Neurodegenerative diseases, such as Alzheimer’s disease (AD) and various types of amyloidosis, are incurable; therefore, understanding the mechanisms of amyloid decomposition is crucial to develop an effective drug against them for future therapies. It has been reported that one out of three people over the age of 85 are suffering from dementia as a comorbidity to AD. Amyloid beta (Aβ), the hallmark of AD, transforms structurally from monomers into β-stranded aggregates (fibrils) via multiple oligomeric states. Astrocytes in the central nervous system secrete the human cystatin C protein (HCC) in response to various proteases and cytokines. The codeposition of Aβ and HCC in the brains of patients with AD led to the hypothesis that cystatin C is implicated in the disease process. In this study, we investigate the intermolecular interactions between different atomic structures of fibrils formed by Aβ peptides and HCC to understand the pathological aggregation of these polypeptides into neurotoxic oligomers and then amyloid plaques. To characterize the interactions between Aβ and HCC, we used a complementary approach based on the combination of small-angle neutron scattering analysis, atomic force microscopy and computational modelling, allowing the exploration of the structures of multicomponent protein complexes. We report here an optimized protocol to study that interaction. The results show a dependency of the sequence length of the Aβ peptide on the ability of the associated HCC to disaggregate it. The development of neurodegenerative disorders  correlates with the extended lifetime of our population. The current progress in medicine only slightly affects the long-term survival of patients diagnosed with Alzheimer’s disease. It is therefore of great importance to seek molecular level mechanisms that can contribute to the design of effective drugs or new therapeutic strategies.1, Down’s syndrome2, or type II diabetes3. Their occurrence in the brains of patients during the development of Alzheimer's disease provided the basis for creation of "the amyloid hypothesis" that associated their presence with the pathogenesis of Alzheimer's disease4. However, the long-term search for potential therapeutic methods aimed at removing amyloid plaques was not successful, which focused the researchers' interest on the neurotoxicity of soluble Aβ a peptide oligomers6. Aβ deposits disrupt neuronal cell homeostasis and the transmembrane flow of ions7. The presence of soluble toxic amyloid oligomers and deposits initiates the process of apoptosis in neuronal cells, causing their death and leading to dementia8. It is known that the pathological variant of the Aβ peptide appears in the extracellular space as a result of the amyloidogenic degradation pathway (proteolytic cleavage by β-secretase and γ-secretase)9 of amyloid precursor protein (APP). The main components of the senile plaques include Aβ 1–40, Aβ 1–38, and Aβ 1–42 fragments. Other, less abundant variants are Aβ 1–37 and Aβ 1–4310. Different oligomeric states of Aβ peptide  are present in the intracellular space. Moreover, this structural diversity endows it with a range of properties, including different lifetimes and neuronal toxicity levels. Recently, the amyloid hypothesis was discussed again by Karran and De Strpper11, who emphasized that Aβ pathology drives Tau pathology, so amyloid plaque should be reduced to a low level, which is important in the context of new, more effective therapies.Amyloid beta (Aβ) peptide deposits are the main component of senile plaques, commonly described as the histological fingerprint of Alzheimer’s disease (AD)12. All amyloidogenic polypeptides can form the same fibril core with a similar molecular arrangement13. It is now commonly known that misfolded oligomers (known as "seeds") or, in particular, Aβ peptides in beta-sheet oligomeric form, lead to a chain nucleation reaction similar to prion peptide infection14. There is also some evidence that Aβ oligomers can induce a similar reaction to Tau protein misfolding15, a biological marker of Parkinson’s disease16.The polymorphism of Aβ fibrils depends on the amino acid sequence of peptides involved in the formation of the “steric zipper” and the “cross-β spine” within the fibril structure17. Intense neuronal HCC immunoreactivity has been observed in cortical neurons of AD patients, particularly in regions most severely affected by the disease, such as the temporal and entorhinal cortical areas and the hippocampal region18. In addition, these conglomerates can be observed, in some cases, even in healthy subjects19. The HCC concentration in CSF is elevated in homeostatic conditions, which might suggest an important protective role in the central nervous system20. Moreover, it has been observed that HCC promotes neural stem‐cell growth. Typically, the concentration of HCC in the CSF of healthy adults oscillates at approximately 5.8 mg/L, which is 5–6 times higher than that in plasma22. Increased HCC concentrations in CSF have been reported in patients with infectious nervous disorders23. The pathogenic form of HCC is manifested by the Leu68Gln mutation, where a hydrophobic amino acid residue is replaced by polar and bulkier one in the hydrophobic core of HCC, which results in development of serious disease—hereditary cystatin C amyloid angiopathy (HCCAA)24.Breaking down amyloid plaques and reducing the formation of Aβ oligomers is a potential way to slow or inhibit the progression of Alzheimer's disease. Therefore, factors capable of breaking down amyloid structures have been intensively researched. Recent studies revealed that Aβ in amyloid plaques in the brain tissue and the cerebrospinal fluid (CSF) of AD patients is accompanied by other proteins, including also human cystatin C (HCC). This small globular protein (MW = 13.3 kDa), secreted by all nucleated cells and present in all body fluids, including the CSF, is involved in controlling the enzymatic activity of cysteine proteases25. The monomeric state of human cystatin C is a native, functional state of this protein in body fluids25. HCC only in the monomeric form is fully effective as an inhibitor of proteases, however, significant amounts of extracellular HCC dimers are present in pathological conditions26.The structure of human cystatin C is flexible and very susceptible to conformational changes and oligomerization through a domain-swapping mechanism see Fig. , forming27. First obtained structure of the wild type HCC (wt HCC) was a domain-swapped dimer which crystallized in two polymorphic crystal forms29. The global conformation of the wt HCC dimer is diverse—the tetragonal form28 forms an extended dimer and the structure of more compact HCC dimer is observed for cubic polymorph25. The structure of HCC dimers with 3D domain swapping was also observed for several point mutants of 30 as well as for N-truncated HCC form31. Unfortunately, the spatial structure of the wt HCC monomer was not characterised so far, however, monomeric structures have been obtained for point mutants in the hinge loop area, which protect molecule against domain swapping  and also for the monomeric form of HCC stab1 variant with an additional engineered disulfide bond introduced (Leu47Cys)-(Gly69Cys)33. Domain swapping is however probably not a critical parameter for cystatin to undergo the oligomerisation. Results obtained by Pearlfein et al.34 for stabilised against domain swapping HCC mutant  showed the possibility of the formation of oligomeric forms, however without domain swapping, since the oligomers retained the inhibitory activity.The tree-dimensional structures of several molecular variants of HCC, were characterized by X-ray crystallography and NMR spectroscopy35 but can also be induced in vitro via X-ray irradiation36. Higher oligomers  were observed and characterized at the microscopic level39. The oligomerization and formation of fibril-like structures of HCC are strongly related to its β-sheet secondary structure and hydrophobic interactions occurring within the protein28.Conformational changes leading to HCC oligomerization are characteristic of the disease state (e.g. as for Leu68Gln variant in HCCAA)41. Kaeser and co-workers42 communicated that HCC overexpression in brains of APP-transgenic mice reduced deposition of cerebral amyloid-β and HCC is able to bind amyloid-β and inhibit Aβ fibril formation. Also Mi et al.43 presented data that HCC binds soluble amyloid-β peptide and inhibits formation of cerebral amyloid in APP-transgenic mice. In a later study performed by Tizon et al.44, it was indicated that the extracellular addition of HCC in presence of preformed oligomeric or fibrillar Aβ forms increased cell survival. They underlined that HCC inhibits Aβ aggregation, however HCC is not able to dissolve preformed Aβ fibrils or oligomers.44 It is also worth to note interesting observation communicated by Wang and co-workers45, that HCC reduced Aβ40 secretion in human brain microvascular endothelial cells. However, in this case HCC induced reduction of Aβ40 level was caused by degradation of β-secretase activity (BACE1).The correlation between Aβ and HCC was supported by the discovery of the direct binding of the proteins into a 1:1 molar high‐affinity complexDespite these interesting from a point of view of potential anti-amyloid therapeutics observations, the mechanism of Aβ-HCC direct and indirect interactions at molecular level as well as the structure of Aβ-HCC complex have not yet been thoroughly fully characterized.47. The results showed a dependency of the sequence length of the Aβ peptide creating the fibril and the ability of the associated HCC to depolymerize them. We discovered that HCC may be a guardian of the CNS and not only inhibit amyloidogenic peptide aggregation but also depolymerize some deposits what could provide protection against further toxic influences on neuronal cells. We