Hepatitis C NS5B polymerase inhibitors: 4,4-Dialkyl-1-hydroxy-3-oxo-3,4-dihydronaphthalene-3-yl benzothiadiazine derivatives

4,4-Dialkyl-1-hydroxy-3-oxo-3.4-dihydronaphthalene-3-yl benzothiadiazine derivatives were synthesized and evaluated as inhibitors of genotypes 1a and 1b HCV NS5B polymerase. A number of these compounds exhibited potent activity against genotypes 1a and 1b HCV polymerase in both enzymatic and cell culture activities. A representative compound also showed favorable pharmacokinetics in the rat.     

Integrating Soil Ecological Knowledge into Restoration Management

The variability in the type of ecosystem degradation and the specificity of restoration goals can challenge restorationists’ ability to generalize about approaches that lead to restoration success. The discipline of soil ecology, which emphasizes both soil organisms and ecosystem processes, has generated a body of knowledge that can be generally useful in improving the outcomes of restoration despite this variability. Here, we propose that the usefulness of this soil ecological knowledge (SEK) for restoration is best considered in the context of the severity of the original perturbation, the goals of the project, and the resilience of the ecosystem to disturbance. A straightforward manipulation of single physical, chemical, or biological components of the soil system can be useful in the restoration of a site, especially when the restoration goal is loosely defined in terms of the species and processes that management seeks to achieve. These single‐factor manipulations may in fact produce cascading effects on several ecosystem attributes and can result in unintended recovery trajectories. When complex outcomes are desired, intentional and holistic integration of all aspects of the soil knowledge is necessary. We provide a short roster of examples to illustrate that SEK benefits management and restoration of ecosystems and suggest areas for future research.     

Physiological significance of high- and low-affinity agonist binding to neuronal and recombinant AMPA receptors

Radioligand binding studies have shown that AMPA receptors exist in two variants that differ about twenty-fold in their binding affinities, with brain receptors being mainly of the low-affinity type and recombinantly expressed receptors having almost exclusively high affinity. However, the physiological correlate of high- and low-affinity binding is not yet known. In this study we examined if physiological experiments similarly reveal evidence for two distinct receptor variants. We therefore measured equilibrium desensitization by glutamate and determined IC(50) values for neuronal receptors and for the homomeric receptors GluR1-4 expressed in HEK293 cells. Contrary to the prediction that these IC(50) values exhibit large differences commensurate with those of high- and low-affinity binding, values for homomeric receptors (1-18 microM) were on an average not different from those of neuronal receptors (3-10 microM). Moreover, simulations with kinetic receptor models suggest that the IC(50) values for neuronal and recombinant receptors correspond to the binding affinity of the low-affinity receptor variant. These findings indicate that the high-affinity binding measured in heterologous expression systems represents an immature receptor variant that does not contribute to the currents recorded from these cells, and that the functional low-affinity receptors are present in such small number that they are effectively masked in binding assays by the high-affinity receptors. Thus, in order to compare experimentally determined saturation binding profiles with those predicted by kinetic receptor models and with dose-response curves from physiological studies, it will be imperative to develop methods for isolating first the low-affinity receptors.     

Adaptive radiation of photosynthetic physiology in the Hawaiian lobeliads: dynamic photosynthetic responses

Hawaiian lobeliads have radiated into habitats from open alpine bogs to densely shaded rainforest interiors, and show corresponding adaptations in steady-state photosynthetic light responses and associated leaf traits. Shaded environments are not uniformly dark, however, but punctuated by sunflecks that carry most of the photosynthetically active light that strikes plants. We asked whether lobeliads have diversified in their dynamic photosynthetic light responses and how dynamic responses influence daily leaf carbon gain. We quantified gas exchange and dynamic light regimes under field conditions for ten species representing each major Hawaiian sublineage. Species in shadier habitats experienced shorter and less numerous sunflecks: average sunfleck length varied from 1.4 ± 1.7 min for Cyanea floribunda in shaded forest understories to 31.2 ± 2.1 min for Trematolobelia kauaiensis on open ridges. As expected, the rate of photosynthetic induction increased significantly toward shadier sites, with assimilation after 60 s rising from ca. 30% of fully induced rates in species from open environments to 60% in those from densely shaded habitats. Uninduced light use efficiency—actual photosynthesis versus that expected under steady-state conditions—increased from 10 to 70% across the same gradient. In silico transplants—modeling daily carbon gain using one species’ photosynthetic light response in its own and other species’ dynamic light regimes—demonstrated the potential adaptive nature of species differences: understory Cyanea pilosa in its light regimes outperformed gap-dwelling Clermontia parviflora, while Clermontia in its light regimes outperformed Cyanea. The apparent crossover in daily photosynthesis occurred at about the same photon flux density where dominance shifts from Cyanea to Clermontia in the field. Our results further support our hypothesis that the lobeliads have diversified physiologically across light environments in Hawaiian ecosystems and that those shifts appear to maximize the carbon gain of each species in its own environment. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Biological treatment and toxicity of low concentrations of oily wastewater (bilgewater)

The biodegradability and toxicity of low concentrations of oily wastewater (bilgewater) were tested under simulated sanitary wastewater treatment conditions. This was done to establish the feasibility of a combined shipboard oily and nonoily wastewater treatment system. The biodegradability of oily wastewater was determined by proxy; 14C-labeled dodecane, toluene, and phenanthrene (representing alkane, aromatic, and polyaromatic compounds, respectively) were mineralized in petroleum fuels and lubricants. We found that low concentrations of oily wastewater components were mineralized, even in the presence of more abundant substrates (such as synthetic graywater, containing vegetable oil, detergent, gelatin, and starch). The toxic effects of diesel fuel and several other components of oily wastewater (such as surfactants and a synthetic lubricant) on a na?ve wastewater assemblage was also tested. In concentrations much higher than would be expected under normal shipboard conditions, we found no evidence of toxic effects of the bilgewater compounds tested. Thus, a combined shipboard bilgewater and sanitary wastewater system might be feasible.     

Evidence of a rheotactic component in the odour search behaviour of freshwater eels

The detection of food odour by the freshwater eels, Anguilla australis and Anguilla dieffenbachii released a behavioural response to flow that resulted in direct upstream movement toward the odour source. Changes in various orientation parameters were observed as eels neared the source. Eels had substantially lower swimming velocities and considerably more variable heading and course angles close to the odour source (≤0·9 m) than further away (>0·9 m). Observed changes in orientation parameters were primarily due to changes in the behaviour of a searcher following odour loss. Cross-stream movements were initiated when the eel moved beyond the lateral margins of the odour plume. The behavioural switch from odour-conditioned rheotaxis to cross-stream casting following odour loss occurred more frequently close to the odour source where the plume was most narrow. Odour-conditioned rheotaxis enables the searcher to move quickly and efficiently toward the odour source without the need to extract directional information from a highly intermittent and complex chemical signal.     

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules ¹. By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes ² and restrict their environment to a more favorable low oxygen pressure ³. As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood.Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria ^4,5. The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor ⁶. Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)^7,8 has also been associated with severe disease ⁹.One of the most recently described P. falciparum cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps ¹⁰. Whether clumping occurs in vivo remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM ¹¹. In addition, the ability of clinical isolate cultures to clump in vitro was directly linked to the severity of disease in Malawian ¹² and Mozambican patients ¹³, (although not in Malian ¹⁴).With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay ¹⁵. Here, we present a method for in vitro platelet-mediated clumping of P. falciparum with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.