Chl-a is a good indicator of phytoplankton biomass and may be used to detect changes in marine ecosystems due to environmental fluctuations. Dramatic climatic changes have been observed in the Arctic during the past decades. The study aimed to give up relations between Chl-a and climatic factors in the Barents Sea. We found an increase in Chl-a over the last four decades, with maximum values in warm periods. High temperature and a decline in sea ice extent were associated with greater Chl-a concentrations. Atmospheric processes estimated through the North Atlantic Oscillation indices strongly affected the surface water temperature, salinity, and Chl-a. Mapping of the Barents Sea showed high concentrations of Chl-a associated with the ice edge in spring and coastal waters in summer. Our study suggests that spatial, seasonal, and temporal variability in Chl-a is controlled by temperature changes, ice extent, and global atmospheric circulation and may be used for future investigations dealing with climatic forcing in the Arctic marine systems.Phytoplankton and other algae form the bases of food webs in aquatic systems as they convert solar energy into chemical compounds that are consumed by the higher trophic levels. Through the photosynthesis process based on chlorophyll a (Chl-a) was estimated in situ with the changes in the surface sea temperature (SST) and salinity (SSS) in the Barents Sea and adjacent waters during the period of 1984–2021. Spatial differences were detected in SST, SSS, and Chl-a. Chl-a increased parallel to SST in the summer-autumn and spring periods, respectively. Chl-a peaks were found near the ice edge and frontal zones in the spring season, while the highest measures were observed in the coastal regions during the summer seasons. SST and Chl-a demonstrated increasing trends with greater values during 2010–2020. Generalized additive models (GAMs) revealed that SST and Chl-a were positively related with year. Climatic and oceanographic variables explained significant proportions of the Chl-a fluctuations, with six predictors  being the most important. GAMs showed close associations between increasing Chl-a and a decline in sea ice extent and rising water temperature. Our data may be useful for monitoring the Arctic regions during the era of global changes and provide a basis for future research on factors driving phytoplankton assemblages and primary productivity in the Barents Sea.The Arctic climate strongly affects phytoplankton production and biomass through several mechanisms, including warming, sea ice retreat, and global atmospheric processes. In order to detect the climatic changes in phytoplankton biomass, long-term variability of chlorophyll  Phytoplankton assemblages in the World Ocean strongly depend on climatic fluctuations, availability of nutrients, and light . Changesa (Chl-a) and productivity indicated different trends and relationships with environmental variables in the upper open ocean systems, including Arctic regions [The Arctic Ocean has experienced clear fluctuations in sea surface temperature at interannual to multidecadal scales due to the impact of the North Atlantic Oscillation ,4. Moreo regions ,9,10. ThThe Barents Sea represents a shallow shelf region with the western boundary defined by the shelf break towards the Norwegian Sea, the eastern boundary confined by Novaya Zemlya, the southern boundary located off the Kola Peninsula, and the northern boundary limited by the continental shelf break towards the deep Arctic Ocean . 2 with an average depth of 230 m and a maximum depth of about 500 m [It has an area of approximately 1.6 million kmut 500 m . Being lut 500 m . The oceut 500 m ,16. Threut 500 m ,13.–2, with the greatest values in AW. The average total annual primary production is approximately 70–100 g cm−2 [Extensive seasonal fluctuations in ice coverage are detected in the Barents Sea, especially in the eastern areas . The ice0 g cm−2 ,19. In g0 g cm−2 . Thus, t0 g cm−2 ,19. Calanus represent the major part of the total zooplankton in the Barents Sea, and they comprise 75–80% of the total biomass [In the AW domain, the phytoplankton growth and development during spring are controlled by thermocline formation due to strong water stratification, while in ArW, the timing of the ice edge phytoplankton bloom is defined by the stability of the water column from melting ice ,21. Rece biomass . Zooplan biomass ,14. Some biomass ,15,16,30 biomass ,23,27,31−3 [Chl-a concentrations are often used as a good proxy to estimate phytoplankton biomass in the marine environment ,33,34. S−3 . Therefo−3 .There was large interannual variability in the satellite-derived surface Chl-a concentrations and magnitude of the spring bloom in the Barents Sea over the 1998–2017 period . Strong The aim of the present study was to analyze long-term variations in the surface Chl-a in the Barents Sea with special attention to large-scale climatic factors. We hypothesize that in situ Chl-a estimations would be associated with variations in environmental factors. To test our hypothesis, we examined links between the surface Chl-a and water temperature, salinity, indices of global atmospheric circulation, temperature/salinity anomalies, and sea ice extent. Using generalized additive models, we studied which factors were responsible for Chl-a variability in the region.In situ data were obtained during 27 cruises in the Barents Sea and adjacent Svalbard waters during the period 1984–2021 . A total2) in April and September. We also used local environmental indices  to reveal their possible impact on Chl-a variability.Seven environmental measures were selected in order to analyze the possible influence of climatic fluctuations on the Chl-a pattern in the Barents Sea: the North Atlantic Oscillation indices , temperature and salinity anomalies (0–200 m) at the Kola section , annual sea ice coverage , sea ice extent ) and updated from recently published sources [The Kola Section is a standard oceanographic transect located between 69°30′ and 77°30′ N in the Barents Sea. Oceanographic studies at this transect have been monitored during the past decade ,49,50. T sources ,16.2), we used data from the Norwegian Polar Institute [Annual sea ice extent data expressed as percentages of the total sea area were provided by the Working Group on the Integrated Assessments of the Barents Sea (WGIBAR) . Monthlynstitute .All data were divided into 5 five groups: spring, summer, autumn, and winter, and combined for all seasons. The normality of the environmental and Chl-a data was checked with the Shapiro–Wilk test. In situ values of Chl-a, SST, SSS, and climatic parameters were found to be normally distributed . Therefoi. An exponential family distribution is specified for Y  along with a link function g  relating the expected value of Y to the predictor variables via an equation such as [Generalized Additive Models (GAMs) with climatic indicators as continuous variables were applied to investigate temporal variability in Chl-a in the Barents Sea. GAMs have been found to be a good tool for analyzing non-linear and non-monotonic relationships between a response variable and fitted predictors . Shortly such as : g(E(Y))In our study, the response variable (Chl-a) might demonstrate linear or non-linear relationships with the explanatory variables . Therefore, we used GAM with both normal and Poisson distributions along with an additive link function (identity) to predict Chl-a fluctuation in relation to environmental variables. Cubic regression splines as the smoothing functions were applied in GAMs with the smoothness determined by estimated predictive accuracy. General algorithms added in the models used any regression-type smoothers as partial residuals. The partial residuals (Chl-a) remove the effects of all other variables; therefore, the Chl-a can be used to model against the effects of climatic predictors. Interannual trends of SST, SSS, and Chl-a (influence of year as a model predictor) were studied with GAMs and linear regressions. We used only values collected at the Kola Section  to avoid spatial influence on the data. Data for this site covered most of the study period and can reflect relevant temporal patterns.www.statsoft.com (accessed on 12 August 2022)). Mean values were calculated with standard errors (±SE). We applied an interpolation to determine the SST, SSS, and Chl-a using ordinary kriging . The contour maps for SST, SSS, and Chl-a were created using MapViewer 6.00 .The GAMs were performed using StatSoft STATISTICA 10 software   . The mea > 0.05) .Temporal variations in the mean values of SST, SSS, and Chl-a are presented in Analysis of summer and combined data for the Kola Section indicated that there was significant temporal variability in SST, SSS, and Chl-a during the study period . Linear Temporal patterns for Chl-a in relation to SST, SSS, and selected climatic indices were evaluated using GAMs. Parametric and non-parametric GAMs revealed that environmental variables explained 25–34% of the total fluctuations in Chl-a in all seasons . Normal-The present study provided an analysis of long-term variations in water temperature, salinity, and Chl-a measured in situ in the surface layer of the Barents Sea. Important limitations of our work must be mentioned. Firstly, our dataset has some temporal gaps for SST, SS, and Chl-a. Secondly, some regions were not fully investigated, especially the northern and north-western areas . Thirdly, we had a restricted dataset for the winter season (only two periods) that was associated with severe environmental conditions preventing successful sampling. However, we were able to investigate spatial and temporal patterns of SST, SSS, and Chl-a in the Barents Sea and adjacent waters using interpolation procedures and GAMs. In general, we confirmed our hypothesis that large-scale atmospheric phenomena, climatic indices , and sea ice extent fluctuations in the region were responsible for temporal variations in Chl-a.Clear climatic variations have been documented during the past decades in the northern hemisphere ,54,55,56Analysis of published data regarding variations in the water temperature and salinity anomalies at the Kola Section ,15,16,49The spatial distributions of SST, SSS, and Chl-a in the Barents Sea demonstrated obvious variability in each sampling season. SST was found to be highest in the western and southern regions during the spring period, and this pattern reflected the influence of warm AW flowing from the Norwegian Sea. Minimal SST was encountered in the northern parts of the sea, where cold currents prevailed. In the summer and autumn seasons, the highest SST was also recorded in CW, especially in the south-eastern and southern Barents Sea. These observations can be explained by seasonal heating and shallowness of the inshore waters ,19. The SSS showed similar spatial distributions as SSTs, with the highest seasonal values in the western and central regions where high-saline AW was present. The northern regions affected by less saline Artic waters had lower salinity partly due to ice melting. Coastal regions with extensive river run-off also demonstrated lower salinity, and this is a common pattern typical for freshwater-affected areas in the Nordic Seas ,14. DespFirst peaks of Chl-a were detected in the spring period in the northern and central parts of the Barents Sea, suggesting earlier phytoplankton bloom in ArW compared to AW, where the outbursts of microalgae were 1–1.5 months later. Our spring averaged estimations were comparable with the results of previous findings that recorded maximum Chl-a values in the Marginal Ice zone of the Barents Sea ,60,61. TOur study provided new data regarding the Chl-a distribution in less-studied and hard-to-access regions of the Barents Sea. We revealed high concentrations of Chl-a in the north-eastern and northern regions during the spring and summer seasons along the ice melting edge. Our study documented a relatively high density of Chl-a near Novaya Zemlya (eastern Barents