Hypoxia-inducible factor 1 subunit alpha (HIF1α) upregulated the transcription of TRA2A by directly binding to its promoter. TRA2A showed a co-expression relationship with HIF1α in PC tissues. Overexpression of TRA2A alleviated the pro-inhibitive functions of HIF1α-inhibition on PC cell proliferation and motility under hypoxia. In conclusion, TRA2A is a crucial downstream gene of HIF1α that accelerates the proliferation and motility of PC cells. TRA2A may be a novel and practical molecular target for investigating the hypoxic response of PC cells.Intratumoral hypoxia is a common feature of pancreatic cancer (PC) and also plays a role in its progression. However, hypoxia-regulated signatures in PC are still not completely understood. This study aimed to identify core hypoxia-associated genes and determine their underlying molecular mechanisms in PC cells. Transformer 2 alpha homolog (TRA2A) was found to be an important hypoxia-associated gene, which was upregulated in PC tissues and in PC cells cultured under hypoxia. High TRA2A expression was associated with advanced stage, poor differentiation, and lymph node metastasis. Under normoxic and hypoxic conditions, knockdown of TRA2A both markedly suppressed PC cell proliferation and motility Abbreviations: TRA2A, transformer 2A protein; PC, pancreatic cancer; HIF1α, hypoxia-inducible factor 1-alpha; GEO, Gene Expression Omnibus; IHC, immunohistochemical staining.  TRA2A was upregulated in PC cells under hypoxia.TRA2A inhibition reduced PC progression under normoxia and hypoxia.TRA2A regulated the AKT pathway in normoxia and hypoxia.HIF1α directly regulated TRA2A expression.Overexpression of TRA2A reversed the effects of HIF1α knockdown.As one of the most aggressive malignancies, pancreatic cancer (PC) is the fourth leading cause of cancer-associated death worldwide . Despiteet al. demonstrated that the hypoxia-related gene fucosyltransferase 11 is highly expressed in PC tissues and accelerates metastasis of PC cells by upregulating the expression of pyruvate dehydrogenase kinase 1 [et al. indicated that HIF1α regulates hypoxia-induced proliferation and invasion by elevating the expression of YEATS domain-containing protein 2 [et al. showed that hypoxic pancreatic stellate cell-derived exosomal miRNAs drive the proliferation and mobility of pancreatic cancer through the PTEN/AKT pathway [Hypoxia commonly exists in solid tumors because of excessive oxygen consumption due to rapid cancer proliferation and insufficient vascular supply . Hypoxiakinase 1 . Similarrotein 2 . Cao et  pathway . HoweverMammalian homologs of TRA2 possess two different gene paralogs that encode TRA2A and TRA2B proteins . TRA2B iIn this study, we aimed to identify the biological functions of TRA2A and explore its association with HIF1α in PC cells. We found that TRA2A is a downstream gene of HIF1α and is involved in the proliferation and motility of PC. TRA2A may be a distinct target for suppressing the accelerating role of the hypoxic environment in PC cells.https://www.ncbi.nlm.nih.gov/gds). Six normoxic and six hypoxic PC samples were identified from GSE9350. A log fold change (FC) >1.0, combined with an adjusted P-value <0.05, was set as the threshold to determine differentially expressed genes. The differentially expressed genes are shown in a volcano plot.The gene expression matrix   was acce2 in Dulbecco’s modified Eagle medium  containing 10% fetal bovine serum . To create a hypoxic microenvironment, PC cells were placed in a three-gas incubator , and the oxygen concentration was set at 1%. The Affiliated Hospital of Guizhou Medical University provided 48 paired clinical PC samples, collected between January 2019 and December 2020, for this study. The Human Research Ethics Review Committee of Guizhou Medical University approved the application of these clinical samples (approved number: 2022–11), which was performed according to the tenets of the Declaration of Helsinki.The PC cell lines PANC-1  and Capan-2  were purchased from the American Type Culture Collection . PANC-1 and Capan-2 cells were cultured at 37°C in 5% COSmall interfering RNAs targeted against TRA2A (si1-TRA2A and si2-TRA2A) and HIF1α (si-HIF1α), and the corresponding negative control (si-NC) were purchased from Sangon Biotech (Shanghai) Co., Ltd. The sequence of siRNA control was ACGUGACACGUUCGGAGAATT, the sequence of si1-TRA2A was UUGGGAUCUGGAUUUGCCCTT, the sequence of si2-TRA2A was GUUGUGUACAAACUGAGGCTT, and the sequence of si-HIF1α was CAAGUAGCCUCUUUCACAA. The TRA2A-overexpressing plasmid, HIF1α-overexpressing plasmid, and its matched control were acquired from Genechem (Shanghai) Co., Ltd. siRNAs and plasmids transfection process was carried out via Lipo 2000 reagent  based on the manufacturer’s guide.Total RNA from PC cells and tissues was isolated using TRIzol reagent . A kit of complete reagent for first-strand cDNA synthesis  was used to reverse transcribe mRNAs into cDNAs. The mRNA expression of target genes was determined using KAPA SYBR® FAST reagent . β-actin was used as a loading control. The primers used were as follows:HIF1α forward primer 5′-ATCCATGTGACCATGAGGAAATG-3′, HIF1α reverse primer 5′-TCGGCTAGTTAGGGTACACTTC-3′, TRA2A forward primer 5′- GGTCAGGATCTCGTAGTCCAT-3′, TRA2A reverse primer 5′- CCTCGACCTGGATTTTGATCTTG-3′, β-actin forward primer 5′-CATGTACGTTGCTATCCAGGC-3′, and β-actin reverse primer 5′-CTCCTTAATGTCACGCACGAT-3′.3 cells/well, followed by culture at 37°C in 5% CO2. After 24-hour and 48-hour incubation, the culture medium was replaced, and 10 µL CCK-8 solution was added to each well. The plate was then placed in a 37°C incubator for 2 h. The absorbance was measured at a wavelength of 450 nm.The cell proliferation rate was determined using the Cell Counting Kit-8 reagent . Briefly, the PC cells were seeded into 96-well-plates at a density of 4 × 10The PC cells were seeded onto 6-well plates, followed by culture until >95% confluence. A 200 µL pipette tip on the cell monolayer was used to generate a scratch lesion. The cells were then washed three times with PBS and a fresh serum-free medium was added. The status of the wound was monitored using an optical microscope from 0 to 24 h.4/well) was placed in an FBS-free medium in the upper transwell chamber  pre-coated with Matrigel . Next, 700 µL medium with 10% FBS was placed in the lower chamber. After 24 h, the top chambers were fixed using 4% paraformaldehyde for 15 min and stained with 1% crystal violet for 20 min. A cotton swab was then used to remove non-invading cells from the upper surface. Invasive cells per field were counted using an optical microscope .Each group of PC cells  was separated using 12% SDS-PAGE gel  and transferred onto PVDF membranes . After blocking using 5% BSA, the membranes were incubated with anti-TRA2A , AKT , p-AKT , PTEN , anti-KI67 , anti-PCNA , anti-HIF-1α  and anti-β-actin . After incubation with a horseradish peroxidase-conjugated secondary antibody  for 2 h, the protein bands were visualized using ECL reagent . Image Pro-Plus software was used to analyze protein expression, and β-actin was used as a loading control.Cells were lysed with RIPA lysis buffer reagent  containing 1% proteinase inhibitor PMSF  on ice for 30 min. After centrifugation at 13,000 6 PANC-1 cells with TRA2A knockdown and negative control cells were subcutaneously injected into the upper-right flank of BALB/c mice(n = 7 in each group). The health status of the mice was monitored daily, while the tumor volume was measured per week. Tumor volume was monitored once a week and determined as follows: (mm3) = (Long × Width2)/2. After 5 weeks, all mice were euthanized, and tumor tissues were extracted to detect the expression of KI67 and PCNA using immunohistochemical staining. The process of animal experiments was approved by Animal Ethics Committee of Guizhou Medical University .For the subcutaneously injected model, 10 female BALB/c nude mice were obtained from the Animal Center of Guizhou Medical University . After adaptive feeding, 2 × 102O2 and 5% bull serum albumin  for 30 min at room temperature. The specimens were incubated with primary anti-TRA2A (1:100), KI67 (1:100), anti-PCNA (1:100), and anti-HIF1α (1:100) antibodies for 12 h and horseradish peroxidase-conjugated secondary antibodies . Next, DAB was used to identify the antigen-antibody complex, and the samples were stained with hematoxylin to visualize the nucleus.The clinical PC and adjacent tissues were fixed with paraformaldehyde , embedded in paraffin , and sectioned into 4 μm thickness. After dewaxing, rehydration, and antigen retrieval with sodium citrate , tumor samples were blocked using HPC cells were crosslinked with 1% formaldehyde for 20 min at room temperature. After breaking the DNA into 200–500 bp fragments via ultrasound, Pierce Magnetic ChIP Kit  combined with HIF1α antibody (1:100) or IgG antibody (1:100) was used to enrich the DNA fragments. The purified immunoprecipitated DNA fragments were analyzed using qRT-PCR.All experiments were performed three times, and the data are shown as the mean ± standard deviation. SPSS 19.0 software (IBM Corp.) was employed to perform all analyses. The difference between two groups was analyzed via Student’s t-test, while one-way exploration of variance (ANOVA) combined with LSD t-test was used to analyze differences between various groups. Co-expression between the two genes was explored using Pearson’s correlation test. *P < 0.05 was regarded as significant.in vivo. Downregulation of TRA2A suppressed the activation of the AKT pathway in both normoxia and hypoxia. HIF1α upregulated the expression of TRA2A by directly binding to its promoter, whereas HIF1α inhibition decreases its expression. TRA2A overexpression reversed the effects of HIF1α knockdown in PC cells.Through the analysis of gene