Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis

In all organisms, the protein machinery responsible for the replication of DNA, the replisome, is faced with a directionality problem. The antiparallel nature of duplex DNA permits the leading-strand polymerase to advance in a continuous fashion, but forces the lagging-strand polymerase to synthesize in the opposite direction. By extending RNA primers, the lagging-strand polymerase restarts at short intervals and produces Okazaki fragments. At least in prokaryotic systems, this directionality problem is solved by the formation of a loop in the lagging strand of the replication fork to reorient the lagging-strand DNA polymerase so that it advances in parallel with the leading-strand polymerase. The replication loop grows and shrinks during each cycle of Okazaki fragment synthesis. Here we use single-molecule techniques to visualize, in real time, the formation and release of replication loops by individual replisomes of bacteriophage T7 supporting coordinated DNA replication. Analysis of the distributions of loop sizes and lag times between loops reveals that initiation of primer synthesis and the completion of an Okazaki fragment each serve as a trigger for loop release. The presence of two triggers may represent a fail-safe mechanism ensuring the timely reset of the replisome after the synthesis of every Okazaki fragment.

Coccidioidomycosis Incidence in Arizona Predicted by Seasonal Precipitation

The environmental mechanisms that determine the inter-annual and seasonal variability in incidence of coccidioidomycosis are unclear. In this study, we use Arizona coccidioidomycosis case data for 1995–2006 to generate a timeseries of monthly estimates of exposure rates in Maricopa County, AZ and Pima County, AZ. We reveal a seasonal autocorrelation structure for exposure rates in both Maricopa County and Pima County which indicates that exposure rates are strongly related from the fall to the spring. An abrupt end to this autocorrelation relationship occurs near the the onset of the summer precipitation season and increasing exposure rates related to the subsequent season. The identification of the autocorrelation structure enabled us to construct a “primary” exposure season that spans August-March and a “secondary” season that spans April–June which are then used in subsequent analyses. We show that October–December precipitation is positively associated with rates of exposure for the primary exposure season in both Maricopa County (R = 0.72, p = 0.012) and Pima County (R = 0.69, p = 0.019). In addition, exposure rates during the primary exposure seasons are negatively associated with concurrent precipitation in Maricopa (R = −0.79, p = 0.004) and Pima (R = −0.64, p = 0.019), possibly due to reduced spore dispersion. These associations enabled the generation of models to estimate exposure rates for the primary exposure season. The models explain 69% (p = 0.009) and 54% (p = 0.045) of the variance in the study period for Maricopa and Pima counties, respectively. We did not find any significant predictors for exposure rates during the secondary season. This study builds on previous studies examining the causes of temporal fluctuations in coccidioidomycosis, and corroborates the “grow and blow” hypothesis

Solid Friction from stick-slip to pinning and aging

We review the present state of understanding of solid friction at low velocities and for systems with negligibly small wear effects. We first analyze in detail the behavior of friction at interfaces between wacroscopic hard rough solids, whose main dynamical features are well described by the Rice-Ruina rate and state dependent constitutive law. We show that it results from two combined effects : (i) the threshold rheology of nanometer-thick junctions jammed under confinement into a soft glassy structure (ii) geometric aging, i.e. slow growth of the real arrea of contact via asperity creep interrupted by sliding. Closer analysis leads to identifying a second aging-rejuvenation process, at work within the junctions themselves. We compare the effects of structural aging at such multicontact, very highly confined, interfaces with those met under different confinement levels, namely boundary lubricated contacts and extended adhesive interfaces involving soft materials (hydrogels, elastomers). This leads us to propose a classification of frictional junctions in terms of the relative importance of jamming and adsoprtion-induced metastability.Comment: 28 page

