p53 Activates the Long Noncoding RNA Pvt1b to Inhibit Myc and Suppress Tumorigenesis.

The tumor suppressor p53 transcriptionally activates target genes to suppress cellular proliferation during stress. p53 has also been implicated in the repression of the proto-oncogene Myc, but the mechanism has remained unclear. Here, we identify Pvt1b, a p53-dependent isoform of the long noncoding RNA (lncRNA) Pvt1, expressed 50 kb downstream of Myc, which becomes induced by DNA damage or oncogenic signaling and accumulates near its site of transcription. We show that production of the Pvt1b RNA is necessary and sufficient to suppress Myc transcription in cis without altering the chromatin organization of the locus. Inhibition of Pvt1b increases Myc levels and transcriptional activity and promotes cellular proliferation. Furthermore, Pvt1b loss accelerates tumor growth, but not tumor progression, in an autochthonous mouse model of lung cancer. These findings demonstrate that Pvt1b acts at the intersection of the p53 and Myc transcriptional networks to reinforce the anti-proliferative activities of p53.

Revisiting the full blood count: Circulating blood cells and their role in coagulation.

There has been an expansion in our understanding of the multifaceted roles of circulating blood cells in regulating haemostasis and contributing to thrombosis. Notably, there is greater recognition of the interplay between coagulation with inflammation and innate immune activation and the contribution of leucocytes. The full blood count (FBC) is a time-honoured test in medicine; however, its components are often viewed in isolation and without consideration of their haemostatic and thrombotic potential. Here, we review how the individual components of the FBC, that is, haemoglobin, platelets and leucocytes, engage with the haemostatic system and focus on both their quantitative and qualitative attributes. We also explore how this information can be harnessed into better management of people with multiple long-term conditions because of their higher risk of adverse clinical events.

Diffusive dynamics of a model protein chain in solution.

A Markov state model is a powerful tool that can be used to track the evolution of populations of configurations in an atomistic representation of a protein. For a coarse-grained linear chain model with discontinuous interactions, the transition rates among states that appear in the Markov model when the monomer dynamics is diffusive can be determined by computing the relative entropy of states and their mean first passage times, quantities that are unchanged by the specification of the energies of the relevant states. In this paper, we verify the folding dynamics described by a diffusive linear chain model of the crambin protein in three distinct solvent systems, each differing in complexity: a hard-sphere solvent, a solvent undergoing multi-particle collision dynamics, and an implicit solvent model. The predicted transition rates among configurations agree quantitatively with those observed in explicit molecular dynamics simulations for all three solvent models. These results suggest that the local monomer-monomer interactions provide sufficient friction for the monomer dynamics to be diffusive on timescales relevant to changes in conformation. Factors such as structural ordering and dynamic hydrodynamic effects appear to have minimal influence on transition rates within the studied solvent densities.

Microscopic theory of a Janus motor in a non-equilibrium fluid: Surface hydrodynamics and boundary conditions.

We present a derivation from the first principles of the coupled equations of motion of an active self-diffusiophoretic Janus motor and the hydrodynamic densities of its fluid environment that are nonlinearly displaced from equilibrium. The derivation makes use of time-dependent projection operator techniques defined in terms of slowly varying coarse-grained microscopic densities of the fluid species number, total momentum, and energy. The exact equations of motion are simplified using time scale arguments, resulting in Markovian equations for the Janus motor linear and angular velocities with average forces and torques that depend on the fluid densities. For a large colloid, the fluid equations are separated into bulk and interfacial contributions, and the conditions under which the dynamics of the fluid densities can be accurately represented by bulk hydrodynamic equations subject to boundary conditions on the colloid are determined. We show how the results for boundary conditions based on continuum theory can be obtained from the molecular description and provide Green-Kubo expressions for all transport coefficients, including the diffusiophoretic coupling and the slip coefficient.

Drug-induced thrombotic thrombocytopenic purpura: A systematic review and review of European and North American pharmacovigilance data.

Many medications have been reported to be associated with thrombotic thrombocytopenic purpura (TTP) through pharmacovigilance data and published case reports. Whilst there are existing data available regarding drug-induced thrombotic microangiopathy, there is no available synthesis of evidence to assess drug-induced TTP (DI-TTP). Despite this lack of evidence, patients with TTP are often advised against using many medications due to the theoretical risk of DI-TTP. This systematic review evaluated the evidence for an association of medications reported as potential triggers for TTP. Of 5098 records available 261 articles were assessed further for eligibility. Fifty-seven reports, totalling 90 patients, were included in the final analysis. There were no cases where the level of association was rated as definite or probable, demonstrating a lack of evidence of any drug causing DI-TTP. This paucity of evidence was also demonstrated in the pharmacovigilance data, where 613 drugs were reported as potential causes of TTP without assessment of the strength of association. This systematic review demonstrates the need for standardised reporting of potential drugs causing TTP. Many reports omit basic information and, therefore, hinder the chance of finding a causative link if one exists.

