Brain endothelial GSDMD activation mediates inflammatory BBB breakdown.

The blood-brain barrier (BBB) protects the central nervous system from infections or harmful substances1; its impairment can lead to or exacerbate various diseases of the central nervous system2-4. However, the mechanisms of BBB disruption during infection and inflammatory conditions5,6 remain poorly defined. Here we find that activation of the pore-forming protein GSDMD by the cytosolic lipopolysaccharide (LPS) sensor caspase-11 (refs. 7-9), but not by TLR4-induced cytokines, mediates BBB breakdown in response to circulating LPS or during LPS-induced sepsis. Mice deficient in the LBP-CD14 LPS transfer and internalization pathway10-12 resist BBB disruption. Single-cell RNA-sequencing analysis reveals that brain endothelial cells (bECs), which express high levels of GSDMD, have a prominent response to circulating LPS. LPS acting on bECs primes Casp11 and Cd14 expression and induces GSDMD-mediated plasma membrane permeabilization and pyroptosis in vitro and in mice. Electron microscopy shows that this features ultrastructural changes in the disrupted BBB, including pyroptotic endothelia, abnormal appearance of tight junctions and vasculature detachment from the basement membrane. Comprehensive mouse genetic analyses, combined with a bEC-targeting adeno-associated virus system, establish that GSDMD activation in bECs underlies BBB disruption by LPS. Delivery of active GSDMD into bECs bypasses LPS stimulation and opens the BBB. In CASP4-humanized mice, Gram-negative Klebsiella pneumoniae infection disrupts the BBB; this is blocked by expression of a GSDMD-neutralizing nanobody in bECs. Our findings outline a mechanism for inflammatory BBB breakdown, and suggest potential therapies for diseases of the central nervous system associated with BBB impairment.

Toxicological safety evaluation of an aqueous lemon balm (Melissa officinalis) extract.

Melissa officinalis (lemon balm) has a long history of safe use as an aromatic herb, flavoring, tea, food supplement, and traditional medicine. An aqueous extract of the leaves of M. officinalis is intended for use as a food ingredient, however the existing safety database does not contain any high quality toxicological studies to support safe consumer exposure. Therefore, a standard tier 1 genotoxicity battery (bacterial reverse mutation and in vitro mammalian cell micronucleus tests) and a 90-day repeated dose oral toxicity study in rats were conducted in accordance with GLP and OECD guidelines. The genotoxicity studies confirmed that aqueous lemon balm extract is not genotoxic at up to the highest concentrations tested (5000 µg/plate or 5000 µg/mL). A non-GLP 14-day dose range finding study was conducted prior to the 90-day study to confirm dietary administration of aqueous lemon balm extract at concentrations of 0, 0.5, 1.6, or 5.0%. The 90-day study was conducted using the established dietary concentrations and no test substance-related adverse effects on clinical, hematological, biochemical, macroscopic, or histopathologic parameters were reported. Thus, the no-observed-adverse-effect-level was determined to be at least 3046.1 and 3720.9 mg/kg body weight/day (the highest doses tested) for male and female rats, respectively.

Systematic immune cell dysregulation and molecular subtypes revealed by single-cell RNA-seq of subjects with type 1 diabetes.

BACKGROUND: Type 1 diabetes mellitus (T1DM) is a prototypic endocrine autoimmune disease resulting from an immune-mediated destruction of pancreatic insulin-secreting ß  cells. A comprehensive immune cell phenotype evaluation in T1DM has not been performed thus far at the single-cell level. METHODS: In this cross-sectional analysis, we generated a single-cell transcriptomic dataset of peripheral blood mononuclear cells (PBMCs) from 46 manifest T1DM (stage 3) cases and 31 matched controls. RESULTS: We surprisingly detected profound alterations in circulatory immune cells (1784 dysregulated genes in 13 immune cell types), far exceeding the count in the comparator systemic autoimmune disease SLE. Genes upregulated in T1DM were involved in WNT signaling, interferon signaling and migration of T/NK cells, antigen presentation by B cells, and monocyte activation. A significant fraction of these differentially expressed genes were also altered in T1DM pancreatic islets. We used the single-cell data to construct a T1DM metagene z-score (TMZ score) that distinguished cases and controls and classified patients into molecular subtypes. This score correlated with known prognostic immune markers of T1DM, as well as with drug response in clinical trials. CONCLUSIONS: Our study reveals a surprisingly strong systemic dimension at the level of immune cell network in T1DM, defines disease-relevant molecular subtypes, and has the potential to guide non-invasive test development and patient stratification.

