Slow Step Research Articles

Filament Formation Induces a Shape Change and Activation of the Nuclease SgrAI

SgrAI is allosteric and is both activated as well as undergoes a change in substrate specificity (cleaving a secondary class of DNA sequences) upon binding to its primary recognition sequence in DNA. We have investigated the allosteric activation of SgrAI and this unusual expansion of DNA sequence specificity using structural, biophysical, and enzyme kinetic methods. 1– 2A variety of biophysical techniques (native gel electrophoresis, analytical ultracentrifugation, ion mobility native mass spectrometry, x‐ray crystallography and electron microscopy) to investigate complexes formed by SgrAI. Single turnover DNA cleavage assays were used to investigate the reaction mechanism of SgrAI, and global modeling to create a computation model of the full reaction pathway including rate constants for each important step. The biophysical characterizations revealed that SgrAI forms run‐on oligomers or filaments when activated, and that these filaments are induced when SgrAI binds to its primary recognition sequence. SgrAI binds tightly to secondary site sequences, which differ from primary by a single base pair, but fails to cleave them and does not induce filament formation. However, SgrAI bound to the secondary site sequences will join filaments formed from SgrAI bound to primary site sequences. Protein‐protein and protein‐DNA contacts between SgrAI/DNA complexes in a filament stabilize a different conformation of SgrAI, hypothesized to be activated due to a reorganization of critical residues in the active site. DNA cleavage kinetics reveal activation of DNA cleavage activity by SgrAI of 200–1000 fold for primary and secondary site sequences upon filament formation. Full global kinetic analysis reveals a slow second order association step of SgrAI addition to a filament, followed by rapid DNA cleavage, then dissociation of SgrAI from the filament, and finally rapid product (i.e. cleaved DNA) release. The slow association step controls SgrAI activity to form filaments preferentially on DNA contiguous with primary site sequences, and incorporation of secondary site sequences into the filament increases the number of DNA cleavages on the contiguous DNA. Hence, this system allows for rapid cleavage of invading phage DNA (with unprotected primary sites).The SgrAI system uses formation of a filamentous form to stabilize an activated conformation. DNA sequence and slow second order association kinetics control where and when this filament is formed in a way that is tuned for optimal performance of its biological role.Support or Funding InformationThis work was supported by the National Science Foundation under Grant No. MCB‐1410355 (to N.H.).

The Filament Forming Mechanism of SgrAI Endonuclease‐Structural and Kinetic Analysis

Filament formation by enzymes has recently emerged as a new paradigm of enzyme regulation 1.Wide‐spread reversible self‐assembly of enzymes in cells has also been shown to occur in all branches of life and diverse metabolic and biological pathways. We use the model system, SgrAI, to investigate the filament forming enzyme mechanism.Filaments formed by SgrAI have been structurally characterized using cryo‐electron microscopy 2 and the full reaction pathway including all rate constants has been determined using single turnover enzyme reactions, FRET, and global kinetic modeling 3– 4.The structural studies uncover the mechanism of activation of enzyme activity in SgrAI by filament formation, as well as the origin of the unusual expansion of substrate specificity in SgrAI within the filament. A conformational change in the SgrAI dimer, stabilized by protein‐protein and protein‐DNA contacts in the filament, leads directly to a shift in the active site, hypothesized to lead to stronger binding of a second catalytic Mg2+ ion. DNA structure and energetics is hypothesized to control substrate specificity and filament formation by SgrAI such that the secondary class of DNA sequences are cleaved by SgrAI only upon joining a filament formed by SgrAI bound to a primary class of DNA sequences. The full kinetic model showed a slow second order step in filament formation which is shown to control enzyme activity such that only invading phage DNA is cleaved, leaving the host genome untouched. This model also shows that the filament forming mechanism is superior in fast activation of enzyme activity upon initial recognition of primary DNA sequences.Using the SgrAI endonuclease as a model system to study filament forming enzymes, we have discovered that the enzyme is activated by stabilization of an activated conformation by contacts made within the filament. Control of the equilibrium between a low activity, non‐filamentous state and the high activity, filamentous state by DNA structural energetics gives rise to the unusual apparent expansion of DNA sequence specificity of SgrAI by filament formation. Simulations using the full kinetic pathway reveal an advantage in the filament mechanism over other non‐filamentous mechanisms in the speed of activation in response to invading phage DNA, giving strong evolutionary advantage to this filament forming mechanism.Support or Funding InformationThis work was supported by the National Science Foundation under Grant No. MCB‐1410355.

