Lectures Session I: Molecular Studies A1 The 5,300-year-old Helicobacter pylori

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Lectures Session I: Molecular Studies

A1 - The 5,300-year-old Helicobacter pylori genome of the Iceman

Frank Maixner1*, Ben Krause-Kyora2*, Dmitrij Turaev3*, Alexander Herbig4,5*, Michael

R. Hoopmann6, Janice L. Hallows6, Ulrike Kusebauch6, Eduard Egarter Vigl7, Peter Malfertheiner8, Francis Megraud9, Niall O´Sullivan1, Giovanna Cipollini1, Valentina Coia1, Marco Samadelli1, Lars Engstrand10, Bodo Linz11, Robert L. Moritz6, Rudolf Grimm12, Johannes Krause4,5#, Almut Nebel2#, Yoshan Moodley13,14#, Thomas Rattei3#, Albert Zink1#
1 Institute for Mummies and the Iceman, EURAC research, Viale Druso 1, 39100 Bolzano, Italy.

2 Institute of Clinical Molecular Biology, Kiel University, Schittenhelmstr. 12, 24105 Kiel, Germany.

3 CUBE - Division of Computational Systems Biology, Department of Microbiology and Ecosystem Science, University of Vienna, Althanstr. 14, 1090 Vienna, Austria.

4 Institute for Archaeological Sciences, University of Tübingen, Rümelinstr. 23, 72072 Tübingen, Germany.

5 Max Planck Institute for the Science of Human History, Kahlaische Str. 10, 07745 Jena, Germany

6 Institute for Systems Biology, 401 Terry Avenue North, Seattle, Washington 98109, USA.

7 Scuola Superiore Sanitaria Provinciale “Claudiana”, Via Lorenz Böhler 13, 39100 Bolzano, Italy.

8 Department of Gastroenterology, Hepatology, and Infectious Diseases, Otto-von-Guericke University, Leipziger Strasse 44, 39120 Magdeburg, Germany.

9 Université de Bordeaux, Centre National de Référence des Helicobacters et Campylobacters and INSERM U853, 146 rue Léo Saignat, 33076 Bordeaux, France.

10 Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, 141 83 Stockholm, Sweden.

11 Department of Veterinary and Biomedical Sciences, Pennsylvania State University, University Park, Pennsylvania 16802, USA.

12 Robert Mondavi Institute for Food Science, University of California, Davis, California 95616, USA

13 Department of Zoology, University of Venda, Private Bag X5050, Thohoyandou 0950, Republic of South Africa

14 Department of Integrative Biology and Evolution, Konrad Lorenz Institute for Ethology, University of Veterinary Medicine Vienna, Savoyenstr. 1a, 1160 Vienna, Austria

*shared first authorship #shared senior authorship

The stomach bacterium Helicobacter pylori is one of the most prevalent human pathogens. It has dispersed globally with its human host, resulting in a distinct phylogeographic pattern that can be used to reconstruct both recent and ancient human migrations. The H. pylori strains found in most Europeans today (hpEurope) have putatively originated from recombination of the two ancestral populations Ancestral Europe 1 and 2 (AE1 and AE2). However, there exist different hypotheses about when and where the hybridization took place, reflecting the complex demographic history of Europeans.

In this study, we screened biopsy samples from the gastrointestinal tract of the Iceman, a 5300-year-old European Copper Age mummy, for the presence of H. pylori. By using metagenomic diagnostics and targeted genome capture, we determined the presence of H. pylori and reconstructed its complete genome. Comparison with contemporary H. pylori sequences and proteomics analysis classified the ancient H. pylori as a cytotoxic type strain that triggered already calprotectin release as a result of host inflammatory immune responses.

Comparative analysis of ancient housekeeping gene fragments with a global multilocus sequence typing (MLST) database and comparative whole-genome analyses assigned the 5,300-year-old bacterium to the population hpAsia2 today commonly found in Central and South Asia. The “Iceman” H. pylori is a nearly pure representative of the bacterial population of Asian origin (AE1) that existed in Europe before hybridization, suggesting that the African population (AE2) arrived in Europe within the past few thousand years, which is much more recent than previously hypothesized.

Correspondence to: frank.maixner@eurac.edu

A2 - Multi-omics study of the Iceman’s stomach content shows main components of a Copper Age meal: fat, wild meat and cereals.