Transimpedance Matrix Can Be Used to Estimate Electrode Positions Intraoperatively and to Monitor Their Positional Changes Postoperatively in Cochlear Implant Patients.

OBJECTIVE: Accurate positioning of the electrode array during cochlear implant (CI) surgery is crucial for achieving optimal hearing outcomes. Traditionally, postoperative radiological imaging has been used to assess electrode position. Transimpedance matrix (TIM) measurements have also emerged as a promising method for assessing electrode position. This involves utilizing electric field imaging to create an electric distance matrix by analyzing voltage variations among adjacent electrodes. This study aimed to investigate the feasibility of using intraoperative TIM measurements to estimate electrode position and monitor postoperative changes. STUDY DESIGN: Retrospective cohort study. SETTING: University Medical center, tertiary academic referral center. PATIENTS: Patients undergoing CI (CI622) surgery between January 2019 and June 2022. INTERVENTION: CI electrode positions and maximal angular insertion depths (maxAID) were determined using X-ray imaging according to Stenvers' projection. The mean gradient phase (MGP) was extracted from the TIM, and a correlation between the MGP and maxAID was examined. A model was then built to estimate the maxAID using the MGP, and changes in electrode location over time were assessed using this model. MAIN OUTCOME MEASURES: Twenty-four patients were included in this study. A positive correlation between the maxAID and the MGP ( R = 0.7, p = 0.0001) was found. The established model was able to predict the maxAID with an accuracy of 27.7 ± 4.4°. Comparing intraoperative and postoperative TIM measurements, a decrease of 24.1° ± 10.7° in maxAID over time was observed. CONCLUSION: TIM measurements are useful for estimating the insertion depth of the electrode and monitoring changes in the electrode's position over time.

Tone Decay Reconsidered: Preliminary Results of a Prospective Study in Hearing-Aid Users with Moderate to Severe Hearing Loss.

Among hearing aid (HA) users, there is a considerable variability in word recognition scores (WRSs). This variability is most pronounced among individuals with moderately severe to severe hearing loss. The variability cannot be adequately explained by factors such as pure-tone audiogram, audiogram type or age. This prospective study was designed to investigate the relationship between tone decay (TD) and WRS in a group of HA users with corresponding pure-tone hearing loss. The study population included 22 patients with hearing loss between 50 and 80 dB HL. Aided WRS, unaided WRS and TD were assessed for both ears. TD was found to be frequency-dependent. TD and WRS were correlated, with up to R = -0.66. The TD test was revealed to be a feasible method for explaining variability in WRS among HA users with hearing loss below 80 dB. This may contribute to improved differential diagnostics. The TD test may thus offer a better understanding of the limitations of HA use in the context of cochlear implant candidacy assessment for HA users.

Pyroptosis inhibiting nanobodies block Gasdermin D pore formation.

Human Gasdermin D (GSDMD) is a key mediator of pyroptosis, a pro-inflammatory form of cell death occurring downstream of inflammasome activation as part of the innate immune defence. Upon cleavage by inflammatory caspases in the cytosol, the N-terminal domain of GSDMD forms pores in the plasma membrane resulting in cytokine release and eventually cell death. Targeting GSDMD is an attractive way to dampen inflammation. In this study, six GSDMD targeting nanobodies are characterized in terms of their binding affinity, stability, and effect on GSDMD pore formation. Three of the nanobodies inhibit GSDMD pore formation in a liposome leakage assay, although caspase cleavage was not perturbed. We determine the crystal structure of human GSDMD in complex with two nanobodies at 1.9 Å resolution, providing detailed insights into the GSDMD-nanobody interactions and epitope binding. The pore formation is sterically blocked by one of the nanobodies that binds to the oligomerization interface of the N-terminal domain in the multi-subunit pore assembly. Our biochemical and structural findings provide tools for studying inflammasome biology and build a framework for the design of GSDMD targeting drugs.

Quantitative proteomics defines mechanisms of antiviral defence and cell death during modified vaccinia Ankara infection.

