P < 0.001) than those with high NXN expression. Biologically, ectopic expression of NXN significantly inhibited the proliferation and metastasis of HCC cells both in vitro and in vivo by suppressing epithelial-mesenchymal transition (EMT). Mechanistically, NXN promoted the ubiquitin-proteasome-mediated degradation of Snail through interaction with DUB3. Further, depletion of Snail abolished NXN-inhibited cell proliferation and metastasis. In summary, NXN suppressed the proliferation and metastasis of HCC by inhibiting DUB3-mediated deubiquitylation of Snail protein. Our study demonstrates that NXN, DUB3 and Snail complex functioned as an important regulatory mechanism of HCC progression and indicates a potential therapeutic approach for the treatment of HCC metastasis.The poor prognosis of hepatocellular carcinoma (HCC) could be attributed to its high metastasis rate. Here, we report the role of nucleoredoxin (NXN), a multifunctional redox-active protein, in HCC metastasis. The expression of NXN in HCC tissues was measured by immunohistochemistry. The role of NXN on HCC proliferation was determined by CCK-8, EdU and colony formation assays in vitro and subcutaneous tumor formation model in vivo. Transwell and wound healing assays and tail vein injection model were performed to assess the function of NXN on HCC metastasis. Co-immunoprecipitation assay was performed to examine the interaction among NXN, Snail and DUB3. Our results showed that NXN was downregulated in HCC tissues compared to adjacent liver tissues. Patients with low NXN expression had shorter overall survival (OS) time ( Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide , 2, and SNAI1) is a key transcriptional repressor of E-cadherin expression that can regulate tumor metastasis [The epithelial-mesenchymal transition (EMT) has important roles in all stages of cancer progression from initiation, invasion and metastasis to colonization –7. EMT itastasis . Many sttastasis –11.We previously found that the methylation signature of genes could predict recurrence in HCC patients . Among tIn this present study, we found that NXN was downregulated in HCC, and its low expression correlated with poor overall survival (OS) in HCC patients. By inhibiting the EMT process, NXN overexpression was found to suppress the proliferation ability and metastatic potential of HCC cells both in vitro and in vivo. By interacting with DUB3, NXN could reduce the interaction ratio of Snail and DUB3 and subsequently promote the ubiquitin-proteasome-mediated degradation of the Snail protein. These findings highlight that NXN played a critical role in HCC metastasis, suggesting NXN as a potential biomarker and therapeutic target for HCC.P = 0.002; Fig. P < 0.001; Fig. P < 0.001 and P = 0.036, respectively; Fig. Investigating the expression of NXN in different tumors and adjacent normal tissues can provide information about its role in cancer. Data from The Cancer Genome Atlas (TCGA) database showed that NXN mRNA expression varied among different cancer types, indicating that it might function differently in the development of diverse tumors Fig. . AnalysiP = 0.027), larger tumor size  and more incomplete tumor capsule  compared to patients with high NXN expression  levels  = −1.85; Fig. By Gene Set Enrichment Analysis (GSEA) in the TCGA dataset, we found that EMT-related genes were significantly enriched in the HCC patients with low NXN expression  pathway enrichment analysis identified that the ubiquitin-mediated proteolysis signaling pathway was significantly enriched Fig. . As the As Snail protein degradation is mediated by the ubiquitin-proteasome pathway, to pinpoint the in-depth mechanism of NXN-induced Snail degradation, we treated NXN overexpression cells with a proteasome inhibitor (MG132). We found that MG132 could significantly restore Snail protein levels in NXN overexpressed Huh7 and PLC-8024 cells, indicating that the ubiquitin-proteasome pathway was involved in the process of Snail degradation induced by NXN Fig. .We evaluated Snail ubiquitination in NXN-overexpressed 293 T, PLC-8024 and Huh7 cells to further validate these findings. As expected, Snail ubiquitination was significantly increased when NXN was overexpressed Fig. . Taken tGiven that Snail could be deubiquitinated and stabilized by DUB3, we explored the relationships among NXN, Snail and DUB3 in HCC cells , 19. Co-We further verified the functional significance of DUB3 in Snail deubiquitylation. After overexpressing DUB3 in 293 T, PLC-8024 and Huh7 cells, the level of Snail protein ubiquitination was significantly reduced Fig. . In DUB3Collectively, these data confirmed that NXN could competitively interrupt the association between DUB3 to bind with Snail and inhibited the deubiquitylation of Snail by DUB3, thereby stabilizing Snail and enhancing HCC metastasis.Next, we examined whether the effects of NXN on the suppression of HCC were Snail-dependent. Snail was depleted (siSnail) in NXN silenced Huh7 cells and was also ectopically expressed in NXN-overexpressed PLC-8024 cells Fig. . CCK-8 aOverall, our study demonstrates that NXN, DUB3 and Snail complex functioned as an important regulatory mechanism of HCC progression and provides a potential therapeutic approach in the intervention of HCC metastasis Fig. .Fig. 7ScMetastasis is one of the major obstacles leading to poor prognosis in HCC patients –23. ElucNXN has been widely documented in regulating physiological processes , 24, 25.Although the physiological functions of NXN have been studied in detail, the function of NXN in cancer remains largely unknown. One study identified that NXN expression was reduced in colon tumor tissue samples and associated with colorectal cancer risk . Other rCancer cell EMT, featured by enhancing cancer cell dissemination, contributes to HCC metastasis , 30. It In summary, our study, for the first time, found that NXN could affect HCC metastasis. NXN, DUB3 and Snail complex functioned as an important regulatory mechanism of HCC progression. These findings could be used as a potential therapeutic approach in the intervention of HCC metastasis.A total of 276 pairs of pathologically confirmed HCC and adjacent non-tumor liver tissues were obtained from patients who received hepatectomy at the Sun Yat-sen University Cancer Center  from April 2008 to September 2014. The median follow-up time was 76 months. Written informed consent was obtained from each patient. This study complied with the 1975 Declaration of Helsinki standards, and the experiments were approved by the Ethics Committee of SYSUCC (No. L102012020090M).HCC specimens were selected and re-embedded into new paraffin blocks for TMA. The TMA blocks were cut into 4 μm sections and underwent IHC staining. The rabbit polyclonal antibodies used were anti-NXN . Based on IHC, positive staining was quantified and classified into 4 categories: ≤25% for 1; 26 to 50% for 2; 51 to 75% for 3; and ≥76% for 4. Staining intensity was graded as negative (scored as 0), weak (1), moderate (2) or strong (3). Two pathologists independently reviewed all scores, and expression levels were defined by the sum of the grades for the percentage of positive staining and intensity. The median of the IHC score was chosen as the cut-off value for the high (>6) and low (≤6) NXN groups.HCC cell lines  and human embryonic kidney cells (293 T cells) were purchased from the Cell Bank of Shanghai Institute of Biological Science  with STR (short tandem repeat) appraisal certificates. Cells were maintained in Dulbecco’s Modified Eagle medium  supplemented with 10% fetal bovine serum  at 37 °C in 5% CO2.Three experiments were performed to analyze the cell proliferation ability. EdU cell proliferation staining was performed using an EdU kit . Briefly, indicated cells were incubated with the EdU buffer for 3 h, fixed by 4% polyformaldehyde and stained the nuclear with Hoechst. The results were observed and captured using the fluorescence microscope .In the Cell Counting Kit-8  assay, 1000 cells were seeded in 96-well plates with six repeated wells for each experimental condition. Two hours after replacing the supernatant with a fresh medium containing CCK-8 reagent in a 10:1 ratio, the absorbance was measured by Biotek Epoch 2 machine  at 450 nm.For the colony formation assay, 500–800 indicated stable cells were plated per well in 6-well plates. Fourteen days later, the cells were fixed in methanol and stained with 0.1% crystal violet. The colonies were photographed and counted to contrast with each other.4 to 1.5 × 105 indicated cells for the migration assay and 8 × 104 to 2 × 105 indicated cells for the invasion assay were plated in the upper chamber in serum-free medium. Fresh media containing 20% FBS was placed in the bottom chamber. After 12–48 h, the cells were stained and photographed under a microscope.Transwell chamber  with polycarbonate membranes was used in the migration assay, and a chamber with matrigel  was used in the invasion assay. Then, 5 × 10For scratch wound healing assay, cells were plated in six-well plates and incubated in the regular condition until the cells reached the full confluence of the plates. A wound was created using a sterile 100 µL pipette tip, and the detached cells were removed by phosphate buffer saline (PBS). Then, the cells were incubated with serum-free DMEM for an indicated time. Images at 0, 24, and 48 h after scratching were taken, and the closure of the wound area was evaluated using ImageJ software.All animal research procedures were performed according to the Animal Care and Use Ethics Committee of SYSUCC, and careful attention was paid to minimizing the animals’ suffering. Male nude mice  were purchased at 3–4 weeks of age and maintained under specific-pathogen-free (SPF) conditions.6 cells) were subcutaneously injected into mice. The body weight and tumor size were monitored every three days. Eleven or thirteen days after inoculation, all mice (n = 4 per group) were euthanized for further analyses. Tumor volume (V) was measured using the formula: V = (L × W2)/2 .To evaluate the inhibitory effect of NXN on tumor growth in vivo, 200 µL Hepa1–6 cells (2 × 106 Hepa1–6 cells were injected into the tail veins of mice (n = 5 per group). After fifteen days, the mice were sacrificed, and the tumor nodules formed on the lung surfaces were counted.To establish the lung metastasis model, 1 × 10For the Co-IP assay, cells were lysed with RIPA lysis buffer (Thermo Fisher Scientific). Primary anti-NXN or anti-IgG (negative control) antibodies were incubated with the lysates at 4 °C overnight. Protein A/G-Agarose beads (Thermo Fisher Scientific) were added to the immune complexes for recovery. After that, the immunoprecipitants were washed and collected. Finally, the immunoprecipitants were subjected to Western blot analysis.t-test or the Mann-Whiney U test was used to compare the values between subgroups. Survival curves were constructed using the Kaplan-Meier method and analyzed by the log-rank test. Significant prognostic factors founded by univariate analysis were entered into a multivariate analysis using the Cox proportional hazards model. All analyses were two-sided, and P values less than 0.05 were considered significant. Statistical analyses were performed using the Statistical Package for Social Sciences version 25  and GraphPad Prism 7.0 software .The experiments were repeated at least three times independently, and the measured data were represented as the mean ± SD. Binary variables were compared by the Chi-squared test. The Student’s checklist tableOriginal Data Filesupplementary figure legends and tablesFigure S1. NXN mRNA expression in human tumor tissues and normal tissues in TCGA database.Figure S2. Overexpression of NXN inhibited proliferation of HCC cells in vitro.Figure S3. Overexpression of NXN inhibited proliferation and metastasis of HCC cells in vivo.Figure S4. The promoting effect of NXN depletion on HCC cells metastasis was rescued by the knock-down of Snail.


