Expression of PD-1 mitigates phagocytic activities TAM in osteosarcoma.

The high expression of programmed death 1 (PD-1) is a hallmark of T cell exhaustion, consequently inhibiting the anti-tumor immunity, tumor-associated macrophages (TAMs) aggravate Osteosarcoma (OS) progression. However, PD-1 expression on TAMs in OS metastasis remains unclear. Here, we used scRNA-Seq of 15500 individual cells from human OS lung metastatic lesion, identified thirteen major cell clusters. Our data revealed that tumor-infiltrating lymphocytes (TILs) OS lung metastatic accompanied by accumulation of exhausted T cells and regulatory T cells (Tregs). CD3+ T cells from human OS lung metastatic exhibited lower proliferation than in primary tissue. Importantly, TAMs mainly comprise immunosuppressive M2 phenotype in OS metastasis. Mechanistically, we found that PD-1 of TAMs inhibits the phagocytic potency, further promoting the progression of OS metastasis. Therefore, the study provides a strong technical support for OS immunotherapy based on PD-1 inhibitors.

Fibrinogen-to-albumin ratio predicts overall survival of hepatocellular carcinoma.

BACKGROUND: Fibrinogen-to-albumin ratio (FAR) has been found to be of prognostic significance for several types of malignant tumors. However, less is known about the association between FAR and survival outcomes in hepatocellular carcinoma (HCC) patients. AIM: To explore the association between FAR and prognosis and survival in patients with HCC. METHODS: A total of 366 histologically confirmed HCC patients diagnosed between 2013 and 2018 in a provincial cancer hospital in southwestern China were retrospectively selected. Relevant data were extracted from the hospital information system. The optimal cutoff for baseline serum FAR measured upon disease diagnosis was established using the receiver operating characteristic (ROC) curve. Univariate and multivariate Cox proportional hazards models were used to determine the crude and adjusted associations between FAR and the overall survival (OS) of the HCC patients while controlling for various covariates. The restricted cubic spline (RCS) was applied to estimate the dose-response trend in the FAR-OS association. RESULTS: The optimal cutoff value for baseline FAR determined by the ROC was 0.081. Multivariate Cox proportional hazards model revealed that a lower baseline serum FAR level was associated with an adjusted hazard ratio of 2.43 (95% confidence interval: 1.87-3.15) in the OS of HCC patients, with identifiable dose-response trend in the RCS. Subgroup analysis showed that this FAR-OS association was more prominent in HCC patients with a lower baseline serum aspartate aminotransferase or carbohydrate antigen 125 level. CONCLUSION: Serum FAR is a prominent prognostic indicator for HCC. Intervention measures aimed at reducing FAR might result in survival benefit for HCC patients.

Low-dose aspirin can inhibit exosomal release induced by radiotherapy in breast cancer and attenuate its inhibitory effect on NK cell proliferation.

BACKGROUND: Breast cancer (BC) seriously threatens women's health. Aspirin plays a key role in the treatment and prognosis of BC. OBJECTIVE: To explore the effect of low-dose aspirin on BC radiotherapy through the mechanism of exosomes and natural killer (NK) cells. METHODS: BC cells were injected into the left chest wall to establish a BC model in nude mice. Tumor morphology and size were observed. Immunohistochemical staining for Ki-67 was used to observe the proliferation of tumor cells. TUNEL was used to detect the apoptosis of cancer cells. Protein levels of exosomal biogenesis- and secretion-related genes (Rab 11, Rab27a, Rab27b, CD63, and Alix) were detected by Western blot. Flow cytometry was used to detect apoptosis. Transwell assays were used to detect cell migration. A clonogenic assay was used to detect cell proliferation. Exosomes of BT549 and 4T1-Luc cells were extracted and observed by electron microscopy. After the coculture of exosomes and NK cells, the activity of NK cells was detected by CCK-8. RESULTS: The protein expression of genes related to exosomal genesis and secretion (Rab 11, Rab27a, Rab27b, CD63, and Alix) in BT549 and 4T1-Luc cells was upregulated under radiotherapy treatment. Low doses of aspirin inhibited exosome release from BT549 and 4T1-Luc cells and alleviated the inhibitory effect of BC cell exosomes on NK cell proliferation. In addition, knocking down Rab27a reduced the protein levels of exosome-related and secretion-related genes in BC cells, further enhancing the promotive effect of aspirin on NK cell proliferation, while overexpressing Rab27a had the opposite effect. Aspirin was combined at a radiotherapeutic dose of 10 Gy to enhance the radiotherapy sensitivity of radiotherapy-tolerant BC cells (BT549R and 4T1-LucR). Animal experiments have also verified that aspirin can promote the killing effect of radiotherapy on cancer cells and significantly inhibit tumor growth. CONCLUSION: Low doses of aspirin can inhibit the release of BC exosomes induced by radiotherapy and weaken their inhibition of NK cell proliferation, promoting radiotherapy resistance.

Pharmacokinetics integrated with network pharmacology to clarify effective components and mechanism of Wendan decoction for the intervention of coronary heart disease.

ETHNOPHARMACOLOGICAL RELEVANCE: Coronary heart disease (CHD), one of the leading causes of mortality in the world among chronic non-infectious diseases, is closely associated with atherosclerosis, which ultimately leads to myocardial injury. Wendan decoction (WDD), a classical famous formula, exerted an intervention effect on CHD according to numerous reports. However, the effective components and underlying mechanisms for the treatment of CHD have not been fully elucidated. AIM OF THE STUDY: An in-depth investigation of the effective components and mechanisms of WDD for the intervention of CHD was further explored. MATERIALS AND METHODS: Firstly, based on our previous metabolic profile results, a quantification method for absorbed components was established by ultra-performance liquid chromatography triple quadrupole-mass spectrometry (UPLC-TQ-MS) and applied to the pharmacokinetics study of WDD. Then the network pharmacology analysis for considerable exposure components in rat plasma was employed to screen key components of WDD. Gene ontology and KEGG pathway enrichment analysis were further performed to obtain putative action pathways. The effective components and mechanism of WDD were confirmed by in vitro experiments. RESULTS: A rapid and sensitive quantification method was successfully applied to the pharmacokinetic study of 16 high-exposure components of WDD at three different doses. A total of 235 putative CHD targets were obtained for these 16 components. Then, 44 core targets and 10 key components with high degree values were successively screened out by the investigation of protein-protein interaction and the network of "herbal medicine-key components-core targets". Enrichment analysis suggested that the PI3K-Akt signaling pathway was closely related to this formula's therapeutic mechanism. Furthermore, pharmacological experiments demonstrated that 5 of 10 key components (liquiritigenin, narigenin, hesperetin, 3,5,6,7,8,3',4'-heptamethoxyflavone, and isoliquiritigenin) significantly enhanced DOX-induced H9c2 cell viability. The cardioprotective effects of WDD against DOX-induced cell death through the PI3K-Akt signaling pathway were verified by western blot experiments. CONCLUSION: The integration of pharmacokinetics and network pharmacology approaches successfully clarified 5 effective components and therapeutic mechanism of WDD for the intervention of CHD.

A screening strategy for bioactive components of Bu-Zhong-Yi-Qi-Tang regulating spleen-qi deficiency based on "endobiotics-targets-xenobiotics" association network.

ETHNOPHARMACOLOGICAL RELEVANCE: Bu-Zhong-Yi-Qi-Tang is a famous traditional Chinese medicine formula that has been prevalent in China for over 700 years to treat spleen-qi deficiency related diseases, such as gastrointestinal and respiratory disorders. However, the bioactive components responsible for regulating spleen-qi deficiency remain unclear and have puzzled many researchers. AIM OF THE STUDY: The current study focuses on efficacy evaluation of regulating spleen-qi deficiency and screening the bioactive components of Bu-Zhong-Yi-Qi-Tang. MATERIALS AND METHODS: The effects of Bu-Zhong-Yi-Qi-Tang were evaluated through blood routine examination, immune organ index, and biochemical analysis. Metabolomics was utilized to analyze the potential endogenous biomarkers (endobiotics) in the plasma, and the prototypes (xenobiotics) of Bu-Zhong-Yi-Qi-Tang in the bio-samples were characterized using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry. Then, these endobiotics were used as "bait" to predict targets based on network pharmacology and to screen potential bioactive components from the absorbed prototypes in the plasma by constructing an "endobiotics-targets-xenobiotics" association network. Further, the anti-inflammatory activities of representative compounds (calycosin and nobiletin) were validated through poly(I:C)-induced pulmonary inflammation mice model. RESULTS: Bu-Zhong-Yi-Qi-Tang exhibited immunomodulatory and anti-inflammatory activities in spleen-qi deficiency rat, as supported by the observation of increased levels of D-xylose and gastrin in serum, an increase in the thymus index and number of lymphocytes in blood, as well as a reduction in the level of IL-6 in bronchoalveolar lavage fluid. Furthermore, plasma metabolomic analysis revealed a total of 36 Bu-Zhong-Yi-Qi-Tang related endobiotics, which were mainly enriched in primary bile acids biosynthesis, the metabolism of linoleic acid, and the metabolism of phenylalanine pathways. Meanwhile, 95 xenobiotics were characterized in plasma, urine, small intestinal contents, and tissues of spleen-qi deficiency rat after Bu-Zhong-Yi-Qi-Tang treatment. Using an integrated association network, six potential bioactive components of Bu-Zhong-Yi-Qi-Tang were screened. Among them, calycosin was found to significantly reduce the levels of IL-6 and TNF-α in the bronchoalveolar lavage fluid, increase the number of lymphocytes, while nobiletin dramatically decreased the levels of CXCL10, TNF-α, GM-CSF, and IL-6. CONCLUSION: Our study proposed an available strategy for screening bioactive components of BYZQT regulating spleen-qi deficiency based on "endobiotics-targets-xenobiotics" association network.

