We expanded our analysis to include previously published data from Maracá and Marajó Island, in the eastern Amazon. Quantitative estimates of the caloric contributions from food groups and their relative nutrients using a Bayesian Mixing Model revealed distinct subsistence strategies, consisting predominantly of plants and terrestrial mammals and variably complemented with aquatic resources. This study offers novel quantitative information on the extent distinct food categories of polyculture agroforestry systems fulfilled the caloric and protein requirements of Late Holocene pre-Columbian populations in the Amazon basin.Archaeological research is radically transforming the view that the Amazon basin and surrounding areas witnessed limited societal development before European contact. Nevertheless, uncertainty remains on the nature of the subsistence systems and the role that aquatic resources, terrestrial mammalian game, and plants had in supporting population growth, geographic dispersal, cultural adaptations and political complexity during the later stages of the pre-Columbian era. This is exacerbated by the general paucity of archaeological human remains enabling individual dietary reconstructions. Here we use stable carbon and nitrogen isotope analysis of bone collagen to reconstruct the diets of human individuals from São Luís Island (Brazilian Amazon coast) dated between  Recent studies reveal a complex picture during the Late Holocene, marked by increased archaeological visibility associated with groups producing distinct material cultures and engaging in diversified subsistence strategies2 with extensive transformation of soils3, vegetation composition4, and landscapes7.Archaeological investigations in lowland South America are radically transforming our understanding of long-term socio-ecological systems in the Neotropics and dismissing the view that the Amazon basin witnessed limited socio-economic development in pre-Columbian times9, with substantial temporal and spatial variability across the basin2. Evidence from coastal areas is relatively limited, but equally diversified. Plant cultivation with extensive raised fields has been documented in seasonally flooded savannahs of the northern Amazon (The Guianas)10, while fishing-based economies supported by water management systems, complemented with plant gathering and possibly some cultivation, occurred in some areas of the eastern Amazon (Marajó Island)12. Numerous shell middens along the coast suggest that food security was also achieved through the exploitation of estuarine and marine resources by groups producing some of the earliest ceramic artefacts in Amazonia13.During the Late Holocene specialized land-use systems based on agriculture or the management of aquatic resources coexisted with more versatile subsistence strategies founded on polyculture agroforestry8, attempts to comprehend their impacts on individual diets have been comparatively limited17. Moreover, while the role of agriculture in supporting population growth and the establishment of sedentary settlements in the recent pre-Columbian era has been the subject of contentious debate11, the importance of aquatic resources has received comparatively little attention18. Aquatic resources are often invoked as economically important food items to both coastal and riverine populations22, but their relative contribution to diet is poorly understood and surrounded by controversial generalizations26. Uncertainties also remain regarding the extent to which plants  and terrestrial animals fulfilled the energy requirements of specialized and mixed subsistence systems. Their potential intensification and role in sustaining dense populations during the later stages of the pre-Columbian era is still a matter of debate2. Stable carbon and nitrogen isotope analysis of bone collagen is a well established approach for deriving dietary information from human, faunal, and plant remains29 and has been employed to reconstruct pre-Columbian subsistence economies across much of South America32. However, due to the paucity of archaeological human remains there have been limited applications on past populations in the Amazon basin until now17, with most studies lacking direct and robust chronological information, as well as contextual isotopic baselines15.Despite the growing interest in the origin and changing nature of pre-Columbian economiesca. 1800 to 1000 cal BP and all were recovered from archaeological contexts associated with distinct ceramic traditions, along with terrestrial mammal, fish, and shellfish remains. The proportional contributions of fish, terrestrial mammals and plants to diet were estimated using Bayesian Stable Isotope Mixing Models (BSIMMs), which provide quantitative estimates of relative caloric and protein contributions from available food groups. Pre-Columbian human remains are rare in the Amazon basin and the obtained results offer the most robust and chronologically secure information on individual diets in pre-Columbian lowland Amazonia to date.Here we offer a novel contribution to unravel pre-Columbian diets in lowland Amazonia and specifically the role of fish as a source of dietary protein. We analyzed the stable carbon and nitrogen isotope composition of bone collagen from five human individuals from archaeological sites  on São Luís Island, located off the Brazilian Amazon coast Fig. . Four inSix human individuals were recovered from the archaeological shell middens of Bacanga n = 1), Paço do Lumiar (n = 1) and Panaquatira (n = 4). The individuals were fragmented and mostly disarticulated, preventing accurate sex and age identification. Four individuals were directly radiocarbon dated using AMS, with the calibrated ages corrected for the average relative contribution of marine carbon to collagen estimated by the BSIMMs  was found in direct association with a ceramic vessel from the Mina tradition, faunal remains, and blocks of ferruginous rocks, and was directly dated to 1840–1700 cal BP (2σ). At Paço do Lumiar, one adult individual (PDL2), dated to 1190–990 cal BP (2σ), was found at the base of the shell midden, with ceramic fragments stylistically comparable to Incised Rim and Mina traditions. At Panaquatira (PNQ), a double burial with one adult (PNQ1A) and one infant (PNQ1B) was found within a ceramic funerary urn with Polychrome decoration, surrounded by stones and shells, emerging from a superficial dark soil overlying the shell midden. The adult (PNQ1A) was dated to 1830–1620 cal BP (2σ). Another adult individual (PNQ2) was found disarticulated and buried with shells and stone tools in a funerary urn with Polychrome decoration, in a dark soil covering the shell midden. This individual did not produce collagen for radiocarbon dating or stable isotope analysis. Another adult individual (PNQ3) was found disarticulated at the base of the shell midden in association with ceramic fragments of Mina tradition and shell beads, and was dated to 1710–1540 cal BP (2σ).13C and δ15N values of terrestrial mammals (n = 12) ranged from − 21.1 to − 19.1 ‰ and from + 4.0 to + 9.4 ‰, respectively  were statistically distinguishable from terrestrial mammals  and ranged from − 12.3 to − 8.9 ‰ and from + 7.5 to + 12.5 ‰, respectively. Some of the remains could be identified as catfish (Ariidae), which is a common taxa in the region35. The high δ13C values observed are typical of fish from marine ecosystems36 and are also comparable with other marine taxa from the southwestern Atlantic Ocean37. Our marine samples show δ13C values considerably higher than modern fish from mangrove environments along the Amazon coast38, suggesting a larger contribution of marine phytoplankton to PNQ’s marine food web. We exclude that fish δ13C values at PNQ could reflect the input of C4 aquatic macrophytes since their contribution to fish diet is negligible in this region39. Fish δ15N values reflect diets based on a mixture of benthic organisms  and detritus38.Faunal material was collected from the site of Panaquatira (PNQ). The δals n = 1 ranged fδ13C and δ15N values from São Luís Island varied considerably within and between taxa  were corrected for the Suess effect (+ 2.13 ‰) (see Methods for details). Maize (Zea mays) had δ13C and δ15N values ranging from − 9.1 to − 8.2 ‰ and from + 2.3 to + 6.3 ‰, respectively. Manioc and macaxeira (both Manihot esculenta) and sweet potato (Ipomoea batatas) had comparable δ13C values, from − 25.3 to − 24.0 ‰, but contrasting δ15N values. A single sweet potato had a δ15N value of + 9.2 ‰, while manioc tubers displayed a larger variability, ranging from − 1.9 to + 6.0 ‰. The reason for the differences in δ15N values among manioc and macaxeira samples is unclear but may arise from the differential use of fertilizers and/or cultivation of macaxeira in soils with high 14N volatilization or mineralization in the context of swidden agriculture40.Modern plant δ13C values for human individuals from São Luís Island ranged from − 16.4 ‰ (PNQ1A and PNQ3) to − 18.3 ‰ (BC1)  to + 12.4 ‰ (PNQ3). There were no substantial differences in the δ15N values between adults and infants, and the lack of sex identification prevented further consideration of the sex and gender dimensions of diet. Nevertheless, it is worth noting that δ15N values for the two infants (PNQ1B and BC1) could also reflect some level of breastfeeding effect41, implying that some young-adult lactating women had even lower δ15N values compared to those reported here. Overall the isotope results indicate mixed subsistence strategies with substantial contribution of C3 plants, terrestrial mammalian game, marine fish and possibly maize .The C1) Fig. . The δ15δ13C and δ15N values consistent with mixed contributions from freshwater fish, C3, and C4 plant protein12. The precise chronology of these samples is unknown12, but they may be attributed to stratified chiefdom-level societies (Marajoara Phase) which supposedly intensified the exploitation of freshwater resources at ca. 1600–650 BP, a process that supported or catalyzed considerable social complexity around the control of aquatic resources in the region43. Freshwater fish was also frequently consumed by Late Holocene individuals (n = 17) from Gruta das Caretas and Gruta do Pocinho in Maracá, on the north bank of the Amazon river, dating to ca. 500 BP. The bulk collagen δ13C and δ15N values for these individuals were interpreted as resulting from diets based on freshwater resources, together with C3 plants, and possibly also some contributions from C4 or CAM crops15.The stable isotope values for the individuals from São Luís Island show significant differences compared to Late Holocene pre-Columbian groups that inhabited the eastern Amazon basin Fig. . A numbeδ13C values at São Luís Island were significantly higher compared with those from the Maracá region , but not significantly different from those of Marajó Island . Differences between São Luís Island and Maracá can be attributed to the consumption of marine resources at São Luís Island. The high δ13C values observed at Marajó Island, instead, could indicate the contribution of maize to dietary proteins12. The δ15N values at São Luís were significantly higher compared to both Maracá  and Marajó Island , suggesting a greater consumption of terrestrial or aquatic animal protein at São Luís and/or regional isotopic