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We developed a mathematical model of the cerebral venous outflow for the simulation of the average blood flows and pressures in the main drainage vessels of the brain. The main features of the model are that it includes a validated model for the simulation of the intracranial circulation and it accounts for the dependence of the hydraulic properties of the jugular veins with respect to the gravity field, which makes it an useful tool for the study of the correlations between extracranial blood redistributions and changes in the intracranial environment. The model is able to simulate the average pressures and flows in different points of the jugular ducts, taking into account the amount of blood coming from the anastomotic connections; simulate how the blood redistribution due to change of posture affects flows and pressures in specific points of the system; and simulate redistributions due to stenotic patterns. Sensitivity analysis to check the robustness of the model was performed. The model reproduces average physiologic behavior of the jugular, vertebral, and cerebral ducts in terms of pressures and flows. In fact, jugular flow drops from ~11.7 to ~1.4 ml/s in the passage from supine to standing. At the same time, vertebral flow increases from 0.8 to 3.4 ml/s, while cerebral blood flow, venous sinuses pressure, and intracranial pressure are constant around the average value of 12.5 ml/s, 6 mmHg, and 10 mmHg, respectively. All these values are in agreement with literature data. [ABSTRACT FROM AUTHOR]

This review article describes the occurrence, general properties, and substrate specificity of the flavoenzymes belonging to the glucose-methanol-choline oxidoreductase superfamily and the l-α-hydroxyacid dehydrogenase family. Most of these enzymes catalyze the oxidations of hydroxyl groups, yielding carbonyl moieties. Over the years, carbanion, hydride transfer, and radical mechanisms have been discussed for these enzymes, and the main experimental evidences supporting these mechanisms are presented here. Regardless of the chemical nature of the organic substrate (i.e., activated and non-activated alcohols), a hydride transfer mechanism appears to be the most plausible for the flavoenzymes acting on CH-OH groups. The reaction of most of these enzymes likely starts with proton abstraction from the substrate hydroxyl group by a conserved active site histidine. Among the different approaches carried out to determine the chemical mechanisms with physiological substrates, primary substrate and solvent deuterium kinetic isotope effect studies have provided the most unambiguous evidences. It is expected that the numerous studies reported for these enzymes over the years will be instrumental in devising efficient industrial biocatalysts and drugs.

The article comments on the study "Current Standard Technique for Modern Flexible Ureteroscopy: Tips and Tricks" by G. Giusti and others.

Enzymes are potent catalysts that increase biochemical reaction rates by several orders of magnitude. Flavoproteins are a class of enzymes whose classification relies on their ability to react with molecular oxygen (O 2 ) during catalysis using ionizable active site residues. Pseudomonas aeruginosa D-arginine dehydrogenase (PaDADH) is a flavoprotein that oxidizes D-arginine for P. aeruginosa survival and biofilm formation. The crystal structure of PaDADH reveals the interaction of the glutamate 246 (E 246 ) side chain with the substrate and at least three other active site residues, establishing a hydrogen bond network in the active site. Additionally, E 246 likely ionizes to facilitate substrate binding during PaDADH catalysis. This study aimed to investigate how replacing the E 246 residue with leucine affects PaDADH catalysis and its ability to react with O 2 using steady-state kinetics coupled with pH profile studies. The data reveal a gain of O 2 reactivity in the E 246 L variant, resulting in a reduced flavin semiquinone species and superoxide (O 2 • -) during substrate oxidation. The O 2 • - reacts with active site protons, resulting in an observed nonstoichiometric slope of 1.5 in the enzyme's log (k cat /K m ) pH profile with D-arginine. Adding superoxide dismutase results in an observed correction of the slope to 1.0. This study demonstrates how O 2 • - can alter the slopes of limbs in the pH profiles of flavin-dependent enzymes and serves as a model for correcting nonstoichiometric slopes in elucidating reaction mechanisms of flavoproteins., Competing Interests: Conflict of interest The authors declare no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Commercial food and l-amino acid industries rely on bioengineered d-amino acid oxidizing enzymes to detect and remove d-amino acid contaminants. However, the bioengineering of enzymes to generate faster biological catalysts has proven difficult as a result of the failure to target specific kinetic steps that limit enzyme turnover, k cat , and the poor understanding of loop dynamics critical for catalysis. Pseudomonas aeruginosa d-arginine dehydrogenase ( Pa DADH) oxidizes most d-amino acids and is a good candidate for application in the l-amino acid and food industries. The side chain of the loop L2 E 246 residue located at the entrance of the Pa DADH active site pocket potentially favors the closed active site conformation and secures the substrate upon binding. This study used site-directed mutagenesis, steady-state, and rapid reaction kinetics to generate the glutamine, glycine, and leucine variants and investigate whether increasing the rate of product release could translate to an increased enzyme turnover rate. Upon E 246 mutation to glycine, there was an increased rate of d-arginine turnover k cat from 122 to 500 s -1 . Likewise, the k cat values increased 2-fold for the glutamine or leucine variants. Thus, we have engineered a faster biocatalyst for industrial applications by selectively increasing the rate of the Pa DADH product release.