Advanced data post-processing method for rapid identification and classification of the major triterpenoids of Alismatis rhizoma by ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry.

INTRODUCTION: Alismatis rhizoma (AR), a distinguished diuretic traditional Chinese herbal medicine, is widely used for the treatment of diarrhea, edema, nephropathy, hyperlipidemia, and tumors in clinical settings. Most beneficial effects of AR are attributed to the major triterpenoids, whose contents are relatively high in AR. To date, only 25 triterpenoids in AR have been characterized by LC-MS because the low-mass diagnostic ions are hardly triggered in MS, impeding structural identification. Herein, we developed an advanced data post-processing method with abundant characteristic fragments (CFs) and neutral losses (NLs) for rapid identification and classification of the major triterpenoids in AR by UPLC-Q-TOF-MSE . OBJECTIVE: We aimed to establish a systematic method for rapid identification and classification of the major triterpenoids of AR. METHODS: UPLC-Q-TOF-MSE coupled with an advanced data post-processing method was established to characterize the major triterpenoids of AR. The abundant CFs and NLs of different types of triterpenoids were discovered and systematically summarized. The rapid identification and classification of the major triterpenoids of AR were realized by processing the data and comparing with information described in the literature. RESULTS: In this study, a total of 44 triterpenoids were identified from AR, including three potentially new compounds and 41 known ones, which were classified into six types. CONCLUSION: The newly established approach is suitable for the chemical profiling of the major triterpenoids in AR, which could provide useful information about chemical constituents and a basis for further exploration of its active ingredients in vivo.

miR-137-LAPTM4B regulates cytoskeleton organization and cancer metastasis via the RhoA-LIMK-Cofilin pathway in osteosarcoma.

Osteosarcoma (OS) is a rare malignant bone tumor but is one leading cause of cancer mortality in childhood and adolescence. Cancer metastasis accounts for the primary reason for treatment failure in OS patients. The dynamic organization of the cytoskeleton is fundamental for cell motility, migration, and cancer metastasis. Lysosome Associated Protein Transmembrane 4B (LAPTM4B) is an oncogene participating in various biological progress central to cancer biogenesis. However, the potential roles of LAPTM4B in OS and the related mechanisms remain unknown. Here, we established the elevated LAPTM4B expression in OS, and it is essential in regulating stress fiber organization through RhoA-LIMK-cofilin signaling pathway. In terms of mechanism, our data revealed that LAPTM4B promotes RhoA protein stability by suppressing the ubiquitin-mediated proteasome degradation pathway. Moreover, our data show that miR-137, rather than gene copy number and methylation status, contributes to the upregulation of LAPTM4B in OS. We report that miR-137 is capable of regulating stress fiber arrangement, OS cell migration, and metastasis via targeting LAPTM4B. Combining results from cells, patients' tissue samples, the animal model, and cancer databases, this study further suggests that the miR-137-LAPTM4B axis represents a clinically relevant pathway in OS progression and a viable target for novel therapeutics.

Characterization of volatile organic compounds with anti-atherosclerosis effects in Allium macrostemon Bge. and Allium chinense G. Don by head space solid phase microextraction coupled with gas chromatography tandem mass spectrometry.

