Pump Regulation Research Articles

Multiple substrate recognition by yeast diadenosine and diphosphoinositol polyphosphate phosphohydrolase through phosphate clamping.

The yeast diadenosine and diphosphoinositol polyphosphate phosphohydrolase DDP1 is a Nudix enzyme with pyrophosphatase activity on diphosphoinositides, dinucleotides, and polyphosphates. These substrates bind to diverse protein targets and participate in signaling and metabolism, being essential for energy and phosphate homeostasis, ATPase pump regulation, or protein phosphorylation. An exhaustive structural study of DDP1 in complex with multiple ligands related to its three diverse substrate classes is reported. This allowed full characterization of the DDP1 active site depicting the molecular basis for endowing multisubstrate abilities to a Nudix enzyme, driven by phosphate anchoring following a defined path. This study, combined with multiple enzyme variants, reveals the different substrate binding modes, preferences, and selection. Our findings expand current knowledge on this important structural superfamily with implications extending beyond inositide research. This work represents a valuable tool for inhibitor/substrate design for ScDDP1 and orthologs as potential targets to address fungal infections and other health concerns.

Insight into Sodium Pump Regulation by FXYD Proteins

The sodium pump (Na+/K+-ATPase, NKA) is a membrane transport ATPase responsible for establishing the vital gradients of sodium and potassium across the plasma membrane of all mammalian cells. NKA activity is regulated by FXYD proteins, a family of transmembrane proteins that serve as auxiliary subunits of the sodium pump. in the heart, NKA activity is modulated by phospholemman (FXYD1), but the structural determinants of this regulatory mechanism are incompletely understood. We used time-resolved and steady-state FRET techniques to study the stoichiometry and affinity of fluorescently labeled NKA and FXYD1. Progressive acceptor photobleaching revealed multiple acceptor-labeled FXYD1 bound to donor-labeled NKA. Complementary global analysis of time-correlated single photon counting experiments revealed presence of medium- and high-FRET species within the population of NKA-FXYD1 complexes. To explore the quaternary structure of the regulatory complex, we obtained fluorescence lifetime data from hundreds of cells spanning a wide range of protein expression level. Over this range, the high FRET species increased with increasing protein concentration at the expense of the medium-FRET population. We attribute these subpopulations to NKA-NKA dimers decorated with either one FXYD1 (at low concentration) or two (at high concentration). Moreover, acceptor photobleaching experiments revealed FRET between donor- and acceptor-labeled NKA α subunits consistent with NKA oligomerization. in addition, we quantified FRET between NKA α and β essential subunits to monitor changes in the dynamic equilibrium between open (E2) and closed (E1) states of NKA. Treatment of NKA with the specific inhibitor ouabain induced a shift in the dynamic equilibrium of NKA toward a higher FRET state, suggesting that the α and β n-termini moved closer together. The experiments reveal new insight into the regulation of NKA by FXYD proteins and by NKA oligomerization.

Quorum-Sensing Regulation of Antimicrobial Resistance in Bacteria.

Quorum sensing is a cell-to-cell communication system that exists widely in the microbiome and is related to cell density. The high-density colony population can generate a sufficient number of small molecule signals, activate a variety of downstream cellular processes including virulence and drug resistance mechanisms, tolerate antibiotics, and harm the host. This article gives a general introduction to the current research status of microbial quorum-sensing systems, focuses on the role of quorum-sensing systems in regulating microbial resistance mechanisms, such as drug efflux pump and microbial biofilm formation regulation, and discusses a new strategy for the treatment of drug-resistant bacteria proposed by using quorum quenching to prevent microbial resistance.

Insight into Sodium Pump Regulation in the Failing Human Heart

Insight into Sodium Pump Regulation in the Failing Human Heart

Multi-cancer V-ATPase molecular signatures: A distinctive balance of subunit C isoforms in esophageal carcinoma.

