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The Micro-Pocket Fission Detector (MPFD) is a small-form-factor real-time fission chamber. MPFD performance has been simulated in the Advanced Test Reactor Critical Facility located at Idaho Nation...

Aim: The molluscum contagiosum virus (MCV) expresses several immune evasion molecules that inhibit activation of NF-κB. Presumably, inhibition of inflammatory responses mediated by NF-κB allows MCV to cause persistent infections. Materials & methods: MC163-IKK-α interactions were detected by immunoprecipitations. Results: Here, we identify a novel MCV inhibitor of NF-κB. Ectopic expression of the MC163 protein resulted in a significant decrease in TNF-α-induced NF-κB activation. However, MC163 had no detectable effect on mitochondrial antiviral-signaling protein-induced activation of the IFN-β-promoter. MC163 dampened NF-κB activation induced via the overexpression of either IKK-α or IKK-β suggesting MC163 targets the IKK complex. Conclusion: Our data highlight a previously unknown function for the MC163 protein and may represent an additional strategy used by MCV to subvert host immune responses.

Microstrip electrodes fabricated using Schott Borofloat® 33 as the electrode substrate were recently characterized in order to optimize the performance of 6Li-based suspended foil microstrip neutron detectors. The Schott Borofloat® 33 microstrip electrode capacitance was measured to be 67 pF before characterization efforts were conducted using a collimated 241Am alpha-particle source positioned between the microstrip electrode and a planar drift electrode. Characterization studies were performed to define counting and gas multiplication curves as well as study the electrical stability of the microstrip electrode substrate. Electrical stability was observed for approximately 23 h after the initial hour testing. A non-uniform drift electric field strength distribution was observed by measuring the pulse amplitude from a collimated 241Am alpha-particle source positioned at five discrete positions along the span of the drift electric field region between the microstrip and drift electrodes. All measurements were conducted within a sealed aluminum enclosure backfilled with P-10 gas.

6Li foils, each 75-µm thick, were positioned between a Schott Borofloat® 33 microstrip electrode and a planar drift electrode to construct suspended foil microstrip neutron detectors. MCNP6 simulations of two detector configurations, one containing a single 6Li foil and the other containing five 6Li foils, indicated expected maximum intrinsic thermal-neutron detection efficiencies of 18.36% and 54.08%, respectively. For comparison, the intrinsic thermal-neutron detection efficiency as a function of thermal-neutron beam position along the foil span was experimentally measured for both detector configurations. A non-uniform intrinsic thermal-neutron detection efficiency distribution was observed along the span of the 6Li foil(s) between the microstrip and drift electrodes. Maximum intrinsic thermal-neutron detection efficiencies of 12.58 ± 0.15% and 29.75 ± 0.26% for the single and five 6Li foils were measured, respectively. Gamma-ray rejection ratios of 6.46 × 10−5 ± 4.32 × 10−7 and 7.96 × 10−5 ± 4.65 × 10−7 were also measured, respectively, for a 137Cs exposure rate of 50 mR h−1. All measurements were conducted with the 6Li foil(s) contained within a sealed aluminum enclosure pressurized with 10 psig of P-10 gas.

Micro-Pocket Fission Detectors (MPFDs) can be used to monitor a neutron flux within the core of a nuclear reactor. When assembled into an array, MPFDs can be used to simultaneously monitor the neutron flux at multiple locations within the reactor. One permutation of the MPFD is designed to be deployed into an iron wire flux port, typical for most research and commercial reactors. A single 5-node MPFD array was fabricated at deployed into a dry flux port within the central thimble of the Kansas State University TRIGA Mk. II research nuclear reactor. The sensitivity of each MPFD node at operating voltages of +50 V, +100 V, and +200 V was observed and an optimal operating voltage of +100 V was identified. No cross-talk was observed between instrument channels during real-time neutron flux measurement. Linear power tracking and real-time response to reactor power were both observed between 10 kWth and 100 kWth reactor power.

