TRPM4) of these analyzed SNPs reached nominal significance regarding an association with SIDS in the overall analysis . In the stratified analysis, further 8 variants in ABCC8 (encoding SUR1) or TRPM4 showed pronounced associations. However, none of the results remained significant after correction for multiple testing. This preliminary study has provided the first evidence for a genetic role of the SUR1-TRPM4 complex in the etiology of SIDS, and we suggest that our initial results should be evaluated by further studies.Increasing evidence suggests that brain edema might play an important role in the pathogenesis of sudden infant death syndrome (SIDS) and that variants of genes for cerebral water channels might be associated with SIDS. The role of the sulfonylurea receptor 1 (SUR1)–transient receptor potential melastatin 4 (TRPM4) non-selective cation channel in cerebral edema was demonstrated by extensive studies. Therefore, we hypothesized that variants at genes of the SUR1-TRPM4 channel complex might be linked to SIDS. Twenty-four polymorphisms in candidate genes involved in the SUR1-TRPM4 non-selective cation channel were investigated in 185 SIDS cases and 339 controls. One (rs11667393 in The online version contains supplementary material available at 10.1007/s00414-022-02819-9. Sudden infant death syndrome (SIDS), the leading cause of death in infants aged 1 month to 1 year in developed countries, is the sudden death of an infant under 1 year of age that remains unexplained after a thorough case investigation, including performing a complete autopsy, examining the death scene, and reviewing the clinical history . HoweverAQP4 variant genotypes , an increased brain-to-body weight ratio was found, and thus a putative association of AQP4 variants and cerebral edema in SIDS [Previous relevant studies have shown increased brain weight and/or cerebral edema in some cases of SIDS –8, altho in SIDS . In a re in SIDS .+ channel could assemble with AQP4 to form a complex (water-ion transport coupling) that plays an important role in cerebral edema [2+ concentration increases [ABCC8 gene) is a member of the ATP-binding cassette transporter family, with its activity modulated by the intracellular ATP/ADP ratio [2+-regulated non-selective ion channel [ABCC8 and TRPM4) [In addition to constitutively expressed transporters like AQP4, Kir4.1 as an ATP-sensitive Kal edema . Opdal eal edema . Like Kincreases –22. It hncreases . The SURDP ratio ; transie channel . Preclin channel , 26. Addd TRPM4) –29.ABCC8 and TRPM4 genes may lead to an increased risk to develop cerebral edema and thus infants susceptible to SIDS and investigated 185 SIDS cases and 339 controls for 24 polymorphisms in candidate genes involved in the SUR1-TRPM4 non-selective cation channel relevant to the development of cerebral edema.Therefore, we hypothesized that variants located in the n = 185) was enrolled from the Institute of Legal Medicine, Hannover Medical School, and its branch (Oldenburg). The enrollment standards based on the San Diego definition of SIDS [n = 46), 73.9% of cases were prone positions. The control cohort (n = 339) comprised 164 males, 146 females, and 29 gender-unrecorded individuals. It comprised 33 children and 306 healthy adults. The 33 included children in controls were in the first year of life but died due to an explicit cause of death but not SIDS . Owing to anonymization exact age information was not available in controls. The local ethics committee of Hannover Medical School approved this study.The SIDS cohort n = 85 was enABCC8 and TRPM4 was on account of previously published studies in which a potential association with a specific disease or a possible influence on gene expression was reported. In brief, the selection criteria were as follows: (1) the minor allele frequency (MAF) ≥ 0.05 in Caucasians; (2) SNPs were not in high linkage disequilibrium (LD) (R2 < 0.8); (3) SNPs were reported in published studies or in the GTEx portal  to influence related gene expressions.The selection of candidate SNPs of ABCC8 gene, 10 in the TRPM4 gene) were included in the analysis. The detailed information of these 24 SNPs is given in Table A total of 24 candidate SNP loci  following the manufacturer’s instructions. All DNA samples were kept at − 20 °C for long-period storage. As described in our previous studies , genotypχ2) tests were applied to check the Hardy–Weinberg equilibrium (HWE) in controls using an online HWE calculator . Two SNPs (rs4802581 and rs60105962) deviating from HWE were eliminated for the subsequent analysis. For the 19 remaining variants, a 2 × 2 chi-square (χ2) test or a Fisher exact test was utilized to test the association of SNPs and SIDS using dominant, and recessive models and a linear-by-linear model of the χ2 test (same to the Cochrane-Armitage trend test) was used under the additive model. Odds ratios (ORs), 95% confidence intervals (CIs), and corresponding p values were calculated.Two SNPs (rs10766397 and rs8104571) had low call rates (< 95%) and were excluded for further analyses. Among the remaining 22 SNPs, one SNP (rs34271662) was excluded owing to the failure of clustering. Chi-square (n = 99), females (n = 59)), age group (0–4 months (n = 104), 2–4 months (n = 75), 4–8 months (n = 51), 8–12 months (n = 9)), time of death season (spring (n = 40), summer (n = 30), spring + summer (n = 70), autumn (n = 35), winter (n = 40), autumn + winter (n = 75)), and sleeping positions (prone position (n = 34), other positions (n = 12)) as shown previously [For the stratified analysis, study subjects were categorized into 4 different groups according to the risk factors for SIDS. These were (1) gender  among the European population were included for the haplotype analysis. Haplotype analysis was performed by Haploview 4.2  in the study subjects and by LDlink (https://ldlink.nci.nih.gov/) in the European population .SNPs in linkage with each other  of the chi-square analysis are listed in Table TRPM4 gene) was below the significance threshold of α = 0.05 .We successfully genotyped 19 variants at ABCC8 and TRPM4 on the hazards of SIDS by age, gender, time of death season, and sleep positions. In the stratified results, 14 nominally significant results (p < 0.05) were obtained. The only one associated locus in the main analysis, rs11667393, showed no specific associations in any one subcategory. However, rs4148622 in ABCC8 indicated significant differences between cases and controls in four subgroups , and rs7950189 in ABCC8 in two subgroups (“age 8–12 months” and “other sleep positions”). Both rs3760662 and rs11083962 in the TRPM4 gene were associated with SIDS in two subgroups (“Summer” and “Spring + Summer”). Variants rs7105832 and rs3819521 located in ABCC8 showed associations with SIDS in one subgroup (“age 4–8 months”). In the “Winter” subcategory, rs7112138 from ABCC8 was indicated to be related to SIDS. SNP rs3758953 at the ABCC8 gene had a single association in the category “Prone sleep position.” None of the above-mentioned findings in the stratified analysis remained statistically significant after Bonferroni correction at α = 0.0001, but these results may still suggest a biological relevance for the etiology of SIDS.The stratified analysis was performed to evaluate the effects of the variants located in ABCC8 and TRPM4 was estimated using Haploview 4.2 and LDlink. The related haplotype blocks were respectively screened in ABCC8 and TRPM4 . The haplotype-based association analysis provided evidence that the haplotype TCAG in ABCC8 may be relevant for SIDS (p = 0.040)  were included in the block. TRPM4 haplotype AAT was significantly associated with SIDS (p = 0.031)  were reported to influence the gene expression in tissue from the CNS.To assess a potential functional relevance for the gene expression, cis-eQTL target gene expression was employed using the online data from Genotype-Tissue Expression (GTEx) project. Eight of the nine SNPs with nominally statistical significance (except rs3758953 from ABCC8 and TRPM4).It has been postulated that disturbances in the brain water balance may be involved in the pathogenesis of at least some SIDS cases. Previous studies have uncovered that SNP loci for genes involved in ion channels and water channels  might be associated with SIDS –14, althTRPM4 and the other six in ABCC8. The only nominally significant SNP in the main analysis was located in TRPM4, and the other 8 genetic variants were identified in the stratified analysis (ABCC8 (6 variants) and TRPM4 (2 variants)).Overall, there was evidence that 9 of the 24 candidate SNP loci could be associated with SIDS, though 8 of them only in subgroups. Three putative risk SNPs were located within TRPM4 were associated with SIDS , suggesting that this SNP locus could be an influential predisposing factor. However, in a study on cerebral edema secondary to traumatic brain injury (TBI), no association for this locus was shown [TRPM4 in several human tissues, suggesting that this variant may affect the TRPM4 gene expression. The increased expression of TRMP4 could aggravate the cation inward flow into the cells of the nervous system [The G allele or GG/AG genotypes of polymorphism rs11667393 located in as shown . Accordis system . In a sis system . RegrettABCC8), as it was associated with SIDS in multiple stratified analyses. It was reported by Jha et al. that the wild allele at this SNP could be used for predicting 3-month outcomes of cerebral edema after severe TBI [ABCC8 in the brain tissues according to the eQTL analysis from the GTEx database. Therefore, one would expect to find an accumulation of the G allele in SIDS. However, the opposite was the case: the frequency of the G allele was lower in SIDS. Nevertheless, higher mRNA levels do not necessarily indicate increased protein expression, as the probe used for the eQTL analysis might just have recognized one splicing isoform: Rs4148622 and its proxy loci (rs2355017 and rs4757516) act as possible splicing variants and might influence the spatial structure of the SUR1 protein, possibly impairing the assembly of the subunits of the SUR1-TRPM4 complex [Regarding the 8 SNP loci with statistical significance in the stratified analysis, the locus of greatest interest was rs4148622 , potentially leading to sudden infant death instead of genuine SIDS [Besides a potential role in brain edema, SUR1 and TRPM4 as important ion channels are expressed in several human organs such as the heart, brain, and pancreas. Therefore, their physiological roles are diverse and thus could contribute to the individual SIDS risk in more ways than just via brain edema: Loss-of-function variants in ine SIDS –39. In aine SIDS . In a poABCC8  and TRPM4 (rs11083962) studied herein reached significant results in different subgroups; however, no previous studies reporting their related association with other diseases could be found. Nevertheless, for these loci was demonstrated by the eQTL analysis that they influence gene expression and thus may make carriers of these loci more susceptible to SIDS. The haplotype analysis demonstrated that the haplotypes in ABCC8 and TRPM4, consisting of risk alleles of partial SNPs, were associated with SIDS. Although the frequency of risk haplotypes is low (< 5%), the results suggest that variants may combine to modulate the risk of SIDS.Some of the variants in genes Several potential limitations in this study should be mentioned. Firstly, all nominal significant results, after multiple testing corrections, no longer reached the required level of significance . Therefore, future studies involving larger sample sizes should be performed to validate our findings. Secondly, as limited information on their biological functions is available for some loci tested herein, it is needed to be careful when interpreting their potential roles in SNP-mediated SIDS etiology. Thirdly, some well-known SIDS risk factors linked to cerebral edema like maternal smoking and alcohol consumption have not been included in the present study as related records were not available to us.In summary, we investigated for the first time the association of genetic variants involved in the SUR1-TRPM4 non-selective cation channel genes related to brain edema with SIDS. Despite these promising results, further studies with a larger sample size are supposed to confirm our results. Even more so, it would be informative if functional studies are conducted in the future to determine the function of risk genotypes and to be better able to interpret their impact on the pathogenesis of SIDS.Supplementary file1 (DOCX 19 kb)Supplementary file2 (DOCX 31 kb)Below is the link to the electronic supplementary material.


