Structure/function analysis of the essential protein Rad4TopBP1 in S. pombe

Rad4TopBP1, a multiple BRCT domain protein, is essential for initiation of replication and participates in checkpoint responses following genotoxic treatment during the S and G2 phases of the cell cycle. Rad4 interacts with several proteins that are involved in initiation of DNA replication (i.e. Sld2) or activation of Chk1 and/or Cds1 checkpoint kinases (i.e. Rad9, Crb2, and Rad3). However, it remained unclear how Rad4 and its homologues are regulated to coordinate these diverse functions. This PhD project presents a comprehensive structure/function analysis of the fission yeast Rad4TopBP1. In order to obtain separation of function mutants of Rad4, a hydroxylamine random mutagenesis genetic screen was performed. However, we were not able to separate a checkpoint activation function from replicative function. Rad4 being phosphorylated in a Cdc2-dependent manner, the role of Rad4 phosphorylation by Cdc2 was investigated. A mutant strain containing multiple mutations at Rad4 Cdc2 consensus phosphorylation sites does not exhibit significant sensitivity to DNA damage or HU. In addition, Rad4 Cdc2 phosphorylation sites do not play a role in DNA re-replication. There is no significant phenotypic effect observed after DNA damage in S. pombe strains expressing a Rad4 protein deleted for a putative domain (RXL motif) interaction with cyclin, or harboring mutations in putative sumoylation motifs, or C-terminus truncation. In higher eukaryotes, TopBP1 binds and activates the ATR-ATRIP complex via an ATR-activating domain (AAD) in order to activate a checkpoint function. We identified a potential AAD in C-terminal of Rad4 in S. pombe. I show that Rad4 physically associates with Rad3 in vitro in an AAD-dependent manner. S. pombe strains mutated in the AAD show a slight sensitivity to DNA damage and HU. The rad4 AAD mutants do not completely prevent Rad3-mediated G2/M checkpoint activation after DNA damage. The sensitivity in a rad4 AAD mutant increases when damage occurs in S-phase, when histone H2A phosphorylation is defective. I established an artificial checkpoint induction system in the absence of exogenous lesions by targeting checkpoint proteins onto chromatin in S. pombe. Interestingly, Rad4 AAD is essential for checkpoint activation in this system. Because this checkpoint activation is independent of ssDNA-RPA formation, the data suggest that the AAD plays a role in chromatin-mediated checkpoint maintenance/amplification. Altogether, this pathway seems to play an important role in S-phase when DSBs resection is limited

The Rad4TopBP1 ATR-Activation domain functions in G1/S phase in a chromatin-dependent manner

DNA damage checkpoint activation can be subdivided in two steps: initial activation and signal amplification. The events distinguishing these two phases and their genetic determinants remain obscure. TopBP1, a mediator protein containing multiple BRCT domains, binds to and activates the ATR/ATRIP complex through its ATR-Activation Domain (AAD). We show that Schizosaccharomyces pombe Rad4TopBP1 AAD–defective strains are DNA damage sensitive during G1/S-phase, but not during G2. Using lacO-LacI tethering, we developed a DNA damage–independent assay for checkpoint activation that is Rad4TopBP1 AAD–dependent. In this assay, checkpoint activation requires histone H2A phosphorylation, the interaction between TopBP1 and the 9-1-1 complex, and is mediated by the phospho-binding activity of Crb253BP1. Consistent with a model where Rad4TopBP1 AAD–dependent checkpoint activation is ssDNA/RPA–independent and functions to amplify otherwise weak checkpoint signals, we demonstrate that the Rad4TopBP1 AAD is important for Chk1 phosphorylation when resection is limited in G2 by ablation of the resecting nuclease, Exo1. We also show that the Rad4TopBP1 AAD acts additively with a Rad9 AAD in G1/S phase but not G2. We propose that AAD–dependent Rad3ATR checkpoint amplification is particularly important when DNA resection is limiting. In S. pombe, this manifests in G1/S phase and relies on protein– chromatin interactions

Overview of the Reporting Sources of Developmentallydelayed Children in Taiwan Between 2011 and 2012

