Matricaria chamomilla L. , twenty mitochondrial haplotypes (genetic distances 0.028–0.693) were identified. Three of the accessions  were monomorphic. The highest genotypic variability was found for the Croatian accession ‘PG029’ with nine mitochondrial haplotypes (mitotypes) and the Argentinian ‘Argenmilla’ with seven mitotypes. However, most of the mitotypes detected in these accessions were infrequent in our sample set, thus disclosing an unusual high amount of substitutions within the mitochondrial genome of these accessions. The mitotypes with the highest frequency in the examined dataset were MT1 (n = 27), MT9 (n = 23) and MT17 (n = 20). All of the frequent mitochondrial lines are distributed not only over several accessions but also over several geographical origins. The origins often build a triplet with on average two to three concurrent lines. The most distantly related accessions were ‘Mat19’ and ‘Camoflora’ (0.539), while ‘PNOS’ and ‘Margaritar’ (0.007) showed the lowest genetic distance.The online version contains supplementary material available at 10.1007/s13353-020-00602-3. Matricaria chamomilla L.  (Germplasm Resources Information Network (GRIN) M. chamomilla L. is just at the beginning. Uniparentally inherited markers of the chloroplast or mitochondrial genome proved adequate to enable investigations of migration routes of populations or the exploration of geographical backgrounds and common ancestors that date back several years  and next-generation sequencing (NGS) were used recently to appraise genetic diversity of different chamomile accessions by exploiting a multitude of SNP data of the nuclear and mt genome  reconstructing their breeding history and (II) estimate diversity between/among accessions and therefore test the applicability of the SNP markers in terms of distant inter-cultivar crossings.Matricaria chamomilla L. were cultivated in the greenhouse at the University of Veterinary Medicine, Vienna , in 2017. Five, ten or 15 individuals per accession , and total genomic DNA was isolated using a modified CTAB (cetyltrimethylammonium bromide) extraction protocol . A LASTZ alignment of the resulting individual mt consensus sequences and Diplostephium hartwegii (KX063855) as reference sample generated with Geneious 9.1.5  .The final reaction volume of 10 μl contained 1× HRM mastermix , 150 nM of the forward and reverse primer each, and 2 ng genomic DNA. All samples and ‘no template controls’ (NTC) were analysed in duplicates.The HRM analysis with pre-amplification was performed with an initial phase of 14 min at 95 °C, 45 cycles of 95 °C/ annealing temperature depending on the primer pair  guidelines , artificial heteroduplexes had to be produced to separate the melting curves of alleles A and T. Therefore, a spike DNA with the known haplotype A was added to the mastermix, resulting in identical HRM curves and melt curves with only one peak for samples with the haplotype A and heteroduplexes and two peaks in the melt analysis for haplotype T samples.m per run and ΔTm were calculated for each primer combination (Table The mean standard deviation of Ton Table .Table 2CN/(N-1), evenness, E.5   (Nei The number of multilocus genotypes (MLG) standardized by sample numbers, the Shannon-Wiener Index (H) Shannon , StoddarExp) Nei  were comMLG and Nei’s genetic distance were used for construction of a minimum spanning network. All statistical analyses were calculated using R 3.3.0  were analysed with 36 SNP markers in an HRM analysis in order to obtain a better insight in the composition of varieties. Thirty-one markers of this core marker set were selected out of the 102 SNPs detected previously in the mt genome sequences of eleven chamomile accessions  validation of the genotyping technique, (ii) verification of the accuracy of the SNPs used for genotyping.With the analysed dataset, we could therefore find fifteen of the originally detected mitotypes by Ruzicka and Novak ; five neIn accordance with the low amount of substitutions (eleven) originally found in the coding regions of the chamomile mt genome  was calculated for the locus Mr_mt158868, which detects a G/T transversion. Mr_mt026887 (G/T) and Mr_mt187230 (A/C) showed the lowest ΔTm values (0.32).ΔTexp and evenness and the highest Simpson’s index and therefore provided highest information content. In general, Simpson’s index ranged from 0.013 (Mr_mt154969) to 0.612 (Mr_mt130788) (mean = 0.268), the expected heterozygosity Hexp ranged from 0.013 to 0.616 (mean = 0.269) and evenness ranged from 0.327 to 0.985 (mean = 0.676) (Table A) was calculated across all markers. The average IA was 4.79 with a p value of 0.001 and ṝd of 0.15 , ‘Bona’ (E.5 0.922/λ 0.72) and ‘Argenmilla’ (E.5 0.667/ λ 0.729). Thus, the accessions with the highest genotypic richness with identified eight, seven and four MLGs , respectively, were the Croatian accession ‘PG029’ (eMLG 6.45), the Argentinian ‘Argenmilla’ (eMLG 5.48) followed by ‘Bona’ from the Slovak Republic (eMLG 4)  showed a variability of 65.7% between the accessions and 34.3% within accessions. Thus, the mt genome of chamomile delivered a high variation for clarifying the relatedness of the accessions but also displayed a high genetic diversity within. Six of the accessions revealed an HOnly three of the accessions (‘Camoflora’ 2×), ‘Mat19’ (2×) and ‘Manzana’ (4×)) were monomorphic , ‘Camoflora’ (0.539) and ‘Soroksári’ (0.538), respectively Table . The cloAll of the mitotypes were distributed over several countries despite of the ten mitotypes represented by single individuals of one accession only . The mitotypes with the highest frequency in chamomile were MT1, MT9 and MT17 , respectively nine (‘PG029’) mitotypes were identified though most of them with low frequencies. Although the results might be biased by different sample volumes in the countries , it is obvious that Croatia and Argentina retrieve the highest variability caused by these two accessions.Several hypotheses are possible to explain the high number of point mutations found within accessions of chamomile: (I) spontaneous mutations, which would explain the low diversity found between some of the mitotypes; (II) use of several plant lines to produce cultivars or traded landraces; (III) exchange of plant material between breeders to establish new chamomile lines in other countries; (IV) cultivation of imported chamomile plants in historical times; (V) migration of chamomile populations into several countries and establishment of those chamomile populations with identical mother lines, which were independently used for producing new cultivars; (VI) contamination of seed material of cultivated chamomile with seeds of wild growing chamomile.The fact that most of the accessions are divided into two or three main mitotypes with high frequencies corroborates the hypotheses of several promising lineages that were used for producing the cultivars and landraces (thinkable for e.g. ‘Soroksári’ and ‘Mat16’) or the extensive exchange of plant material between breeders (e.g. Romanian and Polish accessions). It is known that the Romanian ‘Margaritar’ originates from the Polish ‘Zloty Lan’ and chamomile plants collected in the wild in Poland Das . This coThe high number of monotypic mitotypes in the accessions ‘PG029’ and ‘Argenmilla’ could better be explained by contamination of the seed material or a higher tendency of spontaneous point mutations in these accessions than by extensive plant exchange alone. Nevertheless, ‘Argenmilla’ and ‘PG029’ also were more distinct to many of the other chamomile accessions in the GBS study of Otto et al. .An increased mt mutation rate was detected in an infertile line of onion, where it co-occurs with CMS  (XLSX 12 kb)Online Resource 2Diplostephium hartwegii, individual sample names and the resulting definition of mitotypes (MT). SNPs and MT are consecutively named according to a previous study (Ruzicka and Novak Polymorphic sites investigated with HRM analysis, alignment position corresponding to sequence coordinates of


Our work can be extended to provide electrolyte-related structure-property relationships in redox-active polymers and functionalised electrodes for pseudocapacitive energy storage.Altering electrochemical interfaces by using electrolyte effects or so-called “electrolyte engineering” provides a versatile means to modulate the electrochemical response. However, the long-standing challenge is going “beyond cyclic voltammetry” where electrolyte effects are interrogated from the standpoint of the interfacial properties of the electrode/electrolyte interface. Here, we employ ferrocene-terminated self-assembled monolayers as a molecular probe and investigate how the anion-dictated electrochemical responses are translated in terms of the electronic and structural properties of the electrode/monolayer/electrolyte interface. We utilise a photoelectron-based spectroelectrochemical approach that is capable of capturing “snapshots” into (1) anion dependencies of the ferrocene/ferrocenium (Fc/Fc Conveying electrochemistry in terms of the electrode/electrolyte interfacial properties remains challenging. Here, the authors employ a surface-bound molecular probe and photoelectron spectroscopy to peer into the anion-dictated and potential-induced interfacial electronic and structural properties. The motivation stems from the desire to augment the functionality of devices by controllably altering key parameters such as the redox potential, reaction pathway, reversibility and stability. However, achieving this requires an intimate knowledge of the interfacial properties as this affects the charge transfer process and thus, the overall electrochemical response. This fuels the desire to obtain structure–property relationships that sufficiently encompasses the intricacies of the electrochemical environment at the electrode/electrolyte interface6. With relevance to bio sensing7, redox-induced micro-actuators8 and molecular electronics9, the corresponding Fc termini can switch between neutral Fc (ferrocene) and cationic Fc+ (ferrocenium) redox states via single-electron transfer and ion-pairing reactions with anions: (Fc)SAM + X−aq ⇆ (Fc+–X−)SAM + e−11. Therefore, the anion should play an important role in the Fc/Fc+ redox process and has been shown to influence the apparent formal