Reduction of inhibitor titres by infusion of FVIII gene transduced tolerogenic dendritic cells in haemophilic mice

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72521/1/j.1365-2516.2009.01996_2.x.pd

Induction of serine racemase expression and D-serine release from microglia by amyloid β-peptide

BACKGROUND: Roles for excitotoxicity and inflammation in Alzheimer's disease have been hypothesized. Proinflammatory stimuli, including amyloid β-peptide (Aβ), elicit a release of glutamate from microglia. We tested the possibility that a coagonist at the NMDA class of glutamate receptors, D-serine, could respond similarly. METHODS: Cultured microglial cells were exposed to Aβ. The culture medium was assayed for levels of D-serine by HPLC and for effects on calcium and survival on primary cultures of rat hippocampal neurons. Microglial cell lysates were examined for the levels of mRNA and protein for serine racemase, the enzyme that forms D-serine from L-serine. The racemase mRNA was also assayed in Alzheimer hippocampus and age-matched controls. A microglial cell line was transfected with a luciferase reporter construct driven by the putative regulatory region of human serine racemase. RESULTS: Conditioned medium from Aβ-treated microglia contained elevated levels of D-serine. Bioassays of hippocampal neurons with the microglia-conditioned medium indicated that Aβ elevated a NMDA receptor agonist that was sensitive to an antagonist of the D-serine/glycine site (5,7-dicholorokynurenic acid; DCKA) and to enzymatic degradation of D-amino acids by D-amino acid oxidase (DAAOx). In the microglia, Aβ elevated steady-state levels of dimeric serine racemase, the apparent active form of the enzyme. Promoter-reporter and mRNA analyses suggest that serine racemase is transcriptionally induced by Aβ. Finally, the levels of serine racemase mRNA were elevated in Alzheimer's disease hippocampus, relative to age-matched controls. CONCLUSIONS: These data suggest that Aβ could contribute to neurodegeneration through stimulating microglia to release cooperative excitatory amino acids, including D-serine

Baseline Survey of Root-Associated Microbes of \u3cem\u3eTaxus chinensis\u3c/em\u3e (Pilger) Rehd

Taxol (paclitaxel) a diterpenoid is one of the most effective anticancer drugs identified. Biosynthesis of taxol was considered restricted to the Taxus genera until Stierle et al. discovered that an endophytic fungus isolated from Taxus brevifolia could independently synthesize taxol. Little is known about the mechanism of taxol biosynthesis in microbes, but it has been speculated that its biosynthesis may differ from plants. The microbiome from the roots of Taxus chinensis have been extensively investigated with culture-dependent methods to identify taxol synthesizing microbes, but not using culture independent methods.,Using bar-coded high-throughput sequencing in combination with a metagenomics approach, we surveyed the microbial diversity and gene composition of the root-associated microbiomefrom Taxus chinensis (Pilger) Rehd. High-throughput amplicon sequencing revealed 187 fungal OTUs which is higher than any previously reported fungal number identified with the culture-dependent method, suggesting that T. chinensis roots harbor novel and diverse fungi. Some operational taxonomic units (OTU) identified were identical to reported microbe strains possessing the ability to synthesis taxol and several genes previously associated with taxol biosynthesis were identified through metagenomics analysis

