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New Windsor, United States

Hoffnagle G.F.,TRC
Proceedings of the Air and Waste Management Association's Annual Conference and Exhibition, AWMA | Year: 2011

The EPA BART regulations (40 CFR Part 51.308) require that each State develop a State Implementation Plan to reduce emissions from major sources of SO x and NO x so that reasonable further progress can be made toward reducing visibility impairment in the nation's national parks and wilderness areas. Measured and modeled data in BART analysis for a fairly isolated power plant were compared to demonstrate the model accuracy. Modeling was carried out with CALPUFF using protocols provided by EPA and the Federal Land Managers. The modeling predicted total extinction in the range of actual values for the power plant by itself excluding all other sources except "background". The emphasis in the BART program was on control of SO 2 and NO x emissions. There was so little impact of nitrates at Mount Zirkel on visibility that reductions in NO x emissions do not and cannot improve visibility, i.e., additional controls will not change or improve the visibility at Mount Zirkel. This is an abstract of a paper presented at the 104th AWMA Annual Conference and Exhibition 2011 (Orlando, FL 6/21-24/2011).

Ricker M.C.,Auburn University | Stolt M.H.,University of Rhode Island | Donohue S.W.,TRC | Blazejewski G.A.,U.S. Department of Agriculture | Zavada M.S.,East Tennessee State University
Soil Science Society of America Journal | Year: 2013

Riparian zones are important catchment-scale depositional environments that receive episodic influx of sediment and C from watershed sources. The specific impacts of upland disturbances on riparian soil development and soil organic carbon (SOC) dynamics are still largely unknown. The goal of our study was to understand the role of riparian soils in retaining C at the landscape and catchment scales. We quantified SOC pools to a depth of 1 m at 29 headwater riparian sites in southern New England. Riparian SOC pools ranged from 117 to 495 Mg C ha-1, with a mean pool of 246 Mg C ha-1. On average, >50% of the total SOC was stored below 30 cm. Riparian SOC pools differed significantly between soils formed in relatively fast accreting environments (those that contain buried surface horizons; 277 Mg C ha -1) and those in slow accreting environments where buried horizons were absent (188 Mg C ha-1). Catchment-scale analysis of SOC distribution indicated that riparian zones, on average, occupy 8% of the total watershed area yet store as much as 20% of the total catchment SOC. These results suggest that even though riparian zones occupy a small percentage of the overall watershed, these areas are an important component of the landscape for storage of SOC deposited as a result of catchment-scale disturbances. © Soil Science Society of America.

Moustafa S.F.,Central Metallurgical Research and Development Institute | Kaitbay S.H.,Helwan University | Abdo G.M.,TRC
Defect and Diffusion Forum | Year: 2010

Elemental powders of tungsten, nickel, iron and cobalt of compositions corresponding to (W-3.2Ni- 0.8%Fe), (W-3.5Ni-1.5%Fe), and (W-4.5Ni-1.0Fe-1.5%Co) were mechanically alloyed in a tumbler rod mill for 2 hrs. Mechanically alloyed powders were liquid phase sintered at 1500°C for 90 min in vacuum. The sintered materials were heated up to 1150-1200°C in vacuum atmosphere, followed by quenching in water to suppress the impurity segregated at grain boundary. The sintered materials were subjected to cold-working by swaging from 8-30% reduction in area. The swaged specimens were age-hardened at 700°C. Full characterization for both the elemental powders and the sintered tungsten alloys were performed using optical microscopy, SEM analysis, EDS quantitative analysis, X-ray diffraction, hardness and compression testing. This paper will discuss the effects of the elemental powders characterization and the liquid phase sintering parameters on the microstructure and strength of these three tungsten heavy alloys. © (2010) Trans Tech Publications.

