS, is one of the enterprise business goals.

Phase 1 (DRE), Activities 1.1, 1.2 -First Architecture (V1) with Main Functional Components

The architecture shown in Figure 2 is derived from the BPMN workflows for the VRS, applying the rules shown in Table 1.

The idea is to organize this first configuration by introducing components by grouping the functional activities accomplishing business goals in the stakeholders' lanes; all components are considered at a high abstraction level. Architectural configurations are represented as UML 2.0 logic views [START_REF] Krutchen | Architectural Blueprints -The "4+1" View Model of Software Architecture[END_REF]; note that the UML notation we used here does not show components' interfaces. Components are associated to each BPMN stakeholder's lane, to accomplish business goals and sub-goals, in the following way:

• A component can group lane's activities (performed from a human or a system), representing a sub-goal. • Only functionalities and their cooperation are concerned.

Phase 1 (DRE), Activity 1.3 -Second Architecture (V2) with Functional and Non-Functional Components

Non-functional components represent properties or features not directly perceived by the user (see Figure 3). However Notice that Usability and Efficiency are priority quality properties for the User Interface (UI), but they will not be considered here because they depend on the UI particular design and not on the system architecture. The Maintainability (Modifiability) quality property has not yet been satisfied in the V2 configuration, and it will appear when the architectural style is considered.

Phase 2 (DD), Activity 2.1, Third Architecture (V3) with Domain Style

The choice of an architectural style depends on the domain. Each V2 component has been placed in the V3 configuration as a sub-component of the components of the style (see Figure 4). Notice that some components are divided into several subcomponents in order to satisfy the style; that is why a Base component is introduced to populate the Data Layer.

Each layer is considered as a component; hence we have UI as Presentation Layer, System as Process Layer, Data Base as Data Layer; finally the Communication Layer communicates the different layers and exterior systems/services. Notice that the UI requirement Maintainability (Modifiability) is usually solved by the MVC (Model, View Controller) pattern, and it is split into two components MVC-ClientSide and MVC-ServerSide, to interface Presentation and Process layers (see Figure 4). The Process Layer corresponds to the VRS business goal and it contains all the remaining functional and nonfunctional components of the V2 configuration with the same connections. Component Access Rights Policy is introduced to solve security for access control, and Signature Hierarchy to satisfy the authenticity imposed by the signature rights. Administrative Tasks is in charge of managing and maintaining the vacation request information and a database is introduced to satisfy persistency. In the Data Base Layer four functional sub-components have been added to satisfy NFR requirements, Administrative DB, Employee Rights DB, Access Rights DB, and Signature Rights DB. Correctness is required by data base schemas, persistency to store/get the process results and portability since they are presumably relational databases and do not work directly with object-oriented languages like Java. In the Communication Layer the UI can connect to the Network for e-mail and other external services through a usual browser and to communicate with the Process Layer; Security (authenticity, confidentiality, integrity) in message transmission should be realized by Network Security, and Reliability (Availability) for system access is realized by Network Reliability; they are also priority quality properties that have been added as non-functional components to the Communication Layer since they can jeopardize the whole Web-based system functioning.

Notice that this third step could have been applied at the beginning of the process since the domain style is known; however we have decided to apply it here to ease repeatability with small incremental steps.

Phase 2 (DD), Activity 2.2 -Fourth

Architecture (V4) with Variability Model, the RA Software variability is the ability of a system to be efficiently extended, changed, customized or configured for use in a particular context (Van Grup, 2000). In SPL the term variability model [START_REF] Pohl | SPL Engineering: Foundations, Principles, and Techniques[END_REF] is used to organize variant elements in such a way that they can be reused at the moment of deriving concrete products from the RA. Variation Points are elements (placeholders) of the variability model; in our case they may be functional or non-functional components and they are denoted here by <<name>> as UML stereotypes (see Figure 5). A variation point denotes a set of components called variants; the connector of a variation point denotes a set of connectors, each one belonging to a variant.

The activity 2.2 of our process is the identification of the variation points and their variants from the architecture obtained in V3. In order to achieve this, other similar existing products should have been studied or future products to be built should be considered to cope with the SPL evolution, and new planned functionalities could also have been added, see activities 0.1.3 and 0.1.4 of Phase 0.1. If this study is not done, variation points and their variants are identified directly on the V3 architecture, establishing some criteria, leading to the configuration shown in Figure 5, the RA for a VRS family of systems. All RA abstract functional and non-functional components should be traced and concretized to the corresponding reusable code modules; if no reusable module can be found, it should be adapted or constructed to provide all assets to perform the concrete product derivation from the RA and guarantee complete functional suitability. The Variability Model in the V4 last configuration is defined by the following specific activities: a. Describe the Context as an external system, to proceed with the Application Engineering lifecycle of the SPLE model; the RA variation points should be instantiated to derive the SPL concrete products. This system is in charge of putting into operation or "implement" a concrete product on the client demand, according to a configuration and/or customization process. This part is still an on-going work. b. Identify functional and non-functional components that can have different choices to accomplish their goals; mark them as stereotyped variation point components. c. For each variation point, identify possible variants, such as for example the Hibernate tool to solve Oracle portability to Java objects. d. Describe the dependencies and constraints among choices of variants.

RELATED WORKS

Many works are found in the literature about SPL development and industrial adoption, but few treat the whole SPLE process. In this work we are more concerned with the Domain Requirements life cycle of SPLE, where the RA is built. Three literature reviews will be discussed.

The work of [START_REF] Chen | Variability Management in SPL: An Investigation of Contemporary Industrial Challenges[END_REF] treats variability management for the RA evolution; this topic has been studied for almost 20 years. An empirical study using focus group of SPL experts in different fields (industry, consultancy, academy, etc.) were questioned to collect data; two of the first contributions to variability management research and practice were the known Feature-Oriented Domain Analysis (FODA) [START_REF] Kang | Feature-Oriented Domain Analysis (FODA) Feasibility Study[END_REF] method and the Synthesis approach [START_REF] Kasunic | Synthesis: A reuse-based software development methodology[END_REF] focused on a family of similar systems for a business area. This approach arises for SPL from evidence that, within a class of systems, the understanding of similarities provides significant leverage for constructing a great variety of high-quality systems cheaply and reliably. With Synthesis, a domain is conceived as a realization of the declared business objectives of the specific organization. Business goals determine the types of systems to build and customers. These goals are set on the expertise already available within the organization, as a result of experience in building systems in the past. Among the technical issues, many challenges were considered, such as: -Requirements: complexity management in feature modelling that affects maintainability, the lack of standards in feature representations that affects understandability, and the extraction of commonality and variability from existing poor documentation. -Architecture design: extract the variability from technical artefacts of different similar products and build a common architecture for those products. Decisions are not documented properly and are hard to be retraced. -Evolution: when requirements change, in some cases, the existing architecture does not support the required variability in the new requirements. In an extreme case where the SPL started with one single product, evolving the architecture towards an SPL was seen as an issue. With respect to non-technical aspects, several issues were considered, such as the Business Model that does not encourage reuse. In conclusion, it was found that variability management relies mainly on the technical aspects, the standardization of variability representation and modelling, the documentation, and management.

