The Escherichia coli NarL receiver domain regulates transcription through promoter specific functions

BACKGROUND: The Escherichia coli response regulator NarL controls transcription of genes involved in nitrate respiration during anaerobiosis. NarL consists of two domains joined by a linker that wraps around the interdomain interface. Phosphorylation of the NarL N-terminal receiver domain (RD) releases the, otherwise sequestered, C-terminal output domain (OD) that subsequently binds specific DNA promoter sites to repress or activate gene expression. The aim of this study is to investigate the extent to which the NarL OD and RD function independently to regulate transcription, and the affect of the linker on OD function. RESULTS: NarL OD constructs containing different linker segments were examined for their ability to repress frdA-lacZ or activate narG-lacZ reporter fusion genes. These in vivo expression assays revealed that the NarL OD, in the absence or presence of linker helix α6, constitutively repressed frdA-lacZ expression regardless of nitrate availability. However, the presence of the linker loop α5-α6 reversed this repression and also showed impaired DNA binding in vitro. The OD alone could not activate narG-lacZ expression; this activity required the presence of the NarL RD. A footprint assay demonstrated that the NarL OD only partially bound recognition sites at the narG promoter, and the binding affinity was increased by the presence of the phosphorylated RD. Analytical ultracentrifugation used to examine domain oligomerization showed that the NarL RD forms dimers in solution while the OD is monomeric. CONCLUSIONS: The NarL RD operates as an on-off switch to occlude or release the OD in a nitrate-responsive manner, but has additional roles to directly stimulate transcription at promoters for which the OD lacks independent function. One such role of the RD is to enhance the DNA binding affinity of the OD to target promoter sites. The data also imply that NarL phosphorylation results in RD dimerization and in the separation of the entire linker region from the OD. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12866-015-0502-9) contains supplementary material, which is available to authorized users

Synthesis Process and Properties of V5+-Doped LiFePO4/C

Olivine structure LiFe1−xVxPO4/C (x = 0.02, 0.04, 0.06) composite materials as the cathode for lithium ion batteries were synthesized by carbon-thermal reduction method, using Fe(NO3)3 · 9H2O, LiH2PO4, NH4VO3, and C6H12O6 (glucose) as raw materials. The X-ray diffraction (XRD), scanning electronic microscope (SEM) laser particle size analysis, specific surface area tester, and electrochemical performance testing were used to study its structure, morphology, and electrochemical properties. The results showed that the diffraction peaks of the prepared materials correspond to the single phase of LiFePO4/C and can be indexed as the olivine structure. Particle diameter of LiFe1−xVxPO4/C (x = 0.04) was uniform. Specific surface areas of materials are all increased. An electrochemical test showed that LiFe1−xVxPO4/C (x = 0.04) demonstrated a better electrochemical capacity of 141.065 mAh · g−1 at 0.1C rate, and which had an increase of 10.77% than the un-doped sample. After 20 cycles, charge and discharge specific capacity almost had no attenuation

Prognostic and therapeutic value of disruptor of telomeric silencing-1-like (DOT1L) expression in patients with ovarian cancer

Abstract Background Epigenetics has been known to play a critical role in regulating the malignant phenotype. This study was designed to examine the expression of DOT1L (histone 3 lysine 79 methyltransferase) and H3K79 methylation in normal ovarian tissues and ovarian tumors and to explore the function of DOT1L and its underline mechanisms in ovarian cancer. Methods The expression of DOT1L and H3K79 methylation in 250 ovarian tumor samples and 24 normal ovarian samples was assessed by immunohistochemistry. The effects of DOT1L on cell proliferation in vitro were evaluated using CCK8, colony formation and flow cytometry. The DOT1L-targeted genes were determined using chromatin immune-precipitation coupled with high-throughput sequencing (ChIP-seq) and ChIP-PCR. Gene expression levels were measured by real-time PCR and immunoblotting. The effects of DOT1L on tumor growth in vivo were evaluated using an orthotopic ovarian tumor model. Results DOT1L expression and H3K79 methylation was significantly increased in malignant ovarian tumors. High DOT1L expression was associated with International Federation of Gynecology and Obstetrics (FIGO) stage, histologic grade, and lymphatic metastasis. DOT1L was an independent prognostic factor for the overall survival (OS) and progression-free survival (PFS) of ovarian cancer, and higher DOT1L expression was associated with poorer OS and PFS. Furthermore, DOT1L regulates the transcription of G1 phase genes CDK6 and CCND3 through H3K79 dimethylation; therefore, blocking DOT1L could result in G1 arrest and thereby impede the cell proliferation in vitro and tumor growth in vivo. Conclusions Our findings first demonstrate that DOT1L over-expression has important clinical significance in ovarian cancer and also clarify that it drives cell cycle progression through transcriptional regulation of CDK6 and CCND3 through H3K79 methylation, suggesting that DOT1L might be potential target for prognostic assessment and therapeutic intervention in ovarian cancer

