Na+/Na. However, its poor electronic conductivity needs to be addressed to make this material attractive for practical applications among other anode choices. Here, we report a safe, controllable and affordable method using urea that significantly improves the rate performance of NTO by producing surface defects such as oxygen vacancies and hydroxyl groups, and the secondary phase Na2Ti6O13. The enhanced electrochemical performance agrees with the higher Na+ ion diffusion coefficient, higher charge carrier density and reduced bandgap observed in these samples, without the need of nanosizing and/or complex synthetic strategies. A comprehensive study using a combination of diffraction, microscopic, spectroscopic and electrochemical techniques supported by computational studies based on DFT calculations, was carried out to understand the effects of this treatment on the surface, chemistry and electronic and charge storage properties of NTO. This study underscores the benefits of using urea as a strategy for enhancing the charge storage properties of NTO and thus, unfolding the potential of this material in practical energy storage applications.Na A safe, controllable and affordable method using urea at mild temperatures to synthesise a series of sodium titanate samples with different levels of oxygen vacancies is reported. The formation of oxygen vacancies leads to the reduction of Ti4+ to Ti3+ ions in NTO, together with the formation of hydroxyl groups and a secondary phase, Na2Ti6O13.The urea‐treated samples showed superior electrochemical performance at high rates with respect to pristine NTO. The increasing demand for electrochemical energy storage devices has resulted in rapid development and utilisation of Li‐ion batteries (LIBs) in recent years.2Ti3O7 (NTO) phase as a SIB anode material in the last decade.−1) at a low and safe average potential of 0.3 V vs. Na+/Na.+ ions in NTO is commonly accepted to proceed through a two‐phase reaction mechanism that leads to the formation of the end‐discharge product Na4Ti3O7 (with the corresponding reduction of 2/3 Ti4+ ions to Ti3+).  °C (Eq. ):[21]2 gas then removes oxygen atoms from NTO creating anionic vacancies in the structure, as observed with EPR and XPS , genera23Na MAS NMR data , which may be explained by two different reaction mechanisms; HCNO is reported to decompose to NH3 and CO2 when H2O is present in the medium through a hydrolysis process [Eq. Furthermore, PXRD and tructure.10, 40 Figure S, which m2O is formed upon reduction of NTO (Eq. 7), it is likely that this reaction (Eq. 8) occurs. The generation of CO2 could result in the formation of Na2CO3, either by the reaction of Na2O (formed upon decomposition of NTO) with CO2, or through an exchange of Na+ in NTO with H+ ions from H2O.+ reacts with the OH− group of H2O, which in turn further reacts with CO2 to form Na2CO3.Since H+/Na during cycles 1 and 2 at 0.1 C (17.7 mA g−1) cycling rate. Almost identical load curves were observed for all the samples, except for an additional slope at 0.8–0.6 V vs. Na+/Na  in sample 20U, attributed to the insertion of Na+ ions into the Na2Ti6O13 impurity, observed with PXRD and 23Na NMR  cycling, cycling voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements were carried out to investigate the electrochemical properties of the as‐prepared electrodes. Figure S10 shows the galvanostatic charge/discharge voltage profiles of samples 0–20U in the voltage range 0.01–2.5 V vs. Na+/Na corresponds to the irreversible reaction of Na+ ions with the carbon additive.[+/Na are related to two‐phase transitions corresponding to Na2Ti3O7→Nax3−Ti3O7 and Nax3−Ti3O7→Na4Ti3O7 reactions,+ ions with concomitant reduction of Ti4+ to Ti3+ .[+/Na is observed, which corresponds to the extraction of Na+ from the Na4Ti3O7 structure.[+/Na is observed upon discharge, suggesting that the Na2Ti3O7→Nax3−Ti3O7 pathway process no longer occurs and, hence, Na2Ti3O7 transforms directly to Na4Ti3O7.7 Both NTO and urea‐treated samples show a very similar first discharge and charge capacities of ca. 420 mAh g−1 and 260 mAh g−1, respectively, with a low coulombic efficiency of ca. 60 % . The poor coulombic efficiency observed in the first cycle is explained by the irreversible reaction of Na+ ions with carbon (ca. 0.6 V vs. Na+/Na)[+/Na.[2CO3  does not seem to have a detrimental effect on the overall electrochemical stability of samples 10U and 20U. These results are in accordance with the data reported by Tsiamtsouri et al., who showed that Na2CO3 did not have an effect on the cycling stability.During the first discharge process, the plateau at 0.6 V vs. Naadditive.6, 11 2Ti6O13 do not lead to an improvement in the electrochemical performance at relatively low cycling rates. By contrast, galvanostatic cycling at 1 C (177 mA g−1) and 2 C (354 mA g−1) cycling rates  and 272 mAh g−1 (2 C) (cf. 281 (1 C) and 210 mAh g−1 (2 C) for 0U). 5U and 10U show initial capacities of 299 (1 C), 238 mAh g−1 (2 C) and 306 (1 C), 252 mAh g−1 (2 C), respectively. By the end of cycle 100, 20U shows a discharge capacity of 154 mAh g−1 (1 C) and 145 mAh g−1 (2 C), in contrast to 106 mAh g−1 (1 C) and 90 mAh g−1 (2 C) for 0U.Overall, a capacity retention upon charge of ca. 40 % was observed after 100 cycles. This could be the result of the formation of an unstable SEI layer and the continuous increase of the charge‐transfer resistance, commonly observed in previous studies.69, 70s Figure  show a m−1, respectively. Once the rate decreases from 10 C to 0.1 C, the discharge capacity of all the electrodes increases significantly, reaching approximately 80 % of their initial capacity. The capacity is kept fairly constant in the following five cycles, suggesting good structural stability and reversibility.[3+ and Ti‐OH species) and the formation of Na2Ti6O13 which enable high Na+ ion/e− mobility. These results outperform many of the NTO systems reported in the literature (Table S3).Figure sibility.9, 16 −1 in the voltage range 0.01–2.5 V vs. Na+/Na. In a typical CV for NTO at a low scanning rate, the first cathodic sweep involves the reaction of Na+ ions with the carbon additive at 0.36 V vs. Na+/Na and the insertion of Na+ into the layered NTO structure at 0.08 V vs. Na+/Na, concomitant with the reduction of Ti4+ to Ti3+ ions.[+/Na and becomes more severe at deep discharge voltages (0.01 V).[+ ions from the NTO structure is observed.[+ ions becomes more sluggish.+ ions is more facile than the insertion process.To gain further insight into the electrochemical behaviour of the different materials studied, CV and EIS measurements were conducted. Figure S12 shows the CV curves for samples 0–20U at various scan rates from 0.05 to 0.3 mV si3+ ions.11, 71+ ions, DNa+, can be determined using the Randles–Sevcik relationship (Eq. S2). The calculated intercalation DNa+increases with increasing defect content (i.e. 0U (1.2×10−11 cm2 s−1), 5U (1.7×10−11 cm2 s−1), 10U (2.7×10−11 cm2 s−1) and 20U (4.1×10−11 cm2 s−1)). Mott‐Schottky plots obtained from EIS recorded at a frequency of 500 Hz , 1.09×1023 cm−3 (5U), 4.14×1023 cm−3 (10U) and 2.02×1024 cm−3 (20U) using Eq. S3. Furthermore, EIS data were collected during the first discharge process at different states of charge (open circuit voltage (OCV≈2.5 V), 1.0, 0.4, 0.2 and 0.01 V vs. Na+/Na) at the 0.1 C cycling rate. The Nyquist plots derived from the EIS measurements on samples 0U and 20U in the frequency range from 100 MHz to 50 mHz are shown in Figures Rs at high frequencies, a charge‐transfer resistance (RCT) along with a constant phase element (CPE), a Warburg impedance (ZW) and a capacitance (C). The intercept at the Z’ axis in the high‐frequency range (Rs) is dominated by the resistance of the electrolyte to ion transport; the high‐medium frequency semicircle (RCT) refers to the charge‐transfer resistance for electrons and Na+ ions across the electrode‐electrolyte interface;CPE corresponds to the electrical double layer capacitor on the interface between the electrode and electrolyte; and the slope observed in the low‐frequency domain is attributed to the Warburg impedance (ZW) and corresponds to the Na+ ion diffusion in the bulk of the electrode, followed by the chemical capacitance of the electrode (C) at very low frequencies.RCT) are shown in Table S4. In the OCV state, the RCT of 20U is 40.0 Ω, much lower compared to 0U (i.e. 104.2 Ω). This suggests that the charge transfer process is enhanced when defects are generated in the sodium titanate structure, in accordance to the enhanced electrochemical performance observed at high cycling rates  and in the urea treatment (723 K).The stability of NTO is first analysed by comparing its thermodynamic stability with those of other stable phases reported for the Na‐Ti‐O system. This is achieved by obtaining stable atomic configurations of NTO and 19 stable compositions of Nat Figure . However2O and TiO2. As the temperature increases, calculations start to place the free energy of Na2Ti6O13 below the convex hull. This suggests that Na2Ti6O13 becomes thermodynamically stable at elevated temperature, allowing the decomposition of NTO into Na2Ti6O13 during the urea treatment. Further calculations reveal that, under urea treatment at 450 °C (=723 K), the decomposition of NTO can cause changes in the Gibbs free energy (ΔG) from −6.7 to 193.4 meV atom−1 . The calculated ΔG reveals that most decomposition processes where Na2Ti6O13 forms require energy smaller than the thermal energy imposed during the urea treatment, that is, kT=62.3 meV atom−1, where k is the Boltzmann constant and T is the absolute temperature (723 K). This further supports the formation of Na2Ti6O13 during the urea treatment as observed in the PXRD and 23Na NMR data . Figure d to NTO.39, 402Ti6O13 as the most plausible phase formed during the urea treatment, we next focussed on the effect of Na2Ti6O13 on the electrochemical performance in samples 5–20U. One effective way to do this is to analyse the band structures of the constituents Na2Ti3O7 and Na2Ti6O13 that affect the electrical conductivity of anodes. Previous DFT works have calculated the band structure and associated electronic properties of NTO.[2Ti6O13 using an HSE06 hybrid functional that provides a more accurate description of the electronic structures of Ti‐containing materials  is relatively lower than that of NTO (4.47 eV), which could partially explain the decrease in the bandgap of NTO after the urea treatment, as observed by diffuse reflectance UV‐VIS spectroscopy , where a bandgap of 3.63 eV in the 20U sample was obtained compared to 3.88 eV for pristine NTO. In the case of a composite of NTO and Na2Ti6O13, the effective bandgap measured during the experiments can be further lowered, as the energetic alignment of the VBM and CBM in each phase also affects the size of the effective bandgap.2Ti6O13, we projected the band structures onto atomic orbitals of Na, Ti and O and then aligned them with respect to the vacuum level, as illustrated in Figures 2Ti6O13 to be lower than NTO by 0.07 and 0.19 eV, respectively, causing an overall band structure of Na2Ti6O13 to lie below NTO. This alignment in band structures further decreases the effective bandgap at the interface such that the bandgap of the NTO composite anode is reduced by 0.19 eV, compared to NTO. The decrease in bandgap is in good agreement with the UV‐vis spectroscopy analysis , which showed a decrease of the bandgap by 0.25 eV after the urea treatment of NTO. Further examination of Figure 4+ to Ti3+ and, in turn, further decrease the bandgap of NTO anode. The decrease in bandgap could result in better electronic conductivity and, consequently, in an enhancement of the rate performance. In conclusion, the above calculations suggest that the enhanced anode performance of the urea treated samples is attributed to the synergetic effect of the Na2Ti6O13 secondary phase and O vacancy generated upon urea treatment.Having identified Nas of NTO.9, 74  Figures .