Zoonoses are among the greatest threats to human health, with many zoonotic pathogens believed to have emerged following the Neolithic transition. Palaeomicrobiological investigations of the zooarchaeological record hold potential to uncover the reservoirs, host ranges, and host adaptations of zoonotic pathogens in the past, but face challenges in identifying promising specimens and pathogen DNA preservation. We perform palaeopathological and genetic examinations of 346 skeletal elements from domesticated and wild animals collected from 34 Eurasian sites dating across the last six millennia. We identify 116 signatures of 29 ancient (opportunistic) pathogens and find support that palaeopathological lesions provide guidance for specimen selection. For two pathogen species, Erysipelothrix rhusiopathiae and Streptococcus lutetiensis, we confirm their ancient authenticity using phylogenetics, showcasing an approach to explore the relationship between ancient low-coverage genomes and their modern-day relatives. Our work presents a pathway to understanding prehistoric zoonotic diseases by integrating zooarchaeological, palaeopathological, and genetic data.

Human parvovirus B19 (B19V) is an ubiquitously spread, exclusively human pathogen, mainly posing risks to children, as well as pregnant and immunocompromised individuals. Despite evidence of B19V infection of human populations as far back as 7,000 years, the evolutionary history of B19V remains poorly understood. In this study, we present B19V genomic data from the remains of 53 globally distributed individuals spanning more than 8,000 years, including 7 children. Our findings suggest that the most recent common ancestor of all present B19V lineages existed around 12,000 years ago, at the end of the last Ice Age. Additionally, we identified an extinct Eurasian clade that participated in the recombination event that led to the emergence of B19V genotype 2 (GT-2). We date this event to ∼3,200-1,800 BP, potentially in the greater Mediterranean area. Our study shows aspects of how ancient parvovirus variants arose, disseminated, and impacted human health through time.

The infection of humans by the causal agent of Plague, Yersinia pestis , has been attested as far back as 5,500 BP. Although the specific patho-mechanism and ultimate origin of the disease caused by these prehistoric genomes remains unclear, the bacterium spread through Europe, likely during the Late Neolithic to Bronze Age (LNBA). In this study, we analysed 9 genomic samples originating from 8 different human individuals dating to around 4950 cal BP from the site of Grotta della Spinosa, Tuscany, Italy. Metagenomic screening of these samples reveals one individual (GSP013) to be co-infected by Yersinia pestis, Erysipelothrix rhusiopathiae, and Hepatitis B virus (HBV). At least three further individuals from the site were infected with HBV, indicating its wider presence within the community. The phylogenetic placement of Y. pestis in GSP013 shows that this strain is closely related to the earliest LNBA Caucasus genomes of the bacterium, basal to later European diversity. This represents the earliest evidence of Y. pestis in the Italian peninsula (and Southern Europe more widely) to date, predating previously discovered genomes by at least 200 years. Furthermore, we retrieved 60 newly reported ancient genomes of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum from animals and humans, dating back from 8,300 BP to 100 BP. Of these new genomes, 15 of which stem from individuals known to be infected by Y. pestis . This contributes to our understanding of Y. pestis transmission in prehistoric Europe and possible reservoirs, and offers insights into disease dynamics in communities during the 3rd millennium BCE.

