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Inspired by the bone-seeking property of radioactive strontium (Sr) isotopes, we have prepared casein-hydroxyapatite-(aminomethyl)phosphonic acid (CS-HAP-AMPA) composite microspheres that share sim...

ZrO2-based metal oxides are widely used in heterogeneous catalysis. For heterogeneous catalysis, dispersibility is a challenging job. This article reports a facile synthesis method for preparing Zr...

AXL is a member of the TAM (TYRO3, AXL, MER) subfamily of receptor tyrosine kinases. It is upregulated in a variety of cancers and its overexpression is associated with poor disease prognosis and acquired drug resistance. Utilizing a fragment-based lead discovery approach, a new indazole-based AXL inhibitor was obtained. The indazole fragment hit 11, identified through a high concentration biochemical screen, was expeditiously improved to fragment 24 by screening our in-house expanded library of fragments (ELF) collection. Subsequent fragment optimization guided by docking studies provided potent inhibitor 54 with moderate exposure levels in mice. X-ray crystal structure of analog 50 complexed with the I650M mutated kinase domain of Mer revealed the key binding interactions for the scaffold. The good potency coupled with reasonable kinase selectivity, moderate in vivo exposure levels, and availability of structural information for the series makes it a suitable starting point for further optimization efforts.

Western flower thrips (WFT), Frankliniella occidentalis, has become an important pest of vegetables worldwide, due to its economic damage to crop production. In order to control WFT, chemical insecticides are widely used. However, WFT has developed a high resistance against many kinds of insecticides. Na+, K+-ATPase, playing an important role in the ionic transmission across the membrane, is commonly considered to be the target of several xenobiotic compounds. However, whether the Na+, K+-ATPase can be used as one of the target sites for controlling WFT is still unknown. In this study, resistance levels of WFT to four insecticides (chlorpyrifos, beta cypermethrin, abamectin, and thiamethoxam) were measured. It was found that all four insecticides exhibited significant inhibitory effects on WFT, especially on nymphs. The activity of Na+, K+-ATPase was estimated after the treatment of four insecticides. Additionally, mRNA expression levels of three Na+, K+-ATPase α-subunit isoforms (X1, X2 and X3) were detected using RT-qPCR. The transcription profile of three Na+, K+-ATPase α-subunit isoforms were diverse after treatment by these four insecticides, which indicated that these isoforms might play different roles in the tolerance to insecticides. The results suggested that Na+, K+-ATPase can obviously be inhibited by these four classes of insecticide, and may serve as the new target for controlling WFT.

Western flower thrips (WFT), Frankliniella occidentalis, has become an important pest of vegetables worldwide, due to its economic damage to crop production. In order to control WFT, chemical insecticides are widely used. However, WFT has developed a high resistance against many kinds of insecticides. Na

Currently, insecticides are considered as the primary approach for controlling western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae). However, the heavy use of insecticides resulted in high insect resistance and serious environmental pollution. Given its characteristics of ease of operation and environmental friendliness, insect control using high temperature is receiving considerable renewed research interest. However, although the combination of insecticides and high temperature to control F. occidentalis has been studied before, few studies have focused on the short-term effect of such treatment. In a laboratory study, F. occidentalis adults and second-instar nymphs were exposed to 45°C for 2 h. Then, their susceptibility to acetamiprid, spinosad, methomyl, and beta-cypermethrin was tested after different periods of recovery time (2–36 h). Additionally, the specific activity of three detoxification enzymes (esterase, glutathione S-transferase, and cytochrome p450 (CYP) monooxygenase) of the treated insects was determined. The results indicated that the fluctuation of susceptibility to insecticides and detoxification enzyme activity during F. occidentalis recovery from heat shock are related. Furthermore, several recovery time points (2, 30, and 36 h) of significant susceptibility to four tested insecticides compared with the control were found during the treatment of adults that were heat-shocked. Recovery time points of higher susceptibility compared with the control depended on different insecticides during the second-instar nymph recovery from heat shock. Interestingly, the fluctuation of CYP monooxygenase activity exhibited a trend that was similar to the fluctuation of susceptibility to insecticides (especially spinosad) during the recovery from heat shock of adults. In addition, the glutathione S -transferase and CYP monooxygenase activity trend was similar to the trend of susceptibility to spinosad during the recovery from heat shock of second-instar nymphs. Our results provide a new approach for controlling F. occidentalis using the combined heat shock and insecticide. This effectively enhances the control efficiency of heat shock and significantly reduces the application of insecticides.