Frank Maixner1*, Dmitrij Turaev2*, Ben Krause-Kyora3*, Amaury Cazenave-Gassiot4,5*, Marek Janko6,7*, Michael R. Hoopmann8, Mark Sartain8, Gea Guerriero9, Niall O´Sullivan1, Matthew Teasdale10, Giovanna Cipollini1, Alice Paladin1, Marco Samadelli1, Umberto Tecchiati11, Andreas Putzer12, Mine Palazoglu13, John Meissen13, John Baines14, Bum Jin Kim15, Hyun-Joo An15, Eduard Egarter-Vigl16, Peter Malfertheiner17, Andreas Keller18, Robert W. Stark6,7, Markus Wenk4,5, David Bishop19, Dan Bradley10, Oliver Fiehn13, Lars Engstrand, Robert L. Moritz8, Philip Doble19, Andre Franke3, Almut Nebel3, Klaus Oeggl20, Thomas Rattei2#, Rudolf Grimm21#, Albert Zink1#
1 Institute for Mummies and the Iceman, EURAC research, Viale Druso 1, 39100 Bolzano, Italy.

2 CUBE - Division of Computational Systems Biology, Department of Microbiology and Ecosystem Science, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria.

3 Institute of Clinical Molecular Biology, Kiel University, Schittenhelmstrasse 12, 24105 Kiel, Germany.

4 SLING, Life Sciences Institute, National University of Singapore, Singapore

5 Department of Biochemistry, National University of Singapore, Singapore

6 FB Material- und Geowissenschaften, TU Darmstadt, Petersenstrasse 32, 64287 Darmstadt, Germany

7 Center of Smart Interfaces, TU Darmstadt, Petersenstrasse 32, 64287 Darmstadt, Germany

8 Institute for Systems Biology, 401 Terry Avenue North, Seattle, Washington 98109, USA.

9 Environmental Research and Innovation (ERIN), Luxembourg Institute of Science and Technology (LIST), Esch/Alzette, Luxembourg

10 Smurfit Institute of Genetics, University of Dublin, Trinity College, Dublin 2, Ireland

11 Responsabile del Laboratorio di Archeozoologia della Soprintendenza Provinciale ai Beni culturali di Bolzano – Alto Adige, Ufficio Beni archeologica, 39100 Bolzano, Italia

12 South Tyrol Museum of Archaeology, Museumstrasse 43, 39100 Bolzano, Italy

13 Department of Molecular and Cellular Biology & Genome Center University of California, Davis, USA

14 Max Planck Institute for Evolutionary Biology, August-Thienemann-Strasse 2, D-24306, Plön, Germany

15 Cancer Research Institute & Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, Korea

16 Scuola Superiore Sanitaria Provinciale “Claudiana”, Via Lorenz Böhler 13, 39100 Bolzano, Italy

17 Department of Gastroenterology, Hepatology, and Infectious Diseases, Otto-von-Guericke University, Leipziger Strasse 44, 39120 Magdeburg, Germany

18 Chair for Clinical Bioinformatics, Saarland University, Medical Faculty, Saarbrücken, Germany

19 Elemental Bio-imaging Facility, University of Technology Sydney, Broadway, New South Wales, 2007, Australia.

20 Institute of Botany, Sternwarterstrasse 15, University of Innsbruck, 6020 Innsbruck, Austria

21 Robert Mondavi Institute for Food Science, University of California, Davis, California 95616, USA
*shared first authorship #shared senior authorship

Human evolution is closely linked to dietary changes and food processing. This is clearly observed with the transition from hunter-gatherer lifestyle to agriculture, which gave rise to cultivation of crops, animal husbandry and permanent settlements. The more stable availability of food boosted ancient population growth. However, changes in diet had drawbacks for health such as increased rates of caries. Added to this, permanent large settlements with adoption of agriculture promoted the spread of density-dependent infectious diseases. This study aims to reconstruct the dietary choices of ancient populations and the consequences these may have had for their health.

The detection of the Iceman´s stomach content provided the unique opportunity to fully reconstruct the main components of a Copper Age meal. Initial macro- and microscopic analysis revealed that the material is extraordinarily well preserved and contains large amounts of fat residues. By using a combined multi-omics approach targeting biomolecules (ancient DNA, proteins, metabolites, and lipids), we obtained a molecular “fingerprint” of the Iceman’s diet preceding his death.

This presentation will focus on the results obtained by analyzing the DNA, proteins and metabolites detected in the stomach content, while the results of the lipid analysis are presented by Cazenave-Gassiot and co-workers. The molecular data we present shows the presence of four components in the Iceman´s last meal: ibex and red deer meat supplemented with cereals and traces of bracken. Our multi-omics study provides important insights into the general life and nutritional habits of a Copper Age individual in the Alpine area.

