Mechanism of METTL14 and m6A modification of lncRNA MALAT1 in the proliferation of oral squamous cell carcinoma cells.

OBJECTIVES: Methyltransferase-like 14 (METTL14) plays an epigenetic role in various cancer through N6-methyladenosine (m6A) modification. This study sought to analyze the mechanism of METTL14 in oral squamous cell carcinoma (OSCC) cell proliferation. METHODS: Expression levels of METTL14, lncRNA metastasis associated with lung adenocarcinoma transcript 1 (lncRNA MALAT1), microRNA (miR)-224-5p, and histone lysine demethylase 2A (KDM2A) in OSCC tissues (N = 40), and cell lines (FaDu, SCC-25, CAL-27, and SCC-15) were detected. Cell viability and colony formation capacity were assessed. m6A level, stability, and subcellular localization of lncRNA MALAT1 were determined. Nude mouse xenograft tumor assay was performed to confirm the role of METTL14 in vivo. RESULTS: METTL14 and lncRNA MALAT1 were upregulated, and miR-224-5p was downregulated in OSCC tissues and cells. Silencing METTL14 repressed OSCC cell viability and colony formation. Overexpression of MALAT1 and KDM2A or miR-224-5p downregulation reversed the inhibition of silencing METTL14 on OSCC cell proliferation. METTL14 induced m6A modification of MALAT1 to upregulate MALAT1. MALAT1 is comparatively bound to miR-224-5p to promote KDM2A transcription. In vivo, METTL14 promoted tumor growth via regulating MALAT1/miR-224-5p/ KDM2A. CONCLUSIONS: Overall, our findings verified the therapeutic role of silencing METTL14 in OSCC treatment through the MALAT1/miR-224-5p/KDM2A axis.

Rapid and sensitive analysis of trace ß-blockers by magnetic solid-phase extraction coupled with Fourier transform ion cyclotron resonance mass spectrometry.

A rapid and sensitive method for analyzing trace ß-blockers in complex biological samples, which involved magnetic solid-phase extraction (MSPE) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), was developed. Novel nanosilver-functionalized magnetic nanoparticles with an interlayer of poly(3,4-dihydroxyphenylalanine) (polyDOPA@Ag-MNPs) were synthesized and used as MSPE adsorbents to extract trace ß-blockers from biological samples. After extraction, the analytes loaded on the polyDOPA@Ag-MNPs were desorbed using an organic solvent and analyzed by FTICR-MS. The method was rapid and sensitive, with a total detection procedure of less than 10 min as well as limits of detection and quantification in the ranges of 3.5-6.8 pg/mL and 11.7-22.8 pg/mL, respectively. The accuracy of the method was also desirable, with recoveries ranging from 80.9% to 91.0% following the detection of analytes in human blood samples. All the experimental results demonstrated that the developed MSPE-FTICR-MS method was suitable for the rapid and sensitive analysis of trace ß-blockers in complex biological samples.

CdS QDs: Facile synthesis, design and application as an "on-off" sensor for sensitive and selective monitoring Cu2+, Hg2+ and Mg2+ in foods.

A new yellow CdS quantum dots (CdS QDs) with 59% quantum yield were synthesized successfully by one-pot method using thioglycolic acid as stabilizer, thiourea and sodium sulfide as double reducing agents. Based on CdS QDs, a novel "on-off" sensor for detecting Cu2+, Hg2+ and Mg2+ in foods has been constructed in the presence of glutathione (GSH). The fluorescence intensity of CdS QDs is improved about 1.5-fold by GSH, then quenched linearly by Cu2+, Hg2+ and Mg2+ owning to the electron transfer or ligand competition. Interestingly, without GSH, CdS QDs has no fluorescence response to Hg2+ at all, and with GSH, linear ranges of Cu2+ and Mg2+ is not only broader, but sensitivities are improved 16.9-fold for Cu2+ and 8.5-fold for Mg2+. GSH-CdS QDs sensor discloses a more sensitive, efficient and selective methodology for monitoring Cu2+, Hg2+ and Mg2+ in further heavy metal pollution and food safety.

