Baculovirus superinfection: a probable restriction factor on the surface display of proteins for library screening

In addition to the expression of recombinant proteins, baculoviruses have been developed as a platform for the display of complex eukaryotic proteins on the surface of virus particles or infected insect cells. Surface display has been used extensively for antigen presentation and targeted gene delivery but is also a candidate for the display of protein libraries for molecular screening. However, although baculovirus gene libraries can be efficiently expressed and displayed on the surface of insect cells, target gene selection is inefficient probably due to super-infection which gives rise to cells expressing more than one protein. In this report baculovirus superinfection of Sf9 cells has been investigated by the use of two recombinant multiple nucleopolyhedrovirus carrying green or red fluorescent proteins under the control of both early and late promoters (vAcBacGFP and vAcBacDsRed). The reporter gene expression was detected 8 hours after the infection of vAcBacGFP and cells in early and late phases of infection could be distinguished by the fluorescence intensity of the expressed protein. Simultaneous infection with vAcBacGFP and vAcBacDsRed viruses each at 0.5 MOI resulted in 80% of infected cells coexpressing the two fluorescent proteins at 48 hours post infection (hpi), and subsequent infection with the two viruses resulted in similar co-infection rate. Most Sf9 cells were re-infectable within the first several hours post infection, but the reinfection rate then decreased to a very low level by 16 hpi. Our data demonstrate that Sf9 cells were easily super-infectable during baculovirus infection, and super-infection could occur simultaneously at the time of the primary infection or subsequently during secondary infection by progeny viruses. The efficiency of super-infection may explain the difficulties of baculovirus display library screening but would benefit the production of complex proteins requiring co-expression of multiple polypeptides.

Femtosecond pulse shaping using differential evolutionary algorithm and wavelet operators

Evolutionary meta-algorithms for pulse shaping of broadband femtosecond duration laser pulses are proposed. The genetic algorithm searching the evolutionary landscape for desired pulse shapes consists of a population of waveforms (genes), each made from two concatenated vectors, specifying phases and magnitudes, respectively, over a range of frequencies. Frequency domain operators such as mutation, two-point crossover average crossover, polynomial phase mutation, creep and three-point smoothing as well as a time-domain crossover are combined to produce fitter offsprings at each iteration step. The algorithm applies roulette wheel selection; elitists and linear fitness scaling to the gene population. A differential evolution (DE) operator that provides a source of directed mutation and new wavelet operators are proposed. Using properly tuned parameters for DE, the meta-algorithm is used to solve a waveform matching problem. Tuning allows either a greedy directed search near the best known solution or a robust search across the entire parameter space.

Interpreting Differential Temperature Trends at the Surface and in the Lower Troposphere

Estimated global-scale temperature trends at Earth's surface (as recorded by thermometers) and in the lower troposphere (as monitored by satellites) diverge by up to 0.14°C per decade over the period 1979 to 1998. Accounting for differences in the spatial coverage of satellite and surface measurements reduces this differential, but still leaves a statistically significant residual of roughly 0.1°C per decade. Natural internal climate variability alone, as simulated in three state-of-the-art coupled atmosphere-ocean models, cannot completely explain this residual trend difference. A model forced by a combination of anthropogenic factors and volcanic aerosols yields surface-troposphere temperature trend differences closest to those observed.

Spectral theory of some non-selfadjoint linear differential operators

We give a characterisation of the spectral properties of linear differential operators with constant coefficients, acting on functions defined on a bounded interval, and determined by general linear boundary conditions. The boundary conditions may be such that the resulting operator is not selfadjoint. We associate the spectral properties of such an operator $S$ with the properties of the solution of a corresponding boundary value problem for the partial differential equation $\partial_t q \pm iSq=0$. Namely, we are able to establish an explicit correspondence between the properties of the family of eigenfunctions of the operator, and in particular whether this family is a basis, and the existence and properties of the unique solution of the associated boundary value problem. When such a unique solution exists, we consider its representation as a complex contour integral that is obtained using a transform method recently proposed by Fokas and one of the authors. The analyticity properties of the integrand in this representation are crucial for studying the spectral theory of the associated operator.