Correspondence to: frank.maixner@eurac.edu

A3 - Whole mtDNA sequencing in Alpine populations and the genetic history of the Neolithic Tyrolean Iceman

Valentina Coia1, Giovanna Cipollini1, Paolo Anagnostou2, Frank Maixner1, Alberto G Carballa3, Giovanni Destro Bisol2, Antonio Salas3, Albert Zink1
1 EURAC Institute for Mummies and the Iceman Piazza Angela Nikoletti, 14 Bolzano Italy,

2 University of Rome la Sapienza, Department of Environmental Biology, Italy

3 Universidade de Santiago de Compostela, Unidade de Xenética, Departamento de Anatomía Patolóxica e Ciencias Forenses

The Tyrolean Iceman is an extraordinarily well-preserved natural mummy that lived south of the Alpine ridge ~5,200 years before present (ybp), during the Copper Age. Despite studies that have investigated his genetic profile, the relation of the Iceman´s maternal lineage with present-day mitochondrial variation remains elusive. Studies of the Iceman have shown that his mitochondrial DNA (mtDNA) belongs to a novel lineage of haplogroup K1 (K1f) not found in extant populations. We analyzed the complete mtDNA sequences of 42 haplogroup K bearing individuals from populations of the Eastern Italian Alps - putatively in genetic continuity with the Tyrolean Iceman- and compared his mitogenome with a large dataset of worldwide K1 sequences (> 1070). Our results allow a re-definition of the K1 phylogeny, and indicate that the K1f haplogroup is absent or very rare in present-day populations. We suggest that mtDNA Iceman´s lineage could have disappeared during demographic events starting in Europe from ~5,000 ybp. Finally, we compared our results with published data on mtDNA and Y-chromosome from modern and ancient DNA studies. We propose a scenario that could explain the apparent contrast between the phylogeographic features of maternal and paternal lineages of the Tyrolean Iceman within the context of the demographic dynamics happening in Europe from 8,000 ybp.

Correspondence to: valentina.coia@eurac.edu

A4 - Lipidomics analysis of the Ötzi’s last meal.

Amaury Cazenave Gassiot1, Mark Sartain2, Robert Moritz2, Rudolf Grimm3, Markus Wenk1, Frank Maixner4, Albert Zink4
1 National University of Singapore 28 Medical Drive, Centre for Life Sciences, #03-03 Singapore Singapore,

2 Institute for System Biology, Seattle, U.S.A

3 Agilent Technologies, Santa Clara, U.S.A.

4 EURAC - Institute for Mummies and the Iceman, Bolzano, Italy

Ötzi, the Tyrolean Iceman, was found in the Tyrolean Alps in 1991. One of the oldest natural mummies ever discovered, his body was perfectly preserved for more than 5,300 years in an Alpine glacier. The Iceman is a so-called “wet mummy”: humidity was retained in his cells while he was naturally mummi?ed by freeze-drying. The body tissues are therefore still well preserved, making them suitable for modern scienti?c. Previous analyses of the Iceman’s tissues have provided information regarding his health condition and the way he died.Recently, new radiological analyses led to the discovery of the Iceman’s stomach, which was once thought to have decomposed. New samples were isolated with the aim to shed more light on Ötzi’s lifestyle. To this end, a multi-omics approach was chosen to fully characterise these samples.As part of this multi-omics study, we have conducted lipidomics analysis using state-of-the-art mass spectrometry techniques. Lipids were isolated using well-established organic solvent-based extraction and analysed using liquid chromatography and un-biased high-resolution mass spectrometry on a Q-ToF instrument. The analyses revealed a large number of preserved lipid molecular species, mostly triglycerides. Separate analyses by another team identified very similar profiles. MS/MS analyses revealed that fatty acids esterified to these triglycerides are mostly un- or mono-unsaturated.The Iceman’s stomach content lipid profiles were then compared to contemporary samples from the Tyrolean region, namely muscle and adipose tissues from Capra ibex and Cervus elaphus, and milk and cheese from Capra hircus. Although, species specific identification is not possible from lipid profiles alone, the distribution of triglycerides and their constituting fatty acids is consistent with the consumption of animal muscle and adipose tissue.

