Acknowledgments. We thank E. Haudebourg and A. Aubert from the PAPPSO plateform, T. Meylheuc from the Mima2 plateform for their technical assistance, and O. Triballat Institute for its financial support.

Reference : Boulay M, Al Haddad M, Rul F. Streptococcus thermophilus growth in soya milk: Sucrose consumption, nitrogen metabolism, soya protein hydrolysis and role of the cellwall protease PrtS. Int J Food Microbiol. 2020 Dec 16;335:108903. doi: 10.1016/j.ijfoodmicro.2020.10890

 Contact: francoise.rul@inrae.fr  S. thermophilus LMD-9 grows rapidely in soya milk, consumes sucrose and produces lactate, a molecule of interest for matrix acidification and for gut physiology. The first proteomic map in soya of S. thermophilus presented here underlines the prevalence of nitrogen metabolism.  As in milk, the cell-wall protease PrtS is involved in growth of S.

thermophilus. Its abitiy to hydrolysis soya proteins could help to improve digestibility of soya based food and provide health benefits, such as reducing the allergenicity of soya proteins or modulating the production of bioactive peptides.

Conclusions -Perspectives

Context and objectives

Fermented plant-based food is booming in our diet and soya products fermented by lactic acid bacteria (LAB) are appealing because of their potential health and nutritional benefits.

Whereas the physiology of one of the main starters, Streptococcus thermophiles, is well characterized in dairy matrices, its behaviour, metabolic activities and techno-functional properties are less documented in soya. Here, we characterized the growth of S. thermophilus in soya milk, identified the carbon source it uses and explore its global metabolism, with a focus on nitrogen metabolism.

Experimental design

S. thermophilus LMD-9

wt and DprtS (constructed by gene interruption with a Ery K7)

• acidification: pH and lactic acid measurements (HPLC) • growth: enumerations on M17 lactose (1g/L)

• sugars: glucose, fructose, lactose, sucrose measurements (HPLC, Aminex HPX-87H column)

• proteolytic profile: SDS-PAGE (4-12%)

• milk (powder, 10% reconstituted, Sigma)

• soya milk (SojaSun, Triballat-Noyal) 37°C 10 6 CFU/ml in

•

  proteomic analysis (coll. PAPPSO Plateform INRAE; http://pappso.inra.fr/): -cell-envelope and cytosolic protein fractions of exponential bacterial cultures -separation by LC-MS/MS (UltiMateTM 3000 RSLCnano system coupled to a LTQ-Orbitrap DiscoveryTM mass spectrometer in CID mode -protein identification: X! Tandem software -comparison with protein databases of S. thermophilus LMD-9 (GenBank v. 2013, 1710 entries), soya (Uniprot v. 16-11-14; 150,681 entries), and potential contaminants -Filtration: X! Tandem Pipeline -a peptide E-value of 0.01, a protein E-value of -4, and the presence of 3 peptides per protein minimum. ] -pH 1-S. thermophilus LMD-9 rapidely acidifies soya milk, by lactate production  High and rapid acidification pH 5h  4.98 (milk: 4.83) V max  x 1,5 soya/milk  Correlation between pH drop and lactate production  Higher production of lactate in milk / soya:  related to higher bacterial population in milk Sugar dosages in soya culture supernatants  4 mM decrease of sucrose ( 1.36 g/L) 3-Proteins related to nitrogen metabolism predominates in soya cultures of S. thermophilus Amino acid transport and metabolism [J] Translation, ribosomal structure and biogenesis [G] Carbohydrate transport and metabolism [M] Cell wall/membrane/envelope biogenesis [F] Nucleotide transport and metabolism [S] Function unknown [C] Energy production and conversion [P] Inorganic ion transport and metabolism [O] Posttranslational modification, protein turnover, chaperones [R] General function prediction only [D] Cell cycle control, cell division, chromosome partitioning [K] Transcription [U] Intracellular trafficking, secretion, and vesicular transport [I] Lipid transport and metabolism [V] Defense mechanisms [T] Signal transduction mechanism [L] Replication, recombination and repair [H] Coenzyme transport and metabolism % of total protein identified COG name • Proteomic analysis of LMD-9 soya culture  identification of 328 proteins  19% of proteins are related to amino acid (AA) transport and metabolism  including the cell-wall protease PrtS, AA and peptide transporters for potential use of of soya proteins by their hydrolysis and by transport of peptides  9% of proteins are related to carbohydrate metabolism  including proteins for potential transport and metabolism of sucrose, the main sugar of soya milk (PstI, PstH, ScrA, ScrB) 2-S. thermophilus LMD-9 grows in soya milk, via sucrose consumption 4-The cell-wall PrtS contributes to S. thermophilus LMD-9 growth in soya milk and to soya protein hydrolysis • DprtS mutant: final population decreased by 2,8 fold compared to wt strain  PrtS is involved in S. thermophilus growth in soya milk  More restricted proteolysis in absence of PrtS protease  Probable involvement of other bacterial proteases in soya protein hydrolysis • Proteolytic profiles of soya culture supernatants (SDS-PAGE)

Micalis Institute, INRAE, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas, France


C Gasser

P Garault

C Chervaux

V Monnet

J.-M Faurie

Françoise Rul

Carbon metabolism in Streptococcus thermophilus regulation and diversity within the species

• A lactic acid bacterium widely used in dairy industry.

• Well adapted to the dairy environment thanks to a high efficient use of lactose.

Streptococcus thermophilus

Context

Streptococcus thermophilus…

• What are the sugars consumed in S. thermophilus?

• How is sugar metabolism regulated?

• Is there a diversity?

Objectives

• Screen and comparison of genomes of 39 S. thermophilus strains for potential carbon metabolism (CM) genes.

• Growth and acidification assessment (M17 medium) • Glucose is transported by the sucrose transporter ScrA in LMD-9 strain (data not shown).

•
• Lactose represses the promoter activity of its own transporter gene and that of sucrose transporter gene scrA.

1

  Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, 78350 Jouy-en-Josas, France 2 Danone Nutricia Research, Avenue de la Vauve, 91120 Palaiseau, France * Present address: Global Bioenergies, rue Henri Desbruères, 91000 Evry, France Carbon metabolism in Streptococcus thermophilus: regulation and diversity within the species A low genetic diversity of carbohydrate metabolism genes in S. thermophilus in contrast with a high phenotypic diversity, that could be related to different gene regulations • The regulatory effect of glucose on carbohydrate metabolism genes is unexpected and complex; it is not explained yet. Reference: Gasser et al. Co-utilization of saccharides in mixtures: moving toward a new understanding of carbon metabolism in Streptococcus thermophilus, Int J Food Microbiol, in revision. • Used in new fermented products (dairy matrices supplemented with sugar or in plant-based food), containing a variety of carbohydrates in mixes.

•

  scrA and lacS promoter activities in presence of 2 sugars scrA scrA • S. thermophilus ferments 4 carbohydrates, conducting to different growth level according to strains and sugars. • S. thermophilus is able to co-consume several sugars: lactose & sucrose / sucrose & glucose.


C Gasser

P Garault

C Chervaux

V Monnet

J.-M Faurie

F Rul

Lactose Glucose

Streptococcus thermophilus is a bacterium widely used in the production of yogurts and cheeses, where it efficiently ferments lactose, the saccharide naturally present in milk. However, when used in sweetened dairy products or plant-based products, S. thermophilus may encounter other saccharides (i.e. alone or in mixtures). To date, S. thermophilus growth and metabolic capacities in such contexts as well as carbon metabolism regulation mechanisms remain poorly characterized.

Context

• Glucose maintains its repressive effects in mixed-saccharide media on lacS and scrA promoter activities

• lacS promoter activity is no more repressed in a DccpA mutant, whatever the lactose or glucose concentration (10, 25 or 50 g/L)

• Is there genetic and/or phenotypic diversity of sugar use in S. thermophilus?

• What are the sugars consumed? "dairy-like" mix "plant-like" mix time (h)

• All promoters of CM genes tested are active in presence of lactose, glucose or sucrose and the activities of only lacS and scrA ones are modulated by the nature of the sugar Promoter activity for 8 carbohydrate metabolism genes in S. thermophilus LMD-9 grown in presence of high concentration of lactose, sucrose, or glucose (50 g/L).

-lacS: lactose permease; galR: galactose regulator; scrA: sucrose/glucose transporter; scrB: sucrose hydrolase; fruR: fructose regulator; yxfA: potential glucose permease; manX: potential glucose PTS transporter; ccpA: CCR general regulator.

-Experiments were performed in triplicate. Different letters indicate significant differences in promoter activity for a given gene (ANOVA and Tukey-Kramer post-hoc tests, p<0.05).

Regulation of carbon metabolism genes

Promoter activity for lacS and scrA genes in S. thermophilus LMD-9 grown in presence of different concentrations of glucose or lactose (50, 25, 10, or 5 g/L).

