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Prubířská kapelka je speciální nádobka, ve které probíhá tzv. kupelace, tj. konečný krok stanovení obsahu drahých kovů v rudách, popř. slitinách. V raném novověku byly kapelky zhotovovány ze směsi kostního popela a dřevěného popela. Cílem této práce byla výroba kapelek podle raně novověkého způsobu popsaného v knize Lazara Erckera z roku 1574. Takto vyrobené kapelky byly odzkoušeny a také analyzovány. Pomocí elektronového mikroskopu byly zhotoveny obrazové materiály popelů, řezů kapelkami a získaných stříbrných zrnek. Pomocí energiově disperzní rentgenové spektroskopie bylo určeno prvkové složení popelů používaných k výrobě kapelek. Byla zkoušena také pevnost vyrobených kapelek jak po vysušení, tak po vyžíhání., Cupel is a special vessel, which is used for cupellation – the last step of determination of precious metals in ores or alloys. In Early Modern Age cupels were made of bone ash and wood ash mixture. The goal of this paper was the manufacturing of cupels according to Ercker’s recipe. These cupels were tested and also analyzed. Figures of ashes, cross-sections of cupels and silver grains were acquired by electron microscopy. Elemental composition of ashes used to cupels manufacturing was determined by energy dispersive X-ray spectrometry. The strength of dried and annealed cupels was also tested.

The modelling of high pressure grinding rolls is described by the population balance model, a mass balance which includes several functions that are related to the mineral characteristics, material kinetics and operative conditions of the device. The breakage distribution function is one of these functions and refers to the way in which the daughter particles are generated by the process of comminution. The piston-die press is presented as a methodology to determine the breakage distribution function of two different materials, from the mechanical response point of view: altered granite and a cal-silicate material. The aim is to determine the relation between the operative conditions and the mineral characteristics in order to explain and predict the breakage function parameters. The materials were characterised using XRD and single compression strength tests. The altered granite is a brittle material, which generates more fines under single compression conditions compared to bed compression conditions, mainly due to the mineral composition and the response of the material to the breakage action. The cal-silicate material shows a normal trend in its breakage behaviour. As is expected, the mineralogical characterisation is a useful tool to predict the values of the parameters of the breakage distribution function., Peer Reviewed, Postprint (published version)

Grade and metallurgical sampling can be difficult in sheeted vein gold deposits because of the heterogeneous gold particle distribution and deportment. Metallurgical samples should be undertaken early in the value chain to assess ore characteristics. Mineralogical characterisation is required to optimise subsequent grade and metallurgical sampling protocols. A protocol is presented which responds to the coarse-gold nature of mineralisation and allows maximum data from drill core to support the resource update and prefeasibility study. A change from 1 m half-core samples to 2 m whole core whole assay samples in the indicated resource area of the San Antonio deposit in South America showed a 20% increase in grade from 5.5 g/t Au to 6.6 g/t Au. Preliminary results of sampling at the site indicate two grade recovery domains and three comminution domains. Within the sheeted vein zone, gravity-only recovery may achieve around 40-50% gravity-recoverable gold whereas in the high-grade flat veins recovery is less than 15%. The prefeasibility study is reviewing mining scenarios across early start-up/low capital expenditure selective operation, through to a bulk mine operation. Strategic considerations will include selective campaign mining, contribution to a cluster of small operations in the region, and static versus toll milling or portable process plant with continuous gravity recovery options., Grade and metallurgical sampling can be difficult in sheeted vein gold deposits because of the heterogeneous gold particle distribution and deportment. Metallurgical samples should be undertaken early in the value chain to assess ore characteristics. Mineralogical characterisation is required to optimise subsequent grade and metallurgical sampling protocols. A protocol is presented which responds to the coarse-gold nature of mineralisation and allows maximum data from drill core to support the resource update and prefeasibility study. A change from 1 m half-core samples to 2 m whole core whole assay samples in the indicated resource area of the San Antonio deposit in South America showed a 20% increase in grade from 5.5 g/t Au to 6.6 g/t Au. Preliminary results of sampling at the site indicate two grade recovery domains and three comminution domains. Within the sheeted vein zone, gravity-only recovery may achieve around 40-50% gravity-recoverable gold whereas in the high-grade flat veins recovery is less than 15%. The prefeasibility study is reviewing mining scenarios across early start-up/low capital expenditure selective operation, through to a bulk mine operation. Strategic considerations will include selective campaign mining, contribution to a cluster of small operations in the region, and static versus toll milling or portable process plant with continuous gravity recovery options.

250 aminopyrazoles, a new class of c-jun-N-terminal kinase (JNK) inhibitors, have been synthesized and the biochemical IC50 has been determined for JNK3, JNK2, JNK1, and p38. In addition, these compounds have been tested in cell-based assays that monitor the inhibition of c-jun phosphorylation and some drug metabolism and pharmacokinetic (DMPK) properties have been measured. Moreover, two additional classes of JNK inhibitors have also been generated as backups. 80 compounds from the pyridopyrimidinone class have been synthesized and tested in biochemical and cell based assays, and approximately 25 compounds from the amino acid transporter analog class have been made and tested in biochemical assays. The goal of this work is to find JNK3 isoform selective inhibitors. Eight novel aminopyrazoles have been developed with JNK3 selectivity 20-fold, three novel compounds have been developed with JNK3 selectivity 50-fold, one novel compound has been developed with JNK3 selectivity 200-fold, and two compounds have cell-based IC50s 1 mM. SR-11935, a highly selective JNK2/3 isoform inhibitor from the aminopyrazoles class has been optimized for potency, selectivity, pharmacokinetics, and brain penetration and has been tested in vitro to see if it protects motor neurons from Tg SOD1 G93A mice from astrocyte-mediated toxicity. SR-11935 demonstrated near 100% protection of motor neurons from astrocytemediated toxicity at 50 nM indicating the high potency and in vitro efficacy of this JNK2/3 isoform selective inhibitor. In addition, SR-3306 and SR-11935 have been tested for efficacy in vivo in transgenic G93A SOD1 mice. Preliminary results show that SR-3306 and SR-11935, an aminopyrimidine and aminopyrazole, respectively, are well tolerated with no adverse effects after once daily dosing for 90 days at 30 mg/kg, and 40 mg/kg, respectively., The original document contains color images.

