EXECUTER1- and EXECUTER2-dependent transfer of stress-related signals from the plastid to the nucleus of Arabidopsis thaliana

Shortly after the release of singlet oxygen ((1)O2), drastic changes in nuclear gene expression occur in the conditional flu mutant of Arabidopsis that reveal a rapid transfer of signals from the plastid to the nucleus. In contrast to retrograde control of nuclear gene expression by plastid signals described earlier, the primary effect of (1)O2 generation in the flu mutant is not the control of chloroplast biogenesis but the activation of a broad range of signaling pathways known to be involved in biotic and abiotic stress responses. This activity of a plastid-derived signal suggests a new function of the chloroplast, namely that of a sensor of environmental changes that activates a broad range of stress responses. Inactivation of the plastid protein EXECUTER1 attenuates the extent of (1)O2-induced up-regulation of nuclear gene expression, but it does not fully eliminate these changes. A second related nuclear-encoded protein, dubbed EXECUTER2, has been identified that is also implicated with the signaling of (1)O2-dependent nuclear gene expression changes. Like EXECUTER1, EXECUTER2 is confined to the plastid. Inactivation of both EXECUTER proteins in the ex1/ex2/flu triple mutant is sufficient to suppress the up-regulation of almost all (1)O2-responsive genes. Retrograde control of (1)O2-responsive genes requires the concerted action of both EXECUTER proteins within the plastid compartment.     

H(2)O(2)-induced left ventricular dysfunction in isolated working rat hearts is independent of calcium accumulation

Reactive oxygen species (ROS) and intracellular Ca²⁺ overload play key roles in myocardial ischemia-reperfusion (IR) injury but the relationships among ROS, Ca²⁺ overload and LV mechanical dysfunction remain unclear. We tested the hypothesis that H₂O₂ impairs LV function by causing Ca²⁺ overload by increasing late sodium current (I_Na), similar to Sea Anemone Toxin II (ATX-II). Diastolic and systolic Ca²⁺ concentrations (d[Ca²⁺]_i and s[Ca²⁺]_i) were measured by indo-1 fluorescence simultaneously with LV work in isolated working rat hearts. H₂O₂ (100 μM, 30 min) increased d[Ca²⁺]_i and s[Ca²⁺]_i. LV work increased transiently then declined to 32% of baseline before recovering to 70%. ATX-II (12 nM, 30 min) caused greater increases in d[Ca²⁺]_i and s[Ca²⁺]_i. LV work increased transiently before declining gradually to 17%. Ouabain (80 μM) exerted similar effects to ATX-II. Late I_Na inhibitors, lidocaine (10 μM) or R56865 (2 μM), reduced effects of ATX-II on [Ca²⁺]_i and LV function, but did not alter effects of H₂O₂. The antioxidant, N-(2-mercaptopropionyl)glycine (MPG, 1 mM) prevented H₂O₂-induced LV dysfunction, but did not alter [Ca²⁺]_i. Paradoxically, further increases in [Ca²⁺]_i by ATX-II or ouabain, given 10 min after H₂O₂, improved function. The failure of late I_Na inhibitors to prevent H₂O₂-induced LV dysfunction, and the ability of MPG to prevent H₂O₂-induced LV dysfunction independent of changes in [Ca²⁺]_i indicate that impaired contractility is not due to Ca²⁺ overload. The ability of further increases in [Ca²⁺]_i to reverse H₂O₂-induced LV dysfunction suggests that Ca²⁺ desensitization is the predominant mechanism of ROS-induced contractile dysfunction.     

Coordination of {Mo142} Ring to La3+ Provides Elliptical {Mo134La10} Ring with a Variety of Coordination Modes