In this paper, we introduce a revised model to estimate the corrected initial number of each life stage in field life tables of insect pest populations, with a case example of the diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae). To validate our revised model, we used life table data of P. xylostella from 9 field surveys conducted by the corresponding author in 1996. In these surveys, P. xylostella infestations had been monitored in fields of flowering Chinese cabbage, long white radish, butter cabbage, Shanghai pak choi, and Chinese kale to investigate the impact of different pest management methods on the pest populations. In the past, the numbers of individuals per life stage on any given sampling date were calculated based on sampling data of that particular date and an average temperature determined for the entire sampling period. In several cases, this approach produced unrealistic survival rates (above 100%) for certain life stages in the resulting life tables. This problem continued even after the model was adjusted by calculating the duration of each life stage based on the actual temperature measured on each sampling date (instead of using one average temperature for the entire period). With temperature being an important factor that (i) affects the development time of insect life stages and (ii) can be determined easily during field surveys, we previously hypothesized that including corrected initial numbers of each life stage on each sampling date based on an average of the temperature between the given sampling date and its preceding sampling date will result in realistic and precise life tables. Furthermore, to estimate accurate survival rates, we here hypothesized that (i) it is important to adjust and correct the numbers of life stages on a given sampling date by including the numbers of preceding life stages from a preceding sampling date (not from the given sampling date) in the model; and (ii) the development time of the preceding life stage will determine which sampling date needs to be included in the calculation. By re-constructing and comparing 9 life tables of P. xylostella populations according to the previous and the revised new model, we confirmed these hypotheses. The revised model will allow a precise and realistic evaluation of control efforts against diamondback moth and other insect pest infestations in agriculture.

Fertilizer with different ration of nitrogen (N) to phosphorus (P) could influence performance of plants including crops and vegetables. Spodoptera exigua is an important agriculture pest that has caused serious economic loss especially recent decades. In the present study, we explored that the response of host plants and S. exigua after S. exigua caterpillars damaged on the way of different level intensity and frequency under five fertilizer treatments with different ratio of N to P. The results indicated that fertilizer can significantly influence the interaction between host plants and caterpillars. Compensatory growth was detected under definite fertilizer with N : P = 3 : 1. Higher N proportion in fertilizer appears to contribute to maintain similar stem biomass in defoliated seedlings to control ones. Root biomass had almost no significant difference between defoliation and control, but it can not compensate under definite fertilizer. Higher proportion of N in fertilizer also contributed to enhance antioxidant enzymes activities: antioxidase catalase (CAT), superoxide dismutase (SOD), at low-level intensity caterpillars on host plants.However, when the herbivorous intensity was increasing, higher proportion of P in fertilizer seemed to play more important role on increase the activities of CAT and SOD. Higher phosphorus likely contributes to enhance acetylcholin esterase (AChE) activity at lower degree of defoliation, but higher nitrogen proportion could result in higher AChE activity at higher degree of defoliation. Higher content of nitrogen in definite range contributed to reduce the carboxylesterase (CarE) activity at high intensity within short-term defoliation. However, when defoliation intensity enhanced, the difference of CarE activity between fertilizer categories was small.

BACKGROUND All previously and currently used methods for effectiveness evaluation of control measures for the diamondback moth (DBM) do not simultaneously take the actual damage and population size into consideration. Here, we propose a new method, the cumulative damage index method, in which the number of larvae and their amount of food consumption are simultaneously included in the calculation of the theoretical cumulative damage index (T) and actual cumulative damage index (A). Evaluation was based on the reduced degree of damage calculated according to indexes T and A. RESULTS Based on the new method, the corrected effectiveness of the combined use of biological measures, chemical insecticides, Bacillus thuringiensis (Bt) and P. xylostella granulosis virus (PxGV) on DBM was 35.85, 2.37, 12.50 and 11.77% respectively. Under the action of natural factors, the Population Developmental Index (I) of DBM was 5.1 ± 1.4; under the integrated actions of natural factors and these four types of measure, index I of DBM was 0.34 ± 0.1, 6.1 ± 1.5, 2.1 ± 0.5 and 1.1 ± 0.3 respectively. The sole effectiveness of Trichogramma spp. when integrated with other natural factors, integrated biological measures and chemical insecticides was 21.43 ± 1.69%, 45.27 ± 4.09% and 20.68 ± 2.60% respectively. CONCLUSIONS There was some difference between the effectiveness evaluated by the new method and index I, and the actual damage caused by DBM could be reflected well by index A. The new method is more scientifically appropriate and practical for effectiveness evaluation than existing methods. © 2014 Society of Chemical Industry

The western flower thrips (WFT), Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), an invasive insect pest in China, causes great economic losses worldwide. To understand the effects of heat shock on WFT population dynamics, we constructed life tables to quantify the effects of heat shock on the growth rate, survival, and fecundity of WFT offspring. WFT adults and 2nd instars were heat shocked at 45 °C, and the treatment was repeated after a 24-h recovery period. Heat-shocked and control insects were reared on the leaves of purple cabbage (Brassica oleracea L.; Brassicales: Brassicaceae), and various lifetable and development parameters of their F1 offspring were evaluated. The duration of the immature period of the offspring of heat-shocked adults was not significantly shorter than that of the control, whereas the duration of the immature period of the offspring of the heatshocked 2nd instars was longer than that of the other treatments, indicating that heat-shock of second instar...

