The Secret of the Parchments

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And I showed it to, I think it was my aunt. I showed it to her and she just saw certain poems. Nobody sees the same. I can keep it on the table because nobody is able to read it, the secrets. M: They were all confused.

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One saw it was a song, one saw it was a letter. They were surprised. So I thought something was wrong about that, so that I should not show it to anybody else. It caused laughter, surprise. I just folded the paper and put it away in my drawer. Our Lady told Mirjana to choose a priest that would be responsible for revealing the secrets.

Mirjana chose Fr. Petar Ljubicic. Ljubicic was interviewed by a Friend of Medjugorje for Radio Wave in In part of the interview, Fr. Petar was asked about what he knew about the parchment. Personally I never did. Some of her cousins saw it…And Our Lady then said to Mirjana do not show this to anyone yet, until the time of the revelation comes. Mirjana, during the war, was in Sarajevo. When she returned, she forgot the parchment and left it in Sarajevo.

A year or two ago, a soldier brought it to her with all her belongings that she left behind during the war. Again, something I would say is miraculous. She comes to call the world to conversion for the last time. Afterwards, I will not appear any more on this earth. Radio Wave interview with Fr. Petar Ljubicic by A Friend of Medjugorje, Mej v 3. A Friend of Medjugorje. Caritas of Birmingham. Community of Caritas. Medjugorje Pilgrimages. The 10 Secrets.

the secret of the parchments Manual

We are generating a PDF, please wait Click on the link below to Send us Your Feedback on this Writing. Sign up for MejList Free. Search the Message. Raman spectroscopy, already used to provide the chemical identification of pigments in cultural heritage, was applied to rapidly identify the pigments in the purple spots without altering samples structure. Lastly, the novel Light Transmission Analysis LTA technique, was used to quantify the structural damage suffered by the native collagen in damaged areas.

The NGS metagenomic analysis revealed different prokaryotic communities in the damaged and undamaged areas of the document, with Pseudonocardiales mostly present in the undamaged areas, and Gamma-Proteobacteria mainly Vibrio exclusively present in the damaged ones. Ubiquitous and environmental bacteria were observed in both sets of samples.

In the purple spots, the Raman spectroscopy identified rhodopsin-like pigments: purple transmembrane protein containing retinal produced mainly by Halobacteria whose genetic traces, however, were not found in the parchment. The Light Transmission Analysis technique bared the collagen structural damage in the purple areas: bacteria cause the thinning out of the interfibers collagen texture and some deterioration of the fibre sheath, the more chemically stable kind of collagen. Basing on these results, a biodeterioration model was hypothesized and proposed. This model consists in a microbial succession, regarded as the putative responsible of the purple spot damage of parchments.

During an ecological succession, the community structure of the site changes over time, so that species substitution takes place. The succession starts by the activity and growth of pioneer species. The process occurs in every kind of ecosystems and its course depends on the quality and quantity of available resources. The ecological succession putative responsible of the purple spot damage of parchments still remains unsolved in some points. The aims of this study were: to solve the jigsaw puzzle of the purple spot biodegradation process dynamics, to validate the proposed successional model and to deepen the understanding of the parchment degradation process.

To this end, new data on the microbes responsible of the purple spots and their effects were gathered from three dramatically damaged parchments. These documents were chosen due to the deep and widespread purple spot damage, leading even to the loss of significant portion of the documents. In these extremely damaged conditions, we supposed that the pioneer species could have been more largely present at the beginning of the document history, so that they would have been more likely identified.

D, and are still conserved in their original envelope, along with other documents. Their conservation state is truly poor Fig. Since , they are conserved in the Vatican Secret Archives, under controlled environmental conditions of relative humidity and temperature. The documents were analysed by the same multidisciplinary approach used in the previous study: NGS metagenomic analysis Illumina platform , Raman spectroscopy and LTA analysis.

The three parchments were identified as A—C. Three documents belonging to a collection of dramatically damaged parchments of the Vatican Secret Archives were chosen for the bacterial and haloarchaeal community characterization. These documents were considered impossible to be restored and are archived as Faldone Patrizi A Scritture , sommarii , ed altro relativo a cause tanto attive quanto passive.

Scriptures, summaries, and more about active or passive causes. From to ].

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The analysed samples were collected in sterile conditions from hardly purple stained or quite uncoloured areas in sterile conditions. From each document, three replicates were selectively collected from both purple and uncoloured less damaged areas as control. Due to the dramatic and advanced state of biodeterioration of the documents, the three white pieces were chosen in the better preserved and less damaged area, as far as possible from the purple spots; in the most damaged parchment C , the control samples were collected in an uncoloured area even if it was not clearly differentiated from the purple spotted one.

