Statistical inference of in vivo properties of human DNA methyltransferases from double-stranded methylation patterns

DNA methyltransferases establish methylation patterns in cells and transmit these patterns over cell generations, thereby influencing each cell's epigenetic states. Three primary DNA methyltransferases have been identified in mammals: DNMT1, DNMT3A and DNMT3B. Extensive in vitro studies have investigated key properties of these enzymes, namely their substrate specificity and processivity. Here we study these properties in vivo, by applying novel statistical analysis methods to double-stranded DNA methylation patterns collected using hairpin-bisulfite PCR. Our analysis fits a novel Hidden Markov Model (HMM) to the observed data, allowing for potential bisulfite conversion errors, and yields statistical estimates of parameters that quantify enzyme processivity and substrate specificity. We apply this model to methylation patterns established in vivo at three loci in humans: two densely methylated inactive X (Xi)-linked loci (FMR1 and G6PD), and an autosomal locus (LEP), where methylation densities are tissue-specific but moderate. We find strong evidence for a high level of processivity of DNMT1 at FMR1 and G6PD, with the mean association tract length being a few hundred base pairs. Regardless of tissue types, methylation patterns at LEP are dominated by DNMT1 maintenance events, similar to the two Xi-linked loci, but are insufficiently informative regarding processivity to draw any conclusions about processivity at that locus. At all three loci we find that DNMT1 shows a strong preference for adding methyl groups to hemi-methylated CpG sites over unmethylated sites. The data at all three loci also suggest low (possibly 0) association of the de novo methyltransferases, the DNMT3s, and are consequently uninformative about processivity or preference of these enzymes. We also extend our HMM to reanalyze published data on mouse DNMT1 activities in vitro. The results suggest shorter association tracts (and hence weaker processivity), and much longer non-association tracts than human DNMT1 in vivo.     

Detection and quantification of Lyme spirochetes using sensitive and specific molecular beacon probes

Background  

Lyme disease, caused by Borrelia burgdorferi, affects a large number of people in both the USA and Europe. The mouse is a natural host for this spirochete and is widely used as a model system to study Lyme pathogenesis mechanisms. Since disease manifestations often depend upon the spirochete burden in a particular tissue, it is critical to accurately measure the bacterial number in infected tissues. The current methods either lack sensitivity and specificity (SYBR Green), or require independent analysis of samples in parallel to quantitate host and bacterial DNA (TaqMan). We have developed a novel molecular beacon-based convenient multiplex real-time quantitative PCR assay to identify and detect small numbers of B. burgdorferi in infected mouse tissues.     

Antibody targeting of camptothecin-loaded PLGA nanoparticles to tumor cells

Antibody targeting of drug substances can improve the efficacy of the active molecule, improving distribution and concentration of the drug at the site of injury/disease. Encapsulation of drug substances into polymeric nanoparticles can also improve the therapeutic effects of such compounds by protecting the molecule until its action is required. In this current study, we have brought together these two rationales to develop a novel immuno-nanoparticle with improved therapeutic effect against colorectal tumor cells. This nanoparticle comprised a layer of peripheral antibodies (Ab) directed toward the Fas receptor (CD95/Apo-1) covalently attached to poly(lactide-co-glycolide) nanoparticles (NP) loaded with camptothecin. Variations in surface carboxyl density permitted up to 48.5 microg coupled Ab per mg of NP and analysis of nanoparticulate cores showed efficient camptothecin loading. Fluorescence visualization studies confirmed internalization of nanoconstructs into endocytic compartments of HCT116 cells, an effect not evident in NP without superficial Ab. Cytotoxicity studies were then carried out against HCT116 cells. After 72 h, camptothecin solution resulted in an IC 50 of 21.8 ng mL (-1). Ab-directed delivery of NP-encapsulated camptothecin was shown to be considerably more effective with an IC 50 of 0.37 ng mL (-1). Calculation of synergistic ratios for these nanoconstructs demonstrated synergy of pharmacological relevance. Indeed, the results in this paper suggest that the attachment of anti-Fas antibodies to camptothecin-loaded nanoparticles may result in a therapeutic strategy that could have potential in the treatment of tumors expressing death receptors.     

Translation elongation factor eEF1A binds to a novel myosin binding protein-C-like protein

Eukaryotic translation elongation factor 1A (eEF1A) is a guanine-nucleotide binding protein, which transports aminoacylated tRNA to the ribosomal A site during protein synthesis. In a yeast two-hybrid screening of a human skeletal muscle cDNA library, a novel eEF1A binding protein, immunoglobulin-like and fibronectin type III domain containing 1 (IGFN1), was discovered, and its interaction with eEF1A was confirmed in vitro. IGFN1 is specifically expressed in skeletal muscle and presents immunoglobulin I and fibronectin III sets of domains characteristic of sarcomeric proteins. IGFN1 shows sequence and structural homology to myosin binding protein-C fast and slow-type skeletal muscle isoforms. IGFN1 is substantially upregulated during muscle denervation. We propose a model in which this increased expression of IGFN1 serves to down-regulate protein synthesis via interaction with eEF1A during denervation.     

Acetylcholine receptors at the neuromuscular junction: Developmental change in receptor turnover

The turnover of acetylcholine receptors labeled with ¹²⁵I-labeled α-bungarotoxin was measured in the developing posterior latissimus dorsi muscle of the chick. The degradation rates for acetylcholine receptors at the neuromuscular junction and in extrajunctional regions of the muscle cell were determined. One week after hatching, the rates of junctional and extrajunctional receptor degradation are identical ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 30}\text{hr}$). Three weeks weeks after hatching, however, the rate of junctional receptor degradation is considerably slower ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≥ 5}\text{days}$) and different than the rate of extrajunctional receptor degradation ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 30}\text{hr}$). Thus, receptors which are localized at the neuromuscular junction early in embryonic life only become stable several weeks after hatching.     

Structure, function, and motility of vacuoles in filamentous fungi

Current information on the structure and function of motile tubular vacuoles in Pisolithus tinctorius and other fungi is reviewed. The use of fluorochromes to label the vacuole lumen is evaluated and observations on the structure and motility of vacuoles in P. tinctorius are differentiated from possible artifacts. The styryl dyes FM4-64 and MDY-64, used in yeast to demonstrate endocytosis, show little or no labeling of internal membranes in undamaged P. tinctorius cells. This agrees with our data showing that other probes for endocytosis such as Lucifer yellow CH are not taken up by hyphal tip cells. Overall, the observations do not support endocytosis in hyphal tips. It has been suggested that tubular vacuole systems carry out longitudinal transport, and evidence in favor of this hypothesis is evaluated. New data are presented to show that many of the large vacuoles in subapical cells are attached to the plasma membrane and are relatively immobile, while video sequences show movement of fluorochrome in pulses along a series of several large vacuoles, all interconnected via tubules. Tubular vacuoles from thick sections of hyphae processed under anhydrous conditions are shown by X-ray microanalysis to contain relatively high levels of P and K, as seen previously in the larger vacuoles. These results provide further evidence for a role of the tubular vacuoles in longitudinal transport of P. Copyright 1998 Academic Press.     

Patterning of muscle acetylcholine receptor gene expression in the absence of motor innervation

The patterning of skeletal muscle is thought to depend upon signals provided by motor neurons. We show that AChR gene expression and AChR clusters are concentrated in the central region of embryonic skeletal muscle in the absence of innervation. Neurally derived Agrin is dispensable for this early phase of AChR expression, but MuSK, a receptor tyrosine kinase activated by Agrin, is required to establish this AChR prepattern. The zone of AChR expression in muscle lacking motor axons is wider than normal, indicating that neural signals refine this muscle-autonomous prepattern. Neuronal Neuregulin-1, however, is not involved in this refinement process, nor indeed in synapse-specific AChR gene expression. Our results demonstrate that AChR expression is patterned in the absence of innervation, raising the possibility that similarly prepatterned muscle-derived cues restrict axon growth and initiate synapse formation.     

Modulation of the redox potential of the [Fe(SCys)(4)] site in rubredoxin by the orientation of a peptide dipole.

Rubredoxins (Rds) may be separated into two classes based upon the correlation of their reduction potentials with the identity of residue 44; those with Ala44 have reduction potentials that are approximately 50 mV higher than those with Val44. The smaller side chain volume occupied by Ala44 relative to that occupied by Val44 has been proposed to explain the increase in the reduction potential, based upon changes in the Gly43-Ala44 peptide bond orientation and the distance to the [Fe(SCys)(4)] center in the Pyrococcus furiosus (Pf) Rd crystal structure compared to those of Gly43-Val44 in the Clostridium pasteurianum (Cp) Rd crystal structure. As an experimental test of this hypothesis, single-site Val44 <--> Ala44 exchange mutants, [V44A]Cp and [A44V]Pf Rds, have been cloned and expressed. Reduction potentials of these residue 44 variants and pertinent features of the X-ray crystal structure of [V44A]Cp Rd are reported. Relative to those of wild-type Cp and Pf Rds, the V44A mutation in Cp Rd results in an 86 mV increase in midpoint reduction potential and the [A44V] mutation in Pf Rd results in a 95 mV decrease in midpoint reduction potential, respectively. In the crystal structure of [V44A]Cp Rd, the peptide bond between residues 43 and 44 is approximately 0.3 A closer to the Fe center and the hydrogen bond distance between the residue 44 peptide nitrogen and the Cys42 gamma-sulfur decreases by 0.32 A compared to the analogous distances in the wild-type Cp Rd crystal structure. The results described herein support the prediction that the identity of residue 44 alone determines whether a Rd reduction potential of about -50 or 0 mV is observed.     

The effects of ionized and nonionized compression garments on sprint and endurance cycling

ABSTRACT: Burden, RJ and Glaister, M. The effects of ionized and nonionized compression garments on sprint and endurance cycling. J Strength Cond Res 26(10): 2837-2843, 2012-The aim of this study was to examine the effects of ionized and nonionized compression tights on sprint and endurance cycling performance. Using a randomized, blind, crossover design, 10 well-trained male athletes (age: 34.6 ± 6.8 years, height: 1.80 ± 0.05 m, body mass: 82.2 ± 10.4 kg, V[Combining Dot Above]O2max: 50.86 ± 6.81 ml·kg·min) performed 3 sprint trials (30-second sprint at 150% of the power output required to elicit V[Combining Dot Above]O2max [pV[Combining Dot Above]O2max] + 3 minutes recovery at 40% pV[Combining Dot Above]O2max + 30-second Wingate test + 3 minutes recovery at 40% pV[Combining Dot Above]O2max) and 3 endurance trials (30 minutes at 60% pV[Combining Dot Above]O2max + 5 minutes stationary recovery + 10-km time trial) wearing nonionized compression tights, ionized compression tights, or standard running tights (control). There was no significant effect of garment type on key Wingate measures of peak power (grand mean: 1,164 ± 219 W, p = 0.812), mean power (grand mean: 716 ± 68 W, p = 0.800), or fatigue (grand mean: 66.5 ± 6.9%, p = 0.106). There was an effect of garment type on blood lactate in the sprint and the endurance trials (p < 0.05), although post hoc tests only detected a significant difference between the control and the nonionized conditions in the endurance trial (mean difference: 0.55 mmol·L, 95% likely range: 0.1-1.1 mmol·L). Relative to control, oxygen uptake (p = 0.703), heart rate (p = 0.774), and time trial performance (grand mean: 14.77 ± 0.74 minutes, p = 0.790) were unaffected by either type of compression garment during endurance cycling. Despite widespread use in sport, neither ionized nor nonionized compression tights had any significant effect on sprint or endurance cycling performance.     

Methane indicator values for peatlands: a comparison of species and functional groups

Previous studies have shown a correspondence between the abundance of particular plant species and methane flux. Here, we apply multivariate analyses, and weighted averaging, to assess the suitability of vegetation composition as a predictor of methane flux. We developed a functional classification of the vegetation, in terms of a number of plant traits expected to influence methane production and transport, and compared this with a purely taxonomic classification at species level and higher. We applied weighted averaging and indirect and direct ordination approaches to six sites in the United Kingdom, and found good relationships between methane flux and vegetation composition (classified both taxonomically and functionally). Plant species and functional groups also showed meaningful responses to management and experimental treatments. In addition to the United Kingdom, we applied the functional group classification across different geographical regions (Canada and the Netherlands) to assess the generality of the method. Again, the relationship appeared good at the site level, suggesting some general applicability of the functional classification. The method seems to have the potential for incorporation into large‐scale (national) greenhouse gas accounting programmes (in relation to peatland condition/management) using vegetation mapping schemes. The results presented here strongly suggest that robust predictive models can be derived using plant species data (for use in national‐scale studies). For trans‐national‐scale studies, where the taxonomic assemblage of vegetation differs widely between study sites, a functional classification of plant species data provides an appropriate basis for predictive models of methane flux.