Diffusive hidden Markov model characterization of DNA looping dynamics in tethered particle experiments

In many biochemical processes, proteins bound to DNA at distant sites are brought into close proximity by loops in the underlying DNA. For example, the function of some gene-regulatory proteins depends on such DNA looping interactions. We present a new technique for characterizing the kinetics of loop formation in vitro, as observed using the tethered particle method, and apply it to experimental data on looping induced by lambda repressor. Our method uses a modified (diffusive) hidden Markov analysis that directly incorporates the Brownian motion of the observed tethered bead. We compare looping lifetimes found with our method (which we find are consistent over a range of sampling frequencies) to those obtained via the traditional threshold-crossing analysis (which can vary depending on how the raw data are filtered in the time domain). Our method does not involve any time filtering and can detect sudden changes in looping behavior. For example, we show how our method can identify transitions between long-lived, kinetically distinct states that would otherwise be difficult to discern

Influence of tobacco use on postoperative opiate analgesia requirements in patients undergoing coronary artery bypass graft surgery

Introduction. The objective of this study was to test the hypothesis that tobacco use status is independently associated with postoperative opioid requirements in patients undergoing coronary artery bypass grafting (CABG) when important demographic variables such as age and gender are taken into account. Methods. A retrospective chart review of patients who underwent CABG surgery over a one year period at Mayo Clinic in Rochester, MN was performed. Tobacco users (N=69) were compared to nonusers (N=345) with regards to opiate requirements and the occurrence of severe pain during the first 48 hours postoperatively. For comparison, all postoperative opiates were converted to oral morphine equivalents (OME). Adjusted analysis for age and gender was also performed. Results. Tobacco users were younger than nonusers (P < 0.001), and a greater proportion of former users were male compared to never users (P = 0.003). Tobacco users had greater mean opiate requirements 401 ± 284 than nonusers 314 ± 240 mg OME, (P = 0.009). However, the association between tobacco use and greater postoperative opiate requirements lost significance after adjustment for age and gender. Tobacco use was not associated with increased risk of the development of severe pain (P = 0.51). Conclusions. Although current tobacco users undergoing CABG surgery utilize more opioid analgesics in the first 48 hours following extubation than nonusers of tobacco, when adjusted for age and gender, tobacco use was not independently associated with differences in postoperative opioid use

Awareness regarding eye donation among stakeholders in Srikakulam district in South India

Background There is a huge need for the availability of transplantable donor corneas worldwide to reduce the burden of corneal blindness due to corneal opacity. Voluntary eye donation depends on the awareness levels of various stakeholders in the community. This study aimed to assess the awareness level regarding eye donation among various stakeholders in Srikakulam district in the state of Andhra Pradesh, India. Methods 355 subjects were selected from the district using multi stage random sampling. A pre tested semi structured questionnaire was used to collect information regarding each individual’s awareness, knowledge, and perception regarding eye donation. Each response was scored individually and a total score was calculated. Univariate and multivariate regression analysis was used to determine the factors associated with willingness towards eye donation and increased awareness levels. Results Of the 355 subjects interviewed, 192 (54%) were male and 163 (46%) were female. The mean age of the stakeholders was 35.9 years (SD ±16.1) and all the study subjects were literate. Ninety-three percent of subjects were aware of the concept of eye donation. Knowledge levels were similar among the teaching community and persons engaged in social service, but lower among students (p < 0.05). Among the stakeholders, there was considerable ambiguity regarding whether persons currently wearing spectacles or suffering from a chronic illnesses could donate their eyes. Older age group (p < 0.001), female gender (p < 0.001) and education (p < 0.001) were associated with increased knowledge levels. 82% of the subjects were willing to donate their eyes and this was unaffected by gender or geographical location (rural vs urban). Conclusions Awareness levels and willingness to donate eyes are high among the stakeholders in Srikakulam district in India. The services of stakeholders could be utilized, in conjunction with other community based eye donation counselors, to promote awareness regarding eye donation among the general population

DNA bending and unbending by MutS govern mismatch recognition and specificity

DNA mismatch repair is central to the maintenance of genomic stability. It is initiated by the recognition of base–base mismatches and insertion/deletion loops by the family of MutS proteins. Subsequently, ATP induces a unique conformational change in the MutS–mismatch complex but not in the MutS–homoduplex complex that sets off the cascade of events that leads to repair. To gain insight into the mechanism by which MutS discriminates between mismatch and homoduplex DNA, we have examined the conformations of specific and nonspecific MutS–DNA complexes by using atomic force microscopy. Interestingly, MutS–DNA complexes exhibit a single population of conformations, in which the DNA is bent at homoduplex sites, but two populations of conformations, bent and unbent, at mismatch sites. These results suggest that the specific recognition complex is one in which the DNA is unbent. Combining our results with existing biochemical and crystallographic data leads us to propose that MutS: (i) binds to DNA nonspecifically and bends it in search of a mismatch; (ii) on specific recognition of a mismatch, undergoes a conformational change to an initial recognition complex in which the DNA is kinked, with interactions similar to those in the published crystal structures; and (iii) finally undergoes a further conformational change to the ultimate recognition complex in which the DNA is unbent. Our results provide a structural explanation for the long-standing question of how MutS achieves mismatch repair specificity

Regionalizing the infrastructure turn : a research agenda

An interdisciplinary ‘infrastructure turn’ has emerged over the past 20 years that disputes the concept of urban infrastructure as a staid or neutral set of physical artefacts. Responding to the increased conceptual, geographical and political importance of infrastructure – and endemic issues of access, expertise and governance that the varied provision of infrastructures can cause – this intervention asserts the significance of applying a regional perspective to the infrastructure turn. This paper forwards a critical research agenda for the study of ‘infrastructural regionalisms’ to interrogate: (1) how we study and produce knowledge about infrastructure; (2) how infrastructure is governed across or constrained by jurisdictional boundaries; (3) who drives the construction of regional infrastructural imaginaries; and (4) how individuals and communities differentially experience regional space through infrastructure. Analysing regions through infrastructure provides a novel perspective on the regional question and consequently offers a framework to understand better the implications of the current infrastructure moment for regional spaces worldwide

Testing for Fictive Learning in Decision-Making Under Uncertainty

We conduct two experiments where subjects make a sequence of binary choices between risky and ambiguous binary lotteries. Risky lotteries are defined as lotteries where the relative frequencies of outcomes are known. Ambiguous lotteries are lotteries where the relative frequencies of outcomes are not known or may not exist. The trials in each experiment are divided into three phases: pre-treatment, treatment and post-treatment. The trials in the pre-treatment and post-treatment phases are the same. As such, the trials before and after the treatment phase are dependent, clustered matched-pairs, that we analyze with the alternating logistic regression (ALR) package in SAS. In both experiments, we reveal to each subject the outcomes of her actual and counterfactual choices in the treatment phase. The treatments differ in the complexity of the random process used to generate the relative frequencies of the payoffs of the ambiguous lotteries. In the first experiment, the probabilities can be inferred from the converging sample averages of the observed actual and counterfactual outcomes of the ambiguous lotteries. In the second experiment the sample averages do not converge. If we define fictive learning in an experiment as statistically significant changes in the responses of subjects before and after the treatment phase of an experiment, then we expect fictive learning in the first experiment, but no fictive learning in the second experiment. The surprising finding in this paper is the presence of fictive learning in the second experiment. We attribute this counterintuitive result to apophenia: “seeing meaningful patterns in meaningless or random data.” A refinement of this result is the inference from a subsequent Chi-squared test, that the effects of fictive learning in the first experiment are significantly different from the effects of fictive learning in the second experiment

Transcriptional Infidelity Promotes Heritable Phenotypic Change in a Bistable Gene Network

Bistable epigenetic switches are fundamental for cell fate determination in unicellular and multicellular organisms. Regulatory proteins associated with bistable switches are often present in low numbers and subject to molecular noise. It is becoming clear that noise in gene expression can influence cell fate. Although the origins and consequences of noise have been studied, the stochastic and transient nature of RNA errors during transcription has not been considered in the origin or modeling of noise nor has the capacity for such transient errors in information transfer to generate heritable phenotypic change been discussed. We used a classic bistable memory module to monitor and capture transient RNA errors: the lac operon of Escherichia coli comprises an autocatalytic positive feedback loop producing a heritable all-or-none epigenetic switch that is sensitive to molecular noise. Using single-cell analysis, we show that the frequency of epigenetic switching from one expression state to the other is increased when the fidelity of RNA transcription is decreased due to error-prone RNA polymerases or to the absence of auxiliary RNA fidelity factors GreA and GreB (functional analogues of eukaryotic TFIIS). Therefore, transcription infidelity contributes to molecular noise and can effect heritable phenotypic change in genetically identical cells in the same environment. Whereas DNA errors allow genetic space to be explored, RNA errors may allow epigenetic or expression space to be sampled. Thus, RNA infidelity should also be considered in the heritable origin of altered or aberrant cell behaviour

Tuning the translational freedom of DNA for high speed AFM

Direct observation is arguably the preferred way to investigate the interactions between two molecular complexes. With the development of high speed atomic force microscopy it is becoming possible to observe directly DNA protein interactions with relevant spatial and temporal resolutions. These interactions are of central importance to biology, bio-nanotechnology but also functional biologically inspired materials. Critically, sample preparation plays a central role in all microscopy studies and minimal perturbation of the sample is desired. Here, we demonstrate the ability to tune the interactions of DNA molecules with the surface such that an association strong enough to enable high resolution AFM imaging while providing sufficient translational freedom to allow the relevant protein DNA interactions to take place, can be maintained. Furthermore, we describe a quantitative method for measuring the DNA mobility, which also allows the dissection of the different contributions to the overall movement of the DNA molecules. We find that for weak surface association, a significant contribution to the movement arises from the interaction of the AFM tip with the DNA. In combination, these methods enable the tuning of the surface translational freedom of DNA molecules to allow the direct study of a wide range of nucleo-protein interactions by high speed atomic force microscopy

The nucleotide addition cycle of RNA polymerase is controlled by two molecular hinges in the Bridge Helix domain

Abstract Background Cellular RNA polymerases (RNAPs) are complex molecular machines that combine catalysis with concerted conformational changes in the active center. Previous work showed that kinking of a hinge region near the C-terminus of the Bridge Helix (BH-HC) plays a critical role in controlling the catalytic rate. Results Here, new evidence for the existence of an additional hinge region in the amino-terminal portion of the Bridge Helix domain (BH-HN) is presented. The nanomechanical properties of BH-HN emerge as a direct consequence of the highly conserved primary amino acid sequence. Mutations that are predicted to influence its flexibility cause corresponding changes in the rate of the nucleotide addition cycle (NAC). BH-HN displays functional properties that are distinct from BH-HC, suggesting that conformational changes in the Bridge Helix control the NAC via two independent mechanisms. Conclusions The properties of two distinct molecular hinges in the Bridge Helix of RNAP determine the functional contribution of this domain to key stages of the NAC by coordinating conformational changes in surrounding domains.</p