Molecular cloning and characterization of an alpha1,3 fucosyltransferase, CEFT-1, from Caenorhabditis elegans

We report on the identification, molecular cloning, and characterization of an alpha1,3 fucosyltransferase (alpha1,3FT) expressed by the nematode, Caenorhabditis elegans . Although C. elegans glycoconjugates do not express the Lewis x antigen Galbeta1-- >4[Fucalpha1-->3]GlcNAcbeta-->R, detergent extracts of adult C.elegans contain an alpha1,3FT that can fucosylate both nonsialylated and sialylated acceptor glycans to generate the Lexand sialyl Lexantigens, as well as the lacdiNAc-containing acceptor GalNAcbeta1-->4GlcNAcbeta1-- >R to generate GalNAcbeta1-->4 [Fucalpha1-->3]GlcNAcbeta1-->R. A search of the C.elegans genome database revealed the existence of a gene with 20-23% overall identity to all five cloned human alpha1,3FTs. The putative cDNA for the C.elegans alpha1,3FT (CEFT-1) was amplified by PCR from a cDNA lambdaZAP library, cloned, and sequenced. COS7 cells transiently transfected with cDNA encoding CEFT-1 express the Lex, but not sLexantigen. The CEFT-1 in the transfected cell extracts can synthesize Lex, but not sialyl Lex, using exogenous acceptors. A second fucosyltransferase activity was detected in extracts of C. elegans that transfers Fuc in alpha1,2 linkage to Gal specifically on type-1 chains. The discovery of alpha-fucosyltransferases in C. elegans opens the possibility of using this well-characterized nematode as a model system for studying the role of fucosylated glycans in the development and survival of C.elegans and possibly other helminths.      

Analysis of repetitive element DNA methylation by MethyLight

Repetitive elements represent a large portion of the human genome and contain much of the CpG methylation found in normal human postnatal somatic tissues. Loss of DNA methylation in these sequences might account for most of the global hypomethylation that characterizes a large percentage of human cancers that have been studied. There is widespread interest in correlating the genomic 5-methylcytosine content with clinical outcome, dietary history, lifestyle, etc. However, a high-throughput, accurate and easily accessible technique that can be applied even to paraffin-embedded tissue DNA is not yet available. Here, we report the development of quantitative MethyLight assays to determine the levels of methylated and unmethylated repeats, namely, Alu and LINE-1 sequences and the centromeric satellite alpha (Satα) and juxtacentromeric satellite 2 (Sat2) DNA sequences. Methylation levels of Alu, Sat2 and LINE-1 repeats were significantly associated with global DNA methylation, as measured by high performance liquid chromatography, and the combined measurements of Alu and Sat2 methylation were highly correlative with global DNA methylation measurements. These MethyLight assays rely only on real-time PCR and provide surrogate markers for global DNA methylation analysis. We also describe a novel design strategy for the development of methylation-independent MethyLight control reactions based on Alu sequences depleted of CpG dinucleotides by evolutionary deamination on one strand. We show that one such Alu-based reaction provides a greatly improved detection of DNA for normalization in MethyLight applications and is less susceptible to normalization errors caused by cancer-associated aneuploidy and copy number changes.     

DNA methylation analysis by digital bisulfite genomic sequencing and digital MethyLight

Alterations in cytosine-5 DNA methylation are frequently observed in most types of human cancer. Although assays utilizing PCR amplification of bisulfite-converted DNA are widely employed to analyze these DNA methylation alterations, they are generally limited in throughput capacity, detection sensitivity, and or resolution. Digital PCR, in which a DNA sample is analyzed in distributive fashion over multiple reaction chambers, allows for enumeration of discrete template DNA molecules, as well as sequestration of non-specific primer annealing templates into negative chambers, thereby increasing the signal-to-noise ratio in positive chambers. Here, we have applied digital PCR technology to bisulfite-converted DNA for single-molecule high-resolution DNA methylation analysis and for increased sensitivity DNA methylation detection. We developed digital bisulfite genomic DNA sequencing to efficiently determine single-basepair DNA methylation patterns on single-molecule DNA templates without an interim cloning step. We also developed digital MethyLight, which surpasses traditional MethyLight in detection sensitivity and quantitative accuracy for low quantities of DNA. Using digital MethyLight, we identified single-molecule, cancer-specific DNA hypermethylation events in the CpG islands of RUNX3, CLDN5 and FOXE1 present in plasma samples from breast cancer patients.     

Ascorbalamic acid: An ascorbigen-like plant constituent yielding in hot acid 3-(2-furoyl)alanine

Ascorbalamic acid (C₉H₁₃NO₈) was isolated from Brassica olerocea L. MS study of various methylated derivatives suggested a structure (Ia) derivable by CC coupling of C-3 of alanine with C-2 of ascorbic acid, followed by lactone → lactam rearrangement. Other derivatives provided supporting evidence, as did study of the reaction of L-3-chloroalanine with L-ascorbic acid in vitro. On treatment with hot 6 M HCl, ascorbalamic acid yielded L-aspartic acid and 3-(2-furoyl)alanine. For identification of the latter, DL-3-(2-furoyl)alanine and its N-2,4-dinitrophenyl and N-acetyl methyl ester derivatives were synthesized. Unlike ascorbigens, ascorbalamic acid is probably present in the living plant. It seemed to be present in all crucifers examined, but to have a capricious distribution in other orders. During permethylation, rearrangements of ester groups were observed, both with ascorbalamic acid and with pyrrolidonecarboxylic acid as a model.     

Population interaction structure and the coexistence of bacterial strains playing ‘rock–paper–scissors’

The simplest example of non‐transitive competition is the game rock–paper–scissors (RPS), which exhibits characteristic cyclic strategy replacement: paper beats rock, which in turn beats scissors, which in turn beats paper. In addition to its familiar use in understanding human decision‐making, rock–paper–scissors is also played in many biological systems. Among other reasons, this is important because it potentially provides a mechanism whereby species‐ or strain coexistence can occur in the face of intense competition. Kerr et al. (2002, Nature 418: 171–174) use complementary experiments and simulations to show that RPS‐playing toxic, resistant, and susceptible E. coli bacteria can coexist when interactions between the strains are spatially explicit. This raises the question of whether limited interactions associated with space are sufficient to allow strain coexistence, or whether space per se is crucial. I approach this question by extending the Kerr et al. model to include different (aspatial) population network structures with the same degree distributions as corresponding spatial lattice models. I show that the coexistence that occurs for some parameter combinations when simulated bacterial strains compete on lattices is absent when they compete on random regular graphs. Further, considering small‐world networks of intermediate ‘quenched randomness’ between lattices and random regular graphs, I show that only small deviations from pure spatial interactions are sufficient to prevent strain coexistence. These results emphasize the explicit role of space, rather than merely limited interactions, as being decisive in allowing the coexistence of toxic, resistant, and susceptible strains in this model system.     

Narratives of Agency and Capability from Two Adolescent Girls in Post-conflict Liberia

Between 1989 and 2003, Liberia experienced a brutal civil war characterized by ethnic killings, sexual violence and the use of child soldiers. Five years after the war ended, half the population of Liberia was under 18 years old. Understanding the needs of these youth is thus essential to the recovery of the nation. This study focuses on the narratives of two female adolescents, selected from 75 in-depth individual interviews with post-conflict Liberian youth conducted in 2012. A narrative analysis approach was employed to examine each interview for multiple layers of meaning. The aim of the study was to elucidate factors that may enable post-conflict youth to reclaim a sense of agency and return to normal developmental tasks. The study explores the ways in which these youth navigate complicated power dynamics in the post-conflict setting and how gender impacts their experiences of their own agency and capability. The dynamics between the participants and the interviewer are explored to further illustrate how power dynamics manifest. These narratives support the involvement of youth in projects that help others as an avenue for promoting agency and resilience for themselves.