Final Report

Forest products provide essential resources for human civilization, including energy and materials. In processing forest products, however, unwanted byproducts, such as volatile organic compounds (VOCs) and hazardous air pollutants (HAPs) are generated. The goal of this study was to develop a cost effective and reliable air pollution control system to reduce VOC and HAP emissions from pulp, paper and paperboard mills and solid wood product facilities. Specifically, this work focused on the removal of VOCs and HAPs from high volume low concentration (HVLC) gases, particularly methanol since it is the largest HAP constituent in these gases. Three technologies were developed and tested at the bench-scale: (1) A novel composite material of activated carbon coated with a photocatalyst titanium dioxide (TiO{sub 2}) (referred to as TiO{sub 2}-coated activated carbon or TiO{sub 2}/AC), (2) a novel silica gel impregnated with nanosized TiO{sub 2} (referred to as silica-titania composites or STC), and (3) biofiltration. A pilot-scale reactor was also fabricated and tested for methanol removal using the TiO{sub 2}/AC and STC. The technical feasibility of removing methanol with TiO{sub 2}/AC was studied using a composite synthesized via a spay desiccation method. The removal of methanol consists of two consecutive operation steps: removal of methanol using fixed-bed activated carbon adsorption and regeneration of spent activated carbon using in-situ photocatalytic oxidation. Regeneration using photocatalytic oxidation employed irradiation of the TiO{sub 2} catalyst with low-energy ultraviolet (UV) light. Results of this technical feasibility study showed that photocatalytic oxidation can be used to regenerate a spent TiO{sub 2}/AC adsorbent. A TiO{sub 2}/AC adsorbent was then developed using a dry impregnation method, which performed better than the TiO{sub 2}/AC synthesized using the spray desiccation method. The enhanced performance was likely a result of the better distribution of TiO2 particles on the activated carbon surface. A method for pore volume impregnation using microwave irradiation was also developed. A commercial microwave oven (800 W) was used as the microwave source. Under 2450 MHz microwave irradiation, TTIP was quickly hydrolyzed and anatase TiO2 was formed in a short time (< 20 minutes). Due to the volumetric heating and selective heating of microwave, the solvent and by-products were quickly removed which reduced energy consumption and processing time. Activated carbon and TiO{sub 2}/AC were also tested for the removal of hydrogen sulfide, which was chosen as the representative total reduced sulfur (TRS) species. The BioNuchar AC support itself was a good H{sub 2}S remover. After coating TiO{sub 2} by dry impregnation, H{sub 2}S removal efficiency of TiO{sub 2}/AC decreased compared with the virgin AC due to the change of surface pH. Under UV light irradiation, H{sub 2}S removal efficiency of TiO{sub 2}/AC composite doubled, and its sulfate conversion efficiency was higher than that of AC. The formation of sulfate is preferred since the sulfate can be removed from the composite by rising with water. A pilot-scale fluidized bed reactor was designed to test the efficiency of methanol oxidation with TiO{sub 2}/AC in the presence of UV light. TiO{sub 2}/AC was prepared using the spray desiccation method. The TiO{sub 2}/AC was pre-loaded with (1) methanol (equivalent to about 2%wt) and (2) methanol and water. When the TiO{sub 2}/AC loaded with methanol only was exposed to UV light for one hour in the reactor, most of the methanol remained in the carbon pores and, thus, was not oxidized. The TiO{sub 2}/AC loaded with methanol and water desorbed about 2/3 of the methanol from its pores during fluidization, however, only a small portion of this desorbed methanol was oxidized. A biofilter system employing biological activated carbon was developed for methanol removal. The biofilter contained a mixed packing with Westvaco BioNuchar granular activated carbon, perlite, Osmocote slow release ammonium nitrate pellets, and Agrasoke water crystals in a 4:2:1:1 ratio by volume. The biofilter was inoculated with a bacterial culture collected from a Florida pulp and paperboard plant. A non-inoculated biofilter column was also tested. Use of a biological inoculum enriched from biofilm in the pulp and paper process has the potential to enhance the performance of a GAC biofilter. During testing, packing material was removed from the inlet and oulet of the biofilters and analyzed for genetic diversity using molecular techniques. The biofilter inoculated with specifically-enhanced inoculum showed higher bacterial diversity for methylotrophs and all bacteria, as compared to a non-inoculated biofilter. Mixed methylotrophic cultures, selected as potential biofilter inocula, showed increased methanol removal with highest concentrations of nitrogen provided as nitrate

Pyk2 deficiency potentiates osteoblast differentiation and mineralizing activity in response to estrogen or raloxifene

Bone remodeling is controlled by the actions of bone-degrading osteoclasts and bone-forming osteoblasts (OBs). Aging and loss of estrogen after menopause affects bone mass and quality. Estrogen therapy, including selective estrogen receptor modulators (SERMs), can prevent bone loss and increase bone mineral density in post-menopausal women. Although investigations of the effects of estrogen on osteoclast activity are well advanced, the mechanism of action of estrogen on OBs is still unclear. The proline-rich tyrosine kinase 2 (Pyk2) is important for bone formation and female mice lacking Pyk2 (Pyk2-KO) exhibit elevated bone mass, increased bone formation rate and reduced osteoclast activity. Therefore, in the current study, we examined the role of estrogen signaling on the mechanism of action of Pyk2 in OBs. As expected, Pyk2-KO OBs showed significantly higher proliferation, matrix formation, and mineralization than WT OBs. In addition we found that Pyk2-KO OBs cultured in the presence of either 17β-estradiol (E2) or raloxifene, a SERM used for the treatment of post-menopausal osteoporosis, showed a further robust increase in alkaline phosphatase (ALP) activity and mineralization. We examined the possible mechanism of action and found that Pyk2 deletion promotes the proteasome-mediated degradation of estrogen receptor α (ERα), but not estrogen receptor β (ERβ). As a consequence, E2 signaling via ERβ was enhanced in Pyk2-KO OBs. In addition, we found that Pyk2 deletion and E2 stimulation had an additive effect on ERK phosphorylation, which is known to stimulate cell differentiation and survival. Our findings suggest that in the absence of Pyk2, estrogen exerts an osteogenic effect on OBs through altered ERα and ERβ signaling. Thus, targeting Pyk2, in combination with estrogen or raloxifene, may be a novel strategy for the prevention and/or treatment of bone loss diseases

Restriction landmark genomic scanning (RLGS) spot identification by second generation virtual RLGS in multiple genomes with multiple enzyme combinations.