Introduction: Allium macrostemon Bge. (AMB) and Allium chinense G. Don (ACGD) are both edible Allium vegetables and named officinal Xiebai (or Allii Macrostemonis Bulbus) in East Asia. Their medicinal qualities involve in lipid lowering and anti-atherosclerosis effects. And steroidal saponins, nitrogenous compounds and sulfur compounds are like the beneficial components responsible for medicinal functions. Sulfur compounds are the recognized main components both in the volatile oils of AMB and ACGD. Besides, few researches were reported about their holistic chemical profiles of volatile organic compounds (VOCs) and pharmacodynamic effects. Methods: In this study, we first investigated the lipid-lowering and anti-atherosclerotic effects of volatile oils derived from AMB and ACGD in ApoE -/- mice with high fat and high cholesterol diets. Results: The results showed the volatile oils of AMB and ACGD both could markedly reduce serum levels of TG, TC, and LDL-C (p < 0.05), and had no alterations of HDL-C, ALT, and AST levels (p > 0.05). Pathological results displayed they both could obviously improve the morphology of cardiomyocytes and the degree of myocardial fibrosis in model mice. Meanwhile, oil red O staining results also proved they could apparently decrease the lesion areas of plaques in the aortic intima (p < 0.05). Furthermore, head space solid phase microextraction coupled with gas chromatography tandem mass spectrometry combined with metabolomics analysis was performed to characterize the VOCs profiles of AMB and ACGD, and screen their differential VOCs. A total of 121 and 115 VOCs were identified or tentatively characterized in the volatile oils of AMB and ACGD, respectively. Relative-quantification results also confirmed sulfur compounds, aldehydes, and heterocyclic compounds accounted for about 85.6% in AMB bulbs, while approximately 86.6% in ACGD bulbs were attributed to sulfur compounds, ketones, and heterocyclic compounds. Multivariate statistical analysis showed 62 differentially expressed VOCs were observed between AMB and ACGD, of which 17 sulfur compounds were found to be closely associated with the garlic flavor and efficacy. Discussion: Taken together, this study was the first analysis of holistic chemical profiles and anti-atherosclerosis effects of AMB and ACGD volatile oils, and would benefit the understanding of effective components in AMB and ACGD.

Inhibition of estrogen sulfation by Xian-Ling-Gu-Bao capsule.

Xian-Ling-Gu-Bao capsule (XLGB) is a widely prescribed traditional Chinese medicine used for the treatment of osteoporosis. However, it significantly elevates levels of serum estrogens. Here we aimed to assess the dominant contributors of sulfotransferase (SULT) enzymes to the sulfation of estrogens and identify the effective inhibitors of this pathway in XLGB. First, estrone, 17ß-estradiol, and estriol underwent sulfation in human liver S9 extracts. Phenotyping reactions and enzyme kinetics assays revealed that SULT1A1, 1A2, 1A3, 1C4, 1E1, and 2A1 all participated in estrogen sulfation, with SULT1E1 and 1A1 as the most important contributors. The incubation system for these two active enzymes were optimized with Tris-HCl buffer, DL-Dithiothreitol (DTT), MgCl2, adenosine 3'-phosphate 5'-phosphosulfate (PAPS), protein concentration, and incubation time. Then, 29 compounds in XLGB were selected to investigate their inhibitory effects and mechanisms against SULT1E1 and 1A1 through kinetic modelling. Moreover, in silico molecular docking was used to validate the obtained results. And finally, the prenylated flavonoids (isobavachin, neobavaisoflavone, etc.) from Psoralea corylifolia L., prenylated flavanols (icariside II) from Epimedium brevicornu Maxim., tanshinones (dihydrotanshinone, tanshinone II-A,) from Salvia miltiorrhiza Bge., and others (corylifol A, corylin) were identified as the most potent inhibitors of estrogen sulfation. Taken together, these findings provide insights into the understanding regioselectivity of estrogen sulfation and identify the effective components of XLGB responsible for the promotion of estrogen levels.

Total femur replacement with 18 years of follow-up: A case report.

