Patients and Methods. Patients (N = 90) with nonmetastatic breast cancer aged between 60 and 65 years old were prospectively recruited in this study, compromising a cohort of subjects rece iving 5 years or 10 years of adjuvant endocrine therapy. Patients' DFS was compared between these two groups. Patients' body composition including muscle and fat using CT scans, muscle strength, and gait speed was evaluated in these two groups.  Our study aims to investigate the association of extended adjuvant endocrine therapy with disease-free survival (DFS), muscle mass, muscle strength, and visceral adipose tissue in patients with nonmetastatic breast cancer and the effect of extended endocrine therapy on body composition. P=0.038). Patients receiving 10 years of adjuvant endocrine therapy had less skeletal muscle and more visceral fat compared with patients receiving 5 years of adjuvant endocrine therapy. The skeletal muscle index was 50.3 ± 1.6 cm2/m2 versus 46.5 ± 1.3 cm2/m2 in the 10 years or 5 years of adjuvant endocrine therapy group (P=0.042). The visceral fat was 28.9 ± 2.9 cm2/m2 versus 55.0 ± 3.2 cm2/m2 in the 10 years or 5 years of adjuvant endocrine therapy group (P=0.011). The muscle strength, gait speed, and FFQ results in the two groups not reaching statistical difference.  Dietary behavior was recorded with the food frequency questionnaire (FFQ). Patients' age, body weight, and body mass index (BMI) did not differ between the two groups. An extended adjuvant endocrine therapy into 10 years could translate into DFS benefit (123.8 vs. 102.2 months,  In conclusion, breast cancer patients with 10 years of adjuvant endocrine therapy had DFS benefit, but with more muscle loss and adipose tissue deposits compared to patients receiving 5 years of adjuvant endocrine therapy. Breast cancer is the first most common malignancy in females worldwide . The aveThe adverse events of endocrine therapy could be decreased muscle mass and increased fat tissue. Accelerated muscle loss, also called sarcopenia, has been proved to be correlated to the menopausal transition and thus linked to declining estrogen levels . EstrogePrevious studies proved that hormones significantly influence body visceral adipose tissue distribution and visceral adipose differentiation . Fat tisThus, our study aims to investigate the association of extended adjuvant endocrine therapy with DFS, muscle mass, muscle strength, and visceral adipose tissue in patients with nonmetastatic breast cancer. Our hypothesis is that 10 years of adjuvant endocrine therapy could prolong the DFS, but decrease the muscle mass and muscle strength and increase adipose tissue at the same time.90 patients with nonmetastatic breast cancer aged between 60 and 65 years old were prospectively recruited in this study, compromising a cohort of subjects that receive at least 5 years of adjuvant endocrine therapy. Patients were recruited from July 2017 to March 2020. We selected patients with a random number table. Inclusion criteria are breast cancer patients receiving already 5 years or 10 years of adjuvant endocrine therapy, with age of 60–65 years. Patients were not subjected to radiotherapy. Exclusion criteria included patients with diabetes, metabolic syndrome, tuberculous, rheumatoid arthritis, gout, systematic inflammatory disease, cerebrovascular disease, liver or renal insufficiency, cardiac insufficiency, autoimmune disease, or other severe systemic diseases. All patients were not allowed to receive steroids prior to this study. The study was approved by the Ethics Committee of Hubei Ezhou Central Hospital. All patients signed the written informed consent.We recorded patients' baseline clinical data, including age, weight, body mass index (BMI), skeletal muscle index, visceral adipose index, tumor size, lymphatic invasion, number of metastatic lymph nodes, estrogen receptor (ER) status, progesterone receptor (PR) status, human epidermal growth factor receptor 2 (Her-2) status, adjuvant endocrine therapy, previous chemotherapy, target therapy, and surgery method .2 (cm2/m2). Visceral adipose index = visceral adipose area/height2 (cm2/m2).We evaluated body composition including skeletal muscle mass and visceral adipose area at the third lumbar vertebrae (L3) level using abdominal CT scan using sliceOmatic software  both at baseline and after 5 y or 10 y of endocrine treatment. The skeletal muscle was colored with typical Hounsfield units (HU) ranging from −29 to 150 and visceral adipose tissue was colored with HU ranging from −150 to −50 . The norMuscle strength was measured using handgrip strength. For females, handgrip strength less than 20 kg was regarded as abnormal . A commoPatients' dietary behavior was assessed using the food frequency questionnaire (FFQ). Patients were asked to do a 24-hour dietary recall. Patients' energy intake, protein, fat, and carbohydrate intake were recorded.t test with P < 0.05 considered as statistically significant. Patients' dietary behaviors between the two groups were also compared using Student's t test.We did all statistical analysis using SPSS 23 . Descriptive statistical analyses of baseline characteristics were conducted for all continuous variables by mean values (standard deviation) and categorical variables by numbers (percentage). Comparisons of skeletal muscle index, visceral adipose index, muscle strength, gait speed between patients with 5 years of adjuvant endocrine therapy and 10 years of adjuvant endocrine therapy were made using Student's P=0.521). The average weight in 5 years of endocrine therapy group was 72.2 kg versus 72.6 kg in the 10 years of adjuvant endocrine therapy group (P=0.529). The average BMI in 5 years of endocrine therapy group was 26.1 kg/m2 versus 26.3 kg/m2 in the 10 years of adjuvant endocrine therapy group (P=0.809), both falling within the overweight range proposed by the World Health Organization.Patients' age, body weight, and BMI did not differ between the two groups . PatientP=0.038)  .2/m2 versus 28.9 ± 2.9 cm2/m2 in the 5 years of adjuvant endocrine therapy group    . The visP=0.011) .P=0.329) (P=0.798)  . The gaiP=0.798) .P=0.412). Patients' protein intakes were 65.1 ± 9.9 g in the 5-year endocrine therapy group versus 64.1 ± 9.7 g in the 10-year endocrine therapy group (P=0.221). Patients' fat intakes were 50.9 ± 5.2 g in the 5-year endocrine therapy group versus 48.7 ± 8.2 g in the 10-year endocrine therapy group (P=0.756). Patients' carbohydrate intakes were 296.7 ± 43.1 g in the 5-year endocrine therapy group versus 301.1 ± 31.8 g in the 10-year endocrine therapy group (P=0.109).Patients took a 24-hour dietary recall using the FFD . PatientOur study was the first to evaluate the impact of extended endocrine therapy on patients' DFS, body composition, muscle strength, and gait speed. We proved that patients receiving 10 years of endocrine had DFS benefit, which is in accordance with a previous study . HoweverMuscle maintenance is a very important issue for cancer patients. Numerous studies have indicated that cancer patients with muscle loss had worse survival –32. In p2 had a 2-fold higher risk of mortality rate in comparison with breast cancer patients with normal BMI  [Adipose tissue contributes to cancer development and is negatively associated with prognosis in several cancer types –37. TherR, 2.12) . Other sR, 2.12) , 40.Estrogen and progesterone are the two primary female sex hormones. They are produced mainly in ovaries and produced or converted into forms of estrogen in fat tissue. Estrogen balance is essential for achieving and maintaining muscle and fat loss. Estrogen significantly facilitates adipose tissue depots and its function . EstrogeOur study also has limitations. Our sample size is not large, which means the conclusions still need to be validated in a larger-scale clinical trial. Our study is an observational cohort prospective study; thus, we could not rule out confounding factors that could impact muscle or fat, such as exercise. However, we tried our best to do a FFQ, which rule out the impact of dietary on muscle and fat. Patients' baseline characteristics including age, sex, and BMI did not reach significant difference. However, patients with the same BMI may have different adipose tissue compartments and muscle mass. Our study was the first to look further into the impact of endocrine therapy on body composition in breast cancer patients.In conclusion, breast cancer patients with 10 years of adjuvant endocrine therapy had better DFS, but with more muscle loss and adipose tissue deposits compared with patients receiving 5 years of adjuvant endocrine therapy.