Advancements in nuclear reactor core modeling and computational capability have encouraged further development of in-core neutron sensors. Micro-Pocket Fission Detectors (MPFDs) have been fabricated and tested previously, but successful testing of these prior detectors was limited to single-node operation with specialized designs. Described in this work is a modular, four-node MPFD array fabricated and tested at Kansas State University (KSU). The four sensor nodes were equally spaced to span the length of the fuel-region of the KSU TRIGA Mk. II research nuclear reactor core. The encapsulated array was filled with argon gas, serving as an ionization medium in the small cavities of the MPFDs. The unified design improved device ruggedness and simplified construction over previous designs. A 0.315-in. (8-mm) penetration in the upper grid plate of the KSU TRIGA Mk. II research nuclear reactor was used to deploy the array between fuel elements in the core. The MPFD array was coupled to an electronic support system which has been developed to support pulse-mode operation. Neutron-induced pulses were observed on all four sensor channels. Stable device operation was confirmed by testing under steady-state reactor conditions. Each of the four sensors in the array responded to changes in reactor power between 10 kWth and full power (750 kWth). Reactor power transients were observed in real-time including positive transients with periods of 5, 15, and 30 s. Finally, manual reactor power oscillations were observed in real-time.

The molluscum contagiosum virus (MCV) uses a variety of immune evasion strategies to antagonize host immune responses. Two MCV proteins, MC159 and MC160, contain tandem death effector domains (DEDs). They are reported to inhibit innate immune signaling events such as NF-κB and IRF3 activation, and apoptosis. The RxDL motif of MC159 is required for inhibition of both apoptosis and NF-κB activation. However, the role of the conserved RxDL motif in the MC160 DEDs remained unknown. To answer this question, we performed alanine mutations to neutralize the arginine and aspartate residues present in the MC160 RxDL in both DED1 and DED2. These mutations were further modeled against the structure of the MC159 protein. Surprisingly, the RxDL motif was not required for MC160's ability to inhibit MAVS-induced IFNβ activation. Further, unlike previous results with the MC159 protein, mutations within the RxDL motif of MC160 had no effect on the ability of MC160 to dampen TNF-α-induced NF-κB activation. Molecular modeling predictions revealed no overall changes to the structure in the MC160 protein when the amino acids of both RxDL motifs were mutated to alanine (DED1 = R67A D69A; DED2 = R160A D162A). Taken together, our results demonstrate that the RxDL motifs present in the MC160 DEDs are not required for known functions of the viral protein.

Hepatitis C virus (HCV) infection is a common risk factor for the development of liver cancer. The molecular mechanisms underlying this effect are only partially understood. Here, we show that the HCV protein, nonstructural protein (NS) 5B, directly binds to the tumor suppressor, NORE1A (RASSF5), and promotes its proteosomal degradation. In addition, we show that NORE1A colocalizes to sites of HCV viral replication and suppresses the replication process. Thus, NORE1A has antiviral activity, which is specifically antagonized by NS5B. Moreover, the suppression of NORE1A protein levels correlated almost perfectly with elevation of Ras activity in primary human samples. Therefore, NORE1A inactivation by NS5B may be essential for maximal HCV replication and may make a major contribution to HCV-induced liver cancer by shifting Ras signaling away from prosenescent/proapoptotic signaling pathways. CONCLUSION HCV uses NS5B to specifically suppress NORE1A, facilitating viral replication and elevated Ras signaling. (Hepatology 2017;65:1462-1477).

A series of experiments are reported which serve as milestones in the development of the MPFD technology. Each test involved reactor pulsing, a controlled power excursion that results from rapid removal of control rods. During a reactor pulse, the power level increases by several orders of magnitude in a fraction of a second resulting from prompt-critical neutron generation in the core. Preliminary reactor pulse testing was conducted at the Kansas State University TRIGA Mk. II nuclear reactor (KSU). The MPFD was used to track reactor pulses of $2.77 and $2.50 worth of reactivity insertion with measured Full Widths at Half Maximum (FWHM) of 13.2 and 16.1 ms compared to theoretical FWHM of 12.2 and 14.4 ms using the Fuchs-Nordheim Model. At the University of Wisconsin Madison Nuclear Reactor (UWNR), a set of 7 detectors, each with 4 independent chambers was deployed as part of the NEUP–NEAMS collaboration between UWNR and KSU. Nine of the time-synchronized chambers were able to track six reactor pulses to provide a spatial map of neutron flux. Finally, a two-chamber MPFD was deployed inside the Idaho National Laboratory Transient Reactor Test Facility (TREAT) reactor core and successfully monitored the power excursion of a 15 GW reactor pulse with an estimated neutron flux of ~1018 n/cm2/s. These preliminary tests highlight the multi-faceted capability of the MPFD as an instrument in different research and power reactors.

