Intracerebral accumulation of glutaric and 3-hydroxyglutaric acids secondary to limited flux across the blood-brain barrier constitute a biochemical risk factor for neurodegeneration in glutaryl-CoA dehydrogenase deficiency

Glutaric acid (GA) and 3-hydroxyglutaric acids (3-OH-GA) are key metabolites in glutaryl co-enzyme A dehydrogenase (GCDH) deficiency and are both considered to be potential neurotoxins. As cerebral concentrations of GA and 3-OH-GA have not yet been studied systematically, we investigated the tissue-specific distribution of these organic acids and glutarylcarnitine in brain, liver, skeletal and heart muscle of Gcdh-deficient mice as well as in hepatic Gcdh-/- mice and in C57Bl/6 mice following intraperitoneal loading. Furthermore, we determined the flux of GA and 3-OH-GA across the blood-brain barrier (BBB) using porcine brain microvessel endothelial cells. Concentrations of GA, 3-OH-GA and glutarylcarnitine were significantly elevated in all tissues of Gcdh-/- mice. Strikingly, cerebral concentrations of GA and 3-OH-GA were unexpectedly high, reaching similar concentrations as those found in liver. In contrast, cerebral concentrations of these organic acids remained low in hepatic Gcdh-/- mice and after intraperitoneal injection of GA and 3-OH-GA. These results suggest limited flux of GA and 3-OH-GA across the BBB, which was supported in cultured porcine brain capillary endothelial cells. In conclusion, we propose that an intracerebral de novo synthesis and subsequent trapping of GA and 3-OH-GA should be considered as a biochemical risk factor for neurodegeneration in GCDH deficiency.     

Microbial community composition in soils of Northern Victoria Land, Antarctica

Biotic communities and ecosystem dynamics in terrestrial Antarctica are limited by an array of extreme conditions including low temperatures, moisture and organic matter availability, high salinity, and a paucity of biodiversity to facilitate key ecological processes. Recent studies have discovered that the prokaryotic communities in these extreme systems are highly diverse with patchy distributions. Investigating the physical and biological controls over the distribution and activity of microbial biodiversity in Victoria Land is essential to understanding ecological functioning in this region. Currently, little information on the distribution, structure and activity of soil communities anywhere in Victoria Land are available, and their sensitivity to potential climate change remains largely unknown. We investigated soil microbial communities from low- and high-productivity habitats in an isolated Antarctic location to determine how the soil environment impacts microbial community composition and structure. The microbial communities in Luther Vale, Northern Victoria Land were analysed using bacterial 16S rRNA gene clone libraries and were related to soil geochemical parameters and classical morphological analysis of soil metazoan invertebrate communities. A total of 323 16S rRNA gene sequences analysed from four soils spanning a productivity gradient indicated a high diversity (Shannon-Weaver values > 3) of phylotypes within the clone libraries and distinct differences in community structure between the two soil productivity habitats linked to water and nutrient availability. In particular, members of the Deinococcus/Thermus lineage were found exclusively in the drier, low-productivity soils, while Gammaproteobacteria of the genus Xanthomonas were found exclusively in high-productivity soils. However, rarefaction curves indicated that these microbial habitats remain under-sampled. Our results add to the recent literature suggesting that there is a higher biodiversity within Antarctic soils than previously expected.     

Spatial Analysis of Antibiotic Resistance Along Metal Contaminated Streams

The spatial pattern of antibiotic resistance in culturable sediment bacteria from four freshwater streams was examined. Previous research suggests that the prevalence of antibiotic resistance may increase in populations via indirect or coselection from heavy metal contamination. Sample bacteria from each stream were grown in media containing one of four antibiotics—tetracycline, chloramphenicol, kanamycin, and streptomycin—at concentrations greater than the minimum inhibitory concentration, plus a control. Bacteria showed high susceptibilities to the former two antibiotics. We summarized the latter two more prevalent (aminoglycoside) resistance responses and ten metals concentrations per sediment sample, by Principal Components Analysis. Respectively, 63 and 58% of the variability was explained in the first principal component of each variable set. We used these multivariate summary metrics [i.e., first principal component (PC) scores] as input measures for exploring the spatial correlation between antibiotic resistance and metal concentration for each stream sampled. Results show a significant and negative correlation between metals PC scores versus aminoglycoside resistance scores and suggest that selection for metal tolerance among sediment bacteria may influence selection for antibiotic resistance differently in sediments than in the water column. Our most important finding comes from geostatistical cross-variogram analysis, which shows that increasing metal concentration scores are spatially associated with decreasing aminoglycoside resistance scores—a negative correlation, but holds for contaminated streams only. We suspect our field results are influenced by metal bioavailability in the sediments and by a contaminant promoted interaction or “cocktail effect” from complex combinations of pollution mediated selection agents.     

Open-label comparative clinical study of chlorproguanil-dapsone fixed dose combination (Lapdap) alone or with three different doses of artesunate for uncomplicated Plasmodium falciparum malaria

The objective of this study was to determine the appropriate dose of artesunate for use in a fixed dose combination therapy with chlorproguanil−dapsone (CPG−DDS) for the treatment of uncomplicated falciparum malaria.

Methods

Open-label clinical trial comparing CPG−DDS alone or with artesunate 4, 2, or 1 mg/kg at medical centers in Blantyre, Malawi and Farafenni, The Gambia. The trial was conducted between June 2002 and February 2005, including 116 adults (median age 27 years) and 107 children (median age 38 months) with acute uncomplicated Plasmodium falciparum malaria. Subjects were randomized into 4 groups to receive CPG–DDS alone or plus 4, 2 or 1 mg/kg of artesunate once daily for 3 days. Assessments took place on Days 0−3 in hospital and follow-up on Days 7 and 14 as out-patients. Efficacy was evaluated in the Day 3 per-protocol (PP) population using mean time to reduce baseline parasitemia by 90% (PC90). A number of secondary outcomes were also included. Appropriate artesunate dose was determined using a pre-defined decision matrix based on primary and secondary outcomes. Treatment emergent adverse events were recorded from clinical assessments and blood parameters. Safety was evaluated in the intent to treat (ITT) population.

Results

In the Day 3 PP population for the adult group (N = 85), mean time to PC90 was 19.1 h in the CPG−DDS group, significantly longer than for the +artesunate 1 mg/kg (12.5 h; treatment difference −6.6 h [95%CI −11.8, −1.5]), 2 mg/kg (10.7 h; −8.4 h [95%CI −13.6, −3.2]) and 4 mg/kg (10.3 h; −8.7 h [95%CI −14.1, −3.2]) groups. For children in the Day 3 PP population (N = 92), mean time to PC90 was 21.1 h in the CPG−DDS group, similar to the +artesunate 1 mg/kg group (17.7 h; −3.3 h [95%CI −8.6, 2.0]), though the +artesunate 2 mg/kg and 4 mg/kg groups had significantly shorter mean times to PC90 versus CPG−DDS; 14.4 h (treatment difference −6.4 h [95%CI −11.7, −1.0]) and 12.8 h (−7.4 h [95%CI −12.9, −1.8]), respectively. An analysis of mean time to PC90 for the Day 14 PP and ITT populations was consistent with the primary analysis. Treatment emergent, drug-related adverse events were experienced in 35.3% (41/116) of adults and 70.1% (75/107) of children; mostly hematological and gastroenterological. The nature and incidence of adverse events was similar between the groups. No dose-related changes in laboratory parameters were observed. Nine serious adverse events due to any cause occurred in five subjects including two cases of hemolysis believed to be associated with drug treatment (one adult, one child). One adult died of anaphylactic shock, not associated with investigational therapy.

Conclusions

CPG–DDS plus artesunate demonstrated advantages over CPG–DDS alone for the primary efficacy endpoint (mean time to PC90) except in children for the 1 mg/kg artesunate dose. Based on a pre-defined decision matrix, the primary endpoint in the child group supported an artesunate dose of 4 mg/kg. Secondary endpoints also supported a 4 mg/kg artesunate dose to take forward into the remainder of the development program.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00519467","term_id":"NCT00519467"}}NCT00519467     

When can refuges mediate the genetic effects of fire regimes? A simulation study of the effects of topography and weather on neutral and adaptive genetic diversity in fire‐prone landscapes

Understanding how landscape heterogeneity mediates the effects of fire on biodiversity is increasingly important under global changes in fire regimes. We used a simulation experiment to investigate how fire regimes interact with topography and weather to shape neutral and selection‐driven genetic diversity under alternative dispersal scenarios, and to explore the conditions under which microrefuges can maintain genetic diversity of populations exposed to recurrent fire. Spatial heterogeneity in simulated fire frequency occurred in topographically complex landscapes, with fire refuges and fire‐prone “hotspots” apparent. Interannual weather variability reduced the effect of topography on fire patterns, with refuges less apparent under high weather variability. Neutral genetic diversity was correlated with long‐term fire frequency under spatially heterogeneous fire regimes, being higher in fire refuges than fire‐prone areas, except under high dispersal or low fire severity (low mortality). This generated different spatial genetic structures in fire‐prone and fire‐refuge components of the landscape, despite similar dispersal. In contrast, genetic diversity was only associated with time since the most recent fire in flat landscapes without predictable refuges and hotspots. Genetic effects of selection driven by fire‐related conditions depended on selection pressure, migration distance and spatial heterogeneity in fire regimes. Allele frequencies at a locus conferring higher fitness under successional environmental conditions followed a pattern of “temporal adaptation” to contemporary conditions under strong selection pressure and high migration. However, selected allele frequencies were correlated with spatial variation in long‐term mean fire frequency (relating to environmental predictability) under weak dispersal, low selection pressure and strong spatial heterogeneity in fire regimes.     

Transport of the Harmful Bloom Alga Aureococcus anophagefferens by Oceangoing Ships and Coastal Boats

It is well established that cyst-forming phytoplankton species are transported in ships' ballast tanks. However, there is increasing evidence that other phytoplankton species which do not encyst are also capable of surviving ballast transit. These species have alternative modes of nutrition (hetero- or mixotrophy) and/or are able to survive long-term darkness. In our studies of no-ballast-on-board vessels arriving in the Great Lakes, we tested for the presence of the harmful algal bloom species Aureococcus anophagefferens (brown tide) in residual (i.e., unpumpable) ballast water using methods based on the PCR. During 2001, the brown tide organism was detected in 7 of 18 ballast water tanks in commercial ships following transit from foreign ports. Furthermore, it was detected after 10 days of ballast tank confinement during a vessel transit in the Great Lakes, a significant result given the large disparity between the salinity tolerance for active growth of Aureococcus (>22 ppt) and the low salinity of the residual ballast water (~2 ppt). We also investigated the potential for smaller, recreational vessels to transport and distribute Aureococcus. During the summer of 2002, 11 trailered boats from the inland bays of Delaware and coastal bays of Maryland were sampled. Brown tide was detected in the bilge water in the bottoms of eight boats, as well as in one live-well sample. Commercial ships and small recreational boats are therefore implicated as potential vectors for long-distance transport and local-scale dispersal of Aureococcus.     

Binding of the C6-zinc cluster protein, AFLR, to the promoters of aflatoxin pathway biosynthesis genes in Aspergillus parasiticus

AFLR is a Zn₂Cys₆-type sequence-specific DNA-binding protein that is thought to be necessary for expression of most of the genes in the aflatoxin pathway gene cluster in Aspergillus parasiticus and A. flavus, and the sterigmatocystin gene cluster in A. nidulans. However, it was not known whether AFLR bound to the promoter regions of each of the genes in the cluster. Recently, A. nidulans AFLR was shown to bind to the motif 5′-TCGN₅CGA-3′. In the present study, we examined the binding of AFLR to promoter regions of 11 genes in the A. parasiticus cluster. Based on electrophoretic mobility shift assays, the genes nor1, pksA, adhA, norA, ver1, omtA, ordA, and, vbs, had at least one 5′-TCGN₅CGA-3′ binding site within 200 bp of the translation start site, and pksA and ver1 had an additional binding site further upstream. Although the promoter region of avnA lacked this motif, AFLR bound weakly to the sequence 5′-TCGCAGCCCGG-3′ at −110 bp. One region in the promoter of the divergently transcribed genes aflR/aflJ bound weakly to AFLR even though it contained a site with at most only 7 bp of the 5′-TCGN₅CGA-3′ motif. This partial site may be recognized by a monomeric form of AFLR. Based on a comparison of 16 possible sites, the preferred binding sequence was 5′-TCGSWNNSCGR-3′.     

Phylogenetic characterization of the epibiotic bacteria associated with the hydrothermal vent polychaete Alvinella pompejana.

Alvinella pompejana is a polychaetous annelid that inhabits active deep-sea hydrothermal vent sites along the East Pacific Rise, where it colonizes the walls of actively venting high-temperature chimneys. An abundant, morphologically diverse epibiotic microflora is associated with the worm's dorsal integument, with a highly integrated filamentous morphotype clearly dominating the microbial biomass. It has been suggested that this bacterial population participates in either the nutrition of the worm or in detoxification of the worm's immediate environment. The primary goal of this study was to phylogenetically characterize selected epibionts through the analysis of 16S rRNA gene sequences. Nucleic acids were extracted from bacteria collected from the dorsal surface of A. pompejana. 16S rRNA genes were amplified with universal bacterial primers by the PCR. These genes were subsequently cloned, and the resulting clone library was screened by restriction fragment length polymorphism analysis to identify distinct clone types. The restriction fragment length polymorphism analysis identified 32 different clone families in the library. Four of these families were clearly dominant, representing more than 65% of the library. Representatives from the four most abundant clone families were chosen for complete 16S rRNA gene sequencing and phylogenetic analysis. These gene sequences were analyzed by a variety of phylogenetic inference methods and found to be related to the newly established epsilon subdivision of the division Proteobacteria. Secondary structural model comparisons and comparisons of established signature base positions in the 16S rRNA confirmed the placement of the Alvinella clones in the epsilon subdivision of the Proteobacteria.