Athletes in contact sports who have sustained multiple concussive traumatic brain injuries are at high risk for delayed, progressive neurological and psychiatric deterioration. This syndrome is termed chronic traumatic encephalopathy (CTE) and is also known as dementia pugilistica or punch drunk syndrome. US military personnel and others who have sustained multiple concussive traumatic brain injuries may also be at risk for this condition. Currently, there are no methods to identify progressive tau pathology in living humans. Hypothesis: Aggregated forms of hyperphosphorylated tau protein formed acutely in the setting of traumatic brain injury can seed further aggregation of intracellular tau in nearby cells, leading to delayed propagation of tau pathology and neurodegeneration. Objective: To develop standardized, high-throughput blood and cerebrospinal fluid assays for aggregated forms of tau responsible for propagation of tau pathology after traumatic brain injury. Progress to date: To date, none of the attempts to model progressive tau pathology after repetitive concussive TBI in mice has been optimal. Ongoing efforts include development of more sensitive methods to detect tau, and combinations of repetitive concussive TBI with binge ethanol administration. Substantial progress towards increasing the sensitivity of cell-based assays for tau aggregation activity has been made, and additional antibody-based tau detection methods for blood samples are in development., The original document contains color images.

Our hypothesis is that reversible alterations in histones are a determining factor for low FANCD2 expression in ovarian surface epithelial (OSE) cells in women with a familial risk for ovarian cancer, and that cells with reduced FANCD2 levels are hypersensitive to the genotoxic effects of estrogen, therefore predisposing OSE to malignant transformation. During the study we have screened a large number of normal (no familial risk), high risk (with familial history of this disease), and ovarian cancer cell lines, and determined levels of FANCD2 protein and mRNA. In the first set of experiments, aimed at determining whether histone modifications (i.e. acetylation and/or methylation) affect FANCD2 levels, we established that Trichostatin A (TSA;10nM for 24 hours) corrects FANCD2 levels but not consistently. In the second set of experiments, aimed at establishing whether the estrogen metabolite 4-OHE2 is genotoxic for cells with low FANCD2 levels we: 1) identified the minimal concentration of 4- OHE2 that is associated with DNA damage in human and murine OSE, and; 2) found that OSE cultures with low FANCD2 exhibit significantly increased DNA damage after exposure to 50 uM 4-OHE2, in comparison with OSE cultures with normal levels of FANCD2 protein expression., The original document contains color images.

The major goals of this study were to 1) gain an understanding of the marine mammal immune response with respect to dive adaptation by investigating the response of marine mammal immune cells to simulated dives (i.e. pressure excursions) and 2) to evaluate the potential for additional stressors to alter the response of marine mammal immune cells to simulated dives, thereby providing information which may aid future efforts to assess the impacts of anthropogenic activities on marine mammal health. The specific objectives of this effort were to 1) investigate the effects of simulated dive exposures on cellular immune function in belugas, 2) evaluate the effects of simulated dive exposures on cellular immune function in belugas following a known stressor event, 3) collect biological samples from wild belugas to compare with aquarium whales, and 4) compare the effects of simulated dive exposures on cellular immune function in seals from stranding (stressor) and release (healthy).

Phlebotomus papatasi vectors zoonotic cutaneous leishmaniasis. Previous expression of recombinant P. papatasi acetylcholinesterase (PpAChE1) revealed 85% amino acid sequence identity to mosquito AChE and identified synthetic carbamates that effectively inhibited PpAChE1 with improved specificity for arthropod AChEs compared to mammalian AChEs. We hypothesized that the G119S mutation causing high level resistance to organophosphate insecticides in mosquitoes may occur in PpAChE1 and may reduce sensitivity to inhibition. We report construction, expression, and biochemical properties of rPpAChE1 containing the G119S orthologous mutation. Targeted mutagenesis introduced the G119S orthologous substitution in PpAChE1 cDNA. Recombinant PpAChE1 enzymes containing or lacking the G119S mutation were expressed in the baculoviral system. Biochemical assays were conducted to determine altered catalytic properties and inhibitor sensitivity resulting from the G119S substitution. A molecular homology model was constructed to examine the modeled structural interference with docking of inhibitors of different classes. Genetic tests were conducted to determine if the G119S orthologous codon existed in polymorphic form in a laboratory colony of P. papatasi. Recombinant PpAChE1 containing the G119S substitution exhibited altered biochemical properties, and reduced inhibition by compounds that bind to the acylation site on the enzyme (with the exception of eserine). Less resistance was directed against bivalent or peripheral site inhibitors, in good agreement with modeled inhibitor docking. Eserine appeared to be a special case capable of inhibition in the absence of covalent binding at the acylation site. Genetic tests did not detect the G119S mutation in a laboratory colony of P. papatasi but did reveal that the G119S codon existed in polymorphic form (GGA + GGC)., Published in Parasites & Vectors, v7 n577, 10 Dec 2014. Sponsored in part by USDA.

Our long-term goal is to broaden the existing panel of endocrine stress assessment techniques for large whales. Few methods exist for assessment of physiological stress levels of free-swimming cetaceans (Amaral 2010, ONR 2010, Hunt et al. 2013). Prior to this grant, we demonstrated that respiratory vapor (blow) sampling is practical and feasible for large whales, and that blow samples contain detectable steroid and thyroid hormones (Hunt et al. 2014). We had also developed a suite of fecal hormone assays for reproductive and stress-related hormones in North Atlantic right whales (NARW; Hunt et al. 2006, Rolland et al. 2005, 2012). However, blow sampling needs further testing before it can enter widespread use, and some additional stress-related hormones have not yet been tested in either feces or blow, particularly the adrenal hormone aldosterone. Our aim in this project is to further develop both techniques - respiratory hormone analysis and fecal hormone analysis - for use in stress assessment of large whales.