differences in resource baselines44. These mixed dietary regimes, large variations in the macronutrient compositions of foods, and uncertainties in food isotopic values and in diet-to-consumer isotopic offsets limit the use of simple linear mixing models. The multiple sources of uncertainty can be more efficiently handled by employing Bayesian Stable Isotope Mixing Models (BSIMMs) that express dietary contribution probability distributions47. To estimate the caloric contributions for the São Luís individuals from four food groups  and for both Maracá region and Marajó Island  we employed the Bayesian mixing model FRUITS45. There is no evidence that individuals from both Maracá region and Marajó Island exploited marine resources, however freshwater fish remains were reported for the site of Teso dos Bichos (Marajó Island), along with plants and a few terrestrial mammals15. The model in FRUITS accounts for the uncertainties in consumer and food isotopic values, macronutrient composition, diet-to-consumer isotopic offsets, and dietary routing. The latter accounts for non-protein contributions (carbohydrates and lipids) to consumer δ13C collagen, necessary for higher-accuracy dietary estimates48. Two dietary estimates were generated: the relative caloric contribution from each food group, and the relative caloric contribution from the protein-only component of each food group. These two estimates differ since the macronutrient composition of the food groups varies. Animal food groups typically have a considerably higher protein content when compared to plant foods, thus, caloric contributions may be predominantly from plants while animal sources simultaneously constitute the main source of protein. In the following sections, caloric estimates/contributions refers to food caloric contributions and protein estimates/contributions refers to the caloric contributions from the protein-only component of the food groups .In general, the 3 plants , followed by maize  and marine fish   and/or C3 plants , and then maize  , followed by marine fish , C3 plants  and/or maize , with considerable overlap among estimates. Freshwater fish/reptiles offered the smallest contribution of all food groups . It is worth noting that one individual  had maize as the prevailing source of calories (average 34 ± 14%). Terrestrial mammals  and/or freshwater fish/reptiles  were the main sources of dietary protein, although with estimates overlapping considerably for most of the individuals. These were then followed by maize  and C3 plants .For individuals from Marajó Island, FRUITS estimates assigned the majority of dietary calories to terrestrial mammals , and/or C3 plants , with some overlap in the estimates. These were followed by maize  and freshwater fish/reptiles , although for some individuals these two food groups also overlapped considerably. Dietary proteins were obtained from freshwater fish/reptiles  and/or terrestrial mammals , followed by contributions from maize  and C3 plants .Similarly, the probability distributions generated by FRUITS for individuals from the Maracá region revealed that the majority of dietary calories were sourced from terrestrial mammals  and/or Cδ13C and δ15N values using a BSIMMs approach, reveal distinct adaptive strategies in the eastern Amazon basin during the Late Holocene. The differential dietary contributions from terrestrial mammal and C3 plants at São Luís Island could not be efficiently resolved by BSIMMs due to the isotopic similarity of these sources and the isotopic equifinality resulting from the mixed contributions from all food groups. However, model estimates adequately distinguished the relative contribution of marine fish from the terrestrial food groups . Despite the proximity to marine resources and contextual zooarchaeological evidence for fishing, model estimates reveal that terrestrial mammals provided greater amounts of dietary proteins to individuals at São Luís Island compared to marine fish. These results call into question the widespread assumption that aquatic resources  were the main economic component, or source of protein, to pre-Columbian populations living in proximity to productive aquatic environments in lowland Amazonia24. However, it is necessary to acknowledge that only a small number of samples were analysed.The results from São Luís Island, along with the review of previously published 12, freshwater fish dominated faunal remains at the site of Teso dos Bichos on Marajó Island, while remains of terrestrial mammalian game were rare. However, for most of the sites on Marajó Island and in the Maracá region there is no available information on faunal remains15. This lack of contextual faunal and botanical data, along with uncertainties associated with the local isotope baselines, limit the possibility of obtaining precise dietary estimates. Model estimates, however, do indicate that the majority of caloric contributions were from terrestrial mammals and plants, and not from freshwater resources. While fish may have been a dominant source of protein, our results suggest that considerable investments were allocated to hunting, forest management, and plant cultivation, as shown by other lines of evidence across the Amazon basin49.Freshwater fish/reptiles and/or terrestrial mammals provided the majority of dietary protein to populations located at the mouth of the Amazon river (Marajó Island and Maracá), but uncertainties remain on the relative contributions from these two food groups. According to Roosevelt4 crop in the Amazon by the Late Holocene50, was not a staple for the sampled individuals, but was likely consumed within the broad spectrum of resources exploited by distinct agroforestry systems. The relatively high consumption of C4 plants (possibly maize) by some individuals at Marajó Island is significant though, and may reopen a contentious debate on the role that plant cultivation had in supporting the emergence of political complexity during the Marajoara Phase12. While we recognize that dietary estimates can be further refined with increasing control of contextual faunal and plant baselines51 and isotopic analyses of single amino acids29, the results presented herein constitute the most robust source of information on individual diets in the pre-Columbian Amazon and corroborate the growing consensus that diversified subsistence economies fuelled cultural, demographic and environmental transformations in the eastern Amazon basin during the Late Holocene.Maize, a widespread C2 and is part of the Maranhense Gulf geological-geomorphological feature which forms a large and complex estuarine system delimited by the bays of São Marcos and São José  and rainy (June to December) seasons, with species from the Amazon forest, caatinga, and cerrado biomes, and extensive restinga and mangrove ecosystems along the coast which are important nurseries for aquatic organisms53. The coastal environment is highly dynamic and shows relatively high biological productivity and biomass with limited seasonal variability due to the combination of semidiurnal macro-tidal regimes, mangrove vegetation, and freshwater input (notably in São Marcos Bay)35. This has favoured fish and shellfish exploitation by groups on the island since at least the Middle and Late Holocene54, during the spread of ceramic artefacts associated with the Mina tradition. The archaeological shell midden of Bacanga contains faunal evidence of fishing in marine and estuarine environments at that time, including taxa of different trophic positions . Marine resources were also exploited by groups using ceramic vessels similar to the Amazonian Incised Rim and possibly the Polychrome tradition during the Late Holocene, the latter associated with dark soils visibly similar to Amazonian Dark Earths55. Evidence of fishing and shellfish collection at such sites are better represented at the shell middens of Paço do Lumiar and Panaquatira55. Fishing is well documented historically among local Tupinambá groups in the seventeenth century58 and currently has major socio-economic and cultural importance on the Island59.São Luís Island, in the state of Maranhão, is approximately 832 kmosé Fig. . Lying iBrandi e Bandeira Consultoria Cultural and the permits for stable isotope analyses were obtained from the Instituto do Patrimônio Histórico e Artístico Nacional . Rib bones were used for collagen extraction from all six human individuals. The regional faunal isotopic baseline was established with bone collagen of fish (n = 11) and terrestrial mammal (n = 12) remains selected from the dark soil overlying the shell midden of PNQ. Faunal samples were from a range of different skeletal elements and only a few terrestrial species could be taxonomically identified, including agouti (Dasyprocta punctata), brocket deer (Mazama gouazoubira), lowland paca (Cuniculus paca), and cavy (Kerodon rupestris). These are predominantly herbivorous animals, feeding on leaves, fruits, tubers, flowers, and bark. Only a few fish remains could be identified as catfish (Ariidae)  and sweet potato (Ipomoea batatas) , and maize  were acquired from a local small scale market by A. Bandeira at São Luís and Baixada Maranhense in 2018. Before sending the samples for isotope analysis at the University of York, the samples were registered in the Sistema Nacional de Gestão do Patrimônio Genético e do Conhecimento Tradicional Associado  according to article 22 of Decreto nº 8.772, of 11 May 2016. These crops are known to have been cultivated by pre-Columbian groups in lowland Amazonia60 and may have constituted a substantial source of dietary energy to populations on São Luís Island (Supplementary information Modern plants n = 11) were sampled and analyzed for their stable carbon and nitrogen isotope compositions to complement the faunal isotopic baseline. Major neotropical crops including manioc  demineralized in a 0.6 M HCl solution at 4ºC for 12—72 h. Samples were then rinsed with deionized water (milli-Q®) and immersed in 0.001 M HCl at 80 °C for 48 h. For most samples the supernatant containing the collagen was filtered using Polyethylene Ezee filters . The samples were then filtered using 30 kDa Amicon® Ultra-4 centrifugal filter units , frozen for 24–48 h at − 20ºC, lyophilized, and weighed into tin capsules (1 mg) for stable isotopic analysis.Collagen was extracted at the BioArCh facilities of the University of York (UK) following the procedure reported inPlant samples, including tubers of manioc and macaxeira (n = 5) and sweet potato (n = 1), and maize kernels (n = 5), were rinsed in deionized water (milli-Q®), dried and frozen for several hours at − 20 ºC. Samples were lyophilized, homogenized using an agate pestle and mortar, and weighed into tin capsules (2 mg) for bulk stable carbon and nitrogen isotopic analysis.