potential (Eo′) and reversibility owing to the influence of anion solvation on the behaviour of ion pairing13. Concomitantly, this implies that the anion should affect the microscopic ion-pair structures14 and the related structural changes including monolayer thickness16 and molecular orientation17. While there are techniques that can probe such structural changes, we will show for the first time, the ability to also discriminate the anion-dictated Fc SAM interfacial electronic properties.To this end, surface-confined redox molecules such as ferrocene-terminated alkanethiol self-assembled monolayers (Fc SAM) on Au Fig.  constitu4. The electrified electrode/electrolyte interface consists of a region with a linear potential gradient (inner Helmholtz plane) followed by the outer Helmholtz plane (diffuse layer) where the potential profile decays monotonically into the electrolyte. In the presence of a SAM, the linear potential portion is dependent on the length of the molecules adsorbed on the electrode surface , Porter et al. showed that the role of interfacial capacitance can be indirectly inferred21. Eggers et al. demonstrated the relationship between the apparent formal potential and the height of the Fc termini relative to a co-adsorbed non-electroactive SAM22. While varying the SAM structure can be an effective approach, methods that can directly interrogate the influence of the interfacial potential distribution have thus far, been very limited.A related aspect is the interfacial energetics as first envisioned by Helmholtz and later expanded by Gouy–Chapman and Sternace Fig. 18–20. Ex23. A workaround is to employ a so-called pseudo in situ UHV-EC approach originally conceived by Hubbard and coworkers29 to link the electrochemical (solution) and XPS/UPS  environments (see Methods). We note that alternatively, true in situ XPS can offer another avenue to explore solid/liquid interfaces36, while each approach has noteworthy trade-offs such as signal-to-noise versus temporal resolution . Based on the photoelectric effect, XPS/UPS detects the kinetic energy of ejected photoelectrons to reveal the electronic and chemical environment of atoms at the sample surface. However, an intrinsic obstacle is the need for vacuum conditions in order to obtain useful detection probabilities3SO2]2N−, denoted as TFSI−) and an inorganic anion (PF6−), which should invariably affect the ion–ion and ion–solvent interactions owing to differences in ionic size, charge density, polarisability and flexibility39. We use a spectroelectrochemical approach consisting of XPS/UPS combined with an electrochemical cell (denoted hereon as EC-XPS/UPS) to electrochemically control the Fc SAM followed by spectroscopic observations into the anion-dictated aspects of the Fc/Fc+ redox process including Fc+–X− ion-pairing, monolayer thickness, Fe oxidation state and valence structure. A key aspect of our findings is related to the TFSI− anion which uniquely restricts the Fc+ reaction yield, thereby creating the conditions where the influence of the electrostatic potential across the electrode/monolayer/electrolyte interface is readily observable via concomitant core and valence-level binding energy shifts. The work presented here underscores how “beyond cyclic voltammetry” approaches consisting of vacuum-based methods can be effective in ascertaining the intricacies of the electrode/electrolyte interface.Herein, we interrogate the Fc SAM molecular probe and show how the anion-dictated electrochemistry is conveyed in terms of the electronic and structural properties of the electrode/monolayer/electrolyte interface. We predominantly focus on two anions, namely a bulky organic anion , the nature of the anion also influences the degree of conversion to Fc+ (reaction yield). Namely, the apparent formal potentials (Table − (0.180 V) followed by PF6− (0.240 V) and ClO4− (0.270 V), respectively. The integral charge from the CVs indicates the degree of conversion to Fc+  in comparison to PF6− and ClO4−, which are similar (4.6–4.7 × 10−10 mol cm−2), while it should be noted that the theoretical Fc SAM coverage is 4.5 × 10−10 mol cm−2 (assuming close-packed Fc with a diameter of 0.66 nm)40. All of the CVs are reversible (negligible anodic-cathodic peak separations) and all of the peak currents increase linearly with scan rate  of Fc SAM on Au111) in 0.1 M NaTFSI, NaPFlO4 Fig.  show tha11 in 0.1y PF6− 0.40 V and y PF6− 0.40 V and E(anodic) and E(cathodic) in Fig. E(anodic) and E(cathodic) can be expected to predominantly yield  Fc and oxidised (cationic) Fc+ redox states, respectively. We primarily focus on contrasting TFSI− and PF6− due to their distinct differences, whereas ClO4− exhibits comparable CV characteristics to PF6−40.Subsequent spectroelectrochemical characterisations of the Fc SAM were performed on the pristine sample and after polarisation at 0.625 and 0 V , XPS shows the emergence of anion-related features resembling TFSI−  corresponding to the conversion back to Fc, thus indicates the reversibility of Fc+–X− ion pair formation. For both TFSI− and PF6−, the XPS-determined stoichiometry 45. Complementary experiments examining competitive ion-pairing  is met by a larger increase in effective monolayer thickness  than PF6− (19.9 ± 0.5 Å), which implies differences in Fc+–X− ion-pair structure/size.The Fc/Fclts Fig.  meaning 4− anions47. Therefore, similar to the case with ClO4−, the formation of 1:1 Fc+–X− contact ion-pairs is attributed to the hydrophobic (weakly solvating) nature of TFSI− and PF6−, which strongly favours ion-pair formation to stabilise the charged Fc+. In addition, the O 1s spectra for PF6− (contains no O atoms) is largely featureless following E(anodic) and E(cathodic), while in the case of TFSI−, O 1s features are prominent only after E(anodic) which is attributed to TFSI− forming the abovementioned Fc+–X− ion pairs. These O 1s spectral observations suggest that the immersion process “unzips” and eliminates weakly-interacting, excess electrolyte and that water is not strongly interacting with the immersed Fc SAM electrode48. We further note that (Na+) cation involvement in terms of charge compensation is found to be negligible at the potentials analysed  studies that have suggested that reversible 1:1 ion-pairing occurs in the presence of ClOE(anodic) yields significant changes where the charged Fc+ are effectively screened by counter anions at the inner Helmholtz plane resulting in a robust adlayer of 1:1 Fc+–X− contact ion-pairs, and concomitantly leads to the increase in monolayer thickness. In terms of anion dependencies, in the case of PF6−, there are exclusively 1:1 Fc+–PF6− ion pairs, whereas in the case of TFSI−, 1:1 Fc+–TFSI− ion pairs coexist with neutral Fc due to steric constraints45. On the other hand, for E(cathodic), the inner Helmholtz planes are structurally similar for both TFSI− and PF6− and consists of weakly-interacting water, while any ions that are present are expected to be solvated owing to the negligible surface charge.In light of the abovementioned observations, we can summarize the structural aspects of the electrode/monolayer/electrolyte interface as schematically shown in Fig. +–X− ion pairing. The tendency of ion-pairing can be empirically expressed as K (ion-pair formation constant) in the following Nernst relation10:Eo is the standard reduction potential and all other parameters have their usual meanings. Therefore, the apparent formal potential should depend on both K and anion concentration [X−]. A plot of the apparent formal potential as a function of anion concentration , meaning that the difference in the vertical intercepts originates from the K parameter. Based on the difference of the vertical intercepts (~60 mV), we can ascertain that the K for TFSI− is 10 times larger than in the case of PF6−. We note that alternatively, the ion-pairing tendencies and apparent formal potentials can be conceptually rationalised by invoking Pearson’s hard-soft acid-base theory to explain Fc+–X− interactions based on the differences in the Lewis basicity of the anion49.However, despite reversible ion pairing, the anion-dictated apparent formal potentials are a reflection of differences in the ion–solvent and ion–ion interaction strengths. The resulting competition between the two dictates the favourability or tendency of Fc50. The sharp symmetry of the Fe 2p3/2 peak at 707.9 eV  ligands, Fc has the electronic configuration (a1′)2(e2′)4 and (a1g)2(e2g)4 for the eclipsed (D5h) and staggered (D5d) conformations, respectively54. The XPS Fe 2p (core level) and UPS valence spectra can be tracked together because the Fc HOMO is localised to the Fe heteroatom causing them to be strongly correlated56.We now move to the electronic aspects of the electrode/monolayer/electrolyte interface. The XPS Fe 2p and UPS valence spectra Fig.  confirms eV Fig.  reflects eV Fig.  ascribedE(anodic) leads to significant spectral changes with anion dependencies  owing to final state effects including multiplet splitting caused by the presence of an unpaired valence electron in the (low spin) paramagnetic Fe(III)55. Note that in TFSI−, the remaining Fe(II) features (707.4 eV) indicate residual Fc. Meanwhile, oxidation is also evident in the valence spectra  and is clearly no longer prominent in the case of PF6−. The HOMO of the oxidised Fc+ should be located at higher binding energy due to factors including increased Coulombic attraction but is likely overlapped by the anion and SAM-related features  at binding energies >2 eV  features in the Fe 2p core level and Fc HOMO  show systematic binding energy shifts with respect to the polarisation potential (E(cathodic) and E(anodic)). Specifically, the Fe(II) peak in the Fe 2p3/2 spectra  and E(anodic), respectively. Similarly, in the valence spectra  and E(anodic), respectively. Taken together, this corresponds to a ~0.8 eV/V relationship between binding energy shift and electrode potential. It should be noted that in terms of the intermediate potentials, EC-XPS/UPS measurements performed at increasing potentials with ClO4−  as shown below:58These systematic XPS/UPS binding energy shifts are attributed to the influence of the electrostatic potential across the electrode/monolayer/electrolyte interfacedel Fig. 19,22 wheEFermi) of the Au substrate