BACKGROUND: Osteosarcoma is one of the most common primary malignant bone tumors and is more common in adolescents. The femur is the most common site of osteosarcoma, and many patients require total femur replacement. We reviewed the relevant literature and case findings, summarized and analyzed this case in combination with relevant literature, and in doing so improved the understanding of the technology. CASE SUMMARY: The case we report was a 15-year-old patient who was admitted to the hospital 15 days after the discovery of a right thigh mass. The diagnosis was osteosarcoma of the right femoral shaft. After completion of neoadjuvant chemotherapy and preoperative preparation, total right femoral resection + artificial total femoral replacement was performed. Then, chemotherapy was continued after surgery. The patient recovered well after treatment, and the function of the affected limb was good. No recurrence, metastasis, prosthesis loosening, dislocation, fracture or other complications were found during 18 years of follow-up. At present, the patient can still work and lives normally. The results of the medium- and long-term follow-up were satisfactory. CONCLUSION: Artificial total femur replacement is a feasible limb salvage operation for patients with femoral malignant tumors, and the results of medium- and long-term follow-up are satisfactory.

LAPTM4B-35 promotes cancer cell migration via stimulating integrin beta1 recycling and focal adhesion dynamics.

Metastasis is the main cause of cancer patients' death despite tremendous efforts invested in developing the related molecular mechanisms. During cancer cell migration, cells undergo dynamic regulation of filopodia, focal adhesion, and endosome trafficking. Cdc42 is imperative for maintaining cell morphology and filopodia, regulating cell movement. Integrin beta1 activates on the endosome, the majority of which distributes itself on the plasma membrane, indicating that endocytic trafficking is essential for this activity. In cancers, high expression of lysosome-associated protein transmembrane 4B (LAPTM4B) is associated with poor prognosis. LAPTM4B-35 has been reported as displaying plasma membrane distribution and being associated with cancer cell migration. However, the detailed mechanism of its isoform-specific distribution and whether it relates to cell migration remain unknown. Here, we first report and quantify the filopodia localization of LAPTM4B-35: mechanically, that specific interaction with Cdc42 promoted its localization to the filopodia. Furthermore, our data show that LAPTM4B-35 stabilized filopodia and regulated integrin beta1 recycling via interaction and cotrafficking on the endosome. In our zebrafish xenograft model, LAPTM4B-35 stimulated the formation and dynamics of focal adhesion, further promoting cancer cell dissemination, whereas in skin cancer patients, LAPTM4B level correlated with poor prognosis. In short, this study establishes an insight into the mechanism of LAPTM4B-35 filopodia distribution, as well as into its biological effects and its clinical significance, providing a novel target for cancer therapeutics development.

Endogenous hydrogen sulfide promotes human preimplantation embryonic development by regulating metabolism-related gene expression.

Hydrogen sulfide (H2S) as an endogenous gaseous signaling molecule had been proved to play a vital role in gametes physiology, covering meiosis, maturation and aging. However, little is known about H2S involvement in embryonic development. The present study explored the positive effect of H2S on human early embryonic development. Results validated that the two H2S producing enzymes, CBS and CSE mRNA and proteins were identified in donated human cleavage and blastocyst-stage embryos. The l-cysteine incubation produced endogenous H2S in human blastocysts. NaHS positively affected in vitro blastulation. Single-cell RNA-seq analysis identified 228 differentially expressed genes (DEGs) after NaHS treatment versus the control. The Gene Ontology (GO) enrichment analysis of DEGs showed that genes for protein modification and metabolism were significantly enriched in the NaHS treatment group. For the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, 2-oxocarboxylic acid metabolism, glycosaminoglycan biosynthesis-keratan sulfate, steroid biosynthesis, carbon metabolism, and biosynthesis of amino acids were significantly enriched. Six DEGs, including Neural EGFL like 1 (NELL1), aconitase 1 (ACO1), phosphoglycerate mutase 1 (PGAM1), TP53 induced glycolysis regulatory phosphatase (TIGAR), UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 2 (B3GNT2), and carbohydrate Sulfotransferase 4 (CHST4) were validate by real-time RT-PCR. These findings suggest that H2S is a positive regulator of early embryonic development and may alter the transcription of embryonic genes for protein modification and metabolism in human embryos.

MicroRNA-106a regulates autophagy-related cell death and EMT by targeting TP53INP1 in lung cancer with bone metastasis.

