Y Chromosome Story-Ancient Genetic Data as a Supplementary Tool for the Analysis of Modern Croatian Genetic Pool.

Due to its turbulent demographic history, marked by extensive settlement and gene flow from diverse regions of Eurasia, Southeastern Europe (SEE) has consistently served as a genetic crossroads between East and West and a junction for the migrations that reshaped Europe's population. SEE, including modern Croatian territory, was a crucial passage from the Near East and even more distant regions and human populations in this region, as almost any other European population represents a remarkable genetic mixture. Modern humans have continuously occupied this region since the Upper Paleolithic era, and different (pre)historical events have left a distinctive genetic signature on the historical narrative of this region. Our views of its history have been mostly renewed in the last few decades by extraordinary data obtained from Y-chromosome studies. In recent times, the international research community, bringing together geneticists and archaeologists, has steadily released a growing number of ancient genomes from this region, shedding more light on its complex past population dynamics and shaping the genetic pool in Croatia and this part of Europe.

Widespread horse-based mobility arose around 2,200 BCE in Eurasia.

Horses revolutionized human history with fast mobility1. However, the timeline between their domestication and widespread integration as a means of transportation remains contentious2-4. Here we assemble a large collection of 475 ancient horse genomes to assess the period when these animals were first reshaped by human agency in Eurasia. We find that reproductive control of the modern domestic lineage emerged ~2,200 BCE (Before Common Era), through close kin mating and shortened generation times. Reproductive control emerged following a severe domestication bottleneck starting no earlier than ~2,700 BCE, and coincided with a sudden expansion across Eurasia that ultimately resulted in the replacement of nearly every local horse lineage. This expansion marked the rise of widespread horse-based mobility in human history, which refutes the commonly-held narrative of large horse herds accompanying the massive migration of steppe peoples across Europe ~3,000 BCE and earlier3,5. Finally, we detect significantly shortened generation times at Botai ~3,500 BCE, a settlement from Central Asia associated with corrals and a subsistence economy centered on horses6,7. This supports local horse husbandry before the rise of modern domestic bloodlines.

Development of an Integrated Bioprocess System for Bioethanol and Arabitol Production from Sugar Beet Cossettes.

Research background: An innovative integrated bioprocess system for bioethanol production from raw sugar beet cossettes (SBC) and arabitol from remaining exhausted sugar beet cossettes (ESBC) was studied. This integrated three-stage bioprocess system is an example of the biorefinery concept to maximise the use of raw SBC for the production of high value-added products such as sugar alcohols and bioethanol. Experimental approach: The first stage of the integrated bioprocess system was simultaneous sugar extraction from SBC and its alcoholic fermentation to produce bioethanol in an integrated bioreactor system (vertical column bioreactor and stirred tank bioreactor) containing a high-density suspension of yeast Saccharomyces cerevisiae (30 g/L). The second stage was the pretreatment of ESBC with dilute sulfuric acid to release fermentable sugars. The resulting liquid hydrolysate of ESBC was used in the third stage as a nutrient medium for arabitol production by non-Saccharomyces yeasts (Spathaspora passalidarum CBS 10155 and Spathaspora arborariae CBS 11463). Results and conclusions: The obtained results show that the efficiency of bioethanol production increased with increasing temperature and prolonged residence time in the integrated bioreactor system. The maximum bioethanol production efficiency (87.22 %) was observed at a time of 60 min and a temperature of 36 °C. Further increase in residence time (above 60 min) did not result in the significant increase of bioethanol production efficiency. Weak acid hydrolysis was used for ESBC pretreatment and the highest sugar yield was reached at 200 °C and residence time of 1 min. The inhibitors of the weak acid pretreatment were produced below bioprocess inhibition threshold. The use of the obtained liqiud phase of ESBC hydrolysate for the production of arabitol in the stirred tank bioreactor under constant aeration clearly showed that S. passalidarum CBS 10155 with 8.48 g/L of arabitol (YP/S=0.603 g/g and bioprocess productivity of 0.176 g/(L.h)) is a better arabitol producer than Spathaspora arborariae CBS 10155. Novelty and scientific contribution: An innovative integrated bioprocess system for the production of bioethanol and arabitol was developed based on the biorefinery concept. This three-stage bioprocess system shows great potential for maximum use of SBC as a feedstock for bioethanol and arabitol production and it could be an example of a sustainable 'zero waste' production system.