Sea), where intense blooms were revealed in frontal zones and cold ArW. In autumn, the maxima of Chl-a were located mainly in CW of the Barents Sea and in the open regions in the west. Autumn bloom is characterized by lower Chl-a values relative to spring estimates. The duration of autumn peaks is also shorter ,40, and We found higher Chl-a density in the south-eastern Barents Sea in the summer and autumn periods. These regions are strongly affected by freshwater run-off from the Pechora River. The run-off from the land to sea areas may carry sufficient amounts of nutrient salts to stimulate phytoplankton blooms in the inshore regions . Arctic During winter, phytoplankton production is low, and the distribution of nutrients in the water column is more homogeneous. Studies focused on the winter Chl-a are scarce, and our investigation provides new insights regarding Chl-a patterns in the Barents Sea. Although our winter dataset was limited, we were able to create a map showing the Chl-a pattern in the western part of the sea. High concentrations of Chl-a were revealed in AW, suggesting a positive influence of the warm inflow on the phytoplankton. The second site where high Chl-a was found in Svalbard coastal waters. The western area near Svalbard represents a region with dominating warm AW that might be favorable for phytoplankton growth. The waters east of Svalbard are a frontal zone with interacting AW and ArW ,14 that Our study revealed a general trend for SST to increase from the end of 20 century. This observation is in line with the global warming processes documented over the past decades ,6,7,12. SSS showed no temporal variations during the study period, although in 2015 and 2016, SSS values were maximal. These high estimates indicate a good correspondence with thermal conditions. The period 2015–2016 was one of the warmest periods in the 21 century in the Barents Sea. Considering a strong association of the surface water heating with the degree of AW inflow ,12,14,16We found that Chl-a tended to increase during the study period. Plankton communities are very sensitive to environmental forcing and exhibit different responses to climatic influence ,22,23,25GAMs obtained in this study confirmed our hypothesis regarding the environmental impacts of the climatic factors on the inter-annual variability of in situ Chl-a in the surface layer. We revealed that a considerable part of Chl-a variations could be explained by SST, SSS, NAO indices, water temperature/salinity anomalies, and ice conditions in the Barents Sea. However, only six predictors were found to contribute significantly to the Chl-a dynamics. The GAMs predicted enhanced Chl-a levels during the periods with positive NAO, increased SST, positive temperature anomalies at the Kola Section, and decreased sea ice extent in April or September. Many previous studies have shown strong correlations between NAO indices and water temperature in the Arctic ,64,65,66Positive NAO phases noted in our study led to an increase in AW inflow, enhanced heating of the surface waters, and earlier ice melting, resulting in better conditions of more area of the open water for phytoplankton growth and, thus, greater Chl-a values. The temperature was found to have a positive effect on phytoplankton production in the Arctic, especially during the early bloom in the spring ,69. HoweThe summer-time stratification in the northern part of the Barents Sea may also increase as a result of greater ice melting and glacial discharge near Franz Joseph Land, Svalbard, and Novaya Zemlya. Slight changes in salinity due to increased inflow of high-saline AW during the periods with positive NAO may also play a role in the stabilization of the water column causing earlier spring outbursts in the Barents Sea during the spring and summer periods. In the northern and central Barents Sea, the major diatom peak does not appear to begin with the return of sufficient daylight, and it appears to follow rapidly upon the ice melting in the Marginal Ice Zone ,41,60. SThere is evidence that positive NAO phases are related to a higher degree of AW advection in the Barents Sea ,14. The Our study suggests negative correlations between the extent of sea ice and Chl-a in the Barents Sea. Ice conditions strongly affect the distribution of Chl-a in the Arctic Ocean and adjacent waters, and this influence is mainly connected with light conditions. The spring bloom is strongly dependent on the retreating of the sea ice that determines the amount of solar radiation in the upper euphotic layer zone ,9,71. InMoreover, ice extent anomalies were found to have direct consequences for the spatial distribution of spring blooms in the Barents Sea . In yearChanges in ice cover recorded in the Arctic during the past decades may be a significant factor affecting ice algae. In the Barents Sea, the total ice algal production is an important source of primary production in the northern ice-covered regions and the Marginal Ice Zone. However, it has been found that under-ice bloom supports only 6% of the total annual primary production in that area . AlthougWe also must emphasize that the models obtained in our study explained only part of Chl-a variability in the Barents Sea, suggesting that other drivers besides climatic factors would be important in determining phytoplankton dynamics in the Barents Sea. Phytoplankton growth and development are controlled by a set of environmental drivers, with light intensity, nutrient availability, and grazing impacts being the most significant ,61,62. Light conditions in the Arctic demonstrate a clear seasonal pattern from the continuous daylight in summer to the periods of darkness during the polar night in winter ,14. ChanThe importance of nutrient supply in phytoplankton growth and primary production has been widely recognized in many studies ,60,61,62Many studies have pointed out the significance of the trophic relationship between phytoplankton and zooplankton in the Arctic ecosystems. The high contribution of zooplankton grazing to the decline of phytoplankton stock and the role of zooplankton ingestion in controlling the Arctic marine food web structure is more evident during the periods of spring and summer blooms. Positive correlations between Chl-a and zooplankton abundance have been reported previously in the Barents Sea ,79,80. ZTherefore, the amount of light penetrating the surface, nutrient concentrations, and zooplankton grazing together with climatic factors must be included in the improved models in order to better predict temporal and spatial trends in Chl-a in the Arctic Ocean and adjacent marginal seas.a levels) and a set of environmental factors, including the North Atlantic Oscillation indices, temperature/salinity anomalies at the Kola Section, and sea ice extent in the Barents Sea. Positive temperature anomalies and a decreasing ice extent have been noted since the 2000s. There were significant spatial and temporal trends in SST, SSS, and Chl-a over the period of 1984–2021. SST and Chl-a tended to increase from the maximum values noted in 2015 and 2016. Spring and summer values of Chl-a were found to be higher compared to the autumn and winter estimations. Our study documented relatively high concentrations of Chl-a in the Marginal Ice zone and near the retreating ice edge in the spring seasons, while maximal estimates of Chl-a were encountered in the coastal regions during the summer seasons. Generalized additive models predicted enhanced values of Chl-a in warm periods. Considering continuous climatic changes in the Arctic associated with the heating of water masses and reducing ice coverage, we may propose a subsequent increase in the total phytoplankton stock and Chl-a in the Barents Sea. Rising phytoplankton productivity may have various effects, such as altering pelagic communities, fluctuations in fish and shellfish stocks, and changes in marine biodiversity. Our study provides a basis for future investigations focusing on the ecosystem fluctuations in the Arctic Ocean and adjacent regions associated with climate forcing. For a better understanding of the ecosystem dynamics and productivity in the Barents Sea, further studies with better space-time resolution are needed.Model approaches are a powerful tool for investigating relations between biological systems and environmental fluctuations. Our study showed a good correspondence between measured variables in situ (surface sea temperature, salinity, and chlorophyll


Here, we used Gene Expression Integration, Human Protein Atlas, and The Cancer Genome Atlas (TCGA) to perform a pan-cancer analysis of the role of TGR5 in all 33 tumors. In all TCGA tumors, the TGR5 gene expression has been assessed, and we found that the high TGR5 gene expression in most cancers is associated with poor prognosis of overall survival for cancers such as glioblastoma multiforme (p = 0.0048), kidney renal papillary cell carcinoma (p = 0.033), lower grade glioma (p = 0.0028), thymoma (p = 0.048), and uveal melanoma (p = 0.004), and then the lower expression of TGR5 was linked with poor prognosis in cervical squamous cell carcinoma and endocervical adenocarcinoma (p = 0.014), malignant mesothelioma (MESO) (p = 0.048), sarcoma (p = 0.018), and skin cutaneous melanoma (p = 0.0085). The TGR5 expression was linked with the immune infiltration level of the macrophage M2_TIDE and was also associated with DNA methylation in ovarian and breast cancers. The regulation of hormone secretion, Rap1 pathway, osteoclast differentiation, and bile acid pathway was involved in the functional mechanism of TGR5. Besides, gene expressions were different in different tumors detected by RT-PCR, and cell activity experiments have also found that TGR5 can increase the activity of renal cell carcinoma and reduce the activity of skin cancer and osteosarcoma cells. In this investigation, the aim was to assess the comprehensive overview of the oncogenic roles of TGR5 in all TCGA tumors using pan-analysis.The relationship between G protein–coupled bile acid receptor 1  and, specifically, cancer has been studied in  A first pan-cancer analysis of TGR5The TGR5 gene was highly expressed in most tumors that were associated with different prognoses.The macrophage M2_TIDE and DNA methylation may associate with the expression of TGR5.The correlation between the oncogenic role of TGR5 and the regulation of hormone secretion, Rap1 signaling pathway, and osteoclast differentiation may play an essential role in the progression of cancer.In recent years, the therapeutic advances of cancer immunotherapy have developed rapidly and achieved remarkable effectiveness in treating a broad range of cancers by the interaction between the immune system and human cancers. Despite the successful application of cancer immunotherapy across various types of human cancers, the majority of patients with tumor have limited or no response to these treatments. Therefore, there is a desperate need in excavating predictive biomarkers to assess the response to those immunotherapy approaches and defining staging and prognosis at an early stage. Previous studies have discovered a few indicators associated with tumor prognosis and progression; however, accurate biomarkers for predicting clinical outcome and patient survival continue to be explored.TGR5 (G protein–coupled bile acid receptor1), also known as GPBAR1, belonging to the G protein–coupled receptor (GPCR) superfamily –7. Our pBecause molecular aberration and the complexity of tumorigenesis have increased unprecedentedly in scale and accessibility, pan-cancer analysis has become very crucial in analyzing the clinical outcome and mechanism. The genomics information of different tumors contained in publicly funded TCGA projects and available Gene Expression Omnibus (GEO) databases allows us to analyze pan-cancer –18. ThusIn this study, we represented the TCGA and GEO databases to generate TGR5 detection to determine gene expression and DNA methylation, immune system infiltration, and cell signaling pathways across 33 