δ13C, δ15N = (Rsample/Rstandard)-163, where R = 13C/12C and 15N/14 N. At York the overall uncertainties on the measurements of each sample were calculated based on the method of Kragten64 by combining uncertainties in the values of the international reference materials and those determined from repeated measurements of samples and reference materials (these are expressed as one standard deviation). Caffeine (IAEA-600), ammonium sulphate (IAEA-N-2), and cane sugar (IA-Cane) international standards were used as reference material in each analytical run. In addition, a homogenised bovine bone was extracted and analysed within the same batch. The in-house collagen standard (bovine control) was also exchanged between laboratories (University of York and University of Bradford) to ensure accuracy.The collagen samples of four human individuals  were analyzed in duplicate by EA-IRMS on a Thermo Finnigan Delta Plus XL in the Department of Archaeological Sciences of the University of Bradford (UK). The collagen sample of PNQ3 and the faunal remains, along with the bulk plant samples, were analyzed in duplicate on a Sercon GSL elemental analyser coupled to a 20–22 mass spectrometer  at the University of York (UK). At both York and Bradford, accuracy was determined by measurements of international standard reference materials within each analytical run. The obtained values were corrected from the isotopic ratio of the international standards, Vienna Pee Dee Belemnite (VPDB) for carbon and air (AIR) for nitrogen, according to the following equation: 67. One fish sample had wt%N and wt%C values outside of the acceptable range proposed by Szpak68 but had an acceptable C:N ratio and isotope values comparable to the other fish remains, thus it was considered reliable.Collagen was successfully extracted from five human individuals and 23 faunal samples Table . The extδ13C and δ15N values between samples were performed using one-way ANOVA or Kruskal–Wallis tests (α = 0.05), after checking for normal distribution with Shapiro–Wilk test for normality (α = 0.05); both tests were performed in PAST 3.x69. The relative contributions of different food groups to human diet were estimated using Bayesian Stable Isotope Mixing Models (BSIMMs) in FRUITS 3.145 based on dietary proxies  and food groups  to account for multiple dietary sources, different macronutrient fractions, metabolic routing and uncertainties for all of these parameters. For each food group the average δ13C and δ15N values of the nutrient fraction  were estimated using offsets between edible fractions and food remains reported in the literature70. In the case of individuals from São Luís Island, the δ13C and δ15N values of the dietary macronutrients (protein and energy) were estimated from the average δ13C and δ15N values of terrestrial mammals (n = 12) and fish (n = 11) found at PNQ, using the following offsets—terrestrial mammals: − 2 ‰ (∆13Cprotein-collagen), − 8 ‰ (∆13Clipids-collagen) and + 2 ‰ (∆15Nprotein-collagen); fish: − 1 ‰ (∆13Cprotein-collagen), − 7 ‰ (∆13Clipids-collagen) and + 2 ‰ (∆15Nprotein-collagen). For plants, bulk δ13C values were first corrected for the Suess effect. A linear regression using δ13C values of atmospheric CO2 between 1962 and 200871 was used to estimate the atmospheric δ13C value for 2018 (− 8.6 ‰), when the plants were collected. The difference between 2018 and the value for 1861 (− 6.48 ‰) was then used to correct the bulk plant δ13C values (+ 2.13 ‰). Plant δ13C values were then corrected for the offsets of − 2 ‰ (∆13Cbulk-protein) and + 0.5 ‰ (∆13Cbulk-carbohydrate), while assuming that the δ15N value of plant protein was the same as the average bulk plant δ15N value. A conservative uncertainty of 1 ‰ was used for most of the δ13C offsets, except for the average δ13C values of fish protein and energy (1.5 ‰). Large uncertainties were used for protein δ15N values of terrestrial C3 plants (6 ‰), terrestrial mammals (2 ‰), maize (2 ‰) and fish (2 ‰) to take into account the isotope variation observed between some of these samples 15 and Marajó Island 12, at the mouth of the Amazon River. The model used for these sites differed from São Luís Island in regards to the faunal isotope baseline. Due to the lack of contextual isotopic baselines, their dietary macronutrients were estimated on the δ13C and δ15N values of modern terrestrial mammals (n = 8) and freshwater resources  sampled from Guiana, Lower Orinoco and Upper Amazon73, and from the modern plants collected at São Luís Island  and macronutrients using the aforementioned offsets , but the carbon isotopes could have derived from carbohydrates and lipids with de novo synthesis of non-essential amino acids28. We assumed that dietary proteins contributed to 74 ± 4% of bulk collagen carbon, while lipids and carbohydrates provided the remaining 26%74. Diet-to-collagen δ13C offset (+ 4.8 ± 2 ‰) and δ15N offset (+ 5.5 ± 2 ‰) were taken from Fernandes et al.74. Dietary estimations were constrained for a conservative acceptable range for protein intake of > 5% and < 45% of the total calories according to physiological studies45. The model inputs and parameters can be found in the Supplementary information A concentration-dependent and routed model was selected for the Bayesian estimations75, with McGill et al.76 variations of Box Plots. For these, the horizontal lines correspond to the median and the hinges to the 25th (Q1) and 75th (Q3) percentiles. The whiskers extend from the hinge to the smallest or largest observation greater than or equal to − 1.5 * IQR (interquartile range) or less than or equal to + 1.5 * IQR, respectively; outliers were excluded. Summary statistics of the dataset were performed using dplyr version 1.0.1 and reported in the Supplementary information Box and whiskers plots summarizing the posteriors of dietary estimates generated using FRUITS were created using the R statistical software environment (version 4.0.2) and the package ggplot2 version 3.3.2).3.275, w4 photosynthetic pathway, was consumed by groups at São Luís Island and at the mouth of the Amazon River during the Late Holocene. This assumption is reasonably supported by the presence of maize in the Amazon basin since the Middle Holocene77, and by increasing evidence of maize in archaeological and lake sedimentary records in the central and northern Amazon during the Late Holocene78.Finally, we assumed that maize, a plant using a C79) and terrestrial  curves in OxCal v4.381, taking into account the average relative contribution of marine carbon to collagen estimated by the BSIMMs. We estimated the local marine radiocarbon reservoir correction value (∆R) at São Luís Island using data from the Marine Reservoir Correction Database82. The ∆R point data from the Marine Reservoir Correction Database was used to generate a smooth surface distribution of marine ∆R values using the Bayesian model AverageR developed within the Pandora & IsoMemo initiatives (https://isomemoapp.com). AverageR is a generalized additive mixed model that employs a thin plate regression spline. Further details on the model can be found in Cubas et al.83. The local ∆R (10 ± 41 14C years) was estimated for a point centred at São Luís Island and a radius of 50 km. Calibrated dates were rounded to 10.The radiocarbon dates from São Luís Island, obtained directly from human individuals using Accelerator Mass Spectrometry (AMS), were calibrated (BP) using a combination of the marine (Marine13Supplementary file1Supplementary file2Supplementary file3


No residual disease and a high-complexity surgery significantly prolonged PFS  and OS . The extent of initial peritoneal dissemination did not affect the prognosis when initially disseminated lesions > 1 cm were resected.We examined whether the extent of initial peritoneal dissemination affected the prognosis of patients with advanced ovarian, fallopian tube, and peritoneal carcinoma when initially disseminated lesions > 1 cm in diameter were removed, regardless of the timing of aggressive cytoreductive surgery. The extent of peritoneal dissemination was assessed by the peritoneal cancer index (PCI) at initial laparotomy in 186 consecutive patients with stage IIIC/IV. Sixty patients underwent primary debulking surgery and 109 patients underwent neoadjuvant chemotherapy followed by interval debulking surgery. Seventeen patients could not undergo debulking surgery because of disease progression during neoadjuvant chemotherapy. The median initial PCI were 17. Upper abdominal surgery and bowel resection were performed in 149 (80%) and 171 patients (92%), respectively. Residual disease ≤ 1 cm after surgery was achieved in 164 patients (89%). The initial PCI was not significantly associated with progression-free survival (PFS;  It has been universally used for the assessment of the prognosis or surgical resectability of gastrointestinal carcinomas. Some reports have shown that PCI is also one of the prognostic factors for ovarian cancer9. A negative correlation has been reported between PCI and complete resection rate10, which is the most significant prognostic factor for ovarian cancer12.Majority of ovarian, fallopian tube, and peritoneal carcinoma have already spread to the peritoneal cavity beyond the pelvis upon diagnosis. Among the several assessment tools for the extent of peritoneal dissemination15. Aggressive surgery is necessary to achieve complete resection in patients with a high PCI score. However, in practice, aggressive surgery is not often performed for patients with a high PCI score at primary debulking surgery (PDS). Only 22% patients with high disease burden received high-complexity surgery in the Gynecologic Oncology Group 182 study3. In addition, complete cytoreduction rate in patients treated with PDS was approximately 20–50% even in highly experienced centers in which aggressive surgery is performed18, except for a few19. Perioperative death or severe perioperative complications in patients with a high PCI score could occur when aggressive surgery with PDS was performed18.Whether aggressive cytoreductive surgery overcome the extent of peritoneal dissemination remains debatable18 because many initially disseminated tumors become invisible after NACT, and only visible tumors can be resected during IDS. Therefore, initially disseminated tumors were not resected in such cases. However, our previous study showed that lesions > 1 cm in diameter before NACT administration may harbor microscopic disease even though the initially disseminated tumor becomes invisible after NACT20. The previous study observed that the median progression-free survival (PFS) was longer in patients who underwent aggressive surgery with resection of lesions measuring > 1 cm before NACT than in those who underwent resection of only visible lesions during IDS20. Therefore, we concluded that initially disseminated tumors that could not be resected during IDS contributed to poor prognosis in patients with a high PCI score.The treatment option for patients who could not achieve complete resection with PDS is neoadjuvant chemotherapy (NACT) followed by interval debulking surgery (IDS). It is unclear whether aggressive surgery overcomes the extent of dissemination for the patients treated with IDS. Usually, aggressive surgery is not performed during IDS in patients with a high initial PCI score before initiation of NACTWe hypothesized that aggressive surgery with resection of the initial > 1 cm dissemination would overcome the high PCI score by selecting PDS or NACT followed by IDS depending on whether cytoreduction to no residual disease is achievable at initial laparotomy before starting treatment. In this study, we examined whether PCI affected the prognosis of patients with International Federation of Gynecology and Obstetrics (FIGO) stage IIIC/IV ovarian, fallopian tube, and peritoneal carcinoma when initially disseminated lesions > 1 cm were resected, regardless