Abundance of Novel and Diverse tfdA-Like Genes,Encoding Putative Phenoxyalkanoic Acid Herbicide-Degrading Dioxygenases,in Soil

Phenoxyalkanoic acid (PAA) herbicides are widely used in agriculture. Biotic degradation of such herbicides occurs in soils and is initiated by α-ketoglutarate- and Fe²⁺-dependent dioxygenases encoded by tfdA-like genes (i.e., tfdA and tfdAα). Novel primers and quantitative kinetic PCR (qPCR) assays were developed to analyze the diversity and abundance of tfdA-like genes in soil. Five primer sets targeting tfdA-like genes were designed and evaluated. Primer sets 3 to 5 specifically amplified tfdA-like genes from soil, and a total of 437 sequences were retrieved. Coverages of gene libraries were 62 to 100%, up to 122 genotypes were detected, and up to 389 genotypes were predicted to occur in the gene libraries as indicated by the richness estimator Chao1. Phylogenetic analysis of in silico-translated tfdA-like genes indicated that soil tfdA-like genes were related to those of group 2 and 3 Bradyrhizobium spp., Sphingomonas spp., and uncultured soil bacteria. Soil-derived tfdA-like genes were assigned to 11 clusters, 4 of which were composed of novel sequences from this study, indicating that soil harbors novel and diverse tfdA-like genes. Correlation analysis of 16S rRNA and tfdA-like gene similarity indicated that any two bacteria with D > 20% of group 2 tfdA-like gene-derived protein sequences belong to different species. Thus, data indicate that the soil analyzed harbors at least 48 novel bacterial species containing group 2 tfdA-like genes. Novel qPCR assays were established to quantify such new tfdA-like genes. Copy numbers of tfdA-like genes were 1.0 × 10⁶ to 65 × 10⁶ per gram (dry weight) soil in four different soils, indicating that hitherto-unknown, diverse tfdA-like genes are abundant in soils.Phenoxyalkanoic acid (PAA) herbicides such as MCPA (4-chloro-2-methyl-phenoxyacetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid) are widely used to control broad-leaf weeds in agricultural as well as nonagricultural areas (19, 77). Degradation occurs primarily under oxic conditions in soil, and microorganisms play a key role in the degradation of such herbicides in soil (62, 64). Although relatively rapidly degraded in soil (32, 45), both MCPA and 2,4-D are potential groundwater contaminants (10, 56, 70), accentuating the importance of bacterial PAA herbicide-degrading bacteria in soils (e.g., references 3, 5, 6, 20, 41, 59, and 78).Degradation can occur cometabolically or be associated with energy conservation (15, 54). The first step in the degradation of 2,4-D and MCPA is initiated by the product of cadAB or tfdA-like genes (29, 30, 35, 67), which constitutes an α-ketoglutarate (α-KG)- and Fe²⁺-dependent dioxygenase. TfdA removes the acetate side chain of 2,4-D and MCPA to produce 2,4-dichlorophenol and 4-chloro-2-methylphenol, respectively, and glyoxylate while oxidizing α-ketoglutarate to CO₂ and succinate (16, 17).Organisms capable of PAA herbicide degradation are phylogenetically diverse and belong to the Alpha-, Beta-, and Gammproteobacteria and the Bacteroidetes/Chlorobi group (e.g., references 2, 14, 29-34, 39, 60, 68, and 71). These bacteria harbor tfdA-like genes (i.e., tfdA or tfdAα) and are categorized into three groups on an evolutionary and physiological basis (34). The first group consists of beta- and gammaproteobacteria and can be further divided into three distinct classes based on their tfdA genes (30, 46). Class I tfdA genes are closely related to those of Cupriavidus necator JMP134 (formerly Ralstonia eutropha). Class II tfdA genes consist of those of Burkholderia sp. strain RASC and a few strains that are 76% identical to class I tfdA genes. Class III tfdA genes are 77% identical to class I and 80% identical to class II tfdA genes and linked to MCPA degradation in soil (3). The second group consists of alphaproteobacteria, which are closely related to Bradyrhizobium spp. with tfdAα genes having 60% identity to tfdA of group 1 (18, 29, 34). The third group also harbors the tfdAα genes and consists of Sphingomonas spp. within the alphaproteobacteria (30).Diverse PAA herbicide degraders of all three groups were identified in soil by cultivation-dependent studies (32, 34, 41, 78). Besides CadAB, TfdA and certain TfdAα proteins catalyze the conversion of PAA herbicides (29, 30, 35). All groups of tfdA-like genes are potentially linked to the degradation of PAA herbicides, although alternative primary functions of group 2 and 3 TfdAs have been proposed (30, 35). However, recent cultivation-independent studies focused on 16S rRNA genes or solely on group 1 tfdA sequences in soil (e.g., references 3-5, 13, and 41). Whether group 2 and 3 tfdA-like genes are also quantitatively linked to the degradation of PAA herbicides in soils is unknown. Thus, tools to target a broad range of tfdA-like genes are needed to resolve such an issue. Primers used to assess the diversity of tfdA-like sequences used in previous studies were based on the alignment of approximately 50% or less of available sequences to date (3, 20, 29, 32, 39, 47, 58, 73). Primers specifically targeting all major groups of tfdA-like genes to assess and quantify a broad diversity of potential PAA degraders in soil are unavailable. Thus, the objectives of this study were (i) to develop primers specific for all three groups of tfdA-like genes, (ii) to establish quantitative kinetic PCR (qPCR) assays based on such primers for different soil samples, and (iii) to assess the diversity and abundance of tfdA-like genes in soil.     

