Discovery of heterocyclic ureas as a new class of raf kinase inhibitors: identification of a second generation lead by a combinatorial chemistry approach

Heterocyclic ureas, such as N-3-thienyl N'-aryl ureas, have been identified as novel inhibitors of raf kinase, a key mediator in the ras signal transduction pathway. Structure-activity relationships were established, and the potency of the screening hit was improved 10-fold to IC(50)=1.7 microM. A combinatorial synthesis approach enabled the identification of a breakthrough lead (IC(50)=0.54 microM) for a second generation series of heterocyclic urea raf kinase inhibitors.     

Characterization of gastrointestinal chitinase in the lizard Sceloporus undulatus garmani (Reptilia: Phrynosomatidae)

Most studies on chitinase activity in lizards have been concerned with Palaearctic (European) and Laurasian (Middle Eastern and Asian) taxa. Several genera of Old World lizards, Anguis, Uromastix, Chamaeleo and Lacerta, have been shown to possess chitinolytic activity. To date, only one New World lizard, Anolis carolinensis, has been reported to exhibit chitinolytic activity. In the present study, chitinase activity was characterized in a second New World taxon, Sceloporus undulatus garmani, a New World, phrynosomatid lizard. Chitinolytic activity was measured by incubating tissue extracts with a radioactive chitin substrate, acetyl-[H³]chitin and determining acid soluble radioactivity as an estimate for chitin hydrolysis. Chitinolytic activity was present in stomach, small intestine and pancreas extracts, with the stomach and pancreas having the highest specific activities. Chitinolytic activity was higher at pH 4.5 than at pH 7.5. The stomach chitinase is immunologically similar to the gastric chitinase previously described for rainbow trout. Western blot analysis showed anti-chitinase cross-reactivity in the extracts of the stomach, but no cross-reactivity in the pancreatic or intestinal extracts, suggesting different isoforms of chitinase. There was no detected lysozyme activity (less than 0.01 mg/ml lysozyme) present in the extracts of the stomach, small intestine and pancreas. The localization of chitinolytic activity in S. u. garmani is in agreement with earlier reptilian reports on the distribution of chitinase.     

Pyridopyrimidine analogues as novel adenosine kinase inhibitors

A novel series of pyridopyrimidine analogues 9 was identified as potent adenosine kinase inhibitors based on the SAR and computational studies. Substitution of the C7 position of the pyridopyrimidino core with C2' substituted pyridino moiety increased the in vivo potency and enhanced oral bioavailability of these adenosine kinase inhibitors.     

Bacterial and Archaeal Phylogenetic Diversity of a Cold Sulfur-Rich Spring on the Shoreline of Lake Erie,Michigan

Studies of sulfidic springs have provided new insights into microbial metabolism, groundwater biogeochemistry, and geologic processes. We investigated Great Sulphur Spring on the western shore of Lake Erie and evaluated the phylogenetic affiliations of 189 bacterial and 77 archaeal 16S rRNA gene sequences from three habitats: the spring origin (11-m depth), bacterial-algal mats on the spring pond surface, and whitish filamentous materials from the spring drain. Water from the spring origin water was cold, pH 6.3, and anoxic (H₂, 5.4 nM; CH₄, 2.70 μM) with concentrations of S²⁻ (0.03 mM), SO₄²⁻ (14.8 mM), Ca²⁺ (15.7 mM), and HCO₃⁻ (4.1 mM) similar to those in groundwater from the local aquifer. No archaeal and few bacterial sequences were >95% similar to sequences of cultivated organisms. Bacterial sequences were largely affiliated with sulfur-metabolizing or chemolithotrophic taxa in Beta-, Gamma-, Delta-, and Epsilonproteobacteria. Epsilonproteobacteria sequences similar to those obtained from other sulfidic environments and a new clade of Cyanobacteria sequences were particularly abundant (16% and 40%, respectively) in the spring origin clone library. Crenarchaeota sequences associated with archaeal-bacterial consortia in whitish filaments at a German sulfidic spring were detected only in a similar habitat at Great Sulphur Spring. This study expands the geographic distribution of many uncultured Archaea and Bacteria sequences to the Laurentian Great Lakes, indicates possible roles for epsilonproteobacteria in local aquifer chemistry and karst formation, documents new oscillatorioid Cyanobacteria lineages, and shows that uncultured, cold-adapted Crenarchaeota sequences may comprise a significant part of the microbial community of some sulfidic environments.Cold, sulfidic springs upwelling into caves (1, 16-19) or exposed at the land surface (14, 15, 31, 39, 47, 50, 51) have recently been shown to harbor unique microbial communities, reflective of the aqueous sulfur chemistry of the upwelling groundwater or of unique cave conditions. Within these spring and cave ecosystems, new and unique Epsilonproteobacteria 16S rRNA gene sequences associated with a limited number of cultured isolates that carry out oxidation of sulfur compounds have been discovered (7). The abundance of Epsilonproteobacteria sequences in these settings and associated biogeochemical research have led to new interest in the role of microbially mediated sulfuric acid speleogenesis as an important limestone dissolution process that may contribute to the development of karst features in limestone bedrock (19). Additionally, in streamlets from sulfidic springs, unique symbioses between uncultured Euryarchaeota, Crenarchaeota, and Epsilonproteobacteria spp. that grow in whitish, macroscopically visible filaments have been described (31, 51). Sulfur cycling was identified as a major means of energy production and maintenance of microbial communities in cold, saline, perennial springs emanating from permafrost in the Arctic (47). Studies of cold, sulfidic springs have therefore provided new insights into microbial metabolism, ecology, and evolution as well as groundwater biogeochemistry and geologic processes.All studies of sulfidic springs to date have focused on terrestrial landscapes typically associated with limestone (CaCO₃) bedrock. Limestone is one of several carbonate sedimentary rocks deposited by ancient seas, which may contain significant amounts of gypsum (CaSO₄·2H₂O) as well as pockets of hydrocarbon deposits, both a source of sulfur. Water that moves for long distances through such rocks evolves through sequential dissolution and precipitation reactions to a geochemistry that bears little resemblance to freshwater. SO₄²⁻ becomes available for microbial reduction to sulfide in aquifer zones where conditions are appropriate. Where spring waters rich in CaCO₃, CO₂, and sulfide emerge at the surface, carbonate deposition and microbially mediated sulfide oxidation occur. These processes result in tufa deposits and the whitish crusts often noted in sulfidic spring outflows (12, 13). Carbonate bedrock underlies large portions of the lower Laurentian Great Lakes. Caves in contact with lake water occur on islands in Lake Erie and along the Bruce Peninsula in Ontario, Canada. A cold, sulfidic spring is located in Ancaster, Ontario, about 5 km from the Lake Ontario shoreline (13). Recently, plumes of high-conductivity sulfidic groundwater, surrounded by whitish filamentous materials and variously colored microbial mats, were reported to occur at a 93-m depth in Lake Huron (2, 49). However, there have been few molecular surveys of Bacteria or Archaea in any Great Lakes environment, and no reports focusing on the molecular phylogenetic diversity of microorganisms associated with these Great Lakes sulfidic environments.Along the western shoreline of Lake Erie and within Monroe County, MI, sinkholes and springs are abundant in the Silurian-Devonian carbonate bedrock, and Ca²⁺ and Mg²⁺ with SO₄²⁻ or HCO₃⁻ dominate groundwater composition (43). In some areas of Monroe County, sulfide in groundwater prohibits its use as a drinking water source. Great Sulphur Spring (GSS) was first described by Sherzer in 1900 (53) and was named for its sulfide-rich water. The spring arises from Silurian-Devonian carbonate bedrock within 0.5 km of the Lake Erie shoreline and is a convenient location for accessing sulfide-rich groundwater and for exploring potential interactions between groundwater and lake water. As part of a larger study of nearshore groundwater interactions with Lake Erie (27) and to better understand the potential role of microorganisms in sulfur chemistry of nearshore groundwater, we evaluated the chemistry and bacterial and archaeal 16S rRNA gene diversity of GSS. Our study documents a unique microbial community for the Laurentian Great Lakes, comprised in large part of new lineages and uncultivated members of the Archaea, Deltaproteobacteria, Epsilonproteobacteria, and Cyanobacteria. These sequences suggest a microbial community structure driven by (possibly H₂S-based) carbon fixation and chemolithotrophy of reduced compounds such as H₂, H₂S, or reduced nitrogen compounds, all consistent with spring geochemistry.     

Thermal Sensitivity of Mitochondrial Respiration Efficiency and Protein Phosphorylation in the Clam Mercenaria mercenaria

The mitochondria of intertidal invertebrates continue to function when organisms are exposed to rapid substantial shifts in temperature. To test if mitochondrial physiology of the clam Mercenaria mercenaria is compromised under elevated temperatures, we measured mitochondrial respiration efficiency at 15°C, 18°C, and 21°C using a novel, high-throughput, microplate respirometry methodology developed for this study. Though phosphorylating (state 3) and resting (state 4) respiration rates were unaffected over this temperature range, respiratory control ratios (RCRs: ratio of state 3 to state 4 respiration rates) decreased significantly above 18°C (p < 0.05). The drop in RCR was not associated with reduction of phosphorylation efficiency, suggesting that, while aerobic scope of mitochondrial respiration is limited at elevated temperatures, mitochondria continue to efficiently produce adenosine triphosphate. We further investigated the response of clam mitochondria to elevated temperatures by monitoring phosphorylation of mitochondrial protein. Three proteins clearly demonstrated significant time- and temperature-specific phosphorylation patterns. The protein-specific patterns of phosphorylation may suggest that a suite of protein kinases and phosphatases regulate mitochondrial physiology in response to temperature. Thus, while aerobic scope of clam mitochondrial respiration is reduced at moderate temperatures, specific protein phosphorylation responses reflect large shifts in function that are initiated within the organelle at higher temperatures.     

A Two-Step Path to Inclusion Formation of Huntingtin Peptides Revealed by Number and Brightness Analysis

Protein aggregation is a hallmark of several neurodegenerative diseases including Huntington's disease. We describe the use of the recently developed number and brightness method (N&B) that uses confocal images to monitor aggregation of Huntingtin exon 1 protein (Httex1p) directly in living cells. N&B measures the molecular brightness of protein aggregates in the entire cell noninvasively based on intensity fluctuations at each pixel in an image. N&B applied to mutant Httex1p in living cells showed a two-step pathway leading to inclusion formation that is polyQ length dependent and involves four phases. An initial phase of monomer accumulation is followed by formation of small oligomers (5-15 proteins); as protein concentration increases, an inclusion is seeded and forms in the cytoplasm; the growing inclusion recruits most of the Httex1p and depletes the cell leaving only a low concentration of monomers. The behavior of Httex1p in COS-7 and ST14A cells is compared.     

Evaluating Different Dimensions of Programme Effectiveness for Private Medicine Retailer Malaria Control Interventions in Kenya

Background

Private medicine retailers (PMRs) are key partners in the home management of fevers in many settings. Current evidence on effectiveness for PMR interventions at scale is limited. This study presents evaluation findings of two different programs implemented at moderate scale targeting PMRs for malaria control in the Kisii and Kwale districts of Kenya. Key components of this evaluation were measurement of program performance, including coverage, PMR knowledge, practices, and utilization based on spatial analysis.

Methodology/Principal Findings

The study utilized mixed quantitative methods including retail audits and surrogate client surveys based on post-intervention cross-sectional surveys in intervention and control areas and mapping of intervention outlets. There was a large and significant impact on PMR knowledge and practices of the program in Kisii, with 60.5% of trained PMRs selling amodiaquine medicines in adequate doses compared to 2.8% of untrained ones (OR; 53.5: 95% CI 6.7, 428.3), a program coverage of 69.7% targeted outlets, and a potential utilization of about 30,000 children under five. The evaluation in Kwale also indicates a significant impact with 18.8% and 2.3% intervention and control PMRs selling amodiaquine with correct advice, respectively (OR; 9.4: 95% CI 1.1, 83.7), a program coverage of 25.3% targeted outlets, and a potential utilization of about 48,000 children under five. A provisional benchmark of 7.5 km was a reasonable threshold distance for households to access PMR services.

Conclusions/Significance

This evaluation show that PMR interventions operationalized in the district level settings are likely to impact PMR knowledge and practices and lead to increased coverage of appropriate treatment to target populations. There is value of evaluating different dimensions of public health programs, including quality, spatial access, and implementation practice. This approach strengthens the potential contribution of pragmatic study designs to evaluating public health programs in the real world.     

Significant Association between Sulfate-Reducing Bacteria and Uranium-Reducing Microbial Communities as Revealed by a Combined Massively Parallel Sequencing-Indicator Species Approach

Massively parallel sequencing has provided a more affordable and high-throughput method to study microbial communities, although it has mostly been used in an exploratory fashion. We combined pyrosequencing with a strict indicator species statistical analysis to test if bacteria specifically responded to ethanol injection that successfully promoted dissimilatory uranium(VI) reduction in the subsurface of a uranium contamination plume at the Oak Ridge Field Research Center in Tennessee. Remediation was achieved with a hydraulic flow control consisting of an inner loop, where ethanol was injected, and an outer loop for flow-field protection. This strategy reduced uranium concentrations in groundwater to levels below 0.126 μM and created geochemical gradients in electron donors from the inner-loop injection well toward the outer loop and downgradient flow path. Our analysis with 15 sediment samples from the entire test area found significant indicator species that showed a high degree of adaptation to the three different hydrochemical-created conditions. Castellaniella and Rhodanobacter characterized areas with low pH, heavy metals, and low bioactivity, while sulfate-, Fe(III)-, and U(VI)-reducing bacteria (Desulfovibrio, Anaeromyxobacter, and Desulfosporosinus) were indicators of areas where U(VI) reduction occurred. The abundance of these bacteria, as well as the Fe(III) and U(VI) reducer Geobacter, correlated with the hydraulic connectivity to the substrate injection site, suggesting that the selected populations were a direct response to electron donor addition by the groundwater flow path. A false-discovery-rate approach was implemented to discard false-positive results by chance, given the large amount of data compared.Massively parallel sequencing has increased our ability to study microbial communities to a greater depth and at decreased sequencing costs to an extent that replication and gradient interrogation are now reasonably attainable. However, this massive throughput has mostly been used in exploratory studies, given the challenges to analysis of the big data sets generated and the relative novelty of the technique. To date, no report of a study that has used this method to describe the microbial community over a large area influenced by complicated hydrogeochemical factors during bioremediation has been published. Here, we used pyrosequencing technology complemented with a hypothesis-based approach to identify bacteria associated with biostimulation of U(VI) reduction at Area 3 of the U.S. Department of Energy''s (DOE''s) Oak Ridge Field Research Center (FRC) at Oak Ridge, TN.The Oak Ridge FRC is one of the most-studied sites for uranium bioremediation (2, 8, 19-22, 27, 37, 45-48). Previously used as a uranium enrichment plant, the site remains contaminated with depleted uranium, nitrate, and acidity. To deal with uranium contamination, dissimilatory metal reduction has been studied as an alternative that reduces risk by converting toxic soluble metals and radionuclides to insoluble, less toxic forms (2, 3, 16, 21, 26, 45). For example, some microbes can use metals such as Cr(VI), Se(VI), and the radionuclides U(VI) and Tc(VII) as final electron acceptors, producing a reduced insoluble species, thus blocking dispersal and reducing bioavailability.The ability to reduce U(VI) to U(IV) has been found in several unrelated phylogenetic groups, i.e., Delta-, Beta-, and Gammaproteobacteria, Firmicutes, Deinococci, and Actinobacteria, among others (42). Most previous studies have focused on the Fe(III)-reducing bacteria (FRB), especially Geobacter, and the sulfate-reducing bacteria (SRB), especially Desulfovibrio. Uranium(VI) reduction for bioremediation purposes has been tested and confirmed in laboratory-scale experiments using serum bottles (13, 18, 48), microcosms (23, 32), sediment columns (14, 43), and in situ field studies (3, 21, 41, 45), with the last one demonstrating the feasibility of U(VI) remediation and the correlation of U(VI) reduction with FRB (3, 6, 18, 31, 41) or SRB (40), or both (8, 19, 49).During field studies at Area 3 of the Oak Ridge site, a hydraulic control system together with ethanol injection successfully promoted U(VI) reduction from 5 μM to levels below U.S. Environmental Protection Agency (EPA) maximum contaminant levels (MCLs) for drinking water (0.126 μM) over a 2-year period (46). Reduction of U(VI) to U(IV) was confirmed by X-ray absorption near edge structure (XANES) (22, 46). Previous microbial surveys of sediments and groundwater from Area 3 wells by the use of 16S rRNA gene clone libraries detected genera known to harbor U(VI)-reducing members, such as Geobacter, Desulfovibrio, Anaeromyxobacter, Desulfosporosinus, and Acidovorax, after U(VI) reduction was established (8, 19). In one study, microbial counts from sediments were correlated with the hydraulic path, suggesting differences in organic carbon availability throughout Area 3 (8). The study that tracked the groundwater microbial communities of four locations of Area 3 over a 1.5-year period during ethanol stimulation found that nitrate, uranium, sulfide, and ethanol were correlated with particular bacterial populations and that the engineering control of dissolved oxygen and delivered nutrients was also significant in explaining the microbial community variability (19). However, the analysis of communities has been focused on limited wells and the community of the entire test area has not been characterized.On the basis of the previous results, we further hypothesized that the hydrological control strategy employed for the remediation of the site constrained the geochemistry of the site by controlling the distribution of organic carbon substrates and other nutrients and that this in turn selected a characteristic microbial community that was distinguishable from its surrounding community. We used massively parallel sequencing of 16S rRNA genes from sediments of 15 wells to characterize the microbial communities along hydrological gradients from the microbiologically active and hydraulically protected inner-loop zone to less active and still contaminated areas outside the treatment area and downgradient. Our sediment-sampling strategy allows a more precise spatial characterization than the use of groundwater samples, where filtering large volumes of water is often required, and also because samples of the attached communities can differ from the planktonic ones, as expected in oligotrophic aquifers (15), such as this site. The deeper sequencing allowed a more extensive survey of the communities, higher confidence in the detection of less dominant but significant members, and a more statistically robust indicator species assessment. We were able to detect groups significantly associated with U(VI) reduction and to explain differences in community structure with hydrogeochemical conditions.