Identifying Patients with Polycythemia Vera at Risk of Thrombosis after Hydroxyurea Initiation: The Polycythemia Vera-Advanced Integrated Models (PV-AIM) Project.

Patients with polycythemia vera (PV) are at significant risk of thromboembolic events (TE). The PV-AIM study used the Optum® de-identified Electronic Health Record dataset and machine learning to identify markers of TE in a real-world population. Data for 82,960 patients with PV were extracted: 3852 patients were treated with hydroxyurea (HU) only, while 130 patients were treated with HU and then changed to ruxolitinib (HU-ruxolitinib). For HU-alone patients, the annualized incidence rates (IR; per 100 patients) decreased from 8.7 (before HU) to 5.6 (during HU) but increased markedly to 10.5 (continuing HU). Whereas for HU-ruxolitinib patients, the IR decreased from 10.8 (before HU) to 8.4 (during HU) and was maintained at 8.3 (after switching to ruxolitinib). To better understand markers associated with TE risk, we built a machine-learning model for HU-alone patients and validated it using an independent dataset. The model identified lymphocyte percentage (LYP), neutrophil percentage (NEP), and red cell distribution width (RDW) as key markers of TE risk, and optimal thresholds for these markers were established, from which a decision tree was derived. Using these widely used laboratory markers, the decision tree could be used to identify patients at high risk for TE, facilitate treatment decisions, and optimize patient management.

Genetic cooperativity in multi-layer networks implicates cell survival and senescence in the striatum of Huntington's disease mice synchronous to symptoms.

MOTIVATION: Huntington's disease (HD) may evolve through gene deregulation. However, the impact of gene deregulation on the dynamics of genetic cooperativity in HD remains poorly understood. Here, we built a multi-layer network model of temporal dynamics of genetic cooperativity in the brain of HD knock-in mice (allelic series of Hdh mice). To enhance biological precision and gene prioritization, we integrated three complementary families of source networks, all inferred from the same RNA-seq time series data in Hdh mice, into weighted-edge networks where an edge recapitulates path-length variation across source-networks and age-points. RESULTS: Weighted edge networks identify two consecutive waves of tight genetic cooperativity enriched in deregulated genes (critical phases), pre-symptomatically in the cortex, implicating neurotransmission, and symptomatically in the striatum, implicating cell survival (e.g. Hipk4) intertwined with cell proliferation (e.g. Scn4b) and cellular senescence (e.g. Cdkn2a products) responses. Top striatal weighted edges are enriched in modulators of defective behavior in invertebrate models of HD pathogenesis, validating their relevance to neuronal dysfunction in vivo. Collectively, these findings reveal highly dynamic temporal features of genetic cooperativity in the brain of Hdh mice where a 2-step logic highlights the importance of cellular maintenance and senescence in the striatum of symptomatic mice, providing highly prioritized targets. AVAILABILITY AND IMPLEMENTATION: Weighted edge network analysis (WENA) data and source codes for performing spectral decomposition of the signal (SDS) and WENA analysis, both written using Python, are available at http://www.broca.inserm.fr/HD-WENA/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Mechanical stress increases brain amyloid ß, tau, and α-synuclein concentrations in wild-type mice.

INTRODUCTION: Exposure to traumatic brain injury is a core risk factor that predisposes an individual to sporadic neurodegenerative diseases. We provide evidence that mechanical stress increases brain levels of hallmark proteins associated with neurodegeneration. METHODS: Wild-type mice were exposed to multiple regimens of repetitive mild traumatic brain injury, generating a range of combinations of impact energies, frequencies, and durations of exposure. Brain concentrations of amyloid ß 1-42 (Aß1-42), total tau, and α-synuclein were measured by sandwich enzyme-linked immunosorbent assay. RESULTS: There was a highly significant main effect of impact energy, frequency, and duration of exposure on Aß1-42, tau, and α-synuclein levels (P < .001), and a significant interaction between impact energy and duration of exposure for Aß1-42 and tau (P < .001), but not for α-synuclein. DISCUSSION: Dose-dependent and cumulative influence of repetitive mild traumatic brain injury-induced mechanical stress may trigger and/or accelerate neurodegeneration by pushing protein concentration over the disease threshold.

On the Relation between Chemical Oscillations and Self-Replication.

One proposed scenario for the emergence of biochemical oscillations is that they may have provided the basic mechanism behind cellular self-replication by growth and division. However, alternative scenarios not requiring any chemical oscillation have also been proposed. Each of the various protocell models proposed to support one or another scenario comes with its own set of specific assumptions, which makes it difficult to ascertain whether chemical oscillations are required or not for cellular self-replication. This article compares these two cases within a single whole-cell model framework. This model relies upon a membrane embedding a chemical reaction network (CRN) synthesizing all the cellular constituents, including the membrane, by feeding from an external nutrient. Assuming the osmolarity is kept constant, the system dynamics are governed by a set of nonlinear differential equations coupling the chemical concentrations and the surface-area-to-volume ratio. The resulting asymptotic trajectories are used to determine the cellular shape by minimizing the membrane bending energy (within an approximate predefined family of shapes). While the stationary case can be handled quite generally, the oscillatory one is investigated using a simple oscillating CRN example, which is used to identify features that are expected to hold for any network. It is found that cellular self-replication can be reached with or without chemical oscillations, and that a requirement common to both stationary and oscillatory cases is that a minimum spontaneous curvature of the membrane is required for the cell to divide once its area and volume are both doubled. The oscillatory case can result in a greater variety of cellular shape trajectories but raises additional constraints for cellular division and self-replication: (i) the ratio of doubling time to oscillation period should be an integer, and (ii) if the oscillation amplitude is sufficiently high, then the spontaneous curvature must be below a maximum value to avoid early division before the end of the cycle. Because of these additional stringent constraints, it is likely that early protocells did not rely upon chemical oscillations. Biochemical oscillations typical of modern evolved cells may have emerged later through evolution for other reasons (e.g., metabolic advantage) and must have required additional feedback mechanisms for such a self-replicating system to be robust against even slight environmental variations (e.g., temperature fluctuations).

The Redox Status of Cancer Cells Supports Mechanisms behind the Warburg Effect.

To better understand the energetic status of proliferating cells, we have measured the intracellular pH (pHi) and concentrations of key metabolites, such as adenosine triphosphate (ATP), nicotinamide adenine dinucleotide (NAD), and nicotinamide adenine dinucleotide phosphate (NADP) in normal and cancer cells, extracted from fresh human colon tissues. Cells were sorted by elutriation and segregated in different phases of the cell cycle (G0/G1/S/G2/M) in order to study their redox (NAD, NADP) and bioenergetic (ATP, pHi) status. Our results show that the average ATP concentration over the cell cycle is higher and the pHi is globally more acidic in normal proliferating cells. The NAD+/NADH and NADP+/NADPH redox ratios are, respectively, five times and ten times higher in cancer cells compared to the normal cell population. These energetic differences in normal and cancer cells may explain the well-described mechanisms behind the Warburg effect. Oscillations in ATP concentration, pHi, NAD+/NADH, and NADP+/NADPH ratios over one cell cycle are reported and the hypothesis addressed. We also investigated the mitochondrial membrane potential (MMP) of human and mice normal and cancer cell lines. A drastic decrease of the MMP is reported in cancer cell lines compared to their normal counterparts. Altogether, these results strongly support the high throughput aerobic glycolysis, or Warburg effect, observed in cancer cells.

Necessary and sufficient conditions for protocell growth.

We consider a generic protocell model consisting of any conservative chemical reaction network embedded within a membrane. The membrane results from the self-assembly of a membrane precursor and is semi-permeable to some nutrients. Nutrients are metabolized into all other species including the membrane precursor, and the membrane grows in area and the protocell in volume. Faithful replication through cell growth and division requires a doubling of both cell volume and surface area every division time (thus leading to a periodic surface area-to-volume ratio) and also requires periodic concentrations of the cell constituents. Building upon these basic considerations, we prove necessary and sufficient conditions pertaining to the chemical reaction network for such a regime to be met. A simple necessary condition is that every moiety must be fed. A stronger necessary condition implies that every siphon must be either fed, or connected to species outside the siphon through a pass reaction capable of transferring net positive mass into the siphon. And in the case of nutrient uptake through passive diffusion and of constant surface area-to-volume ratio, a sufficient condition for the existence of a fixed point is that every siphon be fed. These necessary and sufficient conditions hold for any chemical reaction kinetics, membrane parameters or nutrient flux diffusion constants.

Theoretical Analysis of a Self-Replicator With Reduced Template Inhibition Based on an Informational Leaving Group.

The first non-enzymatic self-replicating systems, as proposed by von Kiedrowski (Angew Chem Int Ed Engl 25(10):932-935, 1986) and Orgel (Nature 327(6120):346-347, 1987), gave rise to the analytical background still used today to describe artificial replicators. What separates a self-replicating from an autocatalytic system is the ability to pass on structural information (Orgel, Nature 358(6383):203-209, 1992). Utilising molecular information, nucleic acids were the first choice as prototypical examples. But early self-replicators showed parabolic over exponential growth due to the strongly bound template duplex after template-directed ligation of substrates. We propose a self-replicating scheme with a weakly bound template duplex, using an informational leaving group. Such a scheme is inspired by the role of tRNA as leaving group and information carrier during protein synthesis, and is based on our previous experience with nucleotide chemistry. We analyse theoretically this scheme and compare it to the classical minimal replicator model. We show that for an example hexanucleotide template mirroring that is used by von Kiedrowski (Bioorganic chemistry frontiers, 1993) for the analysis of the classical minimal replicator, the proposed scheme is expected to result in higher template self-replication rate. The proposed self-replicating scheme based on an informational leaving group is expected to outperform the classical minimal replicator because of a weaker template duplex bonding, resulting in reduced template inhibition.

Redesigning the stereospecificity of tyrosyl-tRNA synthetase.

D-Amino acids are largely excluded from protein synthesis, yet they are of great interest in biotechnology. Unnatural amino acids have been introduced into proteins using engineered aminoacyl-tRNA synthetases (aaRSs), and this strategy might be applicable to D-amino acids. Several aaRSs can aminoacylate their tRNA with a D-amino acid; of these, tyrosyl-tRNA synthetase (TyrRS) has the weakest stereospecificity. We use computational protein design to suggest active site mutations in Escherichia coli TyrRS that could increase its D-Tyr binding further, relative to L-Tyr. The mutations selected all modify one or more sidechain charges in the Tyr binding pocket. We test their effect by probing the aminoacyl-adenylation reaction through pyrophosphate exchange experiments. We also perform extensive alchemical free energy simulations to obtain L-Tyr/D-Tyr binding free energy differences. Agreement with experiment is good, validating the structural models and detailed thermodynamic predictions the simulations provide. The TyrRS stereospecificity proves hard to engineer through charge-altering mutations in the first and second coordination shells of the Tyr ammonium group. Of six mutants tested, two are active towards D-Tyr; one of these has an inverted stereospecificity, with a large preference for D-Tyr. However, its activity is low. Evidently, the TyrRS stereospecificity is robust towards charge rearrangements near the ligand. Future design may have to consider more distant and/or electrically neutral target mutations, and possibly design for binding of the transition state, whose structure however can only be modeled.

Chemical Schemes for Maintaining Different Compositions Across a Semi-permeable Membrane with Application to Proto-cells.

Osmotic pressure arising from a higher total chemical concentration inside proto-cells is thought to have played a role in the emergence and selection of self-replicating proto-cells. We present two chemical schemes through which different equilibrium compositions can coexist on each side of a semi-permeable membrane. The first scheme relies upon the concept of moieties and associated number of degrees of freedom. The second scheme relies upon the concept of siphons and of pass reaction capable of transferring matter from outside a siphon into it. Using simple example reaction networks, we show that both schemes are compatible with stationary proto-cell growth with up-concentration, but suffer from shortcomings. To alleviate these we propose a third scheme derived from the second one by having the pass reaction catalyzed by the membrane surface instead of occurring in bulk solution. This may have proven an intermediate step before having the pass reaction occurring only when the nutrient crosses the membrane. This suggests an evolutionary path for the emergence of active transport.

Minimal conditions for protocell stationary growth.

We show that self-replication of a chemical system encapsulated within a membrane growing from within is possible without any explicit feature such as autocatalysis or metabolic closure, and without the need for their emergence through complexity. We use a protocell model relying upon random conservative chemical reaction networks with arbitrary stoichiometry, and we investigate the protocell's capability for self-replication, for various numbers of reactions in the network. We elucidate the underlying mechanisms in terms of simple minimal conditions pertaining only to the topology of the embedded chemical reaction network. A necessary condition is that each moiety must be fed, and a sufficient condition is that each siphon is fed. Although these minimal conditions are purely topological, by further endowing conservative chemical reaction networks with thermodynamically consistent kinetics, we show that the growth rate tends to increase on increasing the Gibbs energy per unit molecular weight of the nutrient and on decreasing that of the membrane precursor.

Cell cycle progression is regulated by intertwined redox oscillators.

The different phases of the eukaryotic cell cycle are exceptionally well-preserved phenomena. DNA decompaction, RNA and protein synthesis (in late G1 phase) followed by DNA replication (in S phase) and lipid synthesis (in G2 phase) occur after resting cells (in G0) are committed to proliferate. The G1 phase of the cell cycle is characterized by an increase in the glycolytic metabolism, sustained by high NAD+/NADH ratio. A transient cytosolic acidification occurs, probably due to lactic acid synthesis or ATP hydrolysis, followed by cytosolic alkalinization. A hyperpolarized transmembrane potential is also observed, as result of sodium/potassium pump (NaK-ATPase) activity. During progression of the cell cycle, the Pentose Phosphate Pathway (PPP) is activated by increased NADP+/NADPH ratio, converting glucose 6-phosphate to nucleotide precursors. Then, nucleic acid synthesis and DNA replication occur in S phase. Along with S phase, unpublished results show a cytosolic acidification, probably the result of glutaminolysis occurring during this phase. In G2 phase there is a decrease in NADPH concentration (used for membrane lipid synthesis) and a cytoplasmic alkalinization occurs. Mitochondria hyperfusion matches the cytosolic acidification at late G1/S transition and then triggers ATP synthesis by oxidative phosphorylation. We hypothesize here that the cytosolic pH may coordinate mitochondrial activity and thus the different redox cycles, which in turn control the cell metabolism.

Filamentation as primitive growth mode?

Osmotic pressure influences cellular shape. In a growing cell, chemical reactions and dilution induce changes in osmolarity, which in turn influence the cellular shape. Using a protocell model relying upon random conservative chemical reaction networks with arbitrary stoichiometry, we find that when the membrane is so flexible that its shape adjusts itself quasi-instantaneously to balance the osmotic pressure, the protocell either grows filamentous or fails to grow. This behavior is consistent with a mathematical proof. This suggests that filamentation may be a primitive growth mode resulting from the simple physical property of balanced osmotic pressure. We also find that growth is favored if some chemical species are only present inside the protocell, but not in the outside growth medium. Such an insulation requires specific chemical schemes. Modern evolved cells such as E. coli meet these requirements through active transport mechanisms such as the phosphotransferase system.