[77,  Figures . A close4+ to Ti3+ ions in NTO, together with the formation of hydroxyl groups and a secondary phase, Na2Ti6O13. The urea‐treated samples showed superior electrochemical performance at high rates with respect to pristine NTO. The enhanced electrochemical performance agrees with the higher Na+ ion diffusion coefficient, higher charge carrier density and reduced bandgap observed in these samples, without the need of nanosizing and/or complex synthetic strategies. The optimal electrochemical performance among the series is found in sample 20U, which displays initial discharge capacities of 316 mAh g−1 (1 C) and 272 mAh g−1 (2 C) and discharge capacities of 154 mAh g−1 (1 C) and 145 mAh g−1 (2 C) which are significantly higher than those observed in the pristine material (106 mAh g−1 (1 C) and 90 mAh g−1 (2 C)) after 100 cycles. The creation of oxygen vacancies has shown to be a promising strategy to improve the performance of NTO electrodes for SIBs by circumventing their inherent low electronic conductivity. However, a complete understanding of the role of oxygen vacancies in the electrochemical performance of NTO is still very limited at this stage. There are still questions that remain unanswered due to their challenging quantification and spatial distribution. In addition, to fully comprehend the role of oxygen vacancies, other factors including the generation of Ti3+ and Ti‐OH groups and the formation of Na2Ti6O13 must be excluded. Similarly, it is equally important to understand the individual contribution of these components in the overall electrochemical performance of NTO. The method for enhancing the electrochemical performance demonstrated in this study could be extrapolated to other anode materials that suffer from poor Na+ ion/e− kinetics, to potentially foster the practical application of advanced SIB anode materials beyond hard carbons.In summary, we have reported a safe, controllable and affordable method using urea at mild temperatures to synthesise a series of sodium titanate samples with different levels of oxygen vacancies. The formation of oxygen vacancies leads to the reduction of TiThe authors declare no conflict of interest.Nuria Tapia‐Ruiz obtained her Ph.D. from the University of Glasgow (UK) in 2013. She then worked as a Research Fellow in the team of Prof. Bruce at the University of St. Andrews and the University of Oxford (UK) in 2013–2016. Currently, she is a Senior Lecturer in the Department of Chemistry at Lancaster University. Her research interests include the understanding of the structure‐property‐performance relationships in materials for energy storage such as monovalent and multivalent batteries and supercapacitors.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Nlgn1 transcript, which was followed by a clear decrease. Lastly, the absence of NLGN1 decreased neuronal counts in the dentate gyrus, which was not the case in wild-type animals, and worsens impairment in spatial learning following chronic hippocampal Aβo injections. Our findings support that NLGN1 is impacted early during neurodegenerative processes, and that Aβo contributes to this effect. Moreover, our results suggest that the presence of NLGN1 favors the cognitive prognosis during Aβo-driven neurodegeneration.Synapse loss occurs early and correlates with cognitive decline in Alzheimer’s disease (AD). Synaptotoxicity is driven, at least in part, by amyloid-beta oligomers (Aβo), but the exact synaptic components targeted by Aβo remain to be identified. We here tested the hypotheses that the post-synaptic protein Neuroligin-1 (NLGN1) is affected early in the process of neurodegeneration in the hippocampus, and specifically by Aβo, and that it can modulate Aβo toxicity. We found that hippocampal NLGN1 was decreased in patients with AD in comparison to patients with mild cognitive impairment and control subjects. Female 3xTg-AD mice also showed a decreased NLGN1 level in the hippocampus at an early age . We observed that chronic hippocampal Aβo injections initially increased the expression of one specific  Hippocampus-dependent memory for recent facts and events (explicit memory) is among the first type of memory that is affected in the disease because of the neurodegenerative process that rapidly and gradually takes place in the hippocampus at the onset of AD5. More precisely, within the hippocampal network, the perforant path that connects the entorhinal cortex to the dentate gyrus (DG) is one of the earliest and most severely affected pathways in AD7. This suggests that the DG is among sites showing initial signs of synaptic dysfunction, including in glutamatergic transmission8, and that it could represent an area of particular relevance for early interventions.Synapse loss is an early event in the pathogenesis of Alzheimer’s disease (AD) and correlates with cognitive decline of the patients1-42 peptides18. Soluble Aβo start to accumulate in the human brain ~10 to 15 years before clinical symptoms and were shown to induce losses of excitatory synapses and neurons22. The oligomerization of Aβ is rapid and it has been suggested that Aβ toxicity results from multiple small Aβ species present at the same time, rather than from one particular type of soluble oligomer24. Although the toxicity (including synaptotoxicity) of soluble Aβo is well established, specific synaptic components that are altered when Aβo begin to increase in the brain at the onset of AD remain to be identified. Identifying Aβo-driven changes in proteins specifically involved in excitatory synapse functioning is required to understand glutamatergic synapse dysfunction and loss, which likely takes place before neurodegeneration induces substantial and irreversible brain damage permanently affecting cognition and autonomy.Despite decades of research focused on the amyloid-beta (Aβ) peptide, its causal role in AD pathogenesis remains unclear at the molecular level. However, strong evidence suggests that soluble Aβ oligomers (Aβo) are particularly neurotoxic and that their presence can correlate with memory deficits in AD patients and animal models, especially when considering oligomers derived from Aβ27. Neuroligin-1 (NLGN1) predominantly localizes at excitatory post-synaptic densities28, and previous work has linked it to neuropsychiatric and neurological disorders such as autism, schizophrenia and stroke31. Interestingly, NLGN1 has also been shown to be involved in synaptic plasticity, N-methyl-D-aspartate (NMDA) receptor function, memory and sleep regulation33, which are all altered in AD. The relevance of NLGN1 (and NRXNs) in the context of AD neurodegeneration has been emphasized in the last decade35. In fact, a single dose of Aβ1-40 fibrils has been shown to negatively impact NLGN1 in rats, which impaired synaptic function and memory36. In addition, Aβo were shown to bind to NLGN1 in vitro, and interfering with this interaction was observed to modulate synaptic integrity38. Moreover, NLGN1 was recently shown to be decreased in the plasma of patients with AD as well as in the preclinical period39. Nonetheless, to the best of our knowledge, it remains to be determined whether NLGN1 is altered in the hippocampus of AD patients as well as whether it is involved in the specific hippocampal pathology induced in vivo by soluble low-molecular-weight Aβo1-42.Neuroligins (NLGNs) are post-synaptic adhesion proteins that interact with pre-synaptic protein neurexins (NRXNs) and have roles in synapse formation, maturation, maintenance and plasticity1-42 injection in the hippocampus. In addition, we tested whether the absence of NLGN1 aggravates memory impairment and neuronal losses caused by Aβo1-42 using chronic hippocampal Aβo1-42 injections combined to immunohistochemistry and assessments of spatial and working memory. We found that the level of NLGN1 is decreased in the hippocampus of aMCI and AD patients and in young 3xTg-AD female mice, and that hippocampal Aβo1-42 injections decreased neuronal count in the DG and induced spatial learning deficits predominantly in Nlgn1 knockout (KO) mice. Our results provide support to the hypothesis that NLGN1 is impacted early during Aβ pathology and that it modulates cognitive functions during Aβo-driven neurodegeneration.Therefore, we here aimed to fill this knowledge gap using quantifications of the NLGN1 level in the hippocampus of patients with AD as well as in two animal models with Aβ-driven neurodegeneration. Importantly, we assessed the time course of the effect on NLGN1 by performing quantifications also in patients with amnesic mild cognitive impairment (aMCI), in triple transgenic (3xTg-AD) mice of 4, 12 and 18 months, and in mice submitted to 2, 4 and 6 days of Aβo40. AD and aMCI were diagnosed using clinical evaluations as previously described40. Briefly, cognitive status, neurologic and physical examinations were performed annually or biannually with a follow up of at least 2 years before death. All subjects had no comorbidity with substance abuse, head injury, encephalitis, meningitis, epilepsy, stroke, infectious disease or major psychiatric illness. Mini-mental state examination (MMSE) score was used as an indicator of overall cognitive status41, with a lower score being indicative of deficits in memory, attention, orientation and/or language. MMSE score was on average 24.4 and 7.8 in aMCI and AD patients, respectively  available to avoid protein degradation  were provided by the brain bank of the Alzheimer’s Disease Center of the University of Kentucky42, and control non-transgenic mice of the same genetic background  were produced and maintained at the animal facility of the Research Center of the Centre Hospitalier de l’Université Laval as previously described43. Male C57BL/6 J, Nlgn1 KO mice and wild-type (WT) littermates were used for chronic Aβo1-42 injections. C57BL/6 J mice (n = 41) were purchased from Jackson Laboratories and submitted to cannula implantation surgery at 13 weeks (see below). Mice heterozygous for the Nlgn1 mutation  were purchased from Jackson Laboratories, backcrossed with C57BL/6 J mice for >10 generations, and bred at the animal facility of the Research Center of the Hôpital du Sacré-Coeur de Montréal. KO and WT mice were implanted with cannulas for intra-hippocampal Aβo injections at 24 ± 10 weeks. Animals were housed individually and maintained in a 12 h light/12 h dark cycle at a temperature of 24 ± 1 °C with food and water available ad libitum. The experiment involving 3xTg-AD mice was approved by the Animal Welfare Committee of the Université Laval according to guidelines of the Canadian Council on Animal Care. All other experimental procedures were approved by the Comité d’éthique de l’expérimentation animale of the Hôpital du Sacré-Coeur de Montréal (Recherche CIUSSS-NIM) also in accordance with guidelines of the Canadian Council on Animal Care.Male and female 3xTg-AD  mice1-42 and control Aβ1-42 scrambled (AβScr) solutions were performed as previously described12. Briefly, synthetic Aβ1-42 and AβScr  were suspended in hexafluoroisopropanol (HFIP) . The HFIP was evaporated with nitrogen gas and Aβ1-42 peptides were resuspended in DMSO . The solution was eluted through a HiTrap desalting column  previously equilibrated with a Tris-EDTA solution . Aβ1-42 peptides were quantified using the BCA protein assay kit  and stored at −80 °C. The average concentration of the solutions used in this study was 0.28 ± 0.04 μg/μL. The biophysical and biological properties as well as the level of oligomerization of this Aβ preparation have been previously characterized46. The Aβ solution was kept at room temperature for 1 h before injection to induce oligomerization and produce soluble low-molecular-weight Aβo1-42 as previously done12. Soluble Aβ quantification in post-mortem tissue was performed