Synthetic biology open language visual (SBOL Visual) version 2.3

People who are engineering biological organisms often find it useful to communicate in diagrams, both about the structure of the nucleic acid sequences that they are engineering and about the functional relationships between sequence features and other molecular species. Some typical practices and conventions have begun to emerge for such diagrams. The Synthetic Biology Open Language Visual (SBOL Visual) has been developed as a standard for organizing and systematizing such conventions in order to produce a coherent language for expressing the structure and function of genetic designs. This document details version 2.3 of SBOL Visual, which builds on the prior SBOL Visual 2.2 in several ways. First, the specification now includes higher-level "interactions with interactions," such as an inducer molecule stimulating a repression interaction. Second, binding with a nucleic acid backbone can be shown by overlapping glyphs, as with other molecular complexes. Finally, a new "unspecified interaction" glyph is added for visualizing interactions whose nature is unknown, the "insulator" glyph is deprecated in favor of a new "inert DNA spacer" glyph, and the polypeptide region glyph is recommended for showing 2A sequences

Progestogens to prevent preterm birth in twin pregnancies: an individual participant data meta-analysis of randomized trials

<p>Abstract</p> <p>Background</p> <p>Preterm birth is the principal factor contributing to adverse outcomes in multiple pregnancies. Randomized controlled trials of progestogens to prevent preterm birth in twin pregnancies have shown no clear benefits. However, individual studies have not had sufficient power to evaluate potential benefits in women at particular high risk of early delivery (for example, women with a previous preterm birth or short cervix) or to determine adverse effects for rare outcomes such as intrauterine death.</p> <p>Methods/design</p> <p>We propose an individual participant data meta-analysis of high quality randomized, double-blind, placebo-controlled trials of progestogen treatment in women with a twin pregnancy. The primary outcome will be adverse perinatal outcome (a composite measure of perinatal mortality and significant neonatal morbidity). Missing data will be imputed within each original study, before data of the individual studies are pooled. The effects of 17-hydroxyprogesterone caproate or vaginal progesterone treatment in women with twin pregnancies will be estimated by means of a random effects log-binomial model. Analyses will be adjusted for variables used in stratified randomization as appropriate. Pre-specified subgroup analysis will be performed to explore the effect of progestogen treatment in high-risk groups.</p> <p>Discussion</p> <p>Combining individual patient data from different randomized trials has potential to provide valuable, clinically useful information regarding the benefits and potential harms of progestogens in women with twin pregnancy overall and in relevant subgroups.</p

Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology

Modular and orthogonal genetic logic gates are essential for building robust biologically based digital devices to customize cell signalling in synthetic biology. Here we constructed an orthogonal AND gate in Escherichia coli using a novel hetero-regulation module from Pseudomonas syringae. The device comprises two co-activating genes hrpR and hrpS controlled by separate promoter inputs, and a σ54-dependent hrpL promoter driving the output. The hrpL promoter is activated only when both genes are expressed, generating digital-like AND integration behaviour. The AND gate is demonstrated to be modular by applying new regulated promoters to the inputs, and connecting the output to a NOT gate module to produce a combinatorial NAND gate. The circuits were assembled using a parts-based engineering approach of quantitative characterization, modelling, followed by construction and testing. The results show that new genetic logic devices can be engineered predictably from novel native orthogonal biological control elements using quantitatively in-context characterized parts

Detecting intratumoral heterogeneity of EGFR activity by liposome-based in vivo transfection of a fluorescent biosensor

Despite decades of research in the epidermal growth factor receptor (EGFR) signalling field, and many targeted anti-cancer drugs that have been tested clinically, the success rate for these agents in the clinic is low, particularly in terms of the improvement of overall survival. Intratumoral heterogeneity is proposed as a major mechanism underlying treatment failure of these molecule-targeted agents. Here we highlight the application of fluorescence lifetime microscopy (FLIM)-based biosensing to demonstrate intratumoral heterogeneity of EGFR activity. For sensing EGFR activity in cells, we used a genetically encoded CrkII-based biosensor which undergoes conformational changes upon tyrosine-221 phosphorylation by EGFR. We transfected this biosensor into EGFR-positive tumour cells using targeted lipopolyplexes bearing EGFR-binding peptides at their surfaces. In a murine model of basal-like breast cancer, we demonstrated a significant degree of intratumoral heterogeneity in EGFR activity, as well as the pharmacodynamic effect of a radionuclide-labeled EGFR inhibitor in situ. Furthermore, a significant correlation between high EGFR activity in tumour cells and macrophage-tumour cell proximity was found to in part account for the intratumoral heterogeneity in EGFR activity observed. The same effect of macrophage infiltrate on EGFR activation was also seen in a colorectal cancer xenograft. In contrast, a non-small cell lung cancer xenograft expressing a constitutively active EGFR conformational mutant exhibited macrophage proximity-independent EGFR activity. Our study validates the use of this methodology to monitor therapeutic response in terms of EGFR activity. In addition, we found iNOS gene induction in macrophages that are cultured in tumour cell-conditioned media as well as an iNOS activity-dependent increase in EGFR activity in tumour cells. These findings point towards an immune microenvironment-mediated regulation that gives rise to the observed intratumoral heterogeneity of EGFR signalling activity in tumour cells in vivo

Assessing powder flowability at low stresses using ball indentation method: Evaluation of constraint factor

Powders can exhibit different flow behaviour resulting from a combination of physical properties of the material and equipment design. Problems with powder flow are ubiquitous in process industry and become prominent when dealing with fine and cohesive powders. It is therefore of great importance to characterise the flowability of cohesive materials for better process control. Powder flowability is commonly assessed under relatively high preconsolidation loads using shear cell and uniaxial compression methods by which the unconfined yield strength (Y) is evaluated as a function of the applied load. However, these techniques are typically limited to applied stresses greater than 1 kPa and require a relatively large quantity of powder. To overcome these limitations, the recently developed Ball Indentation Method (BIM) is used in this work for assessing powder flow behaviour at low stress levels. The unconfined yield strength (Y) is inferred from the resistance to ball penetration into the surface of a powder bed, based on the method for measurement of hardness (H). This requires the flow resistance, represented by hardness, to be related to the unconfined yield strength by a proportionality factor termed the constraint factor, C, following the analogy with yield stress measurement in continuum solids, i.e. Y=H/C. The constraint factor for silanised glass ballotini, calcium carbonate, α-lactose monohydrate, Avicel and limestone is evaluated and reported here. It is shown that the unconfined yield strength inferred by this method correlates well with those from the uniaxial compression and shear cell measurement. The characterisation of the constraint factor makes it possible to use BIM for powder flowability testing at low stress levels and using a very small powder quantity. This is highly desirable for applications such as capsule filling, tableting and dry powder inhaler devices

Single-nucleotide polymorphisms in the RB1 gene and association with breast cancer in the British population

A substantial proportion of the familial risk of breast cancer may be attributable to genetic variants each contributing a small effect. pRb controls the cell cycle and polymorphisms within it are candidates for such low penetrance susceptibility alleles, since the gene has been implicated in several human tumours, particularly breast cancer. The purpose of this study was to determine whether common variants in the RB1 gene are associated with breast cancer risk. We assessed 15 tagging single-nucleotide polymorphisms (SNPs) using a case–control study design (n⩽4474 cases and n⩽4560 controls). A difference in genotype frequencies was found between cases and controls for rs2854344 in intron 17 (P-trend=0.007) and rs198580 in intron 19 (P-trend=0.018). Carrying the minor allele of these SNPs appears to confer a protective effect on breast cancer risk (odd ratio (OR)=0.86 (0.76–0.96) for rs2854344 and OR=0.80 (0.66–0.96) for rs198580). However, after adjusting for multiple testing these associations were borderline with an adjusted P-trend=0.068 for the most significant SNP (rs2854344). The RB1 gene is not known to contain any coding SNPs with allele frequencies ⩾5% but several intronic variants are in perfect linkage disequilibrium with the associated SNPs. Replication studies are needed to confirm the associations with breast cancer