which is the same entity as the electrode potential 20. The net effect of positive potential yields a lower binding energy, while negative potential yields higher binding energy  in TFSI−, there is an additional S 2p doublet at 169.6 and 168.4 eV . The positioning within the monolayer is also ascribed as a contributing factor that causes the broadening of the C 1s spectra upon E(anodic) attributed to the C atoms located along the alkyl chain  and E(anodic)) resulting in a discrete binding energy shift associated with oxidation, and thus masks the binding energy shift from the electrostatic potential. It is noteworthy to mention that in addition to the Fe heteroatom, the TFSI− anion also exhibits binding energy shifts that are observable at an intermediate potential .It is important to reiterate the role of TFSI61, whereas in our case, we utilise a reversible redox-active surface-bound molecular probe (Fc SAM). This difference has distinct advantages. The Fc SAM molecular probe offers a controlled, systematic approach since the number of Fc/Fc+ redox centres is well-defined throughout the experiments (4.5 × 10−10 mol cm−2) thus, allows the electrochemical response  to be directly correlated and verified with XPS/UPS regarding (1) the degree of Fc+–X− ion-pairing (anion coverage), (2) change of Fc/Fc+ redox centre (Fe oxidation state), and (3) binding energy shifts . This corroborative approach allows for more complete conclusions. Additionally, the construct of the Fc SAM molecular probe is beneficial because the alkyl chain physically and electronically separates the Fc termini from the electrode surface. This circumvents the challenges of existing studies where the influence of the high electric fields on species at the electrode surface can lead to ambiguities in the interpretation of the observed binding energy shifts62.We now discuss the present work in the context of previous UHV-EC-based XPS studies that have reported electrochemically-induced binding energy shifts. These previous studies typically focus on bare metal electrodes restricted to the double-layer charging region63, use of ionic liquids as a coating or electrolyte55, or semiconducting electrodes65, which will alter the energetics of the electrode/monolayer/electrolyte interface. Our work shows a framework that can be extended to interrogate electrolyte effects and interfacial energetics in redox-active polymers66 and in applications relating to functionalised electrodes for pseudocapacitive energy storage67.We add that the approach described here opens the door to other compelling opportunities including the modulation of the SAM structure, organic ions with strategically positioned heteroatoms+–X− ion-pair composition, monolayer thickness and degree of conversion to Fc+ arising from Fc+–X− interactions and steric constraints, which manifests in the apparent formal potential and integral charge transferred as observed by CV. Upon subsequent cathodic polarisation corresponding to the conversion back to Fc, our observations are reversible. We have shown that the Fc SAM molecular probe construct enables a useful corroborative approach where the electrochemical response can be readily correlated to the XPS/UPS results allowing for the unobscured observation of the electrostatic potential at the electrode/monolayer/electrolyte interface. This work is part of our ongoing efforts to exploit UHV-EC methods to obtain a richer description of the electrode/electrolyte interface.Using EC-XPS/UPS as a “beyond cyclic voltammetry” approach enables a unique spectroelectrochemical description of the anion-related electronic and structural properties of the electrode/monolayer/electrolyte interface which are not readily assessable with other methods. Upon anodic polarisation, we can spectroscopically discriminate differences in Fc2SO4 (Kanto) and 30% H2O2 (Wako) in a 3:1 volume ratio) at room temperature for at least 3 h. Caution should be exercised because piranha solution is highly corrosive and vigorously reacts with organics meaning that a fume hood and uncovered vessel should be used. After soaking in piranha solution, the Au substrate was rinsed with copious amounts of Milli-Q water , dried under N2 gas flow and then flame annealed under a butane flame. The clean Au substrates were immersed for at least 12 h in ethanol (Wako) solutions containing 0.1 mM 11-ferrocenyl-1-undecanethiol (Dojindo Laboratories) followed by rinsing with ethanol and drying under a stream of N2 gas. For the mixed SAM/dilution experiments, an as-prepared SAM from 1 mM 1-decanethiol (Aldrich) in ethanol was subsequently submerged in 0.1 mM 11-ferrocenyl-1-undecanethiol (in ethanol) for at least 30 mins followed by rinsing with ethanol and drying under a stream of N2 gas. The Au(111) roughness factor was found to be good agreement with the literature which was determined to be ~1.3, based on the charge for the reduction of Au oxide (444 μC cm−2)68.The cylindrical hat-shaped Au(111) substrates  with a diameter of 6 mm were cleaned by soaking in piranha solution  cell where electrochemical measurements are performed under Ar atmosphere . The cell utilises a hanging meniscus setup connected via PFA  tubing to a syringe containing the electrolyte. The typical EC-XPS/UPS experiment involves (1) Transfer of Fc SAM on Au sample to the electrochemical (EC) chamber under vacuum (2) Venting the EC chamber with Ar (3) Insertion of cell with electrolyte allowing for electrode immersion and electrochemical characterisations (4) Removal of the immersed electrode from the electrolyte solution under potential control (5) EC chamber evacuation and subsequent transfer to the XPS/UPS analysis chamber. The EC chamber evacuation and transfer occurs in ~10 min. The XPS/UPS  consists of a monochromatic Al Kα X-ray source (hv = 1486.6 eV) with a detection angle of 53° relative to the normal. The base pressure of the XPS/UPS analysis chamber was ~10−7 Pa. XPS was performed at room temperature (298 K) using a pass energy setting of 100 eV. The binding energies are referenced to the Au 4f7/2 peak at 84.0 eV. Spectral fitting was done using Advantage 5.52 (Thermo Fisher) software using a Shirley background subtraction with Lorentzian and Gaussian ratio of 70:3055. Scofield sensitivity factors70 were used for quantification. The UV source uses He (I) excitation (21.2 eV). UPS was performed with pass energy of 2 eV with the spectra referenced to the Fermi edge of Au at 0 eV. A bias of −10 V was applied to resolve the secondary electron cutoff. The CV and chronoamperometry electrochemical measurements  were performed at room temperature (25 °C). The electrolytes consisted of 0.1 M NaTFSI (sodium bis(trifluoromethanesulfonyl)imide, >98%, TCI Chemicals), NaPF6  or NaClO4  that were prepared using DI water. The electrolytes were bubbled with Ar for at least 25 min prior to use. The counter electrode consists of a coiled Pt wire and the reference electrode is an AuOx wire with the data converted to Ag/AgCl (sat. KCl) for comparison.Our setup of X-ray and ultraviolet photoelectron spectroscopy combined with and electrochemical cell (EC-XPS/UPS) is similar in working principle to existing UHV-EC reports that involve electrochemistry in a solution environment followed by transfer to a vacuum environment for analysis Fig. 2,24,25,6Supplementary information


Dysf−/−) mouse hearts have an impaired recovery from acute ischemia/reperfusion (I/R) injury ex vivo, the role of dysferlin in mediating the recovery from myocardial injury in vivo is unknown. Here we show that Dysf−/− mice develop adverse LV remodeling following I/R injury secondary to the collateral damage from sustained myocardial inflammation within the infarct zone. Backcrossing Dysf−/− mice with mice lacking signaling through the Toll-Interleukin 1 Receptor Domain-Containing Adaptor Protein (Tirap−/−), attenuated inflammation and abrogated adverse LV remodeling following I/R injury. Subsequent studies using Poloxamer 188 (P188), a membrane resealing reagent, demonstrated that P188 did not attenuate inflammation nor prevent adverse LV remodeling in Dysf−/− mice following I/R injury. Viewed together these studies reveal a previously unappreciated role for the importance of membrane sealing and the resolution of inflammation following myocardial injury.Cardiac myocytes have multiple cell autonomous mechanisms that facilitate stabilization and repair of damaged sarcolemmal membranes following myocardial injury. Dysferlin is a protein which facilitates membrane repair by promoting membrane resealing. Although prior studies have shown that dysferlin-deficient ( Given the importance of the sarcolemma to cell viability, it is not surprising that cardiac myocytes have multiple cell autonomous mechanisms that facilitate stabilization and repair of damaged sarcolemmal membranes. These include sarcolemma phospholipid rearrangements at the site of very small disruptions/injury in order to promote membrane resealing, as well as several endogenous mechanisms that are activated in order to repair damage to preserve muscle cell integrity and viability  that are present on the cell surface of cardiac myocytes and cardiac resident immune cells5.In myocardial ischemia–reperfusion (I/R) injury the integrity of the cardiac sarcolemma is severely stressed during ischemia and reperfusion, which leads to membrane tears, blebbing and rupture, and directly contributes to cardiac myocyte dysfunction and cardiac myocyte cell death8, that has been implicated in mediating rapid membrane repair through its ability to direct intracellular vesicles to sites of membrane injury9. Prior studies from this and other laboratories have shown that hearts from dysferlin-deficient mice have an impaired ability to recover from acute ischemia/reperfusion (I/R) injury ex vivo10. However, the role of dysferlin in chronic models of myocardial injury, wherein the heart is exposed to the dual mechanical stresses imposed by left ventricular (LV) remodeling and activation of the sympathetic nervous system, as well as the stress imposed by activation of the innate immune response, is not known. Here we show that mice lacking dysferlin (Dysf−/− mice) undergo adverse LV cardiac remodeling following I/R injury secondary to increased myocardial inflammation that is mediated