Incorporating a Polyethyleneglycol Linker to Enhance the Hydrophilicity of Mitochondria-Targeted Triphenylphosphonium Constructs.

The targeting of bioactive molecules and probes to mitochondria can be achieved by coupling to the lipophilic triphenyl phosphonium (TPP) cation, which accumulates several hundred-fold within mitochondria in response to the mitochondrial membrane potential (Δψm ). Typically, a simple alkane links the TPP to its "cargo", increasing overall hydrophobicity. As it would be beneficial to enhance the water solubility of mitochondria-targeted compounds we explored the effects of replacing the alkyl linker with a polyethylene glycol (PEG). We found that the use of PEG led to compounds that were readily taken up by isolated mitochondria and by mitochondria inside cells. Within mitochondria the PEG linker greatly decreased adsorption of the TPP constructs to the matrix-facing face of the mitochondrial inner membrane. These findings will allow the distribution of mitochondria-targeted TPP compounds within mitochondria to be fine-tuned.

Low production of mitochondrial reactive oxygen species after anoxia and reoxygenation in turtle hearts.

Extremely anoxia-tolerant animals, such as freshwater turtles, survive anoxia and reoxygenation without sustaining tissue damage to their hearts. In contrast, for mammals, the ischemia-reperfusion (IR) injury that leads to tissue damage during a heart attack is initiated by a burst of superoxide (O2·-) production from the mitochondrial respiratory chain upon reperfusion of ischemic tissue. Whether turtles avoid oxidative tissue damage because of an absence of mitochondrial superoxide production upon reoxygenation, or because the turtle heart is particularly protected against this damage, is unclear. Here, we investigated whether there was an increase in mitochondrial O2·- production upon the reoxygenation of anoxic red-eared slider turtle hearts in vivo and in vitro. This was done by measuring the production of H2O2, the dismutation product of O2·-, using the mitochondria-targeted mass-spectrometric probe in vivo MitoB, while in parallel assessing changes in the metabolites driving mitochondrial O2·- production, succinate, ATP and ADP levels during anoxia, and H2O2 consumption and production rates of isolated heart mitochondria. We found that there was no excess production of in vivo H2O2 during 1 h of reoxygenation in turtles after 3 h anoxia at room temperature, suggesting that turtle hearts most likely do not suffer oxidative injury after anoxia because their mitochondria produce no excess O2·- upon reoxygenation. Instead, our data support the conclusion that both the low levels of succinate accumulation and the maintenance of ADP levels in the anoxic turtle heart are key factors in preventing the surge of O2·- production upon reoxygenation.

The Causes and Consequences of Nonenzymatic Protein Acylation.

Thousands of protein acyl modification sites have now been identified in vivo. However, at most sites the acylation stoichiometry is low, making functional enzyme-driven regulation in the majority of cases unlikely. As unmediated acylation can occur on the surface of proteins when acyl-CoA thioesters react with nucleophilic cysteine and lysine residues, slower nonenzymatic processes likely underlie most protein acylation. Here, we review how nonenzymatic acylation of nucleophilic lysine and cysteine residues occurs; the factors that enhance acylation at particular sites; and the strategies that have evolved to limit protein acylation. We conclude that protein acylation is an unavoidable consequence of the central role of reactive thioesters in metabolism. Finally, we propose a hypothesis for why low-stoichiometry protein acylation is selected against by evolution and how it might contribute to degenerative processes such as aging.

Ester Prodrugs of Malonate with Enhanced Intracellular Delivery Protect Against Cardiac Ischemia-Reperfusion Injury In Vivo.

PURPOSE: Mitochondrial reactive oxygen species (ROS) production upon reperfusion of ischemic tissue initiates the ischemia/reperfusion (I/R) injury associated with heart attack. During ischemia, succinate accumulates and its oxidation upon reperfusion by succinate dehydrogenase (SDH) drives ROS production. Inhibition of succinate accumulation and/or oxidation by dimethyl malonate (DMM), a cell permeable prodrug of the SDH inhibitor malonate, can decrease I/R injury. However, DMM is hydrolysed slowly, requiring administration to the heart prior to ischemia, precluding its administration to patients at the point of reperfusion, for example at the same time as unblocking a coronary artery following a heart attack. To accelerate malonate delivery, here we developed more rapidly hydrolysable malonate esters. METHODS: We synthesised a series of malonate esters and assessed their uptake and hydrolysis by isolated mitochondria, C2C12 cells and in mice in vivo. In addition, we assessed protection against cardiac I/R injury by the esters using an in vivo mouse model of acute myocardial infarction. RESULTS: We found that the diacetoxymethyl malonate diester (MAM) most rapidly delivered large amounts of malonate to cells in vivo. Furthermore, MAM could inhibit mitochondrial ROS production from succinate oxidation and was protective against I/R injury in vivo when added at reperfusion. CONCLUSIONS: The rapidly hydrolysed malonate prodrug MAM can protect against cardiac I/R injury in a clinically relevant mouse model.

