Quantification of class 1 integron abundance in natural environments using real-time quantitative PCR

Integrons are bacterial genetic elements capable of capturing and expressing potentially adaptive genetic material. Class 1 integrons constitute the most intensely studied group of these elements to date, mainly due to their well-established role in the acquisition and dissemination of antibiotic resistance genes in clinical environments. However, virtually nothing is known about the distribution or abundance of class 1 integrons outside of the clinical context. Here we develop a SYBR Green-based real-time quantitative PCR assay capable of quantifying the abundance of class 1 integrons in environmental samples. It was shown that the abundance of the intI1 gene in creek sediment correlates with ecological condition, implying that class 1 integrons provide selective advantages relevant to environmental pressures other than the use of antibiotics. By comparing the quantities of intI1 and 16S rRNA gene in each sample, it was demonstrated that approximately 2.7% of cells potentially harbour a class 1 integron. These findings suggest that class 1 integrons are widespread in natural environments removed from clinical settings and occur in a broader range of host organisms than had previously been assumed on the basis of culture-dependent estimates.     

Investigation of apoptosis in cultured cells infected with equine herpesvirus 1

Many viruses alter different stages of apoptosis of infected cells as a strategy for successful infection. Few studies have addressed mechanisms of equine herpesvirus 1 (EHV-1) strain-induced cell death. We investigated the effect of an abortigenic strain (AR8 strain) on heterologous Madin–Darby bovine kidney cells and homologous equine dermis (ED) cells cell lines. We compared morphologic and biochemical features of early and late apoptosis at different postinfection times. We investigated translocation of phosphatidylserine to the cell surface, nuclear fragmentation and changes in the cytoskeleton using flow cytometry and annexin V/propidium iodide staining, DNA laddering, terminal deoxynucleotidyl transferase UTP nick-end labeling assay and immunofluorescence staining of cytokeratin 18 cleavage. AR8 EVH-1 strain interfered with apoptosis in both cell lines, particularly during the middle stage of the replication cycle; this was more evident in ED cells. Although this antiapoptotic effect has been reported for other alpha herpesviruses, our findings may help elucidate how EHV-1 improves its infectivity during its cycle.     

Gene cassette PCR: sequence-independent recovery of entire genes from environmental DNA.

The vast majority of bacteria in the environment have yet to be cultured. Consequently, a major proportion of both genetic diversity within known gene families and an unknown number of novel gene families reside in these uncultured organisms. Isolation of these genes is limited by lack of sequence information. Where such sequence data exist, PCR directed at conserved sequence motifs recovers only partial genes. Here we outline a strategy for recovering complete open reading frames from environmental DNA samples. PCR assays were designed to target the 59-base element family of recombination sites that flank gene cassettes associated with integrons. Using such assays, diverse gene cassettes could be amplified from the vast majority of environmental DNA samples tested. These gene cassettes contained complete open reading frames, the majority of which were associated with ribosome binding sites. Novel genes with clear homologies to phosphotransferase, DNA glycosylase, methyl transferase, and thiotransferase genes were identified. However, the majority of amplified gene cassettes contained open reading frames with no identifiable homologues in databases. Accumulation analysis of the gene cassettes amplified from soil samples showed no signs of saturation, and soil samples taken at 1-m intervals along transects demonstrated different amplification profiles. Taken together, the genetic novelty, steep accumulation curves, and spatial heterogeneity of genes recovered show that this method taps into a vast pool of unexploited genetic diversity. The success of this approach indicates that mobile gene cassettes and, by inference, integrons are widespread in natural environments and are likely to contribute significantly to bacterial diversity.     

Are Ethnic and Gender Specific Equations Needed to Derive Fat Free Mass from Bioelectrical Impedance in Children of South Asian,Black African-Caribbean and White European Origin? Results of the Assessment of Body Composition in Children Study

Background

Bioelectrical impedance analysis (BIA) is a potentially valuable method for assessing lean mass and body fat levels in children from different ethnic groups. We examined the need for ethnic- and gender-specific equations for estimating fat free mass (FFM) from BIA in children from different ethnic groups and examined their effects on the assessment of ethnic differences in body fat.

Methods

Cross-sectional study of children aged 8–10 years in London Primary schools including 325 South Asians, 250 black African-Caribbeans and 289 white Europeans with measurements of height, weight and arm-leg impedance (Z; Bodystat 1500). Total body water was estimated from deuterium dilution and converted to FFM. Multilevel models were used to derive three types of equation {A: FFM = linear combination(height+weight+Z); B: FFM = linear combination(height²/Z); C: FFM = linear combination(height²/Z+weight)}.

Results

Ethnicity and gender were important predictors of FFM and improved model fit in all equations. The models of best fit were ethnicity and gender specific versions of equation A, followed by equation C; these provided accurate assessments of ethnic differences in FFM and FM. In contrast, the use of generic equations led to underestimation of both the negative South Asian-white European FFM difference and the positive black African-Caribbean-white European FFM difference (by 0.53 kg and by 0.73 kg respectively for equation A). The use of generic equations underestimated the positive South Asian-white European difference in fat mass (FM) and overestimated the positive black African-Caribbean-white European difference in FM (by 4.7% and 10.1% respectively for equation A). Consistent results were observed when the equations were applied to a large external data set.

Conclusions

Ethnic- and gender-specific equations for predicting FFM from BIA provide better estimates of ethnic differences in FFM and FM in children, while generic equations can misrepresent these ethnic differences.     

Contrasting patterns of population structure in commercially fished sawsharks from southern Australian waters

Reviews in Fish Biology and Fisheries - In the waters of southeast Australia, two species of sawshark—the common (Pristiophorus cirratus) and southern (Pristiophorus nudipinnis)...     

High diversity and rapid spatial turnover of integron gene cassettes in soil

Integrons are genetic elements that promote rapid adaptation in bacteria by capturing exogenous, mobile gene cassettes. Recently, a subset of gene cassettes has facilitated the global spread of antibiotic resistance. However, outside clinical settings, very little is known about their diversity and spatial ecology. To address this question, we sequenced integron gene cassettes from soils sampled across Australia and Antarctica. We recovered 44 970 open reading frames that encoded 27 215 unique proteins, representing an order of magnitude more cassettes than previous sequencing efforts. We found that cassettes have extremely high local richness, significantly greater than previously predicted, with estimates ranging from 4000 to 18 000 unique cassettes per 0.3 g of soil. We show that cassettes have a heterogeneous distribution across space, and that they exhibit rapid turnover with distance. Similarity between samples drops to between 0.1% and 10% at distances of as little as 100 m. Together, these data provide key insights into the ecology and size of the gene cassette metagenome.     

Phylogeny and form in fishes: Genetic and morphometric characteristics of dragonets (Foetorepus sp.) do not align

Measures of biodiversity are often hindered by a lack of methodological practices that distinguish cryptic or morphologically similar cohabiting species. This is particularly difficult for marine fishes where direct observations of the ecology and demography of populations are difficult. Dragonets (Foetorepus c.f. calauropomus) were collected as bycatch from research trawls deployed in waters off north-eastern Tasmania, Australia. Morphometric and genetic analyses were conducted on the 43 specimens recovered. Sequence analysis of two mitochondrial loci distinguished three genetic clusters, each having levels of dissimilarity consistent with species-level distinctions between other members of the Callionymidae. While clear morphological distinctions were observed between male and female fish, limited morphometric analyses could not differentiate between members of the three genetic groups. This finding highlights questions about the ability of genetically distinct but morphologically similar groups to occupy the same ecological niche, and points to additional and undescribed hidden biodiversity amongst cryptic species of fish.