Relative Stoichiometry Research Articles

Optimization-based energy management strategy for a fuel cell/battery hybrid power system

This paper addresses the energy management strategy (EMS) for a fuel cell hybrid electric vehicle (FC-HEV). The purpose of this paper is to ensure an optimal power splitting between the fuel cell system (FCS) and the battery pack, taking into account the operating conditions of the FCS. The FCS is a multi-physics system, and consequently, its energetic performances depend on the operating conditions (i.e., temperature, gas relative humidity, gas stoichiometry, pressure and ageing). Specific techniques must be used to reach the best performances of the FCS. In this work, models are identified online by using the adaptive recursive least square (ARLS) method to seek a variation in the FCS performances. Then, an optimization algorithm is used on the updated model to find the best efficiency and power operating points. This process is used into an optimal EMS based on Pontryagin’s minimum principle, for a FC-HEV. The effectiveness of the proposed EMS is demonstrated by conducting studies on two FCSs with different levels of degradation.

Control of relative timing and stoichiometry by a master regulator.

Developmental processes in cells require a series of complex steps. Often only a single master regulator activates genes in these different steps. This poses several challenges: some targets need to be ordered temporally, while co-functional targets may need to be synchronized in both time and expression level. Here we study in single cells the dynamic activation patterns of early meiosis genes in budding yeast, targets of the meiosis master regulator Ime1. We quantify the individual roles of the promoter and protein levels in expression pattern control, as well as the roles of individual promoter elements. We find a consistent expression pattern difference between a non-cofunctional pair of genes, and a highly synchronized activation of a co-functional pair. We show that dynamic control leading to these patterns is distributed between promoter, gene and external regions. Through specific reciprocal changes to the promoters of pairs of genes, we show that different genes can use different promoter elements to reach near identical activation patterns.

Bottom-up and middle-down proteomics have comparable accuracies in defining histone post-translational modification relative abundance and stoichiometry.

Histone proteins are key components of chromatin. Their N-terminal tails are enriched in combinatorial post-translational modifications (PTMs), which influence gene regulation, DNA repair, and chromosome condensation. Mass spectrometry (MS)-based middle-down proteomics has emerged as a technique to analyze co-occurring PTMs, as it allows for the characterization of intact histone tails (>50 aa) rather than short (<20 aa) peptides analyzed by bottom-up. However, a demonstration of its reliability is still lacking. We compared results obtained with the middle-down and the bottom-up strategy in calculating PTM relative abundance and stoichiometry. Since bottom-up was proven to have biases in peptide signal detection such as uneven ionization efficiency, we performed an external correction using a synthetic peptide library with known peptide relative abundance. Corrected bottom-up data were used as reference. Calculated abundances of single PTMs showed similar deviations from the reference when comparing middle-down and uncorrected bottom-up results. Moreover, we show that the two strategies provided similar performance in defining accurate PTM stoichiometry. Collectively, we evidenced that the middle-down strategy is at least equally reliable to bottom-up in quantifying histone PTMs.

Sensitive targeted quantification of ERK phosphorylation dynamics and stoichiometry in human cells without affinity enrichment.

Targeted mass spectrometry is a promising technology for site-specific quantification of posttranslational modifications. However, a major constraint is the limited sensitivity for quantifying low-abundance PTMs, requiring the use of affinity reagents for enrichment. Herein, we demonstrate the direct site-specific quantification of ERK phosphorylation isoforms (pT, pY, pTpY) and their relative stoichiometry using a sensitive targeted MS approach termed high-pressure, high-resolution separations with intelligent selection, and multiplexing (PRISM). PRISM provides effective enrichment of target peptides into a given fraction from complex mixture, followed by selected reaction monitoring quantification. Direct quantification of ERK phosphorylation in human mammary epithelial cells (HMEC) was demonstrated from as little as 25 μg tryptic peptides from whole cell lysates. Compared to immobilized metal-ion affinity chromatography, PRISM provided ∼10-fold higher signal intensities, presumably due to the better peptide recovery of PRISM. This approach was applied to quantify ERK phosphorylation dynamics in HMEC treated by different doses of epidermal growth factor at both the peak activation (10 min) and steady state (2 h). The maximal ERK activation was observed with 0.3 and 3 ng/mL doses for 10 min and 2 h time points, respectively. The dose-response profiles of individual phosphorylated isoforms showed that singly phosphorylated pT-ERK never increases significantly, while the increase of pY-ERK paralleled that of pTpY-ERK. This data supports for a processive, rather than distributed model of ERK phosphorylation. The PRISM-SRM quantification of protein phosphorylation illustrates the potential for simultaneous quantification of multiple PTMs.