To determine the impact of exogenous transforming growth factor beta 1 (TGF-β1) on side population (SP) cells isolated from normal, papillary thyroid cancer and anaplastic thyroid cancer cell lines and from human thyroid tissues.All cell populations were stained with Hoechst 33342 and analysed using dual wavelength flow cytometry to identify SP cells. This SP assay was used to assess the impact of TGF-β1 treatment and withdrawal of treatment on SP percentages. Semi-quantitative and quantitative PCR were used for molecular analysis of cells pre and post TGF-β1 treatment.TGFB1 and its receptors, as well as showing variable expression of CDH1 and CDH2, with expressing of CDH1 being highest and CDH2 being lowest in the normal cell line. Exposure to exogenous TGF-β1 resulted in a reduction in mRNA expression of ABCG2 compared to controls which was significant between control and treated cancer cell lines. SP cells were isolated from primary human thyroid tissues, with numbers being significantly higher in papillary thyroid cancers. Exposure to TGF-β1 decreased the SP percentage in both thyroid cancer cell lines and completely abrogated these cells in the primary papillary thyroid cancer cultures. On withdrawal of TGF-β1 the SP phenotype was restored in the cancer cell lines and SP percentages increased to above that of untreated cells.All cell lines expressed mRNA for both TGF-β1 exposure transiently regulates thyroid cancer SP cells, leading to a reduction in SP percentages, while withdrawal of TGF-β1 results in restoration of the SP phenotype. Transforming growth factor beta (TGFβ) is a pleiotropic cytokine that under normal conditions is involved in a range of cellular process including but not limited to regulating cell growth, proliferation and differentiation. The role of TGF-β in cancer progression is still a matter of debate, as it has been described as having both tumour suppression and tumour promoting potential, depending on stage of cancer progression . MoreoveABCG2 expression. Moreover, removal of TGF-β1 resulted in restoration of ABCG2 expression and SP cell numbers  10 ng/mL TGF-β1 for the N-thy ori-3-1 and 1 ng/ml for BCPAP and SW1736 and incubated for 72 h. Following this, SP analysis was performed as described previously .To study the impact of TGF-β1 reversal on the SP percentage, media containing exogenous TGF-β1 was removed. Cells were then washed with PBS and new pre-warmed media added before being incubated for another 72 h, the cells were then assayed for the presence of SP. In every step, following treatment and reversal of treatment of TGF-β1, untreated cells were used as control samples.For tissue-derived cells, at 80% confluency, plated cells were treated with 1 ng/ml TGF-β1 for 14 days and then assayed for SP percentage as described previously . ControlTo confirm that any changes in SP cell percentage following treatment with TGF-β1, were specifically a result of involvement of TGF-β1, cells of the SW1736 cell line were treated with 3 mM SB-505124 .P-value of <0.05 was considered significant. In this study R2 is a measure of explanatory power in this logistic regression model were R2 is used to estimate whether the regression model can be reliably used or not to explain the relationship between the studied variables. Therefore, this was used to explain the relationship between the percentage of SP in relation to gender, age and nodule size. The regression model was run in a stratified manner looking at the influence of each factor on SP percentage . Although not significant, there was also higher expression of this receptor in the SW1736 cell line above that on the N-thy ori-3-1 cell line. BCPAP also showed higher expression levels of TGFB1 than the N-thy ori-3-1 cells, but this was not significant. There was little difference in expression levels of TGFBR1 between the 3 cell lines.Real-time PCR was used to determine expression of mRNA level for ors Fig. . HoweverCDH1 was expressed at significantly higher levels in the N-thy ori-3-1 cells than in the BCPAP or SW1736 cell lines (p < 0.001). All three cell lines expressed CDH2 with expression being lowest in the N-thy ori-3-1 compared with both cancer cell lines and with the highest level of expression being detected in the SW1736 cell line, but these differences in expression were not significant  for all lines. In the N-thy ori-3-1 cells there was a non-significant reduction in expression of ABCG2 whereas, for both cancer lines expression was significantly reduced when comparing BCPAP to control (p < 0.05) and SW1736 to control (p < 0.005)   cells to treated SW1736 (05) Fig. . To detep < 0.05)  Fig. .p < 0.001) and between PTC and BT SP (p < 0.05), with the % being higher in both cases for SP cells isolated from PTC.SP cells were isolated from primary cultures derived from normal thyroid tissue (NT), benign diseased thyroid tissue (BT) and PTC. In the case of the SP from NT, SP could only be detected in 9 out of 11 samples with SP percentage ranging from 0 to 0.4%, with the mean being 0.12%. A representative FACS plot showing an NT SP cell population of 0.1% is shown Fig. . For SP ABCB1 and ABCG2 and all expressed both transporters, although expression was higher in all cases for ABCG2  when untreated there was a small increase in SP percentage for n = 2 but no change for the third donor (FACS data not shown). For cells derived from PTC .ABCG2 expression. We observed that following treatment mRNA levels of ABCG2 were reduced significantly when comparing both cancer cell lines to control. We then went onto determine if this treatment impacted on SP cell percentage, as would be suggested by the reduction in ABCG2 expression and were able to determine that for both the cancer cell lines there was a reduction in SP percentage, but this was only significant when comparing control and treated SW1736. To determine if this was a reversible loss of the SP phenotype, we then removed the exogenous TGFβ and observed restoration of the SP phenotype and an increase in SP abundance in both cancer cell lines compared to controls. To confirm that these changes were due specifically to the impact of TGF-β1, we treated cells of the SW1736 cell line with a TGF-β1 receptor inhibitor and demonstrated that in the presence of this inhibitor the SW1736 SP was restored. This is in line with a study of MCF7 breast cancer SP cells which also showed a reduction in ABCG2 expression and a reduction in SP numbers following TGF-β treatment, both of which were reversable on removal of exogenous TGF-β1 [Having determined that the thyroid cell line models we were employing expressed markers that predicted TGF-β responsiveness we then wanted to determine if TGF-β1 treatment impacted on s TGF-β1 .We then examined the impact of TGFβ treatment on SP cells of primary PTC and NT and observed that while treatment had little effect on SP of NT , the percentage of SP cells in primary PTC cultures were reduced to below detectable levels using our SP protocol and gating strategy.While therapeutic targeting of TGF-β as a means of treating cancers has as yet not lived up to the results observed in some pre-clinical studies, we suggest limitations might be due in part to cell type being targeted and EMT being partial. For example, in ovarian cancer, higher numbers of SP cells have been reported to be present in ovarian cancer cell lines. When treated with TGF-β1 these SP cell numbers are reduced and driven towards a more mesenchymal cell phenotype. They display an increase in expression of some EMT markers. For example, snail1 an important regulator of EMT.Silencing of snail1 then resulted in the ovarian cancer SP cells showing a change in mRNA expression increasing expression of epithelial markers and decreasing expression of mesenchymal markers, again suggesting that regulation of the EMT process can impact on SP cells’ ability to undergo mesenchymal transformation .Our data supports a transient role for TGF-β in regulating thyroid cancer SP cell behaviour and further points to the need for a better understanding of the complexed role of TGF-β in cancer, including in regulation of cancer stem cells.Supplementary Figure 1Supplementary Figure 2Supplementary Figure Legends