In our work, the use of standards is favoured; we adopt a bottom-up strategy to build the RA from a single product, following the mentioned Synthesis approach, starting from a BPMN specification of business processes, extracted from interviewing industrial stakeholders.

The work of (Mazo el al., 2014) on the ERP domain aims to identify and analyse the different ways to improve ERP engineering issues with methods, techniques and tools provided by SPLE. An ERP system is not a family of applications, but a single application. However, just like a product line, configuration mechanisms are used to satisfy the various requirements from different companies. ERP implementation (customization and configuration) focuses the major research axis in ERP. The goal is to take into account the specific needs of the organization when ERP standards and configurable features cannot achieve them. Variability management and the ability for the system to be configured/customized and adapted to a potentially undefined number of environments are two main challenges. ERP configuration deals with parameter changes and it can take several months and no results can be guaranteed; customization instead refers to interface development or code modification, and it requires to be regularly updated to maintain the system agility. This complexity of ERP systems is maybe the most important obstacle to use ERP systems in an efficient and predictable way. The paradigm was adopted to configure and/or customize ERP systems using different methods with varying approaches. However, from the point of view of ERP customers and users, it is difficult to differentiate between product variability and customization.

With respect to our work, the compliance with laws has been found as a non-solved problem; the configuration of the legal requirements as reusable assets is still an open problem in the HRIS domain; this issue must be handled by the SPLE Application Engineering lifecycle.

The ERP domain and SPLE approach are also treated in (Ouali, 2011), focusing on the evolution issue and on the problem of tool support for these approaches. SPL fits to ERP business, and ERP systems can benefit greatly from the concepts of commonality and variability to enhance maintainability or evolution capability. Four methods are reviewed, Van Group, Ziadi, Delstra and Djebbi, being the Main drawbacks: the lack of sufficient tool support and poor interactivity with their users. The SPL development approaches themselves are not enough automated for deriving automatically a concrete product from the RA; most of them use proprietary notations which can handle problems of standardization and interoperability; they do not cover all aspects of SPLE. Every method tries to focus on a particular part of the SPL construction process. However, in this work the discussion on the experimentation with ERP is very limited, and no case study is provided.

The Office of Management and Budget (OMB, 2012) launched the Human Resources Line of Business (HR LOB) Data Model, an effort to build a set of enterprise architectures in compliance with the USA Federal Enterprise Architecture standards for the human resources business function. It is an interesting and complete work and conceptual elements can be used for the general HRIS domain, however it is data-oriented for enterprise architectures and does not concern directly the RA development for SPL.

The revised works mention standardization as an open problem; the ISO/IEC 26550 standard has been recently formulated to provide a framework for SPLE guidelines (ISO/IEC 26550, 2015). The problem of early consideration of software quality is discussed affirming its importance, but it is left to the late Domain Design phase. The bottom-up strategy is recommended in the SPLE Scoping phase to reduce the effort in the subsequent development phases. In our present work we adopt this approach. A couple of years ago we started to work on a SPL Domain Engineering quality-driven method based on the extractive bottom-up strategy, considering several existing products in the Healthcare Information Systems domain [START_REF] Losavio | Refactoring. Graph for Reference Architecture Design Process[END_REF], [START_REF] Losavio | Quality-based Bottom-up Design of Reference Architecture Applied to Healthcare Integrated Information Systems[END_REF].

CONCLUSIONS

The main outcome of this paper is the definition of a domain engineering method to build incrementally a RA, according to a bottom-up reactive strategy. This method, inspired in (ISO/IEC 25010, 2011), defines a systematic and repeatable practical process to build the RA, and it has been applied to a real industrial case. In addition, we have considered the compliance (functional suitability-appropriateness) to legal requirements (laws and regulations) as a priority quality requirement, since they change often overtime and their management is a time consuming and nonsystematic process in HR systems involving full-time human resources. On the other hand, the traceability among functional, non-functional components and their technical solutions has been clearly established by taking into account quality requirements, early in the SPLE lifecycle.

Among the perspectives, it is clear that more lower level architectural configurations can be produced from Figure 4 to detail the variants of variation points, such as 'e-mail' and 'electronic document' in <<Submit Request>>. However, to solve our second research question, an intermediate layer has to be defined to link the abstract RA components to the reusable modules of code, which are all reusable SPL assets. Moreover, mechanisms based on architectural patterns are under design to determine the choice of variants and the addition of new functionalities, without modifying the existing code. Our present work is a first step towards the idea of offering a "product on demand" for HR management to French territorial communities, by facilitating a configuration/ customization of on-line process of the HR system.

Figure 1 .

 1 Figure 1. Domain Engineering process to build the RA.

  , they are required by the functional components to satisfy completely their goals. Notice that the quality properties treated are part of the Domain Quality Model (ISO/IEC 25010, 2011). The functional components of V2 must satisfy four priority quality properties: -Security (Authenticity) realized by non-functional components Login-Security (authenticity) to provide access to the right kind of user, and Signature Hierarchy (authenticity), also required by functional component Check Signature Rights, to follow the enterprise hierarchy of assigned authority. -Suitability (Appropriateness) is realized by nonfunctional components Compliance with Law (s) and Compliance with Employee Rights; this quality property appears because the employee must fill up the right formulary according to legal requirements and must also be compliant with the employee legal rights. -Appropriateness is required by the Evaluate Case functional component. -Data Reliability is added as a new non-functional component to satisfy Availability-Persistency, since the Administrative Tasks functional component requires storing results of the whole vacation request process.

Figure 3 .

 3 Figure 3. Second architecture with functionalities and quality properties (V2).

Figure 2 .

 2 Figure 2. First architecture with functionalities (V1).

Figure 4 :

 4 Figure 4: Third architecture with domain style (V3).

Figure 5 :

 5 Figure 5: Fourth architecture with Variability Model (V4).

Table 1 .