Morphological and evolutionary insights into the keystone element of the human foot's medial longitudinal arch.

The evolution of the medial longitudinal arch (MLA) is one of the most impactful adaptations in the hominin foot that emerged with bipedalism. When and how it evolved in the human lineage is still unresolved. Complicating the issue, clinical definitions of flatfoot in living Homo sapiens have not reached a consensus. Here we digitally investigate the navicular morphology of H. sapiens (living, archaeological, and fossil), great apes, and fossil hominins and its correlation with the MLA. A distinctive navicular shape characterises living H. sapiens with adult acquired flexible flatfoot, while the congenital flexible flatfoot exhibits a 'normal' navicular shape. All H. sapiens groups differentiate from great apes independently from variations in the MLA, likely because of bipedalism. Most australopith, H. naledi, and H. floresiensis navicular shapes are closer to those of great apes, which is inconsistent with a human-like MLA and instead might suggest a certain degree of arboreality. Navicular shape of OH 8 and fossil H. sapiens falls within the normal living H. sapiens spectrum of variation of the MLA (including congenital flexible flatfoot and individuals with a well-developed MLA). At the same time, H. neanderthalensis seem to be characterised by a different expression of the MLA.

Density separation of petrous bone powders for optimized ancient DNA yields.

Density separation is a process routinely used to segregate minerals, organic matter, and even microplastics, from soils and sediments. Here we apply density separation to archaeological bone powders before DNA extraction to increase endogenous DNA recovery relative to a standard control extraction of the same powders. Using nontoxic heavy liquid solutions, we separated powders from the petrous bones of 10 individuals of similar archaeological preservation into eight density intervals (2.15 to 2.45 g/cm3, in 0.05 increments). We found that the 2.30 to 2.35 g/cm3 and 2.35 to 2.40 g/cm3 intervals yielded up to 5.28-fold more endogenous unique DNA than the corresponding standard extraction (and up to 8.53-fold before duplicate read removal), while maintaining signals of ancient DNA authenticity and not reducing library complexity. Although small 0.05 g/cm3 intervals may maximally optimize yields, a single separation to remove materials with a density above 2.40 g/cm3 yielded up to 2.57-fold more endogenous DNA on average, which enables the simultaneous separation of samples that vary in preservation or in the type of material analyzed. While requiring no new ancient DNA laboratory equipment and fewer than 30 min of extra laboratory work, the implementation of density separation before DNA extraction can substantially boost endogenous DNA yields without decreasing library complexity. Although subsequent studies are required, we present theoretical and practical foundations that may prove useful when applied to other ancient DNA substrates such as teeth, other bones, and sediments.

Lorentz-Boost-Driven Magneto-Optics in a Dirac Nodal-Line Semimetal.

Optical response of crystalline solids is to a large extent driven by excitations that promote electrons among individual bands. This allows one to apply optical and magneto-optical methods to determine experimentally the energy band gap -a fundamental property crucial to our understanding of any solid-with a great precision. Here it is shown that such conventional methods, applied with great success to many materials in the past, do not work in topological Dirac semimetals with a dispersive nodal line. There, the optically deduced band gap depends on how the magnetic field is oriented with respect to the crystal axes. Such highly unusual behavior is explained in terms of band-gap renormalization driven by Lorentz boosts which results from the Lorentz-covariant form of the Dirac Hamiltonian relevant for the nodal line at low energies.

An integrative skeletal and paleogenomic analysis of stature variation suggests relatively reduced health for early European farmers.