Exosomes from human umbilical cord mesenchymal stem cells and human dental pulp stem cells ameliorate lipopolysaccharide-induced inflammation in human dental pulp stem cells.

OBJECTIVE: The aim of this study was to evaluate the effects of exosomes derived from human umbilical cord mesenchymal stem cells (hUCMSCs-Exo) and human dental pulp stem cells (hDPSCs-Exo) on lipopolysaccharide-induced inflammation in human dental pulp stem cells. METHODS: Exosomes were extracted from the supernatant of human umbilical cord mesenchymal stem cells and human dental pulp stem cells. Lipopolysaccharide was used to establish the inflammation model in human dental pulp stem cells. Then human dental pulp stem cells were treated with hUCMSCs-Exo and hDPSCs-Exo, respectively. We examined the proliferation, apoptosis, and secretion of inflammatory cytokines of human dental pulp stem cells in vitro. RESULTS: Exosomes had the capacity to reverse the lipopolysaccharide-induced inhibitory effect on human dental pulp stem cells proliferation and suppress lipopolysaccharide-induced apoptosis. Additionally, exosomes reduced the pro-inflammatory cytokines expression and increased anti-inflammatory cytokine expression. However, compared with hDPSCs-Exo, hUCMSCs-Exo had better ability to reverse inhibition of proliferation, suppress apoptosis and regulate release of inflammatory cytokines in human dental pulp stem cells. CONCLUSIONS: Our data suggest that exosomes could alleviate inflammation in human dental pulp stem cells. hUCMSCs-Exo do show even stronger effects than hDPSCs-Exo.

In situ detection and imaging of lysophospholipids in zebrafish using matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry.

In this paper, a matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) (MALDI-FTICR-MS) imaging method was developed to rapid and in situ detect the spatial distribution of lysophospholipids (LPLs) in zebrafish. The combination of MALDI with ultrahigh-resolution FTICR-MS achieves the MS imaging of LPLs with a mass resolution up to 50 000, which allows accurate identification and clear spatial visualization of LPLs in complex biological tissues. A series of lysophosphatidylcholines (LPCs) was detected using positive ion detection mode, and their concentration differences and spatial distributions were clearly visualized in different parts of zebrafish tissue. The method is rapid, simple, and efficient, being a desirable way to understand the spatial distribution of LPLs in biosome.

Covalent Organic Frameworks-Based Solid-Phase Microextraction Probe for Rapid and Ultrasensitive Analysis of Trace Per- and Polyfluoroalkyl Substances Using Mass Spectrometry.

Rapid and ultrasensitive analysis of trace pollutants in complex matrices is of significance for understanding their environmental behaviors and toxic effects. Here a novel method based on the integration of solid-phase microextraction (SPME) and nanoelectrospray ionization mass spectrometry (nanoESI-MS) was developed for rapid and ultrasensitive analysis of trace per- and polyfluoroalkyl substances (PFASs) in environmental and biological samples. A novel SPME probe with F-functionalized covalent organic frameworks (COFs) coating was designed for highly selective enrichment of trace PFASs from complex samples. After extraction, the loaded COFs-SPME probe was directly appplied to nanoESI-MS analysis under ambient and open-air conditions. The method showed satisfactory linearities between 1 and 5000 ng/L for 14 investigated PFASs in water, with correlation coefficient values no less than 0.9952. The limits of detection and quantification varied from 0.02 to 0.8 ng/L and 0.06 to 3 ng/L, respectively. By using the proposed method, ultrasensitive detection of PFASs in environmental water and whole blood was successfully achieved.

Analysis of trace malachite green, crystal violet, and their metabolites in zebrafish by surface-coated probe nanoelectrospray ionization mass spectrometry.