Effect of nitric oxide on VnfA, a transcriptional activator of VFe-nitrogenase in Azotobacter vinelandii.

The transcriptional activator, VnfA, is necessary for the expression of the structural genes encoding vanadium-dependent nitrogenase in Azotobacter vinelandii. We have previously reported that VnfA harbours a Fe-S cluster as a prosthetic group, presumably a 3Fe-4S type, which is vital for the transcriptionally active VnfA. A plausible effector molecule is a reactive oxygen species (ROS), which disassembles the Fe-S cluster switching the active VnfA to become fully inactive. This finding prompted us to investigate the effect of nitric oxide (NO), another physiologically important radical species on the VnfA activity. Unlike ROS, the VnfA activity was moderately inhibited and converged to 70% of the maximum by NO irrespective of its concentration. The Fe-S cluster of VnfA was found to react with NO to form a dinitrosyl-iron complex, either in the dimeric or monomeric form, dependent on the relative stoichiometry of NO to the Fe-S cluster. The VnfA species harbouring the dinitrosyl-iron complexes in each form exhibited 50% ATPase activity compared to the active VnfA. The findings of this study would open an argument about a biological effect of NO on nitrogenase in light of its transcriptional regulatory system.

Parametric Sensitivity Tests—European Polymer Electrolyte Membrane Fuel Cell Stack Test Procedures

As fuel cells are increasingly commercialized for various applications, harmonized and industry-relevant test procedures are necessary to benchmark tests and to ensure comparability of stack performance results from different parties. This paper reports the results of parametric sensitivity tests performed based on test procedures proposed by a European project, StackTest. The sensitivity of a Nafion®-based low temperature PEMFC stack's performance to parametric changes was the main objective of the tests. Four crucial parameters for fuel cell operation were chosen; relative humidity (RH), temperature, pressure, and stoichiometry at varying current density. Furthermore, procedures for polarization curve recording were also tested both in ascending and descending current directions.

Electrode Reaction and Mass-Transport Mechanisms Associated with the Iodide/Triiodide Couple in the Ionic Liquid 1-Ethyl-3-methylimidazolium Bis(trifluoromethanesulfonyl)imide

The oxidation and reduction processes associated with the iodide/triiodide (I–/I3–) couple have been investigated in the room temperature ionic liquid 1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide using cyclic voltammetry, convolution voltammetry, and chronoamperometry. Analogous to the case in acetonitrile, two processes with a relative electron stoichiometry of 2:1 are observed at a platinum electrode and are assigned to the I–/I3– and I3–/I2 processes at lower and higher potentials, respectively. The electro-oxidation of I– has been simulated using a termolecular charge transfer mechanism: I2 + 2e– ⇌ I– + I– (E°, ks, α), I2 + I– ⇌ I3– (Keq, kf), where E°, ks, α, Keq, and kf are the standard potential, standard heterogeneous electron-transfer rate constant, charge transfer coefficient, equilibrium (stability) constant, and bimolecular (forward) rate constant, respectively. The stability constant of I3– in this ionic liquid (Keq = 2.3 × 106, estimated by simulation) is comparable to that...

Multiple enzymatic digestions and ion mobility separation improve quantification of bacterial ribosomal proteins by data independent acquisition liquid chromatography-mass spectrometry.