Correspondence to: amaury_cazenave@nuhs.edu.sg

A5 - Ötzi meets Nanotechnology - Skin, wound tissue, and stomach content analysis

Marek Janko1, Frank Maixner2, Robert W. Stark1, Albert Zink2
1 Technische Universität Darmstadt Alarich-Weiss-Straße 16 Darmstadt Germany,

2 European Academy of Bolzano, Institute for Mummies and the Iceman, Italy

The Iceman, Europe’s oldest natural mummified human body, has been extremely well-preserved over the millennia.To retrieve additional insights in the preservation of the mummy tissue, into the circumstances of death as well as in dietary habits of the man, two quasi-non-invasive analysis techniques, atomic force microscopy (AFM) and Raman spectroscopy, were used. With these methods, the submicron structure, chemical composition, and nanomechanical properties of small mummified tissue samples were determined.Both techniques were used to analyse the collagen in the Iceman skin, to detect and analyze red blood cells in wound tissue samples and to examine meat remnants from the Iceman stomach content.While AFM revealed intact collagen fibrils, showing the characteristic banding patterns of about 69 nm, Raman measurements showed no significant modifications within the collagen molecular structure. Thus, the micro- and the ultrastructure of the collagen were found to be largely unaltered.Further studies revealed particles with the typical morphology of red blood cells in Iceman wound tissue. Raman spectra approved molecular similarities in comparison to recent red blood cells. Also bands characteristic for fibrin were identified, which suggest that some red blood cells are remnants of a blood clot.Finally, analysing meat from the Iceman stomach content by AFM and Raman spectroscopy allowed for determining the dietary habits of the man. Changes in muscle tissue structure due to heat can be observed microscopically. However, no morphologic and no molecular changes were found in the meat samples extracted from the Iceman stomach content. These results foster the assumption that the meat the Iceman consumed was either raw or dried.

Correspondence to: marekjanko@web.de

A6 - Microbial genomes inside the mummy of the Iceman

Thomas Rattei1, Felix Dunda1, Dmitrij Turaev1, Frank Maixner2, Ben Krause-Kyora3, Albert Zink2,
1 CUBE - Division of Computational Systems Biology, Department of Microbiology and Ecosystem Science, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria

2 Institute for Mummies and the Iceman, EURAC research, Viale Druso 1, 39100 Bolzano, Italy.

3 Institute of Clinical Molecular Biology, Kiel University, Schittenhelmstr. 12, 24105 Kiel, Germany.

The DNA isolated from the mummy of the Tyrolean Iceman is of human, of microbial and (in the intestinal tract) of other origin. Unexpectedly high amount of microbial DNA were even observed in bone and muscle samples. The identity of this DNA, its age and its relation to the life of the Iceman raise many questions. Ancient microbial DNA is likely derived from Iceman’s microbiome and from its bacterial infections. Younger and recent DNA is expected from microbes involved in degradation processes and from the environment.To better understand the microbial DNA from the Iceman mummy we have established and applied a computational workflow for the reconstruction and the analysis of microbial genomes from mummy samples. It is based on assembly and multiple-coverage based binning of whole-genome shotgun sequencing reads, estimation of genome completeness and contamination, analysis of DNA degradation patterns and phenotype prediction. This workflow allowed us to investigate the about 60 most abundant microbial genomes from the mummy. Many of these belong to the genera Clostridium and Pseudomonas and are likely to be involved in the microbial degradation.

Correspondence to: thomas.rattei@univie.ac.at

A7 - Analysis of miRNAs in tissue specimens of the 5,300 year-old Tyrolean Iceman

Andreas Keller
Chair for Clinical Bioinformatics, Saarland University, Medical Faculty, Saarbrücken, Germany

Molecular analyses of ancient specimens have gained increasing importance over the past decades. While most research is carried out in the area of DNA analysis we investigated abundance of small non-coding RNAs, so-called miRNAs, in several specimens of the Iceman. To compare the detected miRNAs to modern tissue patterns we first generated a human miRNA tissue atlas (freely available online at https://ccb-web.cs.uni-saarland.de/tissueatlas/). Then we applied RT-qPCR measurement for seven specimens from the Iceman. Our results suggest that different miRNAs are detected in the different specimens, which partially fit to modern miRNAs in the same tissues. The best example are miRNAs in the Iceman and modern muscle tissue, showing a highly significant overlap (9x10-11). Follow-up studies - also including sequence-based information - will potentially improve our knowledge on miRNAs in ancient specimens further.

Correspondence to: andreas.keller@ccb.ubi-saarland.de

Lectures Session II: Archaeology

A8 - Der Mann aus dem Eis aus archäologischer Sicht. Status quo der archäologischen Forschungsarbeit.

Günther Kaufmann
South Tyrol Museum of Archaeology, Museumstrasse 43, 39100 Bolzano, Ital

Am 19.09.1991 hat das Ehepaar Simon durch Zufall die Mumie am Tisenjoch entdeckt. Nach mehreren Begehungen durch verschiedene Personen wurde vom 3.-5. Oktober 1991 eine erste Nachuntersuchung durch die Universität Innsbruck unter der Leitung von Andreas Lippert durchgeführt. Eine zweite archäologische Nachuntersuchung fand vom 20. Juli bis 25. August 1992 statt, sie oblag dem Amt für Bodendenkmäler in Bozen und wurde von Lorenzo Dal Ri und Hans Nothdurfter sowie Andreas Lippert und Bernardino Bagolini geleitet. Bereits im Juni 1992 hat die Universität Innsbruck ein internationales Symposium zum Thema „Der Mann aus dem Eis“ veranstaltet und im selben Jahr noch den Tagungsband herausgebracht. Darin veröffentlichte Markus Egg erstmals die archäologischen Funde. Nach der Bergung sind die Beifunde in den Restaurierungswerkstätten des Römisch-Germanischen Zentralmuseums in Mainz von Roswitha Goedecker Ciolek restauriert worden. Im Jahr 1995 erschien der Vorbericht von Markus Egg und Konrad Spindler zur Gletschermumie und den Ausrüstungsgegenständen. Letztendlich aus dem Jahr 2009 stammt die große Monographie von Markus Egg und Konrad Spindler zur Kleidung und Ausrüstung der Gletschermumie. Dies sind die Meilensteine der archäologischen Forschung über den Mann aus dem Eis. Dazwischen und danach sind weitere wissenschaftliche Arbeiten erschienen, die sich mit der archäologischen Fundstelle, den Beifunden organischer und nicht organischer Natur befasst haben. Der Vortrag bietet einen Überblick über die archäologische Forschungsgeschichte der letzten 25 Jahre und einen Ausblick auf noch offene Fragestellungen.


Markus Egg, Zur Ausrüstung des Toten vom Hauslabjoch, Gem. Schnals (Südtirol), in: Frank Höpfel / Werner Platzer / Konrad Spindler (Hg.), Der Mann im Eis. Band 1. Bericht über das Symposium 1992 in Innsbruck (Veröffentlichungen der Universität Innsbruck 187), Innsbruck 1992, 254-272.

Markus Egg / Konrad Spindler (mit einem Beitrag von Roswitha Goedecker Ciolek), Die Gletschermumie vom Ende der Steinzeit aus den Ötztaler Alpen – Vorbericht, in: Jahrbuch Röm.-Germ. Zentralmus. Mainz 39/1 (1992), 3-113.

Markus Egg / Konrad Spindler† (mit Beiträgen von Roswitha Goedecker Ciolek und Joachim Lange), Kleidung und Ausrüstung der kupferzeitlichen Gletschermumie aus den Ötztaler Alpen (Monographien Röm.-Germ. Zentralmus. 77), Mainz 2009.

Correspondence to: guenther.kaufmann@iceman.it

A9 - Neuer Hot spot am Tisenjoch? / New hot spot at the Tisenjoch?

Walter Leitner
Universität Innsbruck- Institut für Archäologien Langer Weg 11 Innsbruck Austria,

Immer wieder hat man sich die Frage gestellt ob die Fundstelle am Tisenjoch schon erschöpfend ausgegraben ist. Seit der letzten Ausgrabung im Sommer des Jahre 1992 ist die Felsrinne schon mehrmals zur Gänze ausgeapert, ohne dass weitere Funde zu Tage gekommen sind. Wie aber sieht es mit der unmittelbaren Umgebung aus? Auf Luftbildaufnahmen erkennt man mehrere Schneefelder, die unter Umständen potentielle Suchstellen darstellen könnten. Die sog. Snow-patch-Archäologie könnte hier vielleicht zu einem unerwarteten Ergebnis führen.

On and off the question arose whether the find spot on the Tisenjoch has already been completely excavated. Since the last excavation in summer of 1992, the gully has already been entirely free of snow several times, but no further finds have come to light. But what about the immediate vicinity? On aerial photographs several snowfields can be seen, which could be potential sites for new finds. There the so-called snow-patch archeology could perhaps lead to unexpected results.

Correspondence to: walter.leitner@uibk.ac.at

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