Temperature is known to play a crucial role in the population dynamics of insects. Insects have evolved different mechanisms to resist unfavorable extreme temperatures. In recent years, western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), and onion thrips, Thrips tabaci (Lindeman) (Thysanoptera: Thripidae), have caused significant damage to vegetable crops. Because of global warming and expanding areas of vegetable cultivation, a study of the effects of heat stress on these thrips species is warranted. We exposed the various developmental stages of western flower thrips and onion thrips to temperatures of 41, 43, or 45°C for 2, 6, 12, 24, or 36 h to determine the effects of heat stress on survival. Our results showed that the heat resistance of nonadult western flower thrips was greater than that of the nonadult onion thrips, and that the natural heat resistant ability was the primary factor in heat resistance in western flower thrips. In contrast, the heat resistance of adult onion thrips was greater than that of the adult western flower thrips, which was primarily the result of the ability of searching suitable microenvironment that enabled the onion thrips to mitigate the effects of high temperatures more efficiently than the western flower thrips. Our analysis of the differences in heat resistance between western flower thrips and onion thrips provides important information for the development of thermal treatments for controlling western flower thrips and onion thrips.

The condensation heat transfer of steam-ethanol mixture with different weight fractions was investigated experimentally at atmospheric pressure. The results indicate that the heat transfer coefficients (HTC) and condensation modes, i.e. filmwise condensation (FWC), transition state and dropwise condensation (DWC), varied with the mixture compositions and the vapor-surface temperature differences. The interface effect, in terms of equivalent surface free energy difference between condensate and ultra thin liquid film, was introduced to analyze the variation of condensation modes and heat transfer coefficient. The equivalent surface free energy differences under various vapor conditions and vapor-surface temperature differences are calculated quantitatively. The experimental results show that as equivalent surface free energy difference was gradually increased, the condensation mode alternates from filmwise to transition state and finally to dropwise condensation, with heat transfer coefficient simultaneously increasing. The effect of surface free energy difference was also introduced to analyze the data in literature, and the effect of subcooling on heat transfer coefficient was discussed from the perspective of interface effect. The results show that as the vapor-surface temperature difference was gradually increased, the surface free energy difference increase accordingly and then reaches its peak value. The heat transfer coefficient exhibits the same tendency as equivalent surface free energy.

Fertilizer with different ratios of nitrogen (N) to phosphorus (P) can influence crop plant performance and defense against herbivores. Spodoptera exigua is an important agricultural pest that has caused serious economic loss, especially in recent decades. In the present study, we explored effects of different intensities and durations of S. exigua herbivory on host plant biomass and on S. exigua enzyme activities in response to five fertilizer treatments with different N: P ratios of 1: 5, 1: 3, 1: 1, 3: 1 and 5: 1. The results showed that fertilizer type can significantly influence interactions between caterpillars and its hosts. Compensatory growth of leaf biomass was detected under fertilizer with N: P = 3: 1. Fertilizer with a higher proportion of N appears to maintain stem biomass in defoliated seedlings similar to controls that are not exposed to herbivory. There was no significant difference in root biomass under most conditions. High proportion of N also enhanced the activity of two antioxidant enzymes, catalase (CAT) and superoxide dismutase (SOD) in low density of beet armyworm. However, with increased herbivorous intensity, a higher proportion of P played a more important role in increasing the activities of CAT and SOD. Higher P likely enhanced acetylcholine esterase (AChE) activity at lower degrees of defoliation, but a higher N proportion resulted in higher AChE activity at higher degrees of defoliation. Higher N proportion contributed to reduced carboxylesterase (CarE) activity at high intensity, short-term defoliation. However, when defoliation intensity increased, the difference in CarE activity between fertilizer categories was little. The study explored the interaction between the damage of S. exigua and the biomass accumulation of its host plant Brassica rapa, and the influence of the N/P ratio in plant fertilizer on this interaction. Systematic analysis was provided on the biomass of B. rapa and the activity of metabolic enzymes of S. exigua under different treatments.

Assembly of the E. coli bacteriophage P2 into an icosahedral capsid with T = 7 symmetry is dependent on the gpN capsid protein, the gpQ connector protein and the gpO internal scaffolding protein. In the presence of the P4-encoded protein Sid, the same proteins are assembled into a smaller capsid with T = 4 symmetry. Although gpO has long been expected to act as an internal scaffolding protein, it has not been possible to produce P2 procapsids efficiently in vitro or in vivo due to a failure to express gpO at high levels. In this study, we find that full-length gpO undergoes proteolytic degradation within 1 h of induction of expression. However, a truncated version of gpO lacking the N-terminal 25 amino acids (OΔ25) is stably expressed at high levels and is able to direct the formation of P2 size procapsids. In the presence of Sid, OΔ25 is incorporated into P4 procapsids, showing that Sid overrides the effect of gpO on capsid size determination.

