Neuregulin 4 (Nrg4) cooperates with melatonin to regulate the PRL expression via ErbB4/Erk signaling pathway as a potential prolactin (PRL) regulator.

Neuregulin-4 (Nrg4) and melatonin play vital roles in endocrine diseases. However, there is little discussion about the function and potential mechanism of Nrg4 and melatonin in prolactin (PRL) regulation. The human normal pituitary data from Gene Expression Profiling Interactive Analysis (GEPIA) database was used to explore the correlation between NRG4 and PRL. The expression and correlation of NRG4 and PRL were determined by Immunofluorescence staining (IF) and human normal pituitary tissue microarray. Western Blot (WB) was used to detect the expression of PRL, p-ErbB2/3/4, ErbB2/3/4, p-Erk1/2, Erk1/2, p-Akt and Akt in PRL-secreting pituitary GH3 and RC-4B/C cells treated by Nrg4, Nrg4-small interfering RNA, Erk1/2 inhibitor FR180204 and melatonin. The expression of NRG4 was significantly positively correlated with that of PRL in the GEPIA database and normal human pituitary tissues. Nrg4 significantly increased the expression and secretion of PRL and p-Erk1/2 expression in GH3 cells and RC-4B/C cells. Inhibition of Nrg4 significantly inhibited PRL expression. The increased levels of p-Erk1/2 and PRL induced by Nrg4 were abolished significantly in response to FR180204 in GH3 and RC-4B/C cells. Additionally, Melatonin promotes the expression of Nrg4, p-ErbB4, p-Erk1/2, and PRL and can further promote the expression of p-Erk1/2 and PRL in combination with Nrg4. Further investigation into the function of Nrg4 and melatonin on PRL expression and secretion may provide new clues to advance the clinical control of prolactinomas and hyperprolactinemia.

The integrated bioinformatic analysis identifies immune microenvironment-related potential biomarkers for patients with gestational diabetes mellitus.

Background: Gestational diabetes mellitus (GDM), a transient disease, may lead to short- or long-term adverse influences on maternal and fetal health. Therefore, its potential functions, mechanisms and related molecular biomarkers must be comprehended for the control, diagnosis and treatment of GDM. Methods: The differentially expressed genes (DEGs) were identified using GSE49524 and GSE87295 associated with GDM from the Gene Expression Omnibus database, followed by function enrichment analysis, protein-protein interactions network construction, hub DEGs mining, diagnostic value evaluation and immune infiltration analysis. Finally, hub DEGs, the strongest related to immune infiltration, were screened as immune-related biomarkers. Results: A hundred and seven DEGs were identified between patients with GDM and healthy individuals. Six hub genes with high diagnostic values, including ALDH1A1, BMP4, EFNB2, MME, PLAUR and SLIT2, were identified. Among these, two immune-related genes (PLAUR and SLIT2) with the highest absolute correlation coefficient were considered immune-related biomarkers in GDM. Conclusion: Our study provides a comprehensive analysis of GDM, which would provide a foundation for the development of diagnosis and treatment of GDM.

The relationship between serum γ-glutamyltransferase (GGT) and diabetic nephropathy in patients with type 2 diabetes mellitus: a cross-sectional study.

The relationship between serum γ-glutamyltransferase (GGT) and renal dysfunction is controversial. In this study, we examined the relationship of serum GGT to diabetic nephropathy (DN) in patients with type 2 diabetes mellitus (T2DM). A total of 577 patients with T2DM were enrolled and their basic information and laboratory data were collected and analyzed. The prevalence of DN increased with the elevated serum GGT tertiles. The level of serum GGT in the DN group was higher than in the non-DN groups. Multivariate logistic analysis showed that high GGT was independent risks for DN (OR = 1.041, 95% CIs 1.023-1.059). And the OR of log-transformed serum GGT for DN was 6.190 (95% CIs 4.248-9.021). The OR of DN across increasing tertiles of serum GGT were 1.00, 3.288 (1.851-5.840), and 5.059 (2.620-9.769) (P for trend < 0.001). Stratified receiver operating characteristic (ROC) analysis by gender showed that the area under ROC curve (AUC) value for GGT was 0.781 (0.732-0.825, P < 0.05) in male and was 0.817 (0.761-0.864, P < 0.05) in female. Compared with female, GGT in male showed lower sensitivity (52.86% vs. 82.05%) and higher specificity (90.32% vs. 55.26%). And the AUC value for GGT was greater than creatinine (Cr) and estimated glomerular filtration rate (eGFR) in male and smaller than Cr and eGFR in female, respectively. In Conclusion, there was an independently positive relationship between serum GGT levels and DN, which suggested that elevated GGT was a potential indicator for risk of DN. There were gender differences in the predictive property of GGT for DN.

