The results of the characterization by XRD, FTIR, SEM, EDS, TG/DTG, VSM and Surface Acidity showed that the obtained bifunctional catalyst has been successfully synthesized. The study of the reaction parameters, such as reaction temperature (140–180 °C), reaction time (1–5 h), molar ratio MeOH : oil (25 : 1–45 : 1) and catalyst loading (2–10% m m−1) was performed in the conversion of WCO into biodiesel via transesterification. The reactional behavior showed the following optimal reaction conditions: reaction temperature of 180 °C, reaction time of 3 h, molar ratio MeOH : oil of 45 : 1 and catalyst loading of 6%. Based on the results, biodiesel with a maximum ester content of 95.2% was obtained using the WO3/CuFe2O4 magnetic catalyst under the optimal reaction conditions. The magnetic catalyst showed excellent catalytic and magnetic performance and it was applied in five reaction cycles with ester content above 80%. Biodiesel properties were found in accordance with ASTM limits. This research provided the development of a stable and reusable WO3/CuFe2O4 bifunctional catalyst for potential application in biodiesel production.This study aims to synthesize a WO 3/CuFe2O4 catalyst through a wet impregnation method and use it as a new magnetic acid catalyst in the transesterification process of waste cooking oil (WCO).This study aims to synthesize a WO The results presented a ester conversion of 72.5% in the following reaction conditions: reaction temperature of 180 °C, MeOH : oil molar ratio of 30 : 1, catalyst loading of 5% and reaction time of 5 h.23In addition, among the class of heterogeneous catalysts, the catalysts with acidic properties stand out because it is possible to apply them, in esterification and transesterification reactions, in the presence of residual raw materials without causing inconveniences, such as soap production, reactor corrosion, 24,25 Several materials have been applied as catalysts or catalytic supports in the production of biodiesel, such as magnetites (Fe3O4), hematites (Fe2O3) and ferrites (MFe2O4), where M is the transition metal.25,26Among the heterogeneous catalysts magnetic materials have been attracting attention due to the fact that their magnetic properties facilitate the separation step of the catalyst from the reaction medium , through the application of a magnetic field, which would reduce the costs employed in the separation stage and consequently the total costs of the process.et al. studied biodiesel production through the use of chicken fat and methanol. The catalyst used in the reaction consisted of the impregnation of calcium oxide (CaO) in copper ferrite (CuFe2O4) and the biodiesel obtained ester content of 94.52% in the following optimal reaction conditions: reaction temperature of 70 °C, MeOH : oil molar ratio of 15 : 1, 3% catalyst loading and reaction time of 4 h.8Seffati 3 impregnated in cooper ferrite (CuFe2O4), in the production of biodiesel using waste cooking oil and methanol. The effects of the variables present in the transesterification reaction were investigated in the catalytic activity of the catalyst, such as reaction temperature, reaction time, molar ratio MeOH : oil and catalyst loading. It is noteworthy that biodiesel synthesis by magnetic acid catalyst using CuFe2O4 as catalytic support has not been reported in the literature yet.This study aims to study the application of a heterogeneous magnetic acid catalyst, composed of WO2.2.1.4H6O4·H2O, Dinâmica®, 98%) and iron nitrate (iii) (Fe(NO3)3·9H2O, Neon®, 98%) were used as precursors in the synthesis of CuFe2O4. Dihydrate sodium tungstate  was used in the catalyst preparation stage. Sodium hydroxide  and hydrochloric acid  were used to determine the surface acidity of the catalyst. Methyl alcohol  and waste cooking oil (WCO), collected from the restaurant of the Federal University of Pará, were used in the transesterification reaction. Methyl heptadecanoate  and heptane  were used in chromatographic analysis. Ethanol  was used in the catalyst washing process. All reagents used were analytical. Copper acetate II (CuC2.2.2.2.1.2O4) was prepared using the coprecipitation method according to the methodology adapted by Seffati et al.8 Initially, the required amount of CuC4H6O4·H2O and Fe(NO3)3·9H2O at molar ratio 1 : 2 (Cu : Fe) were dissolved into 150 mL of distilled water followed by mechanical agitation at room temperature during 30 min. Then, a solution of NaOH 4 mol L−1 was added drop by drop to adjust the pH of the mixture to 12. At the end of the precipitation process, the system was kept under mechanical agitation at 65 °C for 4 h. Thus, the obtained product was washed with distilled water several times until the washing water achieve neutral pH (pH = 7), and finally, the material was dried in an oven at 80 °C for 12 h and calcined at 500 °C for 3 h (10 °C min−1) in order to obtain the CuFe2O4.Copper ferrite , where the catalyst with 35% of WO3 on the support was chosen as the best catalyst . 3/CuFe2O4.In the process of synthesis of the magnetic acid catalyst, the method of wet impregnation was applied by using the same tungsten precursor and calcination temperature previously described by Kaur 2.3.θ > 75° analysis range. Fourier Transform Infrared Spectroscopy (FTIR): Prestige 21 Model Shimadzu Spectrometer, spectral range of analysis was 2500–400 cm−1, resolution of 4 cm−1 and 32 scans. Scanning Electron Microscopy (SEM): Tescan microscope, Vega 3 LMU model. Energy Dispersion X-Ray Spectroscopy (EDS): Oxford micro-analysis system, AZTec Energy X-Act model, resolution 129 eV. Thermogravimetric Analysis (TG/DTG): Shimadzu equipment, model DTG-60H, temperature range from 25 to 800 °C (heating rate of 10 °C min−1), nitrogen flow of 50 mL min−1, alumina crucible. Vibrant Sample Magnetometry (VSM): Microsense magnetometer, EZ9 model, room temperature, applied magnetic field from −20 000 Oe to 20 000 Oe. Surface Acidity: a mass sample of 0.1 g was placed into 20 mL of standardized NaOH 0.1 mol L−1 solution, remaining under agitation for 24 h at room temperature. Subsequently, the sample was centrifuged and the supernatant was tilted with standardized HCl 0.1 mol L−1 solution in the presence of phenolphthalein as an indicator.28The materials were characterized by several techniques such as X-Ray Diffraction (XRD): Bruker diffractometer, model D2 PHASER, Cu radiation (Kα = 1.54 Å), 40 kV, 30 mA, 5° < 22.4.−1). After the reaction, the catalyst was separated by the application of the external magnetic field to the reaction system. The reaction products were transferred to a funnel, separated and washed with 500 mL of distilled water (60 °C) for removal of residual alcohol and glycerol. Finally, the biodiesel samples were stored for further analysis.The reactions were performed in a PARR 5000 Multireactor reactor with fixed agitation at 700 rpm, the following reaction conditions were investigated: reaction temperature (140–180 °C); reaction time (1–5 h); MeOH : oil molar ratio (25 : 1–45 : 1) and catalyst loading  and capillary column CP WAX 52 CB  was used. Methyl heptadecanoate was used as an internal standard and heptane as a solvent. In addition, the oven temperature programming from 170 °C to 250 °C (same FID temperature) was used at a rate of 10 °C min−1, helium gas was used as a mobile phase with flow of 1.0 mL min−1, and the injection volume was 1 μL of sample. The ester content (EC) was calculated according to AT is the sum of the peaks total area; AIS is the peak area of the internal pattern; CIS is the solution concentration of the internal standard (mg L−1); CB100 is biodiesel's concentration after dilution (mg L−1).The methyl ester content of biodiesel samples was determined by gas chromatography according to the methodology adapted from the European standard (EN 14103) proposed by Silva The main physicochemical properties of biodiesel were determined by a standard method of the American Society for Testing and Materials (ASTM). The kinematic viscosities, analyzed at 40 °C, were determined for the biodiesel samples synthesized according to the ASTM D445 method, using a viscometer model Cannon-Fenske . The density, analyzed at 20 °C, was measured by ASTM D6890 method on a KEM DAS-500 automatic densimeter. The acid value was evaluated conforming to the ASTM D664 method. The flash point was estimated employing the ASTM D093 method on an automatic TANAKA APM 7 Pensky-Martens flash point. The cold filter plugging point was determined using ASTM D6371 methodology on a TANAKA equipment AFP-102 model. The corrosiveness to copper was appraised by ASTM D130 in a copper corrosion bath from Koehler. The oxidative stability was determined using a Rancimat, model 743 from Metrohm, in accordance with EN 14112.2.6.The reuse study was conducted for the magnetic catalyst under optimal transesterification reaction conditions. After each reaction cycle, the catalyst was separated from the reaction medium by the application of the external magnetic field, washed once with heptane (10 mL) and twice with ethyl alcohol (25 mL) and dried in an oven at 80 °C for 12 h.The process of recalcination of the magnetic catalyst was proposed by observing the decrease of the ester content values during the reaction cycles. This study was conducted in two stages. First, the catalyst was reproduced and washed after each reaction cycle, as described above. Then, the catalyst was thermally reactivated by calcination at 500 °C for 3 h, in a muffle oven, to eliminate organic components from the surface of the material.3.3.1.2O4 support and WO3/CuFe2O4 magnetic catalyst were characterized by XRD, FTIR, SEM/EDS, TGA/DTGA, VSM and Surface Acidity techniques.The CuFe3.1.1.2O4, WO3 and WO3/CuFe2O4 materials are presented in 2O4 phase (red line) exhibited peaks at 2θ = 18.44°, 29.98°, 35.67° and 62.49°, which were well correlated to crystallographic planes (111), (220), (311) and (440), with cubic system and spatial group Fd̄3m (ICDD 01-077-0010), confirming the formation of CuFe2O4 with reverse spinel structure, in which Fe3+ ions are situated in an equivalent way in the tetrahedral and octahedral sites, Cu2+ is only in octahedral sites.30,31 In addition, the diffractogram also showed peaks at 2θ = 38.87° and 48.95° corresponding to the planes (111) and (−202) of CuO with monoclinic system (ICDD 03-065-2309). According to Nikolić, the formation of this impurity may be related to the oxidation of Cu2+ ions during the synthesis process carried out under atmospheric conditions.32The X-ray diffraction patterns of CuFe3 (black line), the presence of well-formed peaks with high intensity was identified in 2θ = 23.18°, 23.64°, 24.45°, 26.65°, 28.70°, 33.30° and 34.20°, which were directly indexed to crystallographic planes (001), (020), (200), (120), (111), (021) and (220) of this oxide with monoclinic system (ICDD 01-075-2072).27 The presence of phases WO3 and CuFe2O4 (blue line) in the magnetic catalyst was confirmed in the XRD pattern, whose reduction in intensity of the main peaks revealed the probable dispersion of WO3 in CuFe2O4. Additionally, the presence of peaks in 2θ = 29.98° and 25.67° was identified, which were well indexed to the CuFe2O4 phase and 2θ = 38.87° to the CuO phase, both phases derived from the magnetic support CuFe2O4.For the sample containing the crystalline phase WO3.1.2.2O4 magnetic support and the WO3/CuFe2O4 bifunctional catalyst are presented in 2O4 indicated absorption bands around 462, 1537 and 1684 cm−1. The band at 462 cm−1 is attributed to metal–oxygen elongation , specific to spinel ferrite.8 The bands in 1537 and 1684 cm−1 are related to the stretching vibration O–H and the deformation of the group –OH, respectively, and the absorption of H2O on the metal surface.33 Moreover, all CuFe2O4 spectrum absorptions are present in the WO3/CuFe2O4 magnetic catalyst spectrum, however, new absorption vibrations occur at 719 and 931 cm−1 referring to the stretch bands of the O–W–O and W PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O connections, respectively.34,35 Thus, the analysis of the data from the FTIR spectra presented suggest the efficiency of the ferrite synthesis process and the impregnation of the WO3 species in the magnetic support