Configurational entropy, transition rates, and optimal interactions for rapid folding in coarse-grained model proteins.

Under certain conditions, the dynamics of coarse-grained models of solvated proteins can be described using a Markov state model, which tracks the evolution of populations of configurations. The transition rates among states that appear in the Markov model can be determined by computing the relative entropy of states and their mean first passage times. In this paper, we present an adaptive method to evaluate the configurational entropy and the mean first passage times for linear chain models with discontinuous potentials. The approach is based on event-driven dynamical sampling in a massively parallel architecture. Using the fact that the transition rate matrix can be calculated for any choice of interaction energies at any temperature, it is demonstrated how each state's energy can be chosen such that the average time to transition between any two states is minimized. The methods are used to analyze the optimization of the folding process of two protein systems: the crambin protein and a model with frustration and misfolding. It is shown that the folding pathways for both systems are comprised of two regimes: first, the rapid establishment of local bonds, followed by the subsequent formation of more distant contacts. The state energies that lead to the most rapid folding encourage multiple pathways, and they either penalize folding pathways through kinetic traps by raising the energies of trapping states or establish an escape route from the trapping states by lowering free energy barriers to other states that rapidly reach the native state.

TimeLapse-seq: adding a temporal dimension to RNA sequencing through nucleoside recoding.

RNA sequencing (RNA-seq) offers a snapshot of cellular RNA populations, but not temporal information about the sequenced RNA. Here we report TimeLapse-seq, which uses oxidative-nucleophilic-aromatic substitution to convert 4-thiouridine into cytidine analogs, yielding apparent U-to-C mutations that mark new transcripts upon sequencing. TimeLapse-seq is a single-molecule approach that is adaptable to many applications and reveals RNA dynamics and induced differential expression concealed in traditional RNA-seq.

Global Profiling of Cellular Substrates of Human Dcp2.

Decapping is the first committed step in 5'-to-3' RNA decay, and in the cytoplasm of human cells, multiple decapping enzymes regulate the stabilities of distinct subsets of cellular transcripts. However, the complete set of RNAs regulated by any individual decapping enzyme remains incompletely mapped, and no consensus sequence or property is currently known to unambiguously predict decapping enzyme substrates. Dcp2 was the first-identified and best-studied eukaryotic decapping enzyme, but it has been shown to regulate the stability of <400 transcripts in mammalian cells to date. Here, we globally profile changes in the stability of the human transcriptome in Dcp2 knockout cells via TimeLapse-seq. We find that P-body enrichment is the strongest correlate of Dcp2-dependent decay and that modification with m6A exhibits an additive effect with P-body enrichment for Dcp2 targeting. These results are consistent with a model in which P-bodies represent sites where translationally repressed transcripts are sorted for decay by soluble cytoplasmic decay complexes through additional molecular marks.

The NBDY Microprotein Regulates Cellular RNA Decapping.

Proteogenomic identification of translated small open reading frames in humans has revealed thousands of microproteins, or polypeptides of fewer than 100 amino acids, that were previously invisible to geneticists. Hundreds of microproteins have been shown to be essential for cell growth and proliferation, and many regulate macromolecular complexes. One such regulatory microprotein is NBDY, a 68-amino acid component of the human cytoplasmic RNA decapping complex. Heterologously expressed NBDY was previously reported to regulate cytoplasmic ribonucleoprotein granules known as P-bodies and reporter gene stability, but the global effect of endogenous NBDY on the cellular transcriptome remained undefined. In this work, we demonstrate that endogenous NBDY directly interacts with the human RNA decapping complex through EDC4 and DCP1A and localizes to P-bodies. Global profiling of RNA stability changes in NBDY knockout (KO) cells reveals dysregulated stability of more than 1400 transcripts. DCP2 substrate transcript half-lives are both increased and decreased in NBDY KO cells, which correlates with 5' UTR length. NBDY deletion additionally alters the stability of non-DCP2 target transcripts, possibly as a result of downregulated expression of nonsense-mediated decay factors in NBDY KO cells. We present a comprehensive model of the regulation of RNA stability by NBDY.

Molecular theory of Langevin dynamics for active self-diffusiophoretic colloids.

Active colloidal particles that are propelled by a self-diffusiophoretic mechanism are often described by Langevin equations that are either postulated on physical grounds or derived using the methods of fluctuating hydrodynamics. While these descriptions are appropriate for colloids of micrometric and larger size, they will break down for very small active particles. A fully microscopic derivation of Langevin equations for self-diffusiophoretic particles powered by chemical reactions catalyzed asymmetrically by the colloid is given in this paper. The derivation provides microscopic expressions for the translational and rotational friction tensors, as well as reaction rate coefficients appearing in the Langevin equations. The diffusiophoretic force and torque are expressed in terms of nonequilibrium averages of fluid fields that satisfy generalized transport equations. The results provide a description of active motion on small scales where descriptions in terms of coarse grained continuum fluid equations combined with boundary conditions that account for the presence of the colloid may not be appropriate.

From single particle motion to collective dynamics in Janus motor systems.

The single-particle and collective dynamics of systems comprising Janus motors, solvent, and reactive solute species maintained in nonequilibrium states are investigated. Reversible catalytic reactions with the solute species take place on the catalytic faces of the motors, and the nonequilibrium states are established either by imposing constant-concentration reservoirs that feed and remove reactive species or through out-of-equilibrium fluid phase reactions. We consider general intermolecular interactions between the Janus motor hemispheres and the reactive species. For single motors, we show that the reaction rate depends nonlinearly on an applied external force when the system is displaced far from equilibrium. We also show that a finite-time fluctuation formula derived for fixed catalytic particles describes the nonequilibrium reactive fluctuations of moving Janus motors. Simulation of the collective dynamics of small ensembles of Janus motors with reversible kinetics under nonequilibrium conditions is carried out, and the spatial and orientational correlations of dynamic cluster states are discussed. The conditions leading to the instability of the homogeneous motor distribution and the onset of nonequilibrium dynamical clustering are described.

Expanding the Nucleoside Recoding Toolkit: Revealing RNA Population Dynamics with 6-Thioguanosine.

RNA-sequencing (RNA-seq) measures RNA abundance in a biological sample but does not provide temporal information about the sequenced RNAs. Metabolic labeling can be used to distinguish newly made RNAs from pre-existing RNAs. Mutations induced from chemical recoding of the hydrogen bonding pattern of the metabolic label can reveal which RNAs are new in the context of a sequencing experiment. These nucleotide recoding strategies have been developed for a single uridine analogue, 4-thiouridine (s4U), limiting the scope of these experiments. Here we report the first use of nucleoside recoding with a guanosine analogue, 6-thioguanosine (s6G). Using TimeLapse sequencing (TimeLapse-seq), s6G can be recoded under RNA-friendly oxidative nucleophilic-aromatic substitution conditions to produce adenine analogues (substituted 2-aminoadenosines). We demonstrate the first use of s6G recoding experiments to reveal transcriptome-wide RNA population dynamics.

Water transport and desalination through double-layer graphyne membranes.

Non-equilibrium molecular dynamics simulations of water-salt solutions driven through single and double-layer graphyne membranes by a pressure difference created by rigid pistons are carried out to determine the relative performance of the membranes as filters in a reverse osmosis desalination process. It is found that the flow rate of water through a graphyne-4 membrane is twice that of a graphyne-3 membrane for both single and double-layer membranes. Although the addition of a second layer to a single-layer membrane reduces the membrane permeability, the double-layer graphyne membranes are still two or three orders of magnitude more permeable than commercial reverse osmosis membranes. The minimum reduction in flow rate for double-layer membranes occurs at a layer spacing of 0.35 nm with an AA stacking configuration, while at a spacing of 0.6 nm the flow rate is close to zero due to a high free energy barrier for permeation. This is caused by the difference in the environments on either side of the membrane sheets and the formation of a compact two-dimensional layer of water molecules in the interlayer space which slows down water permeation. The distribution of residence times of water molecules in the interlayer region suggests that at the critical layer spacing of 0.6 nm, a cross-over occurs in the mechanism of water flow from the collective movement of hydrogen-bonded water sheets to the permeation of individual water molecules. All membranes are demonstrated to have a high salt rejection fraction and the double-layered graphyne-4 membranes can further increase the salt rejection by trapping ions that have passed through the first membrane from the feed solution in the interlayer space.

