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.

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.

Footprint area analysis of binary imaged Cupriavidus necator cells to study PHB production at balanced, transient, and limited growth conditions in a cascade process.

Statistical distribution of cell and poly[3-(R)-hydroxybutyrate] (PHB) granule size and number of granules per cell are investigated for PHB production in a five-stage cascade (5CSTR). Electron microscopic pictures of cells from individual cascade stages (R1-R5) were converted to binary pictures to visualize footprint areas for polyhydroxyalkanoate (PHA) and non-PHA biomass. Results for each stage were correlated to the corresponding experimentally determined kinetics (specific growth rate µ and specific productivity π). Log-normal distribution describes PHA granule size dissimilarity, whereas for R1 and R4, gamma distribution best reflects the situation. R1, devoted to balanced biomass synthesis, predominately contains cells with rather small granules, whereas with increasing residence time τ, maximum and average granule sizes by trend increase, approaching an upper limit determined by the cell's geometry. Generally, an increase of intracellular PHA content and ratio of granule to cell area slow down along the cascade. Further, the number of granules per cell decreases with increasing τ. Data for µ and π obtained by binary picture analysis correlate well with the experimental results. The work describes long-term continuous PHA production under balanced, transient, and nutrient-deficient conditions, as well as their reflection on the granules size, granule number, and cell structure on the microscopic level.

Assessing childhood stress in early mediaeval Croatia by using multiple lines of inquiry.

ABSTRACT: Childhood stress, using both subadult and adult remains from early mediaeval (8th-11th c. CE) sites on the eastern Adriatic coast is analysed in this report. A total of 242 individuals (83 subadults, 69 adult females, and 90 adult males) were assessed for the presence of linear enamel hypoplasia, cribra orbitalia, sub-periosteal new bone formation, and scurvy. In addition, the dietary profile of nine subadults was assessed by the analysis of carbon and nitrogen stable isotopes from bulk collagen. Over three quarters of individuals with preserved permanent dentition (44/56 or 78.6%) exhibited evidence of linear enamel hypoplasia, while analysis of the onset of this condition shows that all defects formed between 1.2 and 5.6 years of age. Cribra orbitalia was identified in 60 out of 190 individuals with preserved frontal bones (31.6%). Sub-periosteal new bone formation was recorded in 42.6% of analysed subadults (29/68) with fourteen cases still active at the time of death. Additionally, scurvy was identified in three subadults. The isotopic study of carbon and nitrogen suggests that diet of the analysed subadults was based on terrestrial C3 resources, with a varying input from C4 and a low intake of marine resources. The presented study strongly indicates that a large majority of the analysed individuals suffered from poor health during their childhood as indicated by the high frequencies of linear enamel hypoplasia, cribra orbitalia and sub-periosteal new bone formation.

Dental health and diet in early medieval Ireland.

OBJECTIVE: With the aim to get a better picture of dental health, diet and nutrition in early medieval Ireland a population-based study focusing on several attributes of oral health in adult individuals was conducted. The study focused on possible differences between sexes and age groups in terms of frequency and distribution of studied pathologies in order to determine whether these differences result from different diets, cultural practices or are age-related. DESIGN: Permanent dentitions belonging to adult individuals from five Irish early medieval sites were examined for the evidence of caries, ante-mortem tooth loss, abscesses, calculus, alveolar bone resorption and tooth wear. All pathologies were analysed and presented by teeth and alveoli. RESULTS: A total of 3233 teeth and 3649 alveoli belonging to 167 individuals (85 males and 82 females) were included into the analysis. Males exhibited significantly higher prevalence of abscesses, heavy wear and alveolar bone resorption, while females exhibited significantly higher prevalence of calculus. All studied dento-alveolar pathologies showed a strong correlation with advanced age, except calculus in females. Additionally, dental wear associated with habitual activities was observed in two females. CONCLUSION: The results of the present study confirm the data gained by written sources and stable isotopes analyses suggesting the diet of the early Irish was rich in carbohydrates with only occasional use of meat. Furthermore, significant differences between the sexes in terms of recorded pathologies strongly suggest different nutritional patterns with females consuming foods mostly based on carbohydrates in comparison to males. The observed sex-differences might also occur due to differences between male and female sex such as reproductive biology and pregnancy, a somewhat different age distributions, but also as a result of different cultural practices between the sexes.