Each bar represents the ratio of the maximum RLU/OD600nm value for a given set of experimental conditions to the maximum value in the condition with 50 g/L of glucose.

lacS promoter scrA promoter

• Glucose and lactose repress promoter activity for transporter-encoding genes lacS and scrA in a concentration-dependent manner, whereas sucrose not

• The 39 strains possess genes coding for the potential use of 5-6 carbohydrates  low genetic diversity for carbohydrate metabolism genes

  in presence of single or mix sugars • How is sugar metabolism regulated? Questions Bacterial growth, sugar consumption and glycolytic flux  growth of the 4 strains having an intact fruA gene  no/low growth for most strains Glucose Fructose Galactose Lactose Sucrose   growth of all strains -high final biomass  high variability of growth • S. thermophilus LMD-9 first consumes  lactose over sucrose  sucrose over glucose Quantity of sugar consumed -produced (g/L) Quantity of sugar consumed -produced (g/L)

(•

  sequence and gene organization conserved) Lactose -Galactose Fructose fruA : pseudogene for 36 strains over 39In silico analysis scrR : pseudogene for 3Screen and comparison of genomes of 39 S. thermophilus strains for potential carbon metabolism (CM) genes• Growth and acidification (M17 medium) is higher in presence of lactose or glucose, compared to sucrose  decreases as lactose or glucose concentration decrease  is not affected by sucrose concentration Contact: francoise.rul@inrae.fr• the promoter profil activity for scrA is similar in a DscrR mutant than in the wild-type strain, in glucose, lactose or sucrose (whatever their concentration) transporter ScrA is necessary for growth in LMD-9 strain in presence of glucose or sucrose as a single carbon source (no growth in a DscrA mutant)  ScrA is responsible for the transport of glucose and sucrose in LMD-9 strain  An explanation for the effect of glucose on scrA promoter activity modulation? S. thermophilus is able to co-consume several sugars when mixedCarbohydrate metabolism regulation principally involves lactose and sucrose transporter gene expression via the general regulator CcpA  Catabolic repression in S. thermophilus LMD-9 does not strictly represses the consumption of less preferred sugars; instead, saccharides are jointly consumed  Regulatory profiles for lacS and scrA genes according to sugar concentration and glycolytic flux are similar in LMD-


Andreea Apetrei
email: apetrei-a@chu-caen.fr

Arnaud Molin

Nicolas Gruchy

Manon Godin

Claire Bracquemart

Antoine Resbeut

Gaëlle Rey

Gwenaël Nadeau

Nicolas Richard

A novel synonymous variant in exon 1 of GNAS gene results in a cryptic splice site and causes pseudohypoparathyroidism type 1A and pseudo-pseudohypoparathyroidism in a French family

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

A novel synonymous variant in exon 1 of GNAS gene results in a cryptic splice site and causes pseudohypoparathyroidism type 1A and pseudo-pseudohypoparathyroidism in a French family Andreea Apetrei 1 , Arnaud Molin 1 , Nicolas Gruchy 1 , Manon Godin 1 , Claire Bracquemart 1 , Antoine Resbeut 1 , Gaëlle Rey 2 , Gwenaël Nadeau 2 , Nicolas Richard 1

Introduction

Subjects and Methods Results Discussion and Conclusions

GNAS complex locus

*

The main imprinted sense and antisense transcripts from the GNAS complex locus.

*Gαs is predominantly expressed from the maternal allele in the renal proximal tubules, parathyroid, thyroid, pituitary gland and ovaries and has a biallelic expression in the other tissues.

Mmethylated region

Adapted from Pulix M., Plagge A. ( 2020) Imprinted Genes and Hypothalamic Function

GNAS -associated phenotype

The main disease subtypes caused by inactivating GNAS gene mutations. §

The resistance to parathormone (PTH) is characterized by high serum PTH, hypocalcemia and hyperphosphatemia and it can be associated with resistance to other hormones, such as thyroid stimulating hormone (TSH).

GNAS sequencing

GNAS exons 1-13 were sequenced and interpreted following the routine protocol.

In silico splicing prediction algorithms (SPiP and Alamut) were used.

GNAS expression analysis

Quantification was used to assess the expression of GNAS in cDNA in the proband and in a healthy, sex and aged-paired control. Primers spanned the coding region of exons 1 and 2.

Transcript study and sequencing RT-PCR using a gene-specific primer localized in the exon 5 was followed by a PCR using primers localized in exons 1 and 2. A capillary electrophoresis analysis allowed to isolate and characterize the PCR products before sequencing.

GNAS sequencing

A heterozygous variation Chr20(GRCh37):g.57466889C>A, NM_001077488.2:c.108C>A p.(Val36=) localized in the exon 1 of GNAS gene, absent from the local and international databases was identified in III.4., inherited from her father (II.3):

Chromatogram from DNA sequencing of a nucleotide substitution in the proband (III.4)

GNAS expression analysis

The expression quantification showed a reduction of GNAS expression by 35% in comparison with a normal control:

Relative GNAS expression level measured by qRT-PCR using oligo(dT)18 primer, normalized to GAPDH expression level in the proband (III.4) and a healthy control. A second RT-PCR using a gene-specific primer allowed to amplify the expected PCR product and 2 additional products in smaller quantities, a shorter one and a longer one, respectively corresponding to the wild-type, the mutant allele and a heteroduplex (a mixture of wild-type and mutant allele) product:

Capillary electrophoresis performed on the cDNA obtained by RT-PCR using GNAS gene specific primer. The sequencing of these PCR products showed a 34bp deletion in the mutant allele.

#

  Described Albright's Hereditary Osteodystrophy (AHO) features are obesity, round face, short stature, brachydactyly type E, subcutaneous ossification and mild mental retardation. The crossed alleles correspond to the inactivated alleles of the GNAS gene.

3N

  -normal level *At the low limit of the normal range c.108C>A

CAAGCAGC TACCGGGCCACGCACCGCCTGCTGCTGCTGGGTAA

Wild-type

Mutant

CTAGGTGCTGGAG

Exon 1

Normal splicing of intron 1 NM_001077488.2:c.108C>A p.(Val36=)

GNAS

New splice donor site Aberrant splicing of intron 1 34bp deletion

We describe the first family with PHP1a/PPHP presenting a pathogenic synonymous variant in the GNAS gene, responsible for a splicing defect leading to the premature splicing of the first exon and to a probably unstable RNA or truncated protein:

This observation supports the increasing interest in the identification of splicing variants, particularly those predicted as synonymous, which are often systematically categorized as benign on this sole element.

Splice-prediction algorithms could be added to the bioinformatic pipelines for massive parallele sequencing data in routine analysis to improve the detection of such variations.


 Contact: francoise.rul@inrae.fr  S. thermophilus LMD-9 grows rapidely in soya milk, consumes sucrose and produces lactate, a molecule of interest for matrix acidification and for gut physiology. The first proteomic map in soya of S. thermophilus presented here underlines the prevalence of nitrogen metabolism.  As in milk, the cell-wall protease PrtS is involved in growth of S.

thermophilus. Its abitiy to hydrolysis soya proteins could help to improve digestibility of soya based food and provide health benefits, such as reducing the allergenicity of soya proteins or modulating the production of bioactive peptides.

Conclusions -Perspectives

Context and objectives

Fermented plant-based food is booming in our diet and soya products fermented by lactic acid bacteria (LAB) are appealing because of their potential health and nutritional benefits.

Whereas the physiology of one of the main starters, Streptococcus thermophiles, is well characterized in dairy matrices, its behaviour, metabolic activities and techno-functional properties are less documented in soya. Here, we characterized the growth of S. thermophilus in soya milk, identified the carbon source it uses and explore its global metabolism, with a focus on nitrogen metabolism. • acidification: pH and lactic acid measurements (HPLC) • growth: enumerations on M17 lactose (1g/L)

• sugars: glucose, fructose, lactose, sucrose measurements (HPLC, Aminex HPX-87H column)

• proteolytic profile: SDS-PAGE (4-12%)

• milk (powder, 10% reconstituted, Sigma)

• soya milk (SojaSun, Triballat-Noyal) 37°C 10 6 CFU/ml in

  We thank E. Haudebourg and A. Aubert from the PAPPSO plateform, T. Meylheuc from the Mima2 plateform for their technical assistance, and O. Triballat Institute for its financial support.Reference : Boulay M, Al Haddad M, Rul F. Streptococcus thermophilus growth in soya milk: Sucrose consumption, nitrogen metabolism, soya protein hydrolysis and role of the cellwall protease PrtS.Int J Food Microbiol. 2020 Dec 16;335:108903. doi: 10.1016/j.ijfoodmicro.2020.10890

M

  . Boulay, M. Al Haddad and F. Rul Micalis Institute, INRAE, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas, France Experimental design S. thermophilus LMD-9 wt and DprtS (constructed by gene interruption with a Ery K7)

•

  proteomic analysis (coll. PAPPSO Plateform INRAE; http://pappso.inra.fr/): -cell-envelope and cytosolic protein fractions of exponential bacterial cultures -separation by LC-MS/MS (UltiMateTM 3000 RSLCnano system coupled to a LTQ-Orbitrap DiscoveryTM mass spectrometer in CID mode -protein identification: X! Tandem software -comparison with protein databases of S. thermophilus LMD-9 (GenBank v. 2013, 1710 entries), soya (Uniprot v. 16-11-14; 150,681 entries), and potential contaminants -Filtration: X! Tandem Pipeline -a peptide E-value of 0.01, a protein E-value of -4, and the presence of 3 peptides per protein minimum. ] -pH 1-S. thermophilus LMD-9 rapidely acidifies soya milk, by lactate production  High and rapid acidification pH 5h  4.98 (milk: 4.83) V max  x 1,5 soya/milk  Correlation between pH drop and lactate production  Higher production of lactate in milk / soya:  related to higher bacterial population in milk Sugar dosages in soya culture supernatants  4 mM decrease of sucrose ( 1.36 g/L) 3-Proteins related to nitrogen metabolism predominates in soya cultures of S. thermophilus Amino acid transport and metabolism [J] Translation, ribosomal structure and biogenesis [G] Carbohydrate transport and metabolism [M] Cell wall/membrane/envelope biogenesis [F] Nucleotide transport and metabolism [S] Function unknown [C] Energy production and conversion [P] Inorganic ion transport and metabolism [O] Posttranslational modification, protein turnover, chaperones [R] General function prediction only [D] Cell cycle control, cell division, chromosome partitioning [K] Transcription [U] Intracellular trafficking, secretion, and vesicular transport [I] Lipid transport and metabolism [V] Defense mechanisms [T] Signal transduction mechanism [L] Replication, recombination and repair [H] Coenzyme transport and metabolism % of total protein identified COG name • Proteomic analysis of LMD-9 soya culture  identification of 328 proteins  19% of proteins are related to amino acid (AA) transport and metabolism  including the cell-wall protease PrtS, AA and peptide transporters for potential use of of soya proteins by their hydrolysis and by transport of peptides  9% of proteins are related to carbohydrate metabolism  including proteins for potential transport and metabolism of sucrose, the main sugar of soya milk (PstI, PstH, ScrA, ScrB) 2-S. thermophilus LMD-9 grows in soya milk, via sucrose consumption 4-The cell-wall PrtS contributes to S. thermophilus LMD-9 growth in soya milk and to soya protein hydrolysis • DprtS mutant: final population decreased by 2,8 fold compared to wt strain  PrtS is involved in S. thermophilus growth in soya milk  More restricted proteolysis in absence of PrtS protease  Probable involvement of other bacterial proteases in soya protein hydrolysis • Proteolytic profiles of soya culture supernatants (SDS-PAGE)


Plates

Paul Beliën

Sabine Bourgey

Curtis Clay

Georges Depeyrot

Arianna D'ottone

David Fanning

Jean-Baptiste Giard

Elizabeth Hahn

Mary Hinton

Dominique Hollard

Ursula Kampann

Ian Leins

Attilio Mastrocinque

Olga Palagia

Markus Peter

François Planet

Roberto Russo

Philip Skingley

Massimiliano Tursi

Giuseppe Ucciardello

Hans Voegtli

HADRIEN RAMBACH AND ALAN WALKER THE "HEIFER" AUREI OF AUGUSTUS

This article is devoted to a rare, beautiful and intriguing series of gold coins of Augustus (27 bc -Ad 14) that bear a head of the emperor and the legend CAESAR on the obverse, and a heifer with the legend AVGVSTVS on the reverse 1 The most recent listing of this coinage is in a general book on aurei by Calicó, but it gives no indication as to the location (and number) of examples. Six types can be identified, as summarized in the table below.