of the timing of cytoreductive surgery.The median initial PCI was 17 . The initial PCI was significantly associated with primary site, histology, timing of cytoreductive surgery, residual disease, and surgical complexity score. The initial PCI was not associated with performance status and FIGO stage.p < 0.01).Sixty patients (32%) underwent PDS. One hundred and nine patients underwent IDS after NACT. Seventeen patients did not undergo IDS because of disease progression during NACT (no debulking surgery). The median initial PCI of the patients who underwent PDS, IDS, and no debulking surgery were 8, 19, and 22, respectively. The initial PCI of the patients who underwent PDS was significantly lower than that of the patients who underwent IDS (n = 145) and splenectomy with or without distal pancreatectomy (n = 98), and 171 patients (92%) underwent bowel resection, including rectosigmoid colon (n = 163), large bowel other than the rectosigmoid colon (n = 90), and small bowel (n = 20). The surgical complexity score had a median value of 12 and was significantly higher in the patients who underwent IDS than in those who underwent PDS .Of the 186 patients, 149 patients (80%) underwent upper abdominal procedure, including right diaphragm resection , residual disease measuring 0.1–1 cm in 16 (9%), and residual disease > 1 cm or no debulking surgery in 22 (12%). Among the 60 patients who underwent PDS, cytoreductive surgery was associated with no residual disease in 47 (78%), residual disease measuring 0.1–1 cm in 11 (18%), and residual disease > 1 cm in 2 patients (3%). Among the 109 patients who underwent IDS, cytoreductive surgery was associated with no residual disease in 101 (93%), residual disease measuring 0.1–1 cm in 5 (5%), and residual disease > 1 cm or no debulking surgery in 3 patients (3%).The 30-day and 3-month mortality rates after cytoreductive surgery were 0% and 0.005%, respectively.Distribution of residual disease in 60 patients who underwent PDS was as follows: among 36 patients with PCI scores 1–10, cytoreductive surgery was associated with no residual disease in 31 (86%), residual disease measuring 0.1–1 cm in 3 (8%), and residual disease > 1 cm in 2 patients (6%). Among 20 patients with PCI scores 11–20, cytoreductive surgery was associated with no residual disease in 15 (75%), residual disease measuring 0.1–1 cm in 5 (25%), and residual disease > 1 cm in 0 patients (0%). Among 4 patients with PCI scores 21–39, cytoreductive surgery was associated with no residual disease in 1 (25%), residual disease measuring 0.1–1 cm in 3 (75%), and residual disease > 1 cm in 0 patients (0%).Distribution of residual disease in 109 patients who underwent IDS was as follows: cytoreductive surgery was associated with no residual disease in all 13 patients (100%) with PCI scores 1–10. Among 54 patients with PCI scores 11–20, cytoreductive surgery was associated with no residual disease in 52 (96%), residual disease measuring 0.1–1 cm in 1 (2%), and residual disease > 1 cm in 1 patient (2%). Among 42 patients with PCI scores 21–39, cytoreductive surgery was associated with no residual disease in 36 (86%), residual disease measuring 0.1–1 cm in 4 (10%), and residual disease > 1 cm in 2 patients (5%).The surgical complexity score in 60 patients who underwent PDS was as follows: among 36 patients with PCI scores 1–10, high-complexity surgery (score of 8–18) was performed in 14 (39%), moderate-complexity surgery (score of 4–7) in 15 (42%), and low-complexity surgery (score of 1–3) in 7 patients (19%). Among 20 patients with PCI scores 11–20, high-complexity surgery was performed in 17 (85%) and moderate-complexity surgery in 3 patients (15%). All 4 patients (100%) with PCI scores 21–39 underwent high-complexity surgery.The surgical complexity score in 109 patients who underwent IDS was as follows: among 13 patients with PCI scores 1–10, high-complexity surgery was performed in 9 (69%) and moderate-complexity surgery in 4 patients (31%). Among 54 patients with PCI scores 11–20, high-complexity surgery was performed in 50 (93%), moderate-complexity surgery in 3 (6%), and low-complexity surgery in 1 patient (2%). Among 42 patients with PCI scores 21–39, high-complexity surgery was performed in 38 (90%), moderate-complexity surgery in 2 (5%), and low-complexity surgery in 2 patients (5%).After a median follow-up duration of 40 months , the median PFS for the entire cohort was 28 months, and the median overall survival (OS) was not reached.p = 0.18 and p = 0.39, respectively). Kaplan–Meier curves were drawn for subgroup analyses of patients who underwent PDS, for those who underwent IDS, and for those who received chemotherapy without IDS (no debulking surgery). We observed that the PCI was not associated with PFS and OS in the subgroups of patients who underwent PDS and IDS . We observed that the PCI was not associated with PFS and OS in patients without residual disease Fig. a–d. In cp = 0.13) and with the OS   and the OS .Univariate Cox regression analysis showed that the initial PCI was not significantly associated with the PFS  and with the OS . Multivariate analysis showed that absence of residual disease significantly prolonged the PFS , and absence of residual disease and a high surgical complexity score significantly prolonged the OS .Multivariate Cox regression analysis Table  confirmeThis study showed that PCI did not affect the prognosis of patients with FIGO stage IIIC/IV ovarian, fallopian tube, and peritoneal carcinoma when initially disseminated lesions > 1 cm in diameter were resected, regardless of the timing of cytoreductive surgery. No residual disease after surgery and high-surgical complexity score affected the prognosis. In other words, aggressive surgery could overcome the extent of peritoneal dissemination when patients with a high PCI score undergo high-complexity surgery by selecting PDS or IDS, depending on whether cytoreduction to no residual disease is achievable at initial laparotomy before starting treatment.19. However, studies have shown that the PCI score was negatively associated with the completeness of cytoreduction and residual disease after PDS10. Moreover, the rate of complications with PDS was higher than that with IDS24. Conversely, the use of NACT/IDS enabled the surgeons to accomplish a safe and high-complexity surgery in patients with high extent of peritoneal dissemination because NACT reduces the massive ascites and the tumor volume, improves the general condition, and decreases the difficulty of complex surgery. In this study, high-complexity surgery was performed on 71% of the entire cohort; this led to high rate (80%) of complete cytoreduction rate. Our results showed that not only PDS but also selective use of NACT/IDS allowed many patients to receive complete cytoreduction safely, regardless of the initial PCI.In this study, the timing of cytoreductive surgery was determined at initial laparotomy on the based on the feasibility of achieving no residual disease. As a result, the number of the patients who did not underwent PDS was more than twice as large as the number of the patients underwent PDS. Contrary to our study, the timing of cytoreductive surgery was usually primary at many institutions where aggressive cytoreductive surgery was performed26. The difference between this study and others lies in the surgical policy for IDS. In the present study, all disseminated tumors > 1 cm in diameter identified at initial laparotomy was removed even if the initially disseminated tumor was no longer visible at the time of IDS after NACT. In other words, at IDS, we performed the surgical procedures that would be performed to achieve residual tumor < 1 cm if PDS were to be performed. A drawback of the conventional IDS is that the tumors > 1 cm diameter identified at initial laparotomy would not be resected during IDS if they become invisible due to a good response to NACT. This may lead to a risk of early recurrence or relapse, similar to residual disease > 1 cm diameter after PDS. Our previous study showed that microscopic disease remains present especially in the rectosigmoid colon, transverse mesentery, greater omentum, right diaphragm, paracolic gutters, and vesicouterine pouch even if the tumors become invisible during IDS20. Similar to ours, Lim et al. reported that detached scars had residual cancer cells that assumedly included cancer stem cells28. The surgical removal of all the tumors > 1 cm identified at initial laparotomy could overcome tumor biology and tumor burden.In this study, the surgical complexity score of the patients who underwent IDS was higher than those of the patients who underwent PDS because initial PCI of the patients who underwent IDS was higher than those of the patients who underwent PDS. On the contrary, several studies showed that the surgical complexity and the rate of extra-gynecologic surgery were lower with IDS than with PDS32. For colorectal cancer, patients with high score of PCI were reported to have poor prognosis36 because PCI directly affect the rate of no residual disease after surgery, frequency of postoperative complications, and prognosis36. Similar results have been reported for gastric and appendix cancers.Unlike gastrointestinal carcinoma, another reason that PCI did not affect prognosis in this study is the high response rate to chemotherapy for ovarian cancer. In this study, 87% patients did not have disease progression during NACT and could undergo IDS. In other reports, the response rates to chemotherapy in FIGO stage III/IV ovarian carcinoma is higher in colorectal carcinoma (73–90 vs 15–62%)This study had some limitations such as the single-institution analysis, the small number of patients, and a higher percentage of IDS cases compared with that of PDS cases. The selection criteria for performing PDS or IDS varies among institutions. Notably, 42 of 56 patients with a high PCI score (21–39) were included in the NACT/IDS group; therefore, it is reasonable to conclude that in addition to aggressive surgery, chemotherapy may have affected the prognosis. In this study, 73% of patients with a high PCI score who could not undergo PDS underwent high-complexity surgery following the administration of NACT. Therefore, optimal patient selection for PDS or IDS can enable high-complexity surgery with favorable prognosis even in patients with a high PCI score.In conclusion, the extent of initial peritoneal dissemination did not affect the prognosis for patients with FIGO stage IIIC/IV ovarian, fallopian, and peritoneal carcinoma when initially disseminated lesions > 1 cm was resected, regardless of the timing of cytoreductive surgery. No residual disease after surgery and a high-surgical complexity led to favorable prognosis. Aggressive cytoreductive surgery with selective use of IDS could overcome the extent of peritoneal dissemination.This study was approved by the Institutional Review Board of Chiba University Graduate School of Medicine. All methods were carried out in accordance with relevant guidelines and regulations. Patients with FIGO 2014 stage IIIC/IV ovarian, fallopian tube, and peritoneal carcinoma who were consecutively treated at Chiba University Hospital from January 2008 to December 2017 were included. Written informed consent was obtained from all patients before surgery. Of the 208 patients during the study