BackgroundRestriction landmark genomic scanning (RLGS) is one of the most successfully applied methods for the identification of aberrant CpG island hypermethylation in cancer, as well as the identification of tissue specific methylation of CpG islands. However, a limitation to the utility of this method has been the ability to assign specific genomic sequences to RLGS spots, a process commonly referred to as "RLGS spot cloning."ResultsWe report the development of a virtual RLGS method (vRLGS) that allows for RLGS spot identification in any sequenced genome and with any enzyme combination. We report significant improvements in predicting DNA fragment migration patterns by incorporating sequence information into the migration models, and demonstrate a median Euclidian distance between actual and predicted spot migration of 0.18 centimeters for the most complex human RLGS pattern. We report the confirmed identification of 795 human and 530 mouse RLGS spots for the most commonly used enzyme combinations. We also developed a method to filter the virtual spots to reduce the number of extra spots seen on a virtual profile for both the mouse and human genomes. We demonstrate use of this filter to simplify spot cloning and to assist in the identification of spots exhibiting tissue-specific methylation.ConclusionThe new vRLGS system reported here is highly robust for the identification of novel RLGS spots. The migration models developed are not specific to the genome being studied or the enzyme combination being used, making this tool broadly applicable. The identification of hundreds of mouse and human RLGS spot loci confirms the strong bias of RLGS studies to focus on CpG islands and provides a valuable resource to rapidly study their methylation

Effects of Radiopaque Double Antibiotic Pastes on the Proliferation, Alkaline Phosphatase Activity and Mineral Deposition of Dental Pulp Stem Cells

Objective The aim of this study was to investigate the effects of two radiopaque agents, barium sulfate (BaSO4) or zirconium oxide (ZrO2) in double antibiotic paste (DAP), on the proliferation and mineral deposition of human dental pulp stem cells (DPSC). Materials and methods Radiopaque antimicrobial medicaments composed of methylcellulose (MC) thickening polymer with BaSO4 or ZrO2 and either 1 or 5 mg/mL DAP (equal portions of metronidazole and ciprofloxacin) were used to investigate DPSC proliferation after 3 days, and alkaline phosphatase (ALP) activity and mineral deposition after 7 and 14 days. Radiopaque agents without DAP and Ca(OH)2 were used as controls. Results MC-BaSO4 DAP and MC-ZrO2 DAP at 1 or 5 mg/mL had no adverse effect on DPSC proliferation, compared to the media and MC controls. MC-ZrO2 (DAP-free) greatly increased ALP activity after 7 days. DPSC mineral deposition was modestly reduced at 7 days by MC-BaSO4 DAP and MC-ZrO2 DAP, but not by DAP-free radiopaque agents, and was most reduced by 5 mg/mL DAP in the 14-day cultures. Conclusions MC-BaSO4 or MC-ZrO2 medicaments containing up to 5 mg/mL of DAP supported the proliferation and early osteogenic differentiation of DPSC. Low DAP concentrations and short culture times led to more favorable effects on ALP activity and mineral deposition by DPSC. The findings suggest that radiopaque agents added for the purpose of detecting whether medicaments occupy the full extent of the root canal may have clinical applications. Radiopaque antibiotic medicaments containing low DAP concentrations may be an alternative to Ca(OH)2 for regenerative endodontic procedures

BMP signaling mediates glioma stem cell quiescence and confers treatment resistance in glioblastoma.

Despite advances in therapy, glioblastoma remains an incurable disease with a dismal prognosis. Recent studies have implicated cancer stem cells within glioblastoma (glioma stem cells, GSCs) as mediators of therapeutic resistance and tumor progression. In this study, we investigated the role of the transforming growth factor-β (TGF-β) superfamily, which has been found to play an integral role in the maintenance of stem cell homeostasis within multiple stem cell systems, as a mediator of stem-like cells in glioblastoma. We find that BMP and TGF-β signaling define divergent molecular and functional identities in glioblastoma, and mark relatively quiescent and proliferative GSCs, respectively. Treatment of GSCs with BMP inhibits cell proliferation, but does not abrogate their stem-ness, as measured by self-renewal and tumorigencity. Further, BMP pathway activation confers relative resistance to radiation and temozolomide chemotherapy. Our findings define a quiescent cancer stem cell population in glioblastoma that may be a cellular reservoir for tumor recurrence following cytotoxic therapy

Assessing architectural evolution: A case study

This is the post-print version of the Article. The official published can be accessed from the link below - Copyright @ 2011 SpringerThis paper proposes to use a historical perspective on generic laws, principles, and guidelines, like Lehman’s software evolution laws and Martin’s design principles, in order to achieve a multi-faceted process and structural assessment of a system’s architectural evolution. We present a simple structural model with associated historical metrics and visualizations that could form part of an architect’s dashboard. We perform such an assessment for the Eclipse SDK, as a case study of a large, complex, and long-lived system for which sustained effective architectural evolution is paramount. The twofold aim of checking generic principles on a well-know system is, on the one hand, to see whether there are certain lessons that could be learned for best practice of architectural evolution, and on the other hand to get more insights about the applicability of such principles. We find that while the Eclipse SDK does follow several of the laws and principles, there are some deviations, and we discuss areas of architectural improvement and limitations of the assessment approach