Although the exceptionally high artistic quality of these coins dates them to the golden age of Roman history, the exact date of issue is uncertain. The laurel wreath could "celebrate a victory" -vitulari in Latin, a word quite close to vitulus ("bullock") 3 . A naval victory -perhaps Actium? -is a possibility because of the traditional association of the bull with Neptune 4 . Several dates have been suggested: 27-20 bc (MATTingLy 1923); about 25 bc (KEnT 1984); 21 bc, for the decennalia of Actium (LAffrAnchi 1952); 20 bc (HiLL), or even 19-15 bc (cALicó 2003).

The mint is also uncertain 5 . The portrait style is of Greek workmanship rather than Roman, prompting two principal hypotheses. These coins may have been issued in the east, probably in Asia Minor, and perhaps at Pergamum, or Apamea, or Ephesus, or Laodicea ad Mare. Mattingly (1923) suggested Samos, where Augustus was in residence during 21-20 bc. [START_REF] Gabrici | La numismatica di Augusto[END_REF] attributed these coins 3 See M. grAnT, Complex symbolism and new mints c. 14 bc, NC.9, 1949, p. 28; also ID., The Six Main Aes Coinages of Augustus (Edinburgh 1953), pp. 112 sqq. 4 The god was nicknamed taureos because bulls were sacrificed to him and because the seashore provided good pasture.

5

The few examples known are all in Western collections (apparently none in Greek and Turkish museums). Until a group is found in an archaeological context, the absence of find-spots prevents any mint-identification, especially considering the wide circulation of aurei within the Empire. The die axes (almost all 12 o'clock) do not add any information either.

to Athens, as Myron's statues of oxen were in the Agora 6 . Sinope, in the Black Sea region, has also been proposed because gold was found nearby 7 . The few examples known are all in Western collections, none with a find-spot; if such coins were to be found in an archaeological context, it might finally give concrete elements as to where they were minted, rather than relying on historical hypotheses and style discussions. However, this series could have been minted in Rome, with dies engraved by an important Hellenistic artist 8 . Yet although the reverses are uniform in style, suggesting that one artist was responsible for them all, the obverse dies are the work of two different engravers, one who depicted the emperor with a large, broad head, and one who preferred a narrow bust with sharply defined features.

In this portrait of him as a laurel-wreathed victor, Augustus is depicted as a young Apollo-like emperor, of the same age but more regal than the marble Prima Porta Augustus. Artistically, few Roman coins can compare with this portrait, the equal of any masterpiece of the Hellenistic Age. The image matches that of marble busts of Augustus: he is depicted with a compact mouth and chin, a long nose, high cheeks and a broad forehead. The portrait has much in common with the finest Aegypto Capta issues struck for Augustus in the east (c. 28-27 bc). Furthermore, both these aurei and the Aegypto Capta issues are in two distinct styles: one of high artistic quality and another more pedestrian one 9 . Interestingly, the thin, elongated neck of Augustus on this coin is reminiscent of Caesar's latest portraits. This may possibly be a deliberate visual echo, designed to highlight the transmission of power. Also, the heifer is depicted walking, and her peaceful attitude may perhaps be intended to evoke the earliest Roman coinage: the cast aes signatum with a bull 10 . Could the choice of the heifer type thus be a way for the young Augustus to suggest an ancient Roman lineage for himself and his coinage?

The type 5 shows a heifer with a raised head, whilst type 6 -much rougher in styleshows a bull 11 (clearly identified by its horns and also with a raised head). The style of type 6 is much closer to that of a silver issue12 , than to the types 1-4. Those coins of types 5-6 may be a completely separate issue from types 1-4, both There is a series of aurei of Augustus that show a bull charging with its head down in the attitude of the marble bull of the Kerameikos from the tomb of Dionysios of . The bull on the aurei Calicó 172a is close in type to the small bronze bulls found on the Acropolis during the Greek excavations of 1885-1890, which were supposedly similar to the votive bull of the Council of the Areopagus (which stood in the northeast corner of the court west of the Parthenon: G. P. STEVEnS, The Setting of the Periclean Parthenon, Hesperia Suppl. 3, 1940, fig. 17). This bull has also been linked to Caesar, and to the Legio IV Macedonica (the bull was its symbol), which he founded in 48 bc for the war with Pompey (on bulls as legionary emblems: S. wEinSTocK, Divus Julius (Oxford 1971), pp. 119-121). And for the silver coinage with the bull, Adrien Blanchet has suggested that it may be a reuse of the ancient coinage of Thurium, in honour of Augustus's family's origins (Le taureau des monnaies d'Auguste, Macon 1947, p. 5).

The high quality and distinctive style of engraving of the heifer issues undoubtedly indicate that they had a special significance, and that this heifer has a particular meaning. It may perhaps represent one of four (supposedly identical) bronze statues of heifers cast by the sculptor Myron that are supposed to have been commissioned after the peace-treaty of Nicias with Sparta, in March 421 bc, and to have been dedicated on the Athenian acropolis around 420-417 13 . The artist had taken part in the 90 th Olympiad, together with Polycleitus, Pythagoras and Scopas. These heifers were his most celebrated work, and according to Ovid, "those cattle, true to life, are a masterpiece by Myron" 14 . Pliny explained indeed the specificity of the artist's work: "Myron was born at Eleutherae and likewise a pupil of Hagelades. He was most renowned for his statue of a cow, praised in some famous poems -for many people owe their reputation not to their own talent but to someone else's . . . He was apparently the first to extend the representation of reality, and a more talented artist than Polycleitus -paying more precise attention to a system of proportional parts" 15 .

Johann Wolfgang von Goethe wrote: "About 400 bc, a Greek sculptor named Myron made a bronze cast of a cow. Cicero claims to have seen the statue in Athens, and in the seventh century Procopius saw it in Rome. Thus, for over a thousand years the work had attracted attention. Although considerable information concerning this statue has come down to us, none of it is much help in forming a clear image of the original" 16 . Indeed, very many ancient epigrams were written about those sculptures, and they remain rather vague as to their appearance. Although there were at least four bronze sculptures, which one assumes were identical, the poems tend to refer to as single 'ox'. Smith mistakenly concluded that the cow and the four oxen of Augustus were different works17 : it is in fact a simple case of poetic license, in which the writers refer to the model sculpted by Myron, rather then the actual objects, the bronze casts. These ancient texts relate many anecdotes that insist on the bronzes' striking realism (see below, and also LiSSArrAguE / fronTiSi-ducroux): a calf tries to suck the heifer and starves, a bull tries to mount her, a lion is tempted to bite her, a thief tries to steal her, shepherds were confused, and so on. It was said that the artist himself -having lost the sculpture in a herd -had to chase the living cows away in order to identify his creation, or that the bronze was not in fact a statue, but an actual cow with bronze-like aged skin. The humour of these epigrams justifies them all being quoted in their entirety (here in annex).

13 Because these sculptures were in Athens (and later in Rome), the cow horn (diam. 11 cm) and ear (23 cm long) that were excavated in Olympia can obviously not be Myron's (P. BoL, Grossplastik aus Bronze in Olympia (Berlin 1978), p. 112, pls 24-25, no. 134).

14 Ovid,De Ponto,iv.1.34. 15 Pliny, Nat. hist. XXXIV.57-58. 16 J.W. von Goethe, "On Myron's Cow", Über Kunst und Altertum, 1818, vol. II, pp. 9-10: "Myron, ein griechischer Bildner, verfertigte ungefähr vierhundert Jahre vor unserer Zeitrechnung eine Kuh von Erz, welche Cicero zu Athen, Procopius im siebenten Jahrhundert zu Rom sah, also dass über tausend Jahre dieses Kunstwerk die Aufmerksamkeit der Menschen auf sich gezogen. Es sind uns von demselben mancherlei Nachrichten übriggeblieben; allein wir können uns doch daraus keine deutliche Vorstellung des eigentlichen Gebildes machen…".

These statues were still in Athens in the time of Cicero 18 . Despite Corso's hypothesis that they were taken to Rome by Nero (who stayed in Greece in 66-68) 19 , it seems certain that they were shipped to the city after Octavian's victory at Actium (on September 31 bc), to adorn an altar in his Temple of Apollo on the Palatine (dedicated in 28 bc), next to the Forum Boarium 20 . There, according to Sextus Propertius, "round the altar, stood four of Myron's cattle, carved statues of oxen, true to life" 21 . These cows were later used by Vespasian to decorate his Temple of Pax in Ad 74, when he celebrated his victory against the "Great Revolt" (or First Jewish War, 66-73), and they are depicted on aurei of Vespasian and of his son 22 . Procopius attests that they were still in the Forum of Peace in Rome in 537-538 23 , soon before the sack of Rome by Totila in 546. They were carried to the appropriately named Forum Tauri in Constantinople at a later, unknown date, where Julian, Praetorian Prefect of Egypt in 530-531 Ad, saw them (they were then still a novelty). Constantine Manasses saw them in Constantinople in 1150 24 , and John Tzetzes between 1144 and 1170 25 , but they seem to have disappeared after the fall of the city to the Crusaders in 1204; it is most likely that they -like so many other bronze statues in Constantinople -were melted down to make coins.