To calculate the overall incidence of uveal melanoma in Germany and to compare incidences in different German states. In addition, we computed the overall and cancer-specific survival rates nationwide.Incidence data for the period between 2009 and 2015, covering the entire German population, was collected through the German Center for Cancer Registry. ICD-O-3 topography codes C69.3-C69.4 and histology codes for melanoma subtypes were used to collect the incidence data. Confidence Intervals with a level of 95% (95% CI) were calculated for rates. Survival was calculated using the Kaplan–Meier. The log-rank test was used for survival comparisons.This study comprised 3654 patients with uveal melanomas, including 467 (12.8%) with iridial and ciliary body tumors. The overall age-standardized incidence rate (ASIR) was 6.41 person per million. Generally, the ASIR was higher in males than females (6.67 (95% CI 6.37–6.98) vs. 6.16 (95% CI 5.88–6.45 per million). Higher crude incidence rates were noted in the northeastern states (12.5 per million (95% CI 10.5–14.7) in Mecklenburg-Vorpommern) compared with the southwestern states (2.1 per million (95% CI 1.7–2.6) in Hessen). The 5-year overall survival stood at 47%, while the cancer-specific survival stood at 84%. Multivariate analysis showed that women, younger patients, and patients living in Berlin achieved significantly higher overall survival.Overall ASIR of uveal melanoma in Germany indicates that the disease is more common in males and that it follows the same geographical distribution previously noted in central European countries, with the highest incidence in northern parts of Germany.The online version contains supplementary material available at 10.1007/s00417-021-05317-7. Uveal melanoma is the most common adulthood primary ocular malignancy . It mostData from 2009 to 2015, covering the entire population of Germany, were gathered from the German population-based cancer registries through the German Center for Cancer Registry at the Robert Koch Institute together with the Association of Population Cancer Registries in Germany (GEKID) .n = 10) were recoded as having choroidal disease. This was done with the knowledge of previous practices where there was often miscoding of this group of patients [n = 13) or 5 (n = 4) [Patients were identified as having uveal melanoma by using the ICD-O-3 topography codes, including choroid (ICD-O-3 topography code C69.3), the ciliary body  code (C69.4), and histology codes for melanoma and malignant behavior codes. Patients initially coded as suffering from retinal melanomas (patients . We used (n = 4) . For pranorthern states. Conversely, the states of Hesse, Rhineland-Palatinate, Baden-Württemberg, Bavaria, Saarland, Saxony, and Thuringia were grouped as southern states. In order to examine the burden of disease in the regions of the former East Germany, the states of Brandenburg, Sachsen, Thuringia, Mecklenburg-West Pomerania, and Saxony-Anhalt were grouped as eastern states. Data from the formerly divided Berlin was represented separately. Data for each group were then further subdivided by age and gender, and further analysis was conducted accordingly. Further information on data collection and analysis can be found elsewhere [Population estimates as well as the German Standard Population Report of the 2011 Census, both provided by the Federal Statistical Office, were used to calculate crude (CR) and age-standardized incidence rates (ASIR) . We plotlsewhere .p-value of 0.05 or lower was considered significant for two-tailed tests. We calculated confidence intervals for 95% level [The IBM SPSS version 27 was used to conduct the descriptive statistical analysis . Microso5% level , 18. Dir5% level . Annual 5% level . We alsop-value = 0.055) or patients with choroidal versus ciliary body tumors   were choroidal melanomas (Table % were ch.8) Fig. . The detn = 709). Postoperative histopathological prefixes for TNM were added for 14% of patients (n = 541). TNM T was reported in 45% of all patients (n = 1252), including 9.7% (n = 354) as TNM Tx. Out of all patients reported with TNM T1-4, patients with T3 stood at 32.2% (n = 403), followed those with T2   patients. Of these, 79 (31.2%) only received radiation therapy, 60 (23.7%) underwent operations, and 39 (15.4%) were treated with both irradiation and a surgical intervention . Similarly, patients with choroidal melanomas showed better overall survival compared to those diagnosed with ciliary body melanomas . Furthermore, women showed significantly better cancer-specific survival . Patients with choroidal melanomas showed a slightly better survival compared with those with ciliary body and iris  , younger age patients (p < 0.001), patients from Berlin (p = 0.001), and patients with choroidal tumors (p < 0.001) showed significantly higher overall survivals. In the cancer-specific survival model, those from Berlin (p < 0.001) showed significantly better survival , reported incidence rates ranging from < 2 per million in southern Spain and Italy to > 8 in Norway and Denmark in the north, respectively . HoweverThe highest reported population-based incidence worldwide was of Australian men. It stood at a rate of 10.9 per million, while the lowest population-based incidence was reported in Japan with 0.3 per million . The menContrary to the increasing incidence of skin melanoma in the USA and other countries, uveal melanoma has shown varying annual incidence trends. For example, while Sweden has experienced a declining incidence of uveal melanoma over the years , Canada Older age at diagnosis as well as diagnoses of ciliary body tumors was associated with worse overall survival. Previous studies have reported increased incidences of uveal melanomas as well as worse prognoses in older age groups , 27. TheBetter survival rates for women were a notable finding of our study. This, as a number of studies have suggested, could be attributed to women’s hormonal profile, varied genetic predispositions compared with men, and/or differences in occupational factors including amount of sun exposure , 33–35. In general, the survival of patients from Berlin showed both higher overall and cancer-specific survivals, while those from both eastern and westerns states of Germany showed similar 5-year overall and cancer-specific survivals. On the other hand, graphs showed lower overall survivals after the period of 5 years, indicating a possible affection of health status on the long term, the effect of the age distribution between eastern and western states, or other factors that were not considered in this study. However, our analysis did not yield any differences in the distribution of uveal melanomas between different regions of Germany by age.8720/3 malignant melanoma histological subtype. This may give an impression that pathological studies were either not carefully done or were erroneously reported by the registrars. On the contrary, this highlights the dependency on clinical diagnosis for the management of uveal melanomas, currently the cornerstone of the diagnosis of these tumors [Most of the patients in our study were reported to have been diagnosed with the non-specified e tumors . Clinicae tumors . This ise tumors .The low DCO (not presented) rate indicates a relatively high level of reliable data that can be used in further research. Both show the accuracy of the reporting system in the deliverance of accurate information at presentation or during management, allowing a chance for further follow-up. On the other hand, the discrepancy between the method of confirming diagnoses and histopathological staging indicates a gap in the reporting of the histopathological staging of uveal melanomas. Establishing a central cancer registry for uveal melanomas can help more accurately complementing the national general cancer registry.This study is the first to report on the nationwide incidence of uveal melanomas in Germany. As with all registry-based studies, it has limitations. Some differences between states can be attributed to the degree of registry completion. Two of the southern registries (Hessen and Baden-Wuerttemberg) were founded in 2007–2009 and, therefore, are still being built up. Moreover, variations in the details reported by registries, including treatment and outcome, could have resulted from a lag time between diagnoses and reporting times. Furthermore, the small number of patients within some groups and states may result in inaccurate subgroup and trend analyses. Further efforts should be invested in follow-up studies and training ophthalmologists and cancer registrars on reporting uveal melanomas and other cancers as well.In conclusion, the age-standardized incidence of uveal melanoma in Germany was 6.41 per million. Men showed higher standardized incidences and lower survivals than women. Patients with choroidal disease showed higher survivals than those with a ciliary body or iridial tumors. Patients from the former East Germany showed similar 5-year survival rates to those from the former West Germany.Supplementary file1 (DOCX 2976 KB)Below is the link to the electronic supplementary material.


Cerebral atherosclerotic stenosis (CAS) is a significant factor in the development of acute ischemic stroke (AIS). Previous studies have reported that cytokines are involved in atherosclerotic diseases, although the relationship between serum levels of the chemokine RANTES  and the presence of CAS remains unclear.In total, 127 participants (65 non-AIS controls and 62 patients with AIS) were involved in this study. CAS was defined as the presence of ≥ 50% stenosis in major intracranial or extracranial artery by a Digital Substraction Angiography (DSA) examination, and we classified all participants into four groups according to stroke and CAS status. Serum concentrations of 8 cytokines, including RANTES, were measured by the Human ProcartaPlex Multiplex Immunoassay Kit.p = 0.016) and stroke group , and multivariate logistic regression analysis showed that the RANTES level is independently associated with CAS in both the non-AIS group (adjusted odds ratio (OR), 1.07; 95% CI, 1.02–1.12, P = 0.004) and stroke group .Seventy-eight participants (61.41%) had CAS, of which 39 cases with AIS and 39 case with non-AIS. Patients with CAS had higher RANTES levels compared to non-CAS patients in both the non-AIS group (10.54 ± 0.80 vs. 13.20 ± 0.71, Patients with CAS have higher levels of serum RANTES than non-CAS patients regardless of stroke status suggesting that RANTES may play an important role in the formation of CAS.The online version contains supplementary material available at 10.1186/s12883-023-03079-9. Cerebrovascular disease is a major health problem worldwide and one of the three major causes of death , 2. In cThe chemokine regulated on activation, normal T-cell expressed and secreted (RANTES) is involved in the migration of inflammatory cells to the inner membrane and plays an important role in the formation of plaque . In partIn this study, we aimed to assess the relationship between the concentration of serum RANTES and the presence of CAS, which may serve as a biomarker for identifying patients with high risk of CAS.We prospectively recruited acute ischemic stroke (AIS) and non-AIS control patients who were hospitalized in the Hospital from July 2019 to December 2019. AIS was diagnosed according to clinical symptoms and imaging (magnetic resonance/computer tomography) within 14 days of symptom onset. Non-AIS patients were hospitalized due to dizziness and headache, and did not suffer an acute stroke in the past 6 months. Exclusion criteria was (1) age ≤ 30 years; (2) incomplete neuroimaging data and laboratory tests; (3) prior thrombolysis or thrombectomy; (4) atrial fibrillation, cardioembolism and serious peripheral arterial disease; (5) recent local and systemic infectious disease(s); (6) intracranial and extracranial arterial stenosis caused by arterial dissection, arteritis, moyamoya disease, and muscle fiber dysplasia; and (7) a history of malignant tumors, severe liver or kidney dysfunction, or systemic disease. This study has been approved by the Tongji Hospital Ethics Committee (No. TJ-IRB20210107) and conducted according to the Declaration of Helsinki. All participants provided informed consent.Cerebrovascular images were obtained from all participants, and CAS was defined and determined by 50–99% stenosis or occlusion by digital subtraction angiography (DSA) examinations in at least one major arterial segment, including the intracranial or extracranial segment of the carotid artery (ICA) or vertebral artery (VA), M1 middle cerebral artery (MCA) and basilar artery (BA). A significantly stenotic vessel segment is considered a responsible CAS when an infarction lesion occurs in its blood supply area. In the current study, we assessed the location and severity of responsible CAS.Clinical data were collected from the clinical record and included age, gender, history of smoking and drinking, and medical history . Hypertension was defined as systolic blood pressure (SBP) ≥ 140 mmHg and/or diastolic blood pressure (DBP) ≥ 90 mmHg and/or prior treatment with antihypertensive medication . DiabetePeripheral blood samples were collected within 24 h of admission with vacutainer tubes containing coagulant accelerator  and immediately centrifuged at 1,278 g for 15 min at 4 °C. Serum samples were stored at − 80 °C until analysis. The concentrations of serum cytokines  were analyzed using the Human ProcartaPlex Multiplex Immunoassay Kit  according to the manufacturer’s instructions.P < 0.05 was considered statistically significant. Categorical variables are represented as frequencies (percentages), whereas continuous variables are represented as mean ± standard error of the mean (SEM) or median [interquartile range]. Non-AIS vs. AIS groups or non-CAS vs. CAS groups were compared using a chi-square test, two-sided t-test or Mann–Whitney U-test according to their distributions. The Kruskal–Wallis H test was utilized to explore the association between serum RANTES levels and CAS features. Multivariate logistic regression analyses were used to determine the relationship between serum RANTES levels and CAS. Odds ratio (OR) was adjusted by variables that had a significant clinical correlation  and variables with P < 0.05 by univariate logistic regression analysis. Finally, receiver operating characteristic (ROC) curve analysis was used to determine whether serum RANTES predicts CAS, and the area under the ROC curve (AUC) is reported.Statistical analyses were performed using IBM SPSS 22.0 software , and One hundred twenty-seven participants were included in the study analysis (65 51.18%] AIS patients and 62 [48.82%] non-AIS controls). Thirty-nine (30.71%) AIS patients and 39 (30.71%) non-AIS controls with at least one segment of CAS, while 26 (20.47%) AIS patients and 23 (18.11%) non-AIS controls were absent of CAS. Baseline characteristics are illustrated in Table .18% AIS p < 0.05), and in patients with AIS, those with CAS had higher levels of RANTES and IP-10. Non-AIS controls with CAS, however, had higher levels of RANTES and IL-23 . In AIS patients, RANTES was significantly associated with CAS , and the adjusted odds ratio of RANTES was statistically significant . In non-AIS patients, RANTES was significantly associated with CAS  participants had CAS, including 50 (64.1%) patients with single CAS, 23 (29.5%) patients with 2 CAS, and 5 (6.4%) patients with ≥ 2 CAS (Table S2). RANTES concentration was not significantly different among participants with differing stenosed cerebral vessels. The effect of serum RANTES levels on location and severity of responsible CAS was also analyzed, although was not significantly associated.In the current study, we demonstrate that patients with AIS have elevated serum RANTES levels compared to patients with non-AIS. Although the AIS and no-AIS subjects were not matched for age, importantly, the multivariate regression analysis excluded the impact of this difference on our results. Interestingly, RANTES appears independently associated with CAS and aids in CAS identification regardless of stroke status. Indeed, the concentration of RANTES as a marker in the atherosclerosis process may distinguish individuals with an increased risk of CAS and may be a potential target for future drug intervention.Regarding the correlation between RANTES and ischemic stroke, our results are similar to previous studies that the serum RANTES levels increase in AIS , 20. HowAtherosclerosis is increasingly recognized as a chronic inflammatory disease. Indeed, inflammation plays an important role in all stages of atherosclerosis and provides a significant pathophysiological mechanism for the formation and rupture of atherosclerotic plaque. Previous studies have reported RANTES in atherosclerotic plaques –28 and dIn this study, we found that in addition to RANTES, other cytokines, such as MMP-1, IP-10 and IL-23, were also different between groups. As previously reported, elevated MMP-1 is associated with AIS . This reThe present study has some limitations. First, this study has a small sample size and is conducted exclusively Chinese patients, which may lead to some selection bias and lack of generalizability outside the Asian population. Therefore, the results need to be interpreted carefully and verified further. Second, the use of antiplatelet drugs and statins after ischemic stroke may affect the concentration of RANTES. However, we minimized the potential effects of stroke and related drugs on RANTES levels by dividing the participants into AIS and non-AIS groups. Finally, we could not present the dynamic change of RANTES levels among patients with acute ischemic stroke in this study since we only measured the serum RANTES levels at a single time point.In conclusion, patients with CAS had higher levels of serum RANTES compared to patients with non-CAS, which suggests that the RANTES level might play a role in the physiopathological mechanism of CAS formation, although additional studies are needed to verify this result.YPG, QQK and YZ. collected the clinical data. YPG, JZ and HH. processed statistical data. YPG, ZYY and DH. drafted and revised the manuscript. XL. designed and guided the study. All authors read and approved the final manuscript.Additional file 1:Table S1. Multivariate logistic regression analysis showing the predictors for the CAS. Table S2. CAS characteristics of patients with and without AIS


The non-photosynthetic chlorophyte alga Polytomella sp. SAG 198.80 was found to be able to assimilate butyrate fast. To decipher the metabolic pathways implicated in butyrate assimilation, quantitative proteomics study was developed comparing Polytomella sp. cells grown on acetate and butyrate at 1 gC.L−1. After statistical analysis, a total of 1772 proteins were retained, of which 119 proteins were found to be overaccumulated on butyrate vs. only 46 on acetate, indicating that butyrate assimilation necessitates additional metabolic steps. The data show that butyrate assimilation occurs in the peroxisome via the β-oxidation pathway to produce acetyl-CoA and further tri/dicarboxylic acids in the glyoxylate cycle. Concomitantly, reactive oxygen species defense enzymes as well as the branched amino acid degradation pathway were strongly induced. Although no clear dedicated butyrate transport mechanism could be inferred, several membrane transporters induced on butyrate are identified as potential condidates. Metabolic responses correspond globally to the increased needs for central cofactors NAD, ATP and CoA, especially in the peroxisome and the cytosol.Volatile fatty acids found in effluents of the dark fermentation of biowastes can be used for mixotrophic growth of microalgae, improving productivity and reducing the cost of the feedstock. Microalgae can use the acetate in the effluents very well, but butyrate is poorly assimilated and can inhibit growth above 1 gC.L Other permeases may also be involved in these processes  into the cell. Two different mechanisms have been described in yeast, (i) passive transport rocesses . The rolrocesses  but the rocesses . At pH v acetate . Therefoartments . HoweverDesulfosarcina cetonica (Rhodospirillum rubrum (Candida ingens (Yarrowia lipolytica (via the monocarboxylate transporter (MCT), a 45-kDa plasma membrane protein with 12 transmembrane segments that symports H+ with the butyrate anion . It was grown on synthetic media referred to as HAP (acetate) or HBP (butyrate), based on Tris-Acetate-Phosphate (TAP) medium used for the green alga C. reinhardtii (−1 leading to an ammonium (NH4+) concentration of 7.5 mM, 0.6 mM of MgSO4 and 0.3 mM of CaCl2. To adjust nutrients to the Redfield C:N:P ratio of 106:16:1, corresponding to 83.3:12.6:0.8 mM for 1 g carbon per liter (1.0 gC.L−1), the proper amount of 1 M NH4Cl (5 ml) and 1 M K2HPO4 (0.8 ml) stock solutions were added. Hutner’s trace elements were used at 1 ml.L−1  and pH medium was adjusted to 7.0 (HCl) prior to sterilization at 121°C for 20 min. After cooling, 100 μl.L−1 was added of a stock of vitamin B1 (50 mM), biotin (1 mM) and cyanocobalamin (1 mM), sterilized over a 0.2 μm filter. Precultures were maintained on HAP medium containing 20 mM HEPES and were used to inoculate HAP and HBP media to initial optical density at 750 nm (OD750) = 0.05. The inoculum was prepared by collecting preculture cells in their exponential phase via centrifugation at 2500 g for 10 min, and resuspending them in Phosphate Buffered Saline to a final OD750 = 5. Cultures were done in 500 ml Erlenmeyer flasks filled with 200 ml medium under dim light at 25°C and without agitation.nhardtii , in whic1 ml.L−1 . As carb−1 dry weight (DW) deduced from OD750 determination. To calculate the biomass DW from OD750 values, a correlated factor was used, determined to be 1.0774 (R2 = 0.977). This correlation factor was obtained from a calibration curve gathering 70 data points collected during separated experiments, at various growth stages (stationary phase excluded) and growth conditions (HAP or HBP medium). To determine biomass DW during these experiments, between 5 and 25 ml (depending on growth stage) biomass was centrifuged . Supernatant was discarded, and pellet rinsed with one volume of phosphate buffer saline. Biomass was centrifuged again, supernatant discarded and pellet resuspended in 10 ml distilled wated. The biomass was transferred in a pre-dried and pre-weighed aluminium crucible and dried overnight at 105°C.Biomass growth was followed by measuring optical density at 750 nm (OD750), using a Helios Epsilon spectrophotometer. OD750 was measured by placing 1 ml of liquid culture in a cuvette and comparing to distilled water. The sample was diluted when necessary so that OD750 < 0.6. Biomass production was expressed in g.L−1). The GC system consisted in a Perkin Clarus 580 model equipped with capillary column Elite-FFAP crossbond®carbowax® (15 m) maintained at 200°C and with N2 as the gas vector (flow rate of 6 ml·min−1) with a flame ionization detector (FID) maintained at 280°C .For VFA measurements, samples from fresh cultures were immediately centrifuged, the supernatant filtered over 0.2 μm cut-off filters and frozen at −25°C until analyzed. The VFA concentrations were determined by gas chromatography. A 500 μl aliquot of supernatant was mixed with 500 μl of internal standard solution . The resulting reagent was stored in the dark. First, 2 ml H2SO4 (98%) were added in the tubes containing microalgae cells. The tubes were heated 10 min at 100° C and cooled on ice. The reaction was initiated by addition of 5 ml phosphovanillin reagent prior incubation for 15 min at 37° C. Tubes were periodically shaken by inversion. After cooling, optical density of suspensions was measured at 530 nm with an Aqualytic® spectrometer and compared to distilled water. Calibration curves were obtained using canola oil.Samples from fresh cultures were immediately centrifuged, the supernatants were discarded and the pellets stored at −25°C until used. Prior to analysis, the pellet was thawed, resuspended in 100 μl distilled water and added to 10 ml glass tubes for either lipid or sugar measurement. Total lipid concentrations in the algal samples were determined by the phosphovanillin method . Phospho2SO4 (98%). Two mille liter of anthrone reagent were added in the tubes containing microalgae. Tubes were cooled down on ice and then incubated at 100° C for 10 min. After cooling, absorbance was measured at 625 nm with an Aqualytic® spectrometer and compared to distilled water. Calibration curves were obtained using glucose solution.Total sugars were measured by the anthrone method . Anthronx (gdw.L−1.d−1) and the specific growth rate xμ (d−1) were calculated according to The biomass productivity PfX and 0X are the biomass concentrations at t = final (h) and at t = 0 h.where Y (gdw.gsub−1) was estimated according to The biomass yield fS and 0S are substrate concentrations (g.L−1) at t = final (h) and at t = 0 h.Where Statistical analysis was performed using GraphPad Prism V 8.0.2.Polytomella sp. grown either on acetate or butyrate  were stacked in the top of a 12% SDS-PAGE resolving gel before in-gel digestion using modified trypsin , as previously described , and the corresponding reversed databases. Trypsin/P was chosen as the enzyme and two missed cleavages were allowed. Precursor and fragment mass error tolerances were set at, respectively, at 10 ppm and 25 mmu. Peptide modifications allowed during the search were: Carbamidomethyl , Acetyl  and Oxidation . The Proline software  through concomitant searches against a homemade software  was usedStatistical analysis was performed using the ProStaR software .via the PRIDE . Statistical testing was conducted with limma, whereby differentially expressed (DE) proteins were sorted out using a log2 (fold change) cut-off of 0.6 and a value of p cut-off of 0.004, allowing to reach a false discovery rate < 1% according to the Benjamini-Hochberg method.Intensity-based absolute quantification iBAQ,  values wPolytomella sp. was described previously  and Chlorella sorokiniana (Uniprot UP000239899) to obtain equivalent enzyme identities in these two model microalgae. For subcellular localization predictions, DeepLoc  homology searches at KEGG DeepLoc  and Pred DeepLoc  were usePolytomella sp. was grown on either acetate or butyrate at a fixed C concentration of 1.0 gC.L−1 . For several Chlorella strains it was reported that butyrate assimilation, when it occurred, was slower than acetate assimilation  was obtained in both conditions  on acetate and 55.6 ± 3.1 mg.L−1 on butyrate.A similar maximum biomass yield of 1.07 gnditions . This innditions . In the y starch . Lipid aPolytomella sp. relies on intracellular carbon reserves to survive in absence of external carbon source, growth on butyrate may ultimately negatively impact its survival in oligotrophic conditions. Accumulation of high levels of starch during the exponential growth phase is another distinctive trait of Polytomella sp. as compared to green species, which tend to accumulate storage compounds especially when growth conditions become suboptimal.Although this was not directly measured, the reduced cellular sugar content in the butyrate condition suggests an increase in protein content that may be required to actively metabolize this substrate. Since Polytomella sp. growing on acetate and butyrate were sampled during the exponential phase  between the acetate and butyrate conditions, the above global trend is confirmed but a more detailed picture emerges . RelativPolytomella sp., MapMan was used to project the FC values of individual proteins onto the different metabolic pathways. In section 3.5 these pathways will be discussed in detail. In the category lipid metabolism, the highest FC values are for enzymes of fatty acid synthesis, fatty acid degradation and the glyoxylate cycle  enzyme  enzyme  but alsoC. reinhardtii where it occurs in the mitochondria (The amino acid metabolic pathway most affected by the C-source is the formation and subsequent degradation of branched chain amino acids (BCAAs). It appears to take place largely in the peroxisome based on the subcellular targeting predictions of the corresponding proteins, unlike in its close relative chondria . It is nchondria , which sPolytomella sp. cells growing on butyrate relative to acetate. The most pronounced enrichment of enzymes involved in butyrate assimilation is found in the peroxisomes, small spherical organelles (0.2–1.5 μm) that lack DNA and are surrounded by a single membrane that derives from the endoplasmic reticulum   . The propathways .Polytomella enzyme using BLAST (45–50% ID), including medium chain acyl-CoA synthase and even bacterial 3-methylmercaptopropionyl-CoA ligases. We thus changed the annotation of the enzyme to fatty acyl-CoA synthase (FACS). This enzyme is the prime candidate for the production of butyryl-CoA in Polytomella sp., pending further experimental confirmation. A true LACS isoenzyme was also detected , but since its expression level is much lower than FACS, this enzyme is unlikely to act as the first step of butyrate assimilation. Two acyl CoA oxidases (ACX) were also highly butyrate-specific, with a FC value of 0.001 for ACX4 and 0.021 for its paralog ACX. ACX4 was predicted to be localized in the plastid but homologs in other algae including C. reinhardtii retrieved by BLAST searches (~51% ID) are annotated as peroxisomal  (via phosphate butyryltransferase (EC 2.3.1.19) + butyrate kinase (EC 2.7.2.7)  . Convers2.7.2.7)  or butyr2.7.2.7) . The pre2.7.2.7) . In cont2.7.2.7) .C. reinhardtii, the glyoxylate cycle allows growth on acetate  and isocitrate lyase  are induced on butyrate, but at a lower level than citrate synthase  and malate dehydrogenase    . AconitaFC 0.03)  but noneFC 0.03) . CIS2 isFC 0.03) ; Table 1FC 0.03) . PolytomX5-H/QA) . In C. rrobodies . Earlierytosolic . Since tCA cycle .via a malate/OAA transporter  it can currently not be excluded that MDH2 is cytosolic, and if so, this would indicate an increased need for malate/oxaloacetate shuttle activity to sustain increased production and export of citrate from the peroxisome. A cytosolic localization for MDH, ACO and ICL occurs in yeast and does not impact glyoxylate cycle function  fumarate 10-fold lower, but hardly any citrate was formed  into malate, which can be later exported to the cytosol  via a mansporter . Export nsporter . Despitefunction . The facs formed . Convers by ACLY . In our C. reinhardtii (A0A2K3CWL4/Cre15.g637761) which was confirmed to be involved in the import of activated long-chain fatty acids from the cytosol to the peroxisomal matrix, similar to the yeast peroxisomal ABC transporters PXA1 and PXA2  in the cytosol. Another transporter identified is homologous to the mitochondrial dicarboxylate/tricarboxylate transporter DTC (FC 0.6) in A. thaliana , an oligomeric complex of approximately 150 kDa in the inner mitochondrial membrane which constitute the sole entry point into the mitochondria of pyruvate, produced by glycolysis or from malate. The upregulation of this carrier is of fundamental importance in establishing the metabolic programming of a cell . Once inthaliana . DTCs trPolytomella sp. genome. However, a member of the formate/nitrite transporter (TC 2.A.44) family  was found to be induced by butyrate. FNT transporters transport monovalent anions and are not strictly selective as they can use nitrite or formate but also larger organic anions such as lactate and acetate  superfamily and homologous to bacterial acetate/succinate channels were found to be induced by acetate in C. reinhardtii and possibly implicated in intracellular acetate transport  was found in the proteome induced by butyrate at FC 0.636 (but with p > 0.004).Furthermore, two general substrate transporters of the Major Facilitator Superfamily were identified . MFS transporters can only transport small solutes in response to chemiosmotic ion gradients and can be found anywhere in the cell, so their proposed placement in the peroxisomal membrane for transport of carboxylates is speculative . The mon acetate . It may ransport . In the + -ATPase  that exports cellular protons , was found markedly induced on butyrate (FC 0.003), while a second CAT1 ortholog was far less induced. No clear ortholog was found for the C. reinhardtii ER-localized CAT2. Catalase upregulation probably relates to the β-oxidation of butyrate, where it allows the detoxification of H2O2 produced by ACX into H2O and O2. Using density gradients,  are important in cellular signaling, but stress conditions may cause increased ROS production and result in oxidative damage . Various2O2 . The butoxisomes  and simiadients,  found thoxisomes .2O2 to H2O. Other GAC enzymes were also induced on butyrate, such as glutathione S-transferase , while glutathione reductase (GR) was not. The GAC can be found in different cellular compartments such as plastids, mitochondria, peroxisomes and the cytosol,  was found highly induced on butyrate. PRXs are important for cellular redox signaling and antioxidant defense as they detoxify organic hydroperoxides (R-O-OH) that can be formed from the reaction of organic molecules with H2O2 .The localization of PRX2 and M/FSD1 is uncertain as they lack typical targeting signal and were predicted to be cytosolic by DeepLoc. Intriguingly, the levels of plastid type alternative oxidase , which functions in plastid redox homeostasis by oxidizing plastoquinol to reduce O2 to H2O in photosynthetic organisms  was highly induced by butyrate. It is part of the glutathione-ascorbate cycle (GAC) that uses electrons from NAD(P)H for the reduction of Hcytosol, . Since nith H2O2 . Anotherrganisms , and thee system .2O2 levels that exert a positive regulatory influence  is predicted to be mitochondrial, the Polytomella ortholog is predicted to be peroxisomal  into acetyl-CoA, so the degradation of isoleucine into MS (via propionyl-CoA) does not demand the presence of MMSA in the peroxisome when MS is imported into mitochondria. Mitochondrial MMSA can convert MS into acetyl-CoA which can be further utilized without problems. In the case of valine degradation, the absence of MMSA in the peroxisome would block the pathway at the level of the conversion of MMSA to propionyl-CoA, and in that case MMS would have to be exported to the mitochondria. Here, MMSA would convert MMS into propionyl-CoA, but its fate in the mitochondria would not be obvious: the enzymes necessary for its conversion to succinyl-CoA (propionyl-CoA carboxylase producing (S)-methylmalonate-CoA, methylmalonyl-CoA epimerase producing (R)-methylmalonate-CoA and methylmalonyl-CoA mutase to convert it to succinyl-CoA) could not be identified in the Polytomella genome. This is similar to the case of P. tricornutum where the epimerase step was not detected , somewhat induced by butyrate, was found to be a promiscuous enzyme that produces also thioesters of malate, fumarate and glutarate among others , which serves as precursor for fatty acid synthesis (see 3.5.6). It is noted that even if ALD5/ACSF3 are not located in the mitochondria, the products can be imported into the organelle. Alternatively, the BCAA degradation pathway may be streamlined when MMSA is dually targeted to both peroxisome and mitochondria, which is known for other enzymes such as CAT , whereby sugars and lipids may serve to produce amino acids such as BCAAs. We propose that butyrate is metabolized and yields acetyl-CoA and further di/tricarboxylic acids at a slower rate than acetate, which is compensated for by the degradation of BCAAs to produce acetyl-CoA and possibly succinyl-CoA that feed into carbon metabolism. A factor in the induction of the BCAA degradation pathway specifically may be the fact that it shares several enzymes with the β-oxidation of butyrate .In the non-sulfur purple bacterium butyrate . Interesrivation . In thesPolytomella caeca can grow on acetaldehyde (1 mM) as sole external carbon source  and produces the acetyl-CoA that is a direct substrate for ACC. Biotin has been described to exert regulatory influences in cell signaling, for example the upregulation of glucose metabolism (A clear upregulation is found of two of the four enzymes of the type II fatty acid synthesis (FAS) system, which uses an acyl carrier protein (ACP): 3-oxoacyl-ACP reductase  and enoyl-ACP reductase . In o plants . Fatty ao plants . The FAso plants . Fatty ao plants . Four ouo plants . The mtFtabolism , which wvia consecutive protein phosphorylations by serine and threonine protein kinases  rather than to specific metabolic pathways. Among the over-represented categories are “RNA processing” and “Protein biosynthesis, modification and homeostasis.” This includes heat-shock proteins, protein kinases, maturation-, elongation-, and assembly factors. Although butyrate induced more proteins compared to acetate, there may also be downregulation signals produced in response to specific metabolic needs imposed by butyrate. A specific perception of an acetate-linked metabolite and associated signal cascades may also be involved. Some of the proteins suppressed by butyrate may suggest the implication of a mitogen-activated protein kinase (MAPKs) signal transduction pathway, which modulates important cellular processes such as proliferation, stress responses, apoptosis and immune defense  kinases .We note the induction of a protein of uncertain function, being either protein kinase (MAP3K-RAF) or dual specificity kinase splA isoform B (FC 4.663). It may suggest activation of a signal transduction pathway for positive regulation of gene transcription from a receptor on the cell surface . The preOf note are the three plastidial small heat shock proteins  for which little data exist in microalgae but in plants play a central role in the protection against stress damage, in the folding, intracellular distribution, and degradation of proteins, as well as in signal transduction chains . The HSPThe mitochondrial organization/maturation factor  is the most induced protein compared to butyrate. Its function is uncertain, but may relate to protein translation or post-translational maturation of cytochrome c oxidase. Finally, although most enzymes involved in amino acid synthesis were mildly induced by butyrate, a few enzymes involved in production of aromatic amino acids and methionine were more abundant on acetate (FC ~2.5). Methionine is a direct precursor of S-adenosylmethionine (SAM), an important posttranslational regulator of many cellular processes, including autophagy, the recycling of cellular components in response to stress . ButyratPolytomella sp., and the relation with butyrate metabolism in other organisms is discussed as well as the potential implications of these findings for the capacities for butyrate assimilation of other -green- algae. In addition, a major step has been made in our understanding of peroxisomes in Polytomella sp. and in relation to its close relative C. reinhardtii.The key hypotheses that have been proposed in the past concerning limitations in the trophic metabolism of microalgae relate to cell permeability, toxic products formed from the substrate, lack of enzymes necessary for effective dissimilation of the substrate or their improper cellular location, lack of transcriptional control, effect of low-intensity light in stimulating heterotrophic growth and respiratory deficiency, etc. . In thisvia peroxisomal β-oxidation resulting in acetyl-CoA and di/tricarboxylates for cellular use via the glyoxylate cycle. We found that multiple transporters are induced to facilitate the metabolic interplay between peroxisome and other cell compartments. Although no monocarboxylate transporter was identified for butyrate transport, a formate/nitrite transporter was put forward as candidate for this function. We hypothesize that butyrate causes a major antioxidant defense response related to the production of H2O2 and NADH in β-oxidation. An increased turnover of BCAAs to propionyl-CoA and acetyl-CoA was suggested, which may, together with an overproduction of pyruvate from glycolysis, serve amino acid or cofactor production. Butyrate lowers accumulation of carbohydrates and lipids while fatty acid synthesis was found induced, probably in the mitochondria. This all may serve organellar reorganization (peroxisomes) and the production of cofactors for several central metabolic enzymes to accommodate butyrate utilization. In contrast, acetate utilization seems to stimulate activities that relate to the biosynthesis and homeostasis of proteins.Based on our data, we propose that butyrate is assimilated Polytomella sp. a good model for the study of VFA metabolism, but the high starch levels even during the exponential growth is another distinctive trait that allows continuous cultivation on dark fermentation effluents with potential for biofuel production. However, dark fermentation effluents are much more complex substrates than the model media used in this study, with variable composition depending on the fermentation process. As such, potential effects of dark fermentation effluents on biomass growth remain to be studied. Our proteomics approach revealed in many instances the induction on butyrate of multiple proteins belonging to the same pathway or similar metabolic activities suggest their importance in butyrate metabolism. Other omics and biochemical approaches should be employed to further explore the specificities of butyrate vs. acetate as a carbon source. In particular, metabolomics and fluxomics should be used to reveal the assimilation pathways and quantify metabolic fluxes. This study allows to better define the main issues that remain to be tackled in order to understand why butyrate is problematic especially for green algae, which relate to the import of VFAs into the cell as well as the role and regulation of β-oxidation and associated antioxidant activities in relation to photosynthetic metabolism. As a non-photosynthetic alga, Polytomella can serve as a metabolic reference for efficient butyrate assimilation, to which other (green) algae may be compared. Special focus should be on the root causes of the poor use of butyrate especially by green algae, that this study allows to define, i.e., the identity of butyrate transporter and the role of antioxydant reponses. Since Polytomella sp. does not seem to appear to possess novel metabolic capacities per se, it should be considered that this alga owes its fast butyrate assimilation in some way to the loss of another major metabolic capacity: photosynthesis.Its fast butyrate assimilation makes The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/JL performed experiments, data acquisition, data curation, formal analysis, and writing of the original draft. AA contributed to conceptualization, experiments, data acquisition, data curation, formal analysis, validation, review, and editing of the original draft. SB performed experiments, data acquisition, data curation, and formal analysis. YC performed data acquisition, data curation, formal analysis, validation, review, and editing of the original draft. OV performed data acquisition, data curation, formal analysis, validation, review, and editing of the original draft. JPS contributed to supervision, funding acquisition, validation, review, and editing of the original draft. RL designed original experimental plan and performed experiments, data acquisition, data curation, formal analysis, supervision, funding acquisition, validation, review, and editing of the original draft. All authors contributed to the article and approved the submitted version.JL received a PhD fellowship from European Union from the Occitanie region, France, with complementary funding from FEDER. This study was funded by the National Institute of Agriculture, Alimentation and Environment (INRAE), the CNRS, and was supported by the FermALip project, funded by the Carnot institute 3BCAR as well as by the “Initiative d’Excellence” program from the French State . The proteomic experiments were partially supported by Agence Nationale de la Recherche under projects ProFI  and by GRAL, a program from the Chemistry Biology Health (CBH) Graduate School of University Grenoble Alpes (ANR-17-EURE-0003).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.


