Ancient Egyptian embalming substances and organic coatings applied to coffins and burial furnishings served both sacred and functional roles. However, their molecular characterization remains incomplete despite advances in analytical chemistry, highlighting the need for deeper approaches to refine compositional frameworks. This study investigates organic residues from the New Kingdom (2nd millennium BCE) funerary assemblage of Kha (outer bandages and middle and inner coffins), Merit (inner coffin), and a coeval little jar, all housed at the Museo Egizio in Turin, Italy. Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-high resolution mass spectrometry (LC-HRMS) were combined to determine whether the substances were bituminous or resinous and to detail their composition. The analyses revealed resinous compounds, with no detectable evidence of bitumen, and a diverse organic matrix dominated by plant-derived biomarkers. Distinct molecular profiles emerged between body-related materials and coffin coatings, supporting function-specific formulations. Notably, 18-β-glycyrrhetinic acid (liquorice-derived compound) and pinolenic acid (pine nut oil marker) were assigned, providing the earliest molecular evidence to date for the use of liquorice in a New Kingdom funerary context. Overall, these findings reveal function-related funerary preparations and selective ingredient use, underscoring the potential of integrated HRMS workflows to detect unconventional markers and advance the study on ritual practices.

The aim of our paper is to present and discuss in detail the pathological lesions suggestive of tuberculosis observed in a skeleton (MMG3-75) that was excavated from the 16th-century-CE mass grave No. 3 of the Mohács National Memorial Site (Sátorhely, southwestern Hungary). The differential diagnoses of the observed bony changes, with special attention to the clival alterations, are presented. During the macromorphological, radiological, and digital microscopic examination of MMG3-75, the skull base showed mild cortical erosion and multiple, well-circumscribed osteolytic lesions at the clivus clearly evidenced by 3D imaging. In addition, endocranial granular impressions and abnormal blood vessel impressions were observed in multiple locations on the inner skull surface. Based on the differential diagnosis of the clival changes and their co-occurrence with endocranial alterations indicative of tuberculous meningitis (granular impressions and abnormal blood vessel impressions), they were most likely due to tuberculous involvement of the skull base. Additional aDNA analysis provided no evidence for the presence of Mycobacterium tuberculosis DNA in MMG3-75. To the best of our knowledge, MMG3-75 is the first reported archaeological case of tuberculous clival osteomyelitis with associated meningitis, giving us a unique insight into the occurrence of an extremely rare manifestation of tuberculosis in mediaeval Hungary.

The Iceman mummy, a 5300-year-old natural alpine glacier mummy, provides a unique opportunity to study ancient microbial ecosystems. However, disentangling the mummy’s endogenous microbiome from modern environmental contaminants introduced during three decades of conservation remains a significant challenge. By integrating culture-dependent and culture-independent approaches, including amplicon sequencing, shotgun metagenomics and de novo metagenomic assembly, as well as isolate-level genomics, we performed a comprehensive characterization of the Iceman’s microbial landscape. We identified three distinct microbial drivers: endogenous post-mortem succession, ancient glacier-derived relicts, and modern anthropogenic introduction. Metagenomic analysis of internal tissues revealed anaerobic bacteria, including ancient gut taxa, including such as Romboutsia hominis, Clostridium moniliforme, Eubacterium sp., Ruminococcus bromii, Kineothrix sp., Treponema succinifaciens, Enterousia sp., and Huintestinicola butyrica. These taxa, characterized by ancient DNA (aDNA) damage profiles (C to T deamination frequency), show high similarity to ancestral, non-Westernized human gut communities, providing a rare baseline for Copper Age intestinal ecosystems. Conversely, we identified a shift in the external mycobiome, marked by the recent proliferation of psychrophilic yeasts, including Glaciozyma watsonii, Mrakia robertii, Phenoliferia glacialis, and Goffeauzyma sp. While internal bacterial communities remained stable, these external yeast populations showed increased relative abundance and reduced DNA damage signatures between 2010 and 2019, indicating active, modern colonization. Furthermore, strain-level analysis of Pseudomonas sp. 5C2 confirmed that specific environmental strains have successfully colonized the mummy, persisting across multiple tissue sites with minimal genetic divergence. Our study demonstrates that the Iceman is not a static relic but a dynamic biological interface. The coexistence of ancient, endogenous gut microbes and modern, psychrophilic colonizers highlights the potential for ongoing microbial activity even at sub-zero temperatures. These findings underscore that maintaining strict environmental parameters is essential to prevent these specialized microbial communities from transitioning from latent persistence to active microorganisms.

Abstract Streptococcus pyogenes, or Group A Streptococcus (GAS), is a human pathogen responsible for a range of diseases, from mild infections to severe illnesses. Despite its significance in modern clinical settings, little is known about the pathogen’s evolutionary history or its presence in ancient human populations. Here, we present genomic evidence of S. pyogenes in the pre-Columbian Americas. We analysed a tooth from a naturally mummified individual dating to the Late Intermediate Period (1283–1383 cal AD), housed in the National Museum of Archeology (MUNARQ) in La Paz, Bolivia. Mitochondrial DNA analysis confirmed the host’s Native American ancestry. Shotgun metagenomic sequencing and de-novo assembly enabled the near-complete reconstruction of an ancient S. pyogenes genome displaying close similarity to contemporary strains linked to pharyngitis. The genome contains core virulence genes, but prophages lack streptococcal pyrogenic exotoxins. Phylogenetic analyses place the strain at the base of modern S. pyogenes diversity, and Bayesian analyses indicate that most extant lineages diversified globally within the past ~5,500 years. Our results push back the confirmed presence of S. pyogenes in the Americas by several centuries and suggest that the pathogen circulated among Indigenous populations prior to the European contact.

In Kampehl, a village northwest of Berlin, a mummified body is preserved and exhibited in a church annexe. It is said to represent the remains of Christian Friedrich von Kahlbutz (1651-1702), a local nobleman. According to local legend, he swore before a court that his body would not decay if he were the culprit in an alleged case of murder. We offer the first comprehensive investigation of the mummy using anthropological and historical methods. A CT scan of the mummy revealed an intact skeleton without signs of injury or major disease, minimal remnants of inner organs, a pencil inside the thoracic cavity, likely introduced as a hoax, and a coin-shaped metallic object inside the oral cavity. Sex and age of the body match the historical individual, as does radiocarbon dating. Results of palaeogenetic investigations are limited due to poor endogenous DNA quality. Historical research could establish the place of birth of Christian Friedrich von Kahlbutz as likely being near Lüneburg, i.e., outside Brandenburg. He had military training before living the life of a feudal lord in Kampehl, where he married and had twelve children. Even without a name on the coffin, the identification of the mummy with this historical individual seems plausible. There is only indirect evidence for a historical trial for murder against him. All findings speak for natural mummification, which is not unheard of in the region, as is the placing of a coin into the mouth of a deceased individual as burial object.

Fungal mitochondrial genomes are critical for understanding phylogenetics, evolution, and ecology of the Kingdom Fungi, yet they remain underrepresented in public databases. To address this, we developed a workflow to recover mitochondrial genomes from 12,902 fungal short read sequencing data housed in the Sequence Read Archive (SRA) records, assembling complete circular genomes from 2,695 species. This effort expanded fungal mitochondrial genome diversity by nearly 2.3X particularly in understudied phyla such as Mucoromycota (11X increase) and Zoopagomycota (8X increase). The new dataset contains novel yet undescribed mitochondrial genomes at numerous taxonomic levels, including 15 classes, 64 orders, 178 families, and 544 genera. Taxonomic analysis revealed broad ecological representation among the top-assembled species, including human pathogens (e.g., Cryptococcus tetragattii), plant pathogens (e.g., Melampsora larici-populina), edible mushrooms (e.g., Suillus luteus), and industrial fungi. By leveraging the not yet fully exploited SRA sequencing data, this study fills critical gaps in fungal mitochondrial genomics, tripling the currently known mitochondrial genome diversity of the Kingdom Fungi, and provides an extensive resource for phylogenetic and evolutionary research.

The Eastern Italian Alps played a crucial bridging role between Mediterranean and Northern alpine populations since Prehistory. However, few prehistoric individuals from that region have been genomically analysed so far. Among them, the Iceman (Copper Age, 3368-3108 BC) showed a relatively high Anatolian-Neolithic-related ancestry and low Hunter-Gatherers (HGs)-related ancestry. To investigate how the genomic structure of alpine groups varied over time and to contextualize the Iceman, we analysed 47 alpine individuals dated from the Mesolithic (6380-6107 BC) to Middle Bronze Age (1601-1295 BC). The Mesolithic genome reveals genetic admixture between Western and Eastern HGs that occurred from ~13700 - 8300 BC. Most individuals from the Neolithic onwards present a genomic structure resembling that of the Iceman, supporting genetic continuity. Few individuals carry different ancestries, such as the Steppe-related ones appearing ~2400 BC. Finally, the study suggests local and non-local admixture events between HGs and Neolithic farmers from this alpine area.