The analysis steps for processing sequence data were developed based on state-of-the-art tools for metagenomic. In summary, full. The dataset was normalized to the sample with the lowest number of reads sample AP1 at the common depth of 14, reads per sample, by random sub-sampling, in order to standardize samples differences. A representative sequence from each OTU cluster was chosen and used for taxonomic identification and phylogenetic alignment. Prior to tree building, the alignment was filtered to remove positions, which are phylogenetically uninformative.

Chimeras and singletons were identified and removed from the alignment, so that non-chimeric sequences were aligned and used to create a phylogenetic tree. Diversity metrics were calculated for each sample; the taxonomic and phylogenetic assignment was used to compare the types of community.

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To show the differences among the sample set, PCoA plots were generated. The free software QIIME was used to analyse the bacterial and haloarchaeal community structure and composition on the normalized OTU dataset: i rarefaction curves were created to assess sampling efficiency Fig.

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Raman analyses were performed on purple damaged samples 1 by chemically extracting pigments from purple spots, and on Halobacterium harvested colonies; reddish colonies were analysed directly without any pre-treatment. Due to heterogeneity of the parchment supports and the high entity of damage, less damaged areas were difficult to be focalized, providing fluorescence as the dominant response. In order to assess the deterioration state of the collagen both in purple and uncoloured less damaged areas, the induced hydrothermal denaturation of the collagen has been analysed by means of the LTA technique, integrated by the microscopy continuous monitoring, as in Migliore et al.

In this study, the signal amplitude was recorded for the whole process of hydrothermal denaturation, every 0. The sequences were assigned to a total 1, OTUs from the purple samples and from the unstained ones , out of which were common to the two batches. By random subsampling, the entire dataset was normalized to the lowest common depth of 14, sample AP1 sequence reads per sample, in order to standardize differences among samples. S1 , Supporting Materials. PCoA plot Fig. It showed that the microbial composition of the two sets of samples overlap. No clear patterns of separation are shown. Each sample is identified as: parchment A, B, C , purple damaged P or uncoloured less damaged samples U and replicate 1, 2, 3.

The Venn diagram Fig. Unique or shared OTUs in purple damaged P and uncoloured less damaged samples U of the three parchments. The number and percent of OTUs are also reported. The total number of OTUs is 1, S2 , Supporting Materials. Both Halobacteria and Bacteria were found. Halophylic Archaea accounted for 3, sequences assigned to one single OTU and found in both set of samples; Bacteria accounted for , sequences, assigned to 1, OTUs. Haloarchaeal and bacterial sequence distribution, in purple damaged P, in the purple box and uncoloured less damaged U, in the warm yellow box samples in the three parchment samples A—C documents.

They are more frequent in the purple damaged samples than in the less damaged uncoloured ones, other than in the document A. The main bacterial phyla were Actinobacteria, Proteobacteria and Firmicutes. All of these microorganisms are observed in both the sample sets, reported in Fig. Distribution of taxa at order level in the purple damaged left and uncoloured less damaged right samples of the three parchments, as percentage of sequences found in the samples from the Faldone Patrizi A The distribution of taxa, at order level Fig. On the contrary, the early colonizers - marine bacteria, as the Vibrionales - are found at very low percentage.

The second most represented class is Proteobacteria; which showed a slight prevalence in the less damaged batch [ P The decreasing frequency order in the Class was: Gamma-Proteobacteria [ The Delta- and Epsilon- subclasses were practically absent. Enterobacteriales, Xhantomonadales, Pseudomonadales Gamma-Proteobacteria ; Burkholderiales Beta-Proteobacteria and Sphingomonadales Alpha-Proteobacteria were the more frequently encountered orders. Firmicutes [ P 8.

Raman spectra acquired from Halobacterium salinarum Fig. These bands are attributed to the bacterioruberin, the main carotenoid component responsible for the colour of the red archaea; the signal results very intense because of the resonance condition due to the use of green laser source Other less intense signals are found and could be attribute to bacteriorhodopsin contribution to the spectrum. Raman spectra.

Raman spectra of extracted pigments from parchments are reported in Fig. In particular Fig. The signals of bacterioruberin are almost present in every samples. It is important to underline that bacteriorhodopsin, rhodopsin-like pigments, and retinal isomers exhibit almost overlapping Raman spectra. Samples from the highly damaged areas, c , were also analysed but the results were neither reproducible, nor univocal and are not shown here because of their unsoundness. The grey curves represent the variation rate of the signal generated by the unscattered transmitted light, as a function of temperature and express the intensity of the hydrothermal denaturation process.