to obtain the bifunctional character of the catalyst.The FTIR spectra of the CuFe3.1.3.2O4 and WO3/CuFe2O4 was examined by SEM analysis . The interaction between magnetic particles is responsible for the morphological nature (clusters) of these materials as reported in previous studies.36 The SEM micrograph referring to CuFe2O4 , copper (Cu) and oxygen (O) present on the surface of the magnetic support at percentage concentrations (m m−1) of 46.3%, 27.2% and 26.4%, respectively. The elemental composition presented Fe/Cu ratio of 1.7, a value relatively close to that desired in the synthesis of CuFe2O4 (Fe/Cu = 2.0). The copper ferrite presented the elements evenly distributed on the surface of the material, as shown by the elemental surface mapping in 3/CuFe2O4 catalyst. In the analysis, the presence of the tungsten element (W) on the surface of the catalytic material was verified, in addition to the characteristic elements of CuFe2O4, corroborating the XRD and FTIR analysis data presented earlier. The W element content of 35.4%  shows the efficiency of the impregnation process, while the increase in the content of element O, in about 26.0% (ferrite) to 30.0% , indicates the formation of the species WO3. According to 3.1.4.2O4 and catalyst WO3/CuFe2O4 non-calcined. The results showed two main stages of mass loss for the materials CuFe2O4 and WO3/CuFe2O4. For the CuFe2O4 magnetic support (− ion (acetate) from the metallic precursor.38,39 A small mass loss, 0.6%, was observed in the temperature range of 674–694 °C, and may be related to the phase transition from tetragonal structure to cubic structure, indicating the development of a more stable phase for ferrite CuFe2O4.38,40 However, the heat treatment at 500 °C made the material CuFe2O4 stable. support , the fir3/CuFe2O4 catalyst  obtained by VSM analysis , remaining magnetization (Mr) and coercivity (Hc). Based on the results obtained, the CuFe2O4 support (red curve) and the WO3/CuFe2O4 catalyst (blue curve) showed saturation magnetization values of 22.70 emu g−1 and 12.04 emu g−1, respectively, when a field of ±20 000 Oe was applied at room temperature. It is observed that the Ms value of the WO3/CuFe2O4 catalyst decreased in relation to CuFe2O4 due to the incorporation of the non-magnetic species WO3 in the magnetic support structure, causing a 46.9% reduction in the magnetic activity of the catalyst. However, catalyst magnetization remains effective for the separation process. According to the literature, magnetic catalysts such as CaFe2O4–CaFe2O5–Fe3O4, MgO/MgAl0.4Fe1.6O4 and CaO@Sr2Fe2O5–Fe2O3 were applied in the transesterification process and presented Ms values at magnitudes 0.217 emu g−1, 2.02 emu g−1and 11.09 emu g−1, respectively.42–44 In addition, the hysteresis M–H graph shows the curves in the regular sigmoidal format typical of CuFe2O4, and presents Mr and Hc values equal to 4.80 emu g−1 and 236.75 Oe, respectively. These results classify the ferrite synthesized in this study as mild ferromagnetic and indicate a certain resistance to demagnetizations.45,46 The WO3/CuFe2O4 catalyst also demonstrated ferromagnetic behavior and a higher resistance to demagnetizations by presenting Mr and Hc values equal to 4.76 emu g−1 and 750.67 Oe, respectively.The magnetic properties investigated were saturation magnetization  is another essential fuel property which is an indirect measure of fuel volatility.56 The FP measure of biodiesel reached 155 °C, indicating security for storage and portability. The cold filter plugging point (CFPP) is a parameter used to determine the minimum temperature at which fuel filters clog in automotive engines due to partial solidification of fuel.13 The biodiesel showed CFPP of 0 °C, inferring that the biodiesel could be used in cold weather countries. In the corrosiveness to copper analysis, the biodiesel presented a value of 1a, suggesting that the biofuel will not cause damage to the engine's metallic components. Similar value was obtained by Gonçalves et al.20 All results confirm that the waste cooking oil has been successfully converted into biodiesel using WO3/CuFe2O4 magnetic acid catalyst and conform to ASTM D6751 standard. The biodiesel oxidative stability value of 4.80 h is greater than the minimum limit of 3 h defined by ASTM D6751.Biodiesel obtained from waste cooking oil under optimal reaction conditions, using the magnetic acid catalyst WO3.4.55 The magnetic catalyst WO3/CuFe2O4 was evaluated by carrying out several reaction cycles under the optimal condition of transesterification reaction, as shown in 3/CuFe2O4 magnetic catalyst was reused for five reaction cycles and showed a reduction of its catalytic efficiency of approximately 40%. Two reasons may be related to the loss of catalytic activity: (1) partial leaching of the active sites and (2) deposition of organic matter on the catalyst surface. The first hypothesis was verified by analyzing the EDS of the catalyst recovered after the fifth reaction cycle  to 3.38 mmol H+ g−1 , confirming the leaching of tungsten species from the magnetic catalyst surface.The reuse and recovery capacity are characteristics that make the heterogeneous catalyst more economically feasible for the biodiesel production process.on cycle , the ana3/CuFe2O4 material during transesterification cycles, the catalyst presented ester content values of 95.2 and 57.7% for the first and fifth reaction cycles, respectively. These values are above the values of ester contents of the biodiesel obtained in the reaction performed without the presence of catalyst (18.7%), and only in the presence of the CuFe2O4 support (21.4%), indicating efficiency of the active component WO3 for the biodiesel production process. These results are correlated with the Surface Acidity of the CuFe2O4 support (2.71 mmol H+ g−1) and the catalyst WO3/CuFe2O4 (7.43 mmol H+ g−1). The intrinsic acidity of CuFe2O4 was not sufficient to promote the transesterification process. The impregnation with tungsten increased the surface acidity value through the new Brønsted acid sites present in the catalyst, which enable the catalytic activity of the catalyst by its interaction with the carbonyl group in the transesterification process.27 Catalyst recovery was 88.05 ± 2.92%, which proves the efficiency of the magnetic separation process employed. Furthermore, the VSM analysis of the catalyst before and after the fifth reaction cycle is displayed in Ms) from 12.04 emu g−1 to 19.12 emu g−1. This behavior possibly occurs due to leaching of the non-magnetic component (WO3) of the magnetic support surface (CuFe2O4) during reaction cycles, which makes the Ms value of the reused catalyst closer to the Ms value of the copper ferrite (22.70 emu g−1).Despite the decrease in catalytic efficiency of the WO3/CuFe2O4 catalyst was to perform a heat treatment by calcination at 500 °C/3 h of the catalyst after each reaction cycle. The reuse study of the catalyst using heat treatment is presented in 57 In addition, heat treatment played a key role in assigning greater stability and maintenance of active species in the magnetic support.In view of the previous statements, the strategy adopted to improve the catalytic efficiency of the WO3/CuFe2O4 was carried out to evaluate the impact of leached species in the oxidation process of the obtained esters . The magnetic catalyst developed in this study obtained a biodiesel with an ester content of 95.2% under the optimal reaction condition . The catalyst maintained its catalytic efficiency leading biodiesel ester content above 80% after five reaction cycles and the magnetic property was maintained as well, making separation by magnetic decanting feasible. Thus, WO3/CuFe2O4 catalyst can be considered promising for the biodiesel production process for its excellent catalytic performance, easy separation and good stability.The present work evaluated the application of a new heterogeneous magnetic acid catalyst in the transesterification of the waste cooking oil in methyl biodiesel. The WOHiarla Cristina Lima dos Santos: conceptualization, methodology, investigation, visualization, formal analysis, writing – original draft. Matheus Arrais Gonçalves: methodology, resources, investigation. Alexandre da Cas Viegas: methodology, investigation. Bruno Apolo Miranda Figueira: methodology, investigation Patrícia Teresa Souza da Luz: methodology, investigation. Geraldo Narciso da Rocha Filho: supervision. Leyvison Rafael Vieira da Conceição: project administration, conceptualization, visualization, supervision, project administration, writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.RA-012-D2RA06923G-s001


Hydrogen (H2 on microglia.Mice were used to establish a postoperative pain model using plantar incision surgery. Mechanical allodynia was measured using the von Frey test. Cell signaling was assayed using gelatin zymography, western blotting, immunohistochemistry, and immunofluorescence staining. Animals or BV-2 cells were received with/without ASK1 and Trx1 inhibitors to investigate the effects of H2 increased the expression of Trx1 in the spinal cord and in BV-2 cells. H2 treatment mimicked NQDI1 in decreasing the phosphorylation of ASK1, p38 and JNK. It also reduced MMP-9 activity, downregulated pro-IL-1β maturation and IBA-1 expression in the spinal cord of mice, and ameliorated postoperative pain. The protective effects of H2 were abolished by the Trx1 inhibitor, PX12. In vitro, in BV-2 cells, H2 also mimicked NQDI1 in inhibiting the phosphorylation of ASK1, p38, and JNK, and also reduced MMP-9 activity and decreased IBA-1 expression induced by LPS. The Trx1 inhibitor, PX12, abolished the protective effects of H2 in BV-2 cells.Plantar incision surgery increased MMP-9 activity and ASK1 phosphorylation in the spinal cord of mice. MMP-9 knockout and the ASK1 inhibitor, NQDI-1, attenuated postoperative pain. H2 can be used as a therapeutic agent to alleviate postoperative pain through the Trx1/ASK1/MMP9 signaling pathway. MMP-9 and ASK1 may be the target molecules for relieving postoperative pain.For the first time, the results of our study confirm that H Postoperative pain is the most common health problem caused by tissue and nerve lesions after surgical injury. Approximately 80% of patients who experience surgical injury have acute pain postoperatively, and this progresses to severe chronic pain in over 20% of patients , 2. HoweNeuroinflammation mediated by glia, especially microglia, induces central sensitization and plays a crucial role in the pathogenesis of pain . StudiesWe explored signaling molecules upstream of MMP-9 to identify key targets for the inhibition of neuroinflammation. Apoptosis signal-regulating kinase 1 (ASK1), a member of the mitogen-activated protein kinase (MAP3K) family, activates JNK, MAPK, and p38 MAPK to induce the expression of MMP9 and amplify the inflammatory cascade; hence, it participates in the induction and maintenance of neuroinflammation , 13. Mea2) alleviates lung injury during sepsis by upregulating Trx1 ). PX12 was purchased from MedChem Express (USA) (dissolved in DMSO). Antibodies for thioredoxin (#2429S), p-p38 (Thr180/Tyr182) (#9215S), p-JNK (Thr183/Tyr185) (#9255S), and p-ERK1/2 (Thr202/Tyr204) (#4377S) were purchased from Cell Signaling Technology . Antibodies for p-ASK1 (Thr845) (sc-109911) and β-actin (sc-4778) were purchased from Santa Cruz Biotechnology . Antibodies for IBA-1 (ab178847) and MMP-9 (ab58803) were purchased from Abcam . The antibody for IL-1β was purchased from R&D systems (USA). Elisa kit  were purchased from MULTI SCIENCES. Lipopolysaccharide (LPS) and DMSO were purchased from Sigma . All other chemicals were purchased from Sigma Chemical Co. .2 dissolved in normal saline for 12 h under high pressure (0.4 Mpa) and stored at 4 °C in an aluminum bag under normal pressure. To ensure that the hydrogen concentration was greater than 0.8 ppm, HRS was freshly prepared each time, and assessed using gas chromatography ENH-1000  and Methylene Blue .The procedures of saturated hydrogen-rich saline (HRS) were prepared as previously described . They we2 at 37 °C in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 U/ml). A total of 105 cells were seeded in 6-well plates overnight