Mass spectrometry-based quantification of ribosomal proteins (r-proteins) associated with mature ribosomes and ribosome assembly complexes is typically accomplished by relative quantification strategies. These strategies provide information on the relative stoichiometry of proteins within the complex compared to a wild-type strain. Here we have evaluated the applicability of a label-free approach, enhanced liquid chromatography–mass spectrometry (LC–MSE), for absolute “ribosome-centric” quantification of r-proteins in Escherichia coli mature ribosomes. Because the information obtained in this experiment is related to the number of peptides identified per protein, experimental conditions that allow accurate and reproducible quantification of r-proteins were found. Using an additional dimension of gas-phase separation through ion mobility and the use of multiple endoproteinase digestion significantly improved quantification of proteins associated with mature ribosomes. The actively translating ribosomes (polysomes) contain amounts of proteins consistent with their known stoichiometry within the complex. These measurements exhibited technical and biological reproducibilities at %CV less than 15% and 35%, respectively. The improved LC–MSE approach described here can be used to characterize in vivo ribosome assembly complexes captured during ribosome biogenesis and assembly under different perturbations (e.g., antibiotics, deletion mutants of assembly factors, oxidative stress, nutrient deprivation). Quantitative analysis of these captured complexes will provide information relating to the interplay and dynamics of how these perturbations interfere with the assembly process.

Remineralization of phytoplankton-derived organic matter by natural populations of heterotrophic bacteria

The relative lability, elemental stoichiometry, and remineralization rates of various particulate organic matter (POM) substrates by natural heterotrophic marine microorganisms were investigated. POM was harvested from laboratory cultures of a marine diazotroph (Trichodesmium IMS101), a cosmopolitan diatom (Thalassiosira weissflogii), a common marine cyanobacteria (Prochlorococcus MED4), and from surface waters off the Oregon coast. These POM resources were used as inoculants in a field experiment conducted at the Hawaii Ocean Time-series Station ALOHA in the North Pacific Subtropical Gyre. POM from these various sources was added to seawater collected from below the surface mixed layer, incubated in the dark, and remineralization rates were quantified via high-frequency measurement of soluble phosphorus (P) and nitrogen (N) concentrations over a 6-d period. Rapid solubilization and near complete remineralization of particulate P (PP) occurred in all treatments where cultured POM was used, with lesser relative mobilization of P from a ‘natural’ POM sample isolated from surface seawater off the Oregon coast. Soluble P pools, likely consisting of surface-adsorbed inorganic P and inorganic P liberated from cells during harvesting of biomass accounted for 28% of natural PP pools and 80±32% of cultured PP. 31P nuclear magnetic resonance (NMR) confirmed that PP was predominately present as orthophosphate in all POM types. By the end of the incubation period, all added P from cultured material had been converted to dissolved inorganic P. This finding may be a caveat of our utilization of laboratory cultures and natural POM which has been exposed to high inorganic P concentrations (0.8–5.0μmolL−1), albeit it is consistent with previous reports of significant contributions of surface-adsorbed P to total particulate P. In contrast, over the course of these experiments, only 37–40% of added N had been remineralized to ammonium (NH4+). In general, N remineralization rates of cultured material increased with the amount of N added (per gram of dry material). The net yield of bacterial cells was also positively correlated to the initial amount of C and N added. Most notably, when corrected for non-biological turnover (i.e. removal of soluble pools), the N:P remineralization ratio of cultured material (8.5±1.3) was independent of the N:P of added organic material (5–23).

Observation of chemical separation of In3Sb1Te2 thin film during phase transition

We investigated the chemical states of In3Sb1Te2 (IST) thin film using high-resolution X-ray photoelectron spectroscopy (HRXPS) with the synchrotron radiation during in-situ annealing in ultra-high vacuum. To obtain the oxygen-free amorphous IST (a-IST), we performed the mild Ne+ ion sputtering. And also we confirmed the relative a-IST stoichiometry to be 54%:17%:29% based on HRXPS data. At the first and second phase transition temperatures of 350 and 400°C, we observed the dramatic changes of chemical states from a-IST to InSb and the mixture of crystalline-IST and InTe, respectively. There was a depletion of Sb atoms on the surface after annealing at 750°C. We assume that Sb atom is a key for the phase transition in IST. However, chemical state of the Sb in IST is unstable during the phase transition and it will be caused with the non-reversible process by this structural instability.

