MEGA SAMPLES VOL-106
α2,3-linked sialic acid is the main form of platelet sialylation and is commonly linked to the penultimate galactose (Gal) or N-acetylgalactosamine (GalNAc) on complex N-glycans and O-glycans.7 The sialylation level on platelets is reduced in conditions such as cold storage of platelets, sepsis, and a subset of immune thrombocytopenia. 10,11 It has been reported that desialylated platelets express terminal galactose and are cleared by hepatocytes through interactions with hepatic asialoglycoprotein receptor.12-14 This mechanism is considered to regulate normal platelet homeostasis and contributes to thrombocytopenia under pathological conditions. Our recent study revealed that Küpffer cells, rather than hepatocytes, phagocytose desialylated or O-glycan-deficient platelets.7 Glycosylation is also important for megakaryocytopoiesis and platelet production. Mice with constitutive or inducible global loss of O-glycans exhibited thrombocytopenia due to defects in terminal megakaryocyte differentiation and platelet production, demonstrating that Oglycosylation is critical for thrombocytogenesis.15,16
MEGA SAMPLES VOL-106
Purification of megakaryocytes from the bone marrow, megakaryocyte counts of bone marrow paraffin-embedded sections, proplatelet formation assay, and assay of colony-forming unit-megakaryocyte are detailed in the Online Supplementary Data.
Freshly isolated megakaryocytes or platelets were washed with phosphate-buffered saline, and the pellet was re-suspended in cell lysis buffer (Cell Signaling Technology) containing protease inhibitor (1:100 dilution, Cell Signaling Technology). Further details are given in the Online Supplementary Data.
There is a broad consensus that mega-structures represent some kind of public buildings [2, 8, 10, 31] and that they reflect the social organisation and the fused character of Tripolye giant-settlements [8, 10, 11, 32]. However, there is disagreement regarding the actual activities which were performed in such buildings . Partly, this is due to the still very limited evidence related to the function of these buildings from excavations. So far questions concerning the temporal development of such buildings in relation to the formation of mega-sites and their spatial variations within in the large Tripolye distribution area remain unanswered.
The main aim of this paper is to clarify what the mega-structures actually are and how these buildings can contribute to the reconstruction of the social organization in Tripolye settlements and the understanding of the mega-site phenomenon. Before we will discuss these questions in the last section of this article, different aspects need to be explored:
The first Tripolye mega-structures were identified in 2009 during high-resolution magnetic surveys . The application of improved survey techniques led to the discovery of rectangular constructions bigger than normal houses located mainly in non-built spaces of the domestic sites that were not detectable in former magnetic surveys because of their lower resolution.
In Maidanetske, thirteen magnetic features distributed across the entire settlement were identified as mega-structures according to the above mentioned three criteria (Fig 1). Mega-structure 3 was chosen for excavation in 2016 to investigate a representative mega-structure within the ring-corridor of the settlement. For comparison of mega-structures with domestic houses, the dwellings 44 and 59 from Maidanetske were used, which were excavated in the same manner [34, 37].
The evaluation of the architectural remains, distribution patterns of daub, and negative imprints of timbers allows the reconstruction of the general building design of the investigated mega-structure 3. This evaluation was based on the field documentation which included excavations plans, photos, and the recording of location and masses of daub and movable finds either with point coordinates or in a grid of 1 x 1 m cell size. During excavation, daub was systematically documented to reconstruct both architectural attributes and wood resources [37, 45, 46]. This documentation included the mapping of daub fragments and a typological classification of daub pieces based on their material attributes and on the direction and dimension of imprints of structural (wooden) elements. To determine the architectural construction, the location of wood imprints was mapped differentiated according to the diameter of logs and the width of split wood planks.
Mapping of artefact distributions was performed in order to identify activity zones within the excavated mega-structure 3. Refitting of ceramic vessels and mapping of pottery fragmentation were used for the consideration of taphonomic formation processes and interpretations of primary and secondary artefact distributions.