believe that understanding the mechanisms of the complexation of human cystatin C with Aβ will provide more insight into possible ways to stop or delay this pathogenic process.At this point, it should be considered whether human cystatin C is able to block the formation or break down of existing amyloid Aβ fibrils. The aforementioned presence of HCC in amyloid deposits could potentially indicate such an effect. Therefore, the aim of our study was to visualize interactions in solution between HCC and fibrils formed by selected amyloid beta peptides using a combination of small-angle neutron scattering (SANS), atomic force microscopy (AFM) and modelling. For our study we selected two Aβ peptides, Aβ 1–42, the most abundant peptide variant found in amyloid deposits and Aβ 3–28 peptide, also forming stable fibrils. This second short synthetic peptide contains central hydrophobic region (residues 16–22) important in oligomerisation process and lysine K284HCO3 in water with pH 10.5 (adjusted with ammonium hydroxide) and buffer B is 80% acetonitrile in buffer A. The column was operated by a Shimadzu chromatography system . After purification, the obtained peptides were lyophilized, and their level of purity was verified with analytical chromatography using an XTerra RP8 column  operated by a Nexera-i chromatography system (Shimadzu). We were able to produce high-quality peptides with a purity above 96%.All peptides were synthesized by means of solid-state synthesis using an automatic, microwave-assisted peptide synthesizer  and purified with RP-HPLC. An XBridge Prep C8, 5 µm OBD (Waters) column was used, and peptides were eluted with an appropriate gradient of buffers A and B, where buffer A is composed of 5 mM NH48. First, a solution containing 1 mg of Aβ 1–42 or Aβ 3–28 peptide per 1 mL of ice-cold HFIP (Sigma Aldrich) was prepared and sonicated twice for 15 min in a bath sonicator (Ultrasonic Bath Elmasonic) on ice. After the sonication step, the peptide/HFIP solution was divided into small aliquots and evaporated, and vials with dry peptide films were stored at − 20 °C for future experiments. For experiments presented here, these fractions were freshly prepared and stored no longer than the 5 days before using.To trigger Aβ peptide disaggregation into its monomeric form, hexafluoro-2-propanol (HFIP) was used. HFIP is a strong acid that effectively disrupts the secondary structure of aggregated peptides and causes them to unfold; therefore, we used a modification of a previously published protocol of Aβ disaggregation49. To obtain fibrils of Aβ peptides, a solution of the monomeric form of Aβ peptide (1 mg/mL) was incubated for up to 48 h at 40 °C under gentle agitation. The oligomeric (fibrillary) state of Aβ 1–42 and Aβ 3–28 peptides was assessed using atomic force microscopy. The aggregated peptide samples were then used for SANS kinetics studies and imaging by atomic force microscopy (AFM).The fibrillary Aβ 1–42 or Aβ 3–28 peptides were the main objects of our study; therefore, a standard fibrillation protocol was applied50. The DNA containing the HCC wild-type gene, ampicillin resistance gene, and temperature promoter was transformed and expressed in Escherichia coli BL21 (DE3) competent cells . For the expression of 2H-labelled protein, a modified protocol described by Marley et al.51 was used. As described in a previous publication40, transformed E. coli culture was incubated at 32 °C until the OD600 = 0.4. Next, the bacterial suspension was transferred into M9 minimal medium52 prepared with heavy water (D2O) as a solvent and cultured until the OD = 0.6. Then, the incubation was continued at 42 °C for another 3 h. The isolation and purification of the expressed HCC wild-type proteins were performed with a two-step procedure involving the use of ion-exchange chromatography and size exclusion chromatography53.Initially, the standard HCC expression and purification protocol was tested2O-based media. Ampicillin resistance plasmids are not recommended in high cell density culture protocols with deuterated media because the secreted beta-lactamase degrades antibiotics and causes overgrowth of plasmid-free cells54. Therefore, another approach was undertaken. To optimize protein production, we successfully applied the “high-yield expression” procedure proposed by Cai et al.55, where HCC overexpression occurred at a high bacterial growth level  and at a low culture volume. The newly designed and more robust protocol for HCC production was applied. The pET-24(a) plasmid containing the HCC gene, kanamycin resistance gene, and IPTG-controlled expression promoter was transformed into Shuffle T7B (New England Biolabs) E. coli cells—a strain with enhanced capacity to correctly fold proteins with multiple disulfide bonds  within its cytoplasm. To mitigate the extreme sensitivity of the Shuffle strain to the level of oxygen dissolved in D2O media, different media were tested, and TB medium was selected. The transformed bacterial cells were cultured on LB agar plates at 18 °C for 48 h. Next, a single colony was selected and transferred to 25 mL of LB medium (H2O) containing 50 mg/mL kanamycin and cultured overnight at 30 °C with agitation. The medium was then centrifuged at low rpm (4668 g), and the cell pellet was resuspended in fresh TB medium containing 80% D2O and 20% deionized water (v/v) as a solvent with 100 mg/L kanamycin. The cell culture was then incubated at 30 °C with shaking (220 rpm). The OD600 was measured every 2 h, and induction using 0.2 mM IPTG was performed when the culture optical density reached OD = 5–6. Then, the cells were incubated at 18 °C and harvested after another 20 h. The bacterial pellet was resuspended in lysis buffer  and sonicated  for 5 min  in an ice bath. Next, the suspension was centrifuged for 30 min at 14000 rpm, and the crude lysate was purified with ion-exchange chromatography with HiTrap Q (the flow-through was collected). The purified fractions were then dialyzed against 20 mM ammonium bicarbonate buffer (pH 7.8) and lyophilized. The lyophilized protein was then again diluted in ammonium bicarbonate buffer and purified with size exclusion chromatography with a Superdex 75 10/300 GL column (Cytiva). The presence of D-HCC protein in all the fractions obtained after each purification step was monitored using SDS–PAGE electrophoresis.The protein produced with the described procedure exhibited a high level of deuteration and purity; however, the protocol was not particularly efficient due to the incompatibility of ampicillin with D1H)- or deuterium (2H)-labelled HCC) were performed on the D22 beamline at the Institute Laue-Langevin (ILL), Grenoble (France). Approximately 400 µL of sample was used to fill 1 mm thick banjo-type quartz cuvettes (Hellma 120-000-1-40), which were then closed without introducing any air bubbles. Cuvettes were placed in a tumbling rack to avoid fibril precipitation through the measurement  in double logarithmic plot to power law (I ≈ q−α), where q is a scattering vector and α is a scattering exponents59. Especially the relation between α and specific types of structures  was analysed, similarly as was used previously in analysis of other fibrillar or polymeric systems62.The analysis of the collected data was performed with the use of GRASP,63). The model strongly correlates with the collected experimental data. To visualize the Aβ 3–28 fibril, we adjusted the sequence of the Aβ 1–40 PDB structure and performed molecular dynamics (MD) simulations with Unress64 to optimize the new structure. Unress is a coarse-grain method allowing rapid MD simulation for relatively large molecular complexes. To perform the molecular docking of monomeric Aβ peptides to the HCC structure, we used CabsDock software65, allowing the docking of a flexible amino acid chain to the receptor–protein 3D structure. The restraints of the interaction between Aβ and HCC reported by NMR studies66 were enforced in the simulation. The molecular structures were visualised using PyMOL Molecular Graphics System .To visualize and compare the experimental results with the theoretical data, we chose a model of Aβ fibrils  optimized for biological imaging. Aβ peptide fibrils and their mixtures with human cystatin C were studied using a Tap150-G silicon soft tapping mode AFM probe (BudgetSensors Innovative Solutions Bulgaria Ltd.) and the intermittent (air) contact mode. The visualization and analysis of the topographic images of the samples were carried out using Gwyddion 2.60 modular software for SPM data analysis67.The fibrillary samples of Aβ 1–42 or Aβ 3–28 peptides and their mixtures with human cystatin C in a molar ratio of 1:1 were diluted  in deionized water, and then these solutions were deposited (sample volume approximately 10 µL) onto a freshly prepared mica surface . The samples deposited on mica were dried for a few hours under the cover at room temperature. For AFM imaging, we applied a modified procedure, used previously by us for imaging of human cystatin C oligomersIn our study, we used a combination of experimental methods and molecular modelling to describe the influence of HCC on Aβ 1–42 and Aβ 3–28 peptide fibrils, present the morphology of the complexes formed by selected Aβ peptides and HCC as a result of their interactions and visualize the Aβ peptide fibril disaggregation process induced by HCC.E. coli (Shuffle T7B strain) was implemented. This allowed us to produce approximately 5 mg of purified D-HCC from 400 millilitres of culture. Detailed results of the production and purification of D-HCC are summarized in the Supplementary data , as well as only one structure of Aβ amyloid fibrils purified from Alzheimer's brain tissue obtained using cryo-electron microscopy (PDB: 6SHS63). Amyloid fibrils can also be easily detected and characterized with atomic force microscopy72. However, amyloid peptides are very hard to characterize due to