The Micro-Layered Fast-Neutron Detector (MLFD) is a proton-recoil fast-neutron scintillator that uses lucite as the hydrogen-rich neutron converter and silver-doped zinc sulfide (ZnS:Ag) as the inorganic scintillator medium. The MLFD is constructed by placing alternative layers of neutron converter and scintillation medium in order to maximize its light collection. A 43-mm long MLFD has previously shown a high detection efficiency of 9.2% for a bare 252Cf source, and exhibits acceptable gamma-ray suppression with pulse height discrimination. Pulse-shape discrimination of neutrons and gamma-rays yielded a figure-of-merit of 4.56. Modeling in Geant4 has shown that lengthening the MLFDs up to 200 mm can further increase the neutron detection efficiency. These results indicate that the MLFD can potentially be used in applications such as active and passive interrogation, and Special Nuclear Material (SNM) searches. However, the light collection device used thus far has been photomultiplier tubes (PMTs), which prove to be infeasible at longer lengths of the MLFD. To construct a compact hand-held neutron detection system, the PMTs would require to be replaced with silicon photomultipliers (SiPMs). Thus, this paper reports the performance of the MLFD coupled to SiPMs when irradiated with neutrons and gamma-rays. The results show that the MLFD-SiPM system is highly insensitive to gamma-rays (pulses not discernible from electronic noise), and simply setting a threshold to reject electronic noise is sufficient to register only neutron events. This indicates that the MLFD-SiPM system would be a compact, highly efficient fast-neutron detector in a gamma-ray environment.

A nonlinear reactivity model with thermal-hydraulic feedback is presented for analysis of PWR fuel with various cladding materials. The simplified, batch-wise neutronic model requires batch reactivities and migration areas as predetermined functions of burnup, temperature (fuel and coolant), and any design parameters having neutronic significance (e.g., enrichment or cladding composition). Here, such functions are found by using symbolic regression, a unique approach that finds both a functional form and any model coefficients simultaneously. Core thermal-hydraulics are modeled using single-channel, axially-averaged values. The models were implemented in an open-source, Python-based tool, used here to the analyze fuel with Zr-based cladding having outer, protective layers of either FeCrAl or SiC, two materials proposed for use in next-generation, accident-tolerant fuel.

The synthesis of a series of 5-carba-pterocarpens derivatives involving the cyclization of α-aryl-α-tetralones is described. Several compounds demonstrated potent activity and selectivity in vitro against HCV replicon reporter cells. The best profile in Huh7/Rep-Feo1b replicon reporter cells was observed with 2h (EC50 = 5.5 μM/SI = 20), while 2e was the most active in Huh7.5-FGR-JC1-Rluc2A replicon reporter cells (EC50 = 1.5 μM/SI = 70). Hydroxy groups at A- and D-rings are essential for anti-HCV activity, and substitutions in the A-ring at positions 3 and 4 resulted in enhanced activity of the compounds.