period, 22 patients who did not undergo exploratory laparotomy before NACT because of poor general condition (performance status ≥ 3 or ileus) and/or those aged ≥ 80 years, or who did not be evaluated in whole abdominal cavity due to adhesion were excluded. Therefore, this study included 186 patients in whom the extent of peritoneal dissemination upon initial laparotomy was assessed.21. In brief, the selection of primary or interval debulking surgery was decided during the initial laparotomy which was performed as early as possible after a patient's first visit. The patients was selected for IDS when disseminated tumor burden explored at initial laparotomy was too high to achieve no residual disease; gastrectomy, resection of the hepatic hilum or head of the pancreas, massive intestinal resection, or total colectomy was required; and/or massive ascites caused coagulopathy. At that time, patients underwent ovarian, fallopian tube, or omental biopsies, followed by NACT. Patients other than those mentioned above were selected for primary debulking surgery including upper abdominal surgery and bowel resection, to achieve no residual disease after surgery.The treatment protocol used in this study is described in our previous reports2. The initial PCI groups were classified into three groups based on the PCI scores: 1–10, 11–20, 21–39. When the patients were selected for IDS, the margins of the disseminated tumor (> 1 cm) were marked with a non-absorbable, 3–0 black silk suture after diagnostic biopsy of ovary, fallopian tube or omentum was performed21.The extent of peritoneal dissemination was assessed during initial laparotomy according to the peritoneal cancer index (PCI)21. An implantable port system placed in the abdominal cavity at initial laparotomy was used for collection of peritoneal washing cytology, which was performed every 3 to 4 weeks during NACT. IDS was performed when the peritoneal washing cytology was negative and/or the serum CA-125 had decreased to 15 IU/mL or the serum CA-125 level stopped decreasing when the peritoneal washing cytology remained positive. If the disease progressed during NACT, we did not perform IDS. During IDS, the regions in which the disseminated tumors > 1 cm at the initial laparotomy was removed even when initial tumors were invisible, using the non-absorbable suture marked at initial laparotomy as landmarks.The timing of IDS was described at our previous reports23. The surgical complexity score was classified into three groups: the score of 1–3 was low, 4–7 was moderate, and 8–18 was high23.The complexity of surgery performed were scored according to the surgical complexity score2) and carboplatin (AUC 2–3) for 3 weeks were administered. For the NACT, weekly paclitaxel and carboplatin were administered until the conditions mentioned above20. The median number of NACT cycles was 5. For adjuvant chemotherapy after IDS, six cycles of gemcitabine (500 mg/m2) and irinotecan (50 mg/m2) on days 1 and 8 every 3 weeks were administered. After December 2013, upon its approval for use by the Japanese public insurance, bevacizumab (15 mg/kg) every 21 days for 21 cycles was administered as the first-line therapy.For the adjuvant chemotherapy after PDS, six cycles of weekly paclitaxel . Univariate and multivariate Cox proportional hazards regression analyses were performed to analyze the prognostic factors. All statistical analyses were performed using JMP ver. 11. The Kaplan–Meier survival curves were drawn using IBM SPSS Statistical ver. 22 . For all statistical tests, the differences were considered significant at p < 0.05.The association between the initial PCI and the clinical factors were analyzed using the Mann–Whitney


Platinum group metals (PGMs) palladium, platinum, and ruthenium represent the key materials for automotive exhaust gas treatment. Since there are no adequate alternatives, the importance of these metals for the automotive industry is steadily rising. The high value of PGMs in spent catalysts justifies their recycling. Therefore, it is really important to recovery platinum group metals from aqueous solutions. Of the many PGMs recovery procedures, adsorption is a process with a good efficiency, but an important role is played by the adsorbent material used into the process. In order to improve the adsorption properties of materials were developed new methods for chemical modification of the solid supports, through functionalization with different extractants. In present paper a new adsorbent material (Chitosan-DB18C6) was used for PGMs recovery. The new adsorbent material was produced by impregnating Chitosan with dibenzo-18-crown-6-ether using Solvent Impregnated Resin (SIR) method. The crown ethers were chosen as extractant due to their known ability to bind metallic ions, whether they are symmetrically or unsymmetrically substituted. In order to determine the PGMs recovery efficiency for new prepared adsorbent material the equilibrium and kinetic studies were performed. Also, to study the PGMs adsorption mechanism the experimental data were modelled using pseudo-first-order and pseudo-second order kinetic models. Experimental data were fitted with three equilibrium isotherm models: Langmuir, Freundlich and Sips. The results proved that new adsorbent material (Chitosan-DB18C6) is an efficient adsorbent for PGMs recovery from aqueous solutions. Due to their specific chemical and physical properties platinic group metals 2 are widely used in different industries: electronic industry, catalysts in oil processing industry, medical field and jewelry production3.Platinic metals group (PGMs) it is a group containing 6 chemical elements which have similar physical and chemical properties. This group is also divided into three different subgroups: iridium, platinum  groups2. High cost of PGMs is related with the diminishing availability of the natural resources concomitant with the difficulties related to the mining of such elements5. Therefore, from viewpoint of full utilization of resources it is quite important the effective recovery of PGM’s metals from both natural ore and industrial waste. Recovery of such metals from spent systems can represent an efficient way to reduce the dependence on this limited raw material7.In this moment around 90% from the entire quantity of platinic metals comes from two countries – Russia and South Africa, rest of the world being dependent on imports8, ion exchange9, membrane separation10, complexation11, retention on porous polymers12, coprecipitation, sequential distillation, precipitation and adsorption which is presented in the literature as the method with higher efficiency for recovery of PGM’s from aqueous solutions15.For effective recovery were developed several methods, such as: solvent extraction19. Based on technical and economical points of view a special attention is granted to the materials with good adsorbent properties. From the cost as well as technical perspective, during the last decade, chitosan was preferred for removal and recovery of precious metallic ions16.From all these recovery methods, adsorption seems to be the most suitable method for the recovery of PGM’s from diluted solutions, due to low cost and high efficiency20, iron oxide21, activated graphite22, zeolites23, and adsorbent materials obtained by synthesis or functionalization with pendant groups containing N, S or P atoms, having selective properties for these groups of metals27.In the current stage of development are selected the materials with good adsorbent properties which represent a good technique for removal of PGM’s from solutions with low concentrations due to low cost and higher efficiency. In this context where used adsorbent materials such as: chitosan28. Presence of N into the chitosan structure leads at the improvement of adsorbent properties of metallic ions by electrostatic attractions19. Some limitation appears because of his solubility into the acidic media. In order to overcome this limitation, the structure of chitosan can by modified by functionalization with different pendant groups, leading at improvement of its adsorbent properties for PGM’s ions.Aim of present study was the synthesis, followed by the test of adsorptive properties of new materials obtained by functionalization of chitosan with crown ethers. It is well known that chitosan is a linear polysaccharide formed from fragments of glucosamine (deacetylate unit) and N-acetyl-glucosamine (acetylated unit) randomly distributed, being a non-toxic adsorbent used for removal of metallic ionsOne possible compound used for functionalization of chitosan are crown ethers which represent a macrocyclic polyether organic compound. It is known that the crown ethers can easily link metallic ions forming complex compounds.21. Also was evidenced the higher affinity of adsorbent material for one of these ions.In present work the chitosan adsorbent has been functionalized with dibenzo-18-crown ether 6 (DB18C6). During functionalization process the N atoms from chitosan structures form new hydrogen bonds with the oxygen atoms from crown ether leading at a new complex adsorbent which present a higher selectivity for metallic ions. Functionalization process was carried out by using solvent impregnated resin (SIR) method, and further used for recovery of Pd(II), Pt(IV) and Ru(III) by adsorption5, purchased from Yuhuan Ocean Biology, China) in order to obtain a ratio extractant:suport = 0.1:1. Complete chitosan functionalization was obtained for a contact time of 24 h, after that wet adsorbent material was dried for 24 h at 323 K. Functionalized chitosan has been characterized by:scanning electron microscopy (SEM) coupled with energy dispersive X-ray spectroscopy (EDX) using a FEI Quanta FEG 250 scanning electron microscope;Fourier transform infrared spectroscopy using a Bruker Platinum ATR-QL Diamond spectrometer;Surface area was determined by using Brunauer, Emmet, Teller (BET) method, using an Quantachrome Nova 1200e analyzer.23.also was determined the point of zero charge (pZc)pZc of synthesized adsorbent material has been determined by putting in contact 0.1 g Ch-DB18C6 adsorbent material with 25 mL KCl 0.1 M at 298 K, all systems were agitated at 200 rotations per minute using a thermostatic bath. KCl solutions pH has been adjusted between 2 and 12 by using NaOH solutions with concentrations between 0.05 and 2 N, or HNO3 solutions with concentrations between 0.05 and 2 N. Afterwards, all samples were filtered and solution pH has been determined using a pH-meter Mettler-Toledo SevenCompact.In order to prepare a new adsorbent material by functionalization of chitosan with DB18C6 (designed as Ch-DB18C6) were weighed exactly 0.1 g of extractant—dibenzo-18-crown ether 6 acid  which further was dissolved in 25 mL nitrobenzene . Obtained solution was placed in contact with 1 g of chitosan  method. In order to determine the total pore volume was considered the last recorded point from the adsorption isotherm.In order to obtain information about adsorption process was studied the impact of various factors such as pH, temperature, contact time and initial concentration on the recovery of metallic ions on functionalized chitosan. pH influence is linked to the form of metallic ion into the solution and by the ionic form of the adsorbent functional groups.