Summary The coronavirus nucleocapsid (N) protein packages viral genomic RNA into a ribonucleoprotein complex. Interactions between N proteins and RNA are thus crucial for the assembly of infectious virus particles. The 45 kDa recombinant nucleocapsid N protein of coronavirus infectious bronchitis virus (IBV) is highly sensitive to proteolysis. We obtained a stable fragment of 14.7 kDa spanning its N-terminal residues 29–160 (IBV-N29-160). Like the N-terminal RNA binding domain (SARS-N45-181) of the severe acute respiratory syndrome virus (SARS-CoV) N protein, the crystal structure of the IBV-N29-160 fragment at 1.85 A resolution reveals a protein core composed of a five-stranded antiparallel β sheet with a positively charged β hairpin extension and a hydrophobic platform that are probably involved in RNA binding. Crosslinking studies demonstrate the formation of dimers, tetramers, and higher multimers of IBV-N. A model for coronavirus shell formation is proposed in which dimerization of the C-terminal domain of IBV-N leads to oligomerization of the IBV-nucleocapsid protein and viral RNA condensation.

The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) interacts with cellular receptors to mediate membrane fusion, allowing viral entry into host cells; hence it is recognized as the primary target of neutralizing antibodies, and therefore knowledge of antigenic determinants that can elicit neutralizing antibodies could be beneficial for the development of a protective vaccine. Here, we expressed five different fragments of S, covering the entire ectodomain (amino acids 48 to 1192), as glutathione S -transferase fusion proteins in Escherichia coli and used the purified proteins to raise antibodies in rabbits. By Western blot analysis and immunoprecipitation experiments, we showed that all the antibodies are specific and highly sensitive to both the native and denatured forms of the full-length S protein expressed in virus-infected cells and transfected cells, respectively. Indirect immunofluorescence performed on fixed but unpermeabilized cells showed that these antibodies can recognize the mature form of S on the cell surface. All the antibodies were also able to detect the maturation of the 200-kDa form of S to the 210-kDa form by pulse-chase experiments. When the antibodies were tested for their ability to inhibit SARS-CoV propagation in Vero E6 culture, it was found that the anti-SΔ10 antibody, which was targeted to amino acid residues 1029 to 1192 of S, which include heptad repeat 2, has strong neutralizing activities, suggesting that this region of S carries neutralizing epitopes and is very important for virus entry into cells.

Proteolytic cleavage of the structural proteins is an important part of the maturation process for most bacteriophages and other viruses. In the double-stranded DNA bacteriophages this cleavage is associated with DNA packaging, capsid expansion, and scaffold removal. To understand the role of protein cleavage in the expansion of bacteriophages P2 and P4, we have experimentally cleaved P4 procapsids produced by overexpression of the capsid and scaffolding proteins. The cleavage leads to particle expansion and scaffold removal in vitro. The resulting expanded capsid has a thin-shelled structure similar, but not identical, to that of mature virions.

Elevated levels of the phosphorylated m7G cap binding protein, eukaryotic initiation factor 4E (eIF4E) are associated with neoplasia. An observed oncogenic signaling effect upon activation of Erk1/2 or p38 MAPKs in cells is the phosphorylation of eIF4E specifically at Serine 209 by downstream kinase MAP kinase-interacting kinase 1 and 2 (Mnks). Therefore, inhibiting Mnks could be a potential therapeutic approach that selectively targets cancer cells without introducing toxicity to normal cells. To measure the target engagement and efficacy of Mnk inhibitors, we have established a sensitive method to quantitatively detect phospho-eIF4E and non-phospho-eIF4E. Here, we report using NanoPro 100 system, an automated capillary-based immunoassay platform that could separate the phosphorylated and non-phosphorylated eIF4E isoforms based on different isoelectric points. Primary antibodies against both non-phospho eIF4E and Serine 209 phosphorylated eIF4E were used for identification and verification of eIF4E isoforms in protein samples obtained from cell lysate, blood samples and tumor samples. Two major isoforms of eIF4E with isoelectric point at 5.38 for phospho-eIF4E and 5.88 for non-phospho-eIF4E were identified in eIF4E-overexpressing K562 cells. siRNA knockdown of eIF4E expression reduced both isoforms, while treatment with Mnk inhibitor only reduced phospho-eIF4E level. The measurement and quantification of the relative changes of phosphorylated eIF4E by this method can potentially be used in monitoring the efficacy of Mnk inhibitors in xenograft and future clinical trials. Citation Format: Zhiyuan Ke, Sifang Wang, Vithya Manoharan, Susmitha Vuddagiri, Esther Ong, Sharon Lim, Sin Tiong Ong, Kanda Sangthongpitag, Nacro Kassoum, Jeffrey Hill, May Ann Lee. Identification and quantification of isoforms of eukaryotic initiation factor 4E as biomarker in Mnk inhibitor-treated mouse model by capillary-based immunoassay platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3819. doi:10.1158/1538-7445.AM2017-3819