Ancient DNA is a valuable data source for the understanding of our past. However, to effectively interpret this data, it is essential to know the age of the samples from which the DNA is obtained. Although the field of palaeogenomics has been recognised for its robust open data sharing practices, dating information associated with analysed samples is not reported consistently across palaeogenomic studies, nor is it included as metadata in most genetic data repositories. Here, we describe the addition of standardised precise dating information for ancient microbial genomes into the AncientMetagenomeDir metadata repository of published ancient metagenomic samples. This extension currently includes dating information for over 700 ancient microbial genomic datasets, of which 333 are dated using historical, contextual, or stratigraphic methods, and 405 are radiocarbon dated. We quantitatively assess the quality of radiocarbon date reporting and find that, despite established reporting conventions, radiocarbon dating information is often reported inconsistently across ancient metagenomic studies. This new resource provides ancient microbial researchers with standardised dating information that facilitates more accurate and consistent analysis of metagenomic sequencing data. The dataset also highlights the need for greater standardisation of radiocarbon date reporting in original publications in order to allow effective reuse of this and future ancient microbial data. ### Competing Interest Statement The authors have declared no competing interest. Max Planck Society, https://ror.org/01hhn8329 Merton College Graduate Archaeology Scholarship The Clarendon Fund SciLifeLab and Wallenberg Data Driven Life Science Program, KAW 2020.0239 European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation Programme, SEACHANGE, grant agreement no. 856488 Leverhulme Trust Fund Early Career Research Fellowship, ECF-2022-532 Swiss National Science Foundation Ambizione grant, PZ00P1_223787 Social Sciences and Humanities Research Council [Doctoral - Vanier Canada Graduate Scholarship, 2024] Pierre Elliott Trudeau Foundation Simon Fraser University Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany´s Excellence Strategy, EXC 2051 – Project-ID 390713860, “Balance of the Microverse” Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), 460129525 (NFDI4Microbiota FlexFund: “EnterArchaeo” Werner Siemens Stiftung (“Paleobiotechnology”) Carolyn Weinberg and the Radcliffe Institute for Advanced Study

Data related directly to the First Plague Pandemic [FPP] (541–750 CE) in the Byzantine Empire has been gathering for over a century. Initially, only textual evidence was used, resulting in varied interpretations. In recent decades, however, a wider range of materials from natural sciences and archaeology has been included in FPP reconstructions. The methods used to analyze and interpret these data differ across disciplines and have varying levels of margin of error. The cross-disciplinary use of such data, still in early development, has produced interpretations that are sometimes debated, mainly due to a lack of familiarity and ongoing communication among relevant fields. Even within the same dataset, like written sources, the persistent use of significantly different approaches has resulted in various FPP reconstructions. Our goal is to change that by reexamining both the original data from the eastern and central Mediterranean from the mid-sixth to the mid-eighth century and the methods behind them. We explore new data and apply methods from a broad range of disciplines to identify gaps and uncertainties and suggest ways to address them.

Salmonella enterica subsp. enterica is an extremely diverse bacterial pathogen causing frequent infections and foodborne disease among human populations. More than 1500 different bacterial strains (serovars) have been described, many with a wide host range. A small number of serovars are adapted to infect specific hosts: of these, serovars Typhi and Paratyphi A, B, and C cause primate-specific systemic infections (typhoid and paratyphoid fever). Although Paratyphi C is one of the rarest human-specific serovars today, it was once widespread, and all ancient Salmonella genomes published to date belong to or are ancestral to this lineage. Here, we present 53 new ancient Salmonella genomes spanning Eurasia and dating between 3500 BCE and 1300 CE. This rich genomic dataset allows us to reconstruct the evolutionary history of this pathogen in unprecedented detail. We identify multiple extinct prehistoric lineages that caused infections throughout Eurasia. Multiple lineage replacement events are observed throughout prehistoric and historic times, and Bayesian phylogenetic analysis is used to date and identify host adaptation events within this lineage. We find that host-adapted sublineages Paratyphi C, Choleraesuis, and Typhisuis continued to evolve host specificity independently from each other. We reconstruct signals of convergent host adaptation in the studied lineages and other host-adapted strains by analysing shared pseudogenes and recurrent gene gain and loss events. This analysis demonstrates a role for host interactions as a particular target of selection, highlighting the gradual adaptation of this S. enterica lineage to humans that coincides with the intensification of animal husbandry in pastoralist and sedentary farming societies. ### Competing Interest Statement The authors have declared no competing interest.