−1.In present paper has been studied the influence of solution pH on Pd(II), Ru(III) and Pt(IV) on produced functionalized chitosan. Studies were carried out by contacting 25 mL solution with 0.1 g of adsorbent material for a contact time of 2 h at 298 K, varying the pH from 0.5 to 10, for a metallic ions initial concentration of 10 mg L−1 was added. In order to establish time influence samples were mixed for different times  and at different temperatures . All samples were stirred at 200 rot min−1.In order to establish the influence of contact time and temperature on the adsorption capacity of functionalized chitosan, 0.1 g of adsorbent material has been weighted over which 25 mL solution with metallic ions content (Pd(II), Ru(III), Pt(IV)) with concentration C0 = 10 mg L−1. All these solutions were prepared by dilution from stock one for Pd (5 wt. % Pd(II) chloride in 10 wt.% HCl) and Pt (Pt(IV) chloride 57.7%), respectively from Ru(III) chloride hydrate. Adsorption process has been performed at pH = 2 –98 K form 3 h. After adsorption residual concentration was measured using ICPOS technique performed on a 5100 VDV Agilent technologies with a double pass spray chamber and OneNeb nebulizer. For determination of each element were used the first two most intense spectra lines with 3 replicates and 5 s integration time for each.In order to establish the effect of initial concentration of metallic ions on adsorption capacity were prepared Pd (II), Ru (III) and Pt(IV) solution with concentrations 10, 25, 50, 75, 100, 150 and 175 mg LEquations used to describe adsorption equilibrium and adsorption kinetic are presented in Table e − qt) versus t. When the experimental data were fitted using pseudo-second-order kinetic model, the parameters associated with this model were determined from the linear dependence between t/qt versus t. From line equation were determined the speed constant associated with pseudo-second-order model (k2) and pseudo-second-order model calculated adsorption capacity .Determination of pseudo-first order speed constant and calculated adsorption capacity were determined from the linear dependence between ln; A, Arrhenius constant (g min mg−1); Ea, activation energy (kJ mol−1); T, absolute temperature (K); R, ideal gas constant (8.314 J mol−1 k−1).Further for considered metallic ions adsorption on Ch-DB18C6 were determined activation energy values from Arrhenius equation:2 versus 1/T.Determination of activation energy was possible by using the speed constant obtained from the pseudo-second-order model, which better fit the experimental data. Activation energy was calculated from the slope of linear dependence of lnk34:0, free Gibbs energy standard variation (kJ mol−1); ΔH0, enthalpy standard variation (kJ mol−1); ΔS0, entropy standard variation (J mol−1 k−1); T, absolute temperature (K).In order to establish if the metallic ions adsorption onto the adsorbent surface is a spontaneous process has been evaluated the value of free Gibbs energy from Gibbs–Helmholtz equationd versus 1/T (linear form of van’t Hoff equation), where Kd is the equilibrium constant, being calculated as ratio between equilibrium adsorption capacity (qe) and equilibrium concentration (Ce).Standard variations of enthalpy and entropy were evaluated from linear dependence of ln KFor further practical application of such adsorbent materials it is important to evaluate their regenerative capacity. In order to optimize the cost of such processes, by reuse of adsorbent material, and to obtain good efficiency is necessary that the regenerative process occurs easy, whit a faster desorption of metallic ions in higher quantity.In order to evaluate the maximum number of adsorption/desorption cycles, Pt(IV), Pd(II), Ru(III) ions were adsorbed and desorbed until the adsorption on used adsorbent material was not any more possible.Desorption was performed by mixing 1 g of used CH-DB18C6 containing Pt(IV), Pd(II), and Ru(III) ions with 25 mL HCl 5%, obtained mixture has been shaken at 200 rotation per minute at room temperature for 3 h. After that, filtered adsorbent material has been rinsed with distilled water, and dried at room temperature. These steps were repeated several times until all metallic ions were irreversibly fixed onto the surface of adsorbent material. In this way has been establish the maximum number of usage cycles. Process efficiency was determined by counting the quantity of adsorbed / desorbed metallic ions.After adsorption the exhausted adsorbent material containing important quantities of Pd(II), Ru(III) and Pt(IV) have undergone a thermal decomposition at 600 K, for 240 min, into a controlled atmosphere oven (Nabertherm LHT407GN Furnaces). Produced sample has been analyzed using scanning electron microscopy (SEM) coupled with energy dispersive X-ray spectroscopy (EDX) (Quanta FEG 250 scanning electron microscope).SEM technique has been used in order to analyze the chitosan surface before and after functionalization with DB18C6 . Based on recorded isotherms were calculated the specific parameters for pure and functionalized material  and initial value (pHi)  Fig. .Figure 5Analyzing data presented in Fig. 40.pH effect on studied metallic ions adsorption is presented in Fig. 2 groups found on chitosan surface13 and, in same time, these species be complexed inside of the ring of crow ether41 which explain its good adsorption capacity.These ionic species can be adsorbed onto the surface of –NHDecrease of adsorption capacity at pH higher than 3 can be explained if we consider the lower concentration of chloride ions into the solution which lead at some decrease into the concentration of absorbable species.44.All these observations are in consensus with data found in the literature, which confirm that the adsorption of studied metallic ions better occur at low pH, when the competitive adsorption is reducedOther important parameter for adsorptive processes is represented by the contact time at different temperatures. Contact time influence for adsorption of Pd(II), Ru(III) and Pt(IV) ions is presented in Fig. Obtained experimental data proved that the contact time increase leads at increase of maximum adsorption capacity for all three metallic ions. Maximum adsorption capacity was reached when the contact time was around 180 min, further increase of contact time having no notable influence on maximum adsorption capacity. In order to increase the precision for any further experiment the contact time was set at 180 min. Further for establishing the mechanism of the studied adsorption process, obtained experimental data were modeled using two different kinetic models. In Fig. In order to validate the model which, describe studied adsorption process, were analyzed the values of regression coefficient and of the maximum adsorption capacities. When the regression coefficient value is closer to 1 can assume that the model is better described by the kinetic mechanism for the studied adsorption process. Based on data presented in Table Also, can observe that the maximum adsorption capacity estimated based on this model is closer to the experimental maximum adsorption capacity.3.These observations can indicate that the adsorptions of Pd(II), Ru(III) and Pt(IV) ions on produced adsorbent is a process similar to a chemical adsorption. Can predict that such adsorptions involve certain valence bonds between free electrons of metallic ions complex anions and the surface of Ch-DB18C6 adsorbent material46.Adsorption of studied metallic ions onto the Ch-DB18C6 it is a two stage process: (i) in first stage occurs an instantaneous adsorption on adsorbent surface, (ii) second stage can be a trapped adsorption when the limiting step is the particle diffusion2 and 1/T were calculated the values of Ea, presented in Table In order to confirm the nature of studied adsorption processes were evaluated the values of activation energy using Arrhenius equation , Ru(III) and Pt(IV) adsorptions are greater than 40 kJ mol−1 which confirm that these adsorption processes are chemical one47.Values of EFurther, has been investigated temperature effect on Pd(II), Ru(III), Pt(IV) adsorptions by conducting experiments at three different temperatures . From obtained experimental data can observe that temperature increase has a beneficial effect for all three studied adsorption processes.d and 1/T,  initial concentration of 150 mg L−1, and 30.4 mg g−1 for Ru(III) initial concentration of 175 mg L−1.Obtained experimental data proved that the maximum adsorption capacities increase with increases of initial concentrations until a maximum value is reached. When maximum adsorption capacities were obtained can conclude that the adsorbent material has been saturated and any further increase into the initial concentrations has no further effect on adsorption capacities. During experiments has been obtained a maximum adsorption capacity of: 22.4 mg g-1 for Pt(IV) initial concentration of 175 mg LIn order to understand and describe adsorption mechanism of studied ions onto the new produced adsorbent material experimental data were fitted using Freundlich, Langmuir and Sips models Fig. . Based oAnalyzing data presented in Table In Table After extraction of metallic ions from aqueous solutions, exhausted Ch-DB18C6 adsorbent material was calcinated at 873 K in controlled atmosphere in order to recover metallic particles. This treatment was made by heating samples with a speed of 5 K per minute and kept at 873 K for minimum 240 min.After calcination obtained samples were characterized by recording EDX spectra Fig. .Figure 1Based on EDX spectra presented in Fig. In present paper was investigated the possibility to reuse the Ch-DB18C6 material used adsorbent by establishing the sorption/desorption cycles number. Sorption / desorption studies were repeated with significant results for 5 times for Pt (IV) and Ru (III) and 3 times for Pd(II) as can be observed from data depicted in Fig. Present study confirms that DB18C6 crown ether was successful used as extractant for functionalization of chitosan by SIR method. Obtained adsorbent material has been used for further recovery of PGM’s. Chitosan functionalization has been evidenced by using physic-chemical characterization of new produced adsorbent material. In this context were recorded FT-IR and EDX spectra, SEM micrographs, and was determined the value of pHpzc, which proved the preparation of desired adsorbent materials.Obtained experimental results proved that the PMG’s ions adsorption depends on solution pH, contact time, temperature and initial concentration of the metallic ions into the solution.From obtained experimental data was observed that CH-DB18C6 adsorbent presents the highest adsorption capacity for Ru(III) ions. For all studied systems were performed kinetic, thermodynamic, and equilibrium studies in order to establish metallic ions adsorption mechanism.Based on obtained experimental data was established that the studied adsorptions are better described by pseudo-second-order model. Concomitant obtained experimental data are well fitted by Sips isotherm for all studied metallic ions. Based on obtained information can conclude that the PGM’s recovery by adsorption is an endothermal, spontaneous process influenced by temperature. Starting from exhausted adsorbent material it is possible to recover pure metals by thermal decomposition into controlled atmosphere.