expression profiles in GEO, we found that TRA2A expression was upregulated in PC cells under hypoxia. TRA2A expression was higher in PC tissues than in adjacent tissues, as well as in PC cells cultured under hypoxia. High TRA2A expression was associated with advanced stage, poor differentiation, and lymph node metastasis. TRA2A knockdown reduced cell proliferation and mobility under both normoxia and hypoxia as well as reduced PC cell proliferation Gene expression data (GSE9350) containing six normoxic and six hypoxic PC samples were downloaded from the GEO database, and 1446 differentially expressed genes (DEGs) were identified ). AdditiTwo siRNAs targeted against TRA2A were adopted to suppress the expression of TRA2A under normoxic conditions. The results showed that both effectively inhibited TRA2A expression ). TRA2A in vivo effects of TRA2A knockdown were also determined. We found that tumor tissues with TRA2A knockdown showed a lower growth rate and tumor weight than the negative control (The  control ). We fur control ).FigurePANC-1 and Capan-2 cells were transfected with si-NC, si1-TRA2A, and si2-TRA2A and then cultured under normoxic and hypoxic environments. TRA2A knockdown significantly inhibited TRA2A mRNA and protein levels under hypoxic ). The CCThrough GSEA analysis of TRA2A in PC based on the RNA-seq data of TCGA, we found that TRA2A was positively associated with the activation of the AKT pathway ). InhibiP < 0.001; P < 0.001; To further investigate the regulatory networks of TRA2A, we analyzed the relationship between TRA2A and HIFs. The motif sequence of HIF1α was obtained from the JASPAR database ), and a Furthermore, we co-transfected HIF1α siRNA and TRA2A-overexpression plasmid in PANC-1 and Capan-2 cells under hypoxia ). The CCHypoxia is commonly observed in the solid tumor microenvironment, including PC. The proliferation and metastasis of tumor cells can be promoted by hypoxia . SeveralIn the present study, using bioinformatics, TRA2A in PC cells was found to be upregulated in hypoxia compared with normoxia. TRA2A was also upregulated in PC tissues compared with adjacent tissues, and was associated with poor clinical traits. Through related experiments, we confirmed the increase in TRA2A expression in PC cells and PC tissues cultured under hypoxia. Therefore, TRA2A was considered to be a key hypoxia-related gene that may drive PC progression.Previous studies on the expression of TRA2A and its significance have been extensive. Xu et al. confirmed that high expression of TRA2A indicates poor prognosis in patients with hepatocellular carcinoma . SanchezAKT signaling is a key factor involved in the etiology of PC and is commonly activated in PC. Activation of AKT signaling contributes to PC cell proliferation, metastasis, and differentiation ,23. PrevHypoxia exerts a significant effect on many cancers, and hypoxia-inducible elements are key mediators of hypoxia reactions . HIFs, sTRA2A is a core target gene of HIF1α, which has the potential to drive the proliferation and mobility of PC cells by mediating the activation of the AKT pathway.Click here for additional data file.


It is found that this layer induces the formation of stable solid electrolyte interphase, which significantly improves the structural and electrochemical stability of LAGP toward metallic Li. As a result, the optimized symmetrical cell exhibits a long lifetime of 360 h with an areal capacity of 0.2 mAh cm−2 and a current density of 0.2 mA cm−2. This strategy provides new insights into the stabilization of the solid electrolyte/Li interface to boost the development of ASSLB.Lithium aluminum germanium phosphate (LAGP) solid electrolyte is receiving increasing attention due to its high ionic conductivity and low air sensitivity. However, the poor interface compatibility between lithium (Li) metal and LAGP remains the main challenge in developing all-solid-state lithium batteries (ASSLB) with a long cycle life. Herein, this work introduces a thin aluminum oxide (Al Currentectrode) . Replaciectrode) ,8,9. Criectrode) . Variousa3Zr2O12 , sulfide Zr, Sn) , as well Zr, Sn) ,15,16. Activity >−4 S cm−1titution . LAGP SEtitution , sinterititution , sol-geltitution , to hot-titution , etc. Cotitution ,22,23.Li/LAGP interfacial incompatibility remains the main challenge for the further development of ASSLBs. Due to the high Fermi energy level of Li metal, germanium (Ge) in LAGP would be irreversibly degraded upon being contacted with Li metal at the Li/LAGP interface, resulting in an increased impedance versus time . When an−2. Xiong et al. [2O3) on the surface of Li1.3Al0.3Ti1.7(PO4)3 (LATP), and the Li/LATP interface was significantly stabilized at 0.01 mA cm−2. Currently, most Li/LAGP/Li symmetrical cells show a long cycle time at a low current density of 0.1 mA cm−2 [Li et al.  introducg et al.  created g et al.  adopted  mA cm−2 ,35,36,37 mA cm−2 .−4 S cm−1 at room temperature. Furthermore, an ultrathin Al2O3 coating layer is deposited in the LAGP pellet surface via the ALD technique, which physically isolates the contact between Li metal and LAGP SE and enhances the electrochemical stability of LAGP toward metallic Li. The Li symmetrical cell with Al2O3-coated LAGP SE showed an excellent lifetime of 360 h at a current density of 0.2 mA cm−2 and capacity density of 0.2 mAh cm−2, which is higher than most previously reported Li/LAGP/Li cells with interlayer modification.In this work, the effect of sintering temperature on the structure and ionic conductivity of LAGP is investigated, and 850 °C is found as the optimal sintering temperature to reach an ionic conductivity of 2.4 × 101.5Al0.5Ge1.5(PO4)3 were purchased from the Shanghai Institute of Ceramics . The precursors for ALD were trimethylaluminium (TMA) and deionized water (H2O). Lithium hexafluorophosphate , ethylene carbonate , and diethyl carbonate  were used as received for coin cell assembly.LAGP powders with a stoichiometric formula of Li−1 in a tube furnace. The thickness of the as-prepared LAGP pellets was about 0.8 mm.LAGP pellets were made by a conventional dry-pressing method . To begin, 0.2 g LAGP of powder was placed into a stainless-steel die with 12 mm diameter and pressed at 200 MPa uniaxial pressure at room temperature for 2 min. The obtained pellets were then transferred into a ceramic crucible. The sintering process was conducted at 800–900 °C in the air for 6 h at a heating rate of 2° min2O3 layer was coated on LAGP in a commercial ALD reactor . The coating process was carried out at 100 °C by following a TMA pulse/TMA purge/H2O pulse/H2O purge sequence in each cycle. After 50 cycles, Al2O3 films were deposited on LAGP noted as LAGP@Al2O350.The AlThe morphologies and microstructures of LAGP powder and pallets were observed and analyzed by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS). X-ray diffraction (XRD) was used to analyze the crystal structure of LAGP. Surface elemental analysis was investigated by X-ray photoelectron spectroscopy (XPS).σ) of LAGP was measured by Equation (1):L and A are the thickness and effective area of LAGP, respectively, and R is the resistance obtained by EIS) testing within a frequency range of 0.01–106 Hz with an AC amplitude of 5 mV.Electrochemical impedance spectroscopy EIS measurements were carried out at 25 °C and elevated temperatures (30–60 °C). The LAGP pellets were Au-coated on both sides as electrodes and clamped for conduction. The ionic conductivity  were added to each Li/LAGP/Li cell. Symmetrical Li/LAGP/Li cells were tested in NEWARE battery cycler  at room temperature and 40 °C with current densities ranging from 0.02 to 0.2 mA cm−2 with a plating-striping time of 1 h. The cycled Li/LAGP batteries were disassembled in the argon-filled glove box and then characterized by SEM and XPS to collect surface composition information.CR2032 cells were assembled in an argon-filled glovebox workstation with Hbulk) and along the grain boundary (σgb). With the sintering temperature increasing, unit cell volumes would grow to form wider Li-ion migration channels, thus leading to higher bulk conductivity σbulk [gb) mainly depends on grain-grain ceramics contact and density. High sintering temperatures above 850 °C would possibly induce cracks due to grain strain, decrease ceramics density, and slow down Li-ion transport at the grain boundary [ty σbulk ,39. The boundary ,40,41. C2(PO4)3 (JCPDS PDF No. 80-1924). 6 Hz at room temperature. LAGP T850 possesses a smaller electrochemical impedance (283 Ω) than LAGP T800 (390 Ω) and T900 (392 Ω), thus leading to higher ionic conductivity of 2.4 × 10−4 S cm−1  as illustrated in −4 to 7.3 × 10−4 S cm−1. LAGP T850 will be selected as a reference for further electrochemical stability tests with Li metal due to its highest ionic conductivity.XRD patterns of LAGP pellets sintered at 800–900 °C are shown in −2 and rapidly rises to 3 V at only 0.04 mA cm−2. The large overpotential can be attributed to the unstable Li/LAGP interface and Li dendric deposition [2O3 layer is deposited on the surface of LAGP, as illustrated in 2O3 layer is controlled at 5 nm based on our previous study on the P2-Na0.66(Mn0.54Co0.13Ni0.13)O2 cathode [2O350 from the top view and cross-section. Both demonstrate a compact and flat surface without pores. There is no noticeable change found in the surface and cross-section morphology.position . Herein, cathode . Figure 2O3 layer. As shown in 2O350, suggesting the successful deposition of Al2O3 film on the LAGP surface. The prominent peak in Li 1s XPS spectra  of cycled bare LAGP (2O2 in the SEI layer. The surface Li 1s spectrum (0 s etching) obtained from LAGP@Al2O350 shows higher binding energies of 53.5 and 53.0 eV for LiF and Li2O2  [xPFy ratio becomes larger for the LAGP@Al2O350 after 1060s etching, which also indicates the compositional variation in the SEI layer along with the depth. The XPS results suggest that the SEI layers formed on the bared LAGP and Al2O3 coated LAGP possess different compositions. The Al2O3 coating induces an Al-containing SEI layer [Meanwhile, a XPS test was performed to collect surface elemental information of cycled LAGP and LAGP@Alare LAGP c can be nd Li2O2 d. It sho EC:DEC) . The LiFEI layer ,49, form2O3 film was deposited on the surface of LAGP pellet by the ALD method to address the issue of Li/LAGP interface incompatibility. The optimized Li/LAGP@Al2O350/Li cell maintained a stable voltage profile up to 0.2 mA cm−2 and exhibited a 360-h cycling duration at the capacity density of 0.2 mAh cm−2. Remarkably, this cell showed a superior cumulative Li plated capacity of 72 mAh cm−2 at 0.2 mA cm−2 current density. SEM and XPS characterizations suggest that Al2O3 film induced the formation of an Al-rich SEI layer to regulate Li dendrite deposition and contributes to a uniform and dense interface after long Li plating/stripping cycles.In summary, a thin Al