All previously and currently used methods for effectiveness evaluation of control measures for the diamondback moth (DBM) do not simultaneously take the actual damage and population size into consideration. Here, we propose a new method, the cumulative damage index method, in which the number of larvae and their amount of food consumption are simultaneously included in the calculation of the theoretical cumulative damage index (T) and actual cumulative damage index (A). Evaluation was based on the reduced degree of damage calculated according to indexes T and A.Based on the new method, the corrected effectiveness of the combined use of biological measures, chemical insecticides, Bacillus thuringiensis (Bt) and P. xylostella granulosis virus (PxGV) on DBM was 35.85, 2.37, 12.50 and 11.77% respectively. Under the action of natural factors, the Population Developmental Index (I) of DBM was 5.1 ± 1.4; under the integrated actions of natural factors and these four types of measure, index I of DBM was 0.34 ± 0.1, 6.1 ± 1.5, 2.1 ± 0.5 and 1.1 ± 0.3 respectively. The sole effectiveness of Trichogramma spp. when integrated with other natural factors, integrated biological measures and chemical insecticides was 21.43 ± 1.69%, 45.27 ± 4.09% and 20.68 ± 2.60% respectively.There was some difference between the effectiveness evaluated by the new method and index I, and the actual damage caused by DBM could be reflected well by index A. The new method is more scientifically appropriate and practical for effectiveness evaluation than existing methods. © 2014 Society of Chemical Industry.

We have previously reported that the 3C-like proteinase of the coronavirus infectious bronchitis virus (IBV) is responsible for processing of the 1a and 1a/1b polyproteins to three mature products of 24, 10, and 100 kDa (Liuet al.,1994, 1997; Ng and Liu, 1998). The C-terminal cleavage site of the 100-kDa protein was defined to be the Q891(1b)-S892(1b)dipeptide bond encoded by nucleotides 15,129 to 15,134 (Liu and Brown, 1995). In this report, other cleavage sites of the 3C-like proteinase in the polyprotein encoded by the ORF 1b region were mapped by coexpression, deletion, and site-directed mutagenesis studies. Using two ORF 1b-specific antisera, V58 and V17, three more Q-S(G) dipeptide bonds, encoded by nucleotides 16,929 to 16,934, 18,492 to 18,497, and 19,506 to 19,511, respectively, were demonstrated to be the cleavage sites of the 3C-like proteinase. Cleavage at these four positions would result in the release of four mature products with molecular masses of approximately 68, 58, 39, and 35 kDa. Among them, the 39- and 35-kDa proteins were specifically identified in IBV-infected cells. Taken together with the 100-kDa protein previously identified, these results suggest that the ORF 1b region of IBV mRNA1 may be able to encode five mature products.

It is well known that heat transfer in dropwise condensation (DWC) is superior to that in filmwise condensation (FWC) by at least one order of magnitude. Surfaces with larger contact angle (CA) can promote DWC heat transfer due to the formation of “bare” condensation surface caused by the rapid removal of large condensate droplets and high surface replenishment frequency. Superhydrophobic surfaces with high contact angle (> 150°) of water and low contact angle hysteresis (

A droplet model is proposed with respect to molecular clustering to describe the state of steam molecules before condensing on the cooled solid surface in steam condensation process, and also the model is used to account for the mechanism of steam dropwise condensation in the presence of non-condensable gases (NCG). The mathematical model is presented based on the Dillmann and Meier’s homogeneous nucleation theory. Correction of surface tension term is conducted to match the physical and chemical characteristics of condensation process, and the heat transfer model considering the effect of interfacial effects was used to calculate the mean temperature of clusters. The model predicted results of Gibbs free energy at different subcooling degrees and different saturated temperatures were given. And the ratio of heat transfer coefficient with to that without NCG with different fractions of NCG was also obtained. The results show that the non-condensable gases reduce the condensation rates in the same way as shown by experimental results in the literature. That confirms the validity of the model.

Roughness-induced superhydrophobic surface was applied to promote dropwise condensation (DWC) on a vertical plate in the presence of non-condensable gas (NCG). The DWC heat transfer characteristics were investigated and the wetting behaviors of the condensate droplets were observed visually. The experimental results have shown that the roughness-induced superhydrophobic surface would enhance the heat transfer characteristics of steam condensation in the presence of NCG with high concentration. The underlined mechanism is analyzed in terms of the droplet wetting modes.Copyright © 2010 by ASME