Although iodic acid (HIO3) is widespread and forms particles more efficiently than sulfuric acid, its gas-phase formation mechanism remains unresolved. Here, in CLOUD atmospheric simulation chamber experiments that generate iodine radicals at atmospherically relevant rates, we show that iodooxy hypoiodite, IOIO, is efficiently converted into HIO3 via reactions (R1) IOIO + O3 → IOIO4 and (R2) IOIO4 + H2O → HIO3 + HOI + (1)O2. The laboratory-derived reaction rate coefficients are corroborated by theory and shown to explain field observations of daytime HIO3 in the remote lower free troposphere. The mechanism provides a missing link between iodine sources and particle formation. Because particulate iodate is readily reduced, recycling iodine back into the gas phase, our results suggest a catalytic role of iodine in aerosol formation.Iodine is a reactive trace element in atmospheric chemistry that destroys ozone and nucleates particles. Iodine emissions have tripled since 1950 and are projected to keep increasing with rising O 3) forms aerosols very efficiently, but its gas-phase formation mechanism is not well understood. Atmospheric simulation chamber experiments, quantum chemical calculations and kinetic modelling have now revealed that HIO3 forms as an early iodine oxidation product from hypoiodite. The mechanism explains field measurements and suggests a catalytic role for iodine in particle formation.Iodic acid (HIO Iodine nucleation rates exceed those of H2SO4 (in excess NH3) at comparable concentrations of iodic acid (HIO3)10. Furthermore, HIO3 growth rates of nanoparticles are both charge- and dipole-enhanced, exceeding the neutral collision rate11.Iodine is a trace constituent of the atmosphere that is particularly efficient at forming new particles. While sulfuric acid , iodine emissions have been increasing due to human activity. Iodine is primarily emitted from oceans by the reaction of O3 with iodide (I−) dissolved in surface waters, which liberates volatile iodine species (hypoiodous acid (HOI) and iodine (I2)) to the atmosphere15. This marine source is enhanced as a result of O3 pollution on local and hemispheric scales17 as well as the thinning of sea ice18, and now accounts for iodine emissions of ~3 Tg yr−1 (refs. 20). Over the past 70 years, iodine concentrations have tripled in ice-core records in Greenland18, Alpine glaciers17 and tree-ring records in Tibet21.Currently, iodine particle formation is rarely represented in atmospheric models—such models form most particles from the nucleation of H3 loss, but has, as of yet, not been suggested for particle formation. Iodine in the lower stratosphere has a 6–15 and 400–1,000 times higher O3 destruction potential per atom than bromine and chlorine22. Extremely low mixing ratios of iodine oxide (IO) radicals ; IO = 10−13 volume mixing ratio) can therefore affect the lifetime of climate-active gases 24. This chemical reactivity extends to heterogeneous reactions involving aerosol iodide (I−)15 and iodate (IO3−) (refs. 26 and references therein), which is the thermodynamically most stable form of iodine. The efficient multiphase chemistry of IO3− is markedly different from that of inert aerosol sulfate (SO42−), which accumulates without further chemical conversion until it is scavenged from the atmosphere by wet or dry deposition.Iodine is highly reactive and participates in catalytic reaction cycles that enhance its atmospheric impact. A catalytic role is well known for O28, and HIO3 has been detected in coastal marine air29, the Arctic and Antarctic boundary layer32, various continental sites10 and in the lower free troposphere33. Several precursors for HIO3 have been suggested: hydrated iodine atoms34, hydrated IO radicals34, iodine dioxide (OIO) radicals35 and larger iodine oxides . However, these mechanisms remain speculative and have not been demonstrated experimentally, leaving atmospheric HIO3 observations unexplained. Recent field observations of iodine-induced nucleation over remote oceans31 and of IO3− in stratospheric aerosols22 suggest a widespread role of iodine particle formation, but the conundrum of the missing HIO3 source mechanism blocks our ability to connect iodine sources to particle formation in atmospheric models.Iodine is ubiquitous in the atmosphere3) and associated long wall-loss lifetime , precursor gas-phase concentrations do not need to be increased above atmospheric levels , 40% relative humidity  and 40 ppbv O3 (<1–80 parts per billion by volume (ppbv)). The chemistry is driven by photolysis of I2, which is measured by cavity-enhanced differential optical absorption spectroscopy . HIO3 is measured quantitatively by NO3−-CIMS, and HOI by Br−-MION-CIMS. Both instruments also allow insights into the evolution of other iodine species  and chamber dilution (~2 h). Established iodine chemistry only contains a single reaction predicted from theory35 that could form HIO3 from OIO + OH. This reaction does not form HIO3 in the HOx-free conditions when I2 is photolysed by green light10. Even if OH radicals were present, they would be predominately scavenged by other species. The model base case does not form any HIO3 or HOI under the experimental conditions probed  within a few minutes of the onset of I2 photolysis by green light (grey lines). While zero HIO3 and zero HOI are predicted by the base case model (current state of the art), the extended model achieves excellent agreement with regard to the measured concentrations and the timing of HIO3 and HOI formation. The extended model also improves the closure of timing and concentrations measured for OIO, IOIO and I2O4. Measured HIO3 concentrations reach a steady state after ~8 min, consistent with the wall-loss lifetime of other sticky molecules3 measured at CLOUD . The timing of IO radicals is predicted very well from both the base case and the extended model, reflecting the high level of trust in the gas-phase chemical kinetics during the early stages of the iodine photolysis experiments. Interestingly, iodine oxide clusters IxOy  larger than IOIO are formed too late to explain the rapid formation of HIO3 as an early generation product . Here, pHIO3 is calculated from HIO3 concentration measurements and the well-known loss rates to the chamber walls, and pI is calculated from the photolysis of I2. The HIO3 yield, defined as the ratio of pHIO3 and pI, is a function of the experimental conditions and varies between 10 and 20%. This variability is most pronounced for low pI (<105 molec cm−3 s−1) and is quantitatively explained by the wall loss of HIO3 precursors becoming progressively more relevant at lower gas concentrations. We corroborated that HIO3 formation from I atoms is a multistep process by carrying out an experiment with enhanced stirring (by two fans at the top and bottom of the chamber), thereby decreasing the wall accommodation lifetime of HIO3 from the standard ~8 min to ~2 min, while holding all other parameters constant. The HIO3 concentration decreased by more than one order of magnitude, indicating that the HIO3 suppression exceeds that expected from a change in lifetime alone  species are HIO3 precursors34 at CLOUD. If such IxOy were the precursor, the HIO3 yield would not be constant, but would increase progressively with pI, and pHIO3 would follow a higher-order rate law , which can bias reaction pathways to favour the formation of larger IxOy species , resulting in a slight dependence of the pHIO3-to-pIOIO ratio on O3. That slight dependence is captured by the extended model  = 1). In contrast, a pronounced O3 sensitivity would be expected if IO⋅H2O or OIO were HIO3 precursors  methods M062X/aug-cc-pVTZ-PP, followed by coupled-cluster single-point energy corrections; equences  and R2)3, H2O an−1 (with the exception of the OIO BDE), which translates into approximately one order of magnitude uncertainty in rate constants.Figure k1 ≥ 1.5 × 10−13 molec−1 cm3 s−1 is supported within the error margins of theory and maintains the quantitative conversion of IOIO into HIO3 even at low O3 concentrations , IO radicals  and particle surface area measurements at the Maïdo observatory41 to assess the relevance of CLOUD findings in the real world. The observatory is located in the southern Indian Ocean on Réunion Island at an elevation of 2,200 m, and is frequently exposed to lower free tropospheric air (mornings) and anabatic orographic flows from the ocean (afternoons). The laboratory conditions at CLOUD closely match the conditions at the Maïdo observatory (Supplementary Table −3 s−1) and temperature (~283 K).The laboratory-derived mechanism can explain field measurements of HIO3 in the field and laboratory on a common IO radical concentration axis. pHIO3 is calculated from HIO3 concentrations and the condensation sink surface area, assuming a steady state. IO radical concentrations are measured directly at the Maïdo observatory, and taken from the extended model at CLOUD. The solid line shown in Fig. 3 from CLOUD  via reactions . IxOy species may also contribute locally in coastal hotspots with extremely high iodine concentrations. Freshly nucleated iodine particles are composed almost entirely of HIO3 (ref. 10); HIO3 is a strong acid  that dissociates to form IO3−. IO3− is known to undergo reduction reactions that ultimately form more volatile iodine species , which are re-emitted to the gas phase (step 4). Field observations and laboratory experiments show that IO3− is reduced via iron redox chemistry, H2O2, nitrite, photosensitized reactions, photolysis and numerous other species (refs. 26 and references therein), with the overall effect of recycling iodine to the gas phase. The HIO3 formation mechanism thus completes a catalytic iodine reaction cycle, by which a single iodine atom can repeatedly form HIO3, driving particle formation. For each HIO3 molecule produced from I, three O3 molecules are