A comparative study of the antioxidant scavenging activity of green tea, black tea and coffee extracts: A kinetic approach

The antioxidant activities of three beverages, coffee, black tea and green tea, along with their major components, were investigated in terms of their reaction with the stable radical 2,2′-diphenyl-2-picrylhydrazyl (DPPH). We used a kinetic approach in parallel with quantification methods based on a fixed end-point to determine the scavenging efficiency of compounds abundant in these beverages during their reaction with DPPH using a stopped-flow spectrophotometer-based method. Ascorbic acid, (+)-catechin, (−)-epigallocatechin, tannic acid, and caffeic acid were selected as model antioxidants to study in coffee, black tea and green tea. We applied a second-order model to demonstrate similarities in the kinetics behavior of beverages and related compounds. Our findings showed the slopes k2′((mol/L)-1s-1) and k2max′((mol/L)1s-1) exhibited similar and correlated values; we suggest the variation in k2′ as a function of time is more informative about antioxidant properties than reaction with DPPH alone. We also used IC100 to test the reliability of the relative stoichiometry using a new comparative parameter “n”, which was calculated as: n=c0DPPHIC100 (mol/L(mol/L)−1, (mol/L)mlmg−1 or molg−1).

Determination of Protein–Ligand Binding Constants of a Cooperatively Regulated Tetrameric Enzyme Using Electrospray Mass Spectrometry

This study highlights the benefits of nano electrospray ionization mass spectrometry (nanoESI-MS) as a fast and label-free method not only for determination of dissociation constants (KD) of a cooperatively regulated enzyme but also to better understand the mechanism of enzymatic cooperativity of multimeric proteins. We present an approach to investigate the allosteric mechanism in the binding of inhibitors to the homotetrameric enzyme fructose 1,6-bisphosphatase (FBPase), a potential therapeutic target for glucose control in type 2 diabetes. A series of inhibitors binding at an allosteric site of FBPase were investigated to determine their KDs by nanoESI-MS. The KDs determined by ESI-MS correlate very well with IC50 values in solution. The Hill coefficients derived from nanoESI-MS suggest positive cooperativity. From single-point measurements we could obtain information on relative potency, stoichiometry, conformational changes, and mechanism of cooperativity. A new X-ray crystal structure of FBPase tetramer binding ligand 3 in a 4:4 stoichiometry is also reported. NanoESI-MS-based results match the current understanding of the investigated system and are in agreement with the X-ray structural data, but provide additional mechanistic insight on the ligand binding, due to the better dynamic resolution. This method offers a powerful approach for studying other proteins with allosteric binding sites, as well.

Perfectly AB-alternating copolymers via alternating diene metathesis polymerization: one-step synthesis, characterization and properties

Perfectly alternating copolymers with functional phosphate groups were synthesized via reacting diacrylates with terminal dienes using alternating diene metathesis polymerization, showing a controllable polymerization characteristic which was evidenced by changing the relative stoichiometry of the two monomers. The compositional analysis and the sequence structural information of the polymers obtained from 1H NMR spectra demonstrated that the resulting copolymers possessed strictly alternating structure. In addition, the thermal and flame-retardant properties of the alternating copolymers were investigated, indicating favorable thermal degradation and flame retardancy which is an important feature for future applications. This special combination of the benefits of metathesis polymerization and phosphorus chemistry is a highly versatile system for materials in many applications. © 2013 Society of Chemical Industry

Water distribution and removal along the flow channel in proton exchange membrane fuel cells

Distribution expressions of total gas pressure and partial water vapor pressure along the channel direction were established based on lumped model by analyzing pressure loss in the channel and gas diffusion in the layer. The mechanism of droplet formation in the flow channel was also analyzed. Effects of the relative humidity, working temperature and stoichiometry on liquid water formation were discussed in detail. Moreover, the force equilibrium equation of the droplet in the flow channel was deduced, and the critical flow velocity for the water droplet removal was also addressed. The experimental results show that the threshold position of the liquid droplet is far from the inlet with the increase of temperature, and it decreases with the increase of the inlet total pressure. The critical flow velocity decreases with the increase of the radius and the working pressure.