Since apoptosis is impaired in malignant cells overexpressing prosurvival Bcl-2 proteins, drugs mimicking their natural antagonists, BH3-only proteins, might overcome chemoresistance. Small molecule inhibitors of Bcl-X(L) function have been discovered from diverse structure classes using rational drug design as well as high-throughput screening (HTS) approaches. However, most of the BH3 mimetics that have been identified via screening based on fluorescence polarization displayed an affinity for their presumed protein targets that is far lower than that of BH3-only proteins. Therefore, it is important to establish a simple and inexpensive secondary platform for hit validation which is pertinent to current efforts for developing compounds that mimic the action of BH3-only proteins as novel anticancer agents. These considerations prompted us to explore the differential scanning fluorimetry (DSF) method that is based on energetic coupling between ligand binding and protein unfolding. We have systematically tested known Bcl-X(L)/Bcl-2 inhibitors using DSF and have revealed distinct subsets of inhibitors. More importantly, we report that some of these inhibitors interacted selectively with glutathione S-transferase tagged Bcl-X(L), whereas certain inhibitors exhibited marked selectivity towards native untagged Bcl-X(L). Therefore, we propose that the affinity tag may cause a significant conformational switch in the Bcl-X(L), which results in the selectivity for certain subsets of small molecule inhibitors. This finding also implies that the previous screens involving tagged proteins need to be carefully reexamined while further investigations must ensure that the right conformation of protein is used in future screens.

Equine arteritis virus (EAV) is an enveloped positive-sense RNA virus belonging to the Arteriviridae family, which also includes the porcine pathogen PRRSV and is genetically and structurally related to the coronaviruses. EAV is an important equine pathogen that has caused significant economic losses to the horse-breeding industry and has been difficult to control. The EAV virion consists of a genome-containing nucleocapsid core made of nucleocapsid (N) protein surrounded by a lipid envelope containing several membrane proteins. Here, the crystal structure of the capsid-forming domain of the EAV N protein is presented at 2.0 A resolution. The dimeric N-­protein structure is similar to the previously determined structure of the N protein from PRRSV, with most differences localized to the terminal helices and flexible loops. The N protein is organized as dimers of dimers in the crystal, which may reflect the arrangement of the protein in the viral nucleocapsid.

Various mutations in the Wnt pathway contribute to aberrant activation of Wnt signaling, which is implicated in multiple cancers. Besides the mutation of Adenomatous polyposis coli (APC) or beta-catenin which causes the continuous activation of Wnt signaling by stabilizing beta-catenin, there are mutations of other genes such as RNF43 that regulate Wnt signaling at the level of the ligand or cell surface receptor. Inhibition of the secretion of all human Wnts by blocking their palmitoleoylation by the O-acyltransferase, Porcupine (PORCN), could be an alternative therapeutic approach. Here we have developed a compound with a novel pharmacophore that can inhibit PORCN activity and hence Wnt signaling in nanomolar concentration. In vivo efficacy study demonstrated that the compound is highly efficacious in preventing tumor growth in genetic modification model MMTV-WNT1 mouse. We have identified a subset of pancreatic cancer, cholangiocarcinoma and mucinous ovarian cancer cell lines that are sensitive to the compound. Most of these cell lines harbor mutations in RNF43. Xenograft tumor models derived from the cancer cell lines were found to be sensitive to the compound. Our results demonstrate that inhibiting the Wnt/beta-catenin pathway by targeting PORCN with small-molecule inhibitors in a subset of cancers with mutation in RNF43 is a feasible and nontoxic strategy to overcome the problem of redundancy of Wnts, thereby, providing new option for therapy in diseases with up regulated Wnt expression. Citation Format: Zhiyuan Ke, Babita Madan, Shermaine Q.y. Lim, Sifang Wang, Jenefer Alam, Soo Yei Ho, Duraiswamy Athisayamani Jeyaraj, Kakaly Ghosh, Yun Shan Chew, Li Jun Ding, Vithya Monoharan, Vishal Pendharkar, Esther Ong, Jeffrey Hill, Kanda Sangthongpitag, Thomas Keller, May Ann Lee, David M. Virshup. A novel Porcupine inhibitor is effective in the treatment of cancers with RNF43 mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4449. doi:10.1158/1538-7445.AM2015-4449

We have determined the crystal structure of the core (C) protein from the Kunjin subtype of West Nile virus (WNV), closely related to the NY99 strain of WNV, currently a major health threat in the U.S. WNV is a member of the Flaviviridae family of enveloped RNA viruses that contains many important human pathogens. The C protein is associated with the RNA genome and forms the internal core which is surrounded by the envelope in the virion. The C protein structure contains four alpha helices and forms dimers that are organized into tetramers. The tetramers form extended filamentous ribbons resembling the stacked alpha helices seen in HEAT protein structures.

Bacteriophage P4 is a satellite virus of bacteriophage P2, which has acquired the ability to utilize the structural gene products of P2 to assemble its own capsid. The normal P2 capsid has a T = 7 icosahedral structure comprised of the gpN-derived capsid protein, whereas the capsid produced under the control of P4 has a smaller, T = 4 structure. The protein responsible for this size determination is the P4-coded gene product Sid, which forms an external scaffold on the P4 procapsid. Using an in vitro assembly system, we show that gpN and Sid can coassemble into procapsid-like particles, indistinguishable from those produced in vivo, in the absence of any other gene products. The fidelity of the assembly reaction is enhanced by the inclusion of PEG and has a pH optimum between 8.0 and 8.5. Analysis of the assembly properties of truncated versions of Sid and gpN suggests that the amino-terminal part of Sid is involved in gpN binding, while the carboxyl-terminal part forms trimeric Sid–Sid interactions, and that the first 31 amino acids of gpN are required for binding to Sid as well as for size determination.