Enzymes require flexible regions to adopt multiple conformations during catalysis. The mobile regions of enzymes include gates that modulate the passage of molecules in and out of the enzyme's active site. The enzyme PA1024 from Pseudomonas aeruginosa PA01 is a recently discovered flavin-dependent NADH:quinone oxidoreductase (NQO, EC 1.6.5.9). Q80 in loop 3 (residues 75-86) of NQO is ∼15 Å away from the flavin and creates a gate that seals the active site through a hydrogen bond with Y261 upon NADH binding. In this study, we mutated Q80 to glycine, leucine, or glutamate to investigate the mechanistic significance of distal residue Q80 in NADH binding in the active site of NQO. The UV-visible absorption spectrum reveals that the mutation of Q80 minimally affects the protein microenvironment surrounding the flavin. The anaerobic reductive half-reaction of the NQO-mutants yields a ≥25-fold increase in the K d value for NADH compared to the WT enzyme. However, we determined that the k red value was similar in the Q80G, Q80L, and wildtype enzymes and only ∼25% smaller in the Q80E enzyme. Steady-state kinetics with NQO-mutants and NQO-WT at varying concentrations of NADH and 1,4-benzoquinone establish a ≤5-fold decrease in the k cat /K NADH value. Moreover, there is no significant difference in the k cat /K BQ (∼1 × 10 6  M -1 s -1 ) and k cat (∼24 s -1 ) values in NQO-mutants and NQO-WT. These results are consistent with the distal residue Q80 being mechanistically essential for NADH binding to NQO with minimal effect on the quinone binding to the enzyme and hydride transfer from NADH to flavin., Competing Interests: Conflict of interest The authors declare they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Numerous studies demonstrate that enzymes undergo multiple conformational changes during catalysis. The malleability of enzymes forms the basis for allosteric regulation: residues located far from the active site can exert long-range dynamical effects on the active site residues to modulate catalysis. The structure of Pseudomonas aeruginosa d-arginine dehydrogenase ( Pa DADH) shows four loops (L1, L2, L3, and L4) that span the substrate and the FAD-binding domains. Loop L4 comprises residues 329-336, spanning over the flavin cofactor. The I335 residue on loop L4 is ∼10 Å away from the active site and ∼3.8 Å from N(1)-C(2)═O atoms of the flavin. In this study, we used molecular dynamics and biochemical techniques to investigate the effect of the mutation of I335 to histidine on the catalytic function of Pa DADH. Molecular dynamics showed that the conformational dynamics of Pa DADH are shifted to a more closed conformation in the I335H variant. In agreement with an enzyme that samples more in a closed conformation, the kinetic data of the I335H variant showed a 40-fold decrease in the rate constant of substrate association ( k 1 ), a 340-fold reduction in the rate constant of substrate dissociation from the enzyme-substrate complex ( k 2 ), and a 24-fold decrease in the rate constant of product release ( k 5 ), compared to that of the wild-type. Surprisingly, the kinetic data are consistent with the mutation having a negligible effect on the reactivity of the flavin. Altogether, the data indicate that the residue at position 335 has a long-range dynamical effect on the catalytic function in Pa DADH.