by a standard three-step serial extraction procedure as described elsewhere for these samples40. The Aβ quantification was not available for 4 CTRL, 6 aMCI and 4 AD patients , installed on a stereotaxic apparatus and maintained under anesthesia using 1% isoflurane. Stereotaxic coordinates of the two cannulas implanted just above the DG were defined as 2.3 mm posterior to the bregma, ±1.5 mm from midline and 1.8 mm below the skull surface. Three screws were inserted into the skull to stabilize cannulas fixed to the skull with dental cement. Mice were given buprenorphine (0.1 mg/kg) immediately after the surgery and the day after to ensure sufficient analgesia.The cannula implantation surgery was adapted from the procedure previously described47. In brief, one week after recovery, freely moving mice were injected with Aβ solutions or an equal volume of control AβScr or vehicle  at a constant rate of 0.2 μl/min via cannulas and PE50 tubing (Plastics One) connected to a Hamilton syringe pump system . The tubing was left in place for 5 min after each injection and cannulas were capped after tubing removal to prevent reflux of the injected solution. Injections were performed during the first half of the light phase and were repeated for 2, 4 or 6 consecutive days.The injections were conducted as previously described38. For mouse experiments, hippocampal tissues were homogenized in cold RIPA buffer 1X  with 0.5% CHAPS  and phosphatase and protease inhibitors . Samples were sonicated, gently agitated at 4 °C for 1 h, and centrifuged at 14000 g for 20 min at 4 °C. The supernatant containing total proteins was collected and the protein concentration was quantified using the Pierce BCA protein assay kit. Thirty μg of proteins per sample were separated by electrophoresis using Bolt 4-12% Bis-Tris gels  and transferred onto a PVDF membrane . Membranes were incubated 1 h with Odyssey blocking buffer (Type PBS)  at room temperature and incubated overnight at 4 °C with anti-NLGN1 antibodies , anti-PSD95 antibodies , or anti-GAPDH antibodies  diluted in Odyssey blocking buffer (Type PBS) containing 0.2% Tween 20 . Membranes were then washed and incubated with corresponding secondary antibodies  diluted in Odyssey blocking buffer (Type PBS) containing 0.2% Tween 20 and 0.01% SDS for 1 h at room temperature. Membranes were revealed using the Odyssey CLx  with Image Studio 3.1 software. For some blots, chemiluminescence was visualized using an ECL detection kit . Quantitative densitometric analysis was performed using ImageJ and expressed relative to GAPDH. At least one control sample was loaded on each blot as a calibrator for quantification. Full length blots are presented in Supplementary Information.Proteins extracted from human hippocampi represented the membrane fraction as detailed elsewhere50. Briefly, hippocampal mRNA was extracted using the RNeasy Lipid Tissue mini kit according to manufacturer’s instructions . Reverse transcription was performed with SuperScript II Reverse transcriptase  following the manufacturer’s instructions including 10 min at 25 °C and 60 min at 42 °C. QPCR was performed using 384-well plates with a mix of RNAase free water, TaqMan Fast Advanced Master Mix , and primers and probes for each gene . Three endogenous control genes  were quantified to normalize expression level of Nlgn1 variants and of related mRNA. Gene expression was measured using a Viia 7 thermal cycler (Thermo Fisher) with the following cycling conditions: 2 min at 50 °C, 20 sec at 95 °C, 40 times 1 sec at 95 °C and 20 sec at 60 °C. The analysis was performed with Expression Suite (Life Technologies), which calculated gene expression relative to endogenous controls and to one sample of the control group (modified delta delta Ct).mRNA extraction, reverse transcription and qPCR procedures were performed as previously described12. Briefly, pregnant Long-Evans female rats were ordered from Charles River Laboratories and sacrificed at embryonic day 18. Hippocampi were dissected, transferred to a tube containing HBSS , HEPES , penicillin/streptomycin/amphotericin  and 3 mM sodium pyruvate  at 37 °C, and were treated with 0.25% trypsin , agitated at 900 rpm for 15 min at 37 °C, and dissociated by trituration. Cells were platted in 12-well plates  coated with poly-D lysine hydrobromide  and containing minimum essential medium  supplemented with 10% horse serum , 3 mM sodium pyruvate, 25 mM D-Glucose , 2 mM GlutaMax , and 1% penicillin/streptomycin/amphotericin. Plates were incubated at 37 °C with 5% CO2 overnight, and the initial culture medium was replaced the day after with Neurobasal medium  supplemented with 2% B27 , 2 mM GlutaMax, and 0.5% penicillin/streptomycin/amphotericin. Primary hippocampal cells were cultured for 7 days before starting treatments. For Western blot analysis, cells were washed with cold 10 mM PBS, scratched off the wells with 140 uL of cold RIPA 1X buffer containing CHAPS and phosphatase and protease inhibitors, and rapidly stored at −80 °C. Protein extraction and Western blotting were performed as described above.The primary neuronal culture procedure was carried out as described previouslyueous One Solution Cell Proliferation Assay  according to the manufacturer’s protocol. Briefly, the CellTiter 96 AQueous One Solution reagent was added to the Neurobasal medium containing the cells (v/v ratio of 1:5) and after 1.5 h of incubation at 37 °C the absorbance was recorded at 490 nm. The tested conditions were normalized to values obtained with water-treated wells.Cell viability was measured using the CellTiter 96 AQNlgn1 KO mice and wild-type (WT) littermates similar to previously described52. It was done in an opaque plastic arena (45 × 45 cm) with the floor covered with litter that was mixed after each trial to eliminate olfactory cues. The four walls contained different visual cues and two different objects were placed in adjacent corners of the arena. The walls and objects were cleaned with 70% ethanol between each mouse. During the first day, mice explored the arena and objects for 5 min. Twenty-four hours later, one of the two objects was moved to the opposite corner of the arena and mice were reintroduced in the arena for 5 min. The number of interactions with each object, defined as a touch of the snout or sniffing of the object by the mouse, was counted by an experimenter blind to the genotype and treatment. The discrimination index (DI) was calculated for the number of interactions according to the following formula: (moved object - fixed object)/(moved object + fixed object).The SOR task was conducted 24 hours after the last hippocampal injection  to extract time spent in the new arm and the average time spent in the other two arms. The walls were cleaned with 70% ethanol and the litter was mixed between each mouse to avoid olfactory cues.A two-trial Y-maze task was performed 48 hours after the SOR test . Starting location of the mice was different for each trial. During the probe trial on the fifth day, the platform was removed and mice had 60 sec to explore the pool. The time spent in the target quadrant that previously contained the platform and in the other quadrants, as well as the number of times the mouse crossed the area around the platform (2x the diameter of the platform) were calculated with the Smart software. A final cued task of 3 trials was also performed to verify visual acuity and motivation to escape from water. During this test, mice had a maximum of 30 sec to reach a visible platform that was moved in a different quadrant between each trial. Data were acquired in real time with the Smart software.The MWM task was conducted 48 hours after the Y-maze test  at −20 °C and 10 μm coronal slices were mounted on SuperFrost Plus slides . Hippocampal sections were performed at ±100 μm from the middle of the injection site. Brain slices were mounted in series on 6 slides, with 4 slices per slide. Slides were fixed for 15 min in 10% formalin  and washed with PBS . Sections were immersed in a blocking solution , 0.3% Triton X-100  for 60 min at room temperature and incubated overnight at 4 °C with anti-NeuN antibodies  diluted in PBS 1X, 1% BSA  and 0.3% Triton X-100. The slides were washed, and incubated with a secondary antibody  for 90 min at room temperature and protected from light. Slides were then washed, dried and one drop of Prolong Gold Antifade DAPI  was applied on each section before being covered with a coverslip .Neurons were marked with the NeuN antibody for neuronal counting purposeA Zeiss AxioImager 2 fluorescence microscope, a monochrome camera, and the StereoInvestigator software (MBF Biosciences) were used to acquire IF images. Wavelength filters of 488 nm and 350 nm were used to excite the fluorochromes of the secondary antibody and of DAPI. Neuronal counting was performed using the Neurolucida Software (MBF Biosciences). The 20X objective was used to precisely identify the depth of the injection site. Two mice were excluded from neuronal count and memory assessments because the cannula was positioned outside the target region. Using the 40X objective, a 500 μm wide area was defined in the DG under the injection site as well as in CA2-CA3 distant to the injection site. Since there was little or no cell overlay using 10 μm sections and that NeuN labeling was well defined, the function Detect Cells of the Neurolucida program could discriminate between individual cells. This function made it possible to make a reliable cell count in the area under the injection site and the distant area, which has been validated by manual counting by a blind experimenter. Three to four sections per slide were counted; the average of these sections was calculated and analyzed according to genotype and treatment.Statistical analyses of multiple samples were performed using appropriate analyses of variance (ANOVAs). One-way ANOVA was used for human sample analysis; two-way ANOVAs were used to analyze results of 3xTg-AD mice, cell culture, chronic injections, IF, and most variables of the SOR and MWM tests; three-way ANOVA was used for the MWM training phase. Significant effects were decomposed, when appropriate, using planned comparisons. Student t tests were used for some analyses of the SOR and the Y-maze. Pearson’s correlations were performed to quantify the association between soluble Aβ, MMSE and NLGN1 level. The sizes of the different samples were determined on the basis of previous studies in the field. Statistical analyses were performed using GraphPad Prism 6.0 and/or JMP 14. Significance threshold was set at 0.05 and the levels of statistical significance are identified with *p < 0.05, **p < 0.01, ***p < 0.001. Data are reported as mean ± SEM. The number of samples used for each experiment is reported in figure legends.56. We found that aMCI individuals have lower levels of NLGN1 in the hippocampus compared to age-matched CTRL without cognitive impairment . This commonly used AD model shows beta-amyloid plaques and neurofibrillary tangles with intracellular Aβ accumulation in the hippocampus at 4 months that correlates with cognitive deficits1-42 could directly impact NLGN1 level in vitro, and whether this is linked to neuronal survival. NLGN1 protein level and cell viability were measured in primary culture of hippocampal neurons treated with 2 μM of Aβo1-42 for 48 or 72 hours. As expected from our previous study12, Aβo1-42 significantly decreased neuronal viability by about 40% after 72 hours of treatment  and unchanged after 48 hours  hippocampal injections in WT mice. In addition, we assessed whether Aβo1-42 affect Nlgn1 in a transcript variant-dependent manner, which can be done by measuring mRNA expression using different probe sets targeting the presence or absence of the two NLGN1 inserts50. Two days of Aβo1-42 injections significantly increased the hippocampal expression of amplicons