Our previous studies have shown that sulbactam can play a neuroprotection role in hippocampal neurons by upregulating the expression and function of glial glutamate transporter-1 (GLT-1) during ischemic insult. Here, using rat global cerebral ischemia model, we studied in vivo the role of p38 mitogen-activated protein kinases (MAPK) in the sulbactam-induced GLT-1 upregulation and neuroprotection against ischemia. The hippocampal CA1 field was selected as observing target. The expressions of phosphorylated-p38 MAPK and GLT-1 were assayed with western blot analysis and immunohistochemistry. The condition of delayed neuronal death (DND) was assayed with neuropathological evaluation under thionin staining. It was shown that administration of sulbactam protected CA1 hippocampal neurons against ischemic insult accompanied with significantly upregulation in the expressions of phosphorylated-p38 MAPK and GLT-1. The time course analysis showed that sulbactam activated p38 MAPK before the GLT-1 upregulation in either normal or global cerebral ischemic rats. Furthermore, inhibiting p38 MAPK activation by SB203580 blocked the GLT-1 upregulation and neuroprotection induced by sulbactam. The above results suggested that p38 MAPK, at least partly, participated in the sulbactam-induced brain tolerance to ischemia mediated by GLT-1 upregulation in rats. The homeostasis of the glutamate concentration in the synaptic gap is mainly dependent on GLT-13. Our previous studies showed that upregulation of GLT-1 by cerebral ischemic pretreatment could alleviate ischemic injury on CA1 hippocampal pyramidal neurons5. Therefore, modulating expression and function of GLT-1 might provide a new way to prevent and therapy the cerebral ischemic disease.A variety of studies have shown that cerebral ischemia and hypoxia may cause a great deal of glutamate release to the synaptic gap during ischemic stimulation and the glutamate excitotoxicity is a considerable mechanism involved in ischemic neuronal damage8. However, in view of the side effects such as bacterial resistance and dysbacteriosis caused by extensive use of antibiotics, it is greatly limited using ceftriaxone to prevent and therapy the cerebral ischemic damage. Sulbactam is an atypical β-lactam antibacterial and structurally similar to ceftriaxone, but has minor antibacterial activity. It might be potentially valuable for clinical application in prevent and therapy brain ischemic disease if sulbactam has anti-ischemic effect as well. Recent studies of our team showed that pretreatment with sulbactam could improve GLT-1 protein expression as well in CA1 hippocampus of rats, and enhance the viability of neurons against the subsequent global cerebral ischemia9. However, the mechanism about the upregulation of GLT-1 during the process of sulbactam pretreatment is not yet fully known.Rothstein et al. have reported the role of ceftriaxone in promoting GLT-1 expression and enhancing its ability to clear the glutamate from synaptic gap, which was of great benefit to alleviate disorders associated with over-accumulation of glutamate such as ischemic neuronal damage12. Recently, we found in vitro study that activation of p38 MAPK mediated the sulbactam-induced GLT-1 upregulation and neuronal protection against ischemic insult13. Hereby, we established the present study in vivo to further confirm the role of p38 MAPK activation in the sulbactam-induced GLT-1 upregulation and neuronal protection against ischemic insult using a rats’ global cerebral ischemia model.The p38 MAPK involves in many physiological and pathophysiological processes, such as cell death and survivalAdult rats  were ordered from the Experimental Animal Center of Hebei Medical University. All the rats were fed in a room with an alternation of 12 h light and dark, took food and water freely. The animal care and experimental protocols were carried out according to the ‘ARRIVE’ guidelines and accepted by the Committee of Ethics on Animal Experiments of Hebei Medical University. All efforts were conducted to reduce the suffering and quantity of animals.For this purpose, it was conducted to compare the time courses of the upregulations between phosphorylated-p38 MAPK and GLT-1 with the pretreatment of sulbactam and to explore the effect of inhibiting p38 MAPK with SB203580, a specific p38 MAPK inhibitor, on the upregulation of GLT-1 induced by sulbactam pretreatment. Seventy-five rats were separated into 7 groups randomly: ① sham, ② cerebral ischemia, ③ sulbactam + sham, ④ sulbactam + cerebral ischemia, ⑤ SB203580 + sulbactam + sham, ⑥ SB203580 + sulbactam + ischemia and ⑦ SB203580 + sham groups.The protocol of treatment for each group is shown in Fig. In groups of ⑤–⑦(n = 5 in each group), SB203580, a specific inhibitor of p38 MAPK, was first administrated via beforehand implanted cannula 30 min before sulbactam administration. Then the rats were treated in the same protocols with the group of ③, ④ and sham, respectively, and sacrificed by decapitation under isoflurane anesthesia at 48 h after the administration of sulbactam when the upregulation of GLT-1 is relative obvious according to our preliminary experiment.Twenty-five rats were separated into the following 5 groups randomly (n = 5 in each group): ①Sham, ②cerebral ischemia, ③sulbactam + ischemia, ④SB203580 + sulbactam + ischemia, and ⑤SB203580 + sham groups. The treatments in groups of ①–④ were the same with the corresponding group in part 1, except the administration of sulbactam or normal saline was conducted 2 days before the global cerebral ischemia or its sham operations. In group ⑤ SB203580 was administrated via beforehand implanted cannula 2 days before the sham operations for global cerebral ischemia. Rats were sacrificed by decapitation under isoflurane anesthesia on 7 day after the global cerebral ischemia or its sham operations to evaluate the condition of DND by neuropathological evaluation . A hole was drilled in the specified position of skull  using a dental drill with high speed  according to the rat stereotaxic atlas of brain. A cannula with an inner core  was vertically inserted into the right lateral cerebral ventricle (3.7–4.0 mm depth to the skull surface) after incised the dura carefully, and fixed on the skull using dental cements. The rats were kept in a single cage at least 5 days after the implanting for recovery.14. To the benefit of rat’s recovery from surgical stimulation, the ischemic model was established by two steps. First, the bilateral vertebral arteries of the rats were electrocoagulated under isoflurane anesthesia. A dorsal surgical incision of 1.5 cm overlying the atlas directly was performed. After exposed the atlas carefully, a preheated electrocautery needle was used to electro-coagulate bilateral vertebral arteries permanently via the alar foramen. After two days recovery, the rats’ bilateral common carotid arteries were exposed under anesthesia of isoflurane and clamped by clips for 8 min to occlude the blood flow in them to produce global cerebral ischemia, which usually result in DND of pyramidal neurons of hippocampus CA1 region15. Because it has been reported that systemic anesthesia can protect cerebral neurons against ischemic insult16, to exclude the effect of the anesthetic on the neuronal survival after ischemic insult and to evaluate the occurrence of global cerebral ischemia, the clamping of the bilateral common carotid arteries was performed when the rats awaked from the anesthesia of isoflurane. To prevent pain during the process free of the isoflurane anesthesia, the wound was continuously treated with procaine. Changes on consciousness, pupils, and light reflex were examined to evaluate the condition of the rats. During the global cerebral ischemia, the consciousness of the rat completely lost, thus the neurological behavioral deficits could not be observed. Only rats with complete loss of consciousness and light reflex with pupils dilated completely after the clamping were used for further treatments. The success rate of the model in this study is about 70–80%. After the occlusion, the clips were removed to resume the blood flow of the bilateral common carotid arteries. The consciousness and other neurological functions of the rats completely recovered after the blood flow was restored. The wound was sutured after the treatments. A temperature controllable electric-blanket was used to keep rats’ body temperature at around 37 °C throughout the process. A temperature probe is placed inside the animal's anus to detect the body temperature and the device automatically regulated the temperature of the blanket to maintain the body temperature around 37 °C.Four-vessel occlusion method was used to establish the rat model of global cerebral ischemia according to the reports beforeThe sham operations for the global cerebral ischemia included all the treatments mentioned above except occluding the bilateral vertebral arteries and common carotid arteries.Instead of the inner core, a needle was inserted in the guide cannula. At the determined time point, SB203580  or sulbactam  or NS (10 μl) was respectively administrated using a micro syringe connecting to the needle over a 20-min period. To avoid overflowing, additional 10 min was set to keep the needle in the cannula after the administration.18. After centrifugation, the protein concentration in the supernatant was measured with Lowry method. Before loading to the gel, samples were mixture with loading buffer and heated at 95–100 °C for 5 min to denaturate the protein. Sample with 60 μg protein was loaded in each lane on a 10% SDS–polyacrylamide gel for electrophorse and then transferred to a PVDF membrane. After blocked the nonspecific antigen binding in TBST containing 5% skimmed milk powder for 60 min, membranes were incubated with primary antibodies at 4 °C for 12 h and secondary antibodies at 37 °C for 1 h subsequently (the details about the antibodies are followed in the supplementary files). For GLT-1, the membranes were further incubated with HRP-conjugated streptavidin at 37 °C for 1 h. The blots were detected by Amersham Imager 600  and integrated optical density (IOD) was detected by AlphaEase FC gel image analysis software. Relative changes in expressions of phosphorylated-p38 MAPK protein and GLT-1 protein were represented by the IOD ratio aimed bands to that of β-actin.Rats were executed at the determined time mentioned above. The CA1 region of the right hippocampus was isolated and homogenized in 10 volume lysis buffer2O2 at room temperature for 15 min to remove the influence of endogenous peroxide. Sequentially, the sections were incubated with primary antibodies for 24 h at 4 °C after 1 h incubation with 10% normal goat serum. Then sections were incubated with secondary antibodies at 37 °C for 50 min after washing with PBS 3 times. HRP-conjugated streptavidin was used as third antibody and DAB substrate kit was used to develop the peroxidase activity (the details about the antibodies are followed in the supplementary files). To ensure consistency of reaction conditions, the sections for comparison were subjected to the same immunohistochemistry run. For quantitative analysis of the immunostaining, 5 sections were randomly selected in each rat to measure the mean optical density of immunostaining using a image analysis software .Rats were sacrificed at the determined time mentioned above. The left part of the brain was removed and paraffin sections of 5-μm thickness were prepared firstly. After xylene dewaxing and gradient alchohol hydration, sections were incubated with 3% H19, conditions of the pyramidal neurons in CA1 subfield of hippocampus were assessed by histological grade (HG) and neuronal density (ND). HG including the following 4 grades: ① grade 0, without neuronal death; ② grade I, only scattered neuronal death; ③ grade II, massive neuronal death; ④ grade III, practically all neurons dead. ND was determined by counting the numbers of intact pyramidal neurons within one optical field of high magnification in the CA1 hippocampus. Total of 3 fields were counted for each section and 3 sections were counted per animal. The average value of intact neuron numbers in the optical fields of the CA1 hippocampus was counted and presented with cell number per millimeter in the CA1 hippocampus for the ND value.Rats were sacrificed 7 days after the global cerebral ischemia or its sham operations. Paraffin brain sections with thionin stain were prepared as usual. Using the methods established