various cancers. Furthermore, we also analyzed the molecular mechanism of TGR5 in the skin and renal cancer cell lines. Our study preliminarily unveiled the application of TGR5 as a predictive biomarker of tumor staging and prognosis, which warrants further investigation.http://timer.cistrome.org/) and the ONCOMINE database (www.oncomine.org) to analyze the relationship between different tumor tissues and adjacent normal tissues, as well as the TGR5 expression in tumors  to acquire box plots of the Genotype-Tissue Expression (GTEx) database under the settings of p-value cutoff = 0.01, log2FC (fold change) cutoff = 1, and “match TCGA normal and GTEx data.” The total isoform usage changes of TGR5 in 33 types of tumors have also been analyzed in the GEPIA2 tool. The expression of TGR5 in the different pathological stages of all TCGA tumors had been investigated in the HEPIA2 tool. Mutation of the GPBAR1 gene was documented by oncogenomic analysis.We used the Tumor Immune Estimate Resource  website  + 1] for log scale, matching TCGA and GTEx normal data, and adding all cancer tissue names.We also used the GEPIA2 tool to analyze overall survival (OS) saliency maps and disease-free survival (DFS) of TGR5 survival data in all tumors. Cutoff values (50% and 50%) were used as expression thresholds to separate the high-expression and low-expression cohorts, and the logarithmic sequence test was used in the hypothesis test .https://www.cbioportal.org/) was applied to analyze the protein structure, mutation type, mutation site information, copy number change (CAN), and the three-dimensional structure frequency of changes by checking all TCGAs.The cBioPortal tool  in the STRING website (https://string-db.org/). The most important parameters are defined as follows: the minimum required interaction rating [low confidence (0.150)], the effectiveness of the network edge, and the maximum number of interactions to be displayed (no more than 50 first shells) and active interaction sources.We used the protein–protein interaction network to study the interaction between protein names (TGR5) and organisms  transformed expression data were applied for the box or violin plots.We obtained violin plots of the TGR5 expression in different pathological stages  of all TCGA tumors The GEPIA2 tool was used to select the top 100 TGR5-related genes from all TCGA data sets based on tumors and normal tissues. Then, we performed a Pearson gene correlation analysis on TGR5 and selected genes, calculated the p-value and correlation coefficient (R), and displayed them in the corresponding fields of the graph. The heat map of the selected gene expression profile showed the partial correlation (color) and p-value in the Spearman degree correlation test after purity correction.https://www.r-project.org/) was used in this analysis. Two-tailed p < 0.05 was considered to be statistically significant  pathway analysis. The enriched pathways were visualized with the “tidyr” and “ggplot2” R packages. The R language software  (nificant .https://www.proteinatlas.org/).In order to understand the expression level of the TGR5 protein, we performed immunohistochemical staining of the TGR5 protein in colorectal cancer, breast cancer, lung cancer, and pancreatic cancer and then analyzed these data using the Human Protein Atlas  and SMART network (http://www.bioinfo-zs.com/smartapp/) to analyze the DNA methylation levels of different TGR5 probes in all TCGA tumors. We also examined the methylation of the CpG aggregation of the TGR5 gene, the correlation of DNA methylation, the change of copy number variation, and different tumor stages. Take the beta value of each sample, and take the p correlation value of the Pearson correlation coefficient (R) according to Benjamin–Hochberg. MethSurv data (https://biit.cs.ut.ee/methsurv/) were also used to predict the methylation level of TGR5 DNA.We used MEXPRESS  and human renal cancer cell lines  were cultured in McCoy’s 5a medium  containing 10% FBS, ampicillin, and streptomycin at 37°C in a humidified atmosphere of 95% air and 5% CO2. Human renal cancer cell lines  were purchased from the Institute of Cell Biology, Chinese Academy of Sciences.Human skin basal cell carcinoma cell lines  were grown in Dulbecco’s modified eagle medium (DMEM)  containing 10% FBS  in an incubator with 5% COAll cell lines have been used for more than 20 generations. The middle part was replaced the day before transfection. When the cells reach 70%–90% growth density, the cells were treated with TGR5 agonist (INT777) and si-TGR5 Sirna synthetic transfection . At the same time, non-interfering siRNA transfection was used as a negative control. The transfection process was carried out in strict accordance with the kit instructions, and the cells transfected for 48 h were collected for the next step of detection.As mentioned above, the Cell Counting Kit-8 (CCK-8) was used to assess cell viability . MG-63 BTotal RNA was extracted from cells using TRIzol (Invitrogen). qRT-PCR was performed in triplicate using SYBR Green reagents  with specific primers for TGR5  and glyceraldehyde-3-phosphate dehydrogenase .All measured values are expressed as mean ± standard deviation, and a p value of ≤ 0.05 is considered statistically significant. A repeated-measures two-way ANOVA was used in this study. GraphPad Prism 8.02  was used to analyze the data using one-way ANOVA and Tukey’s multiple.We first investigated the TGR5 expression across TCGA tumors based on the TIMER2 tool. The gene expression of TGR5 was analyzed in the Oncomine data set over a cancer-wide range S13. We We divided tumors into high and low TGR5 expression groups and analyzed the correlation between TGR5 gene expression and survival scores obtained by TCGA and GEO data sets. As shown in Turning now to the experimental evidence on survival analysis using the Kaplan–Meier plotter tool, it can be seen that the lower expression of the TGR5 gene is correlated with poor relapse-free survival (RFS) , KICH, LUAD, LUSC, and THYM but lower expression in tumors of acute myeloid leukemia (LAML), PAAD, and CHOL than normal tissue. Besides, we further found that a low expression of TGR5 also has better OS in GBM, KIRP, LGG, THYM, and UVM but a high expression related with better OS in CESC, MESO, SARC, and SKCM, which means that TGR5 may be an important indicator for predicting the prognosis of cancer patients. Besides, TGR5 also plays different roles in different tumors, which is also consistent with our research results –39. Our In vivo, TGR5 deficiency in mice promoted diethylpromazine-induced hepatocyte death, compensatory proliferation, gene expression of certain inflammatory cytokines, matrix metalloproteinases, and liver carcinogenesis than wild-type mice. In vitro, TGR5 activation strongly inhibits hepatocellular carcinoma proliferation and migration by inhibiting STAT3 signaling and its DNA-binding activity6,7. Therefore, TGR5 receptor could be a new potential biomarker for the diagnosis and treatment of hepatocellular carcinoma in the future. Besides, TGR5 activation can inhibit the proliferation and migration of gastric cancer cells by inhibiting STAT3 and NF-κB signaling pathways8. In HCT116 cells, SW480 cells, and DSS-induced CRC mice, ursodeoxycholic acid (UDCA), as one of the main active components of bile, inhibits the malignant progression of colorectal cancer through TGR5-mediated cAMP-PKA-RhoA signaling pathway antagonizing YES-associated protein9. However, in lung cancer, binding and activation of TGR5 in H1299 lung cancer cells can increase the content of cAMP and the phosphorylation levels of protein kinase A (PKA)10. TGR5 activation strongly inhibited JAK2-STAT3 signaling in vitro and in vivo. The activation of TGR5 in the mesangial membrane of non–small cell lung cancer cells mediates JAK2-STAT3 signaling pathway, which exacerbates the development and migration of lung tumor cells6. Tgr5-induced cAMP-PKA-CREB and JAK2-STAT3 signaling pathways are promising therapeutic strategies and predict the efficacy of lung cancer treatmentThe role of TGR5 in different tumor tissues is obviously different. For example, TGR5 can increase the activity of renal cell carcinoma but reduce the activity of skin cancer and osteosarcoma. In many studies, TGR5 is one of the most common therapeutic targets against hepatocellular carcinoma by regulating energy homeostasis and glucose metabolism. For breast cancer, we investigate the data set of TCGA-BRCA and found a correlation between high TGR5 gene expression in tumors and clinical stages, a good RFS prognosis for BRCA. Overexpression of TGR5 has antiproliferation and pro-apoptosis effects on breast cancer cell adipogenesis . HoweverAccording to our studies, the high TGR5 gene expression in lung and gastric cancers raises the possibility of poor OS and positive correlation of TMB. TGR5 promotes the growth and migration of non–small cell lung cancer cells through the JAK2-STAT3 signaling pathway . Cao et Abnormal DNA methylation is considered to be a key event in the occurrence and development of cancer and related to chromatin remodeling and abnormal gene expression in many malignant tumors . In our The role and underlying mechanism of TGR5 in regulating immune infiltration play an important role in multiple diseases . We alsoOverall, this study from pan-cancer analysis strengthens the idea of a statistical correlation of TGR5 with gene expression, clinical prognosis, genetic alteration, tumor mutation burden, immune cell infiltration, and microsatellite instability for all TCGA tumors. Therefore, this study provides broad molecular signatures for further functional and therapeutic studies of TGR5 and also represents a systemic approach to characterize key proteins in cancer.The original contributions presented in the study are included in the article/Conception and design: ZYG and TK. Acquisition, analysis, and interpretation of the data: ZYG, SFL, ZW, QGZ, LYL, and ZQG. Drafting and writing: XJ and KT. Final approval of the article: ZYG, SFL, ZW, QGZ, LYL, KT and ZQG. All authors contributed to article and approved the submitted version.This work was supported by grants from the Clinical Research Project of Shanghai Tenth People’s Hospital (YNCR2C027) and Research Fund of Shanghai Tongren Hospital, Shanghai Jiaotong University School of Medicine (No: TRYJ2021JC02) and Tongren Xinxing (TRKYRC-xx202215).We would like to thank all participants in this study.