consumed. The re-emission of reduced iodine species thus constitutes a multiphase reaction cycle that destroys O3.The gas-phase formation mechanism of HIOeactions  and R2)3 we pres45. Among the primary radioactive elements, 132Te, 137Cs and 103Ru abundances were found to correlate with the aerosol volume distribution, whereas 131I correlated with the aerosol surface area distribution instead. These empirical observations hint at efficient recycling occurring on timescales of hours to days, consistent with rapid HIO3 formation. Notably, although the reactive uptake of HOI on aerosols is known to be fast46, this reaction de facto removes halides from aerosols to the gas phase. A gas-phase source of HIO3 adds iodine to particles and, in conjunction with iodine recycling, provides a plausible explanation for the correlation of particulate 131I with the aerosol surface area distribution at the molecular level. Particulate IO3− is the primary reservoir of total (gas and particle) iodine in the stratosphere22. Whether HIO3 forms in the stratosphere—and controls iodine partitioning between the gas and particle phases—deserves further study. The HIO3-formation mechanism fills a gap in the representation of the geochemical iodine cycle in current atmospheric models.That iodine recycling controls the iodine partitioning between the gas and particle phases is corroborated by field measurements of the size-resolved iodine activity in radioactive fallout19. This deserves re-evaluation in light of efficient HIO3 formation even at low concentrations, the catalytic role of iodine in particle formation, and the increased global iodine source in recent decades. Iodine particle formation is probably already relevant on global scales today, and will become even more important in view of decreasing global sulfur emissions and increasing iodine emissions in a future climate.Iodine particle formation has heretofore been considered to have only limited global importance50 in Geneva, Switzerland as part of the CLOUD12 and CLOUD13 campaigns during 2017 and 2018. The CLOUD chamber is a temperature-controlled, electropolished stainless-steel reaction vessel with a volume of 26.1 m3. Experiments were carried out at temperatures of 283 and 263 K. The chamber was operated as a continuous-flow reactor, and ultra-pure N2 and O2 at 250–300 l min−1 were continuously replenished at a pressure of 1 atm, resulting in an air exchange time of ~80 min. Two fans at the top and bottom of the chamber established near-homogeneous mixing (mixing time ~2 min). Trace gases were added at the bottom of the chamber. I2 was produced from sublimating iodine crystals , and concentrations inside the chamber were varied in the range 0.5 pptv < [I2] < 330 pptv . O3 was generated from UV irradiation of dry synthetic air, and the chamber was humidified using ultrapurified water, resulting typically in [O3] = 40 ppbv (range < 1–80 ppbv) and RH = 40% (<3–90%).Laboratory experiments were carried out at the CERN CLOUD chamber3 following the selective photolysis of I2 using green light (light-emitting diodes (LEDs) centred at 523 nm, I2 photolysis frequencies 3 and humidity  coupled to a chemical ionization unit, using dibromomethane (CH2Br2) as the reagent gas. The CH2Br2 is fed into the sheath flow of the inlet and illuminated by a soft X-ray source. The produced bromide anions are directed into the sample flow by a negative electric field, and cluster with neutral molecules (I2) in the sample air. The overall uncertainty of the resulting I2 time series is estimated to be better than 30%52. The I2 constraint imposed to the model assimilates the lower bound of the measured I2 time series (within the 30% uncertainty), which results in the best closure between measured and predicted HIO3. Iodine radical production rates, pI, are calculated from the photolysis rate of I2 concentrations.I3 was measured by a NO3−-CIMS system comprising an APi-TOF coupled to a chemical ionization unit that uses nitric acid as the reagent gas. It is used extensively for detecting H2SO4, highly oxygenated organic molecules and HIO3. Details of the instrument used in the present study are provided in ref. 53. The NO3−-CIMS has an ion filter integrated into its sampling line to avoid confusion with ions and charged clusters from the CLOUD chamber. It thus measures only neutral molecules and clusters in CLOUD. The uncertainty of the HIO3 measurement is estimated to be 50%.HIO2SO4 loss rates. The loss to walls is the well-defined dominant sink of HIO3. Experiments that formed a large particle surface area  competitive to chamber wall loss were discarded in this study to avoid introducing uncertainty due to the other less-well-defined sinks for HIO3 and other iodine species. The HIO3 production rates were calculated from measured concentrations under the assumption of a steady state. Periods with rapid changes of HIO3 concentration are not considered in, for example, Fig. The characteristic time for the deposition of sticky molecules to the chamber walls is 440 s with standard mixing by the fans  used in the model is enhanced over the accommodation coefficient for individual collisions57. Extended Data Fig. 2, O3 and H2O, photolysis frequencies , temperature and the aforementioned loss mechanisms. HOI is both lost to the walls and produced on the chamber walls through heterogeneous chemistry14, which also proceeds in dark conditions. This study did not make an attempt to describe the uptake and release of HOI at the molecular level. An empirical uptake efficiency of 25%, relative to H2SO4, establishes closure in regard to the temporal evolution and concentrations of HOI  program.For the reactants, intermediates, transition states and products in Fig. 60. In the simulation, IOIO + O3 was modelled to directly lead to IOIO4 using the MesmerILT method with a pre-exponential value of 2.7 × 10−14 molec−1 cm3 s−1, which corresponds to the transition-state-theory-derived bimolecular rate. The unimolecular isomerization reactions of intermediate complexes were treated using the SimpleRRKM method with Eckart tunnelling. The MesmerILT method with a pre-exponential value of 2.0 × 10−10 molec−1 cm3 s−1 was used for the bimolecular reaction of IOIO4 with H2O, with the latter set as the excess reactant with a defined initial concentration. All intermediate complexes were assigned as ‘modelled’ with Lennard–Jones potentials of σ = 6.5 Å and ϵ = 300 K. These are identical to those used by Galvez and colleagues for their iodine systems61. MESMER uses the exponential down (ΔEdown) model for simulating the collisional energy transfer; a value of 225 cm−1 was used in the simulations, which is within the 175–275 cm−1 range recommended by MESMER for nitrogen bath gas.Final product fractions were calculated using the MESMER program−1 (with the exception of the BDE of OIO). Previous quantum chemical calculations on iodine oxide reactions47 are included in Table The energetics of ozonolysis reactions are difficult to calculate accurately using single-reference methods. The inherent uncertainties are probably even more pronounced for complex iodine-containing systems. Although no experimental values are available for the gas-phase ozonolysis reaction of iodine systems, proton affinities (PAs) and BDEs of simple molecules such as HI, HOI, IO and OIO are available. Table 41, Réunion island, southern Indian Ocean . The observatory is located at 2,200 m above sea level and is frequently exposed to lower free tropospheric air (mornings) and flows from the ocean (afternoons). Near-instrument altitude volume mixing ratios of IO radicals were retrieved from CU MAX-DOAS scattered sunlight observations. The retrieval63 leverages the high sensitivity of the limb viewing geometry to the atmospheric layers nearest to the instrument altitude, allowing for the parameterization of aerosol effects on the observed light path. Gas-phase HIO3 was measured directly by a NO3−-CIMS system using a methodology similar to that used in the laboratory experiments. The instrument was calibrated in the field in its actual field campaign sampling configuration by in situ-produced H2SO4, which resulted in a calibration factor of c = 1.7 × 1010 molec cm−3. This same calibration factor was used for all quantifications, so the determined concentrations here represent lower limits. The uncertainty of the determined [HIO3] was estimated similarly as [H2SO4], at −50% and +100% following the work in ref. 64. Particles were size-selected by a differential mobility particle sizer and counted with a condensation particle counter to determine the available particle surface area. The box modelling constraints are described in Supplementary Section 65 were used to determine the photolysis frequencies of individual iodine species.The field data were collected during an intensive operating period in April 2018 at the Maïdo observatoryAny methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41557-022-01067-z.Supplementary InformationSupplementary discussion, Figs. 1–11 and Tables 1–11.


