Cytochrome P450 26A1 regulates the clusters and killing activity of NK cells during the peri-implantation period.

Cytochrome P450 26A1 (CYP26A1) plays a vital role in early pregnancy in mice. Our previous studies have found that CYP26A1 affects embryo implantation by modulating natural killer (NK) cells, and that there is a novel population of CYP26A1+ NK cells in the uteri of pregnant mice. The aim of this study was to investigate the effects of CYP26A1 on the subsets and killing activity of NK cells. Through single-cell RNA sequencing (scRNA-seq), we identified four NK cell subsets in the uterus, namely, conventional NK (cNK), tissue-resident NK (trNK) 1 and 2, and proliferating trNK (trNKp). The two most variable subpopulations after uterine knockdown of CYP26A1 were trNKp and trNK2 cells. CYP26A1 knockdown significantly downregulated the expression of the NK cell function-related genes Cd44, Cd160, Vegfc, and Slamf6 in trNK2 cells, and Klra17 and Ogn in trNKp cells. Both RNA-seq and cytotoxicity assays confirmed that CYP26A1+ NK cells had low cytotoxicity. These results indicate that CYP26A1 may affect the immune microenvironment at the maternal-foetal interface by regulating the activity of NK cells.

Cytochrome P450 26A1 Modulates the Polarization of Uterine Macrophages During the Peri-Implantation Period.

Uterine M1/M2 macrophages activation states undergo dynamic changes throughout pregnancy, and inappropriate macrophages polarization can cause adverse pregnancy outcomes, especially during the peri-implantation period. Our previous studies have confirmed that Cytochrome P450 26A1 (CYP26A1) can affect embryo implantation by regulating uterine NK cells and DCs. The aim of this study was to investigate whether CYP26A1 regulates the polarization of uterine macrophages in early pregnancy. Here, we observed that Cyp26a1 was significantly upregulated in M1 as compared with M2 of uterine macrophages, Raw264.7 and iBMDM. Knockdown of CYP26A1 in mice uterine significantly decreased the number of embryo implantation sites and the proportion of CD45+F4/80+CD206 - M1-like uterine macrophages. Primary uterine macrophages treated with anti-CYP26A1 antibody expressed significantly lower levels of M1 markers Nos2, Il1b, Il6 and Tnf-a. In CYP26A1 knockout Raw264.7 cells, the protein levels of M1 markers TNF-α, IL-6 and CD86 were significantly decreased as compared with the wild type cells. Moreover, CYP26A1 deficiency decreased the ability to produce nitric oxide and increased the phagocytosis capacity of Raw264.7 cells under M1 stimulation state. The re-introduction of CYP26A1 partially reversed the polarization levels of M1 in CYP26A1 knockout Raw264.7 cells. CYP26A1 may regulate the polarization of uterine macrophages to M1 through Stap1 and Slc7a2. In summary, these results indicate that CYP26A1 plays a significant role in macrophage polarization, and knockdown of CYP26A1 can cause insufficient M1 polarization during the peri-implantation period, which has adverse effects on blastocyst implantation.

A novel Cytochrome P450 26A1 expressing NK cell subset at the mouse maternal-foetal interface.

Cyp26a1 had important roles in mouse embryo implantation and was highly expressed in some of NK cells at the human maternal-foetal interface in early pregnancy. However, the regulatory effect of Cyp26a1 on NK cells remains poorly understood. Through qPCR and flow cytometric assays, we found that Cyp26a1 was expressed by mouse uterine NK cells but not spleen NK cells during the peri-implantation period and there was a group of NK cells that highly expressed Cyp26a1, that is Cyp26a1+ NK cell subset. single cell-population transcriptome sequencing on Cyp26a1+ NK and Cyp26a1- NK cell subsets was performed. We found that there were 3957 differentially expressed genes in the Cyp26a1+ NK cell subset with a cut-off of fold change ≥2 and FDR < 0.01, 2509 genes were up-regulated and 1448 genes were down-regulated in Cyp26a1+ NK cell subset. Moreover, cytokine-cytokine receptor interaction signalling pathway and natural killer cell-mediated cytotoxicity signalling pathway were enriched according to KEGG pathway enrichment analysis. We further found that the expression of Gzma and Klrg1 was significantly increased and Fcgr4 was significantly decreased when inhibiting Cyp26a1. Our experimental results show that there is a novel NK cell subset of Cyp26a1+ NK cells in mouse uterus and Cyp26a1 can regulate the gene expression of Gzma, Klrg1 and Fcgr4 in the Cyp26a1+ NK cells.