Preparation of thin U- and Th-coated 0.3 mm diameter Pt working electrodes by the cyclic potential sweep method is described. Uranyl- and thorium hydroxide layers were electrodeposited from ethanol solutions containing 0.02 M natural uranyl and 0.02 M natural thorium nitrate, each with 3.6 M ammonium nitrate. The cell for electrodeposition was specially developed in order to accommodate the small working electrodes for this research by including a working electrode probe, 3-D translation stage, and microscope. The source material deposition was analyzed using digital microscopy and scanning electron microscopy, and confirmed using x-ray fluorescence measurements. The appropriate potential range for electrodeposition was determined to be −0.62 V to −0.64 V for a 0.3 mm diameter Pt working electrode placed 1 cm from the counter electrode. Smooth, uniform deposition was observed near the central region of the working electrode, while surface cracking and crystalline formations were found near the edge of the working electrode. The final procedure for sample substrate preparation, electrolytic solution preparation and electrodeposition are described.

This essay will address the problem of the identity of Immanuel in Isaiah 7:14 by surveying the major views regarding the identity of the child. The historical background and the larger context of Isaiah 7 through 9 will be explored along with the meaning of almah and its use in Isaiah 7 and Matthew 1:23. Various articles, commentaries, and sources will be cited and documented to explain and defend the position regarding the identity. This essay will survey major views, critique and defend major viewpoints, and provide a conclusion of the issues treated in the problem surrounding the text.

Investigation of a series of 2,N-bisarylated 2-ethoxyacetamides resulted in the identification of four inhibitors 5, 20, 24, 29 with single-digit micromolar in vitro efficacy against two drug-resistant Plasmodium falciparum strains. These compounds are analogs of structurally-related 1,3-bisaryl-2-propen-1-ones (chalcones), the latter showing efficacy in vitro but not in a malaria-infected mouse. The 2,N-bisarylated 2-ethoxyacetamides (e.g., 2, 5, 20) were shown to possess significantly greater stability in the presence of metabolizing enzymes than the corresponding 1,3-bisaryl-2-propen-1-ones (e.g., 1, 3, 18).

Neutron sensors capable of real-time measurement of neutrons in high-flux environments are necessary for tests aimed at demonstrating the performance of experimental nuclear reactor fuels and materials in material test reactors (MTRs). In-core Micro-Pocket Fission Detectors (MPFDs) have been studied at Kansas State University for many years. Previous MPFD prototypes were successfully built and tested with promising results. Efforts are now underway to develop advanced MPFDs with radiation-resistant, high-temperature materials capable of withstanding irradiation test conditions in high performance material and test reactors. Stackable MPFDs have been designed, built, and successfully demonstrated as in-core neutron sensors. Advances in the electrodeposition and measurement of neutron reactive material, along with refinements to composition optimization simulations, have enhanced the capabilities of contemporary MPFDs.

To combat emerging coronaviruses, developing safe and efficient platforms to evaluate viral protease activities and the efficacy of protease inhibitors is a high priority. Here, we exploit a biosafety level 2 (BSL-2) chimeric Sindbis virus system to evaluate protease activities and the efficacy of inhibitors directed against the papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus (SARS-CoV), a biosafety level 3 (BSL-3) pathogen. We engineered Sindbis virus to coexpress PLpro and a substrate, murine interferon-stimulated gene 15 (ISG15), and found that PLpro mediates removal of ISG15 (deISGylation) from cellular proteins. Mutation of the catalytic cysteine residue of PLpro or addition of a PLpro inhibitor blocked deISGylation in virus-infected cells. Thus, deISGylation is a marker of PLpro activity. Infection of alpha/beta interferon receptor knockout (IFNAR −/− ) mice with these chimeric viruses revealed that PLpro deISGylation activity removed ISG15-mediated protection during viral infection. Importantly, administration of a PLpro inhibitor protected these mice from lethal infection, demonstrating the efficacy of a coronavirus protease inhibitor in a mouse model. However, this PLpro inhibitor was not sufficient to protect the mice from lethal infection with SARS-CoV MA15, suggesting that further optimization of the delivery and stability of PLpro inhibitors is needed. We extended the chimeric-virus platform to evaluate the papain-like protease/deISGylating activity of Middle East respiratory syndrome coronavirus (MERS-CoV) to provide a small-animal model to evaluate PLpro inhibitors of this recently emerged pathogen. This platform has the potential to be universally adaptable to other viral and cellular enzymes that have deISGylating activities. IMPORTANCE Evaluating viral protease inhibitors in a small-animal model is a critical step in the path toward antiviral drug development. We modified a biosafety level 2 chimeric virus system to facilitate evaluation of inhibitors directed against highly pathogenic coronaviruses. We used this system to demonstrate the in vivo efficacy of an inhibitor of the papain-like protease of severe acute respiratory syndrome coronavirus. Furthermore, we demonstrate that the chimeric-virus system can be adapted to study the proteases of emerging human pathogens, such as Middle East respiratory syndrome coronavirus. This system provides an important tool to rapidly assess the efficacy of protease inhibitors targeting existing and emerging human pathogens, as well as other enzymes capable of removing ISG15 from cellular proteins.

