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Post by Admin on Sept 5, 2023 23:48:36 GMT
Vlad the Impaler, the notorious medieval ruler of Wallachia, has many myths and legends associated with him – including being the inspiration for Count Dracula. A new study is only going to add to his mystique, as it suggests that Vlad probably had skin and respiratory conditions that could have left him crying tears of blood. A team of Italian, Romanian and Israeli researchers have just published their findings in the journal Analytical Chemistry. They examined three letters written by Vlad to the rulers of the city of Sibiu, one from 1457 and two from 1475. One of these letters is even personally signed by Vlad, who was Voivode of Wallachia on three occasions: for a couple of months in 1448, from 1456 to 1462, and from 1475 until his death at the end of 1476 or early 1477. This letter written by Vlad the Impaler in 1475 contains proteins that suggest he suffered from respiratory problems and bloodied tears. Image courtesy Analytical Chemistry The researchers even translated one of the letters from 1475: We, Vladislav Dracul, voivode of the Transalpine regions, publicly notify and recognize/by the present witnesses, who are all responsible, that the illustrious master Thomas/Altemberger, master of the people of the town of Sibiu, for himself and the other people of/said town and of the town of Braşov, in order to pay the twentieth-part [tax] as by written/command of our gracious lord and king, effectively gave and allotted to us two hundred/Hungarian florins. About those two hundred florins, we free the said master and the consuls of the/aforementioned towns, making them unencumbered and entirely released by the power and/testimony of this document. Written in Bălcaciu, in the day of St. Coloman martyr, in the year of the lord/1475. The two letters from 1475 have been kept in Sibiu’s city archive for over 500 years, apparently in ideal conditions. This allowed the researchers to make detailed scientific tests on them, as they did with the letter from 1457. To uncover the letters’ secrets, the researchers used a specialized plastic film called EVA, or ethylene-vinyl acetate, to extract any proteins or small molecules from the paper without damaging it. These extracts were then analyzed with mass spectrometry, allowing researchers to characterize thousands of different peptides. Of these, the team focused on those with the most advanced deamidation, a form of protein degradation that occurs with age. The most degraded proteins were likely the oldest, and therefore, they are the most likely to be from Vlad compared to newer, less-degraded proteins that could have originated from other people handling the letters more recently. The article, “Count Dracula Resurrected: Proteomic Analysis of Vlad III the Impaler’s Documents by EVA Technology and Mass Spectrometry,” by Maria Gaetana Giovanna Pittalà, Antonella Di Francesco, Annamaria Cucina, Rosaria Saletti, Gleb Zilberstein, Svetlana Zilberstein, Tudor Arhire, Pier Giorgio Righetti, and Vincenzo Cunsolo, appears in Analytical Chemistry. Click here to read it. pubs.acs.org/doi/10.1021/acs.analchem.3c01461
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Post by Admin on Sept 6, 2023 20:36:12 GMT
Count Dracula Resurrected: Proteomic Analysis of Vlad III the Impaler’s Documents by EVA Technology and Mass Spectrometry The interest of scientists in analyzing items of World Cultural Heritage has been exponentially increasing since the beginning of the new millennium. These studies have grown considerably in tandem with the development and use of sophisticated and sensitive technologies such as high-resolution mass spectrometry (MS) and the non-invasive and non-damaging technique, known under the acronym EVA (ethylene-vinyl acetate). Here, we report the results of the MS characterization of the peptides and proteins harvested by the EVA technology applied to three letters written in 1457 and 1475 by the voivode of Wallachia, Vlad III, also known as Vlad the Impaler, or Vlad Dracula. The discrimination of the “original” endogenous peptides from contaminant ones was obtained by monitoring their different levels of deamidation and of other diagenetic chemical modifications. The characterization of the ancient proteins extracted from these documents allowed us to explore the environmental conditions, in the second half of the 15th century, of the Wallachia, a region considered as a meeting point for soldiers, migrants, and travelers that probably carried not only trade goods and cultural traditions but also diseases and epidemics. In addition, the identification of many human peptides and proteins harvested from the letters allowed us to uncover more about Vlad Dracula the Impaler. Particularly, the experimental data show that he probably suffered from inflammatory processes of the respiratory tract and/or of the skin. In addition, proteomics data, although not exhaustive, suggest that, according to some stories, he might also have suffered from a pathological condition called hemolacria, that is, he could shed tears admixed with blood. It is worth noting that more medieval people may have touched these documents, which cannot be denied, but it is also presumable that the most prominent ancient proteins should be related to Prince Vlad the Impaler, who wrote and signed these letters. The data have been deposited to the ProteomeXchange with the identifier ⟨PXD041350⟩.