The lack of drinkable water is one of the most significant risks for the future of the humanity. Estimates show that in the near future, this risk will become the origin of massive migrations leading to humanitarian disaster. As consequence, the development of solutions to provide water is becoming ever more critical, and a significant effort is devoted to identifying new sources of water. Among the developed strategies, fog harvesting, which takes advantage of atmospheric water to provide potable water, is a solution of interest due to its potential in sustainable development. Unfortunately, this approach suffers from low yield. In the present work, we take inspiration from living species to design and elaborate surfaces with high potential for water harvesting applications. This work takes advantage of 3D-printing and post-printing functionalization to elaborate a strategy that allows modelling, printing, and functionalization of surfaces to yield parahydrophobic behavior. The roughness and surface morphology of the prepared surfaces were investigated. These characteristics were then related to the observed wettability and potential of the functionalized interfaces for water harvesting applications. This work highlights significant variations in surface wettability via surface modification; strong hydrophobic behavior was observed via modification with linear carboxylic acids particularly for surfaces bearing vertical blades . Climate change has many consequences for living species. Currently, one of the most significant problems associated with the evolving climate and environment is the associated decrease in drinkable water resources. Recent analyses estimate that by 2025, a significant portion of the global population will suffer from a lack of water . Since wSome creative solutions have been identified which provide potable water to regions most impacted by this growing challenge. One of the more efficient strategies developed has been the desalinization of sea water ,3. This One approach to improve the yield and effectiveness of this strategy is to find inspiration in nature . It is wConsidering cacti examples, the capacity to harvest water is correlated with two parameters. The first of those parameters is the macrostructures present on each plant that increases the surface area available for water harvesting, mainly spines for cacti. These structures increase the specific surface area available for water harvesting and as consequence increase the overall harvesting yield. The second parameter is the wettability. Cacti spines have strong hydrophobic character and high water adhesion, this combination of properties is known as the petal effect or parahydrophobicity ,23,24. These two parameters contribute to the overall mechanism of fog harvesting, which can be briefly described as follows. Due to the high affinity between the surface of the spines and water, a water droplet will accumulate on these structures. As the volume of the water droplet becomes significantly large, the adhesion strength becomes too weak and the droplet can no longer be supported and is driven along the spines due to parallel grove on the spines that allow a directional collection . ConsideIn this work, we propose using a platform that combines strategies which separately control surface morphology and wettability. First, to control macroscopic surface morphology, we employ three-dimensional printing (3D-printing). 3D-printing is a technology with significant potential in a wide range of emerging fields including engineering with conductive polymers, desalination, and solar evaporators ,28,29,30The surfaces were modelled using Fusion 360 from Autodesk .Plate with vertical blade (PVB): PVB was designed as a 2.5 × 2.5 × 2 cm square plate decorated with vertical blades of 3 mm height, 0.65 mm width, and separated by space of 1.3 mm.Grid with vertical blade (GVB): GVB was designed as a 2.5 × 2.5 × 2.5 cm square grid with vertical blades of 0.62 mm width separated by space of 1.25 mm.Large mesh grid (LMG): LMG was designed as a 2.5 × 2.5 × 0.25 cm square grid with mesh of 2.5 mm.Narrow mesh grid (NMG): NMG was designed as a 2.5 × 2.5 × 0.25 cm square grid with mesh of 1 mm.Surfaces were printed using a 3D-printer . The filament used for printing was Cuivre 80 Ultra (Volumic Ultra). This filament is made of polylactic acid (PLA) and loaded with 80% of colloidal copper. The nozzle used for printing is a 25 µm brass nozzle. Extrusion temperature was 195 °C and reception plate temperature was 60 °C. 4)2S2O8 (0.13 M) and then placed on a shaker at room temperature for 2 h. During this period, the colorless solution became blue, confirming the release of Cu2+ ions. After this period, the surfaces were washed three times in water and once in ethanol. After washing, the surfaces were directly used for the next step without drying.Step 1: The surfaces were immersed in a solution of NaOH (2.5 M) and (NH−1) and slowly agitated at room temperature for 2 h. After this period, the surfaces were washed three times in ethanol. The surfaces were then dried in an oven overnight at 70 °C.Step 2: The oxidized surfaces were immersed in carboxylic acid solution in ethanol . Samples were observed with gold coating and at an accelerating voltage of 10 kV. The samples were coated with gold using Quorum Q150R S Sputter Coater .Contact angle measurements were performed on non-functionalized and post-functionalized surfaces. All measurements were performed 5 times to determine standard deviation. The apparent contact angles were obtained with a contact angle system OCA TBU100 (dataphysics). The apparent contact angles were measured using the sessile drop method with deionized water (droplet volume of 2 µL). The goal of this research is to prepare parahydrophobic surfaces with various macroscopic morphologies designed to increase specific surface area and improve the water harvesting capability of the interfaces. To reach this goal, we employed 3D-printing technology. This approach allows us to print surfaces suitable for functionalization with a precise morphology.The first step is to design, and 3D print a surface that can be post-functionalized. Taking inspiration from nature, we chose surfaces with macroscopic substructures that increase the specific surface area and therefore increase the water harvesting capability of the surface. Four surfaces were designed as plates and grids, specifically a plate with vertical blades (PVB) A, a gridSince the printed surfaces need to be suitable for post-functionalization, these designs were printed from a commercially available composite filament comprised of 80% colloidal copper and poly-lactic acid. The printed surfaces are presented in It is well reported in the literature that copper can be used for post-functionalization for various applications including the control of surface wettability ,40,42,432 via immersion in a solution of ammonium persulfate (0.013 M) and sodium hydroxide (0.25 M) for two hours and at room temperature. The oxidized surface reported as Cu(OH)2 was then washed two times in water and once in ethanol. For the second step, the Cu(OH)2 surface was immersed in the functionalization solution for two hours at room temperature without further treatment. The functionalization solution contained 10 mg/mL of carboxylic acid in absolute ethanol. A wide variety of carboxylic acids were employed in this work (The printed surfaces (reported as Cu surface) were functionalized with various carboxylic acids following a two-step strategy. In the first step, copper is oxidized from metallic copper to Cu(OH)his work  to evalu2), hexanoic acid (C6), decanoic acid (C10), myristic acid (C14) and stearic acid (C18)). As negative controls, carboxylic acids with polar side chains and an associated lower hydrophobicity were considered acetic acid and glycolic acid, Methoxy and Gly, respectively). Finally, due to their low associated surface energy, fluorinated and perfluorinated carboxylic acids were also employed for functionalization . After functionalization, all functionalized surfaces were rinsed three times in ethanol and then dried 24 h in oven at 70 °C prior to further analysis and characterization.To engineer hydrophobic properties, carboxylic acids with linear and non-polar chains were selected. The carboxylic acids were chosen with a wide variety of chain lengths to explore the impact of the side chain length on the final surface properties 2, LMG-Cu(OH)2 and NMG-Cu(OH)2 surfaces. Those Ra variations can be linked to changes in morphologies due to swelling when the surfaces are immersed in solution or contraction that occurs when the surfaces are dried. Additionally, the aggressive conditions used to convert