Windecker A.,TRC | Ruder A.,Energy Circle
Transportation Research Part D: Transport and Environment | Year: 2013

This paper presents in-service data collected from over 300 alternative fuel vehicles and over 80 fueling stations to help fleets determine what types of applications and alternative fuels may help them reduce their environmental impacts and fuel costs. The data were compiled in 2011 by over 30 organizations in New York State using a wide variety of commercial vehicle types and technologies. Fuel economy, incremental vehicle purchase cost, fueling station purchase cost, greenhouse gas reductions, and fuel cost savings data clarifies the performance of alternative fuel vehicles and fuel stations. Data were collected from a range of vehicle types, including school buses, delivery trucks, utility vans, street sweepers, snow plows, street pavers, bucket trucks, paratransit vans, and sedans. CNG, hybrid, LPG, and electric vehicles were tracked. © 2013 Elsevier Ltd.

The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. FS5, FS4, FS13, FS14, M93-047, UACC-903, and UACC-1273 cells were maintained in RPMI (Invitrogen), supplemented with 10% FBS, 100 units per millilitre penicillin and streptomycin, and 4 mM L-glutamine. WM35, WM793, WM164, WM1799, and 1205 LU cells were maintained in MCDB153 (Sigma)/L-15 (Cellgro) (4:1 ratio) supplemented with 2% FBS and 1.6 mM CaCl (tumour growth media). WM983b and WM3918 cells were maintained in DMEM (Invitrogen), supplemented with 5% FBS, 100 units per millilitre penicillin and streptomycin, and 4 mM L-glutamine. YUMM1.7 cells were maintained in DMEM F-12 (HEPES/glutamine) supplemented with 10% FBS, 1% NEAA, and 100 units per millilitre penicillin and streptomycin. Fibroblasts were maintained in DMEM, supplemented with 10% FBS, 100 units per millilitre penicillin and streptomycin, and 4 mM L-glutamine. Keratinocytes were maintained in keratinocyte SFM supplemented with human recombinant Epidermal Growth Factor 1-53 (EGF 1-53) and Bovine Pituitary Extract (BPE) (Invitrogen). Cell lines were cultured at 37 °C in 5% CO and the medium was replaced as required. Cell stocks were fingerprinted using an AmpFLSTR Identifiler PCR Amplification Kit from Life Technologies at The Wistar Institute Genomics Facility. Although it is desirable to compare the profile with the tissue or patient of origin, our cell lines were established over the course of 40 years, long before acquisition of normal control DNA was routinely performed. However, each short tandem repeat profile is compared with our internal database of over 200 melanoma cell lines, as well as control lines, such as HeLa and 293 T. Short tandem repeat profiles are available upon request. Cell culture supernatants were tested for mycoplasma using a Lonza MycoAlert assay at the University of Pennsylvania Cell Center Services. Organotypic three-dimensional skin reconstructs were generated as previously described30. In each insert, 6.4 × 104 fibroblasts were plated on top of the acellular layer (BD 355467 and Falcon 353092) and incubated for 45 min at 37 °C in a 5% CO tissue culture incubator. DMEM containing 10% FBS was added to each well of the tissue culture trays and incubated for 4 days. Reconstructs were then incubated for 1 h at 37 °C in HBSS containing 1% dialysed FBS (wash media). Washing media were removed and replaced with reconstruct media I. Keratinocytes (4.17 × 105) and melanoma cells (8.3 × 104) were added to the inside of each insert. Media were changed every other day until day 18 when reconstructs were harvested, fixed in 10% formalin, paraffin embedded, sectioned, and stained. Quantification of the invasion was performed using ImageJ software (available at http://imagej.nih.gov/ij/; developed by W. Rasband). Tissue-culture-treated 96-well plates were coated with 50 μl 1.5% Difco Agar Noble (Becton Dickinson). Melanoma cells were seeded at 5 × 103 cells per well and allowed to form spheroids over 72 h. Spheroids were harvested and embedded as previously described using collagen type I (GIBCO, A1048301). For spheroids incubated with fibroblast conditioned media, fibroblasts were seeded onto 75 cm2 flasks at 7 × 105 to 9 × 105 per flask depending on growth rate. Sixteen hours later, media were replaced and incubated for 72 h. Media from young