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Post by Admin on Sept 8, 2023 5:52:04 GMT
Preface The story of Count Dracula, the most famous feeding on the blood of the living to live forever, is certainly one of Europe’s bloodiest legends, described by the Irish writer Bram Stoker in his book of 1897. (1) Although the nature of this inspiration has been contentious, some scholars and historians theorized that the monstrous legend in Stoker’s famous novel would partly be inspired by a historical figure: The Romanian prince Vlad III, Voivode of Wallachia during the 1400s, also known as Vlad the Impaler, or Vlad Drăculea (the Son of the Dragon). (2) The theory that Vlad III and the Dracula of Stoker’s novel were the same person was developed and popularized by historians Radu Florescu and Raymond T. McNally in their book “In Search of Dracula” (The New York Graphic Society, 1972), but according to different historians and literary scholars, the two Draculas do not really have much in common. On the other hand, the historical figure who shares a name with the literary icon, though there is no real evidence of his alleged vampirism, was no less fearsome. This Prince of Wallachia, in the Carpathian region, reigned with terror over his territory in the 15th century. Vlad Dracula the Impaler (Vlad Tepes in Romanian) was a brutal and sadistic prince-like military leader famous for torturing his foes. Local legends say that he impaled many Ottoman enemies and a lot of people he suspected of plotting against him. By some estimates, he was responsible for the deaths of more than 80,000 people in his lifetime, a large percentage of them by impalement. Centuries later, the sinister reputation of Vlad the Impaler took on a new life when Stoker came across the name Dracula in an old history book, learned that it could also mean “devil” in Walachia, and gave the name to his fictional . However, today, Vlad III is something of a national hero in Romania, where he is remembered for defending his people from foreign invasions, whether Turkish soldiers or German merchants. In light of this preface, it is undeniable that, thanks to the popularization of the image of Bram Stoker’s Dracula, Prince Vlad III himself has also become very famous and immortal, although not in the same way as his literary counterpart. Introduction The interest of scientists in analyzing items (paintings, documents, ceramics, fossils, etc.) of World Cultural Heritage has been exponentially increasing since the beginning of the new millennium. Over the past two decades, studies of ancient biomolecules (mainly, but not only, proteins) have opened an important window into the past, improving our understanding, for example, of the evolutionary history, (3) the diet habits of our ancestors, (4) the past human diseases, (5) or the knowledge of the materials used in a painting. (6) These studies have grown considerably in tandem with the development and use of increasingly sophisticated and sensitive technologies and analytical tools. Particularly, the increased number of scientific publications about the characterization of ancient proteins is strictly linked to the impressive technological development, in terms of performance and sensitivity, of mass spectrometry (MS) together with the improvements of protein databases and bioinformatics tools, that has expanded the application of MS-based approaches also to the World Cultural Heritage. Modern mass spectrometers are able to detect and characterize many proteins with extremely high sensitivity even in very complex mixtures, and on very limited amounts of sample, typically less than a few micrograms of crude extract. On the other hand, it is well known that one of the most important issues of the analysis of ancient samples is represented by the contamination of exogenous proteins, mainly constituted by those coming from the post-discovery of the item or from the proteomic analysis laboratory. Therefore, the challenge of discerning contaminant proteins from endogenous ancient molecules becomes of fundamental importance. The deamidation process of asparagine and mainly of glutamine residues (showing a slower deamination rate) is the most applied marker of aging in many archaeological and paleontological studies. (7,8) In addition, the different patterns of degradation and diagenetic chemical modifications (DCMs) affecting ancient proteins, as opposed to potential modern contaminants, are used as authentication and validation criteria for the results. (9,10) Another important challenge is related to the inestimable value of World Cultural Heritage samples, which should be analyzed without damage or contamination. Therefore, the sampling techniques adopted have to be minimally destructive or non-destructive. Indeed, although modern high-performance mass spectrometers require very low amounts of sample, most of the actual protocols of analyses remain invasive, requiring the destruction of part of the ancient items under study; a practice that is (obviously) discouraged by most museums. Consequently, during the last decade, different minimally invasive methods have been developed to harvest and investigate ancient samples. (11−14) A promising non-invasive technique, which at present appears to allow full exploration of any item in the Cultural Heritage arena without any damage or contamination, is known under the acronym EVA (ethylene-vinyl acetate). (15) It consists of a plastic film of ethylene-vinyl acetate studded with strong cation and anion exchangers as well as with C18 and C8 hydrophobic resins which, when applied to any type of surface, is able to extract proteins and small molecules. Then, the harvested molecules can be eluted and characterized via MS. This non-invasive approach coupled with MS-based methods has been applied with promising results to documents, clothing items, and ancient tissue remains. (16−18) Here, we report the results of the MS characterization of the peptides and proteins harvested by the EVA technology applied to three letters of the 15th century that were written to the burghers of Sibiu (Romania) by a man describing himself as “prince of the Transalpine regions” and signed the letters as Vlad Dracula. The characterization of proteins extracted from these three letters provided a window into Wallachia (part of today’s southern Romania) of the second half of the 15th century, a period of military turbulence, also characterized by diseases and epidemics─and not only. The identification of some human peptides and proteins harvested from the letters investigated allowed us to uncover more about Vlad Dracula the Impaler. As described by the papal legate Nicholas of Modrussy, (19) “he was not very tall, but very stocky and strong, with a cruel and terrible appearance, a long straight nose, distended nostrils, a thin and reddish face in which the large wide-open green eyes were framed by bushy black eyebrows, which made them appear threatening.” Two green eyes that, according to some stories, sometimes cried tears of blood, a physical condition, today known as hemolacria, which causes a person to produce tears that are partially composed of blood.