Morpho-Structural, Electrical and Magnetic Behavior of Chemically Synthesized Multiferroic GdMnO₃ Ceramic.

The observations of room temperature ferroelectricity and ferromagnetism for GdMnO₃ (GMO) ceramics have been reported in this paper. The nanocrystalline GMO powder was synthesized by chemical route and then sintered in the form of bulk pellet at 850 °C for 24 h by following slow step sintering schedule. Bulk GMO sample is observed to be polycrystalline in nature having orthorhombic perovskite structure and Pbnm space group. Well connected grains are observed with higher percentage of porosity in the micrographs obtained from field emission scanning electron microscope. The dielectric anomaly has been observed for the sintered sample at 338 K and a wide ferroelectric hysteresis loop has also been observed at room temperature. Room temperature dielectric constant (ε) value of the sample is high (~2736) at the frequency of 100 Hz. The zero field cooled (ZFC) and field cooled (FC) magnetizations shows the antiferromagnetic behavior around 12 K. Sample shows a weak ferromagnetic behavior at 3 K.

Compact expressions for R1ρ relaxation for N-site chemical exchange using Schur decomposition

The rotating-frame spin relaxation rate constant, R1ρ, is a powerful probe of macromolecular chemical and conformational dynamics in relaxation dispersion, CEST, and DEST NMR experiments. The R1ρ relaxation rate constant is given by the absolute value of the largest (least negative) eigenvalue of the Bloch-McConnell evolution matrix; however, estimation of this eigenvalue require inversion of 3 N × 3 N dimensional matrices, in which N is the number of interconverting sites or states for a given nuclear spin in a molecule. The Schur complement is used to reduce the problem of calculating the characteristic polynomial of a 3 N × 3 N matrix to that of calculating the characteristic polynomial of a 3 × 3 matrix. The resulting expressions for N-site chemical exchange are more numerically tractable, because the largest matrix inversion also is of dimension 3 × 3. In addition, the simplifications offered by the Schurr complement conveniently illustrate the effects of fast or slow kinetic steps within an N-site kinetic topology.

Statistical model of forecasting indicators in one area of sustainable development

This study is based on the seventh goal of sustainable goals development and under the title of clean and affordable As a result of the continuing increase in the number of the world's population, the dramatic rise in energy demand, especially in the production of the renewable energy worldwide, was observed . The Arab region has the highest levels of solar radiation. Many Arab countries are still taking slow steps to develop their renewable energy capacity, which has led many Arab countries today to seek to meet this demand through renewable energy solutions and improved energy efficiency. The field of study presented in this research includes a proposal for the statistical model to analyze the prediction in the field of solar energy, which is one of the most important areas of renewable energy, by providing a comparison between the time series model and the simple regression model to predict data which has been obtained in the field of solar energy.

Adiposity and Slow Stepping Rate Are Related to Under-Estimating Steps per Day in African American Women

Purpose: The purpose of this study was twofold: (1) to determine if the Digi-Walker SW-200 (SW-200), New Lifestyles NL-2000 (NL-2000), and Omron HJ-303 (HJ-303) yield similar daily step counts compared to the StepWatch-3; and (2) to determine if pedometer error is influenced by adiposity and/or stepping rate in African American women. Methods: 60 participants (28.0 ± 9.8 y) wore the devices for three weekdays. ANOVAs were performed to determine if body mass index (BMI) and device were related to steps per day, and to determine if BMI and device were related to error. Stepwise linear regressions were performed to determine which variables contributed to pedometer error. Results: StepWatch-3 counted significantly more steps than all other devices within each BMI category (p < .01). The NL-2000 had significantly less error in the normal (−13.4%) and overweight (−14.9%) groups compared to the SW-200 (−26.2% and −33.3%) and HJ-303 (−32.5% ad −31.5%) (p < .05). The SW-200 had significantly more error in the obese group (−50.7%) compared to the NL-2000 (−17.1%) and HJ-303 (−26.0%) (p < .05). NL-2000 error was not related to any variables while the SW-200 error was related to waist circumference (WC) and the HJ-303 error was related to percentage of slow steps. Conclusion: In African American women adiposity is more strongly related to more pedometer error in a device using a spring-levered mechanism (SW-200). Accumulating steps at a slow rate is related to more pedometer error when using a device with a step filter (HJ-303).