Creatinine-to-cystatin C ratio as a marker of sarcopenia for identifying osteoporosis in male patients with type 2 diabetes mellitus.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is associated with the increased incidence rate of sarcopenia and osteoporosis. Serum creatinine-to-cystatin C ratio (CCR) is a novel and simple tool which can be used as an index of sarcopenia. This study aims to investigate the association between CCR and osteoporosis as well as bone mineral density (BMD) in T2DM patients. METHODS: Four hundred eighteen T2DM patients were recruited, including 166 females and 252 males. General information, BMD data and laboratory data were collected. The correlation between CCR, BMD, bone metabolism markers and osteoporosis was explored by spearman correlation, receiver-operating characteristic (ROC) curve analysis and multiple regression analysis. RESULTS: Spearman correlation analysis showed that there was a positive correlation between CCR and BMD as well as serum calcium in male patients (r = 0.181-0.381, P < 0.01), but such correlation was not found in the female group. In multivariate regression analysis, it was found that there was a significant correlation between CCR and BMD of total lumbar spine, hip as well as femoral neck in male patients. ROC curve showed that the optimal cut-off value of CCR for predicting osteoporosis in male patients was 6.73 with the sensitivity of 88% and specificity of 63%. CONCLUSION: In male T2DM patients, CCR was negatively correlated with osteoporosis and positively correlated with BMD.

Preoperative geriatric nutritional risk index is an independent prognostic factor for postoperative survival after gallbladder cancer radical surgery.

BACKGROUND: Currently, the surgical outcomes of gallbladder cancer (GBC) are not always satisfactory. The geriatric nutritional risk index (GNRI) can effectively assess nutritional status. This study intends to investigate whether the preoperative GNRI can predict the prognosis of GBC. METHODS: 202 consecutive GBC patients who underwent treatment from 2010 to 2017 were selected and analyzed retrospectively. By using the univariate and multivariate Cox regression analyses on overall survival (OS) and recurrence-free survival (RFS), the preoperative GNRI of GBC patients was evaluated. RESULTS: Among the 202 patients, the GNRI of the 86 patients (42.6%) was less than 98. The patients with low preoperative GNRI had the median OS of 26 months, which was less than the median OS of 39 months among those patients who had higher preoperative GNRI (P < 0.001). Univariate analysis showed that low GNRI was related to short survival time (HR 3.656, 95% CI 2.308-5.790, P < 0.001). In addition, the results of multivariate analysis revealed that, the patients with low GNRI showed a lower OS (HR 2.207, 95% CI 1.131-4.308, P = 0.020) and RFS (HR 2.964, 95% CI 1.577-5.571, P = 0.001) than those patients with higher GNRI. CONCLUSION: GNRI is an independent indicator of poor prognosis in GBC patients after GBC radical surgery.

Fabrication of high-precision reflective volume Bragg gratings.

Reflecting Bragg gratings (RBGs) recorded in photo-thermo-refractive (PTR) glasses have been widely used in narrowing and stabilization of the laser emission spectrum. As the center wavelength of RBGs determines the final output wavelength of lasers, it is necessary to carefully control the center wavelength of RBGs during the fabrication process. In this paper, the fabrication process of high-precision RBGs was investigated. We developed a two-step method and demonstrated both theoretically and experimentally that it is effective and can be used to guide the fabrication process of high-precision RBGs. The experimental results show that the center wavelength of the fabricated RBG deviates from the target center wavelength within ±10 pm.

Triptolide inhibits Wnt signaling in NSCLC through upregulation of multiple Wnt inhibitory factors via epigenetic modifications to Histone H3.