Bone metastasis is one of the most serious complications in lung cancer patients. MicroRNAs (miRNAs) play important roles in tumour development, progression and metastasis. A previous study showed that miR-106a is highly expressed in the tissues of lung adenocarcinoma with bone metastasis, but its mechanism remains unclear. In this study, we showed that miR-106a expression is dramatically increased in lung cancer patients with bone metastasis (BM) by immunohistochemical analysis. MiR-106a promoted A549 and SPC-A1 cell proliferation, migration and invasion in vitro. The results of bioluminescence imaging (BLI), micro-CT and X-ray demonstrated that miR-106a promoted bone metastasis of lung adenocarcinoma in vivo. Mechanistic investigations revealed that miR-106a upregulation promoted metastasis by targeting tumour protein 53-induced nuclear protein 1 (TP53INP1)-mediated metastatic progression, including cell migration, autophagy-dependent death and epithelial-mesenchymal transition (EMT). Notably, autophagy partially attenuated the effects of miR-106a on promoting bone metastasis in lung adenocarcinoma. These findings demonstrated that restoring the expression of TP53INP1 by silencing miR-106a may be a novel therapeutic strategy for bone metastatic in lung adenocarcinoma.

Prognostic nomogram for predicting 5-year overall survival in Chinese patients with high-grade osteosarcoma.

This study aimed to construct a widely accepted prognostic nomogram in Chinese high-grade osteosarcoma (HOS) patients aged ≤ 30 years to provide insight into predicting 5-year overall survival (OS). Data from 503 consecutive HOS patients at our centre between 12/2012 and 05/2019 were retrospectively collected. Eighty-four clinical features and routine laboratory haematological and biochemical testing indicators of each patient at the time of diagnosis were collected. A prognostic nomogram model for predicting OS was constructed based on the Cox proportional hazards model. The performance was assessed by the concordance index (C-index), receiver operating characteristic curve and calibration curve. The utility was evaluated by decision curve analysis. The 5-year OS was 52.1% and 2.6% for the nonmetastatic and metastatic patients, respectively. The nomogram included nine important variables based on a multivariate analysis: tumour stage, surgical type, metastasis, preoperative neoadjuvant chemotherapy cycle, postoperative metastasis time, mean corpuscular volume, tumour-specific growth factor, gamma-glutamyl transferase and creatinine. The calibration curve showed that the nomogram was able to predict 5-year OS accurately. The C-index of the nomogram for OS prediction was 0.795 (range, 0.703-0.887). Moreover, the decision curve analysis curve also demonstrated the clinical benefit of this model. The nomogram provides an individualized risk estimate of the 5-year OS in patients with HOS aged ≤ 30 years in a Chinese population-based cohort.

Non-coding RNAs in necroptosis, pyroptosis and ferroptosis in cancer metastasis.

Distant metastasis is the main cause of death for cancer patients. Recently, the newly discovered programmed cell death includes necroptosis, pyroptosis, and ferroptosis, which possesses an important role in the process of tumor metastasis. At the same time, it is widely reported that non-coding RNA precisely regulates programmed death and tumor metastasis. In the present review, we summarize the function and role of necroptosis, pyrolysis, and ferroptosis involving in cancer metastasis, as well as the regulatory factors, including non-coding RNAs, of necroptosis, pyroptosis, and ferroptosis in the process of tumor metastasis.

Granulocyte-Macrophage Colony Stimulating Factor in Single Blastocyst Conditioned Medium as a Biomarker for Predicting Implantation Outcome of Embryo.

Background: It is highly desirable to develop new strategies based on secretomics to more accurately selection of embryos with the highest developmental potential for transfer. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been reported to promote embryo development and pregnancy establishment. However, the predictive value of GM-CSF in single blastocyst selection remains unclear. This study is to determine the concentration of GM-CSF in human single-blastocyst conditioned medium (SBCM) and to evaluate its association with embryo quality and pregnancy outcome. Methods: The patients with ≤38 years of age receiving the first cycle of assisted reproductive therapy were included in this study. The patients who had <4 top-quality embryos formed by the fertilized two pronuclear zygotes on day 3 were excluded. A total of 126 SBCM samples (SBCMs) were included, of which blastocysts from 77 SBCMs were later transferred in subsequent frozen-thawed embryo transfer. The concentrations of GM-CSF were detected by single-molecule array (SIMOA) and analyzed for their possible association with embryo quality and pregnancy outcomes. The top-quality embryo (TQ), positive HCG (HP), clinical pregnancy (CP), and ongoing pregnancy (OP) rates were determined and compared between groups divided based on GM-CSF concentrations. Results: The detection rate of GM-CSF was found to be 50% in all SBCMs. There were significant differences in TQ rate, HP rate, CP rate and OP rate among high concentration group, medium concentration group and low concentration group. Both GM-CSF alone or GM-CSF combined with the morphological score (MS) had a greater AUC of ROC curve than that of MS alone to predict the pregnancy outcome, and GM-CSF combined with MS had the highest AUC. Conclusions: The concentration of GM-CSF in SBCM was detected at fg/ml levels, which was associated with embryo quality and pregnancy outcome. Collectively, GM-CSF may be used as a biomarker for prediction of pregnancy outcome and selection of embryos with high developmental potential for transfer in assisted reproductive technology (ART).