Traditional Chinese Medicine Treatment as Adjuvant Therapy in Completely Resected Stage IB-IIIA Non-Small-Cell Lung Cancer: Study Protocol for a Multicenter, Double-Blind, Randomized, Placebo-Controlled Trial.

Adjuvant chemotherapy (AC) has been proven to yield an approximately 5% improvement in 5-year survival for patients with early-stage non-small-cell lung cancer. With such small gains in survival, the optimal treatment regimen remains to be established. Traditional Chinese medicine (TCM) treatment in combination with AC is frequently used in China. The efficacy and safety of this integrated approach should be scientifically evaluated. We present the rationale and study design of the Combined Adjuvant Chemotherapy and Traditional Chinese Medicine (ACTCM) trial (ChiCTR-IPR-16009062). The ACTCM trial, a prospective multicenter double-blind randomized placebo-controlled study, will recruit 312 patients overall from 5 clinical research centers in China. Within 6 weeks of the thoracic surgery, eligible participants with stages IB-IIIA non-small-cell lung cancer will be randomly assigned in a 1:1 ratio to either the treatment or control group. Patients in the treatment group will receive AC combined with TCM herbal treatment for 4 cycles, then TCM herbal plus injection treatment for 4 cycles. Patients in the control group will receive AC combined with TCM placebo for 4 cycles and then TCM placebo for 4 cycles. Treatment will be discontinued if disease progression or unacceptable toxicity occurs. The primary end point is 2-year disease-free survival. Secondary end points include disease-free survival and quality of life. Other end points are TCM symptoms, performance status, and safety of the regimens. Recruitment started in October 2016 and is ongoing.

Cytochrome P450 26A1 modulates uterine dendritic cells in mice early pregnancy.

Cytochrome P450 26A1 (CYP26A1) plays important roles in the mice peri-implantation period. Inhibiting its expression or function leads to pregnancy failure. However, little is known about the underlying mechanisms involved, especially the relationship between CYP26A1 and immune cells. In this study, using Cyp26a1-specific antisense morpholigos (Cyp26a1-MO) knockdown mice model and pCR3.1-Cyp26a1 vaccine mice model, we found that the number of uterine CD45+ CD11c+ MHCIIlo-hi F4/80- dendritic cells (DCs) was significantly decreased in the treated mice. The percentage of mature DCs (CD86hi ) was obviously lower and the percentage of immature DCs (CD86lo ) was remarkably higher in uterine DCs in the treatment group than that of the control group. Further experiments found that ID2, a transcription factor associated with DCs development, and CD86, a DC mature marker molecule, were both significantly reduced in mice uteri in the treated group. In vitro, ID2 and CD86 also decreased in bone marrow-derived DCs under Cyp26a1-MO treatment. These findings provide novel information that CYP26A1 might affect the embryo implantation via modulating the differentiation and maturation of uterine DCs.

Upregulated lncRNA-UCA1 contributes to progression of lung cancer and is closely related to clinical diagnosis as a predictive biomarker in plasma.