Selective Delivery of Dicarboxylates to Mitochondria by Conjugation to a Lipophilic Cation via a Cleavable Linker.

Many mitochondrial metabolites and bioactive molecules contain two carboxylic acid moieties that make them unable to cross biological membranes. Hence, there is considerable interest in facilitating the uptake of these molecules into cells and mitochondria to modify or report on their function. Conjugation to the triphenylphosphonium (TPP) lipophilic cation is widely used to deliver molecules selectively to mitochondria in response to the membrane potential. However, permanent attachment to the cation can disrupt the biological function of small dicarboxylates. Here, we have developed a strategy using TPP to release dicarboxylates selectively within mitochondria. For this, the dicarboxylate is attached to a TPP compound via a single ester bond, which is then cleaved by intramitochondrial esterase activity, releasing the dicarboxylate within the organelle. Leaving the second carboxylic acid free also means mitochondrial uptake is dependent on the pH gradient across the inner membrane. To assess this strategy, we synthesized a range of TPP monoesters of the model dicarboxylate, malonate. We then tested their mitochondrial accumulation and ability to deliver malonate to isolated mitochondria and to cells, in vitro and in vivo. A TPP-malonate monoester compound, TPP11-malonate, in which the dicarboxylate group was attached to the TPP compound via a hydrophobic undecyl link, was most effective at releasing malonate within mitochondria in cells and in vivo. Therefore, we have developed a TPP-monoester platform that enables the selective release of bioactive dicarboxylates within mitochondria.

Stable mitochondrial CICIII2 supercomplex interactions in reptiles versus homeothermic vertebrates.

The association of complex I (CI), complex III (CIII) and complex IV (CIV) of the mitochondrial electron transport chain into stable high molecular weight supercomplexes (SCs) has been observed in several prokaryotes and eukaryotes, but among vertebrates it has only been examined in mammals. The biological role of these SCs is unclear but suggestions so far include enhanced electron transfer between complexes, decreased production of the reactive oxygen species (ROS) O2- and H2O2, or enhanced structural stability. Here, we provide the first overview on the stability, composition and activity of mitochondrial SCs in representative species of several vertebrate classes to determine patterns of SC variation across endotherms and ectotherms. We found that the stability of the CICIII2 SC and the inclusion of CIV within the SC varied considerably. Specifically, when solubilized by the detergent DDM, mitochondrial CICIII2 SCs were unstable in endotherms (birds and mammals) and highly stable in reptiles. Using mass-spectrometric complexomics, we confirmed that the CICIII2 is the major SC in the turtle, and that 90% of CI is found in this highly stable SC. Interestingly, the presence of stable SCs did not prevent mitochondrial H2O2 production and was not associated with elevated respiration rates of mitochondria isolated from the examined species. Together, these data show that SC stability varies among vertebrates and is greatest in poikilothermic reptiles and weakest in endotherms. This pattern suggests an adaptive role of SCs to varying body temperature, but not necessarily a direct effect on electron transfer or in the prevention of ROS production.

Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS.