by Kitagawa et al. and Kato et alUsing SPSS 16.0 to do statistical analysis and all data were shown as mean ± standard deviation (SD) except HG. One-way ANOVA combined with Student–Newman–Keuls test as a multiple comparison method was used to test differences between groups. Kruskal–Wallis ANOVA combined with Dunn's test as a multiple comparison method was used to compare the differences between groups in HG. It was considered statistically significant with a p-value of less than 0.05. The power analysis indicates that the number of animals in each group meet the research needs.Western blot results displayed that there were basic expressions of phosphorylated-p38 MAPK protein in CA1 hippocampus in the sham rats. After sulbactam treatment, the phosphorylated-p38 MAPK expression significantly, but moderately upregulated by about 1.5-fold compared with sham level. In global cerebral ischemic rats, the phosphorylated-p38 MAPK expression obviously upregulated by more than two fold of sham level, while about 1.5-fold after the treatment of sulbactam Fig. A. The GLIn order to illustrate the relationship between the phosphorylated-p38 MAPK and GLT-1 upregulation after sulbactam pretreatment, we further compared the time sequence of their expression and found it was quite different. The expression of phosphorylated-p38 MAPK showed the most significant upregulation at 6 h after sulbactam treatment among the three observed time points and then began to fall down, while the expression of GLT-1 began to upregulate at 12 h and showed obvious upregulation at 48 h after administration of sulbactam among the three observed time points either in sham and ischemic rats. This phenomenon indicated that activation of p38 MAPK was earlier than the upregulation of GLT-1 protein expression after administration of sulbactam.Immunohistochemistry showed that there were basic expressions of phosphorylated-p38 MAPK and GLT-1 showing by light brown particles in pexiform layer, pyramidal layer and molecular layer in the CA1 hippocampus  belong to the serine/threonine protein kinase family and mainly consist of four members including ERK, JNK, p38 MAPK and BMK. There are 4 isoforms of p38 MAPK, named p38α, p38β (1 and 2), p38γ and p38δ. The isoforms of p38α, p38β1 and p38β2 are widely present in various tissues. The isoform of p38γ is only present in skeletal muscle cells, and p38δ is mainly present in glandular tissues. Some reports showed that both p38α and p38β isoforms of p38 MAPK were activated together in neurons or astrocytes in OGD and cerebral ischemic models although there were some differences in time course of the activation10. Previous studies have shown that activation of p38 MAPK mediates cell death. For instance, in a mouse model of focal cerebral ischemia, p38 MAPK activation was significantly increased both in the neurons and astrocytes of the ischemic region29. Inhibiting p38 MAPK over-activation with SB203580 or SB239063 significantly reduced DND in hippocampal CA1 region induced by cerebral ischemia30. However, recent studies showed that mild or moderate activation of p38 MAPK was neuroprotective in ischemia. For example, cerebral ischemic preconditioning could protect hippocampal neurons against ischemia and induced a moderate activation of p38 MAPK. Furthermore, inhibition of the moderate activation of p38 MAPK blocked the neuroprotection of cerebral ischemic preconditioning. These findings suggested that moderate or mild activation of p38 MAPK took part in the acquirement of cerebral ischemic tolerance after cerebral ischemic pretreatment32. Similar phenomena were also found in the heart, in which the cardioprotective effect induced by glycoprotein IIb/IIIa inhibitor could be blocked by inhibiting p38 MAPK phosphorylation33. Therefore, it is generally agreed that the activation of p38 MAPK play a dual role in the process of ischemia based on its activation level35, e.g., the over-activation of p38 MAPK resulted from fatal ischemia usually leads to neuronal damage in the brain36, while moderate or mild p38 MAPK activation induced by sublethal ischemia normally mediates the neuroprotective effects38. The present study showed that global cerebral ischemia obviously activated p38 MAPK by more than 2 folds of sham level accompanied with serious pyramidal DND in the CA1 hippocampus. This finding was consistent with previous studies that ischemic insult induced p38 MAPK over-activating which involved in the ischemic neuronal injury. Meanwhile, we found that pretreatment with sulbactam induced a significant but mild or moderate upregulation of p38 MAPK with magnitude of about 1.5 folds of sham levels. This phenomenon was similar with that observed in cerebral ischemic preconditioning18, and suggested that the moderate activation of p38 MAPK might be a mechanism involved in the neuronal protection of sulbactam.The p38 MAPK involves in various pathophysiological processes, such as cell death and survival13, and suggested that p38 MAPK signal pathway might act as the upstream mechanism for the upregulation of GLT-1 expression after sulbactam treatment.Considering the beneficial effect of moderate or mild p38 MAPK activation in the neuroprotection mentioned above, we focused in the present study to explore the role of p38 MAPK activation in the upregulation of GLT-1 level and neuroprotection against ischemic insult induced by sulbactam. It was found that sulbactam pretreatment up-regulated both phosphorylated-p38 MAPK mildly or moderately and GLT-1 expression in the CA1 hippocampus in both normal and global cerebral ischemic rats, but the increased activation of p38 MAPK was significantly earlier than the upregulation of GLT-1 after sulbactam treatment. The results are similar with our recent study in vitro in which cultured astrocytes showed upregulation in the p38 MAPK activating before upregulating the GLT-1 protein expression after sulbactam incubation31. In cultured astrocytes, sulbactam induced up-regulation of phosphorylated-p38 MAPK earlier than GLT-1 up-regulation13. These findings support the above suggestion. However, the present study could not provide double labeled immunofluorescence evidence to clearly indicate that the astrocytic p-p38 MAPK expression was upregulated, which indeed was a deficiency of this study and should be improved in the future study.As for the cell types of the p38 MAPK expression in immunohistochemical figures  could rapidly transferred into the nucleus, and regulate the activation of CREB and NF-κB by MAPKAPK-2 and MSK1/29, the pretreatment of sulbactam has certain clinical potential as well in some predictable ischemic insults such as TIA and cardiac surgery and attack which induce global brain ischemia. In these conditions, the pretreatment of sulbactam that can enhance neuronal tolerance to ischemia might be beneficial for reducing the possible neuronal damage caused by global cerebral ischemia. The current study indicated the role of p38 MAPK activation in the sulbactam-induced cerebral ischemic tolerance mediated by GLT-1 upregulation in global cerebral ischemic rats and provide further experimental evidence for the application of sulbactam in the situations above.Although our previous studies showed that only sulbactam pretreatment enhances neuronal resistance to ischemia, but little effect in post-treatmentTaken together, it is concluded that p38 MAPK, at least partly, participates in the sulbactam-induced cerebral ischemic tolerance mediated by GLT-1 upregulation in global cerebral ischemic rats.Supplementary Information 1.Supplementary Information 2.


To examine the structural basis of this reduced efficiency, we sought to obtain the structure of the “product” complex of DNA polymerase (pol) β with the (syn)8OG:dA base pair at the primer terminus by soaking the binary complex crystals with a hydrolysable dCTP analogue complementary to the template base G. Crystallographic refinement of the structure revealed that the adenine of the (syn)8OG:dA base pair had been expelled from the primer terminus and a dCMP was inserted opposite 8OG in a reverse orientation; another uninserted molecule of the analogue was bound to the templating base G. This leads to an abortive complex that could form the basis of oxidatively-induced pol β stalling.Adenine frequently pairs with the Hoogsteen edge of an oxidized guanine base (8OG) causing G to T transversions. The ( Vinod Batra and Samuel Wilson report the structure of the complex of DNA polymerase β with the (syn)8OG:dA base pair at the primer terminus. Through crystallographic refinements, they uncover structural rearrangements that deems the complex abortive and results in oxidatively-induced pol β stalling. Most DNA polymerases insert 8-oxodGTP by virtue of Hoogsteen base pairing opposite the adenine base, and this insertion can be with a relatively high catalytic efficiency. OGG1-initiated base excision repair (BER) removes 8OG from genomic DNA preventing mutagenesis  generated internally by endogenous chemical processes and through external sources. Oxygen free radicals cause oxidation of guanosine leading to the production of 8-oxodGTP. The mutagenic potential of 8-oxodGTP has been widely investigated and the results have been extensively reviewedn − 1 position) were grown; the templating base in the single-nucleotide gapped DNA was the natural dG base These crystals were then soaked with a hydrolysable dCTP analog (dCMPP(CH2)P) with the hope of obtaining a structure resembling the product structure. The rate of incorporation of dCMPP(CH2)P is slower than that of natural dCTP because of the higher pKa5. The soaking experiments were conducted at concentrations which are several fold higher than the in vivo physiological concentration of the analog to obtain near 100% occupancy of the analog in the crystals because of its slower rate of diffusion in the crystals. This property of “in crystallo” slower rate of incorporation of substrate can provide additional information(s) about the reaction intermediates/transition state. Diffraction quality data at 2.0 Å resolution were collected on these crystals (Table 6) with rmsd of 0.295 Å P molecule is bound in the normal fashion, the reverse polarity of the primer terminus O3′, renders the O3′-Pα distance (12.7 Å) incompatible for catalysis.A second dCTP molecule is bound in the active site opposite the natural dG base in the templating strand Fig. . Althoug2)P binds opposite the template strand dG base, stabilizing the abortive ternary complex.Postulated steps in formation of this abortive ternary complex structure Fig.  are summ7.The stalled DNA synthesis complex at different steps, as described in Fig. The structure reported here supports the notion that strand slippage is a fundamental structural mechanism by which abortive complexes can be formed when a polymerase encounters an oxidatively induced 8OG lesions. While the results described illustrate the capacity of pol β and the substrate system to form and stabilize the abortive complex under the conditions used, more work will be required to understand the significance of this complex in vivo.Escherichia coli and purified8. The binary complex crystals with 8OG:dA at the primer terminus and a G as the templating base at the gap were grown as previously described6. The sequence of template with 8OG at n − 1 position was as follows: 5′-CCG ACG GCG CAT CAG C-3′. The primer (10-mer) sequences were 5′-GCT GAT GCG A-3′. Annealing with primer created a 8OG:dA base pair at primer (terminus) after annealing. The templating base was dG. Downstream primer was phosphorylated and was 5′-GTC CC-3′. All the oligonucleotides were purchased from Integrated DNA Technologies, Inc. Binary complex crystals were grown in 50 mM imidazole, pH 7.5, 20% PEG3350, 90 mM sodium acetate. These crystals were then soaked in artificial mother liquor  with a hydrolysable analog (5 mM dCMPP(CH2)P) and 12% ethylene glycol as cryoprotectant. Diffraction quality data were collected on the soaked crystal as described below.Human DNA polymerase β was overexpressed in ®-007HF (Rigaku Corporation) rotating anode generator. Data were integrated and reduced with HKL2000 software9. Ternary substrate complex structures was determined by molecular replacement with a previously determined structure of pol β complexed with 8OG:dA base pair at the primer terminus and incoming dCMPCF2PP (PDB ID 3RJH)6.Data were collected at 100 K on a CCD detector system mounted on a MiraMaxFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary