Rapid, easy and catalyst-free preparation of magnetic thiourea-based covalent organic frameworks at room temperature for enrichment and speciation of mercury with HPLC-ICP-MS.

The complex and cumbersome preparation of magnetic covalent organic frameworks (COFs) nanocomposites on a small scale limits their application. Herein, a rapid and easy route was employed for the preparation of magnetic thiourea-based COFs nanocomposites. COFs were coated on Fe3O4 nanoparticles at room temperature without a catalyst within approximately 30 min. This method is suitable for the large-scale preparation of magnetic adsorbent. Using the as-prepared magnetic adsorbent (Fe3O4@COF-TpTU), we developed a simple, efficient, and sensitive magnetic solid-phase extraction-high performance liquid chromatography-inductively coupled plasma-mass spectrometry (MSPE-HPLC-ICP-MS) for the enrichment and determination of mercury species, including Hg2+, methylmercury (MeHg), and ethylmercury (EtHg). The effects of the experimental parameters on the extraction efficiency, including solution pH, adsorption and desorption time, composition and volume of the elution solvent, salinity, coexisting ions, and dissolved organic matter, were comprehensively investigated. Under optimised conditions, the limits of detection in the developed method were 0.56, 0.34, and 0.47 ng L-1 with enrichment factors of 190, 195, and 180-fold for Hg2+, MeHg, and EtHg, respectively. The satisfactory spiked recoveries (97.0-103%) in real water samples and high consistency between the certified and determined values in a certified reference material demonstrate the high accuracy and reproducibility of the developed method. The as-proposed method with simple operation, high sensitivity, and excellent anti-matrix interference performance was successfully applied to the enrichment and determination of trace levels of mercury species in the natural samples with complicated matrices, such as underground water, surface water, seawater and biological samples.

Metal sulfide nanoparticle-based dual barcode-triggered DNAzyme cascade for multiplex miRNA detection in a single assay.

Single miRNAs are not specific and accurate enough to meet the strict diagnosis requirements in practice. Therefore, simultaneous monitoring of multiplexed miRNA in biological samples can not only improve the accuracy and specificity of bioassays but also avoid the squandering of valuable biological specimens. Herein, we designed a metal sulfide nanoparticle-based dual barcode-triggered DNAzyme cascade strategy for the sensitive and simultaneous multiplex miRNA detection in a single assay. Firstly, the capture probes (H1, H2) specifically recognize targets (miRNA-21, miRNA-141), exposing the stem of H1 and H2. Then, with the introduction of a detection probe (CuS-H3, ZnS-H4), the exposed H1 and H2 catalyze the hairpin assembly (CHA) reaction, realizing target miRNA recycling, and forming H1/H3-CuS and H2/H4-ZnS complexes. Subsequently, the formed H1/H3-CuS and H2/H4-ZnS complexes are encoded on magnetic beads through the biotin/streptavidin interaction. The CuS and ZnS nanoparticles captured by magnetic beads release thousands of Cu2+ and Zn2+via the cation exchange reaction. Finally, the released Cu2+ and Zn2+ specially activate the DNAzyme of the catalytic and molecular beacon (CAMB) system. The CAMB system affords an amplified fluorescence signal output by cycling and regenerating the metal ion-dependent DNAzyme to realize multiple enzymatic turnovers. Benefiting from target recycling, nanoparticle amplification, and catalytic and molecular beacon amplification, there is substantial amplification and the target miRNAs can be detected at 0.06 fM (miRNA-21) and 0.048 fM (miRNA-141) in a single assay. Furthermore, the high selectivity and accuracy of the assay were proved by practical analysis of different cancer cells, which exhibited good practicability in multiplex miRNA detection in clinical sera. The results indicate that the proposed strategy holds great potential for the sensitive detection of multiplex cancer biomarkers and offers the opportunity for future applications in clinical diagnosis.

Alterations of Cytokine Profiles in Patients With Recurrent Implantation Failure.

