Journal of Dermatology and Pigmentation Research

Study of the Red Colour of Ste Marie-Madeleine’s (3-63) Hair by Scanning Electron Microscopic Characterization of its Melanosomes

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Published Date: August 28, 2017.

Study of the Red Colour of Ste Marie-Madeleine’s (≈3-63) Hair by Scanning Electron Microscopic Characterization of its Melanosomes

Gerard Lucotte1* and Thierry Thomasset2

 1Institute of Molecular Anthropology, Paris, France

2Laboratory of Physico-Chemical Analysis, UST of Compiegne, France

*Corresponding author: Gerard Lucotte, Institute of Molecular Anthropology, Paris, France, E-mail: lucotte@hotmail.com.

Citation: Lucotte G, Thomasset T (2017) Study of the Red Colour of Ste Marie-Madeleine’s (≈3-63) Hair by Scanning Electron Microscopic Characterization of its Melanosomes. J Derma Pigm Res 1(1): 108.

Abstract

 

We investigated the red colour of Ste Marie-Madeleine’s (≈3-63) hair by Scanning Electron Microscopic (SEM) and EDX analysis. The hairs under study, that appear red to the naked eye, were obtained from a lock kept at the St Maximin basilica. The hairs are dried out and their scales were abraded; so, residual melanosomes appear as concentrated under the crests of the hair cortex. We can distinguish by SEM the two types of melanosomes: melanosomes of type 1 (the eumelanin melanosomes), of little size (about 1–2 µm), are grains of rice shaped; melanosomes of type 2 (the phaeomelanin melanosomes) are bigger (> 2–3 µm) and generally round in form. By counting the two sorts of melanosomes, we obtain a total proportion of approximately 35% of melanosomes of type 2 per unit of surface. This calculated proportion of type 2 melanosomes corresponds to that of the red-brown hairs, as observed in optical microscopy.

Keywords: Ste Marie-Madeleine’s hair; Red-Brown colour; Types 1 and 2 melanosomes; Melanosomes counting; SEM Studies

Introduction

 

Ste Marie-Madeleine (≈3-63) is the most abundantly cited (at least twelve citations, without taking into account some repeats) woman of the four Gospels. According to the French “tradition des Saints de Provence”, she landed to the French (the Gaule at this era) Mediterranean shores, in a region corresponding to the current part of Les Saintes-Maries-de-la-Mer. She (and her companions) attained further the towns of Marseilles and Aix-en-Provence. Thereafter, she withdrew for thirty years to the cave of La-Sainte-Baume, where she died (in 63); she was buried in the currently named village of Saint-Maximin-la-Sainte-Baume.

Some relics (bones and hairs) of Marie-Madeleine were kept in the Saint-Maximin basilica, where a large lock (red in colour) of Marie-Madeleine’s hair is arranged in a dedicated reliquary.

We have obtained some hairs from this lock, for scientific purposes (microscopic examination and chemical analysis). Genetic studies, by DNA extraction of one of them that had a capillary bulb, showed that it corresponds to the chromosomal XX formula of a woman [1]. Here in, detailed studies in electron microscopy are presented to explain the observed red-colour of these hairs.

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Experimental

 

Several hairs (that are all hair fragments) were extracted from the lock by the basilica priest, and were loaded further on sterile scotch tapes. They were all studied in optic and electronic microscopy. The longest of them (about 7 cm of length) is hair number 2; because it is representative of all the other hairs examined, we choose to concentrate on this one the studies concerning the observed (visible to the naked eye) red colour.

Optical microscopy of the hairs was realized using confocal stereoscopic micrography.

The SEM (Scanning Electron Microscopy) apparatus used is a FEI model Quanta FEG. Elemental analysis was achieved by using EDX (Energy Dispersive X-ray spectroscopy), this SEM microscope being equipped with the probe model X-flash 6/30; both LFD (Large Field Detector) and CBS (Circular Back Scattering) were used, the last one to detect heavy elements.

Each elemental analysis is given in the form of a spectrum, with kiloelectrons / Volts (ke/V) on the abscissa and elemental peaks heights in ordinates. Estimating peaks heights, it is possible to obtain some semi-quantitative results.