Modern Yersinia pestis genomes show the greatest diversity of the plague pathogen in Central Eurasia. This region is now widely linked to the origins of the Y. pestis lineages responsible for two historic plague pandemics: one starting with the so-called "Justinianic Plague" of the mid-sixth century and the other with the "Black Death" of the mid-fourteenth century. These pandemics have mostly been studied in the Mediterranean region and Europe. Although the beginning of the latter is clearly defined both geographically and temporally, the early spread of the former has received less attention, despite being the focus of several competing hypotheses. Here, we build on recent discoveries of Y. pestis in late antique human remains from Central Eurasia and Europe. These findings identified an early victim of the Y. pestis lineage in Central Eurasia, centuries before it appeared in Europe during the Justinianic Plague. We contextualize these analyses with (I) what we can reconstruct from archaeological, written, and paleoclimate evidence about the demographic, economic, environmental, and mobility (human and animal) histories of the region in the earliest centuries CE, and (II) written evidence for epidemic disease from the region and neighboring areas, which may be linked to the spread of the plague before, during, and after the Justinianic Plague. Specifically, we examine sources to establish and evaluate hypotheses about how, why, and if the plague spread from Central Eurasia, ultimately causing the Justinianic Plague and the "First Plague Pandemic," and how significantly Eurasian populations were impacted over these centuries. Despite extensive source analysis, limited information, especially palaeogenomic data, prevents us from definitively pinpointing the immediate origin of the First Plague Pandemic. Still, most evidence strongly suggests that the Y. pestis lineage originated from Central Eurasia.

Migration of immune cells to sites of inflammation is a critical step in the body's response to infections but also during autoimmune flares. Chemokine receptors, members of the GPCR receptors, are instrumental in directing specific cell types to their target organs. Herein, we describe a highly potent small molecule antagonist of the chemokine receptor CCR6, which came out of fine-tuned structural elaborations from a proprietary HTS hit. Three main issues in the parent chemical series-cytotoxicity, phototoxicity, and hERG, were successfully solved. Biological characterization demonstrated that compound 45 (IDOR-1117-2520) is a selective and insurmountable antagonist of CCR6. In vivo proof-of-mechanism studies in a mouse lung inflammation model using a representative compound from the chemical class of 45 confirmed that the targeted CCR6+ cells were efficiently inhibited from migrating into the bronchoalveoli. Finally, ADMET and physicochemical properties were well balanced and the preclinical package warranted progress in the clinic.

Although dozens of ancient Yersinia pestis genomes and a vast corpus of documentary data are available, the origin and spread of consecutive outbreaks of the Second Plague Pandemic in Europe (14th–18th c.) are still poorly understood. For the majority of ancient genomes, only radiocarbon dates spanning several decades are available, hampering an association with historically recorded plague outbreaks. Here, we present new genomic evidence of the Second Pandemic from 11 sites in England, Estonia, the Netherlands, Russia, and Switzerland yielding 11 Y. pestis genomes with >4-fold mean coverage dating to between 1349 and 1710. In addition, we present a novel approach for integrating the chronological information retrieved from phylogenetic analysis with their respective radiocarbon dates, based on a novel methodology offering more precise dating intervals. Together with a fine-grained analysis of documentarily recorded plague outbreaks, this allows us to tentatively associate all available Y. pestis genomes of the Second Pandemic with historically documented plague outbreaks. Through these combined multidisciplinary analytical efforts, our newly sequenced genomes can be attributed to the Black Death in Cambridge (England), the pestis tertia or pestis quarta in the late 14th century (Estonia), previously unknown branches emerging in the 15th century (Estonia, the Netherlands and England), and a widespread pandemic in Eastern Europe around 1500 (western Russia), which all seem to have originated from one or multiple reservoirs located in Central Europe. While the latter continued to harbour a major Y. pestis lineage at least until the 1630s, represented by new genomes of the Thirty Years’ War plague (Switzerland), another lineage consecutively spread into Europe between the 17th and 18th century from the Ottoman Empire, as evidenced by a genome associated with the Great Northern War plague (Estonia). By combining phylogenetic analysis with a systematic historical reconstruction based on textual sources and an innovative phylogenetically informed radiocarbon modelling (PhIRM), we offer a new groundbreaking interdisciplinary approach that solves several fundamental methodological challenges associated with phylogenetic and spatio-temporal reconstruction of historical pandemics.

Protocol for the purification of extracts, modified from Dabney et al. (2013) PNAS (doi: 10.1073/pnas.1314445110).

The CXCR3 chemokine receptor is a G protein-coupled receptor mainly expressed on immune cells from the lymphoid lineage, including activated T cells. Binding of its inducible chemokine ligands CXCL9, CXCL10, and CXCL11 leads to downstream signaling events and the migration of activated T cells to sites of inflammation. Herein, we report the third part of our CXCR3 antagonist program in the field of autoimmunity, culminating in the discovery of the clinical compound ACT-777991 (8a). A previously disclosed advanced molecule was exclusively metabolized by the CYP2D6 enzyme, and options to address the issue are described. ACT-777991 is a highly potent, insurmountable, and selective CXCR3 antagonist that showed dose-dependent efficacy and target engagement in a mouse model of acute lung inflammation. The excellent properties and safety profile warranted progress in the clinics.

The chemokine receptor CXCR3 allows the selective recruitment of innate and adaptive inflammatory immune cells into inflamed tissue. CXCR3 ligands are secreted after exposure to pro-inflammatory cytokines. Upon binding to CXCR3 ligands, CXCR3 expressing T-lymphocytes migrate toward sites of inflammation and can promote tissue damage. Therefore, antagonizing this receptor may provide clinical benefits for patients suffering from autoimmune diseases characterized by high concentrations of CXCR3 ligands. Herein, we report the second part of our CXCR3 discovery program where we explored the benzimidazolo-thiazole core scaffold. The optimization of potency and the mitigation of an hERG liability are described. Further pharmacokinetic considerations led to the identification of the potent CXCR3 antagonist ACT-672125 (29). The compound showed good physicochemical properties and safety profile. In a proof-of-mechanism model of lung inflammation, ACT-672125 inhibited the recruitment of CXCR3 expressing T cells into the inflamed lung in a dose dependent manner.

Abstract Objectives Contemporary archeological theory emphasizes the economic and social complexity of Eurasian steppe populations. As a result, old notions of “nomadic” cultures as homogenously mobile and economically simple are now displaced by more nuanced interpretations. Large part of the literature on diet and mobility among Eurasian pastoralists is focused on the Bronze and Iron Ages. The underrepresentation of more recent contexts hampers a full discussion of possible chronological trajectories. In this study we explore diet and mobility at Tunnug1 (Republic of Tuva, 2nd–4th century CE), and test their correlation with social differentiation. Materials and Methods We compare demographic patterns (by age‐at‐death and sex) of carbon, nitrogen, and sulfur stable isotope ratios (δ13C, δ15N, and δ34S) among 65 humans and 12 animals from Tunnug1 using nonparametric tests and Bayesian modeling. We then compare isotopic data with data on perimortal skeletal lesions of anthropic origin and funerary variables. Results Our analyses show that: (1) diet at Tunnug1 was largely based on C4 plants (likely millet) and animal proteins; (2) few individuals were nonlocals, although their geographic origin remains unclarified; (3) no differences in diet separates individuals based on sex and funerary treatment. In contrast, individuals with perimortal lesions show carbon and nitrogen stable isotope ratios consistent with a diet incorporating a lower consumption of millet and animal proteins. Discussion Our results confirm the previously described socioeconomic variability of steppe populations, providing at the same time new data about the economic importance of millet in Southern Siberia during the early centuries CE.

The study of human pathogens, their genomes and their evolution has been revolutionized by the introduction of ancient DNA techniques both in the lab and in silico. Today, palaeogenomic research can reconstruct microbial genomes starting from as much as a couple of reads detected during screenings. With every year, the number of organisms and genomes increases, and with it the field’s power to answer a wide range of questions pertaining to the evolution and the dynamics of studied pathogens.

DNA hybridization-capture techniques allow researchers to focus their sequencing efforts on preselected genomic regions. This feature is especially useful when analysing ancient DNA (aDNA) extracts, which are often dominated by exogenous environmental sources. Here, we assessed, for the first time, the performance of hyRAD as an inexpensive and design-free alternative to commercial capture protocols to obtain authentic aDNA data from osseous remains. HyRAD relies on double enzymatic restriction of fresh DNA extracts to produce RNA probes that cover only a fraction of the genome and can serve as baits for capturing homologous fragments from aDNA libraries. We found that this approach could retrieve sequence data from horse remains coming from a range of preservation environments, including beyond radiocarbon range, yielding up to 146.5-fold on-target enrichment for aDNA extracts showing extremely low endogenous content (<1%). Performance was, however, more limited for those samples already characterized by good DNA preservation (>20%-30%), while the fraction of endogenous reads mapping on- and off-target was relatively insensitive to the original endogenous DNA content. Procedures based on two instead of a single round of capture increased on-target coverage up to 3.6-fold. Additionally, we used methylation-sensitive restriction enzymes to produce probes targeting hypomethylated regions, which improved data quality by reducing post-mortem DNA damage and mapping within multicopy regions. Finally, we developed a fully automated hyRAD protocol utilizing inexpensive robotic platforms to facilitate capture processing. Overall, our work establishes hyRAD as a cost-effective strategy to recover a set of shared orthologous variants across multiple ancient samples.