Serum cytokine profile and T helper (Th)1/Th2 cell balance are related to the success of embryo implantation, although not yet firmly linked to recurrent implantation failure (RIF), a repeated failure to achieve clinical pregnancy following multiple high-quality embryo transfer. In this prospective study, comprehensive bioinfomatic analysis and logistic regression analysis were used to compare the serum cytokine profiles of 41 RIF patients with those of 29 subjects with first-cycle successful pregnancy in the mid-luteal phase and to assess the alterations of cytokine profiles in patients with clinical pregnancy at five weeks post-transplantation. We found several elevated pro-inflammatory cytokines, decreased anti-inflammatory cytokines, and increased Th1/Th2 cytokine ratios in RIF patients compared to control subjects. Specifically, the receiver operating characteristic (ROC) curve generated using multiple indicators provides a high predictive value for diagnosing RIF (area under the curve [AUC] = 0.94, 95% confidence interval [CI] 0.87-1.00, P < 0.0001), with a sensitivity of 96.55% and a specificity of 87.50%. Meanwhile, at five weeks post-transplantation, patients in both groups diagnosed with clinical pregnancy exhibited increased levels of several cytokines compared with pre-pregnancy levels, and a gradual shift in Th1/Th2 balance toward Th2. These findings suggest that inflammatory serum cytokines and the predominance of Th1 cells likely contribute to RIF and possibly reflect the immune environment at the maternal-fetal interface, suggesting their value as outcome indicators in assisted reproductive therapy.

Engineering an Au nanostar-based liquid phase interfacial ratiometric SERS platform with programmable entropy-driven DNA circuits to detect protein biomarkers in clinical samples.

Developing sensing platforms that simultaneously integrate high sensitivity and accuracy has been a promising but challenging task for the detection of protein biomarkers in clinical samples. Herein, we engineered an Au nanostar-based liquid phase interfacial ratiometric SERS platform with programmable entropy-driven DNA circuits to detect the protein biomarker Mucin 1 (MUC1) in clinical samples.

METTL3-Mediated m6A RNA Modification Regulates Corneal Injury Repair.

Limbal stem cells are essential for continuous corneal regeneration and injury repair. METTL3-catalyzed N6-methyladenosine (m6A) mRNA modifications are involved in many biological processes and play a specific role in stem cell regeneration, while the role of m6A modifications in corneal injury repair remains unknown. In this study, we generated a limbal stem cell-specific METTL3 knockout mouse model and studied the role of m6A in repairing corneal injury caused by alkali burn. The results showed that METTL3 knockout in the limbal stem cells promotes the in vivo cell proliferation and migration, leading to the fast repair of corneal injury. In addition, m6A modification profiling identified stem cell regulatory factors AHNAK and DDIT4 as m6A targets. Our study reveals the essential functions of m6A RNA modification in regulating injury repair and provides novel insights for clinical therapy of corneal diseases.

SERS-based copper-mediated signal amplification strategy for simple and sensitive detection of telomerase activity.

Simple and sensitive detection of telomerase activity is of vital importance for both early diagnosis and therapy of malignant tumors. Inspired by DNA-biobarcode amplification reported by Chad A. Mirkin, we developed a facile DNA-biobarcode-like SERS-based copper-mediated signal amplification strategy for sensitive detection of telomerase activity. In this strategy, a duplex DNA constructed by hybridization of a copper oxide nanoparticle (CuO NP)-labeled reporting sequence (RS) with the telomerase primer sequence (TS) is ingeniously designed, and anchored on the magnetic bead (MB) to build the CuO NPs-encoded magnetic bead (MB-CuO NPs) detection probe. Upon selective sensing of telomerase, telomerase elongation reaction and structure change of TS products make the CuO NP-RS displace and separate from MB. The separated CuO NPs are dissolved into a mass of Cu2+, which prompt monodisperse dopamine-functionalized AgNPs (D-AgNPs) signal probe into aggregation, resulting in color changes and significantly enhancing of SERS signal. The SERS signal increases with the increase of Cu2+, which is directly proportional to the telomerase. Benefiting from the transformation of CuO NP to Cu2+ with a high amplification effect, this strategy could realize the telomerase activity measurement down to 3 HeLa cells and a dynamic range of 10-10000 cells. It shows a significant improvement of sensitivity without need for other enzymes and elaborate design, which escapes from the complicated manipulations and design in polymerase chain reaction (PCR) and DNA amplification techniques. Moreover, with this strategy, telomerase activities of different cell lines and telomerase inhibitors screening were successfully performed. Significantly, it can also be utilized for visual detection of telomerase, which validates the potential on-site application and its application as point-of-care testing (POCT) for efficient monitoring. Given the high-performance for telomerase analysis, the strategy has a promising application in biological detection and clinical diagnosis, as well as point-of-care tests.