Furthermore, it establishes role of TRIP13 in colorectal cancer metastasis and identifies COL6A3, TREM2, SHC3, and KLK7 as its downstream targets. Additionally, TRIP13 activates EGFR‐AKT pathway  Overexpression of TRIP13, a member of the AAA‐ATPase family, is linked with various cancers, but its role in metastasis is unknown in colorectal cancer (CRC). In the current study, we investigated the role TRIP13 in experimental metastasis and its involvement in regulation of WNT/β‐catenin and EGFR signaling pathways. Evaluation of formalin‐fixed paraffin‐embedded (FFPE) and frozen tissues of adenomas and CRCs, along with their corresponding normal samples, showed that TRIP13 was gradually increased in its phenotypic expression from adenoma to carcinoma and that its overexpression in CRCs was independent of patient's gender, age, race/ethnicity, pathologic stage, and p53 and microsatellite instability (MSI) status. Moreover, liver metastases of CRCs showed TRIP13 overexpression as compared to matched adjacent liver tissues, indicating the biological relevance of TRIP13 in CRC progression and metastasis. TRIP13 knockdown impeded colony formation, invasion, motility, and spheroid‐forming capacity of CRC cells irrespective of their p53 and MSI status. Furthermore, xenograft studies demonstrated high expression of TRIP13 contributed to tumor growth and metastasis. Depletion of TRIP13 in CRC cells decreased metastasis and it was independent of the p53 and MSI status. Furthermore, TRIP13 interacted with a tyrosine kinase, FGFR4; this interaction could be essential for activation of the EGFR‐AKT pathway. In addition, we demonstrated the involvement of TRIP13 in the Wnt signaling pathway and in the epithelial–mesenchymal transition. Cell‐based assays revealed that miR‐192 and PNPT1 regulate TRIP13 expression in CRC. Additionally, RNA sequencing of CRC cells with TRIP13 knockdown identified COL6A3, TREM2, SHC3, and KLK7 as downstream targets that may have functional relevance in TRIP13‐mediated tumor growth and metastasis. In summary, our results demonstrated that TRIP13 promotes tumor growth and metastasis regardless of p53 and MSI status, and indicated that it is a target for therapy of CRC. CINchromosomal instabilityCRCcolorectal cancerEMTepithelial–mesenchymal transitionFFPEformalin‐fixed, paraffin‐embeddedLEFlymphoid enhancer factorMSmicrosatelliteMSImicrosatellite instableMSSmicrosatellite stableNSGNOD/SCID/IL2γ receptor‐nullNTnontargetingSACspindle assembly checkpointTCFT‐cell factorTRIP13thyroid hormone receptor interactor 13UABUniversity of Alabama at Birmingham1Colorectal cancer (CRC) is the third leading cause of cancer deaths in the United States . ChemothAPC mutations, oncogenic β‐catenin in the WNT signaling pathway   vs female (n = 47); Supplementary Fig. n = 3), 41–60 years (n = 26), 61–80 years (n = 47) and 81–100 years (n = 19); Supplementary Fig. TRIP13 overexpression in CRCs was independent of the pathologic stage and the race, age, and gender of patients.Publicly available Oncomine 3 [Oncomine™ Platform  data showed high expression of RCs Fig. ] 40]. G. GTRIP13ab.edu/)  showed eion Fig. . Furtherlon Fig. . TCGA da2)] Fig. ; gender 3.2P‐values 6.10e−10; Supplementary Fig. P‐value 1.10e−8). Differences between tumor and normal samples were measured across the tumor and demographic variables and p53 (wt vs. mutation) and MS status (MSI‐H and MSS). TRIP13 IHC scoring was higher for tumors, and no modifying effect was evident within the categories of each variable  showed that miR‐192, miR‐215, and miR‐543 interact with the 3′‐untranslated region (3′‐UTR) of TRIP13 mRNA and may regulate TRIP13 expression. HCT116p53‐wt,MSI cells were transfected with miR‐192 or with another miRNA, miR‐215 or miR‐543, to determine the predicted miRNA that specifically suppresses TRIP13 expression. Western blot analysis showed lower levels of TRIP13 after miR‐192 overexpression  as compared to matched normal colon tissues , carbohydrate antigen 19‐9 (CA19‐9), and tumor stage (pTNM) . FurtherWe initiated the current investigation by analyzing TCGA data on expression of TRIP13 and found that, as reported for other studies , higher KRAS, BRAF, and PTEN) are not available. However, well‐characterized CRC cells for p53 status  and MS status are available. Moreover, most CRCs exhibit p53 mutations (more than 50%) and MSS (about 85% of sporadic CRCs) [To evaluate the oncogenic potential of TRIP13 in CRCs, we used lentiviral TRIP13 shRNA transfected into CRC cells that exhibit different p53 and MS status. Although multiple genetic alterations contribute to the development of CRCs, CRC cell lines that demonstrate wt, mutated, or null status for key molecules  . In addip53‐wt,MSI and HT29p53‐mut,MSS CRC cells that metastasized to liver, lungs, kidney, and bone, showed the involvement of TRIP13 in distant metastasis of CRC. TRIP13 knockdown by shRNA decreased the capacity of CRC cells to spread to lungs, liver, kidney, and bone; the inhibition was independent of the p53 and MS status. In hepatocellular carcinoma (HCC) cells, TRIP13 knockdown increases levels of the epithelial marker, E‐cadherin, and decreases the mesenchymal markers, vimentin, and snail. Thus, in HCC cells, TRIP13 promotes metastasis via inducing the EMT [Among patients with CRC, distant metastasis is the primary cause of cancer‐related deaths . CRC fre the EMT . In the We showed, in CRC cells, that TRIP13 was negatively regulated by miR‐192 at the post‐transcriptional level by binding to the 3′‐UTR of TRIP13 mRNA. Additionally, we observed that miR‐192 overexpression in CRC cells lowered cell proliferation, colony formation, invasion, and spheroid‐forming capacity. However, in prostate cancer cells, miR‐515‐5p inhibits cell migration and invasion through targeting TRIP13 at its 3′‐UTR . MoreoveFurther mechanistic investigations unraveled that PNPT1 as an interacting partner and a regulator of TRIP13 protein and in silico data analyses of CRC tissues and protein expression profiles of CRC cells have indicated PNPT1 as an oncogene in CRC. PNPT1, a 3′,5′‐exoribonuclease, is an interferon inducible gene, induces the expression of cytokines such as IL‐6, IL‐8, RANTES, and MMP3 . A previRAS mutation status, as HCT116p53‐wt,MSI and SW480p53‐mut,MSS cells exhibited mutations in KRAS at the G13D and G12V codons, respectively, and both cell lines showed lower EGFR phosphorylation after TRIP13 knockdown. A prior study demonstrates that TRIP13 binds EGFR and activates its signaling pathway [Understanding tumor biology is relevant for improving therapeutic strategies. For cancers, EGFR overexpression and constitutive activation are related with a poor prognosis . In the  pathway . However pathway  and colo pathway . Thus, t pathway . Constit pathway . The pre pathway . Further pathway . Our resOur RNA sequencing studies with TRIP13 knockdown cells identified downstream molecular targets, including COL6A3 (collagen/extracellular matrix), TREM2 (inflammation), SHC3 (MVP/MEK/ERK activation), and KLK7 . Prior studies have implicated these TRIP13 targets in the progression of various cancers, including COL6A3 , 45 and Based on the crystal structure of TRIP13, a small molecule inhibitor, DCZ0415, was designed to target this protein . As esta5In conclusion, our work showed that expression of TRIP13 is regulated by miR‐192 and that its overexpression in CRCs is independent of tumor stage and patient race/ethnicity, age, and gender and p53 and MS status. Depletion of TRIP13 caused less cell proliferation, colony formation, cell motility, invasion, spheroid‐forming capacity, tumor growth, and distant metastasis regardless of p53 and MSI status. TRIP13 depletion also caused suppression of EGFR activation and WNT/β‐catenin signaling. Additionally, COL6A3, KLK7, TREM2, and SHC3 were identified as downstream targets of TRIP13. In sum, we conclude that TRIP13 promotes CRC metastasis and that this enzyme is a potential target for treatment of CRC.UM, SV, and SA made conceptualization; UM, SV, SA involved in methodology; SA, H‐GK, BVSKC, NG, PB, AE, SAD investigated the study; UM, MJH, SV, PKD provided the resources; MB and DSC made statistical and bioinformatics data analysis; SA, UM, SV prepared original draft; UM, SV, SA reviewed and edited the manuscript; UM supervised the study.The authors declare no conflict of interest.Fig. S1. TRIP13 mRNA expression in CRCs.Fig. S2. TRIP13 protein overexpression in CRCs is independent of pathologic stage, patient's race, gender and age.Fig. S3. Wound‐healing assay performed to assess migration of CRC cells.Fig. S4. TRIP13 knockdown lowers CRC metastasis.Fig. S5. TRIP13 knockdown decreases metastasis of HT29p53‐mut,MSS cells.Fig. S6. TRIP13 knockdown decreases metastasis of HCT116p53‐wt,MSI cells.Fig. S7. Heat‐map showing overexpression of TRIP13 downstream targets.Table S1. List of shRNA sequences used in this study.Table S2. List of qPCR Primer sequences used in this study.Table S3. List of antibodies used in this study.Click here for additional data file.