and divided into the following groups (n = 4/group): sham; LPS (1 μg/ml); LPS + H2 ; H2 alone; LPS + NQDI1 (10 μM); and LPS + H2 + PX12 (8 μM) groups. NQDI1 was added after LPS stimulation. PX12 was added 0.5 h before H2 treatment.Mice were randomly assigned to the following groups: sham, plantar incision (PI), PI + HRS ; HRS treatment alone, PI + NDQI-1 ; and NDQI-1 treatment alone groups, PI + HRS + PX12 . In the HRS treatment groups, the mice received injections twice daily, and in the NDQI-1 treatment groups, the mice received injections once daily. HRS or NDQI-1 were injected 6 h before plantar incision surgery. PX12 was injected 0.5 h before HRS administration. Each group consisted of 12 mice. The experimental groups and the timing of analysis are presented in Additional file 1: Table S1. BV-2 cells were kept in humidified 5% COBefore baseline testing, all animals were placed in the testing environment for at least 2 days for acclimatization. The first researcher was responsible for numbering the animal groups and drugs, as well as for data analysis. The second researcher administered the drugs to the groups according to the assigned numbers, while the third researcher performed the behavioral tests. The staff involved were blinded. Mechanical sensitivity was detected using von Frey hairs . The animals were placed in boxes with an elevated metal mesh floor for habituation 30 min before testing. A series of von Frey hairs (the pressure intensity of mice between 0.16 g and 0.008 g) with logarithmically incrementing stiffness were used to perpendicularly stimulate the plantar surface of each hind paw. Each mouse was tested three times and the average of the threshold was measured.Thermal hyperalgesia was detected using an analgesia meter . Briefly, each mouse was placed on a 55 °C hot-plate apparatus. The heat source was focused on a portion of the hind paw, which was flush against the glass, and a radiant thermal stimulus was delivered to that site. The stimulus was then shut off when the hind paw moved (or after 20 s to prevent tissue damage). The intensity of the heat stimulus was kept constant throughout all the experiments. The elicited paw movement occurred at a latency of between 9 and 14 s in control animals. Thermal stimuli were delivered three times to each hind paw at 5- to 6-min intervals. Behavioral tests were performed blindly.2O).The lumbar spines (L4–L5) of mice were collected and analyzed at 24 h or 5 days after surgery. Mice were anesthetized, and spinal cord segments were rapidly dissected and homogenized in 1% Nonidet P-40 lysis buffer. The solubilized proteins were then resolved on gels (8% polyacrylamide gel containing 0.1% gelatin). After electrophoresis, each gel was incubated with 50 mL of developing buffer for 48 h (37.5 °C) in a shaking bath. Finally, the gels were stained with Coomassie Brilliant Blue , and equal amounts of protein per lane were separated using 8–15% sodium dodecyl sulfate–polyacrylamide gel, and transferred to polyvinylidene fluoride membranes . After being blocked with 5% bovine serum albumin and 5% skim milk for 2 h at room temperature, the membranes were incubated overnight at 4 °C with primary antibodies and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h at room temperature. The primary antibodies used included thioredoxin (1:500), p-p38 (1:1000), p-JNK (1:1000), p-ERK1/2 (1:1000), p-ASK1(1:1000), IL-1b (1:500), and IBA-1 (1:1000). For loading control, the blots were probed with an antibody for β-actin (1:5000). The filters were then developed by enhanced chemiluminescence reagents  with secondary antibodies, Sigma . Finally, data were acquired using the Molecular Imager  and analyzed using the associated software Quantity One 4.6.5 .Lumbar spine samples (L4–L5) were collected and analyzed at 24 h or 5 days after surgery. Cell samples were collected and analyzed 24 h after HThe lumbar spines (L4–L5) of the mice were collected and analyzed 5 days postoperatively. After deep anesthesia, the animal was perfused transcardially with normal saline followed by 4% paraformaldehyde. The lumbar segment L4 and/or L5 were dissected and postfixed in the same fixative. The embedded blocks were sectioned to a thickness of 20 μm. The sections of each group  were then incubated with rabbit antibody for IBA-1 , mouse antibody for MMP-9 , secondary antibody  at room temperature. After being washed three times with PBS, all slides were processed blindly and then studied under a confocal microscope  to observe morphological details after staining. Images were randomly coded and fluorescence intensities were analyzed using Image Pro Plus 6.0 software . The average green and red fluorescence intensity of each pixel was normalized to the background intensity in the same image.The lumbar spines (L4–L5) of the mice were collected and analyzed at 24 h or 5 days after surgery. The samples were sectioned to 5 μm thickness, then incubated with the first antibodies for thioredoxin (1:100), mouse antibodies MMP-9 (1:100) and rabbit antibodies for IBA-1(1:300) in 10% donkey serum and 0.3% Triton-X100. After quenching endogenous peroxidase activity, the slides were washed in PBS and incubated with HRP conjugated secondary antibody for 2 h. Diaminobenzidine was used as a chromogen and counterstaining was performed with hematoxylin. Two independent pathologists evaluated all immunohistochemistry staining sections. The score for each slide was measured as the cross-product of the value of immunostaining intensity and the value of the proportion of positive-staining cells. The intensity of immunostaining was divided into four grades: 0, negative; 1, weak; 2, medium; 3, strong. The proportion of positive-staining cells was also divided into four grades: 1, 0 − 25%; 2, 26 − 50%; 3, 51 − 75%; and 4, > 75%. The score was calculated using the following formula: total score = intensity score × proportion score.t tests and one-way ANOVA. The differences in latency over time among groups were tested with two-way ANOVA. Bonferroni post hoc comparisons was performed between multiple groups. A criterion value of P < 0.05 was considered statistically significant.GraphPad Prism 6 software  was used to conduct all statistical analysis. Continuous variables were presented as means ± SEM. Normally distributed alteration of protein detected expression and changes in behavioral responses were tested with Student’s −/−) were measured. As shown in Fig. To explore the role of MMP-9 in the process of plantar incision-induced postoperative pain, the activity of MMP-9 and the mechanical threshold of mice  promotes ubiquitination and degradation of ASK1 and inhibits ASK1-mediated apoptosis in a redox activity-independent manner , the exp2 in BV-2 cells in vitro. LPS was used to establish a cellular inflammation model. First, the ASK1 inhibitor, NQDI1, was used to explore the importance of ASK1 inhibition in reducing MMP9 activity. As shown in Fig. We further verified the protection mechanism of H2 and found that H2 could mimicked the protective effect of NQDI1. As shown in Fig. 2 decreased the phosphorylation and expression of ASK1, and reduced the activity and expression of MMP-9.Secondly, we measured the effect of H2 on Trx1 upstream of ASK1. As shown in F2 increased the expression of Trx1 compared to its expression in the LPS group. The Trx1 inhibitor, PX12, was used to assess the importance of TRX1 in vitro. We found that H2 could decrease the phosphorylation of ASK1, p-38, and JNK, and reduce the expression of IBA-1 compare to its expression in the LPS group, which were abolished by PX12  H2 decreased MMP-9 activity via the Trx1/ASK1 signaling pathway and reduced microglial activation in BV-2 cells.In this study, the main findings were as follows: (1) MMP-9 knockout delayed and relieved the development of postoperative pain induced by plantar incision surgery; (2) ASK1 inhibition decreased MMP-9 activity and expression, and attenuated postoperative pain; (3) HMMPs are involved in the pathogenesis of neuropathic pain. The mechanism of MMPs activation involves modifying cysteine residues  and dissociating the cysteine residue from the zinc-binding site of MMPs , 27. Sin2 have been confirmed to alleviate hyperpathia and activate autophagy in neuropathic pain models [2 on p-ASK1, p-p38, and p-JNK (Fig. 2 decreased MMP-9 activity and expression, inhibited the expression of IL-1β, IL-6, and TNF-α (FHRS and Hn models , 30. In 2 regulates the phosphorylation of ASK1, p38, and JNK. Trx1 is an endogenous 12 kDa multifunctional protein with two redox-active half-cysteine residues -Cys-Gly-Pro-Cys- [2 could increase the expression of Trx1 (Fig. 2 reduces postoperative pain. To further investigate the protection effects of H2, we collected and analyzed BV-2 cells after H2 treatment. We found that H2 could mimic the ASK1 inhibitor NQDI1 as it decreases the phosphorylation of ASK1, p38, and JNK, and reduces MMP-9 activity and expression (Fig. 2 were abolished by the Trx1 inhibitor, PX12, in BV-2 cells (Fig. 2 could attenuate postoperative pain by regulating the Trx1/ASK1/MMP-9 signaling pathway, and provides further details regarding the mechanism of H2 therapy.Furthermore, we explore how HPro-Cys- . It has Pro-Cys- , 32. ExpPro-Cys- , 34. TrxPro-Cys- . FurtherPro-Cys- . Since TPro-Cys- ; moreoverx1 Fig. A, D, E arx1 Fig. G, these ion Fig. A–F. We alls Fig. G–N. Thes2 has been shown to be safe with few adverse effects. Compared to vitamin E and superoxide dismutase, H2 is a selective antioxidant that reduces cytotoxic oxygen radicals [2, drinking hydrogen water, injection of hydrogen saline, and direct incorporation of molecular hydrogen by diffusion, including eye drops, baths, and cosmetics, are the main methods of ingesting or consuming H2 [2 suppresses not only the initial brain injury, but also its progressive damage [2 has beneficial effects including the promotion of microglia M2 polarization and the reduction of inflammation [2.Hradicals . Inhalatuming H2 . Inhalate damage . H2 has ammation , 23. Intammation , oral ad2 therapy. We only considered the response of male mice to postoperative pain, while ignoring the difference in magnetic responses to pain. We will explore this in future research.One of the limitations of the research is that we did not consider gender differences in H2 attenuates postoperative pain induced by plantar incisions by inhibiting the ASK1/JNK/p38/MMP9 signaling pathway and microglial activation, and this may be related to Trx1 (Fig. 2 may be a potential drug for the treatment of postoperative pain.In summary, we demonstrated that Hrx1 Fig. . Our fin