A 28-electron reduced C_2h-Mo-blue 34Ǻ outer ring diameter circular ring, [Mo₁₄₂O₄₂₉H₁₀(H₂O)₄₉(CH₃CO₂)₅(C₂H₅CO₂)]^30- (≡{Mo₁₄₂(CH₃CO₂)₅(C₂H₅CO₂)}) comprising eight carboxylate-coordinated (with disorder) {Mo₂} linkers and six defect pockets in two inner rings (four and three for each, respectively), reacts with La³⁺ in aqueous solutions at pH 3.5 to yield a 28-electron reduced elliptical C_i-Mo-blue ring of formula [Mo₁₃₄O₄₁₆H₂₀(H₂O)₄₆{La(H₂O)₅}₄{La(H₂O)₇}₄{LaCl₂(H₂O)₅}₂]^10- (≡{Mo₁₃₄La₁₀}), isolated as the Na₁₀[Mo₁₃₄O₄₁₆H₂₀(H₂O)₄₆{La(H₂O)₅}₄{La(H₂O)₇}₄{LaCl₂(H₂O)₅}₂]·144 H₂O Na⁺ salt. The elliptical structure of {Mo₁₃₄La₁₀} showing 36 and 31 Å long and short axes for the outer ring diameters is attributed to four (A-D) modes of LaO₉/LaO₇Cl₂ tricapped-trigonal-prismatic coordination (TTP) geometries. Two different LaO₂(H₂O)₇ and one LaO₂(H₂O)₂Cl₂ TTP geometries (as A-C modes) for each of two inner rings result from the coordination of all three defect pockets of the inner ring for {Mo₁₄₂(CH₃CO₂)₅(C₂H₅CO₂)}, and two LaO₄(H₂O)₅ TTP geometries (as D mode) result from the displacement of two (acetate/propionate-coordinated) binuclear {Mo₂} linkers with La³⁺ in each inner ring. The isothermal titration calorimetry (ITC) of the ring modification from circle to ellipsoid, showing the endothermic reaction of [La³⁺]/[{Mo₁₄₂(CH₃CO₂)₅(C₂H₅CO₂)}] = 6/1 with ΔH = 22 kJ⋅mol^-1, ΔS = 172 J⋅K^-1⋅mol^-1, ΔG = −28 kJ⋅mol^-1, and K = 9.9 × 10⁴ M^-1 at 293 K, leads to the conclusion that the coordination of the defect pockets to La³⁺ precedes the replacement of the {Mo₂} linkers with La³⁺. ¹³⁹La- NMR spectrometry of the coordination of {Mo₁₄₂(CH₃CO₂)₅(C₂H₅CO₂)} ring to La³⁺ is also discussed.     

Ion-Enhanced Etching Characteristics of sp2-Rich Hydrogenated Amorphous Carbons in CF4 Plasmas and O2 Plasmas

The sp²-rich hydrogenated amorphous carbon (a-C:H) is widely adopted as hard masks in semiconductor-device fabrication processes. The ion-enhanced etch characteristics of sp²-rich a-C:H films on ion density and ion energy were investigated in CF₄ plasmas and O₂ plasmas in this work. The etch rate of sp²-rich a-C:H films in O₂ plasmas increased linearly with ion density when no bias power was applied, while the fluorocarbon deposition was observed in CF₄ plasmas instead of etching without bias power. The etch rate was found to be dependent on the half-order curve of ion energy in both CF₄ plasmas and O₂ plasmas when bias power was applied. An ion-enhanced etching model was suggested to fit the etch rates of a-C:H in CF₄ plasmas and O₂ plasmas. Then, the etch yield and the threshold energy for etching were determined based on this model from experimental etch rates in CF₄ plasma and O₂ plasma. The etch yield of 3.45 was observed in CF₄ plasmas, while 12.3 was obtained in O₂ plasmas, owing to the high reactivity of O radicals with carbon atoms. The threshold energy of 12 eV for a-C:H etching was obtained in O₂ plasmas, while the high threshold energy of 156 eV was observed in CF₄ plasmas. This high threshold energy is attributed to the formation of a fluorocarbon layer that protects the a-C:H films from ion-enhanced etching.     

Detailed Inspection of γ-ray,Fast and Thermal Neutrons Shielding Competence of Calcium Oxide or Strontium Oxide Comprising Bismuth Borate Glasses

For both the B₂O₃-Bi₂O₃-CaO and B₂O₃-Bi₂O₃-SrO glass systems, γ-ray and neutron attenuation qualities were evaluated. Utilizing the Phy-X/PSD program, within the 0.015–15 MeV energy range, linear attenuation coefficients (µ) and mass attenuation coefficients (μ/ρ) were calculated, and the attained μ/ρ quantities match well with respective simulation results computed by MCNPX, Geant4, and Penelope codes. Instead of B₂O₃/CaO or B₂O₃/SrO, the Bi₂O₃ addition causes improved γ-ray shielding competence, i.e., rise in effective atomic number (Z_eff) and a fall in half-value layer (HVL), tenth-value layer (TVL), and mean free path (MFP). Exposure buildup factors (EBFs) and energy absorption buildup factors (EABFs) were derived using a geometric progression (G–P) fitting approach at 1–40 mfp penetration depths (PDs), within the 0.015–15 MeV range. Computed radiation protection efficiency (RPE) values confirm their excellent capacity for lower energy photons shielding. Comparably greater density (7.59 g/cm³), larger μ, μ/ρ, Z_eff, equivalent atomic number (Z_eq), and RPE, with the lowest HVL, TVL, MFP, EBFs, and EABFs derived for 30B₂O₃-60Bi₂O₃-10SrO (mol%) glass suggest it as an excellent γ-ray attenuator. Additionally, 30B₂O₃-60Bi₂O₃-10SrO (mol%) glass holds a commensurably bigger macroscopic removal cross-section for fast neutrons (Σ_R) (=0.1199 cm⁻¹), obtained by applying Phy-X/PSD for fast neutrons shielding, owing to the presence of larger wt% of ‘Bi’ (80.6813 wt%) and moderate ‘B’ (2.0869 wt%) elements in it. 70B₂O₃-5Bi₂O₃-25CaO (mol%) sample (B: 17.5887 wt%, Bi: 24.2855 wt%, Ca: 11.6436 wt%, and O: 46.4821 wt%) shows high potentiality for thermal or slow neutrons and intermediate energy neutrons capture or absorption due to comprised high wt% of ‘B’ element in it.     

L-type Ca channel facilitation mediated by H2O2-induced activation of CaMKII in rat ventricular myocytes

The Ca²⁺-dependent facilitation (CDF) of L-type Ca²⁺ channels, a major mechanism for force-frequency relationship of cardiac contraction, is mediated by Ca²⁺/CaM-dependent kinase II (CaMKII). Recently, CaMKII was shown to be activated by methionine oxidation. We investigated whether oxidation-dependent CaMKII activation is involved in the regulation of L-type Ca²⁺ currents (I_Ca,L) by H₂O₂ and whether Ca²⁺ is required in this process. Using patch clamp, I_Ca,L was measured in rat ventricular myocytes. H₂O₂ induced an increase in I_Ca,L amplitude and slowed inactivation of I_Ca,L. This oxidation-dependent facilitation (ODF) of I_Ca,L was abolished by a CaMKII blocker KN-93, but not by its inactive analog KN-92, indicating that CaMKII is involved in ODF. ODF was not affected by replacement of external Ca²⁺ with Ba²⁺ or presence of EGTA in the internal solutions. However, ODF was abolished by adding BAPTA to the internal solution or by depleting sarcoplasmic reticulum (SR) Ca²⁺ stores using caffeine and thapsigargin. Alkaline phosphatase, β-iminoadenosine 5′-triphosphate (AMP-PNP), an autophosphorylation inhibitor autocamtide-2-related inhibitory peptide (AIP), or a catalytic domain blocker (CaM-KIINtide) did not affect ODF. In conclusion, oxidation-dependent facilitation of L-type Ca²⁺ channels is mediated by oxidation-dependent CaMKII activation, in which local Ca²⁺ increases induced by SR Ca²⁺ release is required.     

Investigating the Impact of Cu2+ Doping on the Morphological,Structural, Optical,and Electrical Properties of CoFe2O4 Nanoparticles for Use in Electrical Devices

This study investigated the production of Cu²⁺-doped CoFe₂O₄ nanoparticles (CFO NPs) using a facile sol−gel technique. The impact of Cu²⁺ doping on the lattice parameters, morphology, optical properties, and electrical properties of CFO NPs was investigated for applications in electrical devices. The XRD analysis revealed the formation of spinel-phased crystalline structures of the specimens with no impurity phases. The average grain size, lattice constant, cell volume, and porosity were measured in the range of 4.55–7.07 nm, 8.1770–8.1097 Å, 546.7414–533.3525 ų, and 8.77–6.93%, respectively. The SEM analysis revealed a change in morphology of the specimens with a rise in Cu²⁺ content. The particles started gaining a defined shape and size with a rise in Cu²⁺ doping. The Cu_0.12Co_0.88Fe₂O₄ NPs revealed clear grain boundaries with the least agglomeration. The energy band gap declined from 3.98 eV to 3.21 eV with a shift in Cu²⁺ concentration from 0.4 to 0.12. The electrical studies showed that doping a trace amount of Cu²⁺ improved the electrical properties of the CFO NPs without producing any structural distortions. The conductivity of the Cu²⁺-doped CFO NPs increased from 6.66 × 10⁻¹⁰ to 5.26 × 10⁻⁶ ℧ cm⁻¹ with a rise in Cu²⁺ concentration. The improved structural and electrical characteristics of the prepared Cu²⁺-doped CFO NPs made them a suitable candidate for electrical devices, diodes, and sensor technology applications.     

The Effects of Ru4+ Doping on LiNi0.5Mn1.5O4 with Two Crystal Structures

Doping of Ru has been used to enhance the performance of LiNi_0.5Mn_1.5O₄ cathode materials. However, the effects of Ru doping on the two types of LiNi_0.5Mn_1.5O₄ are rarely studied. In this study, Ru⁴⁺ with a stoichiometric ratio of 0.05 is introduced into LiNi_0.5Mn_1.5O₄ with different space groups (Fd3¯m, P4₃32). The influence of Ru doping on the properties of LiNi_0.5Mn_1.5O₄ (Fd3¯m, P4₃32) is comprehensively studied using multiple techniques such as XRD, Raman, and SEM methods. Electrochemical tests show that Ru⁴⁺-doped LiNi_0.5Mn_1.5O₄ (P4₃32) delivers the optimal electrochemical performance. Its initial specific capacity reaches 132.8 mAh g⁻¹, and 97.7% of this is retained after 300 cycles at a 1 C rate at room temperature. Even at a rate of 10 C, the capacity of Ru⁴⁺-LiNi_0.5Mn_1.5O₄ (P4₃32) is still 100.7 mAh g⁻¹. Raman spectroscopy shows that the Ni/Mn arrangement of Ru⁴⁺-LiNi_0.5Mn_1.5O₄ (Fd3¯m) is not significantly affected by Ru⁴⁺ doping. However, LiNi_0.5Mn_1.5O₄ (P4₃32) is transformed to semi-ordered LiNi_0.5Mn_1.5O₄ after the incorporation of Ru⁴⁺. Ru⁴⁺ doping hinders the ordering process of Ni/Mn during the heat treatment process, to an extent.     

Theoretical investigation of action potential duration dependence on extracellular Ca in human cardiomyocytes

Reduction in [Ca²⁺]_o prolongs the AP in ventricular cardiomyocytes and the QT_c interval in patients. Although this phenomenon is relevant to arrhythmogenesis in the clinical setting, its mechanisms are counterintuitive and incompletely understood. To evaluate in silico the mechanisms of APD modulation by [Ca²⁺]_o in human cardiomyocytes. We implemented the Ten Tusscher-Noble-Noble-Panfilov model of the human ventricular myocyte and modified the formulations of the rapidly and slowly activating delayed rectifier K⁺ currents (I_Kr and I_Ks) and L-type Ca²⁺ current (I_CaL) to incorporate their known sensitivity to intra- or extracellular Ca²⁺. Simulations were run with the original and modified models at variable [Ca²⁺]_o in the clinically relevant 1 to 3 mM range. The original model responds with APD shortening to decrease in [Ca²⁺]_o, i.e. opposite to the experimental observations. Incorporation of Ca²⁺ dependency of K⁺ currents cannot reproduce the inverse relation between APD and [Ca²⁺]_o. Only when I_CaL inactivation process was modified, by enhancing its dependency on Ca²⁺, simulations predict APD prolongation at lower [Ca²⁺]_o. Although Ca²⁺-dependent I_CaL inactivation is the primary mechanism, secondary changes in electrogenic Ca²⁺ transport (by Na⁺/Ca²⁺ exchanger and plasmalemmal Ca²⁺-ATPase) contribute to the reversal of APD dependency on [Ca²⁺]_o. This theoretical investigation points to Ca²⁺-dependent inactivation of I_CaL as a mechanism primarily responsible for the dependency of APD on [Ca²⁺]_o. The modifications implemented here make the model more suitable to analyze repolarization mechanisms when Ca²⁺ levels are altered.     

The adverse cardiopulmonary phenotype of caveolin-1 deficient mice is mediated by a dysfunctional endothelium

