Potential Role of a Phenolic Acid in the Uremic Syndrome

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Simulation and analysis of in vitro DNA evolution

We study theoretically the in vitro evolution of a DNA sequence by binding to a transcription factor. Using a simple model of protein-DNA binding and available binding constants for the Mnt protein, we perform large-scale, realistic simulations of evolution starting from a single DNA sequence. We identify different parameter regimes characterized by distinct evolutionary behaviors. For each regime we find analytical estimates which agree well with simulation results. For small population sizes, the DNA evolutional path is a random walk on a smooth landscape. While for large population sizes, the evolution dynamics can be well described by a mean-field theory. We also study how the details of the DNA-protein interaction affect the evolution.Comment: 11 pages, 11 figures. Submitted to PNA

Thermal denaturation of fluctuating finite DNA chains: the role of bending rigidity in bubble nucleation

Statistical DNA models available in the literature are often effective models where the base-pair state only (unbroken or broken) is considered. Because of a decrease by a factor of 30 of the effective bending rigidity of a sequence of broken bonds, or bubble, compared to the double stranded state, the inclusion of the molecular conformational degrees of freedom in a more general mesoscopic model is needed. In this paper we do so by presenting a 1D Ising model, which describes the internal base pair states, coupled to a discrete worm like chain model describing the chain configurations [J. Palmeri, M. Manghi, and N. Destainville, Phys. Rev. Lett. 99, 088103 (2007)]. This coupled model is exactly solved using a transfer matrix technique that presents an analogy with the path integral treatment of a quantum two-state diatomic molecule. When the chain fluctuations are integrated out, the denaturation transition temperature and width emerge naturally as an explicit function of the model parameters of a well defined Hamiltonian, revealing that the transition is driven by the difference in bending (entropy dominated) free energy between bubble and double-stranded segments. The calculated melting curve (fraction of open base pairs) is in good agreement with the experimental melting profile of polydA-polydT. The predicted variation of the mean-square-radius as a function of temperature leads to a coherent novel explanation for the experimentally observed thermal viscosity transition. Finally, the influence of the DNA strand length is studied in detail, underlining the importance of finite size effects, even for DNA made of several thousand base pairs.Comment: Latex, 28 pages pdf, 9 figure

Investigating the missing data mechanism in quality of life outcomes: a comparison of approaches

Background: Missing data is classified as missing completely at random (MCAR), missing at random (MAR) or missing not at random (MNAR). Knowing the mechanism is useful in identifying the most appropriate analysis. The first aim was to compare different methods for identifying this missing data mechanism to determine if they gave consistent conclusions. Secondly, to investigate whether the reminder-response data can be utilised to help identify the missing data mechanism. Methods: Five clinical trial datasets that employed a reminder system at follow-up were used. Some quality of life questionnaires were initially missing, but later recovered through reminders. Four methods of determining the missing data mechanism were applied. Two response data scenarios were considered. Firstly, immediate data only; secondly, all observed responses (including reminder-response). Results: In three of five trials the hypothesis tests found evidence against the MCAR assumption. Logistic regression suggested MAR, but was able to use the reminder-collected data to highlight potential MNAR data in two trials. Conclusion: The four methods were consistent in determining the missingness mechanism. One hypothesis test was preferred as it is applicable with intermittent missingness. Some inconsistencies between the two data scenarios were found. Ignoring the reminder data could potentially give a distorted view of the missingness mechanism. Utilising reminder data allowed the possibility of MNAR to be considered.The Chief Scientist Office of the Scottish Government Health Directorate. Research Training Fellowship (CZF/1/31

Microcalorimetry and spectroscopic studies on the binding of dye janus green blue to deoxyribonucleic acid

The interaction of the phenazinium dye janus green blue (JGB) with deoxyribonucleic acid was investigated using isothermal titration calorimetry and thermal melting experiments. The calorimetric data were supplemented by spectroscopic studies. Calorimetry results suggested the binding affinity of the dye to DNA to be of the order of 105 M-1. The binding was predominantly entropy driven with a small negative favorable enthalpy contribution to the standard molar Gibbs energy change.The binding became weaker as the temperature and salt concentration was raised. The temperature dependence of the standard molar enthalpy changes yielded negative values of standard molar heat capacity change for the complexation revealing substantial hydrophobic contribution in the DNA binding. An enthalpy–entropy compensation behavior was also observed in the system. The salt dependence of the binding yielded the release of 0.69 number of cations on binding of each dye molecule. The non-polyelectrolytic contribution was found to be the predominant force in the binding interaction. Thermal melting studies revealed that the DNA helix was stabilized against denaturation by the dye. The binding was also characterized by absorbance, resonance light scattering and circular dichroism spectral measurements. The binding constants from the spectral results were close to those obtained from the calorimetric data. The energetic aspects of the interaction of the dye JGB to double stranded DNA are supported by strong binding revealed from the spectral data

miR-1289 and “Zipcode”-like Sequence Enrich mRNAs in Microvesicles

Despite intensive studies, the molecular mechanisms by which the genetic materials are uploaded into microvesicles (MVs) are still unknown. This is the first study describing a zipcode-like 25 nucleotide (nt) sequence in the 3′-untranslated region (3′UTR) of mRNAs, with variants of this sequence present in many mRNAs enriched in MVs, as compared to their glioblastoma cells of origin. When this sequence was incorporated into the 3′UTR of a reporter message and expressed in a different cell type, it led to enrichment of the reporter mRNA in MVs. Critical features of this sequence are both a CUGCC core presented on a stem-loop structure and a miRNA-binding site, with increased levels of the corresponding miRNA in cells further increasing levels of mRNAs in MVs

1968

Maintenance of Golf Carts by Thomas Pepe (page 1) Why a Golf Course Superintendent Should Play Golf by Stephen Skowronski (2) Tree Pruning by Martin Walsh (3) Golf Course Labor: A Dilemma by Robert Barber (5) Turf Problems by Alexander M. Radko (A-1) 1967 Turfgrass Problems by Lee Record (A-3) Southern Turfgrass Production and Problems by Ralph W. White Jr. (A-5) Canadian Turf Grass Production and Problems by David Moote (A-8) Turf Research Abroad by C.R. Skogley (A-13) Turf Research at Home by Victor B. Younger (A-14) Turfgrass Research - An Industrial Approach by J. A. Simmons (A-16) Cutting Labor Costs in Turfgrass Managemnt by Tom Mascaro (A-24) The Reluctant human by John W. Denison (A-28) The Problem Drinker - Management Responsibility by G.E. Osburn (A-31) Contemporary Design Standards by Geoffrey S. Cornish (A-34) Construction - Superintendents\u27 Viewpoint by Robert E. Grant (A-36) Construction b Contract and the Role of the Superintendent by David Canavan (A-39) Seed Production by Robert J. Peterson (A-41) Cemetery Turf Maintenance on a High and Low Budget by Stanley Sosenski (A-45) Ten Steps to a Good Lawn by John Zak (A-49) Review of Herbicides for Turf Weed Control by Alvin A. Baber (A-53

Mechanism of transcription initiation and promoter escape by E. coli RNA polymerase

To investigate roles of the discriminator and open complex (OC) lifetime in transcription initiation by Escherichia coli RNA polymerase (RNAP; α 2 ββ'ωσ 70 ), we compare productive and abortive initiation rates, short RNA distributions, and OC lifetime for the λP R and T7A1 promoters and variants with exchanged discriminators, all with the same transcribed region. The discriminator determines the OC lifetime of these promoters. Permanganate reactivity of thymines reveals that strand backbones in open regions of longlived λP R -discriminator OCs are much more tightly held than for shorter-lived T7A1-discriminator OCs. Initiation from these OCs exhibits two kinetic phases and at least two subpopulations of ternary complexes. Long RNA synthesis (constrained to be single round) occurs only in the initial phase (<10 s), at similar rates for all promoters. Less than half of OCs synthesize a full-length RNA; the majority stall after synthesizing a short RNA. Most abortive cycling occurs in the slower phase (>10 s), when stalled complexes release their short RNA and make another without escaping. In both kinetic phases, significant amounts of 8-nt and 10-nt transcripts are produced by longer-lived, λP R -discriminator OCs, whereas no RNA longer than 7 nt is produced by shorter-lived T7A1-discriminator OCs. These observations and the lack of abortive RNA in initiation from short-lived ribosomal promoter OCs are well described by a quantitative model in which ∼1.0 kcal/mol of scrunching free energy is generated per translocation step of RNA synthesis to overcome OC stability and drive escape. The different length-distributions of abortive RNAs released from OCs with different lifetimes likely play regulatory roles. RNA polymerase | open complex lifetime | transcription initiation | abortive RNA | hybrid length M any facets of transcription initiation by E. coli RNA polymerase (RNAP; α 2 ββ′ωσ 70 ) have been elucidated, but significant questions remain about the mechanism or mechanisms by which initial transcribing complexes (ITC) with a short RNA-DNA hybrid decide to advance and escape from the promoter to enter elongation mode, or, alternately, to stall, release their short RNA, and reinitiate (abortive cycling). For RNAP to escape, its sequencespecific interactions with promoter DNA in the binary open complex (OC) must be overcome. The open regions of promoter DNA in the binary OC are the −10 region (six residues, with specific interactions between σ 2.2 and the nontemplate strand), the discriminator region (typically six to eight residues with no consensus sequence, the upstream end of which interacts with σ 1.2 ), and the transcription start site (TSS, +1) and adjacent residue (+2), which are in the active site of RNAP What drives promoter escape? Escape involves disrupting all the favorable interactions involved in forming and stabilizing the binary OC as well as σ-core interactions. Escape from these interactions is fundamentally driven by the favorable chemical (free) energy change of RNA synthesis, but this energy must be stored in the ITC in each step before escape. Proposed means of energy storage as the length of the RNA-DNA hybrid increases include the stresses introduced by scrunching distortions of the discriminator regions of the open strands in the cleft (2, 5, 6) and by unfavorable interactions of the RNA-DNA hybrid with the hairpin loop of σ 3.2 (7-10). Scrunching of the discriminator region of the template strand is proposed to be most significant for Significance The enzyme RNA polymerase (RNAP) transcribes DNA genetic information into RNA. Regulation of transcription occurs largely in initiation; these regulatory mechanisms must be understood. Lifetimes of transcription-capable RNAP-promoter open complexes (OCs) vary greatly, dictated largely by the DNA discriminator region, but the significance of OC lifetime for regulation was unknown. We observe that a significantly longer RNA:DNA hybrid is synthesized before RNAP escapes from long-lived λP R -promoter OCs as compared with shorter-lived T7A1 promoter OCs. We quantify the free energy needed to overcome OC stability and allow escape from the promoter and elongation of the nascent RNA, and thereby predict escape points for ribosomal (rrnB P1) and lacUV5 promoters. Longer-lived OCs produce longer abortive RNAs, which likely have specific regulatory roles

Mechanism of transcription initiation and promoter escape by E. coli RNA polymerase

To investigate roles of the discriminator and open complex (OC) lifetime in transcription initiation by Escherichia coli RNA polymerase (RNAP; α 2 ββ'ωσ 70 ), we compare productive and abortive initiation rates, short RNA distributions, and OC lifetime for the λP R and T7A1 promoters and variants with exchanged discriminators, all with the same transcribed region. The discriminator determines the OC lifetime of these promoters. Permanganate reactivity of thymines reveals that strand backbones in open regions of longlived λP R -discriminator OCs are much more tightly held than for shorter-lived T7A1-discriminator OCs. Initiation from these OCs exhibits two kinetic phases and at least two subpopulations of ternary complexes. Long RNA synthesis (constrained to be single round) occurs only in the initial phase (<10 s), at similar rates for all promoters. Less than half of OCs synthesize a full-length RNA; the majority stall after synthesizing a short RNA. Most abortive cycling occurs in the slower phase (>10 s), when stalled complexes release their short RNA and make another without escaping. In both kinetic phases, significant amounts of 8-nt and 10-nt transcripts are produced by longer-lived, λP R -discriminator OCs, whereas no RNA longer than 7 nt is produced by shorter-lived T7A1-discriminator OCs. These observations and the lack of abortive RNA in initiation from short-lived ribosomal promoter OCs are well described by a quantitative model in which ∼1.0 kcal/mol of scrunching free energy is generated per translocation step of RNA synthesis to overcome OC stability and drive escape. The different length-distributions of abortive RNAs released from OCs with different lifetimes likely play regulatory roles. RNA polymerase | open complex lifetime | transcription initiation | abortive RNA | hybrid length M any facets of transcription initiation by E. coli RNA polymerase (RNAP; α 2 ββ′ωσ 70 ) have been elucidated, but significant questions remain about the mechanism or mechanisms by which initial transcribing complexes (ITC) with a short RNA-DNA hybrid decide to advance and escape from the promoter to enter elongation mode, or, alternately, to stall, release their short RNA, and reinitiate (abortive cycling). For RNAP to escape, its sequencespecific interactions with promoter DNA in the binary open complex (OC) must be overcome. The open regions of promoter DNA in the binary OC are the −10 region (six residues, with specific interactions between σ 2.2 and the nontemplate strand), the discriminator region (typically six to eight residues with no consensus sequence, the upstream end of which interacts with σ 1.2 ), and the transcription start site (TSS, +1) and adjacent residue (+2), which are in the active site of RNAP What drives promoter escape? Escape involves disrupting all the favorable interactions involved in forming and stabilizing the binary OC as well as σ-core interactions. Escape from these interactions is fundamentally driven by the favorable chemical (free) energy change of RNA synthesis, but this energy must be stored in the ITC in each step before escape. Proposed means of energy storage as the length of the RNA-DNA hybrid increases include the stresses introduced by scrunching distortions of the discriminator regions of the open strands in the cleft (2, 5, 6) and by unfavorable interactions of the RNA-DNA hybrid with the hairpin loop of σ 3.2 (7-10). Scrunching of the discriminator region of the template strand is proposed to be most significant for Significance The enzyme RNA polymerase (RNAP) transcribes DNA genetic information into RNA. Regulation of transcription occurs largely in initiation; these regulatory mechanisms must be understood. Lifetimes of transcription-capable RNAP-promoter open complexes (OCs) vary greatly, dictated largely by the DNA discriminator region, but the significance of OC lifetime for regulation was unknown. We observe that a significantly longer RNA:DNA hybrid is synthesized before RNAP escapes from long-lived λP R -promoter OCs as compared with shorter-lived T7A1 promoter OCs. We quantify the free energy needed to overcome OC stability and allow escape from the promoter and elongation of the nascent RNA, and thereby predict escape points for ribosomal (rrnB P1) and lacUV5 promoters. Longer-lived OCs produce longer abortive RNAs, which likely have specific regulatory roles

Achieving Optimal Growth through Product Feedback Inhibition in Metabolism

Recent evidence suggests that the metabolism of some organisms, such as Escherichia coli, is remarkably efficient, producing close to the maximum amount of biomass per unit of nutrient consumed. This observation raises the question of what regulatory mechanisms enable such efficiency. Here, we propose that simple product-feedback inhibition by itself is capable of leading to such optimality. We analyze several representative metabolic modules—starting from a linear pathway and advancing to a bidirectional pathway and metabolic cycle, and finally to integration of two different nutrient inputs. In each case, our mathematical analysis shows that product-feedback inhibition is not only homeostatic but also, with appropriate feedback connections, can minimize futile cycling and optimize fluxes. However, the effectiveness of simple product-feedback inhibition comes at the cost of high levels of some metabolite pools, potentially associated with toxicity and osmotic imbalance. These large metabolite pool sizes can be restricted if feedback inhibition is ultrasensitive. Indeed, the multi-layer regulation of metabolism by control of enzyme expression, enzyme covalent modification, and allostery is expected to result in such ultrasensitive feedbacks. To experimentally test whether the qualitative predictions from our analysis of feedback inhibition apply to metabolic modules beyond linear pathways, we examine the case of nitrogen assimilation in E. coli, which involves both nutrient integration and a metabolic cycle. We find that the feedback regulation scheme suggested by our mathematical analysis closely aligns with the actual regulation of the network and is sufficient to explain much of the dynamical behavior of relevant metabolite pool sizes in nutrient-switching experiments