Nlgn1NA (p < 0.04) and Nlgn1C (p < 0.02) in comparison to control injections , Nlgn1NB (p < 0.05) and Nlgn1C (p < 0.01)  Fig. . The Nlgons Fig. . Also, h 2, 4 or  days hipin vivo damage. We first investigated the implication of NLGN1 specifically in Aβo-driven memory impairment using assessments of spatial and working memory, selected for their dependency on the hippocampus59. This was done using Nlgn1 KO mice and WT littermates submitted solely to 4 days of injections of Aβo1-42 or AβScr , WT mice injected with Aβo1-42 (p = 0.01) and KO mice injected with AβScr (p = 0.02), indicating memory for spatial location , suggesting impaired spatial memory . When all trials of the 4 training days are considered separately . The first trial of each day was proposed to reflect memory retention57, for which deficits could be induced by Aβo1-42 independent of the genotype. Most importantly, Nlgn1 KO mice injected with Aβo1-42 showed a significant overall higher latency to the platform than all other groups , which is also illustrated in Fig. 1-42 on spatial learning in the absence of NLGN1.Spatial memory was further tested in the MWM. As expected for the learning phase, the latency to reach the platform progressively decreased over training days Fig. . This deely Fig. , Aβo1-421-42 and control injections  are indicative of spatial memory. Beforehand, we verified that the swimming speed did not significantly differ between WT and KO mice with Aβoons Fig. . This coant Fig. . Taken t1-42 injections. This was assessed using the same mice as above tested for spatial and working memory. Neuronal loss was measured in the DG in an area located under the injection site  exposure to Aβo1-42. Neuronal count in a region distant to the injection site  revealed no significant difference between the four groups  in the hippocampus of aMCI patients53. Our observation that the NLGN1 decrease is of greater magnitude than the PSD95 decrease emphasizes the particular relevance of considering the role of NLGN1 in neurodegeneration. Such a role is also supported by the observation of a NLGN1 mutation affecting its function in a patient with AD35. Moreover, the finding that NLGN1 level in the aMCI/AD hippocampus correlates with cognitive function supports the idea that synaptic dysfunction adequately predicts cognitive decline in AD22. Even if the exact mechanisms underlying NLGN modification in aMCI and AD remains to be identified, plasmatic NLGN1 or even hippocampal NLGN1 measured via imaging may be considered as an early biomarker of AD.To our knowledge, our study is the first to show that NLGN1 level in the hippocampus is decreased in aMCI individuals and AD patients. This result is in line with the recent report that NLGN1 is decreased in the plasma of AD patients and in individuals with less severe cognitive symptoms57. This model generally shows signs of neuronal death, human Aβ precursor protein (hAPP) deregulation, and Tau pathology later in life61, but these may all contribute to NLGN1 decrease together with alterations in Preselinin-1 in this model. The observation that only the females show this early decrease likely resides in the worst pathology observed in 3xTg-AD females in comparison to 3xTg-AD males. Indeed, 3xTg-AD females have repeatedly been reported to have, in particular, higher hAPP and Aβ levels than males63. Alternatively, a compensatory mechanism linked to the encoding of other NLGNs by sex chromosomes65, could also contribute to the sex-dependent decrease in NLGN1. Of interest is also that we found a genotype-independent decrease in NLGN1 level with age in male mice . This age-dependent decrease in hippocampal NLGN1 could definitely contribute to the main risk factor for neurodegeneration, namely age. More precisely, a progressive decrease in NLGN1 in the hippocampus may increase the susceptibility of this key brain region to synaptic and cell loss, such as under exposure to Aβo.Importantly, we observed that the decrease in hippocampal NLGN1 protein is also an early event occurring in a recognized mouse model of AD . Indeed, decreased NLGN1 was found at 4 months, an age at which cognitive symptoms are just starting to manifest and have been attributed to intraneuronal Aβ1-42 reduces NLGN1 level in cultured hippocampal neurons, similar to a recent study66. From these data, we cannot exclude a direct effect of neuronal death on NLGN1 level because the NLGN1 decrease paralleled the neuronal viability decrease. Still, these findings are revealing a synaptic component linked to the established neurotoxicity of Aβo as well as their correlation with cognitive functioning in AD patients and animal models18, given the roles of NLGN1 in cognition and its cellular correlates64. Of note is that Aβ1-40 fibrils, which are generally considered less neurotoxic than Aβo19, have also been shown to negatively impact NLGN1 protein level in the rat hippocampus36. The precise manner by which Aβo are decreasing the level of NLGN1, as well as the contribution of neuronal death, remains a question of particular relevance to evaluate the potential for NLGN1 to become a therapeutic target. Previous reports have indicated that Aβo are directly interacting with NLGN1 in vitro38. Such a direct interaction may contribute to an increased NLGN1 protein degradation. Alternatively, the Aβo-induced NLGN1 decrease could result from changes in Nlgn1 gene expression linked to epigenetic modifications such as what has been observed for Aβ fibrils36.Our discovery that NLGN1 level in the hippocampus of aMCI individuals and AD patients significantly correlates with soluble Aβ could suggest a specific contribution of the Aβ pathology in decreasing NLGN1 level. We indeed specifically found that exposure to AβoNlgn1 gene expression is indeed supported by our findings in chronic hippocampal injections. Importantly, the different durations of Aβo treatment we have used allowed for the observation of a bi-phasic time course comprising an initial increase followed by a decrease. Moreover, this expression pattern was found only for specific Nlgn1 variants and not for other variants or other related genes . In fact, the initial increase was observed for Nlgn1 without insert in splice site A, and the following decrease for Nlgn1 without insert in sites A and B. These changes were substantial enough to be also captured by the common probe, but strongly suggest that the expression of Nlgn1 without insert is primarily affected by Aβo. In contrast with other isoforms, the isoform of NLGN1 not containing any insert is located equally at glutamatergic and gamma-aminobutyric acid (GABA) synapses67. This could suggest that the negative impact on NLGN1 could play a role in GABAergic synapse dysfunctions observed in AD and animal models69, as well as in glutamatergic synapse modifications21. Besides, the initial increase in Nlgn1 expression after 2 days of Aβo exposure might belong to a neuroprotective pathway similar to other pathways driven by Aβ in the hippocampus70. NLGN1 was already shown to protect against oxidative stress in a non-mammalian model71 and to express neuroprotective functions in hippocampal neurons72. Alternatively, lower Nlgn1 expression at 4 days of Aβo exposure may result from an increased expression with the vehicle injection, which could also be linked to a neuroprotective role of NLGN1 in response simply to intra-hippocampal injections. This effect would again be very specific to Nlgn1 lacking any insert and would be counteracted by Aβo exposure. The fact that a modulation by chronic Aβo hippocampal injections could not be detected at the protein level likely results from the absence of an isoform-specific antibody. Indeed, a change in NLGN1 isoform without insert could be masked by the absence of change in NLGN1 with insert B, which transcript is highly expressed in the hippocampus67.An impact of Aβo on 1-42. Firstly, Nlgn1 KO mice injected with Aβo1-42 were the only group showing memory impairment in SOR. A similar finding was made in the MWM training phase, during which Nlgn1 KO mice injected with Aβo1-42 showed a longer overall latency to reach the platform. Deficits have been observed in KO mice with a lower exposure/dose than those generally inducing impairments in SOR and the MWM task in WT animals74. Nlgn1 KO mice were previously shown to have only a very subtle deficit in the MWM and to have a preserved locomotion33, which are in line with our observations of very few deficits in vehicle-injected KO mice. The fact that KO mice injected with Aβo1-42 were impaired specifically in the MWM training phase, whereas Nlgn1 KO mice are not33, also emphasizes a more specific implication of NLGN1 in modulating Aβo-induced deficits in learning or in memory acquisition. Of note is that this effect of NLGN1 seems restricted to spatial memory given that working memory assessed using the Y-maze was not affected by chronic hippocampal injections of Aβo. Although a longer exposure of the hippocampus to Aβo was shown to impair memory using a similar task12, the lack of effect in the current study is likely resulting from working memory involving to a great extent the cerebral cortex76. The manner by which NLGN1 modulates spatial learning deficits mediated by Aβo will need to be determined in future studies. Yet, the literature could point to mechanisms related to direct physical interactions between NLGN1 and Aβo38 potentially sequestering toxic oligomers, or linked to NLGN1 cleavage that occurs in response to sustained synaptic activity72.In the second part of our study, we provide data supporting a modulatory role of NLGN1 on spatial learning deficit and neuronal loss mediated by exposure of the hippocampus to AβoNlgn1 KO mice. The absence of NLGN1 thus seems to increase the neurotoxicity of Aβo by inducing neuronal loss following an intermediate exposure  whereas neuronal loss is generally obtained from longer exposure  or higher doses74, similar to memory deficits. It should be noted, however, that the NeuN staining used here is not a direct indication of neuronal death, but that other accepted markers of neurodegeneration such as Caspase-3 and Fluoro-Jade B77 could not be used in the present study due to the sacrifice of animals being performed multiple days after exposure to allow for the evaluation of memory. Nevertheless, our finding supporting that NLGN1 can oppose Aβo-driven neurotoxicity in the DG is in line with observations that a soluble fragment of NLGN1 diminishes the negative impact of Aβo on excitatory synaptic transmission in hippocampal slices38 and that NLGN1 is part of a pathway counteracting Aβo-induced synaptotoxicity in cultured hippocampal neurons66.Secondly, neuronal count in the DG was decreased only in in vitro and in vivo models, that this change is likely driven, at least in part, by Aβo and contributing to cognitive decline. In parallel, our data expose a bi-directional relationship between NLGN1 and Aβo, in which the accumulation of Aβo1-42 negatively impacts NLGN1, and decreased NLGN1 renders neurons/synapses more vulnerable to Aβo1-42 translating in memory deficits and neuronal loss. Our findings support a potential for strategies enhancing NLGN1 to alleviate symptoms of AD, which reconciles with previous research78, including with that reporting NRXN-NLGN mutations in AD patients35. On the contrary, our study does not support approaches opposing NLGN1 as a therapeutic strategy in AD such as the use of anti-NLGN1 antibody37. The current research provides additional insight in the development of early diagnostic tools and treatments of aMCI and AD, which is of crucial importance to benefit the quality of life of millions of AD patients worldwide.We here reveal that NLGN1 is decreased in the hippocampus of patients with prodromal signs of and established neurodegeneration in a symptomatology-dependent manner and, using Supplementary Fig. 1 to 8.