Streptomyces fildesensis strain INACH3013, a psychrotrophic bacterium isolated from Northwest Antarctic soil, was reported. The genome sequence totaling 9,306,785 bp resulted from 122 contigs characterized by a GC content of 70.55%.The draft genome sequence of  Streptomyces fildesensis strain INACH3013, a psychrotrophic bacterium isolated from Northwest Antarctic soil, was reported. The genome sequence totaling 9,306,785 bp resulted from 122 contigs characterized by a GC content of 70.55%.The draft genome sequence of  Streptomyces species are aerobic, spore-forming, filamentous Gram-positive bacteria with large and linear chromosomes characterized by a high GC content   with agitation (180 rpm) for 5 days at 12°C ± 1°C. Genomic DNA was isolated from a single colony using a DNeasy blood and tissue kit , according to the manufacturer’s procedure. The taxonomic affiliation of the strain was confirmed by partial sequencing of the 16S rRNA gene (NCBI accession number KJ624755). The total DNA was processed by using a TruSeq DNA PCR-free library preparation kit and HiSeq 2000 paired-end sequencing . The corresponding genome sequencing generated 9,445,886 short reads with an average length of 100 nucleotide bases. The sequence reads were filtered using Trimmomatic v0.39 (de novo genome assembly was performed using SPAdes 3.14.1 with default parameters (N50 value of 152,215 bp, with the largest contig of 681,566 bp. The S. fildesensis strain INACH3013 genome sequence had an estimated completion of 99.28% (itation 10 rpm forJAAIKO000000000. The version described in this paper is the first version, JAAIKO010000000. The SRA accession number is SRX7554417.This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number


Concordance between CSF biomarkers and amyloid-PET was higher in FTD patients compared to AD cases. None of the AD patients presented both negative Aβ biomarkers. Conclusions: CSF Aβ42/40 ratio significantly increased the diagnostic accuracy of CSF biomarkers. On the basis of our current and previous data, we suggest a flowchart to guide the use of biomarkers according to clinical suspicion: due to the high PPV of both amyloid-PET and CSF analysis including Aβ42/40, in cases of concordance between at least one biomarker and clinical diagnosis, performance of the other analysis could be avoided. A combination of both biomarkers should be performed to better characterize unclear cases. If the two amyloid biomarkers are both negative, an underlying AD pathology can most probably be excluded.Background: The aims of this study were to compare the diagnostic accuracy, sensitivity, specificity, and positive and negative predictive values  of different cerebrospinal fluid (CSF) amyloid biomarkers and amyloid-Positron Emission Tomography (PET) in patients with a clinical diagnosis of Alzheimer’s disease (AD) and Frontotemporal Dementia (FTD); to compare concordance between biomarkers; and to provide an indication of their use and interpretation. Methods: We included 148 patients (95 AD and 53 FTD), who underwent clinical evaluation, neuropsychological assessment, and at least one amyloid biomarker . Thirty-six patients underwent both analyses. One-hundred-thirteen patients underwent Apolipoprotein E (ApoE) genotyping. Results: Amyloid-PET presented higher diagnostic accuracy, sensitivity, and NPV than CSF Aβ Alzheimer’s disease (AD) is the most prevalent type of dementia and it has an increasing incidence worldwide . AD progUntil 2007, AD diagnosis was based on clinical symptoms and cognitive examination, while a defined diagnosis was possible only by postmortem pathological confirmation  whose pr1–42) [42/40) [11C]Pittsburgh compound-B (PIB), [18F]Florbetapir, [18F]Florbetaben, or [18F]Flutemetamol, which allows one to directly visualize fibrillary amyloid-β deposits in brain tissue Florbetaben (FBB)—Bayer-Piramal; [18]Flutemetamol (FMM)—General Electric). Images were rated as either positive or negative according to the criteria defined by the manufacturers.Fifty-eight patients (39 AD and 19 FTD) underwent amyloid-PET. Amyloid-PET imaging was performed according to standard national and international guidelines , with an1–42, t-tau, and p-tau were measured with ELISA kits , and INNOTEST HTAU AG.). Cut-offs for normal values were: Aβ1–42 > 600 pg/mL; t-tau < 300 pg/mL; and p-tau < 60 pg/mL [1–42, Aβ42/40 ratio, t-tau, and p-tau were measured using a chemiluminescent enzyme immunoassay (CLEIA) analyzer LUMIPULSE G600 ). Cut-offs for normal values were: for Aβ1–42 > 670 pg/mL; Aβ42/40 ratio > 0.062; t-tau < 400 pg/mL; and p-tau < 60 pg/mL [The CSF samples were collected by lumbar puncture, then immediately centrifuged and stored at −80 °C until performing the analysis. Two different methods of analysis were used to establish CSF measurements. In 59 patients (39 AD and 20 FTD), Aβ60 pg/mL . For 67 60 pg/mL . ReagentA standard automated method  was used to isolate DNA from peripheral blood samples. ApoE genotypes were investigated by high-resolution melting analysis (HRMA) . Two sethttp://bioinfo.ut.ee/primer3-0.4.0/primer3/) [All the coding exons and the intron/exon boundaries of the familial AD genes  and frontotemporal dementia genes (GRN and MAPT) were amplified by polymerase chain reaction (PCR) using primers designed with Primer3 software (rimer3/) . The anap < 0.05.Patient groups were characterized using means and standard deviations (SD). We tested for the normality distribution of the data using the Kolmogorov–Smirnov test. Depending on the distribution of our data, we used t-tests or non-parametric Mann–Whitney U tests for between-group comparisons and Pearson’s correlation coefficient or non-parametric Spearman’s ρ (rho) to evaluate correlations between groups’ numeric measures. We used a Chi-square test to compare categorical data and calculated the effect size by Cohen’s d for numeric measures and Cramer’s V for categorical data. All statistical analyses were performed with SPSS software v.25  and the computing environment R 4.0.3 . The significance level was set at Raw data that support the findings of this study are not presented due to ethical and patient data security reasons but will be shared upon request from any qualified investigator.2 = 7.077 p = 0.008) , age at CSF analysis, age at amyloid-PET, family history of dementia, sex, years of education, MMSE, and HDRS. Proportion of ApoE ε4+ was higher in the AD group compared to FTD (χ= 0.008) . In two 2 = 48.42 p < 0.001).Seventy-seven AD (81.05%) and 12 FTD (22.64%) patients were Aβ+. Aβ+ pathology proportion was higher in AD compared to FTD (χ1–42 (χ2 = 22.83 p < 0.001); 31 AD (75.60%) and 5 FTD (17.85%) had positive Aβ42/40 (χ2 = 22.24 p < 0.001). Fifty-one AD (64.55%) and 9 FTD (19.14%) were CSF Aβ+ (χ2 = 24.36 p < 0.001). Aβ1–42 and Aβ42/40 were both positive in 28 AD patients (70.00%) and in 4 FTD patients (14.81%).Concerning CSF biomarkers, 48 AD (60.75%) and 8 FTD (17.02%) had positive Aβ2 = 27.37 p < 0.001).Out of 58 patients who underwent amyloid-PET, 35 AD (89.74%) and 4 FTD subjects (21.05%) had positive scans for amyloid deposition (χ2 = 21.23 p < 0.001).Considering the group of patients who underwent both CFS biomarkers analysis and amyloid-PET, 23 AD (100%) and 4 FTD (30.76%) were Aβ+  . CSF Aβ p = 0.034)  .1–42 was available for 10 cases: from a qualitative point of view, CSF Aβ1–42 levels were lower than 700 pg/mL in 9 cases, only slightly higher than the cut-off values. CSF Aβ42/40 ratio was available for three cases: similarly, two of them had CSF Aβ42/40 ratio values less than 0.7, slightly higher than the cut-off values. Moreover, they underwent CSF analysis more than 3 months before amyloid-PET. On the other hand, the only CSF+/PET- case underwent CSF analysis 2 months before amyloid-PET.As regards the AD CSF-/PET+ patient subgroup, CSF Aβ1–42 and Aβ42/40 was 89.55% (95% C.I. 82.33–96.88) in the whole cohort, 92.25% (95% I.C. 84.34–100) in AD patients, and 85.19% (95% C.I. 71.75–98.59) in FTD patients . When we considered the subgroup of patients who underwent both CFS biomarkers analysis and amyloid-PET, the concordance was 88.89% (95% C.I. 78.62–99.16).p = 0.065), which was more frequent in Aβ+ FTD patients.In order to assess if the discordance between amyloid biomarkers and clinical diagnosis was influenced by demographic features, we divided patients into two groups: the first including Aβ+ AD and Aβ- FTD (concordant cases), the second including Aβ- AD and Aβ+ FTD (discordant cases). There were no statistically significant differences between the two groups with respect to demographic features, MMSE, and ApoE ε4 prevalence. We also found no differences when we considered AD and FTD patients separately for this analysis. Considering Aβ+ FTD patients, we found a trend to significance for the ApoE ε4 allele , but not than CSF Aβ42/40 ratio  and CSF Aβ    , but not the Aβ42/40 ratio . We did not detect any differences in specificity between amyloid-PET and CSF Aβ biomarkers  and CSF Aβ   and CSF Aβomarkers  (Table 25–66.21)   processing, Aβ production [1–42 measures soluble forms of Aβ, and a low concentration suggests that significant parenchymal deposition has occurred, whereas amyloid imaging directly identifies fibrillar Aβ in brain tissue [Another aim of our study was to compare the concordance between the amyloid-PET and CSF Aβ biomarkers in the group of patients who underwent both the analyses. Regarding AD patients, we found a very low concordance 39%), which was different from previous works showing higher levels of concordance: Leuzy et al. reported a concordance around 60% , Jung etoduction ,62,63, aoduction . Moreoveoduction ,66. Finaoduction . On the oduction . False-noduction , amyloidoduction . Moreove9%, whichn tissue .1–42 and of the Aβ42/40 ratio (when available), slightly higher than cut-off values. These data are in accordance with previous reports, which suggested that amyloid-PET scans could have clinical utility in addition to CSF examination, where results are borderline and diagnostic uncertainty remains [Despite previous works describing a higher prevalence of CSF+/PET- discordant cases compared to CSF-/PET+ ,57,58, o remains .Nevertheless, our results showed that none of the patients with a clinical diagnosis of AD presented both negative Aβ biomarkers, in accordance with previous works showing that no subjects diagnosed with AD during life had a non-AD pathological diagnosis .In our cohort, concordance between CSF Aβ biomarkers and amyloid-PET was higher in FTD patients than in AD patients, since the two biomarkers were both negative in 70% of cases. According to a multicenter European memory clinic study , the conInterestingly, we found a trend toward significance for the ApoE ε4 allele in FTD patients with Aβ positivity. It has been proposed that the presence of an ApoE ε4 allele could increase AD co-pathology across neurodegenerative diseases .In the FTD group in which both amyloid biomarker analyses were performed, all Aβ+ cases had a clinical diagnosis of sv-PPA. It is well known that PPA is a group of neurodegenerative diseases with heterogeneous neuropathologic causes. A recent work showed that Aβ positivity was detected in 25% of sv-PPA cases and in 40% of patients with mixed PPA with overlapping linguistic features for sv-PPA and lv-PPA (s/lv-PPA) . A recenOn the basis of our current and previous data , we woul42/40 ratio is positive, underlying Aβ pathology can be reasonably suspected and amyloid-PET might be avoided. Similarly, if amyloid-PET detects cortical amyloid deposition, CSF analysis cannot be performed. In conclusion, as also reported in a recent study [If CSF analysis including Aβnt study , the mat○If CSF shows Aβ-, amyloid-PET should be performed, also considering previous results highlighting an advantage of amyloid-PET when used as a second biomarker .