We have studied a thermophilic bacteria GWE1 belonging to Geobacillus genus able to biosynthesize Se0 nanoparticles. Elemental analysis and composition of the particles were verified using transmission electron microscopy and energy-dispersive X-ray analysis. The nanoparticles have a defined spherical shape and are composed by Se0. Previous experiments showed that the presence of the whole microorganism for the reduction of selenite was not necessary. We have isolated an intracellular NADPH/NADH-dependent reductase which mediates selenium nanoparticles synthesis under aerobic conditions. The enzyme was purified and identified as a 1-pyrroline-5-carboxylate dehydrogenase by mass spectroscopy MALDI-TOF TOF technique. FT-IR analysis was utilized to characterize the chemical surface of Se nanoparticles. These results strongly suggest that the biosynthesis of Se nanoparticles is mediated by NADH-dependent enzymes. This ecologically-friendly process allows an efficient control of size, shape and composition of the generated particles. Additionally biosynthesized nanoparticles display good surface bioactivity., The original document contains color images.

The overall goal of the study is to examine whether altered vitamin D status (as measured by serum metabolites and by functional polymorphisms within genes related to vitamin D transport, metabolism and activity) is associated with increased risk of aggressive prostate cancer, and may explain some of the racial disparity seen in aggressive prostate cancer. The majority of project activities as outlined in the Statement of Work Tasks and Milestones are complete. The study team has been organized and participates in monthly conference calls (or more frequent as needed) to discuss study progress and data collection and analyses. IRB approval was obtained from all local institutions and the DoD HSRRB. All assays have been performed and data have been merged and cleaned. Statistical analyses are underway. With the large representation of African Americans in this investigation, the proposed research has tremendous potential to provide insights into a chronically underserved population carrying an unequal burden of disease.

Platelets (PLTs) participate in hemostasis and save lives following trauma. PLTs for transfusion are maintained at room temperature (RT, 22 deg C), limiting viability to 5 days because of metabolic compromise and high risk of bacterial contamination. RT storage may result in weaker clots, delaying hemorrhage control, whereas cold storage (4 deg C) could permit longer PLT shelf life and result in a more hemostatic product. In this study, we characterized the effect of storage temperature on shear-induced PLT aggregation, clot formation, and strength. PLTs obtained from phlebotomized blood or by apheresis were stored at RT or 4 deg C for 5 days, and PLT aggregation and clot strength were assessed at 37 deg C. We studied PLT aggregation at steady and complex patterns of shear rates (500 2,500 per second) by flow cytometry, and the kinetics of clot formation and strength were measured using turbidity and dynamic mechanical analysis, respectively. PLT aggregation was higher in 4 deg C-stored samples on Day 5 compared with fresh or RT-stored samples at all shear rates tested (fresh vs. 4 deg C and RT vs. 4 deg C, p G 0.05). PLTs stored at 4 deg C for 5 days formed significantly stronger clots compared with fresh or RT-stored samples as quantified by turbidity and elastic moduli measurements (fresh vs. 4 deg C and RT vs. 4 deg C, p 0.05). Our results show that cold-stored PLTs are more responsive to aggregation stimuli and form stronger clots, presumably because of thicker fibrin strands. These data suggest that the superior functionality of cold-stored PLTs may support faster hemostasis for acutely bleeding in trauma patients compared with RT-stored PLTs., Published in the J Trauma Acute Care Surgery, v77 n3 supp2 pS88-S93, 2014. Prepared in collaboration with the Department of Biomedical Engineering, University of Texas at San Antonio.

Background: Macrophages are important in wound defense and healing. Dakin s solution (DS), buffered sodium hypochlorite, has been used since World War I as a topical antimi crobial for wound care. DS has been shown to be toxic to host cells, but effects on immune cells are not well documented. Materials and methods: DS at 0.5%, 0.125%, and ten fold serial dilutions from 0.25% 0.00025% were evaluated for cellular toxicity on murine macrophages (J774A.1). The effect of DS on macrophage adhesion, phagocytosis, and generation of reactive oxygen species was examined. Macrophage polarization following DS exposure was determined by gene expression using quantitative real time polymerase chain reaction. Results: Concentrations of DS 0.0025% reduced macrophage viability to 5% in exposure times as short as 30 s. Similarly, phagocytosis of Staphylococcus aureus, Pseudomonas aeru ginosa, and Aspergillus flavus were significantly reduced at all tested concentrations by macrophages pretreated with DS. H2O2 production was reduced by 8% 38% following treatment with 0.00025% 0.125% DS. Macrophage adherence was significantly increased with 0.0025% DS after 15 min of exposure compared with controls. Quantitative real time polymerase chain reaction demonstrated that DS exposure resulted in classical macro phage activation, with increased expression of inducible nitric oxide synthase 2, inter feron g, and interleukin (IL) 1b. Conclusions: DS at clinically used concentrations (0.025% 0.25%) was detrimental to macrophage survival and function. For optimal clinical use, understanding the impact of DS on macrophages is important as depletion may result in impaired pathogen clearance and delayed healing. These findings indicate that 0.00025% DS is a safe starting dose; however, optimal use of DS requires further validation with in vivo models., Prepared in collaboration with the Infectious Disease Service, MCHE MDI, Brooke Army Medical Center, JBSA Fort Sam Houston, TX. Published in the Journal of Surgical Research, v192 p692-699, 18 Jul 2014.

Acute traumatic coagulopathy (ATC) has been linked to an increase in activated protein C (aPC) from 40 pM in healthy individuals to 175 pM. aPC exerts its activity primarily through cleavage of active coagulation factor Va (fVa). Platelets reportedly possess fVa which is more resistant to aPC cleavage than plasma fVa; this work examines the hypothesis that normal platelets are sufficient to maintain coagulation in the presence of elevated aPC. Coagulation responses of normal plasma, fV deficient plasma (fVdp), and isolated normal platelets in fVdp were conducted: prothrombin (PT) tests, turbidimetry. and thromboelastography (TEG), including the dose response of aPC on the samples. PT and turbidimetric assays demonstrate that normal plasma is resistant to aPC at doses much higher than those found in ATC. Additionally, an average physiological number of washed normal platelets (200,000 platelets/mm expn 3) was sufficient to eliminate the anti-coagulant effects of aPC up to 10nM, nearly two orders of magnitude above the ATC concentration and even the steady-state pharmacological concentration of human recombinant aPC, as measured by TEG. aPC also demonstrated no significant effect on clot lysis in normal plasma samples with or without platelets. Although platelet fVA shows slightly superior resistance to aPC's effects compared to plasma fVA in static models, neither fVA is sufficiently cleaved in simulations of ATC or pharmacologically-delivered aPC to diminish coagulation parameters. aPC is likely a correlative indicator of ATC or may play a cooperative role with other activity altering products generated in ATC., Published in PLoS One, v9 n6 article ID e99181, 12 Jun 2014.

XXX aminopyrazoles, a new class of c-jun-N-terminal kinase (JNK) inhibitors, have been synthesized and the biochemical IC50 has been determined for JNK3, JNK2, JNK1, and p38. In addition, these compounds have been tested in cell-based assays that monitor the inhibition of c-jun phosphorylation and some drug metabolism and pharmacokinetic (DMPK) properties have been measured. Moreover, two additional classes of JNK inhibitors have also been generated as backups. XXX compounds from the pyridopyrimidinone class have been synthesized and tested in biochemical and cell based assays, and XXX compounds from the amino acid transporter analog class have been made and tested in biochemical assays. The goal of this work is to find JNK3 isoform selective inhibitors. Eight novel aminopyrazoles have been developed with JNK3 selectivity 20-fold, three novel compounds have been developed with JNK3 selectivity 50-fold, one novel compound has been developed with JNK3 selectivity 200-fold, and two compounds have cell-based IC50s 1 mM. SR-11935, a highly selective JNK2/3 isoform inhibitor from the aminopyrazoles class has been optimized for potency, selectivity, pharmacokinetics, and brain penetration and has been tested in vitro to see if it protects motor neurons from Tg SOD1 G93A mice from astrocyte mediated toxicity. SR-11935 demonstrated near 100% protection of motor neurons from astrocytemediated toxicity at 50 nM indicating the high potency and in vitro efficacy of this JNK2/3 isoform selective inhibitor. In addition, SR-3306 and SR-11935 have been tested for efficacy in vivo in transgenic G93A SOD1 mice. Preliminary results show that SR-3306 and SR-11935, an aminopyrimidine and aminopyrazole, respectively, are well tolerated with no adverse effects after once daily dosing for 90 days at 30 mg/kg, and 40 mg/kg, respectively., The original document contains color images.

Tembusu virus (TMUV) is an important emerging arthropod-borne virus that may cause encephalitis in humans and has been isolated in regions of southeast Asia, including Malaysia, Thailand, and China. Currently, detection and identification of TMUV are limited to research laboratories, because quantitative rapid diagnostic assays for the virus do not exist. We describe the development of sensitive and specific conventional and real-time quantitative reverse transcription polymerase chain reaction assays for detecting TMUV RNA in infected cell culture supernatant and Culex tarsalis mosquitoes. We used this assay to document the replication of TMUV in Cx. tarsalis, where titers increased 1,000- fold 5 days after inoculation. These assays resulted in the detection of virus-specific RNA in the presence of copurified mosquito nucleic acids. The use of these rapid diagnostic assays may have future applications for field pathogen surveillance and may assist in early detection, diagnosis, and control of the associated arthropod-borne pathogens., Published in the American Journal of Tropical Medical Hygeine, v91 n4 p666-671, 2014. Funded in part by Military Infectious Disease Research Program Project U0176_09_RD. Prepared in collaboration with the US Army Medical Research and Materiel Command, Fort Detrick, MD.

Task 1: Engineer Laminin-collagen IV-entactin-perlecan (LCEP) binding domains as fusions to the growth and neurotrophic factors ( protein factors ). Generate LCEP composite fiber scaffolds incorporating protein factors exhibiting either constant surface density or longitudinal density gradients. Match fiber stiffness with that of autograft (G*1 MPa). Characterize the bioactivity of scaffold-bound protein factors using cell signaling assays (0-15 months); performance site: USMA. Task 2: Evaluate each protein factor (singly & in selected combinations) in the organotypic model system for preferential sensory or motor axon extension. Use confocal microscopy to track axon extension of labeled sensory or motor neurons from spinal cord slices (motor) or dorsal root ganglia (DRG) (sensory). 20 Thy1-YFP mice will be used for this task and subtasks. (12-24 mo.); sites: JHU & USMA Task 3: Evaluate top performing compositions from Task 2 in a rat femoral nerve repair model in which sensory and motor axons intermingle proximally and are segregated into discrete cutaneous and muscle branches distally. 80 Sprague-Dawley rats will be used for this task and subtasks. (24-36 mo.); work performed at JHU., The original document contains color images.

Twenty-five multi-drug resistant organisms (MDROs) of high military importance were sequenced using the Illumina HiSeq platform to identify drug resistance and virulence mechanisms contributing to their success as pathogens in the wound environment. The genomes were annotated and submitted to GenBank. Novel bioinformatics pipelines have been developed to (1) identify highly variable genomic regions, (2) classify antibiotic resistance islands, and (3) determine diversity of gene clusters associated with virulence in A. baumannii. Three top priority MDROs and three commensal microbes were chosen and obtained from WRAIR MRSN and ATCC for in vitro co-culturing in confrontation assays aimed at investigating alterations in gene expression, particularly antibiotic resistance genes and virulence factors. Confrontation assays were also carried out to assess gene regulation when the MDROs were co-cultured with cultured human skin cells. RNA-seq analysis of the bacteria-bacteria and bacteria-human confrontation assays are currently in progress. Manuscripts are in preparation describing the findings from this study., The original document contains color images.

Traumatic brain injury (TBI) is a major contributor to morbidity and mortality experienced by those soldiers subjected to improvised explosive devices (IED) as well as in military and civilian high speed collisions. Traumatic cerebral vasospasm (TCV) is a major contributor to morbidity and mortality experienced by those TBI patients. Aggressive neurosurgical treatment motivated by early diagnosis appears to improve the clinical outcome for these patients. Early transcranial Doppler (TCD) measurements of blood flow speed within major cerebral arteries produces the initial diagnosis, hence motivates the rapid treatment of TCV. However, the skill necessary to deploy TCD limits its availability relative to its need. PhysioSonics, Inc, a local company in Seattle, created an automatic TCD system (called Presto or aTCD) that minimizes the skill necessary to perform TCD assays. Our long-term goal is to optimize Presto for patients in vasospasm, through optimization of its spectral Doppler envelope analyzer, its headset and supporting software so it can track blood flow in the internal carotid artery as well as middle cerebral artery., The original document contains color images.

The US military has placed major emphasis on developing therapeutics against nerve agents (NA). Current efforts are hindered by the lack of effective in vitro cellular models to aid in the preliminary screening of potential candidate drugs/antidotes. The development of an in vitro cellular model to aid in discovering new NA therapeutics would be highly beneficial. In this regard, we have examined the response of a differentiated hybrid neuronal cell line, NSC-34, to the NA VX. VX-induced apoptosis of differentiated NSC-34 cells was measured by monitoring the changes in caspase-3 and caspase-9 activity post-exposure. Differentiated NSC-34 cells showed an increase in caspase-3 activity in a manner dependent on both time (17 23 h postexposure) and dose (10 100 nM). The maximal increase in caspase-3 activity was found to be at 20-h post-exposure. Caspase-9 activity was also measured in response to VX and was found to be elevated at all concentrations (10 100 nM) tested. VX-induced cell death was also observed by utilizing annexin V/propidium iodide flow cytometry. Finally, VX-induced caspase-3 or -9 activities were reduced with the addition of pralidoxime (2-PAM), one of the current therapeutics used against NA toxicity, and dizocilpine (MK-801). Overall the data presented here show that differentiated NSC-34 cells are sensitive to VX-induced cell death and could be a viable in vitro cell model for screening NA candidate therapeutics., The original document contains color images. Published in Toxicology Mechanisms and Methods, v24 n7 p488-494, 11 Sep 2014.

We have demonstrated that a low band gap D-A conjugated polymer P3, that absorbs in the NIR (800 nm), can be fabricated into spherical nanoparticles (nano-P3) using Pluronic F127 as a soft template. Nano-P3 (67 nm) was found to be very stable in aqueous media. A heating efficiency curve showed that it took 7 g of nano-P3 to change the temperature of the solution by 20 deg C after one minute. This number is at a lower concentration than other donor acceptor polymer nanoparticles, such as nano-PCPDTBSe and nano-PCPDTBT, which may be due to P3 having a 20% greater molar absorptivity than PCPDTBSe. The nanoparticles, in the absence of NIR light, showed no significant toxicity towards CT26 colorectal cancer cells at concentrations of 5-200 g/mL. Cell viability assays showed that in the presence of NIR light, nano-P3 was shown to generate significant heating to destroy colorectal cancer cells at very low nanoparticle conentration ( 15 g/mL). Using the lowest possible concentration for photothermal ablation is advantageous for translating D-A ECPNs to clinical applications., The original document contains color images.

The promise of PARP-inhibitors in the management of epithelial ovarian cancer is tempered by the fact that approximately 50% of patients with homologous recombination (HR)-proficient tumors do not respond well to these agents. Using a bioinformatics approach, we identified that heat shock protein 90 inhibitors(HSP90i) may suppress HR and thus revert HR-proficient to HRdeficient tumors. Analysis of publicly available gene expression data showed that exposure of HR-proficient cancer cell lines to HSP90i 17-AAG(17-allylamino-17-demethoxygeldanamycin) downregulated HR, ATM and Fanconi Anemia pathways. In HRproficient EOC cells, 17-AAG suppressed HR as assessed using the RAD51 foci formation assay and this was further confirmed using the Direct Repeat-GFP reporter assay. Furthermore, 17-AAG downregulated BRCA1 and/or RAD51 protein levels, and induced significantly more H2AX activation in combination with olaparib compared to olaparib alone. Finally, sublethal concentrations of 17-AAG sensitized HR-proficient EOC lines to olaparib and carboplatin but did not affect sensitivity of the HR-deficient OVCAR8 line arguing that the 17-AAG mediated sensitization is dependent on suppression of HR. These results provide a preclinical rationale for using a combination of olaparib/17-AAG in HR-proficient EOC., The original document contains color images.

Air and ground crews transfer significant amounts of jet fuel and, as a result of transfers, breathe its volatile emissions. Working on flight lines also exposes maintainers to jet fuel exhaust. Little is known concerning levels of exposure and corresponding response biomarkers associated with human jet fuel exposure. The objective of this study was screening blood for detectable jet fuel components and potential biomarkers. As collaborative research the Japan Air Self-Defense Force measured JP-4 or JP-8 components in blood and the U.S. Air Force analyzed for biomarkers. This report describes methods and results for examining ten proinflammatory cytokines in serum. Blood was collected from volunteers at USAF and JASDF air bases in Japan only once pre-shift, post-shift, and the following morning. Concentrations of 5 cytokines (GM-CSF, IFN- , IL-2, IL-1 and IL-1 ) fell below assay range. No changes were observed with IL-6, IL-10, and IL-12 p70. TNF- at Kadena AB showed a significant increase post-shift for JP-8-exposed subjects, but levels did not differ from control subjects. IL-8 levels in JP-4-exposed subjects were higher than those in Tachikawa control subjects (no active runway) at the post-shift time point. IL-8 levels in JP-8-exposed subjects were also higher than levels in control subjects (at the recovery time point). IL-8 levels in JP-4-exposed subjects were higher than levels in JP-8-exposed subjects at all three time points. Data might indicate a low level of inflammation. However, cytokines may not be sensitive markers for jet fuel exposure or exposure was too low to trigger inflammatory effects., Prepared in collaboration with the Henry M. Jackson Foundation for the Advancement of Military Medicine, Wright-Patterson AFB, OH, the Aeromedical Laboratory, Japan Air Self-Defense Force Tachikawa, Tokyo, Japan, the 18 MDOS/SGOL, Kadena AB, Okinawa, Japan, and the 374 AMDS/SGPF, Yokota AB, Tokyo, Japan.

The goal of this project is to develop biopsy based assays to assess the probability that patients with a negative biopsy or with a prostate cancer (CaP) Gleason score 6 (GS6) biopsy actually have significant CaP of Gleason score 7 or higher which was missed during the biopsy evaluations due to insufficient sampling. In 2014, we considerably expanded the size of the discovery samples which will improve the robustness of biomarkers and developed methodologies for improving the detection of epigenetic modification in rare cells. Also, we tested several methods for multiplex analyses of transcriptome and DNA methylation changes. Further, we have secured additional funding which will allow us to expand transcriptome and epigenome profile of indolent and significant prostate tumors. Finally, we have applied for major grant initiatives which if funded, will help expanding the scope of this study through the use of nano-scale devices and quantum dot based assays. We are expecting the publications of our research results in the coming year., The original document contains color images.

Citrullination, catalysed by peptidylarginine deiminase (PAD), is a post-translational modification of arginine to citrulline, which contributes to the pathogenesis of rheumatoid arthritis (RA). We show that citrullinated epithelial-derived neutrophil-activating peptide 78/CXCL5 (cit-ENA-78/CXCL5) is significantly higher in RA synovial fluids (SFs) compared to osteoarthritis (OA) and other inflammatory rheumatic diseases (OD) SFs, and its concentration correlates with RA disease activity. Citrullinated chemokine concentrations were measured by enzyme linked immunosorbent assay (ELISA) in RA and normal (NL) sera and in RA, osteoarthritis (OA), and other inflammatory rheumatic disease (OD) synovial fluids (SFs). The correlation between the citrullinated chemokine levels and clinical data was analyzed. A strong correlation was found between the amount of citrullinated ENA-78/CXCL5 and C-reactive protein or erythrocyte sedimentation rate (ESR) in RA SFs. These results indicate that citrullinated ENA- 78/CXCL5 may have novel inflammatory properties in RA pathogenesis., The original document contains color images.

Interest in developing androgen receptor (AR) inhibitors with novel mechanism of action is slowly increasing since commercial anti-androgens (Bicalutamide, Flutamide, Nilutamide and Enzalutamide) face therapeutic limitations. Current therapies fail over a period of time because they all target hormone binding pocket on AR to which the receptor has already developed effective resistance mechanisms. One of the promising strategies to combat drug resistance is to develop the inhibitors that target an alternative binding pocket of the AR, called Binding Function 3 (BF3). In the current study, we report indole chemical series, identified through systematic in silico screen, as leading AR BF3 inhibitors. The most potent inhibitor (compound VPC-13566) demonstrated excellent anti-androgen potency, anti-PSA activity and abrogates androgen-induced proliferation of LNCaP and Enzalutamide-resistant prostate cancer cell lines. Moreover, it demonstrated clear reduction of tumour growth in tumor xenograft models in mice. Based on these results new derivatives have been developed to improve stability and better efficacy in in-vivo models. These findings provide evidence that targeting AR BF3 pocket using small molecule inhibitors is a viable therapeutic approach for patients with advanced prostate cancer., The original document contains color images.

We used a novel method to discover tumor antigens with the intent to develop a diagnostic assay for lung cancer. We isolated intratumoral B lymphocytes (ITLs), cloned their immunoglobulin genes, and used the expressed antibodies to identify tumor antigens. All of the antibodies produced from ITLs in our study were polyreactive. One antibody that preferentially recognized tropomyosin 4 was identified; this antibody was confirmed in the serum of the same cancer patient, and in a small pilot study, the tropomyosin 4 antibody was found to be specific for the diagnosis of lung cancer, but was not very sensitive (20%). We are currently modifying our approach to identify those ITLs that show evidence of clonal expansion prior to the cloning and expression of recombinant antibodies. This will increase the likelihood that the recombinant antibodies produced are the result of proliferation and differentiation in response to tumor antigens. This will allow us to concentrate our cloning, antigen identification, and validation efforts on those antibodies that are more likely to demonstrate high affinity monoreactivity against tumor antigens. Isolation of single ITLs and cloning of their immunoglobulin genes has the potential to be a useful approach for identifying diagnostic biomarkers or novel molecular targets., The original document contains color images.

During this research period, molecular modeling and biochemical studies were conducted to assess the roles of different acidic residues in the binding of the Src homology 2-containing protein tyrosine phosphatase (SHP2) to its substrates. A peptide derived from this substrate was shown to be a selective inhibitor of SHP2. SHP1 was used as a control, and the peptide did not act on this enzyme. Molecular modeling revealed key binding determinants at the atomic level present in SHP2 that could not be recapitulated in SHP1. These modeling observations were tested using mutation of the peptide. Indeed, when the peptide was mutated to abolish these interactions, inhibitory capacity was abolished entirely. Systematic mutation of the peptide in silico further demonstrated that the substrate needed to bind both of the positively-charged amino acid sidechains adjacent to the active site to facilitate the highest binding stability. Mutation of any of the acidic amino acids next to the phosphotyrosine on the substrate produced significant defects in binding as predicted by molecular docking. These findings are now being parlayed into study of the peptide-based inhibitor on cells, which is work that will continue into the coming year., The original document contains color images.

A quantitative assay was developed for the direct measurement of polymer film degradation from bacterial colonies on agar plates. Small (1 mm diameter) colonies of Pseudomonas protegens Pf-5 (formerly Pseudomonas fluorescens Pf-5) were used for this work. Interactions between the Pf-5 colonies and thin polyurethane (PU) coatings on ZnSe coupons were evaluated for degradation using infrared spectroscopy. Three different coatings were analyzed and were formed from 1) a colloidal, aqueous e based polyester PU (Impranil' DLN); 2) an organic solvent e based polyester PU (Irogran); and 3) an organic solvent e based polyether PU (AS-P108). Over a 24 h time period at 30 C, citrate exposed Pf-5 cultures rapidly degraded Impranil coatings, consistent with analogous zone clearing assays. However, the Irogran and AS-P108 PU's, which are not directly compatible with zone clearing assays, showed no measureable degradation by the coating assay under identical conditions. These results demonstrated the capability to evaluate any variety of polymer formulation as solid films under identical biological conditions. The results also show that rapid microbial degradation of colloidal polyurethanes such as Impranil are not necessarily representative of activity towards other PU materials., Prepared in collaboration with the Chemistry Division, US Naval Research Laboratory, Washington, D.C., and Nova Research, Incorporated, Alexandria, VA. Published in International Biodeterioration & Biodegradation, v95 p311-319, 2 Oct 2014. Sponsored in part by AFOSR.