Malachite green (MG) and crystal violet (CV) are the typical triphenylmethane dyes, which are recalcitrant molecules exerting mutagenic and carcinogenic effects on living organisms. Characterization of the residues of MG, CV, and their metabolites in biological organisms is of importance, especially for in vivo and in situ characterization. In this study, a method for determination of trace MG, CV, and their leuco metabolites in zebrafish by surface-coated probe nanoelectrospray ionization mass spectrometry (SCP-nanoESI-MS) was developed. A microscale solid-phase microextraction (SPME) probe was developed and used for extraction and enrichment of trace MG, CV, and their leuco metabolites in zebrafish after exposure. After that, the loaded SPME probe was directly employed for nanoESI-MS analysis under ambient and open-air conditions. Under the optimum conditions, the method demonstrated good linearity, with correlation coefficient values (r2) no less than 0.9925. The limits of detection and quantification were 0.014-0.023 ng mL-1 and 0.046-0.077 ng mL-1, respectively. By using the proposed method, the bioaccumulation of MG and CV in zebrafish was investigated, and the distribution of MG, CV, and their leuco metabolites in different organs of zebrafish was studied. MG, CV, and their leuco metabolites were all found in zebrafish tissues including brain, muscle, heart, and kidney after exposure, with highest concentration in intestine followed in ovary.

Ratiometric Fluorescent Paper-Based Sensor Based on CdTe Quantum Dots and Graphite Carbon Nitride Hybrid for Visual and Rapid Determination of Cu2+ in Drinks.

A simple and effective ratiometric fluorescence sensor of CdTe QDs/GCNNs for on-site and rapid analysis of Cu2+ has been established by mixing physically CdTe QDs and graphite carbon nitride (GCNNs). Two emissions peaks of CdTe QDs at 572 nm and GCNNs at 436 nm are both excitated at 340 nm. Under a UV lamp, fluorescent of traffic yellow CdTe QDs is linearly quenched by Cu2+ (as the detection signal), while blue GCNNs remains unchanged (as the reference), resulting in a distinguishable color change gradually from pink yellow to blue. The limit of detection (LOD) of this new sensor for Cu2+ is as low as 0.47 ng mL-1 with 1.4 % RSD. The established method has been successfully applied to detection of Cu2+ in various drinks with satisfactory results. Moreover, a paper-based sensor, which has been prepared by soaking cellulose acetate membrane in CdTe QDs/GCNNs sensor solution, has a wide semiquantitative detection range for Cu2+ (0.01 ~ 5.0 µg mL-1 ). It has realized successfully on-site and rapid determination of Cu2+ in red wine without any pretreatment procedure and is of great promotion and application value in determination of Cu2+ in liquid samples.

A microscale solid-phase microextraction probe for the in situ analysis of perfluoroalkyl substances and lipids in biological tissues using mass spectrometry.

The simultaneous analysis of perfluoroalkyl substances (PFASs) and lipids in biological tissues is of importance, especially for in situ and microscale analysis, because it provides significant information to understand the relevance of content, composition, and distribution of lipids to the bioaccumulation of PFASs as well as lipid metabolism affected by the biotoxicity of PFASs. In this study, we report the development of a novel ambient mass spectrometry method for the rapid, in situ, and microscale analysis of PFASs and lipids simultaneously in biological tissues for the investigation of their biological correlation. A microscale solid-phase microextraction (SPME) probe with a probe-end diameter of several-µm was employed for in situ and microscale sampling of biological tissues after PFAS exposure. The SPME probe showed a desirable capacity for the enrichment of PFASs and lipid species simultaneously. After sampling and extraction, the loaded SPME probe was directly applied for nanoESI-MS analysis under ambient and open-air conditions. A high-resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometer operated in the field-induced mode was introduced to record mass spectra using fast polarity switching between positive and negative ion detection. Most of the lipid species were recorded in the positive ion mass spectrum, and PFASs were recorded in the negative ion mass spectrum. By using the developed method, the in situ analysis of PFASs and lipids in the muscle, brain, heart, kidney, liver, and intestine of zebrafish was realized. In addition, simultaneously imaging PFASs and lipids in individual Daphnia magna was successfully achieved for the investigation of their biological correlation.

Production of an aberrant splice variant of CCL5 is not caused by genetic mutation in the mammary glands of mastitis­infected Holstein cows.