○42/40 ratio is suggested.If amyloid-PET is negative for cortical deposition, CSF analysis including AβIn case of a mismatch between clinical diagnosis and one amyloid biomarker, we suggest performing the other analysis, due to the low NPV of both CSF and amyloid-PET. In particular:In conclusion, if both the analyses show Aβ-, an underlying amyloid pathology could most probably be excluded, and a revision of clinical diagnosis should be considered. On the contrary, if the second analysis detects Aβ+, and matching between clinical diagnosis and biomarkers is achieved, amyloid pathology can be reasonably suspected.In case of a clinical diagnosis of AD  Figure a:If CSFIf CSF analysis or amyloid-PET shows Aβ-, an underlying amyloid pathology could most probably be excluded. Thus, the performance of the other biomarker is not required, due to the high concordance between these two analyses in FTD cases.If CSF or amyloid PET detects Aβ+, some factors should be taken into account, in particular ApoE genotyping, age, and a deep neuropsychological evaluation of language to exclude a diagnosis of PPA. Moreover, the presence of comorbidity and co-pathology should be considered, as also suggested by other recent reports .In case of a clinical diagnosis of FTD  Figure b:If CSFSeveral studies have highlighted that biomarkers strongly improved the diagnostic accuracy of clinical diagnostic criteria, and it has been suggested that their combination seems to add accuracy to clinical evaluation . However42/40 ratio was not available in all cases. Moreover, the subgroup of patients who underwent both the analyses was relatively small, making it difficult to perform multivariate analysis to correct for possible confounding factors. Furthermore, CSF and amyloid-PET were not performed at the same time. Another limitation was that FTD patients who underwent both amyloid biomarkers had amyloid-PET performed before CSF analysis, so we cannot exclude that differences in the proportion of Aβ positivity between AD and FTD were influenced by the time between the two analyses. Moreover, APOE genotyping was not available for all patients.The main limitation of our study was the lack of autopsy confirmation of Aβ pathology. We only tested the associations between amyloid biomarkers and clinical diagnosis and the concordance between CSF Aβ biomarkers and amyloid-PET, and it is possible that some subjects were clinically misdiagnosed with AD. However, we detected GRN pathogenetic mutations in two cases with nfv-PPA, so we can diagnose these patients with PPA with defined pathology . SecondlHowever, our study presents some remarkable strengths. First of all, a relatively large number of patients of our cohort underwent at least one amyloid burden biomarker. Another strength of this study is that it was based on a clinical setting cohort; therefore, it provides realistic information on the use and interpretation of amyloid biomarkers. Third, all patients were evaluated by an extensive neuropsychological battery and their clinical diagnosis was well defined.42/40, in cases of concordance between at least one amyloid biomarker and clinical diagnosis, performance of the other analysis could be avoided; on the other hand, a combination of both amyloid-PET and CSF biomarkers should be performed to better characterize unclear cases of dementia. If the two biomarkers of amyloid burden are both negative, an underlying AD pathology could most probably be excluded.In conclusion, due to the high PPV of both amyloid-PET and CSF analysis including Aβ


Daily antenatal multiple micronutrient (MM) compared with iron folic acid (IFA) supplementation from early pregnancy improved birth outcomes and maternal micronutrient status in rural Bangladesh, but effects on newborn status are unknown.We examined cord blood micronutrient biomarkers in relation to antenatal MM and IFA supplementation and maternal gestational micronutrient status in rural Bangladeshi newborns.In a double-blinded, cluster-randomized trial of antenatal IFA or MM (with the same IFA content), we analyzed cord blood plasma from 333 singleton births, and corresponding maternal plasma at 32.5 ± 2.6 wk of gestation, for ferritin (iron stores), folate, cobalamin (vitamin B-12), retinol (vitamin A), 25-hydroxyvitamin D , α-tocopherol (vitamin E), zinc, thyroglobulin, and free thyroxine (iodine status). Intervention effects and associations were determined using linear regression, exploring maternal status as a mediator of intervention effects on cord biomarkers.P < 0.05); ferritin and retinol changed by +2.0%; 95% CI: −8.9, 14.3%; P = 0.72; and +3.5%; 95% CI: −0.4, 7.3%; P = 0.07, respectively. Ferritin, folate, cobalamin, zinc, and thyroglobulin averaged 1.57–6.75 times higher and retinol, α-tocopherol, and 25(OH)D 0.30–0.84 times lower in cord than maternal plasma, suggesting preferential maternal–fetal transfer of iron, folate, cobalamin, and zinc; limited transfer of fat-soluble vitamins; and high fetal iodine demand.The MM intervention increased cord ferritin , 25(OH)D , and zinc . Cord folate , cobalamin , 25(OH)D , α-tocopherol , zinc , thyroglobulin  and thyroxine  increased per 1-SD increment in maternal status D, and zinc, while maternal and newborn folate, vitamins B-12, D, and E, zinc, and iodine biomarkers were positively related. Despite limited effects of MM, better maternal micronutrient status was associated with improved micronutrient status of Bangladeshi newborns. This trial was registered at  Micronutrients play critical roles in human reproduction, supporting gametogenesis, fertilization, embryogenesis, and placental development, function, redox balance, and vascularization . The essIn a cluster-randomized trial in rural Bangladesh, we demonstrated that a daily antenatal multiple micronutrient (MM) supplement, containing 15 micronutrients in approximately 1-RDA amounts , reducedIn the Bangladesh trial, women in their early pregnancies  were anemic (20%), with deficiencies of iron (4.0%); folate (2.5%); vitamins A (6.7%), B-12 (35%), D (64% insufficiency), and E (58%); zinc (13%); and iodine (2.6%) , typically 10 wk of gestation, and at 32 wk of gestation, as described  of 1.20, based on prior experience. Participants in the cord blood collection substudy were similar to those who participated in the larger substudy, and comparisons of both the cord blood study . The intervention effect is presented both as the mean difference (95% CI) in biomarker concentrations in their units of measurement and as the percentage (95% CI) difference in the MM relative to the IFA group to compare the magnitude of impact across biomarkers. For retinol, α-tocopherol, and thyroxine the concentration difference is the β-coefficient (95% CI) associated with the intervention. For log10-transformed biomarkers, the concentration difference was calculated by back-transforming regression parameters and 95% CIs to the arithmetic scale. The percentage difference associated with MM for variables on the arithmetic scale was calculated as 100 × β1(95% CI)/β0, since β1 is the difference from IFA (β0) associated with MM. Finally, percentage difference for log10-transformed variables took advantage of the multiplicative nature of the logarithmic scale (β1(95% CI) could be utilized to calculate the fold difference in biomarkers associated with MM compared with IFA, then expressed as a percentage (95% CI). Finally, logistic regression was used to test the prevalence of newborn deficiency by intervention group when those outcomes are reported.Data on micronutrient status biomarkers are presented as mean ± SD or, to best capture the central tendencies and asymmetry of skewed distributions, geometric mean . Quantile normal plots, measures of skewness and kurtosis, and improved homoscedasticity of residual plots in regression analysis justified logic scale , whereby10-tranformed biomarkers, the fold change in biomarker per any 1-SD increment in maternal status is calculated by 10β1(95% CI), where β1 is the change in cord blood biomarker per increment in maternal biomarker, expressed as a percentage. For retinol, α-tocopherol, and thyroxine, for which associations are not on a multiplicative scale, the percentage increase associated with a change in maternal status was calculated from 0 (the mean) to +1-SD of the maternal biomarker distribution as 100 x β1(95% CI)/β0, with β1 the change in the cord blood per 1-SD gain in maternal biomarker and β0 the value for the cord blood indicator at the mean of the maternal distribution.Associations of cord with maternal micronutrient biomarkers at 32 wk of gestation are depicted with scatterplots and best-fit lines by intervention group. In exploratory analyses, associations between maternal and cord biomarkers, as outcomes, were tested with linear regression. Distributions of maternal biomarkers were standardized to mean±SD = 0 ± 1 to compute the percentage difference in cord blood per 1-SD increment in the maternal biomarker. For logFor biomarkers for which both intervention and maternal status effects were observed [25(OH)D and zinc], models that included both as determinants of newborn status were examined using the “medeff” command to ascertain extent to which the intervention effect was mediated through improved maternal micronutrient status .z-score at birth) were also examined. They did not change fundamental findings of maternal–newborn associations and are only shown as supplementary output when the additional variables were statistically significant. No adjustments were made to maternal biomarkers  based on AGP concentrations to account for inflammation because associations between AGP and all micronutrient biomarkers were lacking at 32 wk of gestation.Models including variables that could influence newborn micronutrient biomarkers , to serve as a proxy for the transfer of nutrients from mother to fetus in late gestation.P < 0.05 was considered statistically significant. Robust SEs to account for cluster randomization were calculated in all regression models.Analyses were conducted using Stata 13.0 (StataCorp), and Supplementary Table 1, demonstrating a benefit of the MM intervention on maternal retinol, 25(OH)D, α-tocopherol, and zinc, albeit with largely overlapping distributions, at 32 wk of gestation.Data on maternal characteristics at the onset of pregnancy and newborn status of the 333 participants are presented in P = 0.03), higher in the MM than the IFA newborns, and mean folate was 5 nmol/L, or 7.5%, lower, although not significantly (P = 