Protein Engineering of the Antitumor Enzyme PpADI for Improved Thermal Resistance

Arginine deiminase (ADI, EC 3.5.3.6) is a potential antitumor drug for the treatment of arginine‐auxotrophic tumors such as hepatocellular carcinomas (HCCs) and melanomas. Studies in human lymphatic leukemia cell lines have confirmed the anti‐angiogenic activity of ADI. Activity and thermal resistance limit the efficacy of ADI in treatment of auxotrophic tumors. Previously, we reengineered ADI from Pseudomonas plecoglossicida (PpADI) for improved activity under physiological conditions (37 °C, PBS buffer, pH 7.4) by two rounds of directed evolution and combination of beneficial substitutions through site‐directed mutagenesis. The best variant, PpADI M6 (K5T/D38H/D44E/A128T/E296K/H404R), showed a 64.7‐fold improvement in k_cat value and a 37.6 % decreased S_0.5 value under physiological conditions. However, M6 lost rapidly its activity (half‐life of ～2 days at 37 °C). Here we report the re‐engineering of PpADI M6 for improved thermal resistance by directed evolution in order to increase its half‐life under physiological conditions. Directed evolution and recombination of the two most beneficial positions yielded variant PpADI M9 (K5T/D38H/D44E/A128T/V140L/E296K/F325L/H404R), for which the T_m value increased from 47 (M6) to 54 °C (M9); this corresponds to an increased half‐life from ～2 days (M6) to ～3.5 days (M9) under physiological conditions. Structure analysis of the homology model of M9 showed that the beneficial substitutions V140L and F325L likely promote the formation of tetrameric PpADI, which has greater thermal resistance than dimeric PpADI.     

Construction of a Fuzzy and Boolean Logic Gates Based on DNA

Logic gates are devices that can perform logical operations by transforming a set of inputs into a predictable single detectable output. The hybridization properties, structure, and function of nucleic acids can be used to make DNA‐based logic gates. These devices are important modules in molecular computing and biosensing. The ideal logic gate system should provide a wide selection of logical operations, and be integrable in multiple copies into more complex structures. Here we show the successful construction of a small DNA‐based logic gate complex that produces fluorescent outputs corresponding to the operation of the six Boolean logic gates AND, NAND, OR, NOR, XOR, and XNOR. The logic gate complex is shown to work also when implemented in a three‐dimensional DNA origami box structure, where it controlled the position of the lid in a closed or open position. Implementation of multiple microRNA sensitive DNA locks on one DNA origami box structure enabled fuzzy logical operation that allows biosensing of complex molecular signals. Integrating logic gates with DNA origami systems opens a vast avenue to applications in the fields of nanomedicine for diagnostics and therapeutics.     

A survey of total hydrocyanic acid content in ready-to-eat cassava-based chips obtained in the Australian market in 2008

Cassava (Manihot esculenta Crantz) is a widely consumed food in the tropics that naturally contains cyanogenic glycosides (cyanogens, mainly composed of linamarin, acetone cyanohydrin, and hydrocyanic acid). If cassava is not adequately processed to reduce the level of cyanogens prior to consumption, these compounds can lead to the formation of hydrocyanic acid in the gut. Exposure to hydrocyanic acid can cause symptoms ranging from vomiting and abdominal pain to coma and death. In 2008, a survey of ready-to-eat (RTE) cassava-based snack foods was undertaken to determine levels of cyanogens measured as total hydrocyanic acid. This survey was undertaken in response to the New South Wales Food Authority being alerted to the detection of elevated levels of cyanogens in an RTE cassava-based snack food. This survey took 374 samples of RTE cassava chips available in the Australian marketplace. Significant variation in the levels of total hydrocyanic acid were observed in the 317 samples testing positive for cyanogens, with levels ranging from 13 to 165 mg of HCN equivalents per kg (mean value, 64.2 mg of HCN eq/kg for positive samples). The results from this survey serve as a timely warning for manufacturers of RTE cassava chips and other cassava-based snack foods to ensure there is tight control over the levels of cyanogens in the cassava ingredient. Evidence from this survey contributed to an amendment to the Australia New Zealand Food Standards Code, which now prescribes a maximum level for hydrocyanic acid in RTE cassava chips of 10 mg of HCN eq/kg, which aligns with the Codex Alimentarius Commission international standard for edible cassava flour.     

