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1. chinaXiv:201605.01421 [pdf]

The Effect of Ionizing Radiation on mRNA Levels of the DNA Damage Response Genes Rad9, Rad1 and Hus1 in Various Mouse Tissues

Zhang, Zhenya; Li, Kaiming; Cai, Zeyuan; Fang, Yu; An, Lili; Hu, Zhishang; Hang, Haiying; Cai, Zeyuan; Hu, Zhishang
Subjects: Biology >> Biophysics >> Biology

Rad9, Rad1 and Hus1 are essential genes conserved from yeast to humans. They form a heterotrimer complex (9-1-1 complex) that participates in the cell cycle checkpoint activation and DNA damage repair in eukaryotic cells. Rad9, Rad1 and Hus1 deficient cells are hypersensitive to ionizing radiation and mouse cells deleted for anyone of the three genes are highly sensitive to the killing by gamma rays. We propose that ionizing radiation-induced transcription of these genes is a mechanism by which cells respond to radiation-induced damage. In this study we used quantitative real-time RT-PCR(qPCR) to analyze the mRNA levels of Rad9, Rad1 and Hus1 in various tissues isolated from mice that were either mock irradiated or exposed to 10 Gy gamma radiation. Our results indicated that the mRNA levels of Rad9, Rad1 and Hus1 genes were very different among these tissues, and we found high natural levels of mRNA in the spleen, lung, ovary and testis of mice before exposure to radiation. The mRNA levels of the three genes were well correlated across these tissues, being high, medium or low in each of the tissues simultaneously. The mRNA levels of the three genes were analyzed at 2, 6, 12, 24 and 48 h after irradiation. In most tissues Rad9 was strongly induced at 2 and 12 h time points and Hus1 was strongly induced at 2, 12 and 48 h time points, but Rad1 was minimally induced in most of the tissues with the exception of slightly higher levels in the heart and lung tissues at the 48 h time point. These results suggest that the regulation mechanisms for the mRNA levels of the three genes in response to ionizing radiation are complex and not well orchestrated. We also detected the induction of Rad9 and Hus1 proteins in the heart and liver of the animals after irradiation, and found that Rad9 protein levels were highly induced in both the heart and liver, while the Hus1 protein levels were significantly induced only in the liver, suggesting that Rad9 and Hus1 protein levels are not regulated in a coordinated manner in response to irradiation. We then went on to measure the mRNA levels of the three genes and the Rad9 and Hus1 protein levels in the mouse liver cell line (NCTC 1469) in response to irradiation in vitro. All three genes in the cultured cells were minimally induced at mRNA level, obviously different from the highly dynamic induction in liver. Rad9 and Hus1 were significantly induced at the protein level, but the induced Rad9 protein levels were higher than the Hus1 levels. Taken together, the good correlation of the mRNA levels of Rad9, Hus1 and Rad1 genes across different tissues isolated from the animals that were mock irradiated and the lack of correlation in mRNA as well as protein levels after irradiation suggest that the 9-1-1 complex has evolved to play various physiological roles in tissues rather than dealing with high doses of gamma radiation or other genotoxic agents. (C) 2015 by Radiation Research Society

submitted time 2016-05-12 Hits2740Downloads1449 Comment 0

2. chinaXiv:201605.01399 [pdf]

Particle Shape Effects on Subvisible Particle Sizing Measurements

Cavicchi, Richard E.; Carrier, Michael J.; Cohen, Joshua B.; Boger, Shir; Montgomery, Christopher B.; Ripple, Dean C.; Hu, Zhishang
Subjects: Biology >> Biophysics

Particle analysis tools for the subvisible (<100 m) size range, such as light obscuration, flow imaging (FI), and electrical sensing zone (ESZ), often produce results that do not agree with one another, despite their general agreement when characterizing polystyrene latex spheres of different sizes. To include the effect of shape in comparison studies, we have used the methods of photolithography to create rods and disks. Although the rods are highly monodisperse, the instruments produce broadened peaks and report mean size parameters that are different for different instruments. We have fabricated a microfluidic device that simultaneously performs ESZ and FI measurements on each particle to elucidate the causes of discrepancies and broadening. Alignment of the rods with flow causes an oversizing by FI and undersizing by ESZ. FI also oversizes rods because of the incorrect edge definition that results from diffraction and imperfect focus. We present an improved correction algorithm for this effect that reduces discrepancies for rod-shaped particles. Tumbling of particles is observed in the microfluidic ESZ/FI and results in particle oversizing and breadth of size distribution for the monodisperse rods. (c) 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:971-987, 2015

submitted time 2016-05-12 Hits1416Downloads724 Comment 0

3. chinaXiv:201605.01323 [pdf]

The Use of Index-Matched Beads in Optical Particle Counters

Hu, Zhishang; Ripple, Dean C.
Subjects: Biology >> Biophysics

In this paper, we demonstrate the use of 2-pyridinemethanol (2P) aqueous solutions as a refractive index matching liquid. The high refractive index and low viscosity of 2P-water mixtures enables refractive index matching of beads that cannot be index matched with glycerol-water or sucrose-water solutions, such as silica beads that have the refractive index of bulk fused silica or of polymethylmethacrylate beads. Suspensions of beads in a nearly index-matching liquid are a useful tool to understand the response of particle counting instruments to particles of low optical contrast, such as aggregated protein particles. Data from flow imaging and light obscuration instruments are presented for bead diameters ranging from 6 mu m to 69 mu m, in a matrix liquid spanning the point of

submitted time 2016-05-11 Hits1164Downloads531 Comment 0

4. chinaXiv:201605.01188 [pdf]

An Interlaboratory Comparison of Sizing and Counting of Subvisible Particles Mimicking Protein Aggregates

Ripple, Dean C.; Montgomery, Christopher B.; Hu, Zhishang
Subjects: Biology >> Biophysics

Accurate counting and sizing of protein particles has been limited by discrepancies of counts obtained by different methods. To understand the bias and repeatability of techniques in common use in the biopharmaceutical community, the National Institute of Standards and Technology has conducted an interlaboratory comparison for sizing and counting subvisible particles from 1 to 25 m. Twenty-three laboratories from industry, government, and academic institutions participated. The circulated samples consisted of a polydisperse suspension of abraded ethylene tetrafluoroethylene particles, which closely mimic the optical contrast and morphology of protein particles. For restricted data sets, agreement between data sets was reasonably good: relative standard deviations (RSDs) of approximately 25% for light obscuration counts with lower diameter limits from 1 to 5 m, and approximately 30% for flow imaging with specified manufacturer and instrument setting. RSDs of the reported counts for unrestricted data sets were approximately 50% for both light obscuration and flow imaging. Differences between instrument manufacturers were not statistically significant for light obscuration but were significant for flow imaging. We also report a method for accounting for differences in the reported diameter for flow imaging and electrical sensing zone techniques; the method worked well for diameters greater than 15 m. (c) 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:666-677, 2015

submitted time 2016-05-11 Hits1149Downloads603 Comment 0

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