Simple Biochemical Pathways far from Steady State Can Provide Switchlike and Integrated Responses

Covalent modification cycles (systems in which the activity of a substrate is regulated by the action of two opposing enzymes) and ligand/receptor interactions are ubiquitous in signaling systems and their steady-state properties are well understood. However, the behavior of such systems far from steady state remains unclear. Here, we analyze the properties of covalent modification cycles and ligand/receptor interactions driven by the accumulation of the activating enzyme and the ligand, respectively. We show that for a large range of parameters both systems produce sharp switchlike response and yet allow for temporal integration of the signal, two desirable signaling properties. Ultrasensitivity is observed also in a region of parameters where the steady-state response is hyperbolic. The temporal integration properties are tunable by regulating the levels of the deactivating enzyme and receptor, as well as by adjusting the rate of accumulation of the activating enzyme and ligand. We propose that this tunability is used to generate precise responses in signaling systems.     

Simple Biochemical Pathways far from Steady State Can Provide Switchlike and Integrated Responses

Covalent modification cycles (systems in which the activity of a substrate is regulated by the action of two opposing enzymes) and ligand/receptor interactions are ubiquitous in signaling systems and their steady-state properties are well understood. However, the behavior of such systems far from steady state remains unclear. Here, we analyze the properties of covalent modification cycles and ligand/receptor interactions driven by the accumulation of the activating enzyme and the ligand, respectively. We show that for a large range of parameters both systems produce sharp switchlike response and yet allow for temporal integration of the signal, two desirable signaling properties. Ultrasensitivity is observed also in a region of parameters where the steady-state response is hyperbolic. The temporal integration properties are tunable by regulating the levels of the deactivating enzyme and receptor, as well as by adjusting the rate of accumulation of the activating enzyme and ligand. We propose that this tunability is used to generate precise responses in signaling systems.     

Signaling complexes control the chemotaxis kinase by altering its apparent rate constant of autophosphorylation

Autophosphorylating histidine kinase CheA is central to signaling in bacterial chemotaxis. The kinase donates its phosphoryl group to two response regulators, CheY that controls flagellar rotation and thus motility and CheB, crucial for sensory adaptation. As measured by coupled CheY phosphorylation, incorporation into signaling complexes activates the kinase ～1000‐fold and places it under control of chemoreceptors. By the same assay, receptors modulate kinase activity ～100‐fold as a function of receptor ligand occupancy and adaptational modification. These changes are the essence of chemotactic signaling. Yet, the enzymatic properties affected by incorporation into signaling complexes, by chemoreceptor ligand binding or by receptor adaptational modification are largely undefined. To investigate, we performed steady‐state kinetic analysis of autophosphorylation using a liberated kinase phosphoryl‐accepting domain, characterizing kinase alone, in isolated core signaling complexes and in small arrays of core complexes assembled in vitro with receptors contained in isolated native membranes. Autophosphorylation in signaling complexes was measured as a function of ligand occupancy and adaptational modification. Activation by incorporation into signaling complexes and modulation in complexes by ligand occupancy and adaptational modification occurred largely via changes in the apparent catalytic rate constant (k_cat). Changes in the autophosphorylation k_cat accounted for most of the ～1000‐fold kinase activation in signaling complexes observed for coupled CheY phosphorylation, and the ～100‐fold inhibition by ligand occupancy or modulation by adaptational modification. Our results indicate no more than a minor role in kinase control for simple sequestration of the autophosphorylation substrate. Instead they indicate direct effects on the active site.     

PROBING THE CIRCADIAN OSCILLATOR OF A MAMMAL BY TWO-PULSE PERTURBATIONS

When organisms are maintained under constant conditions of light and temperature, their endogenous circadian rhythms free run, manifesting their intrinsic period. The phases of these free-running rhythms can be shifted by stimuli of light, temperature, and drugs. The change from one free-running steady state to another following a perturbation often involves several transient cycles (cycles of free-running rhythm drifting slowly to catch up with the postperturbation steady state). Although the investigation of oscillator kinetics in circadian rhythms of both insects and mammals has revealed that the circadian pacemaker phase shifts instantaneously, the phenomenon of transient cycles has remained an enigma. We probed the phases of the transient cycles in the locomotor activity rhythm of the field mouse Mus booduga, evoked by a single light pulse (LP), using LPs at critically timed phases. The results of our experiments indicate that the transient cycles generated during transition from one steady state to another steady state do not represent the state of the circadian pacemaker (basic oscillator) controlling the locomotor activity rhythm in Mus booduga. (Chronobiology International, 17(2), 129–136, 2000)     

The role of neuronal death during the development of topographically ordered projections: a computational approach

At the time of synaptogenesis typically 50% of the neurons die. The biological role of this is still unclear, but there is evidence in the visual system that many neurons projecting to topographically inappropriate parts of their target are eliminated to improve the accuracy of the mapping. The signaling that determines neuronal survival involves electrical activity and trophic factors. Based on these observations, we have elaborated a computational model for the self-organization of a two-layered neural network. We observe changes in the topographical organization between the two layers. In layer 1, a traveling wave of electrical activity is used as input. Activity transmission to layer 2 can generate, according to a Hebbian rule, a retrograde death signal that is compensated by a trophic survival signal generated by the target cells. Approximately 50% of the neurons die, and we observe refinement in the topography between the two layers. In alternative versions of the model, we show that an equivalent reorganization can occur through Hebbian synaptic modification alone, but with less precision and efficiency. When the two mechanisms are combined, synaptic modification provides no further improvement over that produced by neuronal death alone. This computational study supports the hypothesis that neuronal death during development can play a role in the refinement of topographical projections in the nervous system. Received: 9 November 1998 / Accepted in revised form: 14 April 1999     

Retroactive signaling in short signaling pathways

In biochemical signaling pathways without explicit feedback connections, the core signal transduction is usually described as a one-way communication, going from upstream to downstream in a feedforward chain or network of covalent modification cycles. In this paper we explore the possibility of a new type of signaling called retroactive signaling, offered by the recently demonstrated property of retroactivity in signaling cascades. The possibility of retroactive signaling is analysed in the simplest case of the stationary states of a bicyclic cascade of signaling cycles. In this case, we work out the conditions for which variables of the upstream cycle are affected by a change of the total amount of protein in the downstream cycle, or by a variation of the phosphatase deactivating the same protein. Particularly, we predict the characteristic ranges of the downstream protein, or of the downstream phosphatase, for which a retroactive effect can be observed on the upstream cycle variables. Next, we extend the possibility of retroactive signaling in short but nonlinear signaling pathways involving a few covalent modification cycles.     

On the number of steady states in a multiple futile cycle

The multisite phosphorylation-dephosphorylation cycle is a motif repeatedly used in cell signaling. This motif itself can generate a variety of dynamic behaviors like bistability and ultrasensitivity without direct positive feedbacks. In this paper, we study the number of positive steady states of a general multisite phosphorylation–dephosphorylation cycle, and how the number of positive steady states varies by changing the biological parameters. We show analytically that (1) for some parameter ranges, there are at least n + 1 (if n is even) or n (if n is odd) steady states; (2) there never are more than 2n − 1 steady states (in particular, this implies that for n = 2, including single levels of MAPK cascades, there are at most three steady states); (3) for parameters near the standard Michaelis–Menten quasi-steady state conditions, there are at most n + 1 steady states; and (4) for parameters far from the standard Michaelis–Menten quasi-steady state conditions, there is at most one steady state.      

Operating regimes of signaling cycles: statics, dynamics, and noise filtering

A ubiquitous building block of signaling pathways is a cycle of covalent modification (e.g., phosphorylation and dephosphorylation in MAPK cascades). Our paper explores the kind of information processing and filtering that can be accomplished by this simple biochemical circuit. Signaling cycles are particularly known for exhibiting a highly sigmoidal (ultrasensitive) input–output characteristic in a certain steady-state regime. Here, we systematically study the cycle's steady-state behavior and its response to time-varying stimuli. We demonstrate that the cycle can actually operate in four different regimes, each with its specific input–output characteristics. These results are obtained using the total quasi–steady-state approximation, which is more generally valid than the typically used Michaelis-Menten approximation for enzymatic reactions. We invoke experimental data that suggest the possibility of signaling cycles operating in one of the new regimes. We then consider the cycle's dynamic behavior, which has so far been relatively neglected. We demonstrate that the intrinsic architecture of the cycles makes them act—in all four regimes—as tunable low-pass filters, filtering out high-frequency fluctuations or noise in signals and environmental cues. Moreover, the cutoff frequency can be adjusted by the cell. Numerical simulations show that our analytical results hold well even for noise of large amplitude. We suggest that noise filtering and tunability make signaling cycles versatile components of more elaborate cell-signaling pathways.     

Investigating the Role of 4‐Tert Butylpyridine in Perovskite Solar Cells

The majority of hole‐transporting layers used in n‐i‐p perovskite solar cells contain 4‐tert butylpyridine (tBP). High power‐conversion efficiencies and, in particular, good steady‐state performance appears to be contingent on the inclusion of this additive. On the quest to improve the steady state efficiencies of the carbon nanotube‐based hole‐transporter system, this study has found that the presence of tBP results in an extraordinary improvement in the performance of these devices. By deconstructing a prototypical device and investigating the effect of tBP on each individual layer, the results of this study indicate that this performance enhancement must be due to a direct chemical interaction between tBP and the perovskite material. This study proposes that tBP serves to p‐dope the perovskite layer and investigates this theory with poling and work function measurements.     

The structure and formation of the egg-shell of Syphacia obvelata Rudolphi (Nematoda: Oxyurida)

The egg of Syphacia obvelata is a flattened elipsoid. The egg-shell consists of 5 layers: external uterine layer, internal uterine layer, vitelline layer, chitinous layer and lipid layer. An operculum is present at one pole of the egg. The opercular groove consists of a break in the uterine layers and the modification of the chitinous layer by the deposition of lipoprotein material. On the curved side of the egg the uterine layers are modified to form alternate ridges and depressions. Discrete spaces are present in the internal uterine layer between the ridges. These are open to the exterior via pores in the external uterine layer. The structure of the uterine layers is quite different on the flattened side of the egg. The morphology of the reproductive system and the formation of the egg-shell is described. It is suggested that the complex structure of the uterine layers of oxyurids forms by a self-assembly process.     

Phosphorylation of Pkp1 by RIPK4 regulates epidermal differentiation and skin tumorigenesis

Tissue homeostasis of skin is sustained by epidermal progenitor cells localized within the basal layer of the skin epithelium. Post‐translational modification of the proteome, such as protein phosphorylation, plays a fundamental role in the regulation of stemness and differentiation of somatic stem cells. However, it remains unclear how phosphoproteomic changes occur and contribute to epidermal differentiation. In this study, we survey the epidermal cell differentiation in a systematic manner by combining quantitative phosphoproteomics with mammalian kinome cDNA library screen. This approach identified a key signaling event, phosphorylation of a desmosome component, PKP1 (plakophilin‐1) by RIPK4 (receptor‐interacting serine–threonine kinase 4) during epidermal differentiation. With genome‐editing and mouse genetics approach, we show that loss of function of either Pkp1 or Ripk4 impairs skin differentiation and enhances epidermal carcinogenesis in vivo. Phosphorylation of PKP1's N‐terminal domain by RIPK4 is essential for their role in epidermal differentiation. Taken together, our study presents a global view of phosphoproteomic changes that occur during epidermal differentiation, and identifies RIPK‐PKP1 signaling as novel axis involved in skin stratification and tumorigenesis.     

Efficient Indium‐Doped TiOx Electron Transport Layers for High‐Performance Perovskite Solar Cells and Perovskite‐Silicon Tandems

In addition to a good perovskite light absorbing layer, the hole and electron transport layers play a crucial role in achieving high‐efficiency perovskite solar cells. Here, a simple, one‐step, solution‐based method is introduced for fabricating high quality indium‐doped titanium oxide electron transport layers. It is shown that indium‐doping improves both the conductivity of the transport layer and the band alignment at the ETL/perovskite interface compared to pure TiO₂, boosting the fill‐factor and voltage of perovskite cells. Using the optimized transport layers, a high steady‐state efficiency of 17.9% for CH₃NH₃PbI₃‐based cells and 19.3% for Cs_0.05(MA_0.17FA_0.83)_0.95Pb(I_0.83Br_0.17)₃‐based cells is demonstrated, corresponding to absolute efficiency gains of 4.4% and 1.2% respectively compared to TiO₂‐based control cells. In addition, a steady‐state efficiency of 16.6% for a semi‐transparent cell is reported and it is used to achieve a four‐terminal perovskite‐silicon tandem cell with a steady‐state efficiency of 24.5%.     

COMPETITION AND ACCOMMODATION BETWEEN APICAL LAYERS AND THEIR DERIVATIVES IN THE ONTOGENY OF CHIMERAL SHOOTS OF PELARGONIUM X HORTORUM

Two mutant plastogenes in all possible chimeral combinations were followed in Pelargonium X hortorum Bailey (geranium) shoots. The part of stem, leaf, or other structure derived from each apical layer was clearly apparent on a cell to cell basis. Shoots typically were composed of derivatives of three apical layers but we found derivatives of as many as four apical layers in some leaves and of five layers in some stems. In chimeras with one of the mutants, Dpl W₁, the amount of tissue derived from the various apical layers was the same, whether the layer was mutant or wild type. We suggest that there are independent apical layers and cell lineages derived from them in nonchimeral shoots, and that their contribution in normal ontogeny is like that of the layers in Dpl W₁ chimeras. In chimeras carrying the second mutant, Dpl W₂, there was much less tissue derived from mutant than from wild-type apical layers. The phenotypic expression of the plastogenes was unchanged by their transmission through male or female gametes. Comparisons of the ontogeny of geranium plants carrying the W₁ or W₂ mutant suggested that, while there was competition between the apical layers and between their derivatives, the genome imposed a definite harmonious interaction or accommodation which led to a final normal morphology of all plant parts and organs through quite different ontogenetic pathways.     

Structure and Elemental Composition of the Cyst Wall of Echinosphaerium nucleofilum Barrett (Heliozoea,Actinophryida)1

The structure of the cyst wall of the heliozoon Echinosphaerium nucleofilum has been investigated using light microscopy, scanning and transmission electron microscopy, and X-ray microanalysis. The cyst wall is a composite structure of seven or eight layers. These are: an enveloping gelatinous layer; a layer of siliceous spheroidal bodies; an electron-dense supporting membrane; a broad electron-lucent zone; an electron-dense layer; a layer of helicoidally packed material; and one or two layers with a granular appearance lying next to the plasma membrane of the encysted organism. The structure of the cyst wall closely resembles that of Actinophrys sol, confirming the close relationship of these actinophryid heliozoa while emphasizing their distinctiveness from other amoeboid protista.     

Poly(ethylene oxide) layers grafted to dopamine-melanin anchoring layer: stability and resistance to protein adsorption

In this study, we propose substrate-independent modification for creating a protein-repellent surface based on dopamine-melanin anchoring layer used for subsequent binding of poly(ethylene oxide) (PEO) from melt. We verified that the dopamine-melanin layer can be formed on literally any substrate and could serve as the anchoring layer for subsequent grafting of PEO chains. Grafting of PEO from melt in a temperature range 70-110 °C produces densely packed PEO layers showing exceptionally low protein adsorption when exposed to the whole blood serum or plasma. The PEO layers prepared from melt at 110 °C retained the protein repellent properties for as long as 10 days after their exposure to physiological-like conditions. The PEO-dopamine-melanin modification represents a simple and universal surface modification method for the preparation of protein repellent surfaces that could serve as a nonfouling background in various applications, such as optical biosensors and tissue engineering.     

Radial construction of an arterial wall

Some of the most serious diseases involve altered size and structure of the arterial wall. Elucidating how arterial walls are built could aid understanding of these diseases, but little is known about how concentric layers of muscle cells and the outer adventitial layer are assembled and patterned around endothelial tubes. Using histochemical, clonal, and genetic analysis in mice, here we show that the pulmonary artery wall is constructed radially, from the inside out, by two separate but coordinated processes. One is sequential induction of successive cell layers from surrounding mesenchyme. The other is controlled invasion of outer layers by inner layer cells through developmentally regulated cell reorientation and radial migration. We propose that a radial signal gradient controls these processes and provide?evidence that PDGF-B and at least one other signal contribute. Modulation of such radial signaling pathways may underlie vessel-specific differences and pathological changes in arterial wall size and structure. VIDEO ABSTRACT:     

ROLE OF THE LIGHT-DARK CYCLE AND MEDIUM COMPOSITION ON THE PRODUCTION OF COCCOLITHS BY EMILIANIA HUXLEYI (HAPTOPHYCEAE)1

Production of coccoliths by cells of Emiliania huxleyi (Lohmann) Hay and Mohler was measured during exposure of the cells to two diel light-dark cycles (16:8 h). During the light period about eight coccoliths per cell were formed at a constant rate of one coccolith per 2 h. Cells divided during the first half of the dark period. No coccolith production took place during the dark period. With electron microscopy we found early-stage, coccolith-production compartments in cells after mitosis while still in the dark. No calcification was observed in these compartments. Cells grown on enriched seawater (Eppley's medium) tended to produce enough coccoliths to cover the cell in a single layer. When these cells reached the stationary phase coccolith production stopped. Coccolith production was induced by removal of extracellular coccoliths. Cells grown on medium containing 2% of the nitrate and phosphate of Eppley's medium tended to produce coccoliths in the stationary phase. This resulted in the formation of multiple layers of coccoliths. The multiple covering was restored after decalcification of stationary cells. Formation of multiple layers of coccoliths may help the cells reach deeper, nutrient-rich water by increasing the sinking rate of the cells.     

Regeneration of N.W. African Pinus halepensis forests following fire

Pinus halepensis forests of N.W. Algeria are subjected to frequent fires. During the fire the aboveground parts of plants are completely burned but only a few species are killed. Most perennial herb and shrub species survive owing to their underground organs and regenerate vegetatively in the next moist period. The semi-shrubs regenerate both vegetatively and from seeds. The most intensive growth of the shrub layer occurs during the first 2 years and in the 5th year, it reaches a height of 1–1.5 m. Pinus halepensis is completely killed by the fire and it regenerates from seeds only. The regeneration is retarded during the first 2–3 years, apparently by competition of the rapidly developing shrubs and semi-shrubs with P. halepensis. In the following years, there is a more rapid increase in both density and height, although by the 5th year after fire, the height does not exceed 0.5 m. The young trees overtop the shrub layer between 10 and 15 years after fire. The increase in density and cover supress the lower layers, in particular the herb layer. The reduction in density of trees in the following decades enables the herb layer to reconstitute its composition and cover.This process of regeneration resembles forest growth cycles rather than a secondary succession. The shrub and herb layers maintain their identity as they are mostly formed of the same individuals as before the fire; they merely regenerated their aboveground organs. Only the tree layer regenerates anew after the fire.