Modified vaccinia Ankara (MVA) virus does not replicate in human cells and is the vaccine deployed to curb the current outbreak of mpox. Here, we conduct a multiplexed proteomic analysis to quantify >9000 cellular and ~80% of viral proteins throughout MVA infection of human fibroblasts and macrophages. >690 human proteins are down-regulated >2-fold by MVA, revealing a substantial remodelling of the host proteome. >25% of these MVA targets are not shared with replication-competent vaccinia. Viral intermediate/late gene expression is necessary for MVA antagonism of innate immunity, and suppression of interferon effectors such as ISG20 potentiates virus gene expression. Proteomic changes specific to infection of macrophages indicate modulation of the inflammatory response, including inflammasome activation. Our approach thus provides a global view of the impact of MVA on the human proteome and identifies mechanisms that may underpin its abortive infection. These discoveries will prove vital to design future generations of vaccines.

In-depth analysis of human virus-specific CD8+ T cells delineates unique phenotypic signatures for T cell specificity prediction.

Following viral infection, the human immune system generates CD8+ T cell responses to virus antigens that differ in specificity, abundance, and phenotype. A characterization of virus-specific T cell responses allows one to assess infection history and to understand its contribution to protective immunity. Here, we perform in-depth profiling of CD8+ T cells binding to CMV-, EBV-, influenza-, and SARS-CoV-2-derived antigens in peripheral blood samples from 114 healthy donors and 55 cancer patients using high-dimensional mass cytometry and single-cell RNA sequencing. We analyze over 500 antigen-specific T cell responses across six different HLA alleles and observed unique phenotypes of T cells specific for antigens from different virus categories. Using machine learning, we extract phenotypic signatures of antigen-specific T cells, predict virus specificity for bulk CD8+ T cells, and validate these predictions, suggesting that machine learning can be used to accurately predict antigen specificity from T cell phenotypes.

Diphtheria toxin activates ribotoxic stress and NLRP1 inflammasome-driven pyroptosis.

The ZAKα-driven ribotoxic stress response (RSR) is activated by ribosome stalling and/or collisions. Recent work demonstrates that RSR also plays a role in innate immunity by activating the human NLRP1 inflammasome. Here, we report that ZAKα and NLRP1 sense bacterial exotoxins that target ribosome elongation factors. One such toxin, diphtheria toxin (DT), the causative agent for human diphtheria, triggers RSR-dependent inflammasome activation in primary human keratinocytes. This process requires iron-mediated DT production in the bacteria, as well as diphthamide synthesis and ZAKα/p38-driven NLRP1 phosphorylation in host cells. NLRP1 deletion abrogates IL-1ß and IL-18 secretion by DT-intoxicated keratinocytes, while ZAKα deletion or inhibition additionally limits both pyroptotic and inflammasome-independent non-pyroptotic cell death. Consequently, pharmacologic inhibition of ZAKα is more effective than caspase-1 inhibition at protecting the epidermal barrier in a 3D skin model of cutaneous diphtheria. In summary, these findings implicate ZAKα-driven RSR and the NLRP1 inflammasome in antibacterial immunity and might explain certain aspects of diphtheria pathogenesis.

Widespread effects of DNA methylation and intra-motif dependencies revealed by novel transcription factor binding models.

Several studies suggested that transcription factor (TF) binding to DNA may be impaired or enhanced by DNA methylation. We present MeDeMo, a toolbox for TF motif analysis that combines information about DNA methylation with models capturing intra-motif dependencies. In a large-scale study using ChIP-seq data for 335 TFs, we identify novel TFs that show a binding behaviour associated with DNA methylation. Overall, we find that the presence of CpG methylation decreases the likelihood of binding for the majority of methylation-associated TFs. For a considerable subset of TFs, we show that intra-motif dependencies are pivotal for accurately modelling the impact of DNA methylation on TF binding. We illustrate that the novel methylation-aware TF binding models allow to predict differential ChIP-seq peaks and improve the genome-wide analysis of TF binding. Our work indicates that simplistic models that neglect the effect of DNA methylation on DNA binding may lead to systematic underperformance for methylation-associated TFs.

ASC oligomer favors caspase-1CARD domain recruitment after intracellular potassium efflux.