Genetic mutations, including single nucleotide polymorphisms (SNPs), result in aberrant alternatively splicing of gene and involves in susceptibility of inflammatory diseases, including bovine mastitis. C­C motif chemokine ligand 5 (CCL5) is an immune­associated gene, but its alternative splicing (AS) mechanism of gene expression has not yet been understood. The present study identified the splice variant of CCL5 and the compared differential expression of various transcripts between healthy and mastitic mammary gland tissue from cows. A novel transcript lacking exon 2 with a deletion of 112 bp (referred to as CCL5­AS) was identified in the mammary gland. The expression of CCL5­AS was lower compared with CCL5­reference in the healthy and mastitic mammary tissues. A total of two novel SNPs (g.1647 C>T and g.1804 G>A) were identified in exon 2 of CCL5. Using the splicing mini­gene reporter assay in bovine mammary epithelial cells (MAC­T) and 293T cells, it was confirmed that the production of CCL5­AS was not caused by the two SNPs. The present findings suggested that alternative splicing is one of the mechanisms of CCL5 expression regulation and is involved in mastitis infection, but that genetic mutation was not responsible for the generation of the abnormal transcript of CCL5 in cows.

Analyte-triggered cyclic autocatalytic oxidation amplification combined with an upconversion nanoparticle probe for fluorometric detection of copper(II).

The authors describe an upconversion nanoparticle-based (UCNP-based) fluorometric method for ultrasensitive and selective detection of Cu2+. The UCNPs show a strong emission band at 550 nm under near-infrared excitation at 980 nm. The principle of the strategy is that gold nanoparticles (AuNP) can quench the fluorescence of UCNP. In contrast, the addition of L-cysteine (Cys) can induce the aggregation of AuNP, resulting in a fluorescence recovery of the UCNPs. On addition of Cu2+, it oxidizes Cys to cystine and is reduced to Cu+. The Cu+ thusformed can be oxidized cyclically to Cu2+ by dissolved O2, which catalyzes and recycles the whole reaction. Thus, the aggregation of AuNP is inhibited and the fluorescence recovered by Cys is quenched. Under the optimal condition, the quenching efficiency shows a good linear response to the concentrations of Cu2+ in the 0.4-40 nM range. The limit of detection is 0.16 nM, which is 5 orders of magnitude lower than the U.S. Environmental Protection Agency limit for Cu2+ in drinking water (20 µM). The method has been further applied to monitor Cu2+ levels in real samples. The results of detection are well consistent with those obtained by atomic absorption spectroscopy. Graphical abstract Gold nanoparticles (AuNP) as a high efficient fluorescence quenching reagent of upconversion nanoparticles (UCNP) were used in a fluorometric method for detection of Cu2+ based on a cyclic catalytic oxidation amplification strategy.

Differential expression of interferon-gamma, IL-4 and IL-10 in peripheral blood mononuclear cells during early pregnancy of the bovine.

Maternal systemic immune response is regulated by conceptus-derived signals through peripheral blood mononuclear cells (PBMCs) via blood circulation during early pregnancy in cattle. In this study, the PBMCs from day 18 in non-pregnant cows and days 14, 18 and 30 in pregnant cows were used to explore the expression of interferon-gamma (IFN-γ), interleukin 4 (IL-4) and IL-10, and the plasma progesterone (P4) concentration was also determined. The results showed that the expression levels of mRNA and the protein of IFN-γ were lower and that IL-4 and IL-10 were higher in the PBMCs from the pregnant cows than in those of non-pregnant cows. From this study, early pregnancy induced a lower Th1 immunity (IFN-γ) and a higher Th2 immunity (IL-4 and IL-10) in the PBMCs, which may be related to interferon-tau and P4, thereby contributing to successful pregnancy in cattle.

Association of the expression of Th cytokines with peripheral CD4 and CD8 lymphocyte subsets after vaccination with FMD vaccine in Holstein young sires.