Moreover, we show experimentally that gas-phase HOIO2 is not necessary for the formation of HOIO2-containing particles. These insights help to explain new particle formation in the relatively dry polar regions and, more generally, provide for the first time a thermochemically feasible molecular mechanism from ocean iodine emissions to atmospheric particles that is currently missing in model calculations of aerosol radiative forcing.Emitted from the oceans, iodine-bearing molecules are ubiquitous in the atmosphere and a source of new atmospheric aerosol particles of potentially global significance. However, its inclusion in atmospheric models is hindered by a lack of understanding of the first steps of the photochemical gas-to-particle conversion mechanism. Our laboratory results show that under a high humidity and low HO “How iodine-bearing molecules contribute to atmospheric aerosol formation is not well understood. Here, the authors provide a new gas-to-particle conversion mechanism and show that clustering of iodine oxides is an essential component of this process while previously proposed iodic acid does not play a large role.” The characterization of global iodine sources4 and the demonstration of its ubiquitous presence in the marine boundary layer (MBL)6, the free troposphere8, and the stratosphere9 have enabled an assessment of the important role of iodine in O3 depletion in past, present, and future climate scenarios15. Iodine oxide-driven new particle formation was first observed in mid-latitude coastal locations18. Its high nucleation and growth rates may enable new iodine particles to survive fast scavenging by background aerosol19 and to develop into sea mist and fog20. Iodine oxide particles (IOPs) have also been observed in the polar MBL23. Field observations in diverse locations with completely different meteorological and chemical conditions suggest a wider relevance of this phenomenon in the global atmosphere.The impacts of iodine on tropospheric and stratospheric chemistry were anticipated by seminal work published in the 80s and 90s26. For example, both high temperature and humidity slow down IOP growth and reduce IOP number density relative to colder/drier conditions26. The I2O3 and I2O4 molecules have been observed in the laboratory and linked to IOP formation in dry conditions27. However, the O/I ratio measured in the particles collected has been shown to be closer to I2O526. I2O5 is the anhydride form of iodic acid (HOIO2), which is the proposed composition of liquid IOPs in the humid MBL28. Recently, field observations of IO3− and HIO3− containing cluster anions by chemical ionization–atmospheric pressure interface–time-of-flight mass spectrometry (CI-API-ToF-MS) have been reported23. These observations, which employ nitrate (NO3−) and acetate (CH3COO−) reagent ions for reaction with the analytes, have been interpreted as resulting from atmospheric gas-phase HOIO2 and molecular cluster formation via HOIO2 addition steps. Well-established gas-phase kinetics and thermochemistry do not indicate any straightforward route to HOIO2. The OIO + OH reaction is predicted to yield HOIO229, but the concentration of OH in the MBL is far too low to explain IOP formation. It has been proposed that water plays an important role in generating HOIO2 and IOPs30, although a strong anticorrelation between particle production probability and water vapor has been observed in the field31. In order to form HOIO2, H2O must be able to intercept some of the initial products of the reaction between atomic iodine and O3, i.e., IOx  or I2Oy. The bimolecular reactions of IOx with H2O are precluded by their large endothermicities, and termolecular reactions form only weakly bound clusters32. A reaction of any of these H2O…IOx adducts with O3 to form oxyacids requires substantial bond rearrangement and barriers are expected. Recent ab initio studies have found a substantial barrier in the potential energy surface (PES) of the I2O5 + H2O ↔ 2HOIO2 reaction33, precluding not only formation of HOIO2 but also a nucleation mechanism depending on the formation of I2O5 from the HOIO2 self-reaction30.Laboratory studies have investigated the effect of different environmental variables on nm-sized IOPs2 does not require gas-phase HOIO2, implying a limited role of oxyacids in IOP formation, which is instead initiated by clustering of IxOy. Based on our experiments and calculations, an iodine gas-to-particle conversion mechanism is proposed.Here, we present results from flow tube kinetic experiments in order to fill the knowledge gap between gas-phase atmospheric iodine molecules and IOPs. Our results show that water reacts very slowly with atomic iodine and iodine oxides, and that forming particulate HOIO3 and I2 at 10 Torr of N2, and 10.5 eV photoionization (PI). This “dry” spectrum shows positive ion peaks generated by near-threshold PI (Supplementary Table + (m/z = 127), IO+ (143), OIO+ (159), I2+ (254), I2O+ (270), I2O2+ (286), I2O3+ (302), and I2O4+ (318). A peak corresponding to I2O5+ (m/z = 334) is absent. The predicted vertical ionization potential of I2O5 is 11.4 eV, but this molecule is not observed with the 11.6 eV PI beam either, as shown in Fig. 3O7+ (493), I5O12+ (827), I7O17+ (1161), I9O22+ (1495), I11O27+ (1829), and I13O32+ (2163). Minor peaks which indicate up to four missing oxygen atoms are also observed. Starting from I3O7+ and moving to higher m/z, the peaks are separated by I2O5 units nOIO+. Molecular clusters with even numbers of iodine atoms  are also detected but are very minor.We have carried out laboratory flow tube kinetic experiments to look at IOP formation and evaporation  = 2%) at 10 Torr results in a limited reduction of the OIO+ and IxOy+ signals n(HIO3)0–223) are observed, and no clusters with an even number of iodine atoms emerge. The calculated ionization potential of HOIO2 is 11.3 eV, yet again no signal is observed on a time scale of 4 ms when the PI beam is tuned to 11.6 eV  a2O) and minimizing the effect of competitive reactions. At 350 Torr and x(H2O) = 2%, for PLP-PI delays of up to 5 ms, the peaks observed at low pressure in the mass spectrum remain, and oxyacid clusters do not appear  and m/z = 335 (HI2O5+) do not exhibit kinetic change and are decoupled from photolysis-induced chemistry. Thus, they probably result from photofragmentation of large H2O…IxOy clusters or are produced from deposits around the ToF-MS skimmer.Observing slow reactions requires increasing the concentration of the excess reactant HO and min2-forming reactions can be obtained from kinetic modeling of our BBP experiments, and are discussed below. Separate flow tube measurements of atomic iodine by resonance fluorescence (ROFLEX)35 were performed to study the removal of I atoms in the presence of MBL-representative concentrations of O3 and H2O at 760 Torr. A slight enhancement of the I atom removal by O3 in the presence of water was observed  = (2.9 ± 1.0) × 10−19 cm3 s−1 molecule−1 for x(O3) = 92 ppbv and k(I + H2O + O3) = (2.0 ± 0.5) × 10−18 cm3 s−1 molecule−1 for x(O3) = 184 ppbv.We have also performed high-pressure steady-state experiments using continuous BBP. In these experiments, a residence time of a few seconds combined with continuous generation of I atom and high pressure should help in enhancing the concentration of slow-reaction products. Yet again, oxyacid products are not observed under these conditions Fig. . Upper l2 inlet under dry conditions . Thus, the observed HOIO2 and HOI cannot be products of gas-phase high-temperature IxOy + H2O reactions, but evaporation products of IOPs formed upstream in the presence of water. Under dry conditions, the oxyacid evaporation signals reduce drastically. The other effect of heating IOPs is the expected thermal decomposition of solid/liquid-phase iodine oxides into molecular oxygen and iodine37 .Visual inspection of the BBP experiment through a viewport revealed a plume of smoke formed at the IIO2 Fig. . BBP of 38 are considered as a sign of fragmentation, especially if the IO and OIO traces track the growth of larger molecules and clusters. Data at high IOx concentration clearly suffer from this problem  = 334 (I2O5), while I2O5 itself does not appear to form. Thus, I2O2, I2O3, and I2O4 are considered to be the precursors to iodine oxide molecular clusters. However, they do not appear to aggregate stepwise to form clusters. The ion clusters detected have the specific elemental composition In+12On+m5+ , i.e., odd numbers of iodine atoms. By looking at Supplementary Table 4Oy species may fragment to smaller ions, they cannot fragment to I3Oy+, and therefore we assume that the latter corresponds to I3Oy molecules formed in the flow tube.The I2Oy form generally weak adducts with themselves, and this may explain why the I4Oy signals are negligible and, as a consequence, clusters with even numbers of iodine atoms do not form32. The most stable predicted adduct is I4O8, formed from the I2O4 self-reaction. However, our ab initio calculations suggest that this reaction also has an exothermic bimolecular channel making I3O7. Alternative routes to I3Oy are I2Oy + IOx (y ≠ 3) followed by ozone-oxidation steps. Ozone has been found not to be required to form IOPs as long as an alternative source of oxygen atoms is available, but its presence leads to enhanced particle formation26. Thus, it could help in increasing the O/I ratio within each peak progression if the peaks with lower O/I form first from I2Oy + IOx.There are many possible reactions that may generate the observed peaks in the PI mass spectra. Thermochemical data are required to eliminate irrelevant reactions from the chemical mechanism Table . Reactio2O3, in particular, does not form stable adducts with other IOx and I2Oy (including itself)32, and we have not found any exothermic bimolecular reaction between I2O3 and IOx or I2Oy nOIO), e.g.:The rapid kinetics and smoke formation are astonishing, considering that the pressure is relatively low and that all reactions are second order. Removal rates of ~50 sces Fig.  may be o3. HOIO2-containing clusters23 have not been observed, and therefore no attempt has been made to model them.In addition, third-body reactions at their high-pressure limit form aggregates, e.g.:2 are reactions of IOx and I2Oy with water listed in Table 2Oy + H2O . The gas-phase I2O2 reaction  rule out I2Oy reactions in the air-water interface (see “Methods”).Possible sources of gas-phase HOIO and HOIO2. Moreover, the co-products of these reactions HOI and HOIO are very weak or absent from the BBP mass spectra obtained at 10.5 eV. This suggests that these reactions are slow and cannot compete under our experimental conditions with the reactions removing IxOy in the “dry” experiments. This, however, does not rule out the possibility that under atmospheric conditions, the situation reverses as a result of the low ambient IOx  and higher water concentrations. Thus, upper limits to the rate constants of these reactions need to be determined as a first step to establish the relative importance of the oxide and oxyacid clusters. This has been done by kinetic modeling of our long-residence time BBP steady-state experiments at 10.5 eV and 11.6 eV PI energy, where each process in Table m/z = 140–200 Da range. It is assumed that the PI cross section of each iodine oxyacid is equal to that of the nearest oxide. The upper limit of each rate constant k(IxOy + H2O) is taken to be that for which the simulated mass