Results

 

Figure 1 shows optical microscopy photographs of two hair portions of hairs numbers 1 and 2. Their colours are red, as observed to the naked eye for the lock (a fine yellow binding deposit of silver runs longitudinally along each of them). Their thicknesses are about 62 µm and 81 µm, respectively.

Figure 1: Optical images of two hair portions (1: hair number 1; 2: hair number 2) ; magnification is about 10 × for 1, and 25 × for 2 (S indicate the yellow binding of silver).

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Very useful information in the field of forensic medicine concerning hairs was obtained with the help of optical microscopy [2]: particularly the scales (the basic scale structure, their forms, their overlapping, their imbrications…), that are diagnostic characters. In the present study we have studied the scale pattern of Marie-Madeleine’s hairs using electron microscopy. The SEM photograph of figure 2 shows the aspect of a typical hair portion of the hair number 2. The corresponding scale pattern is greatly abraded: there are generally no well-visible scales other than their points; transversal scale rows are very rarely observed; when they exist, scales are completely flattened on the subjacent cortex (that indicates that the cuticle had completely disappeared).

Figure 2: SEM photograph and EDX analysis of a hair portion of hair number 2. Above: SEM photograph (in LFD), 1000 × C1 and C2, and C3 and C4 indicate the locations of the crests of cortex visible at the periphery of the hair. Arrow points show residual scale points in successive rows (spaced of about 5 μm, which is a characteristic of the human hair), located on C3 and C4. The black dot indicates the point where EDX analysis is realized. Below: spectrum at the black dot; C: carbon; O: oxygen ; S (two peaks): sulphur; K: potassium; Ca (two peaks): calcium.

 

It is useful to compare the scale pattern observed to that of the wear gradation of hairs recommended by the L’Oreal firm [3]. Table 1 gives the successive steps of wearing adopted; Marie-Madeleine hair scale pattern corresponds mostly to grades 3 to 5; this denotes much worn out hairs. As expected, the current appearance of Marie-Madeleine’s hair reflects their decay over a period of potentially two-thousand years.

Table 1: Successive grades of the hair wear (from L’Oreal information).

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Another distinctive feature of Marie-Madeleine’s hairs is their dryness: the hair diameter is narrowed, and its topologic initial surface limited to the levels of the tops of the longitudinal crests of the cortex.

The corresponding spectrum is typical to that of one hair [4]: there is an elevated peak of carbon, accompanied by a much lower peak of oxygen (both constituting the organic part (of the hair). The relatively important peak of sulphur [5] corresponds mainly to the keratin (containing amino-acids cystine, cysteine and methionine, and disulfide bridges). There are little peaks (traces) of calcium and of potassium, that denote some mineral deposits at the hair surface.

Figure 3 photograph shows, at highest magnification, two separated examples of the two types of melanosomes, located in an area of the hair number 2 lacking of scales (consequently, they are observed directly on the cortex surface). There are two adjacent melanosomes (numbered 1) of the first sort, which are elongated in form (grains of rice shaped), with lengths of 2.4 and 1.9 µm. There is also one example (numbered 2) of a melanosome of the second sort; this melanosome is bigger (3.2 µm of maximal length) than the previous, and of a round form.

Figure 3: SEM photograph and EDX analyses of melanosomes. Above: SEM photograph (in CBS), 5000 × ; 1 (1’, 1’’ and 1’’’ are other examples of melanosomes of type 1) and 2 (2’ is another example of melanosome of type 2) indicate, respectively, the two eumelanin and the phaeomelanin melanosomes (with their dimensions, in μm); black points indicate the target where EDX analysis are realized. Above: spectras at points 1 and 2. Al: aluminium; Si: silicium.

 

There are two sorts of melanosomes in human hairs (Table 2): the melanosomes of type 1 are little grains, “grains of rice shaped”, containing the eumelanin pigment; they are black in colour. The melanosomes of type 2 are bigger, of round or more irregular forms, containing the pheomelanin pigment; these melanosomes are yellow to red in colour [6,7].

Table 2: The two different sorts of melanosomes (From L’Oreal information).