Early Intervention (EI) services, as defined in The Bye-laws of Children and Youth Welfare Law, provided for 1.2 % of the nation’s infants, toddlers and preschool children between 2011 and 2012; however, the proportion was higher in some counties or cities and lower in others. In order to elucidate factors that may influence reporting rates, we analyzed the reporting sources from 23 counties/cities between 2011 and 2012. We analyzed registry data of newly reported cases between 2011 and 2012, published by the Department of Statistics, Ministry of Interior of Taiwan. The reporting sources were categorized into eight types, and the percentage of cases reported by each source was calculated. The statistical relationship between these variables and the reporting rates were analyzed with suitable methods. P value < 0.05 was regarded as statistically significant. The estimated 2-year average reporting rate of new cases was 11.97‰. The reporting rate was significantly higher among children living in counties compared with children living in cities (P = 0.0007). The reporting rate was also significantly higher among children living in low urbanized areas as compared with children living in highly urbanized areas (P = 0.0067). The proportion of medical organization reported cases was the highest of all the reporting sources (39.99%). Higher reporting rates from householders, guardians and health centers positively affected the total reporting rates (P = 0.0499 and P=0.0151, respectively). In conclusion our study shows that many sources contribute to the notification of children at risk or with developmental delay, with implications for regular surveillance and screening children development by people involved with them. Incorporating more efficient developmental screening tools, including parent-concerned based screening questionnaires during health screening, with additional staff to do the screening, may increase the proportion of children with possible developmental delay being notified

Autologous serum therapy in recalcitrant laser-assisted in situ keratomileusis-induced neurotrophic epitheliopathy

AbstractBackground/PurposeTo evaluate the efficacy of autologous serum eye drops for patients with recalcitrant laser-assisted in situ keratomileusis (LASIK)-induced neurotrophic epitheliopathy (LINE) unresponsive to conventional treatment, and to determine the possible predisposing risk factors of these patients.MethodsWe enrolled 10 consecutive patients (20 eyes) undergoing femtosecond-assisted myopic LASIK surgery presenting with recalcitrant LINE for > 1 year. Another 340 patients (713 eyes) receiving femtosecond-assisted myopic LASIK without recalcitrant LINE were set as controls. Possible risk factors associated with recalcitrant LINE were investigated. Twenty percent autologous serum treatment was prescribed to 20 eyes. The efficacy of autologous serum was assessed with ocular surface conditions, tear function, and the change of best-corrected visual acuity.ResultsAge older than 30 years [odds ratio (OR) = 7.74; 95% confidence interval (CI), 1.74–34.50], flap thickness < 110 μm (OR = 3.47; 95% CI, 1.22–9.73), and a flap diameter < 8.5 mm (OR = 5.38; 95% CI, 1.95–14.85) pose higher risks in femtosecond laser-assisted myopic LASIK. All eyes (100%) achieved remission after autologous serum treatment. The visual acuity before treatment was 0.49 ± 0.41 in LogMAR, and the visual acuity after treatment was 0.14 ± 0.22 in LogMAR. Time to achieve remission was 8.26 ± 11.87 weeks. Mean relapse-free survival after discontinuing autologous serum was 47 weeks.ConclusionRisk factors of recalcitrant LINE in femtosecond laser-assisted myopic LASIK were identified as older age, a thinner flap (<110 μm), and a small flap diameter (<8.5 mm). Autologous serum eye drops can effectively improve corneal surface conditions and postoperative visual acuity

Efficacy and safety of nanohybrids comprising silver nanoparticles and silicate clay for controlling Salmonella infection

Developing effective and safe drugs is imperative for replacing antibiotics and controlling multidrug-resistant microbes. Nanoscale silicate platelet (NSP) and its nanohybrid, silver nanoparticle/NSP (AgNP/NSP), have been developed, and the nanohybrids show a strong and general antibacterial activity in vitro. Here, their efficacy for protecting Salmonella-infected chicks from fatality and septicemia was evaluated. Both orally administrated NSP and AgNP/NSP, but not AgNPs alone, effectively reduced the systemic Salmonella infection and mortality. In addition, quantitative Ag analyses demonstrated that Ag deposition from AgNP/NSP in the intestines was less than that from conventional AgNPs, indicating that the presence of NSP for immobilizing AgNPs reduced Ag accumulation in tissue and improved the safety of AgNPs. These in vivo results illustrated that both NSP and AgNP/NSP nanohybrid represent potential agents for controlling enteric bacterial infections