Sudden heating of H3PO4-loaded coconut shell in CO2 flow to produce super activated carbon and its application for benzene adsorption

Super activated carbons (SACs) can be widely used in adsorption and separation, but it remains challenging to find a facile method to prepare such SACs. In this research, we proposed to synthesize SACs via suddenly heating H3PO4-loaded coconut shell in CO2 flow, without using a traditional slow heating step. The utilization of sudden heating instead of slow heating was favorable for saving preparation time and controlling heating process. Moreover, the SAC prepared by sudden heating at impregnation ratio of 1 in CO2 flow showed a high specific surface area of 2648 m2/g. The SAC made at impregnation ratio of 2 showed a large pore volume of 1.762 cm3/g, superior benzene adsorption capacity (1419 mg/g), and excellent recyclability (no benzene adsorption capacity loss over 7 adsorption/desorption cycles). Both H3PO4 and CO2 contributed to the development of SACs, but the latter contributed more. This research provides a new route to prepare SACs with high application potentials in benzene adsorption.

Intermolecular Trifluoromethyl-Hydrazination of Alkenes Enabled by Organic Photoredox Catalysis.

A metal-free and redox-neutral trifluoromethyl-hydrazination method of alkenes is described. With a commercially available photocatalyst and low-cost reagents, both conjugated and isolated alkenes could be converted to β-trifluoromethyl hydrazines in good to excellent yields. The versatile hydrazine group can be either used for heterocycle synthesis or cleaved to afford a corresponding amine. Mechanistic studies suggested this reaction goes through a radical addition cascade pathway, featuring a fast and irreversible trifluoromethylation and a relatively slow radical hydrazination step.

Oxidation of procainamide by diperiodatocuprate(III) complex in aqueous alkaline medium: a comparative kinetic study

A comparative study of uncatalysed and ruthenium(III) catalyzed oxidation of procainamide(PAH) was carried out spectrophotometrically using diperiodatocuprate(III) in aqueous alkali. Stoichiometry observed was 1:2 (PAH: DPC). The reaction was of first order in both catalyzed and uncatalyzed cases, with respect to [DPC] & [Ru(III)], less than unit order in [PAH] and [alkali]. Negative fraction in [periodate]. Other kinetic parameters viz., ionic strength, dielectric constant, temperature effect and intervention of free radical were also studied. The main oxidation products were characterized by different techniques. The activation parameters with respect to slow step of the mechanism, also the thermodynamic quantities were determined.

Stochastic yield catastrophes and robustness in self-assembly.

A guiding principle in self-assembly is that, for high production yield, nucleation of structures must be significantly slower than their growth. However, details of the mechanism that impedes nucleation are broadly considered irrelevant. Here, we analyze self-assembly into finite-sized target structures employing mathematical modeling. We investigate two key scenarios to delay nucleation: (i) by introducing a slow activation step for the assembling constituents and, (ii) by decreasing the dimerization rate. These scenarios have widely different characteristics. While the dimerization scenario exhibits robust behavior, the activation scenario is highly sensitive to demographic fluctuations. These demographic fluctuations ultimately disfavor growth compared to nucleation and can suppress yield completely. The occurrence of this stochastic yield catastrophe does not depend on model details but is generic as soon as number fluctuations between constituents are taken into account. On a broader perspective, our results reveal that stochasticity is an important limiting factor for self-assembly and that the specific implementation of the nucleation process plays a significant role in determining the yield.

Enabling the Direct Enzymatic Dehydration of d-Glycerate to Pyruvate as the Key Step in Synthetic Enzyme Cascades Used in the Cell-Free Production of Fine Chemicals

A high degree of dependence on fossil fuels is one of the major problems faced by modern societies. d-Glucose and glycerol have emerged in recent years as prospective replacements for fossil fuels used in the production of high-value chemicals, and cell-free bioproduction routes are expected to play a crucial role in such processes. Recently, several synthetic cascades used for the cell-free biotransformations of d-glucose and glycerol to pyruvate and beyond have been described. However, these were limited by the very slow dehydration step of d-glycerate to pyruvate catalyzed by a dehydratase from Sulfolobus solfataricus (SsDHAD), making this step by far the major bottleneck. By combining the vast number of available genomes with a sequence-based discovery approach, we have identified signature sequences leading to the discovery of two distinct classes of dehydratases which exhibit promising activity and total turnover number (TTN) toward d-glycerate. In particular, the dehydratase from Paralcaligenes ureilyticus (PuDHT) demonstrated >100-fold higher activity and TTN for d-glycerate in comparison to SsDHAD. In addition, PuDHT showed exceptionally high activity and TTN toward d-gluconate. The replacement of SsDHAD by PuDHT in our model cascade from d-glucose to ethanol enhanced the production rate 10-fold, reaching a 92% theoretical yield at 50 °C. PuDHT was also suitable for the conversion of glycerol to pyruvate at ambient temperature, leading to a >5-fold improvement in production rate in comparison to the system utilizing SsDHAD at 50 °C and attaining 97% of the theoretical yield. PuDHT was also compatible when crude glycerol was used as the substrate, and it no longer caused a bottleneck in the enzymatic cascade.

Studies on the oxidative degradation of paracetamol by a μ-oxo-diiron(III) complex

In higher organisms, metalloenzymes like cytochrome P450, containing a Fe(III) metal center, play an active role in metabolism of paracetamol (APAP). Here, we have chosen a mimicking μ-oxo-diiron complex, [Fe(III)2(μ-O)(phen)4(H2O)2]4+(1, phen = 1,10-phenanthroline), to study spectrophotometrically the kinetics of the redox interactions with APAP. In acidic buffer media (pH = 3.4–5.1), APAP quantitatively reduces 1 following first-order reaction kinetics. Each molecule of 1 accepts two electrons from APAP and is reduced to ferroin [Fe(phen)3]2+. On oxidation, APAP produces N-acetyl-p-benzoquinone imine (NAPQI), which on hydrolysis results in a mixture of benzoquinone, quinone oxime, acetamide, and acetic acid. In reaction media due to successive deprotonations, 1 exists in equilibrium with the species [Fe(III)2(μ-O)(phen)4(H2O)(OH)]3+(1a) and [Fe(III)2(μ-O)(phen)4(OH)2]2+(1b) (pKa= 3.71 and 5.28, respectively). The kinetic analyses suggest for an unusual reactivity order as 1 < 1a ≫ 1b. The mechanistic possibilities suggest that although 1 is reduced by concerted electron transfer (ET) – proton transfer (PT) mechanism, 1a and 1b may be reduced by a concerted PT–ET mechanism where a slow proton-abstraction step is followed by a rapid ET process. It seems that the initial activation of the bridging μ-oxo group by a proton-abstraction results in the higher reactivity of 1a.

Dynamic performance and control strategy of CO2-mixture transcritical power cycle for heavy-duty diesel engine waste-heat recovery

Carbon dioxide mixture transcritical power cycle (CMTPC) is considered as a new promising technology for waste heat recovery (WHR), while there are still critical challenges arising from the transient fluctuations of heat sources when it comes to the heavy-duty diesel engines (HDDEs) applications. This paper presents a dynamic model of CMTPC systems, which is carefully validated against experimental data. Constant temperature, constant pressure and optimal control strategies are proposed to realize stable and optimal operation. The dynamic performance of the system with different control strategies is predicted under slow step change and transient change conditions. The results demonstrate that under slow step change conditions, the stability of optimal control system and constant pressure control system is better than that of constant temperature control system. In addition, the net power output keeps maximum under the optimal control because of the compromise between operating pressure and temperature. However, under transient conditions the control performance of optimal control is not as obvious as under slow step change conditions. Compared with open loop system, the improvement of system power by optimal control under slow step change is 2.31%, while that is only 0.07% under transient change condition. By contrast, it is found that the constant pressure control achieves the best power improvement of 0.58% under transient conditions because of the satisfactory control effect on operating pressure and relatively small fluctuations of expander inlet temperature. In a word, different control strategies behave obviously different performance under various conditions.

Comparative study of the products from the pyrolysis of raw and hydrolyzed baru wastes

Plant biomass is a complex mixture formed mainly by cellulose, hemicellulose, and lignin, being of great importance as a source of renewable assets of great interest to industries, due to millions of tons/year of wastes produced and their great potential to yield a wide range of high added-value products. In this study, processes and physicochemical characterizations were developed for baru wastes (mesocarp and endocarp). This biomass was subjected to acid hydrolysis and slow pyrolysis steps in order to yield biobased products, among them bio-oil (> 28%) and biochar (> 48%). Analytical techniques such as mid-infrared spectroscopy and gas chromatography coupled to mass spectrometry, surface area and pore size, thermal analysis, and proximate and elemental analysis, helped in the elucidation, identification, and quantification of such compounds. Based on these results, it was possible to conclude that baru wastes have potential for application in several branches of industry, since compounds like vanillin (3.2%), syringol (9%), guaiacol (29%), creosol (22%), and palmitic (32%), and levulinic (4%) acids were found in significant amounts.

Abnormal Gait and Neuromuscular Dysfunction Analysis in Patients with Lumbar Disc Herniation

Abstract The lumbar disc herniation (LDH) has become one of the most common orthopedic diseases. But the effects of LDH on the gait and neuromuscular activities have not been well understood. The purpose of this study was to examine the biomechanical and neuromuscular characteristics in LDH during walking. Sixteen patients with LDH and seven healthy subjects participated in the experiment. All the subjects were required to walk straight along a 5-meters line drawn on the ground at their usual speed. During walking, gait kinematic signals and surface electromyography (sEMG) signals were recorded simultaneously. Seven gait parameters (step length, stride length, step width, cadence, double support phase, single support phase and speed) were extracted for gait assessment. Root mean square (RMS) and median frequency (MDF) of the sEMG signals were extracted for evaluation of neuromuscular function. The bivariate correlation analysis was applied to examine the correlation between gait and muscle activation parameters. The patients with LDH showed decreased cadence, increased muscular activation and increased correlation between single support phase and muscle activation. The LDH can result in slower steps and increased muscle activation, indicating decreased gait flexibility and increased energy consumption in patients with LDH during walking. This study sheds light on the abnormal gait and neuromuscular dysfunction in the LDH patients. The methodology developed in this study may facilitate functional evaluation of LDH.

Алгебраїчні шаблони вразливостей бінарного коду

Detection of software systems’ vulnerabilities is an actual problem in the IT industry nowadays. The approaches to the solution of this problem, based on the symbolic methods, became very popular and promising during the last decade. The article describes an approach to the vulnerabilities detection in the binary code, based on the formal methods of symbolic modeling and algebraic matching. In the article, the formalization of representation of binary code and vulnerabilities based on the behavior algebra, and the method of creation of formal patterns of vulnerabilities are proposed. The behavior algebra used for the representation of the formal binary code behavior, as well as for describing the vulnerabilities behavior. However, while the representation of the binary code in the terms of behavior algebra could be automated, creation of the vulnerabilities description requires development of the correct and effective methodology. Using the behavior algebra representation, the task of vulnerabilities detection can be solved in two steps – relatively fast algebraic matching, and the symbolic modeling itself, based on the data provided by the algebraic matcher. By the development of the vulnerabilities description in the terms of behavior algebra, and the algebraic matching algorithm the speed of detection of vulnerabilities in the binary code can be increased. The methodology of development of the vulnerabilities description in the terms of the behavior algebra has been proposed. The advantage of the algebraic approach is that the code vulnerabilities can be found more precisely, and the vulnerability description in the terms of behavior algebra can take in account different possible kinds of it. Also, the experiments with the implementation prototype shown that the “two-level” vulnerability detection system is faster than “pure” symbolic modeling: the fast matching step is executed first, and the slow modeling step is executed next on the results, provided by the matching step. Prom ble s in programming 2020; 1: 47-54

Kinetics and mechanism of quinolinium chlorochromate mediated oxidation of sugar alcohols in Bronsted acid media

Abstract Kinetics of oxidation of certain sugar alcohols have been studied by quinolinium chloro chromate (QCC) in sulphuric, perchloric, and hydrochloric acid media at different temperatures. Reactions obeyed second order kinetics with a first order in [QCC] and [Alcohol] at constant acidity. Kinetic data were analyzed Zucker-Hammett, Bunnett, and Bunnett-Olsen criteria. On the basis of observed results two plausible mechanisms are proposed one without involving water molecule in slow step, and the other with the involvement of water molecule in the slow step as proton abstracting agent.

Molecular mechanism of translational stalling by inhibitory codon combinations and poly(A) tracts.

Inhibitory codon pairs and poly(A) tracts within the translated mRNA cause ribosome stalling and reduce protein output. The molecular mechanisms that drive these stalling events, however, are still unknown. Here, we use a combination of invitro biochemistry, ribosome profiling, and cryo-EM to define molecular mechanisms that lead to these ribosome stalls. First, we use an invitro reconstituted yeast translation system to demonstrate that inhibitory codon pairs slow elongation rates which are partially rescued by increased tRNA concentration or by an artificial tRNA not dependent on wobble base-pairing. Ribosome profiling data extend these observations by revealing that paused ribosomes with empty A sites are enriched on these sequences. Cryo-EM structures of stalled ribosomes provide a structural explanation for the observed effects by showing decoding-incompatible conformations of mRNA in the A sites of all studied stall- and collision-inducing sequences. Interestingly, in the case of poly(A) tracts, the inhibitory conformation of the mRNA in the A site involves a nucleotide stacking array. Together, these data demonstrate a novel mRNA-induced mechanisms of translational stalling in eukaryotic ribosomes.

Autocatalyzed oxidation of amino acid, L-Citrulline by diperiodatocuprate(III) complex in aqueous alkaline medium: a kinetics and mechanistic approach

Autocatalysed oxidation of important amino acid, L-Citrulline(L-Cit) by coordination complex, diperiodatocuprate(III) in aqueous alkaline medium at a constant ionic strength of 0.05 mol dm−3 was studied spectrophotometrically. Autocatalysis was observed by one of the products formed, i.e., Cu (II). 1:4 stoichiometry (L-Cit:DPC) exhibited between the reaction of L-Cit and DPC in aqueous alkaline medium. The path is of first-order in [DPC], less than unit order in [L-Cit] and independent in [OH-]. Periodate has a retarding effect on the rate of reaction. Ionic strength has a negligible effect on the reaction rate. The main reaction products were identified by spot tests and spectroscopic analysis. A composite mechanism involving uncatalysed and an autocatalysed reaction path (via free radical) was proposed. The activation parameters for the slow step of the mechanism and also the thermodynamic quantities for different steps of the mechanism were determined and discussed. SYNOPSIS The oxidation of L-Citrulline by diperiodatocuprate(III) complex has been studied. Autocatalysis was observed by one of the products formed, i.e., Cu (II). 1:4 stoichiometry (L-Cit:DPC) exhibited between the reaction of L-Cit and DPC. A composite mechanism involving uncatalysed and an autocatalysed reaction path (via free radical) was proposed.

Kinetics and mechanism of quinolinium dichromate mediated oxidation of sugar alcohols in Bronsted acid media

AbstractBronsted acid catalyzed oxidation of certain sugar alcohols (polyols) has been studied by quinolinium dichromate (QDC) using aqueous sulfuric, perchloric, and hydrochloric acids at different temperatures. At constant acidity, reaction kinetics revealed the second‐order kinetics with a first order in [Alcohol] and [QDC]. Zucker‐Hammett, Bunnett, and Bunnett‐Olsen criteria were used to analyze acid‐dependent rate accelerations. Bunnett‐Olsen plots of (log k + Hν) versus (Hν + log [H+]), and (log k) versus (Hν + log [H+]) afforded slope values (ϕ and ϕ*, respectively) > 0.47, suggesting that a water molecule acts as a prton transfer agent in the slow step of the mechanism in the oxidation of alcohols by QDC in the presence of aqueous sulfuric, perchloric, and hydrochloric acids.