Gino Fornaciari

Microsatellite instability and mismatch repair gene inactivation in sporadic pancreatic and colon tumours.

Genomic instability has been proposed as a new mechanism of carcinogenesis involved in hereditary non-polyposis colorectal cancer (HNPCC) and in a large number of sporadic cancers like pancreatic and colon tumours. Mutations in human mismatch repair genes have been found in HNPCC patients, but their involvement in sporadic cancer has not been clarified yet. In this study we screened 21 pancreatic and 23 colorectal sporadic cancers for microsatellite instability by ten and six different microsatellite markers respectively. Microsatellite alterations were observed at one or more loci in 66.6% (14/21) of pancreatic cancers and in 26% (6/23) colon tumours, but all the pancreatic and half of the colon samples showed a low rate of microsatellite instability. All the unstable samples were further analysed for mutations in the hMLH1 and hMSH2 genes and for hypermethylation of the hMLH1 promoter region. Alterations in the hMLH1 gene were found only in colorectal tumours with a large presence of microsatellite instability. None of the pancreatic tumours showed any alteration in the two genes analysed. Our results demonstrate that microsatellite instability is unlikely to play a role in the tumorigenesis of sporadic pancreatic cancers and confirm the presence of mismatch repair gene alterations only in sporadic colon tumours with a highly unstable phenotype.

Reliability in age determination by pulp/tooth ratio in upper canines in skeletal remains

ABSTRACT: Estimation of age of skeletal remains is one of the most complex questions for anthropologists. The most common macroscopic methods are based on dental wear and histological evaluation of bone remodeling. These methods are often qualitative, require great technical expertise, and have proved inexact in the estimation of ages over 50 years. Certain dental methods investigate the apposition of secondary dentine, in the study of tooth cross‐sections, and X‐rays to study width, height, and pulp area. The primary author previously proposed a method of estimating the age of a living person based on the pulp/tooth ratio (PTR) method in the upper canines. The aim of the present study is to verify whether the PTR method can also be used to estimate the age at death of skeletal remains. This paper investigates the study of historical samples of known age as a means to validate the proposed method.

Sequence analysis of bacterial DNA in the colon of an Andean mummy.

We have isolated DNA from 14 tissue samples from the internal organs of an Andean human mummy (10th-11th century A.D.) and have checked the persistence of the original human and bacterial templates using the following main approaches: 1) amino acid racemization test; 2) quantification of mitochondrial DNA copy number; 3) survey of bacterial DNA in the different organs; 4) sequence analysis of bacterial amplicons of different lengths. The results demonstrate that both the original human DNA and the DNA of the bacteria of the mummy gut are preserved. In particular, sequence analysis of two (respectively 100 and 196 bp in length) libraries of bacterial 16s ribosomal RNA gene amplicons from the mummy colon shows that while the shortest amplicons give only modest and biased indications about the bacterial taxa, the longer amplicons allow the identification several species of the genus Clostridium which are typical of the human colon. This work represents a first example of a methodological approach which is applicable, in principle, to many other natural and artificial mummies and might open the way to the study of the structure of the human microbial ecosystem from prehistory to present.

Gut Microbiome of an 11th Century A.D. Pre-Columbian Andean Mummy

The process of natural mummification is a rare and unique process from which little is known about the resulting microbial community structure. In the present study, we characterized the microbiome of paleofeces, and ascending, transverse and descending colon of an 11th century A.D. pre-Columbian Andean mummy by 16S rRNA gene high-throughput sequencing and metagenomics. Firmicutes were the most abundant bacterial group, with Clostridium spp. comprising up to 96.2% of the mummified gut, while Turicibacter spp. represented 89.2% of the bacteria identified in the paleofeces. Microbiome profile of the paleofeces was unique when compared to previously characterized coprolites that did not undergo natural mummification. We identified DNA sequences homologous to Clostridium botulinum, Trypanosoma cruzi and human papillomaviruses (HPVs). Unexpectedly, putative antibiotic-resistance genes including beta-lactamases, penicillin-binding proteins, resistance to fosfomycin, chloramphenicol, aminoglycosides, macrolides, sulfa, quinolones, tetracycline and vancomycin, and multi-drug transporters, were also identified. The presence of putative antibiotic-resistance genes suggests that resistance may not necessarily be associated with a selective pressure of antibiotics or contact with European cultures. Identification of pathogens and antibiotic-resistance genes in ancient human specimens will aid in the understanding of the evolution of pathogens as a way to treat and prevent diseases caused by bacteria, microbial eukaryotes and viruses.

Diffuse idiopathic skeletal hyperostosis in the Medici, Grand Dukes of Florence (XVI century)

Diffuse idiopathic skeletal hyperostosis (DISH) is a common systemic disorder characterised by the ossification of the anterior longitudinal spinal ligament involving at least three contiguous vertebrae and by diffuse extraspinal enthesopathies. The condition is associated with the male sex and with advanced age; its aetiology is uncertain, but seems to be related to obesity and diabetes. The most recent studies in archaeological series demonstrated a relation between high social status and the incidence of DISH. The present study examines two cases of DISH found amongst the members of the Medici family buried in the Basilica of San Lorenzo in Florence. The skeletons of the Grand Dukes Cosimo I (1519–1574) and his son Ferdinand I (1549–1609) showed the typical features of the condition. This result is related to the obesity of the Grand Dukes, attested by the written and artistic sources, and to the protein-based alimentation demonstrated by a paleonutritional study, thus furnishing further evidence to the significance of DISH as a life style.

