Determinants of continuum of care for maternal, newborn, and child health services in rural Khammouane, Lao PDR.

IntroductionThe concept of continuum of care has gained attention as measures to improve maternal, newborn, and child health. However, little is known about the factors associated with the coverage level of continuum of care in Lao PDR. Therefore, this study was conducted 1) to investigate the coverage level of continuum of care and 2) to identify barriers and promoting factors that are associated with mothers' continuation in receiving services in rural Lao PDR.MethodsA community-based, cross sectional study was conducted in a rural district in Khammouane Province, Lao PDR, using a structured questionnaire. The outcome to the express continuum of care was assessed by the modified composite coverage index (CCI) that reflects ten maternal and child health services.ResultsIn total, 263 mothers were included in the final analyses. Only 6.8% of mothers continued to receive all MNCH services. Five factors were shown to have statistically significant associations with modified CCI score: higher educational attainment (B = 0.070, pConclusionsIn this study, we introduced the modified CCI to better explain the utilization of preventive maternal and child health services along with the continuum of care. By utilizing the modified CCI, we identified five factors as determinants of continuum of care. Furthermore, new and modifiable promoting factors were identified for continuum of care: receiving the first antenatal care within the first trimester and family and male involvement. Such demand side actions should be encouraged to improve the continuity of MNCH service use

Sinusoidal model for the annual variation of atmospheric radon concentration in Japan

2nd International Symposium and the 1st IUGS & SCJ International Workshop on Natural Hazard

Establishment of a Wheat Cell-Free Synthesized Protein Array Containing 250 Human and Mouse E3 Ubiquitin Ligases to Identify Novel Interaction between E3 Ligases and Substrate Proteins.

Ubiquitination is a key post-translational modification in the regulation of numerous biological processes in eukaryotes. The primary roles of ubiquitination are thought to be the triggering of protein degradation and the regulation of signal transduction. During protein ubiquitination, substrate specificity is mainly determined by E3 ubiquitin ligase (E3). Although more than 600 genes in the human genome encode E3, the E3s of many target proteins remain unidentified owing to E3 diversity and the instability of ubiquitinated proteins in cell. We demonstrate herein a novel biochemical analysis for the identification of E3s targeting specific proteins. Using wheat cell-free protein synthesis system, a protein array containing 227 human and 23 mouse recombinant E3s was synthesized. To establish the high-throughput binding assay using AlphaScreen technology, we selected MDM2 and p53 as the model combination of E3 and its target protein. The AlphaScreen assay specifically detected the binding of p53 and MDM2 in a crude translation mixture. Then, a comprehensive binding assay using the E3 protein array was performed. Eleven of the E3s showed high binding activity, including four previously reported E3s (e.g., MDM2, MDM4, and WWP1) targeting p53. This result demonstrated the reliability of the assay. Another interactors, RNF6 and DZIP3-which there have been no report to bind p53-were found to ubiquitinate p53 in vitro. Further analysis showed that RNF6 decreased the amount of p53 in H1299 cells in E3 activity-dependent manner. These results suggest the possibility that the RNF6 ubiquitinates and degrades p53 in cells. The novel in vitro screening system established herein is a powerful tool for finding novel E3s of a target protein

Summary of hit E3 ubiquitin ligases (E3s).

<p>E3s for which the relative luminescence signals in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156718#pone.0156718.g004" target="_blank">Fig 4A</a> were higher than 8 are listed. E3s indicated in bold character have been previously reported to interact with p53.</p

Model assay using p53 and MDM2.

<p>(A) Expression of recombinant proteins of p53 and MDM2 with the wheat cell-free system. Wild-type (WT) and mutant p53 lacking the amino acid residues required for binding with MDM2 (∆I) were synthesized as N-terminal biotinylated form. Wild-type MDM2 and its mutant with a single amino acid substitution in the catalytic core cysteine residue (C/S) were synthesized as N-terminal FLAG-tagged protein. Biotinylated and FLAG-dihydrofolate reductase (FLAG-DHFR) were used as negative controls for p53 and MDM2, respectively. The total translation mixture of each protein was centrifuged, and the whole translation mixture (W) and supernatant (S) were subjected to SDS-PAGE followed by immunoblot analysis (IB) using the antibodies indicated below. (B) Binding assay of biotinylated p53 and FLAG-MDM2 using a conventional immunoprecipitation (IP) assay. Fifteen microliters of crude biotinylated p53 (WT or ∆I) and FLAG-MDM2 were mixed and precipitated with anti-FLAG-antibody. Immunopreciptates were detected with anti-FLAG antibody and anti-biotin antibody. FLAG-DHFR was used as controls for FLAG-MDM2. (C) <i>In vitro</i> ubiquitination assay using biotinylated p53 and FLAG-MDM2. The crude translation mixtures of biotinylated p53 (WT or ∆I) and FLAG-MDM2 (WT or C/S) were mixed and then the reaction mixture containing ATP and HA-tagged ubiquitin (HA-Ubi) with or without UbcH5b was added. After the ubiquitination reaction, biotinylated p53 was pulled down with streptavidin (STA) magnet beads and subjected to SDS-PAGE followed by immunoblot analysis using anti-HA-antibody (for ubiquitin) and anti-biotin antibody (for p53).</p