"Where am I?" Impact of display and content variables on spatial presence and comprehension in virtual environments

Forgetting immediate physical reality and having awareness of one�s location in the simulated world is critical to enjoyment and performance in virtual environments be it an interactive 3D game such as Quake or an online virtual 3d community space such as Second Life. Answer to the question "where am I?" at two levels, whether the locus is in the immediate real world as opposed to the virtual world and whether one is aware of the spatial co-ordinates of that locus, hold the key to any virtual 3D experience. While 3D environments, especially virtual environments and their impact on spatial comprehension has been studied in disciplines such as architecture, it is difficult to determine the relative contributions of specific attributes such as screen size or stereoscopy towards spatial comprehension since most of them treat the technology as monolith (box-centered). Using a variable-centered approach put forth by Nass and Mason (1990) which breaks down the technology into its component variables and their corresponding values as its theoretical basis, this paper looks at the contributions of five variables (Stereoscopy, screen size, field of view, level of realism and level of detail) common to most virtual environments on spatial comprehension and presence. The variable centered approach can be daunting as the increase in the number of variables can exponentially increase the number of conditions and resources required. We overcome this drawback posed by adoption of such a theoretical approach by the use of a fractional factorial design for the experiment. This study has completed the first wave of data collection and starting the next phase in January 2007 and expected to complete by February 2007. Theoretical and practical implications of the study are discussed.

Differential effect of beetroot bread on postprandial DBP according to Glu298Asp polymorphism in the eNOS gene: a pilot study.

Our objective was to investigate whether the presence of Glu298Asp polymorphism in the endothelial NO synthase (eNOS) gene differentially affects the postprandial blood pressure response to dietary nitrate-rich beetroot bread. A randomised, single-blind, controlled, crossover acute pilot study was performed in 14 healthy men (mean age: 34±9 years) who were retrospectively genotyped for Glu298Asp polymorphism (7GG; T carriers 7). Volunteers were randomised to receive 200 g beetroot-enriched bread (1.1 mmol nitrate) or control bread (no beetroot; 0.01 mmol nitrate) on two separate occasions 10 days apart. Baseline and incremental area under the curve of blood pressure and NOx (nitrate/nitrite) were measured for a 6-h postprandial period. A treatment × genotype interaction was observed for diastolic blood pressure (P<0.02), which was significantly lower in T carriers (P<0.01) after consumption of beetroot bread compared with control bread. No significant differences were observed in the GG group. The beneficial diastolic blood pressure reduction was observed only in the T carriers of the Glu298Asp polymorphism in the eNOS gene after consumption of nitrate-rich beetroot bread. These data require confirmation in a larger population group.

Differential interference effects of negative emotional states on subsequent semantic and perceptual processing

Past studies have revealed that encountering negative events interferes with cognitive processing of subsequent stimuli. The present study investigates whether negative events affect semantic and perceptual processing differently. Presentation of negative pictures produced slower reaction times than neutral or positive pictures in tasks that require semantic processing, such as natural or man-made judgments about drawings of objects, commonness judgments about objects, and categorical judgments about pairs of words. In contrast, negative picture presentation did not slow down judgments in subsequent perceptual processing (e.g., color judgments about words, size judgments about objects). The subjective arousal level of negative pictures did not modulate the interference effects on semantic or perceptual processing. These findings indicate that encountering negative emotional events interferes with semantic processing of subsequent stimuli more strongly than perceptual processing, and that not all types of subsequent cognitive processing are impaired by negative events.