Opiates are an important component for drug testing due to their high abuse potential. Proper urine opiate interpretation includes ruling out poppy seed ingestion; however, detailed elimination studies after controlled poppy seed administration with known morphine and codeine doses are not available. Therefore, we investigated urine opiate pharmacokinetics after controlled oral administration of uncooked poppy seeds with known morphine and codeine content. Participants were administered two 45 g oral poppy seed doses 8 h apart, each containing 15.7 mg morphine and 3mg codeine. Urine was collected ad libitum up to 32 h after the first dose. Specimens were analyzed with the Roche Opiates II immunoassay at 2000 and 300 μg/L cutoffs, and the ThermoFisher CEDIA(®) heroin metabolite (6-acetylmorphine, 6-AM) and Lin-Zhi 6-AM immunoassays with 10 μg/L cutoffs to determine if poppy seed ingestion could produce positive results in these heroin marker assays. In addition, all specimens were quantified for morphine and codeine by GC/MS. Participants (N=22) provided 391 urine specimens over 32 h following dosing; 26.6% and 83.4% were positive for morphine at 2000 and 300 μg/L GC/MS cutoffs, respectively. For the 19 subjects who completed the study, morphine concentrations ranged from

An updated method of preparing thin, uranium-coated, 0.25-mm diameter platinum electrodes using the cyclic potential sweep method is described. Sixty-four samples were prepared using a 0.2 M UO2(NO3)2 solution with 19.695% enriched uranium. Improvements were made to the sample preparation, electrodeposition, and analysis methods, producing smoother samples with more uniform surface features than prior work. Alpha-particle spectrometry was used to determine the mass of uranium deposited on each sample. The average mass of uranium deposited on the metallic electrode from 100 potential sweep cycles was 0.987 × 10−7 g with a standard deviation of 0.860 × 10−7 g. Removing the outliers reduces the average deposited mass to 0.741 × 10−7 g with a standard deviation of 0.410 × 10−7 g. A smooth, uniform deposition was observed over the entire electrode after deposition and no cracking or crystalline formations were observed. This improved method of electrodeposition remains an attractive method of depositing fissile material onto small metallic substrates for the fabrication of Micro-Pocket Fission Detectors.

Hepatitis C virus (HCV) NS5B polymerase is an important and attractive target for the development of anti-HCV drugs. Here we report on the design, synthesis and evaluation of twenty-four novel allosteric inhibitors bearing the 4-thiazolidinone scaffold as inhibitors of HCV NS5B polymerase. Eleven compounds tested were found to inhibit HCV NS5B with IC50 values ranging between 19.8 and 64.9 RM. Compound 24 was the most active of this series with an IC50 of 5.6 mu M. A number of these derivatives further exhibited strong inhibition against HCV lb and 2a genotypes in cell based antiviral assays. Molecular docking analysis predicted that the thiazolidinone derivatives bind to the NS5B thumb pocketII (TP-II). Our results suggest that further optimization of the thiazolidinone scaffold may be possible to yield new derivatives with improved enzyme- and cell-based activity. (C) 2013 Elsevier Masson SAS. All rights reserved.

