Recombinant human lubricin (rhPRG4) was expressed by transfected CHO- S cells at a level of secretion that may be scalable and thus enable production of a GMP protein for clinical use. A purification bioprocess was created that achieved a purity level of approximately 95%. A total of 5 bands visualized on SDS-PAGE are all product related following excision and analysis by LC-MS. Full- length and truncated rhPRG4 constructs are being tested for chondroprotective ability. The full-length construct significantly lowers friction between pressurized discs of articular cartilage. The coefficient of friction in the presence of rhPRG4 was 0.03 whereas for a saline control 0.07. Study of the 5 other constructs and confirmation of reduction in levels of chondrocyte apoptosis attributable to lower friction is ongoing. The posttranslational glycosylations on rhPRG4 appear to be (1-3)GalNac-Gal (2,3) NeuAc judging by sequential enzymatic deglycosylation and molecular weight shift on SDS-PAGE. Western blotting of rhPRG4 with mAb 9G3, which reacts with the glycosylations in the lubricin mucin domain, also confirms that the glycosylations are consistent with lubricin secreted by human synovial fibroblasts. Analytical ultracentrifugation shows that there are 5 distinct species in rhPRG4, 3 of which are major forms: monomer, dimer and a higher order tetramer. This biophysical form of analysis may also prove useful in a quality assurance program in GMP protein production as the relative amounts of each major form can be monitored.

关键词：关节炎；软骨；细胞凋亡

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Numbers dendritic cells (DCs) increased 100-fold in pancreata of mice with acute pancreatitis to account for nearly 15% of intrapancreatic leukocytes. Intrapancreatic DCs acquired a distinct immune phenotype in mice with acute pancreatitis; they expressed higher levels of major histocompatibility complex II and CD86 and increased production of interleukin- 6, MCP 1, and TNFa. DC over-expansion exacerbated disease. However, DCs were also required for pancreatic viability; the exocrine pancreas died in mice that were depleted of DCs and challenged with caerulein or L-arginine. All mice with pancreatitis that were depleted of DCs died from acinar cell death within 4 days. These data suggest that DC have simultaneous paradoxical pro-inflammatory and protective effects in pancreatitis. Further investigations are warranted.

关键词：树突状结构；胰腺炎；细胞（生物学）

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To realize three-dimensional optical imaging using transillumination images, we developed a novel technique to suppress the scattering effect in a transillumination image and reconstruct a three-dimensional image of the internal light-absorbing structure of animal body from two-dimensional images. The problem of the depth-dependence of PSF was solved by the calculation of the projection image in the filtered back-projection method. The feasibility and the effectiveness of the proposed technique were verified in the experiments with tissue-equivalent phantom and a living mouse. Using the proposed technique, the high-absorption organs such as kidneys and the bottom parts of the liver were reconstructed in a 3D image of mouse abdomen.

关键词：透照成像；散射抑制；反褶积

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The purpose of the report is to determine in a mouse model of high and low fear whether a protein expressed in the lateral amygdala is more abundance in the high fear mice following Pavlovian fear conditioning. These data provide important insights into the micro network mechanisms in the barin that may lead to different levels of fear response in individuals. Understanding the circuit level cellular and molecular mechanisms that underlie individual variability in far learning is critical for the development of effective treatment of fear-related illnesses such as PTSD. These data suggest that inhibiting a specific protein in a precise brain region in mice may reduce high fear memory to the level of low fear memory. This information may begin to provide foundations for the understanding and eventual treatment of pathological fear. Using a mouse model of very high and very low fear memory to understand cellular mechanisms in specific brain regions involved in forming fear memories may help identify novel ways to predict individuals at risk for fear related illness. This can potentially lead to targeted treatments for fear-related disorders such as PTSD.