Dysregulation of the Wnt signaling cascades is implicated in multiple disorders. There are 19 human Wnts that mediate signaling through diverse downstream pathways. To achieve maximum benefit from inhibition of Wnt signaling, targeting all of these pathways may be useful. The secretion and biological activity of all human Wnts requires palmitoylation mediated by Porcupine (PORCN), an endoplasmic reticulum-localized membrane bound O-acyltransferase. Several small molecule inhibitors of PORCN have been developed. Here we report a novel pharmacophore with derivatives that are nanomolar inhibitors of Wnt signaling. By a number of criteria, these compounds potently inhibit PORCN catalytic activity and hence suppress downstream Wnt-activated signaling pathways. The compounds effectively reduce autocrine Wnt signaling activity in selected cancer cell lines. The inhibitory activity is stereospecific, as an (R) enantiomer is inactive. Compounds with good oral bioavailability were tested for their in vivo activity and found to be highly efficacious in reversing tumor growth in both MMTV-WNT1 mice and of tumor xenografts. Treated tumors showed marked nuclear exclusion and decreased cytoplasmic staining of beta-catenin compared to vehicle controls. Importantly the treatment modulated downstream markers of Wnt signaling. No signs of toxicity were observed in mice at therapeutically effective doses. These results and our published results on C59 demonstrate that inhibiting the Wnt/beta-catenin pathway by targeting PORCN with small-molecule inhibitors is a feasible and nontoxic strategy. Use of porcupine inhibitors overcomes the problem of redundancy of Wnts, thereby, providing new options for therapy in diseases with high Wnt activity. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C248. Citation Format: Babita Madan, Zhiyuan Ke, Shermaine Q.y. Lim, Jenefer Alam, Soo Yei Ho, Duraiswamy A. Jeyaraj, Kakaly Ghosh, Yun Shan Chew, Jamal Aliyev, Li Jun Ding, Vishal Pendharkar, Sifang Wang, Kanda Sangthongpitag, Thomas Keller, May Ann Lee, David M. Virshup. Novel PORCN inhibitors are safe and effective in the treatment of WNT-dependent cancers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C248.

Droplet jumping from condensing surfaces induced by droplet coalescence during dropwise condensation of mixed steam on a superhydrophobic surface can significantly enhance condensation heat transfer of mixed steam with non-condensable gas. This phenomenon was visually observed and theoretically analyzed in the present paper. The dynamic evolution of droplet and the velocity distribution inside the droplet during coalescence were simulated using multiphase lattice Boltzmann method. The energy distribution released by droplet coalescence was calculated statistically, and the jumping height induced by droplet coalescence on a superhydrophobic surface was predicted based on the energy conservation method. The theoretical predictions obtained by the modified model proposed in this paper agree well with the experimental observations.

Abstract 180 Background and Aims. The main pathogenic molecular events associated with myeloproliferative neoplasms (Polycythemia Vera, Essential Thrombocytosis, and Primary Myelofibrosis) are mutations in Janus kinase 2 (JAK2) or in the thrombopoietin receptor that arise in the hematopoietic stem/progenitor cells. Both type of mutations lead to constitutive activation of the JAK2 signaling pathways. The approved JAK2 inhibitor (Ruxolitinib) is not expected to be selective for the mutant JAK2/receptor signaling or to completely suppress the multiple signaling pathways activated by the aberrant JAK2 signaling. We postulate that myeloproliferative neoplasms can be treated more effectively if we target the constitutive JAK2 signaling by a JAK2 inhibitor together with another kinase inhibitor targeting a specific pathway that is co-activated by the aberrant JAK2 signaling. This should increase targeting specificity, reduce JAK2 inhibitor dosages, and minimize potential side effects of these drugs. To this end, we constructed cell line models of myeloproliferative neoplasms and tested the models using a JAK2 inhibitor in combination with a panel of kinase inhibitors to identify combination pairs that give the best synergism. The synergistic pair was further confirmed in mouse models of myeloproliferative neoplasms. Methods. Mouse Ba/F3 cells were engineered to express either JAK2 WT, or JAK2 V617F, or TpoR W515L, or TpoR JAK2 WT, or TpoR JAK2 V617F, or Bcr-Abl. The effect of two JAK2 inhibitors (Ruxolitinib and TG101348) in combination with a panel of 15 various kinase inhibitors (one JNK, one B-Raf, one ROCK-1, one TIE-2, one PI3K, two CDK, two MAPK, three p38, and three mTOR inhibitors). An 8×8 constant ratio Latin square design were used for testing inhibition of cell proliferation/survival in these cell line models. Calculations were carried out using the Chou-Talalay method to determine which drug-pair demonstrated synergism in inhibiting cell growth. Further eight PI3K inhibitors were acquired and tested when we found strong synergism between the JAK2 inhibitors and the PI3K inhibitor ZSTK474 in the first panel. The engineered Ba/F3 cells were also inoculated into female BALB/c nude mice to generate the JAK2 mutant mouse model. These mice were treated intravenously with Ruxolitinib and the PI3K inhibitor GDC0941. Blood profile and physical parameters of the mice were measured for 14 days post treatment. Bone marrow cells from mice reconstituted with bone marrow from JAK2 V617F knock-in mice were plated for colony formation in the presence or absence of Ruxolitinib and the PI3K inhibitor GDC0941. Primary Epo-independent colonies from CD34+ cells of one PV patient were assessed in two independent experiments in the presence or absence of combination drugs. Results. Out of 15 kinase inhibitors tested, three PI3K inhibitors (ZSTK474, GDC0941 and BEZ235), synergized with JAK2 inhibitors (Ruxolitinib and TG101348) in inhibiting cell growth. The combination index was less than 0.5 in all 8×8 dose combination ratios. The JAK2-PI3K inhibitors combination was specific for JAK2 signaling as growth of Ba/F3 cells expressing Bcr-Abl (at equivalent STAT5 activation levels) was unaffected by this combination treatment. Balb/c mice inoculated with Ba/F3 cells expressing TpoR JAK2 V617F were found to have increased spleen weight due to proliferation of autonomous cells. Our combination treatment using Ruxolitinib and GDC0941 could drastically reduce spleen weight compared to treatment with either compound alone. Endogenous erythroid colony forming unit (CFU-E) and burst forming unit (BFU-E) formation from JAK2 V617F knock-in bone marrow cells was reduced significantly by the combined use of Ruxolitinib and GDC0941 compared to individual drugs. Similarly, Epo-independent BFU-E colony formation from peripheral CD34+ cells of one JAK2 V617F-positive PV patient was reduced significantly by the drug combination. Conclusions. Our findings of strong synergy between the JAK2 inhibitors and PI3K inhibitors suggested that we may be able to administer these drugs at lower concentrations than when the drugs are used individually. It provides a framework for combination trials using compounds in these two classes in patients with myeloproliferative neoplasms. Disclosures: No relevant conflicts of interest to declare.

This study focuses on three main myeloproliferative neoplasms, namely, polycythaemia vera, essential thrombocytosis, and primary myelofibrosis. The main pathological causes of these three diseases are a mutation in the JAK2 protein or a mutation in the thrombopoietin receptor that arises in the hematopoietic stem/progenitor cells. Both type of mutations lead to constitutive activation of the JAK2 signaling pathways. Current JAK2 inhibitors in clinical trials are not expected to be selective and specific enough to suppress the multiple signaling pathways activated by the aberrant JAK2 signaling. Hence, we are seeking a combination treatment using a specific JAK2 inhibitor with another specific kinase inhibitor to inhibit the multiple signaling pathways activated by the constitutively active JAK2. Mouse Ba/F3 cells were engineered to express either JAK2 WT, or JAK2 V617F, or TpoR W515L, or TpoR JAK2 WT, or TpoR JAK2 V617F, or Bcr-abl. The effect of JAK2 inhibitors in combination with a panel of kinase inhibitors in an 8×8 constant ratio drug combination design were used to test for the inhibition of cell viability in the cell models. Calculation of drug synergism was carried out using the Chou-Talalay method. We found that JAK2 inhibitors synergized with PI3K inhibitors. The JAK2-PI3K inhibitors combination was specific for JAK2 signaling as survival of Ba/F3 cells expressing Bcr-abl was unaffected by this combination treatment. Balb/c mice inoculated with Ba/F3 cells expressing TpoR JAK2 V617F were found to have increased spleen weight. Our combination treatment could drastically reduce spleen weight compared to treatment with individual compound alone. The combination treatment also resulted in significant survival benefit in Balb/c mice inoculated with Ba/F3 cells expressing TpoR JAK2 V617F compared to untreated mice or mice given either compound alone. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B239.

The double-stranded DNA bacteriophage P4 has a T = 4 icosahedral arrangement of the gpN capsid protein derived from the P2 helper phage. The precursor procapsids in addition contain an external scaffold made up of the P4-encoded Sid protein. High yields of pure P4 procapsids have been obtained by coexpressing the gpN and Sid proteins from a chimeric plasmid. Biochemical measurements show that the ratio of gpN to Sid in the procapsids is 2:1, corresponding to 120 copies of Sid per procapsid particle. A reconstruction of the P4 procapsid, made from 213 particle images to an effective resolution of about 21 Å, greatly improves on the previously determined P4 procapsid structures. The structure shows a T = 4 capsid shell and a unique tandem arrangement of 120 copies of chilli-shaped Sid monomers, which form trimers and dimers on the procapsid surface.