the nucleation reaction, which can start very rapidly. Moreover, the difficulties in the description of structural mechanisms of Aβ aggregation result from the complexity of fibril polymorphism. Registering the structure of the complexes formed between Aβ fibrils and other biomolecules with high-resolution methods  is close to impossible because of the high level of sample polydispersity. Therefore, for the structural characteristics of Aβ fibrils, the cryoEM model of Aβ 1–40 fibrils was used. The atomic model of the Aβ 3–28 structure was obtained by a homology modelling approach using Aβ 1–40 as a template. The model was optimized using molecular dynamics (MD) simulation by the Unress64 method. Both these structures and their structural parameters  are presented in Fig. Currently, there are only a few spatial structures of Aβ peptides registered with NMR spectroscopy methods 63 corresponds well with a cross-section function of the ellipsoid model fitted for the experimental scattering profile  alone as well as in mixtures with human cystatin C. Samples were then incubated for approximately 9 h and further investigated by AFM. The incubation time was chosen to correlate with the full time of the SANS kinetic experiment. The representative results of AFM studies are presented in Fig. 19. However, based solely on the results of AFM and SANS studies, we cannot conclusively confirm the direct deposition of HCC on Aβ 1–42 fibrils.The aim of these experiments was to track and visualize the disaggregation process of the Aβ 3–28 and Aβ 1–42 peptide fibrils in the presence of HCC. The data obtained by AFM topography studies of aggregates formed after overnight incubation of both types of fibrils with human cystatin C are shown in Fig. The most important observation from AFM imaging is that the fibrils of the Aβ 3–28 variant appear to be disaggregated after incubation with human cystatin C. The characteristic morphology  disappears completely see Fig. D. They a1. Aβ fibrils are already widely characterized by a variety of methods, such as TEM63 NMR77, AFM78, and thioflavin assays79. However, structural studies of Aβ peptides and their binding to other molecules are difficult due to the demanding nature of the sample. The method of choice, SANS with the use of contrast match80, turns out to be a great experimental method for tracking structural changes in amyloid fibrils, including the interactions between fibrils and other biomolecules81. Combining the SANS methodology with other techniques, such as AFM, we are able to obtain a full picture of the possible interaction and impact of different biomolecules on the aggregation of amyloid-beta peptides.Aβ peptides are the major component of senile plaques and are the fingerprint of Alzheimer's disease81. The senile plaques of the Aβ peptide are composed of Aβ 1–40, Aβ 1–38, Aβ 1–42, Aβ 1–37, and Aβ 1–43. The shorter variant 1–15/16 also appears due to the cleavage of APP; however, it does not take part in forming aggregates82. Aβ 1–28 was characterized as a predominantly helical part of the transmembrane domain83.The different Aβ peptides are produced in sequential cleavage by secretase enzymes in the process of APP degradation84. The results obtained in this study indicate that, during interaction with amyloid fibrils, HCC not only inhibits aggregation85. This is one of the probable reasons why HCC was discovered as a codeposit of senile plaques19. In addition, HCC can penetrate and depolymerize fibrils of the Aβ 3–28 variant, which means that the aggregation can be reversed. We suspect that HCC, as a part of senile plaques, may take part in the disaggregation of fibrils or old aggregates. However, it is worth referring here to the studies conducted by Tizon et al., which showed that the extracellular addition of HCC and preformed Aβ oligomers or fibrils increased cell survival. HCC inhibits Aβ aggregation, unfortunately it is not able to dissolve formed Aβ fibrils or oligomers44.The shorter Aβ 3–28 variant sequence  causes the fibrils to be less hydrophobic, less condensed, more exposed to interactions, and more sensitive to the structural transition. The length of Aβ peptides and conformational state can determine neurotoxicity, aggregation tendency or interactions with other biomolecules86. The intramolecular interaction between these amino acids driven by a combination of hydrophobic and electrostatic interactions is important. It was shown that Zn2+ ions can break an Asp23-Lys28 salt bridge87. In the modelled fibril structure, the Glu22 and Asp23 amino acids can interact by electrostatic interactions when Lys28 stabilizes the loop conformations. Moreover, fibril formation is probably driven by the turn motif of Gly25, Ser26, Asn27, the hydrophobic interaction of Val24, and the side chain of Lys28. In the presented fibril structure Aβ 1–42, Lys28 stabilizes the loop together with Val40 by hydrophobic interaction; however, in the case of variant Aβ 3–28, the C-end is missing, and Lys28 is exposed. Fibril depolymerization of variant 3–28 observed during our experiments can be associated with the fact that Lys28 is not involved in structure stabilization but is exposed to electrostatic and hydrophobic interactions with HCC  and a compact conformation (PDB code: 1G96). Therefore, it should be assumed that the abovementioned flexibility of the HCC conformation allows the exposure of HCC hydrophobic areas that can interact with the surface of the Aβ peptide fibrils. On the other hand, the N-terminal fragment of HCC is highly flexible and could also be involved in the penetration and disruption of the Aβ fibril structure. The structure of such a flexible, unstructured N-terminal HCC fragment was proposed by Perlenfein et al.33. The phenomenon of disaggregation of 3–28 Aβ fibrils by HCC may be driven by disruptions of the hydrophobic forces and dynamic codestabilization between those molecules. Therefore, it can be assumed that the flexible N-terminal fragment of the HCC structure may interact with some amyloid beta fibrils, inducing their disaggregation. Additionally, both of the described effects can probably act synergistically.Analysing the model of the Aβ 3–28 fibril, we can see that its core is stabilized by hydrophobic amino acids. HCC molecules can also, under certain conditions, undergo conformational changes to expose a more hydrophobic interface. The structure of HCC in its native monomeric form is held together by hydrophobic forces17, (ii) which of the known, naturally occurring sequences of Aβ peptides (besides Aβ 1–42) are resistant to disaggregation by HCC and whether this resistance can depend on different structures of the steric zipper formed by those peptides.Further investigation of the disaggregation properties of HCC towards amyloid fibrils formed by other variants of Aβ peptide can provide more answers about the molecular mechanisms underlying the protective properties of HCC. Of extreme interest would be to answer some newly arisen questions: (i) whether the process of disaggregation of the amyloid fibrils by HCC occurs naturally and whether it depends on the level of expression of HCC in CSF94. Recently several small molecules, designed using a joint pharmacophore space (JPS) method, were successfully used in the disassembling of Aβ oligomers and protofibrils95. The observed in this study changes were similar to our AFM imaging results, showing the disappearance of Aβ 3–28 fibrils induced by HCC. Particularly the gradual disaggregation of Aβ fibril structures by the drug candidates (eg. AC0201) reported by Jin et al.95 and formation of oligomers or aggregates was visible there. Many biomolecules have been investigated in clinical trials of Aβ peptide targeting therapy and the reduction of amyloid deposits through degradation and removal. There are also studies aimed at inducing such processes as the inhibition of protein aggregation through the participation of antibodies and enzyme inhibitors97. To date, scientists have designed several inhibitors of Aβ and Tau aggregation, including the promising anti-Aβ monoclonal antibody aducanumab, which is now a treatment endorsed by the Food and Drug Administration (FDA)98. We believe that antibodies or peptides inspired by proteins such as HCC may be of therapeutic importance for the treatment and elimination of amyloidogenic peptides or the digestion of already present senile plaques. Designing new disaggregation strategies is crucial because they are effective in combating and possibly causing regression of the disease when the brain of the individual is in an advanced pathological state.The process of disaggregating amyloid beta peptide fibres can be induced by the presence of various molecules, ranging from small molecules such as surfactants to large protein molecules75. It is also worth noting that Kaeser et al. observed that cystatin C overexpression in brains of transgenic mice reduced the deposition of β-amyloid42. However, Chen et al.99 observed that elevated level of cystatin C in serum of AD patients correlated with the disease progress. Moreover Mi et al.100 reported the association of soluble forms of HCC with Aβ 1–42. Therefore, the continuation of further work leading to a full understanding of the impact of HCC on the amyloidogenic processes is necessary.Summing up the results of our research, we would like to emphasize that they are part of the current trend in the characterization of HCC interactions with Aβ peptides. The growing interest in cystatin as an Aβ binding agent dates back to the early twenty-first century when Levy and co-workers identified it in deposits with amyloid-beta protein in the brain of Alzheimer disease patientsTherefore, we hope that our observations on the effect of HCC on the disaggregation of the Aβ 3–28 peptide will become an inspiration for the search for new methods of therapy for Alzheimer's disease.Supplementary Figures.


BAZ2A promotes migration and invasion in prostate cancer. Two chemical probes, the specific BAZ2‐ICR, and the BAZ2/BRD9 cross‐reactive GSK2801, interfere with the recognition of acetylated lysines in histones by the bromodomains of BAZ2A and of its BAZ2B paralog. The two chemical probes were tested in prostate cancer cell lines with opposite androgen susceptibility. BAZ2‐ICR and GSK2801 showed different cellular efficacies in accordance with their unequal selectivity profiles. Concurrent inhibition of BAZ2 and BRD9 did not reproduce the effects observed with GSK2801, indicating possible off‐targets for this chemical probe. On the other hand, the single BAZ2 inhibition by BAZ2‐ICR did not phenocopy genetic ablation, demonstrating that bromodomain interference is not sufficient to strongly affect BAZ2A functionality and suggesting a PROTAC‐based chemical ablation as an alternative optimization strategy and a possible therapeutic approach. In this context, we also present the crystallographic structures of BAZ2A in complex with the above chemical probes. Binding poses of TP‐238 and GSK4027, chemical probes for the bromodomain subfamily I, and two ligands of the CBP/EP300 bromodomains identify additional headgroups for the development of BAZ2A ligands. It contributes to migration, invasion and stemness of metastatic and recurrent PCa , also inhibiting BAZ2B with similar potency . The only other BAZ2 chemical probe available is GSK2801, with KD = 257 and 136 nM for BAZ2A and BAZ2B, respectively,  and TAF1L (KD = 3.2 μM) bromodomains.The Structural Genomics Consortium developed several chemical probes for a variety of bromodomains , differently in this respect from the clean BAZ2‐ICR profile .GSK2801 slowed PC3 and 22Rv1 cellular growth also in a 3D setting. BAZ2‐ICR instead did not significantly affect PC3 spheroids, while interestingly impacting those deriving from 22Rv1 cells Figure . BRD9 inThe observed limited efficacy of BAZ2‐ICR, together with the suspected GSK2801 off‐target effect, could be overcome with the development of BAZ2A‐directed degraders. These would chemically phenocopy the genetic ablation effects as well as improve specificity looking for optimal surface complementarity between BAZ2A and ubiquitin ligases , to the pt rotamer , folding on the side of BAZ2‐ICR and reducing its solvent accessible area. The Glu1820 carboxylic group is at about 4 Å distance from the probe benzonitrile and methyldiazole tails, these last interacting between them through π–π stacking. In the context of largely conserved interactions, the above comparison supports the similar affinity of the BAZ2‐ICR probe for BAZ2A and BAZ2B. The methyldiazole tail could be extended with a linker/E3 recruiter moving out of the pocket in the region delimited by Glu1820 and Asn1823 side chains.The co‐crystal structure of BAZ2A in complex with BAZ2‐ICR shows a binding pose almost superimposable to that previously reported for a BAZ2‐ICR analogue bound to BAZ2B Figure . Slight GSK2801 interacts with BAZ2A slightly less efficiently than in BAZ2B , also active on BPTF and BRD9 with KD = 120 nM and 1.1 μM, respectively,   are relatively more similar to GSK4027; these interactions, not displacing any of the structural water molecules, are also characterized by an adverse entropic contribution in the context of a much more favorable enthalpic change, as also seen for the vast majority of bromodomain inhibitors. Indeed, the change in sign of the entropic term observed for TP‐238 binding to BAZ2A (compared to BPTF and CECR2) closely resembles the effect reported for crotonyllysine binding to the TAF1 bromodomain (compared to Kac), also displacing an additional water molecule from the binding pocket  moving toward the terminal positively‐charged UP39 piperazine ring, however at a distance >5 Å. A second UP39 molecule is bound to the BAZ2A bromodomain. The acetylbenzene headgroup drives the interaction with a hydrogen bond between the acetyl oxygen and the Asn1823 main chain nitrogen and through the stacking with the central methylbenzoate ring of the other UP39 molecule. This generates a four‐layer sandwich, similar to that observed in the complex with BAZ2‐ICR, with the two UP39 molecules stacked in between side chains of Trp1816 and Leu1826. The tail of the second UP39 molecule protrudes away from the bromodomain and is stabilized by additional interactions with a BAZ2A symmetry‐related copy. This four‐layer organization is also observed in the different CBP/BAZ2 cross‐reactive inhibitor UZH23, described in the supplementary material (Figure Compound UP39 was developed as a CBP bromodomain inhibitor with a l Figure , and in l Figure , 2022. T3This work first demonstrates that the activities of GSK2801 and BAZ2‐ICR are not superimposable and the effects associated with the use of GSK2801 do not appear to derive from the sole interference with BAZ2 and BRD9 bromodomains. We then show that the sole interference with the BAZ2A bromodomain by BAZ2‐ICR does not reproduce the effects observed by its genetic ablation. BAZ2 chemical probes could however be evolved in PROTACs to improve their cytostatic and anti‐metastatic efficacies; the crystallographic structures here disclosed identify suitable derivatization points to move out of the BAZ2A Kac pocket allowing to present BAZ2A differently to E3 ligases and increasing the probability of obtaining effective degraders. Finally, structures of BAZ2A in complex with TP‐238, GSK4027 and UP39 provide additional headgroup and peculiar binding modes for the development of new BAZ2 inhibitors.4BAZ2A and BAZ2B bromodomains were produced and their activity evaluated by AlphaScreen as previously reported (Dalle Vedove et al., Giulia Cazzanelli: Conceptualization; Investigation; Writing ‐ review & editing; Formal analysis; Methodology. Andrea Dalle Vedove: Investigation; Formal analysis; Methodology. Eleonora Parolin: Investigation. Vito Giuseppe D'Agostino: Investigation; Formal analysis. Andrea Unzue: Resources. Cristina Nevado: Resources. Amedeo Caflisch: Conceptualization; Writing ‐ review & editing; Supervision; Funding acquisition; Resources. Graziano Lolli: Conceptualization; Writing ‐ original draft; Writing ‐ review & editing; Formal analysis; Supervision; Funding acquisition; Resources.The authors declare no competing financial interest.Data S1: Supporting InformationClick here for additional data file.


Diabetes mellitus is one of the chronic metabolic diseases whose control remains a challenge. Its increased incidence was mainly attributed to increased environmental contamination. Therefore, this study aims to investigate the effect of feeding clover microgreen (CM) on a diabetes model with or without aflatoxin exposure. Rats were distributed into 8 groups. G1 was a control group. G2 was fed CM. G3 was administered aflatoxin orally. G4 was fed clover and administered aflatoxin. G5 was diabetic rats. G6 was diabetic rats fed CM. G7 was diabetic rats administered aflatoxin. G8 was diabetic rats administered aflatoxin and fed CM. Phytochemical analysis of the CM showed that gardenin was the most common compound. The administration of aflatoxin aggravated diabetes. The groups fed CM showed a decreased glucose concentration compared to the unfed groups. Liver and kidney function parameters were improved by CM. The histopathological alteration of the pancreas, liver, and kidneys was relieved in CM‐fed groups. The area % of insulin in islets of Langerhans was increased in CM‐fed groups. Feeding CM also enhanced the oxidative stress biomarkers. In conclusion, CM improved all evaluated parameters in diabetic rats either exposed to aflatoxin or not compared to the control. Diabetes mellitus is one of the chronic metabolic diseases whose control remains a challenge. Its increased incidence was mainly attributed to increased environmental contamination. Therefore, this study aims to investigate the effect of feeding clover microgreen (CM) on a diabetes model with or without aflatoxin exposure. According to the World health organization, 422 million people suffer from diabetes in 2014 with an increased prevalence in low‐ and middle‐income countries than in high‐income countries.Long‐lasting uncontrolled hyperglycemia in type 2 diabetes affects vital organs such as the kidney, retina, and nervous system, which subsequently affect the quality of life and life expectancy  also known as red clover is a herbaceous plant. Various classes of phytoconstituents have been reported in different parts of the plant including pterocarpans, flavones, phenolic acids, clovamides, and essential oil  (CM) is a member of the Leguminosae/Fabaceae family. Its microgreens (CM) were grown in an open field and harvested at the fully expended green cotyledons stage which was 18 days from seed soaking, washing, and hulling  was incubated at 25–29°C for 9 days on slants of Czapek's agar media  were housed in plastic cages (3 rats per cage) at 25 ± 2°C and humidity 50%–60%. Rats were acclimated for 2 weeks and had free access to a pelleted diet and water. This study was approved by the Institutional Animal Care and Use Committee, Faculty of Veterinary Medicine, Cairo University (Vet CU01102020224) and was carried out per the guidelines of Care and Use of Laboratory Animals stated by the National Institutes of Health, USPHS.2.4Rats were fed a high‐fat diet for 2 weeks and then injected with a low dose of streptozotocin  to induce diabetes type 2  (Merck KGaA) while kits for insulin hormone and liver profile were products of Thermo Fisher Scientific, Waltham, MA, and Biodiagnostic, respectively.2.7Forty‐eight male albino rats were distributed into 8 groups (6 rats each). G1 rats were non‐diabetic and fed the control diet. G2 rats were non‐diabetic and fed a CM diet. G3 rats were non‐diabetic, fed the control diet, and orally given aflatoxin (30 μg/kg) 3 days per week  following the manufacturer's protocol .2.12Primary antibodies against insulin were used to detect insulin in paraffin‐embedded tissue sections of the pancreas  after antigen retrieval using citrate buffer PH6. The avidin‐biotin‐peroxidase complex kit  was used to detect the primary antibody. The brown color was developed using 3,3′‐Diaminobenzidine as a substrate. Image analysis of pancreas photographs using Image J software was used to measure the area % of positive insulin in beta cells of the pancreatic islets in three photos/rats in each group at a 400× magnification power.2.13Oxidative stress biomarkers were assessed in liver homogenate. Superoxide dismutase (SOD) activity was determined according to Nishikimi et al.  method. Reduced glutathione (GSH) was determined based on the reduction of 5.5 dithiobis (2‐nitrobezoic acid) (DTNB) with glutathione (GSH) to produce a yellow compound. The colored chromogen was directly proportional to GSH concentration which can be measured at 405 nm by its absorbance.2O2 solution. Initial absorbance at 240 nm was read after 20 s against a reference cuvette