fibroblasts were combined and media from aged fibroblasts were combined. These conditioned media were added to the top of the collagen plug containing the spheroids. Quantitation of invasive surface area was performed using NIS Elements Advanced Research software. Spheroids were generated and embedded as described above. Spheroids were stained using a LIVE/DEAD Viability/Cytotoxicity Kit (L3224, Invitrogen). Briefly, spheroids were washed with PBS and stained with calcein AM/Ethidium homodimer-1. The dyes were diluted in PBS and 300 μl of the solution was added on the spheroid wells for 1 h at 37 °C. The spheroids were washed in PBS and imaged using a Nikon TE2000 Inverted Microscope. Quantitation of fluorescence intensity was performed using NIS Elements Advanced Research software. Matrigel (BD Biosciences, 354234) was diluted in PBS (1:3,000 dilution). One hundred and fifty microlitres of this mixture was pipetted into each insert of the invasion assay plate (Corning, 3422). The plate was incubated at 37 °C for 2 h and then dried at room temperature (25 °C) overnight under sterile conditions. Melanoma cells were pre-treated for 48 h in six-well plates. After 48 h, the cells were harvested and 1.5 × 105 cells were added to each transwell. High concentration serum media (RPMI with 20% FCS, tumour growth media with 10% FCS) were added to the outside (bottom) of the well. The plates were incubated at 37 °C until cells had migrated to the bottom of the well. The migrated cells were fixed in 95% ice-cold methanol and stained with crystal violet (0.5%) for 10 min. The stain was washed and the wells were left to dry. Cells were imaged and quantified using ImageJ software. In a 24-well plate, 5,000 cells in triplicate were plated per day of measurement. Every 2–3 days, cells were counted using a haemocytometer and the total cell number in the well was recorded and plotted on GraphPad Prism. Cells were seeded onto glass cover slips at 1 × 104 to 4 × 104 cells per well, and incubated overnight. After treatment, cells were fixed using 95% methanol. Primary antibodies were diluted as stated above in blocking buffer and incubated overnight at 4 °C. Cells were washed in PBS and incubated with the appropriate secondary antibody (1:2,000, Invitrogen) for 1 h at room temperature. Cells were then washed in PBS and mounted in Prolong Gold anti-fade reagent containing DAPI (Invitrogen). Images were captured on a Leica TCS SP5 II scanning laser confocal system. All antibodies are described in the Supplementary Information. Patient samples were collected under IRB exemption approval for protocol EX21205258-1. Paraffin embedded sections were rehydrated through a xylene and alcohol series, rinsed in H O and washed in PBS. Antigen retrieval was performed using target retrieval buffer (Vector Labs) and steamed for 20 min. Samples were then blocked in a peroxidase blocking buffer (Thermo Scientific) for 15 min, followed by Protein block (Thermo Scientific) for 5 min, and incubated in appropriate primary antibody diluted in antibody diluent (S0809, Dako) at 4 °C overnight in a humidified chamber. For mouse samples to be incubated with anti-mouse antibody, samples were blocked for 1 h in Mouse on Mouse (M.O.M.) Blocking Reagent (MKB-2213, Vector Labs). After washing in PBS, samples were incubated in biotinylated anti-rabbit or polyvalent secondary antibody (Thermo Scientific) followed by streptavidin-HRP solution at room temperature for 20 min. Samples were then washed in PBS and incubated in 3-amino-9-ethyl-l-carboazole (AEC) chromogen and counterstained with Mayer’s haematoxylin for 1 min, rinsed in cold H O, and mounted in Aquamount. Total protein lysate (50–65 μg) was run on a 4–12% NuPAGE Bis Tris gel (Invitrogen). Proteins were then transferred onto PVDF membrane using an iBlot system, and blocked in 5% milk/TBST for 1 h. All primary antibodies were diluted in 5% milk/TBST and incubated over night at 4 °C. The membranes were washed in TBST and probed with the corresponding HRP-conjugated secondary antibody (0.2–0.02 μg ml−1 of anti-mouse, streptavidin, or anti-rabbit). Proteins were visualized using ECL prime (Amersham), or Luminata Crescendo (Millipore). All clones used are described in the Supplementary Information. All short hairpin RNA (shRNA) was obtained from the TRC shRNA library available through the Molecular Screening Facility at The Wistar Institute. Lentiviral production was performed as described in the protocol developed by the TRC library (Broad Institute). Briefly, 293 T cells were co-transfected with shRNA vector and lentiviral packaging plasmids (pCMV-dR8.74psPAX2, pMD2.G). The supernatant containing virus was harvested at 36 and 60 h, combined and filtered through a 0.45 μm filter. For transduction, the cells were layered overnight with lentivirus containing 8 μg ml−1 polybrene. The cells were allowed to recover for 24 h and then selected using 1 μg ml−1 puromycin. Cells (1.5 × 103 per well) were plated in a 96-well plate and treated with PLX4720. After 48 h, cells were incubated with MTS dye (20 μl per well) for 2 h. Absorbance was determined at 490 nm using an EL800 microplate reader (BioTek). The percentage cell proliferation was calculated by converting the experimental absorbance to percentage of control and plotted versus drug concentration. The values were then analysed using a nonlinear dose–response analysis in GraphPad Prism. Transcriptional profiling was determined using Illumina Sentrix BeadChips. Total RNA was used to generate biotin-labelled cRNA using the Illumina TotalPrep RNA Amplification Kit. In short, 0.5 μg of total RNA was first converted into single-stranded complementary DNA (cDNA) with reverse transcriptase using an oligo-dT primer containing the T7 RNA polymerase promoter site and then copied to produce double-stranded cDNA molecules. The double-stranded cDNA was cleaned and concentrated with the supplied columns and used in an overnight in vitro transcription reaction where single-stranded RNA (cRNA) was generated incorporating biotin-16-UTP. A total of 0.75 μg of biotin-labelled cRNA was hybridized at 58 °C for 16 h to Illumina’s Sentrix Human HT-12 v3 Expression BeadChips (Illumina). Each BeadChip has around 48,000 transcripts with approximately 15-fold redundancy. The arrays were washed, blocked, and the labelled cRNA was detected by staining with streptavidin-Cy3. Hybridized arrays were scanned using an Illumina BeadStation 500X Genetic Analysis Systems scanner and the image data extracted using the Illumina GenomeStudio software, version 1.1.1). Data are available in the GEO database (accession number GSE57445). Microarray expression data were quantile normalized and probes that showed low expression levels (detection P value > 0.05) across all samples were removed from the analysis. Expression values for each cell line were tested separately in multiple linear regression model with fibroblast age and experiment batches as predictor variables. Matlab version 8.0 ‘regress’ function was used to calculated P values for each probe for association with fibroblast age. False discovery rate was estimated using Benjamini-Hochberg procedure; only probes that showed a false discovery rate < 5% in all three cell lines were considered significant. Heat map was plotted using average expression values for three groups of age (young, middle, and aged) normalized to aged group (100%). All animal experiments were approved by the Institutional Animal Care and Use Committee (112503Y_0) and were performed in a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care. From preliminary studies, we observed significant differences (more than 1.8 standard deviations, a very large effect size) in some of the outcomes between young and aged groups. As few as five samples in each group in this study afforded 80% power at a two-sided α of 0.05 to detect a difference of about 1.8 standard deviations in a continuous outcome between young and aged groups, but we increased the sample size slightly to account for potential loss of mice due to health issues associated with ageing. Male C57BL6 mice at 6–8 weeks (young) and 52 weeks (aged) were purchased from Taconic. YUMM 1.7 (1 × 106 cells per 100 μl PBS) or B16F10 (2.5 × 105 per 100 μl PBS) were injected into the tail vein of C57BL6 mice. Alternatively, Yumm1.7 cells were overexpressed with mCherry plasmid (pLU-EF1-MCS-mCherry) using lentivirus. The cells were sorted for mCherry and 1 × 106 cells per 100 μl PBS were injected into tail vein of young C57BL6 mice. After 4 weeks, the mice were euthanized, lungs were harvested, and metastases counted. Lungs were fixed in paraffin and stained with haematoxylin and eosin. Alternatively, lungs were harvested and imaged for presence of metastatic melanoma cells using a Perkin-Elmer IVIS 200 whole body imager. For experiments requiring rsFRP2, mouse rsFRP2 (1169-FR-025/CF, R&D) was diluted in 50 μl PBS and injected at a concentration of 200 ng per mouse twice a week. The levels of sFRP2 were monitored by submandibular blood withdrawal every 2 weeks. All animal experiments were approved by the Institutional Animal Care and Use Committee (112503X_0) and were performed in a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care. From preliminary studies, we observed significant differences (more than 1.8 standard deviations, a very large effect size) in some of the outcomes between young and aged groups. As few as five samples in each group in this study afforded 80% power at a two-sided α of 0.05 to detect a difference of about 1.8 standard deviations in a continuous outcome between young and aged groups, but we increased the sample size slightly to account for potential loss of mice due to health issues associated with ageing. YUMM1.7 (2.5 × 105 cells) were suspended in Matrigel (500 μg ml−1) and injected subcutaneously into young (6 week) and aged (52 week) C57/BL6 mice (Taconic). When resulting tumours reached 200 mm3, mice were fed either AIN-76A chow or AIN-76A chow containing 417 mg kg−1 PLX4720. Tumour sizes were measured every 3–4 days using digital callipers, and tumour volumes were calculated using the following formula: volume = 0.5 × (length × width2). Time-to-event (survival) was determined by a fivefold increase in baseline volume (~1,000 mm3) and was limited by the development of skin necrosis. Upon the occurrence of necrosis, mice were euthanized. For subsequent experiments involving sFRP2 manipulation, Yumm1.7 cells overexpressing mCherry were used. One million cells were suspended in PBS and subcutaneously injected into either 6-week-old or 52-week-old male C57/BL6 mice (Taconic). For treatment with rsFRP2, the mice were injected with recombinant protein (200 ng ml−1) through the tail vein every 2 days as described above. For the experiments performed with sFRP2 blocking antibody (clone 80.8.6, MABC539, EMD Millipore), the mice were treated with 1 mg kg−1 antibody (either sFRP2 or isotype control, Biolegend, 400264) once a week through tail vein injections. WM35 and FS5 melanoma cells were seeded in 12-well plates and treated with conditioned media for 48 h. The cells were harvested in ice-cold PBS. The comet assay was performed using CometSlides (Trevigen). Briefly, 75 μl of a 2 × 105 cell suspension was mixed with 500 μl 1% low melting point agarose. Fifty microlitres of cell/agarose mixture was dropped into the wells and allowed to solidify. Slides were incubated in lysis buffer (1.2 M NaCl, 100 nM EDTA, 0.1% Sarkosyl, pH 10.0) for 1 h at 4 °C. Slides were then electrophoresed at 25 V for 12 min in alkaline buffer (0.03 M NaOH, 2 mM EDTA, pH 8.0). After fixation in 70% ethanol, comets were visualized by staining with SYBR Green (Fisher). The extent of DNA damage was measured as the artificial Olive Moment using Cometscore software downloaded from http://www.tritekcorp.com. Five thousand melanoma cells were seeded in a 96-well plate in triplicate. The cells were then treated with conditioned media from young and aged fibroblasts as well as DMEM as control. Hydrogen peroxide was added at 1 mM as control. After 72 h, ROS were measured using a Cell Meter Fluorimetric Intracellular Total ROS Activity Assay Kit (22901, AAT Bioquest) according to the manufacturer’s protocol. The plates were measured using a PerkinElmer EnVision Xcite Multilabel plate reader using the filters for excitation/emission (Ex/Em) = 520/605 nm. Alternatively, samples were imaged using PerkinElmer Operetta and the fluorescent signal was quantified with Harmony 3.0 software. The cell number was determined by Hoescht staining (Hoechst 33342, Invitrogen) and used to normalize the total fluorescence obtained from ROS staining. Topflash vectors were obtained from Addgene (M51 Super 8x FOPFlash/TOPFlash mutant, 12457; M50 Super 8x TOPFlash, 12456). WM35 cells were plated to achieve 70% confluency in six-well plates. Cells were co-transfected with pTK-RLuc (Green Renilla Luciferase) along with either Topflash or Fopflash vectors. After 5 h of transfection, cells were treated as required. After 48 h, cells were harvested and luciferase activity was measured using a Dual-Luciferase Reporter (DLR) Assay System (Promega, E1910). The firefly luciferase signal from each well was normalized to its Renilla luciferase signal. Topflash/fopflash signal was determined from each treatment and graphed using Graphpad/Prism. NAC was obtained from Sigma (A9165) and dissolved in sterile distilled H O (stock 1 M). Cells were treated for 48 h and analysed. After optimization, 10 mM final concentration was used for subsequent experiments. Melanoma cells were seeded into T25 flasks and incubated for 72 h with 6.5 ml conditioned fibroblast media prepared as described above. Cells were then washed in PBS, harvested with TrypLE Express and fractionated using the cellular fractionation kit (NE-PER, Fisher) as per the manufacturer’s protocol. Cell lysates were then separated on an SDS–polyacrylamide gel electrophoresis gel and visualized using standard western blotting procedures. Nunc MaxiSorp ELISA plates (ebiosciences) were coated with 50 μl of 3 μg ml−1 sFRP2 (ab137560, Abcam) overnight at 4 °C. Plates were washed in PBS containing 0.1% Tween and blocked in ELISA diluent (00-4202-56, eBioscience) for 2 h. Serum was diluted 1:100 before addition to the plates and incubated overnight at 4 °C. The next day, the plates were washed in PBS containing 0.1% Tween20 and incubated with detection antibody (MAB6838, R&D Systems) for 1 h at room temperature. Plates were washed and incubated with secondary antibody for 1 h. After washing, 100 μl TMB (00-4201-56, eBioscience) was added to the plates and incubated for 15 min The reaction was stopped using 50 μl of 2 N H SO and absorbance was measured at 450 nm. Fibroblasts were plated into 12-well dishes, incubated for 48 h, washed with PBS, and fixed in 2% formaldehyde/0.2% glutaraldehyde. Cells were then incubated in staining solution (150 mM NaCl, Sigma), 2 mM MgCl (Sigma), 5 mM K Fe(CN) (Millipore), 5 mM K Fe(CN) (Millipore), 40 mM Na PO (Sigma) pH 5.5, 20 mg ml−1 X-gal (Applichem, Darmstadt, Germany) at 37 °C overnight. Stain was removed and cells were stored in 70% glycerol before being imaged. All primers are listed in the Supplementary Information. Mouse tissue was snap frozen in liquid nitrogen immediately after harvesting. Ten milligrams of the lung tissue was homogenized and RNA was extracted using Trizol (Invitrogen) and RNeasy Mini kit (Qiagen) as described previously. One microgram of RNA was used to prepare cDNA using iscript DNA synthesis kit (1708891, Bio-Rad). cDNA was diluted 1:5 before use in further reactions. Each 20 μl well reaction comprised 10 μl Power SYBR Green Master mix (4367659, Invitrogen), 1 μl primer mix (Final concentration 0.5 μM), and 1 μl cDNA. Standard curves were generated for all primers and each set of primers was normalized to an 18 s primer pair. For in vitro studies, a Student’s t-test or Wilcoxon rank-sum (Mann–Whitney) test was performed for two-group comparisons. Estimate of variance was performed and parameters for the t-test were adjusted accordingly using Welch’s correction. An ANOVA or Kruskal–Wallis test with post-hoc Bonferroni’s or Holm–Šídák’s adjusted P values was used for multiple comparisons. For dose–response analysis, Spearman’s correlation was calculated. For in vivo studies, the indicated sample size for each experiment was designed to have 80% power at a two-sided α of 0.05 to detect a difference of large effect size of about 1.25 between two groups on a continuous measurement. The fold change in tumour volume at each time point after treatment relative to baseline was calculated and then the fold change in treatment group relative to the age-matched control group was used with a mixed-effect model to evaluate the treatment effect between age groups. Stata 12.0 (StataCorp) was used for data analysis for in vivo studies and human samples. For other experiments, Graphpad/Prism6 was used for plotting graphs and statistical analysis. Significance was designated as follows: *P < 0.05; **P < 0.01; ***P < 0.001. Extended statistical analyses for patient data are provided in the Supplementary Information.

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