In this study, a simple and effective ratiometric fluorescence method has been developed for carbaryl detection, utilizing red emissive carbon dots (R-CDs). The underlying principle of this proposed strategy relies on the rapid hydrolysis of carbaryl under an alkaline condition and production of 1-naphthol with blue-emission at 462 nm. Furthermore, the as-synthesized R-CDs (Em. 677 nm), serve as a reference, enhancing the visual tracking of carbaryl through the transformation of fluorescent color from red to blue. The concentration of carbaryl exhibits a commendable linear correlation with the ratio of fluorescence intensity, ranging from 0 to 20 μg/mL (R2 = 0.9989) with a low detection limit of 0.52 ng/mL. Additionally, the described methodology can be used for the enzyme-free visual assay of carbaryl, even in the presence of other carbamate pesticides and metal ions, in tap water and lake water samples with excellent accuracy (spiked recoveries, 94%–106.1%), high precision (relative standard deviation (RSD) ≤ 2.42), and remarkable selectivity. This fast and highly sensitive naked-eye ratiometric sensor holds immense promise for carbaryl detection in intricate environments and food safety fields.

Cervical cancer causes malignant tumors in females and threatens the physical and mental health of women. Current research shows that persistent infection of high-risk HPV is the main cause of cervical cancer. However, not all cervical cancer is caused by HPV infection, which may also be related to other factors, such as nutritional status and lifestyle. This study focuses on the effect of alcohol consumption on the methylation status of cervical cancer from the perspective of methyl donors. We established a mouse tumor-bearing model with cervical cancer SiHa cells, and at the same time, we cultured SiHa cells in vitro. Different concentrations of ethanol were administered to the model mice and SiHa cells. Then, we detected the levels of the methyl-donor folate and methionine and their metabolite homocysteine levels in mice serum, tumor tissues, and SiHa cells. Furthermore, we determined the expression of the members of the DNA methyltransferase family (DNMT1, DNMT3a, and DNMT3b) in tumor tissue by immunohistochemistry. qRT-PCR and Western blotting analysis were used to detect the mRNA and protein levels of members of the DNA methyltransferase family in cervical cancer SiHa cells. Our results show that the levels of the methyl donor (folate and methionine) decreased with the increase of ethanol concentration (p < 0.05), and the homocysteine level increased significantly (p < 0.05). In SiHa cells, the mRNA and protein levels of the DNMT family members and their receptors were significantly higher than those in the control group (p < 0.05). Collectively, these results suggest that ethanol could influence DNMT expression by inducing methyl donor consumption, thereby causing cervical cancer cells to exhibit genome-wide hypomethylation.

Polycystic ovarian syndrome (PCOS) is often accompanied by overweight/obesity and insulin resistance. The dysfunctions of ovarian granulosa cells (GCs) are closely linked with the pathogenesis of PCOS. Fat mass and obesity-associated gene (FTO), an N6-methyladenosine (m6A) demethylase, has been reported to be implicated in the risks and insulin resistance of PCOS. However, the roles of FTO in the development of GCs along with its m6A-related regulatory mechanisms are poorly defined. Cell proliferative ability was detected by MTT assay. Cell apoptotic rate was measured via flow cytometry. Insulin resistance was assessed by GLUT4 transport potential. The mRNA and protein levels of FTO and flotillin 2 (FLOT2) were determined by RT-qPCR and western blot assays, respectively. FLOT2 was screened out to be a potential FTO target through differential expression analysis for the GSE95728 dataset and target prediction analysis by POSTAR2 and STARBASE databases. The interaction between FTO and FLOT2 was analyzed by RNA immunoprecipitation (RIP) assay. The effect of FTO upregulation on FLOT2 m6A level was measured by methylated RIP (meRIP) assay. FLOT2 mRNA stability was examined by actinomycin D assay. FTO overexpression facilitated cell proliferation, hindered cell apoptosis, and induced insulin resistance in GCs. FTO promoted FLOT2 expression by reducing m6A level on FLOT2 mRNA and increasing FLOT2 mRNA stability. FLOT2 loss weakened the effects of FTO overexpression on cell proliferation/apoptosis and insulin resistance in GCs. FTO induced the dysfunctions of GCs by upregulating FLOT2, suggesting that FTO/FLOT2 might play a role in the pathophysiology of PCOS.