Historians and classicists usually state that there is no visual evidence for the appearance of Myron's heifer. The hypothesis that it is depicted on these coins has been rejected on the basis of the varied postures of the heifer on the reverses26 . It is true that the heifer on type 5, and even more so the bull on type 6, raises its head. But type 4 (heifer right) is a direct inversion of types 1-3 (heifer left), and the posture of the animal on these dies is identical, with a raised rear, face down (but not bent), walking, the tail between the legs, and so on. There are of course variations in the proportions -compare, for example, coins 1.10 and 1.11 -but this is a result not of different models but of the dies being by different engravers. Moreover, the coinage of Vespasian, although poorly engraved, shows the heifer in a comparable position. These coins of Augustus most likely depict this celebrated ancient sculpture. Münzenzeitung, vol. 12.2, Feb. 1977, p. 85. See cALicó nos 617-618, 621-623, 733-736. 23 Procopius ,De Bello Gothico VIII.21.14. 24 Constantine Manasses, Beiblatt col. 75. Quoted from: Constantine Manasses, Ekphrasis, ed. by L. STErnbAch, Jhb. Oest. arch. (1902).

25

John Tzetzes, Chiliades VIII,hist. 194,.

Abstract

This essay is devoted to the study of a series of Roman gold coins, struck under Augustus, with the type of a heifer on the reverse. The authors aimed at making a corpus of the few surviving examples, which allows to identify various types, and a number of different dies. Those suggest that the emission was of importance, despite the great rarity of the coins nowadays (only 21 examples listed for all 5 varieties). The authors also tried to summarise the different hypotheses made as to the date and place of emission. The type is believed to depict the bronze sculptures by Myron, and ancient epigrams relating to it are quoted.

Zusammenfassung

Eine besondere Serie von aurei des Kaisers Augustus zeigt auf dem Revers eine Färse. Die Autoren präsentieren ein Corpus der bekannten Exemplare und der verwendeten Stempel, mit denen die unterschiedlichen Typen geprägt wurden. Die Zahl der Stempel legt den Schluss nahe, dass die Prägung ein beachtliches Ausmass hatte, obschon diese Münzen heute sehr selten sind (von den 5 Varianten sind insgesamt nur 21 Exemplare bekannt). Die unterschiedlichen Hypothesen zur Datierung und Lokalisierung dieser Emission werden ebenso diskutiert wie die These, dass auf der Rückseite die berühmte Bronzestatue des Myron dargestellt ist. In einem Anhang sind die antiken Quellen zusammengestellt, welche diese Skulptur erwähnen.

Hadrien J. Rambach

Alan S. Walker 34 Campden Hill Towers Nomos AG London W11 3QW

Zähringerstrasse 27 England Postfach 2664 coinadvisor@yahoo.co.uk CH-8022 Zürich awalker@nomosag.com

• The lead and stone hold me fast, but, otherwise, thanks to thee, sculptor Myron, I would be nibbling lotus and rushes. (G.A. 9.723, the heifer addressing the sculptor) • I think the heifer will low. Of a truth it is not Prometheus alone who moulds living creatures, but thou too, Myron. (G.A. 9.724, the poet addressing the sculptor) • The heifer, I think, will low, and if it delays it is the fault of the senseless bronze, not Myron's.

(G.A. 9.728)

in date and place.

	6	See below, and BMC p. CXXIV.
	7	See C. H. V. SuThErLAnd, The Emperor and the coinage (London 1976), p. 58.
	8	giArd (1976) suggested the name of Augustus's renowned gem-engraver Dioscorides,
		who lived with his sons in the empire's capital city.
	9	The Aegypto Capta issues have a Capricorn beneath the bust on the finer style pieces and
		lack it on the more normal examples.
	10	See B. ThurLow / I. VEcchi, Italian Cast Coinage (Dorchester 1979), nos AS3 and AS 18.
	11

  Verrine orations, IV.60.135. Die Kuh des Myron auf einem Aureus des Titus, Helvetische

	19	See corSo 1994, p. 78.
	20	The Forum Boarium was the cattle market, see MiLLEr 2009, p. 189.
	21	Sextus Propertius, The Love Elegies, II.31.
	22	See O.P. wEngEr,

18

Cicero,

  Perhaps Anacreon 582-485 bc, more likely Hellenistic • Herdsman, pasture thy herd far from here, lest taking Myron's heifer to be alive thou drive it off with the rest. (G.A. 9.715, the poet addressing the herdsman) • Myron pretended this heifer to be the work of his hands, but it was never formed in the mould, but turned into bronze owing to old age.

	(G.A. 9.716)
	Evenus 30
	• Either a complete hide of bronze clothes here a real cow, or the bronze has a soul inside it.
	(G.A. 9.717)
	• Perhaps Myron himself will say this: "I did not mould this heifer, but its image."
	(G.A. 9.718)
	Anonymous and undated
	• I am Myron's little heifer, set up on a base. Goad me, herdsman, and drive me off to the herd.
	(G.A. 9.713, the heifer addressing the herdsman)
	• Why, Myron, didst thou set me here by the altars? Wilt thou not lead me into the house?
	(G.A. 9.714, the heifer addressing the sculptor)
	• Myron was looking for his own cow among the others, and found it with difficulty by
	driving the rest away.
	(G.A. 9.725)
	• The cow, its mother, moulded this heifer giving birth to it, but the hand of Myron did
	not mould it, but gave birth to it.
	(G.A. 9.726)
	• The horned cow would have spoken, though made of bronze, if Myron had worked
	entrails inside it.
	(G.A. 9.727)
	• Let someone attach me to the solid plough and put a yoke on my neck, for as far as
	depends on thy art, Myron, I will plough.
	(G.A. 9.729, the heifer addressing the sculptor)
	• Myron placed me, the heifer, here, but the herdsmen throw stones at me thinking I have strayed.
	(G.A. 9.731, the heifer addressing a passer-by)
	• Stranger, it was Myron who moulded this cow, on which this calf fawns as if it were
	alive, taking it for its mother.
	(G.A. 9.733, the poet addressing a passer-by)

30

Either Evenus of Ascalon, or Evenus of Athens/Paros, or Evenus Grammaticus of Sicily.

02_Heifer.indd 55 13.02.13 14:42

  Thou strikest the bronze heifer. Art deceived thee much, herdsman: Myron did not add life. (G.A. 9.737, the poet addressing the herdsman) • Thou wast bronze, deceptive heifer, and the husbandman came up to thee dragging a plough and carrying a yoke. He far excels all other artists, Myron, who by his labour made thee alive, just like a labouring cow. (G.A. 9.741, the poet addressing the heifer) Demetrius of Bithynia, date uncertain • If a calf sees me, it will low; a bull will mount me, and the herdsman drive me to the herd. (G.A. 9.730) Marcus Argentarius, first half of the 1 st -century Ad • Stranger, if thou seest my herdsman, give him this message, that the sculptor Myron tied me up here. (G.A. 9.732, the heifer addressing a passer-by) Geminus, possibly first half of the 1 st -century Ad • It is the base to which it is attached that keeps back the heifer, and if freed from this it will run off to the herd. For the bronze lows. See how much alive the artist made it. If you yoke a fellow to it, perhaps it will plough. (G.A. 9.740) Philippus of Thessalonica, middle of the 1 st -century Ad • Take off from my neck, husbandman, the collar, and free me from the iron furrow-cutter; for Myron did not make my bronze into flesh, but his art gave me the aspect of being alive, so that often I even wished to low. He did not, however, let me go to work, but tied me to a base. (G.A. 9.742, the heifer addressing the herdsman) Ausonius 31 , c. Ad 310-395 • I am a heifer, wrought in bronze by the chisel of Myron my creator: nay, I think I was not wrought but born, so does the bull make for me, so does the heifer by my side low, so the calf athirst seeks my udders. Doest wonder that the herd mistakes me? The master of the herd himself oft reckons me with his grazing beast. (Aus. 68) • Why thrustest thou at the cold udders of a brazen dam, O calf, and seekest milky liquid from bronze? That also would I supply had Heaven made me within as Myron without.

	• A calf died beside thy heifer, Myron, deceived into thinking that the bronze had milk
	inside.
	(G.A. 9.735, the poet addressing the sculptor)
	• Alack! Myron, thou didst not have time to complete thy casting, but the bronze hardened
	before thou couldst put life into it.
	(G.A. 9.736, the poet addressing the sculptor) 1.1 1.2 1.3 1.6 1.7 1.8 1.1 1.2 1.3 1.6 1.7 1.8 • (Aus. 69) 1.11 (1.11) 1.12 1.11 F 1.12	1.4 1.9 1.4 1.9 2.1 2.1	1.5 1.10 1.5 1.10 3.1 3.1 3.1

31

The poems by the fourth-century Roman poet Ausonius are quoted from the Loeb edition: Ausonius, H. G. White transl., vol. II

(London 1921), epigrams 68-75, pp. 194- 199

. Another translation, with commentary, can be found in: Ausonius, Epigrams, ed. by N.M. KAy (London 2001), pp. 198-209, epigrams 63-71. 02_Heifer.indd 56 13.02.13 14:42 02_Heifer.indd 59 13.02.13 14:42 02_Heifer.indd 60 13.02.13 14:42

Based on this number of dies, we can expect the total issue to have been of about 200,000-250,000 aurei.02_Heifer.indd 41 13.02.13 14:42

02_Heifer.indd 42 13.02.13 14:42

Perhaps struck in Samos, see SuThErLAnd 1974. 02_Heifer.indd 43

13.02.13 14:42

W. SMiTh, Dictionary of Greek and Roman Antiquities (Boston

1870), p. 1131. 02_Heifer.indd 44 13.02.13 14:42

See corSo 1994, pp. 69-70. 02_Heifer.indd 45 13.02.13 14:42

02_Heifer.indd 46 13.02.13 14:42

02_Heifer.indd 48 13.02.13 14:42

02_Heifer.indd 49 13.02.13 14:42

02_Heifer.indd 50 13.02.13 14:42

02_Heifer.indd 52 13.02.13 14:42

02_Heifer.indd 54 13.02.13 14:42

02_Heifer.indd 58 13.02.13 14:42

Catalogue

 Type 1 Specimens 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 1 , vol. 131.4, Winter 2010, pp. 589-634).