Study overview.

<p>cDNA clones encoding the E3 ubiquitin ligases (E3s) used in this study were divided into two categories. The clones in category B could be used directly for split-primer PCR, but those in category A were inserted into vectors that cannot be used directly for split-primer PCR and thus were transferred into the pDONR221 vector by using the Gateway system. The names of the vectors and the origins of the clones are indicated.</p

RNF6-dependent ubiquitination and degradation of p53 in cells.

<p>(A) Effect of RNF6 on p53 expression level. H1299 cells were co-transfected with a fixed amount of p53-V5 plasmid (25 ng) and an increased amount of FLAG-RNF6 plasmid (100 to 400 ng). Cell lysates from each reaction were subjected to SDS-PAGE followed by immunoblot analysis using anti-V5 antibody and anti-FLAG antibody. The band intensity of p53 in each reaction was quantified using imageJ software. Relative intensities normalized with the reaction of empty vector were indicated below the blot detected with anti-V5 antibody. (B) Co-transfection of p53-V5 with the wild-type (WT) or activity-deficient mutant (C/S) RNF6. (C) Stability of p53 in the presence of wild-type or C/S mutant of RNF6 was investigated by a pulse-chase assay. H1299 cells co-transfected with p53-V5 and wild-type or C/S mutant of FLAG-RNF6 were treated with 100 μg/ml of cycloheximide (CHX) for indicated time periods. The amount of p53-V5 detected by immunoblot analysis was quantified as same procedure as (A), and relative intensities normalized with the reaction without cycloheximide were indicated below the blot. (D) Endogenous RNF6 in H1299 cells were knockdown by siRNAs targeting RNF6, and overexpressed p53 was detected by immunoblot analysis (upper). The intensity of p53 band in each lane was quantified and intensites relative to the control were indicated below. Efficiency of RNF6 knockdown was confirmed with reverse transcription-quantitative PCR (lower).</p

Comprehensive screening to identify p53-binding E3 ligases.

<p>(A) AlphaScreen assay to detect the binding between p53 and 258 E3s including eight negative controls. In this experiment, binding of all E3s to biotinylated p53 and biotinylated DHFR was measured, and the relative value was calculated as follows: value from E3 and p53 / value from E3 and DHFR. Green arrowheads indicated the negative controls. (B) <i>In vitro</i> ubiquitination assay using p53-binding E3s obtained in (A). p53-V5 was mixed with each E3, and the ubiquitination assay was carried out using His-ubiquitin. Left panel, p53-V5 was precipitated with anti-p53 antibody and detected with anti-ubiquitin antibody. Right panel, His-ubiquitin was pull-down with Ni-sepharose, and ubiquitinated p53 was detected with anti-p53 antibody.</p

Development of AlphaScreen assay.

<p>(A) AlphaScreen assay to detect binding between p53 and MDM2. Crude translation mixtures of biotinylated p53 (WT or ∆I) and FLAG-MDM2 (0.75 μL each) were used for the assay. FLAG-DHFR was used as a control for MDM2. (B) A reaction mixture containing p53 and MDM2 prepared under the same conditions as those in (A) was mixed with 2, 10, or 50 μM Nutlin-3. Biotinylated FLAG-peptide was used as a control to measure the interference of Nutlin-3 in the AlphaScreen assay. (C) Validation of the quality of the AlphaScreen assay. Z′ factor was calculated from the reaction of wild-type p53 and MDM2 (positive control, n = 40, square) and the reaction of p53 ∆I mutant and MDM2 (negative control, n = 40, circle). In (A) and (B), all data are expressed as mean values of three independent experiments with error bars indicating standard deviations.</p