by Toll-Interleukin 1 Receptor (TIR) Domain-Containing Adaptor Protein (TIRAP) signaling in the heart.We have shown previously that tumor necrosis factor (TNF), a pro-inflammatory cytokine released by cardiac myocytes and immune cells in response to ischemic tissue injury, confers cytoprotective responses in the heart that are mediated, at least in part, through the upregulation of an emergency response gene termed dysferlin8. To extend these studies in vivo, wherein the extent of tissue injury can be modulated by the autonomic nervous system and the immune system, we performed closed-chest ischemia reperfusion studies in wild-type (WT) and Dysf−/− mice. Figure Dysf−/− hearts when compared to WT mouse hearts. LV end-diastolic volume (EDV)  was not significantly different (p > 0.999) in WT and Dysf−/− mice, the infarct size by Masson’s trichrome staining was significantly greater (p = 0.003) in Dysf−/− hearts when compared to WT at 2 weeks  was significantly increased (p = 0.02) in the hearts of Dysf−/− mice when compared to WT hearts  Fig. B (p < 0.DV) Fig. C  Domain-Containing Adaptor Protein (TIRAP), which is involved in bridging the myeloid differentiation primary response gene 88 (MyD88) to the receptor complex for TLR2 and TLR4 signaling in response to bacterial infection or tissue injury, in order to generate Dysf−/−/Tirap−/− double knock out mice. The rationale for these studies was two-fold: first, we and others have shown the release of damage associated molecular patterns (DAMPs) by necrotic cells leads to increased cardiac inflammation that is mediated, at least in part, through Toll-like receptor 4 (TLR4) signaling pathways5; second, a prior study in dysferlin null mice showed that there was less inflammation in mice that lacked MyD8811, a canonical adaptor protein that interacts with TIR domain-containing proteins, and which mediates inflammatory signaling pathways through TLR signaling pathways12. The salient finding shown in Fig. Dysf−/−/Tirap−/− mice at 2 or 4 weeks of age. Both LV EDV  and ESV  were not significantly different than the values observed in WT hearts. Moreover, LV EF  and LVM  were not significantly different in the Dysf−/−/Tirap−/− hearts when compared to WT . Consistent with these observations, the extent of EBD uptake was not significantly different (p > 0.999) in Dysf−/−/Tirap−/− and WT hearts  for the adverse LV remodeling in the Dysf−/− mice leads to increased skeletal muscle inflammation13 and demonstrating a role for TLR2 in I/R induced inflammation14, we hypothesized that membrane fragility contributed to the adverse LV remodeling in Dysf−/− hearts secondary to increased inflammation that was mediated through TIRAP-dependent signaling pathways that were activated by the release of DAMPs from damaged and/or dying cells. Figure Dysf−/− and WT hearts 2 weeks after closed-chest I/R injury; Fig. Dysf−/− hearts compared to WT hearts. We also observed a significant (p = 0.01) increase in Ly6G+ cells  and Ly6G+ (p > 0.999) cells within the infarct zone of Dysf−/−/Tirap−/− hearts when compared to WT hearts.Based on prior studies demonstrating that increased membrane fragility in 16, we also performed experiments, wherein we reconstituted the bone marrow of Dysf−/− mice with WT mice donor cells, as well as reciprocal experiments wherein we reconstituted the bone marrow of WT mice with Dysf−/− donor cells, and then subjected these chimeric mice to I/R injury 8 weeks after they were reconstituted. These studies showed that 2 weeks after I/R injury there was no difference in the AAR, LV EDV, LV ESV, LV EF, nor LV mass in Dysf−/− → WT chimeric mice when compared to WT → WT chimeric mice  was cardioprotective in Dysf−/− mouse hearts subjected to I/R injury ex vivo10, we sought to determine whether treatment with P188 would rescue the phenotype of the Dysf−/− mice subjected to I/R injury in vivo. We first confirmed that in our hands P188 (10 µM) improved LV functional recovery (p = 0.007) in Dysf−/− mouse hearts subjected to 30 min of no-flow ischemia, followed by 60 min of reperfusion ex vivo  in WT hearts treated with P188 when compared to diluent treated hearts. As we have reported previously8, there were small but statistically significant differences in LV developed pressure between WT  and Dysf−/−  mouse hearts following I/R injury (p = 0.035 by ANOVA).The findings that the increase in myocardial inflammation and adverse LV remodeling observed in Dysf−/− mice ex vivo, we treated WT and Dysf−/− mice with P188 in vivo immediately after closed chest I/R injury. For these studies P188 (or diluent) was administered via the jugular vein immediately after reperfusion, followed by an i.p. injection of diluent or P188 6 h later. Subsequently, the mice were given daily i.p injections of diluent or P188 up until the time of terminal sacrifice at 14 days. Figure Dysf−/− (p > 0.999) hearts treated with either diluent or P188. Consistent with our earlier studies . Infarct size was not significantly different in the P188 treated WT mice (p > 0.999) or the P188 treated Dysf−/−mice (p > 0.999), relative to diluent treated WT and Dysf−/− mice.After confirming that P188 was cardioprotective in Dysf−/− mouse hearts 2 weeks after I/R injury. Figure Dysf−/− and WT hearts, whereas Fig. Dysf−/− hearts when compared to diluent and P188 treated WT mouse hearts. Figure Dysf−/− and WT hearts 2 weeks after closed-chest I/R injury, and Fig. Dysf−/− hearts when compared to diluent treated WT mouse hearts. There was also a significant increase (p = 0.022) in the number of Ly6G+ cells in the P188 treated Dysf−/− hearts when compared to P188 treated WT mouse hearts. Although there was a numerical increase in the number of CD68+ and Ly6G+ cells in the P188 treated WT hearts when compared to diluent treated hearts, these changes were not statistically significant . There was no significant difference in the number of CD68+ (p = 0.365) and Ly6G+ (p > 0.999) cells in the diluent and P188 treated Dysf−/− hearts.To determine whether P188 had an effect on I/R-induced inflammation, we quantified the number of CD68+ and Ly6G+ cells in diluent and P188 treated WT and 19, we asked whether P188 provoked increased IL-1β secretion and increased phagocytosis in ipMACs. Figure As a first step towards understanding why P188 was cytoprotective ex vivo, but had no effect in vivo, we performed studies with P188 and cultured intraperitoneal macrophages (ipMACs). Based on a prior study which showed that P188 activates immune cellsDysf−/− mice, when compared to WT controls  that engage TLR2/TLR4 receptors on cardiac myocytes and resident immune cells within the infarct zone, leading to sustained inflammation. Similar findings have been observed in skeletal muscle of Dysf−/− mice20. Third, given that increased membrane permeability has been proposed as one of the mechanisms for increased tissue injury and inflammation in the Dysf−/− mice, we treated the Dysf−/− mice with P188, a non-ionic tri-block copolymer that has been shown to seal membranes, at the time of reperfusion. As shown in Figs. 10, treatment with P188 did not decrease myocardial inflammation, nor attenuate adverse cardiac remodeling following I/R injury in vivo.The observation that the increased inflammation within the infarct zone of und Fig. , suggest23. Dysferlin is a type II transmembrane protein belonging to the ferlin family of proteins which have been shown to play an important role in membrane repair by facilitating Ca2+-mediated trafficking of vesicles to the site of membrane injury24. While the absence of dysferlin is known to lead to skeletal muscle damage in limb girdle muscular dystrophy type 2B and Miyoshi myopathy, the effects of dysferlin in the heart are less well known. Two prior studies have shown that Dysf−/− mice have impaired ability to recover LV function following I/R injury ex vivo25. A prior in vivo study by Han et al.17 demonstrated that infarct size and LV ejection fraction were not significantly different in WT and Dysf−/− mice following acute coronary artery ligation. Our results significantly expand upon these acute studies by demonstrating that the longer term sequelae of ischemic myocardial injury in Dysf−/− mice results in significant infarct expansion secondary to TIRAP-dependent increased inflammation within the infarct zone. Our findings in the heart are consistent with prior studies that showed that skeletal muscle performance was improved in dysferlin-deficient mice when crossed with Myd88-deficient mice (TIRAP interacts directly with MyD88)20. Uaesoontrachoon and colleagues hypothesized that breaches in the skeletal muscle membrane in dysferlin-deficient mice resulted in the release of endogenous danger signals that bind to cognate TLR ligands on muscle and immune cells, which in turn activate downstream processes that lead to the recruitment of inflammatory cells that worsen the initial damage caused by the dysferlin gene defect11.Sarcolemmal integrity is preserved through several cardiac myocyte cell autonomous mechanisms that either stabilize the membrane through phospholipid rearrangements at the site of tissue injury, or directly promote membrane resealing. Thus far, studies have shown that a number of different molecules play an integral role in maintaining membrane integrity including dysferlin, the dystrophin glycoprotein complex (DGC), Mitsugumin 53 (TRIM72), thrombospondin 4, synaptogamins, SNARE proteins, the endosomal sorting complex required for transport and GRAF128. P188 is composed of a central hydrophobic chain of polyoxypropylene flanked by two hydrophilic chains of polyoxyethylene, and is currently approved by the Food and Drug Administration as an anti-viscosity agent. The most well documented characteristic of P188 is its ability to repair damaged cell membranes by mechanisms that are not entirely clear. It is believed that P188 inserts into areas of low membrane tension (perhaps areas of membrane damage) due to its amphiphilic and hydrophobic properties29. P188 has been shown to reduce infarct size following I/R injury in dogs and pigs when administered acutely32. Moreover, a 30-min intravenous infusion of high dose P188 (460 mg/kg) was shown to acutely (~ 40 h) improve LV ejection fraction and decrease LV end-systolic volume when administered to rats 8 weeks after acute