Oocyte maturation is a prerequisite for successful fertilization and embryo development. Incomplete oocyte maturation can result in infertility. Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) has been found to be implicated in oocyte maturation and embryo development. However, the cellular and molecular mechanisms of UCH-L1 underlying oocyte maturation have not been fully elucidated. In the present study, we observed that the introduction of UCH-L1 inhibitor LDN-57444 suppressed first polar body extrusion during mouse oocyte maturation. The inhibition of UCH-L1 by LDN-57444 led to the notable increase in reactive oxygen species (ROS) level, conspicuous reduction in glutathione (GSH) content and mitochondrial membrane potential (MMP), and blockade of spindle body formation. As a conclusion, UCH-L1 inhibitor LDN-57444 suppressed mouse oocyte maturation by improving oxidative stress, attenuating mitochondrial function, curbing spindle body formation and down-regulating extracellular signal-related kinases (ERK1/2) expression, providing a deep insight into the cellular and molecular basis of UCH-L1 during mouse oocyte maturation.

Food enriched with organic selenium is considered as a good source for selenium supplementation. In the current research, we cultivated Pleurotus citrinopileatus with medium containing different levels of sodium selenate, evaluated the antioxidant activity, and sequenced the transcriptome of the Se-enriched mushroom. Selenium content in Se-enriched mushroom is dependent on selenium level in the surroundings. The ABTS total radical scavenging ability was not significantly different between selenium enriched mushroom and the control, though the total phenol content was increased in Se-enriched mushroom. Transcriptome sequencing showed a total of 1036 differentially expressed genes, including 987 upregulated genes and 49 downregulated genes. These differentially expressed genes are involved in 20 metabolic pathways, most of which are involved in carbon metabolism and amino acid biosynthesis, while many differentially expressed genes are in growth, plasma membrane, and protein binding. It needs to be noted that the sulfur metabolism and ABS transporters, which are closely related with selenium metabolism and transportation, are particularly enriched. The mushroom P. citrinopileatus has strong ability to uptake selenium from the surroundings, which modifies many biological pathways. This paper provides a theoretical reference for the development of Se-enriched fungal foods.

IntroductionReports suggest that lncRNAs have implications in the development of several diseases including cancer. It is therefore believed that lncRNAs may act as therapeutic targets for cancer treatment. The treatment of ovarian cancer is mainly obstructed by lack of biomarkers and efficient drug targets. Against this backdrop, this study was undertaken to reveal the role and therapeutic implications of lncRNA CCAT2 in ovarian cancer.Material and methodsExpression analysis was carried out with quantitative real-time polymerase chain reaction (qRT-PCR). Transfections were carried out using Lipofectamine 2000 reagent. Cell counting kit 8 (CCK-8) assays were used to determine the cell viability. AO/EB and annexin V/propidium iodide (PI) were used for detection of apoptosis. Wound healing and transwell assays were used to determine cell migration and invasion. The expression of the proteins was estimated by western blotting.ResultsThe results showed that the expression of CCAT2 was significantly overexpressed in ovarian cancer tissues and cell lines. Si-RNA mediated silencing of CCAT2 resulted in the decrease of proliferation rate and colony formation potential of the OVACAR-3 cells via induction of apoptotic cell death, which was also accompanied by cleavage of PARP, downregulation of Bcl-2 and upregulation of Bax, caspase-3 and caspase-9. Suppression of CCAT2 enhanced the chemosensitivity of the OVACAR-3 cells to cisplatin and also decreased their migration and invasion.ConclusionsThe findings of this study revealed that lncRNA CCAT2 suppression inhibits the proliferation, drug sensitivity, and metastasis of ovarian cancer and may prove beneficial in ovarian cancer management.