Food and disease at the Renaissance courts of Naples and Florence: A paleonutritional study

A paleonutritional study of the Medici Grand Dukes in Florence (16th-17th centuries) and of the Aragonese Princes in Naples (15th-17th centuries) was performed. Carbon and nitrogen stable isotope analysis of bone collagen showed high values of delta(15)N at the level of carnivores and demonstrated a diet that was very rich in meat. delta(13)C values are in accordance with an intake of fish, especially for the Aragonese series from Southern Italy, which can be estimated at 14-30% for the Medici and 12-40% for the Aragonese. Adherence to medieval fasting is likely to have been the main reason for this dietary change.

Taxonomic and predicted metabolic profiles of the human gut microbiome in pre-Columbian mummies.

Characterization of naturally mummified human gut remains could potentially provide insights into the preservation and evolution of commensal and pathogenic microorganisms, and metabolic profiles. We characterized the gut microbiome of two pre-Columbian Andean mummies dating to the 10-15th centuries using 16S rRNA gene high-throughput sequencing and metagenomics, and compared them to a previously characterized gut microbiome of an 11th century AD pre-Columbian Andean mummy. Our previous study showed that the Clostridiales represented the majority of the bacterial communities in the mummified gut remains, but that other microbial communities were also preserved during the process of natural mummification, as shown with the metagenomics analyses. The gut microbiome of the other two mummies were mainly comprised by Clostridiales or Bacillales, as demonstrated with 16S rRNA gene amplicon sequencing, many of which are facultative anaerobes, possibly consistent with the process of natural mummification requiring low oxygen levels. Metagenome analyses showed the presence of other microbial groups that were positively or negatively correlated with specific metabolic profiles. The presence of sequences similar to both Trypanosoma cruzi and Leishmania donovani could suggest that these pathogens were prevalent in pre-Columbian individuals. Taxonomic and functional profiling of mummified human gut remains will aid in the understanding of the microbial ecology of the process of natural mummification.

Plasmodium falciparum immunodetection in bone remains of members of the Renaissance Medici family (Florence, Italy, sixteenth century).

Medical accounts and ancient autopsy reports imply that tertian malarial fevers caused the death of four members of the Medici family of Florence: Eleonora of Toledo (1522-1562), Cardinal Giovanni (1543-1562), don Garzia (1547-1562) and Grand Duke Francesco I (1531-1587). All members of the Medici family hunted in the endemic malarial areas of Tuscany, such as the marshy areas surrounding their villas and along the swampy Maremma and were, therefore, highly exposed to the risk of being infected by Falciparum malaria. To determine if the original death certificates issued by the court physicians were correct, we carried out immunological investigations and then compared the biological results to the historical sources. Bone samples were examined for the presence of Plasmodium falciparum histidine-rich- protein-2 (PfHRP2) and P. falciparum lactate dehydrogenase (PfLDH) using two different qualitative double-antibody immunoassays. Our findings provide the first modern laboratory evidence of the presence of P. falciparum ancient proteins in the skeletal remains of four members of the Medici family. We confirm the clinical diagnosis of the court physicians, using modern methods. Finally, this study demonstrates that immunodetection can be successfully applied not only to mummified tissues but also to skeletal remains, thus opening new paths of investigation for large archaeological series.

The use of mercury against pediculosis in the Renaissance: the case of Ferdinand II of Aragon, King of Naples, 1467-96.

The hair samples of Ferdinand II of Aragon (1467–1496), King of Naples, whose mummy is preserved in the Basilica of San Domenico Maggiore in Naples, showed a high content of mercury, with a value of 827ppm. Furthermore, examination using a stereomicroscope and a scanning electron microscope (SEM) of head and pubic hairs of Ferdinand II, revealed a lice infestation. The reasons for the massive presence of the mercury in the kings hair are discussed and contemporary literature regarding the use of this metal in medical therapies and in cosmetic practices is analysed. As a result, the high value of mercury in the hair of Ferdinand II can be attributed to antipediculosis therapy, applied as a topic medicament. This case represents an important finding for the history of medicine, because demonstrates that in the Renaissance mercury was applied locally not only to treat syphilis, as well attested by direct and indirect sources, but also to prevent or eliminate lice infestation.

Hyperostosis frontalis interna (HFI) and castration: the case of the famous singer Farinelli (1705-1782)

The famous castrato singer Farinelli (1705–1782) was exhumed by our research group in July 2006 for the purpose of gaining some insight into his biological profile through a study of his skeletal remains. Farinelli was castrated before puberty to preserve the treble pitch of the boy’s voice into adult life. His powerful and sweet voice became legendary. In spite of its bad preservation state, the skeleton displayed some interesting characteristics that are probably related to the effects of castration, including long limb‐bones, persistence of epiphyseal lines and osteoporosis. In particular, the frontal bone was affected by severe hyperostosis frontalis interna (HFI). This condition consists in a symmetrical thickening of the inner table of the bone. The epidemiology of HFI shows that it is relatively common in postmenopausal women but very rare in men. Men affected by this pathology suffer from diseases, syndromes or treatments causing androgen deficiency. In the case of Farinelli, castration was probably responsible for the onset and development of this lesion.