0.07). Among other nutrients, only 25(OH)D  and zinc  were increased in cord blood by the MM intervention. Differences in vitamin B-12, retinol, vitamin E, and iodine status indicators by intervention group were all associated with P > 0.05, although point estimates for vitamins B-12 and E in particular were positive. AGP did not differ by intervention .Cord plasma biomarker concentrations are shown in P = 0.10 for difference by logistic regression), 67.9% and 66.2%, respectively, had retinol <0.70 µmol/L (P = 0.76), with 5.2% and 10.6% <0.35 µmol/L (P = 0.11); 82.8% and 75.5% had cord α-tocopherol < 7.5 µmol/L (P = 0.29). For 25(OH)D, 77.1% of newborn values in the IFA group and 64.2% in the MM group (P = 0.010) were <50 nmol/L, and 24.8% compared with 10.8% (P = 0.001) were <30 nmol/L, respectively, by logistic regression. Percentiles for each micronutrient biomarker are presented in Supplementary Table 2.Using published cutoffs, 4.6% of IFA and 1.8% of MM newborns had ferritin <34 µg/L  and those for free thyroxine were weakest (<2% difference). Maternal status explained ∼45% of the variability in cord blood 25(OH)D and ∼26% of the variability in folate and vitamin B-12 (as R2). There was an intermediary association of maternal status with α-tocopherol and thyroglobulin, both explaining >6% of the variability in newborn values, despite the ∼20% change in thyroglobulin but only an 8.7% change in α-tocopherol per 1-SD shift in maternal status. The association of maternal zinc with cord zinc concentrations was small yet significant (P = 0.015), with an ∼2% increase in cord blood zinc concentration per 1-SD shift in maternal status.Supplementary Table 3), both the MM intervention  and maternal zinc concentrations  remained independent determinants of cord blood zinc, with 11.3%  of the total effect on newborn status explained by mediation through maternal status. For 25(OH)D, only maternal status retained significance as a determinant of newborn 25(OH)D , with an estimated 62.4%  of the intervention effect mediated through improved maternal status.In mediation models (Supplementary Table 4) include a positive association of newborn retinol with size at birth, a positive association of AGP with retinol and α-tocopherol in cord blood, and a stronger effect of early than late pregnancy thyroxine on newborn thyroxine. Maternal and fetal AGP were not associated with each other .Additional findings , and zinc and iodine status. Adaptive mechanisms seemed to ensure a modicum of fetal nutriture among most poorly nourished mothers. Findings are further explored for each nutrient.An unexpected impact of the MM intervention was 12% higher cord ferritin despite the same iron content of the IFA and MM supplements. Along with modestly reduced late pregnancy ferritin observed in a larger group of MM mothers , this suMaternal and newborn folate were strongly associated, with cord folate double that of folate in mothers. Cord folate of Bangladeshi infants was similar to that observed in folic acid supplementation studies, which also showed that cord folate predominantly comprises the reduced, active metabolites rather than unmetabolized folic acid , 29, a dMaternal and cord blood cobalamin were also strongly associated, with approximately double the concentration in cord blood, as previously observed  and reviUnlike the B-vitamins, fat-soluble vitamins were lower in newborn than maternal circulation, as previously observed . Cord reSimilarly, cord α-tocopherol was only 30% of that in maternal circulation but was associated with maternal status, in agreement with known aspects of maternal–fetal vitamin E metabolism , 50. TraCord 25(OH)D was related to the MM intervention, strongly associated with maternal concentrations, and ∼80% of maternal values, which are responsive to supplementation , 55. SimThe MM intervention and maternal status were independently associated with newborn zinc status, with higher zinc in cord blood than maternal circulation. With progression of pregnancy, affinity of serum albumin for zinc declines , possiblNewborn thyroxine and thyroglobulin did not differ by intervention, and both were positively associated with maternal concentrations. Thyroxine from the mother, which is transferred in proportion to maternal production, is required for fetal brain development early in pregnancy . DespiteAlthough determining intervention effects was our primary aim, biomarker associations from exploratory analyses enhance understanding of micronutrient pathways in the maternal–newborn dyad; unmeasured biological mechanisms are likely at play where the contribution of intervention or maternal status to characterizing cord biomarkers is small. We were not able to measure biomarkers for all of the micronutrients in the MM supplement or all metabolites of interest, and there is a limited ability to characterize infant status with cord blood micronutrient biomarkers, including AGP. However, our findings provide a basis for interpreting newborn data from similar contexts in future studies. Finally, we cannot eliminate the possibility that participants represented a biased sample given that they were derived from a limited geographic location and time period within the larger randomized trial, although intervention groups were comparable across a variety of factors.Integration of findings from the JiVitA-3 trial shows that an antenatal MM supplement confers important public health benefits in rural Bangladesh, including reduced preterm birth and low birth weight through longer gestation  rather tnqaa223_Supplemental_FileClick here for additional data file.


Haematococcus pluvialis; adonixanthin is an intermediate product of astaxanthin. It is known that both astaxanthin and adonixanthin possess radical scavenging activity and can confer a protective effect on several damages. In this study, we clarified the antitumour effects of astaxanthin and adonixanthin using glioblastoma models. Specifically, astaxanthin and adonixanthin showed an ability to suppress cell proliferation and migration in three types of glioblastoma cells. Furthermore, these compounds were confirmed to transfer to the brain in a murine model. In the murine orthotopic glioblastoma model, glioblastoma progression was suppressed by the oral administration of astaxanthin and adonixanthin at 10 and 30 mg/kg, respectively, for 10 days. These results suggest that both astaxanthin and adonixanthin have potential as treatments for glioblastoma.Several antitumour drugs have been isolated from natural products and many clinical trials are underway to evaluate their potential. There have been numerous reports about the antitumour effects of astaxanthin against several tumours but no studies into its effects against glioblastoma. Astaxanthin is a red pigment found in crustaceans and fish and is also synthesized in  Glioblastoma is one of the most lethal types of brain tumour; it arises from glial cells ,2. The sIn this study, we focused on a xanthophyll carotenoid, astaxanthin, and its intermediate product, adonixanthin  9]. Ast. Ast9]. The purpose of this study was to clarify whether astaxanthin and adonixanthin have antitumour effects against glioblastoma following their oral administration. Furthermore, we aimed to verify whether orally administered astaxanthin or adonixanthin can be absorbed by the brain tissue. We investigated the antitumour mechanisms of astaxanthin and adonixanthin using glioblastoma cells.Robust cell viability is an important characteristic of tumour cells. We performed a cell viability assay using the murine glioblastoma cell line GL261 and the human glioblastoma cell line U251MG. Both astaxanthin and adonixanthin showed antitumour effects against GL261 and U251MG cells, in a concentration-dependent manner A,B. In GIn addition to cell viability, cell migration is also important for tumour enlargement. We performed a wound healing assay using the murine glioblastoma cell line GL261 and the human glioblastoma cell line U251MG. Astaxanthin and adonixanthin suppressed cell migration by 8.84 ± 2.09% and 13.74 ± 4.01% in GL261, respectively A,B. AstaWe performed immunoblotting to reveal the mechanisms of the antitumour effects of astaxanthin and adonixanthin. In general, the phosphorylation of ERK1/2 and Akt are accelerated during tumour progression in many types of tumour ,26. The In previous reports, the presence of ROS was shown to promote tumour progression via the phosphorylation of ERK1/2 and Akt ,28. Thertrans- or cis-astaxanthin in the serum was 6.04 ± 1.16 and 13.35 ± 3.78 ng/mL, respectively. Astaxanthin was not detected in the serum in pretreatment samples . Adonixanthin is an intermediate compound between zeaxanthin and astaxanthin [Both astaxanthin and adonixanthin obtained from axanthin ,43. ThesThe human glioblastoma cell line U251MG was purchased from European Collection of Authenticated Cell Cultures . The murine glioblastoma cell line GL261 was kindly provided by Dr. Saio, Graduate School of Health Sciences, Gunma University. The human glioblastoma cell line U87MG was obtained from American Type Culture Collection (ATCC). These cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with low glucose  supplemented with 10% foetal bovine serum , 100 units/mL penicillin, and 100 mg/mL streptomycin at 37 °C in 5% CO₂. Cells were passaged by trypsinization and used within 10 passages.3 cells/well with DMEM supplemented with 10% FBS and then incubated for 24 h, after which the culture medium was changed to DMEM containing 10% FBS. Then, astaxanthin, adonixanthin or temozolomide  were added to the culture. Cell proliferation was determined using the CCK-8 assay according to the manufacturer’s instructions . After each incubation, 10 μL of CCK-8 solution were added to each well. Plates were incubated for 3 h for 37 °C, and the absorbance was read at 450 nm with a reference wavelength of 630 nm using a Varioscan Flash 2.4 microplate reader .U251MG, GL261, or U87MG cells were seeded onto 96-well plates at a density of 2 × 103 cells/well with DMEM supplemented with 10% FBS and then incubated for 24 h, after which the culture medium was changed to DMEM containing 10% FBS. Then, astaxanthin, adonixanthin, or temozolomide  were added to the culture. After 72 h of culture, the culture medium was changed to DMEM containing 10% FBS and treated with BrdU at 10 µM for 3 h. After that, immunocytochemistry was performed according to the protocol of anti-BrdU antibody .GL261 cells were seeded onto 96-well plates at a density of 2 × 10The cell migration assay was conducted as previously described .4 cells per well) were plated in a 12-well plate  with culture medium supplemented with 10% FBS. After 24 h of incubation, the medium was changed to DMEM containing 1% FBS. After 6 h, wounds were scratched by a P1000 pipette tip and washed with phosphate-buffered saline (PBS) to eliminate cell debris. Then, fresh medium was added with 10 µM astaxanthin or adonixanthin. Pictures were taken at 48 h and these scratched areas were measured using an All-in-One Fluorescence Microscope . The cell migration rate was measured the area of the wound before migration (S0) and after migration (S1) and calculated