BackgroundV-ATPases are hetero-oligomeric enzymes consisting of 13 subunits and playing key roles in ion homeostasis and signaling. Differential expression of these proton pumps has been implicated in carcinogenesis and metastasis. To elucidate putative molecular signatures underlying these phenomena, we evaluated the expression of V-ATPase genes in esophageal squamous cell carcinoma (ESCC) and extended the analysis to other cancers.MethodsExpression of all V-ATPase genes were analyzed in ESCC by a microarray data and in different types of tumors available from public databases. Expression of C isoforms was validated by qRT-PCR in paired ESCC samples.FindingsA differential expression pattern of V-ATPase genes was found in different tumors, with combinations in up- and down-regulation leading to an imbalance in the expression ratios of their isoforms. Particularly, a high C1 and low C2 expression pattern accurately discriminated ESCC from normal tissues. Structural modeling of C2a isoform uncovered motifs for oncogenic kinases in an additional peptide stretch, and an actin-biding domain downstream to this sequence.InterpretationAltogether these data revealed that the expression ratios of subunits/isoforms could form a conformational code that controls the H+ pump regulation and interactions related to tumorigenesis. This study establishes a paradigm change by uncovering multi-cancer molecular signatures present in the V-ATPase structure, from which future studies must address the complexity of the onco-related V-ATPase assemblies as a whole, rather than targeting changes in specific subunit isoforms.FundingThis work was supported by grants from CNPq and FAPERJ-Brazil.

Optimize the cost of paying for electricity in the water supply system by using accumulating tanks

The article considers the method of optimizing the pumps in the water supply system of the first water rise station in the zones of the day, where there is a different system of payment for electric energy. To assess the regulatory capacity of pumps on a temporary parameter, the authors of the article propose to use existing water tanks in the water supply system, which act as a buffer, smoothing the unevenness of water consumption. The studies have revealed that the comprehensive optimization of pumps on the criterion of minimizing specific electricity consumption by lifting water and optimizing the operation of pumps in the zones of the day allows to significantly improve energy efficiency and reduce the cost of extraction and transportation of water to the consumer. In the article, the authors examine an algorithm that allows us to assess the economic potential of pump regulation in the real-world conditions of the system.

A regulated multiple-output high-voltage charge pump IC for implantable neural stimulators

In this paper, a single-input multiple-output high-voltage charge pump integrated circuit (IC) for neural stimulation applications is presented. The proposed circuit, implemented using 0.18-μm standard low-voltage CMOS process, reliably generates three high-voltage outputs of 12.8 V, 9.6 V, and 6.4 V from a single 2.8 V-input source while regulating the output with load current up to 1 mA. In addition, an input voltage modulated regulation scheme is employed for good efficiency and regulation performance. The designed IC, which includes three charge pump cores and regulation circuits, occupies 0.8 mm2 of core die area.

Multi-Cancer V-ATPase Molecular Signatures: A Distinctive Balance of Subunit C Isoforms in Esophageal Carcinoma

Background: V-ATPases are hetero-oligomeric enzymes consisting of 13 subunits and playing key roles in ion homeostasis and signaling. Differential expressions of these proton pumps have been implicated in carcinogenesis and metastasis. To elucidate putative molecular signatures underlying these phenomena, we evaluated the V-ATPase genes expression in esophageal squamous cell carcinoma (ESCC) and extended the analysis to other cancers. Methods: Expression of all V-ATPase genes were analyzed in ESCC by a microarray data and in different types of tumors available from public databases. Expression of C isoforms was validated by qRT-PCR in paired ESCC samples. Findings: A differential expression pattern of V-ATPase genes was found in different tumors, with combinations in up- and down-regulation leading to an imbalance in the expression ratios of their isoforms. Particularly, a high C1 and low C2 expression pattern accurately discriminated ESCC from normal tissues. Structural modeling of C2a isoform uncovered motifs for oncogenic kinases in an additional peptide stretch, and an actin-biding domain downstream to this sequence. Interpretation: Altogether these data revealed that the expression ratios of subunits/isoforms could form a conformational code that controls the pump regulation and interactions related to tumorigenesis. This study establishes a paradigm change by uncovering multi-cancer molecular signatures present in the V-ATPase structure, from which future studies must address the complexity of the onco-related V-ATPase assemblies as a whole, rather than targeting changes in single subunit/isoforms moved by standard cutoffs. Funding Statement: This work was supported by grants from Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) and Fundacao de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ, Brazil). This study was financed in part by the Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior - Brasil (CAPES) - Finance Code 001 (PhD fellowship to JCVCS and Post-doctoral fellowship to EPC). JCVCS received Postdoctoral fellowship from Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF) and FFF received a PhD fellowship from FAPERJ. Declaration of Interests: The authors state that they have no conflicts of interest. Ethics Approval Statement: Human subjects: The use of the human samples was approved by the Ethic Committee of the institution (INCA - 116/11). All patients, who kindly agreed to participate in the study, signed an informed consent form prior to study enrollment.