Endometrial injury increases expression of hypoxia-inducible factor and angiogenesis in the endometrium of women with recurrent implantation failure.

RESEARCH QUESTION: The aim of this study was to compare expression of hypoxia-inducible factor 1-alpha (HIF-1α), angiogenesis and apoptosis in endometrial tissue near the implantation window of women with recurrent implantation failure (RIF) and in fertile control women, and to describe possible mechanisms of endometrial injury. DESIGN: A controlled clinical study was conducted. Endometrial tissue specimens were obtained from 20 women undergoing IVF who had had at least three previous failed treatment cycles; normal endometrial specimens were obtained from 10 fertile control women. RESULTS: HIF-1α expression was down-regulated in the endometrium of women with RIF compared with that of control women. In addition, micro-vessel density (MVD) was much lower in the endometrium of women with RIF than in that of the control women. Apoptosis was significantly reduced in the endometrium of the RIF group compared with the control group. Endometrial injury increased HIF-1α expression and MVD in endometrial samples of the RIF group, but apoptosis was not significantly altered. CONCLUSIONS: HIF-1α expression, MVD and endometrial apoptosis were reduced in the peri-implantation endometrium of women with RIF. This suggests that altered endometrial HIF-1α expression and angiogenesis may contribute to implantation failure.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) inhibits human ovarian cancer cell proliferation.

PURPOSE: The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, mediates a broad spectrum of biological processes, including ovarian growth and ovulation. Recently, we found that an endogenous AhR ligand (ITE) can inhibit ovarian cancer proliferation and migration via the AhR. Here, we tested whether 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, an exogenous AhR ligand) may exert similar anti-ovarian cancer activities using human ovarian cancer and non-cancerous human ovarian surface epithelial cells. METHODS: Two human ovarian cancer cell lines (SKOV-3 and OVCAR-3) and one human ovarian surface epithelial cell line (IOSE-385) were used. Cell proliferation and migration activities were determined using crystal violet and FluoroBlok insert system assays, respectively. AhR protein expression was assessed by Western blotting. Expression of cytochrome P450, family 1, member A1 (CYP1A1) and member B1 (CYP1B1) mRNA was assessed by qPCR. Small interfering RNAs (siRNAs) were used to knock down AhR expression. RESULTS: We found that TCDD dose-dependently suppressed OVCAR-3 cell proliferation, with a maximum effect (~70% reduction) at 100 nM. However, TCDD did not affect SKOV-3 and IOSE-385 cell proliferation and migration. The estimated IC50 of TCDD for inhibiting OVCAR-3 cell proliferation was 4.6 nM. At 10 nM, TCDD time-dependently decreased AhR protein levels, while it significantly increased CYP1A1 and CYP1B1 mRNA levels in SKOV-3, OVCAR-3 and IOSE-385 cells, indicating activation of AhR signaling. siRNA-mediated AhR knockdown readily blocked TCDD-mediated suppression of OVCAR-3 cell proliferation. CONCLUSION: Our data indicate that TCDD can suppress human ovarian cancer cell proliferation via the AhR signaling pathway and that TCDD exhibits an anti-proliferative activity in at least a subset of human ovarian cancer cells.

Distinct roles of HIF1A in endothelial adaptations to physiological and ambient oxygen.