Pseudomonas aeruginosa PAO1 d-2-hydroxyglutarate (D2HG) dehydrogenase (PaD2HGDH) oxidizes D2HG to 2-ketoglutarate during the vital l-serine biosynthesis and is a potential therapeutic target against P. aeruginosa. PaD2HGDH, which oxidizes d-malate as an alternative substrate, has been demonstrated to be a metallo flavoprotein that requires Zn 2+ for activity. However, the role of Zn 2+ in the enzyme has not been elucidated, making it difficult to rationalize why nature employs both a redox center and a metal ion for catalysis in PaD2HGDH and other metallo flavoenzymes. In this study, recombinant His-tagged PaD2HGDH was purified to high levels in the presence of Zn 2+ or Co 2+ to investigate the metal's role in catalysis. We found that the flavin reduction step was reversible and partially rate limiting for the enzyme's turnover at pH 7.4 with either D2HG or d-malate with similar rate constants for both substrates, irrespective of whether Zn 2+ or Co 2+ was bound to the enzyme. The steady-state pL profiles of the k cat and k cat /K m values with d-malate demonstrate that Zn 2+ mediates the activation of water coordinated to the metal. Our data are consistent with a dual role for the metal, which orients the hydroxy acid substrate in the enzyme's active site and rapidly deprotonates the substrate to yield an alkoxide species for hydride transfer to the flavin. Thus, we propose a catalytic mechanism for PaD2HGDH oxidation that establishes Zn 2+ as a cofactor required for substrate orientation and activation during enzymatic turnover., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Pseudomonas aeruginosa couples the oxidation of d-2-hydroxyglutarate (D2HG) to l-serine biosynthesis for survival, using d-2-hydroxyglutarate dehydrogenase from P. aeruginosa (PaD2HGDH). Knockout of PaD2HGDH impedes P. aeruginosa growth, making PaD2HGDH a potential target for therapeutics. Previous studies showed that the enzyme's activity increased with Zn 2+ , Co 2+ , or Mn 2+ but did not establish the enzyme's metal composition and whether the metal is an activator or a required cofactor for the enzyme, which we addressed in this study. Comparable to the human enzyme, PaD2HGDH showed only 15% flavin reduction with D2HG or d-malate. Upon purifying PaD2HGDH with 1 mM Zn 2+ , the Zn 2+ :protein stoichiometry was 2:1, yielding an enzyme with ∼40 s -1 k cat for d-malate. Treatment with 1 mM EDTA decreased the Zn 2+ :protein ratio to 1:1 without changing the kinetic parameters with d-malate. We observed complete enzyme inactivation for the metalloapoenzyme with 100 mM EDTA treatment, suggesting that Zn 2+ is essential for PaD2HGDH activity. The presence of Zn 2+ increased the flavin N 3 atom pK a value to 11.9, decreased the flavin ε 450 at pH 7.4 from 13.5 to 11.8 mM -1  cm -1 , and yielded a charged transfer complex with a broad absorbance band >550 nm, consistent with a Zn 2+ -hydrate species altering the electronic properties of the enzyme-bound FAD. The exogenous addition of Zn 2+ , Co 2+ , Cd 2+ , Mn 2+ , or Ni 2+ to the metalloapoenzyme reactivated the enzyme in a sigmoidal pattern, consistent with an induced fit rapid-rearrangement mechanism. Collectively, our data demonstrate that PaD2HGDH is a Zn 2+ -dependent metallo flavoprotein, which requires Zn 2+ as an essential cofactor for enzyme activity., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