Ovarian reserve reflects both the quantity and quality of oocytes available for procreation and is affected by many known and unknown factors. ABO blood type is related to several infertility processes, but it is unclear whether and how ABO blood type affects ovarian reserve.The purpose of the study was to explore the correlation between ABO blood types and ovarian reserve in infertile Chinese Han women under 40 years of age undergoing the in vitro fertilization (IVF)/ intracytoplasmic sperm injection (ICSI)-embryo transfer (IVF/ICSI-ET) treatment.Women aged < 40 years who underwent IVF/ICSI-ET at our institution and had a documented ABO blood type were eligible for this study. In this study, patients were divided into two groups according to the diminished ovarian reserve (DOR) group  and the non-diminished ovarian reserve (non-DOR) group . The relationship between ovarian reserve and ABO blood group was determined by correlation analysis.n = 57, 30.5%) and A  and in the non-DOR group with A  and O  were the most frequent in the non-DOR group.In this retrospective cohort study, clinical data were collected from 1690 Chinese Han women treated with IVF/ ICSI-ET in hospital records between April 2019 and March 2020 in the affiliated hospital of Southwest Medical University, located in Luzhou, China. The differences in age, duration of infertility, BMI, FSH, FSH / LH, and p (DOR vs non-DOR) for each parameter (DOR vs non-DOR) were statistically significant, and the differences in LH and E2 were not statistically significant. ABO blood groups were most prevalent in the DOR group with O  and A  and in the non-DOR group with A  and O . ABO blood groups were most prevalent in the DOR group with O (In this retrospective cohort study, we confirmed the lack of a significant association between ABO blood type and ovarian reserve. Further studies are needed to clarify whether there is any prognostic correlation between ABO blood group and ovarian reserve in women undergoing IVF/ICSI-ET. Infertility is a common concern for couples , 2. InfeDiminished ovarian reserve is defined by a decreased quantity or quality of ovarian follicles in women. It is a condition between normal reproductive physiology and premature ovarian insufficiency. Although the criteria for diminished ovarian reserve have not been defined and vary from region to region , it is bThere has been interest in the relationship between infertility and ABO blood groups. Many studies have investigated the association of ABO blood groups with diminished ovarian reserve (DOR), but have obtained contradictory results. An increase in live birth rates has been found for blood group B by Goldsammler et al. . In subsTo date, the relationship between ABO blood groups and ovarian reserve is complex and study findings are sometimes contradictory and therefore additional studies are necessary to reconcile these conflicting findings. Accordingly, in the present study, we sought to assess the potential association between ABO blood groups and ovarian reserve in a cohort of women undergoing IVF/ICSI-ET.A total of 1690 Chinese Han women diagnosed with infertility were included in this retrospective cohort study at the Reproductive Medicine Center of Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan, from April 2019 to March 2020. Inclusion Criteria: (a) Ethnicity was Han. (b) No history of alcoholism, smoking, etc. (c) Both couples were chromosomally normal. (d) These infertile women were diagnosed with tubal factor infertility, including hydrosalpinx and proximal tubal obstruction. (e) Patients with clinical information. Exclusion Criteria: (a) Chromosomal abnormalities in one or both spouses. (b) Women with PCOS. Patients enrolled in the study were divided into two groups: diminished ovarian reserve (DOR) group  and non-diminished ovarian reserve (non-DOR) group  based on patients’ AMH and AFC. The flow chart and selection process of participants are detailed as shown in Fig. 2), progesterone (P), and ABO blood group. Serum levels of sex hormone were measured by using an automated chemiluminescence enzyme immunoassay analyzer . The serum AMH level was measured by using the electrochemiluminescence immunoassay kit . Serum follicle stimulating hormone(FSH), luteinizing hormone(LH), FSH/LH, estradiol(E2), and progesterone(P) levels were measured between day 2 and day 5 of the menstrual cycle. Serum follicle stimulating hormone(FSH), luteinizing hormone(LH), FSH/LH, estradiol(E2), and progesterone(P) levels were measured between day 2 and day5 of the menstrual cycle. AFC  was measured using transvaginal ultrasound .Clinical information included age, duration of infertility, BMI (Body mass index), follicle stimulating hormone(FSH), luteinizing hormone(LH), FSH/LH, estradiol normality test. The Mann-Whitney test was used to compare the two groups. Count data were expressed as percentages (%) and analyzed by the χ 2 test. Statistical Analysis SPSS 19.0  statistical software was used for statistical processing. A P < 0.001). There were significant differences between the DOR and non-group in terms of duration of infertility, BMI, basal FSH, basal FSH/LH, and basal P (P < 0.05). All statistical results are shown in Table The median age was higher in the DOR group than in the non-group 35 vs. 31), with a statistically significant difference , and those with AMH ≥ 1.1 ng/mL and AFC ≥ 6 were assigned to non-DOR (n = 1279). The proportions of blood type O  were higher in DOR group than non-DOR group. There were more patients with type O than other blood types in the DOR group aged less than 40 years (34.8%), whereas there were more patients with type A than other blood types in the non-DOR group (33.5%). Overall, blood type O was the most prevalent in the DOR group (34.8%), followed by blood type A (29.7%), blood type B (27.3%), and blood type AB (8.3%). In parallel, blood type A was the most prevalent (33.5%), followed by blood type O (32.8%), blood type B (24.1%), and blood type AB (9.7%) in the non-DOR group aged less than 40 years. There were no significant differences in the proportions of blood types between the groups . The above results indicate that the blood type is not related to ovarian reserve. The detailed results are shown in Table As shown in Table PO = 0.552, PA = 0.177, PB = 0.193, PAB = 0.286, respectively). The above results suggest that blood group is not related to ovarian reserve capacity after excluding infertile women over 35 years. The detailed results are shown in Table As shown in Table This retrospective analysis used AMH and AFC values to determine normal ovarian reserve and DOR and concluded that there was no relationship between blood type and ovarian reserve in infertile women. Furthermore, after adjusting for age (< 35 years), history of EMS, and ovarian surgery, the correlation between blood type and ovarian reserve remained stable with no significant difference.DOR, defined as a decreased quantity and quality of oocytes, affects nearly 10% of women seeking fertility treatment . MultiplP = 0.278) and progesterone (P = 0.002). Harzif et al. showed that ovarian reserve capacity decreases with age  and A  in the DOR group, while A  and O  accounted for the most in the non-DOR group in women < 35 years, respectively. Li et al. found that age was an independent factor affecting ovarian function, especially in those > 40 years, ovarian function decreased more than those < 40 years (28.4 vs. 65.2%) [In our study, we found that the most common ABO blood types were O [. 65.2%) . Yu et a. 65.2%) . After a. 65.2%) . A meta-. 65.2%) . TherefoIn conclusion, a growing number of studies have confirmed the existence of a relationship between ABO blood group and ovarian reserve function. However, the results vary depending on the study design, population, sample size, ovarian function predictors and specific cut-off values. Therefore, broader sample size prospective studies are needed to confirm whether blood type independently affects ovarian reserve function and to elucidate the mechanisms through more in-depth, even molecular and genetic studies.There were several strengths of our study. This study is the first to examine the region’s blood group and ovarian reserve in infertile population. This allows us to better understand the situation related to infertility in this region. A relatively large number of patients were included in this study compared to other clinical follow-ups, allowing for highly detailed information on comorbidities, hospitalization parameters and treatments, which contributed to the validity of this study. Age factors that may have influenced the results were excluded. Nevertheless, there are some potential limitations of this study. This study is a retrospective single-center analysis. Although all patients at our reproductive center who underwent assisted reproductive technologies (ART) during this period were included, the number of patients was relatively small. Our study was conducted in infertile women and is not representative of women with normal fertility. The participants in our study were solely from the Chinese Han ethic group and may not be representative of other ethnical groups. The relationship between ABO blood group and ovarian reserve was not elucidated.It was found that AMH was correlated with age, infertility duration, BMI, FSH, FSH/LH, and P. Furthermore, AFC was correlated with age, FSH, and FSH/LH. However, this study found no significant correlation between ovarian reserve and ABO blood group in infertile women. Whether blood type independently affects ovarian reserve function requires a broader sample size prospective study to clarify the mechanism by more in-depth or even molecular and genetic studies.


Quantifying the pulmonary vascular obstruction index (PVOI) is essential for the management of patients with pulmonary embolism or chronic thromboembolic pulmonary hypertension (CTEPH). The reference method for quantifying the PVOI with planar lung ventilation/perfusion (V/Q) scintigraphy is the Meyer score, which was validated using pulmonary angiography as a reference standard. However, it is complex to use in daily practice. In contrast, a rapid and fast quantification method consists in estimating the PVOI based on the number of segmental perfusion defects. However, the accuracy of this method has never been evaluated. In this study, we aimed to compare PVOI quantification on planar V/Q scintigraphy assessed by a segmental visual scoring (SVS) to the Meyer score.The eligible study population consisted of consecutive patients who underwent planar V/Q scan for CTEPH screening. A central review was performed by three nuclear medicine physicians. PVOI was assessed by summing the number of segmental perfusion defects or equivalent (2 sub-segments = 1 segment = 5%) and by Meyer’s method. The two interpretations were performed 6 months apart. A Spearman rank correlation coefficient was calculated to evaluate correlation between the two measurement methods. An intra-class correlation (ICC) was calculated to assess agreement. A Bland et Altman plot analysis was used to evaluate agreement between the two measurements.A total of 226 V/Q scans were interpreted. Spearman rank correlation coefficient between SVS and Meyer was 0.963 (95%CI 0.952–0.971) for mismatched perfusion defects and 0.963 (95%CI 0.953–0.972) for perfusion defects regardless of ventilation. Intra-class correlation (ICC) for agreement was 0.978 (95%CI 0.972–0.983) for mismatched perfusion defects and 0.968 (95%CI 0.959–0.976) for perfusion defects regardless of ventilation. In Bland & Altmann analysis, the mean difference between the SVS method and the Meyer score was 0.42 and 0.61 for the mismatched or matched evaluation, respectively.Our study shows a high correlation, and low differences in PVOI quantification when using a segmental visual scoring (SVS) as compared to the Meyer score. The SVS has the great advantage to be easy and rapid to apply in daily practice. Lung ventilation/perfusion (V/Q) scintigraphy is a well-established test for the diagnosis of acute pulmonary embolism (PE) , 2. It iIn patients with PE or CTEPH, there is a growing interest in quantifying the pulmonary vascular obstruction index (PVOI), i.e., the percentage of the whole lung volume with perfusion defects. Indeed, a high pulmonary vascular obstruction index (PVOI) measured at the time of PE diagnosis was associated with an increased risk of residual obstruction , 5 and rThe reference method for quantifying the PVOI with planar lung V/Q scintigraphy is the Meyer score, which was validated using pulmonary angiography as a reference standard . Each loIn contrast, another method based on a segmental visual scoring (SVS) is commonly used by nuclear medicine physicians. In this method, the score is obtained by adding 5% for each segmental perfusion defects or equivalent (2 sub-segments = 1 segment) which correspond to the mean percent volume of a segment in a 20-segment model (100 divided by 20). This approach, which does not take into account differences in segments size, has the advantage to be easier and faster to use in routine. However, to the best of our knowledge, how the SVS reliably estimates the perfusion score has never been evaluated.In this study, we aimed to compare PVOI quantification on planar ventilation/perfusion (V/Q) lung scintigraphy assessed by a segmental visual scoring (SVS) to the Meyer score.We reviewed the same 226 planar V/Q scans from a previous study that assessed the accuracy of various interpretation criteria of planar V/Q scintigraphy for the screening of CTEPH . The eliPlanar V/Q lung scans were performed according to the Société Française de Médecine Nucléaire guidelines on lung scintigraphy protocol . Images A retrospective central review of all planar V/Q lung scintigraphy was performed by three nuclear physicians with different level of expertise, blinded to clinical results and to final diagnosis. Interpretation was determined via consensus reading.Initially, the number, extent  and type (matched or mismatched with ventilation images) of perfusion defects were reported for each planar V/Q lung scintigraphy. A defect was visually defined as segmental if it involved more than 75% of a segment and sub-segmental if it involved less than 75% , 16. SmaFor the purpose of the present study, the same three nuclear medicine physicians reviewed all scans and calculated the PVOI based on the Meyer score . This inFor each planar V/Q lung scintigraphy, PVOI assessed by summing the number of segmental perfusion defects or equivalent (2 sub-segments = 1 segment = 5%) was computed on first reading. Then, PVOI assessed by Meyer method was computed on second reading 6 months later.Because of a non-normal distribution of values, a Spearman rank correlation coefficient was calculated to evaluate correlation between the two measurement methods and plot graphs were drawn. An intra-class correlation (ICC) was calculated to assess agreement of the two methods based on a two way random effect model. A Bland et Altman plot analysis was used to evaluate agreement between the two measurements across the range of possible values. The mean difference (μ) and the standard-deviation (σ) of the differences between the two measurements were plotted against their average, and limits of agreements were defined as μ ± 1.96 * σ for both matched and mismatched evaluations, as recommended by B&A .All these statistical tests were performed for mismatched perfusion defects, and for perfusion defects regardless of the ventilation .Statistical analysis was done using R version 4.0– © 2009–2021 RStudio, Inc.A total of 226 patients with newly diagnosed PH, who underwent V/Q planar scintigraphy for screening of CTEPH at the Brest University Hospital between January 2004 and January 2019 were analyzed. Out of them, 56 (25%) were eventually diagnosed with CTEPH. Among the 56 patients, 29 patients (39%) had no PE history. Among 170 patients (75%) diagnosed with non-CTEPH, 92 were classified in group 1 of PH classification (41%), 24 in group 2 (10%), 40 in group 3 (18%), 4 in group 5 (2%), and 10 were classified as having mixed causes PH  (4%).r = 1) and blue line as local polynomial regression fitting of SVS method on Meyer score, for mismatched perfusion defects or perfusion defects regardless of ventilation, respectively.Spearman rank correlation coefficient between SVS and Meyer was 0.963 (95%CI 0.952–0.971) for mismatched perfusion defects and 0.963 (95%CI 0.953–0.972) for perfusion defects regardless of the ventilation. Intra-class correlation (ICC) for agreement was 0.978 (95%CI 0.972–0.983) for mismatched perfusion defect and 0.968 (95%CI 0.959–0.976) for perfusion defects regardless of the ventilation. Plot graph  illustraConcerning the Bland & Altmann analysis , the meaIn this series of 226 patients with PH who underwent planar scintigraphy for CTEPH screening, a high correlation was found between the SVS method and the Meyer score for the quantification of the PVOI, with a correlation coefficient of 0.963 (95%CI 0.952–0.971). This result supports the use of the SVS method to quantify the PVOI in daily clinical practice.When quantifying the PVOI based on mismatched perfusion defect, correlation and agreement between SVS and Meyer scores were excellent with a Spearman rank correlation coefficient of 0.963 (95%CI 0.952–0.971) and an ICC agreement of 0.978 (95%CI 0.972–0.983). On Bland & Altman graph , the meaWhen quantifying the PVOI based on perfusion only images (regardless of ventilation), correlation and agreement coefficients were still good with a Spearman rank correlation of 0.963 (95%CI 0.953–0.972) and an ICC agreement of 0.968 (95%CI 0.959–0.976). However, the limits of agreement between the SVS method and the Meyer score for perfusion defects regardless ventilation was wider on Bland et Altman . In factFor both mismatched and perfusion only approaches, plot graphs , which iAlthough the Meyer score was validated using pulmonary angiography as a reference standard  and was In contrast, the segmental visual scoring (SVS) has the advantage to follow the principles of interpretation of the V/Q scan for PE or CTEPH, based on a segmental approach of perfusion defects. Indeed, the PIOPED and EANM criteria used for PE diagnosis are based on the number of sub-segmental and segmental defects , 15, 16.What are the clinical implications of our findings? PVOI quantification has been described to be of particular relevance in various clinical scenarios. In patients with PE, PVOI at diagnosis or after a minimum of 3 months of anticoagulant therapy has been described as an independent risk factor of PE recurrence, and may be used to adapt the duration of anticoagulant therapy , 8, 9. IOur study has some limitations. First, we performed a consensus reading and did not assess interobserver reproducibility. However, principles of interpretation of the SVS method, based on the recognition of mismatched perfusion defects are well-known by nuclear medicine physicians. In contrast, the Meyer score with its lobar approach is unusual, and Wan et al. , reporteOur study shows a high correlation and low differences in PVOI quantification on V/Q planar scintigraphy when using a segmental visual scoring (SVS) as compared to the Meyer score. The SVS has the great advantage to be easy to learn and apply in daily practice. As PVOI quantification may have a prognostic implication in thrombo-embolic diseases, it could be systematically calculated using the SVS and provided in lung scan reports.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the French National PH Registry (authorization number: 842063). The patients/participants provided their written informed consent to participate in this study.RLP, CT, PL, PS, and CO performed the material preparation, data collection, and analysis. RLP and PL wrote the first draft of the manuscript. All authors contributed to the study conception and design, commented on previous versions of the manuscript, and read and approved the final manuscript.


Bifidobacterium increased significantly at 3 days after calving. Both challenges increased the intestinal abundance of fiber-associated taxa, e.g., Butyrivibrio and unclassified Ruminococcaceae. NMR analyses of rumen and duodenum samples identified up to 60 metabolites out of which fatty and amino acids, amines, and urea varied in concentrations triggered by the two challenges. Correlation analyses between these parameters indicated a close connection and dependency of the microbiome with its host. It turns out that the combination of phylogenetic with metabolite information supports the understanding of the true scenario in the forestomach system. The individual stages of the production cycle in dairy cows reveal specific criteria for the interaction pattern between microbial functions and host responses.Little is known about the interplay between the ruminant microbiome and the host during challenging events. This long-term study investigated the ruminal and duodenal microbiome and metabolites during calving as an individual challenge and a lipopolysaccharide-induced systemic inflammation as a standardized challenge. Strong inter- and intra-individual microbiome changes were noted during the entire trial period of 168 days and between the 12 sampling time points.  The modern dairy cow is confronted with a multitude of challenges. This, among others, includes feed changes, pathogens, heat stress, and the transition from dry period to high-performance milk production. These impair the cow’s health and hence, productivity as well as product quality. Ketosis, metritis, milk fever, and displaced abomasum might go along with or even originate from a disrupted forestomach and gut microbial community and might appear as a decrease in microbial diversity, richness, and functionality . The gutThe transition from the dry period to lactation is one of the most critical phases in a cows’ life. The metabolism focuses on milk production with high energy and protein requirements at limited energy intake, making the cow more susceptible to internal and external challenges and often leading to inflammatory conditions . The reqIt is still unclear, which factors contribute to the robustness of individual cows, and how they manage the balance between high productivity at good fitness and therefore contribute to high farm profitability. The response of the microbiome and its possible function toward stimulating the host physiology and metabolism during host challenging situations are also less studied so far. Therefore, scoping into the rumen as the major site of energy conversion and the duodenum as the first site of host nutrient uptake could enlighten these responses to challenging periods.This study hypothesizes that modern dairy cows respond to the challenge of calving, the subsequent feed change, and a LPS-induced standardized inflammation by dynamic adaptation of ruminal and duodenal bacteriomes. These responses either support or intensify the severity of the reaction toward stress. Additionally, the role of supplemented L-carnitine for the bacterial consortium in the matrixes, rumen, and duodenal fluid, across a particularly long period of time, is elucidated. This study connects detected metabolites with bacterial abundances and selected host health parameters to pinpoint possible interactions.p = 0.009) from an average of 6.9 ± 0.3 (SD) to 5.8 ± 0.6 (p = 0.03). LPS challenge significantly increased pH in RUM and DUO . The pH values in DUO increased from −14 (2.4 ± 0.2) to shortly after calving .The animal trial covered 168 days and used eight double fistulated dairy cows, which resulted in a total of 143 samples from rumen (RUM) and duodenum fluid DUO; . From −4.8 ± 0.6 . This sip < 0.0001], Firmicutes , and Actinobacteria  were the dominant phyla in RUM and DUO samples  of 68 RUM and 75 DUO samples were taxonomically assigned to 16 phyla, 65 families, and 102 genera. Bacteroidetes [average (av.): RUM = 51% ± 13 (SD), DUO = 40% ± 12;  samples , 2.p ≤ 0.05, ap and pp time points . DUO samples at −42 were significantly different from +100, 12hL, 24hL, +118, and +126, respectively  due to higher abundances of Succiniclasticum and Ruminobacter .The L-carnitine supplementation was not affecting bacterial communities in both matrixes ; therefoe points . RUM −42p = 0.008, DUO: p = 0.05) and increased, induced by LPS challenge . Significant negative correlations were observed between α-diversities and abundances of certain bacterial taxa such as Olsenella, unclassified Lachnospiraceae and Roseburia abundances in both sample types (Shannon diversity (α-diversity) varied throughout the entire trial period on average between 4.0 (+14) to 6.2 (12hC) in RUM and 4.4 (+14) to 5.8 (12hL) in DUO, demonstrating slightly greater diversity fluctuations in RUM . Diversile types .Bifidobacterium increased significantly at 72hC in both matrixes . Thereafter, Bifidobacterium abundance decreased to antepartum (ap) abundance level. Olsenella species increased significantly after calving and feed adaptation in RUM  and DUO . At LPS challenge time points, a decrease in abundance was observed, which was significant in RUM .The overall taxonomic composition showed fluctuations in abundances in both sampling sites along the animal trial caused by calving, changes in feeding regime, and LPS challenge. Abundances of Clostridiales abundances significantly decreased in DUO after calving and feed change  and significantly increased shortly after the LPS challenge . Unclassified Ruminococcaceae increased significantly with calving and/or feed change in both matrixes  and in DUO samples during LPS challenge . Unclassified Lachnospiraceae increased in RUM significantly with the calving and/or feed-changing event . Fibrobacter was significantly higher abundant in DUO than in RUM (p = 0.02). Butyrivibrio in both matrixes significantly decreased with calving and/or feed change  and significantly increased with LPS challenge in DUO .Unclassified p = 0.008, p = 0.01). After calving and feed change, unclassified Gammaproteobacteria clearly dominated this phylum in DUO which was not the case in RUM samples. Here, the LPS challenge triggered a very clear increase of unclassified Gammaproteobacteria. This genus dominated the total sequenced Proteobacteria at the end of the trial, summing more than 95% in RUM and 85% in DUO. In RUM, a significant decrease of Ruminobacter is observed from −42 to +14 (p = 0.02) and an increase induced by the LPS challenge . In both matrixes, Succinimonas completely vanished after feed calving and feed change . RUM Succinivibrio significantly decreased from −14 to +14 (p ≤ 0.05) but recovered from +100 on.Proteobacteria showed similar abundances in RUM and DUO and higher genera diversity and evenness before calving and feed change. This phylum significantly decreased after calving and feed change  and DUO (N = 67) samples were significantly grouping apart from each other , which was confirmed by parallel GC–MS measurements of the samples (p ≤ 0.05).In total, 60 host and bacteria-derived metabolites were measured using NMR spectroscopy . RUM (N  samples  compared samples . RUM prop ≤ 0.05) and not significantly different between DUO samples of control (CON) and carnitine-supplemented (CAR) animals . In DUO at −42, 12hL, +118, and + 126, aspartate, proline, and histidine strongly increased, compared to all other time points (p ≤ 0.006). Free RUM glucose increased significantly after calving and/or feed change (−14 to +14) and decreased during LPS challenge  and decreased during LPS challenge  and decreased during LPS challenge significantly , trimethylamine, and propionate (p < 0.0001) as well as pimelate, alanine, and acetone (p < 0.0001). In DUO, glycine (p = 0.0004), isovalerate (p = 0.001), carnitine (p = 0.01), and isobutyrate (p = 0.006) negatively correlated with high microbial diversities. In rumen, 3-phenylpropionate correlated positively with high α-diversities (p = 0.02).Most metabolites in both matrixes correlated negatively with high α-diversities, such as in RUM samples valerate (p < 0.0001) as well as with DUO glycine, propionate, and isobutyrate concentrations  were the second most common across time; however, Bacteroides genus was found to correlate with all 60 metabolites analyzed at least once across the whole trial period. Choline, in turn, was the most common metabolite correlator for bacteria, 86 out of 102 genera correlated with this metabolite, whereby formate was the most frequently repeating correlator across all time points and both matrixes, mostly with Fusobacterium, Megasphaera, Ruminobacter, SR1 genera i.s., unclassified Succinivibrionaceae and unclassified Bacteroidia. Together with unclassified Succinivibrionaceae and SR1 genera i.s., Olsenella correlated most frequently and positively with the lactate concentration in RUM and DUO. The most frequent metabolite:genus combinations in both sample types were Fumarate:SR1 genera i.s, Ribose:SR1 genera i.s, Aspartate:unclassified Succinivibrionaceae and Ribose:unclassified Succinivibrionaceae.Correlating a great number of metabolites and genera and two matrixes at 12 different time points brings up a wealth of significant correlating combinations. Counting significant combination repeats between genera and metabolites, such as “SuccinivibrioFormate,” across all time points resulted into insights of those combinations appearing the most. The higher the repeat number of the combination across all time points, the more meaningful this combination may be to the host and the ecosystem. Unclassified Olsenella was positively and unclassified Bacteroidales negatively associated with L-carnitine in both matrixes. In RUM, Roseburia (p < 0.0001) and Syntrophococcus (p < 0.0005) were positively and unclassified Proteobacteria negatively (p < 0.03) correlating with L-carnitine. In DUO, unclassified Eubacteriaceae (p < 0.04) and unclassified Succinivibrionaceae (p < 0.004) were positively associated with L-carnitine. Choline, carnitine, and trimethylamine are known to be involved in trimethylamine-N-oxide (TMAO) formation and at various time points correlating significantly with Olsenella, Pseudoscardovia, and unclassified Veillonellaceae abundances , accompanied by reduced levels of its fermentation product lactate  values around 0 or negative. In contrast, animal 306 was observed with considerably high Bifidobacterium abundances, and only had some skin issues, but no infectious diseases or claw infections. The fecal microbiome of this animal could be grouped to the third microbiome cluster (C-Bifi) of the previous study, which was characterized by medium milk yields, positive REI values, lowest BCS and body weights (BW) at high milk protein and milk fat yields  and increased in the rumen when concentration in the diet was increased. Interestingly animals with highest concentration of free ruminal glucose throughout the trial were those assorted to the animal cluster “C-Clos,” associated with longer days in illness, significantly higher milk somatic cell counts and blood mean platelet volumes at significantly lower dry matter intakes compared to the other two clusters within a related study including the same animals  higher choline concentrations found in DUO, either deriving from the microbial activity or a host-derived synthesis of choline in between the rumen and duodenum, as no rumen-protected choline was fed , in Braunschweig, Germany in accordance with the German Animal Welfare Act approved by the LAVES  (AZ33.19-42502-04-16/2378).antepartum . This consisted of 80% roughage and 20% concentrate until calving (day 0) and contained the supplements in the concentrate feed. From the day of calving up to day 14 postpartum , the amount of concentrate was gradually increased up to a ratio of 50:50. This regimen was continued until the end of the trial. Roughage comprised 70% maize silage and 30% grass silage; water was offered ad libitum. Details about the animal experimental setup and feeding are published for the calving period , funded by the German Research Foundation (DFG) including 59 multiparous German Holstein-Friesian dairy cows , 2021. Wvia fistula (inner diameter: 10 cm), using a manual pump, which was thoroughly washed with water between samplings and animals, and fitted with a sieve that filtered larger feed particles. The first spills of pumped rumen fluid were discarded. Duodenal fistulae (inner diameter: 2 cm), placed before the pancreatic and bile duct, were equipped with simple screwing caps. After opening them, the first spill of duodenal fluid was discarded and the further fluid was collected. In total, 73 rumen fluid and 75 duodenal fluid samples with each 30 ml per time point were collected, pH was measured right after sampling , and samples were immediately stored at −80°C until further processing. During the trial, animal 039 was euthanized right after time point +14 due to a multifactorial inflammation, and animal 248 died shortly after the samples at time point 12hL were taken after an inflammatory shock. However, samples deriving from these animals have been included in this study.Rumen (RUM) and duodenum (DUO) samples were taken regularly at 7 am after milking and at 12 time points between day −42 and day +126, including the sampling time points 12, 24, and 72 h after calving  and an LPS-induced inflammatory challenge at day +111  for soil protocol with minimal changes as described previously . DNA quamplicons . PCR ampRaw sequence reads obtained from Illumina MiSeq system  were analyzed using QIIME2 v 20192O, 99.9% Sigma-Aldrich, Germany) containing 5 mM 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt . Data were processed using Chenomx NMR Suite 8.2 software  and permutational multivariate analysis of variance (PERMANOVA). This approach was used to evaluate similarity between different groups . The similarity percentages (SIMPER) tool was applied to find the main contributors of differences between groups. In JMP® Pro 15.2.1, Shapiro test was used to test the normality of distribution of bacteria and metabolites and Wilcoxon/Kruskal–Wallis test was used to test for significance. Barplots and boxplots were drawn using JMP® Pro 15.2.1 software. The same software was used for the Pearson’s product–moment correlation coefficient measures between metabolites and genera.PRIMER-E , accession number PRJEB41061. NMR data are available from the corresponding author upon request.Sequences were submitted to European Nucleotide Archive . The authors acknowledge support by the High Performance and Cloud Computing Group at the Zentrum für Datenverarbeitung of the University of Tübingen, the state of Baden-Württemberg through bwHPC and the German Research Foundation (DFG) through grant no INST 37/935-1 FUGG.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.