Cu to Cu(OH)2 may degrade PLA (saponification), particularly at the edge of the blades or meshes. The partial saponification of PLA during oxidation has already been reported in the literature and can be confirmed by simple IR observation  the roughness measurements are performed on the edge of the macroscopic structures. Here, the more interesting point is not the variations in roughness between the different functionalization but more the fact that for all designs a significant increase in roughness is observed after oxidation of the Cu surfaces confirming the impact of the oxidative treatment on the PLA. Raw copper surfaces present Ra of 2.8 ± 0.3 µm, 3.0 ± 0.5 µm, 7.4 ± 0.9 µm and 4.0 ± 1.4 µm, respectively, for PVB-Cu, GVB-Cu, LMG-Cu and NMG-Cu surfaces. Whereas oxidized surfaces present Ra of 9.8 ± 1.7 µm, 7.8 ± 1.1 µm, 11.3 ± 1.3 µm and 13.8 ± 2.4 µm, respectively, for PVB-Cu(OH)ervation  44]. Fo. Fo2, GVTo confirm the observations made on roughness, scanning electronic microscopy was also performed .2 surface  between 120° and 140° depending on the surface design 2: θ = 146.2° ± 2.8, LMG Cu(OH)2: θ = 139.3° ± 1.3 and NMG Cu(OH)2: θ = 144.7° ± 1.0). Only the GVB Cu(OH)2 surface presents a lower contact angle value, but still remains strongly hydrophobic with θ = 128.9° ± 1.2. For surfaces functionalized with linear carboxylic acids, an increase in hydrophobicity is observed after functionalization. No significant differences are observed between the different linear carboxylic acids employed, and globally all carboxylic acid functionalization display strong hydrophobic properties. Only surfaces functionalized with acetic acid were “less” hydrophobic than the others, having apparent contact angles (θ) between 145° and 155°. Respectively, the contact angles are θ = 150.1° ± 0.4, θ = 154.4° ± 1.1, θ = 144.0° ± 1.2 and θ = 149.6° ± 2.0 for PVB C2, GVB C2, LMG C2 and NMG C2 surfaces. All other surfaces functionalized with C6, C10, C14 and C18 present contact angles near 155° or even higher n higher B. All va2F4, F4, F6) present lower contact angle values compared to that observed for linear carboxylic acids. This may be a consequence of the associated reactivity of fluorinated acids and overall lower degree of functionalization of the surface compared to aliphatic carboxylic acids. Despite this, the fluorinated surfaces remain strongly hydrophobic with apparent contact angles between 145° and 155° depending on the chain used for functionalization and the printed design of the surface. As examples, surfaces functionalized with C2F4 show, respectively, apparent contact angles of θ = 147.0° ± 2.6, θ = 156.0° ± 1.1, θ = 145.4° ± 2.9 and θ = 150.7° ± 0.3 for PVB C2F4, GVB C2F4, LMG C2F4 and NMG C2F4 surfaces.Globally, the fluorinated and perfluorinated chains (Cθ) between 130° and 145° depending on the surface macro-structure . Interestingly, it is important to note that all surfaces (except the surfaces modified with Methoxy) present significantly higher hydrophobic feature compared with not functionalized surfaces. This highlights the importance of controlling the surface macroscopic structures and the advantage provided by employing a 3D-printing strategy.As expected, the surfaces functionalized with carboxylic acids bearing polar chains present lower hydrophobicity since the polarity of their side chain affords interaction with water. All the surfaces functionalized with glycolic acid (Gly) present hydrophilic features and no contact angle could be measured due to the spreading of the water into the macro-structures of the surfaces C. While To confirm that the prepared surfaces are parahydrophobic , dynamic contact angle measurements were completed ,23,24. ATo show the potential of the developed surfaces for future applications in fog harvesting, it is necessary that the affinity of water to the surface is overcome as the water droplet grows, meaning the water should eventually be released to enable subsequent collection. To investigate this behavior, a water droplet of 100 µL was deposited onto the surfaces and the surfaces were then tilted .With such a large droplet volume, the surfaces no longer demonstrate parahydrophobic behavior nor having strong adhesion, as all droplets roll off the surface even at small tilt angles (less than 20°). This confirmation allows us to consider that the prepared surfaces will be candidates of interest for future fog harvesting investigations.In the present work, we utilize 3D-printing and post-functionalization to create parahydrophobic surfaces. Four surface shapes were designed to increase the specific surface area, then 3D printed and finally post-functionalized with a wide range of carboxylic acids . Surface roughness was characterized after functionalization on the edge of the surface structuration. Roughness measurements were consistent, and one design remain mostly the same regardless the functionalization. Surface morphologies were observed using SEM. These observations on functionalized confirmed the roughness measurements and showed that the functionalized surfaces present thin nano-crystallization as described for such kind of functionalization in the literature ,45,46. M


Herein, we exploited single-cell Ca2+ imaging to assess the signal transduction pathways whereby histamine generates intracellular Ca2+ signals in the human fetal lung fibroblast cell line, WI-38. WI-38 fibroblasts were loaded with the Ca2+-sensitive fluorophore, FURA-2/AM, and challenged with histamine in the absence and presence of specific pharmacological inhibitors to dissect the Ca2+ release/entry pathways responsible for the onset of the Ca2+ response. Histamine elicited complex intracellular Ca2+ signatures in WI-38 fibroblasts throughout a concentration range spanning between 1 µM and 1 mM. In accord, the Ca2+ response to histamine adopted four main temporal patterns, which were, respectively, termed peak, peak-oscillations, peak-plateau-oscillations, and peak-plateau. Histamine-evoked intracellular Ca2+ signals were abolished by pyrilamine, which selectively blocks H1R, and significantly reduced by ranitidine, which selectively inhibits H2R. Conversely, the pharmacological blockade of H3R and H4R did not affect the complex increase in [Ca2+]i evoked by histamine in WI-38 fibroblasts. In agreement with these findings, histamine-induced intracellular Ca2+ signals were initiated by intracellular Ca2+ release from the endoplasmic reticulum through inositol-1,4,5-trisphosphate (InsP3) receptors (InsP3R) and sustained by store-operated Ca2+ channels (SOCs). Conversely, L-type voltage-operated Ca2+ channels did not support histamine-induced extracellular Ca2+ entry. A preliminary transcriptomic analysis confirmed that WI-38 human lung fibroblasts express all the three InsP3R isoforms as well as STIM2 and Orai3, which represent the molecular components of SOCs. The pharmacological blockade of InsP3 and SOC, therefore, could represent an alternative strategy to prevent the pernicious effects of histamine on lung fibroblasts in asthmatic patients.Histamine is an inflammatory mediator that can be released from mast cells to induce airway remodeling and cause persistent airflow limitation in asthma. In addition to stimulating airway smooth muscle cell constriction and hyperplasia, histamine promotes pulmonary remodeling by inducing fibroblast proliferation, contraction, and migration. It has long been known that histamine receptor 1 (H1R) mediates the effects of histamine on human pulmonary fibroblasts through an increase in intracellular Ca Asthma is a heterogeneous disease, generally characterized by chronic inflammation of the airways, defined by a clinical history of respiratory symptoms, such as wheezing, shortness of breath, chest tightness, and cough that vary in intensity and frequency, along with variable expiratory airflow . About 3The complex network of inflammatory responses in the pathophysiology of asthma involves the release of inflammatory mediators, such as cytokines, chemokines, proteases, and histamine . HistamiIn vitro studies demonstrated that histamine stimulated lung fibroblast collagen synthesis (2+ concentration ([Ca2+]i) in human valvular myofibrobasts , while the extracellular Ca2+ concentration is 1000-fold higher (> 1 mM) and the Ca2+ concentration in the primary intracellular Ca2+ store, the endoplasmic reticulum (ER), fluctuates between 100–800 µM , H1R, which activates phospholipase C (PLC). PLC hydrolyzes a membrane phospholipid, phosphatidylinositol 4, 5 bisphosphate (PIP2), to produce inositol-1, 4, 5-trisphosphate (InsP3). InsP3 triggers Ca2+ mobilization from the ER through InsP3 receptors (InsP3R) that are located on the ER membrane and can in turn activate adjacent ryanodine receptors (RyR) through the process of Ca2+-induced Ca2+ release (CICR)  (2+ channels (SOC), which are the main Ca2+ entry pathway in non-excitable cells. In the ER, Stromal Interaction Molecules (STIM1/2) act as sensors of ER Ca2+ concentration that, after a reduction in intraluminal Ca2+, multimerize and translocate towards peripheral ER cisternae to functionally interact with the Ca2+ permeable Orai channels on the plasma membrane (2+ has been termed store-operated Ca2+ entry (SOCE) and mediates agonist-induced Ca2+ influx in human fibroblasts isolated from several tissues, including lungs (An elevation in i also in human lung fibroblasts  and cultured to 75% confluence in DMEM (Dulbecco’s Modified Eagle Medium) culture medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in an atmosphere of 95% O2 and 5% CO2. Fibroblasts from passages 5–10 were seeded on coverslips for 24 h and then incubated for 48 h in medium devoid of serum.Human fetal lung fibroblast cell lines were purchased from American Type Culture Collection, Collection WI-38 , Ca2+ was replaced with 2 mM NaCl and 0.5 mM EGTA was added as a Ca2+ chelator. Osmolarity was measured with an osmometer  solutions were adjusted to pH 7.4 with NaOH.Physiological saline solution (PSS) with the following composition (in mM) 150 NaCl, 6 KCL, 1.5 CaCl2 1 MgCl2, 10 glucose, 10 HEPES was used for this study. To obtain Ca2+ measurement in fibroblasts has been previously described , the filter rotating wheel, as well as to draw the regions of interest of the fluorescent signal to be measured, a customized software, previously validated, running in LINUX environment, was used. The signal measurement was captured every 3 s and the images obtained were stored on a hard disk and subsequently converted into 340/380 ratio images using ImageJ software . An increase in 340/380 is indicative of an elevation in [Ca2+]i  gel agarose, stained with ethidium bromide, and acquired with the iBrightTM CL1000 Imaging System . The molecular weight of the PCR products was compared with the DNA molecular weight marker VIII .Total RNA was isolated from fibroblasts using QIAzol Lysis Reagent , and reverse transcription was performed as described in . ReverseFor the acquisition of fluorescence values, ImageJ software was used, and Origin Pro 2021 and GraphPad Prism 8.0 were used for graphing and statistical analysis of the results.p ≤ 0.05) were statistically analyzed by the nonparametric Mann-Whitney test for two groups and Kruskal–Wallis for more than two groups. For normal data, an unpaired Student t-test for two groups and ANOVA for more than two groups were used. A value of p ≤ 0.05 was considered statistically significant.Data were expressed as mean ± standard error (SE). Non-Gaussian data, identified by the D'Agostino and Pearson omnibus normality test (Y is the response (relative to the Ca2+ transient amplitude), [Histamine] is the histamine concentration and the mean maximal effective concentration (50EC) is the [Histamine] that induced 50% of the maximal response.Histamine concentration-response data were adjusted by the following 2+]i was measured simultaneously in several individual fibroblasts from the same population. A 22.5% of WI-38 fibroblasts displayed spontaneous Ca2+ oscillations, as also reported in human cardiac fibroblasts ; the second in a peak followed by cyclic increases and decreases in [Ca2+]i, termed peak-oscillations  are shown in 2+]i at very low concentrations, such as 100 and 300 nM. The Ca2+ response to histamine appeared at 1–3 µM; at these concentrations, the Ca2+ signal arising in most WI-38 cells displayed a single peak in response to agonist stimulation. However, at 10 µM histamine, the Ca2+ peak was followed by a short train of consecutive Ca2+ oscillations. The number of oscillations over 60 min of histamine application was increased at histamine concentrations ranging from 30 µM up to 1 mM. The non-cumulative concentration-response curve of histamine-induced elevation in [Ca2+]i is depicted in 2+ response (peak). The maximum increase in the peak amplitude was observed at concentrations higher than 300 µM, whereas raising histamine concentration up to 1 mM did not significantly augment the magnitude of the response. Slight stimulation occurred at 3 µM, while no effect was detectable at concentrations lower than 1 µM (100 nM and 300 nM). The concentration of histamine required to produce a half maximal response (EC50), which was calculated by fitting the concentration-response curve as described in Materials and Methods, was 4.96 μM  was calculated  to the same fibroblast leads to receptor desensitization.Homologous desensitization is a feature of GqPCRs, including H1R . Figure plitudes , as wellplitudes , were si2+ signal in WI-38 human lung fibroblasts, specific HR antagonists were used: for H1Rs pyrilamine (100 μM), for H2Rs ranitidine (50 μM), for H3R clobenpropit (50 μM) and, finally, for the H4R receptor, NJ7777120 (10 μM). After 30 min preincubation and in the continuous presence of the histaminergic antagonists, histamine (300 µM) was applied as indicated by the green bars. In order to confirm cell viability, arachidonic acid (AA) 50 µM was applied in cells in which histamine failed to induce an increase in [Ca2+]i  and presence of the different antihistaminergic receptors antagonists  are summarized. These results indicate that, for the [Ca2+]i elevation to take place in the lung fibroblast cell line, WI-38, the activation of mainly H1R and, to a lesser extent, H2R is necessary, whereas H3R and H4R seem to play no role.In order to elucidate the HR subtype through which histamine triggers an intracellular Ca [Ca2+]i . Pharmac+ signal , H2R blo+ signal , while H+ signal  and H4R + signal  had no s2+ signaling. H3R and H4R are canonically coupled to Gαi/o proteins  was not statistically different from untreated cells  U73122 (10 μM), a selective inhibitor of PLC  prevented the Ca2+ response to histamine  U733r of PLC ; and 3) r of PLC . The res+ signal , whereasresponse . Likewisistamine . These r2+ stores contributes to histamine-evoked Ca2+ signals upon PLC activation, we evaluated the involvement of InsP3R, which provides the main pathway for ER Ca2+ release in fibroblasts    and a sp, 10 μM) . The resr nickel  or nifedr nickel , respect2+ entry pathway sustaining the Ca2+ response to chemical stimuli in human lung fibroblasts  and Gd3+ (10 μM) abrogated the plateau and Ca2+ oscillations in 79 and 40 cells exposed to La3+ and Gd3+, respectively  subfamily, i.e., TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6  peak, 2) peak and oscillations, 3) peak plateau and oscillations, and 4) peak and plateau. Spatial and temporal heterogeneity of the Ca2+ signature can be a hallmark of the Ca2+ response evoked by both mechanical  transient, by repetitive Ca2+ oscillations, c) a sustained Ca2+ plateau or d) a sustained plateau overlapped by repetitive Ca2+ oscillations  in human microvascular endothelial cells   . It is ldulation . The prilate RyR  or inhib RyR  peak-oscillations, 64.7%, and mode 3) peak-plateau-oscillations, 13.39%, entail the occurrence of intracellular Ca2+ oscillations. In agreement with this observation, STIM2 and Orai3 can enhance the percentage of cells showing intracellular Ca2+ oscillations upon GqPCR stimulation (2+, InsP3R3 may provide a constant release of Ca2+ that stimulates InsP3R1 and InsP3R2 to rhythmically release ER stored Ca2+, whereas SOCE maintains the Ca2+ response by ensuring ER Ca2+ refilling (2+ transients transition into a sustained plateau either when InsP3R3 expression increases (2+ release through InsP3R3 is enhanced by the tight coupling with the ER-embedded protein, Jaw1 (3R isoform, as well as cell-to-cell variability in their expression, subcellular distribution, or posttranslational regulation, could contribute to pattern a heterogenous array of Ca2+ signatures in WI-38 adult lung fibroblast (2+ waves because our experiments were performed in fibroblasts devoid of serum for 48 h, which causes cell cycle arrest in G0 phase (2+ dynamics is not due to cell cycle asynchrony (A preliminary qRT-PCR analysis of the Caroblasts , in whicllations . Intriguepletion , which mence see  but not roblasts . Conversroblasts . Quite s+ influx . In agreroblasts . It is wmulation . Furtherefilling . Howeverncreases  or when in, Jaw1 . Therefobroblast . ConversG0 phase . Similarynchrony .2+]i in the widely employed human pulmonary fibroblast cell line, WI-38. The Ca2+ signal is mainly triggered by H1R and can adopt multiple signatures, the most common of which encompasses intracellular Ca2+ oscillations, which have long been known to stimulate gene expression, proliferation, contraction and migration in human pulmonary fibroblasts (2+ response to histamine is triggered by ER Ca2+ release through InsP3R and maintained over time by SOCE activation. These data suggest that the Ca2+ handling machinery could provide an alternative molecular target to prevent the pernicious effects of histamine on lung fibroblasts in asthmatic patients, as recently suggested also for pulmonary hypertension (Streptococcus pneumoniae-induced lung injury (2+ signaling in lung fibroblasts. A limitation of the present study is that we did not use lung fibroblasts from an asthmatic model or from patients with asthma. Future work will have to compare the effect generated by histamine in lung fibroblasts from normal airways and in lung fibroblasts from asthmatic airways.In conclusion, the present investigation showed that histamine induces a dose-dependent increase in [Caroblasts . The Ca2rtension , Streptog injury , and astg injury . Much re