Human culture, biology, and health were shaped dramatically by the onset of agriculture ∼12,000 y B.P. This shift is hypothesized to have resulted in increased individual fitness and population growth as evidenced by archaeological and population genomic data alongside a decline in physiological health as inferred from skeletal remains. Here, we consider osteological and ancient DNA data from the same prehistoric individuals to study human stature variation as a proxy for health across a transition to agriculture. Specifically, we compared "predicted" genetic contributions to height from paleogenomic data and "achieved" adult osteological height estimated from long bone measurements for 167 individuals across Europe spanning the Upper Paleolithic to Iron Age (∼38,000 to 2,400 B.P.). We found that individuals from the Neolithic were shorter than expected (given their individual polygenic height scores) by an average of −3.82 cm relative to individuals from the Upper Paleolithic and Mesolithic (P = 0.040) and −2.21 cm shorter relative to post-Neolithic individuals (P = 0.068), with osteological vs. expected stature steadily increasing across the Copper (+1.95 cm relative to the Neolithic), Bronze (+2.70 cm), and Iron (+3.27 cm) Ages. These results were attenuated when we additionally accounted for genome-wide genetic ancestry variation: for example, with Neolithic individuals −2.82 cm shorter than expected on average relative to pre-Neolithic individuals (P = 0.120). We also incorporated observations of paleopathological indicators of nonspecific stress that can persist from childhood to adulthood in skeletal remains into our model. Overall, our work highlights the potential of integrating disparate datasets to explore proxies of health in prehistory.

Production of Different Biochemicals by Paenibacillus polymyxa DSM 742 From Pretreated Brewers' Spent Grains.

Brewers' spent grains (BSG) are a by-product of the brewing industry that is mainly used as feedstock; otherwise, it has to be disposed according to regulations. Due to the high content of glucose and xylose, after pretreatment and hydrolysis, it can be used as a main carbohydrate source for cultivation of microorganisms for production of biofuels or biochemicals like 2,3-butanediol or lactate. 2,3-Butanediol has applications in the pharmaceutical or chemical industry as a precursor for varnishes and paints or in the food industry as an aroma compound. So far, Klebsiella pneumoniae, Serratia marcescens, Clostridium sp., and Enterobacter aerogenes are being used and investigated in different bioprocesses aimed at the production of 2,3-butanediol. The main drawback is bacterial pathogenicity which complicates all production steps in laboratory, pilot, and industrial scales. In our study, a gram-positive GRAS bacterium Paenibacillus polymyxa DSM 742 was used for the production of 2,3-butanediol. Since this strain is very poorly described in literature, bacterium cultivation was performed in media with different glucose and/or xylose concentration ranges. The highest 2,3-butanediol concentration of 18.61 g l-1 was achieved in medium with 70 g l-1 of glucose during 40 h of fermentation. In contrast, during bacterium cultivation in xylose containing medium there was no significant 2,3-butanediol production. In the next stage, BSG hydrolysates were used for bacterial cultivation. P. polymyxa DSM 742 cultivated in the liquid phase of pretreated BSG produced very low 2,3-butanediol and ethanol concentrations. Therefore, this BSG hydrolysate has to be detoxified in order to remove bacterial growth inhibitors. After detoxification, bacterium cultivation resulted in 30 g l-1 of lactate, while production of 2,3-butanediol was negligible. The solid phase of pretreated BSG was also used for bacterium cultivation after its hydrolysis by commercial enzymes. In these cultivations, P. polymyxa DSM 742 produced 9.8 g l-1 of 2,3-butanediol and 3.93 g l-1 of ethanol. On the basis of the obtained results, it can be concluded that different experimental setups give the possibility of directing the metabolism of P. polymyxa DSM 742 toward the production of either 2,3-butanediol and ethanol or lactate.

Twenty-first century bioarchaeology: Taking stock and moving forward.