Fetoplacental endothelial cells reside under physiological normoxic conditions (∼2-8% O2) in vivo. Under such conditions, cells are believed to sense O2 changes primarily via hypoxia inducible factor 1 α (HIF1A). However, little is known regarding the role of HIF1A in fetoplacental endothelial function under physiological normoxia. We recently reported that physiological chronic normoxia (PCN; 20-25 day, 3% O2) enhanced FGF2- and VEGFA-stimulated proliferation and migration of human umbilical vein endothelial cells (HUVECs) via the MEK/ERK1/2 and PI3K/AKT1 pathways compared to standard cell culture normoxia (SCN; ambient O2: ∼21% O2). Here, we investigated the action of HIF1A in regulating these cellular responses in HUVECs. HIF1A adenovirus infection in SCN-cells increased HIF1A protein expression, enhanced FGF2- and VEGFA-stimulated cell proliferation by 2.4 and 2.0-fold respectively, and promoted VEGFA-stimulated cell migration by 1.4-fold. HIF1A adenovirus infection in SCN-cells did not affect either basal or FGF2- and VEGFA-induced ERK1/2 activation, but it decreased basal AKT1 phosphorylation. Interestingly, HIF1A knockdown in PCN-cells via specific HIF1A siRNA transfection did not alter FGF2- and VEGFA-stimulated cell proliferation and migration, or ERK1/2 activation; however, it inhibited FGF2-induced AKT1 activation by ∼50%. These data indicate that HIF1A differentially regulates cell proliferation and migration, and ERK1/2 and AKT1 activation in PCN- and SCN-HUVECs. These data also suggest that HIF1A critically regulates cell proliferation and migration in SCN-, but not in PCN-HUVECs.

Estradiol 17ß and its metabolites stimulate cell proliferation and antagonize ascorbic acid-suppressed cell proliferation in human ovarian cancer cells.

Estradiol 17ß (E2ß) and ascorbic acid (AA) have been implicated in cancer progression. However, little is known about the actions of biologically active metabolites of E2ß, 2-hydroxyestradiol (2OHE2), 4-hydroxyestradiol (4OHE2), 2-methoxyestradiol (2ME2), and 4-methoxyestradiol (4ME2) synthesized sequentially by cytochrome P450, family 1, subfamily A (CYP1A1) and B (CYP1B1), polypeptide 1, and catechol-O-methyltransferase (COMT) on ovarian cancer. Herein, we examined the expression of CYP1A1, CYP1B1, COMT, and estrogen receptor α (ERα) and ß (ERß) in human ovarian surface epithelial (IOSE-385) and cancer cell lines (OVCAR-3, SKOV-3, and OVCA-432). We also investigated the roles of E2ß, 2OHE2, 4OHE2, 2ME2, and 4ME2 in cell proliferation, and their interactive effects with AA on ovarian cells. We found the expression of CYP1A1, CYP1B1, COMT, ERα, and ERß in most cell lines tested. Treating cells with physiological concentrations of E2ß and its metabolites promoted (13%-42% of the control) IOSE-385 and OVCAR-3 proliferation. The ER blockade inhibited IOSE-385 (∼76%) and OVCAR-3 (∼87%) proliferative response to E2ß but not to its metabolites. The ERα blockade inhibited (∼85%) E2ß-stimulated OVCAR-3 proliferation, whereas ERß blockade attenuated (∼83%) E2ß-stimulated IOSE-385 proliferation. The AA at ≥250 µmol/L completely inhibited serum-stimulated cell proliferation in all cell lines tested; however, such inhibition in IOSE-385, OVCAR-3, and OVCA-432 was partially (∼10%-20%) countered by E2ß and its metabolites. Thus, our findings indicate that E2ß and its metabolites promote cell proliferation and antagonize the AA-suppressed cell proliferation in a subset of ovarian cancer cells, suggesting that blocking the actions of E2ß and its metabolites may enhance AA's antiovarian cancer activity.