The structure of the described magnetic compound was characterized by different physicochemical techniques including XRD, EDS, AAS, SEM, FT-IR, X-ray elemental mapping, TGA, and VSM analyses. The prepared magnetic nanoparticles exhibit excellent catalytic activity in synthesizing bis (pyrazolyl)methanes and oxidation of sulfide derivatives under green conditions. The heterogeneous nature of the catalyst was confirmed via the hot filtration experiment. Further, ZnFe2O4@SiO2@APTES@DHBS-Cu showed high efficiency and reusability that could be reused for at least five consecutive runs.In this paper, we report the synthesis of ZnFe In catalytic studies, the recovery and reusability of the catalyst due to its adaptability to environmental concerns are important features of the catalytic process that has received much attention in recent years7. The recovery and reusability of catalysts is an important challenge in modern research because the employed catalysts are often very cost-effective or the obtained products are often very valuable from the economical and medicinal points of view10. Recently, heterogeneous catalysts containing an organic–inorganic material hybrid, have received much attention due to having both the advantages of homogeneous and heterogeneous catalysts15. Magnetically separable nanomaterials could be considered as a one of the most important classes of materials with unique physicochemical properties that have attracted the attention of a wide variety of researchers18. Regarding the catalytic support materials, spinel ferrite compounds have great potential in industry and technology as green heterogeneous catalysts in various organic functional group transformations and as catalytic supports22. Magnetic materials such as zinc ferrite due to their unique and non-toxic magnetic properties23, good biological compatibility24, and adjustable magnetic properties25, have been used in various fields including cancer therapy, drug delivery26, and targeting27. Magnetic nanoparticles have properties such as high active surface area, recyclability, chemical, and thermal stability, which are introduced as separated types of heterogeneous catalysts30. Zinc ferrite (ZnFe2O4) has the same properties as homogeneous catalysts and is also facilely extracted from the reaction by an external magnet and does not require more rigorous methods such as filtration or centrifugation. In comparison to homogeneous catalysts, heterogeneous ones have been exclusively studied because of their easy recovery and separation from the reaction mixture31. The use of magnetically separable catalysts is a well-favored and fascinating strategy to bridge the split between heterogeneous and homogenous catalysis processes32. As a main member of the ferrite family, ZnFe2O4 has promising potential for use as novel catalytic support. Additionally, the surface hydroxyl groups over them facilitate their surface modifications with a wide variety of organic compounds.In the past decade, some specific efforts have been made to design effective magnetic nanoparticles instead of immobilized precious metals on mesoporous silica materials34. Furthermore, some of the sulfoxides play leading roles as therapeutic factors such as antibacterial35, anti-ulcer36, antimicrobial37, etc. Sulfide derivatives can be easily oxidized in the presence of a large wide variety of catalysts40. Several of these catalysts are not suitable for selective sulfoxidation reactions because of various reasons such as over oxidation to sulfones, low yields, toxicity, use of costly reagents, and low selectivity of products42.One of the most essential organic processes is the oxidation of sulfides because the corresponding sulfoxides are valuable mediators for the synthesis of chemical and biological molecules43.The five-membered ring containing two Nitrogen functional groups on positions 1 and 2 generates one of the most important heterocyclic ring systems—pyrazoles. In recent years, pyrazoles and their derivatives have received great attention due to a broad spectrum of biological and pharmacological activities2O4@SiO2@APTES@DHBS-Cu MNPs as a green, novel, reusable, and eco-friendly nanocatalyst for the synthesis of bis (pyrazolyl)methanes derivatives and oxidation of sulfide to the sulfoxides. ZnFe2O4@SiO2@APTES@DHBS-Cu has a super magnetic property and thus can be simply separated from the reaction mixture using an external magnet.Herein, we report ZnFeAll required materials for the synthesis of catalyst, oxidation of sulfides, synthesis of bis (pyrazolyl) methanes, solvents, and reagents have been purchased from Fluka or Merck.2O4 magnetic nanoparticles, 2.94 g of iron (II) chloride tetrahydrate (FeCl2.4H2O), and 3.00 g of Zinc nitrate hexahydrate (Zn (NO3)2.6H2O) were mixed and vigorously stirred in 100 mL of deionized water. In the next step, 5 g NaOH in 50 mL deionized water was solved and added to the reaction mixture dropwise. Subsequently, the mixture reaction was stirred for 1 h under a nitrogen gas (N2) atmosphere. The final product was separated by a magnet, and washed three times, with hot ethanol (30 mL) and deionized water (30 mL). Finally, polycrystalline spinel magnetite nanoparticles (ZnFe2O4) were dried at 65 °C44.At first, for the synthesis of ZnFe2O4 was dispersed in a mixture of EtOH (50 mL), 5.0 mL of ammonia solution, 10 mL of H2O, followed by the addition of 2.65 g of Polyethylene glycol (PEG-400) and 3 mL of tetraethyl orthosilicate (TEOS). After that, the reaction mixture was stirred at 25 °C for 34 h. Eventually, the product was separated by a magnet and washed with EtOH (30 mL) and deionized water (30 mL) five times, and dried at 25 °C.In the second step, 1.0 g of the obtained ZnFe2O4@SiO2@APTES@DHBS-Cu MNPs were readily synthesized according to the route depicted in propylamine, 1 g obtained ZnFe2O4@SiO2 nanoparticles were added to 30 mL of a toluene solution and then 2 mL of 3-(Triethoxysilyl)propylamine was added dropwise to this mixture. The reaction mixture was stirred at reflux condition for 23 h. Then, the resulting solid was filtered, washed with ethanol (30 mL) and water (30 mL) several times, and dried at room temperature. An appropriate amount of ZnFe2O4@SiO2@APTES (1 g) was dispersed in deionized water (100 mL) by sonication for 20 min. Subsequently, 2.5 mmol of 3,5-dichloro-2-hydroxybenzenesulfonyl chloride (DHBS) was added and the reaction mixture was stirred at 85 °C for 23 h. The reaction was performed under a nitrogen gas (N2) atmosphere. Finally, to prepare ZnFe2O4@SiO2@APTES@DHBS-Cu, the obtained ZnFe2O4@SiO2@APTES@DHBS (1 g) was dispersed in 30 mL by EtOH sonication for 20 min. Subsequently, 2.5 mmol of Copper (I) chloride was added to the reaction mixture which was stirred under the nitrogen gas (N2) atmosphere at reflux conditions (90 °C) for 24 h a day. Afterward, the reaction mixture was cooled at 25 °C and, then the final ZnFe2O4@SiO2@APTES@DHBS-Cu (MNPs) were isolated, using a magnet, from the reaction mixture, washed by EtOH (30 mL) and distilled water (30 mL) , to remove the residual impurities and, eventually, and then dried at 75 °C for 20 h45.The novel ZnFe in Fig. . In the 2O4@SiO2@APTES@DHBS-Cu (0.03 g) was added, and the reaction mixture was refluxed in ethanol for 60 min. Completion of the following reaction has been analyzed via TLC. After the reaction mixture was diluted with hot ethanol to dissolve the organic products, the catalyst was separated using an external magnet and the resultant unrefined bis (pyrazolyl) methane products, were further purified through recrystallization in an ethanol , ethyl acetoacetate (2 mmol), and substituted aromatic aldehydes (1 mmol) and ZnFe mmol, et2O2 33% (0.3 mL) was poured into the round-bottomed flask containing ZnFe2O4@SiO2@APTES@DHBS-Cu MNPs (0.02 g). The resulting mixture was stirred under solvent-free conditions at 25 °C. After the completion of the reaction, the ZnFe2O4@SiO2@APTES@DHBS-Cu MNPs were separated by a magnet, and the products were extracted with water and ethyl acetate. Finally, the combined organics were dried over anhydrous sodium sulfate (2 g). Evaporation of the solvent gave the pure corresponding sulfoxides an excellent yield  and H1H NMR : 7.22–7.65  ppm.Sulfinyldibenzene: 1H NMR :2.7 , 7.48–7.93  ppm.Methylsulfinyl)benzene: 1H NMR : 0.94, 1.40 , 1.70 , 2.66  ppm.1-(Butylsulfinyl)butane: 4,4′-((4-Tolyl)methylene)bis(3-methyl-1-phenyl-1H-pyrazol-5-ol): 2.29 , 2.41 , 4.79 , 7.09–7.69 , 7.72  ppm.1H NMR : δ 2.25 , 3.75 , 4.77 , 6.77–7.95 , 8.57–8.61  ppm.4,4′-((4-Methoxyphenyl)methylene)bis(3-methyl-1-phenyl-1H-pyrazol-5-ol): 2O4, (b) ZnFe2O4@SiO2, (c) ZnFe2O4@SiO2@APTES, (d) ZnFe2O4@SiO2@ APTES@DHBS and (e) ZnFe2O4@SiO2@APTES@DHBS-Cu  are presented in Fig. −1 are assigned to the stretching vibrations of the zinc–oxygen and the iron-oxygen bonds, respectively. In Fig. −1 are respectively assigned46. Figure 2O4 (MNPs) and the alkoxysilane molecules of TEOS as the first layer. Absorbed peaks at 3444 and 2956 cm−1 were specified as hydroxide stretching vibration mode. The three absorption peaks around 1074, 584 cm−1, and 471 cm−1 indicated the presence of Si–O–Si asymmetric and symmetric stretching vibrations and bending vibration mode of Si–O–Si, as well as a small peak around 1658 cm−1, was assigned to hydroxide stretching vibration of Silicon-hydroxy and twisting vibration of adsorbed H–O–H in a silica shell47. In Fig. 2O4@SiO2@ APTES, the bands in the range of 2879 cm−1 correspond to the bending vibration of CH2 confirming the attachment of APTES chain molecules49. The bands at 1442 cm−1 correspond to NH due to the deformation of the hydrogen-bonded amine groups respectively. NH2 stretching vibrations are present at51 3466 cm−1. In the spectra of ZnFe2O4@SiO2@APTES@DHBS (d) the peak in the 1327 cm−1 is attributed to the appearance of sulfone groups (–SO2–) and the peaks in the 1425 and 1605 cm−1 correspond to the carbon–carbon double bond (C=C) in the aromatic ring. Moreover, a certain band in the 3437 cm−1 is due to aromatic O–H45. In Fig. 2O4@SiO2@APTES@DHBS-Cu MNPs vibration bands, and only a slight difference in the intensity of bands can be noticed. These observations demonstrate the immobilization of Cu on the surface of ZnFe2O4@SiO2@APTES@DHBS MNPs  ZnFe2O4@SiO2@APTES@DHBS-Cu after recovery, which confirmed the stability of this catalyst under reaction conditions.A comparison of FT-IR spectra of the catalyst after recycling is shown in Fig. 2O4 and ZnFe2O4@SiO2@APTES@DHBS-Cu samples are shown in Figs. 2O4@SiO2@APTES@DHBS-Cu NPS confirm that the particles have a crystalline structure in the cubic spinel phase54.X-ray diffraction (XRD) analysis was used to investigate and identify the structure of the zinc ferrite sample. XRD patterns of ZnFe2O4@SiO2@APTES@DHBS-Cu are shown in Fig. 2O4@SiO2@APTES@DHBS-Cu, the weight loss of about 3% below 200 °C is due to the removal of physically adsorbed water and surface hydroxyl groups. In addition, according to the TGA curves, observed the weight loss was about 12% at 200–650 °C for ZnFe2O4@SiO2@APTES@DHBS-Cu that is contributed to the thermal decomposition of immobilized organic components on the ZnFe2O4 surface45.The TGA results of ZnFe2O4@SiO2@APTES@DHBS-Cu. In another investigation, EDX analysis confirmed the existence of silicon, oxygen, chlorine, sulfur, Iron, Carbon, Nitrogen, and Copper elements in the synthesized magnetic nanoparticles and confirm the synthesis of ZnFe2O4@SiO2@APTES@DHBS-Cu. The existence of Copper peaks in this spectrum indicates the loading of Cu onto the ZnFe2O4@SiO2@APTES@DHBS.The EDX analysis is brought in Fig. 2O4@SiO2@APTES@DHBS-Cu after recovery, which confirmed the stability of this catalyst under optimal reaction conditions.Figure 2O4@SiO2@APTES@DHBS-Cu and their distribution , ethyl acetoacetate (2 mmol), and 4-chloro benzaldehyde 1 mmol). First, the effect of solvents was considered. Surprisingly, EtOH (2 mL) was the most suitable solvent to reach the desired product  methane derivatives to identify the generality and the high proficiency of catalytic system  is considered faster than the one with electron-donating benzaldehydes .After optimization, we examined various electron-withdrawing and electron-releasing substituted benzaldehydes in ZnFe2O4@SiO2@APTES@DHBS-Cu nanocatalyst has been presented in Fig. 2O4@SiO2@APTES@DHBS-Cu nanocatalyst for the attack of lone pair of nitrogen form phenylhydrazine to form intermediate pyrazolone (I). In the next step, the activated aromatic aldehyde by ZnFe2O4@SiO2@APTES@DHBS-Cu nanocatalyst undergoes a tandem reaction with intermediate (II) (which is the tautomer of intermediate (I)) leading to intermediate (III) after removal of an H2O molecule. The next step is a Michael addition of another intermediate of (III) to (II) to form intermediate (IV). In the last step, after the tautomeric proton shift, afford bis(pyrazolyl)methanes.Based on our previous studies, a proposed possible mechanism for synthesis of bis (pyrazolyl)methanes using ZnFe2O4@SiO2@APTES@DHBS-Cu was tested in the oxidation of sulfide. The reaction of the methyl phenyl sulfide with H2O2 (as a green oxidizing agent) was chosen as a model reaction (for optimization). The effect of various solvents with different polarities , H2O) as well as solvent-free conditions, was checked. The best reaction yield was gained under solvent-free conditions at 30 min (Table 2O4@SiO2@APTES@DHBS-Cu even after 360 min (Table 2O4 and ZnFe2O4@SiO2@APTES@DHBS (0.02 g) for the oxidation of sulfides. Meanwhile, the best yield of products was obtained in the presence of ZnFe2O4@SiO2@APTES@DHBS-Cu (0.02 g).In another part of this research project, the catalytic activity of ZnFe2O4@SiO2@APTES@DHBS-Cu NPs. In all cases, sulfoxides were produced in high yields at short reaction times, which showed efficiency and excellent catalytic activity of described catalyst. As shown in Table After the optimization, the oxidation of different sulfides was investigated in the presence of ZnFe2O4@SiO2@APTES@DHBS-Cu has been presented in Fig. 2O4@SiO2@APTES@DHBS-Cu as a magnetic nanocatalyst is to form the active oxidant complex. Based on this mechanism, the transfer of oxygen to sulfur leads to the formation of sulfoxide.Based on previous studies, a suggested and possible mechanism for the sulfoxidation reaction catalyzed by ZnFe2O4@SiO2@APTES@DHBS-Cu in the synthesis of bis (pyrazolyl) methane compounds hot filtration experiment was performed using 4-chlorobenzaldehyde as a model substrate under the optimal reaction conditions. After half time of the reaction (30 min), the reaction was terminated, and the yield was found to be 53%. In another experiment, the reaction was designed again, and at half time of reaction, the catalyst was separated and the reaction mixture was stirred further without the catalyst. Herein, we observed that low conversion (< 4%) of the product happened through the heating of the catalyst-free mixture for another 30 min. It can be concluded that nanocatalyst is completely heterogeneous.To approve the heterogeneous nature of the ZnFe2O4@SiO2@APTES@DHBS-Cu was studied by atomic absorption spectroscopy (AAS). Based on AAS analysis, the amount of copper in fresh and reused catalysts were 3.6 × 10–4 mol g−1 and 3 × 10–4 mol g−1 respectively, which shows that Cu leaching into reaction media from the ZnFe2O4@SiO2@APTES@DHBS-Cu framework is very low.Also, copper leaching of ZnFe2O4@SiO2@APTES@DHBS-Cu in the oxidation of sulfides (A) and synthesis of bis (pyrazolyl)methanes (B) using methyl phenyl sulfide and 4-chlorobenzaldehyde respectively as a model substrates. These reactions were performed under optimized conditions to test the reusability of the ZnFe2O4@SiO2@APTES@DHBS-Cu methanes and oxidation of sulfides in model reactions with the previously reported methods  was synthesized. To specify the physicochemical features of the nanocatalyst, various techniques; including, FT-IR, SEM, EDX, XRD, TGA, Map, and VSM analysis were used. This study reported a novel route for the synthesis of an extensive range of bis (pyrazolyl)methanes and sulfoxides with excellent yields. This catalyst can upgrade yields and reaction times in the synthesis of bis (pyrazolyl)methanes and oxidation of sulfides to the sulfoxides compared to many reported methods, with meager leaching amounts of supported catalyst into the reaction mixture. The simple and easy manufacturing method of this catalyst, along with its ability to recover and reuse, makes it economical. The wondrous features of the mentioned organic reactions are high novelty, short reaction times, no use of harmful solvents, simple synthetic procedure, high yields of reactions, facile filtration, and reusability of the catalyst. Furthermore, the synthesized nanocatalyst could be separated easily using an external magnet and reused five times without considerable loss of its activity (Supplementary Information In this study, a new type of magnetically recoverable nanocatalyst (ZnFeSupplementary Information.


Spiroplasma eriocheiris, a helical wall-less bacterium, has five MreB homologous (SpeMreB1-5) that probably contribute to swimming motility. Here, we investigated the structure, ATPase activity and polymerization dynamics of SpeMreB3 and SpeMreB5. SpeMreB3 polymerized into a double-stranded filament with possible antiparallel polarity, while SpeMreB5 formed sheets which contained the antiparallel filament, upon nucleotide binding. SpeMreB3 showed slow Pi release owing to the lack of an amino acid motif conserved in the catalytic centre of MreB family proteins. Our SpeMreB3 crystal structures and analyses of SpeMreB3 and SpeMreB5 variants showed that the amino acid motif probably plays a role in eliminating a nucleophilic water proton during ATP hydrolysis. Sedimentation assays suggest that SpeMreB3 has a lower polymerization activity than SpeMreB5, though their polymerization dynamics are qualitatively similar to those of other actin superfamily proteins, in which pre-ATP hydrolysis and post-Pi release states are unfavourable for them to remain as filaments.MreB is a bacterial protein belonging to the actin superfamily. This protein polymerizes into an antiparallel double-stranded filament that determines cell shape by maintaining cell wall synthesis.  Th. Thi relith K174 f. Therefi release rate of SpeMreB3 D147E was higher than that of the wild-type. Pi release by SpeMreB3 K174T and SpeMreB3 S176D was similar to that of the wild-type, whereas Pi release by SpeMreB3 K174T/S176D was as high as 1.2 nM/s at 3 µM protein concentration . The Pi release rates of SpeMreB5 T160A, T160K and D162S were at least 5.6-fold slower than those of the wild-type . These results indicate that the amino acid motif ‘E … T - X - [D/E]' is important for Pi release from SpeMreBs and that the Thr–Asp/Glu pair plays a role in ATPase activity that is distinct from that the first glutamate in the motif.To elucidate the role of these residues in SpeMreBs ATPase activity, we designed four SpeMreB3 variants  and three SpeMreB5 variants ). All the variants formed filamentous structures . The P… T - X - [D/E]' is conserved in MreB family proteins, we analysed the amino acid sequences of MreBs from all bacterial phyla (g). The motif was conserved in 95.8% of MreB family proteins in non-Spiroplasma species and in 98.2% of Spiroplasma MreBs, except for MreB3 . In all known Spiroplasma MreB3 ' motif is important for Pi release by MreB family proteins, except for Spiroplasma MreB3.To determine whether ‘E al phyla g. The mo. 2.4i release assay, while no filamentous structure was observed by EM in the Nf condition , suggesting that the amorphous aggregation affected the measurements. Therefore, we searched for a solution in which the proteins did not form aggregates without Mg-ATP but polymerized in the presence of Mg-ATP. We found that a solution containing 20 mM Tris–HCl pH 8.0, 1 M NaCl, 200 mM L-arginine-HCl pH 8.0 and 5 mM DTT (buffer S) was suitable for sedimentation assays .To evaluate SpeMreB3 and SpeMreB5 polymerization activity, we measured their critical concentrations, which reflect the minimum concentration required for polymerization and the steady-state filament amounts, by sedimentation assays. However, significant amounts of proteins precipitated even without nucleotides in standard buffer used for EM observation and PTo estimate the time required to a reach steady state, protein samples in buffer S were incubated with 2 mM Mg-ATP for 1, 3 and 6 h and centrifuged. No significant differences were found in pellet amounts , indicating that SpeMreB polymerization reached a steady state within 1 h. Therefore, sedimentation assays were conducted after incubation for 1 h.c; electronic supplementary material, figure S9D, figure 4c; electronic supplementary material, figure S9C, figure 4c).The critical concentration of SpeMreB5 was estimated to be 0.14 ± 0.07 µM which is comparable to that of actin polymerized in a standard buffer for the actin polymerization, KMEI buffer (0.12–0.24 µM), and of walled-bacterial MreBs in buffers akin to KMEI buffer (0.5 µM)  were not significantly different from those of their respective wild-types . The critical concentration of SpeMreB5 was lowest in the presence of ATP and was two- and three-fold higher in the presence of AMPPNP and ADP, respectively . Although some intra-protofilament interactions along the filament axis were observed in Nf-SpeMreB3, the interaction area is smaller than that in SpeMreB3-AMPPNP . This difference is derived from domain closure upon AMPPNP binding (figure 2d), suggesting that this conformational change is necessary to form filaments in solution. In contrast to our model, the ATP hydrolysis-deficient variants used in this study except for SpeMreB3 S176D showed lower or similar critical concentrations compared with the corresponding wild-types  forms a major intra-protofilament interaction that is attenuated upon Pi release ' is mostly conserved in MreB family proteins, except for Spiroplasma MreB3 (g), suggesting that the ATP hydrolysis mechanism proposed here is conserved in most MreB family proteins.Our Pn CcMreB d, suggesi of ATP d. This ric water i 47]. T. Ti relema MreB3 g, sugges. 