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Spinal cord injury (SCI) is a devastating type of neurological disorder of the central nervous system (CNS) with high mortality and disability. The pathological processes of SCI can usually be described as two stages, namely, primary and acute secondary injuries. Secondary injury produces more significant exacerbations of the initial injury. Among all the mechanisms of secondary damage, infection and inflammatory responses, as the principle culprits in initiating the second phase of SCI, can greatly contribute to the severity of SCI and numerous sequelae after SCI. Therefore, effectively antagonizing pro-inflammatory responses may be a promising treatment strategy to facilitate functional recovery after SCI. Olfactory ensheathing cells (OECs), a unique type of glial cells, have increasingly become potential candidates for cell-based therapy in the injured CNS. Strikingly, there is growing evidence that the mechanisms underlying the anti-inflammatory role of OECs are associated with the immune properties and secretory functions of these cells responsible for anti-neuroinflammation and immunoregulatory effects, leading to maintenance of the internal microenvironment. Accordingly, a more profound understanding of the mechanism of OEC immunological functions in the treatment of SCI would be beneficial to improve the therapeutic clinical applications of OECs for SCI. In this review, we mainly summarize recent research on the cellular and molecular immune attributes of OECs. The unique biological functions of these cells in promoting neural regeneration are discussed in relation of the development of novel therapies for CNS injury. Spinal cord injury (SCI) is a kind of servere neurological disease generally caused by a variety of traumas or diseases that usually result in complete or incomplete neural function deficiency. Among all the directly or indirectly causal external factors resulting in SCI, traumatic factors, such as traffic accidents, falls and sports/recreation, are the most common aetiologies of SCI –3. AdditIn general, the pathophysiological types of SCI are characterized as acute, secondary and chronic phases , 8. PrimOwing to the complexity of the CNS and the inhospitable environment in and around the lesion site in SCI, combination strategies to promote tissue regeneration are currently being pursued . When coOlfactory ensheathing cells (OECs) are a specialized type of glial cell population in the olfactory nervous system that accompany and envelop bundles of primary olfactory axons , 29. TheAlthough OECs are a specialized type of glial cells that bridge the boundary between the peripheral and central olfactory systems, and exhibit numerous molecular and cellular properties of both Schwann cells and astrocytes, they are obviously distinguishable from both Schwann cells and astrocytes by differential molecular expression characteristics , 44. Ear2+-binding proteins that participate in regulating numerous intracellular events including protein phosphorylation, cell differentiation and proliferation, and is extensively expressed in Schwann cells  is expressed in OECs during the development of the ONL, but its obviously declines, and cannot be detected in OECs in either the inner ONL or the peripheral olfactory system in adulthood , 48, 59.in vivo and in vitro, as well as in the olfactory mucosa and bulb of neonatal and adult animals. Although no experimental data are available to support the antigenic difference between these two temporally and spatially different sources of OECs, a currently prevalent notion that is widely held is that the antigenic heterogeneity in OECs mainly results from the existence of OEC subpopulations, contamination with astrocyte-like cells, different development phases, various culture conditions, and the number of OEC passages  in their extending processes , 69. In in vitro , 70. In in vitro . These din vitro . The resin vitro , 74. Thiin vitro , 67, 74.2′,3′-Cyclic nucleotide 3′-phosphohydrolase (CNPase), an enzyme ubiquitously localized to noncompact myelin areas, is a critical antigen in OECs . CNPase in vivo and in vitro. This mainly depends on the context, such as the developmental stage , animal species and culture conditions , phosphatidylserine receptors and mannose receptors -mediated phagocytosis for damaged “self” and invading “non-self” clearance  can be continuously renewed throughout the lifespan , 84. In eceptors , 86–88, eceptors , 80, 86.learance . Apart feceptors , 90. Morin vitro , 89–91. turmeric , which aturmeric . In compvia integrin receptors  impairment in the acute phase, which is accompanied by the infiltration of immune cells and accumulation of cytokines near the injury site  and the underlying LP are continuously exposed to a variety of potentially infectious environmental agents. However, most microbial invasion does not occur from the olfactory mucosal surfaces ury site . Infiltrury site –131. Durury site –134, seeury site , 136. Houry site , 138. Thury site  observatury site . In the ury site , 139. Inury site , 141. Itury site , 143, suury site , 31. Henury site , 145. Usury site , 147. Foury site –151. Somury site , 153. Foury site –156. Astury site , 158. Moammation , 160. Alvia polarization of M1 microglia to M2 microglia, leading to neural survival and axonal regeneration , Natural Science Foundation of Shaanxi Province , and Xi’an Science and Technology Research Project [2019114913YX004SF037(3)].The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Oxygen measurements have been interpreted to indicate that the ocean’s oxygen-deficient zones (ODZs) are expanding under global warming2. However, models provide an unclear picture of future ODZ change in both the near term and the long term6. The paleoclimate record can help explore the possible range of ODZ changes in warmer-than-modern periods. Here we use foraminifera-bound nitrogen (N) isotopes to show that water-column denitrification in the eastern tropical North Pacific was greatly reduced during the Middle Miocene Climatic Optimum (MMCO) and the Early Eocene Climatic Optimum (EECO). Because denitrification is restricted to oxygen-poor waters, our results indicate that, in these two Cenozoic periods of sustained warmth, ODZs were contracted, not expanded. ODZ contraction may have arisen from a decrease in upwelling-fuelled biological productivity in the tropical Pacific, which would have reduced oxygen demand in the subsurface. Alternatively, invigoration of deep-water ventilation by the Southern Ocean may have weakened the ocean’s ‘biological carbon pump’, which would have increased deep-ocean oxygen. The mechanism at play would have determined whether the ODZ contractions occurred in step with the warming or took centuries or millennia to develop. Thus, although our results from the Cenozoic do not necessarily apply to the near-term future, they might imply that global warming may eventually cause ODZ contraction.Dissolved oxygen (O By using foraminifera-bound nitrogen isotopes, it is shown that, during two warm periods of the Cenozoic, oxygen-deficient zones contracted rather than expanded, suggesting that global warming may not necessarily lead to increased oceanic anoxia. The global deoxygenation trend has been attributed to the decreased oxygen solubility and enhanced upper-ocean stratification expected from global warming, with predictions that global oxygen concentration will continue to decrease over the next decades and centuries, affecting marine ecosystems7. However, there is controversy as to the future of the ODZs in particular, with predictions of both expansion and contraction6.Observations indicate that oxygen concentrations have been decreasing in coastal and open-ocean waters over the past five decades, substantially expanding the volume of the ocean’s ODZs2 concentrations have decreased and global climate has cooled8. Against this background of Cenozoic cooling, there were two distinct periods of prolonged warm climate: the MMCO, about 6 °C warmer than today, and the EECO, about 13 °C warmer than today9. The MMCO and the EECO were characterized by higher CO2  and reduced or no continental ice12. These two climate optima present an opportunity to investigate the response of ocean oxygen concentrations to prolonged warmth13.Earth’s climate has evolved in response to tectonic and orbital forcing over the Cenozoic era, the past 66 million years (Myr). Overall, atmospheric CO2 concentration of less than approximately 5 µmol kg−1, based on standard measurements). Bacterial reduction of nitrate to N2 (‘denitrification’) requires suboxia and discriminates against 15N. Consequently, denitrification in the ODZs of the ocean water column causes an elevation in the 15N/14N ratio, or δ15N, of nitrate in the regions of the ODZs14, also increasing the nitrate δ15N of the global ocean15 (δ15N = ((15N/14N)sample/(15N/14N)atmN2 − 1)) × 1,000‰). The isotopic signal of ODZ denitrification is incorporated into newly formed biomass in the surface ocean, the by-products of which accumulate in deep-ocean sediments. Thus, the N isotopes provide a tool for reconstructing past changes in ODZ-hosted denitrification16. Organic matter that is native to and trapped within the mineral matrix of planktic foraminifer shells (foraminifer-bound organic matter) has been developed as an archive of the N isotopic history of surface ocean productivity17. Foraminifer-bound organic matter is isolated from the sedimentary environment by the mineral matrix, preserving the original unaltered N isotopic signal even over millions of years18. In areas of complete nitrate consumption, foraminifer-bound δ15N records the δ15N of the nitrate in the underlying pycnocline19. Pycnocline nitrate δ15N bears regional signals of water-column denitrification and of N2 fixation, the dominant source of biologically available  N to the ocean20. Pycnocline nitrate δ15N will also vary with the δ15N of mean ocean nitrate. Mean ocean nitrate δ15N, in turn, is largely controlled by the relative proportions of denitrification occurring in the water column versus the sediments15.The ratio of N isotopes in the ocean is sensitive to oxygen deficiency, or ‘suboxia’ (a dissolved O15N of foraminifer-bound organic matter (FB-δ15N) to investigate changes in the marine N cycle over the Cenozoic. We present new species-specific FB-δ15N data since the Miocene, as well as genus-specific measurements across the early Miocene, Oligocene and late Eocene epochs from two sediment cores, one located in the Pacific (ODP Site 872) and the other in the Atlantic (DSDP Site 516)  compilation that includes new measurements as well as previously published data  using a single temperature proxy , with a greater decline in the Pacific (of around 11‰) than in the Atlantic (about 6‰) , known as the Middle Miocene Climate Transition, again with a greater change in the Pacific than in the Atlantic. Finally, during the late Miocene (from 10 Ma to 5 Ma), the records decouple: the Pacific record shows a strong FB-δ15N increase followed by a decline at 3 Ma, whereas the Atlantic record first shows a small decrease and then remains stable until the present.The new data show that Atlantic and Pacific FB-δ6‰) Fig. . FB-δ15N15N and a decrease in the FB-δ15N difference between the Pacific and the Atlantic  and of the Central American Seaway (CAS) starting around 9.2 Ma (ref. 26). In a model experiment, CAS closure has been observed to increase regenerated CO2 storage in the Pacific interior27, presumably enhancing suboxia in the eastern Pacific. This and other effects of the CAS closure may help to explain the increases in Pacific FB-δ15N and Δδ15NP-A as well as the relative stability of South Atlantic FB-δ15N after 9.2 Ma . With warming, the loss of land ice and other processes might cause a sea level rise12, encouraging sedimentary denitrification29. Such a change might be argued to contribute to the low δ15N of the MMCO and the EECO. However, a sea-level-driven change in sedimentary denitrification would produce similar δ15N declines in the Atlantic and Pacific basins. Therefore, the larger decline from extremely high δ15N at Sites 872 and 1209 cannot be explained solely by enhanced sedimentary denitrification; rather, a reduction in water-column denitrification is required18.Global ocean nitrate δ30. Second, circulation changes in the ocean interior, such as enhanced Southern Ocean deep-water formation, may increase oxygen supply6. Our TEX86-based reconstructions of SST and ΔSSTL-H are consistent with either mechanism .Climate models point to several processes that work to shrink the ODZs under global warming, offering potential explanations for our finding of reduced water-column denitrification during the MMCO and EECO. First, a decrease in upwelling-supported biological export production in the equatorial and tropical Pacific would reduce oxygen demand in the subsurfaceism Fig. . On the 33, which should have caused a reduction in the supply of nutrients to the surface. These findings are consistent with reduced low-latitude export production and, thus, reduced oxygen demand as the cause of ODZ contraction  concentration of the ocean. However, the modern biological pump is weaker than this limit, largely because of ventilation of the deep ocean by the Southern Ocean36, which tends to fill the ocean interior with unused nutrients, reducing oxygen consumption in the ocean interior due to the ‘missed opportunity’ for nutrient-fuelled biological productivity at the Southern Ocean surface. Therefore, the decline in water-column denitrification during the MMCO and