The mechanistic target of rapamycin (mTOR) functions in two distinct complexes: mTORC1, and mTORC2. mTORC1 has been implicated in the pathogenesis of flaviviruses including dengue, where it contributes to the establishment of a pro-viral autophagic state. Activation of mTORC2 occurs upon infection with some viruses, but its functional role in viral pathogenesis remains poorly understood. In this study, we explore the consequences of a physical protein-protein interaction between dengue non-structural protein 5 (NS5) and host cell mTOR proteins during infection. Using shRNA to differentially target mTORC1 and mTORC2 complexes, we show that mTORC2 is required for optimal dengue replication. Furthermore, we show that mTORC2 is activated during viral replication, and that mTORC2 counteracts virus-induced apoptosis, promoting the survival of infected cells. This work reveals a novel mechanism by which the dengue flavivirus can promote cell survival to maximize viral replication. Mechanistic target of rapamycin (mTOR) is a ubiquitous, essential serine/threonine kinase that functions in several key aspects of cell biology [reviewed in ]. mTOR emTORC2 is composed of mTOR, rictor, mLST8, and SIN1 . mTORC2 Given the important role of mTORC1 in regulating cellular metabolism, it is not surprising that several viruses have evolved mechanisms to modulate mTORC1 signaling . NumerouIn the case of dengue, virus-induced modulation of mTORC1 has been suspected due to the importance of autophagy in dengue infection , althougHere, we describe a role for mTORC2 in promoting cell survival during dengue infection. We find that the dengue non-structural protein 5 (NS5) interacts with mTORC1 and mTORC2 complexes, and that dengue infection leads to the activation of mTORC2 signaling. We report that inactivation of mTORC2 signaling leads to a decrease in viral replication and an increase in virus-induced apoptosis and cell death. These findings suggest a mechanism by which dengue counteracts apoptosis to maintain cell survival and maximize viral replication.bona fide dengue-host protein-protein interactions, defined a high-confidence protein interaction network including a predicted interaction between the dengue NS5 protein and mTOR  or rictor (a component of mTORC2) or, as a control, a nonspecific scrambled oligo-sequence . TransduWe next assessed the effect of reduced mTOR, raptor (mTORC1), or rictor (mTORC2) protein levels on dengue serotype 2 (DENV2) replication by infecting respective knockdown cells and quantifying the amount of infectious virus released  or an isolate of DENV-3 (H87), or DENV-4 (H241). In all cases, we observed an increase in the phosphorylation of AKT ser473 . In each case, increased viral replication was observed in cells depleted for raptor while decreased viral replication was observed in cells depleted for rictor  . To asseA, D, and E, p < 0.001 for activated caspase 3 level, p < 0.01 for cell viability). Interestingly, in contrast to what we observed in dengue infected cells, staurosporine treatment increased cell death and apoptosis at similar levels in the scramble control, rictor, and raptor knockdown cells, and there were no statistically significant differences in the uninfected populations  and used at 1:1000 for western blotting: Raptor clone 24C10, Rictor clone D16H9, phospho-Rictor thr1135 clone D30A6, mTOR 7C10, AKT clone 11E7, phospho-AKT ser473 clone D9E (used at 1:2000), p70 S6K clone 49D7, phospho-p70 S6K thr389 clone 108D2, LC3A/B clone D3U4C, and B-actin clone 13E5. The dengue 2 E protein antibody PA5-32246 was used at 1:10,000 for western blotting . Secondary detection for western blotting used anti-rabbit HRP antibody diluted 1:10,000 . Polyclonal affinity purified NS3 and NS5 proteins were produced as previously described . The panThe lentiviral shRNA plasmids pLKO.1 scramble, Raptor_2, mTOR_2, and Rictor_2 were gifts from David Sabatini . pMD2.G and psPAX2 were gifts from Didier Trono (Addgene plasmids 12259 and 12260). Dengue NS5-GFP fusion construct was generated by inserting the dengue New Guinea C NS5 coding sequence from pDVW601  into pAC2, in medium composed of MEM supplemented with 10% FBS, 1× non-essential amino acids, 50 units/mL of penicillin and 50 µg/mL of streptomycin. Medium was replenished frequently during experiments to avoid signaling changes caused by nutrient or growth factor depletion. The Aedes albopictus derived cell line C6/36 was propagated at 28°C in 5% CO2 in medium composed of MEM supplemented with 10% FBS, 1× non-essential amino acids, 50 units/mL of penicillin and 50 µg/mL of streptomycin. The B lymphocyte hybridoma cell line D1-4G2-4-15 was obtained from the ATCC , and was maintained in ATCC Hybri-Care Medium supplemented with 10% FBS and 1.5 g/L sodium bicarbonate.HEK293FT, HepG2, Huh7 and Vero cells were obtained from the ATCC  and cultured at 37°C in 5% COHEK293FT cells were plated to 70-80% density in antibiotic free medium. Transfection complexes were prepared by mixing plasmid DNA with polyethyleneimine  in a 1:4 mass ratio. After 15 min incubation at room temperature DNA complexes were added dropwise to the cell cultures.2, 25 mM NaCl, and 1× protease/phosphatase inhibitor cocktail . Lysates were clarified by centrifugation for 10 min at 13,000 × g at 4°C. Affinity capture beads were immediately added to the clarified lysate and incubated for 10 min at room temperature with rotation. Beads were then washed 3× with lysis buffer, and bound protein complexes eluted with 1.1× LDS sample buffer for 10 min at 70°C. For SDS-PAGE and western blot analysis, 10× reducing agent (Thermo-Fisher Scientific) was added and samples were heated for an additional 10 min at 70°C. SDS-PAGE and western blot analysis were performed as described below. Gel staining was performed using Sypro Ruby fluorescent gel stain (Thermo-Fisher Scientific) according to the manufacturer’s instructions.To prepare affinity-capture beads, the dimerized GFP nanobody LaG-16–G4S–LaG-2 (green lobster), mTOR IgG, and non-specific IgG were covalently linked to magnetic beads (Dynabeads M-270 epoxy Thermo Fisher Scientific) as previously described . Brieflyg for 4 h at 4°C. Lentiviral pellets were resuspended in OptiMEM and stored at -80°C until use. For lentiviral transductions, viral stocks were diluted to the desired concentration with OptiMEM and 0.8 µg/mL polybrene and added to cells. At 48 h post-transduction, cells containing stably integrated constructs were selected using 2 µg/mL puromycin. Experiments were performed on cell lines that were maintained and passaged for no more than 3 weeks before discarding and establishing fresh cell lines.To generate lentiviral vector stocks, shRNA constructs were cotransfected with pMD2.G and psPAX2 into HEK 293T cells. Supernatants were harvested, passed through 0.45 µm filters, layered on 20% sucrose cushions, and centrifuged at 100,000 × in vitro-transcribed RNA into Vero cells, followed by no more than 5 passages in C6/36 cells. Virus was propagated by interchangeably infecting 80% confluent C6/36 or Vero monolayers with low-passage stock virus at a MOI of 0.01, and harvesting infectious supernatants 5-7 days post-infection. Infectious supernatants were cleared of cellular debris by centrifugation and filtration through 0.2µm PVFD membrane then stored at -80°C until use. Virus stocks and experimental infectious supernatants were titrated using a flow cytometry approach which has been described elsewhere ). To prepare whole cell extracts for the immunoprecipitation load control, SDS lysis buffer  was used instead of Triton X-100 lysis buffer. Protein concentration was determined using BCA assay and a BSA standard curve, and equivalent amounts of protein were mixed with 4× LDS sample buffer and 10× reducing agent (Thermo-Fisher Scientific), followed by denaturation at 70°C for 10 min. Proteins were then resolved on 4-12% Bis-Tris gels (for lower molecular weight proteins) or 3-8% Tris-Acetate gels (for higher molecular weight proteins)  and run in MOPS or Tris-Acetate running buffer respectively. Proteins were transferred to PVDF membrane and blocked in 5% milk/TBS-T for 1-2 h. Primary antibodies were diluted in 5% BSA/TBS-T, and incubated overnight at 4°C. Membranes were washed and incubated with anti-rabbit HRP antibody for 1-2 h at room temperature. Membranes were then washed with TBS-T, exposed to chemiluminescent substrate, and imaged using a digital CCD platform . Band densitometry was performed using ImageJ software.Cells were fixed with 4% paraformaldehyde/PBS for 15 min at room temperature, permeabilized with 0.1% Triton X-100/PBS for 10 min at room temperature and blocked with 5% normal goat serum in 0.05% Tween-20/PBS. Cells were then stained with phalloidin-Alexafluor 488 (Thermo Fisher Scientific) for 1 h at room temperature and mounted using medium containing DAPI.For viability analysis, cells were trypsinized, washed with PBS, and stained with a cell impermeable amine reactive dye  according to the manufacturer’s instructions. Cells were fixed and permeabilized using the BD Cytofix/Cytoperm kit according to the manufacturer’s instructions (BD Biosciences). Permeabilized cells were stained with fluorophore-conjugated antibodies as indicated in the text. Cells were analyzed on a BD LSR-II cytometer, and data were analyzed using FlowJo software.The original contributions presented in the study are included in the article/CC, FM, JO, NB, AK, and JA have made substantial, direct, and intellectual contributions to the work, and have approved it for publication. All authors contributed to the article and approved the submitted version.FM was supported by a postdoctoral fellowship of the Canadian Institutes for Health Research. This study was supported by grant R01GM101183 to AK and grants R21AI124266 and P41GM109824 to JA from the National Institutes of Health.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Ka value between pH 6.0 and 7.0, suggesting that chaperone effects can be differently affected by physiological pH variations.Proteins can self-assemble into amyloid fibrils or amorphous aggregates and thereby cause disease. Molecular chaperones can prevent both these types of protein aggregation, but to what extent the respective mechanisms are overlapping is not fully understood. The BRICHOS domain constitutes a disease-associated chaperone family, with activities against amyloid neurotoxicity, fibril formation, and amorphous protein aggregation. Here, we show that the activities of BRICHOS against amyloid-induced neurotoxicity and fibril formation, respectively, are oppositely dependent on a conserved aspartate residue, while the ability to suppress amorphous protein aggregation is unchanged by Asp to Asn mutations. The Asp is evolutionarily highly conserved in >3000 analysed BRICHOS domains but is replaced by Asn in some BRICHOS families. The conserved Asp in its ionized state promotes structural flexibility and has a p Activities of the BRICHOS domain against amyloid fibril formation and amyloid-induced neurotoxicity are affected by pH. DNAJB6b and HSPB1.7,8 Amyloidogenic polypeptides can be expressed as proproteins which are subjected to proteolytic cleavage to release the amyloid forming fragments.9–11 Some amyloid-generating proproteins contain the domain BRICHOS, initially found in dementia-associated integral membrane protein 2B , cancer-relevant Chondromodulin-1, and ILD-related prosurfactant protein C (proSP-C).12,13 The BRICHOS domain is suggested to promote the correct folding and prevent amyloid formation of the amyloid-prone region of its proprotein during biosynthesis,14–16 and is itself released by proteolysis.17–19Proteins and peptides can self-assemble into highly ordered fibrillar structures as well as into smaller oligomers with less well-defined structures but apparently stronger ability to cause toxicity to cells. These phenomena are linked to amyloid diseases, which encompass about forty severe human diseases including interstitial lung disease (ILD), Alzheimer's disease (AD) and type 2 diabetes (T2D).19–21 and has thus emerged as a model compound in studies of amyloid fibril formation.22–25 For instance, recombinant human (rh) BRICHOS domains recognise and bind to amyloid fibrils of amyloid β-peptide  and islet amyloid polypeptide , and reduce their cellular toxicity,25–27 bind the smallest emerging toxic Aβ oligomers,24 bind to amyloid fibrils of Huntingtin (linked to Huntington's disease) and α-synuclein (linked to Parkinson disease),28 and can be used to efficiently reduce Aβ neurotoxicity in mouse hippocampal slice preparations and in vivo in animal models of AD.27,29–33 This broad anti-amyloid spectrum makes BRICHOS a potential candidate for amyloid disease prevention and even treatment.Recently, the BRICHOS domain has been shown to have chaperone activities against fibril formation and neurotoxicity of “alien” client peptides associated with human diseases but not part of BRICHOS containing proproteins,14,34,35 but the underlying pathogenic mechanisms are largely unknown. The rh BRICHOS domain from proSP-C is an efficient inhibitor of amyloid toxicity of Aβ42 in mouse hippocampal slice preparations and a Drosophila fruit fly model, reducing the generation of toxic Aβ42 oligomers, but it is not very competent to reduce the overall amyloid fibril formation rate.20,23,31,32 Rh Bri2 BRICHOS, on the other hand, assembles into differently sized species, of which monomers potently prevent Aβ42 neuronal toxicity, dimers strongly suppress Aβ fibril formation and large oligomers inhibit non-fibrillar protein aggregation.20,30,32,36 A Bri2 BRICHOS mutant (R221E), designed to stabilize the monomeric conformation prevents Aβ42 neurotoxicity rather than its overall fibrillization rate.30 A corresponding mutation in