This article presents outcomes from a Workshop entitled "Bioarchaeology: Taking Stock and Moving Forward," which was held at Arizona State University (ASU) on March 6-8, 2020. Funded by the National Science Foundation (NSF), the School of Human Evolution and Social Change (ASU), and the Center for Bioarchaeological Research (CBR, ASU), the Workshop's overall goal was to explore reasons why research proposals submitted by bioarchaeologists, both graduate students and established scholars, fared disproportionately poorly within recent NSF Anthropology Program competitions and to offer advice for increasing success. Therefore, this Workshop comprised 43 international scholars and four advanced graduate students with a history of successful grant acquisition, primarily from the United States. Ultimately, we focused on two related aims: (1) best practices for improving research designs and training and (2) evaluating topics of contemporary significance that reverberate through history and beyond as promising trajectories for bioarchaeological research. Among the former were contextual grounding, research question/hypothesis generation, statistical procedures appropriate for small samples and mixed qualitative/quantitative data, the salience of Bayesian methods, and training program content. Topical foci included ethics, social inequality, identity (including intersectionality), climate change, migration, violence, epidemic disease, adaptability/plasticity, the osteological paradox, and the developmental origins of health and disease. Given the profound changes required globally to address decolonization in the 21st century, this concern also entered many formal and informal discussions.

Screening of Lignocellulolytic Enzyme Activities in Fungal Species and Sequential Solid-State and Submerged Cultivation for the Production of Enzyme Cocktails.

Various fungal species can degrade lignocellulolytic materials with their enzyme cocktails composed of cellulolytic and lignolytic enzymes. In this work, seven fungal species (Mucor indicus DSM 2185, Paecilomyces variotii CBS 372.70, Myceliophthora thermophila CBS 663.74, Thielavia terrestris CBS 456.75, Botryosphaeria dothidea JCM 2738, Fusarium oxysporum f.sp. langenariae JCM 9293, and Fusarium verticillioides JCM 23107) and four nutrient media were used in the screening for effective lignocellulose degrading enzymes. From the seven tested fungi, F. oxysporum and F. verticilliodes, along with nutrient medium 4, were selected as the best medium and producers of lignocellulolytic enzymes based on the determined xylanase (>4 U mg-1) and glucanase activity (≈2 U mg-1). Nutrient medium 4 supplemented with pretreated corn cobs was used in the production of lignocellulolytic enzymes by sequential solid-state and submerged cultivation of F. oxysporum, F. verticilliodes, and a mixed culture of both strains. F. oxysporum showed 6 times higher exoglucanase activity (3.33 U mg-1) after 5 days of cultivation in comparison with F. verticillioides (0.55 U mg-1). F. oxysporum also showed 2 times more endoglucanase activity (0.33 U mg-1). The mixed culture cultivation showed similar endo- and exoglucanase activities compared to F. oxysporum (0.35 U mg-1; 7.84 U mg-1). Maximum xylanase activity was achieved after 7 days of cultivation of F. verticilliodes (≈16 U mg-1), while F. oxysporum showed maximum activity after 9 days that was around 2 times lower compared to that of F. verticilliodes. The mixed culture achieved maximum xylanase activity after only 4 days, but the specific activity was similar to activities observed for F. oxysporum. It can be concluded that both fungal strains can be used as producers of enzyme cocktails for the degradation of lignocellulose containing raw materials, and that corn cobs can be used as an inducer for enzyme production.

Reconstructing genetic histories and social organisation in Neolithic and Bronze Age Croatia.

Ancient DNA studies have revealed how human migrations from the Neolithic to the Bronze Age transformed the social and genetic structure of European societies. Present-day Croatia lies at the heart of ancient migration routes through Europe, yet our knowledge about social and genetic processes here remains sparse. To shed light on these questions, we report new whole-genome data for 28 individuals dated to between ~ 4700 BCE-400 CE from two sites in present-day eastern Croatia. In the Middle Neolithic we evidence first cousin mating practices and strong genetic continuity from the Early Neolithic. In the Middle Bronze Age community that we studied, we find multiple closely related males suggesting a patrilocal social organisation. We also find in that community an unexpected genetic ancestry profile distinct from individuals found at contemporaneous sites in the region, due to the addition of hunter-gatherer-related ancestry. These findings support archaeological evidence for contacts with communities further north in the Carpathian Basin. Finally, an individual dated to Roman times exhibits an ancestry profile that is broadly present in the region today, adding an important data point to the substantial shift in ancestry that occurred in the region between the Bronze Age and today.