Signaling through the inflammasome is important for the inflammatory response. Low concentrations of intracellular K+ are associated with the specific oligomerization and activation of the NLRP3 inflammasome, a type of inflammasome involved in sterile inflammation. After NLRP3 oligomerization, ASC protein binds and forms oligomeric filaments that culminate in large protein complexes named ASC specks. ASC specks are also initiated from different inflammasome scaffolds, such as AIM2, NLRC4, or Pyrin. ASC oligomers recruit caspase-1 and then induce its activation through interactions between their respective caspase activation and recruitment domains (CARD). So far, ASC oligomerization and caspase-1 activation are K+-independent processes. Here, we found that when there is low intracellular K+, ASC oligomers change their structure independently of NLRP3 and make the ASCCARD domain more accessible for the recruitment of the pro-caspase-1CARD domain. Therefore, conditions that decrease intracellular K+ not only drive NLRP3 responses but also enhance the recruitment of the pro-caspase-1 CARD domain into the ASC specks.

Engineered Escherichia coli for the in situ secretion of therapeutic nanobodies in the gut.

Drug platforms that enable the directed delivery of therapeutics to sites of diseases to maximize efficacy and limit off-target effects are needed. Here, we report the development of PROT3EcT, a suite of commensal Escherichia coli engineered to secrete proteins directly into their surroundings. These bacteria consist of three modular components: a modified bacterial protein secretion system, the associated regulatable transcriptional activator, and a secreted therapeutic payload. PROT3EcT secrete functional single-domain antibodies, nanobodies (Nbs), and stably colonize and maintain an active secretion system within the intestines of mice. Furthermore, a single prophylactic dose of a variant of PROT3EcT that secretes a tumor necrosis factor-alpha (TNF-α)-neutralizing Nb is sufficient to ablate pro-inflammatory TNF levels and prevent the development of injury and inflammation in a chemically induced model of colitis. This work lays the foundation for developing PROT3EcT as a platform for the treatment of gastrointestinal-based diseases.

Mitochondrial damage activates the NLRP10 inflammasome.

Upon detecting pathogens or cell stress, several NOD-like receptors (NLRs) form inflammasome complexes with the adapter ASC and caspase-1, inducing gasdermin D (GSDMD)-dependent cell death and maturation and release of IL-1ß and IL-18. The triggers and activation mechanisms of several inflammasome-forming sensors are not well understood. Here we show that mitochondrial damage activates the NLRP10 inflammasome, leading to ASC speck formation and caspase-1-dependent cytokine release. While the AIM2 inflammasome can also sense mitochondrial demise by detecting mitochondrial DNA (mtDNA) in the cytosol, NLRP10 monitors mitochondrial integrity in an mtDNA-independent manner, suggesting the recognition of distinct molecular entities displayed by the damaged organelles. NLRP10 is highly expressed in differentiated human keratinocytes, in which it can also assemble an inflammasome. Our study shows that this inflammasome surveils mitochondrial integrity. These findings might also lead to a better understanding of mitochondria-linked inflammatory diseases.

Impact of in vitro phytohormone treatments on the metabolome of the leafy liverwort Radula complanata (L.) Dumort.

INTRODUCTION: Liverworts are a group of non-vascular plants that possess unique metabolism not found in other plants. Many liverwort metabolites have interesting structural and biochemical characteristics, however the fluctuations of these metabolites in response to stressors is largely unknown. OBJECTIVES: To investigate the metabolic stress-response of the leafy liverwort Radula complanata. METHODS: Five phytohormones were applied exogenously to in vitro cultured R. complanata and an untargeted metabolomic analysis was conducted. Compound classification and identification was performed with CANOPUS and SIRIUS while statistical analyses including PCA, ANOVA, and variable selection using BORUTA were conducted to identify metabolic shifts. RESULTS: It was found that R. complanata was predominantly composed of carboxylic acids and derivatives, followed by benzene and substituted derivatives, fatty acyls, organooxygen compounds, prenol lipids, and flavonoids. The PCA revealed that samples grouped based on the type of hormone applied, and the variable selection using BORUTA (Random Forest) revealed 71 identified and/or classified features that fluctuated with phytohormone application. The stress-response treatments largely reduced the production of the selected primary metabolites while the growth treatments resulted in increased production of these compounds. 4-(3-Methyl-2-butenyl)-5-phenethylbenzene-1,3-diol was identified as a biomarker for the growth treatments while GDP-hexose was identified as a biomarker for the stress-response treatments. CONCLUSION: Exogenous phytohormone application caused clear metabolic shifts in Radula complanata that deviate from the responses of vascular plants. Further identification of the selected metabolite features can reveal metabolic biomarkers unique to liverworts and provide more insight into liverwort stress responses.