In the last decade, it has become clear that epigenetic changes act together with genetic mutations to promote virtually every stage of tumorigenesis and cancer progression. This knowledge has triggered searches for "epigenetic drugs" that can be developed into new cancer therapies. Here we report that triptolide reduced lung cancer incidence from 70% to 10% in a Fen1 E160D transgenic mouse model and effectively inhibited cancer growth and metastasis in A549 and H460 mouse xenografts. We found that triptolide induced lung cancer cell apoptosis that was associated with global epigenetic changes to histone 3 (H3). These global epigenetic changes in H3 are correlated with an increase in protein expression of five Wnt inhibitory factors that include WIF1, FRZB, SFRP1, ENY2, and DKK1. Triptolide had no effect on DNA methylation status at any of the CpG islands located in the promoter regions of all five Wnt inhibitory factors. Wnt expression is implicated in promoting the development and progression of many lung cancers. Because of this, the potential to target Wnt signaling with drugs that induce epigenetic modifications provides a new avenue for developing novel therapies for patients with these tumor types.

JMJD1B Demethylates H4R3me2s and H3K9me2 to Facilitate Gene Expression for Development of Hematopoietic Stem and Progenitor Cells.

The arginine methylation status of histones dynamically changes during many cellular processes, including hematopoietic stem/progenitor cell (HSPC) development. The arginine methyltransferases and the readers that transduce the histone codes have been defined. However, whether arginine demethylation actively occurs in cells and what enzyme demethylates the methylarginine residues during various cellular processes are unknown. We report that JMJD1B, previously identified as a lysine demethylase for H3K9me2, mediates arginine demethylation of H4R3me2s and its intermediate, H4R3me1. We show that demethylation of H4R3me2s and H3K9me2s in promoter regions is correlated with active gene expression. Furthermore, knockout of JMJD1B blocks demethylation of H4R3me2s and/or H3K9me2 at distinct clusters of genes and impairs the activation of genes important for HSPC differentiation and development. Consequently, JMJD1B-/- mice show defects in hematopoiesis. Altogether, our study demonstrates that demethylase-mediated active arginine demethylation process exists in eukaryotes and that JMJD1B demethylates both H4R3me2s and H3K9me2 for epigenetic programming during hematopoiesis.

Triptolide-Assisted Phosphorylation of p53 Suppresses Inflammation-Induced NF-κB Survival Pathways in Cancer Cells.

Chronic inflammation plays important roles in cancer initiation and progression. Resolving chronic inflammation or blocking inflammatory signal transduction may prevent cancer development. Here, we report that the combined low-dose use of two anti-inflammatory drugs, aspirin and triptolide, reduces spontaneous lung cancer incidence from 70% to 10% in a mouse model. Subsequent studies reveal that such treatment has little effect on resolving chronic inflammatory conditions in the lung, but it significantly blocks the NF-κB-mediated expression of proliferation and survival genes in cancer cells. Furthermore, triptolide and aspirin induce distinct mechanisms to potentiate each other to block NF-κB nuclear localization stimulated by inflammatory cytokines. While aspirin directly inhibits IκB kinases (IKKs) to phosphorylate IκBα for NF-κB activation, triptolide does not directly target IKKs or other factors that mediate IKK activation. Instead, it requires p53 to inhibit IκBα phosphorylation and degradation. Triptolide binds to and activates p38α and extracellular signal-regulated kinase 1/2 (ERK1/2), which phosphorylate and stabilize p53. Subsequently, p53 competes with IκBα for substrate binding to IKKß and thereby blocks IκBα phosphorylation and NF-κB nuclear translocation. Inhibition of p38α and ERK1/2 or p53 mutations could abolish the inhibitory effects of triptolide on NF-κB. Our study defines a new p53-dependent mechanism for blocking NF-κB survival pathways in cancer cells.

Role of FEN1 S187 phosphorylation in counteracting oxygen-induced stress and regulating postnatal heart development.