28

The Greek Anthology (here "GA"), from which comes the majority of the epigrams on Myron's heifer, is quoted from the Loeb edition: W. R. Paton transl., vol. III, London 1917, book 9, epigrams 713-742 & 793-798, pp. 392-403 & 428-431).

The epigrams from the Epigrammata Bobiensia are simple translations from poems from the Greek Anthology: EB 10 is GA 9.


domain: scco info shs

text:
John M Wandeto

Birgitta Dresp-Langley
email: birgitta.dresp@unistra.fr

Self-Organizing Artificial Intelligence Captures Landscape Changes Correlated with Human Impact Data

Keywords: SatelliteImages, Landscapes, Urban Environment, Las Vegas, Self Organizing Map (SOM), Quantization Error, Demographic Data, Human Impact

Self-organization is the core principle of all learning in Adaptive Resonance Theory (ART), which has been highly successful in accounting for biological visual learning, or biologically plausible computational modelling of visual processing. Such processing includes the analysis of visual data we may not be able to see consciously, such as changes in fine visual detail in images relating to alterations in natural or urban landscapes, for example. In the course of time, such imaging data may reveal structural changes that are the consequence of measurable human impact or climate change. Capturing such change in time series of satellite images before the human eye can see them makes them available at early stages to citizens, professionals, and policymakers, promotes change awareness, and facilitates early decision making for action. Here, we use unsupervised Artificial Intelligence (AI) that exploits principles of self-organized biological visual learning for the analysis of time series of satellite images. The Quantization Error (QE) in the output of a Self-Organizing Map prototype is exploited as a computational metric of variability and change. Given the proven sensitivity of this neural network metric to the intensity and polarity of image pixel contrast, and its proven selectivity to pixel colour, it is shown to capture critical changes in urban landscapes. This is achieved here in the example of satellite images from two regions of geographic interest in Las Vegas County, Nevada, USA across the years 1984-2008. The SOM-QE analysis is combined with the statistical analysis of demographic data revealing human impacts correlated with the structural changes in the specific regions of interest. By correlating the impact of human activities with the structural evolution of urban environments we further expand SOM-QE analysis, as a parsimonious and reliable AI approach, to the rapid detection of human footprint-related environmental change.

Introduction

Grossberg's Adaptive Resonance Theory proposes neural network models that enable stable brain learning in non-stationary and unexpected worlds [START_REF] Grossberg | Conscious Mind, Resonant Brain: How Each Brain Makes a Mind 2021[END_REF]. These models rely on self-organized learning akin to biological synaptic learning [START_REF] Hebb | The Organization of Behaviour[END_REF][START_REF] Grossberg | Self-organizing neural networks for stable control of autonomous behavior in a changing world[END_REF][START_REF] Dresp-Langley | Seven Properties of Self-Organization in the Human Brain[END_REF]. In biological neural networks, such learning is by nature unsupervised and may be accounted for in terms of competitive winner-takes-all matching principles [START_REF] Carpenter | Distributed Learning, Recognition, and Prediction by ART and ARTMAP Neural Networks[END_REF]. The Self-Organizing Map (SOM) is, by comparison with the ART models, a parsimonious, one may call it minimalist, artificial neural network architecture [START_REF] Kohonen | Self-Organizing Maps[END_REF][START_REF] Kohonen | MATLAB Implementations and Applications of the Self-Organizing Map[END_REF] with inputdriven self-organization using winner-takes-all learning in sensory model neurons that become locally and globally ordered during learning. In our previous studies, we exploited the functional properties of sensitivity to spatial extent, intensity, and colour of local pixel contrasts of the Quantization Error (QE) in the output of a SOM for automatic data classification as a function of finest, mostly invisible, yet, clinically or functionally relevant, local variations in visual or other data time series. This work, extensively and well documented in our prior publications [START_REF] Wandeto | SOM-QE ANALYSIS -a biologically inspired technique to detect and track meaningful changes within image regions[END_REF][START_REF] Liu | Spatiotemporal modeling of grip forces captures proficiency in manual robot control[END_REF][START_REF] Dresp-Langley | Unsupervised classification of cell-imaging data using the quantization error in a self-organizing map[END_REF][START_REF] Dresp-Langley | Human symmetry uncertainty detected by a self-organizing neural network map[END_REF][START_REF] Dresp-Langley | Surgical task expertise detected by a self-organizing neural network map[END_REF][START_REF] Dresp-Langley | Pixel precise unsupervised detection of viral particle proliferation in cellular imaging data[END_REF][START_REF] Wandeto | The quantization error in a self-organizing map as a contrast and colour specific indicator of single-pixel change in large random patterns[END_REF][START_REF] Dresp-Langley | Vision-inspired automatic detection of water level changes in satellite images: the example of Lake Mead[END_REF][START_REF] Wandeto | Ultrafast automatic classification of SEM image sets showing CD4 cells with varying extent of HIV virion infection. 7ièmes Journées de la Fédération de Médecine Translationnelle de Strasbourg[END_REF][START_REF] Wandeto | The quantization error in a self-organizing map as a contrast and colour specific indicator of single-pixel change in large random patterns[END_REF][START_REF] Wandeto | Detection of smallest changes in complex images comparing self-organizing map to expert performance[END_REF][START_REF] Wandeto | Detection of small changes in medical and random-dot images comparing self-organizing map performance to human detection[END_REF], has permitted establishing SOM-QE [START_REF] Wandeto | SOM-QE ANALYSIS -a biologically inspired technique to detect and track meaningful changes within image regions[END_REF] as a reliable computational model of statistical variability in sensed data, directly relating to critical local changes in images of one and the same landscape [START_REF] Dresp-Langley | Vision-inspired automatic detection of water level changes in satellite images: the example of Lake Mead[END_REF], of organs [START_REF] Wandeto | Detection of small changes in medical and random-dot images comparing self-organizing map performance to human detection[END_REF], cells [START_REF] Dresp-Langley | Unsupervised classification of cell-imaging data using the quantization error in a self-organizing map[END_REF][START_REF] Dresp-Langley | Pixel precise unsupervised detection of viral particle proliferation in cellular imaging data[END_REF][START_REF] Wandeto | Ultrafast automatic classification of SEM image sets showing CD4 cells with varying extent of HIV virion infection. 7ièmes Journées de la Fédération de Médecine Translationnelle de Strasbourg[END_REF], and other simulated visual objects at different moments in time [START_REF] Dresp-Langley | Human symmetry uncertainty detected by a self-organizing neural network map[END_REF][START_REF] Wandeto | The quantization error in a self-organizing map as a contrast and colour specific indicator of single-pixel change in large random patterns[END_REF][START_REF] Wandeto | The quantization error in a self-organizing map as a contrast and colour specific indicator of single-pixel change in large random patterns[END_REF][START_REF] Wandeto | Detection of smallest changes in complex images comparing self-organizing map to expert performance[END_REF], and in temporal series of sensor responses from surgical data simulating robotic control by grip forces distributed in the human hands of experts and novices [START_REF] Liu | Spatiotemporal modeling of grip forces captures proficiency in manual robot control[END_REF][START_REF] Dresp-Langley | Surgical task expertise detected by a self-organizing neural network map[END_REF].

Any technique for the determination of the finest changes of visual or other sensed data across time, even when combined with an advanced imaging model, sooner or later requires human classification for an interpretation. This may involve guesswork when the spatiotemporal uncertainty [START_REF] Cattell | The influence of the intensity of the stimulus on the length of the reaction time[END_REF][START_REF] Exner | Ueber die zu einer Gesichtswahrnehmung noetige Zeit[END_REF][START_REF] Green | Signal detection theory and psychophysics[END_REF] in the data or visual image contents is high. It then becomes difficult to rule out subjectivity of the analyst, even when the latter is a skilled expert. To ensure quality decision making, affordable precision software for automatic data classification should combine high accuracy with further advantages relative to speed, objectivity, and reproducibility of the classification. All these have been proven ensured by SOM-QE within the scope of its application [START_REF] Wandeto | SOM-QE ANALYSIS -a biologically inspired technique to detect and track meaningful changes within image regions[END_REF]. In this work here, we show simulations exploiting SOM-QE analysis [START_REF] Wandeto | SOM-QE ANALYSIS -a biologically inspired technique to detect and track meaningful changes within image regions[END_REF] to demonstrate its statistically significant sensitivity to the spatial extent of local pixel contrast translating significant landscape changes correlated with significant increase in human impact, and its ecological footprint as a consequence [START_REF] Dou | Dynamic landscapes and the influence of human activities in the Yellow River Delta wetland region[END_REF], in response to these changes. NASA Landsat images of Las Vegas City Centre and the residential North of Las Vegas from a time period between 1984 and 2008 were submitted to SOM-QE analysis for this study. In the 1980ies Las Vegas City, located in the middle of the Nevada Desert, featured mainly 'The Strip', with a number of smaller casinos and motels.

Subsequently, in a large restructuration project between 1990 and 2007 a large number of the old casinos and motels were demolished. The ensuing reconstruction of Las Vegas City Centre and the subsequent opening of a large number of mega-resorts with casino spaces, tropical landscapes with waterfalls or simulations of urban environments such as Venice, restaurants with world-class chefs, and shows performed by international megastars like Celine Dion transformed Las Vegas City Centre and The Strip entirely. By offering multiple kinds of entertainment, dining gambling, and lodging, attracting millions of visitors from all over the world, Las Vegas City Centre has become one of the largest entertainment poles in the world [START_REF] Luke | Gaming space: casinopolitan globalism from Las Vegas to Macau[END_REF]. Most elements of the project opened in late 2009. This was accompanied by the rapid spread of greater Las Vegas, including the residential North, into the adjacent desert. The population count grew from thousands in 1984 to millions in 2009.