Electronically activating interfacial hydrogen atoms on the TiO2 surface is a common way to control their reactivity. Modulating the potential landscape is another way, but dedicated studies for such an activation are limited. Here we show the single hydrogen atom manipulation, and on-surface facilitated water deprotonation process on a rutile TiO2 (110) surface using low temperature atomic force microscopy and Kelvin probe force spectroscopy. The configuration of the hydrogen atom is manipulated on this surface step by step using the local field. Furthermore, we quantify the force needed to relocate the hydrogen atom on this surface using force spectroscopy and density functional theory. Reliable control of hydrogen atoms provides a new mechanistic insight of the water molecules on a metal oxide surface.The discovery of hydrogen atoms on the TiO 2 is a prominent photocatalyst for overall water splitting, but the on-surface activation of hydrogen atoms is still not fully understood. Here, the authors use atomic force and kelvin probe force microscopy to study the lateral manipulation of hydrogen on a rutile (110) surface.Rutile TiO Detail understanding of this adsorbate and its control at atomistic level is essential for fully elucidating the nature of a deprotonated water reaction on a TiO2 surface23. Moreover, oxidation of hydrogen atoms by oxygen molecule on the rutile TiO2 surface results in the reactive oxygen species (ROS), such as reaction intermediates of water species H2O, HO2, H2O2, and H3O225. Exploring these species requires the fundamental understanding and detailed description of the interaction between oxygen and hydrogen atoms. Ideally, attacking these problems require to access the atomic scale study of a hydrogen atom on this surface, which remains a great challenge owing to the light mass and small size of the hydrogen atom. In particular, hydrogen species on a rutile TiO2 surface were investigated using various experimental techniques, including scanning tunneling microscopy (STM)26. STM provides a unique opportunity for electronically inducing the reactions of a hydrogen atom on this surfaces21. However, STM easily induces the stochastic behavior of molecules owing to its flowing current; therefore, it might be difficult to precisely control in the desired configuration at atomic level18. Moreover, the contrast mechanism of STM is related to the density of electronic states, which is still obscure to investigate the real-space of the atomic configuration26.The hydrogen atom detection on a rutile TiO44. Another fascinating capability of AFM is the possibility to measure directly the interaction forces that induce the adsorbate’s motion and thus, to provide a detailed insight into the interaction force for the target species43. Kelvin probe force spectroscopy (KPFS), owing to its force modulation mechanism, allows us to precisely control the different states of on-surface species signaled by the appearance of a jump in the frequency shift (Δf) vs. bias voltage (Vbias) parabola33. The vertical shift between the two parabolas is strongly influenced by the different local electric fields automatically formed between the tunneling junction33. Especially on the metallic surface, controllable on-surface lateral manipulation of the single molecule47 has been well established and the adsorbate can also be activated48 by means of electric field50. Moreover, on the rutile TiO2 surface, the vertical desorption of hydrogen atom21, reversible migration of hydrogen atom23, the stochastic motion of hydrogen atom induced by inelastic tunneling electrons3, manipulation of oxygen adatom27 and lateral tip induced excitation of the single molecule35 have been clarified. However, the lateral manipulation of the single hydrogen atom to the desired position on rutile TiO2 surface has been poorly reported experimentally up to now10, although it could provide an efficient means of the arrangement of the on-surface water splitting reactions, dissociation dynamics, which critically control the efficiency of heterogeneous catalytic reactions on this surface23. Especially, elucidating the interaction force between tip and water configuration on the TiO2 surface is important for answering the current controversial question of whether a proton atom should remain as one water molecule or two distinct hydroxyls from the mechanical point of view8. For local chemistry, the mechanical sensitivity and stability of hydrogen bond43 are also crucial properties, but studies of these properties remain elusive on this surface.Atomic force microscopy (AFM), as a viable alternative, has been used to provide a precise measurement of the surface configuration, also manipulating atoms and molecules based on its force modulation mechanism2(110)−(1 × 1) surface, and investigate its mechanical properties by the force field mapping. We analysis the manipulation outcome with the local electric field and the density functional theory (DFT). Our results demonstrate that the hydrogen atom can be manipulated along the oxygen row. The force field on the top of these configurations quantifies the possible tilting of the hydrogen atom on these configurations.Here, we use AFM and KPFS to present the manipulation of hydrogen atom and reversible water reaction on a rutile TiO2 surface. By using the KPFS technique, we experimentally demonstrated the water reaction as shown in Fig. 2 (110)−(1 × 1) surface contains twofold-coordinated surface bridging oxygen (Os) atoms and fivefold-coordinated titanium (Ti) atoms that are alternatively aligned1. Moreover, the practical sample preparation in ultrahigh vacuum (UHV) will induce point defects, such as oxygen vacancies (OV) and hydroxyl defects (OsH)1. When the TiO2 surface is exposed to oxygen at room temperature, oxygen will dissociate on this surface and will be adsorbed as an adatom (Oad) on Ti row29. We previously discovered that the Oad has two stable charge states, namely, singly charged (Oad−) and doubly charged (Oad2−). Moreover, we previously found that Oad2− is the most stable on the rutile TiO2 surface29. Figure sH−Oad2−−OsH species initially formed on top of the rutile TiO2 (110) surface obtained by the tip in hole mode 44. Notably, the oxygen adatom is spontaneously charged to Oad2− and adsorbed between two OsH defects29. After AFM imaging −, while the symmetric configuration is initiated by positioning the tip on top of the hydrogen atom and ramping the bias from zero to a certain negative voltage and then back to zero −Os configuration during this manipulation cycle. However, even though we bring back the bias voltage to zero immediately after the Δf jump, OsH−(OadH)−−Os is generally transformed to Os−(OadH)−−OsH −Os configuration may be stochastically very rare compared with the Os−(OadH)−−OsH configuration during this manipulation, possibly because of the low temperature of 78 K8 and the energy barrier for the recombination of two hydroxyls to water is relatively high while the barrier to move them is low owing to the DFT calculations in Du et al. study5. Additionally, considering the charged nature of the protons, it is not surprising that forming a water molecule is much less likely than moving both hydroxyls in concert. Hence, the transition of Os−(OadH)−−OsH → OsH−(OadH)−−Os may generally occur immediately after the Δf jump. Here, we note that even though we always placed the tip same height with symmetrically on the OsH−(OadH)−−Os configuration, we found that the bias voltage of Δf jump slightly fluctuates within a bias range of ±0.15 V as shown in Fig. 32, existence of intrinsic subsurface defect44 or polaron underneath the surface53. Notably, we do not observe the apparent telegraph noise between Os−(OadH)−−OsH and OsH−(OadH)−−Os in KPFS measurement, thereby we safely exclude the possibility of inelastic tunneling electrons mechanism, which reported by Tan et al.3. The initial state shown in Figure adH)− and ramped the bias from zero to a certain negative voltage and then back to zero −−OsH ↔ OsH−(OadH)−−Os.First, we show an experiment for the reversible water reaction on the TiOero Fig. . Figure ero Fig. . NotablyOsH Fig. . In reveero Fig. . Hence, sH−(OadH)−−Os and Os−(OadH)−−OsH, we further perform KPFS measurement as a function of tip-sample distance, ensuring that no tip changes or deformation of the target Os−(OadH)−−OsH could occur33. Fig. s−(OadH)−−OsH was prepared by the same procedure as shown in Fig. sH in Os−(OadH)−−OsH was confirmed by AFM imaging. After AFM imaging, the tip was brought above the OsH and the feedback loop was turned off. The tip was vertically approached on top of the OsH and KPFS measurement was performed. We repeat this procedure by changing the vertical tip height and the lateral tip position on top of the OsH. As we can see in Fig. Vjump) in KPFS becomes large by reducing the tip height. These results qualitatively demonstrate that further bias voltage is required to reversibly manipulate the configuration between OsH−(OadH)−−Os and Os−(OadH)−−OsH at a large tip-sample distance. One has to keep in mind that for a non-zero local contact potential difference (LCPD) between tip and sample, even at zero bias voltage there is a finite electric field across the junction33. Moreover, this LCPD also depends on the tip-sample distance owing to the average effect31. Only at compensated LCPD, that is, at Vbias = VLCPD the local electric field vanishes. Although this is very well known from KPFS, it is usually less considered in pure STM experiments. The quantitative assignment of Vjump is further shown in Fig. VLCPD, where Vjump − VLCPD linearly becomes large by reducing the tip height. Moreover, during the negative bias KPFS, the current was in the noise limit of our current amplifier −−OsH and OsH−(OadH)−−Os. This linear behavior is similar to the experimental evidence for the electric field-stimulated reaction of oxygen adatom27 and desulfurization process on metal surface48. Estimating the exact value of the local electric field might be challenging because the local electric field will also generally be affected by the local bulk condition, such as Ti interstitial and subsurface defects on this surface53, and the exact tip apex shape.To find out more physical mechanism behind the reversible manipulation between OsH in OsH−(OadH)−−Os. In the case of field-induced switching mechanisms, the reverse polarity should lead to the opposite perturbation to the potential landscape on the surface56. Figure sH in OsH−(OadH)−−Os. Figure sH−(OadH)−−Os species prepared by a similar procedure as shown in Fig. sH and the bias was ramped from zero to a certain positive voltage and then back to zero. Figure VLCPD shift and Vjump at +3.8 V. The VLCPD of blue and red curves in Fig. VLCPD shift to larger bias voltage after jump indicates the reduction of the positive charge under the tip33. Figure ad2− appear, suggesting that the two positively charged hydrogens desorbed from the surface. This is in line with the positive VLCPD shift shown in Fig. sH and oxygen row (Os) at Vjump are 0.105 nA and 0.039 nA. This observation agrees with the previous STM results that OsH flows larger tunneling current than Os under the typical positive bias condition26. The observed tunneling current with the positive bias KPFS is contrary to that of negligible tunneling with negative bias KPFS −−Os, OsH−Oad−OsH and Os−(OadH)−−OsH are separated by a potential barrier representing the activation energy required to induce the configuration change. Our DFT calculation in Supplementary Table s−(OadH)−OsH is generally more energetically favorable than