The Iceman, a 5,300-year-old Copper age individual, was discovered in 1991 in the Italian Alps. The Iceman’s genome was decoded for the first time more than 10 years ago. Advances in sequencing technologies since then have now enabled a research team from the Max Planck Institute for Evolutionary Anthropology and Eurac Research to reconstruct his genome in higher resolution. The results of this recent analysis provide a more comprehensive picture of the Iceman’s genetic history and phenotype.

The Subalyuk hominin remains were uncovered in 1932 in a cave of the same name in the Bükk Mountains, near the village of Cserépfalu in Borsod-Abaúj-Zemplén County, Northern Hungary. The remains represent two individuals, an adult and a young child who have been described in a few publications since their discovery, providing substantial anthropological data and general assessments of their Neanderthal affiliation. They were associated with Late Mousterian industry. Thus, the Bükk Mountains gain importance in the discussion concerning the contribution of East Central European sites to the debate on the peopling history of Europe during the Late Middle to Early Upper Palaeolithic transition. In this paper, we summarize the archaeological and chronological context of the two individuals, and publish the first direct dating results that place them among the Last Neanderthals of Central Europe.

Background:Several computed tomographic studies have shown the presence of atherosclerosis in ancient human remains. However, while it is important to understand the development of atherosclerotic cardiovascular disease (ASCVD), genetic data concerning the prevalence of the disease-associated single nucleotide polymorphisms (SNPs) in our ancestors are scarce. Objective:For a better understanding of the role of genetics in the evolution of ASCVD, we applied an enrichment capture sequencing approach to mummified human remains from different geographic regions and time periods. Methods:Twenty-two mummified individuals were analyzed for their genetic predisposition of ASCVD. Next-generation sequencing methods were applied to ancient DNA (aDNA) samples, including a novel enrichment approach specifically designed to capture SNPs associated with ASCVD in genome-wide association studies of modern humans. Findings:Five out of 22 ancient individuals passed all filter steps for calculating a weighted polygenic risk score (PRS) based on 87 SNPs in 56 genes. PRSs were correlated to scores obtained from contemporary people from around the world and cover their complete range. The genetic results of the ancient individuals reflect their phenotypic results, given that the only two mummies showing calcified atherosclerotic arterial plaques on computed tomography scans are the ones exhibiting the highest calculated PRSs. Conclusions:These data show that alleles associated with ASCVD have been widespread for at least 5,000 years. Despite some limitations due to the nature of aDNA, our approach has the potential to lead to a better understanding of the interaction between environmental and genetic influences on the development of ASCVD.

The early Iron Age (800 to 450 BCE) in France, Germany and Switzerland, known as the 'West-Hallstattkreis', stands out as featuring the earliest evidence for supra-regional organization north of the Alps. Often referred to as 'early Celtic', suggesting tentative connections to later cultural phenomena, its societal and population structure remain enigmatic. Here we present genomic and isotope data from 31 individuals from this context in southern Germany, dating between 616 and 200 BCE. We identify multiple biologically related groups spanning three elite burials as far as 100 km apart, supported by trans-regional individual mobility inferred from isotope data. These include a close biological relationship between two of the richest burial mounds of the Hallstatt culture. Bayesian modelling points to an avuncular relationship between the two individuals, which may suggest a practice of matrilineal dynastic succession in early Celtic elites. We show that their ancestry is shared on a broad geographic scale from Iberia throughout Central-Eastern Europe, undergoing a decline after the late Iron Age (450 BCE to similar to 50 CE).

Analyzing taxonomic diversity and identification in diverse ecological samples has become a crucial routine in various research and industrial fields. While DNA barcoding marker-gene approaches were once prevalent, the decreasing costs of next-generation sequencing have made metagenomic shotgun sequencing more popular and feasible. In contrast to DNA-barcoding, metagenomic shotgun sequencing offers possibilities for in-depth characterization of structural and functional diversity. However, analysis of such data is still considered a hurdle due to absence of taxa-specific databases. Here we present taxonize-gb, a command-line software tool to extract GenBank non-redundant nucleotide and protein databases, related to one or more input taxonomy identifier. Our tool allows the creation of taxa-specific reference databases tailored to specific research questions, which reduces search times and therefore represents a practical solution for researchers analyzing large metagenomic data on regular basis. Taxonize-gb is an open-source command-line Python-based tool freely available for installation at https://pypi.org/project/taxonize-gb/ and on GitHub https://github.com/msabrysarhan/taxonize_genbank. It is released under Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0).

