Simultaneous sequencing of genetic and epigenetic bases in DNA.

DNA comprises molecular information stored in genetic and epigenetic bases, both of which are vital to our understanding of biology. Most DNA sequencing approaches address either genetics or epigenetics and thus capture incomplete information. Methods widely used to detect epigenetic DNA bases fail to capture common C-to-T mutations or distinguish 5-methylcytosine from 5-hydroxymethylcytosine. We present a single base-resolution sequencing methodology that sequences complete genetics and the two most common cytosine modifications in a single workflow. DNA is copied and bases are enzymatically converted. Coupled decoding of bases across the original and copy strand provides a phased digital readout. Methods are demonstrated on human genomic DNA and cell-free DNA from a blood sample of a patient with cancer. The approach is accurate, requires low DNA input and has a simple workflow and analysis pipeline. Simultaneous, phased reading of genetic and epigenetic bases provides a more complete picture of the information stored in genomes and has applications throughout biomedicine.

Effects of substitutions in the CXXC active-site motif of the extracytoplasmic thioredoxin ResA.

The thiol-disulfide oxidoreductase ResA from Bacillus subtilis fulfils a reductive role in cytochrome c maturation. The pK(a) values for the CEPC (one-letter code) active-site cysteine residues of ResA are unusual for thioredoxin-like proteins in that they are both high (>8) and within 0.5 unit of each other. To determine the contribution of the inter-cysteine dipeptide of ResA to its redox and acid-base properties, three variants (CPPC, CEHC and CPHC) were generated representing a stepwise conversion into the active-site sequence of the high-potential DsbA protein from Escherichia coli. The substitutions resulted in large decreases in the pK(a) values of both the active-site cysteine residues: in CPHC (DsbA-type) ResA, DeltapK(a) values of -2.5 were measured for both cysteine residues. Increases in midpoint reduction potentials were also observed, although these were comparatively small: CPHC (DsbA-type) ResA exhibited an increase of +40 mV compared with the wild-type protein. Unfolding studies revealed that, despite the observed differences in the properties of the reduced proteins, changes in stability were largely confined to the oxidized state. High-resolution structures of two of the variants (CEHC and CPHC ResA) in their reduced states were determined and are discussed in terms of the observed changes in properties. Finally, the in vivo functional properties of CEHC ResA are shown to be significantly affected compared with those of the wild-type protein.

The active-site cysteinyls and hydrophobic cavity residues of ResA are important for cytochrome c maturation in Bacillus subtilis.

ResA is an extracytoplasmic membrane-bound thiol-disulfide oxidoreductase required for cytochrome c maturation in Bacillus subtilis. Previous biochemical and structural studies have revealed that the active-site cysteinyls cycle between oxidized and reduced states with a low reduction potential and that, upon reduction, a hydrophobic cavity forms close to the active site. Here we report in vivo studies of ResA-deficient B. subtilis complemented with a series of ResA variants. Using a range of methods to analyze the cellular cytochrome c content, we demonstrated (i) that the N-terminal transmembrane segment of ResA serves principally to anchor the protein to the cytoplasmic membrane but also plays a role in mediating the activity of the protein; (ii) that the active-site cysteines are important for cytochrome c maturation activity; (iii) that Pro141, which forms part of the hydrophobic cavity and which adopts a cis conformation, plays an important role in protein stability; (iv) that Glu80, which lies at the base of the hydrophobic cavity, is important for cytochrome c maturation activity; and, finally, (v) that Pro141 and Glu80 ResA mutant variants promote selective maturation of low levels of one c-type cytochrome, subunit II of the cytochrome c oxidase caa(3), indicating that this apocytochrome is distinct from the other three endogenous c-type cytochromes of B. subtilis.

Childhood adversity, poly-substance use, and disordered eating in adolescent Latinas: mediated and indirect paths in a community sample.

OBJECTIVE: Although many studies have identified associations between childhood maltreatment and later substance use and disordered eating, few have examined causal or explanatory pathways, and whether victim characteristics predict the development of one versus the other of these outcomes remains uninvestigated. Furthermore, relatively little childhood maltreatment research has examined Latino samples. To address these gaps, this study examined among Latina adolescents the effects of sexual abuse, physical/emotional abuse, neglect, disconnection from family, and parental alcohol problems on poly-substance use and disordered eating, and whether five individual characteristics explain or differentiate these outcomes. METHOD: Data from a community sample of 361 Latina adolescents were analyzed using structural equation modeling. RESULTS: Physical/emotional abuse predicted poly-substance use and weight concerns, and these associations were mediated by impaired current attachment. Disconnection from family predicted bulimic behaviors, and this association was mediated by dysphoria. One indirect path also emerged: Disconnection from family predicted low social conformity, and low social conformity predicted poly-substance use. Childhood sexual abuse did not uniquely predict any adverse outcome or individual characteristic examined. CONCLUSIONS: Dysphoria and impaired current attachment appear to play important roles in the development of substance use and disordered eating in Latina adolescents when physical/emotional abuse or disconnection from family predicts these outcomes. Dysphoria and low social conformity may differentiate the development of bulimic behaviors and poly-substance use, respectively, when family disconnection predicts these outcomes.