Panax ginseng is a natural medicine that has been used globally for a long time. Moreover, several studies have reported the effective activity of ginseng in treating malignancies. Various agents containing ginseng were widely used as an antitumor treatment nowadays. Lung cancer is the most common fatal cancer in China, and lung adenocarcinoma is the most common histological type of non-small cell lung cancer (NSCLC). What's worse, many patients may have a failed response to conventional therapy including chemotherapy, radiotherapy, or molecule-targeted therapy due to drug resistance. Apoptosis is a highly ordered cellular suicidal process that plays an essential role in maintaining normal homeostasis. The pharmacological mechanism of many antineoplastic drugs involves triggering of apoptotic process. In several recent studies, ginsenosides are regarded as major active components of ginseng that have the potential to control lung cancer. Most of these results have proved that ginsenosides induce apoptosis in lung cancer cells through many different signaling pathways such as PI3K/Akt, NF-κB, EGFR, and so on. This study is aimed at reviewing the signaling pathways that underlie ginsenosides-triggered apoptotic process and encourage further studies to target promising agents against lung cancer treatment.

Wound healing is a complicated event that develops in three overlapping phases: inflammatory, proliferative, and remodeling. MicroRNAs (miRNAs) have been proved to play an important role in the healing process of skin trauma, and alteration of specific miRNA expression during different phases may be associated with abnormal wound healing. In this study, we determined the variation of miR-23b expression after trauma in normal mice and in cultured cells exposed to lipopolysaccharide. We further demonstrated that excessive miR-23b could significantly accelerate wound healing in vivo. Up-regulation of miR-23b decreases infiltration of inflammatory cells, as evidenced by pathologic staining. Meanwhile, miR-23b could significantly inhibit the expression of pro-inflammatory cytokines, including TNF-α, IL-1β, IL-6, and Ccl2, and significantly increase anti-inflammatory factor IL-10. Furthermore, miR-23b could also promote α-SMA expression in a fiber pattern and increase the expression of Col1a1 and Col3a1. Importantly, we also showed that miR-23b could inhibit inflammation to promote wound healing by targeting apoptotic signal-regulating kinase 1 (ASK1). Notably, knockdown of ASK1 could reduce inflammation factor expression in vitro. Together, our data reveal that miR-23b is a potent therapeutic agent for cutaneous wound healing that shortens the period of inflammatory responses and promotes keratinocyte migration for the re-epithelialization of wound sites.

As a progressive age-related neurodegenerative disorder, Alzheimer's disease (AD) is a global health concern. Despite the availability of psychological testing, neuroimaging, genetic testing, and biochemical assays of cerebrospinal fluid, convenient and accurate blood biomarkers for the prediction, diagnosis, and preclinical studies of AD are still lacking. The present study aims to longitudinally evaluate the feasibility of β-amyloid proteins, α2-macroglobulin (α-2M), complement factor H (CFH), and clusterin as blood biomarkers of AD. Using APP/PS1 transgenic and wild-type mice, cognitive impairment and amyloid plaque counts in the brain were evaluated over a range of ages using the Morris water maze test and immunohistochemistry methods, respectively. Serum Aβ40, Aβ42, α-2M, CFH, and clusterin levels were measured by enzyme-linked immunosorbent assay and correlated with progression of AD. APP/PS1 transgenic mice presented progressive AD characteristics at the ages of 3, 6, 9, and 12 months. Serum Aβ42 levels and Aβ42/Aβ40 ratios increased significantly in transgenic 3- and 6-month-old mice compared with controls. Serum CFH levels decreased significantly in 3- and 6-month-old transgenic mice compared with controls. Meanwhile, serum clusterin levels increased significantly in 12-month-old transgenic mice compared with controls. The α-2M level was not significantly different between transgenic and wild-type mice. The APP/PS1 transgenic mouse is a model of familial AD. The present study indicated that the serum Aβ42 level, Aβ42/Aβ40 ratio, and CFH level are potential biomarkers in preclinical and early stages of AD, whereas serum clusterin level is a potential biomarker in the late stage of AD.

