Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera

DESIGNING NEW BIOMATERIALS: Modifying a Spider Silk Gene For Efficient Bacterial Expression for Industrial Production

Spider silks have remarkable physical properties due to a combination of strength and elasticity. In addition, spider silks are biocompatible and biodegradable. Our laboratory has shown that the strength of products, such as fibers, produced with other silk proteins correlates with the size of the silk protein. The aciniform silk protein (AcSp1), has been shown to produce the thinnest and strongest fibers of all the natural spider silks. Aciniform silk is composed of a nonrepetitive amino-terminal region, 14 repeats of approximately 200 amino acids each, and a nonrepetitive carboxy-terminal region. We have been able to produce different variants of this gene. All AcSp1 protein variants were able to express in E. coli. The bacteria expression of the AcSp1 protein is low and the protein is expressed not only as a full length polypeptide but also as fragments of the protein. We identified a sequence in the amino-terminal region of the first repeat of the AcSp1 gene that acts as an early termination sequence. Our objective is to modify this region on the gene to study changes in the expression efficiency of AcSp1

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized λ DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2)

Effects of anisotropic interactions on the structure of animal groups

This paper proposes an agent-based model which reproduces different structures of animal groups. The shape and structure of the group is the effect of simple interaction rules among individuals: each animal deploys itself depending on the position of a limited number of close group mates. The proposed model is shown to produce clustered formations, as well as lines and V-like formations. The key factors which trigger the onset of different patterns are argued to be the relative strength of attraction and repulsion forces and, most important, the anisotropy in their application.Comment: 22 pages, 9 figures. Submitted. v1-v4: revised presentation; extended simulations; included technical results. v5: added a few clarification

Real-time single-molecule observation of rolling-circle DNA replication

We present a simple technique for visualizing replication of individual DNA molecules in real time. By attaching a rolling-circle substrate to a TIRF microscope-mounted flow chamber, we are able to monitor the progression of single-DNA synthesis events and accurately measure rates and processivities of single T7 and Escherichia coli replisomes as they replicate DNA. This method allows for rapid and precise characterization of the kinetics of DNA synthesis and the effects of replication inhibitors

The role of multiple marks in epigenetic silencing and the emergence of a stable bivalent chromatin state

We introduce and analyze a minimal model of epigenetic silencing in budding yeast, built upon known biomolecular interactions in the system. Doing so, we identify the epigenetic marks essential for the bistability of epigenetic states. The model explicitly incorporates two key chromatin marks, namely H4K16 acetylation and H3K79 methylation, and explores whether the presence of multiple marks lead to a qualitatively different systems behavior. We find that having both modifications is important for the robustness of epigenetic silencing. Besides the silenced and transcriptionally active fate of chromatin, our model leads to a novel state with bivalent (i.e., both active and silencing) marks under certain perturbations (knock-out mutations, inhibition or enhancement of enzymatic activity). The bivalent state appears under several perturbations and is shown to result in patchy silencing. We also show that the titration effect, owing to a limited supply of silencing proteins, can result in counter-intuitive responses. The design principles of the silencing system is systematically investigated and disparate experimental observations are assessed within a single theoretical framework. Specifically, we discuss the behavior of Sir protein recruitment, spreading and stability of silenced regions in commonly-studied mutants (e.g., sas2, dot1) illuminating the controversial role of Dot1 in the systems biology of yeast silencing.Comment: Supplementary Material, 14 page

Health and survival of young children in southern Tanzania

With a view to developing health systems strategies to improve reach to high-risk groups, we present information on health and survival from household and health facility perspectives in five districts of southern Tanzania. We documented availability of health workers, vaccines, drugs, supplies and services essential for child health through a survey of all health facilities in the area. We did a representative cluster sample survey of 21,600 households using a modular questionnaire including household assets, birth histories, and antenatal care in currently pregnant women. In a subsample of households we asked about health of all children under two years, including breastfeeding, mosquito net use, vaccination, vitamin A, and care-seeking for recent illness, and measured haemoglobin and malaria parasitaemia. In the health facility survey, a prescriber or nurse was present on the day of the survey in about 40% of 114 dispensaries. Less than half of health facilities had all seven 'essential oral treatments', and water was available in only 22%. In the household survey, antenatal attendance (88%) and DPT-HepB3 vaccine coverage in children (81%) were high. Neonatal and infant mortality were 43.2 and 76.4 per 1000 live births respectively. Infant mortality was 40% higher for teenage mothers than older women (RR 1.4, 95% confidence interval (CI) 1.1 - 1.7), and 20% higher for mothers with no formal education than those who had been to school (RR 1.2, CI 1.0 - 1.4). The benefits of education on survival were apparently restricted to post-neonatal infants. There was no evidence of inequality in infant mortality by socio-economic status. Vaccine coverage, net use, anaemia and parasitaemia were inequitable: the least poor had a consistent advantage over children from the poorest families. Infant mortality was higher in families living over 5 km from their nearest health facility compared to those living closer (RR 1.25, CI 1.0 - 1.5): 75% of households live within this distance. Relatively short distances to health facilities, high antenatal and vaccine coverage show that peripheral health facilities have huge potential to make a difference to health and survival at household level in rural Tanzania, even with current human resources

A high-throughput de novo sequencing approach for shotgun proteomics using high-resolution tandem mass spectrometry

<p>Abstract</p> <p>Background</p> <p>High-resolution tandem mass spectra can now be readily acquired with hybrid instruments, such as LTQ-Orbitrap and LTQ-FT, in high-throughput shotgun proteomics workflows. The improved spectral quality enables more accurate <it>de novo </it>sequencing for identification of post-translational modifications and amino acid polymorphisms.</p> <p>Results</p> <p>In this study, a new <it>de novo </it>sequencing algorithm, called Vonode, has been developed specifically for analysis of such high-resolution tandem mass spectra. To fully exploit the high mass accuracy of these spectra, a unique scoring system is proposed to evaluate sequence tags based primarily on mass accuracy information of fragment ions. Consensus sequence tags were inferred for 11,422 spectra with an average peptide length of 5.5 residues from a total of 40,297 input spectra acquired in a 24-hour proteomics measurement of <it>Rhodopseudomonas palustris</it>. The accuracy of inferred consensus sequence tags was 84%. According to our comparison, the performance of Vonode was shown to be superior to the PepNovo v2.0 algorithm, in terms of the number of <it>de novo </it>sequenced spectra and the sequencing accuracy.</p> <p>Conclusions</p> <p>Here, we improved <it>de novo </it>sequencing performance by developing a new algorithm specifically for high-resolution tandem mass spectral data. The Vonode algorithm is freely available for download at <url>http://compbio.ornl.gov/Vonode</url>.</p

Differential impact of impaired fasting glucose versus impaired glucose tolerance on cardiometabolic risk factors in multi-ethnic overweight/obese children

We aimed to investigate the prevalence of impaired fasting glucose (IFG) and impaired glucose tolerance (IGT), and their associations with cardiometabolic risk factors, according to ethnicity in a large obese paediatric cohort. A 75-g oral glucose tolerance test was performed in 1,007 overweight/obese Dutch children of multi-ethnic origin, referred to the obesity outpatient clinics of two Dutch hospitals in Amsterdam (mean age, 11.4 ± 3.2 years; 50.7% boys). Anthropometric parameters and blood samples were collected, and cardiometabolic risk factors were assessed. The cohort consisted of Dutch native (26.0%), Turkish (23.7%), Moroccan (18.8%) and children of ‘other’ (31.5%) ethnicity. The prevalence of IFG was significantly higher in Moroccan and Turkish children as compared to Dutch native children (25.4% and 19.7% vs. 11.8%, respectively, P < 0.05). IGT was most frequently present in Turkish and Dutch native children, relative to Moroccan children (6.3% and 5.3% vs. 1.6%, P < 0.05). Besides pubertal status and ethnicity, components of ‘metabolic syndrome’ (MetS) which were associated with IGT, independent of hyperinsulinaemia, were hypertension [odds ratio (OR), 2.3; 95% CI, 1.1–4.9] while a trend was seen for high triglycerides (OR, 2.0; 95% CI, 0.9–4.3). When analyzing components of MetS which were associated with IFG, only low high-density lipoprotein cholesterol was significantly associated (OR, 1.7; 95% CI, 1.2–2.5) independent of hyperinsulinaemia. In conclusion, in a Dutch multi-ethnic cohort of overweight/obese children, a high prevalence of IFG was found against a low prevalence of IGT, which differed in their associations with cardiometabolic risk factors