proSP-C BRICHOS (T187R) generates monomers that bind to the smallest emerging Aβ42 oligomers and is more efficient in vitro than the wildtype against amyloid fibril formation.24 Interestingly, another mutation at the same location in human proSP-C BRICHOS (T187N) leads to ILD with amyloid deposits.37 Thus, understanding the molecular mechanisms that regulate BRICHOS anti-amyloid chaperone activities are of interest from a basic science point of view, and for the development of treatments against amyloid diseases.Mutations in the BRICHOS domain or in the proproteins are associated with different protein misfolding and amyloid diseases,12,30 The physiological function of the Asp is unknown, yet two mutations of D105 in human proSP-C BRICHOS are linked to ILD.14,18,38 Here, we focused on this evolutionarily conserved Asp residue and investigated Asp to Asn mutations of two BRICHOS domains.In the BRICHOS superfamily, the secondary structure elements are highly conserved but the amino acid sequence conservation is rather low, and only one aspartic acid residue  and two cysteine residues that form a disulphide bridge were previously found to be strictly conserved, from analyses of a rather small number of sequences.i.e., ILD and familial dementias, respectively, possess different chaperone activity spectra, and have only ∼17% amino acid sequence identities.13,32,36The observation that mutations of the conserved Asp in the BRICHOS domain can result in amyloid disease prompted us to mutate it to Asn. The BRICHOS domains from proSP-C (D105) and Bri2 (D148) were selected , since tKa of the transition of about 6.1  migrated as a dimer on size-exclusion chromatography (SEC). In contrast, cleavage of differently sized NT*-Bri2 BRICHOS D148N species resulted in rh Bri2 BRICHOS D148N species with similar chromatographic profiles as the corresponding wildtype states  rh Bri2 BRICHOS D148N showed a distribution between dimers and monomers, approximately at a ratio of 1 : 1  and allowed to stabilize for 30 min prior to any recordings. As previously observed,23,32,45 100 nmol L−1 wildtype proSP-C BRICHOS prevented Aβ42-induced degradation of γ oscillations, which were remained at control levels . By contrast, surprisingly, rh proSP-C BRICHOS D105N showed a complete loss of the preventative efficacy at the same concentration . Apparently, mutating the conserved Asp to Asn in proSP-C BRICHOS dramatically impaired its capacity to prevent Aβ42-induced neurotoxicity.We recorded  BRICHOS . γ oscil47 fluorescence to monitor the kinetics of Aβ42 fibril formation in the absence and presence of different concentrations of rh proSP-C BRICHOS  BRICHOS  Fig. S6entration , while t decline . Interesand rmax , which iand rmax . These rγ oscillations30,36 and we therefore tested rh Bri2 BRICHOS D148N monomers. Rh Bri2 BRICHOS D148N monomers (50 nmol L−1) showed reduced potency to prevent Aβ42-induced neurotoxicity compared to wildtype monomers (50 nmol L−1), but did not completely lose its preventative efficacy . In the ThT assay, rh Bri2 BRICHOS D148N monomers were significantly more efficient in inhibiting Aβ42 fibril formation compared to wildtype monomers  and/or primary nucleation (kn) in addition to secondary nucleation (k2). However, the effects on secondary nucleation (k2) of rh wildtype proSP-C and the D105N mutant did not show striking differences (ESI49 and surface plasmon resonance (SPR). With seeding, the fibrillization traces typically follow a concave aggregation behavior  delays Aβ42 fibril formation without changing the total number of oligomers generated, while suppressing the secondary nucleation (k2) efficiently prevents the generation of oligomers, and blocking elongation (k+) significantly increases the number of oligomers formed.23 Of relevance for the results on Aβ42 neurotoxicity of wildtype vs. D105N proSP-C BRICHOS , which shows that the conserved Asp148 is buried and not involved in intersubunit interactions . These observations provide a likely explanation for the different effects the rh Bri2 BRICHOS D148N mutation on anti-amyloid activity versus ability to suppress amorphous protein aggregation.All the so far studied BRICHOS domains are able to suppress amyloid fibril formation, but the activity against amorphous protein aggregation is only found for oligomers of rh Bri2 and Bri3 BRICHOS.ractions . This isractions  Fig. S7c53–55 while the functions of BRICHOS domains in group II are unknown.The unexpected diverse effects of mutating the conserved Asp to Asn in proSP-C and Bri2 BRICHOS inspired us to analyse all so far deposited BRICHOS containing proproteins. Amino acid sequences of 3355 BRICHOS domain precursors were extracted from the SMART database, covering 537 different metazoan species spanning worms to humans . After sInterestingly, 1% of all analysed BRICHOS containing proproteins contain multiple BRICHOS domains along with their corresponding amyloid-prone regions, and these were found in GKN1, proSP-C, CNMD and group II families  Fig. S10Ka between 6 and 7 in both BRICHOS domains studied.In this work, we find that the capacity of the BRICHOS domain to inhibit amyloid-associated neurotoxicity and fibril formation, respectively, is oppositely affected by mutations of a phylogenetically conserved Asp, whereas the capacity to suppress non-fibrillar, amorphous protein aggregation is not affected by Asp to Asn replacement. Moreover, conformational changes occur as a result of Asp to Asn mutations in both proSP-C and Bri2 BRICHOS, and those changes can partly be mimicked by lowered pH and the conserved Asp titrates with an apparent p14,18,38 Based on molecular dynamic simulations, monomeric wildtype proSP-C BRICHOS and a D105N mutant behaved differently: only minor conformational changes were seen in the mutant, but several large-scale changes occurred in the wildtype protein at moderately elevated temperatures, which resulted in a more loosely folded structure.14 D105N mutation in rh proSP-C BRICHOS results in a more ordered conformation, as judged by CD spectroscopy, and apparently more efficient trimer formation, while D148N mutation of rh Bri2 BRICHOS results in transition of monomers to more compact dimers  are linked to ILD.t dimers . Similart dimers . This sut dimers . The diswith Asn .56 It has been shown that the small heat shock protein αB-crystallin uses different interfaces to bind to amyloid and amorphous substrates, respectively, but it is not known how different conformational states of αB-crystallin are regulated to affect activities against different types of substrates.57 The chaperoning capacities of BRICHOS domain against amyloid neurotoxicity and fibril formation can apparently be modulated by a conserved Asp in response to pH changes, suggesting the possibility that the microenvironment may affect BRICHOS function. For example, pH in the secretory pathway span from 7.2 to 5.7.58 The results here are based on in vitro and ex vivo experiments, and further work that addresses the different activities of BRICHOS are motivated.Molecular chaperones or chaperone-like domains have not been studied extensively as regards sensitivity to pH. One exception is clusterin, the activity of which is enhanced at mildly acidic pH, which appears to result from an increase in regions with solvent-exposed hydrophobicity, but independent of any major changes in secondary or tertiary structure.59 3355 BRICHOS sequences were downloaded while the sole bacterial BRICHOS precursor (Paeniclostridium sordellii ATCC 9714) was not included. Incomplete sequences were filtered out, resulting in total of 3190 sequences. Identical sequences were filtered by means of CD-HIT,60 with a threshold of 100% of sequence identity, which gave 3093 amino acid sequences. The hidden markov model profile (HMM) of BRICHOS (PF04089) was extracted from PFAM database.61 The CD-HIT filtered BRICHOS protein sequences were then scanned against the HMM profile using HMMER software v3.3.262 with an E-value cut-off less than 1.0 × 10−5. An in-house python script was written to filter out the significant BRICHOS proteins from the HMM result file and to extract the BRICHOS domain sequences (each BRICHOS amino acid sequence was extended six residues upstream from the BRICHOS domain starting position defined by PFAM). Further, BRICHOS domain sequences less than 68 aa were removed as one rodent BRICHOS domain is just 69 residues and still functional against amyloid fibril formation,63 which eventually generated 2019 BRICHOS sequences. The multiple sequence alignment of the 2019 BRICHOS domain sequences were created using MAFFT alignment server with the default settings,64 and the sequence logo was generated using Weblogo webserver.65 RAxML HPC (v8.2.10)66 was employed for constructing the phylogenetic tree using the PROTGAMMAAUTO model with 100 times bootstrap iterations. The tree shown in this study was visualized using the Interactive Tree of Life (iTOL) server67 and Geneious software. The taxonomy tree common for species that contain BRICHOS precursors were generated by NCBI Taxonomy (https://www.ncbi.nlm.nih.gov/Taxonomy/CommonTree/wwwcmt.cgi) and visualized by Geneious software.From the SMART database,36,39) plasmid as PCR template Bri2 BRICHOS D148N was obtained with QuikChange II XL Site-Directed Mutagenesis Kit , and the DNA sequence was confirmed . Similarly, the amino acid Thr at position 206 was mutated to Trp using KAPA HiFi HotStart ReadyMix PCR Kit  together with the designed complementary primers (5′-CCTATCTGATTCATGAGCACATGGTTATTTGGGATCGCATTGAAAAC-3′ and 5′-GTTTTCAATGCGATCCCAAATAACCATGTGCTCATGAATCAGATAGG-3′) and verified by sequencing (Eurofins Genomics). As described,30,36 the Bri2 BRICHOS variants were expressed in SHuffle T7 E. coli cells. Briefly, the cells were incubated at 30 °C in LB medium with 15 μg mL−1 kanamycin until an OD600nm around 0.9. For overnight expression, the temperature was lowered to 20 °C, and 0.5 mmol L−1  isopropyl β-d-1-thiogalactopyranoside (IPTG) was added. The induced cells were harvested by centrifugation  and the cell pellets were resuspended in 20 mmol L−1 Tris pH 8.0. After 5 min sonication  on ice, the lysate was centrifuged  for 30 min and the protein of interest was isolated with a Ni-NTA column. To remove the His6-NT* part, the target proteins were cleaved with thrombin  at 4 °C overnight and loaded over a second Ni-NTA column. Different species of rh Bri2 BRICHOS variants were isolated and analysed by size-exclusion chromatography (SEC) with Superdex 200 PG, 200 GL or 75 PG columns  using an ÄKTA system  with buffer of 20 mmol L−1 NaPi (Sodium Phosphate) with 0.2 mmol L−1 EDTA at different pHs. For generating rh proSP-C BRICHOS D105N, the PCR primers 5′-CACTGGCCTCGTGGTGTATAACTACCAGCAGCTGCTGATCGC-3′ and 5′- GCGATCAGCAGCTGCTGGTAGTTATACACCACGAGGCCAGTG-3′ were synthesized. With the wildtype proSP-C BRICHOS (corresponding to the solubility tag thioredoxin followed by proSP-C residues 86–19714) plasmid as PCR template, proSP-C BRICHOS D105N was obtained with QuikChange II XL Site-Directed Mutagenesis Kit , and the DNA sequence was confirmed . The expression and purification were performed as described.14,68 Briefly, both wildtype proSP-C BRICHOS and the D105N mutant were expressed in Origami 2 (DE3) pLysS E. coli cells. The cells were grown at 37 °C in LB medium containing 100 μg mL−1 ampicillin until an OD600nm around 0.9. The temperature was lowered to 25 °C and 0.5 mmol L−1  IPTG was added for overnight expression. The cells were harvested by centrifugation at 7000 × g for 20 min, and the cell pellets were resuspended in 20 mmol L−1 Tris pH 8.0. The protein was purified using Ni-NTA column and ion exchange chromatography . Thrombin  was used to remove the thioredoxin tag. The purified rh proSP-C BRICHOS variants were analysed by Superdex 200 GL columns  using an ÄKTA system . The BRICHOS mutants in this study were expressed and purified in parallel with their wildtype counterparts.For generating rh Bri2 BRICHOS D148N, the amplification primers 5′- ATAGTGATCCTGCCAACATTGATAACTTTAACAAGAAACTTACA-3′ and 5′- TGTAAGTTTCTTGTTAAAGTTATGAACAATGTTGGCAGGATCACTAT-3′ were synthesized. With the wildtype NT*-Bri2 BRICHOS . At 100% D2O, 1 mL LB was used