Materials and Methods

satellite images of the geographic regions of interest (ROI), Las Vegas City Center and

Residential North were pre-processed and then submitted to SOM-QE analysis. The original preprocessed input images can be accessed from the Supplementary Materials.

Image input

The original images were extracted from time-lapse animations of Las Vegas County, Nevada, for a reference time period from 1984 to 2008, as captured by NASA Landsat sensors [START_REF]NASA/Goddard Space Flight Center Landsat images from USGS Earth Explorer[END_REF]. VLC [START_REF]The VLC media player source code[END_REF],

an open source media player, was used to generate static images from the time-lapse animations provided. The images are colour-coded for optimal visualization [START_REF] Dresp-Langley | Colour for behavioural success[END_REF], displaying arid desert regions in brownish-gray, building structures in dark-gray and healthy vegetation and green spaces in red pixels. Water is represented by black pixels. Sample copies of two of the pre-processed 25 images for each ROI, Las Vegas City centre (Fig. 1) and residential North (Fig. 2 Before running the SOM-QE analysis on the image time series for the two geographic ROI, the images were pre-processed to ensure they are all identically scaled and aligned within a given series. This was achieved by applying the method of co-registration using StackReg [START_REF] Thévenaz | Pyramid Approach to Subpixel Registration Based on Intensity[END_REF], which is a plug-in for ImageJ, an open source image processing program designed for scientific multidimensional image processing. The last image of the time series was used to anchor the registration. Control for variations in contrast strength between images of a given series was performed after registration. This was achieved by increasing the image contrast and by removing any local variations at different times of image acquisition [START_REF] Wieland | Performance Evaluation of Machine Learning Algorithms for Urban Pattern Recognition from Multi-spectral Satellite Images[END_REF][START_REF] Schneider | From NIH Image to ImageJ: 25 years of image analysis[END_REF]. For each extracted image, contrast intensity (I) normalization was ensured using

Ifinal = (I -Imin/Imax -Imin) x255 (1)
The registered and normalized image taken in 2008 from each ROI was used to train the SOM. Since the original images used colour to emphasize different areas on the maps, pixel-based RGB values are used as input features to the SOM. This ensures a pixel-by-pixel capture of detail and avoids errors due to inaccurate feature calculation, which often occurs with complex images [START_REF] Schneider | From NIH Image to ImageJ: 25 years of image analysis[END_REF].

SOM-QE analysis

The Self-Organizing Map (the prototype for this study here is graphically represented in Fig. 3, for illustration) may be described formally as a nonlinear, ordered, smooth mapping of highdimensional input data onto the elements of a regular, low-dimensional array [START_REF] Kohonen | Self-Organizing Maps[END_REF][START_REF] Kohonen | MATLAB Implementations and Applications of the Self-Organizing Map[END_REF][START_REF] Wandeto | SOM-QE ANALYSIS -a biologically inspired technique to detect and track meaningful changes within image regions[END_REF]. It is assumed that the set of input variables can be defined as a real vector x, of n-dimensionality. A parametric real vector mi of n-dimension is associated with each element in the SOM. Vector mi is a model and the SOM is therefore an array of models. Assuming a general distance measure between x and mi denoted by d(x, mi), the map of an input vector x on the SOM array is defined as the array element mc that matches best (smallest d(x, mi)) with x. During the learning process, the input vector x is compared with all the mi in order to identify mc. The Euclidean distances ||x-mi|| define mc. Models topographically close in the map up to a certain geometric distance indicated by hci will activate each other to learn something from their common input x. Therefore, it is the presence or absence of an active response at a specific map location and not so much the exact input-output signal transformation or magnitude of the response that provides the interpretation of the input. To obtain the initial values for the map size, a trial-and-error process was implemented. Map sizes larger than 4 by 4 produced observations where some models ended up empty, which meant that these models did not attract any input by the end of the training. As a consequence, 16 models were sufficient to represent all the fine structures in the image data.

Neighborhood distance and learning rate were constant at 1.2 and 0.2 respectively. These values were obtained through the trial-and-error method after testing the quality of the first guess, which is directly determined by the value of the resulting quantization error; the lower this value, the better the first guess. It is worthwhile pointing out that the models were initialized by randomly picking vectors from the training image. This allows the SOM to work on the original data without any prior assumptions about any level of organization within the data. This, however, requires starting with a wider neighborhood function and a higher learning-rate factor than in procedures where initial values for model vectors are pre-selected. The approach is economical in terms of computation times, which constitutes one of its major advantages for rapid change versus no change detection on the basis of even larger sets of image data, prior to any further human intervention or decision making. The last image of the series from the time range here was used to train the SOM.

After training, SOM-QE analysis permits determining the QE in the map output for each of the 25 images of the series. The code used for implementing the SOM-QE is available in the "R-badged articles" series [START_REF] Wandeto | SOM-QE ANALYSIS -a biologically inspired technique to detect and track meaningful changes within image regions[END_REF] of the journal Software Impacts, a collection that presents software publications that have been verified for computational reproducibility by CodeOcean, a cloud-based computational reproducibility platform that helps the community by enabling sharing of code and data as a resource for non-commercial use. Certified papers have an attached Reproducibility Badge, a permanent Reproducibility Capsule, and are listed on the CodeOcean website [START_REF] Wandeto | SOM-QE ANALYSIS -a biologically inspired technique to detect and track meaningful changes within image regions[END_REF].

Results

The results from the SOM-QE analysis on the image time series for the two ROI are given in Here, the increase in QE captures the increasing structural variability of the landscapes, directly reflected by an increasing variability in image pixel colors, across these critical years of restructuration of Las Vegas City accompanied by the progressive building development of the Residential North. In a first analysis, these data were submitted to linear regression analysis to assess the statistical significance of the increase in QE across the years.

Linear regression and statistical trend analysis on the QE data

The linear fits to the trend in the QE to increase across the image years are shown in Figure 4. As an estimate of the part of variance in the data that is accounted for by a linear trend, or fit, the regression coefficient r 2 is a direct reflection of the goodness of that fit. The statistical significance of the trend in the data in any given direction, upward or downward, is determined by the probability that the linear adjustment sufficiently differs from zero on the basis of Student's distribution (t). The results from the comparison QE versus year of image acquisition reveal a statistically significant linear trend towards increase in QE as a function of time for both ROI.The results from the linear regression analysis with the slopes and intercepts of the fits and their regression coefficients r 2 are shown in Table 2. The results from the statistical trend analyses with Student's t, Degrees of Freedom (DF) for a given comparison and the associated probability (p), limits are given in Table 3. The regression coefficients r 2 (Table 2) reveal that the linear fit to the QE data for the images of the residential North is a reasonably good one, while the linear model poorly fits the QE data for the images of Las Vegas City. This is consistent with the type of structural change that took place in each of the ROI across the study years. There was a step-by-step reorganization of the City Center, with old casinos and hotel centers disappearing one after the other to be replaced by new ones, and a much smoother, rather progressive development of buildings the in desert regions that have become part of what is now the residential North. The linear trend statistics (Table 3) reveal a statistically highly significant increase in the QE data across the image years for both ROI.

Human impact data

For the reference time period of this study, the Las Vegas Convention and Visitors Authority [START_REF]Las Vegas Convention and Visitors Authority[END_REF], and the Las Vegas Population Review [32] have provided publically archived data that show the increase of human impact across the same years as those from which the satellite images analyzed here were taken. These data are shown in Table 4 in terms of annual population estimates in thousands for Greater Las Vegas, which includes the City and the residential North, and visitors per annum in millions. These data, shown graphically in Figure 5, were also submitted to linear regression and statistical trend analysis.

Figure 5.Linear fits to the human impact data as a function of the year in which the study images were taken.

Linear regression and statistical trend analysis on the human impact data

The results from the comparisons population estimate versus year of image acquisition and annual visitor estimate versus year reveal statistically significant linear trends towards increase as a function of time for both types of human impact data.The linear fits to the trends in these data to increase across the image years are shown in Figure 5. The results from the linear regression analysis with the slopes and intercepts of these fits and their regression coefficients r 2 are shown in Table 5. The results from the statistical trend analyses with Student's t, Degrees of Freedom (DF) for a given comparison and the associated probability (p), limits are given in Table 6. The regression coefficients r 2 (Table 5) reveal that the quality of the linear fits to the human impact data across the years is excellent. The steady increase in population and visitors of Greater Las

Vegas is consistent with the restructurations that took place across these years, creating an increasingly larger offer for state-of-the-art entertainment on the one hand, and a need for more residential development catering for the needs of people providing their workforce for this expanding industry. The linear trend statistics (Table 6) reveal a statistically highly significant increase in the visitors and population data across these critical years.

