Multiple sclerosis (MS) is the most common non-traumatic cause of disability in young people, with vision loss in the disease representing the second largest contributor to disability. In particular, African-American patients with MS are noted to have lower vision than their Caucasian counterparts. In this review, we examine the disparities in eye diseases in the MS population with our gaps in knowledge and discuss the underlying nature of pathological disparities. Disability accumulates by two separate components of the disease. Primarily, there is the relapsing form of the disease, where neurological symptoms often develop subacutely through inflammatory demyelinating plaques of the central nervous system with subsequent disability accumulation from incomplete recovery. Secondarily, there is an increased risk of neurodegeneration in a significant subpopulation of the disease, where neurologic disability accumulates independent of relapses, often termed the progressive form of the disease. Both forms of the disease can overlap and often lead to progressive neurological disability, often including vision loss.4Multiple sclerosis (MS) is a demyelinating disease of the central nervous system and is the most common cause of non-traumatic disability in young people.6 Historical literature in the field indicates that MS occurs more commonly in Caucasian patients; however, recent studies have demonstrated that the prevalence is higher than previously reported in African-American populations and possibly comparable when compared to Caucasian populations.710 The prevalence of MS among those who identify as Hispanic, Latino, and/or Latinx varies, but is lower than what is seen in African-American and Caucasian populations.8 However, this number is expected to grow as these populations now represent the most rapidly growing minority in the United States.Epidemiologically, MS affects 2.8 million people in the world and approximately 1 million people in the United States.12 In addition to rising numbers, it has also been well demonstrated that both African-American patients with MS (AAwMS) and HwMS have more aggressive disease courses and are more likely to suffer severe disability.13 Symptomatically, this disability accumulation manifests with a variety of neurological symptoms, including cognitive dysfunction, gait dysfunction, motor dysfunction, and sensory dysfunction; however, vision loss remains one of the most common symptoms of the disorder. There are some data to suggest that optic neuritis is a more common manifestation of the disease among both HwMS and AAwMS. In this narrative review, we summarize the neuro-ophthalmic manifestations in HwMS and AAwMS, inequities that are present in the literature, as well as underlying possible mechanisms.While Hispanic, Latino, and/or Latinx are often used interchangeably when describing patient ethnicity, it should be noted that these terms have variable meanings. For this paper, we will refer to those who identify as Hispanic, Latino, and/or Latinx as Hispanics with MS (HwMS). Often characterized by the subacute development of vision loss , pain with eye movement, and dyschromatopsia color desaturation, inflammation and demyelination within the optic nerve cause delay of action potentials through the optic nerve, and secondary axonal block with eventual axon loss. While there is a strong association between MS and optic neuritis, there are a wide variety of etiologies to optic neuritis, with up to 31% of patients suffering from recurrent vision loss.Broadly, optic neuritis is one of the most common neuro-ophthalmic diagnoses associated with MS. In approximately 20% of patients, optic neuritis is the initial presentation and is thought to occur in roughly 50% of patients in their lifetime. Visual evoked potentials have provided a technology to measure the space constant of the optic nerves and have robust diagnostic and prognostic value.1719 Optical coherence tomography (OCT) is another noninvasive technology that provides a great spatial resolution of the retina and has been used as a highly sensitive diagnostic evaluation for optic neuritis both in acute and chronic settings.2023 Given the increasing precision of ophthalmic imaging technologies and increased availability, it is becoming an advantageous medium to detect pathological variability between minorities and non-minorities with MS.Notably, given the association of optic neuritis and MS, the visual system has often been used as a window into the mechanisms of disease activity. In this pivotal trial, there was no difference between sex, age, presence of disc edema, or fulfillment for MS at presentation between African Americans and Caucasians, but African-American patients had more frequent severe vision loss, both at presentation and at follow-up. This discrepancy was at least partially attributed to the increased prevalence of the ‘neuromyelitis optica’ presentation of neurological deficits, which occurred in 58% of the AAwMS studied. This hypothesis for the discrepancy remained as anti-aquaporin-4 (AQP4) antibody assays were not commercially available at the time of the study; however, anti-AQP4 was not detected in any of the 116 serological samples from the ONTT that were tested, making this hypothesis less likely. Over time, the differences in vision loss between AAwMS and Caucasians with multiple sclerosis (CAwMS) were felt to reflect the differentiation of risk for ‘opticospinal MS’ in African Americans relative to their Caucasian counterparts. As neuromyelitis optica spectrum disorder (NMOSD) became more differentiated from MS by the commercial availability and utilization of the anti-AQP4 assay, differences in vision loss between AAwMS and CAwMS became partially explained, but not fully accounted for. As serological positivity for the anti-AQP4 assay is now synonymous with NMOSD, a distinct demyelinating disease from MS, whether similar disparities exist for NMOSD remains an active area of research.Historically, differences between African-American and Caucasian patients with optic neuritis have been noted back to the optic neuritis treatment trial (ONTT). This is perhaps partially explained by retinal neurodegeneration in the fellow, non-optic neuritis eye.29 Patients who identify as AAwMS and HwMS have been shown to have higher rates of disability when looking at the Patient-Derived Multiple Sclerosis Severity Score. There is frankly a dearth of neuro-ophthalmic data for HwMS, both in terms of specifics of structural and functional assessments of visual function.Not only did those AAwMS suffer from increased risk of disability accumulation through optic neuritis but also in global disability.31 Understanding the disparities between the neuro-ophthalmic findings in this population yields insight into the different pathological mechanisms.Why AAwMS and HwMS suffer from increased visual disability and inflammatory disease activity is not fully understood. As historical data from MS suggest the disorder is underdiagnosed in racial and ethnic minorities, their disability data are not proportionally represented in the literature prior to recent efforts. Investigations into socioeconomic status have yielded some data to suggest an increased risk of disability accumulation; however, these covariates do not explain racial and/or ethnic disparities. MME could represent the microscopic precursor to macular edema, which in itself is associated with vision loss through a direct maculopathy. Although this direct comparison is controversial, longitudinal data do support this hypothetical spectrum of disease, although such data are limited. While MME is nonspecific, occurring in a multitude of disorders, it has a strong association with optic neuropathies such as optic neuritis and is well described