Differential brain activity during emotional versus nonemotional reversal learning

The ability to change an established stimulus–behavior association based on feedback is critical for adaptive social behaviors. This ability has been examined in reversal learning tasks, where participants first learn a stimulus–response association (e.g., select a particular object to get a reward) and then need to alter their response when reinforcement contingencies change. Although substantial evidence demonstrates that the OFC is a critical region for reversal learning, previous studies have not distinguished reversal learning for emotional associations from neutral associations. The current study examined whether OFC plays similar roles in emotional versus neutral reversal learning. The OFC showed greater activity during reversals of stimulus–outcome associations for negative outcomes than for neutral outcomes. Similar OFC activity was also observed during reversals involving positive outcomes. Furthermore, OFC activity is more inversely correlated with amygdala activity during negative reversals than during neutral reversals. Overall, our results indicate that the OFC is more activated by emotional than neutral reversal learning and that OFC's interactions with the amygdala are greater for negative than neutral reversal learning.

Coalescence of particles by differential sedimentation

We consider a three dimensional system consisting of a large number of small spherical particles, distributed in a range of sizes and heights (with uniform distribution in the horizontal direction). Particles move vertically at a size-dependent terminal velocity. They are either allowed to merge whenever they cross or there is a size ratio criterion enforced to account for collision efficiency. Such a system may be described, in mean field approximation, by the Smoluchowski kinetic equation with a differential sedimentation kernel. We obtain self-similar steady-state and time-dependent solutions to the kinetic equation, using methods borrowed from weak turbulence theory. Analytical results are compared with direct numerical simulations (DNS) of moving and merging particles, and a good agreement is found.

Cardiac protein kinases: the cardiomyocyte kinome and differential kinase expression in human failing hearts

Aims. Protein kinases are potential therapeutic targets for heart failure, but most studies of cardiac protein kinases derive from other systems, an approach that fails to account for specific kinases expressed in the heart and the contractile cardiomyocytes. We aimed to define the cardiomyocyte kinome (i.e. the protein kinases expressed in cardiomyocytes) and identify kinases with altered expression in human failing hearts. Methods and Results. Expression profiling (Affymetrix microarrays) detected >400 protein kinase mRNAs in rat neonatal ventricular myocytes (NVMs) and/or adult ventricular myocytes (AVMs), 32 and 93 of which were significantly upregulated or downregulated (>2-fold), respectively, in AVMs. Data for AGC family members were validated by qPCR. Proteomics analysis identified >180 cardiomyocyte protein kinases, with high relative expression of mitogen-activated protein kinase cascades and other known cardiomyocyte kinases (e.g. CAMKs, cAMP-dependent protein kinase). Other kinases are poorly-investigated (e.g. Slk, Stk24, Oxsr1). Expression of Akt1/2/3, BRaf, ERK1/2, Map2k1, Map3k8, Map4k4, MST1/3, p38-MAPK, PKCδ, Pkn2, Ripk1/2, Tnni3k and Zak was confirmed by immunoblotting. Relative to total protein, Map3k8 and Tnni3k were upregulated in AVMs vs NVMs. Microarray data for human hearts demonstrated variation in kinome expression that may influence responses to kinase inhibitor therapies. Furthermore, some kinases were upregulated (e.g. NRK, JAK2, STK38L) or downregulated (e.g. MAP2K1, IRAK1, STK40) in human failing hearts. Conclusions. This characterization of the spectrum of kinases expressed in cardiomyocytes and the heart (cardiomyocyte and cardiac kinomes) identified novel kinases, some of which are differentially expressed in failing human hearts and could serve as potential therapeutic targets.

Physiological and morphological adaptations of herbaceous perennial legumes allow differential access to sources of varyingly soluble phosphate

The aim of this study was to investigate the capacity of three perennial legume species to access sources of varyingly soluble phosphorus (P) and their associated morphological and physiological adaptations. Two Australian native legumes with pasture potential (Cullen australasicum and Kennedia prostrata) and Medicago sativa cv. SARDI 10 were grown in sand under two P levels (6 and 40 µg P g−1) supplied as Ca(H2PO4)2·H2O (Ca-P, highly soluble, used in many fertilizers) or as one of three sparingly soluble forms: Ca10(OH)2(PO4)6 (apatite-P, found in relatively young soils; major constituent of rock phosphate), C6H6O24P6Na12 (inositol-P, the most common form of organic P in soil) and FePO4 (Fe-P, a poorly-available inorganic source of P). All species grew well with soluble P. When 6 µg P g−1 was supplied as sparingly soluble P, plant dry weight (DW) and P uptake were very low for C. australasicum and M. sativa (0.1–0.4 g DW) with the exception of M. sativa supplied with apatite-P (1.5 g). In contrast, K. prostrata grew well with inositol-P (1.0 g) and Fe-P (0.7 g), and even better with apatite-P (1.7 g), similar to that with Ca-P (1.9 g). Phosphorus uptake at 6 µg P g−1 was highly correlated with total root length, total rhizosphere carboxylate content and total rhizosphere acid phosphatase (EC 3.1.3.2) activity. These findings provide strong indications that there are opportunities to utilize local Australian legumes in low P pasture systems to access sparingly soluble soil P and increase perennial legume productivity, diversity and sustainability.

Differential roles of the PKC novel isoforms, PKCδ and PKCε, in mouse and human platelets

Background Increasing evidence suggests that individual isoforms of protein kinase C (PKC) play distinct roles in regulating platelet activation. Methodology/Principal Findings In this study, we focus on the role of two novel PKC isoforms, PKCδ and PKCε, in both mouse and human platelets. PKCδ is robustly expressed in human platelets and undergoes transient tyrosine phosphorylation upon stimulation by thrombin or the collagen receptor, GPVI, which becomes sustained in the presence of the pan-PKC inhibitor, Ro 31-8220. In mouse platelets, however, PKCδ undergoes sustained tyrosine phosphorylation upon activation. In contrast the related isoform, PKCε, is expressed at high levels in mouse but not human platelets. There is a marked inhibition in aggregation and dense granule secretion to low concentrations of GPVI agonists in mouse platelets lacking PKCε in contrast to a minor inhibition in response to G protein-coupled receptor agonists. This reduction is mediated by inhibition of tyrosine phosphorylation of the FcRγ-chain and downstream proteins, an effect also observed in wild-type mouse platelets in the presence of a PKC inhibitor. Conclusions These results demonstrate a reciprocal relationship in levels of the novel PKC isoforms δ and ε in human and mouse platelets and a selective role for PKCε in signalling through GPVI.

Differential roles for the adapters Gads and LAT in platelet activation by GPVI and CLEC-2.

The results demonstrate that Gads plays a key role in linking the adapter LAT to SLP-76 in response to weak activation of GPVI and CLEC-2 whereas LAT is required for full activation over a wider range of agonist concentrations. These results reveal the presence of a Gads-independent pathway of platelet activation downstream of LAT.

Differential uptake, partitioning and transfer of Cd and Zn in the soil-pea plant-aphid system

The biomagnification of trace metals during transfer from contaminated soil to higher trophic levels may potentially result in the exposure of predatory arthropods to toxic concentrations of these elements. This study examined the transfer of Cd and Zn in a soil−plant−arthropod system grown in series of field plots that had received two annual applications of municipal biosolids with elevated levels of Cd and Zn. Results showed that biosolids amendment significantly increased the concentration of Cd in the soil and the shoots of pea plants and the concentration of Zn in the soil, pea roots, shoots, and pods. In addition, the ratio of Cd to Zn concentration showed that Zn was preferentially transferred compared to Cd through all parts of the system. As a consequence, Zn was biomagnified by the system whereas Cd was biominimized. Cd and Zn are considered to exhibit similar behaviors in biological systems. However, the Cd/Zn ratios demonstrated that in this system, Cd is much less labile in the root−shoot−pod and shoot−aphid pathways than Zn.