Reversible inhibition of murine cytomegalovirus replication by gamma interferon (IFN-γ) in primary macrophages involves a primed type I IFN-signaling subnetwork for full establishment of an immediate-early antiviral state

Activated macrophages play a central role in controlling inflammatory responses to infection and are tightly regulated to rapidly mount responses to infectious challenge. Type I interferon (alpha/beta interferon [IFN-α/β]) and type II interferon (IFN-γ) play a crucial role in activating macrophages and subsequently restricting viral infections. Both types of IFNs signal through related but distinct signaling pathways, inducing a vast number of interferon-stimulated genes that are overlapping but distinguishable. The exact mechanism by which IFNs, particularly IFN-γ, inhibit DNA viruses such as cytomegalovirus (CMV) is still not fully understood. Here, we investigate the antiviral state developed in macrophages upon reversible inhibition of murine CMV by IFN-γ. On the basis of molecular profiling of the reversible inhibition, we identify a significant contribution of a restricted type I IFN subnetwork linked with IFN-γ activation. Genetic knockout of the type I-signaling pathway, in the context of IFN-γ stimulation, revealed an essential requirement for a primed type I-signaling process in developing a full refractory state in macrophages. A minimal transient induction of IFN-β upon macrophage activation with IFN-γ is also detectable. In dose and kinetic viral replication inhibition experiments with IFN-γ, the establishment of an antiviral effect is demonstrated to occur within the first hours of infection. We show that the inhibitory mechanisms at these very early times involve a blockade of the viral major immediate-early promoter activity. Altogether our results show that a primed type I IFN subnetwork contributes to an immediate-early antiviral state induced by type II IFN activation of macrophages, with a potential further amplification loop contributed by transient induction of IFN-β.     

Pathway Thermodynamics Highlights Kinetic Obstacles in Central Metabolism

In metabolism research, thermodynamics is usually used to determine the directionality of a reaction or the feasibility of a pathway. However, the relationship between thermodynamic potentials and fluxes is not limited to questions of directionality: thermodynamics also affects the kinetics of reactions through the flux-force relationship, which states that the logarithm of the ratio between the forward and reverse fluxes is directly proportional to the change in Gibbs energy due to a reaction (Δ_rG′). Accordingly, if an enzyme catalyzes a reaction with a Δ_rG′ of -5.7 kJ/mol then the forward flux will be roughly ten times the reverse flux. As Δ_rG′ approaches equilibrium (Δ_rG′ = 0 kJ/mol), exponentially more enzyme counterproductively catalyzes the reverse reaction, reducing the net rate at which the reaction proceeds. Thus, the enzyme level required to achieve a given flux increases dramatically near equilibrium. Here, we develop a framework for quantifying the degree to which pathways suffer these thermodynamic limitations on flux. For each pathway, we calculate a single thermodynamically-derived metric (the Max-min Driving Force, MDF), which enables objective ranking of pathways by the degree to which their flux is constrained by low thermodynamic driving force. Our framework accounts for the effect of pH, ionic strength and metabolite concentration ranges and allows us to quantify how alterations to the pathway structure affect the pathway''s thermodynamics. Applying this methodology to pathways of central metabolism sheds light on some of their features, including metabolic bypasses (e.g., fermentation pathways bypassing substrate-level phosphorylation), substrate channeling (e.g., of oxaloacetate from malate dehydrogenase to citrate synthase), and use of alternative cofactors (e.g., quinone as an electron acceptor instead of NAD). The methods presented here place another arrow in metabolic engineers'' quiver, providing a simple means of evaluating the thermodynamic and kinetic quality of different pathway chemistries that produce the same molecules.     