the EECO may have been driven by stronger Southern Ocean ventilation of the deep ocean.The oxygen concentration of the deep ocean (and of the ocean interior as a whole) is tightly linked to the ocean’s ‘biological carbon pump’, the storage of respiratory CO38)  show a lower planktic–benthic difference during the MMCO and an increase after the event, coinciding with the increase in Δδ15NP-A , coinciding with the increase in water-column denitrification (high Δδ15NP-A) during the Mi-2 glaciation at about 15.9 Ma  and the Pacific (ODP Site 872), respectively. DSDP Site 516 (Holes Z and F)  was drilled at 1,313 m water depth by the Deep Sea Drilling Project (DSDP) during Leg 72 in the southwest Atlantic50. The site is located on the Rio Grande Rise. ODP Site 872  was cored from a sea mount around the Marshall Islands in the eastern equatorial Pacific during Ocean Drilling Program (ODP) Leg 144 (ref. 51). The unconsolidated sediment consists of abundant, well-preserved planktic foraminifers. Our new data are compiled with previously published FB-δ15N data from Pacific ODP Site 1209 and Atlantic ODP Site 1263, most of which are mixed taxa18. In addition, mixed-taxa FB-δ15N measurements were made in several samples of late Oligocene and early Miocene age from Pacific ODP Site 1211 , which is in close proximity to Site 1209 .The new FB-δ15N of the nitrate supplied from the pycnocline to the surface. Sites 872 and 1209 are located close to the margin of nitrate-bearing surface waters associated with the equatorial Pacific upwelling. This may modify FB-δ15N relative to the δ15N of the subsurface nitrate supply in the region, most probably increasing FB-δ15N (ref. 56). As a consequence, any reduction in equatorial upwelling at times in the past might have modestly decreased FB-δ15N at Site 872.The surface waters above all core sites are environments of complete nitrate consumption and, therefore, should record the δ86 palaeothermometer21.We also report new glycerol dialkyl glycerol tetraethers (GDGTs) measurements from DSDP Sites 516 and 588  as well as ODP Sites 667 , 704 , 730 , 754 , 1146  and 1263 . The new measurements allow us to generate a continuous record of the SST evolution of low-to-middle and high latitudes across the Cenozoic based exclusively on the TEX59 adapted to GTS12 (ref. 60), magnetostratigraphy by Florindo et al.61 and age tie points based on K–Ar dating62 and an iridium spike for the K–Pg boundary63. For ODP Site 872, we used the age model based on biostratigraphic datums65 adapted to the GTS12 timescale60. The age models for ODP Sites 1209 and 1263 based on biostratigraphy are described in Kast et al.18. We revised these age models to the astronomically tuned chronologies recently published by Westerhold et al.66. The age model for ODP Site 1211 is based on biostratigraphy67. The age model for DSDP Site 588 is based on compiled biostratigraphy, magnetostratigraphy and isotope stratigraphy . Ages for ODP Site 667 were determined by linear interpolation of absolute constraints from biostratigraphic  datums69. Ages for ODP Site 704 were calculated by interpolating biostratigraphic  datums and magnetostratigraphic data70, and by aligning the new TEX86 record to the TEX86 records from Sites 1171 and U1318 across the middle Miocene climate transition, which are based on high-resolution benthic δ18O stratigraphy72. Ages for ODP Site 730 were determined by linear interpolation of biostratigraphic  datums73. The age model for ODP Site 754 is based on biostratigraphy 74. The age model for ODP Site 1146 is based on biostratigraphy (foraminifers) reported by Nathan and Leckie75. Ages for ODP Sites 690/689 are based on refs. 76. Ages for ODP Site 1171 are based on ref. 71. All the biostratigraphic datums and palaeomagnetic reversals were calibrated to GTS12 (ref. 60)  from the Miocene, Pliocene and Pleistocene sections of DSDP Site 516 and ODP Site 872 . We used the persulfate oxidation-denitrifier method for FB-δ15N first described for planktic foraminifers by Ren et al.17, with minor adjustments in the protocol. As a first step, 5–7 mg of foraminifer tests were gently crushed, transferred to a disposable, 15-ml polypropylene, conical-base centrifuge tube and chemically treated to remove external N contamination. 10 ml of Na-polyphosphate solution (pH 8) was added to the sample tubes, which were ultrasonicated for 10 s to remove clays. After rinsing the sample three times with Milli-Q water (by filling and decanting), 7 ml of a dithionite–citric acid solution  was added to each sample77. Sample tubes were then placed in a water bath at 80 °C for 30 min. After cooling, all samples were rinsed three times with Milli-Q water. Next, an oxidative cleaning step was performed to remove external organic matter: a potassium persulfate/sodium hydroxide solution  was added and autoclaved at 125 °C for 65 min. After cooling, the samples were rinsed four times with Milli-Q water and dried overnight in an oven at 60 °C. Next, 3–5 mg cleaned shell fragments were weighed and dissolved in 45 μl 4N hydrochloric acid to release organic matter for analysis. After the CaCO3 dissolution step, organic N was oxidized to nitrate by adding 1 ml of a second recipe of basic potassium persulfate solution . Nitrate concentration was measured for each sample by conversion to nitric oxide followed by chemiluminescence detection78. Subsequently, 5 nmol N of nitrate in the sample solution was converted to nitrous oxide using the denitrifier method, and its δ15N was measured by gas chromatography–isotope ratio mass spectrometry82. In contrast to previous studies84, samples were not pH adjusted before injection into the denitrifying bacteria; rather, they were injected without adjustment into 2.75 ml media buffered at pH 6.3 and containing the denitrifying bacteria (see ref. 85 for details). To quantify the precision and accuracy of the corrected isotope values, for each series of 30 samples, a total of nine different in-house (MPIC) foraminifer and coral laboratory standards were analysed. The following were analysed (each in triplicate): a coral standard from the taxon Porites (PO-1) with δ15N of 6.2 ± 0.3‰, a coral standard from the taxon Lophelia with δ15N of 10.1 ± 0.4‰ (ref. 85) and a mixed foraminifer standard (63–315-μm size fraction) from the North Atlantic (MSM58-17-1) with δ15N of 5.84‰ (uncertainty not yet assessed). After calibration with international nitrate isotopic references IAEA-NO3 and USGS-34 and correction for the oxidation blank, the long-term analytical precision for the foraminifer standard was 0.23‰ . The oxidation blank per oxidized sample was typically below 0.2 nmol N.Around 600–800 individual foraminifers from the species 86. After accelerated solvent extraction of the freeze-dried sediment samples, we added 60 μl of an internal standard  for quantification. The extracts were dried in a centrifugal Rocket Evaporator (Genevac) and filtered with a polytetrafluoroethylene filter (0.2-μm pore size) with a 1.4% mixture of hexane:isopropanol (hex:IPA). GDGTs were analysed using a high-performance liquid chromatographer  coupled to a single-quadrupole mass spectrometer detector  following the protocol proposed by Hopmans et al.88, with some small modifications. Normal-phase separation was achieved with one ultra-high-performance liquid chromatographer silica column  maintained at 30 °C. The flow rate of the 1.4% hex:IPA mobile phase was 0.2 ml min−1 and kept constant for the first 25 min, followed by a gradient to 3.5% hex:IPA in 25 min and a column-cleaning step with 10% IPA in hexane. We used a single-ion monitoring of the masses m/z = 1,302.3, 1,300.3, 1,298.3, 1,296.3, 1,292.3, 744.0, 1,050.0, 1,036.0, 1,022.0, 1,020.0 and 1,018.0. The injection volume was 5 μl or 20 μl, depending on the concentration of the samples analysed.5–20 g of freeze-dried sediment per sample was extracted and separated into two fractions at the Martínez-García lab at the MPIC, following the protocol proposed by Auderset et al.15N of extinct foraminifers to that of modern taxa for which the incorporation of the N isotope signal has been studied90. Our new dataset from DSDP Site 516 demonstrates that the FB-δ15N of T. sacculifer, a modern photosymbiotic, surface-dwelling species, overlaps with the FB-δ15N of the extinct species S. dehiscens, as well as with the FB-δ15N of mixed planktic foraminifers during the early Miocene and late Oligocene  differences in their N content. Dentoglobigerina altispira δ15N values, in turn, are similar to those of D. tripartita in the early Miocene. In this way, we can combine measurements from different foraminifer species/genera to obtain a continuous record of FB-δ15N throughout the Cenozoic . However, the sensitivity of Site 1209 to the isotopic signal of denitrification in the eastern tropical Pacific was eventually reduced by its migration, with the change occurring between the middle Eocene and the early Miocene. This is indicated by the similarity of early Miocene FB-δ15N at Site 1211 (a close neighbour of Site 1209) to the FB-δ15N at South Atlantic Site 516, both of which are substantially lower than FB-δ15N at Pacific Site 872 during the early Miocene  middle  and high latitudes (ODP Site 704) and integrated the records with existing TEX86-SST datasets  covering the past 69 Ma (refs. 136). The data and references are summarized in Supplementary Data 52. The categories as follows: high latitudes (above 50° N/S), low latitudes (0–20° N/S), mid latitudes (20–40° N/S), transition (40–50° N/S) and costal upwelling regions  after applying a local regression (LOESS) with a smoothing factor of 0.02 and a 0.2-Myr sampling step . Sediment cores located in transition regions , as well as upwelling-sensitive locations , were not considered in these calculations to minimize the effect of local changes in upwelling intensity and frontal migrations.For the presented latitudinal SST stack, we combined a total of 653 new TEX86 values, we used the calibrations proposed by Kim et al.137 (TEXH86) and Tierney and Tingley138 . Overall, the trends obtained when calculating the latitudinal temperature gradient are the same for both calibrations, but there are slightly larger amplitudes of change in the Bayspar-derived gradient  from our calculation of the latitudinal temperature gradient (355 samples)  branched GDGTs can be produced in situ147 and (2) degradation rates of crenarchaeol are two times higher than those of branched GDGTs148. Thus, in some environments, the BIT index can be high (above 0.4), despite the relatively low terrestrial inputs of brGDGTs. In these cases, samples with BIT > 0.4 have been used for palaeotemperature reconstructions129. In recent years, it has been shown that in situ-produced brGDGTs tend to be characterized by higher abundances of cyclic brGDGTs, resulting in high values for the #ringstetra index, and it has been suggested that #ringstetra values >0.7 are indicative of predominant in situ production of branched GDGTs149. Some of the new samples reported here, as well as some of the samples published in previous studies that are included in our compilation, have BIT values >0.4. However, some of the samples with BIT values >0.4 also have values for #ringstetra index >0.7, indicating that the brGDGTs were produced in situ, rather than transported from terrestrial soils. In the calculation of the latitudinal SST gradient, we exclude samples with BIT > 0.4 if the #ringstetra was <0.7 (178 samples) to avoid potential influences of terrestrial GDGTs on the estimated SST . However, we note that there are no substantial differences in the SST gradient if all the samples with BIT > 0.4 are included in the calculation  and compilation of all published marine TEX86 records. This table contains all GDGTs measurements, calculated indices, temperatures and gradients.TEXSupplementary Data 3Applied age models for ODP/DSDP Sites 516, 588, 667, 690, 704, 730, 738, 754, 1146, 1209, 1211 and 1263 with biostratigraphic  and magnetostratigraphic datums adjusted to the GTS12 timescale.