spectrometric signal at m/z = 144, m/z = 160, and/or m/z = 176 becomes indistinguishable from the background noise of the BBP experimental data. These upper limits can then be compared with rate constants calculated with the master equation solver MESMER39 using the molecular parameters  derived from our ab initio calculations 34. Second, the water concentration is one order of magnitude higher as a result of the higher pressure (the S/N ratio at 10.6 eV was not high enough to perform experiments above 30 Torr). The upper limits are listed in Table 2O2 + H2O and I2O4 + H2O reactions, the rate constant cannot exceed, respectively, 1 × 10−19 cm3 s−1 molecule−1 and 5 × 10−19 cm3 s−1 molecule−1. The upper limit of k(I2O3 + H2O) is 5 × 10−18 cm3 s−1 molecule−1, but owing to the large barrier of the I2O3 + H2O reaction  present, respectively, a submerged barrier of −7.5 kJ mol−1  = 92 ppbv (k(I + H2O + O3) = (2.9 ± 1.0) × 10−19 cm3 s−1 molecule−1) appears to agree with the upper limit determined from the ToF-MS experiments: k(I + H2O + O3) < 2.8 × 10−19 cm3 s−1 molecule−1. However, this is coincidental. The O3 concentration in the ToF-MS experiments was three orders of magnitude larger than in the ROFLEX experiments, indicating that the rate constant in the former should be, if linearly extrapolated from the ROFLEX result, of the order of 2.9 × 10−16 cm3 s−1 molecule−1, i.e., well above the detection limit of ~10−18 cm3 s−1 molecule−1. Thus, the yield of HOIO and HOIO2 from this process is <0.001, implying an oxyacid-formation rate constant of the order of 10−22 cm3 s−1 molecule−1 for atmospherically relevant O3 concentrations. Therefore, the composite I + H2O + O3 reaction is not a sufficiently fast source of HOIO2. This is very important because, as discussed in Supplementary Note xOy: otherwise, oxyacid nucleation needs to proceed through two slow second-order processes to form the first cluster. To explore the relative contribution of iodine oxides and oxyacids to the nucleation of IOPs, we have modeled a BBP flow tube experiment with longer residence time (200 s). Calculated rate constants of oxyacid–oxyacid and oxide–oxyacid aggregation and dissociation rates are listed in Supplementary Table 2O] concentrations between laboratory experiments23 and ambient). Despite the many assumptions involved in this exercise, a conclusion that can be drawn is that molecular clusters descending directly from HOIO and HOIO2 can only become dominant at high water concentration (>5 × 1016 molecule cm−3), and if the direct precursor of these oxyacids is atomic iodine. If the precursors of these oxyacids are oxides—which is more likely—nucleation is started by iodine oxides.The upper limit determined for the rate constant of a hypothetical composite reaction where atomic iodine complexes with water, and the resulting Hids Fig. a, e can  = 2.9 ± .0 × 10−1xOy bimolecular and third-body reactions, both under dry and wet conditions. The mechanism of atmospheric IOP formation proposed in Fig. 2 are constituents of these particles, even though we did not observe them in the gas phase at room temperature, which implies that they are not indispensable IOP gas-phase precursors. This evidences the transformation of the IxOy clusters into oxyacid-containing clusters within the flow tube residence time, which confirms earlier predictions about the composition of I2O5 IOPs in the MBL28. This scenario is represented in Fig. 2O5 units present in the iodine oxide clusters with the composition (I2O5)nOIO. We reported previously that the number and the size of IOPs reduces when they are formed in the presence of water26. Particle capillary collapse following water uptake could not alone explain those observations. The attachment of water molecules to molecular clusters may have the effect of reducing cluster formation and particle coagulation rates. At the molecular cluster level of the mass spectrometric observations discussed here, the addition of water results in a consistent reduction of the intensity of all masses without concomitant formation of products, which we have interpreted as inhibition of photofragmentation of larger clusters. We found previously32 that the key iodine oxide I2O4 forms a relatively stable complex with water, such that under the conditions prevailing in the MBL, a large fraction of I2O4 molecules would be hydrated as I2O4…H2O (> 40%). This could block the I2O4 halogen-bonding sites, and the subsequent necessary geometrical rearrangement for the reaction of I2O4 with itself or with IxOy to proceed may result in barriers in the corresponding PES. The same may occur for larger molecular clusters. It should be noted that IxOy…H2O clusters are not observed in our experiments, which may be because they promptly dissociate following absorption of vacuum ultraviolet (VUV) photons.Our laboratory results strongly support an atmospheric gas-to-particle conversion mechanism where the initial clustering steps are driven by I9. This would be favored by low water concentrations, but it must be noted that if the mixing ratios of IxOy are low, the clustering process slows down compared to photolysis  mixing ratios are in general lower than in macroalgae-rich mid-latitude locations5, and consequently lower IxOy mixing ratios should result in slower IOP formation rates  from I2Oy in the atmosphere  and NO3− (and CH3COO−), and therefore it is likely that I2Oy contributes significantly to the IO3− signal. The apparent paradox is that the IO3− CI-API-ToF measurements23 remains a very useful proxy for tracking iodine nucleation in the field, because it may result from ionization of I2Oy and/or HOIO2 generated by I2Oy + H2O. Nevertheless, connecting the dots between these observations and those of precursors and smaller gas-phase species 5 is crucial to facilitating the development of a chemical mechanism that can be adapted for atmospheric modeling purposes. To our best knowledge, ours is the first complete iodine gas-to-particle conversion mechanism  was introduced in the reactor, depending on the target pressure. I2 molecules were entrained in the carrier flow by passing a smaller flow of carrier gas (10–500 sccm) through a heated, temperature-controlled flask containing I2 crystals. O3 was added by flowing O2 (10–200 sccm) through an electrical discharge. The excimer laser and the lamp beams were passed unfocussed through a quartz viewport along the tube main axis. An atmospheric pressure bubbler was employed to entrain water vapor into the carrier flow at approximately the equilibrium vapor pressure, resulting in a water mixing ratio of ~2% in the flow tube ([H2O] = 2.3 × 1017 molecule cm−3 at 350 torr and 295 K). The pressure in the reactor was set by a throttle valve placed between the reactor and an Edwards 80 roots blower—oil rotary pump combination. The flows were set using MKS calibrated mass flow controllers, and pressure was measured using a set of 10 Torr and 1000 Torr MKS Baratron pressure transducers. O3 and I2 concentrations were measured using a Herriott-type absorption cell situated upstream of the reactor42.A PLP system  gas-phase species was achieved using the older of these two systems27. The newer machine employed in this work has been described in detail in a previous publication46.PI-ToF-Ms systems at Leeds University have been employed extensively to study reaction kinetics relevant to combustion-related and tropospheric organic and halogen chemistryP = 10−4−10−5 Torr) before mass spectrometric detection. Alternatively, a differentially pumped roughing chamber can be inserted between the reactor and the PI chamber for experiments requiring high pressure (P > 20 Torr). In this configuration, the gas is sampled via a 500-μm pinhole into the roughing chamber, forming a high-density jet that is directed toward the 200-μm pinhole skimmer cone. The PI chamber is fitted with viewports, allowing a pulsed laser beam to be directed through the high-density region of the sampled gas jet. VUV ionization is achieved by tightly focusing in a rare-gas cell (Xe) the third harmonic of a Nd:YAG laser (Continuum Surelite 10-II), which produces VUV radiation by frequency tripling 48. The 118.2-nm radiation is delivered at the PI chamber by a waveguide, which is a ¼” glass tube with either constant inner diameter or tapered for enhanced PI efficiency. This technique shows an excellent detection limit for organic molecules such as acetone43 , which is used as a standard for optimizing the performance of the instrument. The conversion efficiency of frequency tripling in a rare gas is low (~10−5), and therefore VUV PI is a viable method for molecules with substantial PI cross-sections (>10−18 cm2 molecule−1). Possible interference from the 355-nm fundamental, if not separated from the VUV third harmonic, needs to be considered. This is important for molecules with UV–vis absorption bands, low ionization energy and high two-photon PI cross-sections. For example, water is detected at m/z = 18  PI beam can also be generated by frequency tripling the second harmonic of a 532-nm pumped dye laser running on DCM dye. The power of this beam is approximately an order of magnitude lower than that of the 10.5 eV beam.The gas in the reactor tube . The mass peaks appear in the time axis of the oscilloscope delayed with respect to the PI laser pulse by a characteristic time of flight. The peaks are then gate-integrated and passed on to a PC for further analysis. A limitation of this method is that strong signals cause detector overload after the corresponding peak during a significant lapse, which appears as a negative signal with respect to the spectrum baseline. Thus, peaks of other species arriving at the detector closely after the species causing a large peak are affected by this interference. Overloads can be eliminated by tuning down the detector gain, but this also reduces the sensitivity to small signals. To prevent detector overload by I+ and I2+, their mass peaks were gated out by pulsed biasing of the ion optics using a house-built dual-gating box.The positive ions resulting from PI of molecules in the sampled gas jet are accelerated toward the ToF-MS (Kore Technology) by a set of ion optics. This machine has been described in detail elsewhereThe digital output is provided to a counting system  by means of a fast comparator. The Kore Technology TDC counts ions within a sequence of flight-time bins (0.5 ns) referred to the PI laser pulse. In this way, a histogram of counts versus time of flight,  is built. The counting method is suitable for capturing small signals (low concentration or low PI efficiency). The signal remains proportional to concentration for low counting rates. Although spectra measured with this technique are not affected by detector overload, for high counting rates, departure from linearity occurs and the peaks appear saturated.Lasers and detectors are synchronized using a delay generator . In every measuring cycle, the delay generator triggers the Nd:YAG laser and after a few microseconds triggers the TDC or the scope to establish the time zero event of each measuring cycle. In addition, the delay generator triggers the excimer laser that initiates chemistry by photolyzing a radical precursor. The delay between the photolysis and the PI laser is scanned in order to sample reaction kinetics. In a typical digital experiment, 1000 digital spectra were accumulated for every reactant concentration and laser pulse delay, which at a 5-Hz