A single-cell atlas identifies pretreatment features of primary imatinib resistance in chronic myeloid leukemia.

Primary resistance to tyrosine kinase inhibitors (TKIs) is a significant barrier to optimal outcomes in chronic myeloid leukemia (CML), but factors contributing to response heterogeneity remain unclear. Using single-cell RNA (scRNA) sequencing, we identified 8 statistically significant features in pretreatment bone marrow, which correlated with either sensitivity (major molecular response or MMR) or extreme resistance to imatinib (eventual blast crisis [BC] transformation). Employing machine-learning, we identified leukemic stem cell (LSC) and natural killer (NK) cell gene expression profiles predicting imatinib response with >80% accuracy, including no false positives for predicting BC. A canonical erythroid-specifying (TAL1/KLF1/GATA1) regulon was a hallmark of LSCs from patients with MMR and was associated with erythroid progenitor [ERP] expansion in vivo (P < .05), and a 2- to 10-fold (6.3-fold in group A vs 1.09-fold in group C) erythroid over myeloid bias in vitro. Notably, ERPs demonstrated exquisite TKI sensitivity compared with myeloid progenitors (P < .001). These LSC features were lost with progressive resistance, and MYC- and IRF1-driven inflammatory regulons were evident in patients who progressed to transformation. Patients with MMR also exhibited a 56-fold expansion (P < .01) of a normally rare subset of hyperfunctional adaptive-like NK cells, which diminished with progressive resistance, whereas patients destined for BC accumulated inhibitory NKG2A+ NK cells favoring NK cell tolerance. Finally, we developed antibody panels to validate our scRNA-seq findings. These panels may be useful for prospective studies of primary resistance, and in assessing the contribution of predetermined vs acquired factors in TKI response heterogeneity.

Inhibition of cellular RNA methyltransferase abrogates influenza virus capping and replication.

Orthomyxo- and bunyaviruses steal the 5' cap portion of host RNAs to prime their own transcription in a process called "cap snatching." We report that RNA modification of the cap portion by host 2'-O-ribose methyltransferase 1 (MTr1) is essential for the initiation of influenza A and B virus replication, but not for other cap-snatching viruses. We identified with in silico compound screening and functional analysis a derivative of a natural product from Streptomyces, called trifluoromethyl-tubercidin (TFMT), that inhibits MTr1 through interaction at its S-adenosyl-l-methionine binding pocket to restrict influenza virus replication. Mechanistically, TFMT impairs the association of host cap RNAs with the viral polymerase basic protein 2 subunit in human lung explants and in vivo in mice. TFMT acts synergistically with approved anti-influenza drugs.

Numerical Study and Experimental Verification of Biomass Conversion and Potassium Release in a 140 kW Entrained Flow Gasifier.

In this study, a Eulerian-Lagrangian model is used to study biomass gasification and release of potassium species in a 140 kW atmospheric entrained flow gasifier (EFG). Experimental measurements of water concentration and temperature inside the reactor, together with the gas composition at the gasifier outlet, are used to validate the model. For the first time, a detailed K-release model is used to predict the concentrations of gas-phase K species inside the gasifier, and the results are compared with experimental measurements from an optical port in the EFG. The prediction errors for atomic potassium (K), potassium chloride (KCl), potassium hydroxide (KOH), and total potassium are 1.4%, 9.8%, 5.5%, and 5.7%, respectively, which are within the uncertainty limits of the measurements. The numerical model is used to identify and study the main phenomena that occur in different zones of the gasifier. Five zones are identified in which drying, pyrolysis, combustion, recirculation, and gasification are active. The model was then used to study the transformation and release of different K species from biomass particles. It was found that, for the forest residue fuel that was used in the present study, the organic part of K is released at the shortest residence time, followed by the release of inorganic K at higher residence times. The release of inorganic salts starts by evaporation of KCl and continues by dissociation of K2CO3 and K2SO4, which forms gas-phase KOH. The major fraction of K is released around the combustion zone (around 0.7-1.3 m downstream of the inlet) due to the high H2O concentration and temperature. These conditions lead to rapid dissociation of K2CO3 and K2SO4, which increases the total K concentration from 336 to 510 ppm in the combustion zone. The dissociation of the inorganic salts and KOH formation continues in the gasification zone at a lower rate; hence, the total K concentration slowly increases from 510 ppm at 1.3 m to 561 ppm at the outlet.