OBJECTIVE: Long non-coding RNAs (lncRNAs) have been shown to play an important regulatory roles in cancer biology, and the lncRNA-UCA1 is upregulated in several cancers such as bladder cancer, breast cancer and colorectal cancer, however, the contributions of UCA1 to non-small cell lung cancer (NSCLC) remain largely unknown. METHODS: Expression levels of lncRNA-UCA1 in tumor tissues and plasma from NSCLC patients was evaluated by quantitative real-time PCR, and its association with overall survival of patients was analyzed by statistical analysis. Moreover, the UCA1 expression correlation between tumor tissues and plasma was demonstrated by linear regression analysis. RESULTS: the results showed that the expression of UCA1 in NSCLC tissues was obviously higher than that observed in pair-matched adjacent nontumourous tissues, (P < 0.001). The agarose gel electrophoretogram of RT-PCR products further confirmed that UCA1 was increased in NSCLC tissues. To assess the correlation of UCA1 expression with Clinicopathological data, we found that the expression level of UCA1 was associate with histological grade (P < 0.001) and lymph node metastasis (P < 0.001). Intriguingly, the expression of UCA1 was significantly increased in plasma from NSCLC patients. The UCA1 expression measurements obtained from plasma and tumor tissues were strongly correlated in 60 patient samples (r = 0.881). By receiver operating characteristic curve (ROC) analysis, plasma UCA1 provided the highly diagnostic performance for detection of NSCLC (the area under the ROC curve (AUC), 0.886; P < 0.001). In conclusion, the current results indicated that Plasma UCA1 could serve as a potential biomarker for diagnosis of NSCLC. UCA1 as a biomarker in clinical application might significantly improve the efficacy of human NSCLC screening.

Integrative proteome analysis of Brachypodium distachyon roots and leaves reveals a synergetic responsive network under H2O2 stress.

The plant oxidative stress response is vital for defense against various abiotic and biotic stresses. In this study, ultrastructural changes and the proteomic response to H2O2 stress in roots and leaves of the model plant Brachypodium distachyon were studied. Transmission electron microscopy (TEM) showed that the ultrastructural damage in roots was more serious than in leaves. Particularly, the ultrastructures of organelles and the nucleus in root tip cells were damaged, leading to the inhibition of normal biological activities of roots, which then spread throughout the plant. Based on two-dimensional electrophoresis (2-DE) and MALDI-TOF/TOF-MS, 84 and 53 differentially accumulated protein (DAP) spots representing 75 and 45 unique proteins responsive to H2O2 stress in roots and leaves, respectively, were identified. These protein species were mainly involved in signal transduction, energy metabolism, redox homeostasis/stress defense, protein folding/degradation, and cell wall/cell structure. Interestingly, two 14-3-3 proteins (GF14-B and GF14-D) were identified as DAPs in both roots and leaves. Protein-protein interaction (PPI) analysis revealed a synergetic H2O2-responsive network.

XRCC1 genetic polymorphisms and sensitivity to platinum-based drugs in non-small cell lung cancer: an update meta-analysis based on 4708 subjects.

OBJECTIVE: To evaluate the correlation between genetic polymorphisms in x-ray repair cross complementing group 1 (XRCC1) and sensitivity to platinum-based chemotherapy drugs in patients with non-small cell lung cancer. METHODS: Reports published before June 2014 were retrieved from the following databases: China Biology Medicine (CBM), China Academic Journal Full-Text Database (CNKI), China Science and Technology Journal Full-Text Database (VIP), Wanfang Data, PubMed and Excerpta Medica dataBASE (EMBASE). After extracting the data and evaluating the quality, meta-analysis was performed using RevMan5.2 software. RESULTS: A total of 29 studies with 4807 patients were included. Two polymorphisms (Arg399Gln and Arg194Trp) were analyzed. Meta-analysis showed that the efficacy of chemotherapy for patients with the TT genotype [TT vs. CC, OR=1.66, 95% OR=1.66, 95 CI (1.30-2.14)] and the CT genotype [CT vs. CC, OR=1.62, 95% CI (1.35-1.93)] at codon 194 of the XRCC1 gene was significantly higher than that for patients with the CC genotype. The efficacy of chemotherapy for patients with mutant (CT+TT) genotypes was significantly higher than for patients with the wild-type (CC) genotype [TT+CT vs. CC, OR=1.63; 95% CI (1.38-1.92)]. The sensitivity to chemotherapy in patients with the AG genotype at codon 399 of the XRCC1 gene was lower than in patients with the GG genotype [AG vs. GG, OR=0.72, 95% CI (0.55-0.92)] in Chinese population. However, we did not found this association in Caucasus population. CONCLUSION: Genetic polymorphisms in the XRCC1 gene are correlated with sensitivity to platinum-based chemotherapy in patients with non-small cell lung cancer.