OsH−Oad−OsH. Based on these results, inducing the local electric field in a specific site with respect to the atomic configuration deforms the potential energy landscape of three different configurations in a characteristic manner and thus changes the configuration, as shown in Fig. 4(b–d)56. This can be achieved by changing the polarity of the bias voltage between the tip and sample, and the lateral position of the tip. In the case of reversible manipulation between Os−(OadH)−−OsH and OsH−(OadH)−−Os −−Os −−Os as shown in Fig. sH−(OadH)−−Os −−Os −−OsH and OsH−(OadH)−−O. Our DFT calculation also supports that an electric field up to −0.5 V/nm would significantly stabilize OsH−Oad−OsH −Os Fig. , the tip−Os Fig. . In this−Os Fig. . However−Os Fig. . It is sOsH Fig. . As showsH−Oad2−−OsH, Os−(OadH)−−OsH and OsH−(OadH)−−Os, now we analyze the hydrogen configurations of three manipulation outcomes based on AFM images and force field images. Figure f(z) mapping, F(z) mapping and AFM images obtained on top of the OsH−Oad2−−OsH, Os−(OadH)−−OsH and OsH−(OadH)−−Os. The Δf(z) mapping experiment was performed as follows. The lateral tip position was fixed at the side of the oxygen row and the Δf(z) was recorded over OsH−Oad2−−OsH, including a long-range force from the TiO2 substrate. Then, the tip was placed on top of the hydrogen atom and KPFS measurement was performed. We repeat this procedure by manipulating among the three configurations of OsH−Oad2−−OsH, Os−(OadH)−−OsH and OsH−(OadH)−−Os. Notably, F(z) was obtained from Δf(z) using Sader Jarvis method57. Fig. sH−Oad2−−OsH, O−(OadH)−−OsH and OsH−(OadH)−−Os configurations. The positions of the line profiles are shown in Fig. s−(OadH)−−OsH and OsH−(OadH)−−Os configurations have symmetric line profiles, and most importantly, the two hydrogen positions in the Os−(OadH)−−OsH configuration indicated by the black and red arrows. Focusing on the light-blue and pink curves, the hydrogen atom on the right (red arrow) has the same position as the oxygen row . However, the hydrogen atom on the left (black arrow) is slightly tilted to the left from the center of Oad2−. The distances from the center of Oad2− to these dips were 320 pm and 116 pm, respectively. Notably, the distance of 320 pm is the half distance between the oxygen row1, which indicates the reliable lateral resolution of the contrast in the AFM images Fig. adH)− and the nearest neighbor oxygen row; therefore, a too small tilt was observed (limited by the convolution of the tip apex shape and sample structure). This assertion is also supported by the similar height of (OadH)− and the nearest neighbor oxygen row. The equivalent net charge of (OadH)− and the nearest neighbor oxygen row result in a similar topographical height owing to the contrast mechanism of the tip in hole mode34. Moreover, this off-center orientation of the hydrogen bond in Fig. 3 and Du et al.5 studies. Hence, give us extra confidence to conclude that the hydrogen atom on the right has similar interactions from (OadH)− and the nearest neighbor oxygen row. Here, we should note that one would expect a small influence of the possible tilting of the hydrogen atom under the attractive force interaction. Because, for example, the AFM imaging shows different bond lengths and atom positions in C60 when changing the Z distance39. Therefore, we perform the frequency shift mapping as shown in Fig. f(z) decrease by reducing the tip height. To allow easy visualization, we draw a constant frequency shift value of −145 Hz and −345 Hz by the circle in Fig. 34. On the other hand, the position of the hydrogen atom is “less negative” than the oxygen atom, which gives them a slight dark contrast. Remarkably, when the tip approaches very close to the surface, the local maximum of ∆f(z) appears on top of the hydrogen atom, which is indicated by the black dotted arrow of the constant frequency of −345 Hz in Fig. 34 as shown in Fig. f(z) mapping in Fig. F(z) mapping obtained from Fig. F(z) decrease by reducing the tip height, owing to the increasement of the attractive force. In Fig. 59. The long-range dominant region of the ∆f (z) curves, which we defined as z > 0.3 nm, was fitted into the inverse-power function of z−s59. The short range part of ∆f (z) was obtained by subtracting the long-range part of ∆f (z) from the raw ∆f (z) curve. Then, the short range part of ∆f (z) curve was numerically converted to FSR (z) using the Sader Jarvis method59. In Fig. F (z) generally decrease by reducing tip-sample distance, which indicates the attractive tip-sample interaction. Especially in Fig. FSR (z) has a similar tendency with F (z). Hence, the atomic contrast of Fig. F  in Fig. FSR, the attractive short range force acting on the tip was about FSR = −0.6 nN, indicated by the pink arrow in Fig. FSR, the results shown in Fig. FSR. In the DFT calculation shown in Supplementary Fig. adH from tilted geometry to upright is estimated to be around 0.440 nN, which is smaller than experimentally measured 0.6 nN. This difference additionally propose that the rearrangement of the hydrogen atom is presumably induced by the attractive force of tip background short range van der Waals interaction or tip dipole, dominates the tip-sample interaction62. Compared to the other works, the magnitude of FSR = −0.6 nN is quantitatively larger than the lateral or vertical force for displacing a physisorbed CO molecule on metal surface38. This is generally in line with the physical aspect of the hydrogen-oxygen atom interaction that the hydrogen bond is generally stronger than the van der Waals interaction64. Hence, suggesting that the deprotonated configurations of Os−(OadH)−−OsH and OsH−(OadH)−−Os are significantly stable under the specific force field.To consider the precise three atomic configurations of OsH defect on a rutile TiO2 surface using an individual single OsH defect, because this OsH defect is also the most fundamental atomic feature on this surface1 and known to provide a critical role in photocatalysis at tremendous condition23. Figure 2 (110) surface partially exposed to oxygen at room temperature obtained using a hole mode tip34. The black spot can be observed in Fig. sH defect34. Notably, an oxygen adatom can be observed on the lower left side as a bright spot. Next, we demonstrate that this OsH defect can be precisely manipulated along the [001] direction by KPFS. After AFM imaging  surface by low-temperature AFM and KPFS. We succeeded in the reliable control and characterization of a hydrogen atom on top of the three different outcomes of OsH−Oad2−−OsH species by using a functionalized tip in hole mode with the KPFS manipulation. The force mapping with an atomic resolution allowing us to preciously determine the hydrogen position; interestingly, one hydrogen atom was tilted forward and another was straight. We believe that the achievement of our large body of work intrinsically provides the opportunity to understand the mechanochemical process of reactive oxygen species, the hydrogen atom and water species, naturally the world’s most important chemical species, on an oxide surface.In summary, we have demonstrated the lateral manipulation of hydrogen atom on the rutile TiO−11 Torr. The temperature of the AFM unit was kept at liquid nitrogen temperature (78 K). The AFM measurements were performed in the frequency modulation (FM) detection mode. The atom tracking method was used to compensate for the thermal drift between the tip and surface during the measurements. The dc bias voltage was applied to the sample. The AFM imaging was performed using constant Δf mode at Vbias = 0 V to avoid the tunneling current to flow. The cantilever was oscillated at resonance frequency keeping the oscillation amplitude constant. We used iridium (Ir)-coated Si cantilever . Metal Ir tips provide stable AFM imaging compared to the bare Si tip. The tip was initially annealed to 600 K and then cleaned by Ar+ sputtering to remove the contamination before experiments. The rutile TiO2 (110)-(1 × 1) sample was prepared by sputtering and annealing to 900 K in several cycles. The sample was exposed to oxygen at room temperature for ~0.5 L and then transferred to the measurement chamber precooled to 78 K. The OsH groups on the TiO2(110) surface were spontaneously created from the dissociation of water molecules  over oxygen vacancy sites by transferring hydrogen atoms to neighboring oxygen bridge sites1. We modify the tip apex by gently poking the tip into the surface using the controlled force distance spectroscopy, until reaching to the sharp tip in hole mode (positively terminated tip). Notably, we can distinguish OsH from the oxygen vacancies using the previous results9.The experiments were carried out using a low-temperature ultrahigh vacuum AFM system. The deflection of the cantilever was measured using the optical beam deflection method. The base pressure was lower than 5.0 × 1065 and the PBE0-TC-LRC-ADMM hybrid density functional67, containing 20% HFX exchange, which was truncated at a distance of 2.5 Å. The cutoff of the finest real-space integration grid is 400 Ry. The primary basis set is MOLOPT68, of DZVP quality for Ti and TZV2P quality for O, with corresponding GTH pseudopotentials70. The auxiliary Gaussian basis for the ADMM method was cFIT11 for Ti and cFIT3 for O. The dispersion interactions were considered within the Grimme D3 method71. The force convergence criterion used for geometry relaxations was 0.01 eV/Å. The TiO2 (110) surface was constructed using slab model consisting of six atomic layers and cell parameters from bulk calculation. The external electric field was simulated by introducing a saw-shaped periodic electrostatic potential along z-axis. In the evaluation of surface geometry under external load, external force was applied on the H atom along z-axis.The DFT calculations were carried out using the CP2K Quickstep packageSupplementary Information


The most accepted hypothesis in Alzheimer’s disease (AD) is the amyloid cascade which establishes that Aβ accumulation may induce the disease development. This accumulation may occur years before the clinical symptoms but it has not been elucidated if this accumulation is the cause or the consequence of AD. It is however, clear that Aβ accumulation exerts toxic effects in the cerebral cells. It is important then to investigate all possible associated events that may help to design new therapeutic strategies to defeat or ameliorate the symptoms in AD. Alterations in the mitochondrial physiology have been found in AD but it is not still clear if they could be an early event in the disease progression associated to amyloidosis or other conditions. Using APP/PS1 mice, our results support published evidence and show imbalances in the mitochondrial dynamics in the cerebral cortex and hippocampus of these mice representing very early events in the disease progression. We demonstrate in cellular models that these imbalances are consequence of Aβ accumulation that ultimately induce increased mitophagy, a mechanism which selectively removes damaged mitochondria by autophagy. Along with increased mitophagy, we also found that Aβ independently increases autophagy in APP/PS1 mice. Therefore, mitochondrial dysfunction could be an early feature in AD, associated with amyloid overload. The study of these neuropathological hallmarks gave birth to the main hypothesis to explain the origin of the disease, but nowadays none of them has been totally validated. The most accepted is the amyloid cascade hypothesis which establishes that Aβ oligomeric accumulation is causing AD3. This only occurs when APP is processed throughout the amyloidogenic pathway, then producing the Aβ peptide. This accumulation may start years early in the disease progression even years before the main clinical symptoms are evident in patients but the reason because this peptide accumulates is unknown4. Along with amyloid accumulation, other possible disease events may occur. Secondary pathological features in AD are evident along with amyloid deposition and they include alterations in the mitochondrial physiology causing energetic deficiency due to mitochondrial damage and functional failure6. Mitochondria in neurons are the main source of energy and for any reason they become less functional in neurodegeneration and particularly in AD, resulting in energetic deficiency with the disease progression. It is not clear if this could be consequence of the pathological conditions and if could be an early event in the disease.Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease affecting more than 50 million people worldwide. Patients suffering AD show deep cognitive impairment along with behaviour disorders as the main clinical symptoms. Preceding AD there is a prodromal stage known as mild cognitive impairment (MCI) in which patients still do not show clinical signs of dementia but they undergo the loss of memory, language and other mental abilities with the disease progression. Neuropathologically, AD is characterised by the presence of brain extracellular deposits of amyloid-β (Aβ) peptide coming from the APP processing, the intraneuronal deposits of hyperphosphorylated tau protein, neuroinflammation, and the neuronal cell death in specific brain areas11. Contrarily, it has been demonstrated that Aβ peptide accumulation can be an autophagy trigger itself so we can speculate that autophagy fails at one point in the disease progression and this could be consistent with the amyloid accumulation in the progression of AD. There are a number of studies showing that in addition of autophagy, Aβ peptide may induce mitophagy in AD. Mitophagy is a mitochondrial quality control that selectively removes damaged or superfluous mitochondria by autophagy12. This has been shown using cellular and animal models of the disease but it is not clear if this could be an early or late event in the pathology18.It is known that excessive production of Aβ peptide can be removed by an autophagy-dependent mechanism and this is confirmed by a number of studiesHere we analysed the effect of Aβ peptide accumulation in the mitochondria of the most affected brain structures in AD, the hippocampus and cerebral cortex from 3, 6 and 12 month-old APP/PS1 mice. We noticed that mitochondrial mass was reduced in both regions from APP/PS1 mice before such event occurs in wild-type (wt) mice. This could be explained as the mitochondrial biogenesis is reduced but also because we found early impairments in the mitochondrial dynamics that ultimately increases mitophagy. Additionally, we found that Aβ overload in APP/PS1 mice increased autophagy highlighting a dual effect of Aβ accumulation in AD. We confirm all these results related to Aβ overload using primary neuronal and SH-SY5Y cellular models. Our results suggest that Aβ overload exert a dual effect increasing mitophagy and autophagy early in the AD pathology and this is maintained in aging.40 levels in cerebral cortex and hippocampus from 12-month-old mice were found significantly increased compared with 3-  and 6-month-old  old mice  Fig. B. We fouRegarding the inner mitochondrial membrane fusion protein Opa1, we found a significant reduction in APP/PS1 cerebral cortex levels early in age (3 month-old) compared with the age-matched wt mice and such significance was maintained as mice with aging  and in absence or presence of the specific vacuolar H+ ATPase (V-ATPase) inhibitor bafilomycin (100 nM). Bafilomycin prevents the maturation of autophagic vacuoles by inhibiting late-stage fusion between autophagosomes and lysosomes as well as lysosomal degradation21. Due to its ability to specifically target V-ATPase, bafilomycin disrupts autophagic flux, and is frequently used to study autophagy and endosomal acidification. Then we used bafilomycin to analyse the accumulation of autophagosome-engulfed mitochondria. Figure 42 incubation increased mitophagy as demonstrated the increase of autophagy markers colocalising with the mitochondria  upon amyloid incubation and bafilomycin increased the autophagosome accumulation in cells but much more under Aβ42 treatment condition  compared with wt mice group in which the reduction in the mitochondrial mass was evident at 12-month-old age. Imbalances in the mitochondrial fusion and fission proteins were found biased towards mitochondrial fission and therefore, to mitophagy. We confirmed the effect of Aβ overload by using rat primary neuronal cell cultures and SH-SY5Y cells subjected to oligomeric Aβ24.Here, we found that most of the proteins studied were found altered earlier in the brain cortex from APP/PS1 mice than in the hippocampus  or even remained unaltered (PGC-1a and Opa1). This could be related with the lower Aβ levels found in the hippocampus from APP/PS1 mice compared with those in the cortex. The increased autophagy activity found in the hippocampus of APP/PS1 mice from 6-month-old onwards might be enough to remove the expected increased amyloid peptide levels in this region. This would avoid the Aβ effects on the mitochondrial dynamics in the hippocampus. A novel explanation linking the damaged mitochondrial activity could be related to the effect of increased APP-derived fragments other than Aβ as was shown in different works from Area-Gomez group25. The reasons by which AD pathology occurs are still far to be elucidated, and a huge subset of factors may contribute to the origin of the disease. Such factors have built additional hypothesis other than amyloid cascade that may explain the origin of AD.Defective mitochondrial dynamic balance has been suggested to be one of the reasons as well as consequence of AD-related pathology. Autophagy in neurons is important under physiological and pathological conditions playing a crucial role for the degradation of Aβ. Although this could represent a controversial issue, a recent work established an interlink mechanism for the mitochondrial dysfunctioning, oxidative stress, autophagy dysregulation, and neuronal cell death in AD26. Reduced mitochondrial mass was also found in brain lysates and hippocampal sections from AD patients29. Consistent with the mentioned studies, we demonstrate here that mitochondrial mass reduction in AD could be consequence of Aβ accumulation and also we probe that in general this would be an early effect in the progression of disease. However it is worth to mention that in the hippocampus the loss of mitochondrial mass could be consequence of other additional disease-triggering events or undetectable but still pathological levels of Aβ as we already found such reduction in the 3-month old mice along with no evidence of amyloid overload.A reduction in the mitochondrial mass is recurrent in neurodegeneration. Neurons may die because energetic collapse due to the lack of power production which is carried out by the mitochondria. Related to AD, it was found a reduction in the amount and volume of mitochondria along with increased mitochondrial fragmentation and impaired mitochondrial transport in Aβ-treated mouse hippocampal neurons30. Our results in the cerebral cortex of APP/PS1 mice showing reduced PGC-1α levels at early age accounted of the reduced mitochondrial mass found in that brain region. This was consistent with the reduced PGC-1α levels and mRNA expression found in other previous studies33. In brains from AD patients, a relationship between reduced PGC-1α levels and damaged mitochondrial biogenesis was also previously demonstrated35. Other works also demonstrated that the reduction in the PGC-1α levels were consequence of Aβ overload using similar cellular models as in our present study37. In those cases authors also showed the link with reduced mitochondrial biogenesis. Hippocampal analysis of PGC-1α however did not account the reduced mitochondrial mass found in this region. Also we would like to mention that reduced PGC-1α levels in brain cortex form APP/PS1 mice were evident at 3-month old mice but the mitochondrial mass was reduced at 6-month old mice. For this reasons, we also searched other possible causes conditioning the mitochondrial mass that could add up to the reduction in case of the hippocampus or exert a compensatory mechanism in case of the brain cortex at early ages in the APP/PS1 mice brains. This could be the increased mitochondrial degradation by mitophagy, which is preceded by imbalances in the mitochondrial fusion/fission proteins such as Mfn1, Mfn2, Opa1 and Drp1 among others. We found a reduction in the levels of fusion proteins Mfn1, Mfn2 and L-Opa1 at early ages in brain regions from APP/PS1 mice. Contrarily, the levels of the fission protein Drp1 were found increased but this observation was not found as early as the reduction in the fusion protein levels. This could be explained because higher levels of amyloid accumulation are necessary to induce effects on Drp1 levels or because this fission protein could exert an early stage compensatory effect avoiding the mitochondrial degradation. This is consistent with the unchanged mitochondrial mass levels in the brain cortex from 3-month-old APP/PS1 mice where a reduction of the CxVβ levels was found from 6-month-old mice onwards. Again, our experiments in cells incubated with oligomerised Aβ confirmed that such effects were due to amyloid overload. Therefore we may conclude that in our AD mouse model, a disequilibrium between the fusion and fission processes exists and this may mimic AD pathology encouraging the mitochondrial fission and then mitophagy.Low amount of mitochondria in brain of APP/PS1 mice may reflect in some cases was found to be consequence of reduced mitochondrial biogenesis as we found in the present study. Mitochondrial biogenesis is controlled by several transcription factors and PGC-1α is the main regulator38 and an increase in the levels of mitochondrial fission proteins (Drp1)39. Also other AD transgenic mice exhibited increased mitochondrial fission processes in their brains42. In neuronal cell cultures from AD mouse models, authors demonstrated these imbalance encouraging the mitochondrial fission were demonstrated with increased fission and reduced fusion mRNA expression and protein levels26. Consequence of these alterations, mitochondrial morphology was found modified as they display small and rounded shape, and increased fragmentation with possible negative consequences in the mitochondrial function. The imbalance of the mitochondrial fusion and fission proteins in AD was previously proposed as an early event of the disease progression43, and these were supported by evidences of changes in the fusion and fission protein levels towards mitochondrial fission in brain tissue from AD patients44.Studies carried out in primary neurons described similar effects on mitochondrial fission and fusion events, in which the treatment with diffuse forms of the Aβ peptide showed a decrease in the levels of mitochondrial fusion proteins 45. We verified the increase in mitophagy in SH-SY5Ys cells after incubation with Aβ, obtaining increased colocalisation of autophagy markers p62 and LC3 with mitochondria, as well as mitochondrial accumulation after applying bafilomycin. Some works agree with our observations, also indicating that the Aβ peptide may be a trigger for mitophagy46. This was