to inoculate 100 mL M9 in 100% D2O enriched with 15N H4Cl and 13C glucose, and was grown over night at 31 °C. After overnight incubation, the 100 mL was added to 900 mL M9 in D2O enriched with 15N and 13C and grown at 30 °C until OD600nm was around 0.8. The temperature was lowered to 20 °C and 0.5 mmol L−1  IPTG was added for overnight expression. The purification was performed as described above.For the NMR experiments, gene fragment encoding human proSP-C BRICHOS was transformed into SHuffle T7 1H–15N TROSY-HSQC experiments were obtained at 37 °C on Bruker 800 MHz spectrometer equipped with a TCI cryogenic probe. Spectra were processed with the software NMRPipe and visualized using Sparky NMR. The concentrations of 2H, 15N,13C-labeled proSP-C was 288 μmol L−1 in 20 mmol L−1 ammonium acetate pH 7.2 and 229 μmol L−1 in 20 mmol L−1 ammonium acetate pH 5.5, both in 90% H2O/10% D2O.2D −1. The bandwidth was set to 1 nm, data pitch 0.5 nm, and scanning speed 50 nm min−1. The spectra shown are averages of five consecutive scans.Circular dichroism (CD) spectra were recorded in 1 mm path length quartz cuvettes at 25 °C from 260 to 185 nm in a J-1500 Circular Dichroism Spectrophotometer  with a protein concentration of 4.2–10 μmol L−1 HEPES/KOH pH 7.5 to 600 nmol L−1  and then equilibrated at 45 °C with and without different concentrations of rh Bri2 BRICHOS D148N oligomer or proSP-C BRICHOS variants. The aggregation kinetics were measured by reading the apparent increase in absorbance at 360 nm under quiescent conditions using a microplate reader .Citrate synthase from porcine heart  was diluted in 40 mmol L−1, calculated for the monomeric subunit) in 20 mmol L−1 NaPi pH 8.0 or pH 6.0 were incubated at 25 °C with 2 μmol L−1 bis-ANS --5,5′-disulfonic acid dipotassium salt) for 10 min, and the fluorescence emission spectra were recorded from 420 to 600 nm after excitation at 395 nm with the Infinite M1000 plate reader . Rh Bri2 BRICHOS T206W monomers were diluted to 2 μmol L−1 by using 20 mmol L−1 NaPi containing 0.2 mmol L−1 EDTA with different pH in the final samples in the range of 6.3–8.0. For tryptophan fluorescence measurements, samples were prepared in duplicates with a volume of 150 μL. Samples were excited at 280 nm (5 μm bandwidth) and fluorescence emission from 300–400 nm  was recorded on black polystyrene flat-bottom 96-well plates (Costar) using a spectrofluorometer (Tecan Saphire 2). For the final fluorescence intensities, the results were corrected by subtracting the background fluorescence of the buffer.Different rh BRICHOS . For each image, the contrast transfer function (CTF) parameters were estimated on boxed out regions  using e2ctf.auto.py program. A reference-free 2D classification based on the selected 10 223 phase-flipped particles (low-pass filtered to 20 Å) was performed using e2refine2d.py. The 2D classes show an approximate 2-fold symmetry, which is consistent with both the results of rh wildtype Bri2 BRICHOS oligomer and the biochemical data. Generated 2D classes were used as the input for building the 3D initial model using e2initialmodel.py. 3D refinement was performed in several rounds using e2refine_easy.py applying D2 symmetry aiming at a final resolution of 15 Å. The first two rounds of 3D refinements were performed with pixel size of 3.52 Å after binning the data by a linear factor 2. In the last round of refinement, the data was resampled to 2.464 Å per pixel. The final map from the first round of refinements was used as model in the second, and the final map from the second round of refinements was used as model in the third. The resolution was determined based on a Fourier shell correlation (FSC) value of 0.143,71 following the gold standard FSC procedure implemented in EMAN2.72Rh Bri2 BRICHOS D148N oligomers after SEC isolation were immediately stored on ice followed by grid preparation. Aliquots (4 μL) were adsorbed onto glow-discharged continuous carbon-coated copper grids  for one min. The grids were subsequently blotted with filter paper, washed with two drops of Milli-Q water, and negatively stained with one drop of 2% (w/v) uranyl acetate for 45 s before final blotting and air-drying. The sample was imaged using a Jeol JEM2100F field emission gun transmission electron microscope  operating at 200 kV. Single micrographs for evaluating the quality of the sample were recorded on a Tietz 4k × 4k CCD camera, TVIPS  at magnification of ×85 200 (1.76 Å per pixel) and 1.0–2.8 μm defocus. A total of 16 micrographs were recorded for single particle analysis. All 16 micrographs were imported to EMAN2 (version 2.3) for further processing.−1 with 20 mmol L−1 sodium phosphate pH 8.0 containing 0.2 mmol L−1 EDTA as running buffer, and the other details were set according to the manufacturer's instructions. After immobilization with a final reponse level of 727 RU, the flow-cells were stabilized over night in running buffer (10 mmol L−1 HEPES pH 7.5 containing 150 mmol L−1 NaCl and 0.2 mmol L−1 EDTA) at a flow rate of 20 μL min−1. For binding analysis, 25 μmol L−1 rh wildtype proSP-C BRICHOS or the D105N mutant in 10 mmol L−1 HEPES pH 7.5 containing 150 mmol L−1 NaCl and 0.2 mmol L−1 EDTA were injected in over the chip surfaces at 25 °C at a flow rate of 20 μL min−1 for 3 min, respectively. 10 mmol L−1 NaOH was used for further chip surface regeneration. For kinetic analysis, different concentrations of rh proSP-C BRICHOS D105N mutant in running buffer, i.e. 0, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 μmol L−1, were individually injected in over the chip surfaces at 25 °C at a flow rate of 20 μL min−1. 30 mmol L−1 NaOH was used for further chip surface regeneration. In all experiments, the response from the blank surface was subtracted from the immobilized surface response, the baselines of the sensorgrams were adjusted to zero and buffer spikes were excluded.Aβ42 monomers were immobilized by amine coupling onto flowcell 4 on a CM5 sensor chip  using a BIAcore 3000 instrument (BIAcore AB). A reference surface was prepared on flowcell 3 using the same coupling protocol but without protein injected. The immobilization was performed at a flow rate of 20 μL minversus BRICHOS concentrations. The baseline of the sensorgrams were adjusted to zero and buffer spikes were excluded for global fits to reflect the binding affinity. Since the response signals of the two lowest protein concentrations  used in kinetic analysis were too weak, only sensorgrams obtained from rh proSP-C BRICHOS D105N mutant ranging from 6.25 μmol L−1 to 100 μmol L−1 were included in the global fits. The dissociation was globally fitted to a biexponential model as described by 24,73R1 is the response signal at the starting time for the dissociation phase t1, and x is between 0 and 1. kd1 and kd2 are the dissociation rate constants for the fast and slow dissociation phases, respectively. The global fitted value kd2 was used to calculate the apparent KD value.Steady state apparent affinities for rh proSP-C BRICHOS D105N mutant to immobilised Aβ42 monomers were estimated by plotting the maximum binding response 24R0 and Rf are the initial and final response signal of the association phase, respectively. kobs is the observed rate constant described by 24,73c is the protein concentration, ka is the association rate constant, and kd from this analysis is related to secondary binding artifacts corresponding to kd1. Linear regression was used to determine ka. The apparent KD value was calculated as ratio of the dissociation rate constant and association rate constant.The association phase was fitted to E. coli (B strain) cells and purified by ion exchange.36 The purified Aβ42 peptides were lyophilized and re-dissolved in 20 mmol L−1 Tris pH 8.0 with 7 mol L−1 Gdn-HCl, and the monomers were isolated in 20 mmol L−1 sodium phosphate pH 8.0 with 0.2 mmol L−1 EDTA by a Superdex 30 column 26/600 . The concentration of monomeric Aβ42 was calculated with using an extinction coefficient of 1424 M−1 cm−1 for (A280–A300). For Aβ42 fibrillization kinetics analysis, 20 μL solution containing 10 μmol L−1 ThT, 3 μmol L−1 Aβ42 monomer and different concentrations of rh BRICHOS at molar ratios 0, 10, 30, 50, 70 or 100% (relative to Aβ42 monomer molar concentration), were added to each well of half-area 384-well microplates with clear bottom , and incubated at 37 °C under quiescent conditions. The ThT fluorescence was recorded using a 440 nm excitation filter and a 480 nm emission filter using a microplate reader . For Aβ42 seeds preparation, 3 μmol L−1 Aβ42 monomers were incubated for about 20 h at 37 °C, and the fibrils were then sonicated in a water bath for 3 min. For seeding of Aβ42 fibrillization, 20 μL solution containing 10 μmol L−1 ThT, 3 μmol L−1 Aβ42, different concentrations of rh BRICHOS at 0, 10, 50, 70 and 100%, and 0.6 μmol L−1 seeds  were added at 4 °C to each well in triplicate of 384-well microplates with clear bottom  and immediately incubated at 37 °C under quiescent conditions. The elongation rate constant k+ in the presence of rh BRICHOS was calculated from the highly seeded experiments. The initial slope of the concave aggregation traces was determined by a linear fit of the first ∼20 min traces. For all the experiments, aggregation traces were normalized and averaged using four or three replicates, and data defining one dataset was recorded from the same plate at the same time, and all the ThT data was from the same batch of Aβ42 peptide.Recombinant Met-Aβ1–42 here referred to as Aβ42, was produced in BL21*(DE3) pLysS 30,36 where the half time τ1/2 and the maximal growth rate rmax were extracted:A the amplitude and F0 the base value.Fibrillization traces of Aβ42 with and without different concentrations of rh BRICHOS were fitted to a sigmoidal equation 23M(t) is the total fibril mass concentration, and the intermediate coefficients are functions of λ and κ, and nC and n2 are the reaction orders for primary and secondary nucleation, respectively:To dissect the molecular mechanism underlying BRICHOS counteracting Aβ42 aggregation, the fibrillization traces were globally fitted by kn, k+, and k2 are for primary nucleation, elongation, and secondary nucleation, respectively. The kinetic data were globally fitted to kn, k+ or k2) is the sole fitting parameter.30,36 To investigate the generation of nucleation units, according to the nucleation rate rn(t):23rn(t) over the reaction.The microscopic rate constants −1 Aβ42 monomer was incubated at 37 °C with 100% rh wildtype proSP-C BRICHOS and the D105N mutant overnight, and the fibrils were collected at 4 °C by centrifugation for 1 h at 22 000 × g. The fibrils were gently resuspended in 20 μL 1 × TBS, of which 2 μL were applied to carbon coated copper grids, and incubated for about 5 min. Excess solution was removed and the girds were blocked by incubation in 1% BSA in 1 × TBS for 30 min, followed by 3 × 10 min washing by 1 × TBS. The grids were then incubated with polyclonal antibody against human proSP-C (SFTPC)  overnight at 4 °C, and washed 3 × 10 min with 1 × TBS. Finally, the grids were incubated with anti-rabbit IgG-gold coupled to 20 nm gold particles  for 2 h at room temperature, and washed 5 × 10 min with 1 × TBS. Excess solution was removed, and 2 μL of 2.5% uranyl acetate was added to each grid (kept about 20 s). Excess solution was removed, and the grids were air-dried at room temperature, and analysed by transmission electron microscopy .Five μmol LAll the animal experiments were carried out in accordance with the ethical permit granted by Norra Stockholm's Djurförsöksetiska Nämnd (dnr N45/13). C57BL/6 mice of either sex  were used in the experiments. Before sacrificed, all the mice were anaesthetized deeply using isoflurane.