Large-scale phosphoproteome analysis in seedling leaves of Brachypodium distachyon L.

BACKGROUND: Protein phosphorylation is one of the most important post-translational modifications involved in the regulation of plant growth and development as well as diverse stress response. As a member of the Poaceae, Brachypodium distachyon L. is a new model plant for wheat and barley as well as several potential biofuel grasses such as switchgrass. Vegetative growth is vital for biomass accumulation of plants, but knowledge regarding the role of protein phosphorylation modification during vegetative growth, especially in biofuel plants, is far from comprehensive. RESULTS: In this study, we carried out the first large-scale phosphoproteome analysis of seedling leaves in Brachypodium accession Bd21 using TiO2 microcolumns combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MaxQuant software. A total of 1470 phosphorylation sites in 950 phosphoproteins were identified, and these phosphoproteins were implicated in various molecular functions and basic cellular processes by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Among the 950 phosphoproteins identified, 127 contained 3 to 8 phosphorylation sites. Conservation analysis showed that 93.4% of the 950 phosphoproteins had phosphorylation orthologs in other plant species. Motif-X analysis of the phosphorylation sites identified 13 significantly enriched phosphorylation motifs, of which 3 were novel phosphorylation motifs. Meanwhile, there were 91 phosphoproteins with both multiple phosphorylation sites and multiple phosphorylation motifs. In addition, we identified 58 phosphorylated transcription factors across 21 families and found out 6 significantly over-represented transcription factor families (C3H, Trihelix, CAMTA, TALE, MYB_related and CPP). Eighty-four protein kinases (PKs), 8 protein phosphatases (PPs) and 6 CESAs were recognized as phosphoproteins. CONCLUSIONS: Through a large-scale bioinformatics analysis of the phosphorylation data in seedling leaves, a complicated PKs- and PPs- centered network related to rapid vegetative growth was deciphered in B. distachyon. We revealed a MAPK cascade network that might play the crucial roles during the phosphorylation signal transduction in leaf growth and development. The phosphoproteins and phosphosites identified from our study expanded our knowledge of protein phosphorylation modification in plants, especially in monocots.

Lack of association between XRCC3 rs861539 (C > T) polymorphism and lung cancer risks: an update meta-analysis.

X-ray repair cross-complementing protein 3 (XRCC3) belongs to DNA double-strand break repair pathway and XRCC3 rs861539 (C > T) polymorphism has been suspected with lung cancer risk. However, results from previous studies are inconclusive and affected by bias. Electronic databases of PubMed, EMBASE, China National Knowledge Infrastructure, and SinoMed were searched. References of relative reviews were also screened. Pooled odds ratios (ORs) and 95 % confidence intervals (CIs) were calculated to estimate the association strength. A number of 18 eligible studies with 6 studies of Asians, 11 of Caucasians, and 1 of African were extracted and analyzed, including 4,896 lung cancer cases and 6,360 controls. No significant correlation between XRCC3 polymorphism and lung cancer risk was observed in homozygote comparison (CC vs. TT; OR=0.877; 95 % CI, 0.659, 1.168), heterozygote comparison (CT vs. TT; OR=0.857; 95 % CI, 0.675, 1.089), dominant model (CC/CT vs. TT; OR=0.862; 95 % CI, 0.663, 1.123), or recessive model (CC vs. CT/TT; OR=1.047; 95 % CI, 0.956, 1.145). Subgroup analyses of ethnicity and controls did not reveal any significant association with lung cancer risk. No publication bias was detected. In this update meta-analysis of 18 studies and 11,256 participants, we find that XRCC3 rs861539 polymorphism does not contribute to lung cancer risk and there is no difference between Asians and Caucasians.

Can determination of circulating endothelial cells and serum caspase-cleaved CK18 predict for response and survival in patients with advanced non-small-cell lung cancer receiving endostatin and paclitaxel-carboplatin chemotherapy? a retrospective study.