laser-repetition rate takes 200 s. The built-in software of the MS-ToF does not handle synchronization of the delay generator and the TDC, and therefore the delays were set manually, at the cost of a poorer time resolution. Once the accumulation was complete, the delay between laser pulses and/or concentration of reagents was changed, and a new accumulation started. Kinetic experiments using the digital output are shown in Fig. 35 in an atmospheric pressure BBP flow tube set up. A 3–15 slm flow of synthetic air carrying molecular iodine (~1010 molecule cm−3) and ozone (~1012 molecule cm−3) was passed to a 4-cm-diameter, 50-cm-long quartz tube where it was irradiated by a Xe arc lamp. The main flow and flow ratios were varied to keep the concentration of I2 and O3 constant while varying the residence time of the gas mixture on the tube. The gas mixture was sampled at the end of the flow tube by using a small aperture into the ROFLEX chamber at 40 Torr. Iodine atomic resonance fluorescence is excited by a temperature-stabilized iodine radiofrequency lamp, and collected perpendicularly by using a VUV-sensitive channel photomultiplier in counting mode. The data collected in these experiments is plotted in Supplementary Fig. Additional experiments to study the influence of water on the removal of atomic iodine were carried out with the Resonance and off-Resonance Fluorescence by Lamp Excitation (ROFLEX) machineThe processed PI-ToF-MS data consist of integrated peak signals  versus laser delay time in the case of PLP experiments, or a single mass spectrum in the case of BBP experiments, which correspond to a single time point, time being the residence time in the flow tube. The data obtained with the ROFLEX machine comprises the iodine atomic resonance fluorescence signal versus residence time in the flow tube.Simple chemical systems can be described by sets of ordinary differential equations (ODEs) that are analytically solvable. In these cases, the observed data can be fitted to analytical expressions using a least-squares method, from which reaction rates can be extracted. This can be done for example in the case of the resonance fluorescence experiments if we assume that the only relevant reactions describing the behavior of atomic iodine are:2, an effective iodine loss rate in the presence of water can be extracted by simply fitting exponential decays to the time traces and calculating the differences between the removal rates with and without water. This, however, would not allow the concentration of O3 to be estimated because IO reactions recycling atomic iodine are ignored:Because of the fast photolytic removal of I50.Multiplexed detection systems like the PI-ToF-MS provide simultaneous data from many molecules. To explain their kinetic traces, complex mechanisms with many reactions need to be constructed  and pressure (1 atm).All gas-phase quantum-mechanical calculations reported in this work were performed using the Gaussian092O2 + H2O ↔ HOIO + HOI and I2O3 + H2O ↔ HOIO2 + HOI reactions in the gas-phase . Normal-mode vibrational frequency analyses were carried out to ensure that the stable minima had all positive vibrational frequencies. Energies were refined at the CCSD(T)54 level of theory with the aug-cc-pVTZ+LANL2DZ basis set. For all reactions, unscaled vibrational frequencies calculated with the M06-2X/aug-cc-pVDZ+LANL2DZ method were used to estimate the zero-point energy (ZPE) corrections.The I30. The droplet system contained 191 water molecules and one I2Oy  molecule. The simulations were carried out in the constant volume and temperature (300 K) ensemble. The integration step was set as 1 femtosecond, which has been proven to achieve sufficient energy conservation in water systems58. The I2O2, I2O3, and I2O4 systems were simulated for 50, 80, and 33 picoseconds, respectively (Supplementary Videos A detailed description of the methods employed in our Born–Oppenheimer Molecular Dynamics (BOMD) simulations can be found in a previous publication59. This may be described as a supplemented (15s12p6d)/[10s9p4d] 6-311G basis, the  contraction scheme being supplemented by diffuse s and p functions, together with d and f polarization functions. The full basis set is given in Table XIII of Glukhovtsev et al.59 Following geometry optimizations of the reactant and product molecules and ions and the determination of their corresponding vibrational frequencies and (harmonic) ZPE, energies relative to the reactants were obtained. Geometry optimizations and energy calculations for most of the molecules listed in Table 60. The additional calculations in this work have been carried out for IxOy with y = 3, 4 and HIOz with z = 2, 3. Ionization energies and reaction enthalpies obtained at B3LYP/6-311 + G (+AE) level of theory compare reasonably well with experimental values and calculations at the higher level reported in the literature 63 is used to calculate the microcanonical association rates from an estimate of the high-pressure limiting-rate coefficient for the molecular association. The microcanonical dissociation rate coefficients are then determined by detailed balance. Collisional energy transfer probabilities are described by using the exponential down model. For further details, see Galvez et al.32 and references therein.To determine rate constants from ab initio data, we have used the Master Equation Solver for Multi-Energy well Reactions (MESMER)Supplementary materialPeer Review FileDescription of Additional Supplementary InformationSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4


Experimental and computational mechanistic investigations reveal that upon H2 activation, the covalently bound pyridonate substituent becomes a datively bound pyridone ligand. Dissociation of the formed pyridone borane complex liberates Piers borane and enables a hydroboration of the allene. The allylboranes generated in this way are reactive towards nitriles. A catalytic protocol for the formation of allylboranes from H2 and allenes and the allylation of nitriles has been devised. This catalytic reaction is a conceptually new way to use molecular H2 in organic synthesis.Here we report the in situ generation of nucleophilic allylboranes from H 2 and allenes is reported. The reaction is catalyzed by pyridonate borane that displays frustrated‐Lewis‐pair reactivity. The allylboranes that are generated in this way are reactive towards nitriles. Mechanistic investigations show that a change in the binding mode of the pyridonate substituent upon hydrogen activation is essential for the formation of the allylboranes.The in situ formation of nucleophilic allylboranes from H Furthermore, a reaction sequence consisting of allylation of an electrophile by the allylborane formed in this way, followed by a protodeborylation mediated by the pyridone 5, would regenerate the pyridonate borane 3. That would enable us to realize an allylation that requires only catalytic amounts of the pyridonate borane 3.Piers borane 3 in benzene and the reaction mixture was exposed to H2 (1.1 bar). This led to the formation of a new borane complex that was assigned to be the pyridone allylborane complex 7 in 66 % yield . However, this reaction did not yield the expected allylimine, but the β‐diketiminate borane complex 9 together with the bispyridone complex 10 =409 nm with a molar attenuation coefficient of ϵ359=6928 cm−1 M−1 (see SI for full spectra).The β‐diketiminate borane complex 9 presumably originates from a nucleophilic attack of the enamine tautomer of an intermediately formed allylimine to acetonitrile. To verify this hypothesis and to check if an allylimine was formed as intermediate en route to 9, the allylborane 8, in situ generated by hydroboration of phenylallene, was reacted with one equivalent acetonitrile. This reaction furnished the ketiminoborane 11, which is the allylation product of acetonitrile, in 83 % yield. The formation of 11 demonstrates that allylborane 8 is indeed nucleophilic 2 fragment is irreversibly bound in 9 which inhibits any further catalytic reactivity. We envisioned that the addition of a strong Lewis acid would enable us to capture the intermediately formed allylimine prior to the β‐diketiminate borane complex formation 3 the formation of the allylimine borane complex 13 from phenylallene, acetonitrile, and dihydrogen in presence of catalytic amounts of the pyridone borane complex 4 (10 mol %) was observed . Thus, the two isomers, fully characterized by NMR and ESI‐MS, were obtained in a 93:7 ratio after column chromatography. The molecular structure of 13 in the solid‐state was further analyzed by SCXRD .To prove that the catalytic activity of s Scheme . Electro20 forms upon coordination of acetonitrile to B(C6F5)3. In agreement with previous investigations, the computations reveal that the catalytic cycle commences with hydrogen activation by the pyridonate borane 3. The pyridone borane complex 4 that is formed in this way dissociates to liberate Piers borane 6. As a stoichiometric amount of B(C6F5)3 is present, we considered that the pyridone 5 coordinates to the B(C6F5)3, which overall renders the liberation of Piers borane 6 thermodynamically more favorable. The hydroboration of the terminal allene requires passing a moderate barrier of 8.4 kcal mol−1 and is according to the computations exergonic by 27.6 kcal mol−1.The mechanism of this novel catalytic transformation was further addressed by dispersion‐corrected Density Functional Theory (DFT) computations at the revDSD‐PBEP86‐D4/def2‐QZVPP//PBEh‐3c level 3 complex 21 and recoordination of 5, a virtually barrier‐less intramolecular proton transfer yields the allylimine complex 26. Based on our experimental findings, we propose that the allylimine complex 26 is the common intermediate for the formation of the β‐diketiminate borane complex 32 and the allylimine B(C6F5)3 complex 28. In the absence of B(C6F5)3, an intramolecular proton transfer from the methyl group of the allylimine to the pyridine nitrogen that requires a Gibbs free activation energy of 21.3 kcal mol−1 yields the pyridone enamine borane complex 29. Dissociation of this complex, thermodynamically favored by formation of the bispyridone complex 10, enables the nucleophilic addition of the enamine to acetonitrile via the six‐membered transition state 30/31TS. While the C−C bond formation is already exergonic, the tautomerization to the diketiminate borane complex 32 provides a further decisive driving force to the reaction. In the presence of B(C6F5)3, the kinetically favored pathway commences with the dissociation of the allylimine complex 26 that is endergonic by 14.4 kcal mol−1. Coordination of the B(C6F5)3 to the free allylimine 27 yields the allylimine B(C6F5)3 complex 28. The coordination of B(C6F5)3 to the allyimine 27 imparts a barrier of 25.3 kcal mol−1 for the tautomerization to the pyridone enamine borane complex 29 and therefore suppresses the formation of the β‐diketiminate borane complex 32. However, according to the computations, the allylimine B(C6F5)3 complex 28 must be regarded as the kinetic product while the β‐diketiminate borane complex 32 is the thermodynamic product of the initial allylation. To verify this computational result experimentally, the isolated allylimine B(C6F5)3 complex 13 was reacted with one additional equivalent MeCN and the pyridonate borane 3 at an elevated temperature of 80 °C  should be addressed to the authors.SupplementaryClick here for additional data file.


