Copy Number Variation in Transcriptionally Active Regions of Sexual and Apomictic Boechera Demonstrates Independently Derived Apomictic Lineages

In asexual (apomictic) plants, the absence of meiosis and sex is expected to lead to mutation accumulation. To compare mutation accumulation in the transcribed genomic regions of sexual and apomictic plants, we performed a double-validated analysis of copy number variation (CNV) on 10 biological replicates each of diploid sexual and diploid apomictic Boechera, using a high-density (>700 K) custom microarray. The Boechera genome demonstrated higher levels of depleted CNV, compared with enriched CNV, irrespective of reproductive mode. Genome-wide patterns of CNV revealed four divergent lineages, three of which contain both sexual and apomictic genotypes. Hence genome-wide CNV reflects at least three independent origins (i.e., expression) of apomixis from different sexual genetic backgrounds. CNV distributions for different families of transposable elements were lineage specific, and the enrichment of LINE/L1 and long term repeat/Copia elements in lineage 3 apomicts is consistent with sex and meiosis being mechanisms for purging genomic parasites. We hypothesize that significant overrepresentation of specific gene ontology classes (e.g., pollen–pistil interaction) in apomicts implies that gene enrichment could be an adaptive mechanism for genome stability in diploid apomicts by providing a polyploid-like system for buffering the effects of deleterious mutations.     

Glucose-6-phosphate as a probe for the glucosamine-6-phosphate N-acetyltransferase Michaelis complex

Glucosamine-6-phosphate N-acetyltransferase (GNA1) catalyses the N-acetylation of d-glucosamine-6-phosphate (GlcN-6P), using acetyl-CoA as an acetyl donor. The product GlcNAc-6P is an intermediate in the biosynthesis UDP-GlcNAc. GNA1 is part of the GCN5-related acetyl transferase family (GNATs), which employ a wide range of acceptor substrates. GNA1 has been genetically validated as an antifungal drug target. Detailed knowledge of the Michaelis complex and trajectory towards the transition state would facilitate rational design of inhibitors of GNA1 and other GNAT enzymes. Using the pseudo-substrate glucose-6-phosphate (Glc-6P) as a probe with GNA1 crystals, we have trapped the first GNAT (pseudo-)Michaelis complex, providing direct evidence for the nucleophilic attack of the substrate amine, and giving insight into the protonation of the thiolate leaving group.     

Decreased thiamine and magnesium levels in the potentiation of the neurotoxicity of lead in occupational lead exposure

The relationship between blood lead (Pb) and serum levels of calcium and of neural nutrients such as thiamine and magnesium (Mg) has been determined in a Nigerian population that is occupationally exposed to Pb. Forty-seven male Pb workers were recruited as test subjects and 25 males unexposed to Pb served as controls. The test subjects were classified into three groups, based on severity of exposure to Pb. Blood lead (BPb) and the serum levels of Mg, thiamine, and calcium were determined in both test subjects and controls. The mean blood Pb level was not significantly higher in Pb workers. In contrast, Mg and thiamine levels were significantly decreased (p<0.05; p<0.01, respectively). However, the calcium level was not significantly lower in test subjects than in controls. Also, there was a significant negative correlation between serum thiamine and blood Pb levels (r=-0.50; p<0.01). Furthermore, there was a significant negative correlation between serum calcium and BPb levels (r=-0.41; p<0.01). This study has shown that relatively low BPb levels can enhance Pb absorption and also potentiate Pb neurotoxicity in the presence of decreased serum thiamine and Mg levels.     

Genetic validation of Aspergillus fumigatus phosphoglucomutase as a viable therapeutic target in invasive aspergillosis

Aspergillus fumigatus is the causative agent of invasive aspergillosis, an infection with mortality rates of up to 50%. The glucan-rich cell wall of A. fumigatus is a protective structure that is absent from human cells and is a potential target for antifungal treatments. Glucan is synthesized from the donor uridine diphosphate glucose, with the conversion of glucose-6-phosphate to glucose-1-phosphate by the enzyme phosphoglucomutase (PGM) representing a key step in its biosynthesis. Here, we explore the possibility of selectively targeting A. fumigatus PGM (AfPGM) as an antifungal treatment strategy. Using a promoter replacement strategy, we constructed a conditional pgm mutant and revealed that pgm is required for A. fumigatus growth and cell wall integrity. In addition, using a fragment screen, we identified the thiol-reactive compound isothiazolone fragment of PGM as targeting a cysteine residue not conserved in the human ortholog. Furthermore, through scaffold exploration, we synthesized a para-aryl derivative (ISFP10) and demonstrated that it inhibits AfPGM with an IC₅₀ of 2 μM and exhibits 50-fold selectivity over the human enzyme. Taken together, our data provide genetic validation of PGM as a therapeutic target and suggest new avenues for inhibiting AfPGM using covalent inhibitors that could serve as tools for chemical validation.     