INTRODUCTION: Early prediction of the efficacy of a combination of an antiangiogenic drug with cytotoxic chemotherapy is a significant challenge. In that regard, circulating endothelial cells (CECs) and cytokeratins (CKs) seem to reflect their roles in both tumor angiogenesis and tumor cell death. METHODS: Patients with advanced, previously untreated non-small-cell lung cancer were randomly assigned to an endostatin treatment group (paclitaxel + carboplatin + endostatin) and a control group (paclitaxel + carboplatin + placebo). A total of 122 patients were evaluated, of whom 107 had measurements of blood CECs, CK8, caspase-cleaved CK18 (ccCK18), and uncleaved CK18 (CK18) before and at weeks 3 and 6 of treatment, respectively. RESULTS: Higher baseline CECs in patients with a tumor response (partial remission + stable disease, p = 0.002 for the entire group; p = 0.000 for the treatment group) were observed. The number of CECs decreased significantly after endostatin treatment (p = 0.000), whereas CK levels increased. Increased levels of ccCK18 and CK18, but not CK8, reached significance (p = 0.001 and p = 0.048, respectively) when compared with the baseline. Tumor response showed a strong correlation with reduction of CECs (p = 0.000) and increase of ccCK18 (p = 0.040) after endostatin therapy. Cutoff values of changes of CECs and ccCK18 for prediction of survival were 0.58/µl and 19.6 ng/ml, respectively. Reduction of CECs and increase of ccCK18 significantly correlated with longer median survival (p = 0.013 and p = 0.016 for progression-free survival; p = 0.009 and p = 0.012 for overall survival, respectively). CONCLUSIONS: CECs and CKs could be biomarkers for selecting patients with non-small-cell lung cancer who will benefit from treatment with endostatin in combination with paclitaxel plus carboplatin.

[A multicenter, randomized, double-blind, placebo-controlled safety study to evaluate the clinical effects and quality of life of paclitaxel-carboplatin (PC) alone or combined with endostar for advanced non-small cell lung cancer (NSCLC)].

OBJECTIVE: To analyze the efficacy and quality of life and safety for paclitaxel and carboplatin (TC) and TC combined with endostar in the treatment of advanced non-small cell lung cancer (NSCLC). METHODS: This is a prospective, multicenter, randomized, double-blind, placebo-controlled clinical study. A total of 126 cases of untreated advanced NSCLC were enrolled in this study. There were 63 patients in the TC control arm and TC combined endostar arm, respectively. All enrolled patients were continuously followed-up for disease progression and death. RESULTS: The objective response rate (ORR) of TC combined with endostar arm was 39.3%, and that of TC control arm was 23.0%, P = 0.078. The progression-free survival rates for TC combined with endostar arm and TC control arm were 78.3% and 58.8%, respectively, in 24 weeks (P = 0.017). The hazard ratio for the risk of disease progression was 0.35 (95%CI 0.13 to 0.90, P = 0.030). The median time to progression (TTP) of the TC combined with endostar arm was 7.1 months and TC arm 6.3 months (P > 0.05). The follow-up results showed that the median survival time (mOS) of the TC + Endostar arm was 17.6 months; (95%CI 13.4 to 21.7 months), and the TC + placebo arm 15.8 months (95%CI 9.4 to 22.9 months) (P > 0.05). The quality of life scores (LCSS patient scale) after treatment of the TC combined with endostar arm was improved, and that of the TC group was improved after completion of two cycles and three cycles of treatment. The quality of life scores compared with baseline after the completion of one cycle treatment was significantly improved for both the TC combined with endostar arm (P = 0.028 and), and TC arm (P = 0.036). It Indicated that TC combined with endostar treatment improved the patient's quality of life in the early treatment. The difference of adverse and serious adverse event rates between the two groups was not significant (P > 0.05). CONCLUSIONS: Compared with TC alone treatmrnt, TC combined with endostar treatment can reduce the risk of disease progression at early time (24 weeks), increase the ORR, and can be used as first-line treatment for advanced NSCLC. The TC combined with endostar treatment has good safety and tolerability, improves the quality of life, and not increases serious adverse effects and toxicity for patients with advanced NSCLC.

[The application of endobronchial ultrasound-guided transbronchial needle aspiration: report of 70 cases].