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There are three sulphur atoms in the pheomelanin formulae and no sulphur at all in that of the eumelanin [8]. We originally hoped to distinguish between the two sorts of melanosomes by EDX analysis; in fact, in the corresponding spectra, the sulphur peaks are only lightly more elevated (compared to those of the oxygen) for pheomelanin melanosomes in comparison to eumelanin melanosomes. That is due to the fact that the EDX probe surface exploring is about 1µm2: in this sort of analysis the eumelanin melanosome is too small to give an adequate EDX measurement, because it overflows on the sulphur of the cortex background (in the examples taken silicium/aluminium and calcium contents are differential between these two sorts of melanosomes, but that is probably due to local differences in mineral deposits). So, it is impossible to distinguish between the two sorts of melanosomes otherwise by examining their sizes and forms.

The above SEM photograph of figure 4, in LFD and at one adequate magnification (1200×), shows details and measurements of some melanosomes located on the first (C1) and the second (C2) longitudinal crests in a portion of the hair number 2. Both types of melanosomes were scattered across the analyzed region: two melanosomes (round and of 2.8 µm) of type 2, located below, on crests C1 and C2; the five melanosomes (“grains of rice shaped” and of 1.1 to 2.1 µm) of type 1, located on crests C1 and C2.

Figure 4: SEM photographs concerning hair portions of the hairs numbers 2 and 8. Above: SEM photograph (in LFD), 1200 ×, of an intermediate segment of hair number 2; C1-C4: the four longitudinal crests. Above: SEM photograph (in LFD), 800 ×, of one extremity of hair number 8 (SD: skin debris); 1: melanosomes of type 1, 2: melanosomes of type 2.

 

Melanosome counting necessitates finding some areas of hairs lacking scales – similar to those found in one hair located on the Face of the Turin Shroud [9] sufficiently intact to contain melanosomes in relatively great numbers and conserved forms. Such an area (at one extremity of the hair number 8) is shown on the below photograph of figure 4: in this area, a total number of 64 melanosomes can be identified; 44 of them are of type 1, and 20 of type 2 (about one melanosome of type 2 per two melanosomes of type 1).

Table 3 gives, for a total number of 488 melanosomes observed in five analyzed hairs (numbers 8–12) areas, the partial counting of melanosomes of the two types; these are numbers of 314 of melanosomes of type 1 for 174 melanosomes of type 2. The global proportion of melanosomes of type 2 on the total is 35.6%.   

Table 3: Counting of melanosomes of the two types in five different hair areas.

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Discussion

 

We found here, by counting melanosomes of the two types at the cortex surface of some peculiar areas of Marie-Madeleine’s hair where the melanosomal pattern is relatively well conserved, that the proportion of melanosomes with pheomelanin (melanosomes of type 2) is about one-third of the total count of the melanosome number.

Hair colour depending of melanosomes (that are located on the cortex surface, scales and cuticle being transparent) is function of: (i) the melanosome sizes (we have seen that melanosomes of type 2 are bigger than those of type 1); (ii) the melanosome numbers, i.e. number of melanosomes per unit of the cortex surface; (iii) the melanosome density, i.e. (related to ii) of the sacing of melanosomes per unit of surface.

Typically [3], dark hairs contain a great amount of eumelanine melanosomes (melanosomes of type 1) and red hairs contain a relatively great amount of pheomelanin melanosomes (melanosomes of type 2). The observed melanosome pattern of Marie-Madeleine’s hairs corresponds to a proportion of one melanosome of type 2 on two melanosomes of type 1; consequently, the melanosome-dependent colouring of these hairs would be predicted to be of dark red colour. It is effectively the hair colour that we observe in optical microscopy (see photographs of figure 1), and also the colour of these hairs seen to the naked eyes.

Reddish brown (or more exactly said red-brown) is also the hair colour of the first-known Marie-Madeleine portrait (Figure 5), which is dated from around 1275. There is no doubt at all that this portrait is that of Marie-Madeleine: she was painted in an iconic fashion (hands and feet positions, general form of the face) ; her naked body is entirely covered (in a manner that looks-like to that of “Marie the Egyptian”) with her very long red-brown hairs ; she holds in her left hand a phylactery on which is written (in latin) “don’t desperate , you whom have the habit to sin : with your example, help up our self in Good”. The perimeter of this Marie-Madeleine representation is entirely covered to charismatic scenes of her own life, according to the Provencal tradition.

