Model-based Analysis of ChIP-Seq (MACS)

We present Model-based Analysis of ChIP-Seq data, MACS, which analyzes data generated by short read sequencers such as Solexa's Genome Analyzer. MACS empirically models the shift size of ChIP-Seq tags, and uses it to improve the spatial resolution of predicted binding sites. MACS also uses a dynamic Poisson distribution to effectively capture local biases in the genome, allowing for more robust predictions. MACS compares favorably to existing ChIP-Seq peak-finding algorithms, and is freely available.     

The EphA3 receptor is expressed in a subset of rhabdomyosarcoma cell lines and suppresses cell adhesion and migration

Elevated expression of the Eph receptor tyrosine kinase EphA3 is associated with lymphocytic leukaemia, but little is known about its expression or function in solid tumours. Out of a panel of cancer cell lines, we found that EphA3 was expressed only on two rhabdomyosarcoma (RMS) cell lines of the embryonal histological subtype and on one of the alveolar RMS subtype, whereas it was not detected on two other cell lines of the alveolar subtype. Other EphA receptors (1-7) were, either not expressed in any, or expressed in all five RMS cell lines. Stimulation of EphA3-expressing TE671 and RD RMS cells with ephrinA5 resulted in loss of adhesion to fibronectin, decreased migration towards the stromal cell-derived growth factor-I (SDF-I), increased EphA3 phosphorylation, and increased Rho GTPase activity. In contrast, ectopic expression of EphA3 in the EphA3 negative CRL2061 cell line resulted in decreased cell adhesion. Finally, suppression of EphA3 expression by siRNA in RD cells results in increased SDF-I-mediated motility. These data indicate that EphA3 expression may define subsets of RMS tumours, and that EphA3 suppresses motility through regulation of Rho GTPases in RMS cells.     

Modular construction of plasmids through ligation-free assembly of vector components with oligonucleotide linkers

We have developed a modular method of plasmid construction that can join multiple DNA components in a single reaction. A nicking enzyme is used to create 5' and 3' overhangs on PCR-generated DNA components. Without the use of ligase or restriction enzymes, components are joined using oligonucleotide linkers that recognize the overhangs. By specifying the sequences of the linkers, desired components can be assembled in any combination and order to generate different plasmid vectors.     

Cerebrospinal Fluid Monoaminergic Metabolites in Wild Papio anubis and P. hamadryas are Concordant with Taxon-specific Behavioral Ontogeny

We used a cross-sectional sample to compare ontogenetic trajectories in the concentrations of monoamine neurotransmitter metabolites in cerebrospinal fluid of wild anubis (Papio anubis, n = 49) and hamadryas (P. hamadryas, n = 54) baboons to test the prediction that they would differ, especially in males, in association with their distinct behavioral ontogenies. Values of all 3 metabolites [3-methoxy-4-hydroxyphenylglycol (MHPG), the norepinephrine metabolite; 5-hydroxyindoleacetic acid (5-HIAA), the serotonin metabolite; and homovanillic acid (HVA), the dopamine metabolite] declined consistently with dentally-calibrated maturation, and few taxon-related differences were apparent among juveniles. Adult females were too few for adequate comparison, but a discriminant function suggested that they might differ by taxon. Adult males of the 2 species differed strikingly from juveniles and from each other. Contrary to our initial hypothesis, adult male anubis had significantly lower HVA and MHPG, and higher 5-HIAA levels, than predicted from the overall, age-related trend, and MHPG continued to decline with age among adults. As young adults, male hamadryas had low 5-HIAA and a high HVA/5-HIAA ratio, while older males [normatively one-male unit (OMU) leaders] showed a reversal in the trend, with 5-HIAA rising and the HVA/5-HIAA ratio tending to fall. We speculate that the results are related to the dispersing and philopatric ontogenies of anubis and hamadryas males, respectively. Adult male anubis, whose fitness depends on building social networks with nonkin, have high relative serotonin activity, commonly associated with greater social circumspection and skill. Young adult male hamadryas, living among agnatic kin and mating opportunistically, exhibit low 5-HIAA levels, generally associated with impulsivity and social irresponsibility. This reverses as a male approaches the age at which he is normatively the leader of a one-male unit (OMU), and his fitness depends on his maintaining stable relationships with other leaders and with females. An erratum to this article can be found at     

A diatom gene regulating nitric-oxide signaling and susceptibility to diatom-derived aldehydes

Diatoms are unicellular phytoplankton accounting for approximately 40% of global marine primary productivity [1], yet the molecular mechanisms underlying their ecological success are largely unexplored. We use a functional-genomics approach in the marine diatom Phaeodactylum tricornutum to characterize a novel protein belonging to the widely conserved YqeH subfamily [2] of GTP-binding proteins thought to play a role in ribosome biogenesis [3], sporulation [4], and nitric oxide (NO) generation [5]. Transgenic diatoms overexpressing this gene, designated PtNOA, displayed higher NO production, reduced growth, impaired photosynthetic efficiency, and a reduced ability to adhere to surfaces. A fused YFP-PtNOA protein was plastid localized, distinguishing it from a mitochondria-localized plant ortholog. PtNOA was upregulated in response to the diatom-derived unsaturated aldehyde 2E,4E/Z-decadienal (DD), a molecule previously shown to regulate intercellular signaling, stress surveillance [6], and defense against grazers [7]. Overexpressing cell lines were hypersensitive to sublethal levels of this aldehyde, manifested by altered expression of superoxide dismutase and metacaspases, key components of stress and death pathways [8, 9]. NOA-like sequences were found in diverse oceanic regions, suggesting that a novel NO-based system operates in diatoms and may be widespread in phytoplankton, providing a biological context for NO in the upper ocean [10].     

Impaired naive and memory B-cell responsiveness to TLR9 stimulation in human immunodeficiency virus infection

Toll-like receptor 9 (TLR9) agonists such as unmethylated bacterial CpG DNAs activate B lymphocytes directly, potentially influencing their function and homeostasis. To assess B-cell responsiveness to TLR9 agonists in human immunodeficiency virus (HIV) disease, we examined the ability of naive and memory B cells to proliferation and to increase surface expression of CD80 in response to CpG oligonucleotides (ODN). CpG ODN induced expression of CD80 similarly in B cells from HIV-infected persons and from healthy controls. In contrast, proliferation responses to CpG ODN were markedly impaired in both naive and memory B-cell subsets from HIV-infected persons. Naive B-cell proliferation defects were related to plasma HIV RNA and, among memory B cells, to the frequencies of CD21-negative cells. Importantly, TLR9 mRNA levels were significantly diminished in freshly prepared naive B cells and especially so in memory B cells from HIV-positive viremic donors, suggesting a possible underlying mechanism for the observed functional impairments. Dose-response studies indicated that optimal induction of CD80 expression was achieved with much lower concentrations of CpG ODN than optimal induction of proliferation. We propose that the relatively low threshold of activation that is required for CD80 induction by CpG ODN might explain the preservation of this response in B cells from HIV-infected persons despite diminished TLR9 expression. Impaired responsiveness to TLR9 agonists may contribute to defects in humoral immunity in HIV infection.     

An effective DNA extraction protocol for brown algae

Successful extraction of total DNA from brown algae, which are generally polysaccharide and polyphenol rich, is often problematic using current methods. Persistent polysaccharide and polyphenolic compounds can hinder further application of modern molecular techniques requisite to molecular‐based evolutionary studies. Our broad and long‐term research goals with fucalean taxa, especially Sargassum, and problems with existing DNA extraction methods were an impetus to develop a reliable DNA extraction method. Initial research established hexadecyltrimethylammonium bromide (CTAB) based total‐DNA methods as the most viable for further empirical development. Several constituents effective at either complexing secondary compounds or creating a reductive extraction environment were increased in concentration or added to the extraction buffer. These seemingly minor changes resulted in the creation of a highly reductive extraction buffer and effective total‐ DNA harvesting technique. We detail these modifications and demonstrate the reliability of the modified protocol with a variety of brown algae and tissue preservation methods. Such DNA is shown to be suitable for a variety of molecular techniques.     

Functional Enhancement of Electrofusion-derived BRIN-BD11 Insulin-secreting Cells After Implantation into Diabetic Mice

Electrofusion-derived BRIN-BD11 cells are glucosesensitive insulin-secreting cells which provide an archetypal bioengineered surrogate β-cell for insulin replacement therapy in diabetes mellitus, 5x10⁶ BRIN-BD11 cells were implanted intraperitoneally into severely hyperglycaemic (>24mmol/l) streptozotocin-induced insulin-treated diabetic athymic nude (nu/nu) mice. The implants reduced hyperglycaemia such that insulin injections were discontinued by 5–16 days (<17mmol/l) and normoglycaemia (<9mmol/l) was achieved by 7–20 days. Implanted cells were removed after 28 days and re-established in culture. After re-culture for 20 days, glucose-stimulated (16.7mmol/l) insulin release was enhanced by 121% (p<0.001) compared to non-implanted cells. Insulin responses to glucagon-like peptide-1 (10⁻⁹mol/l), cholecystokinin-8 (10⁻⁸ mol/l) and L-alanine (10 mmol/l) were increased by 32%, 31% and 68% respectively (p<0.05–0.01). Insulin content of the cells was 148% greater at 20 days after re-culture than before implantation (p<0.001), but basal insulin release (at 5.6 mmol/l glucose) was not changed. After re-culture for 40 days, insulin content declined to 68% of the content before implantation (p<0.01), although basal insulin release was unchanged. However, the insulin secretory responses to glucose, glucagonlike peptide-1, cholecystokinin-8 and L-alanine were decreased after 40 days of re-culture to 65%, 72%, 73% and 42% respectively of the values before implantation (p<0.05–0.01). The functional enhancement of electrofusion-derived surrogate β-cells that were re-cultured for 20 days after implantation and restoration of normoglycaemia indicates that the in vivo environment could greatly assist β-cell engineering approaches to therapy for diabetes.