The chemokine receptor CXCR3 is a seven-transmembrane G-protein-coupled receptor (GPCR) involved in various pathologies, in particular autoimmune diseases. It is activated by the three chemokine ligands CXCL9, CXCL10, and CXCL11 and enables the recruitment of immune cell subsets leading to damage of inflamed tissues. Starting from a high-throughput screening hit, we describe the iterative optimization of a chemical series culminating in the discovery of the selective CXCR3 antagonist ACT-660602 (9j). The careful structural modifications during the lead optimization phase led to a compound with high biological potency in inhibiting cell migration together with improvements of the metabolic stability and hERG issue. In a LPS-induced lung inflammation model in mice, ACT-660602 led to significantly reduced recruitment of the CXCR3+ CD8+ T cell in the bronchoalveolar lavage compartment when administered orally at a dose of 30 mg/kg.

Cenerimod is a potent, selective sphingosine 1-phosphate receptor 1 (S1P1) modulator currently investigated in a Phase IIb study in patients with systemic lupus erythematosus (SLE) (NCT03742037). S1P1 receptor modulators sequester circulating lymphocytes within lymph nodes, thereby reducing pathogenic autoimmune cells (including T and B lymphocytes) in the bloodstream and inflamed tissues, making them an effective therapeutic concept for autoimmune disorders. Although the effect of S1P receptor modulators in reducing circulating lymphocytes is well documented, the precise molecular role of the S1P1 receptor on these cell types is not fully understood. In this study, the mode of action of cenerimod on human primary lymphocytes in different activation states was investigated focusing on their chemotactic behavior towards S1P in real-time, concomitant to S1P1 receptor expression and internalization dynamics. Here, we show that cenerimod effectively prevents T and B cell migration in a concentration-dependent manner. Interestingly, while T cell activation led to strong S1P1 re-expression and enhanced migration; in B cells, an enhanced migration capacity and S1P1 receptor surface expression was observed in an unstimulated state. Importantly, concomitant treatment with glucocorticoids (GCs), a frequently used treatment for autoimmune disorders, had no impact on the inhibitory activity of cenerimod on lymphocytes.

Background The human pathogen Haemophilus influenzae was the main cause of bacterial meningitis in children and a major cause of worldwide infant mortality before the introduction of a vaccine in the 1980s. Although the occurrence of serotype b (Hib), the most virulent type of H. influenzae , has since decreased, reports of infections with other serotypes and non-typeable strains are on the rise. While non-typeable strains have been studied in-depth, very little is known of the pathogen’s evolutionary history, and no genomes dating prior to 1940 were available. Results We describe a Hib genome isolated from a 6-year-old Anglo-Saxon plague victim, from approximately 540 to 550 CE, Edix Hill, England, showing signs of invasive infection on its skeleton. We find that the genome clusters in phylogenetic division II with Hib strain NCTC8468, which also caused invasive disease. While the virulence profile of our genome was distinct, its genomic similarity to NCTC8468 points to mostly clonal evolution of the clade since the 6th century. We also reconstruct a partial Yersinia pestis genome, which is likely identical to a published first plague pandemic genome of Edix Hill. Conclusions Our study presents the earliest genomic evidence for H. influenzae , points to the potential presence of larger genomic diversity in the phylogenetic division II serotype b clade in the past, and allows the first insights into the evolutionary history of this major human pathogen. The identification of both plague and Hib opens questions on the effect of plague in immunocompromised individuals already affected by infectious diseases.