关键词：恐惧；内存；老鼠；脑细胞（生物学）

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Prostate cancer is the second leading cause of cancer death among American men. Although radiotherapy (RT) is one of the two curative treatments for prostate cancer patients, approximately 10% of low-risk cancer patients and 30-60% of high-risk prostate cancer patients experience biochemical recurrence within five years, among them 20% die in 10 years. The proposed research is based on the hypothesis that targeting protein arginine methyltransferase 5 (PRMT5) can sensitize primary and recurrent prostate cancer cells to RT. During the first grant period, we have successfully demonstrated that knockdown of PRMT5 or inhibition of PRMT5 by a specific inhibitor can sensitize prostate cancer cells (LNCaP, DU-145 and PC-3) to radiation in vitro. This radiosensitization is likely due to the involvement of PRMT5 in the regulation of the DNA damage response. These results collectively suggest that targeting PRMT5 can sensitize prostate cancer cells to radiation. We are currently isolating stably integrated clones to inducibly knock down PRMT5 for proposed in vivo experiments. We have also successfully isolated 3 radiation-resistant sublines from DU-145 after 40 Gy of fractionated ionizing radiation. These resistant cell sublines along with previously isolated LNCaP radiation- resistant sublines will be used for PRMT5 targeting experiments. In addition, we found that PRMT5 regulates prostate cancer cell growth in an AR-dependent manner, and this effect is likely medicated by epigenetic regulation of AR transcription. We will continue to pursue this novel and exciting finding.

关键词：电离辐射；前列腺癌；放射疗法

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In this project we set out to establish conditions for differentiation of human neonatal foreskin skin fibroblast-derived iPSCs into prostate epithelium-like cells and identify differences in gene expression between prostate epithelial cells derived from iPSCs of Caucasian (white) and African-American (black) foreskin fibroblasts. We identified and optimized culture conditions that promote prostate epithelial cell-like differentiation of humaniPS clone, IMAR90-4. Our data show that a feeder layer of urogential mesenchymal(UGSM) cells from neonatal mouse of either gender in combination with neonatal human dermal fibroblasts induced a striking morphological changes that resembles epithelila differentiation with formation of lumen-like structures. We show requirement of components of the extracellular matrix that promote epithelial-type differentaition. Immunofluorescence and biochemivcal analyses showed expression of androgen receptor and markers of epithelial differentiation. Analyses of pluripotency marker expression by RT-PCR showed that while human dermal fibroblasts have higher constitutive expression of Nanog, Oct4 and Sox2 compared to UGSM and IMP90 cells. Preliminary studies also showed that black black fibrobalst population has higher constitutive expression of pluripotency markers than cells from white individuals.

关键词：前列腺癌；干细胞；血细胞

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Staphylococcus aureus methicillin-resistant and methicillin- susceptible (MRSA/MSSA) is a leading cause of infections in military personnel, but there are limited data regarding baseline colonization of individuals while deployed. We conducted a pilot study to screen non-deployed and deployed healthy military service members for MRSA/MSSA colonization at various anatomic sites and assessed isolates for molecular differences.

关键词：菌落（生物学）；金黄色葡萄球菌；解剖学

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The vascular endothelial growth factor (VEGF) family of cytokines promotes vascularization, tumorigenesis and metastasis in many cancers. Our goal is to develop computational models that combine mechanistic topological data on the VEGF protein interaction network with gene expression datasets for a large population of prostate cancers. We have assembled databases of prostate cancer gene expression data, and analyzed the data using bioinformatic techniques, identifying key VEGF-based subgroups of prostate cancer plus biomarkers that identify these groups. We have also created new computational models to simulate prostate cancer, based on the individualized gene expression data. These models will be used to simulate therapies that target the pathway. The therapies to be tested include anti-ligands such as bevacizumab but also anti-receptors and small molecules such as tyrosine kinase inhibitors. In this way, we can build on both the successes and the failures of anti-VEGF trials to date in order to develop more effective therapies for prostate cancer. This progress will continue, and we will be able to develop models of therapies including bevacizumab and other drugs, in order to design improved therapeutic approaches (both for individuals and for the population).

关键词：转移；前列腺癌；飞行速度；生长（生理学）

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关键词：激素；军事人员；神经科学；类固醇

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Effective in vitro models of myelination, dysmyelination, and/or remyelination would substantially speed the development and testing of potential therapies for myelin disorders such as multiple sclerosis. Tissues engineered from human induced pluripotent stem (iPS) may be effective at accurately modeling aspects of human physiology for screening of potential therapies. Substantial progress has been made toward demonstrating the feasibility of developing microengineered human neural tissues that can be assessed non-invasively. A population of neurons has been derived from human iPS cell lines, a micropatterned hydrogel culture system has been identified for the support of human neurons, and an optical method for monitoring physiological responses in microengineered tissue constructs has been demonstrated. This works represents a unique combination of enabling technologies, including human iPS cells, microfabrication, and optical neural recording, with the goal of showing how it may be possible to create a high-throughput assay of human neural activity.

关键词：体外分析；生理效应；响应（生物学）

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