The synthesis of a new series of flurbiprofen hydrazide-hydrazones using microwave assisted reactions is described. Substituted aldehydes were condensed with flurbiprofen hydrazide by microwave irradiation to corresponding hydrazones. Synthesis of N’-[(4-bromothiophen-2-yl)methylidene]-2-(2-fluorobiphenyl-4-yl) propanehydrazide (3o) employing microwave assisted process resulted in higher yields, in faster time and with less chemical waste compared to traditional techniques. (2-fluorobiphenyl-4-yl)-N’- (phenylmethylidene)propanehydrazide (3p) and N’-[(2-chloro-6-fluorophenyl) methylidene]- 2-(2-fluorobiphenyl-4-yl)propanehydrazide (3s) inhibited the growth of a leukemia cancer cell line HL-60 (TB) by 66.37% and an ovarian cancer cell line OVCAR-4 by 77.34% (single dose, 10 μM), respectively at the National Cancer Institute (NCI), but had no significant effect on a panel of sixty human tumor cell lines. Flurbiprofen hydrazide-hydrazones were weak inhibitors of hepatitis C virus NS5B polymerase activity with N’-[(5-ethylfuran-2-yl) methylidene]-2-(2-fluorobiphenyl-4-yl)propanehydrazide (3m) being the most active of this series. Binding mode investigations of compound 3m suggested that allosteric pocket (AP)- B may be the potential binding site for flurbiprofen hydrazones and these results will also assist in further derivatization of 3m using the green chemistry approach and improve the potency of S-flurbiprofen hydrazide-hydrazones. KEY WORDS: anticancer activity, E-Z isomerism, flurbiprofen, hepatitis C NS5B polymerase, hydrazide-hydrazone, microwave.

Micro-Pocket Fission Detectors (MPFDs) continue to be an important development in advancing nuclear research capabilities. Commonly, nuclear reactor cores are located far from the location which is desired for read-out electronics. For example, the core of the TRIGA Mk II research nuclear reactor at Kansas State University is submerged under approximately 16 feet of water. Research nuclear reactor facilities often possess unanticipated sources of noise due to the aging facilities, electronic pump and compressor motors, and electromechanical control systems. A robust method to transmitMPFD pulse signals from the MPFD sensors to the readout electronics is under development. Numerous pre-amplifiers were tested to determine the preferred signal processing method for MPFDs. An MPFD emulator was developed which enabled benchtop electronics testing and optimization of the pre-amplifier circuit for future MPFD sensor testing. Finally, an MPFD was constructed and tested at the Kansas State University TRIGA Mk. II nuclear research reactor with the preferred electronics and compared to the MPFD emulator. Emulated pulses were produced with an amplitude of 144 mV and a rise- time of 115 ns using a shaping time of 0.25μs and gain of -1.3. Similarly, neutron-induced pulses were observed with an amplitude of 116 mV and a rise-time of 316 ns using a shaping time of 1.0μs and a gain of -1.3.The improved electronics increased the signal-to-noise ratio for the neutron-induced signal from approximately 2 to approximately 5.

Apoptosis is a powerful host cell defense to prevent viruses from completing replication. Poxviruses have evolved complex means to dampen cellular apoptotic responses. The poxvirus, Molluscum Contagiosum Virus (MCV), encodes numerous host interacting molecules predicted to antagonize immune responses. However, the function of the majority of these MCV products has not been characterized. Here, we show that the MCV MC163 protein localized to the mitochondria via an N-terminal mitochondrial localization sequence and transmembrane domain. Transient expression of the MC163 protein prevented mitochondrial membrane permeabilization (MMP), an event central to cellular apoptotic responses, induced by either Tumor Necrosis Factor alpha (TNF-α) or carbonyl cyanide 3-chlorophenylhydrazone (CCCP). MC163 expression prevented the release of a mitochondrial intermembrane space reporter protein when cells were challenged with TNF-α. Inhibition of MMP was also observed in cell lines stably expressing MC163. MC163 expression may contribute to the persistence of MCV lesions by dampening cellular apoptotic responses.