Potential Determinants for Metabolic Fates and Inhibitory Effects of Isobavachalcone Involving in Human Cytochrome P450, UDP-Glucuronosyltransferase Enzymes, and Efflux Transporters.

Isobavachalcone, a naturally occurring chalcone in Psoralea corylifolia, posses many biological properties including anticancer, antiplatelet, and antifungal. However, its glucuronidation, glucuronides excretion, and drug-drug interaction (DDI) involving in human cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT) enzymes, and efflux transporters (BCRP and MRPs) remains unclear so far. After incubation, three glucuronides were produced by HLM and HIM with total intrinsic clearance (CLint) of 236.71 and 323.40 µL/min/mg, respectively. Reaction phenotyping proved UGT1A1, 1A3, 1A7, 1A8, and 1A9 played important roles in glucuronidation with total CLint values of 62.69-143.00 µL/min/mg. Activity correlation analysis indicated UGT1A1 and UGT1A3 participated more in the glucuronidation. In addition, the glucuronidation showed marked species differences, and rabbits and dogs were probably appropriate model animals to investigate the in vivo glucuronidation. Furthermore, BCRP, MRP1, and MRP4 transporters were identified as the most important contributors to glucuronides excretion in HeLa1A1 cells based on gene silencing method. Moreover, isobavachalcone demonstrated broad-spectrum inhibitory effects against CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, UGT1A1, UGT1A9, UGT2B7 with IC50 values of 1.08-9.78 µM. Except CYP2B6 and CYP2D6, the calculated [I]/Ki values for other enzymes were all greater than 0.1, indicating the inhibition of systemic metabolism or elimination for these enzyme substrates seems likely. Taken together, we summarized metabolic fates of isobavachalcone including glucuronidation and efflux transport as well as inhibitory effects involving in human CYP and UGT enzymes.

Transcriptome Sequencing reveals the expressed profiles of mRNA and ncRNAs and regulate network via ceRNA mediated molecular mechanism of lung adenocarcinoma bone metastasis in Xuanwei.

BACKGROUND: The most ordinary subtype of lung cancer is lung adenocarcinoma (LuAC), which is characterized by strong metastatic ability. And LuAC rates in Xuanwei leads to the poor prognosis and high death rate. In this study, we systematically explored the molecular mechanism of LuAC bone metastasis in Xuanwei by transcriptome sequencing. METHODS: RNA Sequencing was conducted to explore the noncoding RNAs (ncRNAs) expression profiles in primary LuAC and LuAC bone metastasis. We identified differentially expressed mRNAs (DEmRNAs), miRNAs (DEmiRNAs), lncRNAs (DElncRNAs) and circRNAs (DEcircRNAs). Bioinformatics analyses the possible relationships and functions of the LuAC bone metastasis-related competing endogenous RNA (ceRNA). And qRT-PCR was performed to evaluate the expression of these differently expressed genes in serum. RESULTS: A total of 2,141 DEmRNAs, 43 DEmiRNAs, 136 DElncRNAs and 706 DEcircRNAs were identified in the Xuanwei patients with primary LuAC vs. LuAC bone metastasis, respectively. The circRNA/miRNA/mRNA and lncRNA/miRNA/mRNA networks of LuAC in Xuanwei with bone metastasis were built, and the gene expression mechanisms regulated by ncRNAs were unveiled via the ceRNA regulatory networks. We observe that lncRNA (ADAMTS9-AS2, TEX41, DLEU2, LINC00152)-miR-223-3p-SCARB1 and hsa_circ_0000053-miR-196a-5p/miR-196b-5p-HOXA5 ceRNA networks might play an important role in bone metastasis of Xuanwei LuAC. CONCLUSIONS: We comprehensively identified ceRNA regulatory networks of LuAC in Xuanwei with bone metastasis as well as revealed the contribution of different ncRNAs expression profiles. Our data demonstrate the association between mRNAs and ncRNAs in the metastasis mechanism of LuAC in Xuanwei with bone metastasis.