Figure 5: Master of Marie-Madeleine: Ste Marie-Madeleine, with eight scenes of her passion. Painting on wood (164 × 76 cm); gallery of the Academy, Florence (Italy). From left to right, and up to down: the meal at home of Simon; the Lazare’s resurrection: the meeting with Jésus resurrected (Noli me tangere); the prediction to Marseillans; the delightness; an angel’ visit bearing eucharisty; the last communion; Marie-Madeleine’s death (Marie-Madeleine death corpus being surrounded by the St Lazare and St Maximin Bishops).

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The presumed dating (of 1275, which is indicative) of this first Marie-Madeleine painting is interesting to consider because it corresponds approximately to that of the date given by Bernard Gui [10], who was an auricular witness of the facts (Chronics of Popes and Emperors in 1320): “In the year of grace of Jesus-Christ in 1279, the nineteen day of December, the Prince Charles, son of Charles King of Sicily, Count of Provence and then King of Sicily, had looked for the body of Ste Marie-Madeleine with so much solicitude and as much as devotion in this holy oratory in which St Maximin, one of the seventy disciple of the Seigneur Jésus-Christ, venerated in the (diocese of) Aix formerly, had given (to her) one burial place…”

Since the time of the discovery (on 19 of December 1279) of the Marie-Madeleine remains, several pieces were found: almost all of the bones (but the mandible) and an intact leg; the conserved tongue was stuck to the throat; a little piece of flesh adhered to the frontal bone (Noli me tangere). Concerning the hair, her “hairs surrounded the cranium” (they were found in that state when the whole skeleton was discovered).

All the Marie-Madeleine’s remains were conserved, in a first time (on 25 of May 1281), in a splendid reliquary of silver enhanced with gold ornaments; it was made by a famous artist of the time. Marie-Madeleine’s head was then brought to Aix, and placed in a chapel of the Counts of Provence palace. Charles of Provence had proposed to keep the head in a reliquary of gold, some more magnificent than the silver of the previous one. His father, Charles the First of Anjou, had sent from Italy to his son his own royal gold crown, with the order to put it on the head of Marie-Madeleine (that he considered to be the Holy Patroness and the protective of all of his States). This magnificent reliquary (Supplementary Figure 1-1) was finally ready only in 1283, and Marie-Madeleine’s head translation took place (on the 10th of December 1283) in Aix. This gold reliquary will be placed (in 1295) in the Saint-Maximin oratory. In 1511 the spouse of Charles VIII, Anne de Bretagne, remarried with the King Louis XII, offers a new reliquary-bust destined to accommodate Marie-Madeleine’s head. This reliquary disappeared during the french revolution in 1793.

It is important to note that, in these two last reliquaries (as well as in the most modern reliquary – named as the Revoil reliquary – showed today in the Saint-Maximin basilica), Marie-Madeleine’s hairs are only represented: it is not Marie-Madeleine’s hair, but its pattern only. This pattern reminds that: (i) Marie-Madeleine had very long hair; (ii) Marie-Madeleine’s hair was directly put on the cranium, as in their initial positions since that time of the 1279 exhumation.

We know, as certified by inventories, that the Marie-Madeleine’s hairs were shown, kept in other distinctive reliquaries: in the 1578 inventory (S. Razzi), they were conserved “in a flask of crystal”; in the 1780 inventory (the new inventory of the relics and the treasure of Saint-Maximin, on 15 of February 1780), they were kept “in a vase of glass”.

In fact, since the beginning, Marie-Madeleine’s hairs were separated from the cranium and then conserved (Supplementary Figure 1-2) “in a glass recipient.” [11]. In 1793, General Barras presented himself to Saint-Maximin basilica in the goal to recuperate reliquaries. Relics, extracted from their precious reliquaries, were thrown mixed; some of them (including the head, the Noli me tangere and some osseous of the arm), and especially “some part of the hairs”, had be recuperated by the sacristan Joseph Bastide who, with the aim of some parishioner believers, keeps them at home. In 1803 the archbishop of Aix named Antoine Rostand as the delegate commissioner for the elaboration of a verbal-trial concerning the authenticity of the relic’s desacrated in 1793; this share of relics (comprising in particular the head and the Noli me tangere) included in “a flask, 5.4 cm of height, containing the hairs of Marie-Madeleine”.

The hairs under study originate from a lock of hairs [1] contained in a brass-made reliquary of ostensoir form (Figure 6). In accordance to the Saint-Maximin basilica priest, this reliquary was manufactured after the time of the french revolution. The glass situated on one face of this reliquary permit us to well observe the lock of hairs and their colour (Figure 7).

Figure 6: The hair reliquary of ostensoir form (O: a little bone piece).

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Figure 7: Hairs (h) under the glass, surrounding the bone (O).

 

Supplementary Figure 2 shows two glass splinters (cracked in form) that were pulled from the glass of this reliquary. The spectrum of the first of them shows an elevated peak of silicium, and a lower peak of calcium. There are little peaks of sodium, magnesium, aluminium, sulphur, chlorine and potassium. This sample is contaminated by the brass (copper and zinc) of the reliquary, and probably also by organic mineral (some part of the carbon and the oxygen peaks). This spectrum, with relatively high silice (SiO2) and lime (CaCO3) contents, corresponds to that of a modern glass [12] of the type [13] of low alkali glass (with relatively low concentrations of K2O and Na2O).

This sort of glass is solid, very difficult to peel off; we have not found any pieces of this sort of glass on the surface of the various hairs we studied. In the hope to find some other sorts of glass – that could be eventual witness to ancient transparent reliquary material – we have explored systematically to this goal the surface of the Marie-Madeleine’s hairs (20 hair fragments) available to us; only one look-like glass particle was found (Supplementary Figure 3), located on the surface of the hair number 2: it is elongated in form (25 µm long on 5 µm wide), with acute borders. The corresponding spectrum shows an elevated silicium peak, and a very little peak of calcium. The aluminium peak is the second of importance, among the mineral elements; there is a relatively important peak of sodium. In fact this particle looks-like more to one feldspar [14], of the sodic feldspar category (of the albite type: plagioclase).

The story of this lock of hair of Marie-Madeleine is somehow tortuous; but ever these hairs, located in transparent containers, were available to observers. Their distinctive features (long hair, shimmering in aspect, with chestnut-brown to red-brown colours) will inspire for long time the iconography [15]. We think that it is the direct observation of these hairs that determinates their earlier representations (Figure 5).            

In a very recently published catalogue of Marie-Madeleine representations [16], one can see that some of them depict her as a woman with blond hair (The Titien: Noli me tangere, toward 1514), blond venetian hair (Rubens: Meal at the home of Simon the Pharisian, 1618–1620), or blond ash (Crivelli: Marie Madeleine, 1495). However most of them, like to the painting of figure 5, represented Marie-Madeleine with red-brown hair: Supplementary Table 1 characterizes twelve most famous representations of Marie-Madeleine with red-brown hair, all of them being dated between the 15th and the 17th Centuries.

What is the biochemical determinism of the pheomelanin production? The pathway of pheolenanin biosynthesis intervenes in the presence of sulphur, which is present in cells in the form of the amino-acid cysteine, or of glutathione (that, under the action of the glutamyl-transpeptidase enzyme, can liberate one cysteine). The spontaneous reaction of cysteine with Dopaquinone terminates to the formation of the 5-S cysteinyldopa, that polymerises to form the pheomelanins [8].

What determines the yellow-red colour of pheomelanins? Paradoxically, it seems that it is not due to the sulphur. In fact both sorts of melanins had conjugated double-links in their formulas [8]: we know that the more successive conjugated double bonds an organic molecule has, the more it appears as coloured (non-black). Moreover, certain atoms or atom groups (like–OH and–NH2) of one organic molecule can affect which wavelengths are absorbed and thereby influence the perceived colour. While –OH is present both in eumelanin and in pheomelanin molecules, -NH2 are found in pheomelanin only [8].

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Other completely different mechanisms could explain the red colour of the hairs: it was observed that archaeological hair samples, human and animals, frequently exhibit red coloration. Post-mortem (or post-depositional) colour change of hairs may be attributed to such factors as photodegradation of melanosomes in sunlight, or to the oxidation of melanin pigment granules over millennia [17]. Some of these mechanisms can explain (at least partially) the red colour of Marie-Madeleine’s hair (that has a potential age of about two thousand years ago).

In fact little is known about the regulation of human hair pigmentation [3]: it is thus likely that the expression of proopiomelanocortin (POMC) gene products, together with the MC1 receptor (MC1-R) and cognate receptor of α-MSH, play a role in regulating hair pigmentation. MC1-R coupled to a GSα protein activates adenylcyclase upon binding of POMC-derived ACTH, α-MSH or ß-MSH peptides; the activity of the local POMC-MC1-R axis plays certainly a role in the physiological regulation of anagen –associated hair pigmentation. This is confirmed by the facts (i): loss-of-function mutations at the MC1-R are associated with switch from eumelanin to pheomelanin production, resulting in a red to yellow coat colour [18], and (ii) : the major part of red-haired individuals are compound heterozygotes and homozygotes for up to five relatively frequent loss-of-function mutations of the MC1-R gene [19].

The DNA study of Marie-Madeleine’s hair had begun [1]; but mitochondrial DNA (mtDNA) only could be extracted, from the bulb of the hair number 10. To study the MC1-R gene mutations that characterized hair colour, it is necessary to obtain genomic DNA from another sort of biological material. Skin debris, like those shown on the surface of one extremity of hair number 8 (Figure 4), cannot be such a material because they correspond to keratinized cells (without cellular nuclei, and without genomic DNA consequently).But we have observed several other sorts (dandruffs, skin portions, and even scalp parts) of skin fragments, located at the vicinity of some Marie-Madeleine’s hair. Supplementary Figure 4 gives an example of such a skin portion (similar in aspect to that of the Noli me tangere), adherent to hair number 7. The SEM photograph of this portion of skin shows several layers of epidermis (with characteristics pleats), covering a relatively compact dermis fragment. By EDX analysis of this fragment of dermis, the corresponding spectrum shows an elevated peak of carbon (which constitutes, with oxygen and nitrogen, the organic matter), a little peak of sulphur (of keratin) and traces of chlorine and sodium (the ClNa of the sweat).

We hope in the future to extract genomic DNA from that sort of dermis fragment, in the goal to study (among other things) the MC1-R gene mutations determining the observed red colour of Marie-Madeleine’s hair.

Conclusion

 

Marie-Madeleine’s hair, as observed today, is dried out and their scales are abraded; that permits the observation of the residual melanosomes conserved under the crests of their hair cortex. Because melanosomes are too small in size (about 1 µm), they cannot be well observed in optical microscopy nor analysed by EDX for their relative contents in sulphur.

The only possible approach of these melanosomes is that of their morphology: melanosomes of type 1 (eumelanin melanosomes) are “grains of rice shaped”, and melanosomes of type 2 (pheomelanin melanosomes) are generally round in form and their sizes are a little bit greater than those of type 1. By counting the two types of residual melanosomes, we obtain a global portion of about 35% of melanosomes of type 2 per unit of surface; this calculated percentage of pheomelanin melanosomes can explain the red-brown colour of Marie-Madeleine’s hair, as observed to the naked eye.

Since the time of their discovery (by Charles de Provence, in 1281), Marie-Madeleine hairs were always exposed into various transparent recipients; so, they can be observed as such for their colour (independently to their length and to their shimmering aspect). We suggest that the initial (and later) representations of Marie-Madeleine hairs result from the direct observation of these red-brown Marie-Madeleine’s hairs exhumed in 1281.

 Acknowledgements

 

We thank the priest of St-Maximin-la Sainte Baume, who furnished to us Marie-Madeleine’s hairs and the reliquary samples. We thank Jean-David Malnati (BNP Bank) for his continuous financial support. Thank you to Dr Philippe Hallegot for his precious information (given in the GN-MBA conferences) concerning details for the scaling pattern of hairs and for the observations of the two types of melanosomes by SEM. The present study is included in our program of scientific investigations developed at the Institute of Molecular Anthropology concerning the vestimentary Christ relics (Argenteuil Tunic, Turin Shroud, Prum Sandal and so on).

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Conflict of Interest

 

The authors declared that there is no conflict of interest.

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Copyright: © 2017 Lucotte G, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.