in the MS literature.3436One glaring disparity in neuro-ophthalmic disease in MS is microcystic macular edema (MME). While technically a misnomer, MME is defined as cystic, lacunar areas of hyporeflectivity with clear boundaries within the inner nuclear layer (INL) of the retina, shown in  Optic neuritis seems to be highly coincident with, but not sufficient, for the development of MME in MS. Interestingly, most cases of MME are transient, with only 16% remaining over a mean follow-up of 233 days in one study. There are some data to suggest that MME is associated with a higher risk of disease in MS patients, although it is less clear whether this is independent of race and/or ethnicity itself.In the MS population, both MME and macular edema have been described. Of particular concern among AAwMS, the MME incidence is reported to be approximately 12% over a mean follow-up of 4 years, where overall frequency in the entire MS population is estimated to be anywhere from 1% to 6% over a slightly longer follow-up duration. The inflammatory activity of optic neuritis being highly coincident in its development does suggest that inflammation, either chronic or acute, plays a significant role. Analogous to MS, MME is also strongly associated with NMOSD, where its mechanism was posited to involve direct insult to Müeller cells  through a direct astrocytopathy. Interestingly, there are conflicting OCT data to support this hypothesis, perhaps again emphasizing multifocal mechanisms.4042 While transsynaptic degeneration is increasingly appreciated in MS, a transsynaptic retrograde maculopathy is an unlikely explanation for its mechanism given the temporal dynamics of the disease.4345 This, in combination with the lower prevalence of macular edema (1–6%) in contrast to highly prevalent neurosensory retinal loss appreciated with optic neuritis (60%), makes this similar at best a partial explanation.The mechanism(s) of MME in MS is not precisely clear but were hypothesized to reflect dynamic fluid shifts, either through vascular permeability and/or Müller cell dysfunction, or perhaps non-inflammatory changes including mechanical traction or retrograde maculopathy. These risk factors for S1P-associated MME furthered the hypothesis that vascular permeability may be playing a significant role in the pathophysiology of MME for most MS patients. As such, screening for macular edema and MME became integral to S1P prescription, as some patients have developed the adverse event 24 h following therapy initiation. Given the high prevalence of MME noted in AAwMS, initiation of S1P modulation should follow local prescribing regulations, with careful consideration of MME risk, as this finding is transitory in the majority of cases. Whether those with a history of MME that has resolved are at risk of MME and/or macular edema following S1P modulation has not been examined.Most pertinent in the therapeutic realm of MS was the early realization of an increased risk of MME following S1P modulation in patients with MS. The absolute risk of MME was low with fingolimod, the originally FDA-approved S1P modulator, at 1% annual incidence with exposure, with both diabetes and uveitis identified as risk factors for its development.48 In addition, MME has been encountered in S1P modulation in other non-neurological conditions, such as ulcerative colitis, which argues against a direct neuroinflammatory effect underlying its pathogenesis. There are data to suggest that nonselective S1P modulation may be associated with an increase in macular volume, which could represent subclinical macular edema, although this hypothesis was limited by the single-center design, and did not report measures of the INL, where the majority of MME presents. Despite the risk of macular edema associated with S1P modulators, a systematic review indicated that a favorable outcome was observed in select cases with continuation of this therapy with concomitant macular edema treatment when alternative therapies were untenable, although extreme caution is highly encouraged in such cases.The mechanism of MME as an adverse of S1P modulation is similarly unclear. It was originally hypothesized that S1P3 modulation at the orbital vascular endothelial junctions was directly responsible, although macular edema has been reported as an adverse event with ponesimod, a highly selective S1P1 modulator.While MME represents a relatively uncommon neuro-ophthalmic finding in MS, it has significant implications both diagnostically and therapeutically for most patients with MS. A variety of mechanisms have been posited to explain its mechanism(s), shown diagrammatically in  Indeed, neurosensory retinal loss measured in a variety of ways is accelerated in those who identify as African American relative to Caucasian, primarily within the inner retina. In addition, macular volume loss correlates well with global brain volume loss, serum neurofilament light chain levels,51 as well as with visual function, including low contrast visual acuity.5254 Taken cumulatively, there are data to suggest the cross-sectional volume of the macula has significant prognosis value, with lower macular volumes being predictive of an increased risk of disability accumulation at 10 years, regardless of optic neuritis. In this way, measurement of retinal structure and function captures ‘silent progression’ in MS, what is globally being described as progression independent of relapse activity.As MS has both an inflammatory and neurodegenerative component, the visual system has been used to capture quantitative measures of disease activity. Disease activity was primarily appreciated through disease within the optic nerve itself. The retinal nerve fiber layer thickness, an OCT metric often taken as a surrogate for axons within the optic nerve, has captured neurosensory retinal loss, known to occur in patients with MS at an average rate of 0.4 µm/year, whereas healthy controls lose on average 0.2 µm/year.58 In terms of health disparities, there is evidence that obesity may have differential effects in racial subgroups. Investigation into the socioeconomic subgroups independent of race has shown a similar relationship of higher neurosensory retinal loss with lower socioeconomic status measured by a variety of variables, including median household income, education level, and state area deprivation index.52Analogous to brain volumetric studies, it is known that several variables, such as inflammatory disease activity and obesity, can affect neurosensory retinal loss. The quantitative methodologies available to neuro-ophthalmology make the incorporation of immunological, environmental, and socioeconomic covariates into disease models of disability ultimately highly tenable.As the mechanisms of disability accumulation in MS are multifactorial, further study into the dynamics of both structure and function through the visual system yields the opportunity to understand the disease in a more diverse patient population. Indeed, as OCT-based diagnostic criteria are considered potential diagnostics criteria for MS, incorporating ethnically and racially diverse patient cohorts will be critical to their generalization.60 Studies evaluating the specific roles in AAwMS have demonstrated that there is a difference in these previously established genetic risk variants among AAwMS with both HLA-DRB1*15:01 and HLA-DRB1*15:03 inferring an increased risk of developing MS consistent with other populations, but HLA-DQB1*06:02 having no association with increased risk.62 Within certain Latin American populations, HLA-DRB1*15:01, HLA-DRB1*15:03, and HLA-DQB1*06:02 have shown an association with increased risk similar to what is seen in other populations. Studies have also demonstrated that there is a genetic influence on the