Qualities and quantities of objects which were associated with the mega-structure and two normal dwellings provide an estimate for reconstructing activities performed related to these different objects. Generally, similar formation processes of find assemblages are assumed for the compared different types of buildings. Specifically, we assume that possible behavioural differences during abandonment of the houses and the mega-structures are balanced through the inclusion of materials from probably waste disposal areas in the surrounding of the buildings. Additionally, the degree of fragmentation of pottery is used to evaluate the questions if vessels were found in primary or secondary waste disposal contexts.
For botanical macro-remains 214 samples of sediment of 10 litres each were taken in the trench 111 (198 of these samples come from the stratigraphy of the mega-structure 3). Systematic sampling was carried out grid-wise and intensified for every second quadrant (1 m2) for the ancient soil surface. Flotation has been carried out on these samples, using a metal sieve of 300 μm, as previously done for the other samples from the site [45, 47]. Overall 414 charred botanical finds have been retrieved from trench 111, but preservation conditions allowed the taxonomic identification of only 305 of those. The carbonised remains found in trench 111 (S1 Table) include cereal grains (Triticum sp., Hordeum sp., mostly cereals indet.), pulses (Fabaceae) and feather-grass awns (Stipa sp.).
The use group sizes of mega-structures were quantified based on the numerical ratio between mega-structures and dwellings. Therefore, the key-value of the estimates is the determination of the number of dwellings that belonged in purely arithmetical terms to a single mega-structure. Within settlements, we assume the rough contemporaneity all low-level mega-structures visible in the magnetic plans, since this is clearly indicated by the uniform distribution of the buildings. The population estimates are based on the average floor space of 7 m2 per person determined using a cross-cultural ethnographic data-set . For each site the average size of residential buildings was taken into account. We are aware that the values calculated in this schematic way of quantification provide only maximum use group sizes since the contemporaneity of the houses is assumed in the most cases. Nevertheless, the calculations are suitable for identifying regional and chronological trends regarding the size of use groups.
Key parameters of the regional sample of mega-structures such as frequency, architectural design, positioning in settlements, and dimensions were analysed with regard to their variability in space and time. For mega-structure 3 from Maidanetske, the basis for the chronological investigations are 14C data calibrated and analysed using the software OxCal v. 4.3.2 [49, 50]. In the case of the regional data set, 14C calibrations of already established Tripolye periodization schemes were used [51, 52].
In the following section, the architectural design and the internal organization of an average mega-structure in Maidanetske will be evaluated. The extent to which the architecture and spatial organization of this building differs from normal houses is then examined by comparison with houses 44 and 59 from Maidanetske [34, 36, 37]. The extent to which the findings made for Maidanetske mega-structure 3 are representative is finally evaluated on the basis of comparisons with mega-structures in magnetic plans and other archaeologically examined buildings.
Mega-structure 3 in trench 111 was chosen for excavation because the magnetic plan displayed both highly magnetised and low magnetised sections within a clearly demarcated area. The different parts were hypothetically interpreted as roofed and not-roofed areas for different activities. During the excavation it turned out that the area was used for settlement activities already before the construction of the mega-structure.
Overall, the dates fall into a plateau of the calibration curve and the following steep section, covering a long range of about 300 years between 3950 and 3650 BCE. Through application of Bayesian modelling and the use of the function boundary with the assumption of two successive occupation phases and several events, the range of the dates becomes significantly narrowed, roughly into the 38th century BCE (Fig 3 and S1 File). However, the overall probability of this model amounts to only 40% (Amodel = 33.8) due to widely identical dates from the different phases. Higher overall probabilities of more than 100% can only be obtained through exclusion of the potential (too old) outliers Poz-87605, Poz-87609, and Poz-87610. The dating results imply that mega-structure 3 was constructed during phase 3 of the site chronology suggested by Ohlrau . Consequently, mega-structure3 from Maidanetske was build related to the rapid population increase of the 38th century and abandoned at the beginning of phase 4, related to the starting population decrease.
In the magnetic plan of the Maidanetske settlement, the architectural remains of mega-structure 3 appeared as a northwest-southeast aligned anomaly with a floor size of approximately 190 m2 (dimensions 19 x 10 m) (Fig 4). Trench 111, opened over this anomaly, measured 23 x 15 m and the daub package of mega-structure 3 was encountered buried under a Chernozem 0.5 m thick. 041b061a72