Numerical studies on liquid water flooding in gas channels used in polymer electrolyte fuel cells

Water management plays an important role in the development of low-temperature polymer electrolyte fuel cells (PEFCs). The lack of a macroscopic gas channel (GC) flooding model constrains the current predictions of PEFC modeling under severe flooding situations. In this work, we have extended our previously developed one-dimensional channel flooding model for including phase change and liquid water flooding on the anode side. Several case studies are conducted to explore the effects of inlet relative humidity, net water transfer coefficient and GC shape on water film flooding in the GCs on both sides with the assumption that, both GCs are free from water droplets for simplicity. We demonstrate that the present model is capable of capturing key phenomenon of liquid water flooding in GCs. Our primary results show that gas drag force prevents liquid water accumulation in GCs, which dominates the liquid water removal. Both inlet relative humidity and stoichiometry can influence the liquid water flooding in GCs dramatically. At last, it is shown that increasing the upper half corner angle of the GC can mitigate the film water flooding in GCs.

Expression Analysis of MLH3, MLH1, and MSH4 in Maturation Arrest

The expression of DNA mismatch repair (DMMR) genes in patients with maturation arrest (MA) was analyzed. Samples were subjected to mutL homolog 3 (MLH3) mutation analysis by denaturing high-performance liquid chromatography/sequencing and quantification of MMR expression in testicular tissue by real-time polymerase chain reaction (PCR). Microsatellite instability assays were negative. Two missense and 1 intronic mutations were found. The missense mutation 2531C/T (P844L), predicted to affect MLH3 function, was found in 3 MA cases in association with the intronic variant IVS9 + 66G/A. Relative messenger RNA (mRNA) quantification identified 2 patients who overexpressed MLH3, 1 of them also overexpressing mutL homolog 1 (MLH1). The latter also presented the 2531C/T-IVS9 + 66G/A mutation. In conclusion, we suggest that a predominance of MLH3 expression might favor the MLH1/MLH3 complex which then would compete with the MLH1/PMS2 complexes. This could convey disruption of the relative stoichiometry between MLH1/MLH3 and MLH1/PMS2 complexes, thus causing meiosis failure, as MLH1/PMS2 complexes are supposed to replace MLH1/MLH3 during diplonema.

Mass Spectrometry in Biomarker Applications: From Untargeted Discovery to Targeted Verification, and Implications for Platform Convergence and Clinical Application

It has really been only in the last 10 years that mass spectrometry (MS)2 has had a truly important (but still small) impact on biomedical research. Much of this impact can be attributed to proteomics and its more basic applications. Early biomedical applications have included a number of efforts aimed at developing new biomarkers; however, the success of these endeavors to date have been quite modest—essentially having been confined to preclinical applications—and have often suffered from combinations of immature technology and hubris. Now that MS-based proteomics is reaching adolescence, it is appropriate to ask if and when biomarker-related applications will extend to the clinical realm and what developments will be essential for this transition. Biomarker development can be described as a multistage process consisting of discovery, qualification, verification, research assay optimization, validation, and commercialization (1). From a MS perspective, it is possible to “bin” measurements into 1 of 2 categories—those aimed at discovering potential protein biomarkers and those seeking to verify and validate biomarkers. Approaches in both categories generally involve digesting proteins (e.g., with trypsin) as a first step to yield peptides that can be effectively detected and identified with MS. Discovery-based approaches use broad “unbiased” or “undirected” measurements that attempt to cover as many proteins as possible in the hope of revealing promising biomarker candidates. A key challenge with this approach stems from the extremely large dynamic range (i.e., relative stoichiometry) of proteins of potential interest in biofluids such as plasma and the expectation that biomarker proteins of the greatest clinical value for many diseases may very well be present at low relative abundances (2). Protein concentrations in plasma extend from approximately 1010 pg/mL for albumin to approximately 10 pg/mL and below for interleukins and other cytokines. Proteins secreted or leaking into the blood from specific early-stage …

Multi-addressable photochromic terarylene containing benzo[b]thiophene-1,1-dioxide unit as ethene bridge: multifunctional molecular logic gates on unimolecular platform

A photochromic terarylene (BTO) containing a benzo[b]thiophene-1,1-dioxide unit as a central ethene bridge was synthesized and characterized, and exhibits good thermal stability and fatigue resistance both in solution and bulk crystals. When triggered by chemical ions, protons and light, BTO can behave as an absorbance and fluorescence switch, leading to a multi-addressable system. The different color changes of BTO upon adding Cu2+ and Hg2+ ions arise from the relative binding stoichiometry and the association affinities. The titration of the Job plot indicates that BTO forms a 1:1 complex with Hg2+, whereas in the case of Cu2+, the Job plot exhibits a maximum at about a 0.33 mole fraction, indicating that BTO forms a 2:1 complex with Cu2+. Moreover, the conversion yield of BTO can be modulated with chemical ions. Impressively, an increase in conversion yield was observed by adding Hg2+ to the solution of BTO, that is, the conversion yield and cyclization quantum yield are increased from 77.6% and 28.5% (for BTO only) to 99.8% and 43.1% (BTO-Hg2+), respectively. A series of molecular logic gates such as a half-adder, half-subtractor, 4-to-2 encoder, 2-to-4 decoder, and a 1:2 demultiplexer were constructed on the unimolecular platform by employing absorption and emission properties at different wavelengths as outputs, with the appropriate combination of chemical and photonic stimuli, which is a further step towards data processing on the molecular level with potential applications in sensing and labeling as well as for data manipulation.

Gamma Tubulin Phospho Regulation: Insights into Spindle Assembly

The mitotic spindle is composed of spindle poles, chromatin, and pole-to-pole and pole-kinetochore microtubules. Pole-kinetochore microtubules are anchored at the spindle poles, with the opposite end attached to centromeric chromatin. Pole-to-pole microtubules are represented by cross-linked microtubules from the opposite poles, and form the microtubule core bundle (CMB), that plays an important role in stability of pre-anaphase mitotic spindle. The mitotic spindle is a highly coupled mechanical system, whose dynamic behavior changes in response of Cdk1-dependent signaling.In cells, the majority of spindle microtubules are assembled from nucleation sites located at the spindle poles, centrosomes in animal cells and spindle pole bodies in yeast. γ-tubulin is an essential part of γ-TuRC (γ-Tubulin Ring Complex), a template for nucleating the majority of microtubules in the cell. Here, we show the coupling of Cdk1-phosphorylation state of γ-tubulin with microtubule organization during assembly of the mitotic spindle. We use confocal microscopy and high-resolution analysis of fluorescently labeled proteins to characterize in vivo the dynamic behavior of the spindle during its assembly in wild-type cells, phospho-mimicking and phosphor-inhibiting mutants. We reconstructed short (1-2.5 μm) spindles using EM tomography and could establish the relative stoichiometry of γ -TuRC : microtubules in phosphor-mutants of γ-tubulin. Our results suggest that γ−tubulin is a key regulator of number and organization of microtubules. Here, we propose a model, where the phosphorylation state of γ -tubulin contributes to determination of microtubule fate during spindle assembly and stabilizing the spindle prior and during anaphase.

Molecular chaperones DnaK and DnaJ share predicted binding sites on most proteins in the E. coli proteome.

In Escherichia coli, the molecular chaperones DnaK and DnaJ cooperate to assist the folding of newly synthesized or unfolded polypeptides. DnaK and DnaJ bind to hydrophobic motifs in these proteins and they also bind to each other. Together, this system is thought to be sufficiently versatile to act on the entire proteome, which creates interesting challenges in understanding the interactions between DnaK, DnaJ and their thousands of potential substrates. To address this question, we computationally predicted the number and frequency of DnaK- and DnaJ-binding motifs in the E. coli proteome, guided by free energy-based binding consensus motifs. This analysis revealed that nearly every protein is predicted to contain multiple DnaK- and DnaJ-binding sites, with the DnaJ sites occurring approximately twice as often. Further, we found that an overwhelming majority of the DnaK sites partially or completely overlapped with the DnaJ-binding motifs. It is well known that high concentrations of DnaJ inhibit DnaK-DnaJ-mediated refolding. The observed overlapping binding sites suggest that this phenomenon may be explained by an important balance in the relative stoichiometry of DnaK and DnaJ. To test this idea, we measured the chaperone-assisted folding of two denatured substrates and found that the distribution of predicted DnaK- and DnaJ-binding sites was indeed a good predictor of the optimal stoichiometry required for folding. These studies provide insight into how DnaK and DnaJ might cooperate to maintain global protein homeostasis.