High immune response (HIR) cows have a balanced and robust host defense and lower disease incidence, and immune response is more important to consider for selecting young sires than for selecting cows. The protective immune response against foot-and-mouth disease (FMD) virus infection is T-cell-independent in an animal experimental model. However, there is no convenient method to select young sires with a HIR to FMD virus. In this study, 39 healthy Holstein young sires were vaccinated with the trivalent (A, O and Asia 1) FMD vaccine, and T-lymphocyte subsets in peripheral blood lymphocytes (PBLs) were detected using flow cytometric analysis before and after vaccination. The expression of interferon-gamma (IFN-γ), interleukin-2 (IL-2), IL-4, and IL-6 mRNA in PBLs was analyzed after stimulation by lipopolysaccharide (LPS) or Concanavalin A (ConA) after vaccination. According to the percentage of CD4+ lymphocyte and CD4/CD8 ratio after vaccination for selecting the HIR young sires, the results showed that the percentages of CD3+, CD4+, CD3+CD4+ lymphocytes and the CD4/CD8 ratio in the HIR group were higher compared to those in the medium immune response (MIR) and low immune response (LIR) groups before vaccination. Additionally, the percentage of CD4+ lymphocytes and the CD4/CD8 ratio after vaccination were positively associated with the expression level of IFN-γ mRNA in the PBLs after stimulation by LPS. In conclusion, the in vitro expression level of IFN-γ mRNA in the PBLs stimulated by LPS may serve as a parameter for selecting young sires with a HIR to FMD virus.

Hypoxia-inducible factor-1 drives annexin A2 system-mediated perivascular fibrin clearance in oxygen-induced retinopathy in mice.

Oxygen-induced retinopathy (OIR) is a well-characterized model for retinopathy of prematurity, a disorder that results from rapid microvascular proliferation after exposure of the retina to high oxygen levels. Here, we report that the proliferative phase of OIR requires transcriptional induction of the annexin A2 (A2) gene through the direct action of the hypoxia-inducible factor-1 complex. We show, in addition, that A2 stabilizes its binding partner, p11, and promotes OIR-related angiogenesis by enabling clearance of perivascular fibrin. Adenoviral-mediated restoration of A2 expression restores neovascularization in the oxygen-primed Anxa2(-/-) retina and reinstates plasmin generation and directed migration in cultured Anxa2(-/-) endothelial cells. Systemic depletion of fibrin repairs the neovascular response to high oxygen treatment in the Anxa2(-/-) retina, whereas inhibition of plasminogen activation dampens angiogenesis under the same conditions. These findings show that the A2 system enables retinal neoangiogenesis in OIR by enhancing perivascular activation of plasmin and remodeling of fibrin. These data suggest new potential approaches to retinal angiogenic disorders on the basis of modulation of perivascular fibrinolysis.

Feedback regulation of endothelial cell surface plasmin generation by PKC-dependent phosphorylation of annexin A2.

In response to blood vessel injury, hemostasis is initiated by platelet activation, advanced by thrombin generation, and tempered by fibrinolysis. The primary fibrinolytic protease, plasmin, can be activated either on a fibrin-containing thrombus or on cells. Annexin A2 (A2) heterotetramer (A2·p11)(2) is a key profibrinolytic complex that assembles plasminogen and tissue plasminogen activator and promotes plasmin generation. We now report that, in endothelial cells, plasmin specifically induces activation of conventional PKC, which phosphorylates serine 11 and serine 25 of A2, triggering dissociation of the (A2·p11)(2) tetramer. The resulting free p11 undergoes ubiquitin-mediated proteasomal degradation, thus preventing further translocation of A2 to the cell surface. In vivo, pretreatment of A2(+/+) but not A2(-/-) mice with a conventional PKC inhibitor significantly reduced thrombosis in a carotid artery injury model. These results indicate that augmentation of fibrinolytic vascular surveillance by blockade of serine phosphorylation is A2-dependent. We also demonstrate that plasmin-induced phosphorylation of A2 requires both cleavage of A2 and activation of Toll-like receptor 4 on the cell surface. We propose that plasmin can limit its own generation by triggering a finely tuned "feedback" mechanism whereby A2 becomes serine-phosphorylated, dissociates from p11, and fails to translocate to the cell surface.