Prevalence of multiple antibiotic resistance among bacterial isolates from selected poultry waste dumps in Southwestern Nigeria

The prevalence of multiple antibiotic resistant bacteria in the waste dumpsite of ten poultry farms in Southwestern Nigeria was investigated. The susceptibility of 195 organisms isolated from the study sites to eight antimicrobial agents were tested using disc diffusion method and the minimum inhibitory concentration of cloxacillin and amoxicillin determined by the agar dilution method. Resistance to the test antibiotics ranged between 0% for gentamicin and 100% for tetracycline and ampicillin among the organisms. Overall, 70 and 90% of the isolates from Okuku, 65.2 and 95.6% from Ogbomoso, and 46.1 and 84.6% from Oyo had MIC above 512 μg/ml for amoxicillin and cloxacillin. Generally, drugs used in high volumes in the studied farms are the least active against the bacterial isolates. Results of this study shows that poultry waste can serve as environmental reservoirs of multiple antibiotic resistant bacteria and their indiscriminate dumping in the environment can expose surrounding human populations to health risks from drug resistant zoonotic pathogens. Part of the data presented in this paper was the subject of a presentation at the World Congress of Pharmacy and Pharmaceutical Sciences/67TH International Congress of the International Pharmaceutical Federation (FIP), 31 August—6 September 2007, Beijing, People’s Republic of China.     

Genetic and structural validation of Aspergillus fumigatus UDP‐N‐acetylglucosamine pyrophosphorylase as an antifungal target

The sugar nucleotide UDP‐N‐acetylglucosamine (UDP‐GlcNAc) is an essential metabolite in both prokaryotes and eukaryotes. In fungi, it is the precursor for the synthesis of chitin, an essential component of the fungal cell wall. U DP‐N‐a cetylglucosamine p yrophosphorylase (UAP) is the final enzyme in eukaryotic UDP‐GlcNAc biosynthesis, converting UTP and N‐acetylglucosamine‐1‐phosphate (GlcNAc‐1P) to UDP‐GlcNAc. As such, this enzyme may provide an attractive target against pathogenic fungi. Here, we demonstrate that the fungal pathogen Aspergillus fumigatus possesses an active UAP (AfUAP1) that shows selectivity for GlcNAc‐1P as the phosphosugar substrate. A conditional mutant, constructed by replacing the native promoter of the A. fumigatus uap1 gene with the Aspergillus nidulans alcA promoter, revealed that uap1 is essential for cell survival and important for cell wall synthesis and morphogenesis. The crystal structure of AfUAP1 was determined and revealed exploitable differences in the active site compared with the human enzyme. Thus AfUAP1 could represent a novel antifungal target and this work will assist the future discovery of small molecule inhibitors against this enzyme.     

Recycling of aquaculture wastewater using charcoal based constructed wetlands

Abstract

The competitive demand for water makes it a scarce resource for agricultural use. This necessitates wastewater reuse for irrigation and any other agricultural purpose, especially in developing countries where treatment of wastewaters is not a priority. The aim of this study was to evaluate the performance of a charcoal-based constructed wetland (CBCW) in treating aquaculture wastewater. Aquaculture wastewater from a Research Fishpond Farm was treated in a CBCW planted with Sacciolepsis africana and Commelina cyannae for 5?days retention time. Raw wastewater and the treated wastewater from the constructed wetland (CW) was sampled and the physicochemical parameters determined. The performance of the CW in treating aquaculture wastewater was conducted. The result showed that the CBCW was capable of removing 50% TSS, 88% COD, 93% BOD_5, and 100% nitrate nitrogen. The pH and DO of the wastewater before treatment and after treatment ranged from 6.68 to 6.91 and 4.13 to 6.30?mg/l, respectively. Thus, CWs have great potential for the treatment of aquaculture wastewater and prevention of environmental degradation through wastewater treatment, thereby solving the problem of water scarcity for agriculture for optimum food production.