Flap endonuclease 1 (FEN1) phosphorylation is proposed to regulate the action of FEN1 in DNA repair as well as Okazaki fragment maturation. However, the biologic significance of FEN1 phosphorylation in response to DNA damage remains unknown. Here, we report an in vivo role for FEN1 phosphorylation, using a mouse line carrying S187A FEN1, which abolishes FEN1 phosphorylation. Although S187A mouse embryonic fibroblast cells showed normal proliferation under low oxygen levels (2%), the mutant cells accumulated oxidative DNA damage, activated DNA damage checkpoints, and showed G1-phase arrest at atmospheric oxygen levels (21%). This suggests an essential role for FEN1 phosphorylation in repairing oxygen-induced DNA damage and maintaining proper cell cycle progression. Consistently, the mutant cardiomyocytes showed G1-phase arrest due to activation of the p53-mediated DNA damage response at the neonatal stage, which reduces the proliferation potential of the cardiomyocytes and impairs heart development. Nearly 50% of newborns with the S187A mutant died in the first week due to failure to undergo the peroxisome proliferator-activated receptor signaling-dependent switch from glycolysis to fatty acid oxidation. The adult mutant mice developed dilated hearts and showed significantly shorter life spans. Altogether, our results reveal an important role of FEN1 phosphorylation to counteract oxygen-induced stress in the heart during the fetal-to-neonatal transition.-Zhou, L., Dai, H., Wu, J., Zhou, M., Yuan, H., Du, J., Yang, L., Wu, X., Xu, H., Hua, Y., Xu, J., Zheng, L., Shen, B. Role of FEN1 S187 phosphorylation in counteracting oxygen-induced stress and regulating postnatal heart development.

Mutation of DNA Polymerase ß R137Q Results in Retarded Embryo Development Due to Impaired DNA Base Excision Repair in Mice.

DNA polymerase ß (Pol ß), a key enzyme in the DNA base excision repair (BER) pathway, is pivotal in maintaining the integrity and stability of genomes. One Pol ß mutation that has been identified in tumors, R137Q (arginine to glutamine substitution), has been shown to lower polymerase activity, and impair its DNA repair capacity. However, the exact functional deficiency associated with this polymorphism in living organisms is still unknown. Here, we constructed Pol ß R137Q knock-in mice, and found that homozygous knock-in mouse embryos were typically small in size and had a high mortality rate (21%). These embryonic abnormalities were caused by slow cell proliferation and increased apoptosis. In R137Q knock-in mouse embryos, the BER efficiency was severely impaired, which subsequently resulted in double-strand breaks (DSBs) and chromosomal aberrations. Furthermore, R137Q mouse embryo fibroblasts (MEFs) were more sensitive to DNA-damaging reagents, such as methyl methanesulfonate (MMS) and H2O2. They displayed a higher percentage of DSBs, and were more likely to undergo apoptosis. Our results indicate that R137 is a key amino acid site that is essential for proper Pol ß functioning in maintaining genomic stability and embryo development.

A Selective Small Molecule DNA2 Inhibitor for Sensitization of Human Cancer Cells to Chemotherapy.

Cancer cells frequently up-regulate DNA replication and repair proteins such as the multifunctional DNA2 nuclease/helicase, counteracting DNA damage due to replication stress and promoting survival. Therefore, we hypothesized that blocking both DNA replication and repair by inhibiting the bifunctional DNA2 could be a potent strategy to sensitize cancer cells to stresses from radiation or chemotherapeutic agents. We show that homozygous deletion of DNA2 sensitizes cells to ionizing radiation and camptothecin (CPT). Using a virtual high throughput screen, we identify 4-hydroxy-8-nitroquinoline-3-carboxylic acid (C5) as an effective and selective inhibitor of DNA2. Mutagenesis and biochemical analysis define the C5 binding pocket at a DNA-binding motif that is shared by the nuclease and helicase activities, consistent with structural studies that suggest that DNA binding to the helicase domain is necessary for nuclease activity. C5 targets the known functions of DNA2 in vivo: C5 inhibits resection at stalled forks as well as reducing recombination. C5 is an even more potent inhibitor of restart of stalled DNA replication forks and over-resection of nascent DNA in cells defective in replication fork protection, including BRCA2 and BOD1L. C5 sensitizes cells to CPT and synergizes with PARP inhibitors.

Okazaki fragment maturation involves α-segment error editing by the mammalian FEN1/MutSα functional complex.