in which the same amount of distilled water was added instead of H2O2. The time required for initial absorbance to decrease by 0.05 units was noted. Catalase activity in the sample was calculated and expressed in U/mg of protein  activity was estimated by measuring the breakdown of hydrogen peroxide in the reaction mixture. The reaction was started by the addition of 50 1 of suitably diluted supernatant to 3 mL of phosphate buffer HMalondialdehyde (MDA) in an acidic medium at a temperature of 95°C for 30 min reacted to form a reactive product of thiobarbituric acid where the absorbance of the resultant pink product can be determined at 534 nm Aebi, .2.14Statistical analysis of obtained data was analyzed using SPSS software. The homogeneity of variances was tested then the ANOVA test was used to detect significance followed by suitable post hoc tests. The data presented was the mean value ± standard error  followed by 5,7‐Dimethoxy‐4‐methyl coumarin (15.96%) belonging to the large subclass of benzopyrones and having a structure of fused benzene and a‐pyrone ring. Other compounds found include 2‐Hexadecanol (11.15%), 1‐Dodecanol, 3,7,11‐trimethyl (10.3%), 9‐cis‐Retinoic acid (8.59%) (Table 9.11% fol3.2The rats' body weight gain (BWG) was decreased in G5‐8, especially G7, compared to G1. The BWG was improved relatively in G8 (STZ‐Aflatoxin‐CM) compared to G7. G2 receiving CM diet recorded the highest BWG Table .3.3The glucose level of rats was elevated significantly in G3–G8 fed with aflatoxin and/or injected with STZ compared to G1 and G2. G7 recorded the highest glucose level. The glucose level however was lowered in diabetic and/or aflatoxicated groups fed CM compared to their counterparts.The insulin level was significantly lower in G5‐G8 with the lowest decrease in G7 in comparison to the G1 and G2 groups Table .3.4The Beta cells function significantly declined in G3–G8 with the lowest value in the G7 group. Feeding CM in G4, G6, G8 significantly improved the Beta cells function compared to G3, G5, and G7, respectively.However, insulin resistance was significantly high in G3, G5, and G7 groups compared to G1 and G2. However, in G4, and G6 groups fed CM, it was decreased compared to their counterparts and was insignificantly similar to G1 and G2.Insulin sensitivity was significantly reduced in G5 and greatly in G7 compared to G1 and G2. On the contrary, it was enhanced in G6 and G8 groups fed CM and was comparable with the control.The deposition index was significantly reduced in G3–G8 especially in G7 compared to G1 and G2. Nevertheless, G4, G6, and G8‐fed CM recorded a significantly improved deposition index compared to their counterparts Table .3.5Rats in G3–G8 had significantly elevated liver function enzymes and total bilirubin and a significantly decreased total protein compared to G1 and G2. The liver function parameters were the worst in G7. On the reverse, G4, G6, and G8‐fed CM recorded improved liver function parameters except for the total protein which still did not record a significance compared to their counterparts , the area % of insulin‐positive beta cells moderately decreased and the cells were irregularly arranged compared to the control group. In G4, the area % of beta cells were comparable to that of the control group and cells were well arranged. The area % of beta cells in the pancreas of G5 was greatly reduced compared to those of G1. In G6, the area % of beta cells increased significantly compared to that of G5. In G7, the area % of insulin‐positive beta cells recorded the lowest value of all groups. However, in G8, the area % of insulin‐positive beta cells was partially restored  that is used in the current study were considered the optimum for consumption. CM contained high content of Gardenin (29.11%) which possesses antihyperlipidemic and hepatoprotective activities in nonalcoholic fatty liver disease as it inhibits α‐amylase, glutathione S‐transferase, α‐glycosidase  and its derivative retinoic acid (RA) were present in high amounts in CM. They are crucial in the regulation of epithelial cell growth and cellular differentiation. Retinoid is necessary for vision and RA affects the pattern formation in developing and regenerating limbs and prevents the growth of some malignant cells Zhu, .Aflatoxin exposure in the current study decreased body weight and impaired growth which was also reported previously ; writing – original draft . Sara M. Mohamed: Conceptualization . Marwa S. Khattab: methodology, writing‐ reviewing & editing . Ahmed M. M. Abido: Writing – original draft . Emam A. Abdelrahman: supervision. Frederick Sarpong: Validation . Ammar Al‐Farga: writing‐reviewing & editing. Faisal Aqlan: visualizationThis research received no specific grant from any funding agency in the public, commercial, or not‐for‐profit sectors.The authors declared no potential conflicts of interest concerning the research, authorship, and/or publication of this article.This study was approved by the Institutional Animal Care and Use Committee, Faculty of Veterinary Medicine, Cairo University (Vet.CU.IACUC) (Vet CU01102020224) and was carried out following the guidelines of Care and Use of Laboratory Animals stated by the National Institutes of Health, USPHS.We disclose that it is not submitted to a preprint.All have participated and approved the manuscript and agree with submission.This manuscript has not been published elsewhere and is not under consideration by another journal. We believe that the findings of this study are relevant to the scope of your journal and will be of interest to its readership.


Herein, via modulating the CO2 feed mode and quantitative analyzing CO2 utilization with the aid of mass transport modeling, we develop a descriptor denoted as the surface‐accessible CO2 concentration ([CO2]SA), which enables us to indicate the transient state of the local [CO2]/[OH−] ratio and helps define the limits of CO2‐to‐CO conversion. To enrich the [CO2]SA, we developed three general strategies: (1) increasing catalyst layer thickness, (2) elevating CO2 pressure, and (3) applying a pulsed electrochemical (PE) method. Notably, an optimized PE method allows to keep the [CO2]SA at a high level by utilizing the dynamic balance period of CO2 neutralization. A maximum jCO of 368±28 mA cmgeo−2 was achieved using a commercial silver catalyst.Zero‐gap anion exchange membrane (AEM)‐based CO 2 concentration ([CO2]SA) is developed, which enables to indicate the transient state of the local [CO2]/[OH−] ratio and helps define the limits of CO2‐to‐CO conversion. Three general tactics are developed to enrich [CO2]SA for high current electrolysis: (1) increasing the catalyst layer thickness, (2) elevating CO2 pressure, and (3) employing the pulse electrochemical method.A descriptor called the surface‐accessible CO These species crossover to the anode side leading to a ≈50 % loss of CO2.2 utilization of recently reported zero‐gap AEM‐based electrolyzer studies for CO2‐to‐CO conversion..2R and HER needs to be considered. Three modelled linear‐polarization curves of CO2‐to‐CO reaction at different assumed conditions are displayed in Figure jCO at three marked overpotential ranges are controlled by the intrinsic catalyst kinetics, the mixed kinetic‐diffusion, and CO2 mass‐transfer diffusion, respectively. A high local [CO2] favors both an increase in the reaction rate and the limiting jCO. In ideal conditions, the maximum limiting jCO (black curve) will be achieved when the [CO2] is saturated near the catalyst surface, which is ≈34 mM at room temperature in an aqueous electrolyte. However, when CO2 neutralization occurs, the local [CO2] decreases and reaches a new equilibrium, causing a lower limiting jCO (blue curve). By further considering the competing HER that will reduce FECO with an increasing overpotential, the jCO will show a volcano type curve behavior and a lower limiting value at a higher overpotential. This volcano effect has actually been seen in the work by Larrazabal et al. where the jCO is maximal at 200 mA cm−2  and then decreases at higher total current densities.2 neutralization process severely obstructs CO2 mass transfer and is one of the major constraints for better CO2 utilization. Although substantial quantitative analyses have been reported in the ocean acidification field regarding CO2 neutralization,2 electrolysis. To the best of our knowledge, there is still a lack of investigations related to the neutralization chemistry of local CO2 and OH− concentrations at large‐current density conditions and concomitantly developed strategies to detour such internal limitations.Along with changes in operating conditions, CO surface.7a, 8 2 feed mode  in a zero‐gap AEM‐based electrolyzer on CO2 neutralization and the micro‐environment. By combining a series of quantitatively analyzed experimental results with theoretical calculations, we found that surface accessible CO2 concentrations ([CO2]SA), which is a function of the local [CO2]/[OH−] ratio, is a good descriptor for CO2R performance at high current densities where mass transfer issues often dominate.Herein, using a commercial silver catalyst, we systematically explored the effect of changing the CO2]SA in the vicinity of the catalyst, we developed three general strategies: (1) increasing catalyst layer thickness, (2) elevating CO2 pressure, and (3) applying a pulsed electrochemical method. Consequently, a jCO of 368±28 mA cm−2 was achieved through an optimized pulsed‐electrochemical method. Under these conditions, the cell can maintain a FECO of >70 % for 13 h at 500 mA cm−2.To enrich the SA.The increase of 2 neutralization amounts from the experiment  is not considered since an unsubstantial amount of the Faradaic efficiency goes towards this reaction. Applying the chronopotentiometry (CP) method allows for the OH− formation rate (mol s−1 cm−2) to be calculated to predict the local pH. We assumed a fully saturated GDE and uniform catalytic activity throughout the 50 μm catalyst layer.jtotal in the absence of any CO2 neutralization. In general, a larger jtotal allow for a higher alkaline condition at the cathode surface due to the increase of OH− flux, e.g., local pH=13.3 at 100 mA cm−2.2 neutralization (CO2+2OH−→CO32−+H2O), the local pH values at different PCO2 conditions can be further estimated . Their concentrations have a baseline value of ≈100 mM according to the anolyte (100 mM KHCO3). It shows the [HCO3−+CO32−] increases with both the PCO2 and current densities, agreeing with the experimental results  at different conditions are provided. The CO32− are the main carrier ions during the electrolysis and this tendency stands out at larger current densities. This result matches with our previous experiments.