ligation of the left anterior descending artery27. Although we were able to confirm the findings of Martindale et al., which showed that administration of P188 improved recovery of LV function in Dysf−/− subjected to I/R injury ex vivo 10, chronic treatment with P188 did not decrease infarct size, nor the extent of adverse LV remodeling following I/R injury in the Dysf−/− mice. Although the reasons for the discrepancy between the present findings and the prior short term studies are not known, one possibility is that the dose of P188 that we employed was suboptimal for the experimental conditions studied herein. This statement notwithstanding, the dose and dosing strategy used in the present study were based on recommendation by the manufacturer, prior in vivo studies with P18831, as well as a pharmacokinetic study by the manufacturer that demonstrated the feasibility of the dose that was chosen. A second interesting possibility suggested by our in vitro studies is that P188 was less bioavailable within the infarct zone because of phagocytic uptake by macrophages33. While direct correlations between relatively short-term experimental studies in mice and longer-term clinical studies in humans are not appropriate, our data are consistent with the results of the CORE  phase II clinical trial, which did not show a benefit of P188 on the primary end point of mortality, reinfarction or cardiogenic shock in patients with ST-segment myocardial infarction undergoing thrombolytic therapy34.Several studies have used chemical-based membrane stabilizers, such as P188, to protect striated muscle membranesDysf−/− mice at a time when inflammation was completely resolved within the infarct zone of WT mice suggests that the inability to reseal membranes in dysferlin-deficient injured cardiac myocytes results in the sustained release of intracellular molecules (e.g. DAMPs) that engage TLR2/TLR4 receptors on cardiac myocytes and immune cells within the infarct zone, leading to the activation of TIRAP-dependent signaling cascades that foster sustained recruitment of inflammatory cells to the infarct zone, resulting in increased cell death, increased infarct size and adverse LV remodeling. While our studies focused on the role of TIRAP dependent TLR2/4 mediated signaling, we cannot exclude an important role for endosomal TLRs or DNA-sensing receptor cyclic GMP–AMP synthase (cGAS) and signaling effector stimulator of interferon genes (STING) in terms of mediating sustained inflammation following I/R injury in Dysf−/− mouse hearts35. Given the increasing recognition that cytosolic proteins (e.g. troponin) are released from cardiac myocytes in non-ischemic settings36, the results of the present studies also serve the heuristic purpose of focusing future studies on the role of membrane integrity in the development of the chronic inflammation that occurs in the setting of hemodynamic pressure and volume overload39, as well as on the need to better understand the molecular pathways that regulate membrane integrity in health and disease.This study reveals a previously unappreciated role for the importance of membrane sealing and the resolution of inflammation following myocardial injury. The observation that inflammation was sustained within the infarct zone of the hearts of Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health.All studies were performed with the approval of the Institutional Animal Care and Use Committee at Washington University School of Medicine. These investigations conform to the Dysftm1Kcam/J[Dysf−/−], Jackson Labs) were maintained on a C57BL/6 background18. The Tirap−/− mice were a gift from Dr. Ruslan Medzhitov 40. Male Dysf−/− mice were crossed with Tirap−/− mice to generate Dysf−/−/Tirap−/− double knockout mice. The Dysf−/−/Tirap−/− mice were born with the expected Mendelian frequency and had normal life spans. All mice were maintained in a pathogen‐free environment and were fed pellet food and water ad libitum.The dysferlin null mice  injury, using a closed-chest ischemia reperfusion model, as described42. We used 10–12 week old male mice, anesthetized with 1.5% isoflurane, that were subjected to 60 min of closed-chest ischemia, followed by reperfusion for 2–4 weeks. Sham-operated animals underwent the identical procedure with the exception that ischemia was not induced. For experiments using Poloxomer 188 , mice were given 460 mg/kg P188 via jugular vein infusion immediately following reperfusion. Mice were then given a bolus of 2,000 mg/kg P188 i.p. 6 h later. Daily i.p. injections of 460 mg/kg P188 were then given for 14 days at which time echoes were performed and hearts were harvested for histology. The dose was determined based on the literature26, and the dosing strategy was based on prior studies by MAST Therapeutics.WT, Dysf−/− mice were isolated and perfused at a constant pressure of 70 mmHg with modified Krebs–Henseleit buffer as described previously43. After a 20-min stabilization period, hearts were subjected to no-flow ischemia (t = 0 min) for 30 min followed by reperfusion (t = 30 min) for up to 60 min (t = 90 min). For experiments with P188, Krebbs Henseleit buffer was prepared with 10 µM P18810. KH buffer containing P188 was perfused during baseline and during reperfusion. Functional data were recorded at 1 kHz on a data acquisition system . LV developed pressure (LVDP) was calculated as the difference between peak systolic pressure and LVEDP, and the resulting LV functional recovery data are expressed as the percentage of LVDP at baseline.Hearts from 10 to 12 week old male WT and 42.Ultrasound examination of the cardiovascular system was performed using a Vevo 2100 Ultrasound System  equipped with a 30 MHz linear-array transducer, as previously described43. Noninvasive evaluation of the AAR was performed by assessing the segmental wall motion score index (SWMSI) at the time of reperfusion, as described43. Isoflurane  was used during ischemia and Avertin (0.005 ml/g) was used for sedation for imaging at 2 and 4 weeks based on previously established methods for infarct quantitation in vivo42.Mice were imaged by echocardiography at baseline and simultaneously during the imposition of closed-chest ischemia, and then at 2 weeks and 4 weeks after I/R injury to evaluate parameters of LV remodeling as described43.1% Evans Blue dye  in PBS was injected i.p. 18 h prior to harvesting hearts. Hearts were harvested, perfused with 10 ml PBS followed by 10 ml Z-fix and allowed to sit in Z-fix overnight before being transferred to 70% ethanol prior to paraffin embedding44. To estimate the volume fraction (%) of collagen, mid-papillary sections of Masson’s trichrome-stained sections were quantified using color thresholding by ImageJ software , and data were expressed as the average percentage of the left ventricle stained (n = 5–6 per group). To evaluate myocyte size, sections were deparaffinized, rehydrated, and stained with fluorescent rhodamine-conjugated wheat germ agglutinin at 5ug/ml in 1% BSA, 1X TBS . Fluorescence was visualized using a Zeiss confocal microscope, and digital images were analyzed and measured with Zeiss Axiovision software45.Two weeks after I/R injury mice were euthanized and the hearts were removed and processed, paraffin-embedded, and stained with hematoxylin/eosin and Masson’s trichromeWe enumerated the CD68+ and Ly6G+ cells in the heart via 10X tile scanning of the entire heart at the mid-papillary level. Hearts were fixed in 4% PFA overnight and then transferred to 30% sucrose for 24 h prior to embedding in OCT and flash freezing. Prior to immunostaining, frozen sections were washed briefly in PBS and then blocked in 3% BSA/5% horse serum. Sections were then incubated with antibodies to CD68  or Ly6G  followed by Alexa 555 conjugated secondary antibody incubation . Fluorescence was visualized using a Zeiss confocal microscope. Images (~ 70–130 per section) were stitched together using the Axiovision software to form whole mid-papillary section images , necrotic myocardial cell extracts 5, and lipopolysaccharide  for 24 h. IL-1β was measured in stimulated cell culture supernatants using an IL-1β ELISA kit (R&D kit Cat. #MLB00C) according to the manufacturer’s instructions. The phagocytic capacity of stimulated cultures of ipMACs was assessed by measuring phagocytosis of 0.5um Fluoresbrite yellow-green fluorescent polystyrene microspheres . For these experiments ipMACs were plated on coverslips and incubated with microspheres for 2 h, at a ratio of 200 microspheres per cell, and then cultures were fixed with 4% paraformaldehyde. Cells were then incubated with anti CD68 antibody , followed by incubation with goat anti-rat 594 secondary antibody  and mounted with DAPI containing Vectashield mounting media . Fluorescence was imaged via confocal at 20× and quantitated. CD68 positive cells that had internalized more than 5 microspheres were considered positive for phagocytosis. In separate experiments, macrophages were incubated with FITC-labeled P188  for 18 h and the ipMACs imaged using fluoresence confocal microscopy.Intraperitoneal macrophages (ipMACs) were isolated as previously describedp < 0.05 was considered statistically significant43.Data are expressed as means ± SEM. One-way ANOVA with Bonferroni post-hoc analysis (Graphpad Prism) was used to test for differences in EBD uptake, Trichrome staining, cell area, CD68 and Ly6G quantification. For repeated measures analysis, (2-D echocardiography and Langendorff perfusion data), mixed models methodology or 2- way repeated measures ANOVA were used. For the mixed model, group, time and the interaction between group and time were all entered into the model. Time was treated as a categorical variable. The interaction was evaluated to determine if the relationship between group and echo parameter varied with respect to time. Based on the Akaike Information Criteria (AIC) and Shwarz Information Critieria (BIC), an unstructured variance–covariance matrix was used to model the repeated measurements. Group means were based on model results and differences between groups were determined overall and within each time point. All pair-wise comparisons were considered. Test results were adjusted based on Sidak’s method to control the overall type I error rate. A value of Supplementary Information 1.