Development and use of polyethylene passive samplers to detect triclosans and alkylphenols in an urban estuary

To be able to use polyethylene passive samplers (PE) in the field, the partitioning constants between PE and water (K(PEw)) of the compounds examined must be known. The K(PEw)s of triclosan (TCS), methyl-triclosan (MTCS), n-nonylphenol (n-NP), nonylphenol-technical mix (NP-tech), n-octylphenol (n-OP), and t-octylphenol (t-OP) were measured as a function of pH, temperature, and salinity, and a salt effect was calculated for TCS, n-OP, and t-OP. Log K(PEw)s used for calculating dissolved concentrations were taken from 20 °C studies taking salt into account: 3.42 (TCS), 4.53 (MTCS), 4.20 (n-NP), 3.69 (n-OP), and 2.87 (t-OP). The K(PEw) of hydroxyl-group containing compounds were strongly affected by pH, whereas MTCS with its methoxy-group was not. Measured K(PEw)s could not be estimated from octanol-water partitioning constants due to the semipolar makeup of the compounds investigated. Instead, a good correlation (K(PEw) = 0.679 × K(hdw) + 1.033, r(2) = 0.984, p = 0.001) was obtained with hexadecane-water partitioning constants (K(hdw)) predicted from COSMOtherm. During deployments in Narragansett Bay (RI) in the fall of 2009, concentrations of MTCS and t-OP in surface and bottom waters ranged from 40-225 pg L(-1) and 3.5-11 ng L(-1), respectively. These concentrations are far below EC(50) values for rainbow trout. Surface/bottom and bottom/porewater activity ratios were calculated. These indicated surface waters as the main source of MTCS, while surface water as well as sediments were sources of t-OP.     

Thermal, microstructural and textural characterisation of gelatinised corn, cassava and yam starch blends

The functional properties of mixtures of maize, cassava and yam starches and their relationships with microstructural characteristics were investigated. Experiments were performed following the simplex‐centroid design with internal points and restrictions (upper limit) for yam starch proportion. The statistical model used (Scheffé canonical equation) was a powerful tool to predict the pastes behaviour within the limits of the experimental area. Polynomials with second level interactions were applied to obtain the surface response. Viscoelastic attributes of mixtures differed from those of individual starches. As shown by differential scanning calorimetry and microscopical observations, physical properties of the mixtures depended on the type of networks obtained. These networks varied upon yam, corn and cassava starch proportions as each one has a particular characteristic: gelatinisation temperature, granule size, swelling capacity and amylose/amylopectin ratio among others.     

What fraction of young adults are at risk for future smoking, and who are they?

Recent evidence indicates that higher smoking rates among young adults in the United States may be related in part to increased initiation during young adulthood. The tobacco industry, restricted from overtly targeting adolescents, appears to be focusing on young adults. Thus it is important to estimate the percentage and identify the characteristics of the young adult population (aged 18-29 years) at risk for future smoking. This group would include all current established smokers (smoked at least 100 cigarettes in their lifetime), current experimenters, former established smokers, and former experimenters at risk for smoking again, as well as never-smokers who do not rule out future smoking. Using data from the population-based 2002 California Tobacco Survey (N = 9,364 young adults aged 18-29 years), we designated groups of young adults at risk and identified factors associated with risk. Altogether, 86.7+/-1.5% of current and former established smokers were at risk for future smoking, and 59.6+/-4.3% of former established smokers were at risk. Over half (52.2+/-2.0%) of all experimenters but only 9.0+/-1.2% of never-smokers were at risk. Overall, 43.0+/-1.2% of the young adult population was at risk. Although different demographic and other factors were associated with risk among former established smokers, experimenters, and never-smokers, three factors were consistently associated in each group: Younger age, not having a smoke-free home, and going to bars and clubs. We conclude that the group of young adults at risk for future smoking is sizable and presents a fertile target for tobacco industry efforts to promote smoking. Counter-efforts might include promotion of smoke-free environments.     

Stress facilitates consolidation of verbal memory for a film but does not affect retrieval.

The effect of psychosocial stress on distinct memory processes was investigated in 157 college students using a brief film, which enabled comparison of verbal and visual memory by using a single complex stimulus. Participants were stressed either following stimuli presentation (consolidation) or before testing 48 hr later (retrieval) and were compared with no-stress controls. Salivary cortisol was measured before and 20 min after stress. The consolidation group significantly outperformed controls on total and verbal film scores. Stress did not impair retrieval relative to controls. Exploratory analyses revealed a significant correlation between cortisol and verbal scores across all groups (r = .18). Results provide the first evidence of a facilitative effect of a stressor on verbal memory, but failed to replicate retrieval findings. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

A multi-target tracking algorithm for application to adaptive cruise control

This paper presents a Multiple Target Tracking (MTT) Adaptive Cruise Control (ACC) system which consists of three parts; a multi-model-based multi-target state estimator, a primary vehicular target determination algorithm, and a single-target adaptive cruise control algorithm. Three motion models, which are validated using simulated and experimental data, are adopted to distinguish large lateral motions from longitudinally excited motions. The improvement in the state estimation performance when using three models is verified in target tracking simulations. However, the performance and safety benefits of a multi-model-based MTT-ACC system is investigated via simulations using real driving radar sensor data. The MTT-ACC system is tested under lane changing situations to examine how much the system performance is improved when multiple models are incorporated. Simulation results show system response that is more realistic and reflective of actual human driving behavior.