− and CO2 concentration. At 100 mA cm−2, all PCO2 conditions show a similar local [OH−]; while a 2.6 times enhancement of local [OH−] is demonstrated when PCO2 is changed from 100 % to 25 % at 300 mA cm−2. More interestingly, increasing the current density leads to a change of local [CO2] based on partial pressure, which is expected to be 4 times between PCO2 of 100 % and 25 %. In fact, a 5‐times local [CO2] difference is shown at 300 mA cm−2, indicating the additional impact related to CO2 neutralization affects the high current electrolysis and lowers the [CO2]SA. Moreover, the local [CO2]/[OH−] ratios and CO2R/HER ratios are provided in Figure 2]/[OH−] ratio is a good descriptor to reflect the [CO2]SA, which substantially affects the CO2R activity. Comparing with the values of local [CO2] or local [OH−] alone, this descriptor has the advantage to show the combined effect of the micro‐environment, and especially amplifies the dynamic evolution of [CO2] and [OH−] at variable operation conditions. Thus, it is of great significance to maintain the high [CO2]SA by separately regulating the local [CO2] and local [OH−] for overcoming the internal limitation of CO2 neutralization for large current density electrolysis.Mass‐transport modeling is further employed to verify the above assumptions. A one‐dimensional MEA‐based model  containing a gas diffusion electrode, silver catalyst layer (CL), AEM, and anolyte was developed by a COMOSL multi‐physics field simulation. Dissolved COus works.8b, 132] will drop the local [OH−] and thus improve the [CO2]SA on the catalyst for enhancing the JCO, as displayed in Figure 2]SA is closely related to the in situ generated [OH−] per active site and CO2 mass transfer effects  during the electrolysis period. In cases where pulsed electrolysis is used, tr is specific to the given time in the absence of CO2R, thus allowing the CO2 concentration to recover. Accordingly, we utilized three general strategies to enrich [CO2]SA at the catalyst interface: (1) increasing catalyst layer thickness, (2) elevating operating pressure, and (3) applying a pulsed electrochemical technique.Mass‐transport modeling has shown that increasing local SA. The estimated local pH by utilizing the quantitative CO2 neutralization amounts also shows the reduction from 13.5±0.2 to 13.2±0.1, verifying the assumption. Moreover, the mass‐transfer modelling result also verifies this assumption, as shown in Figure S9. It shows that the local [CO2] raises from 25.7 to 29.3 mM when the catalyst layer thickness increases from 50 μm to 100 μm. However, an increase of catalyst layer thickness will also impede the CO2 and H2O mass transport, therefore there is an optimal range depending on the porosity and structure of the catalyst distribution within the gas diffusion layer.Currently, the main way to compare electrolysis performance under different experimental conditions is using a 2‐dimensional electrode area for denoting current density. Although this way is simple and explicit, it neglects the significant function of catalyst structure and loading on the electrolyzer's performance.PCO2 will increase the local [OH−] and concomitantly decrease the local [CO2]. Accordingly, an opposite trend may appear if the CO2 total pressure is increased. We show a simple example of this in Figure jCO is enhanced from 168±14 mA cm−2 to 211±23 mA cm−2. The increased pressure not only increases the reactant gas density for high local [CO2], but also promotes the CO2 consumption amount for neutralization which reduces the predicted local pH (from 13.6±0.1 to 13.4±0.1), as shown in Figure S10. These features result in a high [CO2]SA allowing for better CO2R performance. This demonstration reveals the immense potential of high‐pressure (>10 bar) CO2 electrolysis.[2] would also be helpful. Thus, another general strategy based on a pulsed electrochemical (EC) method was developed.The partial pressure experiments demonstrate the degree to which a decrease of 2] self‐recovery in the MEA‐based electrolyzer. The pulsed method denoted below was operated within the metallic regime of Ag, but at a potential that was either just reductive enough to react with CO2 or slightly anodic of this potential, which eliminated Ag oxidation issues and mitigated double‐layer capacitance build‐up transient effects. As a 2‐electrode device was used, the exact cathodic potentials were hard to gauge.Contrary to previous research that utilized the pulsed EC method to regulate the micro‐structure and chemical state of the electrocatalyst ,ta=1 s) at 3.3 V for the reaction and a recovery time (tr=0.5 s) at 2.0 V. During the recovery time, the current of cell equals to zero, indicating no Faradaic reaction. To evaluate the FE of CO and H2, the concentrations of gas products achieved from the GC data were compensated by the deadtime since the gas flow maintains during the whole pulse period, which reflects the average results including the period where there was no reaction. Remarkably, the PP method allows jCO to increase from 164±24 mA cm−2 to 191±5 mA cm−2 and the FEH2 reduces to <4 % as compared to the CA method. Such improvement comes from the recovery period where no CO2R occurs but the CO2 transfer and neutralization are still continuously happening. This allows [CO2]SA to increase during the recovery period while simultaneously permitting anions  to leave the local environment as they should be driven away from the negatively charged cathode since there still is an electric field (2 V) between the cathode and anode.Two operation methods, (1) pulsed potential (PP) and (2) chronoamperometry (CA), are compared in Figure 2]SA. Figure 2] and local [OH−] change at reacting or recovery periods during the PC method. To simulate the PC method, a pulse period containing a reacting period (ta) of 1 s at an itotal=100 mA cm−2 and a recovery period (tr) of 0.5 s at open circuit conditions was repeatedly applied. This shows that the local CO2 is saturated and local [OH−] is low when no reaction happens (t0). When applying pulse electrolysis, during the ta the local [CO2] would gradually drop to a lower degree until reaching steady‐state, and then during the tr (if the time was long enough) it would return to near the initial saturated level; meanwhile the local [OH−] would vary the opposite way. According to the local CO2 recovery and local [OH−] mitigation, the PC method allows for a higher [CO2]SA during the ta compared to the static CP method. Importantly, the ta and tr values can largely regulate the average [CO2]SA during the ta period. Experimentally, we operated a jtotal of 500 mA cm−2 and optimized the tr by fixing the ta=1 s  is calculated based on the CO2 consumption for the CO2R divided by the CO2 supply during the whole period (tr+ta) or only the ta period, respectively. As expected, the jCO, instantaneous X and FECO increase with the tr. An optimized high jCO of 368±28 mA cm−2 with FECO of 74±6 % and instantaneous X of 42 % ±4 % can be achieved when tr≥0.5 s, which is 1.8 times higher than that of the CP method. Further increasing the duration of tr shows no increase of the CO2R performance implying that the local [CO2] is almost recovered when tr≥0.5 s. It is noted that the total X stays at 27±2 % for all tr conditions. Actually, the total charge passing the electrode as well as the total in situ formed OH− amount should decrease if they are normalized to the whole period with lengthening tr. In this case, the maintained total X verifies the continuous process of CO2 neutralization during tr period, which lowers the local [OH−] and raises local [CO2], resulting in the boost of jCO during the ta period. Moreover, the cell resistances in Figure tr indicating a higher CO32−/OH− ratio in the membrane due to the lower mobility of CO32− than OH− and also a higher CO2 neutralization degree during the whole period.Literature has demonstrated that a pulsed potential method has shown great promise to regulate the micro‐environment. period ta of 1 s tr=0.5 s and the normal chronopotentiometry (CP) method at jtotal=500 mA cm−2 is shown in Figure S11. The jCO was calculated based on the reacting period of the two methods. During the first 13 h, the PC method shows much better stability in comparison to the CP method. It still shows a jCO of ≈350 mA cm−2 with FECO of ≈70 % at 13 h, which is 2.6 times higher than that of CP method . If considering the whole pulse period, the normalized jCO is ≈233 mA cm−2, which is still 1.7 times higher than that of the CP method. Moreover, after 25 h of CO2 electrolysis, the PC method can maintain a total CO2 conversion rate higher than 22 %  in contrast to the CP method of only 14 %. The above results illustrate the bright perspective of the pulse EC method to apply for commercially viable CO2 electrolysis designs.Furthermore, the long‐term stability comparison of the pulse‐current (CP) method with 2 conversion (SPC) as a factor to reflect the CO2 utilization has attracted great attention recently.2 neutralization and provide three strategies to enrich [CO2]SA. These strategies have one general feature: they decrease the [OH−] as well as improve the [CO2]SA by utilizing CO2 neutralization. Therefore, they are not suitable for pursuing high SPC. On the other hand, some advanced cell configurations regarding the electrolyte and membrane designs have been reported to increase the SPC of CO2. Their design principle is to create an acidic environment  or a flow of protons by utilizing a cation‐exchange membrane (CEM) to either directly suppress the CO2 neutralization or in situ release the consumed CO2.Single‐pass CO2 reduction is to precisely measure the local pH and CO2 concentration near the catalyst surface (within micro‐ or nano‐meters).2 reduction by using scanning electrochemical microscopy.2R due to the formation of HCO3− buffering the reaction interface. This kind of experimental results are useful to estimate the real local pH values across the diffusion layer, thus further helping to develop the model and improve the modelling parameters. However, until recently, it is still a challenge to in situ characterize the local pH/CO2 concentration in MEA‐based setups due to the complex interfacial structure. Thus, mass‐transfer modelling and calculations play an important role in supporting the experimental results.2 electrolysis.Another important research area for CO2 neutralization in terms of local [CO2] and local [OH−] near the catalyst layer by regulating the CO2 feed mode  and applied current densities in a zero‐gap AEM‐based electrolyzer. The quantitative analyses and theoretical calculation results together reveal that the [CO2]SA , which has the advantage of amplifying the dynamic evolution of local [CO2] and local [OH−] in the micro‐environment at different operation conditions, is a good descriptor for CO2R performance. A higher [CO2]SA is favorable for electrochemical CO2 to CO conversion.In this work, we systematically explored the change of COPCO2 and jtotal help set the initial local [CO2] and local [OH−], respectively; whereas once electrolysis proceeds, they would chemically balance with each other until reaching steady state. Increasing the initial PCO2 and decreasing the jtotal will both render a lower ratio of CO2 to participate in the neutralization process, leading to a high local [CO2] and a low local [OH−], i.e., improving the [CO2]SA. Moreover, CO2 neutralization is a dynamic process requiring some period of time to reach equilibrium. This phenomenon creates a possibility to utilize the dynamic evolution of the local [CO2] and [OH−] to keep the [CO2]SA at a high level, such that one may overcome the internal limitations of CO2 neutralization. Based on these understandings, three general strategies were developed to enrich [CO2]SA for high current electrolysis: (1) increasing the catalyst layer thickness to reduce the jtotal per active sites, (2) elevating CO2 pressure , and (3) employing pulse electrochemical method to keep the [CO2]SA at a high level. Consequently, a commercial silver baseline catalyst achieved a jCO of 368±28 mA cm−2 with FECO=74±6 % via an optimized pulsed‐electrochemical method.In principle, The authors declare no conflict of interest.1As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


While aqueous organic redox flow batteries (RFBs) represent potential solutions to large-scale grid storage, their electrolytes suffer from short lifetimes due to rapid degradation. We show how an understanding of these degradation processes can be used to dramatically improve performance, as illustrated here via a detailed study of the redox-active biomolecule, flavin mononucleotide (FMN), a molecule readily derived from vitamin B2. Via in-situ nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) we identify FMN hydrolysis products and show that these give rise to the additional plateau seen during charging of an FMN-cyanoferrate battery. The redox reactions of the hydrolysis product are not reversible, but we demonstrate that capacity is still retained even after substantial hydrolysis, albeit with reduced voltaic efficiency, FMN acting as a redox mediator. Critically, we demonstrate that degradation is mitigated and battery efficiency is substantially improved by lowering the pH to 11. Furthermore, the addition of cheap electrolyte salts to tune the pH results in a dramatic increase in solubility (above 1 M), this systematic improvement of the flavin-based system bringing RFBs one step closer to commercial viability. Aqueous organic redox-flow batteries are known to suffer capacity loss via degradation of the redox-active species. Here, the authors use in situ methods to study the electrolyte flavin mononucleotide, identifying a redox mediator mechanism that mitigates capacity loss and a route to prevent its degradation. To date, many different organic molecules including quinones4, viologens6, phenazines8, and alloxazines9 have been investigated as potentially cheap RFB active molecules. Although a few molecules have shown a good performance in alkaline solution (pH 14)8, most organic molecules considered for RFBs generally experience degradation, reducing cell lifetime1. In 2016, Orita et al. 10 reported an RFB comprising flavin mononucleotide (FMN3−) at pH 14 as the anolyte against a potassium hexacyanoferrate K4[FeII(CN)6] catholyte. The cell showed a capacity retention of 99% over the course of 100 cycles. Despite the encouraging capacity retention, an additional FMN reduction plateau appeared during charge, which was assigned to a dimerization process10. The process was not seen on discharge, leading to considerable cell hysteresis. Consequently, the resulting capacity retention and energy efficiency were not good enough for grid-scale storage systems, where even longer long-life times with minimal degradation and high coulombic efficiency are required. More recently, Nambafu et al. attached a 2,2,6,6- tetramethylpiperidinyl-N-oxyl (TEMPO) radical to FMN to form a bifunctional redox active material, which showed improved stability at neutral pH11. However, significant capacity loss was seen within 100 cycles, which was largely ascribed to degradation of the TEMPO functionality.Redox flow batteries (RFBs) are a promising energy storage system for grid-level storage, where low-cost and scalability are essential10. This molecule is also used in the food industry as an orange-red food color additive, utilized in Europe as E101a12; the sodium salt is commonly known as E106 and is found in foods for babies and young children as well as jelly, milk products, and sweet products12.FMN is a commercially available, non-toxic biomolecule, readily derived from vitamin B2, motivating its further study in an RFB3− by coupling in situ NMR and EPR techniques4. We explain how degradation, which we show involves the hydrolysis of FMN3− rather than a dimerization process, leads to the additional charging plateau. We investigate the electrochemical behavior of hydrolyzed FMN3− with in situ NMR and demonstrate that FMN3− acts as a redox mediator, chemically reducing the hydrolyzed product, explaining the lack of an additional plateau on discharge yet good cycling behavior, despite degradation and poor reversibility of hydrolyzed FMN’s redox reactions. Lastly, we provide a strategy to avoid hydrolysis by lowering the pH. The battery performance is improved significantly and the FMN solubility is increased dramatically, by addition of a simple salt.Here, we demonstrate a powerful strategy to study the degradation of FMN3− and 1 M KOH in D2O as the anolyte and 0.2 M K4[Fe(CN)6] with 0.05 M K3[Fe(CN)6] in 1 M KOH in D2O as the catholyte. The cycling data  and a second, sloping plateau or process starting at 1.71 Vcell (γred) during the first charge (reduction of FMN3−); the theoretical capacity of 3.22 Ah L−1 was achieved (here: 3.18 Ah L−1) before the second plateau, γred, was completed (4.62 Ah L−1). On discharge (oxidation of FMN5−) only a single sloping plateau at 1.09 Vcell (αox) was seen, and 2.79 Ah L−1 was recovered. On subsequent cycles, an additional charge plateau centered at 1.41 Vcell (βred) grew in prominence over 90 cycles, resulting in a steady drop in the voltaic efficiency 64−/3−, respectively , which is corroborated by the formation of gas bubbles 6]3−/4− in a 2,6−dihydroxyanthraquinone/K4[Fe(CN)6] study3 and the literature shows that the thermodynamic potential for hydrogen evolution from water at pH 14 is around −1.2 V vs. SHE4. In this case, the water splitting commenced at higher voltages (1.71 Vcell vs. [Fe(CN)6]3−/4−) and we hypothesize that the hydrogen evolution is kinetically inhibited due to the carbon electrodes used in this work. The rapid decrease in this reaction with cycling is tentatively ascribed to a passivating film forming on the carbon paper used as the electrode. The reaction can be completely avoided by using a lower upper cut-off voltage of 1.7 V (see later).Plateaus ely Fig. , no distβred suggests that a new reduction process is occurring, that is distinct from the reduction of FMN3−, or the semiquinone FMN4−•, to FMN5−. This process was seen in the earlier work of Orita et al.10 where they hypothesized that the two charging plateaus result from reduction of the monomer and dimer of FMN3−. They argued that due to the slower kinetics of the dimer oxidation, compared to that of the monomer, the discharge curve does not mirror the charge curve. We, however, propose that this process is associated with a degradation product of FMN3−. To identify the species giving rise to the plateaus observed in the cycling data, a combination of in situ NMR and EPR measurements were performed.The gradual appearance of plateau 3− during battery operation were followed by NMR/EPR spectroscopy (Supplementary Note 3− solution.The redox reactions and degradation of FMN3− (60 mM in 1 M KOH/D2O) anolyte and corresponding in situ 1H NMR and EPR spectra are shown in Fig. cell), the FMN3− ring protons, H9 and H6, were clearly seen in the in situ 1H NMR spectra at 7.25 ppm and 7.33 ppm, respectively, the shifts correlating well to those obtained via ex situ NMR 1- and 2-dimensional NMR measurements (Supplementary Note αred), the alloxazine ring proton signals  in the 1H NMR spectra disappeared immediately . The EPR signal disappeared, indicating that all of the radicals have been reduced. No signals for FMN3− were seen, confirming the complete reduction of FMN3− to FMN5− during plateau αred. The reverse of the trends during the αred plateau was observed in both the NMR and EPR spectra during plateau αox, the 1H NMR signals  disappearing due to the formation of FMN4−•, as confirmed by the appearance of a signal (at 337.8 mT) in the EPR spectra. Note that the signals H6 and H9 did not appear again at the end of αox since the cell was not held long enough at low voltages to remove all the radicals, the data shown here being part of a longer cycling study . The H6* and H9* 1H NMR signals, by contrast, remained . That no EPR signals were seen during plateau βred, indicates that the reduction of the species giving rise to H6* and H9* does not generate any (EPR-observable) radicals. As before, no EPR signal was observed during γred and the intensity of the 1H NMR signals  remained constant.At the end of the ox, the 1H NMR signals for FMN5− disappeared immediately. The signals for H6*” and H9*” disappeared more slowly, their intensity loss again correlating inversely with the appearance of the signals H6* and H9*. The EPR signal for FMN4−• (337.8 mT) was observed again. The in situ 31P NMR spectra also show a similar trend of disappearing signals , a process that can be accelerated at high temperatures14  was observed, confirming that no FMN3− is present in the solution and instead a plateau, solely from the reduction of RQC3− (β*red) at 1.32 Vcell is now seen. In the corresponding 1H NMR spectra, the signals assigned to RQC3−  gradually decreased, eventually disappearing at the end of the β*red process, with the concomitant appearance of two peaks at 6.70 ppm and 6.95 ppm. The shifts of the new peaks matched those of H6*” and H9*” seen in the reduced aged sample. The process at 1.71 Vcell (γred) due to water reduction was again observed, and no change in the 1H NMR signals were observed during this plateau, consistent with this assignment. Of note, no EPR signals were observed during either of the two plateaus β*red and γred  shown in the supporting information is consistent with this proposal  or increasing the time spent at the end of discharge at 0.5 Vcell  was fully reduced to RQC5−  was then added (in Step II) and the solution was allowed to equilibrate for 5 h, the signals for RQC5− slowly disappearing concomitant with an increase in the signals for RQC3− in the 1H-NMR spectra. No signals for either FMN3− or FMN5− were seen throughout Step II, which is ascribed to the oxidation of RQC5− by FMN3−, forming FMN4−• and eventually FMN5−, and the consequent rapid electron transfer processes between the diamagnetic and paramagnetic FMNn− ions. The battery was then charged (Step III), and even though FMN3− had been added to the solution, plateau α (associated with the reduction of FMN3−) was not observed, and instead plateau βred (associated with the reduction of RQC3−) was seen. This is supported by the in situ 1H NMR spectra: the appearance of the signals for RQC5− was inversely correlated with the disappearance of the signals for RQC3−. Signals for FMN5− were observed at the start of plateau βred and their intensity remained unchanged , confirming that an almost complete reduction of FMN3− had already occurred before the start of plateau βred. This is consistent with the reduction of FMN3−to FMN5− during the 5-hour equilibration period (step II), and the oxidation of RQC5− to RQC3−; we propose that this occurs via an intermolecular process, in which FMN3− acts as the redox mediator serving to oxidize RQC5− chemically.We propose that an alternative process occurs during discharge of the full cell that facilitates the re-oxidation of RQCC5− Fig. , Step I,αox plateau was observed, but with a higher capacity (2.36 Ah L−1) than expected if only FMN5− was oxidized to FMN3− . This indicates that another species must also be oxidized during plateau αox, again consistent with redox mediation mechanism: RQC5− is oxidized by FMN3− forming RQC3− and FMN5−, which can then be electrochemically re-oxidized to FMN3−. However, the expected capacity of FMN3− and RQC3− combined (3.22 Ah L−1) was not observed suggesting that the redox mediator reactions are slower than the electrochemical oxidation of FMN5−. This is corroborated by the in situ 1H NMR spectra during step IV, as residual signals for RQC5−, in addition to those for FMN3− and RQC3−, could be observed at the end of plateau αox. During the following charge (step V), plateaus αred and βred were now observed. The same capacity was obtained as during the previous discharge cycle (step IV).During discharge (step IV), the 3−, i.e., adding a shorter rest period  and is accelerated by higher temperatures, the pH of the electrolyte solutions was lowered to minimize hydrolysis and increase capacity retention. Supplementary note 3− and 62 mM KOH in D2O to obtain a pH of 11.4. 1 M potassium chloride (KCl) was added to compensate for the reduced salt concentration (previously 1 M KOH was used). 62 mM KOH was added to the catholyte (K4[Fe(CN)6]/K3[Fe(CN)6] in 1 M KCl in D2O) to match the salt concentration to that in the anolyte. The RFB was cycled over a narrower voltage range (0.5–1.4 Vcell) to avoid the water splitting reaction. Over the course of 98 cycles, the electrochemical data , dimerization would involve a species with ten negative charges.This work shows that the observed asymmetry in the galvanostatic profile of this battery system (and the resulting voltaic efficiency) can be explained by the presence of hydrolyzed FMN3− at pH 14 (1 M KOH) was only 0.1 M10, but at pH 11.4, the addition of 1 M KCl to a solution of 62 mM KOH led to a much-improved FMN3− solubility of 1.8 M  required for a commercial aqueous organic RFB17. Furthermore, these results suggest that other simple salts may similarly help achieve higher solubility in a wider range of electrolytes. Excellent cycling performance was maintained at the lower pH of pH 11 with a higher concentration of 240 mM FMN3− and a higher current density of ±50 mA cm−2 as shown in Fig. SFMN has multiple protonation states, and its solubility is likely to be extremely pH dependent. Thus, the solubility of FMN and the effect of salt concentration was then re-evaluated at a lower pH 63−/ Fe(CN)64−) observed on charging solutions containing the hydrolysis products, in addition to the expected FMN reduction process at 1.02 Vcell. The new plateau, assigned to the two-electron reduction of RQC3− to form RQC5−, was not observed on discharge— unless deep discharging was used , indicating poor reversibility of the redox reaction. Despite this poor reversibility, solutions containing the hydrolysis products showed very little decrease in reversible capacity on galvanostatic cycling. Via a thorough assessment of the intermolecular processes using both NMR and EPR spectroscopy, we showed that the ability of FMN3− to act as a redox mediator was responsible for this phenomenon, and in solutions containing both FMN3− and its hydrolysis products, RQC5− is chemically re-oxidized to reform RQC3− -based RFB is demonstrated in this work. Critically, this insight is then used to propose solutions to mitigate it. Via in- and ex-situ NMR spectroscopy, FMN3−, leading to a noticeable improvement in capacity retention  were purchased from Sigma Aldrich Chemicals. Potassium hydroxide  and potassium chloride  were purchased from Fisher Chemical. The aging process was conducted under air. The hydrolyzed FMN/RQC was synthesized by dissolving FMN in 1 M KOH/D2O and heating for 2 h at 90 °C. The solutions with a specific pH were made by dissolving a specific amount of FMN in 1 M KCl/D2O; the pH was then adjusted by the addition of KOH.Flavin mononucleotide , potassium hexacyanoferrate(II) trihydrate , potassium hexacyanoferrate(III) , ≥99.0%), and D2 active area, which were used without further treatment. Nafion 212 was used as the ion transport membranes. Pre-treatment of the Nafion 212 membranes was performed by first heating the membrane in 80 °C deionized water for 20 min and then soaking it in 5% hydrogen peroxide solution for 35 min. All battery performance experiments were carried out using a potentiostat 4.The hardware of the flow battery was purchased from Scribner Associates. Graphite flow plates with serpentine flow patterns were used for both electrodes. Each electrode comprised of three pieces of Sigracet 39 AA carbon paper (FuelCellStore) with 5 cm−1, for the electrolyte to move through the system. From the electrolyte reservoir to the battery takes 3 s, from the battery to the EPR detection region 3 s, from the EPR to the NMR detection 29 s and from the NMR back to the electrolyte reservoir is 29 s. To minimize heating of the aqueous solution by microwave irradiation, a flat EPR cell  was used. A customized adaptor made of polyether ether ketone (PEEK) connected the flat EPR cell to the 1/16 in. tube. The cell was orientated in the resonator such that the strength of the magnetic field was maximized, and the strength of the electric field was minimized across the sample. The volume of the cell in the excitation region of the microwave was 0.03 mL (2.00 cm × 0.50 cm × 0.03 cm), giving a residence time of 0.13 s for the electrolyte solution at a flow rate of 13.6 mL min−1. Details of the NMR sampling tube were provided in our previous publication4.The setup consists of a flow battery (Scribner), two peristaltic pumps , an electrochemical cycler , a benchtop EPR , and an NMR  spectrometer. The battery and the EPR spectrometer were positioned outside the 5 G line of the NMR magnet. The electrolyte was pumped through the flow battery, then flowed through the EPR and NMR magnets, and finally back to the electrolyte reservoir. The direction of flow was from the bottom to the top of both magnets. PFA tubes (1/16 in.) were used to connect the electrolyte reservoir, the battery, and the EPR and NMR sampling tubes. It took 64 s, at a flow rate of 13.6 mL min4[Fe(CN)6] and 37.5 mM K3[Fe(CN)6] was used as the catholyte. The solvent was D2O, with 1 M KOH as the supporting electrolyte. The flow rate was 13.6 mL min−1.For the coupled in situ EPR and NMR experiment, 30 mL of 60 mM FMN was used as the anolyte, 30 mL of 150 mM KPseudo-2D NMR experiments were performed by direct excitation with a 90° radio-frequency pulse. Each NMR spectrum was acquired by collecting 16 free induction decays (FIDs) with a recycle delay of 5 s. The pulse length for a 90° pulse was 27 μs. All spectra were referenced to the chemical shift of water at 4.79 ppm before battery cycling began.4−• radical anions, the magnetic field was swept from 336.5 to 339 mT. The sweep time was 60 s per single scan. Bm was 0.001 mT, and Q was recorded for each spectrum. Q was 1306 ± 5. The temperature of the resonator was kept at 29 °C, and a time delay of 35 s was added between each scan.For the in situ EPR measurement of FMNCV experiments were carried out using a three-electrode cell. A Thermo Haake DC50-K75 circulator was used to ensure that the voltametric cell was at 25 °C and GL14 screw threads and caps, in combination with rubber O-rings, were used to ensure a tight seal around the electrodes. A rubber septum was used to close the remaining opening, and for addition and removal of samples under inert atmosphere. The cell was flushed with nitrogen prior to measurement and kept under an overpressure of the inert gas during measurement using the ground-glass opening at the top of the cell. The nitrogen was dried over a desiccant column before use. All voltametric experiments used a glassy carbon working electrode embedded in a PEEK housing  and a coiled platinum counter electrode (1.00 mm diameter). The glassy carbon was polished using 0.05 µm polishing alumina paste (Biologic A-001050) on alumina polishing pads (Biologic A-001040), and where necessary using 1 µm polishing diamond paste (Biologic A-002054) on diamond polishing pads (A-001041) with Millipore deionized water. 3 × 100 cycles of figure-eight were used to polish the working electrode, with intermediate sonication steps of one second duration followed by drying under nitrogen gas. The platinum was purchased from Sigma-Aldrich and cleaned using nitric acid and deionized water before use. A biologic VSP 200 potentiate was used, with the potentiostat software, for the experiments described here.−1 was chosen as the comparison scan-rate, providing a compromise between duration of the experiment and sufficient time to allow relaxation and separation of the different redox processes. Cyclic voltammetry was run over long time periods where possible to investigate the stability of the analyte against the supporting electrolyte fully, and to observe if alternative detrimental processes, such as electrode deposition, occurred.Electrolyte and supporting electrolyte solutions were prepared under inert atmosphere, avoiding the use of vacuum grease where possible, prior to measurement. A background CV was run on the supporting electrolyte solution to ensure that it was sufficiently free from contamination prior to use. 20 mVs19 was used for all calculations. The molecules were first optimized at the PBE21/TZVP23 level of theory without any solvation model applied. The optimized coordinates were then used as the initial structure for optimization at the PBE/TZVP level of theory with an applied polarized continuum model (PCM)24 of solvation using the dielectric constant of water. These optimized structures were then further optimized at the B3LYP28/TZVP level of theory, again using the PCM model for water. The energetics and vibrational frequencies were calculated for all species of interest. The absence of imaginary frequencies confirms that the obtained geometry corresponds to a ground state structure.The software package Gaussian 16Supplementary Information