The endothelial cell annexin A2 system and vascular fibrinolysis.

Vascular endothelial cell surface expression of annexin A2 and its binding partner p11 is a key element in maintaining fibrinolytic balance on blood vessel surfaces. In the recent decade, investigators have made significant progress toward understanding the mechanisms that regulate heterotetrameric (A2*p11)(2) receptor translocation from the cytoplasm to the outer cell surface. Accumulating evidence now shows that heterotetrameric (A2*p11)(2) cell surface expression is a dynamic process that modulates plasmin activation during periods of vascular stress or injury, and is independent of the classical endoplasmic reticulum-Golgi pathway. Translocation of heterotetrameric (A2*p11)(2) is facilitated both by src-kinase mediated phosphorylation of A2 at tyrosine 23, and by expression of and partnering with p11. In the absence of A2 both in vivo and in vitro, p11 is expressed at very low levels in endothelial cells, because unpartnered p11 is polyubiquitinated and rapidly degraded through a proteasome-dependent mechanism. A2 directly binds and stabilizes intracellular p11 by masking an autonomous polyubiquitination signal on p11. This modulatory role of A2 binding prevents accumulation of unpartnered p11 within the endothelial cell, and ultimately suggests that the regulation of heterotetrameric (A2*p11)(2) receptor surface expression is precisely attuned to the intracellular level of p11.

Endothelial cell annexin A2 regulates polyubiquitination and degradation of its binding partner S100A10/p11.

The annexin A2 (A2) heterotetramer, consisting of two copies of A2 and two copies of S100A10/p11, promotes fibrinolytic activity on the surface of vascular endothelial cells by assembling plasminogen and tissue plasminogen activator (tPA) and accelerating the generation of plasmin. In humans, overexpression of A2 by acute promyelocytic leukemia cells is associated with excessive fibrinolysis and hemorrhage, whereas anti-A2 autoantibodies appear to accentuate the risk of thrombosis in patients with anti-phospholipid syndrome. Complete deficiency of A2 in mice leads to a lack of tPA cofactor activity, accumulation of intravascular fibrin, and failure to clear arterial thrombi. Within the endothelial cell, p11 is required for Src kinase-mediated tyrosine phosphorylation of A2, which signals translocation of both proteins to the cell surface. Here we show that p11 is expressed at very low levels in the absence of A2 both in vitro and in vivo. We demonstrate further that unpartnered p11 becomes polyubiquitinated and degraded via a proteasome-dependent mechanism. A2 stabilizes intracellular p11 through direct binding, thus masking an autonomous p11 polyubiquitination signal that triggers proteasomal degradation. This interaction requires both the p11-binding N-terminal domain of A2 and the C-terminal domain of p11. This mechanism prevents accumulation of free p11 in the endothelial cell and suggests that regulation of tPA-dependent cell surface fibrinolytic activity is precisely tuned to the intracellular level of p11.

Listeria monocytogenes promotes tumor growth via tumor cell toll-like receptor 2 signaling.

The contribution of bacterial infection to tumorigenesis is usually ascribed to infection-associated inflammation. An alternate view is that direct interaction of bacteria with tumor cells promotes tumor progression. Here, we show that the microenvironment of large tumors favors bacterial survival, which in turn directly accelerates tumor growth by activating tumor cell Toll-like receptors (TLR). Listeria monocytogenes (Lm) survives in the microenvironment of large but not small tumors, resulting in the promotion of tumor growth. Lm did not affect the percentage of regulatory T cells or myeloid suppressor cells in the tumor. Through TLR2 signaling, Lm activated mitogen-activated protein kinases and nuclear factor-kappaB in tumor cells, resulting in the increased production of nitric oxide and interleukin-6 and increased proliferation of tumor cells. All of these effects were abrogated by silencing expression of TLR2, but not TLR4. The interaction of Helicobacter pylori with tumor cells from gastric carcinoma patients resulted in similar effects. These findings provide a new insight into infection-associated tumorigenesis and illustrate the importance of antibiotic therapy to treat tumors with bacterial infiltration.