AMiBA Wideband Analog Correlator

A wideband analog correlator has been constructed for the Yuan-Tseh Lee Array for Microwave Background Anisotropy. Lag correlators using analog multipliers provide large bandwidth and moderate frequency resolution. Broadband IF distribution, backend signal processing and control are described. Operating conditions for optimum sensitivity and linearity are discussed. From observations, a large effective bandwidth of around 10 GHz has been shown to provide sufficient sensitivity for detecting cosmic microwave background variations.Comment: 28 pages, 23 figures, ApJ in press

AMiBA: scaling relations between the integrated Compton-y and X-ray derived temperature, mass, and luminosity

We investigate the scaling relations between the X-ray and the thermal Sunyaev-Zel'dovich Effect (SZE) properties of clusters of galaxies, using data taken during 2007 by the Y.T. Lee Array for Microwave Background Anisotropy (AMiBA) at 94 GHz for the six clusters A1689, A1995, A2142, A2163, A2261, and A2390. The scaling relations relate the integrated Compton-y parameter Y_{2500} to the X-ray derived gas temperature T_{e}, total mass M_{2500}, and bolometric luminosity L_X within r_{2500}. Our results for the power-law index and normalization are both consistent with the self-similar model and other studies in the literature except for the Y_{2500}-L_X relation, for which a physical explanation is given though further investigation may be still needed. Our results not only provide confidence for the AMiBA project but also support our understanding of galaxy clusters.Comment: Accepted by ApJ; 8 pages, 3 figures, 5 table

AMiBA: Broadband Heterodyne CMB Interferometry

The Y. T. Lee Array for Microwave Background (AMiBA) has reported the first science results on the detection of galaxy clusters via the Sunyaev Zel'dovich effect. The science objectives required small reflectors in order to sample large scale structures (20') while interferometry provided modest resolutions (2'). With these constraints, we designed for the best sensitivity by utilizing the maximum possible continuum bandwidth matched to the atmospheric window at 86-102GHz, with dual polarizations. A novel wide-band analog correlator was designed that is easily expandable for more interferometer elements. MMIC technology was used throughout as much as possible in order to miniaturize the components and to enhance mass production. These designs will find application in other upcoming astronomy projects. AMiBA is now in operations since 2006, and we are in the process to expand the array from 7 to 13 elements.Comment: 10 pages, 6 figures, ApJ in press; a version with high resolution figures available at http://www.asiaa.sinica.edu.tw/~keiichi/upfiles/AMiBA7/mtc_highreso.pd

Nanohybrids of Silver Particles Immobilized on Silicate Platelet for Infected Wound Healing

Silver nanoparticles supported on nanoscale silicate platelets (AgNP/NSP) possess interesting properties, including a large surface area and high biocide effectiveness. The nanohybrid of AgNP/NSP at a weight ratio 7/93 contains 5-nm Ag particles supported on the surface of platelets with dimensions of approximately 80×80×1 nm3. The nanohybrid expresses a trend of lower cytotoxicity at the concentration of 8.75 ppm Ag and low genotoxicity. Compared with conventional silver ions and the organically dispersed AgNPs, the nanohybrid promotes wound healing. We investigated overall wound healing by using acute burn and excision wound healing models. Tests on both infected wound models of mice were compared among the AgNP/NSP, polymer-dispersed AgNPs, the commercially available Aquacel, and silver sulfadiazine. The AgNP/NSP nanohybrid was superior for wound appearance, but had similar wound healing rates, vascular endothelial growth factor (VEGF)-A levels and transforming growth factor (TGF)-β1 expressions to Aquacel and silver sulfadiazine