Objective: The aim of this study is to investigate potential evidence of tuberculosis in mummified remains. Materials: The natural mummy of an anonymous friar from the mortuary chapel of the church of Santa Maria della Grazia in Comiso (Sicily) Methods: The mummy was studied through macroscopic examination; tissue sampling was conducted through breaches in the dorsal surface of the thorax. Radiological, histological and immunohistochemical analyses were performed on the pulmonary parenchyma. Results: The mummified remains are those of an adult male approximately 25-45 years old. In the left lung, 7 intra parenchymal calcified nodules were detected. The fibrocalcific nodules showed some lacunae surrounded by fibrous tissue containing amorphous necrotic, most probably caseous, material. Conclusions: These findings are compatible with a chronic infectious-inflammatory disease, likely a calcification of a previous Ghon complex of an apical nodular tuberculosis. Significance: Our study supports the great spread of the disease in the 19th century; a time when it reached its maximum peak in Europe. Limitations: Molecular investigations failed to detect traces of Mycobacterium tuberculosis DNA in the sample. Suggestions for further research: The investigation on the mummies from Comiso is still in progress, and further analyses will potentially provide paleopathological data on this community of Modern Age which could be integrated with historical and archival sources.

Microscopy of mummified visceral tissue from a Medici family member in Italy identified a potential blood vessel containing erythrocytes. Giemsa staining, atomic force microscopy, and immunohistochemistry confirmed Plasmodium falciparum inside those erythrocytes. Our results indicate an ancient Mediterranean presence of P. falciparum, which remains responsible for most malaria deaths in Africa.

By applying Next Generation Sequencing (NGS) methods to ancient human remains, we can learn a lot about our own past. These methods not only allow us to analyse highly fragmented ancient DNA molecules, but also give us a tool for their ancient authentication. An example of what is possible by using NGS methods in paleogenetics is shown here with the “Lady of the Barfüsser Church”, where both ancient human and microbial DNA was studied.

The Tyrolean Iceman is known as one of the oldest human glacier mummies, directly dated to 3350-3120 cali-brated BCE. A previously published low-coverage genome provided novel insights into European prehistory, despite high present-day DNA contamination. Here, we generate a high-coverage genome with low contam-ination (15.33) to gain further insights into the genetic history and phenotype of this individual. Contrary to previous studies, we found no detectable Steppe-related ancestry in the Iceman. Instead, he retained the highest Anatolian-farmer-related ancestry among contemporaneous European populations, indicating a rather isolated Alpine population with limited gene flow from hunter-gatherer-ancestry-related populations. Phenotypic analysis revealed that the Iceman likely had darker skin than present-day Europeans and carried risk alleles associated with male-pattern baldness, type 2 diabetes, and obesity-related metabolic syndrome. These results corroborate phenotypic observations of the preserved mummified body, such as high pigmen-tation of his skin and the absence of hair on his head.

Skeletal remains of two Neanderthal individuals, a 25-35 year-old woman and a 3-4 year-old child, were discovered in a Subalyuk Cave in North-Eastern Hungary. Radiocarbon dating of the female and child remains revealed an age of 39,732-39,076 and 36,117-35,387 cal BP, respectively. Paleopathological studies of these Neanderthal remains revealed probable evidence of skeletal mycobacterial infection, including in the sacrum of the adult specimen and the endocranial surface of the child's skull. Application of PCR amplification to the juvenile cranium and a vertebra gave a positive result (IS6110) for tuberculosis, backed up by spoligotyping. Lipid biomarker analyses of the same two specimens revealed definitive signals for C-32 mycoserosates, a very characteristic component of the Mycobacterium tuberculosis complex (MTBC). A vertebra from the adult provided weak evidence for mycocerosate biomarkers. The correlation of probable skeletal lesions with characteristic amplified DNA fragments and a proven lipid biomarker points to the presence of tuberculosis in these Neanderthals. In particular, the closely similar biomarker profiles, for two distinct juvenile cranial and vertebral bones, strengthen this diagnosis.