TEA domain (TEAD) transcription factors bind to the coactivators YAP and TAZ and regulate the transcriptional output of the Hippo pathway, playing critical roles in organ size control and tumorigenesis. Protein S-palmitoylation attaches a fatty acid, palmitate, to cysteine residues and regulates protein trafficking, membrane localization and signaling activities. Using activity-based chemical probes, we discovered that human TEADs possess intrinsic palmitoylating enzyme-like activities and undergo autopalmitoylation at evolutionarily conserved cysteine residues under physiological conditions. We determined the crystal structures of lipid-bound TEADs and found that the lipid chain of palmitate inserts into a conserved deep hydrophobic pocket. Strikingly, palmitoylation did not alter TEAD's localization, but it was required for TEAD's binding to YAP and TAZ and was dispensable for its binding to the Vgll4 tumor suppressor. Moreover, palmitoylation-deficient TEAD mutants impaired TAZ-mediated muscle differentiation in vitro and tissue overgrowth mediated by the Drosophila YAP homolog Yorkie in vivo. Our study directly links autopalmitoylation to the transcriptional regulation of the Hippo pathway.

The human TEAD family of transcription factors (TEAD1-4) is required for YAP-mediated transcription in the Hippo pathway. Hyperactivation of TEAD's co-activator YAP contributes to tissue overgrowth and human cancers, suggesting that pharmacological interference of TEAD-YAP activity may be an effective strategy for anticancer therapy. Here we report the discovery of a central pocket in the YAP-binding domain (YBD) of TEAD that is targetable by small-molecule inhibitors. Our X-ray crystallography studies reveal that flufenamic acid, a non-steroidal anti-inflammatory drug (NSAID), binds to the central pocket of TEAD2 YBD. Our biochemical and functional analyses further demonstrate that binding of NSAIDs to TEAD inhibits TEAD-YAP-dependent transcription, cell migration, and proliferation, indicating that the central pocket is important for TEAD function. Therefore, our studies discover a novel way of targeting TEAD transcription factors and set the stage for therapeutic development of specific TEAD-YAP inhibitors against human cancers.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx or GPx4; EC1.11.1.12) is a selenoperoxidase that can directly reduce phospholipid and cholesterol hydroperoxides. The mature cytoplasmic GPx4 is a monomeric protein with molecular weight of 19.5 kDa. In this study, human GPx4 (hGPx4) gene was amplified from the complementary DNA library of human hepatoma cell line. Eukaryotic expression plasmid pSelExpress1-leader-GPx4 was constructed and transfected into the eukaryotic cells HEK293T. Expression of hGPx4 was detected by Western blotting, and the target protein was purified by immobilized metal affinity chromatography. The results of the activity and kinetics of the purified protein show that the obtained protein follows a ping-pong mechanism, which is similar to that of native cytosolic glutathione peroxidase (GPx1; EC1.11.1.9). This is the first time that hGPx4 could be expressed and purified from HEK293T cells, and this work will provide an important resource of hGPx4 for its functional study in vitro and in vivo. (c) 2013 IUBMB Life, 65(11):951-956, 2013