It was possible to obtain O—H bond lengths and anisotropic displacement parameters for disordered hydrogen atoms which are in good agreement with the corresponding results of single-crystal neutron diffraction data. These results show that HAR combined with X-ray diffraction can compete with neutron diffraction in detailed studies of polymorphic forms of ice and crystals of other hydrogen-rich compounds.Accurate crystal structures of H 2O, D2O and mixed (50%H2O/50%D2O) ice VI obtained by Hirshfeld atom refinement (HAR) of high-pressure single-crystal synchrotron and laboratory X-ray diffraction data are presented. It was possible to obtain O—H/D bond lengths and ADPs for disordered hydrogen atoms which are in good agreement with the corresponding single-crystal neutron diffraction data. These results show that HAR combined with X-ray diffraction can compete with neutron diffraction in detailed studies of polymorphic forms of ice and crystals of other hydrogen-rich compounds. As neutron diffraction is relatively expensive, requires larger crystals which can be difficult to obtain and access to neutron facilities is restricted, cheaper and more accessible X-ray measurements combined with HAR can facilitate the verification of the existing ice polymorphs and the quest for new ones.Water is an essential chemical compound for living organisms, and twenty of its different crystal solid forms (ices) are known. Still, there are many fundamental problems with these structures such as establishing the correct positions and thermal motions of hydrogen atoms. The list of ice structures is not yet complete as DFT calculations have suggested the existence of additional and – to date – unknown phases. In many ice structures, neither neutron diffraction nor DFT calculations nor X-ray diffraction methods can easily solve the problem of hydrogen atom disorder or accurately determine their anisotropic displacement parameters (ADPs). Here, accurate crystal structures of H It forms different liquid forms such as aerosols and twenty different phases of solid ice. Water is essential for all known forms of life, it interacts with the most important biological substances such as proteins, DNA and polysaccharides, influencing protein folding, DNA base pairing and other crucial phenomena.i.e. ice crystals . The next major breakthrough was made possible by the studies of the Braggs and Max von Laue  as well as two amorphous solid forms . Ice X is not considered a disordered phase since the hydrogen atoms are located half-way between the oxygen atoms. Ice Isd is a stacking disordered form of ice I  . Unfortunately, the restricted access to reciprocal space in DACs significantly reduces the amount of information (the number of available reflections) obtainable from these experiments. As a result, such experiments usually exhibit low data completeness.et al., 2016et al., 2020et al., 2014et al., 2020Although XRD is the most widely used diffraction technique in regular measurements, it has not been the first method of choice in ice phase investigations. XRD-determined covalent bond lengths to hydrogen atoms were typically 10–15% too short for organic molecules  utilizes the concept of transferability of atomic electron densities  ice VI.Both TAAM  K, a = 6.21 Å, c = 5.73 Å] and also by Kuhs et al. , and its hydrogenated variant by Kamb  Å, c = 5.79 (1) Å] and Komatsu et al. (2011p = 0.95 (5) GPa, T = 298 (1), a = 6.1990 (14) Å, c = 5.698 (3) Å].The structure of ice VI is formed from liquid water in the 0.6–2.2 GPa pressure range at 270 K  space group symmetry. Water molecules connected by hydrogen bonds form two separate interpenetrating but non-interconnecting frameworks [see Fig. S2 of the supporting information]. There are two distinct types of water molecules and four distinct types of hydrogen bonds et al., 2019Each molecule of water forms hydrogen bonds to the four neighboring water molecules. In the average unit cell, each hydrogen atom has partial occupancy (0.5) and neighbors another hydrogen in close proximity. In the real structure, only one of the two atoms is present . However, non-typical bond lengths and shapes of ADPs are not uncommon for disordered structures.The bond lengths in the ice VI structure for the 1:1 mixture of normal and heavy water ice VI obtained from HAR using data collected with an in-house source are included in Table 13.2O ice VI and deuterated ice VI structures under pressure, resulted in bond lengths and ADP values which are in good agreement with the corresponding single-crystal neutron diffraction data. The average differences in O—H bond lengths are equal to 0.019 Å for synchrotron data for D2O, 0.016 Å for in-house source data for D2O and 0.031 Å for H2O ice VI. It was also possible to obtain hydrogen ADPs similar to those obtained from neutron diffraction. However, some qualitative differences were visible and for some datasets the ADPs from X-ray data were quite different than those from neutron diffraction. The results show that it is possible to obtain an accurate structure for high-pressure disordered ice from XRD combined with HAR refinement, but this cannot be taken for granted since it has a strong dependence on the quality of the collected data.In summary, we have demonstrated that it is possible to successfully use XRD for accurate determination of the high-pressure, disordered structure of ice VI. This type of structural analysis is challenging, since X-rays are scattered from electrons and, therefore, they ‘see’ hydrogen atoms (with only one electron) less clearly than heavier atoms. The situation is even worse in the case of disordered structures with partial occupancies of hydrogen sites. Fortunately, in the case of ice VI the fractional occupancies of the hydrogen sites are known to be at least 0.5, whereas in other cases such as ice III or V occupancy deviates from 0.5 and must also be refined. An additional challenge is related to high-pressure measurements as they require the use of DACs. This restricts access to the reciprocal space and, as a consequence, diminishes the completeness and redundancy of the collected data. Yet with progress in experimental high-pressure techniques, an increase of data collection quality, and improved methods of experimental data analysis and interpretation, it is now possible to solve these kinds of problems with XRD. This certainly opens new possibilities for exploration of the phase diagram of water. Our results from high-pressure single-crystal synchrotron and in-house source X-ray data collection, combined with HAR, obtained for H4.4.1.2O/50%H2O) ice VI were obtained in a DAC . The pressure chamber was filled with D2O. Pressure in the DAC chamber was gradually increased until the sample froze under isothermal conditions in the polycrystalline form. By increasing the temperature, all crystal grains except one melted. Then the temperature was slowly lowered until this single crystal grew under isochoric conditions to entirely fill the pressure chamber at room temperature.Single crystals of ice VI, deuterated ice VI and mixed  are presented in Table S1 of the supporting information.The experimental details for all experimental data ice VI D4.2.et al., 1975CrysAlis PRO  were recorded. Frames collected in each particular batch were processed together. Finally, data from all 15 batches were merged to form one 4.4.Olex2  D2O_APS_HAR_mod1, D2O_APS_HAR, D2O_APS_IAM, D2O_DESY_HAR, D2O_DESY_IAM, D2O_home_HAR, D2O_home_IAM, H2O_D2O_home_HAR, H2O_D2O_home_IAM, H2O_home_HAR, H2O_home_IAM. DOI: 10.1107/S2052252522006662/lt5051sup2.pdfSupporting figures and tables. DOI: 2160500, 2160502, 2160503, 2160504, 2160505, 2160506, 2160507, 2160508, 2160510, 2189429, 2189430CCDC references:


Guided by multipletsimulation, the excited states at 0.5 and 1.3 eV can be identifiedas the 4T2 excited state ofthe tetrahedral Co2+ and the 3Tg2 excited state of the octahedral Co3+, respectively. The ground states of Co2+ andCo3+ sites are determined to be high-spin 4A2(Td) and low-spin 1Ag1(hO), respectively. It indicatesthat the high-spin Co2+ is the magnetically active sitein Co3O4. Additionally, the ligand-to-metalcharge transfer analysis shows strong orbital hybridization betweenthe cobalt and oxygen ions at the Co3+ site, while thehybridization is weak at the Co2+ site.Wepresent the cobalt2p3d resonant inelastic X-ray scattering(RIXS) spectra of Co The determination of the local electronic structureis a relatively straightforward task for localized materials, butit faces an experimental challenge for a mixed-valence TM compound,especially when the constituting TM ions have a rich overlapping multipletstructure, which is the case for Co3O4 crystallizes in a normal spinel structure(AB2O4) below 850 K.2 TheCo ions in the A sites with tetrahedral (Td) symmetry are divalent (Co2+), whilethose in the B sites with octahedral (hO) symmetry are trivalent (Co3+) with a small trigonaldistortion. An experimental determination of the electronic structureof two inequivalent Co sites is of fundamental importance to elucidatethe magnetic coupling responsible for the antiferromagnetic order(below ∼40 K).4 The Co-site dependence of covalencyis crucial for large exchange anisotropies achieved by a substitutionof Ni or Fe for Co ions and its application to magneto-optical informationstorage.7 The covalency also influences the charge capacity.Co3O4 shows a high charge capacity (700 mAh/g)and a good cycle performance of nanosize negative electrodes, whichmakes Co3O4 an interesting compound for thelithium battery applications.9 Moreover, the role ofCo2+ ions in the oxygen evolution reaction step of wateroxidation is debated.17 A small band gap was found in Co3O4 that isimportant for the application in a photovoltaic cell,20 where the electronic structures of individual sites potentiallyplays an important role.Co3O4 has beencharacterized by the ultraviolet/visible (UV/vis)25 and near-infrared (NIR) absorption.26 These absorption spectra show excitations at 0.82, 0.93, 1.64, and2.81 eV,22 while diverse interpretationsfor these excitations are proposed,25 partly related to the fact that the dd excitations are usually dipoleforbidden.27 Hibberd et al. revealed