Correlation Analysis

In the next analysis, the correlations between the QE distributions from the SOM-QE analysis and the distributions reflecting human impact data in terms of population and visitors per annum statistics and were computed. Correlations are useful because they may indicate a predictive relationship between variables, which then can be further exploited in practice. To that effect we computed Pearson's correlation coefficient R, which mathematically determines statistical covariance. The probability p that the covariance of two observables is statistically significant is determined by the magnitude of the Pearson coefficient, which is directly linked to the strength of correlation, while its sign is directly linked to the direction of the covariance (positive or negative) of two variables. This analysis was performed on the paired distributions for the QE from the image analysis of the residential North as a function of the population data and for the results from the image analysis of Las Vegas City as a function of the average yearly visitor estimates. The results show statistically significant positive correlations between the paired variables in both cases. The correlation statistics, with the Pearson coefficients for a given comparison and the associated DF and probability limits, are shown in Table 7. The correlations were plotted graphically for visualization (Figure 6), showing the SOM-QE from the 25 images of the residential North as a function of the yearly population estimates

Discussion

This study is couched in the larger framework of pixel colour-based approaches to the analysis of Earth images from satellites such as Landsat to study temporal changes relating to the effects of human activities on landscape changes and/or climate change [START_REF] Orheim | Investigating Climate Change by Digital Analysis of Blue Ice Extent on Satellite Images of Antarctica[END_REF][START_REF] Furusawa | Time-series analysis of satellite imagery for detecting vegetation cover changes in Indonesia[END_REF]. Here, the SOM-QE data from analyses of satellite images of the residential North of Greater Las Vegas and the Las Vegas City

Center consistently capture the anthropogenic structural changes in each ROI across the study years. The step-by-step reorganization of the City Center, with old casinos and hotel centers disappearing and replaced by more and larger new ones, and the progressive development of housing and infrastructure in desert regions that are now part of the residential North. The significant positive correlations between the QE distributions from the SOM-QE analysis and the distributions reflecting human impact data in terms of population and visitors per annum allow for adeeper analysis of anthropogenic movements across these critical years. The steady increase in visitors of Las Vegas City reflects the direct anthropogenic response to the increasingly larger offer of state-of-the-art entertainment in the City. This engendered an increasing need for residential development in desert regions to provide housing for an increasing population providing the necessary workforce. During these same years, the water supply to these regions dwindled away progressively, as shown in a previous study applying SOM-QE to satellite images of Lake Mead, the major source of water supply to greater Las Vegas, which includes the residential North, and Las Vegas City. These observations reveal combined effects of climate change and increase in human footprint [START_REF] Camacho | Human-induced changes in landscape configuration influence individual movement routines: lessons from a versatile, highly mobile species[END_REF] on this region, bearing in mind that greater Las Vegas is one of the driest regions in the world [START_REF] Frumkin | Protecting health in dry cities: considerations for policy makers[END_REF]. The analyses provided here open a window for understanding the links between economic development and environmental impact in this particular region. When plotting the total revenues from gaming and leisure activities [37] of Las Vegas City Centre ('The Strip') and the water level statistics for Lake Mead provided by the Hoover Dam Control Room [38] across the years of the reference period (for illustration only here), we see a negative correlation (Pearson's R

(1,24) = -0,82; p<.00) between economic development and resource availability (Figure 7). The results from this study highlight some novel aspects of the full potential of input-driven selforganization in a parsimoniously designed neural network model (AI). SOM-QE provides a highly reliable output metric that scales, in a few minutes and with a to-the-single-pixel precision, local variability in time series of images containing millions of pixels each. Although it is self-organizing, learning in the SOM is explainable and so are its prototype design architecture and the neural workspace. The input data are not biased, and clearly defined physically. The output metric, the QE, initially used as a quality metric and for network quantization [START_REF] Kohonen | Self-Organizing Maps[END_REF][START_REF] Kohonen | MATLAB Implementations and Applications of the Self-Organizing Map[END_REF], is proven a powerful and consistent detector of invisible local changes. When the input to a SOM is constant across time, the QE metric is invariant [START_REF] Kohonen | Self-Organizing Maps[END_REF][START_REF] Kohonen | MATLAB Implementations and Applications of the Self-Organizing Map[END_REF]. When variability in a locally defined dimension of the input data is systematic across time, in a given direction (increase or decrease), the QE will systematically and reliably increase or decrease, as shown in the bulk of our previous work and, once again, here in this

  ) from the years 1984 and 2008 are shown here below.

Figure 1 .

 1 Figure 1. Representation of two of the 25 satellite image extracts of Las Vegas city centre in 1984 (left) and 2008 (right), after preprocessing.

Figure 2 .

 2 Figure 2. Representation of two of the 25 satellite image extracts of Las Vegas residential North in 1984 (left) and 2008 (right), after preprocessing.

Figure 3 .

 3 Figure 3. Graphic representation of the 4x4 SOM prototype with 16 models, indicated by the filled circles in the grey box. Each of these models is compared to the SOM input in an unsupervised winner-take-all learning process. The input vector corresponds to the RGB image pixel space.

Figure 4 .

 4 Figure 4. Linear fits to the QE data as a function of the year in which a study image for a given ROI was taken.

(Fig. 6 ,

 6 top) and the SOM-QE from the 25 images of Las Vegas City as a function of the annual number of visitors across the years of the reference period (Fig.6, bottom).

Figure 6 .

 6 Figure 6. Correlation between the QE values from the image analyses and the pertinent human impact estimate for a given geographic ROI.

Figure 7 .

 7 Figure 7. Revenues from gaming and leisure activities (top) and water level statistics for Lake Mead across the years of the reference study.

Table 1

 1

as a function of the image the year was taken and the type of ROI. They show a general trend towards increase in the QE metric across images for each ROI between 1984 and 2008. The QE in the SOM output is a reliable indicator of variability in pixel color (or contrast intensity; here in this study all images were normalized for contrast intensity and did not display variability across images) as shown in our previous work.

Table 1 .

 1 SOM-QE output as a function of the image year and geographic ROI.

	Year Las Vegas City Residential North
	1984 0,240437503	0,151226618
	1985 0,264341069	0,157865360
	1986 0,271480118	0,155998180
	1987 0,289065099	0,169213765
	1988 0,282803632	0,210600120
	1989 0,286535270	0,213982186
	1991 0,303956828	0,219973707
	1991 0,298541690	0,225406972
	1992 0,301994751	0,214975264
	1993 0,293683986	0,208605453
	1994 0,304328745	0,221313177
	1995 0,298240329	0,212630331
	1996 0,309779114	0,222464495
	1997 0,284870821	0,225816809
	1998 0,291493024	0,223026329
	1999 0,296067339	0,238731572
	2000 0,304491317	0,246826836
	2001 0,311488540	0,254509105

Table 2 .

 2 Fit parameters from the linear regression analysis of the QE data as a function of the

	geographical ROI.
	Linear Fit Parameter	Las Vegas City Residential North
	Slope (b1)	1,554	4,0331
	Intercept (b0)	-2,8077	-7,8286
	r 2	0,4776	0,7995

Table 3 .

 3 Trend statistics as a function of the geographical ROI.

	Trend Parameter Las Vegas City Residential North
	t	88,98	33,45
	DF	(1, 24)	(1, 24)
	p	<.001	<.001

Table 4 .

 4 Human impact data across the years in terms of visitor (in millions) and population (in thousands) estimates per annum.

	Year Visitors (in millions) Population (in K)
	1984 12,8000	191,0000
	1985 14,2000	197,0000
	1986 15,2000	204,0000
	1987 16,2000	226,0000
	1988 17,2000	240,0000
	1989 18,1000	266,0000
	1991 20,9000	276,0000
	1991 21,3000	298,0000
	1992 21,9000	310,0000
	1993 23,5000	330,0000
	1994 28,2000	352,0000
	1995 29,0000	374,0000
	1996 29,6000	406,0000
	1997 30,4000	423,0000
	1998 30,6000	448,0000
	1999 33,8000	466,0000
	2000 35,8000	483,0000
	2001 35,0000	506,0000

Table 5 .

 5 Fit parameters from the linear regression analysis of the human impact data in terms of visitors in millions and population (residents) in thousands per annum.

	Linear Fit Parameter	Visitors Population
	Slope (b1)	1,1828	19,0723
	Intercept (b0)	-2,333	-3766
	r 2	0,9657	0,9955

Table 6 .

 6 Trend statistics as a function of the type of human impact data

	Trend Parameter Visitors Population
	t	15,70	14,20
	DF	(1, 24)	(1, 24)
	p	<.001	<.001

Table 7 .

 7 Pearson correlation statistics as a function of the type of comparison

	Correlation Parameter QE North vs Population QE City vs Visitors
	Pearson's R	0,87	0,71
	DF	(1, 24)	(1, 24)
	p	<.001	<.001

Funding: This research received no external funding.

Acknowledgments:

The authors gratefully acknowledge support from their respective host institutions.

study. Easily implemented, fast, and computationally economic, SOM-QE satisfies all the current criteria for trustworthy and sustainable AI [START_REF] Vetter | Lessons Learned from Assessing Trustworthy AI in Practice[END_REF][START_REF]AI HLEG -High-Level Expert Group on Artificial Intelligence. Assessment List for Trustworthy Artificial Intelligence (ALTAI) for self-assessment[END_REF], within a modest but diverse range of applications.

Supplementary Materials:

The following are available online at www.mdpi.com/link: Folder S1, 50 original pre-processed images from the time series of 25 images for each the two ROI.

Conflicts of Interest:

The authors declare no conflict of interest.


Camille Kropp

Katy Le Corf

Karima Relizani

Kevin Tambosco

Ccori Martinez

Florian Chain

Georges Rawadi

Philippe Langella

Claus Sandrine

Rebeca Martin

The keystone bacterium Christensenella minuta DSM 22607 improves colitis in in vivo preclinical IBD models

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

The keystone bacterium Christensenella minuta DSM 22607 improves colitis in in vivo preclinical IBD models

Camille KROPP * (1,2) , Katy LE CORF (2) , Karima RELIZANI (2) , Kevin TAMBOSCO (1) , Ccori MARTINEZ (2) , Florian CHAIN (1) , Georges RAWADI (2) , Philippe LANGELLA (1) , Sandrine CLAUS (2) and Rebeca MARTIN (1)

1 Micalis Institute, AgroParisTech, INRAE, Université Paris-Saclay, 78350 Jouy-en-Josas, France 2 Ysopia Bioscience, 33000 Bordeaux, France

There is increasing evidence that microbiome-based therapies can correct dysbiosis and reduce inflammation associated with Inflammatory Bowel Diseases (IBD). In particular, the human commensal family of Christensenellaceae bacteria has been reported as missing in several cohorts of Crohn's disease patients, indicating that it may have a role in maintaining microbial symbiosis in this population. To assess its potential for IBD management, we used the reference type species Christensenella minuta DSM 22607 to examine its anti-inflammatory properties.

C. minuta displays strong immunomodulatory properties in vitro and in vivo.

Our findings open the door to intriguing new research questions. Although additional research is needed to better understand the bacterium's effects and their underlying mechanisms, our work underscores for the first time that C. minuta holds promise for treating IBDs and merits further study with a view to developing single-strain biotherapies.

These results have been published in Scientific Reports (DOI: 10.1038/s41598-021-90885-1).

BACKGROUND METHODS

SCFAs determination using Gas chromatography

C. minuta protects against DNBS and TNBS-induced colitis in mice and rats

C. minuta is a high acetate and moderate butyrate producer

C. minuta displayed anti-inflammatory properties by decreasing the secretion of IL-8 through NF-kB inhibition

  preclinical colitis models: a moderate DNBS-induced colitis model in mice and a severe TNBS-induced colitis model in rats.Understand C. minuta mechanism of action kropp@inrae.fr / Camille.kropp@ysopia.bioImmunomodulation in HT-29 cellsIn vivoIn vitro


Birgitta Dresp-Langley

Keywords: dopamine, brain, addiction, stress, anhedonia

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The neurotransmitter dopamine fulfills a critical function in regulating the responses of the mesolimbic system, also known as the reward system, in the neural circuits of the mammalian brain . The reward system governs and regulates responses ranging from pleasure and cravings to disgust and anhedonia, triggered by chemicals and other stimuli and guiding a larger proportion of our behavior than we may be aware of or are ready to admit . In ancient times, the function of the reward system made the difference between life and death, because it made us and other species deploy most of our or their attention and behavioral effort towards things that were important for the survival of the species, such as food, sleep, and sex. Our ancestors did not have a supermarket around the corner but had to hunt for food and often had to deploy a considerable amount of time and energy to find it. An individual who found sweet fruit in the environment, for example, had better consume it quickly, in as large of quantities as possible, before another did. Ripe fruits have the highest sugar content and provide higher amounts of instant energy compared with other foods. The human preference for sweet nutrients may, indeed, be due to the evolutionary advantage that craving and eating high-calorie foods has caused. Responding selectively to survival-relevant stimuli, in the course of evolution, has become hardwired into the brain's reward system . Food, sleep, physical contact, and sex are primary stimuli that reinforce the neural connections of the reward system, and a craving for these primary stimuli is inherent in humans , as well as most other mammals .

The reward system is composed of brain structures that mediate the physiological and cognitive aspects of a reward. This involves neurobiological processes driving the brain's capapcity to associate stimuli such as substances or activities with a pleasant and positive outcome. Reward-associated mechanisms explain why individuals search for initially positive stimuli again and again and why this can ultimately lead to compulsive behaviors and addicions that pose a threat to mental health. Pandemic-related adversities and the stresses they engendered , with the long lockdown periods where people in social isolation had to rely on food for comfort or digital tools to get feedback rewards via the internet, can be seen as major triggers of changes in motivation and compulsive reward-seeking behaviors worldwide. These are linked here to the pathological adaptation of dopamine-mediated reward circuitry, triggered and consolidated through functional links with brain structures controlling the circadian rythm and/or the responses to chronic stress. This offersplausible insight into why environmental adversity pushed individuals of all nations, during and after the pandemic, into compulsive behaviors that couldlead to sleep disorders, anhedonia (from the Greek language: "inability to feel or experience pleasure"), depression, and in extreme cases, suicidal ideation.

COVID-19 Pandemic and Mental Health

In 2022, the World Health Organisation (WHO) issued a brief with facts and figures showing that the COVID-19 pandemic has had a severe impact on the mental health and well-being of people around the world. While many individuals have adapted, others have experienced mental health problems that, only in some cases, are a consequence of COVID-19 infection. The brief stated further that the pandemic also continues to impede access to mental health services and raised concerns about increases in suicidal behavior, stressing the causal role of psychological and environmental factors producing ontological insecurity in individuals and populations. In the wake of the COVID-19 pandemic, experts havewitnessed a significant rise in addictions and related mental illnesses;

[1] [2] [3] [4] [5] [6] [7]
➢ ➢ significant rise in the corresponding anti-depressant prescription uptake; increased risk of suicidal ideation or suicide.

Any potential doubts about the impact of COVID-19 on mental health have been largely put to rest by the most recent papers on the topic . Loneliness, physical exhaustion, sleep disturbances, and anhedonia linked to compulsive or addictive behaviors such as binge-eating , substance abuse , and digital reward craving were identified asbeing linked to suicidal ideation -in particular, in younger individuals-during and after the pandemic. These reflect some of the most negative effects of pandemic-related adversity on mental health worldwide.

Adversity and Vulnerability

Mental health issues arise from a specific context. Recently reported effects of the pandemic have shown, beyond all reasonable doubt, that adversity affects people's capacity tocope with the stresses it produces. Conditions of extreme adversity challenge the stress and immune system responses needed for coping, and beyond a certain threshold, such conditions can trigger behavioral changes as listed and discussed here above; all of them have been functionally linked to the complex brain pathways involving dopamine release, reward mechanisms, and their interactions or regulations .

During the different waves of the pandemic, many individuals had an actual COVID-19 infection, with direct effects on the brain's immune system, stress centers, and the dopamine system, affecting people's mood beyond the actual duration of the illness. Cohort studies examining the psychopathological and cognitive status of COVID-19 pneumonia survivors , for example, have shown that three months after discharge from the hospital, close to 40% still self-rated their symptoms in the clinical range for at least one psychopathological dimension, with persistent depressive symptomatology, while anxiety and insomnia progressively decreased during follow-up. Studies on the impact of the COVID-19 pandemic on individuals with eating disorders have been performed all over the world, showing that the traumatic effects of COVID-19

have exacerbated specific eating disorder-related psychopathologies , pointing towards complex interactions between environmental and personal factors. Healthy siblings of patients with eating disorders have beenfound to present specific psychological vulnerabilities, i.e., specific associations between interpersonal sensitivity and posttraumatic symptoms, including anxiety, depression, and compulsive behaviors . Moreover, having to cope with adversity during childhood increases the risk of mental health problems in adulthood, and it has been suggested that this effect may be mediated by increased striatal dopamine neurotransmission . Also, while resilience may be viewed as an individual's positive adaptation to experiences of adversity , it is also known that conditions of adversity reinforce the already exisiting vulnerability to mental illness and may create a new vulnerabilty duringspecific adverse contexts such as a pandemic or armed conflict . It has been confirmed that stress responses can cause functional changes in the amygdala and the dopaminergic circuits of the brain in relation to stress-induced cortisol release mechanisms . Some of these studies have shown a cortisol-induced increase in serotonin in subjects with major depressive disorder, offering further insight into the functional links between stress, sleep disorders, and depression, where an increase in one brain correlate affects another, thereby creating or increasing the physiological vulnerability to mental illness in individuals under prolonged stress.

Bingeing

The COVID-19 pandemic creatednegative health consequences such as consuming high-fat and sugar foods, increasing body weight , anxiety and depression , anhedonia , or a combination thereof . As a core characteristic of eating disorders and all forms of addiction , bingeing is associated with changes in the activity of the brain's reward system in the process of pathological adaptation of the central nervous system through deregulation of the reward mechanisms generating positive reinforcement. As explained earlier, this deregulation progressively leads to the consolidation of the anti-reward brain state that produces anxiety, anhedonia, and depression. Most people during COVID-19 lockdown reportedly turned to substances or other "rewarding" activities to deal with the stress of isolation and the negative feelings it engendered . A number of factors related to the COVID-19 pandemic may have contributed to the reportedly high incidence of food addictions during and after that period . It has been suggested that people under the influence of the excessive and uncontrolled consumption of extremely palatable foods experience similar highreward sensations as recorded for addicts resorting to classic intoxicants, both at the behavioral and neurobiological levels . As lives and daily routines were transformed by the pandemic and rapidly rising anxiety fueled by social isolation and, often, unemployment, the mental health of millions is at stake. In the United States alone , close to 60% of adults screened declared that the pandemic led them to abuse alcohol and other drugs, binge eating, and/or compulsive internet gambling.

The New Digital Drug

COVID-19 spread across the world ata rapid pace, and to further limit the spread of infection, lockdowns were declared in most parts of the world. People were forced to stay indoors, and the internet was the only source of entertainment. addiction to the internet have been identified as part of the consequences of the COVID-19 pandemic, with a major impact on the mental health of predominantly young people and teenagers . This involves, like all compulsive behavior loops, the brain's reward circuitry, with all the complex interactions between environmental, metabolic, and neurobiological changes in the brain discussed above. Dopamine, and the modulation thereof under conditions of adversity and stress (Figure 1), is the common functional denominator .

Figure 1 .

 1 Figure 1. Behavioral consequences of an anti-reward brain state, with ultimately depleted dopamine levels in the reward centersand increased cortisol levels in the centers that regulate the brain's response in pathological adaptation to chronic stress. Under adverse environmental conditions, such as those generated by the COVID-19 pandemic, healthy rewardseeking has progressively evolved into stress-related compulsory behaviors and/or addictions leading to anhedonia and related mental health issues worldwide.

  This context engendered addictive behavior with measurable negative effects on anxiety and sleep quality, especially among younger individuals . Digital addiction involves theinternet as a channel through which individuals may access whatever content they want (games, social media, shopping, etc.) wherever and whenever they want. The development of the addictive response is digitally facilitated by such instant availability . At an advanced stage, digital addiction is associated with significant and permanent symptomatic psychological, cognitive, and physiological states,

	with a measurable dopamine deficiency and impaired mental health	. Psychological stress	, anxiety and
	depression	, eating disorders	, sleeplessness	, and mood changes, along with suicidal ideation
	, are the most frequently reported. A compilation of cross-national studies on more than 89,000 participants from 31
	nations performed almost ten years ago, well before the COVID-19 pandemic, already suggested a global prevalence
	estimate for digital addiction of 6% worldwide	. Interdependent variables such as sociocultural factors, biological
	vulnerabilities (genetic predisposition and preexisting metabolic disorders), and psychological factors (personality
	characteristics and a negative affect) play a critical role	. Excessive seeking of the new digital drug and