−1 NaCl, 24 mmol L−1 NaHCO3, 25 mmol L−1 glucose, 1.25 mmol L−1 NaH2PO4, 1 mmol L−1 ascorbic acid, 3 mmol L−1 NaPyruvate, 2.5 mmol L−1 KCl, 4 mmol L−1 MgCl2, 0.5 mmol L−1 CaCl2 and 75 mmol L−1 sucrose. Horizontal sections (350 μm thick) of the ventral hippocampi from both hemispheres were sliced with a Leica VT1200S vibratome . The sections were immediately transferred to a submerged incubation chamber containing standard ACSF: 124 mmol L−1 NaCl, 30 mmol L−1 NaHCO3, 10 mmol L−1 glucose, 1.25 mmol L−1 NaH2PO4, 3.5 mmol L−1 KCl, 1.5 mmol L−1 MgCl2 and 1.5 mmol L−1 CaCl2. The chamber was held at 34 °C for at least 20 min after dissection and it was subsequently cooled to room temperature (∼22 °C) for a minimum of 40 min. Proteins (Aβ42 and rh BRICHOS) were first added to the incubation solution for 15 min, and then the slices were transferred to the interface-style recording chamber for extracellular recordings. During the incubation, slices were supplied continuously with carbogen gas  bubbled into the ACSF.The brain was dissected out and placed in modified ice-cold ACSF . The ACSF contained 80 mmol Lγ oscillations) were recorded at 32 °C in an interface-type chamber (perfusion rate 4.5 mL per minute) and elicited by applying kainic acid . The oscillations were stabilized for 20 min before any recordings. No Aβ42, rh Bri2 BRICHOS R221E species or combinations thereof were present in the recording chamber either during γ oscillations stabilization, or during electrophysiological recordings. The interface chamber recording solution contained 124 mmol L−1 NaCl, 30 mmol L−1 NaHCO3, 10 mmol L−1 glucose, 1.25 mmol L−1 NaH2PO4, 3.5 mmol L−1 KCl, 1.5 mmol L−1 MgCl2 and 1.5 mmol L−1 CaCl2.Recordings were performed with borosilicate glass microelectrodes in hippocampal area CA3, pulled to a resistance of 3–5 MΩ, filled with ACSF and placed in stratum pyramidale. Local field potentials . The signals were sampled at 10 kHz, conditioned using a Hum Bug 50 Hz noise eliminator , software low-pass filtered at 1 kHz, digitized and stored using a Digidata 1322A and Clampex 9.6 software .Power spectral density plots (from 60 s long LFP recordings) were calculated using Axograph X  in averaged Fourier-segments of 8 192 points. Oscillation power was calculated from the integration of the power spectral density from 20 to 80 Hz.Q = 1%) followed by D’Agostino & Pearson omnibus normality test. Based on the previous experience with the outliers and overall sample behaviour, sample size was determined based on previous studies performed in an interface-type chamber.23,30,36,46,74,75 Each experimental round was performed with parallel controls (Control KA and Aβ42) from the same animal and randomized preparations . For comparison purposes data from Control KA and Aβ42 was pooled from interleaved slices recorded in these conditions. The number of recorded slices per condition (at least from 3–5 mice) are shown in the corresponding figure legend and source data file. Kruskal–Wallis test followed by Dunn's multiple comparisons were carried out due to the non-parametric nature- or relatively small size of some data. Comparison of the preventative efficacies of rh Bri2-BRICHOS D148N monomers and rh proSP-C BRICHOS D105N was assessed with the Mann Whitney test. Significance levels are * p < 0.05, ** p < 0.01, and ***p < 0.001. The ThT assay data are presented as means ± standard deviation, and the aggregation traces are averaged from four or three replicates.The electrophysiological data is presented as means ± standard errors of the means. Prior statistical analysis all the data was subjected to outlier determination and removal with the ROUT method (The density map of the Bri2 BRICHOS D148N oligomer have been deposited in the Electron Microscopy Data Bank (EMDB) under the accession code EMD-13005. All data and materials related to this paper are available from G. C. (gefei.chen@ki.se).G. C., Y. A. T., X. Z., H. P., H. B., A. L., and N. K. performed experiments. S. H. and G. C. carried out bioinformatic analyses and visualization. G. C., Y. A. T., X. Z., H. B., H. P., A. A., N. K., A. R., H. H., P. K., A. F., and J. J. analysed the data. G. C. and J. J. conceived the study. G. C. and J. J. wrote the paper. All authors discussed the results and commented on the manuscript.The authors declare no competing financial interests.CB-003-D2CB00187J-s001CB-003-D2CB00187J-s002


These engineered a-SnO2 interfaces, preserving all the precursor’s 2D surface-to-volumefeatures, are stable in dry/wet air up to 250 °C, with excellentbaseline and sensor’s signal reproducibility to H2S (400 ppb to 1.5 ppm) and humidity (10–80% relative humidity(RH)) at 100 °C for one year. Specifically, by combined densityfunctional theory and ab initio molecular dynamics, we demonstratedthat H2S and H2O compete by dissociative chemisorptionover the same a-SnO2 adsorption sites,disclosing the humidity cross-response to H2S sensing.Tests confirmed that humidity decreases the baseline resistance, hampersthe H2S sensor’s signal  = Ra/Rg), and increases the limit of detection (LOD). At 1 ppm, the H2S sensor’s signal decreases from an RR of 2.4 ±0.1 at 0% RH to 1.9 ± 0.1 at 80% RH, while the LOD increasesfrom 210 to 380 ppb. Utilizing a suitable thermal treatment, here,we report an amorphization procedure that can be easily extended toa large variety of TMDs and MCs, opening extraordinary applicationsfor 2D layered amorphous metal oxide gas sensors.Two-dimensional (2D)transition metal dichalcogenides (TMDs) andmetal chalcogenides (MCs), despite their excellent gas sensing properties,are subjected to spontaneous oxidation in ambient air, negativelyaffecting the sensor’s signal reproducibility in the long run.Taking advantage of spontaneous oxidation, we synthesized fully amorphous  This phenomenon is further enhancedwhen the sensor’s operating temperature (OT) is increased inthe range of 25–150 °C to compensate for irreversibleadsorption of gas molecules, as frequently experienced in metal oxideand 2D layered sensors.24Two-dimensional(2D) layeredtransition metal dichalcogenide (TMD) and metal chalcogenide (MC)semiconductors, with near atomic-scale thickness, have been extensivelyproposed in the past decade as alternative materials for traditionalnanocrystalline metal oxides (MO) for gas sensing applications.a-MO/TMD and a-MO/MC heterostructures with unexpected applications in the fieldof catalysis and gas sensing.25 On thisaccount, we recently demonstrated by means of experiments and theorythat it is possible to synthesize a-SnO2/SnSe2 heterostructures to detect NO2, H2, NH3, and humidity27 by controlledoxidation in air of 2D exfoliated SnSe2 layers. Besidestheir excellent gas sensing response, these 2D amorphous/2D crystallineheterostructures unfortunately retain some remarkable limitations.The difficulty of controlling the final thickness of the growing a-SnO2 oxide over the 2D crystalline platformand the risk that the a-SnO2 film is notself-passivating, i.e., not protecting the underlying 2D layer fromfurther oxidation, make their practical exploitation challenging.28Spontaneous oxidationof chalcogenides represents, indeed, an excellentopportunity to synthesize new kinds of interfaces comprising thinlayers of amorphous metal oxides grown over 2D layered crystallinematerials, yielding 2 layers(10–30 nm thick) and controlling the oxidation in air at 250°C for two weeks, at temperatures below the crystallization temperatureof a-SnO2 oxide ,we show for the first time that the oxidation process of 2D SnSe2 can be successfully driven to the “core” ofthe flakes, yielding single-phase, fully amorphous 2D a-SnO2, which is stable up to 250 °C and sensitiveto H2S gas (400 ppb to 1.5 ppm) and to humid air (10–80%RH (relative humidity) (RH @ 25 °C)) at a 100 °C operatingtemperature. Layered amorphous metal oxide sensors (LAMOS) like a-SnO2 can be easily manufactured in a thin-filmform by standard spin-coating deposition techniques representing anew interface for chemoresistive gas sensing applications. The amorphizationthermal oxidation process here validated can be extended to a largevariety of TMDs and MCs, opening new opportunities for “LAMOS”interfaces with unexplored surface-science capabilities, probablywell beyond gas sensing applications.Departing from liquid-phase exfoliatedSnSe2 flakes has beenpreliminarily investigated by simultaneous thermogravimetric (TG)and differential thermal analysis (DTA) techniques in air and nitrogenatmospheres by heating as-exfoliated SnSe2 at 5 °C/min,to maximize the gain of the TG signal, to 1050 °C as shown in 2O2 oxide29 and (ii) rules out any sublimation of SeO2 species .Thermal stability of exfoliated SnSe2 atmosphere in the 200–1000°Crange, as shown in 2 of −22.4% (at 612°C) and −67.9% (at 835 °C) can be further attributedto the conversion of SnSe2 to Sn2Se332 and the complete removal of Se and partial sublimationof Sn atoms, respectively,31 as reportedin the literature.Heating in a N2 at250 °C for two weeks has been investigated by XPS analysis. 2 flakes after two weeks of annealing in staticair at 250 °C. Deconvolution of the Sn 3d5/2 core-levelspectrum (2 (orange line) with maximum peak intensity of the j = 5/2 component centered at 487.4 eV33 covering approximately 97% of the whole spectral area and (ii) defectiveSnOx2– (green line) at 486.6 eV34 representing nearly 3% of the entire signal.The surface chemical composition of annealedSnSespectrum 2aand qu35 correspondingto the vertical dashed line of 38 Those located at 532.2 and 532.7 eV are ascribedto SeO240 (≈10%) and adsorbed H2O41 (≈40%), respectively.Finally, the Se 3d core-level spectrum . It may be concludedthat after controlled thermal treatment, the gas-responding surfaceof the annealed SnSe2 flakes comprises almost stoichiometricSnO2 and a negligible amount, approximately close to theinstrumental resolution of the XPS equipment (±1%), of SeO2 phases.Notably, the negligible contribution of the Se 3d signal in theannealed sample is confirmed in the survey spectra of 2 at different annealingtimes and temperatures has been investigated by the grazing incidence(GI)-XRD technique over spin-coated and annealed thin films depositedover silicon substrates. According to 2 exhibitsa major diffraction peak (black line) corresponding to the (001) planeof SnSe2 at 2θ = 14.4° (ICDD card no. 96-154-8806).At 250 °C, with proceeding the annealing time from one week (blueline) to two weeks (magenta line), the (001) peaks almost disappear, has been characterized byHRTEM microscopy and is shown in 2 , extending by approximately 8 nm insidethe flake’s terrace, is also detectable . With annealingat 250 °C for one week  sensor’s signalamplitude, as represented by the relative response ratio (RR = Rair/Rgas), and (ii)recovery of the baseline resistance after gas desorption . Tests have been carried outin the OT range of 25–150 °C in dry-air carrier gas exposingthe film to 1 ppm H2S and 40% relative humidity (40% @25 °C), as shown in n-type response to both H2S and humid-air reducing gases, consistent with preliminary resultson a-SnO2/SnSe2 interfaces28 and metal oxide SnO2 sensors.45The best operating temperature (OT) for H2S and no recovery of theBLR (dashed black lines in the figure) even after 2 h of dry-air purge.With increasing the OTs, both the sensor’s signal and desorptionkinetics improve. The BLR is fully recovered, departing from the 100°C OT, topping the best sensor’s signal response at 150°C. Poor recovery of the BLR in the temperature range of 25–75°C, shown in 46 On this account, to improve gas desorption rates and BLRrecovery, UV–vis light irradiation or increasing the sensor’soperating temperature in the 75–150 °C range has beenproposed for both traditional metal oxide48 and 2D TMD/MD sensors.50 These strategies indeedshow remarkable limitations. UV-light irradiation of 2D TMDs/MDs requiresalmost monolayer thin interfaces ,52 whereas thermal heating at highertemperatures stimulates fast surface oxidation, hampering BLR reproducibilityover the long run. On this account, a-SnO2 annealed at 250 °C can be safely operated in the temperaturerange of 100–150 °C without any risk of further degradationor recrystallization, providing an effective solution for fast BLRrecovery and improved sensor’s signal reproducibility. Watervapor at 40% RH (RH @ 25 °C) behaves like H2S, asshown in Figure S4). Notably, a complete recovery of the baseline is also recordedat 25 °C, possibly on account of a protonic (H+) Grotthusschain-like conduction mechanism induced by physisorbed water at lowertemperatures.53 Departing from the 100°C OT, most of the physisorbed water is removed54 and water vapor responds as a reducing