Rituximab is safe and effective for treating relapsing–remitting multiple sclerosis (RRMS) according to phase II and observational studies. There are limited data on disease activity after discontinuation and dose reduction. The objective of this study was to evaluate the effects on inflammatory disease activity after discontinuation or dose reduction of rituximab in patients with RRMS or clinically isolated syndrome (CIS).In this retrospective observational study, we included all RRMS and CIS patients ever treated with rituximab at the University Hospital of Umeå who had either; (1) discontinued treatment at any time or (2) reduced the dose to a mean of < 1000 mg yearly. The patients served as their own controls by contributing patient years on full dose, reduced dose, and off treatment.p = 0.09), neither regarding proportion MRI scans with new or enlarged T2 lesions  or contrast-enhancing lesions .A total of 225 patients treated with mean (SD) 6256 (2456) mg rituximab during mean (SD) 6.5 (2.0) years were included. There were no differences regarding the annualized relapse rates during full dose versus reduced dose or off treatment  is sufficient to maintain suppression of inflammatory disease activity in patients with stable disease. In relapsing–remitting multiple sclerosis (RRMS), inflammatory disease activity decreases with higher age and longer disease duration –3. The chttp://www.neuroreg.se) at the University Hospital of Umeå. The registry data extraction was performed 7 May 2020, selecting all RRMS or CIS patients ever treated with rituximab at the University Hospital of Umeå. Patients were included if they fulfilled any of the following inclusion criteria; either (a) had discontinued treatment with rituximab at any time (i.e. ≥ 18 months since last infusion) or (b) reduced the treatment dose to a mean of < 1000 mg of rituximab yearly. Exclusion criteria were: (a) patients who was diagnosed with secondary progressive MS (SPMS) within 12 months from starting rituximab treatment, (b) patients who had only received one dose of rituximab, (c) patients who had received a total of < 1000 mg of rituximab, (d) incomplete medical records, (e) no MRI examinations during the observation period or (f) disapproval to participate in research.Figure The study was approved by the local ethics committee in Umeå (2013/445-31) and all patients had provided oral consent to participate in the MS registry.Data were extracted from the Swedish MS registry and patients’ medical records and included age, sex, diagnosis and disease course, date of disease onset, Expanded Disability Status Scale (EDSS) scores, dates and doses of rituximab infusions, dates of MRI examinations, numbers of T2 lesions and CEL(s) on MRI and dates of relapses. The baseline EDSS score and baseline MRI were defined as the examination closest in time within 12 months prior to starting rituximab. For patients lacking an EDSS score or an MRI within this timeframe, the baseline value was obtained from within 3 months after starting rituximab, if available. Date of data censure was 7 May 2020. If a patient had received any other disease-modifying therapy (DMT) during the observation period, the time after administration of that drug was excluded from further analyses. If a patient had converted to SPMS, the year of conversion was noted in the registry. If this had occurred during the observation period, all time from 1 January that year was excluded from further analyses. All patients were followed from 3 months after the first rituximab infusion until the date of data censure or lost to follow-up , whichever came first.Inflammatory disease activity was defined as either relapse, new or enlarged T2 lesions and/or CEL(s) on MRI. Concerning CEL(s), a valid scan was defined as an MRI performed with gadolinium contrast administration, and a positive scan was defined as such an MRI with CEL(s). A clinical relapse was defined as an episode of neurological impairment with a duration of > 24 h without another apparent cause. The patients served as their own controls by contributing with patient years on both full dose, low dose, super-low dose and/or off treatment during the observation period  was compared using the Fisher’s exact test in Win-pepi software version 11.65.Statistical analyses were performed using IBM SPSS Statistics for Windows, Version 26.0 . Normally distributed data were presented as mean (SD) and non-normally distributed data were presented as median (IQR). The independent samples n = 11); one patient had 1 new T2 lesion on full-dose treatment and one patient had 1 new T2 lesion after discontinuation. The remaining nine patients had no disease activity on any dose regimen before lost to follow-up. The baseline EDSS for the study cohort was assessed at a median of 14 (2–43) days prior to the first rituximab infusion and the baseline MRI was performed at a median of 26 (7–71) days prior to rituximab start, excluding those with MRI and EDSS assessments performed after rituximab initiation.Table The distribution of outcomes by treatment period is seen in Table n = 15) with those who did not (n = 210) after dose reduction or discontinuation of rituximab (Table p = 0.01) compared with the group with no disease activity. The latest available EDSS score during the observation period was assessed at a mean of 8.1 (1.9) and 6.0 (2.0) years from baseline for patients with disease activity and patients with no disease activity, respectively. Minimal changes in EDSS (− 0.5 in both groups) were observed during the observation time.We compared the group of patients who regained disease activity (ab Table . More (3n = 1), stable disease (n = 1), dosing interval of 24 months (n = 1) and following a clinical study protocol [n = 4). Among the 15 patients showing signs of disease activity 11 had new or enlarged T2 lesion(s) on MRI (1–2 lesions); two of these patients also had one CEL  and fouIn this retrospective observational study on patients with RRMS or CIS, we evaluated the effects on inflammatory disease activity after discontinuation and dose reduction (to a mean of < 1000 mg yearly) of rituximab treatment. The results indicate that rituximab has long-term effects on inflammatory disease activity and that disease reactivation is rare in previously rituximab treated RRMS and CIS patients who discontinued treatment. Our study also implies that a low-dose maintenance treatment protocol may be sufficient for RRMS and CIS patients with stable disease.Patients who had transitioned to SPMS within 12 months from starting rituximab were excluded from this study as clinicians tend to be cautious and frequently diagnose SPMS retrospectively after a period of diagnostic uncertainty, resulting in a delay of SPMS diagnosis by up to 3 years .A high proportion of patients had a heavy T2 lesion burden and CEL(s) at baseline which suggests that our study population was inflammatory active at inclusion. Our results confirm a low ARR and a low risk for new lesions on MRI during treatment with rituximab, in line with previous studies –11, 13. Pregnancy is one of the most common reasons for discontinuing rituximab , 15 and In the natural history of MS, the inflammatory activity is at its highest early in the disease and generally decreases with age and disease duration , 3. YounA few patients had a very short observation time on low- and super-low dose which results in a misleading dispersion of the yearly rituximab dose Table . The freThis study indicates that rituximab has long-term effects on inflammatory disease activity and that disease reactivation is rare in previously rituximab treated RRMS and CIS patients who discontinued treatment for any reason. It also suggests that treatment with a low-dose protocol of rituximab (< 1000 mg yearly) is sufficient to maintain suppression of inflammatory disease activity in most RRMS and CIS patients with stable disease. These findings could be useful to aid treatment decisions for RRMS patients during the COVID-19 pandemic during which the risk/benefit ratio of immunosuppressive treatment for MS may shift.