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Post by Admin on Sept 9, 2023 5:58:59 GMT
Experimental Details Chemicals All the chemicals employed were used without further purification because they were of the highest purity commercially available. Ammonium bicarbonate (AMBIC), dithiothreitol (DTT), formic acid (FA), and iodoacetamide (IAA) were purchased from Aldrich (St. Louis, Missouri, USA); ammonia was purchased from Carlo Erba (Milan, Italy); sequencing grade modified porcine trypsin was purchased from Promega (Madison, WI, USA); and water and acetonitrile (ACN) (OPTIMA LC/MS grade) for LC/MS analyses were purchased from Fisher Scientific (Milan, Italy). Letters The documents investigated are three letters, made of rag paper, written and signed by the voivode of the Transalpine regions, Vladislav Dracul. Vlad Dracula wrote these three documents in different settlements of Transylvania (Bălcaciu and Braşov) and sent these documents to the city of Sibiu. Its addressees were the rulers of the city of Sibiu and, in particular, the ruler of the city of Sibiu Master Thomas Altemberger. The first letter (Figure 1a) was written in 1475 (archive catalog number is II 365); the second letter (Figure 2a) also written in 1475 (archive catalog number is III 32 N 484) and contains the personal signature of Vlad Dracula in the left bottom part; the third letter (Figure 3a) was written in 1457 (archive catalog number is V 1658). The two documents dated 1475 (Figures 1a and 2) have been kept for their entire 500 years’ history in the archives of the city of Sibiu; they are in excellent condition and were not restored. The letter written in 1457 (Figure 3a) was instead restored in Bucharest in the 20th century. All restoring operations were carried out minimizing the possibility of biological and chemical contamination. There is probably a big difference in the quality of the preservation of documents of 1475 and 1457 because the Sibiu Archive was founded in 1465 and it has been officially operating since that year. So, the two well-preserved documents of 1475 immediately began to be stored in the city’s official archive. The conditions for storing documents include storage in a paper envelope and in separate folders separately from each other. Documents can only be touched with gloves. Each view of a document is recorded on a record sheet, and the history of document views is traced. A complete set of photos of all three Dracula letters (without EVA foils), photographed on both sides, is provided in the Supporting Information. The first letter (Figure 1a), dated August 4, 1475, and translated from Latin to English, reports as follows: Figure 1. (a) First letter (archive catalog number is II 365), dated August 4, 1475, here investigated, also showing the positions of the EVA strips (brownish rectangles) applied to its surface for capturing biological material; (b) mapping of the fluorescence of phenylalanine, tyrosine, and tryptophan under flash UV illumination (see the text).
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Post by Admin on Sept 10, 2023 20:39:51 GMT
Figure 2 Figure 2. (a) Second letter (dated 1475; archive catalog number is III 32 N 484) here investigated. The letter shows the personal signature of Vlad Dracula in the left bottom part; brown patch/tape-like materials are the EVA films applied for capturing biological material; (b) mapping of the fluorescence of phenylalanine, tyrosine, and tryptophan under flash UV illumination (see the text). Figure 3 Figure 3. (a) Third letter (dated 1457; archive catalog number: V 1658) here investigated after restoration, and regions of sampling by EVA diskettes (the four brown patch/tape-like materials); (b) mapping of the fluorescence of phenylalanine, tyrosine, and tryptophan under flash UV illumination (see the text). “We, Vladislav Dracul, voivode of the Transalpine regions, publicly notify and recognize/by the present witnesses, who are all responsible, that the illustrious master Thomas/Altemberger, master of the people of the town of Sibiu, for himself and the other people of/said town and of the town of Braşov, in order to pay the twentieth-part [tax] as by written/command of our gracious lord and king, effectively gave and allotted to us two hundred/Hungarian florins. About those two hundred florins, we free the said master and the consuls of the/aforementioned towns, making them unencumbered and entirely released by the power and/testimony of this document. Written in Bălcaciu, in the day of St. Coloman martyr, in the year of the lord/1475.”
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