Enhanced cellular responses and distinct gene profiles in human fetoplacental artery endothelial cells under chronic low oxygen.

Fetoplacental endothelial cells are exposed to oxygen levels ranging from 2% to 8% in vivo. However, little is known regarding endothelial function within this range of oxygen because most laboratories use ambient air (21% O2) as a standard culture condition (SCN). We asked whether human umbilical artery endothelial cells (HUAECs) that were steadily exposed to the physiological chronic normoxia (PCN, 3% O2) for ∼20-25 days differed in their proliferative and migratory responses to FGF2 and VEGFA as well as in their global gene expression compared with those in the SCN. We observed that PCN enhanced FGF2- and VEGFA-stimulated cell proliferation and migration. In oxygen reversal experiments (i.e., when PCN cells were exposed to SCN for 24 h and vice versa), we found that preexposure to 21% O2 decreased the migratory ability, but not the proliferative ability, of the PCN-HUAECs in response to FGF2 and VEGFA. These PCN-enhanced cellular responses were associated with increased protein levels of HIF1A and NOS3, but not FGFR1, VEGFR1, and VEGFR2. Microarray analysis demonstrated that PCN up-regulated 74 genes and down-regulated 86, 14 of which were directly regulated by hypoxia-inducible factors as evaluated using in silico analysis. Gene function analysis further indicated that the PCN-regulated genes were highly related to cell proliferation and migration, consistent with the results from our functional assays. Given that PCN significantly alters cellular responses to FGF2 and VEGFA as well as transcription in HUAECs, it is likely that we may need to reexamine the current cellular and molecular mechanisms controlling fetoplacental endothelial functions, which were largely derived from endothelial models established under ambient O2.

An endogenous aryl hydrocarbon receptor ligand inhibits proliferation and migration of human ovarian cancer cells.

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor mediates many biological processes. Herein, we investigated if 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE, an endogenous AhR ligand) regulated proliferation and migration of human ovarian cancer cells via AhR. We found that AhR was widely present in many histotypes of ovarian cancer tissues. ITE suppressed OVCAR-3 cell proliferation and SKOV-3 cell migration in vitro, which were blocked by AhR knockdown. ITE also suppressed OVCAR-3 cell growth in mice. These data suggest that the ITE might potentially be used for therapeutic intervention for at least a subset of human ovarian cancer.

Transcriptional and functional adaptations of human endothelial cells to physiological chronic low oxygen.

Endothelial cells chronically reside in low-O2 environments in vivo (2%-13% O2), which are believed to be critical for cell homeostasis. To elucidate the roles of this physiological chronic normoxia in human endothelial cells, we examined transcriptomes of human umbilical vein endothelial cells (HUVECs), proliferation and migration of HUVECs in response to fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGFA), and underlying signaling mechanisms under physiological chronic normoxia. Immediately after isolation, HUVECs were cultured steadily under standard cell culture normoxia (SCN; 21% O2) or physiological chronic normoxia (PCN; 3% O2) up to 25 days. We found that PCN up-regulated 41 genes and down-regulated 21 genes, 90% of which differed from those previously reported from HUVECs cultured under SCN and exposed to acute low O2. Gene ontology analysis indicated that PCN-regulated genes were highly related to cell proliferation and migration, consistent with the results from benchtop assays that showed that PCN significantly enhanced FGF2- and VEGFA-stimulated cell proliferation and migration. Interestingly, preexposing the PCN cells to 21% O2 up to 5 days did not completely diminish PCN-enhanced cell proliferation and migration. These PCN-enhanced cell proliferations and migrations were mediated via augmented activation of MEK1/MEK2/ERK1/ERK2 and/or PI3K/AKT1. Importantly, these PCN-enhanced cellular responses were associated with an increase in activation of VEGFR2 but not FGFR1, without altering their expression. Thus, PCN programs endothelial cells to undergo dramatic changes in transcriptomes and sensitizes cellular proliferative and migratory responses to FGF2 and VEGFA. These PCN cells may offer a unique endothelial model, more closely mimicking the in vivo states.