distinctabsorption peaks for the Co valence (2+ or 3+) and local environment(hO or Td) of Co3O4 using Co L2,3-edge (2p) X-ray absorption spectroscopy (XAS).28 However, due to possible overlap of the multiple Co-sitesignals and the lifetime broadening (∼200 meV), the detailsof the local electronic structure are limited.The electronic structure of Co3O4 using Co 2p3d resonant inelastic X-rayscattering (RIXS). By setting the resonant photon energy to the distinctCo L3-XAS features of the Co2+ and Co3+ sites,28 the site-selective local excitationsare measured. The site-selectivity of RIXS has been discussed in literature.31 The (photon-in–photon-out) transitions in RIXS brings a broadsensitivity to dd excitations of the system that provides a betterdetermination of the local electronic structure including the orbitalhybridization effects.36 This has also been demonstrated using 1s3d RIXS on Co3O4,38 but the intensity was limitedby the absorption cross-section of the quadrupole 1s3d transition.We show that the Co 2p3d RIXS is a good chemical site-selective approachto study accurate details of the ground state and the core excitedstates.In this work,we study the Co-site resolved low-energy local excitationsin Co22.13O4 powder produced by Alfa Aesar.A 10 mm × 0.5 mm (diameter× height) cylindrical pellet Co3O4 wasprepared for the measurements. Co 2p XAS spectra were acquired atthe soft X-ray WERA beamline of the Karlsruhe Research Accelerator(KARA) synchrotron in Germany. The instrumental resolution was calibratedto be ∼280 meV full width at half-maximum (fwhm) at the CoL2,3 edge (∼780 eV). Both the total electron yield(TEY) and the fluorescence yield (FY) methods were employed. The Co2p3d RIXS measurements with linearly vertical (V) and horizontal (H)polarized incident X-rays were performed at the Taiwan Light Source(TLS) beamline 05A of National Synchrotron Radiation Research Center(NSRRC) in Taiwan.39 The experimental energyresolution of the incident photon was ∼700 meV and the combinedresolution of RIXS was ∼90 meV. A grazing incident geometry(∼20°) with the spectrometer at 90° was used. Giventhe energy-compensation principle, the RIXS resolution is much betterthan the incident photon resolution.40 For a perfectmonochromator-spectrometer pair, the photons will be focused at themirror position of the photon source. As soon as the photon lost itsenergy in the path, the focus will not be kept at the same position.Thus, using a position sensitive detector combined with the activemonochromator-spectrometer system, the RIXS resolution can be decoupledfrom the incident photon resolution. To compare the XAS spectra measuredat different facilities, the background signals were subtracted fromthe spectra, as described in the Supporting Information (SI). The subtracted spectra were normalized to the maximumof the Co L3 edge. We calibrated the photon energy of theRIXS to the absorption spectra acquired at the WERA beamline. TheRIXS spectra measured with horizontal (H) polarization were normalizedto the exposure time. Then the spectra with vertical (V) polarizationwere normalized to the H-polarization spectra according to the profileat high energies (above 2.5 eV). The measurements at the KARA-WERAand TLS-05A beamlines were carried out at 50 and 150 K, respectively.We note that the simulated intensity is given as absolutely cross-section,while the experimental intensity is given in normalized units.The experiments were performedon 99.9985% Co2.242 To study the mixed-valence spectra of Co3O4, the Co2+(Td) site and the Co3+(hO) site were calculated separately. We took the initialparameters from literature for a high-spin Co2+ groundstate (4B1 in Dd2 symmetry) and a low-spin Co3+ ground state (1Ag1 in hO symmetry).45 The values were refined by a detailed comparison with the high-resolutionRIXS data and its polarization dependence . The parameters  were extracted from the tight-binding modelwhich spans the Co 3d and O 2p bands by a Wannier projection.The results were analyzedusing a cluster model that includes the Coulomb multiplet interaction,the crystal-field splitting, the spin–orbit coupling, and thecharge transfer between Co 3d and O 2p orbitals. The calculation wascarried out with the program Quanty, which implements a configuration-interactionscheme. section . To eval33.12+ and the Co3+ sites in Co3O4, respectively.28 TheFY spectrum, on the other hand, is rather broad, and the featuresare obscure. This is due to strong saturation and self-absorptioneffects for a bulk sample. In the 2p3d RIXS result  site.5 As a reference, divalent 4B1(Dd2 symmetry) Co2+ RIXS at the L3 edge of K5H[CoW12O40]·xH2O is shown in 44 where thelocal structure is a distorted tetrahedral Co2+. The distortionchanges the ground state symmetry from the 4A2(dT) to the 4B1(Dd2) that gives rise to a strong polarization dependence at the0.5 eV peak in the RIXS spectra. However, the feature B shows no dichroismin Co3O4, which suggests that the distortionis negligibly small on the Co2+ site and supports the 4A2(dT) symmetry of the ground state in the calculation.To gain more insight, 3.23O4 and the crystal-fieldparameters can be determined. In the cluster-model analysis, theseparameters identify the symmetry of the local site and the crystal-fieldenergy (10Dq) is the key factor to identify the energysplitting of the Co 3d orbitals (t2g(t2) andeg(e) orbitals in Oh(dT) symmetry). For a system involvingcharge transfer effects, the crystal-field energy can be decomposedinto two components:36 the ionic crystalfield energy (10Dqionic) and the effectivecrystal field energy induced by the charge transfer effect (10DqCT). The combination of the two (10Dqtot) gives the final splitting between thee/eg and t/t2g orbitals. Dqionic for the Co3+ and Co2+ site, respectively. The 2p3d RIXS probedat 780.0 eV shows three features at 0.9, 1.3, and 1.9 eV, indicatedwith the green arrows in 3+ site. The feature at 0.5 eV shows no polarization dependence,which indicates that the feature is likely the tail contribution ofthe 4T2 feature of the Co2+ site , suggesting that theground state on the Co3+ site in Co3O4 is a robust low-spin singlet 1Ag1. For the Co2+ site, the 10Dqionic ∼ −0.1 eV reproduces theenergy position of the characteristic features at 0.5, 0.9, and 1.9eV in the RIXS data probed at 778.5 eV, which are indicated in Dq value indicates an inversion of the e and t2 manifolds in the dT symmetry.Guidedby the experimental results, the low-energy local excitations characterizethe two Co sites in Co site cf 2. As seeSI. The charge transfer energy Δ is an energy relatedto electron transfer from a ligand to the Co site and V(g)e/V2(g)t are the values for electron hopping. The Udd and Upd values parametrizethe Coulomb interaction, which are set to reference values.43 Since the ligand-to-metal charge transfer also contributes to theenergy splitting of Co 3d states,36 weprovide the 10Dqtot value that is calculatedby the resultant energy splitting by including the charge transfer.To simulate a contraction of Co 3d wave functions by the presenceof the core hole,46 the 10Dqtot value in the intermediate state was reduced from thatin the ground state by ∼15%, see also SI.2+(Td) site, the estimated values are −0.10 eV for 10Dqionic and 1.29 (1.82) eV for Ve (V2t). For the Co3+(hO) site, the estimated values are 0.7 eV for 10Dqionic and 3.03 (1.74) eV for Vge  for changingtheground state symmetry (singlet 1Ag1) of the Co3+ site in Co3O4, but gives a minor correction to the multipletenergies. Because the trigonal distortion gives no visible effecton the spectra, we neglect it in our simulation and the site is referredto as hO for simplicity. We conclude thatthe optimized values agree well with the ab initio estimates exceptfor a small discrepancy in the 10Dqionic value at the Co3+ site, which is possibly due to an underestimateof the covalency in the LDA scheme.47To evaluate the parameters in 2+ and Co3+ siteswith the values listed in 1Ag1) ground state on the Co3+ site is angular isotropic, its intensity is strongly suppressedwhen the scattering angle is about 90° with the H-polarizationcondition. Thus, the intensity at zero energy loss (elastic line)is mainly due to the Co2+ site with the 4A2 ground state. 2+ and Co3+ spectra were adjusted by a 1.8 eV differencein between the main L3 feature to reproduce the experimentalCo L3 XAS  and hybridization (charge transfer effect).Some intensity discrepancies are observed, that is, the calculatedRIXS at 780.5 eV shows much stronger 1.3, 1.7, and 2.5 eV excitedfeatures. These discrepancies can be explained by saturation and self-absorptioneffects  spectra were acquiredfor energy transfer of the dd excitations (A–E) from the RIXSmap to discuss the local excitations in detail. The experimental dataare obtained by fitting the RIXS intensities of these dd excitationscf 4d, andt2+ (blue) and Co3+ (green)sites well matches with the experimental TEY data (gray bars and redline). The contributions of two sites are separated in the incidentenergies that enables to observe site-resolved local excitations byRIXS, as we show in 2+ site and the Co3+ siteexcitations, respectively, while the features C and E show an overlapwith both Co2+ and Co3+ regions. We stress thatthe overlap is essential since, according to our simulation in 49X, α, and θrefer to the emission edge of element, the scattering angle, and thesample rotation angle, respectively. In this work, the experimentalgeometry was set to α = 90° and θ = 20°. The μtot(ωin) and μtot(ωout) are the absorption factor of thephoton-in and photon-out channel in the RIXS process. We multiplieda self-absorption coefficient SX and μX(ωin)). The formula implies that the saturationeffect is stronger for a large absorption factor. Thus, the featuresat ∼780 eV in PFY spectra are to be suppressed. Consequently,the PFY weights on the Co3+ region is largely suppressedfor the feature C  excitedstate on the Co2+ site and 3Tg1(hO) excitedstate on the Co3+ site. In comparison with the infrared4 and 1s3d RIXS,38 those individualsite can be resolved much better. Our result confirms that the Co3O4 is mainly composed by the magnetically activehigh-spin Co2+(Td) and the diamagnetic low-spin Co3+(hO).Thepresent analysis shows that the excited states at 0.5 and 1.3eV are the unique features to identify the 4.23 XAS spectra,which implies that the ligand–metal hybridization is strongin Co3O4.50 The sensitivity of 2p3d RIXS to the dd excitations allows us toaddress the question, and furthermore study the site-dependence ofthe covalency in Co3O4. 