The profile of these curves can be seen as the combination of two peak curves associated with the contributions to the denaturation of material fractions, characterized by collagen with different chemical stability. In particular, the low temperature peak describes the denaturation of the less stable collagen, classified as native N in previous works 1 , 17 , while the high temperature peak is related to the denaturation of a more stable collagen, the so called stabilized S one 17 , 18 , typical of the fibres sheath and characterized by a larger thermal stability.

It has been shown 17 , 18 that the deterioration of the collagen populations leads to a temperature downshift of the related LTA peak 1. In this view the graph of Fig. A quite different situation is observed for the S peaks. On the left, a piece of parchment from Faldone Patrizi A 19, in red light, that shows as dark brown the purple stained areas ; on the right, the corresponding denaturation curves: a uncoloured less damaged and b purple damaged sample, the samples being representative of all the three documents.

The image is representative of the parchment patch from where samples have been withdrawn circled areas. They have been deconvolved in two curves black and red lines representing the contribution to the denaturation by collagen fractions with different thermal stability.

The c samples did not give repeatable results and are not shown. In this study, the purple spot damage of ancient parchments was studied by an interdisciplinary approach, comparing hardly purple vs uncoloured less damaged areas from three dramatically damaged parchments archived as Faldone Patrizi A 19 in the Vatican Secret Archives, dated back to XVI-XVII century A. In a previous work on the parchment roll A. So, we chose extremely damaged documents, on the premise that a possible larger amount of halobacteria in the original brines could have caused a faster and widespread formation of the purple spots, and that the DNA of the putative pioneer species would have been more likely found out.

Actually, in these largely damaged documents, the NGS revealed the presence of Halobacterium salinarum , and the Raman spectroscopy confirmed the presence of bacteriorhodopsin together with bacterioruberin in all the purple samples 1. Bacterioruberin is also produced by some species of Actinobacteria out of Micrococcales and Corynebacteriales order 21 , 22 which have been found also in the documents from Faldone Patrizi A 19 , at low percentage in all samples. Nevertheless, the possibility of them to determine the purple spot-damage had been already ruled out in the previous work, as their number was comparable in the neatly undamaged and damaged samples from A.

As suggested by Marshall et al. By comparing the A. Regarding the prokaryotic biodeteriogens, in these three documents from the Faldone Patrizi A 19 NGS found the traces of halobacteria, with sequences, at different percentages in all samples. Other than haloarchaea, the bacterial taxa in the three documents are almost the same as those found in the A. The roll, however, is still quite well preserved, and the quantitative distribution of taxa in the purple and undamaged areas was dramatically different. This, highlights a significantly different colonization rate between the two set of samples, while analyzed as the complete dataset.

The exiguity of sample material and the variability of microbial community composition, even at this small scale, are responsible of the absence of significant differences within each document. Nevertheless, while comparing the entire dataset, the algorithm is able to highlight the differences. This implies that the environmental and nutritional conditions in the damaged parchment environment select relatively few microbial strains and that, even in the same document, the damaged and undamaged areas offer different environments.

However, in all samples from the Faldone Patrizi A 19 the dominant component of the colonizing microbial community belonged to Proteobacteria, Firmicutes and Actinobacteria. In our previous study we hypothesized a two phases heterotrophic microbial succession, able to degrade the parchment. This work allowed to have a more accurate picture of the parchment succession Fig. Dynamics of the microbial succession in the parchment. In the deterioration process the main actors are in the sequence: haloarchea purple , halotolerant bacteria light blue , actinobacteria gold and fungi green.

The presence of Halobacterium salinarum in the Faldone Patrizi A 19 parchments put in place a first pivotal piece in our biodegradation jigsaw puzzle, confirming the identity of the actual pioneer population. The brining of the hides in ancient times, as nowadays, was a common step of the manufacturing process of both parchments and leathers.

In the brine, the halophilic and halotolerant microbes from the marine salt, can grow and enter into the hides, forming the core of the purple spot damage as it happens in brine-cured leathers They grow inside hides by producing proteolytic and lipolytic enzymes which attack and degrade the parchment collagen matrix Later, maybe when the salt concentration within the parchment lowered, halobacteria lysed, in the hot-spots where they had grown up, releasing bacteriorhodopsin and cellular content and providing a nutrient and energy supplement.