Dynamics of Janus motors with microscopically reversible kinetics.

Janus motors with chemically active and inactive hemispheres can operate only under nonequilibrium conditions where detailed balance is broken by fluxes of chemical species that establish a nonequilibrium state. A microscopic model for reversible reactive collisions on a Janus motor surface is constructed and shown to satisfy detailed balance. The model is used to study Janus particle reactive dynamics in systems at equilibrium where generalized chemical rate laws that include time-dependent rate coefficients with power-law behavior are shown to describe reaction rates. While maintaining reversible reactions on the Janus catalytic hemisphere, the system is then driven into a nonequilibrium steady state by fluxes of chemical species that control the chemical affinity. The statistical properties of the self-propelled Janus motor in this nonequilibrium steady state are investigated and compared with the predictions of a fluctuating thermodynamics theory. The model has utility beyond the examples presented here, since it allows one to explore various aspects of nonequilibrium fluctuations in systems with self-diffusiophoretic motors from a microscopic perspective.

A microscopic model for chemically-powered Janus motors.

Very small synthetic motors that make use of chemical reactions to propel themselves in solution hold promise for new applications in the development of new materials, science and medicine. The prospect of such potential applications, along with the fact that systems with many motors or active elements display interesting cooperative phenomena of fundamental interest, has made the study of synthetic motors an active research area. Janus motors, comprising catalytic and noncatalytic hemispheres, figure prominently in experimental and theoretical studies of these systems. While continuum models of Janus motor systems are often used to describe motor dynamics, microscopic models that are able to account for intermolecular interactions, many-body concentration gradients, fluid flows and thermal fluctuations provide a way to explore the dynamical behavior of these complex out-of-equilibrium systems that does not rely on approximations that are often made in continuum theories. The analysis of microscopic models from first principles provides a foundation from which the range of validity and limitations of approximate theories of the dynamics may be assessed. In this paper, a microscopic model for the diffusiophoretic propulsion of Janus motors, where motor interactions with the environment occur only through hard collisions, is constructed, analyzed and compared to theoretical predictions. Microscopic simulations of both single-motor and many-motor systems are carried out to illustrate the results.

Free energy simulations of amylin I26P mutation in a lipid bilayer.

The amylin peptide in a dioleoylphosphatidylcholine (DOPC) bilayer is studied using united atom molecular dynamics (MD) simulations. Dynamics and transport properties of the peptide and the phospholipid bilayer are investigated. The lateral diffusion of DOPC is in the order of 10(-8) cm(2) s(-1), which is in agreement with the experimental results. The order parameter and density profile for phospholipid molecules in the bilayer are calculated. The secondary structure of amylin peptide shows that the amino acids in two terminals are structureless and two α-helical segments in the peptide are connected through an unstructured link. This structure is similar to the experimental structure in the membrane-mimicking media. Free energy calculations of the Ile26 â†’ Pro mutation in the amylin peptide are performed in the bilayer and in aqueous solution using molecular dynamics simulations and a thermodynamic cycle. It is shown that in the mutated peptide in aqueous solution, the α-helix structure changes to a 5-helix, whereas this configuration is preserved in the bilayer environment. It is interesting that the accessible surface area increases for hydrophobic residues in the bilayer and for hydrophilic residues in aqueous solution as the coupling parameter changes from 0 to 1. These results are significant to understanding the aggregation mechanism of human amylin monomers in membranes to the dimers, trimers, oligomers, and fibrils associated with the type 2 diabetes at the atomic level.

Efficient parameterization of torsional terms for force fields.

A novel method is presented for fitting force-field dihedral angles using an ensemble of structures generated from an ab initio Monte Carlo simulation. Importance sampling is used to achieve an efficient algorithm using a low level of theory to minimize the system at each step with the dihedral angles constrained, followed by dihedral fitting using the single point energies at a higher level of theory. The resulting method is an order of magnitude more efficient than the traditional method of doing a constrained scan over each dihedral independently. Also as the sampling is more uniformly distributed, the full surface is approximated to a greater accuracy. The dihedral fitting is done with a nonlinear optimization method to vary the phase as well as the force constant. The utility of the method is demonstrated by fitting dihedrals of methyl L-lactate, diisopropyl fluorophosphate, isopentenyl phosphate, a leucine dipeptide, and two inhibitors of Signal Transducer and Activator of Transcription 5. The results show that the Monte Carlo scheme is more efficient than constrained scans and is particularly effective at approximating the underlying potential energy surface when the dihedral degrees are coupled.

Structural and spectroscopic characterization of iron(II), cobalt(II), and nickel(II) ortho-dihalophenolate complexes: insights into metal-halogen secondary bonding.

Metal complexes incorporating the tris(3,5-diphenylpyrazolyl)borate ligand (Tp(Ph2)) and ortho-dihalophenolates were synthesized and characterized in order to explore metal-halogen secondary bonding in biorelevant model complexes. The complexes Tp(Ph2)ML were synthesized and structurally characterized, where M was Fe(II), Co(II), or Ni(II) and L was either 2,6-dichloro- or 2,6-dibromophenolate. All six complexes exhibited metal-halogen secondary bonds in the solid state, with distances ranging from 2.56 Å for the Tp(Ph2)Ni(2,6-dichlorophenolate) complex to 2.88 Å for the Tp(Ph2)Fe(2,6-dibromophenolate) complex. Variable temperature NMR spectra of the Tp(Ph2)Co(2,6-dichlorophenolate) and Tp(Ph2)Ni(2,6-dichlorophenolate) complexes showed that rotation of the phenolate, which requires loss of the secondary bond, has an activation barrier of ~30 and ~37 kJ/mol, respectively. Density functional theory calculations support the presence of a barrier for disruption of the metal-halogen interaction during rotation of the phenolate. On the other hand, calculations using the spectroscopically calibrated angular overlap method suggest essentially no contribution of the halogen to the ligand-field splitting. Overall, these results provide the first quantitative measure of the strength of a metal-halogen secondary bond and demonstrate that it is a weak noncovalent interaction comparable in strength to a hydrogen bond. These results provide insight into the origin of the specificity of the enzyme 2,6-dichlorohydroquinone 1,2-dioxygenase (PcpA), which is specific for ortho-dihalohydroquinone substrates and phenol inhibitors.

Derivation of a Markov state model of the dynamics of a protein-like chain immersed in an implicit solvent.

A Markov state model of the dynamics of a protein-like chain immersed in an implicit hard sphere solvent is derived from first principles for a system of monomers that interact via discontinuous potentials designed to account for local structure and bonding in a coarse-grained sense. The model is based on the assumption that the implicit solvent interacts on a fast time scale with the monomers of the chain compared to the time scale for structural rearrangements of the chain and provides sufficient friction so that the motion of monomers is governed by the Smoluchowski equation. A microscopic theory for the dynamics of the system is developed that reduces to a Markovian model of the kinetics under well-defined conditions. Microscopic expressions for the rate constants that appear in the Markov state model are analyzed and expressed in terms of a temperature-dependent linear combination of escape rates that themselves are independent of temperature. Excellent agreement is demonstrated between the theoretical predictions of the escape rates and those obtained through simulation of a stochastic model of the dynamics of bond formation. Finally, the Markov model is studied by analyzing the eigenvalues and eigenvectors of the matrix of transition rates, and the equilibration process for a simple helix-forming system from an ensemble of initially extended configurations to mainly folded configurations is investigated as a function of temperature for a number of different chain lengths. For short chains, the relaxation is primarily single-exponential and becomes independent of temperature in the low-temperature regime. The profile is more complicated for longer chains, where multi-exponential relaxation behavior is seen at intermediate temperatures followed by a low temperature regime in which the folding becomes rapid and single exponential. It is demonstrated that the behavior of the equilibration profile as the temperature is lowered can be understood in terms of the number of relaxation modes or "folding pathways" that contribute to the evolution of the state populations.

Transcriptional noise, gene activation, and roles of SAGA and Mediator Tail measured using nucleotide recoding single-cell RNA-seq.

We describe a time-resolved nascent single-cell RNA sequencing (RNA-seq) approach that measures gene-specific transcriptional noise and the fraction of active genes in S. cerevisiae. Most genes are expressed with near-constitutive behavior, while a subset of genes show high mRNA variance suggestive of transcription bursting. Transcriptional noise is highest in the cofactor/coactivator-redundant (CR) gene class (dependent on both SAGA and TFIID) and strongest in TATA-containing CR genes. Using this approach, we also find that histone gene transcription switches from a low-level, low-noise constitutive mode during M and M/G1 to an activated state in S phase that shows both an increase in the fraction of active promoters and a switch to a noisy and bursty transcription mode. Rapid depletion of cofactors SAGA and MED Tail indicates that both factors play an important role in stimulating the fraction of active promoters at CR genes, with a more modest role in transcriptional noise.