Glutathione peroxidase (GPX) is a crucial antioxidant selenocysteine (Sec) containing enzyme which plays a significant role in protecting cells against oxidative damage by catalyzing the reduction of hydroperoxides with glutathione (GSH). Several methods have been used to generate GPX mimics, however, only a few of these methods involved genetic engineering and none of them have achieved specific site-directed incorporation of Sec without other modifications, which has hampered further structure-function studies. Here, we report for the first time the conversion of human glutathione transferase Zeta (hGSTZ1-1) into seleno-hGSTZ1-1 by means of genetic engineering in eukaryotes. Fluorescence microscopy images of the expression of Seleno-GST-green fluorescent protein chimaera indicated that we successfully achieved the read-through of the UGA codon to specifically incorporate Sec. Therefore, we achieved the conversion of human glutathione transferase Zeta (hGSTZ1-1) into a seleno-GST (seleno-hGSTZ1-1) by means of genetic engineering in eukaryotes. These results show that recombinant selenoproteins with incorporation of specific selenocysteine residues may be heterologously produced in eukaryotes by using a Sec insertion sequence in the 3' untranslated region (3'-UTR) of the mRNA, and the recombinant selenoproteins is single catalytically active residue and well-characterized structure. In this case a novel GPX activity of 2050±225 U/μmol was introduced into hGSTZ1-1 by substitution of serine 15 by Sec 15. This result will lay a foundation for preparing much smaller GPX mimics with higher activity.

As one of the most important antioxidant enzymes, glutathione peroxidase(GPX) protects cells and tissues from oxidative damage, and plays an important role in cardiovascular and cerebrovascular injuries induced by oxidative stress. The antioxidant effect of selenium-containing glutathione S-transferase(Se-GST), a mimic of GPX was investigated on rat cardiomyocytes. To explore the protection function of Se-GST in hydrogen peroxide(H2O2) challenged rat cardiomyocytes, we examined malondialdehyde(MDA), lactate dehydrogenase(LDH), superoxide dismutase(SOD) and cell apoptosis. The results demonstrate exposure of rat cardiomyocytes to H2O2 for 6 and 12 h induced the significant increases of MDA, LDH and apoptosis rate of cardiomyocytes, but pretreatment of rat cardiomyocytes with Se-GST at 0.0005 or 0.001 unit/mL prevents oxidative stress induced by H2O2 with the decreases of cell apoptosis. All the results hint Se-GST has antioxidant activity for oxidative stress challenged rat cardiomyocytes.

Accumulating evidence shows that glutathione peroxidase (GPX, EC.1.11.1.9), one of the most important antioxidant selenoenzymes, plays an essential role in protecting cells and tissues against oxidative damage by catalyzing the reduction of hydrogen peroxide by glutathione. Unfortunately, because of the limited availability and poor stability of GPX, it has not been used clinically to protect against oxidative stress. To overcome these problems, it is necessary to generate mimics of GPX. In this study, we have used directed mutagenesis and the inclusion of a selenocysteine (Sec) insertion sequence to engineer the expression in eukaryotic cells of human glutathione transferase zeta1–1 (hGSTZ1–1) with Sec in the active site (seleno‐hGSTZ1–1). This modification converted hGSTZ1–1 into an active GPX and is the first time this has been achieved in eukaryotic cells. The GPX activity of seleno‐hGSTZ1–1 is higher than that of GPX from bovine liver, indicating Sec at the active site plays an important role in the determination of catalytic specificity and performance. Kinetic studies revealed that the ping–pong catalytic mechanism of Se‐hGSTZ1–1 is similar to that of the natural GPX. Copyright © 2012 John Wiley & Sons, Ltd.

Lipid-bound [VHb(406)] and lipid-free [VHb(402)] wide type Vitreoscilla hemoglobin were separated from E. coli BL21(DE3). The R(Z) is 3.30 for [VHb(406)] and 3.15 for [VHb(402)], respectively. VHb(406) shows a characteristic absorption band at 626 nm, while VHb(402) shows one at 644 nm, and it has more a-helix than VHb(402). Data of Raman spectra experiment shows under the excitation wavelength 488 nm, v(4) vibration of both VHb(402) and VHb(406) had a pure and strong signal at 1375 cm(-1), which proves that iron porphyrin of both the samples is at their trivalence oxidation state. And no matter lipid binds VHb or not, the vinyl of porphyrin is at cis state. The catalase activity of Vitreoscilla hemoglobin was explored with L-dopa and H(2)O(2) as substrates. The results indicate that VHb(406) has more catalase activity than VHb(402), VHb is a cell's oxygen modulator and its catalase ability is modulated by the membrane.