During nuclear DNA replication, proofreading-deficient DNA polymerase α (Pol α) initiates Okazaki fragment synthesis with lower fidelity than bulk replication by proofreading-proficient Pol δ or Pol ε. Here, we provide evidence that the exonuclease activity of mammalian flap endonuclease (FEN1) excises Pol α replication errors in a MutSα-dependent, MutLα-independent mismatch repair process we call Pol α-segment error editing (AEE). We show that MSH2 interacts with FEN1 and facilitates its nuclease activity to remove mismatches near the 5' ends of DNA substrates. Mouse cells and mice encoding FEN1 mutations display AEE deficiency, a strong mutator phenotype, enhanced cellular transformation, and increased cancer susceptibility. The results identify a novel role for FEN1 in a specialized mismatch repair pathway and a new cancer etiological mechanism.

Mammalian DNA2 helicase/nuclease cleaves G-quadruplex DNA and is required for telomere integrity.

Efficient and faithful replication of telomeric DNA is critical for maintaining genome integrity. The G-quadruplex (G4) structure arising in the repetitive TTAGGG sequence is thought to stall replication forks, impairing efficient telomere replication and leading to telomere instabilities. However, pathways modulating telomeric G4 are poorly understood, and it is unclear whether defects in these pathways contribute to genome instabilities in vivo. Here, we report that mammalian DNA2 helicase/nuclease recognizes and cleaves telomeric G4 in vitro. Consistent with DNA2's role in removing G4, DNA2 deficiency in mouse cells leads to telomere replication defects, elevating the levels of fragile telomeres (FTs) and sister telomere associations (STAs). Such telomere defects are enhanced by stabilizers of G4. Moreover, DNA2 deficiency induces telomere DNA damage and chromosome segregation errors, resulting in tetraploidy and aneuploidy. Consequently, DNA2-deficient mice develop aneuploidy-associated cancers containing dysfunctional telomeres. Collectively, our genetic, cytological, and biochemical results suggest that mammalian DNA2 reduces replication stress at telomeres, thereby preserving genome stability and suppressing cancer development, and that this may involve, at least in part, nucleolytic processing of telomeric G4.

Synthesis, structure-activity relationships, and docking studies of N-phenylarylformamide derivatives (PAFAs) as non-nucleoside HIV reverse transcriptase inhibitors.

A series of N-phenylarylformamide derivatives (PAFAs) with anti-wild-type HIV-1 activity (EC(50) values) ranging from 0.3 nM to 5.1 nM and therapeutic index (TI) ranging from 10 616 to 271 000 were identified as novel non-nucleoside reverse transcriptase inhibitors. Among them, compound 13g (EC(50) = 0.30 nM, TI = 184 578), 13l (EC(50) = 0.37 nM, TI = 212 819), 13m (EC(50) = 0.32 nM, TI = 260 617) and 13r (EC(50) = 0.27 nM, TI = 271 000) displayed the highest activity against this type virus nearly as potent as lead compound GW678248. Moreover, all of them were also active to inhibit the double mutant strain A(17) (K103N + Y181C) with EC(50) values of 0.29 µM, 0.14 µM, 0.10 µM and 0.27 µM, respectively. In particular, compound 13m, which showed broad-spectrum anti-HIV activity, was also effective to inhibit the HIV-2 ROD replication within 4.37 µM concentration.

Polyploid cells rewire DNA damage response networks to overcome replication stress-induced barriers for tumour progression.

Mutations in genes involved in DNA replication, such as flap endonuclease 1 (FEN1), can cause single-stranded DNA breaks (SSBs) and subsequent collapse of DNA replication forks leading to DNA replication stresses. Persistent replication stresses normally induce p53-mediated senescence or apoptosis to prevent tumour progression. It is unclear how some mutant cells can overcome persistent replication stresses and bypass the p53-mediated pathways to develop malignancy. Here we show that polyploidy, which is often observed in human cancers, leads to overexpression of BRCA1, p19arf and other DNA repair genes in FEN1 mutant cells. This overexpression triggers SSB repair and non-homologous end-joining pathways to increase DNA repair activity, but at the cost of frequent chromosomal translocations. Meanwhile, DNA methylation silences p53 target genes to bypass the p53-mediated senescence and apoptosis. These molecular changes rewire DNA damage response and repair gene networks in polyploid tumour cells, enabling them to escape replication stress-induced senescence barriers.