iLOV is an engineered flavin-binding fluorescent protein (FbFP) with applications for in vivo cellular imaging. To expand the range of applications of FbFPs for multicolor imaging and FRET-based biosensing, it is desirable to understand how to modify their absorption and emission wavelengths (i.e., through spectral tuning). There is particular interest in developing FbFPs that absorb and emit light at longer wavelengths, which has proven challenging thus far. Existing spectral tuning strategies that do not involve chemical modification of the flavin cofactor have focused on placing positively charged amino acids near flavin's C4a and N5 atoms. Guided by previously reported electrostatic spectral tunning maps (ESTMs) of the flavin cofactor and by quantum mechanical/molecular mechanical (QM/MM) calculations reported in this work, we suggest an alternative strategy: placing a negatively charged amino acid near flavin's N1 atom. We predict that a single-point mutant, iLOV-Q430E, has a slightly red-shifted absorption and fluorescence maximum wavelength relative to iLOV. To validate our theoretical prediction, we experimentally expressed and purified iLOV-Q430E and measured its spectral properties. We found that the Q430E mutation results in a slight change in absorption and a 4-8 nm red shift in the fluorescence relative to iLOV, in good agreement with the computational predictions. Molecular dynamics simulations showed that the carboxylate side chain of the glutamate in iLOV-Q430E points away from the flavin cofactor, which leads to a future expectation that further red shifting may be achieved by bringing the side chain closer to the cofactor.

The jugular venous pulse (JVP) is a one of the crucial parameters of efficient cardiovascular function. Nowadays, limited data are available regarding the response of JVP to exercise because of its complex and/or invasive assessment procedure. The aim of the present work is to test the feasibility of a non-invasive JVP plethysmography system to monitor different submaximal exercise condition. Twenty (20) healthy subjects (13M/7F mean age 25 ± 3, BMI 21 ± 2) underwent cervical strain-gauge plethysmography, acquired synchronously with the electrocardiogram, while they were carrying out different activities: stand supine, upright, and during the execution of aerobic exercise (2 km walking test) and leg-press machine exercise (submaximal 6 RM test). Peaks a and x of the JVP waveform were investigated since they reflect the volume of cardiac filling. To this aim, the Δax parameter was introduced, representing the amplitude differences between a and x peaks. Significant differences in the values of a, x, and Δax were found between static and exercise conditions (p < 0.0001, p < 0.0001, p < 0.0001), respectively. Particularly, the Δax value for the leg press was approximately three times higher than the supine, and during walking was even nine times higher. The exercise monitoring by means of the novel JVP plethysmography system is feasible during submaximal exercise, and it provides additional parameters on cardiac filling and cerebral venous drainage to the widely used heartbeat rate value.

NAD(P)H:quinone oxidoreductases (NQOs) play an essential protective role as antioxidants in the detoxification of quinones in both Prokaryotes and Eukaryotes. NQO from Pseudomonas aeruginosa PAO1 uses FMN to catalyze the two-electron reduction of various quinones with NADH. In this study, steady-state kinetics, kinetic solvent viscosity effects, and rapid reaction kinetics were used to determine which kinetic steps control the overall turnover of the enzyme with benzoquinone or juglone. The rate constant for flavin reduction (k red ) at pH 6.0 was 12.9 ± 0.3 s -1 , and the K d for NADH was at least an order of magnitude lower than 90 μM. With benzoquinone, the k cat value was 11.7 ± 0.3 s -1 , consistent with flavin reduction being almost entirely rate-limiting for overall turnover. With juglone, a k cat value of 10.0 ± 0.5 s -1 was recorded. The normalized plot of the relative solvent viscosity effects on the k cat values established that hydride transfer from NADH to the FMN and quinol product release, with a calculated rate constant (k P-rel ) of 52 s -1 , are partially rate-limiting for the overall turnover of NQO. Kinetic solvent viscosity effects with glucose or sucrose revealed a hyperbolic dependence on the k cat and k cat /K m values with benzoquinone or juglone, respectively, consistent with the presence of a solvent-sensitive internal isomerization of the enzyme-substrate complex (ES). The data demonstrate opposing effects of benzoquinone and juglone on the equilibrium of the NQO ES isomerization with glucose or sucrose. Thus, our study demonstrates how quinol substrate properties alter the equilibrium of NQO ES isomerization., (Copyright © 2022 Elsevier Inc. All rights reserved.)