age of onset in HwMS leading to MS presenting at earlier ages; however, there is conflicting data surrounding this idea in AAwMS.While the pathogenesis of MS is complex, it has been well documented that genetics play a role in the development of MS, with the class II region of human leukocyte antigen (HLA) having the largest effect, in addition to a growing number of almost 200 other genetic loci. In a 2018 study, Amezcua et al. demonstrated regional differences in the development of optic neuritis in HwMS, with those with Native American ancestry in the southwestern United States having higher rates of optic neuritis relative to CAwMS.65 Similar studies have not been replicated in AAwMS.More interesting and specific to this current narrative review, there appears to be a genetic influence on the development of optic neuritis as a presenting symptom in MS among Hispanics based on genetic admixture, the interbreeding of individuals from isolated lineages. The humoral immune system is highly active in both AAwMS and HwMS, as there are increased levels of antibody-secreting cells in peripheral blood samples. Furthermore, it is now known that AAwMS have higher levels of intrathecal humoral inflammatory changes based on higher levels of IgG synthesis rate and IgG index. It has also been demonstrated that the presence of oligoclonal bands is associated with higher levels of disability among this patient population, and so one possible explanation for the disparate disability could relate to this immunological heterogeneity.70 There is further anatomic evidence to support this hypothesis, as there is also an association with cerebral gray matter volume loss and the presence of an elevated cerebrospinal fluid (CSF) IgG index in AAwMS.The answer to why there is an interethnic and interracial difference in disease aggressiveness is likely complex, with influence from genetics, environmental exposures, immunological variations, as well as social determinants of health. Specific to immunological mechanisms, one possible explanation relates to the differing pathological phenotypes of MS. The four classic pathologically determined lesion types, from type I to type IV, are based on different immune system characteristics. Of specific relevance are type II lesions, which are characterized by antibody deposition, and thus directly implicating the humoral immune system in the development of MS lesions.Post hoc analysis of the EVIDENCE trial, which evaluated the efficacy of interferon-β-1a in treatment-naïve MS patients, demonstrated that AAwMS treated with interferon-β-1a suffered from a higher number of exacerbations were more likely to have a relapse, and developed a higher number of new T2 lesions at 48 weeks compared to CAwMS. This differing treatment response was further illustrated in a retrospective review comparing treatment response to interferon-β-1a or interferon-β-1b, glatiramer acetate, as well as natalizumab in AAwMS compared to CAwMS suggesting this relationship is not DMT specific among the early DMTs. There is currently very limited data evaluating DMT exposure in HwMS, making analogous comparisons untenable.Another argument for an immunological disparity can be made by exploring the interracial data on exposure to disease-modifying therapies (DMTs), as AAwMS have been shown to have a less robust therapeutic response to certain DMTs. 75 While early data identified that those with African ethnicity are more likely to have these uveitis, in recent literature, the prevalence of uveitis in MS has decreased to 0.51%. In addition, in the large UK Biobank study, 87.5% with both MS and uveitis identified as White. In the modern era, given the relatively low incidence of uveitis, it is difficult to understand the contribution of both race and ethnicity to this disease pathophysiology. In addition, the uveitis is often anterior or intermediate, for these reasons, is beyond the scope of this narrative review.Historically, there has long been an appreciated relationship between uveitis and MS, where the incidence varied between 10% and 23%. There is a dearth of literature examining ethnic and racial disparities in these findings, which is worth mentioning, particularly as there is literature suggesting that structurally the brainstem regions controlling these functions are selectively vulnerable in those with African-American ancestry. Further study is needed on this topic.Similarly, ocular dysmotility, including internuclear ophthalmoplegia (INO) and wall-eyed bilateral INO, are highly prevalent in MS.et al. found that less than 1% of the 60,000 publications on MS were focused on minority populations. The lack of minority representation in clinical trials is even more concerning, with a recent review highlighting that less than 10% of patients in these trials were representative of minority backgrounds. As mentioned previously, it has been shown that AAwMS have less robust responses to early DMTs, but whether this relationship continues among the more recent DMTs remains unknown. Furthermore, similar relationships are not known to exist between HwMS and CAwMS.Despite the increased prevalence and higher risk for aggressive disease in AAwMS and HwMS, both populations are grossly underrepresented in research efforts. A PubMed review completed in 2014 by Khan  In addition, the results of the FLUENT study, which investigated the change in peripheral lymphocyte dynamics in patients with relapsing forms of MS treated with fingolimod, demonstrated a reduction in both T cells (CD4+ and CD8+) and B cells (CD19+ naïve and memory) in a patient treated with fingolimod. While not directly aimed at evaluating treatment responses in minority MS patient populations, these results highlight that the S1P receptor modulator class of medications could be particularly beneficial in AAwMS given the higher level of B-cell activity noted in the peripheral blood of these patients. Ultimately, we must continue to broaden our understanding of DMT use in these patient populations to have a better understanding as to why these patient populations accumulate greater visual and global disability.Given the increasing attention to representation disparities, there have been more recent efforts to bridge this gap. The CHIMES trial is a phase IV clinical trial evaluating the efficacy and safety of ocrelizumab in minority patients with relapsing forms of MS. The findings from this trial will broaden our knowledge of disparate pathology among AAwMS and HwMS.While MS is the most disabling non-traumatic cause of young people, there are disparities and inequities that require both scientific and social attention. There are a number of lines of evidence that AAwMS and HwMS are at increased risk of disability accumulation. This can be seen in markers of clinical disability, radiographic outcomes, and visual outcomes, shown diagrammatically in Lastly, while there are relationships between the neuro-ophthalmic findings among AAwMS relative to CAwMS, analogous data for HwMS are desperately needed. As this represents a binarization of a variable of interest, a robust relation is likely subject to both type I and type II errors. If interethnic neuro-ophthalmic disease supports a similar disparity, this may provide the data needed for proposed universal mechanisms, and garner a greater understanding of how their participation in future therapy development will affect outcomes.This review has a number of limitations. First and foremost is its narrative format. There is both diverse and comprehensive literature on disparities in MS, and so the narrative format permits the appreciation of a number of topics, but should not be considered exhaustive in its coverage. The authors refer the reader to a number of the references for additional reading.