Periodontal diseases at the transition from the late antique to the early mediaeval period in Croatia.

OBJECTIVE: We tested the hypothesis that the transition from the late antique to the early mediaeval period in Croatia had a negative impact on the periodontal health. METHODS: 1118 skulls were examined for dental calculus, alveolar bone resorption, fenestrations, dehiscences and root furcation involvement. RESULTS: The prevalence of teeth with calculus varied from 40.7% in the LA sample of continental parts of Croatia to 50.3% in the LA sample of Adriatic Croatia. The prevalence of alveolar bone resorption ranged between 21.2% in the EM sample from continental Croatia and 32.3% in the LA sample from Adriatic Croatia. The prevalence of individuals with alveolar bone dehiscences varied from 8.6% in the LA sample from continental Croatia up to 15.0% in the EM sample from Adriatic Croatia. The prevalence of individuals with alveolar bone fenestrations varied from 21.5% in the LA sample from Adriatic Croatia up to 36.2% in the LA sample from continental Croatia. The prevalence of individuals with exposed root bifurcations or trifurcations varied from 9.0% in the EM sample from Adriatic Croatia up to 20.7% in the EM sample from continental Croatia. Statistically significant differences were found between samples. CONCLUSION: The transition from the late antique to the early mediaeval period in Croatia did not have a negative impact on periodontal health. Studies of periodontal health of ancient populations should be performed to provide a better and more reliable reconstruction of living conditions in the past.

Health and disease in a Roman walled city: an example of Colonia Iulia Iader.

The paper presents the results of the bioarchaeological study of a Roman period (3rd-5th century) skeletal sample from Zadar, Croatia with the focus on subadult stress indicators (cribra orbitalia and dental enamel hypoplasia) and indicators of non-specific infectious diseases (periostitis). The total frequency of cribra orbitalia, an indicator of iron deficiency anaemia, in Zadar is 20.1%. Half of the subadult skeletons from Zadar exhibit signs of cribra orbitalia, of which two are in active form. Adults not affected by cribra orbitalia lived on average 4.5 years longer than individuals affected by this pathological change. Total frequency of dental enamel hypoplasia in adults is 61.1% with somewhat higher frequency in females. The frequency of periostitis in subadults (66.7%) is significantly higher than in adults (30.4%). A positive correlation was established between cribra orbitalia and periostitis in males. The presented data suggest relatively low quality of life in Roman Zadar, most probably due to the overcrowding inside the walled city which led to deterioration of sanitary conditions and the occurrence of infectious diseases.

Temporal trends in demographic profiles and stress levels in medieval (6th-13th century) population samples from continental Croatia.

AIM: To analyze and compare the demographic profiles and disease frequencies of early (6th-9th century) and late (10th-13th century) medieval skeletal series from continental Croatia. METHODS: Age and sex distributions in three early (n=277) and six late (n=175) medieval skeletal series were compared. All skeletons were analyzed for the presence of dental enamel hypoplasia, periostitis, trauma, and presence of Schmorl s depressions in vertebral bodies. RESULTS: Data collected from the skeletal series suggested significantly higher stress in the late medieval period. This stress may have affected mortality, as evidenced by significantly higher subadult mortality and shorter adult average life span. Men in the late medieval series, in particular, seem to have been under greater stress. They exhibited significantly higher mortality in the 21-25 years age category, and significantly higher frequencies of periosteal lesions, cranial and postcranial trauma, and Schmorl s depressions. CONCLUSION: The frequencies of all skeletal indicators of stress increased significantly during the late medieval period. This was accompanied by a significant increase in subadult mortality and shortening of the average life span of adult men and women.