d-Arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH) catalyzes the flavin-dependent oxidation of d-arginine and other d-amino acids. Here, we report the crystal structure at 1.29 Å resolution for PaDADH-Y249F expressed and co-crystallized with d-arginine. The overall structure of PaDADH-Y249F resembled PaDADH-WT, but the electron density for the flavin cofactor was ambiguous, suggesting the presence of modified flavins. Electron density maps and mass spectrometric analysis confirmed the presence of both N5-(4-guanidino-oxobutyl)-FAD and 6-OH-FAD in a single crystal of PaDADH-Y249F and helped with the further refinement of the X-ray crystal structure. The versatility of the reduced flavin is apparent in the PaDADH-Y249F structure and is evidenced by the multiple functions it can perform in the same active site., (Copyright © 2021. Published by Elsevier Inc.)

Cerebral venous outflow is investigated in the diagnosis of heart failure through the monitoring of jugular venous pulse, an indicator to assess cardiovascular diseases. The jugular venous pulse is a weak signal stemming from the lying internal jugular vein and often invasive methodologies requiring surgery are mandatory to detect it. Jugular venous pulse can also be extrapolated via the ultrasound technique, but it requires a qualified healthcare operator to perform the examination. In this work, a wireless, user-friendly, wearable device for plethysmography is developed to investigate the possibility of monitoring the jugular venous pulse non-invasively. The proposed device can monitor the jugular venous pulse and the electrocardiogram synchronously. To study the feasibility of using the proposed device to detect physiological variables, several measurements were carried out on healthy subjects by considering three different postures: supine, sitting, and upright. Data acquired in the experiment were properly filtered to highlight the cardiac oscillation and remove the breathing contribution, which causes a considerable shift in the amplitude of signals. To evaluate the proper functioning of the wearable device for plethysmography, a comparison with the ultrasound technique was carried out. As a satisfactory result, the acquired signals resemble the typical jugular venous pulse waveforms found in literature.

Multi-scale calcium (Ca 2+ ) dynamics, exhibiting wide-ranging temporal kinetics, constitutes a ubiquitous mode of signal transduction. We report a novel endoplasmic-reticulum (ER)-targeted Ca 2+ indicator, R-CatchER, which showed superior kinetics in vitro (k off ≥2×10 3  s -1 , k on ≥7×10 6  M -1  s -1 ) and in multiple cell types. R-CatchER captured spatiotemporal ER Ca 2+ dynamics in neurons and hotspots at dendritic branchpoints, enabled the first report of ER Ca 2+ oscillations mediated by calcium sensing receptors (CaSRs), and revealed ER Ca 2+ -based functional cooperativity of CaSR. We elucidate the mechanism of R-CatchER and propose a principle to rationally design genetically encoded Ca 2+ indicators with a single Ca 2+ -binding site and fast kinetics by tuning rapid fluorescent-protein dynamics and the electrostatic potential around the chromophore. The design principle is supported by the development of G-CatchER2, an upgrade of our previous (G-)CatchER with improved dynamic range. Our work may facilitate protein design, visualizing Ca 2+ dynamics, and drug discovery., (© 2021 Wiley-VCH GmbH.)

This study utilizes the FMN-dependent NADH:quinone oxidoreductase from Pseudomonas aeruginosa PAO1 to investigate the effect of introducing an active site negative charge on the flavin absorption spectrum both in the absence and presence of a long-range electrostatic potential coming from solution ions. There were no observed changes in the flavin UV-visible spectrum when an active site tyrosine (Y277) becomes deprotonated in vitro . These results could only be reproduced computationally using average solvent electrostatic configuration (ASEC) QM/MM simulations that include both positive and negative solution ions. The same calculations performed with minimal ions to neutralize the total protein charge predicted that deprotonating Y277 would significantly alter the flavin absorption spectrum. Analyzing the distribution of solution ions indicated that the ions reorganize around the protein surface upon Y277 deprotonation to cancel the effect of the tyrosinate on the flavin absorption spectrum. Additional biochemical experiments were performed to test this hypothesis.

Accurate and reproducible measurement of blood flow profile is very important in many clinical investigations for diagnosing cardiovascular disorders. Given that many factors could affect human circulation, and several parameters must be set to properly evaluate blood flows with phase-contrast techniques, we developed an MRI-compatible hydrodynamic phantom to simulate different physiological blood flows. The phantom included a programmable hydraulic pump connected to a series of pipes immersed in a solution mimicking human soft tissues, with a blood-mimicking fluid flowing in the pipes. The pump is able to shape and control the flow by driving a piston through a dedicated software. Periodic waveforms are used as input to the pump to move the fluid into the pipes, with synchronization of the MRI sequences to the flow waveforms. A dedicated software is used to extract and analyze flow data from magnitude and phase images. The match between the nominal and the measured flows was assessed, and the scope of phantom variables useful for a reliable calibration of an MRI system was accordingly defined. Results showed that the NO-HYPE phantom is a valuable tool for the assessment of MRI scanners and sequence design for the MR evaluation of blood flows. Overview of the NOvel HYdrodynamic Phantom for the Evaluation of MRI flow measurements (NO-HYPE). Left: internal of the CompuFlow 1000 MR pump unit. Right: Setting of the NO-HYPE before a MRI acquisition session. Soft tissue mimicking material is hosted in the central part of the phantom (light blue chamber). Glass pipes pass through the chamber carrying the blood mimicking fluid., (© 2021. The Author(s).)

Proteins are inherently dynamic, and proper enzyme function relies on conformational flexibility. In this study, we demonstrated how an active site residue changes an enzyme's reactivity by modulating fluctuations between conformational states. Replacement of tyrosine 249 (Y249) with phenylalanine in the active site of the flavin-dependent d-arginine dehydrogenase yielded an enzyme with both an active yellow FAD (Y249F-y) and an inactive chemically modified green FAD, identified as 6-OH-FAD (Y249F-g) through various spectroscopic techniques. Structural investigation of Y249F-g and Y249F-y variants by comparison to the wild-type enzyme showed no differences in the overall protein structure and fold. A closer observation of the active site of the Y249F-y enzyme revealed an alternative conformation for some active site residues and the flavin cofactor. Molecular dynamics simulations probed the alternate conformations observed in the Y249F-y enzyme structure and showed that the enzyme variant with FAD samples a metastable conformational state, not available to the wild-type enzyme. Hybrid quantum/molecular mechanical calculations identified differences in flavin electronics between the wild type and the alternate conformation of the Y249F-y enzyme. The computational studies further indicated that the alternate conformation in the Y249F-y enzyme is responsible for the higher spin density at the C6 atom of flavin, which is consistent with the formation of 6-OH-FAD in the variant enzyme. The observations in this study are consistent with an alternate conformational space that results in fine-tuning the microenvironment around a versatile cofactor playing a critical role in enzyme function.

Cardiovascular haemodynamics alters during posture changes and exposure to microgravity. Vascular auto-remodelling observed in subjects living in space environment causes them orthostatic intolerance when they return on Earth. In this study we modelled the human haemodynamics with focus on head and neck exposed to different hydrostatic pressures in supine, upright (head-up tilt), head-down tilt position, and microgravity environment by using a well-developed 1D-0D haemodynamic model. The model consists of two parts that simulates the arterial (1D) and brain-venous (0D) vascular tree. The cardiovascular system is built as a network of hydraulic resistances and capacitances to properly model physiological parameters like total peripheral resistance, and to calculate vascular pressure and the related flow rate at any branch of the tree. The model calculated 30.0 mmHg (30%), 7.1 mmHg (78%), 1.7 mmHg (38%) reduction in mean blood pressure, intracranial pressure and central venous pressure after posture change from supine to upright, respectively. The modelled brain drainage outflow percentage from internal jugular veins is 67% and 26% for supine and upright posture, while for head-down tilt and microgravity is 65% and 72%, respectively. The model confirmed the role of peripheral veins in regional blood redistribution during posture change from supine to upright and microgravity environment as hypothesized in literature. The model is able to reproduce the known haemodynamic effects of hydraulic pressure change and weightlessness. It also provides a virtual laboratory to examine the consequence of a wide range of orthostatic stresses on human haemodynamics.