However, for neutral rings, the maximal value of “n” to which Hückel’s rule applies remains unknown. Large macrocycles exhibiting global ring current can serve as models for addressing this question, but the global ring current are often overshadowed in these molecules by the local ring current of the constituent units. Here, we present a series of furan-acetylene macrocycles, ranging from the pentamer to octamer, whose neutral states display alternating contributions from global aromatic and antiaromatic ring currents. We find that the odd-membered macrocycles display global aromatic characteristics, whereas the even-membered macrocycles display contributions from globally antiaromatic ring current. These factors are expressed electronically , optically (emission spectra), and magnetically , and DFT calculations predict global ring current alternations up to 54 π-electrons.Aromaticity can be assigned by Hückel’s rule, which predicts that planar rings with delocalized  π-electrons are aromatic, whereas those with 4n π-electrons are antiaromatic, but for neutral rings the maximum value of n for which Hückel’s rule applies remains unclear. Here, the size-dependency of Hückel’s rule is studied in a series of furan-acetylene macrocycles whose neutral states display alternating contributions from global aromatic and antiaromatic ring currents. While aromaticity is expressed in energetic, geometric, and optical terms3, it is most commonly quantified in magnetic terms, with either diatropic or paratropic ring currents for aromatic or antiaromatic compounds, respectively5. It is estimated that, for neutral annulenes, Hückel’s rule is effective for ≤30 π-electrons, but theoretically it can also be valid for larger annulenes7. However, distortion from planarity with increased degrees of freedom, as well as increasing numbers of stereoisomers, prevents the experimental validation of this assumption. π-Conjugated macrocycles, which are composed of smaller aromatic rings in a cyclic arrangement, are shape-persistent, often more stable than annulenes, and are expected to show similar trends and thus produce aromatic or antiaromatic global ring currents11.Planar cyclic molecules with π-conjugation display aromaticity or antiaromaticity depending on their number of π-electrons, 412. Consequently, they show aromatic or antiaromatic global ring currents, depending on their size14. For example, Wu’s group demonstrated that for [m]-cycloparaphenyl-methines 27, shorter interring distances24, as well as in magnetic ring currents. The exo angle of furan is predicted to favor macrocycles with significantly smaller sizes than can be obtained from thiophenes28. Macrocyclic furans , photophysical (absorption and fluorescence spectra), and electrochemical properties of these furan-acetylenes indicate a contribution from global antiaromaticity in even-membered macrocycles, whereas odd-membered macrocycles are globally aromatic. Nucleus independent chemical shifts (NICS) calculations support this trend, predicting that the contribution of the global ring current is significant up to 54 π-electrons in neutral Cm. Overall, this work highlights the prospect of furan serving as a probe for global aromatic effects in macrocycles.Here, we introduce a series of furan-acetylene macrocycles, ngs Fig. . Unlike Cm series, comprising both odd- and even-membered macrocycles, we adopted the monomer rather than the oligomer approach. For [m]TA 35, was coupled to triisopropylsilyl-acetylene by Sonogashira coupling using triethylamine as a solvent in the presence of bis(triphenylphosphine)palladium(II) dichloride or copper iodide to obtain 2 with a 78% yield. The hexyl-groups were found to be important for the solubility of both the intermediates during the macrocyclization step and the final macrocycles. Lithiation of 2 with lithium-diisopropylamine followed by dropwise addition of a solution of I2 in tetrahydrofuran afforded 3, and the triisopropylsilyl group was subsequently removed with tetra-n-butylammonium fluoride to afford 4 with an overall yield of 60%.The synthesis of 4 using Sonogashira coupling, we attempted modification of several parameters, including solvent, base, temperature, and reactant concentration (see Supplementary Table 4 (22 mM) to 60 °C with palladium-tetrakis(triphenylphosphine) (10% mol) and copper (I) iodide (3% mol) for four days produced C5–C8 with an overall yield of 35%. The macrocycles were then separated using size exclusion chromatography. The size distribution is in-line with the calculated strain  and highest yield (13%) and C5 displays the highest strain energy  and lowest yield (<1%).For the macrocyclization of ) iodide % mol forC6 and C7 were grown by slow evaporation from hexane and chloroform solutions. The solid-state structures are displayed in Fig. C6 is 26°, and in the solid state it adopts a chair conformation, similar to cyclohexane. C7 is significantly more planar, with an average dihedral angle of 12°, which also corresponds to the abovementioned lower strain energy. The calculated structures for Cm are nearly planar for all sizes, with an average dihedral angle of 1° or less. We note that, as the barrier to rotation around the triple bond is low, the average conformation in solution is also expected to be planar or nearly-planar, as indicated by the calculated structures. The C ≡ C bond lengths do not display any significant changes  between the different members of the Cm series, and this also applies to other bond distances in the π-conjugated backbone. This is consistent with previously reported examples, in which alternating global aromaticity in large (>20 π-electrons) π-conjugated macrocycles was not expressed in the bond distances20.Crystals of Cm in dichloromethane. The oxidation potential  of C5 is 0.25 V higher compared with C6 . C6 displays two distinct reversible oxidation peaks. C7 also exhibits a higher oxidation state compared with C6, although the difference is only 0.07 V (Epa = 0.59 V) and the oxidation potential for C8 is again lower than C7 (Epa = 0.52 V). C6 stands out as the only member of the Cm series that exhibits two distinct reversible oxidation peaks, and we have previously observed similar behavior from the antiaromatic 8-membered furan macrocycle30. Therefore, C6 is the only member that exhibits a significant contribution from global antiaromatic ring current. The antiaromatic character of C6 is quite pronounced, whereas the cyclic voltammogram of C8 is not affected significantly by the global antiaromaticity.Figure Cm are expected to exhibit higher oxidation potentials compared with their even-membered analogs, since oxidation to the cation is expected to result in the loss of aromaticity, and further oxidation to the dication is expected to result in the emergence of an antiaromatic contribution. Thus, the alternating trend of the oxidation potentials is consistent with the expected trend of global aromaticity/antiaromaticity. We note that the redox potential of charged macrocycles was previously suggested as a tool to probe global antiaromaticity36.As a result of their global aromatic character, odd-membered C5 is c.a. 0.3 eV lower than that of C6 , which corresponds to the abovementioned trend in the experimentally observed oxidation potentials. The HOMO of C7 (−5.27 eV) is even lower compared with C6 and the HOMO of C8 (−5.11 eV) is higher compared with C7. Overall, the alternating behavior of the HOMO energy levels with size matches the trends observed experimentally in the electrochemical oxidation. The difference between the oxidation potentials of C6 and C8 can be explained by the difference between their calculated HOMOs, which is 0.3 eV higher for C8. It is interesting to note that macrocycles with formal antiaromatic systems were previously applied as active materials in batteries since they display stable redox states37.The calculated (M06/6-311 G(d)) energy of the highest occupied molecular orbital (HOMO) of Cm with an excess of nitrosonium hexafluoroantimonate (NOSbF6), which was found to be an effective oxidant of macrocyclic furans28. Oxidation of C6 resulted in two sets of spectra, depending on the amount of oxidant added. The first set of spectra, showing a broad absorption band at 1744 nm, and can be attributed to the cation radical, whereas the second set with bands at 1130 nm and 550 nm, can be attributed to the dication , with no apparent absorption bands that can be attributed to cationic or dicationic species. The greater stabilization upon oxidization of C6 and C8 compared with C5 and C7 is further evidence of the aromatic character of dicationic C6 and cationic C8.To study further the stability of the oxidized species, we attempted to chemically oxidize Cm are depicted in Fig. C5 and C6 display relatively sharp absorption bands, with a weak S0→S1 transition. For C5, the vibronic shoulders for the S0→S1 transition can be observed at 415 nm and 431 nm. We note that, although C5 is more rigid than C6, the relative intensity of the S0→S1 transition is stronger (by 15% for C5 and 8% for C6) compared with the S0→Sn transition (n > 1). This indicates the involvement of additional factors in the oscillator strength. C7 and C8 display broader absorption spectra, corresponding to their more flexible structures. In chloroform, the absorption spectra of C6 and C7 exhibit concentration dependence above 10−5 M  for C5 is 490 nm, there is a significant bathochromic shift of 72 nm (0.32 eV) for C6. This also manifests in a clear difference in color upon irradiation in black light . We note that, although the differences are large, this should be taken with caution, as increasing macrocycle size also results in increased flexibility, which in turn affects the emission properties. The fluorescence quantum efficiencies are small (5–6%) for the rigid C5 and C6, and increase significantly for C7 and C8 . In addition, the emission maxima seem to occur in different vibronic states. Nevertheless, time-dependent density functional theory (TD-DFT) calculations display the same alternating trend as observed experimentally, with S1→S0 transitions at longer wavelengths for even-membered macrocycles  level). The green trace in Fig. Cm, showing the same alternating trend as the experimental emission maxima (gray trace). Overall, the emission spectra show alternating behavior, with even-membered rings bathochromically-shifted compared with the odd-membered rings.The emission spectra for Cm series to understand better the alternating behavior of its members. The frontier molecular orbitals for Cm are depicted in Fig. 38.We utilized DFT to calculate the behavior of the C5 and C7) display degenerate HOMO and LUMO levels, and even-membered macrocycles (C6 and C8) displayed degeneracy of the HOMO-1 and LUMO + 1 levels. This is consistent with the trend for expanded porphyrins previously observed by Sessler and Kim39, who suggested optical properties as a parameter for aromaticity, and reported that the difference between aromaticity and antiaromaticity is expressed in red-shifted and weaker emission in globally antiaromatic macrocycles compared with globally aromatic macrocycles.As can be observed in Fig. 40. The HOMO–LUMO gap also alternates between odd and even Cm, with the odd-membered (aromatic) C5 and C7 displaying larger gaps  compared with the even-membered (antiaromatic) C6 and C8 . We note that for even Cm, both the HOMO and LUMO levels have the same number of nodal planes, as expected for antiaromatic compounds on the basis of basic symmetry arguments41.The difference in degeneracy is explained by the localized π-electron character of the structures with antiaromatic contribution compared with the delocalized electron structure of aromatic macrocycles 1H-NMR spectra of Cm show the dependency of the chemical shifts on macrocycle size. In particular, the protons located at the β-position of the furan rings experience an upfield shift of 0.28 ppm for C6 (6.41 ppm) compared with C5 . The calculated chemical shifts at the M06-2X/6-311 G(d) level show the same trend, with the even-membered Cm members experiencing an upfield shift , whereas positive values suggest a paratropic ring current (an antiaromatic system)42. We have chosen to monitor the NICS(1)zz values for the furan rings, since the comparison of ring current for different ring sizes will necessarily result in different NICS values, as the distance from the dummy atom varies. First, the NICS(1)zz values for each member of the Cm series were calculated. Figure C5,C6, and C7 are clearly observed in the interior area of the macrocycles: C5 and C7 show very weak aromatic (negative) values, whereas the larger red area in C6 indicates a contribution from global antiaromatic current. The same trend also applies to C8, although in a lesser extent. We note that the aromatic furan units should induce an external deshielding, which can explain why the internal space of the odd-membered macrocycles is not aromatic zz calculations, plots of the anisotropy of the current (induced) density (ACID) only show global ring currents for the macrocycles in their dicationic states (Cm2+). Unlike Cm, the NICS(1)zz maps for [m]CPPA do not display any visible global aromaticity or antiaromaticity regardless of their size n + 2 rule applies to small annulenes, the critical value of n is still debatable. For example, the aromatic stabilization energy of neutral annulenes is effective up to n = 6–7 (26–30 π-electrons)6. However, although magnetic criteria are perhaps the commonly applied tool for the quantification of aromaticity, the attenuation of chemical shifts with size was not reported for neutral macrocycles. In the current work, we show that, although the properties are clearly attenuated, alternation in magnetic and electronic properties can be observed experimentally in the Cm series up to 48 π-electrons (m = 8). To gain insight into the rate of attenuation for sizes larger than m = 8, the NICS(1) values of Cm members were calculated for m = 5–12. For comparison, we also calculated the NICS(1) for the same sized [m]CPPA series members . This is a significantly larger value than was observed previously for neutral macrocycles in the S0 state. By contrast, alternation in the NICS(1) values of [m]CPPA attenuates rapidly with size, and no effective change can be observed beyond m = 7. The difference between the two systems can be attributed to the lower local aromaticity of furan amplifying the global properties. It is interesting to analyze these results in the context of ‘concealed aromaticity’ as recently coined by Glöcklhofer45. The global properties of Cm are revealed predominantly upon oxidation  and in the excited state (by fluorescence maxima), and can therefore be categorized as both type-I and type-II concealed aromaticity. However, in contrast to the [m]CPPA series, the aromatic character of Cmis also revealed in the neutral state , highlighting the importance of lowering local aromaticity to reveal global properties.The average NICS(1) values for the furan rings are more negative for odd-members n + 2 π-electrons, show a global aromatic character whereas the even membered macrocycles (4n π-electrons) show a global antiaromatic character. These effects are expressed electronically , optically  and magnetically . NICS(1) calculations support the experimental observations. The differences are more pronounced for the smaller macrocycles, C5 and C6, and are attenuated, yet still observable, for C7 and C8. We show that the size-dependency of Hückel’s rule derives from the local aromaticity of the constituent unit. For macrocyclic furan acetylenes, this dependency can theoretically proceed up to 54 π-electrons, whereas for [m]CPPA, the alternation decays rapidly, and the effective size is 42 π-electrons. Overall, this work shows the first example of global aromaticity switching in neutral non-quinoidal macrocycles, and highlights the potential of macrocyclic furans to serve as shape persistent models for large annulenes.In summary, we have introduced a series of π-conjugated furan-acetylene macrocycles in which the low aromaticity of furan manifests as distinct global effects in the neutral state. The odd-membered macrocycles, consisting of 42 columns. 1H and 13C NMR spectra were recorded in solution on Brucker-AVIII 400 MHz and 500 MHz spectrometers using tetramethylsilane as the external standard. The spectra were recorded using chloroform-d as the solvent. Chemical shifts are expressed in δ units. High resolution mass spectra were measured on a high resolution quadrupole time-of-flight liquid chromatograph mass spectrometer (MS) and Waters Micromass GCT_Premier MS using electrospray ionization. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) spectra were acquired using an MALDI-TOF/TOF autoflex speed MS , which is equipped with a smartbeam-II solid-state laser . The instrument was operated in positive ion, reflectron mode. The accelerating voltage was 21.0 kV. The delay time was 130 ns. Laser fluence were optimized for each sample. The laser was fired at a frequency of 2 kHz and spectra were accumulated in multiples of 500 laser shots, with 1500 shots in total. 2-[(2E)-3-(4-tert-Butylphenyl)-2-methylprop2-enylidene] malononitrile (DCTB) matrix solutions were prepared to a concentration of 20 mg mL−1 in dichloromethane (DCM). Sample solutions were prepared to an approximate concentration of 5 mg mL−1 in DCM. Sample and matrix solutions were premixed at a ratio of 1:9 or 1:40 (v/v). A volume of 1 μL of this mixture was disposed on a MALDI steel target plate. After evaporation of the solvent, the target was inserted into the mass spectrometer. Preparative size exclusion chromatography was performed in chloroform solution at room temperature using a Recycling Preparative HPLC LaboACE LC-7080 through JAIGEL-2HR and JAIGEL-2.5HR columns connected in series. 2-Bromo-3-hexylfuran (1) was synthesized according to literature procedures35. Absorption spectra were recorded on an Agilent Technologies Cary 5000 UV-Vis-NIR spectrophotometer. Fluorescence and excitation spectra were performed on Horiba Scientific Fluoromax-4 spectrofluorometer. All spectroscopic experiments were performed using standard quartz cuvettes of path length 1 cm and solutions prepared in spectroscopic grade solvents. The excitation laser used was 330 nm with a pulse width of less than 1.4 ns. The synthetic pathway for 4 is described in the Supplementary Synthesis section  , tetrakis(triphenylphosphine)palladium  and CuI  were dissolved in a mixture of 75 mL of dry toluene and 75 mL of distilled N,N-diisopropylethylamine under an inert atmosphere, heated to 60 °C, and the reaction mixture was stirred for 3 days. Afterward, the reaction mixture was allowed to reach room temperature and was subsequently filtered over celite. The filtrate was evaporated and the residue obtained was purified by flash column chromatography on silica gel using a mixture of DCM and hexane (9:1) as eluent to give a mixture of macrocycles. The mixture was separated using gel permutation chromatography and chloroform as the eluent to give C5 , C6 , C7 , and C8 .2-Ethynyl-3-hexyl-5-iodo-furan (Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3


An omission to the funding section of the original article was made in error. The following sentence has been added: “Open access funding was provided by the École Polytechnique Fédérale de Lausanne.”The publisher apologizes for this mistake. The original article has been updated.


Fagopyri Dibotryis Rhizoma (FDR) is an effective Chinese herbal medicine with a long history of use in China. FDR is effective in heat clearing and detoxifying, promotion of blood circulation, relieving carbuncles, dispelling wind, and removing dampness. Its seeds also have high nutritional value, are rich in protein, and contain a variety of mineral elements and vitamins. Therefore, FDR is considered a natural product with medical and economic benefits, and its chemical composition and pharmacological activity are of interest to scientists. The current review provides an overview of the available scientific information on FDR, particularly its botany, chemical constituents, and pharmacological activities. Various sources of valid and comprehensive relevant information were consulted, including the China National Knowledge Infrastructure, Web of Science, and PubMed. Among the keywords used were “Fagopyri Dibotryis Rhizoma”, “botanical features”, “chemical composition”, and “pharmacological activity” in combination. Various ailments are treated with FDR, such as diabetes, tumor, sore throat, headache, indigestion, abdominal distension, dysentery, boils, carbuncles, and rheumatism. FDR is rich in organic acids, tannins, flavonoids, steroids, and triterpenoids. Experiments performed in vitro and in vivo showed that FDR extracts or fractions had a wide range of pharmacological activities, including antitumor, anti-inflammatory, immunomodulatory, antioxidant, antimicrobial, and antidiabetic. The current review provides an integrative perspective on the botany, phytochemistry and pharmacological activities of FDR. FDR may be used as a medicine and food. Based on its chemical composition and pharmacological effects, the main active ingredients of FDR are organic acids, tannins, and flavonoids, and it has obvious antitumor pharmacological activity against a variety of malignant tumors. Therefore, FDR is worthy of further study and application as a potential antitumor drug. Chinese herbal medicine plays a crucial role in the prevention and treatment of diseases as a drug resource for the traditional medical system and as an important raw material for chemical drugs, international botanicals, and the food industry. A significant amount of evidence suggests that medicinal plants may be used to treat a variety of diseases and for the discovery of novel pharmacologically active molecules. The phytochemicals identified from medicinal plants have provided promising lead compounds for effective new drugs . MedicinFagopyrum dibotrys (D. Don) Hara is a perennial herb of the genus Fagopyrum in the family Polygonaceae, and it is widely distributed in the Sichuan Basin, the hills of Guangdong and Guangxi, and the Yunnan-Guizhou Plateau in China and Thailand, Nepal, India and other countries (Fagopyri Dibotryis Rhizoma (FDR) has been widely used as a folk medicine to cure forms of chronic bronchitis, lung cancer, sore throat, rheumatic disease, dysentery, and enteritis  Hara is a perennial herb that is native to eastern, central and southwestern China, India, Nepal, Vietnam, Thailand, and other countries. The habitat of Fagopyrum dibotrys (D. Don) Hara is 250–3,200 m above sea level in valley wetlands and hillside forests. The rhizomes are black‒brown, stout, and ligneous, and the stems are long and erect, green, or brownish, 40–100 cm high, branched, striate, and glabrous. The petiole is 2–10 cm, and the leaf blade is triangular at 4-12 × 3–11 cm. Both surfaces are papillate, the base is nearly hastate, the leaf margin is entire, and the apex is acuminate. The ocrea is brown, 5–10 mm, membranous, and oblique, and the apex is truncate, not ciliate. Plants have terminal, axillary or corymbose inflorescence. Bracts are ovate-lanceolate, ca. 3 mm, with membranous margins, and an acute apex, each 4-flowered and rarely 6-flowered. Pedicels are in equaling bracts that articulate at the middle. Perianth are white, and tepals are narrowly elliptical, ca. 2.5 mm. Stamens are included. The styles are free, and stigmas are much longer than the persistent perianth, capitate, and opaque. During April-August, the chenes are blackish brown, dull, broadly ovoid, 6–8 mm long, trigonous, sometimes narrowly winged, with smooth to repandous angles, and an acute apex -epicatechin -epicatechin , 3,5-dimcatechin .Flavonoids are widely present in naturally growing plants and refer to a class of compounds with two benzene rings connected by three carbon atoms that create the C6-C3-C6 structure . QuercetTannins are phenolic compounds with complex structures that are widely distributed in plants. Procyanidin b2, procyanidin c1 , procyanβ-sitosterol and β-daucosterol from FDR , phosphorylated-protein kinase B (p-Akt) and phosphorylated-mammalian target of rapamycin (p-mTOR) of the PI3K/Akt pathway (κB) to DNA in the H-RS cell line and inhibited NF-κB-driven genes, including anti-apoptotic proteins  and inhibition of the anti-apoptotic factor B-cell lymphoma-2 (Bcl2). The extract of FDR can also activated caspase-8, caspase-9, and caspase-3 and released mitochondrial cytochrome C . Some exer model . Fr4 proer model . The FDRpression .in vitro via the dual action of PG cells and HUVECs, which inhibited tumor cell metastasis in the blood channel , interleukin-1 and interleukin-6 (IL-6) and inhibition of the expression of nitric oxide synthase, phosphorylation of p38 mitogen-activated protein kinase (p-p38MAPK), extracellular signal regulated kinases 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) (κB transcription factors and activating JNK and p38 MAPK (Various extracts . The effse (JNK) . Differese (JNK) . Protocap38 MAPK . Rhamnetp38 MAPK .κB and activator protein 1 and suppressing the phosphorylation of PI3K/Akt and MAPK, chrysoeriol inhibited the inflammatory response of LPS-stimulated RAW 264.7 cells /Nrf-2 pathways to inhibit arthritis.By inhibiting the TLR4-mediated activation of NF-.7 cells . Gallic .7 cells . The ant.7 cells . Byun et.7 cells  indicateg et al.  demonstrκB and the induced production of TNF-α, interleukin-8, IL-6, transforming growth factor-β1 and precollagen peptide III activity in chronic obstructive pulmonary disease rats, which improves lung tissue inflammation  were more effective than SASP alone in ulcerative colitis (UC), and the mechanism may be the anti-inflammatory and immunomodulatory effects of intervening in UC  pathway . FDR tab pathway . The eff pathway . Some st pathway .via multiple pathways, links, and targets -Epiiability . Procyane damage . Procyane damage . Kim et e damage  confirmeFDR leaf tea has significant antioxidant scavenging ability against DPPH radicals, ABTS radicals and hydroxyl radicals . FDR extβ Hemolyticus Streptococcus and Streptococcus pneumoniae in vitro and inhibited infections caused by strains of Streptococcus pneumoniae in mice in vivo  mice and regulated lipid metabolism and oxidative stress levels in model animals . FDR leain vitro, and its active ingredient is the flavonoid of FDR, which is concentration dependent . Downregulation of transient receptor potential vanilloid 1 (TRPV1) expression also had a therapeutic effect on hyperalgesia in IBS rats -epicatechin and genkwanin show significant antitumor activity. However, whether the antitumor effects of FDR are the result of the joint action of various components and the specific antitumor mechanism are not clear. Therefore, there is a need for in-depth research on the following aspects. According to pharmacodynamic studies, the effective site of the antitumor effect of FDR must be clarified


Dentin hypersensitivity is an oral health concern affecting a large percentage of the world's adult population. Occlusion of the exposed dentinal tubules is among the treatment options available, and silver diammine fluoride (SDF) is an occluding agent used for interrupting or dampening the stimulus of the dental pulp nerves that produce pain. In addition to dentin permeability testing, the evaluation of desensitizing agents occluding dentinal tubules strongly relies on microscopic techniques, such as scanning electron microscopy (SEM). Limitations of SEM are that it provides only surface images that lack detailed information on the depth of penetration and amount of material present within the treated specimen, and it is prone to sample preparation artifacts. Here, we present high-resolution X-ray computed tomography (nano-CT) as a potential method for investigating dentin specimens with occluded tubules. We studied human dentin treated with SDF as an exemplary dentinal occlusion treatment option. We evaluated the silver deposits formed on the dentin surface region near the dentinal tubules and in the tubular regions using cross-section SEM, Energy Dispersive X-ray (EDX) analysis, and nano-CT. The resulting images obtained by SEM and nano-CT had comparable resolutions, and both techniques produced images of the tubules' occlusion. Nano-CT provided three-dimensional images adequate to quantitate tubule size and orientation in space. Moreover, it enabled clear visualization of dentinal tubules in any virtual plane and estimation of the amount and depth of occluding material. Thus, nano-CT has the potential to be a valuable technique for evaluating the occluding effects of virtually any material applied to dentinal tubules, supporting deciding between the best occluding treatment options. The occlusion of dentinal tubules’ apertures by desensitizing agents is the primary strategy currently used by clinicians to prevent the pain11. In vitro models are usually used to understand the occlusion efficacy of desensitizing agents12, assessed by morphological analysis of precipitates formed in the dentinal tubules through microscopic methods17.Silver diammine fluoride (SDF) solution is used for preventing and arresting carious lesions in adults and children20, which can be combined with Energy Dispersive X-ray (EDX) analysis to provide surface morphological information, and the identity and ratio of chemical elements on and in the dentinal tubules21. SEM cross-section imaging provides additional information on occluding material penetration depth within the exposed imaged surface22. Different approaches can expose a cross-section of dentin specimens, such as fracturing the sample18, or using a Focused Ion Beam (FIB)24. These preparation techniques, however, are not free from artifacts. Fracturing the sample may add debris to the tubules that could be mistaken as occluding material, and FIB-SEM produces a three-dimensional image from serial two-dimensional SEM images24 in a process that is often challenging, time-consuming and requires post-acquisition image processing, which is limiting to most users25. Thus, the assessment of occlusion depth and the identification of different occluding materials in treated dentin specimens through morphological data can be difficult, either by limitations related to sample preparation or intrinsic to the microscopic imaging technique itself.Those methods include Scanning Electron Microscopy (SEM)26. Micro-computed tomography (micro-CT) was used to investigate the effect of SDF on carious lesions27 and on the enamel surface28. High-resolution CT (nano-CT) was already used to produce images of the dentin, giving details of the architecture of the dentinal tubules in different species at the nanometer scale34. Additionally, it can often access the distribution of the electron density, enabling quantitative analysis of the features observed in the specimens analyzed34. Nano-CT requires sample preparation that can create artifacts in the area where the specimen is processed, but the region of interest for imaging can remain unaffected and artifacts-free. To the best of our knowledge, nano-CT has not been used for the visualization of the occlusion of dentinal tubules after a treatment. Thus, here we show the potential of this technique for assessing dentinal tubule occlusion. We used two different SDF formulations to illustrate the ability of nano-CT to detect their occlusion efficacy and we discuss nano-CT benefits and disadvantages when compared to traditional microscopic methods.X-ray computed tomography (CT) is a cutting-edge imaging technology able to produce volumetric information that has been successfully applied to image small features in biomaterials19. Briefly, eighteen specimens of 4 mm × 4 mm dentin from human tooth roots were prepared to mimic exposed dentin after gingival recession. Human dentin specimens were prepared at Indiana University from human tooth roots. Ethical approval for use of the extracted teeth was given by the Indiana University Institutional Review Board (NSO 911-07). The specimens were polished and, to open their dentinal tubules, all specimens were immersed in a 17% EDTA solution  for 5 min, then rinsed with running deionized water for 5 s. They were then randomly allocated in equal amounts to three test groups (n = 6 for each group). Specimens in test group 1 were treated with one drop (c.a. 50 μL) of a standard commercial 38% SDF formulation  applied with a micro-brush. The same procedure was done in test group 2, in which the specimens were treated with an experimental SDF formulation  that contains 38% SDF but with a higher viscosity (~ 30 cP at room temperature). In test group 3, specimens were treated with a placebo solution, free from fluoride or silver, that was freshly prepared and certified by an FDA-regulated laboratory independent of the investigators. The solutions applied onto the specimens remained on the exposed dentin surfaces undisturbed for 1 min, then rinsed with running deionized water for 5 s. The specimens were then immersed in artificial saliva  for 2 h35, rinsed again with running deionized water for 5 s and the vials with excess moisture were sealed and shipped to the imaging facility.The samples were prepared as described previouslyA total of four specimens from each of the three groups were prepared as illustrated in the supplementary information Fig. 36. Each cone was then glued onto the top of a metallic pin and imaged using ZEISS Xradia 810 Ultra nanoscale X-ray microscope operating with a chromium X-ray source (5.4 keV). Samples were imaged using absorption-contrast. A total of 901 projections were obtained over 180°, a camera binning of 2  and exposure time of 30 s per projection. Image reconstruction was performed by a filtered back-projection algorithm using the software XMReconstructor integrated into the device. Volumetric data was visualized either in Avizo  or VGStudio (version 2022.2). For the estimation of silver within the tubules, data were processed in Avizo, first matching the contrast between the two datasets  to assure that the signal from silver was comparable between datasets. Then, a median filter was applied for image correction, followed by segmentation using interactive threshold, with the threshold defined by the clear boarders seen in the tomograms . The nano-CT 3D image of the conical specimen treated with placebo solution Fig.  shows tu3) and the volume occupied by the silver-containing particles (4.8 µm3). This information enabled the estimation of the silver percentage in relation to the tubules volume, which is equal to 0.63% for this specimen.Different than the placebo formulation-treated sample, the volumetric images of SDF-treated samples Figs.  and S3 s3) and the volume occupied by the silver-containing particles (6.9 µm3). Assuming that the particles are composed purely by silver, this information enabled the estimation of the silver percentage in relation to the tubules volume, which is equal to 1.68% for this specimen.The volumetric reconstruction of the sample treated with the experimental SDF formulation shows silver agglomeration in the tubules Fig. a, besideWe show in Fig. 38. In part the difference may be because the teeth studied in this earlier work had large natural caries lesions with greater destruction of the tubule architecture than the sound dentin treated with EDTA in this study. With EDX analysis  SEM combined with EDX, and nano-CT. We evaluated silver deposits formed on the dentin surface near the dentinal tubules and in the tubular regions. Our findings show that nano-CT is a useful method for studying dentin. It produces high-resolution 3D images that enable clear visualization of dentinal tubule detail, showing features as small as a few hundred nanometers. Though we used the widely adopted SDF as an example, nano-CT can be used to image all types of materials that produce X-ray contrast against the dentin composition. Our data illustrates that nano-CT is a powerful technique for non-destructive imaging within the volume analyzed, enabling detailed characterization of tubule obstruction. Moreover, it can produce volumetric quantitative data when statistical analysis is of interest, such as when comparing different dentin treatments. Nano-CT enables quantitative evaluation of tubule size and orientation in space, allowing estimation of the volume occupied by occluding particles within the tubules. Thus, nano-CT has the potential to be one alternative methodology for the evaluation of tubular occlusion. We anticipate that it will benefit the evaluation of the occluding effects of virtually any material applied to dentinal tubules, showing the penetration depth of the material within an entire volume of tens of micrometers, thus aiding in the development of more effective interventions for preventing dentin hypersensitivity.Supplementary Video 1.Supplementary Video 2.Supplementary Video 3.Supplementary Information.