2+ site, theionic model (ii) with reduced Slater integrals  yields good agreement with the experimental data. Note thatboth model (i) and (ii) show nice agreement on the 4T2 and 4T1 excited states, which indicates that those features are less sensitiveto the hybridization change. On the other hand, for the Co3+(hO) site, the Slater integrals are reducedto 55% (80%) from the ionic F2dd (F4dd) value to fit the experimentaldata. These unconventional reduction rates for the Co3+(hO) site imply that the differentialscreening effect via a ligand-to-metal charge transfer channel isnot negligible. The cluster model including the ligand-to-metal chargetransfer channel shows good agreement to the experimental results.We discusshere the question whether the ligand–metal hybridization influencesthe local electronic structure based on the ligand-to-metal chargetransfer model. Hibberd et al. have shown in their ionic-model analysisthat the Slater integrals of the ionic Coulomb multiplet need to bereduced substantially to reproduce the Co Ln) with ones with ligand holes (3dn+1L1 and3dn+2L2). In the Co3+ site, the ligand-hole configurations (3d7L1 and 3d8L2) show large weights, which indicates thatthe Co3+ site is strongly hybridized with the ligand states.In contrast, the ligand-hole configuration (3d8L1) contributes only ∼20% to the groundstate in the Co2+ site. This observation suggests thatthe Co3+ site is highly covalent, while the Co2+ site is rather ionic in Co3O4. The differencealso affects the RIXS profile: the ionic Co2+ site showssharp local dd excitations; the covalent Co3+ site exhibitsa broad intense fluorescence-like feature cation was analyzed using the approach described in the SI, the eg orbital forms a strong bondingwith neighboring oxygen 2p orbitals. This bonding is represented bythe d7L configuration in an eg symmetry, yielding the reduced value (50%) of the orbitalcovalency.The orbital covalency of theCoin the SI and is s53O4. The 2p3d RIXS provides good chemical site selectivity to the localelectronic structure, from which we can identify orbital covalenciesof different ions in the compound. The polarization-dependent analysisindicates the symmetry character of the dd excitations, which providesa solid guide to analyze the local electronic structure. By selectingcharacteristic excitations, the PFY spectra are able to give additionalsite dependent information. The result shows the 4T2 excited state of the tetrahedral Co2+ site at 0.5 eV, which is beyond the discriminative power of opticalabsorption. In addition, the 1Ag1 to 3Tg2 excitation of the octahedral Co3+ site at 1.3 eVcan be uniquely identified. The ground state electronic structureof the Co2+ ions and the Co3+ ions are respectivelyhigh-spin 4A2(Td) and low-spin 1Ag1(hO), wherethe high-spin Co2+ must be the magnetically active site.Our result also shows strong ligand–metal hybridization onthe Co3+ site, which indicates that the Co3+ site in Co3O4 is rather covalent. In contrast,the Co2+ site shows weak hybridization implying that Co2+ is more ionic. This chemical site selectivity will helpthe further understanding on the site-dependent catalytic activityand magnetic activity of the spinel oxides.We presentthe Co 2p XAS and 2p3d RIXS experimental results incomparison with cluster model simulations on Co


Bridging bone defects in revision total hip arthroplasty is a challenge to orthopedic surgeons. The eccentric revision cup is a progression of jumbo cup. Our aim is to confirm the optimal screw-hole positions of the eccentric revision cup by morphological measurements of three-dimensional pelvic reconstruction.Eighty CT images were converted to virtual three-dimensional bones. After simulating the surgery procedure, all available screw holes were inserted with the screws in virtual. By measuring the length of the screw in the pelvic bone, we determined the rich bone stock area. Then the screw holes were designed according to the characteristics of bone stock distribution. The peripheral screw-hole cluster and inner screw-hole cluster were studied respectively.For peripheral screw-hole cluster, five screw holes were evenly distributed between point A and point B in the thicker rim. For inner screw-hole cluster, screw hole 1 and screw hole 2 are the recommended inner screw holes.The eccentric revision cup has inherited the strengths of jumbo cup besides several unique advantages, including using the peripheral screws enhancing primary stability; decreasing the shift of hip rotation center and restoring biomechanical function; reducing the risk of dislocation because of the smaller head-cup differences; increasing the contact area between the outer cup and the host bone while maintaining a normal inclination of the inner cup. In this study, we confirmed the optimal screw-hole positions of the eccentric revision cup by surgical simulation and morphological measurement. However, biomechanical tests are still being further explored. Bridging bone defects in revision total hip arthroplasty (rTHA) is a major challenge to orthopedic surgeons . Among tThe eccentric revision cup -built on the strengths of jumbo cup and improved the problems mentioned above- is a progression of jumbo cup Fig. . Each ecIn revision THA, screws are an essential supplement for acetabular shell fixation. Together with the press-fit technique, they increase the primary stability of the cup, which is basic for long-term osseointegration –14. The Eighty  CT images  were obtained from a database composed of normal skeletal adults who underwent pelvic CT scans for non-hip diseases. The basic information was shown in Table The average native acetabular diameter was 54.24 ± 3.98 mm (ranged from 45 to 62 mm). We categorized them into 4 groups by the native acetabular size. Group 1, with diameter from 45 to 51 mm (22 cases); Group 2, with diameter from 52 to 54 mm (19 cases). Group 3, with diameter from 55 to 56 mm (19 cases); Group 4, with diameter from 57 to 62 mm (20 cases). To simulate revision THAs using extra-large acetabular components, for each group, the biggest native acetabular diameter added 12 mm .Our aim is to validate the optimal screw-hole positions of the eccentric revision cup through simulating surgical procedure and morphological measurement. The result of optimal peripheral screw-hole position is shown in Table The lengths of inner screws are shown in Table In this article, we located the rich bone stock area to study the optimal positions of screw holes. For peripheral screw-hole cluster, five screw holes were evenly distributed between point A and point B in the thicker rim. Three holes were located posterior to the symmetrical axis and two located anterior. For inner screw-hole cluster, screw hole 1 and screw hole 2 are the optimal inner screw holes.Bone deficiency is a challenge in rTHA. To solve this problem, several reconstruction strategies are performed. In most patients, stability can be achieved using an uncemented normal-sized hemispheric acetabular shell or a Jumbo cup–22. OtheComparing with other methods, jumbo cup has become a preferable way with its unique advantages. First, it has a larger absolute contact area with the host bone, which is basic for long-term biological fixation. Second, the technique of jumbo cup is a relatively easy way (similar to primary THA). Third, jumbo cup provides an alternative to placing a cup into the superior defect or using augments in some certain cases. However, the elevation of hip rotation center has aroused wide concerns using jumbo cups, 8. A veLimited screw fixation option is another vital problem using jumbo cups. Besides conventional dome screw fixation, the offset COR cup allows peripheral screws to be fixed into the posterior column of the pelvis. HoweverIn summary, the eccentric revision cup has inherited the strengths of jumbo cup besides several unique advantages as follows. First, this design decreases the shift of hip rotation center restoring biomechanical function. Second, the peripheral screw holes are designed in the thicker rim of the eccentric revision cup enhancing primary stability of the cup by locking screws. Third, There are several limitations of this study. First, our study was based on normal pelvic and acetabular anatomy. However, it may not represent the variation in individual anatomy encountered in different revision THA settings. Second, this study was a 3D CT reconstruction morphological study and biomechanical tests are still being further explored. Third, because of individual variation, the peripheral screw holes do not always perfectly match the rich bone stock area in every patient. However, initial stability was achieved with 2 or 3 screw fixations in most jumbo cups. In our design, besides inner screws, one or two peripheral screws may enough for most patients. In the eccentric revision cup, there are 5 screw holes in the thick rim of the cup, which are enough to place screws pursuing the initial stability. Fourth, our study focused on screw-hole design on acetabular cups. However, improved design will not substitute for good surgical techniques. The proper surgical approach, adequate exposure, preparation of the acetabulum, and correct position of the acetabular cup are equally important.Based on surgical simulation and morphological measurement, five peripheral screw holes evenly distribute between point A and point B in the thicker rim; screw hole 1 and screw hole 2 are the recommended inner screw holes. Although this novel acetabular design has many theoretical advantages, we recommend more studies be performed to determine that it achieves durable fixation and improved clinical outcomes before its widespread adoption. The costs and risks associated with new implant designs must still be justified by studies that evaluate implant durability and patient-reported outcome scores.