Mass violence in Copper Age Europe: The massacre burial site from Potocani, Croatia.

OBJECTIVES: To provide a comprehensive analysis of perimortem cranial injuries found on human remains from the Eneolithic (ca. 4200 BCE) mass grave discovered at Potocani, Croatia, to test if the assemblage is a result of a deliberate violent episode on a massive scale. MATERIALS AND METHODS: Standard bioarchaeological analysis, including inventory of the preserved elements, minimum number of individuals, sex determination, age at death, as well as pattern and distribution of trauma, was recorded. RESULTS: A minimum of 41 people are present in the sample. Both sexes and almost all age groups are represented, with a prevalence of children and young adults. Four blunt force antemortem injuries are registered in three adult males and one subadult while perimortem injuries are recorded on 13 crania with a total of 28 injuries. The distribution of perimortem injuries is not patterned with age, sex, or siding, and their location is on lateral, posterior, or superior parts of the crania. No "defensive wounds" or other type of injuries are observed on postcranial elements. DISCUSSION: The injuries, manner of disposal of the bodies, radiocarbon dates, and other available data strongly suggest that the Potocani sample represents a single episode of execution during which the Potocani people were unable to defend themselves. The Potocani massacre is the oldest such example in southeastern Europe and provides additional evidence that indiscriminate violence on a massive scale is not a product of modern societies.

Genome-wide analysis of nearly all the victims of a 6200 year old massacre.

Paleogenomic and bioanthropological studies of ancient massacres have highlighted sites where the victims were male and plausibly died all in battle, or were executed members of the same family as might be expected from a killing intentionally directed at subsets of a community, or where the massacred individuals were plausibly members of a migrant community in conflict with previously established groups, or where there was evidence that the killing was part of a religious ritual. Here we provide evidence of killing on a massive scale in prehistory that was not directed to a specific family, based on genome-wide ancient DNA for 38 of the 41 documented victims of a 6,200 year old massacre in Potocani, Croatia and combining our results with bioanthropological data. We highlight three results: (i) the majority of individuals were unrelated and instead were a sample of what was clearly a large farming population, (ii) the ancestry of the individuals was homogenous which makes it unlikely that the massacre was linked to the arrival of new genetic ancestry, and (iii) there were approximately equal numbers of males and females. Combined with the bioanthropological evidence that the victims were of a wide range of ages, these results show that large-scale indiscriminate killing is a horror that is not just a feature of the modern and historic periods, but was also a significant process in pre-state societies.

Origins and genetic legacy of prehistoric dogs.

Dogs were the first domestic animal, but little is known about their population history and to what extent it was linked to humans. We sequenced 27 ancient dog genomes and found that all dogs share a common ancestry distinct from present-day wolves, with limited gene flow from wolves since domestication but substantial dog-to-wolf gene flow. By 11,000 years ago, at least five major ancestry lineages had diversified, demonstrating a deep genetic history of dogs during the Paleolithic. Coanalysis with human genomes reveals aspects of dog population history that mirror humans, including Levant-related ancestry in Africa and early agricultural Europe. Other aspects differ, including the impacts of steppe pastoralist expansions in West and East Eurasia and a near-complete turnover of Neolithic European dog ancestry.

Metabolically engineered Lactobacillus gasseri JCM 1131 as a novel producer of optically pure L- and D-lactate.

High-quality environmentally-friendly bioplastics can be produced by mixing poly-L-lactate with poly-D-lactate. On an industrial scale, this process simultaneously consumes large amounts of both optically pure lactate stereoisomers. However, because optimal growth conditions of L-lactate producers often differ from those of D-lactate producers, each stereoisomer is produced in a specialised facility, which raises cost and lowers sustainability. To address this challenge, we metabolically engineered Lactobacillus gasseri JCM 1131T, a bioprocess-friendly and genetically malleable strain of homofermentative lactic acid bacterium, to efficiently produce either pure L- or pure D-lactate under the same bioprocess conditions. Transformation of L. gasseri with plasmids carrying additional genes for L- or D-lactate dehydrogenases failed to affect the ratio of produced stereoisomers, but inactivation of the endogenous genes created strains which yielded 0.96 g of either L- or D-lactate per gram of glucose. In this study, the plasmid pHBintE, routinely used for gene disruption in Bacillus megaterium, was used for the first time to inactivate genes in lactobacilli. Strains with inactivated genes for endogenous lactate dehydrogenases efficiently fermented sugars released by enzymatic hydrolysis of alkali pre-treated wheat straw, an abundant lignocellulose-containing raw material, producing 0.37-0.42 g of lactate per gram of solid part of alkali-treated wheat straw. Thus, the constructed strains are primed to serve as producers of both optically pure L-lactate and D-lactate in the next-generation biorefineries.

Radiographic analysis and virtual cleaning of a bioarchaeological remain enclosed in mineral deposits from a limestone cave.

In limestone caves, environmental processes often cause alterations of human or animal skeletal remains, complicating classical analytical methods. Exemplary, a proximal femoral skeletal fragment, enclosed by a thick layer of speleothemic calcite deposits, was discovered during the exploration of the Bedara cave in Zumberak, Croatia. An examination without removal of the surrounding mineral deposits, possibly leading to damage of the specimen, was, therefore, desirable.We describe and discuss the applied techniques, including clinical computed tomography, virtual cleaning by a specially developed segmentation protocol using an open-source DICOM viewer, and virtual visualisation and dimensioning using computer-aided design software, so that this "hidden" specimen could be non-invasively examined in great detail. We also report on the circumstances and origin of the find, the results of radiocarbon dating, and its anatomical and taxonomic identification, according to which, the bone fragment belonged to a wild boar (Sus scrofa) from the timeframe of the Middle Eneolithic Retz-Gajary culture in the region (4,781 ± 35 years before present). This study provides a reference for future paleontological and anthropological analyses, seeking to unlock the enormous potential of anatomical studies of comparable skeletal remains that are either petrified or enclosed in speleothemic deposits.

The Genomic History of the Bronze Age Southern Levant.

We report genome-wide DNA data for 73 individuals from five archaeological sites across the Bronze and Iron Ages Southern Levant. These individuals, who share the "Canaanite" material culture, can be modeled as descending from two sources: (1) earlier local Neolithic populations and (2) populations related to the Chalcolithic Zagros or the Bronze Age Caucasus. The non-local contribution increased over time, as evinced by three outliers who can be modeled as descendants of recent migrants. We show evidence that different "Canaanite" groups genetically resemble each other more than other populations. We find that Levant-related modern populations typically have substantial ancestry coming from populations related to the Chalcolithic Zagros and the Bronze Age Southern Levant. These groups also harbor ancestry from sources we cannot fully model with the available data, highlighting the critical role of post-Bronze-Age migrations into the region over the past 3,000 years.

Author Correction: The spread of steppe and Iranian-related ancestry in the islands of the western Mediterranean.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Differential DNA methylation of vocal and facial anatomy genes in modern humans.

Changes in potential regulatory elements are thought to be key drivers of phenotypic divergence. However, identifying changes to regulatory elements that underlie human-specific traits has proven very challenging. Here, we use 63 reconstructed and experimentally measured DNA methylation maps of ancient and present-day humans, as well as of six chimpanzees, to detect differentially methylated regions that likely emerged in modern humans after the split from Neanderthals and Denisovans. We show that genes associated with face and vocal tract anatomy went through particularly extensive methylation changes. Specifically, we identify widespread hypermethylation in a network of face- and voice-associated genes (SOX9, ACAN, COL2A1, NFIX and XYLT1). We propose that these repression patterns appeared after the split from Neanderthals and Denisovans, and that they might have played a key role in shaping the modern human face and vocal tract.