OBJECTIVE: To evaluate the diagnostic yield and the safety of endobronchial ultrasound-guided transbronchial needle biopsy (EBUS-TBNA) in mediastinal and hilar lymph nodes and lung tumors. METHODS: EBUS-TBNA was performed in 70 patients with thoracic masses or mediastinal-hilar lymphoadenopathy proved by CT scan. RESULTS: From July 2009 to January 2010, 70 patients were included in the study. EBUS-guided TBNA was performed to obtain samples from mediastinal and hilar lymph nodes (120 stations) and lung tumors (11 masses). In 46 cases of newly diagnosed lung cancer, 44 were confirmed by EBUS-TBNA without on site cytology assistance, with 2 false negative cases. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of EBUS-TBNA in the diagnosis of lung cancer were 96%, 100%, 100%, 92% and 97% respectively. Non-caseous granuloma formed by epithelioid cells was found in EBUS-TBNA histological specimen from 5 out of 10 patients with clinically diagnosed sarcoidosis. TBNA cytological smear showed acid-fast bacilli and histology of the lymph node demonstrated coagulatory necrosis from 1 out of 4 tuberculous cases. The procedure was uneventful, and there were no complications. CONCLUSION: EBUS-TBNA is an effective and safe method for the diagnosis of bronchogenic carcinoma and unknown mediastinal-hilar lymphadenopathy.

[Clinical observation of gefitinib for the treatment of 125 cases with advanced non-small cell lung cancer].

OBJECTIVE: To evaluate the efficacy and safety of gefitinib for the treatment of advanced non-small cell lung cancer (NSCLC). METHODS: 125 patients with advanced NSCLC who had failed or not tolerated or refused chemotherapy received 250 mg oral doses of gefitinib once daily until the disease progression or intolerable toxicity. RESULTS: A total of 125 NSCLC patients were studied, the overall response rate (RR) and the disease control rate (DCR) after administration of gefitinib were 35.2% (44/125) and 77.6% (97/125), respectively. The median progression-free survival and the median survival time were 5.8 and 11.2 months, respectively. The one-year survival rate was 40.5%. The response rate was significantly higher in females, adenocarcinoma and nonsmokers than that in males, non-adenocarcinoma and smokers (P < 0.05). The response rate did not show significant differences regarding ECOG score or previous treatment. The median progression-free survival was significantly longer in ECOG PS 0-1 and gefitinib effective patients than that in ECOG PS >or= 2 and gefitinib ineffective patients (P < 0.01). The median survival time was significantly longer in adenocarcinoma, nonsmokers and gefitinib effective patients than that in non-adenocarcinoma, smokers and gefitinib ineffective patients (P < 0.05). The most common side effects were rash (51.2%) and diarrhea (34.4%), but usually were mild. CONCLUSION: Gefitinib is effective and safe in the treatment of advanced NSCLC patients.

[Randomized study of thalidomide combined with vinorelbine and cisplatin chemotherapy for the treatment of advanced non-small cell lung cancer].

OBJECTIVE: To evaluate the efficacy, median time to progression (TTP), quality of life and toxicity in the patients with advanced non-small cell lung cancer (NSCLC), treated with thalidomide plus vinorelbine and cisplatin (NP) or NP alone. METHODS: Sixty six patients with advanced NSCLC were divided randomly into two groups, the trial and control groups. The trial group was treated with vinorelbine 25 approximately 30 mg/m(2) i.v. on D1 and D8, cisplatin 70 approximately 80 mg/m(2) i.v. on D1 (NP regimen), and thalidomide 200 mg orally and daily from D1. The control group received vinorelbine and cisplatin as above described. RESULTS: Of 66 assessable patients, the overall response rate was 51.5% in the trial group and 36.4% in the control group (P = 0.22). The median TTP was 6.0 months for the trial group, and 3.6 months for the control group (P < 0.001). The score of quality of life in trial group was higher than that in the control group, but no significant difference was observed between the two groups (P > 0.05). There were no significant differences in toxicities between the two groups (P > 0.05). CONCLUSION: NP regimen combined with thalidomide can significantly prolong the median time to tumor progression in patients with advanced NSCLC. Thalidomide may have a synergic activity with NP regimen without increased toxicities.