Direct discrimination of different plant populations and study on temperature effects by Fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy was used to characterise highland and lowland populations of Polygonum minus Huds. grown in different controlled environments. A thermal perturbation technique of two-dimensional correlation infrared spectroscopy (2D-IR) correlation spectra was applied to establish differences between the populations. The absorption peaks at 3,480 cm^?1 (hydroxyl group), 2,927 cm^?1 (methyl group), 1,623 cm^?1 (carbonyl group), and 1,068 cm^?1 (C–O group) were particularly powerful in separating the populations. These peaks, which indicate the presence of carbohydrate, terpenes, amide and flavonoids were more intense for the highland populations than lowland populations, and increased in environments with a higher temperature. Wavenumbers (1,634, 669 cm^?1) and (1,634, 1,555 cm^?1) in the 2D-IR correlation spectra provided fingerprint signals to differentiate plants grown at different temperatures. This study demonstrates that IR fingerprinting, which combines mid-IR spectra and 2D-IR correlation spectra, can directly discriminate different populations of P. minus and the effects of temperature.     

<Emphasis Type="Italic">In vitro</Emphasis> conservation of Malaysian biodiversity—achievements,challenges and future directions

Malaysia is fortunate and proud to contain some of the world’s richest biodiversity. In Malaysia, there are an estimated 185,000 species of fauna and 12,500 species of flowering plants, many of which are endemic to tropical forests in this region. Indeed, such diversity is an important and invaluable national asset to safeguard both present and future generations. In vitro conservation offers possible techniques for the preservation of plant germplasm that at present is difficult to maintain or is maintained with limited success. Research at the Universiti Kebangsaan Malaysia (The National University of Malaysia) focuses on the cryopreservation of woody fruit species with seeds that cannot tolerate cryopreservation (recalcitrant or intermediate). Among the plants with recalcitrant seeds are such traditionally important edible tropical fruits as mangosteen, langsat, and rambai (Garcinia mangostana, Lansium domesticum, and Baccaurea motleyana). Citrus aurantifolia, Citrus suhuiensis, Citrus madurensis, Citrus hystrix, and Fortunella polyandra are among the Citrus and Citrus-related species studied. Cryopreservation studies include the Nepenthes species (pitcher plants) of Malaysia. Fundamental research on desiccation and low-temperature tolerance and on the physiology of desiccation are used to understand seed behavior, a prerequisite for the development of successful conservation techniques. At the same time, cryopreservation protocols for several Citrus and forestry species were developed for embryonic axes and adventitious shoots, mainly using rapid dehydration and PVS2 vitrification techniques. There are no successful standard techniques or protocols for species with highly recalcitrant seeds such as Garcinia species. Modification of existing protocols or development of new methods is required, but this can be accomplished only when a detailed understanding of the recalcitrant nature of the seeds or explants is achieved. While we have considerable knowledge concerning the basics of biochemical processes and some molecular data from work on desiccation-tolerant seeds, a great need remains for understanding the cause of the recalcitrance or desiccation sensitivity of these seeds. It may be necessary to use a systems biology approach that exploits the “omics” technologies to generate global molecular data. In combination with bioinformatics for data integration and analyses, this approach would move toward improved modeling of the biological pathways associated with the development of recalcitrant seeds.     

Chemometric analysis of lipase-catalyzed synthesis of xylitol esters in a solvent-free system

Immobilized Candida antarctica lipase B-catalyzed esterification of xylitol and two fatty acids (capric and caproic acid) were studied in a solvent-free system. The Taguchi orthogonal array method based on three-level-four-variables with nine experiments was applied for the analysis and optimization of the reaction parameters including time, substrate molar ratio, amount of enzyme, and amount of molecular sieve. The obtained conversion was higher in the esterification of xylitol and capric acid with longer chain length. The optimum conditions derived via the Taguchi approach for the synthesis of xylitol caprate and xylitol caproate were reaction time, 29 and 18 h; substrate molar ratio, 0.3 and 1.0; enzyme amount, 0.20 and 0.05 g, and molecular sieve amount of 0.03 g, respectively. The good correlation between the predicted conversions (74.18% and 61.23%) and the actual values (74.05% and 60.5%) shows that the model derived from the Taguchi orthogonal array can be used for optimization and better understanding of the effect of reaction parameters on the enzymatic synthesis of xylitol esters in a solvent-free system.