Recently generated caveolin-1 deficient mice (cav-1^−/−) display several physiological alterations such as severe heart failure and lung fibrosis. The molecular mechanisms how the loss of caveolin-1 (cav-1) mediates these alterations are currently under debate. A plethora of studies support a role of cav-1 as a negative regulator of endothelial nitric oxide synthase (eNOS). Accordingly, constitutive eNOS hyperactivation was observed in cav-1^−/−. Given the hyperactivated eNOS enzyme we hypothesized that disturbed eNOS function is involved in the development of the cardiopulmonary pathologies in cav-1^−/−. The present study argues that loss of cav-1 results in enhanced eNOS activity but not in increased vascular tetrahydrobiopterin (BH₄) levels (which acts as an essential eNOS cofactor) thereby causing a stoichiometric discordance between eNOS activity and BH₄ sufficient to cause dysfunctional eNOS signaling. The resultant oxidative stress is largely responsible for major cardiac and pulmonary defects observed in cav-1^−/−. BH₄ donation to cav-1^−/− led to a normalized BH₄/BH₂ ratio, to reduced oxidant stress, to substantial improvements of both systolic and diastolic heart function and to marked amelioration of the impaired lung phenotype. Notably, the antioxidant tetrahydroneopterin which is not essential for eNOS function showed no relevant effect. Taken together these novel findings indicate that dysfunctional eNOS is of central importance in the genesis of the cardiopulmonary phenotype of cav-1^−/−. Additionally, these findings are generally of paramount importance since they underline the deleterious role of an uncoupled eNOS in cardiovascular pathology and they additionally suggest BH₄ as an effective cure.     

Syntheses and a Solid State Structure of a Dinuclear Molybdenum(V) Complex with Pyridine

A mononuclear complex [MoOCl₄(H₂O)]⁻ readily forms a metal−metal bonded {Mo₂O₄}²⁺ core. A high content of pyridine in the reaction mixture prevents further aggregation of dinuclear cores into larger clusters and a neutral, dinuclear complex with the [Mo₂O₄Cl₂(Py)₄] composition is isolated as a product. Solid state structures of two compounds containing this complex, [Mo₂O₄Cl₂(Py)₄]·2.25Py (1) and [Mo₂O₄Cl₂(Py)₄]·1.5PyHCl (2), were investigated by X-ray crystallography.     

Combustion Synthesis and Photoluminescence Properties of Red-Emitting CaAlSiN3:Eu2+ Phosphor for White-LEDs

A combustion synthesis method has been developed for synthesis of Eu²⁺ doped CaAlSiN₃ phosphor and its photoluminescence properties were investigated. Ca, Al, Si, and Eu₂O₃ powders were used as the Ca, Al, Si and Eu sources. The addition of NaN₃, NH₄Cl and Si₃N₄ powders was found to increase significantly the product yield. These powders were mixed and pressed into a compact, which was then wrapped up with an igniting agent (i.e., Mg+Fe₃O₄). The compact was ignited by electrical heating under a N₂ pressure of ≤1.0 MPa. Effects of these experimental parameters on the product yield were investigated and a reaction mechanism was proposed. The synthesized CaAlSiN₃:Eu²⁺ phosphor absorbs light in the region of 200–600 nm and shows a broad band emission in the region of 500–800 nm due to the 4f⁶5d¹ → 4f⁷ transition of Eu²⁺. The sample doped with Eu²⁺ at the optimized molar ratio of 0.04 is efficiently excited by the blue light (460 nm) and generates emission peaking at ~650 nm with peak emission intensity ~106% of a commercially available phosphor, YAG:Ce³⁺(P46-Y3).The internal quantum efficiency of the synthesized phosphor was measured to be 71%, compared to 69% of the YAG:Ce³⁺ (P46-Y3).     

Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria

In exponentially growing yeast, the heme enzyme, cytochrome c peroxidase (Ccp1) is targeted to the mitochondrial intermembrane space. When the fermentable source (glucose) is depleted, cells switch to respiration and mitochondrial H₂O₂ levels rise. It has long been assumed that CCP activity detoxifies mitochondrial H₂O₂ because of the efficiency of this activity in vitro. However, we find that a large pool of Ccp1 exits the mitochondria of respiring cells. We detect no extramitochondrial CCP activity because Ccp1 crosses the outer mitochondrial membrane as the heme-free protein. In parallel with apoCcp1 export, cells exhibit increased activity of catalase A (Cta1), the mitochondrial and peroxisomal catalase isoform in yeast. This identifies Cta1 as a likely recipient of Ccp1 heme, which is supported by low Cta1 activity in ccp1Δ cells and the accumulation of holoCcp1 in cta1Δ mitochondria. We hypothesized that Ccp1’s heme is labilized by hyperoxidation of the protein during the burst in H₂O₂ production as cells begin to respire. To test this hypothesis, recombinant Ccp1 was hyperoxidized with excess H₂O₂ in vitro, which accelerated heme transfer to apomyoglobin added as a surrogate heme acceptor. Furthermore, the proximal heme Fe ligand, His175, was found to be ∼85% oxidized to oxo-histidine in extramitochondrial Ccp1 isolated from 7-d cells, indicating that heme labilization results from oxidation of this ligand. We conclude that Ccp1 responds to respiration-derived H₂O₂ via a previously unidentified mechanism involving H₂O₂-activated heme transfer to apoCta1. Subsequently, the catalase activity of Cta1, not CCP activity, contributes to mitochondrial H₂O₂ detoxification.Cytochrome c peroxidase (Ccp1) is a monomeric nuclear encoded protein with a 68-residue N-terminal mitochondrial targeting sequence (1). This presequence crosses the inner mitochondrial membrane and is cleaved by matrix proteases (2, 3). Mature heme-loaded Ccp1 is found in the mitochondrial intermembrane space (IMS) in exponentially growing yeast (2, 3) but the point of insertion of its single b-type heme is unknown. Under strict anaerobic conditions, Ccp1 is present in mitochondria as the heme-free form or apoform (4). Once cells are exposed to O₂ and heme biosynthesis is turned on, apoCcp1 converts rapidly to the mature holoenzyme by noncovalently binding heme (5).It is well established that mature Ccp1 functions as an efficient H₂O₂ scavenger in vitro (6). Its catalytic cycle involves the reaction of ferric Ccp1 with H₂O₂ (Eq. 1) to form compound I (CpdI) with a ferryl (Fe^IV) heme and a cationic indole radical localized on Trp191 (W191^+•). CpdI is one-electron reduced by the ferrous heme of cytochrome c (Cyc1) to compound II (CpdII) with ferryl heme (Eq. 2), and electron donation by a second ferrous Cyc1 returns CpdII to the resting Ccp1^III form (Eq. 3):Ccp1^III + H₂O₂ → CpdI(Fe^IV, W191^+?) + H₂O[1]CpdI(Fe^IV, W191^+?) + Cyc1^II → CpdII(Fe^IV) + Cyc1^III[2]CpdII(Fe^IV) + Cyc1^II → Ccp1^III + Cyc1^III + H₂O.[3]Because Ccp1 production is not under O₂/heme control (4, 5), CCP activity is assumed to be the frontline defense in the mitochondria, a major source of reactive oxygen species (ROS) in respiring cells (7). Contrary to the time-honored assumption that Ccp1 catalytically consumes the H₂O₂ produced during aerobic respiration (8), recent studies in our group reveal that the peroxidase behaves more like a mitochondrial H₂O₂ sensor than a catalytic H₂O₂ detoxifier (9–11). Notably, Ccp1 competes with complex IV for reducing equivalents from Cyc1, which shuttles electrons from complex III (ubiquinol cytochrome c reductase) to complex IV (cytochrome c oxidase) in the electron transport chain (12).Because CCP activity in the IMS siphons electrons from energy production, an H₂O₂ sensor role for Ccp1 should be energetically more favorable for the cell. Key evidence for a noncatalytic role for Ccp1 in H₂O₂ removal is that the isogenic strain producing the catalytically inactive Ccp1^W191F protein accumulates less H₂O₂ than wild-type cells (10). In fact, this mutant strain exhibits approximately threefold higher catalase A (Cta1) activity than wild-type cells (10) whereas CCP1 deletion results in a strain (ccp1Δ) with negligible Cta1 activity and high H₂O₂ levels (5). Unlike Cta1, which is the peroxisomal and mitochondrial catalase isoform in yeast (13), the cytosolic catalase Ctt1 (14) exhibits comparable activity in the wild-type, Ccp1^W191F, and ccp1Δ strains (10). Given that both Ccp1 and Cta1 are targeted to mitochondria, we hypothesized that Ccp1 may transfer its heme to apoCta1 in respiring cells.Cta1 is nuclear encoded with embedded mitochondrial and peroxisomal targeting sequences (15). Like Ccp1, each monomer noncovalently binds a b-type heme and mature Cta1 is active as a homotetramer. Synthesis of the Cta1 monomer is under O₂/heme control such that the apoenzyme begins to accumulate only during the logarithmic phase of aerobic growth (16). Hence, its O₂/heme independent production (4, 5) allows apoCcp1 to acquire heme while cells are synthesizing apoCta1. This, combined with our observation that Cta1 activity increases in respiring cells producing Ccp1 or Ccp1^W191F but not in ccp1Δ cells (10), led us to speculate that respiration-derived H₂O₂ triggers heme donation from Ccp1 to apoCta1 within mitochondria.What experimental evidence would support heme donation by Ccp1? It has been demonstrated that mutation of the proximal heme Fe ligand, His175, to a residue with weak or no Fe-coordinating ability produces Ccp1 variants (H175P, H175L, H175R, and H175M) that undergo mitochondrial processing but do not accumulate in isolated yeast mitochondria (17). Presumably, reduced heme affinity allows the Ccp1 variants to unfold and cross the outer mitochondrial membrane (17). Hence, we argued that if wild-type Ccp1 donated its heme, the apoprotein would likewise exit mitochondria. Consequently, we examine here age-dependent Ccp1–green fluorescent protein (Ccp1-GFP) localization in live cells chromosomally expressing Ccp1 C-terminally fused to GFP as well as the distribution of wild-type Ccp1 between subcellular fractions. Because weakening or removal of the proximal Fe ligand on His175 mutation reduces heme affinity (17), His175 oxidation in wild-type Ccp1 should have a similar effect, which we investigate here. We further speculated that in the absence of apoCta1 as an acceptor for its heme, more Ccp1 would remain trapped in the IMS so we compare mitochondrial Ccp1 levels in wild-type and cta1∆ cells. Our combined results support triggering of heme donation from Ccp1 to apoCta1 by respiration-derived H₂O₂. Such H₂O₂-activated heme transfer between proteins has not been reported to date and its implications in H₂O₂ signaling are discussed.     

Machine learning–based inverse design for electrochemically controlled microscopic gradients of O2 and H2O2

A fundamental understanding of extracellular microenvironments of O₂ and reactive oxygen species (ROS) such as H₂O₂, ubiquitous in microbiology, demands high-throughput methods of mimicking, controlling, and perturbing gradients of O₂ and H₂O₂ at microscopic scale with high spatiotemporal precision. However, there is a paucity of high-throughput strategies of microenvironment design, and it remains challenging to achieve O₂ and H₂O₂ heterogeneities with microbiologically desirable spatiotemporal resolutions. Here, we report the inverse design, based on machine learning (ML), of electrochemically generated microscopic O₂ and H₂O₂ profiles relevant for microbiology. Microwire arrays with suitably designed electrochemical catalysts enable the independent control of O₂ and H₂O₂ profiles with spatial resolution of ∼10¹ μm and temporal resolution of ∼10° s. Neural networks aided by data augmentation inversely design the experimental conditions needed for targeted O₂ and H₂O₂ microenvironments while being two orders of magnitude faster than experimental explorations. Interfacing ML-based inverse design with electrochemically controlled concentration heterogeneity creates a viable fast-response platform toward better understanding the extracellular space with desirable spatiotemporal control.

Ubiquitous spatiotemporal heterogeneity of natural environments fosters the diverse and fascinating biology that our world embraces, and motivates researchers to mimic natural environments with high spatiotemporal resolution (1–5). Given their close relevance in biochemical metabolisms, dioxygen (O₂) and hydrogen peroxide (H₂O₂) as a surrogate of reactive oxygen species (ROS) are two ubiquitous biologically relevant species in extracellular medium (1, 6). Their extracellular spatial and temporal distributions, particularly at the microscopic scale ranging from 1 μm to 100 μm (7–11), are critical for signal transduction, protein expression, biochemical redox balance, and regulation for cellular metabolism with extensive ecological, environmental, and biomedical implications (Fig. 1A) (1, 3, 8–13). A programmable creation of the spatiotemporal concentration profiles of O₂ and H₂O₂ offers the freedom to mimic, control, and perturb the microenvironments of O₂ and H₂O₂ and hence advance our understanding in microbiology.Open in a separate windowFig. 1.AI-based inverse design of electrochemically generated O₂ and H₂O₂ heterogeneities. (A) The ubiquitous spatiotemporal heterogeneities of O₂ and H₂O₂ in microbiology and the challenges posed in this research topic. (B) The combination of electrochemistry and ML-based inverse design offers a viable approach to mimicking and controlling the heterogeneities of O₂ and H₂O₂ in microbiology. O, oxidant; R, reductant; E_appl (t), the time-dependent electrochemical voltages applied on electrodes. (C) The design of the electrochemically active microwire array electrodes for the generation of O₂ and H₂O₂ gradients; 4e⁻ ORR & 2e⁻ ORR, four-electron and two-electron oxygen reduction reaction into H₂O and H₂O₂, respectively. (D and E) The 45°-tilting images of SEM for the representative microwire arrays used for the training of the ML model (D) and the ones inversely designed for targeted O₂ and H₂O₂ gradients (E); k = (P, D, L), the morphological vector that includes the P, D, and L of the synthesized wire arrays in units of micrometers. (Scale bars, 20 μm.)Despite recent progress (14–18), there remain major technical challenges, particularly in the achievable spatiotemporal resolution and high-throughput design of concentration profiles to suit a plethora of scenarios in microbiology. Approaches based on microfluidics and hydrogels have been able to achieve concentration gradients of O₂ and H₂O₂ through the provision of either O₂/H₂O₂ source (14, 19–21), O₂/H₂O₂ scavenging agents (15, 22, 23), or a combination of both (24) across liquid-impermeable barriers such as agar layers or polymeric thin films (25, 26). Yet such approaches, dependent on passive mass transport and diffusion across more than 10² μm, are inherently incapable of achieving spatial features of less than 100 μm and temporal resolution smaller than ∼10¹ s, the prerequisites to investigate microbiology at cluster or single-cell levels (10–12). Moreover, the large variations of extracellular O₂ and H₂O₂ gradients in different microbial systems demand an inverse design strategy, which, with minimal expenditure, quickly programs a desired concentration profile catering to a specific biological scenario (2–5). The current lack of inverse design protocol impedes the adoption of controllable extracellular heterogeneity to mimic and investigate microbial systems that are of environmental, biomedical, and sustainability-related significance.We envision that the integration of electrochemically generated concentration gradients with inverse design based on machine learning (ML) will address the aforementioned challenges (Fig. 1B). Electrochemistry offers a venue for transducing electric signals into microscopic concentration profiles within ∼10⁰ μm to ∼10² μm away from electrodes’ surface, following the specific electrode reaction kinetics and the mass transport governing equations in the liquid phase (27). Proper designs of electrodes’ microscopic spatial arrangement and electrochemical kinetics lead to concentration gradients that are spatiotemporally programmable by time-dependent electric signals of varying voltages (28). Such benefits of electrochemically generated concentration gradients lead us to employ electrochemistry as a tool to spatiotemporally control the concentration profiles in the extracellular medium. In one example, we found that wire arrays electrochemically active toward O₂ reduction create anoxic microenvironment about 20 μm away from the aerobic external bulk environments, modulate the size and extent of O₂ depletion in the anoxic microenvironment by the wire array’s morphology and applied electrochemical potential (E_appl), and hence enable O₂-sensitive rhizobial N₂ fixation in ambient air powered by renewable electricity (29). Moreover, while not reported before as far as we know, electrochemically generated concentration heterogeneity is commensurate with ML-based inverse design (30, 31), thanks to the mathematically well-defined electrochemical processes that can be numerically simulated (32, 33). We recently reported neural networks, trained by numerically simulated data, that explore the influence of electrode geometry on electrochemical N₂ fixation and achieve optimized morphologies of wire array electrodes untenable without such an ML-based strategy (34). An inverse design for the electrochemically generated gradients will quickly program desirable microenvironments of O₂ and ROS with high spatiotemporal resolutions, thanks to the well-reported electrochemical transformation related to O₂ and H₂O₂ with high electrochemical selectivity (35, 36).In this work, we report an inverse design based on neural networks for independent electrochemical creation of O₂ and ROS microscopic gradients that are relevant, and mimic their extracellular heterogeneities in microbial systems. We hypothesize that careful design of electrocatalysis of O₂ reduction reaction (ORR) can either facilitate four-electron ORR on Pt electrocatalyst for a controllable O₂ spatiotemporal profile or promote two-electron ORR on Au electrocatalyst for a programmable generation of H₂O₂ gradient without significantly perturbing the O₂ one, thanks to their concentration differences in biological mediums (∼10⁻¹ μM to ∼10¹ μM for H₂O₂ and ∼10¹ μM to ∼10² μM for O₂) (2, 7–11). Electrochemically active microwire array electrodes as exemplary model systems (Fig. 1C) are experimentally shown to achieve tunable heterogeneities of O₂ and H₂O₂ independently, with spatial resolution of ∼10¹ μm and temporal resolution of ∼10° s, and are suitable as a platform for independently perturbing biologically relevant O₂ and H₂O₂ profiles in microbial systems. We further established and experimentally validated two neural networks that inversely design the wire array electrodes’ morphologies toward targeted microenvironments of O₂ and H₂O₂, respectively, which is at least one order of magnitude faster than trial-and-error numerical simulation and two orders of magnitude faster than experimental explorations. The demonstrated inverse design of electrochemically generated controlled gradients not only demonstrates a full electrochemical control of concentration profiles in an electrode’s proximity but also establishes an approach of spatiotemporally mimicking and perturbing extracellular space guided by artificial intelligence.