Hepatitis C virus (HCV) NS5B polymerase is a key target for the development of anti-HCV drugs. Here we report on the identification of novel allosteric inhibitors of HCV NS5B through a combination of structure-based virtual screening and in vitro NS5B inhibition assays. One hundred and sixty thousand compounds from the Otava database were virtually screened against the thiazolone inhibitor binding site on NS5B (thumb pocket-2, TP-2), resulting in a sequential down-sizing of the library by 2.7 orders of magnitude to yield 59 NS5B non-nucleoside inhibitor (NNI) candidates. In vitro evaluation of the NS5B inhibitory activity of the 59 selected compounds resulted in a 14% hit rate, yielding 8 novel structural scaffolds. Of these, compound 1 bearing a 4-hydrazinoquinazoline scaffold was the most active (IC 50 = 16.0 μM). The binding site of all 8 NNIs was mapped to TP-2 of NS5B as inferred by a decrease in their inhibition potency against the M423T NS5B mutant, employed as a screen for TP-2 site binders. At 100 μM concentration, none of the eight compounds exhibited any cytotoxicity, and all except compound 8 exhibited between 40 and 60% inhibition of intracellular NS5B polymerase activity in BHK-NS5B-FRLuc reporter cells. These inhibitor scaffolds will form the basis for future optimization and development of more potent NS5B inhibitors.

Poxviruses express proteins that limit host immune responses to infection. For example, the molluscum contagiosum virus MC160 protein inhibits tumor necrosis factor alpha (TNF-α)-induced NF-κB activation. This event correlates with MC160-induced IKK1 protein degradation, suggesting a mechanism for the above-mentioned phenotype. IKK1 is stabilized when it associates with the cellular heat shock protein 90 (Hsp90). Here, Hsp90 overexpression restored IKK1 levels in MC160-expressing cells, suggesting that MC160 competitively interacted with Hsp90. In support of this, further investigation showed that a mutant MC160 protein comprising only the C-terminal region (C protein) immunoprecipitated with Hsp90. In contrast, Hsp90 IP with a mutant MC160 protein consisting of only the N-terminal tandem death effector domains (DEDs) (N protein) was dramatically decreased. Since cells expressing either the N or C mutant MC160 protein remained similarly resistant to TNF-α-induced NF-κB activation, the N mutant protein probably utilized a different mechanism for inhibiting NF-κB. One likely mechanism for the N protein lies in its association with the DED-containing procaspase-8 protein, a cellular apoptosis precursor protein that regulates NF-κB activation. Here, IPs revealed that this association relied on the presence of the DED-containing N terminus of the MC160 protein but not the C-terminal portion. These interactions appear to have relevance with NF-κB activation, since the expression of the viral DEDs strongly inhibited procaspase-8-mediated NF-κB activation, an event not substantially altered by the C protein. Thus, the MC160 protein utilizes at least two distinct mechanisms for impeding NF-κB activation, association with Hsp90 to result in IKK1 protein degradation or interaction with procaspase-8.

Malaria, the most important of the human parasitic diseases, causes about 500 million infections worldwide and over 1 million deaths every year. The search for novel drug candidates against specific parasitic targets is an important goal for antimalarial drug discovery. Recently the antimalarial activity of chalcones has generated great interest. These compounds are small non-chiral molecules with relative high lipophilicity (clogP approximately 5-7), have molecular weights in the range of 300 to 600 g/mol, and possess in vivo efficacy against both P. berghei and P. yeolii. Preliminary data on our on-going chalcone synthesis project indicate that these compounds are active in vitro against P. falciparum, but are rapidly metabolized in liver microsome assays. Structurally-related compounds not including the enone linker are found to be much more metabolically stable and yet have comparable in vitro efficacy. In this study, we have utilized the efficacy data from an in-house on-going chalcone project to develop a 3D pharmacophore for antimalarial activity and used it to conduct virtual screening (in silico search) of a chemical library which resulted in identification of several potent chalcone-like antimalarials. The pharmacophore is found to contain an aromatic and an aliphatic hydrophobic site, one hydrogen bond donor site, and a ring aromatic feature distributed over a 3D space. The identified compounds were not only found to be potent in vitro against several drug resistant and susceptible strains of P. falciparum and have better metabolic stability, but included one with good in vivo efficacy in a mouse model of malaria.