Synthesis, Characterization, and Identification of New <i>in Vitro</i> Covalent DNA Adducts of Divinyl Sulfone, an Oxidative Metabolite of Sulfur Mustard

Divinyl sulfone (DVS) is an important oxidative metabolic product of sulfur mustard (SM) <i>in vitro</i> and <i>in vivo</i>. Although DVS is not a classical blister agent, its high reactivity and toxicity induced by vinyl groups can also cause blisters like SM upon contact with the skin, eyes, and respiratory organs. The purpose of this paper was to identify whether DVS could covalently bind to DNA bases to form new DNA adducts in cells <i>in vitro</i>. A series of adducts were synthesized and characterized using purine, nucleoside, or DNA, separately, as starting materials. The covalent site, pattern, and relative reactivity of adduct formation were identified and discussed in detail. The results showed that five high abundance site-specific DNA adducts, including two monoadducts and three cross-linked adducts, were obtained when DNA was used as a substrate. When HaCaT cells were exposed to 30 μM of DVS, four new DNA adducts containing monoadducts and cross-linked adducts were found and identified in cells, including N3-A monoadduct, N7-G monoadduct, N7G-N7G bis-adduct, and N3A-N7G cross-linked adduct. Among them, the abundance of N3-A monoadduct was 10 times higher than that of the other three adducts. DNA adduct formation with DVS showed significant differences from that observed with SM. The observation of these new DNA adduct <i>in vitro</i> cells revealed that DNA damage could be also induced by DVS

A noninvasive surfactant adsorption test predicting the need for surfactant therapy in preterm infants treated with continuous positive airway pressure

Objective: To determine the diagnostic accuracy of the surfactant adsorption test (SAT) as a predictor for the need for surfactant replacement therapy in neonates with respiratory distress syndrome (RDS). Study design Amniotic fluid samples were collected from 41 preterm neonates with RDS treated with continuous positive airway pressure (CPAP) and 15 healthy control term neonates. Purified porcine surfactant served as a further control. Lamellar bodies and lung ultrasound score were also measured in a subset of the neonates treated with CPAP. Surfactant was administered according to the European guidelines, and clinical data were collected prospectively. Surfactant activity was measured as adsorption at the air/liquid interface and given in relative fluorescent units (RFU). Results: Surfactant activity differed among native porcine surfactant (median, 4863 RFU; IQR, 4405-5081 RFU), healthy term neonates (median, 2680 RFU; IQR, 2069-3050 RFU), and preterm neonates with RDS (median, 442 RFU; IQR, 92-920 RFU; P < .0001). The neonates who failed CPAP had lower surfactant activity compared with those who did not fail CPAP (median, 92 RFU; IQR, 0-315 RFU vs 749 RFU; IQR, 360-974 RFU; P = .0002). Differences between groups were more evident beyond 20-30 minutes of fluorescence; the 30-minute time point showed the highest area under the curve (0.84; P < .001) and the best cutoff level (170 RFU; specificity, 72%; sensitivity, 96%) for the prediction of CPAP failure. Surfactant activity at 30 minutes was significantly correlated with lamellar bodies (r = 0.51, P = .006) and lung ultrasound score (r = -0.39, P = .013). Conclusion: This technique has the potential to be developed into a fast, simple-to-interpret clinical test. The SAT can reliably identify preterm infants with subsequent CPAP failure and shows promise as a screening test for surfactant replacement in preterm neonates

Additional file 1: Table S1. of Prognostic and therapeutic value of disruptor of telomeric silencing-1-like (DOT1L) expression in patients with ovarian cancer

The primer sequences used for PCR. (DOCX 15 kb

Additional file 6: Figure S4. of Prognostic and therapeutic value of disruptor of telomeric silencing-1-like (DOT1L) expression in patients with ovarian cancer

DOT1L regulates the transcription of G1 arrest genes CDK6 and CCND3 through H3K79 dimethylation. (a) and (b) RT-PCR detecting the change of primary G1 arrest genes, D-type cyclins (D1, D2, and D3), cyclin E, Cdks (2, 4, and 6) in mRNA levels in TOV21G cells knocking down of DOT1L with shDOT1L shRNAs lentiviral particles (a) and DOT1L inhibitors, EPZ004777 and SGC0946 (b) (P < 0.05, Student’s t test, data represented as mean±SD). (c) and (d) DOT1L transcriptionally regulates the expression of CDK6 (c) and CCND3 (d) through induction of H3K79 dimethylation in the binding regions of these genes. Chromatin immunoprecipitation (ChIP) PCR assays were performed in a loss-of-function system. TOV21G cells were transfected with shDOT1L shRNAs and shCON lentiviral particles, and ChIP assays using anti-DOT1L and anti-H3K79me2 antibodies were performed with ChIP primers targeting the binding regions of CDK6 and CCND3 (P < 0.05, Student’s t test, data represented as mean±SD). (TIF 815 kb