3.3S. poulsonii MreBs using a heterologous expression system suggested that MreB3 and MreB5 play distinct roles in the cell ,' which is conserved in the catalytic centre of most MreB family proteins . The DNA fragments encoding the SpeMreBs were excised using NdeI and BamHI restriction enzymes and inserted into pET-15b  or pCold-15b, which was constructed from pCold I  by replacing the histidine tag and proteinase digestion site sequences with that of pET-15b. Each construct was transformed into E. coli BL21 (DE3) and C43 (DE3) cells. E. coli carrying the constructed plasmid were grown overnight in LB medium in the presence of 50 µg ml−1 ampicillin at 37°C. The culture was then diluted with fresh medium and incubated at 37°C. When the OD600 value reached 0.4–0.6, IPTG was added to a final concentration of 1 mM and the cultures were incubated for 24 h at 15°C. Cells were harvested, washed twice with PBS , and stored at −80°C until further use.The DNA sequences of SpeMreB1 to SpeMreB5  were cod. 5.2g at 4°C for 30 min). The supernatant was loaded onto a HisTrap HP column , washed with 10 column volumes of buffer A, and eluted with 13 mL of arranged buffer A containing 230 mM imidazole-HCl pH 8.0 at 25°C. The eluted SpeMreBs were further purified using a HiLoad 26/600 Superdex 200 pg column (Cytiva) at 4°C equilibrated with buffer B (20 mM Tris–HCl pH 8.0 at 25°C and 300 mM NaCl). For SpeMreB5 and its variants, the centrifugation strength was decreased to 12 000×g. For purification of SpeMreB3 for the crystallization experiments, buffer C (10 mM Tris–HCl pH 8.0 at 25°C and 150 mM NaCl) was used for gel filtration with a HiLoad 26/600 Superdex 200 pg column instead of buffer B. Protein concentrations were determined from the absorbance at 280 nm measured using NanoDrop One (Thermo Fisher Scientific) with the following absorption coefficients: 0.474 (mg mL−1)−1 cm−1 for SpeMreB3 and its variants and 0.578 (mg mL−1)−1 cm−1 for SpeMreB5 and its variants.The wild-type and its variants of SpeMreB3 were purified by fusion with a 6 × histidine tag at the N-terminus as follows. Cell pellets harvested from 1-L cultures were resuspended in 20–40 mL buffer A  and sonicated with a probe sonicator . The cell lysate was then centrifuged  equilibrated with buffer D (50 mM HEPES-NaOH pH 7.5 at 25°C and 250 mM NaCl) or dialysed with buffer D to replace the buffer. To methylate lysine residues in SpeMreB3, dimethylamine-borane complex (DMAB) (Merck) and formaldehyde (Merck) were added at final concentrations of 20 and 40 mM, respectively [2+-NTA affinity chromatography, the mixture of the sample and 100 units of thrombin (Cytiva) was dialysed overnight at 4°C in buffer D to cleave the histidine tag from SpeMreB3. (2) after 2 h of incubation with 20 mM DMAB and 40 mM formaldehyde, two incubation steps were added before the removal of excess DMAB and formaldehyde. DMAB and formaldehyde were added at concentrations of 40 mM and 80 mM, respectively, and the sample was incubated for 2 h at 4°C. Then, an additional 10 mM DMAB  was added and the sample was incubated overnight at 4°C. In this procedure, although the terminal 7–13 amino acids of methylated SpeMreB3 were cleaved, and small amounts of degraded products appeared even after the final purification step, methylated SpeMreB3 was successfully crystallized. (3) Buffer C was used for gel filtration on a HiLoad 26/600 Superdex 200 pg column instead of buffer B.SpeMreB3 eluted from Niectively ,33, and . 5.4i release rates, SpeMreBs were polymerized in standard buffer . For sedimentation assays, SpeMreBs were polymerized in buffer S . Prior to polymerization, the SpeMreB buffer was replaced from buffer B to the desired buffer in the absence of DTT, MgCl2 and ATP by overnight dialysis at 4°C with a buffer volume 50–100 times higher than that of the sample solution. Monomeric SpeMreBs with a concentration lower than the desired concentration were concentrated using Amicon Ultra 10 K cassettes. The samples were centrifuged to remove aggregates. Then, DTT, MgCl2 and ATP were added to initiate polymerization. All polymerization reactions were performed at room temperature (approximately 25°C).To prepare samples for EM observations and to measure P. 5.5d,e; electronic supplementary material, figure S4J). The subunit repeats in the 2D averaged images were estimated as the distances between the minimal values of the greyscale profiles quantified using ImageJ . To determine each subunit axis of the 2D averaged images, the images were subjected to black and white inversion and binarized with a grey value threshold of 75 (for the SpeMreB3 and SpeMreB5 filaments) and 68 (for the SpeMreB5 five-stranded sheet), in which the densities for intra- and inter-protofilament interaction regions were excluded using ImageJ. Each separated subunit density was subjected to elliptical fitting using ImageJ and the major axis was defined as the subunit axis.The SpeMreBs were polymerized for 3 h, which was long enough to obtain steady state samples for the other MreBs –8,41,51.figure 1. 5.6−1) in buffer C with an equivalent volume of reservoir solution containing 100 mM MES-NaOH pH 6.0, 20% (w/v) PEG-8000 and 200 mM calcium acetate. The crystals belonged to the space group P21 with unit cell dimensions of a = 52.4, b = 68.1, c = 54.6 Å and β = 91.7°. The SpeMreB3 AMPPNP complex crystals used for X-ray data collection were obtained at 20°C from drops prepared by mixing 0.5 µL of protein solution (5 mg mL−1) in buffer C containing 5 mM MgCl2 and 5 mM Li4AMPPNP with an equivalent volume of reservoir solution containing 100 mM acetate-NaOH pH 4.6, 30% (w/v) PEG-4000 and 200 mM ammonium acetate. The crystals belonged to the space group P21 with unit cell dimensions of a = 50.3, b = 56.3, c = 120.5 Å and β = 90.6°.Crystallization screening was performed using the sitting-drop vapour-diffusion technique with the following screening kits: Wizard Classic I-II , Wizard Cryo I-II , PEG/Ion Screen I-II , Crystal Screen I-II (Hampton Research), SaltRx I-II (Hampton Research) and PEG/Ion 400 (Hampton Research), at 4°C and 20°C. Nf-SpeMreB3 crystals used for X-ray data collection were grown at 4°C from drops prepared by mixing 0.5 µL of protein solution  with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) . The crystals were cryoprotected by soaking in a 1 : 9 mixture of glycerol and reservoir. The diffraction data were processed using MOSFLM  and scal. 5.7The SpeMreB3 structures were divided into four subdomains according to the definition of MreBs in walled-bacteria . The first 17 amino acids (M1-P17) and the last eight amino acids (I345-K352) were excluded from subdomain IA because the corresponding regions were not modelled in SpeMreB3-AMPPNP Mol-B and Nf-SpeMreB3, respectively. The centroid of each subdomain was calculated from the backbone atoms without mass-weighting on each atom using UCSF Chimera v. 1.13.1. The centroids were used for calculations of the domain opening and dihedral angles using UCSF Chimera v. 1.13.1 .. 5.8i release was measured using EnzChek kits (Thermo Fisher Scientific), in which the Pi concentration was traced by the absorbance of the reaction product between Pi and 2-amino-6-mercapto-7-methylpurine riboside (MESG), a molecular probe for Pi, at 360 nm (A360) [2, ATP and MESG to SpeMreBs in standard buffer without Mg-ATP. The A360 values of SpeMreBs without MESG and control buffer containing MESG were subtracted from those measured for SpeMreBs with MESG. Data were collected using a Varioskan Flash spectrophotometer (Thermo Scientific), which had an initial measurement delay of approximately 1 min.Pm (A360) ,37–39. R. 5.9x-intercept of a linear fit with the precipitate amounts at the steady state over the total SpeMreB concentration.SpeMreBs with a volume of 200 µL were polymerized for 1–6 h and were centrifuged (100 000 r.p.m. at 23°C for 120 min) in a TLA-100 rotor (Beckman Coulter). The pellet was resuspended in 200 µL water. The supernatant and pellet fractions were subjected to electrophoresis on a 12.5% Laemmli gel and stained with Coomassie Brilliant Blue R-250 to determine the concentration of each fraction. The SpeMreB band intensities were quantified using ImageJ. The concentrations of the supernatant and pellet fractions were estimated as the products of the total SpeMreB concentration and the ratio of each fraction to the sum of the supernatant and pellet fractions. The critical concentration was determined as the