gas as previouslyreported.55According to 2 (yellowregion), (ii) two-weeks/250 °C annealed SnSe2 (greenregion), and (iii) a-SnO2 thin films afterone-year conditioning at 100 °C (blue region). The amorphizationprocess, producing truly stable a-SnO2 oxide, sharply increases the BLR in dry air (yellow-green region),which finally stabilizes, exhibiting excellent reproducibility andstability (±5% of the BLR variation) over the long run (blueregion). One-year recordings of the electrical resistance of a-SnO2 to 1 ppm H2S gas and 40% humidity,shown in Figure S5, confirmed a remarkablereproducibility with an associated uncertainty of the sensor’ssignal to H2S gas and humidity as low as ±0.1 and±0.2, respectively.Baseline resistance reproducibilityover the long run, under sustainedOTs and dry/wet conditions, also represents a key issue for the exploitationof this new kind of interface. a-SnO2 to H2S (400 ppb to 1.5 ppm range) andH2O  in dry-aircarrier gas, respectively. Both H2S and H2Oat a 100 °C OT yield strong interactions with the a-SnO2 surface, indeed with excellent recovery of the BLRfollowing each step of gas/humidity purge. The effect of the 40% RHbackground to the dynamic H2S response shown in Figure S6 at 60 and 80% RH backgrounds) highlightsthat humid water decreases the BLR, though preserving a satisfactoryH2S gas dynamic modulation.2S compete at the same time over the sensor’ssurface represents another key issue of the sensor’s performance.This feature, known as humidity cross-response (CR) to H2S sensing, is shown in 2S, (ii) a second step at a 40% RH background and 1 ppmH2S, and (iii) a third step, equivalent to (i), to checkfor short-term repeatability. Comparing the sensors’ signalsin dry (i) and wet conditions (ii), no relevant changes of the RRs(RR = Ra/Rg) are displayed, considering that 1 ppm H2S yields anRR of 2.4 ± 0.1 in dry air and an RR of 2.3 ± 0.1 in humidair (40% RH), provided an associated sensor’s signal uncertaintyof ±0.1 (measured over a set of five consecutive measurements).Moreover, no substantial changes in the electrical response are displayedcomparing panels (i) and (iii), demonstrating an excellent short-termrepeatability.The assessment of the sensor’ssignal variations when bothhumidity and H2S sensing in thewhole gas/humidity concentration range at a 100 °C OT, as representedby the log–log calibration plots of the sensor’s signal vs H2S gas concentrations and different RH values,is shown in 2S, sensor’ssignal amplitude decreases from an RR of 2.4 ± 0.1 at 0% RH toan RR of 1.9 ± 0.1 at 80% RH. Moreover, congruent with this tendency,water vapor has a negative effect on increasing the H2Slimit of detection (LOD). By numerical extrapolation of the calibrationlines of Section S3), the theoretical LOD increases from 210 ppb at 0% RH to 380 ppbat 80% RH, confirming the inhibiting effect of water vapor upon H2S sensing. The antisynergistic interaction of water vaporupon H2S detection is also confirmed in Figure S7, showing the adsorption/desorption times of 1 ppm H2S as a function of the humidity content. Increasing RH% (asto the direction of the red arrow), the adsorption time of 1 ppm H2S increases from 9 (@ 0% RH) to 20 min (@ 80% RH), while thesensors’ signal amplitude decreases from an RR of 2.4 ±0.1 at 0% RH to an RR of 1.9 ± 0.1 at 80% RH. As a concludingremark, the adsorption–desorption times here reported (orderof minutes) are in most cases much longer than those frequently reportedin the literature (order of seconds). On this account, it should benoted that response times are mostly dependent on the experimentalconditions like the humidity content and gas fluid dynamics insidethe test cell. As to the latter, the theoretical residence time ofthe gas (TRT) given by the ratio between the cell volume [cm3] and the gas flow rate [cm3/min] may significantly differfrom the mean residence time (MRT), representing the actual time tocompletely fill/empty the test cell. In a previous paper utilizingNO2 as a marker gas, we found that the characteristic MRTof our experimental setup is between 4 and 5 min28 with respect to a TRT of only 1 min. Humidityas well influences response time. As shown in 2S almost doubles from dry to 80% RH wet conditions. In conclusion,when comparing response times of different sensors/gases, the experimentalsetup, the humidity content, and operating temperatures should alwaysbe considered and eventually normalized.The humidity cross-response (CR) to HRa/Rg) of selected sensors, calculatedby normalizing literature data to 1 ppm H2S in dry airand at different OTs, are compared in 56 and metal oxide heterostructures,which guarantee the most favorable catalytic efficiency, a-SnO2 performs better than traditional 2D TMD/MD4 sensors operated in the same temperature range (150–200 °C).2D n-n/p-n heterostructures4 operating at room temperature show RRs slightly smallerthan that of a-SnO2, excluding n-n SnSe2/SnO229 with an associated sensor’s signal ashigh as 7.5 to 1 ppm H2S gas. Regarding n-n/p-n 2D heterostructures,which can be classified as “decorated” interfaces sincethey almost comprise 3D crystalline metal oxide nanoparticles grownover 2D TMD/MD flakes, it is not usually appreciated that the naked2D TMD/MD surfaces of the heterostructure, i.e., the ones not protectedby the “decoration”, are likely to be oxidized in dry/wetair in the long run, negatively affecting the reproducibility of theelectrical signal.Gas sensing relativeresponses .73 Three stable geometries, allchemisorbed, whose structure and energetics are shown in Supporting Information. Specifically, 2 thatmolecularly adsorbed H2S attack is not favored.74 The first structure  and to one O atom (dSn–O = 1.68 Å). This mechanism is exothermic by 1.38 eV (following eq S1 in the SI). Bader analysis confirms whatis expected on the presence of a newly formed S–O bond, thatis, a slightly positively charged sulfur atom (+0.19) because of thelarger electronegativity of oxygen.To support the experimentalresults, we carried out DFT atomisticsimulations of He 1, see 7, top is2 in Eads = −4.52 eV) and is characterizedby a sulfur atom bound to two Sn atoms. Such two Sn–S bondsare 2.44 and 2.46 Å, respectively, showing that the enhancedstability may be correlated with the shorter Sn–S bond length.In this case, the Bader analysis reveals an enhanced electron localizationon S (−0.71), a fingerprint of the more marked electronegativityof S .75 The last structure , is still markedly stabilized, comparedto the reactants, with an adsorption energy of −3.65 eV andcharge distribution on S (−0.72) very close to that in 2. Regarding the H(1) and H(2) hydrogen adsorption modes,deriving from the rupture of the H2S molecule, the Badercharge associated to the formation of O–H bonds with the Oatoms at the surface is similar for all the three structures witha positive sign confirming the direction of the charge transfer fromthe adsorbed species to the a-SnO2 surface.The second structure 2 in 7, middleure 3 in 7, bottom2S adsorptionconfiguration over the optimized initial clean a-SnO2 surface shown on the right-hand side of 2S with the formation of arooted S atom, two-fold coordinated with Sn lattice atoms indicatedas (S)2Sn, and two rooted hydroxyls group O).An overall schematization of the most stable H2O,28 water vapor chemisorbsover the a-SnO2 surface with the formationof a rooted hydroxyl, two-fold coordinated with surface Sn atoms indicatedas (OH)2Sn, and one rooted hydroxyl group (OH)O. Corresponding energetics and Bader charges are shown in the lastline of 2O attack  overall Bader charge balance of H2 utilizing operando DRIFT spectroscopy76 confirmed, opposite to our dissociative adsorptionmechanism, that neither physisorption nor dissociative water adsorptionoccurs over the surface of crystalline SnO2 in the temperaturerange of 100–150 °C. On this account, theoretical vs experimentalconditions should always be considered. Our theoretical approach refersto a clean a-SnO2 amorphous interface,while DRIFT experiments specifically apply to sol–gel-preparedcrystalline SnO2. Overall, it may be concluded that regardlessof (i) the preparation conditions, (ii) the crystalline or amorphousnature of the interface, and (iii) the 2D or 3D geometry of the platform,in all cases, humidity substantially affects the resistance of thedevice. Specifically, it seems that the 2D layered nature of TMDsand MDs does not gain substantial advantages to improve humidity cross-interferencecompared to traditional metal oxide sensors. However, theoreticaland practical investigations of the water vapor adsorption mechanismover 2D layered materials are still young, while effective practicalstrategies to promote selectivity have not been implemented yet.Fundamental investigations on the temperatureinteraction of watervapor over crystalline SnOa-SnO2 sensor represents an ideal platform for operando DRIFT spectroscopymeasurements considering the absence of crystalline planes, an opportunitythat rules out any influence of the preparation conditions on theadsorption mechanism of different molecules.77It is our opinion that theoretical and experimental investigationsat lower operating temperatures (OTs) are needed, considering thatboth metal oxide and 2D TMD/MD layered gas sensors are increasinglyoperated between room temperature and a 100 °C OT. On this account,the amorphous seamless texture of the a-SnO2 and the conduction mechanism of nanosheetnetworks. Departing from few-layer gray-colored a-SnO2 flakes, representing the situation in dry air )80 exceptionally topping the field-effect mobility of ≈100cm2/(V s) for amorphous SnO2,81 a junction-limited conduction mechanism where RJ ≫ RS prevails. Thismodel addresses the formation of Schottky barriers between the flakes,modulated by the nature and composition of the adsorbing gas in thesame way as the conduction mechanism of loosely sintered metal oxidenanoparticles in traditional chemoresistive sensors. dry air 9a, as soa-SnO2 metal oxide sensors, by annealing liquid-phase exfoliated2D SnSe2 in air for two weeks at 250 °C. The oxidationprocess of 2D SnSe2 here validated, carried out at temperaturesbelow the crystallization temperature of SnO2 (280 °C),enables the spontaneous substitution of sulfur with oxygen atoms in2D SnSe2. Such layered amorphous a-SnO2 flakes, which are stable up to 250 °C, preserve allthe geometrical features of their 2D precursor counterparts. Thin-filmsensors of amorphous a-SnO2 flakes, fabricatedby spin-coating over patterned electrodes, are sensitive to H2S and humidity at a 100 °C operating temperature, withexcellent baseline resistance recovery and sensor’s signalreproducibility over one-year deployment. We also found that the electricalresponse to H2S and humidity of a-SnO2 is like that of crystalline SnO2 microporous metaloxides, with associated humidity cross-effects on H2S sensingand a reduced sensor’s signal amplitude with increasing thehumidity content. We also demonstrated the hindering effect of watervapor upon H2S sensing by a combined DFT+AIMD computationalapproach, highlighting that both H2O and H2Scompete at the same time, over the same a-SnO2 adsorption site, according to a dissociative chemisorptionmechanism. We additionally indicated a possible conduction mechanismof the a-SnO2 thin-film device, theorizingthe formation of Schottky barriers between the flakes, modulated bythe nature and composition of the adsorbing gas, in the same way asthe conduction mechanism of loosely sintered metal oxide nanoparticlesin traditional chemoresistive sensors.We have reported aninnovative and simple procedure to synthesize“LAMOS”, specifically layered amorphous 2 chalcogenide flakes to yield a-SnO2 gas sensors. Remarkably, this methodologycan be extended to a large variety of TMDs and MCs, opening new opportunitiesfor “LAMOS” interfaces with unexplored surface-sciencecapabilities, probably well beyond gas sensing applications.In conclusion, we havevalidated an effective strategy to offsettypical drift electrical signal phenomena in 2D TMD/MD sensors inducedby spontaneous degradation in dry/wet ambient conditions of the sensor’ssurface. On this account, we validated a “core” oxidation/amorphizationsynthesis of pristine 2D SnSe