The purpose of data collection was to generate isoscapes of oxygen and hydrogen isotopes for South Korea. To achieve the objective, we collected human scalp hair and three different types of water samples: groundwater, stream water and tap water. The data presented in the article are raw isotope data of water and hair samples in tabulated manner and interpolated isoscapes generated using those data. Further information related to the datasets and discussion about them can be found in the related research article entitled “Spatial variations in oxygen and hydrogen isotopes in waters and human hair across South Korea” [1].The data presented here include the results of oxygen (δ Specifications table•The data will contribute to better understanding the geospatial variation in isotopes of water and hair on national scale and are an addition to the global isotope database•Data are useful to other researchers who are interested in using water isotope for generating better global isoscapes and for those who use isotopes of drinking water and hair for provenance discrimination and in forensic analyses.•The data will contribute to better understanding the application of oxygen and hydrogen isotopes as geographic tracer for agricultural products.•The data can be used by other researchers who use isotope signatures of drinking water and human hairs for provenance discrimination and forensic analyses.118O), hydrogen (δ2H) and d-excess values (d = δ2H – (8 × δ18O) 18O, δ2H and d-excess data for groundwater, stream water, tap water and human scalp hair samples collected from different sites in South Korea. To understand the isotopic distribution on national scale, interpolated maps or isoscapes were developed, and spatial distribution of δ18O, δ2H and d-excess values were presented in Gautam et al. (in press) The data presented include oxygen  and hair samples were located across the country, encompassing all the eight administrative provinces of South Korea .2.2In September–October 2010, 56 groundwater, 130 stream water and 91 tap water samples were collected across South Korea, and their GPS coordinates and other metadata were recorded. Sampling emphasized obtaining nation-wide high-resolution datasets for the construction of reliable interpolated isoscapes. For successful sampling that incorporated the maximum variability, the campaign was carried out in urban, suburban and rural areas. Groundwater samples were obtained from local houses in person (offered by residents) and from automated bore wells. Prior to sample collection, water was allowed to flow out for 10 to 15 min to remove factors that might cause fractionation  and to obtain well-mixed groundwater samples. When possible, we collected tap water  samples from the same locations where groundwater sampling was conducted. Samples were collected only from in-use cold water taps, and the tap was run for one min prior to sample collection. Where feasible, the surface water and stream water sampling locations were chosen based on proximity to groundwater and tap water sampling locations. To remove the possibility of evaporative enrichment, samples were collected only from the flowing sections of streams. All water samples were filtered after collection through a 0.45-μm PVDP Millipore® syringe filter  into 60-mL glass vials. Vials were filled to the neck and closed with rubber-lined caps to avoid exchange with the atmosphere. The vials were immediately stored in portable iceboxes with re-usable ice packs (to reduce photodegradation) after collection and transported to the laboratory. In the laboratory, the vials were stored at 4°C until isotopic analyses.2.3Human scalp hairs were sampled from different locations (barbershops) across South Korea (n = 100). Hair samples discarded at the barbershops were collected. The length was not taken into consideration, but it was usually 4-6 cm long and there is a weak possibility that a hair will mix with various lengths of hair. Only undyed hair samples were collected for the study. To ensure that the isotopic signature of hair samples reflected the geographic location of sampling regions, hair samples were obtained from local residents, when possible, who lived in close proximity to the barbershop. Care was taken to select barbershops near the water sampling sites. With this approach, we can assume that the individuals whose hair was collected had access to local water sources. In addition, three sets of discarded hair clippings were collected in the suburban towns to increase the probability that the hair samples were associated with local residents.2.42 gas in a glove bag overnight at 25±0.1°C 2 gas was subsequently extracted and cryogenically purified. For hydrogen isotope analyses, metallic zinc was used to produce hydrogen gas using an automatic online sample preparation system .The oxygen and hydrogen isotope ratios of water and hair samples were measured using Optima isotope ratio mass spectrometer  and dual inlet Isoprime isotope ratio mass spectrometer equipped with an on-line Euro PyrOH preparation system  at the Korea Basic Science Institute (KBSI). To determine oxygen isotopic composition, 0.25 mL water sample was transferred to a 5-mL Labco Exetainer® vial  and equilibrated with COTo remove lipids and other contaminants, scalp hair samples were cleaned several times with acetone  and ultrapure water  according to the treatment procedure recommended by the International Atomic Energy Agency sample/Rstandard) – 1] × 1000, and R = 2H/1H or 18O/16O. The analytical reproducibilities for δ18O and for δ2H were ±0.1‰ and ±1‰ for water samples, respectively, and 0.25‰ and below 2‰ for hair samples, respectively. Deuterium excess values were calculated following Dansgaard d = δ2H – (8 × δ18O).All the isotopic ratios are reported in delta (δ) notation relative to Vienna Standard Mean Ocean Water (VSMOW), where δ (‰) = [. The inverse distance weighted (IDW) interpolation method was used to analyze spatial patterns because IDW is intuitive and efficient . For IDW, we used the default in the ArcGIS that included the inverse of the distance raised to the 2The authors declare that they have no conflict of interest.


Retrospectively reported TV viewing for ≥ 3 h/day at 5–10 years of age was associated with a 34% greater risk of adult T2D . Additional adjustments for adult variables, including adult TV viewing and current BMI attenuated the effect estimates . The present study suggests that TV viewing during early childhood increases risk of T2D in adult women; adult BMI explains part of this association. Further research is required to confirm this observation and understand the mediating pathways.We examined whether regular television (TV) viewing at ages 3–5 and 5–10 years is related to the incidence of type 2 diabetes mellitus (T2D) in adult women. We used data from 34,512 mother-nurse daughter dyads in the Nurses’ Health Study (NHS) II and the Nurses’ Mothers’ Cohort Study. Mothers of NHS II participants completed a questionnaire on their pregnancy with the nurse and her early life experience. During 391,442 person-years of follow-up from 2001 to 2013, 1515 nurses developed T2D. Increasing levels of TV viewing at 3–5 years of age retrospectively reported by the mothers were related to a greater risk of T2D in adulthood: multivariable-adjusted hazard ratios (HRs) for ≤ 1, 2, and ≥ 3 h/day vs. no TV viewing were 1.11 [95% confidence interval (CI) 0.96–1.28], 1.20 (95% CI 1.02–1.41), and 1.35 (95% CI 1.11–1.65),  The proliferation in type 2 diabetes mellitus (T2D) is likely largely due to increases in overweight and obesity, and a lack of physical activity2. Sedentary behavior is recognized as a risk factor for major chronic diseases with the strongest and most persistent associations seen for T2D in adults4. Of particular concern is television (TV) viewing, one of the most prevalent sedentary behaviors among children, given its association with childhood obesity in both cross-sectional and longitudinal studies6. Children spend substantial time watching TV with national data indicating that 3- to 7-year-old children in the United States spend on average 2–3 h per day watching TV, with video and computer games adding an additional hour to children’s screen time7.With 422 million people affected by diabetes worldwide, the past four decades have witnessed the quadrupling of diabetes incidence8, no previous study has considered the role of TV viewing during childhood on the risk of T2D later in life. The perinatal and early life period is of particular importance since it is the phase of life marked by early adaptation in metabolic regulation and therefore represents a vulnerable time window for the development of T2D and heart disease in adult life10. It is also a time when children develop behaviors that may persist into later life.Although the association between TV viewing and the metabolic syndrome has been investigated in adolescence beforeTo our knowledge, our study is the first to examine whether TV viewing at ages 3–5 years or 5–10 years is related to T2D in adulthood among women. We further estimated the magnitude of the associations explained by childhood body fat and body mass index (BMI) and TV viewing in adulthood using mediation models.11. Follow-up questionnaires were mailed to study members every two years updating information on lifestyle factors and health. In March 2001, 35,830 mothers of NHS II participants who were free of cancer participated in the Nurses’ Mothers Cohort Study and answered mailed questionnaires about their pregnancy and early live exposures of their daughters. Further details of the Nurses’ Mothers’ Cohort Study have been provided elsewhere12. We excluded NHS II participants who were adopted or whose adoption status is unknown, those with a diagnosis of type 2 before 2001, type 1 diabetes (T1D), and those with missing data on the exposures leaving 34,512 (TV viewing at age 3–5 years) and 34,337 (TV viewing at age 5–10 years) mother-nurse daughter dyads for the final analysis  and demonstrated fair test–retest reliability (r = 0.30) when the same question was completed one week apart15 that best depicted their body fatness with levels ranging from 1 (represents the most lean) and 9 (represents the most overweight). Somatotypes were combined into five categories. The validity of this measure of body shape in early life have been assessed among 181 participants aged 71–76 years in the Third Harvard Growth Study16. Information on current age of the nurses, race, family history of diabetes, was collected from the NHS II baseline questionnaire. We used biennially updated information about caloric intake, alcohol intake, smoking, physical activity, TV viewing, and BMI during adulthood from the NHS II questionnaires.Mothers of the nurses provided information on maternal, perinatal and early life risk factors of their daughters including: maternal education, pre-pregnancy weight, height, weight gain during pregnancy, smoking during pregnancy, and physical activity during pregnancy; paternal weight and height; nurse daughter’s gestation age at birth, birthweight, and breast feeding. Mothers further provided information about physical activity of their daughters between the ages of 3–5 and 5–10 years by answering the following question at both ages: “How would you best describe your nurse daughter’s activity level compared to other girls of similar age?” Response categories were: “highly physically active,” “active,” “mostly inactive,” and “inactive.” In the NHS II baseline questionnaire, nurses provided information on their body fatness at ages 5 and 10 years by choosing one of nine pictorial body diagrams (somatotypes) developed by Stunkard et al.17: (1) one or more classic symptoms  and fasting plasma glucose concentrations ≥ 7.8 mmol/L or random plasma glucose concentrations ≥ 11.1 mmol/L; (2) two or more elevated plasma glucose concentrations