suggested by studies in which it was shown in mouse primary neuronal cell cultures that exposure to oligomeric Aβ42 induced imbalances in the transcription of genes encoding mitochondrial fission and fusion proteins, increasing mitochondrial fission and autophagy48.The fission of damaged mitochondria leads to their degradation by mitophagy50 while other works reported an increase51. It is possible that these variations within the same pathological conditions in p62 levels are related to the involvement of this protein in other cellular processes as p62 by interaction through its different functional domains or by variation experimental when analysing the levels of this protein in each of these works53.Our results show a dual effect of Aβ peptide overload. Aβ exerts a detrimental effect on mitochondria, possibly compromising their function and processes such as calcium homeostasis, ATP generation, and apoptosis. On the other hand, Aβ overload increased autophagic flux, as we have verified in our in vitro but also in our in vivo AD mouse models in which an increase in autophagic markers p62 and LC3-II was observed. There is some controversy regarding the interpretation of the levels of p62. Some authors have shown a decrease in the levels of this autophagy marker in brains from AD patients42 administration in SH-SY5Ys cells. The administration of bafilomycin in the cell cultures caused the levels of both p62 and LC3-II to increase, more significantly in the presence of Aβ42 indicating higher autophagy flux. Alterations in autophagy have been seen in in vitro and in vivo models of AD, and by analysis of postmortem AD brains54. Increased LC3-II levels have been found in the cerebral cortex and hippocampus of APP/PS1 mice at different ages, suggesting autophagosome accumulation in AD57. In addition to protein levels11, other authors have shown that LC3-II and p62 mRNA levels also increased after treatment with Aβ in vitro9.We have seen an increase of LC3-II upon oligomeric AβTaken together, our results show that the Aβ peptide produces a dual effect as we have shown in our in vitro experiments. This dual effect could be extrapolated to in vivo as we have seen that the autophagy process is exacerbated in APP/PS1 mice at early age and the mitochondrial dynamics imbalances also occur in these stages. Mitochondrial biogenesis and fusion/fission protein levels became altered with advanced age but these features occurred much late compared with the APP/PS1 mice, observation that could be attributable to aging as risk factor affecting mitochondrial activity. Although it has not been corroborated at the mitochondrial level, it is possible that these imbalances in the mitochondrial fusion and fission proteins end up triggering the elimination of damaged mitochondria and, therefore, confirming the double effect of the Aβ peptide in our in vivo model. It should be noted that, at older ages, despite the increase in autophagy, a greater accumulation of the Aβ peptide is observed, suggesting that, despite being able to be eliminated by autophagy, the production of Aβ would exceed elimination levels. This offers the possibility of studying in patients how the dual effect of Aβ can contribute both to its accumulation and to a reduction in energy production, both characteristic features of AD.40 and Aβ42) were confirmed along with time. Both Aβ isoforms result from the amyloidogenic processing of APP and are considered pathogenic. In the brain regions, protein levels of those related to mitochondrial biogenesis, dynamics and autophagy were analysed using biochemistry approaches. To confirm the role of Aβ in the observed alterations in brain tissue, the obtained results were also confirmed and studied in the human dopaminergic neuroblastoma cell line SH-SY5Y and neuronal primary cell cultures, both under incubation with oligomerised Aβ42 as this isoform is declared as the most toxic. In addition, the effect of Aβ on mitophagy was studied in the cell cultures analysing the levels and mitochondrial localisation of the main autophagy markers with the mitochondria. The biochemistry approaches utilised in cells were equivalent to those used in the mice brain tissue but also included immunofluorescence approaches. Below, the reagents, antibodies, animals, cell cultures and techniques utilised in this study are shown in detail.To analyse the effects of Aβ on the mitochondria along with the progression of AD, brain regions of 3-, 6- and 12-month old APP/PS1 mice as Alzheimer’s disease mouse model were obtained and analysed as well as the same regions from wild type mice with equivalent ages. In these models, the levels of Aβ isoforms .AβPrimary antibodies used were as follows: mouse anti-complex V β subunit , rabbit anti-peroxisome proliferator-activated receptor γ co-activator 1 α , rabbit anti- mtTFA , mouse anti-Mitofusin1 , mouse anti-Mitofusin2  mouse anti-mitochondrial dynamin-like GTPase , rabbit anti-Dynamin-related protein 1  mouse anti-β actin HRP , rabbit anti-p62/SQSTM1 , rabbit anti-LC3 . Secondary antibodies used for WB were as follows: goat anti-rabbit HRP secondary antibody , goat anti-mouse HRP secondary antibody . Secondary antibodies used for ICC were as follows: anti-rabbit  or anti-mouse .59 . APP/PS1 mouse model comes from a cross between Tg2576 (overexpressing human APP695) and mutant PS1 (M146L). As wild-type (wt) controls we used age-matched mice not expressing the transgene. For the sacrifice animals were deeply anesthetised. Then, they were perfused transcardially with saline before organ extraction for biochemical analysis. Sample size was estimated for each condition using the SPSS statistics software, assuming a normal distribution in all cases. This sample size is necessary to minimise the number of animals obtaining significant differences between groups and reducing the possible intragroup variability with an established statistical power of 95% confidence. In all cases, variability or standard deviation lower than the mean along with less than 10% of losses (mice deaths) are expected.3-, 6- and 12-month-old male double transgenic APP/PS1 mice were used from our inbred colonyFor rat primary neuronal cell cultures, pregnant Wistar rats (3–5 months) were obtained from the inbred colony of the Hospital Doce de Octubre Research Institute, Madrid, Spain.All animals were handled and cared for according to the Council Directive 2010/63/UE of 22 September 2010 and the revisited ARRIVE guidelines (2020).59. 4 × 105 cells per well were plated on poly-l-lysine coated coverslips in 6-well plates. Cells were maintained at 37 °C in a 5% CO2 humidified atmosphere in Neurobasal A medium  containing 2 mM l-glutamine, and 10% B27 Supplement . 9 days after cell culture preparation, cortical neurons were treated with or without oligomerised Aβ42 (1 μM). All live-cell imaging experiments were performed between d10 and d14 in culture. Human dopaminergic neuroblastoma cell line (SH-SY5Y) was purchased from the European Collection of Cell Cultures  and maintained as previously described60. Unless otherwise stated, SH-SY5Y cells were seeded at a density of 4 × 104 cells/cm2 and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 2 mM l-glutamine, and 1% (v/v) Penicillin/Streptomycin at 37 °C and 5% CO2. Cell cultures were incubated in fresh medium with or without oligomeric Aβ42 (1 μM).Primary cortical neuronal cultures were prepared from rat embryos (E15–16) based on methods previously described40 Aβ42 in cerebral cortex and hippocampus from APP/PS1 and wild type mice were determined using the human specific Aβ40 and Aβ42 ELISA kits , according to the manufacturer’s instructions. The obtained data were normalised to the protein content.Levels of Aβ58. Briefly, protein content from brain tissue or cell lysates was obtained using the NP-40 lysis buffer  and maintained at − 80 °C. The amount of protein was estimated using the BCA method . 20 µg of protein were loaded in SDS-PAGE gradient gels (4–20%). The gels with the separated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes . Each specific protein was determined using its specific primary antibody. Then, the appropriate horseradish peroxidase-conjugated secondary antibodies was used to bind each specific primary antibody and an enhanced chemiluminescence reagent  was used to reveal the immunocomplexes using the ImageQuant LAS 4000 system . Protein loading was monitored using a mouse monoclonal antibody against β-actin. Protein densitometric quantification was carried out using the Image Studio Lite 5.0 software . β-actin levels were used to normalise each protein levels and they are expressed as percentage of the control group. Upon requirements, in some cases blot membranes were cut to carry out simultaneous antibody hybridisations. In other cases blot membranes were covered during the digital exposure to avoid the signal of previous antibody incubations. These two points are specified in the legends of each corresponding figure within the supplementary information where the full-length blots are shown.Proteins were isolated from brain tissue or cell cultures by standard methods4 cells were plated onto sterilised coverslips in 24-well plates. 24 h later, (when cells reached 80% confluence approximately) cells were fixed with 4% paraformaldehyde in 0.1 M Dulbecco's phosphate buffered saline  for 10 min at room temperature. Cells were then washed three times in DPBS, blocked and permeabilised at room temperature for 45 min in 10% horse serum in DPBS with 0.1% Triton-X100 . Primary antibodies were prepared in the blocking/permeabilising solution and cells were incubated at 4 °C overnight. Secondary antibodies were used to reveal the primary antibody staining. Coverslips were mounted onto slides using the ProLong Gold Antifade Mountant with DAPI . Images were acquired with a Zeiss LSM 710 confocal microscope and processed by using the Zen 2009 software. Colocalisation analysis was carried out by using the Volocity software  and it was calculated following the instructions reported by the Software. For p62 and LC3 colocalisation with the mitochondria a number of 6 independent coverslips were analysed and results are referred as % of untreated cells. For experiments showing the p62 or LC3 levels, a number of 8 independent coverslips were analysed. For LC3-II and p62 analysis of mitophagy  experiments, 6 × 10In vivo and in vitro results are shown related to wt mice and untreated cells, respectively. All of them are expressed as the mean ± standard error of the mean (SEM) in percentage. For animal experiments, sample size estimation was carried out using the IBM SPSS Statistics software, Version 21.0. . Statistical analysis and exponential curve fitting were performed using the GraphPad Prism 6.01  software. Grubbs outlier filter was used for all data and Shapiro–Wilk normality test indicated that all results met normality criteria. For two-group comparisons, statistical analysis was carried out using Student’s t-test. For more than two-group comparisons, the one-way Analysis of variance (ANOVA) followed by Fisher’s post hoc test for multiple comparisons was used. For experiments carried out with animals at different ages, the two-way Analysis of variance (ANOVA) followed by Tukey’s correction test was used Statistical significance was set at p < 0.05.All animals were handled and cared for according to the Council Directive 2010/63/UE of 22 September 2010 and the revisited ARRIVE guidelines (2020) and approved by the Autonomous University Ethics Committee for Animal Experimentation (license number: CEI 97-1778-A291).Supplementary Figures.