Real-time hybrid dike breach model: simulating reservoirs of arbitrary shape and volume in laboratory tests

ABSTRACT Dikes and earthen embankments are common parts of modern infrastructure. Dike breaches resulting from extreme weather conditions and a lack of maintenance have caused significant damage to populated and agricultural areas in the past. Despite recent research findings, the effects of the parameters reservoir water volume and reservoir shape have not been properly assessed due to limitations in laboratory channel size and/or a lack of adequate test methodology. Furthermore, repeatability during the breach initiation phase has proved difficult to achieve when applying standard test methods. Therefore, a novel pump regulation method is introduced with the objective of simulating reservoirs of arbitrary size and shape during dike breach tests. This real-time, fully coupled hybrid modelling approach reduces test limitations due to limited laboratory space and considerably improves repeatability during the breach initiation phase. The pump regulation method, its limitations, test results and possible applications in the hydraulic laboratory are presented.

Meet the First Authors.

Meet the First Authors.

Novel approach for quantification of endoplasmic reticulum Ca2+ transport.

The type 2a sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA2a) plays a key role in Ca2+ regulation in the heart. However, available techniques to study SERCA function are either cell destructive or lack sensitivity. The goal of this study was to develop an approach to selectively measure SERCA2a function in the cellular environment. The genetically encoded Ca2+ sensor R-CEPIA1er was used to measure the concentration of Ca2+ in the lumen of the endoplasmic reticulum (ER) ([Ca2+]ER) in HEK293 cells expressing human SERCA2a. Coexpression of the ER Ca2+ release channel ryanodine receptor (RyR2) created a Ca2+ release/reuptake system that mimicked aspects of cardiac myocyte Ca2+ handling. SERCA2a function was quantified from the rate of [Ca2+]ER refilling after ER Ca2+ depletion; then, ER Ca2+ leak was measured after SERCA inhibition. ER Ca2+ uptake and leak were analyzed as a function of [Ca2+]ER to determine maximum ER Ca2+ uptake rate and maximum ER Ca2+ load. The sensitivity of this assay was validated by analyzing effects of SERCA inhibitors, [ATP]/[ADP], oxidative stress, phospholamban, and a loss-of-function SERCA2a mutation. In addition, the feasibility of using R-CEPIA1er to study SERCA2a in a native system was evaluated by using in vivo gene delivery to express R-CEPIA1er in mouse hearts. After ventricular myocyte isolation, the same methodology used in HEK293 cells was applied to study endogenous SERCA2a. In conclusion, this new approach can be used as a sensitive screening tool to study the effect of different drugs, posttranslational modifications, and mutations on SERCA function. NEW & NOTEWORTHY The aim of this study was to develop a sensitive approach to selectively measure sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA) function in the cellular environment. The newly developed Ca2+ sensor R-CEPIA1er was used to successfully analyze Ca2+ uptake mediated by recombinant and native cardiac SERCA. These results demonstrate that this new approach can be used as a powerful tool to study new mechanisms of Ca2+ pump regulation.

Evolutionary Analysis of the Lysine-Rich N-terminal Cytoplasmic Domains of the Gastric H+,K+-ATPase and the Na+,K+-ATPase.

The catalytic α-subunits of both the Na+,K+-ATPase and the gastric H+,K+-ATPase possess lysine-rich N-termini which project into the cytoplasm. Due to conflicting experimental results, it is currently unclear whether the N-termini play a role in ion pump function or regulation, and, if they do, by what mechanism. Comparison of the lysine frequencies of the N-termini of both proteins with those of all of their extramembrane domains showed that the N-terminal lysine frequencies are far higher than one would expect simply from exposure to the aqueous solvent. The lysine frequency was found to vary significantly between different vertebrate classes, but this is due predominantly to a change in N-terminal length. As evidenced by a comparison between fish and mammals, an evolutionary trend towards an increase of the length of the N-terminus of the H+,K+-ATPase on going from an ancestral fish to mammals could be identified. This evolutionary trend supports the hypothesis that the N-terminus is important in ion pump function or regulation. In placental mammals, one of the lysines is replaced by serine (Ser-27), which is a target for protein kinase C. In most other animal species, a lysine occupies this position and hence no protein kinase C target is present. Interaction with protein kinase C is thus not the primary role of the lysine-rich N-terminus. The disordered structure of the N-terminus may, via increased flexibility, facilitate interaction with another binding partner, e.g. the surrounding membrane, or help to stabilise particular enzyme conformations via the increased entropy it produces.

Dynamic exergoeconomic analysis of a heat pump system used for ancillary services in an integrated energy system

The integration of different energy sectors, such as the electricity and heating sector, is an effective way to integrate large shares of renewable energy into the energy system. Heat pumps allow efficient heat production based on electricity. As such, they may be used to provide two different services - the generation of heat and the provision of demand flexibility as ancillary services for the power system. The paper presents a method to assess the impact of providing demand flexibility on the performance of the conversion system based on a dynamic exergoeconomic analysis. A way to allocate the cost of heat and flexibility products based on the difference in exergy destruction was proposed. The method was applied to a case of a groundwater-source heat pump system supplying a district heating island system. It was found that providing demand flexibility causes higher exergy destruction, mainly due to heat losses during storage and the need to reheat the fluid using an electric heater. The major part of the additional exergy destruction was not related to heat pump regulation. When providing flexibility the overall cost of the system increased and according to the proposed allocation, demand flexibility accounted for 12% of the overall cost.

Negative Regulation Gene Circuits for Efflux Pump Control.

Synthetic biologists aim to design biological systems for a variety of industrial and medical applications, ranging from biofuel to drug production. Synthetic gene circuits regulating efflux pump protein expression can achieve this by driving desired substrates such as biofuels, pharmaceuticals, or other chemicals out of the cell in a precisely controlled manner. However, efflux pumps may introduce implicit negative feedback by pumping out intracellular inducer molecules that control gene circuits, which then can alter gene circuit function. Therefore, synthetic gene circuits must be carefully designed and constructed for precise efflux control. Here, we provide protocols for quantitatively modeling and building synthetic gene constructs for efflux pump regulation.

Stabilization of diastolic calcium signal via calcium pump regulation of complex local calcium releases and transient decay in a computational model of cardiac pacemaker cell with individual release channels.

Intracellular Local Ca releases (LCRs) from sarcoplasmic reticulum (SR) regulate cardiac pacemaker cell function by activation of electrogenic Na/Ca exchanger (NCX) during diastole. Prior studies demonstrated the existence of powerful compensatory mechanisms of LCR regulation via a complex local cross-talk of Ca pump, release and NCX. One major obstacle to study these mechanisms is that LCR exhibit complex Ca release propagation patterns (including merges and separations) that have not been characterized. Here we developed new terminology, classification, and computer algorithms for automatic detection of numerically simulated LCRs and examined LCR regulation by SR Ca pumping rate (Pup) that provides a major contribution to fight-or-flight response. In our simulations the faster SR Ca pumping accelerates action potential-induced Ca transient decay and quickly clears Ca under the cell membrane in diastole, preventing premature releases. Then the SR generates an earlier, more synchronized, and stronger diastolic LCR signal activating an earlier and larger inward NCX current. LCRs at higher Pup exhibit larger amplitudes and faster propagation with more collisions to each other. The LCRs overlap with Ca transient decay, causing an elevation of the average diastolic [Ca] nadir to ~200 nM (at Pup = 24 mM/s). Background Ca (in locations lacking LCRs) quickly decays to resting Ca levels (<100 nM) at high Pup, but remained elevated during slower decay at low Pup. Release propagation is facilitated at higher Pup by a larger LCR amplitude, whereas at low Pup by higher background Ca. While at low Pup LCRs show smaller amplitudes, their larger durations and sizes combined with longer transient decay stabilize integrals of diastolic Ca and NCX current signals. Thus, the local interplay of SR Ca pump and release channels regulates LCRs and Ca transient decay to insure fail-safe pacemaker cell operation within a wide range of rates.

Sodium pump regulation of locomotor control circuits.

Sodium pumps are ubiquitously expressed membrane proteins that extrude three Na+ ions in exchange for two K+ ions, using ATP as an energy source. Recent studies have illuminated additional, dynamic roles for sodium pumps in regulating the excitability of neuronal networks in an activity-dependent fashion. We review their role in a novel form of short-term memory within rhythmic locomotor networks. The data we review derives mainly from recent studies on Xenopus tadpoles and neonatal mice. The role and underlying mechanisms of pump action broadly match previously published data from an invertebrate, the Drosophila larva. We therefore propose a highly conserved mechanism by which sodium pump activity increases following a bout of locomotion. This results in an ultraslow afterhyperpolarization (usAHP) of the membrane potential that lasts around 1 min, but which only occurs in around half the network neurons. This usAHP in turn alters network excitability so that network output is reduced in a locomotor interval-dependent manner. The pumps therefore confer on spinal locomotor networks a temporary memory trace of recent network performance.

Predictive control of multi‐pump stations with variable‐speed drives

The growing energy market and increasing energy costs require new solutions in pumping technology aiming to rise the energy efficiency. In the study, a novel centrifugal pump predictive control organisation is proposed. Unlike the traditional speed-oriented pump regulation, an efficiency-oriented approach is realised where the specific power loss in the motor drives are taken into account alongside the general pump and pipeline losses. The offered management strategy and algorithm are based on the prediction of optimal operating points using combined tabulated and analytical performance characteristics of a pumping station. Applying the efficiency distribution diagram, the control map has been designed intended for running the optimal number of pumps at the appropriate speeds. As a result, the system performance within one of the best efficiency regions is maintained now at all the requested head and pressure inputs and pipeline condition instabilities.

Analysis of the Fluoroquinolone Antibiotic Resistance Mechanism of Salmonella enterica Isolates.

Quinolone-resistant Salmonella strains were isolated from patient samples, and several quinolone-sensitive strains were used to analyze mutations in the quinolone resistance-determining region (QRDR) of gyrA, gyrB, parC, and parE and to screen for plasmid-mediated quinolone resistance. Among the 21 strains that showed resistance to nalidixic acid and ciprofloxacin (MIC 0.125-2.0 μg/ml), 17 strains had a mutation in QRDR codon 87 of gyrA, and 3 strains had a single mutation (Ser83 → Phe). Another cause of resistance, efflux pump regulation, was studied by examining the expression of acrB, ramA, marA, and soxS. Five strains, including Sal-KH1 and Sal-KH2, showed no increase in relative expression in an analysis using the qRT-PCR method (p < 0.05). In order to determine the genes involved in the resistance, the Sal-9 isolate that showed decreased susceptibility and did not contain a mutation in the gyrA QRDR was used to make the STM (MIC 8 μg/ml) and STH (MIC 16 μg/ml) ciprofloxacin-resistant mutants. The gyrA QRDR Asp87 → Gly mutation was identified in both the STM and STH mutants by mutation analysis. qRT-PCR analysis of the efflux transporter acrB of the AcrAB-TolC efflux system showed increased expression levels in both the STM (1.79-fold) and STH (2.0-fold) mutants. In addition, the expression of the transcriptional regulator marA was increased in both the STM (6.35-fold) and STH (21.73-fold) mutants. Moreover, the expression of soxS was increased in the STM (3.41-fold) and STH (10.05-fold) mutants (p < 0.05). Therefore, these results indicate that AcrAB-TolC efflux pump activity and the target site mutation in gyrA are involved in quinolone resistance.

Probing the Structural Mechanism of Cardiac Calcium Pump Regulation by Phospholamban with Site-Directed Spin Labeling

Proper calcium transport is critical to muscle function, and the calcium pump sarcoplasmic reticulum Ca-ATPase (SERCA) plays a key role in facilitating muscle relaxation by sequestering calcium ions in muscle cells. Reduced SERCA activity is associated with heart failure, making SERCA an important subject of study for seeking heart failure treatment. The small membrane protein phospholamban (PLB) inhibits cardiac SERCA activity, and phosphorylation of PLB substantially restores SERCA activity, but the structural mechanism behind this process is not well understood. Previous spectroscopic studies on the SERCA-PLB complex have focused on the cytoplasmic domain of PLB, showing a structural shift of the cytoplasmic domain relative to SERCA that accompanies phosphorylation of PLB at Ser-16. Since it has been shown that only expression of PLB's transmembrane domain is necessary for observing SERCA inhibition, in this work, we performed site-directed spin labeling and EPR accessibility measurements on the transmembrane domain. We show that SERCA binding reduces access of the transmembrane domain to the lipid environment, and phosphorylation of PLB alters its topology without dissociation from SERCA.

On the Study of Setting Dynamics of Axial Piston Pumps with Electro Hydraulic Proportional Control

The paper considers recent trends in the development of electrically operated hydraulic drive. The main requirements to the dynamic characteristics of variable axial piston pumps with electric control and the need for search of ways of their improvement are specified. The investigation results show the dynamic characteristics of the process to control electro-hydraulic axial piston pump capacity at different parameters of pulse width modulation (PWM) signal. The regularity between PWM-signal frequency and the time of establishing the proper pump displacement was found. The paper stresses the need for more detailed study of this fact and its usage for the optimization of variable pump regulation speed in order to be applied in electro-hydraulic flow control systems.