So, the absence 1 or the low percentage of the Haloarcheal OTUs are explained by the intrinsic mechanism of the ecological succession. The first phase of the succession, therefore, is predictable as mainly driven by the brining process - according to the Clements model Differently, the second phase attack to the parchment, is driven by a casual Gleasonian model 29 , and puts in place a second piece of the puzzle. Even the second-phase bacteria, depend on the history of each parchment, that is, on the environment where the parchment was kept libraries, bookcases, scholars, etc.

In this study, as in the previous one, Pseudonocardiaceae soil-dwelling, ubiquitous, often able to stand harsh environments have been detected as the major colonizers of all the samples. In the late colonization phases even some human-derived species, like staphylococci and enterobacteria can be found. Therefore, it is not surprising that Actinobacteria mainly Pseudonocardiales have been frequently pointed out as the responsible of the parchment deterioration as, at the end point, they are actually dominant on the damaged parchments, even in the undamaged portions of the documents [1—2,30 and this study].

Concurrently the molecular approaches such as 16S libraries, and even the much more sensitive NGS, take a snapshot of the archaeal and bacterial colonizers which arrived on the parchments over the centuries and, obviously, the more recent are better represented than the old ones.

This explains why the composition of the prokaryotic community found in the Faldone Patrizi A 19 documents accounts for a prevalence of the second phase taxa, as even the halotolerant Gamma-Proteobacteria are quite few and the Actinobacteria definitely dominate the scene. Most probably they can also benefit of the initial lesions produced by the first colonizers to penetrate the document, adding more and more collagen degradation in the already damaged stained spots.

Moreover, Actinobacteria are surely involved in the damage, so as fungi 30 , 31 , due to their ability to deeply attack the collagen, but they participate to the last act of the parchment tragedy, rather than to its causal trigger, at the first beginning. As a final point, by comparing the results on the A.

LTA gives insight on the collagen degradation: the peak temperatures for N and S depicts the thermal stability of the collagen populations, the higher the peak temperature, the higher the stability. Steps of the parchment degradation process. They were identified by the temperatures of hydrothermal denaturation measured in both the A. Downshift of the denaturation temperatures have been already reported in Migliore et al. In this document, however, the width of the T S peak increased with the deterioration, revealing a process that made the stabilized collagen population less homogeneous.

Differently, in the measurements on the Faldone Patrizi A 19 documents Fig. Furthermore, it is worth noting that the thermal stability of the S population for the undamaged samples of the A. I-XVIII parchment roll was much higher than the one of the Faldone Patrizi A 19 documents, and maybe different starting thermal stability can address different dynamics of the deterioration process.

The results on the three Faldone Patrizi A 19 documents seem to constitute a continuum with those found on the A. In the framework of the progressive process of collagen degradation and utilization, intrinsic in the successional process, the samples from the A. I-XVIII document represent the starting point, a well-preserved structure of the parchment the uncoloured samples , and a first degradation step purple samples.

At the starting point, a diffused matrix encompassing the more robust fibres was found, while, as the process of collagen degradation goes on, the matrix is completely absent and only the more robust fibres can be found. Actinobacteria are able to deeply penetrate into the collagen structure and damage even the more robust fibres, by a slow but constant action. The uncoloured samples show comparable collagen stability to the A. Hence, it is possible to hypothesize that a general collagen degradation already happened in the entire document, even in the uncoloured areas.

The purple damaged samples in some cases were so deteriorated see, for instance, the circled area c in Fig. Other less stained samples, surprisingly, displayed a relevant quantitative and qualitative amount of damage in the S collagen, while the N population seemed almost unchanged. This result could be explained in term of a biological attack to the stabilizing crosslinks in the S collagen present in the more robust fibers, representing the frame structures of the parchment 17 , 18 ; probably microbes are able to attack and degrade collagen to a less structured status.

In conclusion, the integrated advanced multidisciplinary approach adopted in this study, was able to validate the successional model hypothesized in the previous study, and to further shed light on the causes and processes involved in historical parchment deterioration, successfully deciphering other pieces of the purple spot degradation jigsaw puzzle.

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The importance of this study is not only limited to the ancient parchment story but also to the nowadays problem of the red-heat deterioration in brine cured hides, that show similar features with purple spots, although a common causative agent has not been demonstrated so far.

Hence, this study opens opportunities of intervention on ancient parchments, but may significantly contribute to the efficiency of leather manufacturing. Migliore, L. Purple spot damage dynamics investigated by an integrated approach on a A. Scientific Reports 7 , Unmasking the measles-like parchment discoloration: molecular and micro-analytical approach.

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