Ischaemia-reperfusion injury occurs when the blood supply to an organ is disrupted and then restored, and underlies many disorders, notably heart attack and stroke. While reperfusion of ischaemic tissue is essential for survival, it also initiates oxidative damage, cell death and aberrant immune responses through the generation of mitochondrial reactive oxygen species (ROS). Although mitochondrial ROS production in ischaemia reperfusion is established, it has generally been considered a nonspecific response to reperfusion. Here we develop a comparative in vivo metabolomic analysis, and unexpectedly identify widely conserved metabolic pathways responsible for mitochondrial ROS production during ischaemia reperfusion. We show that selective accumulation of the citric acid cycle intermediate succinate is a universal metabolic signature of ischaemia in a range of tissues and is responsible for mitochondrial ROS production during reperfusion. Ischaemic succinate accumulation arises from reversal of succinate dehydrogenase, which in turn is driven by fumarate overflow from purine nucleotide breakdown and partial reversal of the malate/aspartate shuttle. After reperfusion, the accumulated succinate is rapidly re-oxidized by succinate dehydrogenase, driving extensive ROS generation by reverse electron transport at mitochondrial complex I. Decreasing ischaemic succinate accumulation by pharmacological inhibition is sufficient to ameliorate in vivo ischaemia-reperfusion injury in murine models of heart attack and stroke. Thus, we have identified a conserved metabolic response of tissues to ischaemia and reperfusion that unifies many hitherto unconnected aspects of ischaemia-reperfusion injury. Furthermore, these findings reveal a new pathway for metabolic control of ROS production in vivo, while demonstrating that inhibition of ischaemic succinate accumulation and its oxidation after subsequent reperfusion is a potential therapeutic target to decrease ischaemia-reperfusion injury in a range of pathologies.

Psi4 1.4: Open-source software for high-throughput quantum chemistry.

PSI4 is a free and open-source ab initio electronic structure program providing implementations of Hartree-Fock, density functional theory, many-body perturbation theory, configuration interaction, density cumulant theory, symmetry-adapted perturbation theory, and coupled-cluster theory. Most of the methods are quite efficient, thanks to density fitting and multi-core parallelism. The program is a hybrid of C++ and Python, and calculations may be run with very simple text files or using the Python API, facilitating post-processing and complex workflows; method developers also have access to most of PSI4's core functionalities via Python. Job specification may be passed using The Molecular Sciences Software Institute (MolSSI) QCSCHEMA data format, facilitating interoperability. A rewrite of our top-level computation driver, and concomitant adoption of the MolSSI QCARCHIVE INFRASTRUCTURE project, makes the latest version of PSI4 well suited to distributed computation of large numbers of independent tasks. The project has fostered the development of independent software components that may be reused in other quantum chemistry programs.

Control of mitochondrial superoxide production by reverse electron transport at complex I.

The generation of mitochondrial superoxide (O2̇̄) by reverse electron transport (RET) at complex I causes oxidative damage in pathologies such as ischemia reperfusion injury, but also provides the precursor to H2O2 production in physiological mitochondrial redox signaling. Here, we quantified the factors that determine mitochondrial O2̇̄ production by RET in isolated heart mitochondria. Measuring mitochondrial H2O2 production at a range of proton-motive force (Δp) values and for several coenzyme Q (CoQ) and NADH pool redox states obtained with the uncoupler p-trifluoromethoxyphenylhydrazone, we show that O2̇̄ production by RET responds to changes in O2 concentration, the magnitude of Δp, and the redox states of the CoQ and NADH pools. Moreover, we determined how expressing the alternative oxidase from the tunicate Ciona intestinalis to oxidize the CoQ pool affected RET-mediated O2̇̄ production at complex I, underscoring the importance of the CoQ pool for mitochondrial O2̇̄ production by RET. An analysis of O2̇̄ production at complex I as a function of the thermodynamic forces driving RET at complex I revealed that many molecules that affect mitochondrial reactive oxygen species production do so by altering the overall thermodynamic driving forces of RET, rather than by directly acting on complex I. These findings clarify the factors controlling RET-mediated mitochondrial O2̇̄ production in both pathological and physiological conditions. We conclude that O2̇̄ production by RET is highly responsive to small changes in Δp and the CoQ redox state, indicating that complex I RET represents a major mode of mitochondrial redox signaling.

Complex I assembly into supercomplexes determines differential mitochondrial ROS production in neurons and astrocytes.

Neurons depend on oxidative phosphorylation for energy generation, whereas astrocytes do not, a distinctive feature that is essential for neurotransmission and neuronal survival. However, any link between these metabolic differences and the structural organization of the mitochondrial respiratory chain is unknown. Here, we investigated this issue and found that, in neurons, mitochondrial complex I is predominantly assembled into supercomplexes, whereas in astrocytes the abundance of free complex I is higher. The presence of free complex I in astrocytes correlates with the severalfold higher reactive oxygen species (ROS) production by astrocytes compared with neurons. Using a complexomics approach, we found that the complex I subunit NDUFS1 was more abundant in neurons than in astrocytes. Interestingly, NDUFS1 knockdown in neurons decreased the association of complex I into supercomplexes, leading to impaired oxygen consumption and increased mitochondrial ROS. Conversely, overexpression of NDUFS1 in astrocytes promoted complex I incorporation into supercomplexes, decreasing ROS. Thus, complex I assembly into supercomplexes regulates ROS production and may contribute to the bioenergetic differences between neurons and astrocytes.

Ischemic preconditioning protects against cardiac ischemia reperfusion injury without affecting succinate accumulation or oxidation.

Ischemia-reperfusion (IR) injury occurs when blood supply to an organ is disrupted and then restored, and underlies many disorders, notably myocardial infarction and stroke. While reperfusion of ischemic tissue is essential for survival, it also initiates cell death through generation of mitochondrial reactive oxygen species (ROS). Recent work has revealed a novel pathway underlying ROS production at reperfusion in vivo in which the accumulation of succinate during ischemia and its subsequent rapid oxidation at reperfusion drives ROS production at complex I by reverse electron transport (RET). Pharmacologically inhibiting ischemic succinate accumulation, or slowing succinate metabolism at reperfusion, have been shown to be cardioprotective against IR injury. Here, we determined whether ischemic preconditioning (IPC) contributes to cardioprotection by altering kinetics of succinate accumulation and oxidation during IR. Mice were subjected to a 30-minute occlusion of the left anterior descending coronary artery followed by reperfusion, with or without a protective IPC protocol prior to sustained ischemia. We found that IPC had no effect on ischemic succinate accumulation with both control and IPC mice having profound increases in succinate compared to normoxia. Furthermore, after only 1-minute reperfusion succinate was rapidly metabolised returning to near pre-ischemic levels in both groups. We conclude that IPC does not affect ischemic succinate accumulation, or its oxidation at reperfusion.

Identification and quantification of protein S-nitrosation by nitrite in the mouse heart during ischemia.

Nitrate (NO3-) and nitrite (NO2-) are known to be cardioprotective and to alter energy metabolism in vivo NO3- action results from its conversion to NO2- by salivary bacteria, but the mechanism(s) by which NO2- affects metabolism remains obscure. NO2- may act by S-nitrosating protein thiols, thereby altering protein activity. But how this occurs, and the functional importance of S-nitrosation sites across the mammalian proteome, remain largely uncharacterized. Here we analyzed protein thiols within mouse hearts in vivo using quantitative proteomics to determine S-nitrosation site occupancy. We extended the thiol-redox proteomic technique, isotope-coded affinity tag labeling, to quantify the extent of NO2--dependent S-nitrosation of proteins thiols in vivo Using this approach, called SNOxICAT (S-nitrosothiol redox isotope-coded affinity tag), we found that exposure to NO2- under normoxic conditions or exposure to ischemia alone results in minimal S-nitrosation of protein thiols. However, exposure to NO2- in conjunction with ischemia led to extensive S-nitrosation of protein thiols across all cellular compartments. Several mitochondrial protein thiols exposed to the mitochondrial matrix were selectively S-nitrosated under these conditions, potentially contributing to the beneficial effects of NO2- on mitochondrial metabolism. The permeability of the mitochondrial inner membrane to HNO2, but not to NO2-, combined with the lack of S-nitrosation during anoxia alone or by NO2- during normoxia places constraints on how S-nitrosation occurs in vivo and on its mechanisms of cardioprotection and modulation of energy metabolism. Quantifying S-nitrosated protein thiols now allows determination of modified cysteines across the proteome and identification of those most likely responsible for the functional consequences of NO2- exposure.

Suppression of reactive oxygen species generation in heart mitochondria from anoxic turtles: the role of complex I S-nitrosation.

Freshwater turtles (Trachemys scripta) are among the very few vertebrates capable of tolerating severe hypoxia and re-oxygenation without suffering from damage to the heart. As myocardial ischemia and reperfusion causes a burst of mitochondrial reactive oxygen species (ROS) in mammals, the question arises as to whether, and if so how, this ROS burst is prevented in the turtle heart. We find that heart mitochondria isolated from turtles acclimated to anoxia produce less ROS than mitochondria from normoxic turtles when consuming succinate. As succinate accumulates in the hypoxic heart and is oxidized when oxygen returns, this suggests an adaptation to lessen ROS production. Specific S-nitrosation of complex I can lower ROS in mammals and here we show that turtle complex I activity and ROS production can also be strongly depressed in vitro by S-nitrosation. We detect in vivo endogenous S-nitrosated complex I in turtle heart mitochondria, but these levels are unaffected upon anoxia acclimation. Thus, while heart mitochondria from anoxia-acclimated turtles generate less ROS and have a lower aerobic capacity than those from normoxic turtles, this is not due to decreases in complex I activity or expression levels. Interestingly, in-gel activity staining reveals that most complex I of heart mitochondria from normoxic and anoxic turtles forms stable super-complexes with other respiratory enzymes and, in contrast to mammals, these are not disrupted by dodecyl maltoside. Taken together, these results show that although S-nitrosation of complex I is a potent mechanism to prevent ROS formation upon re-oxygenation after anoxia in vitro, this is not a major cause of the suppression of ROS production by anoxic turtle heart mitochondria.

Protein CoAlation: a redox-regulated protein modification by coenzyme A in mammalian cells.

Coenzyme A (CoA) is an obligatory cofactor in all branches of life. CoA and its derivatives are involved in major metabolic pathways, allosteric interactions and the regulation of gene expression. Abnormal biosynthesis and homeostasis of CoA and its derivatives have been associated with various human pathologies, including cancer, diabetes and neurodegeneration. Using an anti-CoA monoclonal antibody and mass spectrometry, we identified a wide range of cellular proteins which are modified by covalent attachment of CoA to cysteine thiols (CoAlation). We show that protein CoAlation is a reversible post-translational modification that is induced in mammalian cells and tissues by oxidising agents and metabolic stress. Many key cellular enzymes were found to be CoAlated in vitro and in vivo in ways that modified their activities. Our study reveals that protein CoAlation is a widespread post-translational modification which may play an important role in redox regulation under physiological and pathophysiological conditions.

Charge-Shift Corrected Electronegativities and the Effect of Bond Polarity and Substituents on Covalent-Ionic Resonance Energy.

Bond dissociation energies and resonance energies for HnA-BHm molecules (A, B = H, C, N, O, F, Cl, Li, and Na) have been determined in order to re-evaluate the concept of electronegativity in the context of modern valence bond theory. Following Pauling's original scheme and using the rigorous definition of the covalent-ionic resonance energy provided by the breathing orbital valence bond method, we have derived a charge-shift corrected electronegativity scale for H, C, N, O, F, Cl, Li, and Na. Atomic charge shift character is defined using a similar approach resulting in values of 0.42, 1.06, 1.43, 1.62, 1.64, 1.44, 0.46, and 0.34 for H, C, N, O, F, Cl, Li, and Na, respectively. The charge-shift corrected electronegativity values presented herein follow the same general trends as Pauling's original values with the exception of Li having a smaller value than Na (1.57 and 1.91 for Li and Na respectively). The resonance energy is then broken down into components derived from the atomic charge shift character and polarization effects. It is then shown that most of the resonance energy in the charge-shift bonds H-F, H3C-F, and Li-CH3 and borderline charge-shift H-OH is associated with polarity rather than the intrinsic atomic charge-shift character of the bonding species. This suggests a rebranding of these bonds as "polar charge-shift" rather than simply "charge-shift". Lastly, using a similar breakdown method, it is shown that the small effect the substituents -CH3, -NH2, -OH, and -F have on the resonance energy (<10%) is mostly due to changes in the charge-shift character of the bonding atom.

Assessing the Delivery of Molecules to the Mitochondrial Matrix Using Click Chemistry.

Mitochondria are central to health and disease, hence there is considerable interest in developing mitochondria-targeted therapies that require the delivery of peptides or nucleic acid oligomers. However, progress has been impeded by the lack of a measure of mitochondrial import of these molecules. Here, we address this need by quantitatively detecting molecules within the mitochondrial matrix. We used a mitochondria- targeted cyclooctyne (MitoOct) that accumulates several- hundredfold in the matrix, driven by the membrane potential. There, MitoOct reacts through click chemistry with an azide on the target molecule to form a diagnostic product that can be quantified by mass spectrometry. Because the membrane potential-dependent MitoOct concentration in the matrix is essential for conjugation, we can now determine definitively whether a putative mitochondrion-targeted molecule reaches the matrix. This "ClickIn" approach will facilitate development of mitochondria-targeted therapies.

Effect of Lone Pairs on Molecular Resonance Energy.

In an effort to understand the theoretical parameters of charge-shift bonding, computational experiments have been designed to elucidate the factors effecting molecular resonance energy. Valence bond theory calculations have been used to calculate resonance energies of homonuclear bonds in the series [HnX-XHn]Z, where n = 0-3 and Z = 2n - 6, 2n - 4, 2n - 2, and 2n for X = C, N, O, and F, respectively. It is shown that the resonance energy decreases as the number of lone pairs increases. Calculated orbital contraction coefficients show that this unexpected result is due to the dominance of orbital size over the lone-pair effect. These results are irrespective of the HXX bond angles. It is also shown that the resonance energy increases with decreasing HXX bond angles due to the increase in p character in the bonding orbital.

Using exomarkers to assess mitochondrial reactive species in vivo.

BACKGROUND: The ability to measure the concentrations of small damaging and signalling molecules such as reactive oxygen species (ROS) in vivo is essential to understanding their biological roles. While a range of methods can be applied to in vitro systems, measuring the levels and relative changes in reactive species in vivo is challenging. SCOPE OF REVIEW: One approach towards achieving this goal is the use of exomarkers. In this, exogenous probe compounds are administered to the intact organism and are then transformed by the reactive molecules in vivo to produce a diagnostic exomarker. The exomarker and the precursor probe can be analysed ex vivo to infer the identity and amounts of the reactive species present in vivo. This is akin to the measurement of biomarkers produced by the interaction of reactive species with endogenous biomolecules. MAJOR CONCLUSIONS AND GENERAL SIGNIFICANCE: Our laboratories have developed mitochondria-targeted probes that generate exomarkers that can be analysed ex vivo by mass spectrometry to assess levels of reactive species within mitochondria in vivo. We have used one of these compounds, MitoB, to infer the levels of mitochondrial hydrogen peroxide within flies and mice. Here we describe the development of MitoB and expand on this example to discuss how better probes and exomarkers can be developed. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

The DIAD Approach to Correlative Synchrotron X-ray Imaging and Diffraction Analysis of Human Enamel.

The Dual Imaging and Diffraction (DIAD) beamline at Diamond Light Source (Didcot, U.K.) implements a correlative approach to the dynamic study of materials based on concurrent analysis of identical sample locations using complementary X-ray modalities to reveal structural detail at various length scales. Namely, the underlying beamline principle and its practical implementation allow the collocation of chosen regions within the sample and their interrogation using real-space imaging (radiography and tomography) and reciprocal space scattering (diffraction). The switching between the two principal modes is made smooth and rapid by design, so that the data collected is interlaced to obtain near-simultaneous multimodal characterization. Different specific photon energies are used for each mode, and the interlacing of acquisition steps allows conducting static and dynamic experiments. Building on the demonstrated realization of this state-of-the-art approach requires further refining of the experimental practice, namely, the methods for gauge volume collocation under different modes of beam-sample interaction. To address this challenge, experiments were conducted at DIAD devoted to the study of human dental enamel, a hierarchical structure composed of hydroxyapatite mineral nanocrystals, as a static sample previously affected by dental caries (tooth decay) as well as under dynamic conditions simulating the process of acid demineralization. Collocation and correlation were achieved between WAXS (wide-angle X-ray scattering), 2D (radiographic), and 3D (tomographic) imaging. While X-ray imaging in 2D or 3D modes reveals real-space details of the sample microstructure, X-ray scattering data for each gauge volume provided statistical nanoscale and ultrastructural polycrystal reciprocal-space information such as phase and preferred orientation (texture). Careful registration of the gauge volume positions recorded during the scans allowed direct covisualization of the data from two modalities. Diffraction gauge volumes were identified and visualized within the tomographic data sets, revealing the underlying local information to support the interpretation of the diffraction patterns. The present implementation of the 4D microscopy paradigm allowed following the progression of demineralization and its correlation with time-dependent WAXS pattern evolution in an approach that is transferable to other material systems.