Synthesis and in vitro evaluation of a novel iodinated resiniferatoxin derivative that is an agonist at the human vanilloid VR1 receptor

Using a 'directed' iodination procedure, novel iodo-resiniferatoxin congeners were synthesized from 4-acetoxy-3-methoxyphenylacetic acid and resiniferinol- 9,13,14-ortho-phenylacetate (ROPA). The 2-iodo-4-hydroxy-5-methoxyphenylacetic acid ester of resiniferinol 5 displayed high affinity binding (K(i)=0.71 nM) for the human vanilloid VR1 receptor and functioned as a partial agonist.     

Association of APOE genotype with carotid atherosclerosis in men and women: the Framingham Heart Study

The aim of this study was to determine the association between APOE genotype and carotid atherosclerosis, defined as intimal-medial thickness (IMT) and stenosis, and to assess if other cardiovascular risk factors modify this association. A total of 1,315 men and 1,408 women from the Framingham Offspring Study underwent carotid ultrasound during examination cycle 6 and had complete data on APOE genotype. Three APOE genotype groups were defined: APOE2 (including E2/E2, E3/E2 genotypes), APOE3 (E3/E3), and APOE4 (including E4/E3, E4/E4 genotypes). Carotid IMT and the presence of carotid stenosis > 25% were determined by ultrasonography. In women, the APOE2 group was associated with lower carotid IMT (0.67 vs. 0.73 mm) and lower prevalence of stenosis (odds ratio = 0.49; 95% confidence interval = 0.30-0.81) compared with the APOE3 group. In men, APOE genotype was not associated with carotid IMT or stenosis in the whole group; however, diabetes modified the association between APOE genotype and carotid IMT (P for interaction = 0.044). Among men with diabetes, the APOE4 group was associated with a higher internal carotid artery IMT (1.22 mm) than the APOE3 group (0.90 mm) or the APOE2 group (0.84 mm). The E2 allele was associated with lower carotid atherosclerosis in women, and the E4 allele was associated with higher internal carotid IMT in diabetic men.     

A genome-wide screen identifies the evolutionarily conserved KEOPS complex as a telomere regulator

Telomere capping is the essential function of telomeres. To identify new genes involved in telomere capping, we carried out a genome-wide screen in Saccharomyces cerevisiae for suppressors of cdc13-1, an allele of the telomere-capping protein Cdc13. We report the identification of five novel suppressors, including the previously uncharacterized gene YML036W, which we name CGI121. Cgi121 is part of a conserved protein complex -- the KEOPS complex -- containing the protein kinase Bud32, the putative peptidase Kae1, and the uncharacterized protein Gon7. Deletion of CGI121 suppresses cdc13-1 via the dramatic reduction in ssDNA levels that accumulate in cdc13-1 cgi121 mutants. Deletion of BUD32 or other KEOPS components leads to short telomeres and a failure to add telomeres de novo to DNA double-strand breaks. Our results therefore indicate that the KEOPS complex promotes both telomere uncapping and telomere elongation.     

A Faithful Record of Stressful Life Events Recorded in the Dental Developmental Record of a Juvenile Gorilla

The pattern and rate of dental development are critical components of the life history of primates. Much recent research has focused on dental development in chimpanzees and other hominoids, but comparatively little is known about dental development in Gorilla. To date, dental chronologies for Gorilla are based on a sample of 1 and information about variations in the time and timing of crown initiation and completion is lacking. We provide data on dental development in 1 captive, juvenile, female, western lowland Gorilla gorilla gorilla of known age, sex, life events, and date of death (carefully documented as part of zoo records) that experienced various physical insults during her first year of life. The perfect natural experiment allowed us to test the association of the timing of accentuated stress lines in teeth with significant physiological and psychological events during ontogeny of this juvenile gorilla. We analyzed histological sections from 14 permanent teeth (maxillary and mandibular I1-M2) and assessed crown initiation (CI) and crown formation times (CFT) using short- and long-period incremental lines in both enamel and dentine; they are advanced for all teeth compared to previously published chronology. The data suggest a relatively accelerated pace of dental development in gorillas compared to chimpanzees and fit an emerging pattern of an accelerated life history schedule in gorillas. Data on the timing of major accentuated lines in the developing dentition are tightly associated with exact dates of surgical procedures and follow-up hospital visits as recorded on zoo medical records. Our data highlight the importance of captive individuals with well-documented medical records for studying life history.

Polycell-gel assay of water for spores of Saprolegniaceae (fungi), especially those of the Saprolegnia pathogen of fish

Large aliquot water samples (30 ml) were enriched with nutrients, then gelled in the cold with Polycell (wallpapering paste) and incubated to derive Saprolegniaceae colonies and hence deduced spore counts, from water collections of about 1 litre. The Saprolegnia pathogen of fish was a component of the total recoveries; it was recognised on the basis of its secondary zoospore cyst ornamentation. Windermere water entering The Ferry House fish hatchery gave spore assays of 2 to 28 1^–1 for the pathogen, a significant component of total Saprolegnia, which was 6–73 spores per litre.