P38 kinases mediate NLRP1 inflammasome activation after ribotoxic stress response and virus infection.

Inflammasomes integrate cytosolic evidence of infection or damage to mount inflammatory responses. The inflammasome sensor NLRP1 is expressed in human keratinocytes and coordinates inflammation in the skin. We found that diverse stress signals induce human NLRP1 inflammasome assembly by activating MAP kinase p38: While the ribotoxic stress response to UV and microbial molecules exclusively activates p38 through MAP3K ZAKα, infection with arthropod-borne alphaviruses, including Semliki Forest and Chikungunya virus, activates p38 through ZAKα and potentially other MAP3K. We demonstrate that p38 directly phosphorylates NLRP1 and that serine 107 in the linker region is critical for activation. NLRP1 phosphorylation is followed by ubiquitination of NLRP1PYD, N-terminal degradation of NLRP1, and nucleation of inflammasomes by NLRP1UPA-CARD. In contrast, activation of NLRP1 by nanobody-mediated ubiquitination, viral proteases, or inhibition of DPP9 was independent of p38 activity. Taken together, we define p38 activation as a unifying signaling hub that controls NLRP1 inflammasome activation by integrating a variety of cellular stress signals relevant to the skin.

[Recommendations of the S3 Guideline "Use of Extracorporeal Circulation (ECLS/ ECMO) for Cardiac and Circulatory Failure" of the Association of Scientific Medical Societies in Germany]. / Empfehlungen der S3-Leitlinie (AWMF) "Einsatz der extrakorporalen Zirkulation (ECLS/ECMO) bei Herz- und Kreislaufversagen".

In recent years, the use of mechanical support for patients with cardiac or circulatory failure has continuously increased, leading to 3,000 ECLS/ECMO (extracorporeal life support/extracorporeal membrane oxygenation) implantations annually in Germany. Due to the lack of guidelines, there is an urgent need for evidence-based recommendations addressing the central aspects of ECLS/ECMO therapy. In July 2015, the generation of a guideline level S3 according to the standards of the Association of the Scientific Medical Societies in Germany (AWMF) was announced by the German Society for Thoracic and Cardiovascular Surgery (GSTCVS). In a well-structured consensus process, involving experts from Germany, Austria and Switzerland, delegated by 16 scientific societies and the patients' representation, the guideline "Use of extracorporeal circulation (ECLS/ECMO) for cardiac and circulatory failure" was created under guidance of the GSTCVS, and published in February 2021. The guideline focuses on clinical aspects of initiation, continuation, weaning and aftercare, herein also addressing structural and economic issues. This article presents an overview on the methodology as well as the final recommendations.

Quantitative Tomographic Laser Absorption Imaging of Atomic Potassium during Combustion of Potassium Chloride Salt and Biomass.

Gaseous potassium (K) species play an important role in biomass combustion processes, and imaging techniques are powerful tools to investigate the related gas-phase chemistry. Here, laser absorption imaging of gaseous atomic K in flames is implemented using tunable diode laser absorption spectroscopy at 769.9 nm and a high-speed complementary metal oxide semiconductor (CMOS) camera recording at 30 kfps. Atomic K absorption spectra are acquired for each camera pixel in a field of view of 28 × 28 mm at a rate of 100 Hz. The technique is used to determine the spatial distribution of atomic K concentration during the conversion of potassium chloride (KCl) salt and wheat straw particles in a laminar premixed CH4/air flame with an image pixel resolution of up to 120 µm. Due to axisymmetry in setup geometry and, consequently, atomic K distributions, the radial atomic K concentration fields could be reconstructed by one-dimensional tomography. For the KCl sample, the K concentration field was in excellent agreement with previous point measurements. In the case of wheat straw, atomic K concentrations of around 3 ppm were observed in a cylindrical flame during devolatilization. In the char conversion phase, a spherical layer of atomic K, with concentrations reaching 25 ppm, was found within 5 mm of the particle surface, while the concentration rapidly decreased to sub-ppm levels along the vertical axis. In both cases, a thin (∼1 mm) layer without any atomic K was observed in close vicinity to the particle, suggesting that the potassium was initially not released in its atomic form.