 1 Rules to convert BPMN workflows into architectural components.

	Tasks	BPMN graphical	Rules for BPMN
		representation	conversion to
			architectural components
	User		A user task is represented
	(human)		as 1 functional component
			involving an interface
	Service		A service task is
			represented as 1 system
			functional component
	Receive		They are gateways to
			catch or receive messages;
			they are represented as
	Send		connections between the actor or system
			components
	Abstract		It represents an abstract
			component that will be
			specified later on
	Looped		A looped sub-process is
	Sub-		represented as 1 functional
	process		component involving an
			iterative process
	Sub-		A sub-process is
	process		represented as 1 functional
			component


Xi Liu

Jingkun Li

Andrea Zitolo

Meng Gao

Jun Jiang

Xiangtian Geng

Qianqian Xie

Di Wu

Huizhen Zheng

Xiaoming Cai

Jianmei Lu

Frédéric Jaouen

Ruibin Li

Doped Graphene to Mimic Bacterial NADH Oxidase for One-step NAD + Supplementation in Mammals

Keywords: Doped Graphene, Single Atom Catalyst, Nanotoxicity, Nanozyme, NAD +, NADH, Fe-N4

Nicotinamide adenine dinucleotide (NAD) is a critical regulator of metabolic networks, and declining levels of its oxidized form, NAD + , are closely associated with numerous diseases. While supplementing cells with precursors needed for NAD + synthesis has shown poor efficacy in combatting NAD + decline, an alternative strategy is the development of synthetic materials that catalyze the oxidation of NADH into NAD + , thereby taking over the natural role of NADH oxidase (NOX) present in bacteria. Herein, we discovered that metal-nitrogen doped graphene (MNGR) materials can catalyze the oxidation of NADH into NAD + . Among MNGR materials with different transition metals, Fe-, Co-and Cu-NGR displayed strong catalytic activity combined with > 80% conversion of NADH into NAD + , similar specificity to NOX for abstracting hydrogen from the pyridine ring of nicotinamide, and higher selectivity than 51 other nanomaterials. The NOX-like activity of FeNGR functioned well in diverse cell lines. As a proof of concept of in vivo application, we showed that FeNGR could specifically target the liver and remedy the metabolic flux anomaly in obesity mice with NAD + -deficient cells. Overall, our study provides a distinct insight for exploration of drug candidates by design of synthetic materials to mimic the functions of unique enzymes (e.g., NOX) in bacteria.

INTRODUCTION

Nicotinamide adenine dinucleotide is an essential cofactor extensively expressed in all living cells [START_REF] Zapata-Perez | NAD + homeostasis in human health and disease[END_REF] . Its oxidized and reduced forms (NAD + and NADH, respectively) play a key role in the electron transport chain of metabolic processes, where NAD + serves as an electron acceptor in metabolic redox reactions of cells [START_REF] Kim | Polyunsaturated fatty acid desaturation is a mechanism for glycolytic NAD + recycling[END_REF][START_REF] Palzer | Alphaamino-beta-carboxy-muconate-semialdehyde decarboxylase controls dietary niacin requirements for NAD + synthesis[END_REF] . These reactions are crucial for cells to maintain their elemental biological functions by harvesting energy from fuel molecules such as glucose and fatty acids [START_REF] Roichman | Restoration of energy homeostasis by SIRT6 extends healthy lifespan[END_REF] . The physiological functions of NAD + as an essential coenzyme in metabolic networks have been highly underlined [START_REF] Ralto | NAD + homeostasis in renal health and disease[END_REF] . While the decline in NAD + levels is closely associated with aging [START_REF] Zhang | NAD + repletion improves mitochondrial and stem cell function and enhances life span in mice[END_REF] , metabolic diseases [START_REF] Samuel | Nonalcoholic fatty liver disease as a nexus of metabolic and hepatic diseases[END_REF][START_REF] Hand | Energy flux and its role in obesity and metabolic disease[END_REF] , neurodegenerative disorders [START_REF] Kapogiannis | Disrupted energy metabolism and neuronal circuit dysfunction in cognitive impairment and Alzheimer's disease[END_REF] and cancers [START_REF] Deberardinis | Cellular metabolism and disease: What do metabolic outliers teach us?[END_REF][START_REF] Schwartz | Probabilistic controllability approach to metabolic fluxes in normal and cancer tissues[END_REF] , elevation of this cofactor by supplementing cells with precursors needed for NAD + synthesis has shown considerable benefits in preclinical trials [START_REF] Titov | Complementation of mitochondrial electron transport chain by manipulation of the NAD(+)/NADH ratio[END_REF][START_REF] Canto | The NAD + precursor nicotinamide riboside enhances oxidative metabolism and protects against high-fat diet-induced obesity[END_REF][START_REF] Cameron | Inflammatory macrophage dependence on NAD(+) salvage is a consequence of reactive oxygen species-mediated DNA damage[END_REF][START_REF] Nacarelli | NAD + metabolism governs the proinflammatory senescence-associated secretome[END_REF] . This has aroused great interest in the exploration of efficient and affordable pathways to increase NAD + levels for biomedical applications.

Mammalian cells can produce NAD + via two pathways, namely, the de novo synthesis (DNS) pathway and the salvage pathway [START_REF] Covarrubias | Senescent cells promote tissue NAD + decline during ageing via the activation of CD38(+) macrophages[END_REF] (Figure S1). The former requires seven steps to synthesize NAD + from its main building block, namely, the amino acid tryptophan, and was shown to be poorly efficient in biomedical applications [START_REF] Mehr | De novo NAD + biosynthetic impairment in acute kidney injury in humans[END_REF] . The salvage pathway utilizes vitamin B3 derivatives, including niacinamide (NAM), nicotinic acid (NA) or nicotinamide ribose (NR), bypassing the synthesis of adenine and resulting in the synthesis of NAD + in 2 fewer steps [START_REF] Cardoso | Need for NAD + : Focus on striated muscle laminopathies[END_REF] . However, supplementation of B3 derivatives may lead to NADH accumulation, resulting in side effects such as hepatic pressure, tissue-specific cytotoxicity and even carcinogenesis [START_REF] Yoshino | NAD + intermediates: the biology and therapeutic potential of NMN and NR[END_REF] . In contrast to mammalian cells, a NADH oxidase (NOX) was found in some bacteria to be involved in the regulation of NAD + levels via the direct conversion of NADH into NAD +20 . The advent of synthetic nanomaterials with NOX-like activity holds great interest.

The rapid development of nanotechnologies allows the precise design of nanostructures that mimic the catalytic properties of natural enzymes, namely, nanozymes [START_REF] Gao | Two-dimensional Tin selenide (SnSe) nanosheets capable of mimicking key dehydrogenases in cellular metabolism[END_REF][START_REF] Wu | Engineering Fe-N doped graphene to mimic biological functions of NADPH oxidase in cells[END_REF] . However, most nanozymes can merely catalyse the degradation of peroxides, oxygen and superoxide, while nanozymes with reactivity toward C-H bonds have rarely been reported. Synthetic metalnitrogen-graphene (MNGR) materials originally developed for catalyzing O2 electroreduction were recently investigated in the field of heterogeneous catalysis for the activation of C-H bonds [START_REF] Liu | Discriminating catalytically active FeNx species of atomically dispersed Fe-N-C catalyst for selective oxidation of the C-H bond[END_REF][START_REF] Xie | Atomically dispersed Co and Cu on N-doped carbon for reactions involving C-H activation[END_REF][START_REF] Deng | A single iron site confined in a graphene matrix for the catalytic oxidation of benzene at room temperature[END_REF] . The 3d transition metals are often chosen as the dopants due to their superior efficiency to catalyze reduction and/or oxidation reactions, low toxicity and high abundance in the continental crust. An FeNGR material with single-metalatom sites (Fe-Nx moieties) was shown to selectively catalyze the oxidation of a broad range of aromatic hydrocarbons into their corresponding ketones [START_REF] Liu | Discriminating catalytically active FeNx species of atomically dispersed Fe-N-C catalyst for selective oxidation of the C-H bond[END_REF] , and a CoNGR material with Co-Nx sites was reported to selectively catalyze the one-hydrogen abstraction from propane, resulting in propene [START_REF] Xie | Atomically dispersed Co and Cu on N-doped carbon for reactions involving C-H activation[END_REF] . This ability of MNGR materials with M-Nx sites covalently embedded in graphene sheets to catalytically activate C-H bonds is in line with a similar ability reported for non-pyrolysed metalphthalocyanines comprising a well-defined M-N4 centre [START_REF] Parton | Iron-phthalocyanine immobilized on activated carbon black: A selective catalyst for alkane oxidation[END_REF][START_REF] Pereira Monteiro | Metallophthalocyanines as catalysts in aerobic oxidation[END_REF] .

Metal-phthalocyanines are, however, less robust than MNGR single-atom catalysts, and their synthesis is more complex.

Due to the reported activity of MNGR to activate C-H bonds selectively and efficiently, we hypothesized that they can be potential catalysts to mimic the activity of NOX for catalytic elevation of NAD + in mammalian cells. While MNGR materials have recently been investigated as nanozymes with peroxidase-or catalase-like properties 28-36 , they have not yet been investigated as a NOX-like nanozyme to elevate NAD + .

Given that Fe, Mn, Zn, Cu and Co often serve as the catalytic core of many enzymes, we designed a series of MNGR materials by doping graphene with these transition metal and nitrogen atoms to investigate their catalytic properties toward NADH oxidation (Figure S1). The catalytic products of NADH and O2 in abiotic aqueous solutions were first examined by a colorimetric substrate, liquid chromatography and tandem mass spectrometry (LC-MS/MS). Among five synthetic MNGR materials, iron-, copper-and cobalt-doped NGR (FeNGR, CuNGR and CoNGR) were found to exhibit potent catalytic activity. Focusing on FeNGR due to iron's biocompatibility, the substrate selectivity, stability and kinetics of FeNGR were then extensively assessed under physiological conditions. The biological functions of FeNGR as an alternative to NOX were assessed in diverse mammalian cell lines and NAD + -deficient cell model by detecting the key intermediates in metabolic networks. Finally, as a proof of concept, the FeNGR material was exploited to remedy the metabolic flux anomaly in mice receiving high-fat diet (HFD). In summary, our study reports the discovery of the first nanozyme that mimics the catalytic properties of NOX from bacteria, with proven catalytic elevation of NAD + levels in mammalian cells and animals.

RESULTS

Synthesis and Characterization of MNGR Materials. Five MNGR materials were prepared by changing only the nature of the transition metal in the synthesis. ZnNGR, CuNGR, CoNGR, FeNGR and MnNGR single-atom catalytic materials were prepared via flash pyrolysis at 1050 °C in flowing argon of dry-mixed metal acetate, 1,10-phenanthroline and ZIF-8, according to a method previously developed by us [START_REF] Zitolo | Identification of catalytic sites for oxygen reduction in iron-and nitrogen-doped graphene materials[END_REF][START_REF] Li | Volcano trend in electrocatalytic CO2 reduction activity over atomically dispersed metal sites on nitrogen-doped carbon[END_REF] . These materials were characterized by atomic force microscopy (AFM), Raman spectroscopy, X-ray diffraction (XRD), and Xray absorption spectroscopy (XAS). The characterization data of Fe-, Cu-and Co-NGR, with high catalytic activity, are presented in the main manuscript, while those of Zn-and Mn-NGR (materials with lower activity) are presented in the supplementary materials.

The AFM images showed that the five MNGR materials had a lamellar structure consisting of a monolayer or a few atomic layers of carbon, with a thickness of 1.5-4.5 nm and a lateral size of 200-500 nm (Figure 1A and Figure S2A). The Raman spectra were similar for the five MNGR materials (Figure S3), revealing the G and D bands at 1583 and 1328 cm -1 that are characteristic of the graphene plane and edges of small graphene planes, respectively [START_REF] Tuinstra | Raman spectrum of graphite[END_REF] . These results are in line with the small lateral size of graphene planes observed by AFM. Powder XRD showed only a first diffraction peak at ca.

22° that can be assigned to the reflection (002) of graphite and a second, broader peak at ca. 42° that can be assigned to the convoluted (100) and (101) reflections of graphite (Figure 1B and Figure S2B). The absence of diffraction peaks arising from crystalline metallic structures or other metal-rich crystal-line structures in the as-prepared MNGR materials indicate that the metal atoms are likely atomically dispersed, as supported also by the extended X-ray absorption fine structure (EXAFS) analysis discussed below. The similar signatures of the carbon support in Raman spectra and XRD patterns for the five MNGR indicate that the NGR support is similar in all five materials, with the materials differing mainly in the nature of the metal dopant. We then resorted to XAS for identification of the average local structure, and coordination environment of the transition metal in each MNGR material (Figure 1C-1D and Figure S2C). The Fourier transform (FT) of the EXAFS spectra of the MNGRs at their respective metal K-edge showed a major peak (∼1.5 Å) and a secondary peak (2-3 Å).

These two peaks can be attributed to the backscattering of light atoms (O, N, or C) situated in 1 st and 2 nd coordination spheres of the absorbing metal (Figure 1C). No strong M-M backscattering signal (expected at ~2 Å) was observed in the FT-EXAFS spectra, supporting the atomic dispersion of the metal centers in this series of MNGR materials. This is in line with previous characterizations on MNGR materials prepared identically by us, which have been extensively studied for various electrocatalytic applications [START_REF] Sun | Activity-selectivity trends in the electrochemical production of hydrogen peroxide over single-site metal-nitrogen-carbon catalysts[END_REF] .

M-Nx moieties (one central metal coordinated with multiple nitrogen atoms within one plane and possibly with additional axial oxygen ligand(s)) were then used as model structures to fit the metal K-edge EXAFS spectra. These model structures have successfully explained many structural features of MNGR materials with atomically dispersed transition metal sites achieved via pyrolysis [START_REF] Li | Volcano trend in electrocatalytic CO2 reduction activity over atomically dispersed metal sites on nitrogen-doped carbon[END_REF][START_REF] Chung | Direct atomic-level insight into the active sites of a high-performance PGM-free ORR catalyst[END_REF] . The strong affinity of the metal for nitrogen is known to result in the spontaneous formation of stable M-Nx moieties at high temperature [START_REF] Li | Evolution pathway from iron compounds to Fe-1(II)-N-4 sites through gasphase iron during pyrolysis[END_REF] . The proximity of metal and nitrogen atoms in MNGR was also experimentally supported with aberration-corrected scanning transmission electron microscopy [START_REF] Chung | Direct atomic-level insight into the active sites of a high-performance PGM-free ORR catalyst[END_REF] . The best-fit FT-EXAFS spectra obtained with such model sites showed an average coordination number of ∼4 for Mn-, Co, Cu-, and ZnNGR, which supports the presence of in-plane M-N4 moieties (Table S1). A higher overall coordination number of 5-6 was found for FeNGR, which again includes four nitrogen atoms in plane but also one or two axial ligands, likely oxygen ligands due to the high oxophilicity of Fe (Table S1). The results therefore indicate that the metal cations in MNGR are involved in an M-N4 structure. These M-N4 moieties are covalently embedded in-plane in graphene and resemble the square-planar M-N4 structure of metal macrocycles such as metal-phthalocyanines (MPcs). This is supported by the analogous metal K-edge Xray near edge absorption spectroscopy (XANES) spectra of MNGR and MPcs (Figure 1D As shown in Figure 2A, more than 80% NADH was converted after 2 h under catalysis by CoNGR, CuNGR and FeNGR, while MnNGR showed a moderate effect (62.8%), and merely 21.2% conversion was detected in the presence of ZnNGR. In contrast, undoped GR (prepared by ultrasonic exfoliation of graphite) [START_REF] Kang | Solutionbased processing of monodisperse two-dimensional nanomaterials[END_REF] showed only ca. 5% conversion of NADH, indicating that M-Nx moieties in MNGRs account for their NOX-like catalytic activity rather than the graphene plane structure.

Given the high biocompatibility of iron over cobalt and copper dopants (Figure S5), FeNGR was selected for further exploration and application. To validate the catalytic activity assessed by the colorimetric assay, LC-MS/MS was used to directly detect the targeted catalytic product, NAD + . NADH and NAD + standards were also included for comparison and showed distinct peaks at 2.45 min (m/z =666.1 for NADH2 + ) and 1.73 min (m/z=664.1 for NAD + ), respectively (Figure 2B).

After the reaction of NADH with FeNGR, an intense peak assigned to NAD + was detected, and no peak assigned to NADH was observed. Notably, FeNGR catalysis in a 2 h reaction time resulted in a high NAD + yield (84.4%), which is similar to the effect (87.7%) of protein NOX (Figure 2C). To

      /    m a x m v V S K S
where With the colorimetric assay, we then benchmarked the NOX-like catalytic activity of FeNGR to a pool of 52 synthetic nanomaterials covering 39 chemical elements, 17 crystal phases and 6 morphologies (Figure 2D, Table S2). For a snapshot view of the results, the measured absorbance values after 2 h of reaction time were ranked into three levels, leading to a visual display where high, moderate and negligible levels of NADH oxidation are represented by red, yellow and blue colors, respectively (Figure 2D). Interestingly, only FeNGR displayed dose-and time-dependent catalytic activity among all 52 tested materials, indicating good catalytic selectivity.

Moreover, FeNGR specifically catalyzed hydrogen abstraction from the 1, 4-dihydropyridine-3-carboxamide and had little effect on hydrogens in the benzene, quinoline, pyran and pyrimidine rings (Table S3). These results suggest that FeNGR is a NOX-like nanozyme, displaying the prominent features of enzymes [START_REF] Lyu | Mimicking enzymes: The quest for powerful catalysts from simple molecules to nanozymes[END_REF] , including high catalytic efficiency under physiological conditions, catalytic specificity and substrate selectivity. As shown in Figure 4B, compared to parent HepG2 cells, the ETC-dysfunctional cells displayed a 55.5% decrease in the NAD + : NADH ratio. Accordingly, glucose uptake and ATP production reduced by 60.9% and 42.0%, respectively (compare the 1 st and 3 rd columns in Figure 4C and4E). In contrast, dihydroxyacetone phosphate, an important intermediate that is rapidly exhausted in the natural glycolysis pathway, showed a 35.5% increase (compare the 1 st and 3 rd columns in Figure 4D). These data confirm the successful construction of ETCdeficient cell models. Exposure of such cells to FeNGR significantly elevated the relative ratio of NAD + /NADH from 0.4 to 0.9 (compare the 3 rd and 4 th columns in Figure 4B). Consequently, there were 88.0%, 27.0% and 49.1% increments of glucose uptake, dihydroxyacetone phosphate consumption and ATP production, respectively, in ETC-deficient cells after FeNGR treatment (compare the 3 rd and 4 th columns in Figure 4C,D,E). Interestingly, although the uptake of FeNGR in normal HepG2 cells induced a significant increase in the NAD + /NADH ratio, it had negligible effects on the glucose, dihydroxyacetone phosphate and ATP levels. was retained there for 7 days (Figure 5A). Although corona formation on FeNGR surfaces (Table S6) may affect the catalytic activity, FeNGR maintained > 70% activity in serum over 9 d exposure (Figure S10).

Stability of

Confocal microscopy further revealed the distribution details in liver tissue sections. As shown in Figure 5B, the uptake of FeNGR was mainly by hepatocytes and Kupffer cells, both of which play important roles in hepatic metabolic processes.

The long hepatic bio-persistence and strong cellular internalization capability of FeNGR offered opportunities for catalytic elevation of NAD + in the liver. Meanwhile, the H&E staining images of tissues as well as blood test results showed negligible detrimental effects (Figure S11 and Table S4).

We then examined the effects of FeNGR injected to HFDfed mice via tail vein. Animals that received a chow diet (CD)

were also included for comparison. Significant glucose tolerance was detected in HFD mice after 12 weeks of feeding.

Then, the HFD mice were subjected to intravenous injection of 10 As shown in Table S5, administration of FeNGR to HFD mice resulted in a 2.7-fold decrease in the ED value compared to HFD mice. Principal component analysis (PCA) was also exploited to assess the differences in metabolite expression among the CD (green square), HFD (red triangle) and FeNGR-HFD (blue circle) groups (Figure 5F). The maximum number of PCs was set to 26 for the analysis. As shown in Figure 5F, the cumulative contribution of the first two principal components to the total variance was 89.5%. The PCA plot showed that the HFD group was clearly distinct from the CD group.

Interestingly, after FeNGR treatment, the metabolite levels of HFD mice were normal in six out of eight HFD mice, as revealed by their distribution in the green region of the CD group. These results suggest that FeNGR administration could relieve the metabolic disorder of HFD-fed mice.

DISCUSSION

The elevation of NAD + level has considerable benefits in regulating metabolic flux of mammals. To supplement NAD + in mammals, biological researcher has raised two approaches, namely the natural synthesis and catalytic conversion. According to the synthesis pathway of NAD + , a few intermediates including NAM [START_REF] Mitchell | Nicotinamide improves aspects of healthspan, but not lifespan, in mice[END_REF] , NA [START_REF] Trammell | Nicotinamide riboside is uniquely and orally bioavailable in mice and humans[END_REF] and NR [START_REF] Brakedal | The NADPARK study: A randomized phase I trial of nicotinamide riboside supplementation in Parkinson's disease[END_REF] have been exploited to elevate NAD + levels, bypassing the synthesis of adenine from the main building block, tryptophan. Besides, Mootha's group attempted to further improve the supplement efficiency by engineering protein enzymes, including combined enzymes consisting of bacterial lactate oxidase (LOX) and catalase (CAT) and bacterial water-forming NADH oxidase (NOX) [START_REF] Goodman | Hepatic NADH reductive stress underlies common variation in metabolic traits[END_REF][START_REF] Patgiri | An engineered enzyme that targets circulating lactate to alleviate intracellular NADH:NAD(+) imbalance[END_REF] , for the catalytical elevation of NAD + in cells. To the best of our knowledge, chemical approaches were rarely developed to increase NAD + levels. Given the shortcomings of protein enzymes, such as poor cellular internalizations, low stability in harsh biological fluids, and high cost to scale up, we engineered a synthetic material to mimic the activity of bacterial NOX and elevate NAD + in mammals.

Biosafety is a paramount issue in terms of the clinical translation of nanomedicine and is often controversial [START_REF] Orecchioni | Graphene and the immune system: challenges and potentiality[END_REF][START_REF] Heller | Banning carbon nanotubes would be scientifically unjustified and damaging to innovation[END_REF] . Recent studies indicate that nanomaterials could be designed by tuning their physicochemical properties to acquire desired biological effects [START_REF] Gazzi | other carbon nanomaterials and the immune system: toward nanoimmunity-by-design[END_REF][START_REF] Guiney | Assessing and mitigating the hazard potential of two-dimensional materials[END_REF][START_REF] Zheng | Nanoparticle-induced ferroptosis: detection methods, mechanisms and applications[END_REF] . Although Co, Cu-and Fe-NGRs showed similar NOX-like activity, FeNGR was selected for further exploration in cells and animals because iron is abundantly expressed in biological systems and relatively safe compared to the other two metals. FeNGR was coated with lipidated polyethylene glycol to improve dispersion stability.

The biodistribution and clearance of FeNGR in mice were examined by SPECT/CT after radiolabelling. Consistent with a literature report [START_REF] Jasim | Tissue distribution and urinary excretion of intravenously administered chemically functionalized graphene oxide sheets[END_REF] , the intravenously injected FeNGR rapidly targeted the liver in 6 h, and most of the FeNGR was eliminated in 9 days. The biological fate of nanomaterials is largely determined by their morphology and surface corona [START_REF] Cao | Coronas of micro/nano plastics: a key determinant in their risk assessments[END_REF] . For instance, the comparison of MoS2 biodistribution in transgenic mice with fibrinogen (Fg), apolipoprotein E (ApoE), immunoglobulin M (IgM) or complement 3 (C3) deficiency provides convincing support that ApoE binding on MoS2 nanosheets dictates their accumulation in liver sinusoid and splenic red pulp [START_REF] Ren | Chemical and biophysical signatures of the protein corona in nanomedicine[END_REF][START_REF] Cao | Molybdenum derived from nanomaterials incorporates into molybdenum enzymes and affects their activities in vivo[END_REF] . In this study, twelve apolipoproteins were identified in the 320 corona proteins of FeNGR (Table S6). Given that FeNGR has similar planar structure with MoS2 nanosheets, we speculated that the excellent liver targeting of FeNGR could be attributed to the constituent of apolipoproteins in surface corona. There might be two mechanisms dictating the clearance of FeNGR in vivo. On the one hand, the intravenously injected graphene sheets were reported to be eliminated by both fecal and urinary excretions [START_REF] Jasim | Tissue distribution and urinary excretion of intravenously administered chemically functionalized graphene oxide sheets[END_REF] ; on the other hand, enzymes including peroxides were demonstrated to catalyze the biodegradation of graphene derivatives [START_REF] Martin | Enzymatic degradation of graphene quantum dots by human peroxidases[END_REF][START_REF] Lalwani | Enzymatic degradation of oxidized and reduced graphene nanoribbons by lignin peroxidase[END_REF] . Moreover, we examined the possible pathogenic changes in tissue sections and biochemical indicators in blood from mice receiving intravenous injection of FeNGR. Although the numbers of white blood cells and lymphocytes showed slight increases on the 1 st day, they all recovered on the 7 th day. H&E staining images of heat, liver, spleen, kidney and lung showed no pathogenic changes. To further minimize the risks of detrimental effects in humans from exposure and facilitate the clinical translation of MNGR, the candidate materials should be engineered in a colloidal dispersion state with small-sized individual graphene sheets and hydrophilic groups on surfaces.

Two mechanisms, i.e., coupled electrochemical reaction (CER) and inner-sphere reaction (ISR) have been raised to explain the oxidation reactions catalyzed by MNGRs [START_REF] Ryu | Thermochemical aerobic oxidation catalysis in water can be analysed as two coupled electrochemical half-reactions[END_REF][START_REF] Rojas-Carbonell | Effect of pH on the activity of platinum group metal-free catalysts in oxygen reduction reaction[END_REF] . The CER mechanism could be interpreted as two coupled electrochemical reactions, one for O2 electro-reduction and the other for the substrate electro-oxidation, the coupled half-reactions working formally as a short-circuited electrochemical cell. In this view, a better O2 reduction catalyst then results in a higher driving force for the electrooxidation of the organic molecule.

In contrast, Bates et al proposed that the aerobic oxidation of substrate on MNGR materials is not the sum of two independent electrochemical half-reactions, but rather a direct innersphere reaction between O2 and the organic molecule [START_REF] Bates | Chemical and electrochemical O2 reduction on earth-abundant MNC catalysts and implications for mediated electrolysis[END_REF] . In this mechanism, O2 and the organic molecules co-adsorb on the metal center of the MNGR surface. As a result, the best catalyst for O2 reduction will not necessarily be the most active Taken together, the discovery of MNGR as a NOX-like catalyst provides a promising approach to regulate metabolic flux by the rational design of synthetic nanomaterials. FeNGR and other MNGR could potentially be exploited to treat some metabolic disorders, such as diabetes, fatty liver and hyperlipemia.

ASSOCIATED CONTENT

Supporting Information. The Supporting Information is available free of charge at http://pubs.acs.org.

Experimental procedures, statistical method, Raman, AFM, EXAFS, XANES, K-Edge FT-EXAFS Spectra, kinetics of reaction, catalytic activity with different substrate, cell viability histological H&E staining images, glucose sensitivity, insulin sensitivity and blood test data (PDF).

the O2 electro-reduction performance of pyrolyzed Fe-N-C catalysts.

Catalysts 2015, 5 (3), 1333-1351.

(72) Xu, X.; Xia, Z.; Zhang, X.; Li, H.; Wang, S.; Sun, G. Sizedependence of the electrochemical performance of Fe-N-C catalysts for the oxygen reduction reaction and cathodes of direct methanol fuel cells. Nanoscale 2020, 12 (5), 3418-3423.

Figure 1 .

 1 Figure 1. Characterization of MNGR materials. Characterization of the physicochemical properties of FeNGR, CuNGR and CoNGR by (A) AFM, (B) powder XRD patterns, (C) Fourier transformed EXAFS and (D) XANES spectra. A drop of MNGR material suspension at 50 μg/mL was added to mica plates and dried at room temperature for AFM observation. Ten milligrams of MNGR powder were placed on silicon plates by direct compression for XRD. Pelletized MNGR powders bound with Teflon powder were used for EXAFS and XANES measurements at the metal K-edge. The EXAFS and XANES spectra for the respective metal-phthalocyanine compounds with welldefined metal-N4 coordination are also shown (red curves in C and D).

  and Figure S2D). The most prominent difference in the XANES spectra of MNGR and MPcs is the peak intensities of the pre-edge shoulder, which is the characteristic feature of the square-planar M-N4 structure. The negligible pre-edge shoulder in the XANES spectra of MNGR indicates that the M-N4 moieties formed via pyrolysis are distorted, deviating from the well-defined square-planar M-N4 geometry with four equal M-N bond lengths and four equal N-M-N bond angles in MPcs. Assessment of the Catalytic Features of MNGRs. The MNGRs were subjected to reaction with aqueous NADH for examination of their catalytic activity by a colorimetric substrate, MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), which could rapidly react with NADH within 30 s, resulting in a color change from yellow to purple. This assay was exploited to acquire the standard curve of NADH, by which the percentages of NADH oxidized after a reaction time of 2 h were quantitatively determined by a calibration curve (Figure S4).

Figure 2 .

 2 Figure 2. Abiotic assessment of the NOX-like activity of MNGRs. Assessment of catalytic activities of MNGR materials by (A) colorimetric assay and (B) LC-MS/MS. (C) NAD + yields in NADH oxidation reactions. Nanoparticles of MNGR at 200 μg mL -1 and NOX at 200 mU mL -1 were used to react with NADH for 2 h in PBS (pH 7.4). The residual NADH amount was detected by MTS assay or LC-MS/MS (n=3 independent samples). For LC-MS/MS, the NAD + yield was calculated according to the following equation: NAD + yield (%) = SNAD + /(SNADH +SNAD + ) ×100%, where SNADH and SNAD + represent the molar amounts of NADH and NAD + detected by LC-MS/MS, respectively. (D) Heatmap presenting dose (0-200 μg mL -1 ) and time (0-2 h) responses of 52 nanomaterials towards the catalysis of NADH oxidation. The catalytic activities were assessed by the absorbance changes of the substrate in the MTS assay (see supplementary material, methods section: Assessment of NOX-like activity by colorimetric assay). (E) Comparisons of the stability of FeNGR and NOX in four simulated biofluids. FeNGR (200 μg/mL) and NOX (200 mU/mL) were pretreated in phagolysosomal simulated fluid (PSF), simulated gastric fluid (SGF), simulated intestinal fluid (SIF) and serum for 2 h. The catalytic activities of treated FeNGR or NOX were detected by MTS assay (n=3 independent samples). (F) Durability of FeNGR and NOX. An ultrafiltration membrane was used to separate FeNGR and NOX after each NADH oxidation reaction cycle. The catalytic activity before each cycle was measured by MTS assay (n=3 independent samples).

Figure

  Figure S6, the kinetic curves of NADH oxidation catalyzed by FeNGR and NOX could both be fitted by the Michaelis-Menten equation:

Figure 3 .

 3 Figure 3. Validation of the catalytic activity of FeNGR in cells. (A) Representative confocal images of FeNGR in cells. FeNGR was labelled by FITC conjugated with BSA and denoted as FITC-FeNGR (green). HepG2 cells treated with 10 μg/mL FeNGR for 24 and 48 h were stained with Hoechst 33342 (blue) and anti-LAMP1 (red) to visualize the nuclei and lysosome in cells, respectively (scale bar, 10 μm). Enzymatic activity of FeNGR in HepG2 cells at (B) different concentrations and (C) different incubation times. HepG2 cells were treated with different concentrations of FeNGR (0-10 μg/mL) or NR (10-150 μg/mL) for 24 h (B) or with 10 μg/mL FeNGR and 150 μg/mL NR for 0-192 h. (C). The treated cells were collected and lysed to detect the ratios of NAD + /NADH by an Amplite™ Fluorimetric NAD/NADH ratio assay kit (n = 3 biologically independent cell samples). (D) Catalytic activity of FeNGR in different cell lines. HepG2, MCF-7, Caco-2, THP-1, HeLa and A549 cells were exposed to 10 μg/mL FeNGR for 24 h. Then, the cells were collected and lysed to detect NAD + and NADH by an Amplite™ Fluorimetric NAD/NADH ratio assay kit (n = 3 biologically independent cell samples). The NAD + /NADH ratios reported in this figure are normalized to the value measured for NAD + /NADH for either the control (B and D) or at time 0 (C). * p< 0.05, **p< 0.01 and *** p< 0.001 compared to control cells by two tailed Student t-test.

Figure 4 .

 4 Figure 4. Effects of FeNGR in ETC-deficient cells. (A) Scheme showing key changes in metabolite levels in ETC-deficient cells. (B) The intracellular NAD + /NADH ratio, (C) glucose uptake, (D) dihydroxyacetone phosphate accumulation and (E) ATP generation in ETCdeficient HepG2 cells with or without FeNGR treatment. The ETC-deficient HepG2 cell model was established by 2 μM antimycin A treatment, denoted as antimycin A + in figure B-E. Antimycin A -indicates normal HepG2 cells. Parent and ETC-deficient cells were incubated with 10 μg/mL FeNGR for 72 h before measurement of the NAD + /NADH ratio and dihydroxyacetone phosphate by LC-MS/MS as well as ATP production by Celltiter Glo luminescent cell viability assay. Data are presented as the mean ± S.D. (n = 3 biologically independent cell samples). * p< 0.05, **p< 0.01 and *** p< 0.001 compared to ETC-deficient cells by two tailed Student t-test.

  These results demonstrate that FeNGR helps recovering the metabolism of ETC-deficient cells by catalytic elevation of NAD + . Catalytic Elevation of NAD + in Mice with Metabolic Diseases. In animals, the liver is the core organ to metabolize lipid, carbohydrate and protein. Defective hepatic metabolism has been found to be responsible for severe diseases such as adiposity and diabetes. As a proof of concept for the biomedical application of FeNGR, we explored the possibility of elevating NAD + levels in the livers of mice subjected to a highfat diet (HFD). First, we examined whether FeNGR could target this organ. Radio-and fluorophore-labelled FeNGR ( 125 I-FeNGR and FITC-FeNGR) were prepared to examine the biodistribution by single photon emission computed tomography-computed tomography (SPECT-CT) or confocal microscopy. More than 98.8% labeled FeNGR stably present in a representative biological medium, serum (Figure S9). After intravenous injection of 125 I-FeNGR dispersed in PBS, a large portion of 125 I-FeNGR rapidly targeted the liver within 6 h and

Figure 5 .

 5 Figure 5. Effect of FeNGR in HFD mice. (A) SPECT/CT imaging of 125 I-FeNGR in mice and (B) confocal imaging of FITC-FeNGR in liver sections. Mice were exposed to 10 mg/kg 125 I-FeNGR or FITC-FeNGR by tail vein injection. 125 I-FeNGR in mice was visualized by SPECT-CT (IVIS spectrum, MILabs, Netherlands) for 9 days. Mice that received FITC-FeNGR injection were sacrificed at 24 h to collect livers for fixation in formaldehyde solution and preparation of liver sections. The hepatocytes and Kupffer cells in liver sections were stained with Cy3-labelled albumin and F4/80 antibodies (red), respectively. Cell nuclei were stained with DAPI (blue). The stained sections were visualized by confocal microscopy imaging. (C) Schematic image of animal treatment procedure. (D) Hepatic NAD + /NADH

  one for aerobic oxidation of an organic molecule. Nevertheless, whether the CER or ISR mechanism dictates the NADH oxidation, the MNGR catalyst must have the ability to bind and activate O2. As such, the Co-, Cu-and FeNGR types of materials are known to be far more active towards O2 electroreduction than ZnNGR and MnNGR 68 , and thus their higher activity and selectivity in the present study towards NADH oxidation can be rationally explained, according to either of these mechanisms.While the activity for oxidation of NADH into NAD + normalized per active site is ca. 2 times lower for the present FeNGR than for NOX, the number of active sites per mass of catalyst is ca. 3 times higher for the present FeNGR than for NOX, resulting in comparable effects at the same dose. In the future, optimized syntheses can improve the activity of FeNGR via increased site density, improved site-specific activity, and tuned catalyst morphology. A recent work has shown that FeNGR materials with a site density of 1.9×10[START_REF] Li | Cloning, expression, characterization and homology modeling of a novel water-forming NADH oxidase from Streptococcus mutans ATCC 25175[END_REF] sites/gram can be prepared, ca. 6 times higher than in the present FeNGR[START_REF] Jiao | Chemical vapour deposition of Fe-N-C oxygen reduction catalysts with full utilization of dense Fe-N-4 sites[END_REF] . Although Fe2O3 has been observed operando from FeNGR degradation in proton exchange membrane fuel cell, Fe2O3 is poorly reactive toward NADH oxidation (Figure2D), indicating that FeN4 is the true active site. The sitespecific activity of FeNGR materials might also be improved 13 by the selective formation of Fe-Nx sites with a specific oxidation state and spin state (while the present FeNGR has two main Fe-Nx species, differing both in their Fe oxidation and spin states) or by co-doping the carbon matrix with nitrogen and another light element, such as sulfur[START_REF] Jia | Atomically dispersed Fe-N-4 modified with precisely located S for highly efficient oxygen reduction[END_REF] . Finally, the catalyst morphology may also play an important role for in vivo applications, including effects on the rate of cellular internalization of FeNGR and its elimination from the body. While commercial ZIF-8 was used to prepare the present FeNGR, the synthesis of ZIF-8 with a controlled crystal size is well established (from 30 to 1600 nm) and was shown to translate into precise control of the size of FeNGR particles after pyrolysis[START_REF] Armel | Effect of ZIF-8 crystal size on[END_REF] 72 .CONCLUSIONSIn summary, synthetic CuNGR, FeNGR and CoNGR nanosheets showed significant NOX-like activity. Compared to natural NOX, FeNGR is more robust to four representative biological fluids. FeNGR could be internalized into the cytoplasm to elevate NAD + /NADH ratios, contributing to increments of glucose uptake and ATP production, in ETCdeficient cells. Intravenously injected FeNGR could target livers internalized by hepatic and Kupffer cells and enabled 4.1-fold increases in the NAD + /NADH ratio in the liver tissue of HFD-fed mice. The serum metabolite profiles demonstrated a dramatic relief of metabolic disorders in HFD-fed mice.

  [S] is the concentration of substrate, Km is the Michaelis constant, Vmax is the maximum rate, and v is the initial rate for

	a given [S]. The NADH oxidation catalyzed by FeNGR and
	NOX has similar kinetic constants, with Vmax values of 2.5×
	10 -2 and 4.1× 10 -3 mM•s -1 and Michaelis constants of ca 3.97
	and 0.35 mM, respectively (Figure S6). We exploited the Vmax
	values to calculate the average single-site activity of catalytic
	sites in FeNGR (Fe-N4) and compared it to the active site in
	NOX (tryptophan 47 domain). While the quantification of the
	number of active sites with a cyanide molecular probe indicat-

ed that the Fe-N4 site density on the top surface in this FeNGR is ca. 3.3×10

[START_REF] Yoshino | NAD + intermediates: the biology and therapeutic potential of NMN and NR[END_REF]

sites/g 44 , the number of catalytic domains in NOX can be assessed from its known molecular weight to be 1.12×10

[START_REF] Yoshino | NAD + intermediates: the biology and therapeutic potential of NMN and NR[END_REF]

sites/g (see supplementary material, methods section: Detection of catalytic reaction kinetics). As a result, after normalization by the number of sites in presence during the catalytic test, the single-site activity of FeNGR is 2.4 mole of NAD + per mole of Fe-N4 sites and per second, while the single-site activity of NOX is ca 5.5 mole of NAD + per mole of tryptophan 47 domains and per second (see supplementary material, methods section: Detection of catalytic reaction kinetics).

ACKNOWLEDGMENT

This work was supported by grants from the National Key R&D Program of China, Ministry of Science and Technology of China (2020YFA0710700, 2022YFE0124000), the National Natural Science Foundation of China (21976126), the Natural Science Foundation of Jiangsu Province (BK20211545), and the Project of National Center for International Research on Intelligent Nano-Materials and Detection Technology in Environmental Protection, Soochow University (No. SDGH2202). We acknowledge Synchrotron SOLEIL (Gif-sur Yvette, France) for provision of synchrotron radiation facilities at beamline SAMBA (proposal number 20180635)

AUTHOR INFORMATION

Email: liruibin@suda.edu.cn Frédéric Jaouen -Univ. Montpellier, CNRS, ENSCM, Montpellier, France; Email: frederic.jaouen@umontpellier.fr

Notes

The authors declare no competing financial interest.