Here we present the synthesis of the enzyme DNase 1 stabilized gold nanoclusters (DNase 1:AuNCs) with core size consisting of either 8 or 25 atoms. The DNase 1:Au8NCs exhibit blue fluorescence whereas the DNase 1:Au25NCs are red emitting. In addition to the intense fluorescence emission, the synthesized DNase 1:AuNC hybrid retains the native functionality of the protein, allowing simultaneous detection and digestion of DNA with a detection limit of 2 microg/mL. The DNase 1:AuNCs could be conveniently employed as efficient and fast sensors to augment the current time-consuming DNA contamination analysis techniques., Published in Analytical Chemistry, v86 p7377-7382, 4 Jul 2014. The original document contains color images.

Growth and development of the prostate is highly dependent on androgen, and aberrant androgen and androgen receptor (AR) signaling is the key driver in the pathology and progression of prostate cancer. Blocking androgen synthesis and inhibiting AR function is the first line of therapy for prostate cancer patients. Although this approach is initially effective in suppressing disease progression, castration resistant tumors eventually develop resulting from reactivation of AR activity. A novel approach to overcome resistance to therapy is to identify selective AR modulators (SARMs) that inhibit the expression of genes promoting tumor growth but enable the expression of genes for differentiation. We hypothesize that functionally distinct set of genes have different promoter signature marks that are recognized by AR. Re-testing and validation of several hits obtained from a high-throughput promoter-dependent compound screen of FDA approved drugs identified a lead compound that had a differential effect on AR promoter-element recognition in transient transfection assays and in chromatin precipitation assays, and in the expression of select AR target genes associated with proliferation and differentiation. Cell proliferation assays indicate that the compound is able to inhibit AR-dependent cell growth. Our discovery of this compound in an unbiased screen for SARMs provides proof-of-concept that drugs may be developed that differentially target promoter elements that distinguish pro-proliferation from pro-differentiation genes. Further optimization of transfection protocol and multiplexing of reporters for the high-throughput promoter screen was performed. A second high-throughput screen was done with a 1.6% hit rate that yielded 26 candidate compounds to be tested for secondary assay validation and mechanism of action analysis., The original document contains color images.

Dysregulated estrogen receptor (ER) function underlies many forms of breast cancer. This proposal is aimed at understanding how ER activates its target genes from distal enhancers in a Mediator-dependent fashion. We have hypothesized that ER-Mediator interactions would be critical for signal transduction at ER target genes through establishment of chromatin loops that facilitate long-range enhancerpromoter communication. The main aims of the proposal are thus to establish both cell based and cell-free (in vitro) transcription systems to recapitulate and mechanistically dissect Mediator-dependent ER function from a distal enhancer and further to develop peptidomimetic inhibitors of the ER -MED1 interaction to disrupt enhancer-promoter communication. In this phase of the project we have established an assay system that supports rudimentary ER function from distal binding sites. Using this as a baseline, future years will see further refinement of the in vitro systems so that contributions of Mediator and cohesin can be assessed. We have also begun generating the reagents we will require for our studies. Key reagents that we have obtained thus far include an active core of the Mediator complex that we have reconstituted through overexpression of its subunits using a baculovirus expression system. ER -interacting MED1 and its mutant variants will be incorporated into this core Mediator for further ER -based functional studies. We also have obtained GFP-derivatives of Mediator and TFIID complexes for proposed FRET analyses for looped chromatin. Peptidomimetic synthesis and analyses are also projected to commence shortly., The original document contains color images.

All living organisms shed DNA into the environment. Recent studies have demonstrated that environmental water samples can be effectively assayed for the DNA of target organisms. The DNA contained in these samples is commonly referred to as environmental DNA or eDNA. Because DNA assays can be sensitive to very small amounts of genetic material, eDNA surveys have the potential to detect sites where target organisms occur at very low abundances, potentially even when organisms are too rare for conventional survey methods to effectively detect them. We designed and tested twelve new eDNA markers for aquatic eDNA surveys of North American sturgeon. Eight of the markers are general for all North American sturgeon (i.e., not genus- or species-specific), two are specific to Scaphirhynchus sturgeon, and two are specific to white sturgeon (Acipenser transmontanus). The Scaphirhynchus marker could identify sites where endangered species like the pallid (S. albus) or Alabama (S. suttkusi) sturgeon might occur. Positive detections with the general sturgeon marker combined with the absence of positive detections with the white sturgeon marker could be used to discern if green sturgeon (A. medirostris) might occur at a location. All sturgeon markers were tested for specificity against a battery of 32 non-target fish species common to the Mississippi and Illinois River watersheds and the sensitivity or limit of detection for each marker was determined with assays of increasingly dilute solutions of target DNA. Four of the general sturgeon markers were used to assay 88 water samples from the Lower Mississippi River for sturgeon eDNA and resulted in 3 positive detections. While additional optimization of sturgeonspecific sampling protocols and marker assays is advised prior to use of these markers for sturgeon eDNA surveys, the newly designed markers represent a significant step forward in the use of eDNA in sturgeon monitoring and management.

To determine the identity of a potential threat, an essential step includes the reliable isolation and purification of unique nucleic acids from unknown agents. The ability to provide polymerase chain reaction-ready DNA is necessary for the positive identification of unknown biological threats. At the time of this study, there were several commercially available assays for isolating and purifying nucleic acids from potential threat agents, but these assays were designed to isolate nucleic acids either for specific types of pathogens or from predetermined matrices. There were no commercially available nucleic acid preparation assays that had the flexibility and broad scope necessary for the wide variety of samples and sample matrices that can be encountered in a biodefense or first-responder setting. It was important to evaluate the available technologies and determine which preparation, collection, and preservation method(s) would be most useful in an emergency setting. This study provided an unbiased technical evaluation of commercially available nucleic acid preparation assays, collection methods and devices, and preservation methods and devices that could be used to support Department of Defense and other interagency acquisition decisions. This study was divided into three specific subsections: (1) sample preparation, (2) sample collection, and (3) sample preservation., The original document contains color images. Prepared in collaboration with Excet, Inc., Springfield, VA, the Oak Ridge Inst for Science and Education, Belcamp, MD, Leidos, Inc., Gunpowder, MD, and TMG Biosciences, LLC, Incline Village, NV.

This study evaluates both the effects of physical restraint and use of candidate biomarkers in a CD2F1 male mouse partial-body irradiation model for biological dosimetry diagnostic assays. Mice were irradiated (6-Gy, 250-kVp X ray) to 3/3rd (total body), 2/3rd (gut and torso), 1/3rd (gut only) and 0/3rd (sham) of total body. Blood was sampled for haematology and blood plasma proteomic biomarkers at 1 and 2 d after exposure. Increases in the body fraction exposed showed progressive decreases in lymphocyte counts and increases in the neutrophil-to-lymphocyte ratios with no significant differences in the neutrophil and platelet counts. The radioresponse for plasma biomarker Flt3L showed proportional increases; however, G-CSF and SAA levels exhibited dramatic and non-proportional increases in levels. Physical restraint at 1 d post-exposure increased lymphocyte counts and SAA, decreased neutrophil-to-lymphocyte ratio and Flt3L and showed no effects on neutrophil and platelet counts or G-CSF., Published in Radiation Protection Dosimetry, v159 n1-4 p46-51, 21 Apr 2014. The original document contains color images. Prepared in collaboration with Meso Scale Diagnostics, Rockville, MD. Sponsored in part by the Office of the Assistant Secretary for Preparedness and Response, U.S. Department of Health and Services.

For many diseases conferring rapid protection against an intracellular pathogen is required to prevent pathogen related death. To this end, we have developed a microparticulate vaccine carrier comprised of the novel polymer, acetalated dextran (Ac-DEX). Ac-DEX is an aptly designed polymer for vaccine applications because it s base material is FDA approved dextran and it has acid sensitivity for triggered release inside the phagosome, tunable degradation that can range from hours to months, and enhanced MHC I & II presentation with subunit antigen, compared to other biomaterials. In microparticulate form it can be used to passively target dendritic cells through size exclusion. We have formulated particles encapsulating recombinant protective antigen (rPA) or lysate (Francisella novicida, Burkholderia pseudomallei strain 1026) and the TLR 7/8 agonist resiquimod. Vaccination at 0 and 7 days (sub-Q) results in high levels of antigen specific antibodies for encapsulated formulations, compared to rPA + alum. Additionally, A/J mice (n=10) were aggressively challenged intratracheally with Bacillus anthracis (Sterne Strain) on day 14, 21 and 28, with survival in groups with encapsulated and/or free rPA and resiquimod. Studies were also performed with Ac-DEX microparticles encapsulating F. novicida or B. pseudomallei lysate. Balb/C (n=10 for F. novicida and n=25 for B. pseudomallei) mice were vaccinated on 0 and 7 days (sub-Q) with i.p. challenge on day 14. For the B. pseudomallei evaluation, we had delay onset to death in several groups, with post-mortem liver, spleen and blood in three of the nine experimental groups. Overall we have shown the efficacy of Ac-DEX microparticles for rapid vaccination against two intracellular bacterial pathogens.

Athletes in contact sports who have sustained multiple concussive traumatic brain injuries are at high risk for delayed, progressive neurological and psychiatric deterioration. This syndrome is termed chronic traumatic encephalopathy (CTE), and is also known as dementia pugilistica or punch drunk syndrome. US military personnel and others who have sustained multiple concussive traumatic brain injuries may also be at risk for this condition. Currently, there are no methods to identify progressive tau pathology in living humans. Hypothesis: Aggregated forms of hyperphosphorylated tau protein formed acutely in the setting of traumatic brain injury can seed further aggregation of intracellular tau in nearby cells, leading to delayed propagation of tau pathology and neurodegeneration. Objective: To develop standardized, high-throughput blood and cerebrospinal fluid assays for aggregated forms of tau responsible for propagation of tau pathology after traumatic brain injury. Progress to date: The major year 1 goal for the Brody lab was to determine which mouse model of experimental TBI and which human tau transgenic mouse line would be most useful for these experiments. We have determined that controlled cortical impact in 3xTg-AD mice will be optimal. The major year 1 goal for the Diamond lab was to refine and standardize the tau propagation assay. We have increased the sensitivity of the assay by nearly 1000 fold using a flow-cytometry based assay and established quantitative standard curves., The original document contains color images.

Pre-clinical test results qualify the Cryptosporidium real-time PCR assay as a lead candidate for transition to clinical phase testing. Diagnostic sensitivity results were 96% to 100% in testing conducted under laboratory and field conditions. Current commercially available molecular-based diagnostic assay sensitivity is 95% to 98% representing the standard that must be met or exceeded to qualify as a candidate for FDA clearance. In addition to test activities, a Cryptosporidium spp Detection Kit pre-IDE document was prepared to serve as a point of departure for discussion with the FDA Office of In Vitro Diagnostic Device Evaluation and Safety (OIVD) on guidance and clarification of specific testing requirements for eventual clearance. During the conduct of RDT&E activities a formal GME training program was established by the investigators. The program provides for scholarly and challenging research opportunities in a real-world environment., The original document contains color images. This project was conducted under memorandum of agreement (MOA) between Walter Reed Army Institute of Research (WRAIR), Silver Spring, Maryland & 59th Medical Wing (MDW) Lackland AFB, Texas (MOA 2007 - 2013. Agreement No.: DODI 4000.19

Our long-term goal is to understand the impact of high levels of SNAI repressor proteins in the etiology, progression and pathogenicity of breast cancer. Our central hypothesis is that combinatorial treatment of SNAI-high breast tumor cells with the SNAI inhibitors will not only diminish their aggressiveness but also make these cells sensitive to the inhibition of some of the conventional anticancer agents such as vitamin D and anti-estrogens. Specific Aims: (1) To evaluate the levels of SNAI proteins in relation to the levels of the proteins that impart resistance against vitamin D and anti-estrogens in human breast cancer tissues; (2) To determine the effects of alterations of the levels of SNAI proteins in breast cancer cells on their sensitivity towards vitamin D and/or 4HT; and (3) To evaluate the efficacy of RNA and peptide-based inhibitors of SNAI-protein functions on the aggressiveness, metastatic ability and drug sensitivity of SNAI-high human breast cancer cells in a mouse xenograft model., The original document contains color images.