on different occasions  in the absence of symptoms; or (3) treatment with hypoglycemic medication .In each biennial questionnaire, nurses were asked whether they had been diagnosed with T2D in the past 2 years. Respondents reporting a diagnosis were then mailed a supplemental questionnaire about the results of diagnostic tests, symptoms, and therapy. The diagnosis was confirmed if at least one of the following was reported according to the National Diabetes Data Group criteria18.The American Diabetes Association diagnostic criteria for T2D diagnosis were used from 1998 onward using the fasting glucose concentration cutoff of 7.0 mmol/L. In a validation study, about 98% cases were reconfirmed against blinded physician medical record reviewCox proportional hazards regression models were computed conditioned on age and follow-up cycle to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) of T2D across increasing categories of TV viewing at ages 3–5 years or 5–10 years. To allow for a prospective analysis of T2D risk, participants contributed follow-up time from the return of the NHS II questionnaire in 2001 until their last returned questionnaire, the date of death, T2D diagnosis, or end of follow-up on June 1, 2013. Participants who experienced a competing risk event were censored from further analyses. Women were also censored at the date of type 1 diabetes mellitus (T1D).We tested the proportional hazards assumption by plotting a graph on the log negative log survival against log of survival time. The proportional hazards assumption was considered satisfied since largely parallel lines between different groups of exposure were found , caloric intake (quintiles), alcohol intake (quintiles), smoking , physical activity (quintiles), and TV viewing  during adulthood. If covariate values were missing the previous value was either carried forward for one time interval or a missing indicator category was created.Multivariable-adjusted models were adjusted for perinatal variables, adiposity during childhood, and adult variables of the nurses including physical activity, TV viewing,current BMI, caloric intake, alcohol consumption, and smoking. Adult variables were updated biannually from the NHS II follow-up questionnaires and treated as time-varying covariates in the analytic model. The fully adjusted model had maternal education , maternal pre-pregnancy BMI (quintiles), paternal BMI at nurses’ birth (quintiles), maternal weight gain during pregnancy , maternal smoking during pregnancy , maternal physical activity during pregnancy , gestation age , birth weight , breast feeding , body fatness at ages 5 or 10 years (five groups), physical activity at 3–5 years or 5–10 years , race , family history of diabetes , age of the nurses (continuous), BMI . In addition, we evaluated joint associations of TV viewing during childhood and TV viewing during adulthood with adult T2D risk. To test for a linear trend across categories of exposures, we created a continuous variable using the midpoint of each category. All statistical analyses were performed using the SAS statistical software version 9.4 .We calculated the proportionDuring 391,442 person-years of follow-up, 1515 newly diagnosed cases of T2D occurred between 2001 and 2013.The distribution of early life and adult variables of the nurses across categories of TV viewing at ages 3–5 years and 5–10 years is shown in Table p trend < 0.001, Table p trend = 0.001), body fatness at age 5 years , adult life variables , adult TV viewing , and adult BMI ; and the positive association between TV viewing at ages 3–5 years and later T2D risk was attenuated with their inclusion  of the positive association of TV viewing at ages 3–5 years with adult T2D incidence, respectively. Body fatness at age 3–5 years appears not to be a mediator of this relation (proportion of the childhood TV viewing effect explained by childhood body adiposity < 1%).Based on mediation analysis, adult BMI and TV viewing were responsible for 25.3% (p < 0.001) and 12.5% , as well as adult life factors  revealed attenuated but positive associations between TV viewing at ages 5–10 years and later T2D risk. The associations were further attenuated and no longer significant following additional adjustment for adult TV viewing  and BMI  although a trend for a dose–response relation remained evident. Adult BMI and TV viewing were estimated to mediate 36.1% (p < 0.001) and 15.3% (p < 0.001) of this association, respectively, whereas childhood adiposity appears not to mediate this association (p = 0.13).Long duration of TV viewing ≥ 3 h/day) versus no TV viewing at ages 5–10 years was related to a greater risk of T2D in adult life  and during adulthood were related to a greater risk of adult T2D compared to ≤ 1 h/day of childhood TV viewing and adult TV viewing, although these associations were attenuated following additional adjustment for current BMI Table . By compIn this study, TV viewing in early and later childhood as retrospectively reported by mothers of participants was positively associated with risk of T2D later in life. The association was independent of childhood physical activity and childhood adiposity but appears to be partially explained by adult BMI.21.To the best of our knowledge, no previous study examined the relation of TV viewing during childhood to the risk for T2D later in life. In four previous meta-analyses, a positive association between TV viewing during adulthood and diabetes risk had a summary risk that ranged from 1.20 to 2.1922 and higher caloric intake associated with TV viewing23 can lead to a positive energy balance, thereby promoting obesity and obesity-related metabolic disruption , and the risk for diabetes. Other proposed mechanisms relating watching TV to dysmetabolic effects may include impaired sleep quality24, distraction from habitual food intake control or satiety, and a reduced number of family meals consumed25. Health behaviors acquired in childhood may lead to persistent behavioral and biological adaptations contributing to greater vulnerability to chronic diseases in adulthood.Biologic mechanisms to explain these observations are unclear but may involve pathways related to energy expenditure. Spending hours at a time watching TV can displace time spent being physically active8 using data from the Northern Swedish Cohort reported that watching 'several shows a day' compared with 'one show/week' or less at age 16 years was related to the metabolic syndrome at age 43 years following adjustment for TV viewing at ages 21 and 30 years and other potential confounders , but the association was attenuated after adding BMI at age 30 to the model. Although the current literature suggests a link between sedentary behavior and adiposity among children26, the role of BMI in the relation between sedentary behavior and disease outcomes is not clear yet. Our results indicate a relation of TV viewing in early childhood with T2D in adulthood that is independent of adiposity during childhood; however, additional control for adult variables, in particular adult BMI attenuated this association. Adjusting for adult BMI, however, may not provide the most informative analytic model since adult BMI is an intermediate variable. More research is required to confirm these observations and clarify the mediating pathways.previous longitudinal studyAdult metabolic diseases may originate in childhood, but studying such associations in a longitudinal study is challenging since several decades of follow-up would be necessary. Assessing TV watching in childhood through mothers’ reports makes an unbiased study feasible: while the frequency of TV watching during childhood was retrospectively assessed, prevalent cases of T2D among the daughters were excluded at baseline. Hence, at the time of reporting of their daughters’ childhood behavior mother had no knowledge whether their daughter might develop T2D later excluding the chance of recall bias; any error in the retrospective reporting of TV watching by the mothers would be random in nature. Only incidence cases of T2D diagnosed during the subsequent 12 years were included in our analyses. Therefore, we were able to embed the retrospective assessment of the exposure in a prospective follow-up.27. While the long recall period for the exposures by maternal proxy reports is a challenge, differential misclassification was avoided by our prospective design which included only incident cases occurring after 2001. Further, parents may be the only reliable source of TV watching habits of their children and since TVs were still relatively new during the exposure period examined the reports are likely to have a good validity. Another limitation is that our study population is > 95% Caucasian white. Black and Hispanic children have been reported to watch significantly more TV, have higher rates of overweight and obesity, higher consumption of sugar-sweetened beverages, but lower rates of maternal smoking during pregnancy than non-HispanicWhite, or Asian children30, limiting the generalizability of our findings.Our study has both strengths and limitations. One strength of our study includes its two-generational design, which provides the unique opportunity to examine data collected directly from the mothers and combine them with data independently provided by the nurses. Further strengths of our study are its prospective nature, the large number of study participants, and adjustment for numerous potential confounding variables in early and adult life. Despite our study pointing towards a positive association between TV viewing during childhood and adult T2D, our findings need to be interpreted in the context of certain limitations. Although, we carefully controlled for numerous potential confounding variables in our models, unmeasured confounding or residual confounding may have occurred. Moreover, exposure assessment is based on maternal recall decades later and lack of memory may have led to some degree of non-differential misclassification in our study. The Nurses’ Mothers’ Cohort questionnaire has not directly been validated for the exposures examined herein, but the questions are similar to assessments of TV viewing during adulthood in the NHS II questionnaire reported to have reasonable validity31. Current screen time is a lot more heterogeneous and is not limited to TV viewing only. Thus, our associations in all likelihood may underestimate the influence of extended screen time patterns in current adolescents on their future metabolic health suggesting an even more pressing need to establish intervention programs.The proportion of childhood TV viewing does not represent the longer screen time among children today, which is typical for prospective cohort studies. By 1950, nine percent of American households had a television set, a proportion that had increased to 64.5 percent only five years later and reached 93% in 195532. More research is needed to confirm our findings and to build the evidence base for precise sedentary behavior guidelines beyond broad recommendations to reduce sedentary behavior.Our study identified TV viewing during childhood as a potential risk factor for T2D later in life. This observation supports recommendations to limit TV viewing during childhood to one hour per day as advocated by the American Academy of PediatricsOur study is the first investigation on childhood TV viewing and risk of developing T2D later in life suggesting that long hours spent watching TV during childhood may increase risk for T2D in adulthood. Given that frequent TV viewing at an early age may predict long-term behavior pattern and physiologic adaptation during life, promoting reduction in screen time and improvement of physical activity beginning in early age may be crucial to reduce the long-term risk for diabetes. Future studies are needed to continue this line of evidence.Supplementary Information