S1/(S0−S1) × 100. The control group was then standardized to be 100%. Wound widths at 0 h were created within 1 to 1.5 mm and data were excluded if they were not suitable for that width.GL261 cells and U251 cells  with a protease inhibitor cocktail (Sigma-Aldrich), phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich), and sample buffer . The protein concentration was determined by comparison with a known concentration of bovine serum albumin using the BCA Protein Assay Kit (Thermo Fisher Scientific). The amount of total protein was 2 µg. Equal amounts of protein in sample buffer containing 10% 2-mercaptoethanol were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in 5–20% gradient gels , and the separated proteins were transferred to polyvinylidene difluoride membrane . After blocking for 30 min with Blocking One-P , we incubated the membranes with primary antibodies overnight at 4 °C. The primary antibodies were a rabbit anti-phospho-p44/42 MAPK; ERK1/2 (T202/Y204) 197G2 , a rabbit anti-p44/42 MAPK; ERK1/2 , a rabbit anti-phospho-Akt (S473) 193H12 , a rabbit anti-Akt , a rabbit anti-phospho-p38 MAPK (T180/Y182) , a rabbit anti-p38 MAPK , a rabbit anti-mmp-2 , a rabbit anti-mmp-9 , a rabbit anti-fibronectin , a rabbit anti-cyclin D1 , a mouse anti-p27 , a rabbit anti-Nox4 , a mouse anti-Bcl-2 , and a mouse anti-β-actin antibody .GL261 cell was seeded at 2.5 × 10After that, the membrane was incubated with the following secondary antibodies: a goat anti-rabbit IgG, or a goat anti-mouse IgG antibody . The band intensity was measured using an Immuno Star LD (Wako). Band intensity was measured using an LAS-4000 UV mini Luminescent Image Analyzer  and Multi Gauge Version 3.0 (Fujifilm). The phosphorylation of ERK1/2, Akt, and p38 MAPK was measured by normalizing against total ERK1/2, total Akt, and total p38 MAPK. Equal loading was confirmed using β-actin as controls for phosphoprotein signals.Fibronectin mRNA expression, we performed quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis. GL261 cell was seeded at 2.5 × 104 cells well in 24-well plates with culture medium supplemented with 10% FBS. After 24 h of incubation, the medium was changed to DMEM containing 10% FBS and 0.1% DMSO PBS, 300 μM temozolomide, 10 µM astaxanthin, or 10 µM adonixanthin was added for 48 h. After 48 h of treatment, RNA was isolated from GL261 cells using Nucleo Spin RNA II . RNA concentrations were determined using NanoVue Plus . Single-strand cDNAs were synthesized from the isolated RNAs via reverse transcription with a PrimeScript RT Reagent Kit . Quantitative real-time RT-PCR was performed using TB Green Premix Ex Taq II  and a TP800 Thermal Cycler Dice Real Time System (Takara). All procedures were carried out in accordance with the manufacturer’s instructions. The PCR primer sequences for Fibronectin were as follows: 5′-CGA GGT GAC AGA GAC CAC AA-3′ (forward) and 5′-CTG GAG TCA AGC CAG ACA CA -3′ (reverse). β-actin  was as follows: 5′-CAT CCG TAA AGA CCT CTA TGC CAA C-3′ (forward) and 5′-ATG GAG CCA CCG ATC CAC A-3′ (reverse). The cycling conditions were in accordance with the manufacturer’s protocol. The results are expressed as relative gene expression levels normalized to that of β-actin.To evaluate the effect of astaxanthin and adonixanthin on the expression of 3 cells/well with DMEM supplemented with 10% FBS and then incubated for 24 h, after which the culture medium was changed to DMEM containing 10% FBS. Then, astaxanthin, adonixanthin, or temozolomide  were added to the culture. Cell death was measured by Hoechst 33,342  and propidium iodide (Invitrogen). At 96 h after treatment, the Hoechst 33,342 and propidium iodide were added to the medium to final concentrations of 8.1 and 1.5 µM, respectively, for 15 min. Images of stained cells were captured with a Lionheart™ FX Automated Microscope . The percentage of propidium iodide-positive cells was determined by distinguishing Hoechst 33,342 and propidium iodide fluorescence.GL261 cells were seeded onto 96-well plates at a density of 2 × 102DCFDA; Thermo Fisher Scientific, MA, USA). Six hours after treatment of temozolomide, astaxanthin, or adonixanthin, CM-H2DCFDA was added to the culture medium and incubated at 37 °C for 1 h under shading in GL261. Fluorescence was measured using a Varioscan Flash 2.4 microplate reader  at 485 (excitation)-535 nm (emission). Measurements were performed 0, 30, and 60 min after the addition of CM-H2DCFDA.Intracellular radical activation within GL261 cells was measured with 5-(and-6)-chloromethyl—2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester  guidelines. These experiments were approved by the animal experiment committees of Gifu Pharmaceutical University, Japan . For all experiments, male C57BL/6J mice  and male ICR mice  purchased from Japan SLC, Inc.  were used. Animals were housed at 24 ± 2 °C under a 12-h light-dark cycle. Food and water were available to all animals ad libitum. All experimental procedures and outcome assessments were performed in a blinded manner.5 cells in 2 µL of PBS using a Hamilton microliter syringe at the following coordinates: 1 mm anterior, 2 mm lateral (left of middle) to bregma, at a depth of 3 mm from the dural surface. This protocol was completed using a stereotactic frame.Murine glioblastoma cell (GL261) transplantation was performed as previously described . BrieflyIn the experiment of the murine orthotopic glioblastoma model, oral administration of each astaxanthin (10 and 30 mg/kg) and adonixanthin (10 and 30 mg/kg) was initiated 3 days after intracranial injection of GL261 cells and was continued for 10 days. In the experiment of the brain tissue absorption with oral astaxanthin or adonixanthin, after one week of adaptation, ICR mice were randomly divided into the following three groups: control group, astaxanthin group (50 mg/kg), and adonixanthin group (50 mg/kg). Mice were orally administered each reagent suspended in olive oil (5 mL/kg) by the daily single dose for 10 days. The control group was treated by olive oil alone (5 mL/kg).Since 50 mg/kg is known to be a dose of astaxanthin that does not show adverse effects even with long-term administration, this dose of astaxanthin and adonixanthin was used in the distribution experiment of this study. Moreover, it was reported that the oral administration of astaxanthin at 25 mg/kg inhibited hippocampal inflammation in diabetic mice . Thus, wMice were euthanized and transcranial perfused with cold saline for 2 min at room temperature. After that, the perfusate was changed to 0.1 M phosphate buffer  containing 4% paraformaldehyde  for 3 min. Brains were fixed in 4% paraformaldehyde, embedded in paraffin , cut into 5-µm sections, and processed for haematoxylin-eosin (HE) staining. Pictures were taken using an All-in-One Fluorescence Microscope . We assessed the maximum cross-sectional area of the tumour and tumour volume as described previously .i.p). To separate serum and from blood, we centrifuged at 1700 g for 10 min. After mice were euthanized by exsanguination under deep anaesthesia, tissues, such as brain, were picked. Furthermore, the cerebral cortex, cerebellum, striatum, and hippocampus were separated from the whole brain.In mice, blood samples were collected under anaesthesia by using sodium pentobarbital  for the mobile phase at a flow rate of 1.0 mL/min as described above. This fraction was evaporated to dryness, dissolved in isopropanol: hexane , and subjected to chiral HPLC. Identification of each carotenoid was performed by authentic carotenoids obtained from Paracoccus  by our routine methods [Carotenoid fraction was collected from blood and some tissues by silica gel HPLC using a Cosmosil 5SL-II column with acetone:hexane . We performed the experiments assuming normality and selected an appropriate statistical analysis method depending on the presence or absence of equal variance. Specifically, student’s t-test or Welch’s test was used in the case of equal or non-equal variance under Bonferroni correction in


Radiotherapy, chemotherapy, and surgery have made crucial strides in glioblastoma treatment, yet they often fail; thus, new treatment and new detection methods are needed. Aberrant expression of Nanos3 has been functionally associated with various cancers. Here, we sought to identify the clinical significance and potential mechanisms of Nanos3 in human glioblastoma.Nanos3 expression was studied in nude mouse glioblastoma tissues and glioblastoma cell lines by immunohistochemistry, Western blot, and RT-PCR. Clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 gene editing assay was performed to generate the Nanos3 knockdown glioblastoma cell lines. The effects of Nanos3 on glioblastoma cells proliferation, migration, invasion, chemoresistance, germ cell characteristics, and tumor formation were analyzed by CCK8, transwell, cell survival experiments and alkaline phosphatase staining in vitro and in nude mouse models in vivo. Correlation between the expression of stemness proteins and the expression of Nanos3 was evaluated by Western blot.P < 0.05). Nanos3 knockdown enhanced the sensitivity of glioblastoma cells to doxorubicin (DOX) and temozolomide (TMZ) (P < 0.05), and Nanos3+/− glioblastoma cell lines did not show the characteristics of the germline cells. In addition, Nanos3 deletion inhibited subcutaneous xenograft tumor growth in vivo (P < 0.001). Moreover, the oncogenesis germline protein levels of CD133, Oct4, Ki67, and Dazl decreased significantly in glioblastoma cells following Nanos3 knockdown.We found that Nanos3 was strongly expressed in both glioblastoma cell lines and tissues. Western blot and sequencing assays showed that the Nanos3 knockdown glioblastoma cell lines were established successfully, and we discovered that Nanos3 deletion reduced the proliferation, migration, and invasion of glioblastoma cells in vitro (Both in vitro and in vivo assays suggest that Nanos3, which is a cancer-germline gene, initiates the tumorigenesis of glioblastoma via acquiring the oncogenesis germline traits. These data demonstrate that ectopic germline traits are necessary for glioblastoma growth. Gliomas, the most common primary brain tumor, are associated with a poor median survival time that barely exceeds 12 months despite the development of new effective treatments . The regDrosophila tumors expressing malignant brain tumor (mbt), and some germline genes upregulated in mbt tumors are orthologs of human CG genes such as NANOS1/Nanos [mbt tumors might be relevant to human cancer.Janic et al.  proposedS1/Nanos , 12. TheNanos genes encode a small family of evolutionarily conserved RNA-binding proteins that are required for germ cell development and embryonic patterning in diverse model organisms. The first Nanos family member described was a unique Nanos gene in Drosophila melanogaster, which was identified as a maternal effect gene required for abdomen formation [Nanos genes  have been studied and have been shown to have conserved functions in germline development [Drosophila, ectopic expression of Nanos drives the growth of malignant brain tumors, such as glioblastoma [Nanos has a dynamic role in cancer cell proliferation [The ormation . Nanos hormation –16. The ormation . In humaelopment , 17, 18.elopment . In Drosblastoma . Upregulferation . HoweverDeleted in azoospermia like (Dazl) served as CG gene [+/− cell lines and evaluated whether knockdown of Nanos3 could inhibit tumor growth, migration, invasion, and resistance. In addition, we explored the potential molecular mechanisms linking Nanos3 and the cancer-germline gene in human glioblastoma cells.Bone morphogenetic proteins (BMPs), which are embryonic proteins, are considered potent inhibitors of glioblastoma during development and clonogenicity , 22. It  CG gene  and stem CG gene –33, coul CG gene –33. An i2 in Dulbecco’s modified Eagle medium  containing 10% (v/v) fetal bovine serum, 4 mM glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin and 1% nonessential amino acids . Antibodies against Cas9 , Nanos3 , cyclin D1 , Gapdh  and α-tubulin  were purchased from Abcam . The Cell Counting Kit-8 (CCK-8) reagent (CK04) was purchased from DOJINDO Molecular Technologies, Inc. (Japan). An Alkaline Phosphatase Stain Kit (SK-5300) was purchased from Vector Laboratories, Inc. . Puromycin dihydrochloride (60210ES25) was purchased from Yeasen Biotech Co., Ltd. . Blasticidin S hydrochloride (15205), Doxorubicin hydrochloride (DOX) (D1515), DMH2 (SML 1535), and BMP4 (B2680) were purchased from Sigma Aldrich , and temozolomide (TMZ) was purchased from Merck .The GBM cell lines A172 and U251 were purchased from the Institute of Fudan IBS Cell Center , and the human glioblastoma LN229 cell line was kindly provided by Guoxiang Jin . Normal human astrocytes (NHA) were bought from the KeyGEN Biotech Company . All cells were cultured at 37 °C in 5% CO2 on glass coverslips pretreated with 0.1 mg/ml poly-l-lysine  to promote adherence. After 24 h, the slices were then washed twice with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature. To stain cytoplasmic markers, slices were permeabilized by incubation with 0.3% Triton-X-100 for 5 min at room temperature. Cells were blocked by incubation with 5% bovine serum albumin  for 1 h at room temperature and then incubated overnight at 4 °C with the primary antibodies. Controls were performed with 5% BSA as the primary antibody. After washing with PBS, the cells were incubated for 1 h at RT with an AlexaFluor 488 anti-rabbit IgG secondary antibody . Cell nuclei were stained with DAPI . Coverslips were examined with an inverted fluorescence microscope . All the antibodies used for immunofluorescence staining are shown above.A172, U251, and LN229 cells were used for immunofluorescence analyses. Cells were seeded at a density of 10,000 cells/cmTotal protein was lysed with RIPA lysis buffer  and phenylmethanesulfonyl fluoride  cocktails; then, they were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane . The membrane was blocked for 2 h at room temperature and then incubated with primary antibody overnight at 4 °C. The membrane was then incubated with the HRP-conjugated secondary antibody  for 1 h. The immunoconjugates were detected with an enhanced chemiluminescence substrate  using a chemiluminescence imaging system . Band density was analyzed with ImageJ software. The antibodies used for detecting protein expression are shown above.2O2 solution was applied to quench endogenous peroxidase activity. The slides were then blocked in 10% goat serum  for 1 h. The slides were incubated with the appropriate primary antibodies overnight at 4 °C. After incubation with the primary antibody, all slides were incubated with appropriate HRP secondary antibodies and stained with a DAB kit  and with hematoxylin solution . Finally, slides were dehydrated, cleared and mounted with a permanent mounting medium. Images were acquired under a microscope .Tissue samples were fixed in 10% formalin, paraffin-embedded and cut into 3 μm sections, which were mounted on superfrost plus microscope slides and dried at 60 °C for 1 h. Tissues were deparaffinized by incubating in xylene and rehydrated in an ethanol gradient with decreasing amounts of ethanol until the final wash, which was water. Sodium citrate-hydrochloric acid buffer  was used for antigen retrieval, and a 3% H−ΔΔCt method. The primers used for detecting gene expression were human Nanos3-F: CATTTATTGAGGGCTGACTGGAT; human Nanos3-R: CGGAACTCCTGTGCTTTGTCT; human GAPDH-F: TGCACCACCAACTGCTTAGC; human GAPDH-R: GGCATGGACTGTGGTCATGAG.Total RNA was extracted with Trizol  according to the manufacturer’s instructions, and the concentration of RNA was detected by a NanoDrop2000 spectrophotometer . Total RNA (500 ng) was reverse-transcribed using a PrimeScript™ RT reagent kit . Quantitative RT-PCR was performed using SYBR premix  according to the manufacturer’s instructions. qPCR experiments were run, and the melting curves of the amplified products were used to determine the specificity of amplification. The threshold cycle number for the genes analyzed was normalized to human GAPDH. All reactions were performed in triplicate, and the results were analyzed via the 23 cells per well, and then, they were incubated for 0, 1, 2, 3, 4, 5, 6 and 7 days. Then, 100 µl of reagent comprising 90 µl DMEM and 10 µl of CCK-8 solution was added to each well, and the plates were incubated for 2 h at 37 °C. The absorbance was measured at 450 nm with a microplate reader .GBM cells were seeded in 96-well plates at a density of 1 × 10For alkaline phosphatase (AP) staining, GBM WT cells and Nanos3 deletion cells were treated with a VECTASTAIN ABC-AP kit  according to the manufacturer’s instruction. After AP staining, 10 microscopic fields (20× magnification) of each treatment were randomly selected, and AP-positive colonies were counted.Nanos3 Knockdown in GBM cells was carried out following the protocol of Ran et al. [Nanos3 gene was cloned into a CRISPR/Cas 9 vector: hU6-gRNA-PGK-Puro-T2A-BFP. The lenti-cas9 pSpCas9 (BB)‐2A‐GFP (PX458) plasmid was a gift from Feng Zhang (Addgene plasmid #48138). Lenti-Cas9 was transfected into 70–80% confluent GBM cells using X-tremeGENE 9 DNA Transfection Reagent  and lenti-090 and lenti-091. Puromycin selection was performed for 3–5 days with a final concentration of 1 µg/ml, 3 µg/ml and 2 µg/ml in A172, U251, and LN229 cells, respectively; Lenti-Nanos3 sgRNA-treated cells were then seeded, and another 3–5 days of blasticidin selection was performed using a final concentration of 7 µg/ml, 22 µg/ml, 10 µg/ml in A172, U251, and LN229 cells, respectively. Later, a double selection of clones growing in blasticidin-containing medium and expressing Blue Fluorescent Protein (BFP) was performed. Positive clones were isolated and transferred to 6-well plates; finally, the positive clone was verified by sequencing. Nanos3 deletion was further verified by Western blotting of lysate from the stable cells.CRISPR/Cas9‐mediated n et al. . To gene5) stably expressing Nanos3+/− and GBM cells expressing Nanos3+/+ were subcutaneously injected into 4-week-old female BALB/c nude mice . Tumor growth was assessed by measuring tumor diameters with Vernier calipers once every 6 days. Tumor volumes were calculated according to the equation: tumor volume (mm3) = (length × width × width)/2. The survival of the remaining mice was assessed via Kaplan–Meier analysis. When the experiments were stopped, experimental animals were sacrificed by carbon dioxide inhalation, and tumors were removed for biochemical (frozen tissue) and histological (paraffin fixed tissue) analyses.All animal studies were performed according to the “Guide for the Care and Use of Laboratory Animals” of the National Institutes of Health. GBM cells . Differences between groups were considered statistically significant when To evaluate whether cancer-germline genes are upregulated in human glioblastoma, RT-PCR experiments, Western blot assays and immunohistochemistry staining were performed to detect the expression of germline-associated genes, namely, Nanos3, in human glioblastoma cell lines and glioblastoma tissues Fig. . The gli−/− could inhibit cell proliferation and could not grow in the culture conditions. All of the deletion cell lines were heterozygous (Nanos3+/−), but the expression of Nanos3 was still reduced at both the protein and RNA levels in these cells  [Nanos gene was primarily found to be crucial for anterior–posterior axis polarity, abdomen formation, and germ cell development [Mus musculus [Nanos genes are responsible for germline traits such as survival, which are also important for tumor cells [+/− GBM cell lines via CRISPR/Cas9 knockout technology and the Nanos3+/− GBM cell lines grew stably for generation. Then, we demonstrated that ectopic expression of the germline gene Nanos3 in human glioblastoma brain tumors and its association with tumorigenicity. Moreover, high-level ectopic expression of Nanos3 is an independent marker of overall survival and may therefore be essential for the progression of glioblastoma. Our results confirm this view and show the close similarity between tumor formation and tissue development. Interestingly, Nanos3 is expressed in PGCs at mouse E7.25, and loss of function results in a lack of germ cells and a failure of PGCs fails to migrate into the gonad [D. melanogaster, Nanos was found to be one of the crucial germline genes that were upregulated in a brain tumor model [Nanos was originally discovered and researched in uit fly) . The Nanelopment –41. The musculus . Since Omusculus  proposedor cells . There ior cells . CRISPR/or cells , 45 and or cells  as well or cells –49. In the gonad . Nanos phe gonad , 50. We he gonad . It is whe gonad , 52, andhe gonad , 16. In Nanos3 gene could represent ideal candidates, since it belongs to a unique group of CG genes that primarily contribute to some key process of tumor development, including unlimited proliferation, metastasis, adaption to cellular energetics constraints, and resistance to apoptosis. The observation that Nanos3 gene exerts oncogenic functions may explain the origin of the tumor cells. Further researches on these CG genes may therefore open the way to the development of highly selective anti-cancer therapies.Glioblastoma, the most common neurological tumor, could be inhibited by BMP treatment. Differentiation-inducing properties of BMPs could inhibit the tumorigenicity of GBM cells, making BMPs promising candidates for GBM therapy , 53–55. In summary, our results strongly suggest that Nanos3 may play a crucial role in glioblastoma development and progression. Nanos3, which is a cancer-germline gene, influences glioblastoma progression by enhancing cell proliferation, migration, and invasion. This implies that human cancer-germline genes are suspected to contribute to oncogenesis germline traits such as immortality, migration, invasiveness, and survival, suggesting that Nanos3 may function as an important marker of cancer in the germline and the detection of glioblastoma.Additional file 1: Figure S1. Expression of Nanos3 in glioblastoma cell lines. Figure S2. Colony formation of GBM cells after chemotherapy. Figure S3. The relationship between Nanos3 and BMP pathway. Figure S4. The regulatory relationship between Nanos3 and Dazl. Figure S5. Effect of Nanos3 on glioblastoma cell cycle.


As such, 14 were included. These patients were matched with 14 aseptic and 14 septic THAs with ceramic on a PE bearing, which constituted the control group. The mean serum chrome level was 20.0 ± 15.5 nmol/L and cobalt level 18.4 ± 22.1 nmol/L. The synovial WBC and PMN% varied significantly between MoM bearing group and the aseptic THA ceramic PE group (both p < 0.001), as well as the septic THA group . Furthermore, the septic THA group had significantly higher CRP values than the aseptic MoM group (p = 0.016). Conclusion: MoM bearing shows significantly higher synovial WBC and PMN% when compared to aseptic THA with ceramic on PE bearing above the MSIS cut-off. This is an important consideration when diagnosing periprosthetic joint infection using the MSIS guidelines. Introduction: The effect of different bearings on synovial white blood cell (WBC) count and polymorphonuclear percentage (PMN%) in aspirations remains unclear. Therefore, this study investigates the impact of aseptic Metal-on-Metal (MoM) bearing on synovial fluid. Methods: We searched our arthroplasty registry for aseptic painful THAs with MoM bearings between 2011 and 2018. Then, a case-matched control group was selected with septic and aseptic Total Hip Arthroplasty (THA) with ceramic on a polyethylene (PE) bearing. The matching criteria consisted of gender, age +/−10 years, and time of aspiration (+/−2years). Periprosthetic Joint Infection (PJI) was defined according to the Infectious Diseases Society of America (IDSA), and Musculoskeletal Infection Society (MSIS) using bacterial cultures, sonication and histology. Results: In total, 19 patients who underwent hip aspiration with MoM bearing were identified. Five patients had to be excluded due to insufficient synovial fluid obtained ( In total hip arthroplasty (THA), different bearings are being used, including metal-on-metal (MoM) and ceramic on polyethylene (PE) bearings. In 1988, Weber et al. reintroduced the second generation of MoM bearings to minimize the PE wear complications, which resulted in periprosthetic osteolysis ,3,4,5,6.To exclude other causes of loosening, such as periprosthetic infection (PJI), most guidelines recommend plain films to assess for osteolysis and metal wear, in addition to serum-based infection parameters and joint aspirates, before proceeding with surgery . TherefoHowever, no study to date has investigated the influence of different bearings—metal on metal versus polyethylene on ceramic—on synovial inflammatory markers. This study aims to assess the impact of bearing choice in THAs on synovial and serum inflammatory markers, in both aseptic and septic states.A retrospective chart and radiographic review were performed between November 2015 and 2019. All consecutive patients with aseptic, painful metal-on-metal bearing THAs who underwent hip aspiration and revision surgery due to aseptic loosening were included. The inclusion criteria consisted of patients who are 18 years old or older, serum inflammatory markers including synovial fluid differentiation, synovial blood count (WBC), polymorphonuclear distribution in percentage (PMN%), as well as erythrocyte count and postoperative histology, microbiology cultures and sonication results. Histology was classified according to Morawietz L et al. . For micAll cases were matched to patients with primary aseptic THAs as well as septic total hip arthroplasties. Matching criteria included gender, age plus or minus 10 years, year of surgery plus or minus 2 years. In both aseptic and septic cases, the control group used polyethylene inlays with ceramic femoral heads. Likewise, for the metal-on-metal group, infection was diagnosed according to the MSIS 18 and IDSA criteria as illustrated in t-test, and post hoc least significant difference test (LSD) for descriptive statistics were applied to calculate the aspirate differentiation, cell count, PMN% and monomorphic nuclear cells (percentage) for categorical variables. The mean and standard error of the mean (SE) were used for normally distributed continuous variables. For statistical analysis, Origin Pro 8.0, Microsoft Excel and IBM SPSS Statistics 25 were used. A one-way n = 2/19) were excluded because not enough synovial fluid (less than 2 mL) was obtained. Further, three patients showed bacterial growth after sonication that was initially negative with the standard microbiological cultures. This left 14 patients for inclusion, which were matched with 14 aseptic and 14 septic THAs with ceramic on PE bearing, as previously described. Gender was equally distributed, with seven patients in each . The mean age at time of aspiration was 64.6 ± 12.2 years old for the MoM group, 66.0 ± 10.8 years old for the aseptic group, and 67.5 ± 21 years old for the septic THAs (p = 0.752). A total of 19 patients with metal-on-metal bearing were identified during the studied time period. Two patients , and in three cases, a periprosthetic membrane of indeterminate type formed by connective tissue  was observed. In three patients, histology revealed low-grade infection  but no specific organism was isolated in microbiological cultures. Therefore, after interdisciplinary discussion with the infectious disease specialist, we decided to treat these patients as aseptic according to the MSIS and IDSA criteria. In the MoM group, the mean WBC and CRP in serum were 9.1 ± 2.6 G/L (reference range 3.9–10.2 G/nL) and 8.8 ± 14.2 mg/dL (reference range <5 mg/dL), respectively. The serum chrome level was 20.0 ± 15.5 nmol/L (reference range 1–8 nmol/L). The cobalt level was 18.4 ± 22.1 nmol/L (reference range <12 mmol/L). In the aspirate, the synovial WBC was 3198 ± 2452 lc/µL, including 857 ± 787 lc/µL polymorphonuclear cells. PMN% was reported to be 31.8 ± 20.9%, eosinophil cells at 24 ± 35 lc/µL, and erythrocytes in 126 ± 228 lc/µL. For histology, in nine patients, foreign body particles including macrophages and multinucleated giant cells were detected . The eosinophil cell count was 21 ± 29 lc/µL with 311 ± 302 lc/µL erythrocytes. In all cases, histology as well as microbiology revealed no pathological findings.staphylococcus epidermidis was identified (n = 4/10), followed by one case each of enterococcus faecium, streptococcus sanguinis, staphylococcus aureus, bacillus cereus, pseudomonas acnes, and paenibacillus species (n = 6/10). Additionally, ten cases had a histology that revealed either low-grade  or high-grade infection . All findings are listed in The infected THA group showed higher serum WBC and CRP at 6.7 ± 1.8 G/L and 38.3 ± 38.9 mg/dL, respectively. In joint aspirate, the synovial WBC was 14,946 ± 16,380 lc/µL with 13,361 ± 15,339 lc/µL of polymorphonuclear cells. The PMN% was reported at 77.6 ± 23.0%. The eosinophil and erythrocyte count was slightly higher compared to the other groups at 406 ± 781 and 421 ± 676 lc/µL, respectively. According to MSIS as well as IDSA criteria, all patients met the criteria for PJI. In 10 of 14 cases, microbiology cultures revealed bacterial growth. In most cases, p = 0.048). However, no statistical significances were found for the serum WBC. For the aspirate, synovial WBC varied significantly between the individual groups. It was lowest for the aseptic group, followed by the metal-on-metal group . For the infected THAs, multiple comparisons were significant  with a post hoc Least Significant Difference (LSD) p value of 0.001. Similar findings were observed for the polymorphonuclear cells  at a post hoc LSD p value of <0.001. Also, PMN% was significantly higher for the septic group, followed by the aseptic hip and MoM groups . No significant differences were found between aseptic and metal on metal bearing THAs (p = 0.48).Between the different groups, a couple of significant findings were identified. Serum CRP was significantly higher for the septic compared to the MoM group (p = 0.001) as well as polymorphonuclear cell distribution (p ≤ 0.001). The mean WBC in aseptic MoM according to the MSIS and IDSA criteria was 3198 ± 2452 lc/µL, which exceeds even the threshold recommended by the MSIS [Our study shows that the bearing of total hip arthroplasties has a significant impact on the synovial white blood cell count . Significant differences were found for synovial white blood cells and PMN% for MOMs at 3198 ± 2452 lc/µL and 31.8 ± 20.9%. Aseptic THAs is reported at 711 ± 391 lc/µL and 45.0 ± 21.0%, respectively. Finally, septic group is reported at 14,946 ± 16,380 lc/µL and 77.6 ± 23.0% (p < 0.001), respectively.Within the 14 patients included in our study, mean chrome level (20.0 ± 15.5 nmol/L) and cobalt level (18.4 ± 22.1 nmol/L) was elevated. No correlation between serum leucocytes was found. However, CRP correlated most strongly with the septic group, followed by aseptic THAs and MoM group . It has to be mentioned that the mean synovial WBC in the MoM group outranged the MSIS threshold. All microbiology cultures and sonication remained sterile. In three patients, histology showed an intermediate type of corrosion with suspicion of a low-grade infection—type three, according to Morawietz L et al. These cases were not considered to be PJI and were treated as contamination .This study is limited by its retrospective design, describing only a small cohort of 14 patients in each group. The mean cobalt and chrome levels were elevated and well above the serum reference, in part because of metal particles from wear. This can lead to a culture negative infection which typically ranges between 0% and 42.1% . TherefoSynovial white blood cell count in metal on metal bearing is critical in order to agree on how to interpret findings in order to decide on the subsequent treatment. This study shows that the bearing has a significant impact on the synovial WBC count above the MSIS cut-off. The diagnosis of periprosthetic joint infection requires multiple measures like serum-based or synovial-based inflammatory markers. Furthermore, sonication, microbiological cultures and histology after THA removal also play a role. Therefore, other diagnostic tools should be considered to exclude infection.