This paper presents a detailed assessment of the performance of the CRYO ARM 300 electron microscope in a multiuser cryo-EM facility setting. It is shown that the microscope can be used for reliable automated data collection. The strengths and weaknesses of the hardware are discussed. Single-particle cryo-EM has become an indispensable structural biology method. It requires regular access to high-resolution electron cryogenic microscopes. To fully utilize the capacity of the expensive high-resolution instruments, the time used for data acquisition and the rate of data collection have to be maximized. This in turn requires high stability and high uptime of the instrument. One of the first 300 kV JEOL CRYO ARM 300 microscopes has been installed at the cryo-EM facility BECM at VIB-VUB, Brussels, where the microscope is used for continuous data collection on multiple projects. Here, the suitability and performance of the microscope is assessed for high-throughput single-particle data collection. In particular, the properties of the illumination system, the stage stability and ice contamination rates are reported. The microscope was benchmarked using mouse heavy-chain apoferritin which was reconstructed to a resolution of 1.9 Å. Finally, uptime and throughput statistics of the instrument accumulated during the first six months of the facility operation in user access mode are reported. At the Biological Electron Cryogenic Microscopy (BECM) facility at VUB/VIB, Brussels, one of the first new-generation cryogenic 300 kV microscopes from JEOL, the CRYO ARM 300, has been installed.et al., 2019The microscope is equipped with a 300 kV cold FEG (cFEG), four-lens condenser illumination system, high-contrast objective lens, autoloader for 12 cryo-grids, phase plate, in-column Ω energy filter and K2 direct electron detector (Gatan). In this configuration, the microscope has been used for a period of six months of continuous user access and high-throughput data collection. Although the suitability of the CRYO ARM 300 has been reported for collection of high-resolution data  was loaded onto the stage from the autoloader storage. Images were recorded at a magnification of 60 000 on a K3 detector at intervals of 30 s for a period of 30 min. A total of 100 movie frames with per frame exposure of 0.038 s were acquired for each time point. In both cases the trajectory of the sample movement was determined by movie frame alignment in −2, as defined in RELION2.0 and later) until the end of exposure were used. The total drift was estimated either by measuring the distance between the first late exposure frame and the last frame (referred to as the end-to-end method) or by summing up the absolute shifts between consecutive frames . The specific method used is indicated in the figure legends and the choice of method is explained in Section 3.1.2To calculate the drift, movie frames corresponding to late exposure . The SerialEM scripts initially provided by JEOL application scientists were significantly modified and extended to allow for versatile scenarios of data collection including sparse focusing and ice thickness measurements (see the supporting information).Automated data collection was performed using 2.5.E. coli, cells were grown in Luria–Bertani medium at 37°C, with 0.5% glucose and 50 µg ml−1 kanamycin, to an OD at 600 nm of 0.5. The protein expression was induced with 1 mM IPTG  for 4 h. Subsequent steps were all performed at 4°C. The cells from a 1 l culture were harvested by centrifugation at 6500g for 15 min and resuspended in 20 ml lysis buffer , supplemented with 50 µg ml−1 DNase I (Merck/Sigma–Aldrich) and 1 mg ml−1 lysozyme (Merck/Sigma–Aldrich). After 1 h incubation while stirring, cell debris was spun down at 39 000g for 30 min. The supernatant was heated to 70°C while gently shaking, followed by another round of centrifugation at 4°C. Ammonium sulfate was added to the supernatant to a final concentration of 52.5%(w/v). This suspension was centrifuged at 39 000g for 1 h at 4°C and the pellet was resuspended in 2 ml purification buffer . Overnight dialysis against the purification buffer was used to remove the remaining ammonium sulfate. The dialyzed sample was concentrated to approximately 0.9 ml, with a 50 kDa cut-off Vivaspin 2 Ultrafiltration concentrator (Sartorius) and ran on a size-exclusion Superose 6 10/300GL column  equilibrated in the purification buffer. The peak fractions containing pure apoferritin were concentrated and stored at −80°C after flash freezing in liquid nitrogen. For cryo-EM grid preparation, purified apoferritin at a concentration of approximately 4 mg ml−1 was centrifuged at 50 000 rev min−1 for 10 min at 4°C to remove aggregates. A small amount of sample (3 µl) was applied to 300 mesh UltrAuFoil holey Quantifoil R 2/2 grids that were glow-discharged for 1 min in an ELMO glow discharge system (Cordouan). Grids were blotted for 4 to 6 s with the offset of −1 mm and humidity of 98% at room temperature, after which, they were immediately plunged into liquid ethane using Cryoplunge 3 (Gatan).The plasmid with mouse heavy-chain apoferritin was supplied by Dr H. Yanagisawa at the University of Tokyo. After transformation of the plasmid in BL21 2.6.− pixel−1 s−1 (corresponding to a dose rate per frame of 0.6 e− Å−2) resulting in a total dose of 48 e− Å−2. Coma-free alignment by CTF was performed in SerialEM just before starting data collection. A total of 5377 micrographs were collected with a defocus range between −0.5 and −1.5 µm. After micrograph inspection 2794 micrographs were retained for further processing. The micrographs were selected based on the following criteria: total drift below 50 Å  and early drift below 6 Å. Only micrographs with defocus between −0.2 and −1.5 µm were retained to ensure minimal contrast needed for particle alignment and to avoid CTF aliasing . Refinement for each subset independently resulted in a resolution between 2.2 and 2.3 Å. Following polishing and subset merging, the resolution further improved to 2.1 Å. Refinement of Cs from a nominal value of 2.7 to 2.55 mm resulted in further improvement in resolution to 1.9 Å (1.94 Å). To verify a possible influence of the cFEG flashing on the stability of beam tilt, the dataset was split into subsets acquired with the same beam shift and between two consecutive gun flashes. The beam tilt was refined separately for each subset and the deviation from the average tilt associated with the corresponding direction of beam-image shift was estimated by the refine CTF routine in RELION (v. 3.0). However, this did not increase the resolution. Further aberration correction in RELION  refined in PHENIX using real space refinement  and 1(b)c)d)e)f)] which is used to thermostat the microscope lenses.The microscope room and stability of the environmental conditions are important determinants of the microscope performance; therefore, we provide a brief description of the setup of our microscope below. The CRYO ARM 300 is installed on an isolated concrete slab in a temperature- and humidity-controlled room . Under the assumption that flashing recovers intensity, we applied LOW flashes (flash settings: current 1.4 Å over 0.7 s) every 4.5 h aiming to maintain electron flux variation within 10%. The resulting beam intensity recorded over a period of a 55 h corresponding to the duration of a typical dataset collection is shown in Fig. 2b). We observe that the LOW flash produces somewhat unpredictable changes in the beam intensity resulting in intensity variation of up to 25% within the 55 h time period. Not even a HIGH flash (flash settings: current 2.8 Å over 1.4 s) recovered the beam intensity to the initial level. While this article was under revision, JEOL engineers changed the settings of the LOW flash to make it identical to the HIGH flash following the report by Hamaguchi et al.  between the images are accounted for by maximum-likelihood refinement algorithms, like those implemented in The intensity fluctuations are a technical problem which can most likely be solved in the near future. Otherwise, special care should be taken at the image-processing step to account for the electron dose fluctuations. This in turn requires knowing the electron dose for each recorded image. Here we propose two approaches for doing this. First, to perform regular measurements of the blank beam intensity during data collection. The beam intensity is proportional to the electron dose and will allow estimation of relative electron dose at regular time intervals, between which the values can be interpolated. It might even be sufficient to measure the blank beam intensity only once between consecutive gun flashes since the intensity decay between the flashes appears to be reproducible Figs. 2 and S3 aet al., 2018I0, can be determined from the following equation: I and Ief are the beam intensities without and with the slit measured in the same position on ice, Λ and β are the apparent mean-free path of electrons in ice, and the aperture- and energy-filter limited scattering ]. Interestingly, the gun flashing does not seem to affect the beam tilt per se, since there are no jumps in the beam tilt upon gun flashing. The beam tilt does however change gradually over the time period of 42 h, which may indicate instabilities caused by environmental factors. The observed deviations of ±0.1 mrad are not significant enough to affect 3D reconstruction to a resolution up to around 2 Å.The stability of the beam tilt is critical for high-resolution 3D reconstruction . The fringes spread over a distance of at least 150 to 200 nm from the edge of the aperture, rendering this area unusable for imaging. Consequently, for common imaging conditions with a magnified pixel size of ∼1 Å and a corresponding diagonal of the K2 detector of 530 nm, the required minimal beam diameter is 900 nm, making only 22% of the total beam area suitable for imaging. Thus, the strong fringes reduce the area suitable for single-particle data acquisition within individual holes and consequently reduce the rate of data collection. The extension of fringes can be reduced or fringes can be almost completely eliminated using Kohler illumination , while integration of the trajectory results in drift rates that grow with increasing frame rate. Therefore, here we used the method based on measuring the distance between the first late and the last frames to estimate the stage drift excluding drifts measured after liquid nitrogen refilling, where the drift pattern appeared more complex than a continuous linear drift.The drift rates measured in this work were derived by analysing the trajectory of the movie frames aligned by a)]. In both cases, the drift reduces to ca 2 Å s−1 within around 15 min.The procedures of sample insertion in the microscope column and refilling of the liquid nitrogen (LN) dewars induce strong stage drift that decreases over time. The time needed to stabilize the drift is subtracted from the total time available for data collection and therefore should be as short as possible. We measured the drift of the stage after grid insertion in the column from the autoloader and after refilling of the LN dewars . For data recorded on the K2 detector the typical exposure time per frame was 0.2 s, which translated into an average drift per frame of ∼0.4 Å and maximum of 0.8 Å. Such a stage drift does not have significant influence on the quality of the micrographs to a resolution of at least 2 Å ]. The corresponding average increases in the ice thickness were 1.1 ± 0.6, 0.4 ± 0.5 and 0.2 ± 0.6 nm (n = 15). The total electron dose applied per measurement, including grid positioning, was below 0.5 e− Å−2. Hence, the electron-induced ice thinning was below 1 Å  are shown in Fig. 4b). The gradual increase in the ice thickness can be clearly seen despite the areal spread of the data points. The linear fit of the ice thickness against time indicates an ice growth rate of ca 2.5 Å h−1, or 6 nm per 24 h. This value is within the JEOL specifications for an in-column ice contamination rate of 5 Å h−1. Such an ice contamination rate is not negligible and should be taken into account when data are collected over periods of time exceeding two days from samples with very thin ice and particularly for small particles. However, for the more commonly encountered conditions for which the ice thickness exceeds 50 nm the effect of the ice contamination is not significant. Moreover, with the increasing rate of data collection due to faster detectors and the use of coma-corrected imaging ]. Therefore, a dataset exceeding 5000 images was collected, resulting in a reconstruction to a resolution of 1.9 Å from around 90 000 particles [Table 2b) and 5(c)]. Apart from the higher magnification (80 000), this dataset was collected under settings used for regular data collection including a beam-image shift of 0.55 µm from the hole centre to record four images within 2 µm holes. To reach sub-2 Å resolution reconstruction the Cs value was refined from the nominal value of 2.7 to 2.55 mm. The deviation of the refined values of Cs from the nominal values specified by the manufacturer have also been observed for other microscopes and can indicate specific properties of the particular objective lens as well as incorporate other systematic changes of experimental conditions including altered accelerating voltage , which is a significant deviation from coma-free conditions and occurred despite the coma-free alignment performed before launching the data collection. This appears to be a systematic issue and needs further investigation. Furthermore, we found that the beam-image shift of 0.55 µm induced 0.2 mrad beam tilt (Table S1) which had to be corrected for to reach sub-2 Å resolution. This is significant enough to affect the phases of 3D reconstruction below 2.8 Å resolution ] ] suggests that higher order aberrations have not been adequately corrected. This might be caused by coma induced by beam-image shift applied to align the beam to the hole centres, inducing beam tilts of up to 0.1 mrad which affects resolution starting from 2.2 Å ]. We could see a trend in decreased downtime over time, and during longer periods of microscope use we expect the time useful for data collection to surpass 80%. In a period of six months of constant operation 33 successful datasets were collected, accounting for around 96 000 images and 85 tomograms on 15 different projects.The long-term reliability of the microscope during continuous operation in multiuser settings can be assessed by analysis of the fraction of time used for data collection versus time used for regular maintenance procedures and technical downtime. Usage statistics of our CRYO ARM 300 over a period of six months is summarized in Fig. 74.SerialEM permits efficient and reliable data collection over long periods of time without the need for supervision. The routine data collection protocols deliver datasets that can be reconstructed to resolutions exceeding 2 Å. We conclude that the CRYO ARM 300 is suitable for repetitive reliable multiday data collection in the environment of a multiuser facility. It should also be noted that while cFEG does provide advantages over Schottky FEG for very high-resolution 3D reconstruction, as has been shown by the 1.54 Å reconstruction of apoferritin (Kato et al., 2019Here we characterized the suitability and performance of the CRYO ARM 300 with a K2 detector for automated single-particle cryo-EM data collection. We found that the beam intensity fluctuations of the cFEG due to gun flashing are significant but not detrimental for the quality of single-particle 3D reconstructions. We suggested possible approaches on how to monitor the beam intensity during the data collection which can be integrated into data collection procedure and later allow for more accurate correction of radiation-induced sample damage. We found that the microscope stage is stable enough to permit routine high-resolution automated data collection without the need for significant delays after stage positioning. The ice contamination rate is low enough to enable data collection over a period of several days without significant contrast losses. The contamination rate is particularly low in the autoloader, enabling sample storage for weeks without deteriorating the ice quality. Finally, apoferritin, EMD-10675EMDB reference: Click here for additional data file.10.1107/S2052252520006065/pw5013sup1.zipSerialEM scripts for automated data collection on CryoARM300. DOI: 10.1107/S2052252520006065/pw5013sup2.pdfSupplementary figures. DOI: