The glass transition temperature (gT) of the extracted PHBVs varied between −0.7 and −7.4 °C. Some of the extracted PHBVs showed two melting temperatures (mT) which the lower mT1 ranged between 126.1 °C and 159.7 °C and the higher mT2 varied between 152.1 °C and 170.1 °C. The weight average molar mass of extracted PHBVs was wide ranging from 6.49 × 105 g·mol−1 to 28.0 × 105 g·mol−1. The flexural and tensile properties were also determined. The extracted polymers showed a reverse relationship between the 3HV content and Young’s modulus, tensile strength, flexural modulus, and flexural strength properties.Poly (PHBV) with various 3-hydroxyvalerate (3HV) contents biosynthesized by mixed microbial consortia (MMC) fed fermented dairy manure at the large-scale level was assessed over a 3-month period. The thermal, mechanical, and rheological behavior and the chemical structure of the extracted PHBV biopolymers were studied. The recovery of crude PHBV extracted in a large Soxhlet extractor with CHCl Polyhydroxyalkanoates (PHA) are natural polyesters of hydroxyalkanoate (HA) building blocks that are produced and accumulated intracellularly by numerous bacteria as an energy reserve material in the presence of excess carbon source and/or under limited nutritional conditions ,2,3,4,5.mT) [Improving the flexibility of PHB can be achieved by the introduction of HA comonomers, such as 3-hydroxyvalerate (3HV), to form the copolymer poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV). Compared to PHB, PHBV is less crystalline and as a result is more flexible with improved impact resistance and toughness which makes PHBV suitable for applications such as flexible packaging ,10. The Tm) .Current commercial production of PHBV uses pure bacterial culture and/or genetically modified strains in a sterile environment. Furthermore, commercial production uses refined carbon substrates such as glucose and fatty acids which also increase production costs ,8,11. ReThis study aims were to (i) produce PHBV with different 3HV contents using MMC that are fed with fermented dairy manure liquor over a several month operating period , (ii) de3, 99.8%) was obtained from EMD Millipore, and acetone, petroleum ether, and methanol were technical grade. Two commercial PHBV samples ) were used as reference materials. PHBV rich biomass samples were produced in a pilot-scale bioreactor over eight production periods  consisting of (i) sequencing batch dairy manure fermenter (760 L) to produce volatile fatty acids (VFA), (ii) an enrichment reactor (680 L) which was fed VFAs to produce an MMC culture that can produce PHBV via a feast-famine regime and (iii) PHBV production reactor (450 L) which was fed VFAs to produce and accumulate PHBV [Chloroform (CHCl3 (3.5 L) then filtered under vacuum to remove any residual biomass and the extract was concentrated to dryness (rotary evaporator) to obtain crude PHBV. Each of the crude PHBV samples were dissolved in CHCl3 (100 mL) and added dropwise to cold (4 °C) petroleum ether (1.5 L) with constant stirring to precipitate the PHBV, recovered by filtration and dried. The recovered PHBV was further purified by dissolving in CHCl3 (100 mL) and added dropwise to cold (4 °C) methanol (1.5 L) with constant stirring to precipitate the purified PHBV, recovered by filtration, dried under vacuum, and yield recorded. The crude and purified PHBV yields are given in Purified PHBV was recovered from the PHBV-rich biomass produced at the PHBV pilot facility following the extraction and purification. Frozen PHBV-rich biomass (1–2 kg) of each operational day was lyophilized. To remove lipids, the lyophilized biomass (165–260 g) was acetone washed in three steps  stirring continuously, recovered by vacuum filtration, and dried in air. Lipid-free PHBV-rich biomass was Soxhlet extracted for 24 h with CHClv/v H2SO4 in methanol (2 mL) plus CHCl3  at 100 °C for 4 h. After cooling, water was added (2 mL), the solution mixed, and the CHCl3 layer separated and transferred to a vial. The resulting methyl ester derivatives were analyzed by GC-MS  [Lyophilized biomass samples (20 mg) were methanolyzed in 3% WI, USA) . SeparatnM), weight average molar mass (wM), and polydispersity index (w/MnM) of the PHBV samples (2 mg/mL) were determined by size exclusion chromatography (SEC) using a Jordi DVB linear mixed bed column (7.8 mm × 300 mm2) on elution with CHCl3 (0.5 mL/min) equipped with a Waters model 2414 refractive index detector and a PostNova PN3609 (658 nm) multi-angle light scattering (MALS) detector  at 30 °C. The system was calibrated using a narrow polystyrene standard . Data were analyzed using the NovaSEC v1.5.0 software .Number average molar mass (C=O AI1720/A1740) was determined on normalized spectra that were curve-fitted using Igor Pro 9 software  [A) of each carbonyl band at 1720 cm−1 and 1740 cm−1 were Gaussian curve fitted using a peak width at half height of 10 cm−1 and integrated. Fourier-Transform Infrared Spectroscopy (FTIR) was performed on PHBV samples, in duplicate, using a Nicolet-iS5 spectrometer  equipped with an attenuated total reflectance (ATR) accessory . Spectra were baseline corrected, averaged, and analyzed using Omnic v9.5 software . The carbonyl index  ,26. The TM aluminum pans) using a TA instrument model Q200 DSC  with refrigerated cooling under nitrogen (50 mL/min). The DSC was calibrated using indium and sapphire standards using following manufacturer protocols. Samples were equilibrated at 40 °C (3 min) then ramped at 10 °C/min to 180 °C (3 min) to remove any thermal history, then cooled to −50 °C at −10 °C/min (3 min), and the cycle repeated. The glass transition temperature (gT), melting temperature (mT), and the melting enthalpy (∆fH) were determined from the inflection point and endothermal peak of the second heating scan, respectively. The degree of crystallinity (cX) was determined using the following Equation (1)fH indicates the melting enthalpy of the sample, and ∆0fH shows the melting enthalpy of the pure crystalline polymer (146 J/g for PHBV) [Differential scanning calorimetry (DSC) was performed on purified PHBV  . Data we3, and 1H- and 13C-nuclear magnetic resonance (NMR) spectra were recorded on an Advance Bruker 300 MHz and 500 MHZ spectrometer  at room temperature, respectively. Spectra were analyzed using TopSpin V3.6.2 software . The 1H-NMR spectra obtained for the PHBV samples were used to estimate monomer unit content. The molar fraction of the 3HV unit of the fractionated PHBV samples was estimated from the relative integrated CH3 (V5) resonance. 13C-NMR spectra was used to determine the sequence distribution of 3HV and 3HB in PHBV as previously described [PHBV samples were dissolved in CDClescribed ,28,29.η*) was measured as a function of angular frequency (Ꞷ) in the oscillatory mode at 180 °C using a Bohlin CVO 100 N rheometer  equipped with 25 mm diameter parallel plates, 0.5% strain, and the frequency range of 0.01 Hz to 100 Hz (ω = 0.0628 to 628 rad/s).Compression-molded discs (1.5 mm (h) × 25 mm Ø) were prepared from purified PHBV in a pellet die using a hydraulic press at room temperature. Complex viscosity  were prepared in a pellet die using a hot press (PHI 300 mm × 300 mm) at 180 °C. PHBV discs were cut into 10 mm × 2.5 mm × 0.5 mm sized specimens for testing. Tensile testing was conducted with a gauge length of 10 mm and controlled force of 3 N/min until yield. 3-point bending tests were performed using a span of 10 mm with constant strain of 1% until yield. Experiments were performed on a DMA Q800  at room temperature with at least 5 replicates. Tensile strength, Young’s modulus, flexural strength, and flexural modulus were specified from the stress–strain curve using Universal Analysis v4.5A software .1H-NMR and data was from Guho et al. [3 selectively extracted PHBV with lower 3HV content. Wei et al. [wM of the extracted PHBVs decreased when extraction time exceeded the optimum level [PHBV containing biomass was produced at pilot-scale by MMC fed fermented dairy manure as described by Guho et al. . The PHBo et al. . The lipo et al. . The resi et al.  reportedi et al. . There ium level , and a lum level ,33. Furtum level ,34. wM and nM) and polydispersity index (PDI) of purified PHBV were determined by SEC-MALS and the results were from Guho et al. [PDI varied between 1.06 and 1.43. The higher PDI indicates a broader range of distribution of molar mass. The PDI values of the PHBV in this study were lower than those reported for PHBV (PDI of 2.1–2.2) [3 showed a high PDI of 3.06 [PDI can be used for applications such as controlled drug delivery [PDI values of the PHBV in this study can be attributed to both upstream and downstream parameters. Upstream processing parameters which mentioned earlier will determine the initial HV content and wM of the cultured PHBV. Downstream processing parameters include some polymer removal in the pretreatment step from lyophilized biomass, solvent type and its atomic interactions with solute, heating energy, and time applied in extraction and purification steps. The combination of these upstream and downstream parameters affects HV content, wM, nM, and as a result PDI of the purified polymer [A variety of parameters in both upstream and downstream processing of PHA production can influence the molar mass of the PHA produced. The upstream parameters include the microorganisms present in the MMC, the medium composition, the state of inoculum, and the fermentation conditions. The downstream processing parameters includes pre-treatment of biomass, extraction method, and subsequent polymer purification procedures . The molo et al.  Table 2Mw and Mn2.1–2.2) . In an s of 3.06 . PHBV hadelivery . Lower P polymer ,32,33,34wM (= −152.0 × HV + 42.68) and nM (= −134.27 × HV + 37.939) with the HV content of the extracted PHBVs (R2 ≥ 0.83). Pearson tests were performed between wM and HV content (p = 0.031), and nM and HV content (p = 0.014). Furthermore, similar correlation was seen between wM (= −33.261× HV + 17.475) with the HV content of the PHBV in lyophilized biomass (R2 = 0.91). Pearson test was performed between wM and HV content of the PHBV in lyophilized biomass (p = 0.012). There was no significant relation between nM with HV content, and PDI with HV content of the PHBVs in lyophilized biomass. It was found that there was a negative linear correlation between −1 [−1 and from 1226 to 1275 cm−1 [−1 was assigned to the C–O–C stretching of the amorphous PHBV [−1 (highest intensity) was assigned to a C=O stretching of an ester structure in PHBV [3) and methylene (–CH2) groups appeared at 2975 cm−1 and 2933 cm−1, respectively [C=IO). Curve fitting of the crystalline band at 1720 cm−1 to amorphous band at 1740 cm−1 was used to determine band areas and C=OI (C=OI for purified PHBV ranged from 2.01 to 2.59 (R2 = 0.53) between C=OI and cX by DSC (17–29%) was observed. These results are lower than reported values for PHB (C=OI 6.94 and cX 57.6%) [C=OI 1.6 and cX 53.7%) [Purified PHBV samples were analyzed by FTIR spectroscopy to gain insight into its chemical structure; results are given in −1 ,39. The 275 cm−1 ,39,40,41ous PHBV . The ban in PHBV ,40. The ectively . Analysiand IC=O  [25,26,3 to 2.59 . A poor c 57.6%) , and higc 53.7%) . It has c 53.7%) .gT; melting temperature, mT; crystallization temperature (cT) and degree of crystallization, cX); the results obtained from Guho et al. [gT of the PHBV samples was similar to the results obtained from Guho et al. [mT1 and mT2 of the PHBV samples was similar to the results obtained from Guho et al. [mT1) first. In crystals with lower HV content, the crystallinity ratio is higher, so the crystals will melt at a higher temperature (mT2) [The purified PHBV samples and commercial standards were analyzed by DSC to determine their thermal properties  . Other fre (Tm2) .cX of the purified PHBV obtained from first cooling cycle which was slightly higher than that obtained from Guho et al. [C=OI of all the extracted PHBV samples ranged between 2.01 to 2.59 which was higher than the previously reported value of 0.949 for PHB [cX (61%), by X-ray diffraction, than PHBV (36.2%) and also showed higher value of C=OI by FTIR (3.8 vs. 2.7) [C=OI by FTIR and cX by DSC of the extracted PHBVs gave a poor correlation (R2 = 0.53). There was no correlation between the melting temperature of the polymers and 3HV content. The o et al.  (16.6–29 for PHB . PHB wasvs. 2.7) . The valcT for the extracted PHBVs ranged from 56 to 107 °C (cT (107.1 °C) with a 3HV content of 0.18 while sample PHBV-71 had the lowest cT (56.5 °C) and a higher 3HV content of 0.23. There was a negative linear correlation between cT and 3HV content (R2 = 0.54), and also between cT and cX of the extracted PHBVs (R2 = 0.54). An Increase in 3HV content results in an increase in the amorphous phase and thus leads to a reduction in cX and cT [cT indicates that the complete crystallization takes a longer time [cX (29%), and low cT of 62 °C. This can be attributed to the higher PDI of 1.43 in PHBV-15. Higher PDI indicates a larger size distribution of polymer chains. Shorter chains need less time for crystallization compared to longer chains which results in reduction of cT [cT value ranging from 41 to 82 °C for CHCl3 extracted PHBV with 3HV contents of (20–60%) have been reported [3 had a cT value of 109 °C [The o 107 °C . Sample c and Tc . A lowerger time . PHBV-15on of Tc . A Tc vareported . Anotherf 109 °C . dT) was determined. The dT values of the PHBV biopolymers was between 318 °C and 329 °C, which was higher than those reported in the literature for PHBV (dT = 286 °C) [mT which limits its processability [mT relative to dT and thus improves its processing window for molding and extrusion [onsetT of 177–238 °C, dT 276–296 °C, and end set temperature of 291–309 °C is reported for PHBV (5–20% HV) [d1%T (temperature at which 1% weight loss occurs) of 258 °C and dT 284 °C for PHB is reported [dT was found to decrease with increasing 3HV content (R2 = 0.49), the difference between onsetT and endsetT was found to increase with increasing 3HV content further reinforcing that the thermal stability of PHBV improved [TGA analysis was performed to determine the thermal degradation behavior of the purified PHBV samples . All of  286 °C)  and PHB  286 °C) . For PHBsability . The prextrusion ,31,49,54xtrusion ,31,52,55xtrusion . The Tonr PHBV 5–% HV [Td11H-NMR spectroscopy (The molar HV content in PHBV was determined by troscopy . The moltroscopy .13C-NMR spectroscopy (To determine whether the PHBV samples were random or co-block-polymer a sequence distribution (CSD) was determined by troscopy  based ontroscopy . The cartroscopy  were in The relative peak intensities obtained for the carbonyl (V1 and B1) resonances and the methylene  resonances of PHBV samples were used to determine the mole, diad, and HV-centered triad sequence distributions . In the D  CSD, whereas it is not sensitive to the broadness of CSD [s of CSD .R has a value of 1  Bernoullian, (ii) first-order Markovian, and (iii) a mixture of two Bernoullian random copolymers  was determined for PHBV at 180 °C against shear rate (Ꞷ) as frequency sweeps ; results are shown in η* with Ꞷ showing shear thinning behavior typical for non-Newtonian fluid [η* of PHBV-15 decreased more rapidly than the other PHBV samples (PDI of 1.47 in the polymer [Rheology presents information about of the behavior of polymer melts through measuring the flow (viscosity) as a function of shear rate . From dyan fluid ,57. At han fluid . Thus, lan fluid ,57,58. T samples . This mi polymer ,59,60. SꞶ) [To quantify the shear thinning behavior, the rheological data were fitted to the Power–law  model, which represents viscosity as a function of some power of the shear rate (Ꞷ) ,61. The Ꞷ) ,61. ExchK and n that must be fitted to experimental data. These parameters are determined from the plot double log η*-Ꞷ. The parameter K is known as the consistency coefficient, is the y-intercept of the log η*-log Ꞷ fitted plot. The parameter n is the non-Newtonian or flow behavior index which is the slope of the log η*-log Ꞷ fitted curve. For n < 1 the material is pseudo-plastic, when n = 1 the material is Newtonian, and n > 1 the material is dilatant [R2 was used. All the PHBV samples had R2 ≥ 0.92. The K and n parameters, and complex viscosity at the frequency of Ꞷ = 6.28 rad·s−1 (1 Hz) for PHBV-15 to -84 are summarized in K ranging from 3.27 to 28.07 Pa·s. The n ranged from 0.645 to 0.888 showing pseudo-plastic behavior. Reported values for PHBV (5% HV) at 160 °C at were K = 1.35 × 106 Pa·s and n = 0.0823 [η* of the PHBV samples at 1 Hz ranged from 1.7 Pa·s to 14.57 Pa·s. The PHBV samples with higher HV content showed higher η* at 1 Hz. It is expected an increase in the HV content leads to a reduction in η* [η* and lower PDI which decreases the η* in polymers [The modified model has two parameters of dilatant ,61,62,63= 0.0823 ,61. The on in η* ,64,65. TThe mechanical  properties of materials provide information on how they will behave under load. R2 = 0.68), flexural modulus and HV% (R2 = 0.78), tensile strength and HV% (R2 = 0.69), and Young’s modulus and HV% (R2 = 0.83) were observed. The strength and flexibility of these polymers was comparable to LDPE which makes them applicable for the packaging industry [The PHBV samples were shown to have a flexural strength of 18.1 to 27.5 MPa and moduli of 0.80 to 1.54 GPa. Literature values for flexural strength for PHBV(8% HV) were 34.5 MPa  and 28.2industry . PHBV shindustry . For com% R2 = 0., and Youindustry . Low conindustry .R2 = 0.68), flexural modulus and PDI (R2 = 0.78), tensile strength and PDI (R2 = 0.69), and Young’s modulus and PDI (R2 = 0.83) were observed.The molar mass distribution of polymers, PDI, is known to influence their properties. Polymers with higher PDI contain some shorter chain polymers that can act as a plasticizer and therefore reduce their mechanical properties . A negat3, recovery, and characterization of properties of PHBV with various copolymer compositions biosynthesized by mixed microbial consortia fed fermented dairy manure was performed. The purified PHBV showed a higher range of thermal properties, broader processing window, and more flexible mechanical properties as compared to commercial PHBV (control), which make the produced PHBV suitable for processing applications such as blow and injection molding. Generally, the purified PHBV showed some variations in properties over the 3 months of operation, which may be attributable to as-yet-refined operational strategies and process controls. To reduce variations at scale would require the operations to be carefully monitored and process controlled in future studies.The successful investigation of large lab-scale Soxhlet extraction using CHCl


Purpose: Etomidate is widely used in general anesthesia and sedation, and significant individual differences are observed during anesthesia induction. This study aimed to explore the molecular mechanisms of different etomidate susceptibility at the genetic level.Methods: 128 patients were enrolled in the study. The bispectral index (BIS), mean arterial pressure (MAP) and heart rate (HR) were recorded when the patients entered the operating room for 5 min, before the administration of etomidate, 30 s, 60 s, 90 s, 120 s and 150 s after the administration of etomidate, and the corresponding single nucleotide polymorphisms (SNPs) were analyzed.Results: Significant individual differences were observed in etomidate anesthesia. The results of two-way ANOVA showed that CYP2C9 rs1559, GABRB2 rs2561, GABRA2 rs279858, GABRA2 rs279863 were associated with the BIS value during etomidate anesthesia; UGT1A9 rs11692021 was associated with the Extended Observer’s Assessment of Alertness and Sedation (EOAA/S) score during etomidate anesthesia; GABRB2 rs2561 was associated with MAP. Multiple linear stepwise regression model results showed that CYP2C9 rs1559, GABRA2 rs279858 and GABRB2 rs2561 were associated with the BIS value and UGT1A9 rs11692021 was associated with the EOAA/S score; GABRB2 rs2561 was associated with MAP.Conclusion: GABRA2 rs279858, GABRB2 rs2561, CYP2C9 rs1559 and UGT1A9 rs11692021 are the SNPs with individual differences during etomidate anesthesia. This is the first to study the SNPs of etomidate, which can provide certain evidence for the future use of etomidate anesthesia and theoretical basis for precision anesthesia. Etomidate is widely used in general anesthesia and sedation, especially in patients with cardiovascular disease, because it does not inhibit sympathetic tone or myocardial function  and has It is found that different patients have different sensitivity to etomidate. Keita et al. found that factors such as age could change the sensitivity of patients to etomidate . Howeverin vivo mediated by α2B receptors (Gamma-aminobutyric acid type A (GABAA) receptors have been confirmed to be the target of etomidate . Etomidaeceptors ; They aleceptors . In addieceptors , but it This study was performed in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology . All patients signed informed consent. A total of 128 participants undergoing elective ear nose and throat (ENT) surgery with general anesthesia were recruited from december 2019 to november 2020 at Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. All participants denied blood ties. The inclusion criteria were as follows: 1) patients undergoing elective ENT with general anesthesia; 2) age between 20-55 years old; 3) no history of surgery; 4) no history of drug use and addiction, such as opioids, opium, heroin, marijuana, Meth, ecstasy and K powder, etc; 5) no hypertension, coronary heart disease, diabetes and other medical history; 6) ASA I or II. Exclusion criteria: 1) History of allergy or drug dependence; 2) Pregnancy or breastfeeding; 3) Abnormal liver and kidney function; 4) History of hypertension, diabetes, coronary heart disease and other cardiovascular diseases; 5) Patients with any neurological or psychological disease; 6) Other diseases that may affect the test results, for example, patients with adrenal insufficiency. The preoperative visits were all conducted by the same anesthesiologist.The patients were not given premedication before surgery. After the patients entered the operating room, the non-invasive blood pressure, electrocardiogram, bispectral index (BIS) and blood oxygen saturation of the patient were routinely monitored, and the Extended Observer’s Assessment of Alertness and Sedation (EOAA/S), mean arterial pressure (MAP), heart rate (HR) and BIS values were recorded for 5 min after the patient entered the room (T1), before the administration of etomidate (T2), 30 s (T3), 60 s (T4), 90 s (T5), 120 s (T6) and 150 s (T7) after the etomidate administration. The regular dose of etomidate takes 15–20 s to take effect, the effect reaches its peak in about 1 min, and the duration of action is about 2–3 min . In addiFour milliliter venous blood was collected from each patient 10 min prior to induction of anesthesia. Samples were stored at -80°C and subsequently extracted using the TIANamp Genomic DNA kit. The Snap shot technology system was used to determine genotypes based on detection by MALDI-TOF MS .p < 0.05 was considered as deviation from equilibrium. To further evaluate the independent influence of these clinical factors , multiple stepwise linear regression analysis was applied, with p < 0.05 indicating statistical significance. Two-tailed p < 0.05 was used in the statistical analysis.All statistical analyses were performed by SPSS 27.0 software . Categorical variables were described with percentages, and continuous variables were presented with mean ± standard deviation (SD). Patients were divided into two groups, homozygous for the major allele, heterozygous for the major allele and homozygous for the minor allele . Differen = 20); 2) severe muscle fibrillation led to trial termination (n = 1); 3) HR was too fast after etomidate administration (n = 1); 4) propofol was added to patients who developed irritability after etomidate administration (n = 2); 5) endotracheal intubation was performed immediately after the upper airway was obstructed (n = 1); 6) HR was less than 50 bpm after the etomidate administration, then atropine was given to increase the HR (n = 1). Finally, a total of 102 patients  were collected and genotyped. The age (years) and BMI (kg/m2) of the participants were 37.60 ± 8.62 and 23.88 ± 3.30 respectively. The experimental flow chart was shown in Ultimately, 128 patients were enrolled in the study. 26 patients were excluded due to the following reasons: 1) blood samples or clinical data were not completely recorded , 4.7% ± 13.4% (p = 0.0005), 2.1% ± 12.9% and 0.9% ± 11%, respectively; MAP was 91.84 ± 11.28 mmHg when the patients were quiet for 5 min after entering the operating room, and MAP was 75.87 ± 13.72 mmHg before etomidate anesthesia. MAP at 30 s, 60 s, 90 s, 120 s and 150s were 92.25 ± 11.40 mmHg, 91.70 ± 11.88 mmHg, 91.03 ± 11.65 mmHg, 91.96 ± 11.88 mmHg, and 92.80 ± 11.74 mmHg, respectively. Compared with the basal MAP, the MAP change rate at 30 s, 60 s, 90 s, 120 s, and 150 s were 0.5% ± 2.9%, -0.1% ± 5.0%, -0.8% ± 5.9%, 0.3% ± 7.2% and 1.2% ± 7.1%, respectively . The remaining 40 SNPs had genetic variation in patients and were in compliance with the HWE equilibrium .The sedation depth induced by etomidate was assessed using the BIS and EOAA/S scores. According to the SNP genotyping results, the patients were divided into two groups: 1) homozygous for major allele; 2) heterozygote for major allele and homozygous for minor allele. For BIS, 4 SNPs  showed significant differences at different time points; for EOAA/S score, only one SNP (UGT1A9 rs11692021) had a statistically significant difference .In order to study the effects of multiple factors  and corresponding SNPs on BIS, a multiple linear stepwise regression model was used to perform regression analysis on these factors. The results showed that CYP2C9 rs1559 and GABRA2 rs279858 had statistical significance at T5 (120 s) after intravenous etomidate; GABRB2 rs2561 had statistical significance at T6 (120 s); GABRB2 rs2561 had statistical significance at T7 (150 s). However, there was no significant difference in age, gender and BMI at any time pointe .In order to study the effects of multiple factors  and the corresponding SNPs on EOAA/S scores, a multiple linear stepwise regression model was used to perform regression analysis on these factors. The results showed that gender differences were statistically significant at T3 (30 s); The UGT1A9 rs11692021 was statistically significant at T4 (60 s); Gender and BMI were statistically significant at T5 (90 s); BMI was statistically significant at T6 (120 s) and T7 (150 s) .MAP was used as one of the hemodynamic indexes. According to the SNP genotyping results, the patients were divided into two groups: 1) homozygous for major allele; 2) heterozygote for major allele and homozygous for minor allele. Only one SNP (GABRB2 rs2561) was significantly different in MAP .In order to study the effects of multiple factors  and the corresponding SNPs on MAP, a multiple linear stepwise regression model was used to perform regression analysis on these factors. The results showed that age and GABRB2 rs2561 were statistically significant at T3 (30s), T4 (60s) and T5 (90s); Age, GABRB2 rs2561 and BMI were statistically significant at T6 (120s) and T7 (150s) .In the present study, we found that there were obvious individual differences during the etomidate anesthesia. According to the target of etomidate and its metabolic mechanisms, 48 possible SNPs were screened in this experiment. Among them, CYP2C9 rs1559, GABRA2 rs279858 and GABRB2 rs2561 were associated with BIS values during etomidate anesthesia; UGT1A9 rs11692021 was associated with MOAA/S score; In addition, GABRB2 rs2561 were associated with MAP.Previous studies have confirmed that etomidate mainly acts on GABAA receptors . A type To date, there is no literature on the correlation between etomidate sensitivity and SNPs. However, some studies have reported the relationship between propofol and SNPs, while propofol and etomidate have similar mechanisms of action. Zeng et al. found that GABRB2 rs3816596 and GABRA1 rs4263535 polymorphisms were associated with susceptibility to propofol sedation. ABRA1 rs1157122 and GABRB2 rs76774144 polymorphisms were associated with the degree of blood pressure drop after propofol infusion . A previCYP2C9 and UGT1A9 are the major metabolic genes for many drugs, including anesthetics. Propofol is primarily metabolized by CYP2B6 and CYP2C enzymes. The enzymes SULT1A1 and NQO1 are involved in the later steps of propofol metabolism . A previThe sample size of this study is relatively small. We will further expand the sample size in the follow-up study to enhance the statistical power of the relevant SNP to verify the results of this study. Besides, the genes we selected are only based on the currently known polymorphisms, and many genes that have not yet been discovered due to genetic polymorphisms may have an effect on the effect of etomidate. Moreover, in this study, two-way ANOVA and multiple linear regression models were used to jointly verify the differential SNPs. However, results presented were not adjusted for multiple comparisons. When we adjusted for multiple comparisons, these associations were no longer statistically significant. The reason for this result may be related to the size of the sample. Therefore, we will increase the sample size for further verification in subsequent experiments. However, the current findings still have great inspiration, prompting us to carry out further research.In summary, our study suggested that significant individual differences were observed in etomidate induced anesthesia. GABRA2 rs279858, GABRB2 rs2561, CYP2C9 rs1559 and UGT1A9 rs11692021 are the SNPs with individual differences during etomidate anesthesia. This is the first to study the SNPs of etomidate, which can provide certain evidence for the future use of etomidate anesthesia.


Based on these findings, the ternary W2Ir5B2 boride can be considered hard.In the present study, we perform a systematic examination of the products formed by mixing and heating of tungsten boride and iridium powders at different ratios in a broad temperature range using qualitative and quantitative X-ray analysis and time-of-flight neutron diffraction (TOF-ND), in combination with scanning electron microscopy/energy-dispersive spectroscopy (SEM/EDS) performed at different accelerating voltages. The well-known and unknown ternary W–Ir–B phases were detected. The Vickers microhardness value for the new ternary W Tungsten–iridium- and tungsten–iridium–boron-based materials are commonly demanded in the industry. These materials can be used for fabricating engines, cutting tools, wear-resistant and antioxidant coatings, as well as electrocatalysts due to the outstanding properties of both metals, including high melting points (2447 °C for Ir and 3422 °C for W), high level of mechanical properties, high hardness, and good oxidation resistance ,9,10,11.3Ir (P 42/mnm), WxIr1−x solid solution with a very wide homogeneity range (from 23–25 at.% to 52–57 at.% W) (P 63/mmc), as well as two ordered WIr (P mma) and WIr3 (P 63/mmc) phases, each of them also having a homogeneity range of ~2 at.% W and 6 at.% W, respectively [xIr1−x solid solution. This might cause significant difficulties for interpreting the research results.The active expansion of the W–Ir and especially the W–Ir–B-based materials to new application areas is impeded by the fact that the available data on their composition, structure, and properties are scarce and contradictory. Several intermetallic compounds are known to exist in the Ir–W system, including Wectively ,19,20,212Ir3B6−x (x ≈ 1) [l8Irl3B69 phases [2B phases); no ternary borides were found [According to phase equilibria in the W–Ir–B system, there are only two ternary W (x ≈ 1)  and Wl8I9 phases ,24 in thre found ,25,26, Hre found ,28,29,30re found ), one ca2Ir5B2 boride was measured. Based on these findings, ternary W2Ir5B2 boride can be considered hard. Our findings expand the class of hard materials.In the present study, we performed a systematic examination of the products formed by mixing and heating of tungsten boride and iridium powders at different ratios in a broad temperature range using qualitative and quantitative X-ray analysis and time-of-flight neutron diffraction (TOF-ND), in combination with scanning electron microscopy/energy-dispersive spectroscopy (SEM/EDS) performed at different accelerating voltages. The well-known and unknown ternary W–Ir–B phases were detected. The Vickers microhardness value for the new ternary W50~7.4 μm, D90 = 15 μm. The habitus of particles was plate-like with a thickness of 100–300 nm. According to the EDS analysis, the iridium powder contained tungsten as a contaminant (~0.5 wt.%), which can be explained by the use of lining and grinding media composed of tungsten carbide.Iridium powder  with the particle size D50 ~22 μm  was mechanically pre-treated in a PM 100 CM planetary ball mill  according to the procedure described by Bannykh et al. . The lin2B phase. The initial tungsten boride was mechanically treated in a planetary ball mill. The particle size was found to be equal to D50 ≈ 400 nm. After mechanical treatment, WC (1–2 wt.%) was detected as a contaminant. The cell parameters  and cell volume (V = 147.0 Å3) of the W2B phase were in good agreement with the reference data (ICSD 42528) reported by Havinga et al. [2B. Contrariwise, the cell volume of the WB phase was estimated to be 162.4 Å3, which corresponds to the composition of WB0.96 [The initial tungsten boride  contained 16 wt.% of the WB phase in addition to the dominant Wa et al.  and sliga et al. ,35. Thisf WB0.96 .The W–Ir–B-based materials were obtained by mixing tungsten boride and iridium powders at different ratios in an agate mortar followed by consolidation by uniaxial pressing  at ambient temperature. The molar ratios of tungsten boride and iridium powder are listed in −3 Pa, heated to a given temperature  at a rate of 640 °C/h, and exposed to the given temperature for 1 h. The samples were cooled down under continuous vacuuming at a rate of 300 °C/h (5 °C/min).Next, the pressed powder mixtures were loaded into grafoil boxes and placed into a high-temperature SNVE 1,7.3.1,7/20 vacuum furnace , evacuated to P~1.33∙10The phase composition was studied using a Bruker D8 Advance X-ray powder diffractometer  employing CuKα radiation in the 10° ≤ 2Θ ≤ 110° range. The time-of-flight neutron diffraction (TOF-ND) patterns of the product obtained after heating the powdered 1:1 mixture  were collected using a real-time diffractometer of the IBR-2 reactor  . The surIn order to measure the microhardness of different phases, the powdered products were packed into epoxy resin and polished. Vickers microhardness measurements were performed using a DuraScan-50 hardness testing machine  at a load of 0.245 N (25 gf) in accordance with the ASTM E384-17 standard test method. The dwell time was 10 s.2B and Ir is the emergence of the XRD peaks belonging to iridium boride, IrB1.1, and WxIr1−x intermetallics (2Θ = 57.05°) in the products obtained at 1100 °C. The composition of the intermetallic phase was calculated to be W0.23Ir0.77 in accordance with the Vegard’s rule. For calculation, the reference data by Tylkina et al. [According to the phase equilibria in the binary Ir–B, W–B, and W–Ir systems, the lowest-melting eutectic was formed in the Ir–B system at 1248 °C . Therefoa et al.  and Rauba et al.  were usexIr1−x. The quantitative X-ray analysis showed that the content of the WB phase was 27 wt.% (1200 °C), which is much higher than that in the initial reagent  has a very broad homogeneous range, namely, x = 0.25–0.53.The phase composition of the products obtained at 1200 °C in the 1:1 mixtures was found to also be presented by WB and Wgent see . The addiessling , the pha0.23Ir0.77 (87 wt.%) and WB (4.5 wt.%), but also to IrB1.1 are observed  and W0.5Ir0.5 (WIr) phases have crystal structure types MgCd3 and AuCd, respectively. The XRD peaks of the W0.25Ir0.75 and W0.5Ir0.5 intermetallic phases disappear as the temperature increases (1400 °C and above). This result is not surprising because the W0.33Ir0.67 phase is thermodynamically the most stable [According to the XRD analysis, only the WB and Wanalysis b. Accordt stable .x approaches WB0.96 [xIr1−x:WB ratio is almost constant in the 1300–1600 °C temperature range.As mentioned above, tungsten boride was also found among the products obtained in the 1300–1600 °C temperature range. As the temperature rises, the composition of WBs WB0.96 ; furtherxIr1−x solid solution, low-intensity peaks at d~3.25 and 2.80 Å can be seen in the TOF-ND patterns  and W0.25Ir0.75 , IrB1.1, WB, and W2Ir3B6−x phases  (It follows from the TOF-ND pattern that the peaks belonging to IrBphase(s) .1.1. The EDS spectra were processed in terms of the W/Ir atomic ratio. Surprisingly, for both the light gray and middle gray large areas that clearly differ in contrast, the W:Ir ratio was almost similar and close to 0.27–0.28:0.73–0.72. This can imply that the contrast differed in these areas because of the presence of a third element  in the middle gray areas. Indeed, a hardly noticeable shoulder with a maximum at ~180–200 eV is observed in the EDS spectrum taken from the middle gray area at an accelerating voltage of 20 kV , was also detected; however, it was a very rare occurrence  . Elementcurrence . In addiB2.3±0.2 e. It is B2.3±0.2 . One sho2Ir5B2 is formed at temperatures higher than 1300 °C. No phase with a similar composition has been previously reported for the W–Ir–B system. In our opinion, a key factor contributing to the formation of ternary boride phases via the reaction between iridium and tungsten boride is the emergence of liquid iridium boride at temperatures above 1300 °C. This is a liquid phase, which promotes improved mass transfer and ensures the formation of ternary borides, W2Ir3B6 and W2Ir5B2.Thus, the data on phase and elemental compositions of the products formed by heat treatment of the 3:1 mixtures were obtained. The good reproducibility of the powder XRD, SEM/EDS, and TOF-ND analysis data unambiguously confirms that a novel ternary boride with tentative composition W2Ir5B2 boride were measured at a load of 0.245 N. The results are presented in 0.36Ir0.64 is ~13.1 ± 2.0 GPa (18 tests), whereas the microhardness of hexagonal WIr3 crystals was evaluated to be 10.7 ± 1.6 GPa (104 tests). These microhardness values are in good agreement with the data previously reported by Tylkina et al. for similar compositions [2Ir5B2, was measured for the first time and was equal to 15.5 ± 3.1 GPa (16 tests). Based on this result, the new ternary boride can be considered the hard phase. This value is lower than that reported previously for the ternary Hf–Ir–B boride of similar composition (18.2 ± 1.7 GPa for Hf2Ir5B2) [In this work, the Vickers microhardness values for binary intermetallic areas and new ternary W 2.0 GPa  tests, wositions  (Table 3f2Ir5B2) .xIr1−x phases, IrB1.1, and ternary W–Ir–B borides. A set of analytical techniques unambiguously confirmed that the novel ternary boride with statistical composition W2.2±0.1Ir5B2.3±0.2 was formed together with the known W2Ir3B6−x ternary boride at temperatures above 1300 °C. No phase with a similar composition (W2Ir5B2) has been previously reported for the W–Ir–B system. It was suggested that emergence of liquid iridium boride at temperatures above 1300 °C improves mass transfer and contributes to the formation of a variety of ternary borides, W2Ir3B6 and W2Ir5B2. The Vickers microhardness values for new ternary W2Ir5B2 boride were measured at a load of 0.245 N. The microhardness was found to be equal to 15.5 ± 3.1 GPa. Based on these findings, the new ternary boride can be considered a hard phase.The products of the reaction between iridium and tungsten diboride were studied in a broad temperature range (1000–1600SC) using X-ray powder diffraction analysis and SEM/EDS at different accelerating voltages, and TOF-ND analysis. It was stated that the phase composition of the products varies depending on the experimental conditions and can include the intermetallic WDue to compositional diversity, the phases formed via the reaction between tungsten boride and iridium can have a broad range of physical and chemical properties. This fact offers additional opportunities for designing structural materials with improved properties for high-temperature applications.


The high diffusion coefficient and stable tunneled structure of NaV6O15 facilitate Zn2+ intercalation/extraction and long-term cycle stability.Reversible aqueous zinc-ion batteries (ZIBs) have great potential for large-scale energy storage owing to their low cost and safety. However, the lack of long-lifetime positive materials severely restricts the development of ZIBs. Herein, we report NaV 6O15 microflowers were synthesized as a stable cathode material for aqueous zinc ion batteries, which show a high specific capacity and excellent long-term cycling performance.NaV However, commercial Li-ion batteries cannot solve the problem of large-scale energy storage because of the limited lithium resources, high cost, safety issues, and environmental impact of toxic electrolyte.4–8 Recently, rechargeable aqueous Zn-ion batteries (ZIBs) have been introduced as prospects for large-scale electrochemical energy storage due to their intrinsic safety (no flammable organic electrolytes), easy assembly, low cost, and high ionic conductivity (two orders of magnitude higher than those of organic electrolytes).9–14To alleviate the increasingly severe energy crisis and climate-related challenges, the use of safe, green, reliable, and economical energy resources such as wind, solar, and water power is becoming a focus worldwide.2 and ZnMn2O4.15,16 Among these compounds, sodium or potassium layered vanadate  are candidate cathode materials in ZIBs.17–20 The large lattice spacing in the layered structure and the “pillar” effect of Na+/K+ between the vanadium oxygen interlayers might be responsible for the high capacity and good cycle performance, respectively. However, due to the weak interaction between the triconnected oxygen atoms on the layered surface and the Na+ or K+ ions along with the strong electrostatic interaction between the inserted Zn2+ and the unstable layers, the interlayer spacing in layered vanadate decreases, and the structure is destroyed.20,21 Vanadium-based compounds with tunneled structures  have also been reported as cathode materials for ZIBs.20–22 The stable tunneled structure provides an effective diffusion path for Zn2+ insertion and avoids structural damage, resulting in excellent cycle performance. Mai et al. prepared Na0.33V2O5 nanowires with a high capacity of 367 mA h g−1 at 100 mA g−1 and long-term cycle stability with a capacity retention of over 93% for 1000 cycles.22 Kim et al. synthesized NaV6O15 nanorods that delivered a high discharge capacity of 427 mA h g−1 at 50 mA g−1 and a capacity retention of 65% over 300 cycles at 1 A g−1.23To date, layered vanadium-based compounds have displayed better electrochemical performance than manganese-based compounds such as MnO6O15 microflowers were successfully synthesized as a cathode material for Li/Na-ion batteries.24 Herein, NaV6O15 microflowers are also demonstrated to be a competitive cathode material for ZIBs based on the following parameters: a high initial specific capacity of ∼300 mA h g−1 at 100 mA g−1; 141 mA h g−1 maintained after 2000 cycles at 5 A g−1; and a capacity retention of ∼107%. Meanwhile, the storage mechanism of the cathode was also investigated.In our past work, high-performance NaV2.6O15 microflowers were fabricated by a simple hydrothermal method. NaOH (0.24 g), Na2CO3 (0.053 g), and NH4VO3 (0.234 g) were dissolved together in 40 mL of deionized water under magnetic stirring at 80 °C for 20 min. When the solution was cooled to room temperature, 1 mL of 3% H2O2 was added into the solution. Subsequently, the pH of the solution was adjusted to 1.9 via the dropwise addition of dilute hydrochloric acid (2 M). The orange solution was then transferred to a 50 mL Teflon-lined sealed autoclave and maintained at 200 °C for 24 h. After centrifugation, the yellow precursors were washed three times with distilled water and alcohol followed by drying for 10 h. NaV6O15 powders were finally obtained by heating the precursors at 400 °C in air for 4 h.NaVThe crystal structure of the as-prepared product was evaluated by X-ray diffraction  with a Cu Kα X-ray source. The morphology of the sample was investigated by scanning electron microscopy (SEM) with a Hitachi-4800 scanning electron microscope. X-ray photoelectron spectroscopy (XPS) was conducted using an Escalab250 spectrometer equipped with an Al Kα source. Galvanostatic charge–discharge experiments were performed using a battery testing system (Neware CT-3008) in the voltage window of 0.2–1.6 V. Cyclic voltammetry (CV) and galvano-static intermittent titration technique (GITT) measurements were carried out using a Bio-Logic VSP-300 multichannel.−2. Finally, the film was dried at 60 °C under vacuum for 10 h. To assemble the 2032-type coin cell, we choose 3 M Zn(CH3F3SO3)2 aqueous solution as the electrolyte to obtain good electrochemical performance. Metallic zinc foil was used as the anode, and glass microfiber served as the separator.Our cathode electrode for testing was prepared by mixing 70 wt% active material, 20 wt% acetylene black, and 10 wt% polytetrafluoro-ethylene (PTFE) binder to form a homogeneous mud pie, which was then rolled into a film on a carbon paper. The loading density of active material in the film was approximately 1 mg cm3.6O15 microflowers is shown in 6O15 phase (PDF #24-1155), and no other impurities are detected. An SEM image of microflower-like NaV6O15 with a diameter of ∼5 μm is shown in 6O15. The layered NaV6O15 is composed of VO6 octahedra and VO5 square pyramids along the b-axis, and the layers are bonded with single-connected oxygen atoms; this structure is more stable than those of other layered vanadium oxides.25,26 The interlayer sodium ions along the a-axis act as pillars to further increase the stability of the layered vanadium oxide during Zn insertion/extraction.27,28 The tunneled structure along the b-axis . After the first three cycles, a higher discharge/charge capacity of 348/347 mA h g−1 was obtained with ∼100% coulombic efficiency. The similar discharge/charge profiles compared to the first cycle also demonstrate the good electrochemical stability of the NaV6O15 electrode.6O15 electrodes were also investigated  after approximately 40 cycles. The reason for this is discussed later in detail. After 100 cycles, the capacity was reduced to 225 mA h g−1 with a capacity retention of 57.8% and 98% coulombic efficiency; this might explain the partial dissolution of the active material and poor electronic conductivity.30 At the higher current density of 5 A g−1, an initial discharge capacity of 132 mA h g−1 was obtained. After 2000 cycles, the NaV6O15 electrode still maintained a discharge capacity of 141 mA h g−1 with a high coulombic efficiency of ∼100% and a capacity retention of ∼107%.The rate capabilities of the NaVstigated . The ave6O15 electrode, ex situ XRD and XPS were carried out, respectively. To more clearly observe the phase shift or transformation of NaV6O15 during the discharge/charge process, the electrodes were washed with alcohol under ultrasonic treatment to remove the byproduct before ex situ XRD analysis, as reported previously.17,31ex situ XRD spectra of the NaV6O15 electrode collected at different discharge/charge states in the first cycle at a current density of 100 mA g−1. When the electrode was discharged to 0.5 V, new peaks at 2θ = 6.5°, 12.9°, and 19.5° appeared; these peaks can be assigned to the set of (00l) reflections from a layered ZnxV2O5·nH2O phase.32,33 After discharging to 0.2 V, three obvious reflection peaks attributed to the (002), (104), and (106) planes of the initial NaV6O15 electrode were slightly shifted from 2θ = 12.1°, 29.0°, and 41.4° to higher positions of 12.3°, 29.2°, and 41.6°, respectively. These shifts might be due to the strong electrostatic interaction between intercalated Zn2+ and the tunnel-structured [V6O15]−.34,35 During the subsequent charging process, the ZnxV2O5·nH2O phase disappeared, and the above lattice planes of the NaV6O15 electrode shifted back to the low positions of 12.2°, 29.1°, and 41.5°, respectively, demonstrating the stability of the NaV6O15 structure and the high reversibility of the Zn2+ intercalation/extraction process in the NaV6O15 electrode. To provide further insight into the structural stability of the NaV6O15 electrode during long-term cycling, the structure and morphology of the electrode were respectively evaluated by XRD and SEM after 1000 cycles at a current density of 5 A g−1 , (104), and (106) planes remained, and the morphology of the NaV6O15 nanorods was well maintained, demonstrating the stable structure of the NaV6O15 electrode during long-term cycling.To explore the mechanism of the increased capacity and stable cycle performance of the NaVEx situ XPS was conducted to explore the evolution of the valence states of Zn, Na, and V during Zn insertion/deintercalation in the first cycle. As shown in 3/2) and 1046.5 eV (Zn 2p1/2) appeared, indicating the insertion of Zn2+. When charged to 1.6 V, the intensity of the Zn2+ peak decreased but did not disappear, indicating the incomplete extraction of Zn2+. Meanwhile, the intensity of the Na 1s signal was gradually reduced after discharging to 0.2 V and charging to 1.6 V  to investigate the pseudocapacitive-controlled and diffusion-controlled processes  versus log(v)  , the b vDZn). As shown in −1 for 10 min followed by a 60 min open-circuit step to bring the voltage back to equilibrium. This process was repeated until the voltage reached 0.2 V. The DZn value of the NaV6O15 electrode  corresponding to the current pulse. The value of DZn was calculated to be approximately 10−7 to 10−9 cm2 s−1. The high value of DZn can be attributed to the intrinsic tunneled structure of NaV6O15, which is responsible for the remarkable electrochemical performance of the NaV6O15 electrode.GITT was used to calculate the diffusion coefficient of zinc ions 2·nH2O,46 the NaV6O15 electrode exhibited a competitive energy density and power density (225 W h kg−1 at 65 W kg−1). When the power density was as high as 1100 W kg−1, the energy density remained at a high value of 91 W h kg−1, indicating that the NaV6O15 microflowers have a high-power capability for ZIBs.The Ragone plots of the NaV4.6O15 microflowers with high specific capacity (∼300 mA h g−1 at 100 mA g−1) and excellent cycling stability (141 mA h g−1 maintained after 2000 cycles at 5 A g−1) in aqueous ZIBs. Ex situ XRD and XPS analyses demonstrated that the NaV6O15 electrode has a stable and reversible structure during the zinc intercalation/extraction process, even after long-term cycling. The findings show that NaV6O15 microflowers are a promising cathode material for aqueous ZIBs.We hydrothermally synthesized NaVThere are no conflicts to declare.


The as-prepared sensor exhibited high performance when used as the sensing material for acetone detection at room temperature. Furthermore, the V2C/V2O5 MXene-based sensor exhibited a higher response (S% = 11.9%) toward 15 ppm acetone than pristine multilayer V2CTx MXenes (S% = 4.6%). Additionally, the composite sensor demonstrated a low detection level at ppb levels (250 ppb) at room temperature, as well as high selectivity among different interfering gases, fast response-recovery time, good repeatability with minimal amplitude fluctuation, and excellent long-term stability. These improved sensing properties can be attributed to the possible formation of H-bonds in multilayer V2C MXenes, the synergistic effect of the newly formed composite of urchin-like V2C/V2O5 MXene sensor, and high charge carrier transport at the interface of V2O5 and V2C MXene.High-performance, room temperature-based novel sensing materials are one of the frontier research topics in the gas sensing field, and MXenes, a family of emerging 2D layered materials, has gained widespread attention due to their distinctive properties. In this work, we propose a chemiresistive gas sensor made from V The development of the Internet of Things (IoT) has enabled the integration of several types of active sensors into a single network, allowing users to be warned of impending risk through smart technologies2. One category of sensors, gas sensors , has played a pivotal role in monitoring hazardous gases and volatile organic compounds (VOCs) in industries, indoor areas, and medical environments to improve the safety and security of humans5. Another category, point-of-care smart sensing devices, has garnered attention for achieving real-time diagnoses of diseases6. For example, human breath is a mixture of various gases, such as N2, O2, CO2, water vapor, trace amounts of VOCs , and inorganic gases . These gases are generated either endogenously (in the body) or exogenously 8. In particular, acetone is a useful biomarker for diagnosing diabetes; it is a byproduct of the metabolic process of ketosis and is expelled from the body via waste or breath9. Concentrations of acetone range from 0.2–0.9 parts per million (ppm) in healthy individuals and 0.9–1.8 ppm in diabetic patients10. Local statistics indicated that nearly 17.3% of the United Arab Emirates (UAE) population aged 20–80 had type 2 diabetes in 2017, while nearly 1 million people had type 1 diabetes, ranking the country fifteenth worldwide11. Compared to a conventional blood glucose test, which can be painful, exhale breath analysis is a promising, non-invasive, non-hazardous, and cost-effective approach to detect acetone13. Therefore, novel, high-performance sensing materials are needed for designing sensitive gas sensor devices to detect acetone in the breath. Novel materials have been employed to detect VOCs and toxic gases, including metal-oxide semiconductor (MOXS)-based chemiresistors14, carbon nanotubes (CNTs)10, and graphene-based, two-dimensional (2D) materials15. However, although MOXS gas sensors are frequently employed as effective transducer gas sensors, their high working temperature is a significant practical obstacle3. Room temperature (RT)-operated gas sensing has been targeted as a solution to this challenge. While CNTs and graphene-based 2D materials can operate at RT, their sluggish reaction and low response behaviors impede practical applications17. Therefore, alternative sensing materials that can operate at RT and show enhanced sensing properties are necessary.With the growing awareness of rapid environmental pollution and the importance of health diagnoses, designing smart, sensitive sensors has become a frontier research topic in the gas sensing field18. MXenes have demonstrated their potential in myriad applications, including gas sensors, due to their unusual features, such as their surface functional groups, versatile chemistry, exceptional solubility, high metallic conductivity, and high specific surface area17. Since the discovery of Ti3C2Tx MXene, numerous studies on other MXene materials and their properties have been conducted20. Ti3C2Tx MXene has been widely used for gas sensors because of its high stability, metallic behavior, and easy synthesis21. Vanadium-based MXenes, such as V2CTx, also hold potential for gas sensor applications, and a few recent studies have reported on their gas sensing properties24. Furthermore, a series of reports have examined the fabrication of MXene-based composites27 for improving the efficiency of gas sensors. Moreover, MXene can be directly converted to metal oxides at different thermal conditions29.MXenes are a novel class of emergent 2D transition-metal carbides/nitrides that are typically synthesized by selectively etching Al from the MAX phase2Ox from thermally annealed V2CTx MXene for gas sensing. Considering the above-stated advantages of MXene-based composite materials, we hereby propose a novel strategy for using thermally oxidized, multilayered V2CTx MXene to synthesize V2CTx MXene-derived, urchin-like vanadium oxide (V2Ox) hybrid structures for gas sensing applications. We used a hydrothermal method to synthesize multilayered V2CTx MXenes. These pristine V2CTx MXene materials were transformed into urchin-like vanadium oxide (V2Ox) hybrid structures at different annealing temperatures, and then we investigated the acetone-sensing properties at RT. Compared to the pristine V2CTx MXene, the urchin-like vanadium oxide (V2Ox) hybrid sensor showed improved acetone-sensing performances at ppb-level detection with high selectivity, long-term stability, and good repeatability when utilized as a sensing material. The acetone sensing properties and potential sensing mechanisms are discussed in this manuscript.To our knowledge, no research has been conducted on the synthesis of V2AlC MAX-phase powders  were slowly dropped into the above LiF-HCl solution, and the mixture was stirred for 10 min to achieve complete mixing. This mixture was then sealed in a Teflon-lined steel autoclave and heated at 90 ℃ for 5 days in an oven. Upon completion of the reaction, the etched solution was washed and centrifuged with copious amounts of DI water until the pH of the solution reached ~ 6. A detailed description of the washing procedure is shown in Supplementary Fig.2CTx MXene precipitates were dried at 80 °C for 12 h in a vacuum oven.The synthesis approach used in this study involved mixing 1.5 g of LiF  powder with 30 ml of HCl  in a 100-ml PTFE bottle by stirring. Subsequently, 1.5 g of V2CTx powder was calcined at different temperatures ranging from 300–450 °C at a rate of 1 °C/min. The composition and morphology of the calcined powders were investigated.The as-obtained V2CTx MXene sample was conducted using the METTLER Toledo TGA2 STARe System. The UV–Visible Diffuse Reflectance Spectra (DRS) was measured for V2O5 MXene using Shimadzu UV-3600 diffuse reflectance spectrophotometer .The prepared MXene samples were characterized using powder X-ray diffraction  with a CuKα X-ray (λ = 1.5406) at a scan rate of 1°/min. Scanning and transmission electron microscopies  were used to examine the microstructure of the solid materials. Additionally, an energy-dispersive X-ray attachment was used to evaluate the elemental composition of the prepared materials. X-ray photoelectron spectroscopy (XPS) analysis was also performed using the PHI5000 Version Probe III. Thermogravimetric (TG) and differential thermogravimetric analysis (DTA) of the V2O3) substrate with Pt-interdigitated electrodes (IDEs) , which were measured by a commercially procured humidity meter operated at RT (23 °C).The sensing device was built on a 1 × 1 cm alumina AlO3 substr2CTx MXenes were synthesized by a hydrothermal method using LiF and HCl solution at 90 °C for 5 days, as shown in Fig. 2AlC MAX phase and its exfoliated product, V2CTx MXene materials, were characterized by SEM and TEM, respectively  were present on its surface. After the hydrothermal treatment with LiF-HCl etchant, typical accordion-like V2CTx MXenes with a few or multiple layers were formed 24. No other phase peaks were found in the pattern, indicating the high purity of the prepared V2AlC phase. The intensities of all peaks representing the MAX phase V2AlC decreased in the XRD patterns of the V2CTx phase, confirming the elimination of Al layers from V2AlC. Meanwhile, a broad peak appeared at a low angle (2θ = 7.33°), corresponding to the (002) plane of as obtained accordion-like V2CTx MXene24. The formation of the peak indicated the formation of MXene sheets.The XRD patterns of the V2CTx MXene further, we performed TEM analysis as shown in Fig. 2CTx MXene, which demonstrated the polycrystalline nature of the prepared materials. Figure 2CTx MXene. Figure 2CTx MXene.To investigate the morphology of the V2CTx MXene materials were annealed at different temperatures to produce MXene-derived hybrid structures and evaluate their sensing properties. The V2CTx MXene samples were calcined at 300, 350, and 450 °C in air. Hereafter, these samples are designated as V2C-300, V2C-350, and V2C-450, respectively. Morphological and structural analyses of the calcined V2C materials were also conducted using SEM and XRD. The SEM micrographs shown in Fig. 2CTx MXene ranged from 1–50 μm  was shifted to a higher theta angle as the annealing temperature increased; these findings supported our previous results30. However, compared to that of pristine V2CTx MXene, the intensity of the (002) peak was decreased in the annealed samples, which may have been due to the formation of V2Ox phases. When the annealing temperature increased to 450 °C, the V2CTx MXene was oxidized to urchin-like interconnected networks of V2O5 inherited from the V2CTx MXene. Henceforth, the V2CTx MXene-derived V2C-450 sample will be designated as urchin-like V2CTx/V2O5 MXene. The XRD pattern of V2C-450 corresponded to the diffraction peaks of orthorhombic V2O5 (JCPDS no. 41-1426)31. Other impurities of the V2AlC phase  were evident in the XRD pattern24. The partial oxidation of the V2CTx MXene was further shown using TGA-DTG analysis  and the interaction of V2CTx MXene with O2 molecules from the air during calcination, which resulted in V2Ox34. However, the partial oxidation of V2CTx MXene ceased at 600 °C, after which weight gain was no longer observed.To examine the phase composition of the thermally annealed Vsis Fig. a. We fou35. Thus, to investigate the chemical states in the V2CTx MXene further, we performed XPS analysis, as shown in Fig. 2CTx MXene surface was occupied with V, O, C, and F elements. The synthesis method we utilized played a pivotal role in determining the specific quantities of these groups23. Figure 2p, C1s, and O1s of V2CTx MXene. The high-resolution spectrum of V2p revealed the presence of vanadium, predominately in its V5+ and V4+ forms. The V2p spectra could be fitted by six peaks for V5+ at 517.39 and 525.11 eV, for V4+ at 516.66 and 524.30 eV, and for V3+ at 514.27 and 522.16 eV37. Of these peaks, the peak area of the V5+ oxidation state was higher than the peaks of other oxidation states. Vanadium was mainly present in the high-valence state of V4+ and V5+, which was likely due to the existence of a combination of monolayer oxide/vanadium oxide on the V2CTx MXene surface40. The C1s spectrum , and intercalated water resulting from the partial surface oxidation of V2CTx MXene42.After etching or delamination, MXene surfaces are spontaneously re-occupied with different functional groups, such as OH, O, and F2CTx MXene material  and V4+ at 516.51 and 524.23 eV (minor peaks). The intensity of the V2p-V5+ peak was higher than that of the pristine, non-annealed V2CTx MXene. The O1s XPS spectra of V2CTx/V2O5 MXene . Figure 2CTx MXene and urchin-like V2CTx/V2O5 MXene sensors when tested with acetone vapor (0.25–15 ppm). Compared to conventional metal-oxide-based chemiresistors, MXene shows a positive response (an increase of resistance)43, likely due to its metallic characteristics. We observed a significant increase in the amplitudes of the urchin-like V2CTx/V2O5 MXene sensor compared to pristine V2CTx MXene. Supplementary Fig. 2CTx and urchin-like V2CTx/V2O5 MXene sensors when tested with acetone vapor (0.25–15 ppm).We evaluated our prepared sensor devices, which were based on pristine V2CTx MXene and urchin-like V2CTx/V2O5 MXene sensors showed positive response behaviors. The initial resistance of the urchin-like V2CTx/V2O5 MXene sensor was higher than that of the pristine V2CTx MXene25. We attributed the lower baseline resistance of V2CTx MXene to its excellent metallic properties and intrinsic high electrical conductivity. Our findings verified that V2CTx is the most important part of electrical transportation systems and that V2O5 is a contributor to an improved sensing response. During the gas-sensing experiment, we exposed the sensor device to the target gas for 120 s, followed by purging with synthetic air for 300 s to remove any remaining gas molecules. We used Eq.  to 15-ppm acetone vapor, while the urchin-like V2CTx/V2O5 MXene sensor showed a response of 11.9%, with a low detection limit of 4.76% for 0.25 ppm. The response time and recovery time of a gas sensor are critical parameters for determining how quickly the sensor responds to the target gases. A gas sensor’s response time (τresponse) is the time needed for 90% changes in resistance during exposure to the target gas, whereas the recovery time (τrecovery) is the time required for a 90% return to baseline resistance after the target gas is switched off and synthetic air is turned on. Figure 2CTx/V2O5 MXene sensor toward 15 ppm acetone. The response and recovery times of this sensor toward 15 ppm acetone were 115 s and 180 s, respectively. Selectivity is also an essential factor for a gas sensor in terms of practical application; it is the sensor’s ability to distinguish a target gas from other interfering gases. The selectivity test of the urchin-like V2CTx/V2O5 MXene sensor is shown in Fig. 2, CO, H2S, CO, and C2H4. The urchin-like V2CTx/V2O5 MXene sensor demonstrated the highest response of 47% for 100 ppm acetone, displaying high selectivity against acetone vapor.Both the pristine V2CTx/V2O5 MXene sensor was exposed to six consecutive cycles of acetone vapor (100 ppm) at RT (23 °C). The results indicated good repeatability, with negligible variations in resistance. The stability of a gas sensor is another important parameter for practical applications. To examine stability, the urchin-like V2CTx/V2O5 MXene sensor was tested for three weeks 55. This narrows down the depletion layer and triggers the movement of the charge carrier at the interface and results in high conductivity in the system, as shown in Fig. 60. Upon exposure to the acetone vapor, the pre-adsorbed oxygen species react with acetone molecules to release trapped electrons  MXene was successfully fabricated using a one-step hydrothermal synthesis technique at 90 °C. Multilayered V2CTx MXenes were partially transformed into urchin-type V2O5 structures (V2CTx/V2O5 MXene) at 450 °C calcination temperature. The morphological, structural, and surface properties of both materials were investigated, and both sensors were evaluated for their efficiency in sensing acetone at RT. The dominant metallic characteristics of the V2CTx MXene were reflected in a positive response toward acetone. The as prepared V2CTx MXene-derived, urchin-like V2CTx/V2O5 MXene hybrid sensor showed an improved response (S% = 11.9) toward 15 ppm acetone at RT compared to pristine V2CTx MXene sensor. The sensor demonstrated a ppb-level detection with a low limit of detection (250 ppb). Furthermore, the V2CTx/V2O5 MXene sensor exhibited high selectivity to acetone among different interfering gases, fast response-recovery time (115s/180s), and excellent reproducibility and long-term stability (21 days) at RT.In summary, accordion-type vanadium carbide (VSupplementary Information.


An electrospinning process is used to produce SCFG fibers. In a symmetrical cell, Sm0.2Ce0.8O1.9 (SDC) nanoparticles are infiltrated into the porous fibrous SCFG cathode layer after it was applied to the SDC dense electrolyte. Electrochemical impedance spectroscopy (EIS) analysis reveals that the polarization resistance of the SCFG cathode with fiber morphology is significantly lower than that of the same combination with powder morphology. In addition, it is shown that infiltration of SDC oxygen ion conductor nanoparticles enhanced electrochemical performance. The lowest value of polarization resistance, 0.03 Ω cm2 at 800 °C, is attained by the SCFG with a fibrous structure containing 14 wt% SDC nanoparticles.This paper examines the electrochemical and microstructural features of SrCo Energy has long been one of the most crucial needs in human societies. On the other hand, the widespread use of fossil fuels has created several issues, including a rise in global temperature, the destruction of the ozone layer, the production of toxic gases, acid rain, and water and soil pollution. Therefore, the development of clean energy and renewable energy sources is vital for human civilization. Consequently, fuel cells are one of the sources of clean energy production, as they can directly convert chemicals into electrical energy. Among fuel cells, solid oxide fuel cells (SOFCs) exhibit the highest efficiency and excellent flexibility in terms of fuel type ,2,3,4,5.Accordingly, it is essential to develop intermediate-temperature solid oxide fuel cells (IT-SOFCs) with high efficiency to reduce system costs and enable commercialization. On the other hand, decreasing the operating temperature increases the polarization resistance of the cathode due to a decrease in oxygen reduction reaction (ORR) activity, thereby reducing the overall efficiency of IT-SOFCs. Therefore, to improve the performance of IT-SOFCs, it is essential to develop cathode materials with excellent catalytic activity for an ORR at low operating temperatures ,10,11,123−d), including the SrCoO3−δ family, have been introduced as excellent cathode materials for SOFC application due to their high oxygen ion conductivity and superior electrical conductivity [In addition, the SOFC cathode must possess a high electrical conductivity, thermal expansion compliance, and chemical compatibility with other cell components. Oxides with a defective perovskite structure  has been introduced as a mixed ionic and electronic conductor (MIEC) with sufficient high electrical conductivity of 122 S·m−1 at 800 °C, suitable oxygen permeability, and stability in reducing atmosphere at high temperatures [−6 K−1 in the temperature range of room temperature to 800 °C, which is comparable to the TEC value obtained for the Sm0.2Ce0.8O1.9 (SDC) electrolyte [Among these compounds, SrCoeratures ,20,21. Tctrolyte . As a reElectrochemical reactions occur at the three-phase boundary (TPB) of the SOFCs’ cathode, whereas for mixed ionic-electronic conductors, it is widely spread throughout the cathode layer. Furthermore, the presence of electrolyte particles in composite cathodes generates a more expanded three-phase boundary, which increases oxygen permeability and enhances the cathode’s catalytic activity ,22. In a0.6Sr0.4Co0.2Fe0.8O3−δ [1−xSrxFeO3−δ (BSFO) [0.5Sr0.5CoO3−δ-Gd0.2Ce0.8O1.9 [0.5Fe0.5−xBixO3−δ [1.6Sr0.4NiO4 (LSN) [Recently, electrospinning has been introduced as a promising technique for producing polymer and ceramic fibers with high specific surface area per unit mass and small-sized porosity ,23,24,25e0.8O3−δ , Bi1−xSrδ (BSFO) , Sm0.5Sre0.8O1.9 , BaCe0.5xBixO3−δ , and La1O4 (LSN) , have beO4 (LSN) ,32,33,340.8Sr0.2Co0.2Fe0.8O3−δ tubes impregnated with Ce0.8Gd0.2O1.9 nanoparticles [0.4Sr0.6Co0.2Fe0.7Nb0.1O3−δ (PSCFN) nanofibers [To this end, several recent studies have shown that composite SOFC cathodes with nanofibrous structures produced by electrospinning plus solution infiltration demonstrate higher electrochemical performance than composite electrodes with powder morphologies. Among them, it can refer to GDC infiltrated LSCF nanofibers , La0.8Srarticles , GDC infnofibers , and LSMnofibers . Nano-fi0.8Fe0.1Ga0.1O3−δ (SCFG) fibers. Infiltration of the SCFG fibrous cathodes with different amounts of nano SDC particles is also adopted to fabricate composite cathodes with improved electrochemical properties.In the present work, the electrospinning method is explored for the fabrication of perovskite oxide SrCo3)2, Co(NO3)2·6H2O, Fe(NO3)3·9H2O, and Ga(NO3)3·xH2O (x = 8)  were dissolved in deionized water to prepare the electrospinning solution based on the stoichiometry of SrCo0.8Fe0.1Ga0.1O3−δ compound. The polyvinyl alcohol (PVA) polymer was subsequently dissolved in deionized water before being added to the clear and homogenous nitrate solution. Afterward, the created homogenous solutions were stirred for 5 h at room temperature using a magnetic stirrer. In this instance, the electrospinning solution concentration was 7 g L−1, and the mass ratio of total nitrate powders to PVA was 1:1. The electrospinning solution was then placed in a 10 mL syringe attached to a nozzle with a 21-needle gauge. The electrospinning procedure was then implemented at 27 kV.SCFG fibers were produced through the electrospinning process. Initially, the required amounts of Sr method. For the EIS test, the structure of symmetric cells supported by electrolyte was SCFG-x wt% SDC/SDC/SCFG-x wt% SDC . To prepare symmetrical cells, a slurry containing 65 wt% alpha-terpineol solution, 35 wt% SCFG fibers, and ethyl cellulose powder equal to 5 wt% of the amount of spent cathode fibers was used.−1. The thickness of the applied electrodes was approximately 25 microns, and the cathode’s effective surface area in the obtained symmetrical cells was 0.79 cm2.A homogeneous cathode slurry was prepared by slowly stirring the mixture with a magnetic stirrer for one hour. The resultant cathode slurry was applied to the sintered SDC electrolyte tablets using a spatula. Multiple applications of the slurry to the electrolyte were required to achieve the desired thickness. The cathodes applied to SDC electrolyte tablets were sintered at a temperature of 1000 °C for 2 h at a heating rate of 3 °C min3)3·6H2O  and Ce(NO3)3·6H2O  powders were dissolved in deionized water to prepare the infiltration solution. Citric acid was then added to the solution as a complexing agent, in a molar ratio of 3:1 citric acid: total metal cations. The prepared solution included 0.2 mol L−1 of SDC. Afterward, ethanol equal to 30% v/v of the prepared solution was added to improve the wetting properties of the infiltration solution.SDC nanoparticles were infiltrated into the scaffold of SCFG cathodes to modify their surface. According to the SDC stoichiometry, Sm technique was employed to ensure the formation of SDC and SCFG phases and to check the chemical compatibility between them after heating at 1000 °C for 1 h. The microstructure of the electrospun fibers before/after calcination, as well as different prepared cathodes, were examined using field emission scanning electron microscopy (FESEM). The EIS test of symmetrical cells was conducted at temperatures between 600 and 800 °C in air. The impedance curves under an open circuit were recorded in the frequency range of 0.1–1 MHz, where the applied signal amplitude was 40 mV. Finally, Z-view software (version 3.4) was used to analyze Rp data.The PVA/SCFG precursor fibers were characterized using a thermogravimetric-differential thermal analyzer (TG-DTA) with a heating rate of 10 °C minThe TG-DTA curves of PVA/SCFG precursor fibers from room temperature to 1000 °C are presented in The XRD pattern of the SDC electrolyte powder obtained via the solid-state reaction method a indicatBefore infiltration b, the sup) for variously prepared cathodes in the 600–800 °C temperature range.The polarization resistances of pure and infiltrated fibrous cathodes were evaluated using an electrochemical impedance (AC) spectroscopy test of symmetrical cells under open circuit conditions at temperatures between 600 and 800 °C in air. p [p .2 [2 for the fibrous SCFG cathode. Comparing the fibrous SCFG cathode to the conventional SCFG cathode, the advantageous microstructure with continuous pores and more reaction sites decreases the polarization resistance of the cathode. At 800 °C, the polarization resistance of pure fibrous SCFG cathode was obtained as 0.12 Ω cm2.As expected, the cathode’s electrochemical performance highly depends on its composition and microstructure. Compared to the conventional SCFG cathode results obtained by other researchers , changin2 , whereas2, whereas the TPB is expanded by adding an SDC electrolyte to all areas of the SCFG cathode. At 800 °C, the Rp value decreased to 0.08 Ω cm2 when 7 wt% SDC nanoparticles were infiltrated into the porous body of the fibrous SCFG cathode. By increasing the loading of SDC nanoparticles in the fibrous structure of the SCFG cathode to 14 wt%, the lowest polarization resistance values were obtained in the temperature range of 600–800 °C. Due to the continuous porous channels that facilitate gas flow, the polarization resistance of SDC infiltrated fibrous SCFG cathodes is considerably reduced. However, more loading of 21 wt% SDC nanoparticles into the SCFG cathode caused a slight increase in polarization resistance, which can be interpreted as a decrease in cathode porosity and TPB, or nanoparticles agglomeration.According to the data presented in In this research, a novel composite cathode composed of SCFG fibers, fabricated by an electrospinning process, and decorated with SDC nanoparticles through an infiltration process, is fabricated. The results of the phase characterization and thermal analyses revealed the well crystalline phase formation and excellent high-temperature chemical compatibility of the prepared composite cathodes. Microstructural analyses, carried out by high resolution SEM, revealed the creation of well-distributed SDC nanoparticles on the porous body of the fibrous SCFG structure.p value for the fibrous SCFG cathode was determined to be 0.12 Ω cm2 at a temperature of 800 °C, which is significantly lower than what was obtained for the same chemical composition cathode, prepared by using SCFG fine powder. On the other hand, it was demonstrated that the infiltration of SDC nanoparticles into the SCFG cathode with a fiber structure increases the active sites and enhances the oxygen reduction reaction (ORR) catalytic activity in the cathode. By infiltrating approximately 7, 14, and 21 wt% SDC nanoparticles into the body of the fibrous SCFG cathode, at 800 °C, the Rp value was determined to be 0.08, 0.03, and 0.04 Ω cm2, respectively, which is significantly lower than what was obtained for the pure fibrous SCFG cathode. Consequently, it can be concluded that the electrochemical performance of electrospun electrodes may be improved through a simple infiltration technique and heat treatment, which can facilitate the low-cost commercialization of SOFC technology.The R


We previously reported that primary glioblastoma stem cells (GSCs), which grow as multicellular tumour spheroids, express functional ASIC1a and ASIC3, whereas ASIC2a is downregulated in GSCs. Using a 2.5D migration assay, here we report that acidic pH dramatically increased migration of GSCs of the pro-neural subtype. Pharmacological blockade as well as CRISPR-Cas9-mediated gene knock-out of ASIC1a or stable overexpression of ASIC2a, however, revealed that neither ASIC1a nor ASIC3, nor downregulation of ASIC2a, mediated the aggressive migration at acidic pH. Therefore, we tested the role of two other proteins previously implicated in cancer cell migration: the Ca2+-activated K+ channel KCa3.1 (KCNN4) and phosphoinositide 3-kinase (PI3K). While pharmacological blockade of KCa3.1 did also not affect migration, blockade of PI3K decreased migration at acidic pH to control levels. In summary, our study reveals a strongly enhanced migration of GSCs at acidic pH in vitro and identifies PI3K as an important mediator of this effect.The microenvironment of proliferative and aggressive tumours, such as the brain tumour glioblastoma multiforme (GBM), is often acidic, hypoxic, and nutrient deficient. Acid-sensing ion channels (ASICs) are proton-sensitive Na Glioblastoma, IDH wild-type multiforme (GBM) is a type of glioma. GBM is the most common primary malignant brain tumour, with a survival rate of < 10% 5 years after the initial diagnosis . Surgica+ channels, which, in the CNS, assemble either as homomeric ASIC1a or heteromeric ASIC1a/2a or ASIC1a/2b [Acid-sensing ion channels (ASICs) are proton-gated NaSIC1a/2b . It has SIC1a/2b . These GSIC1a/2b . GSCs clSIC1a/2b , whereasSIC1a/2b , 30. ASISIC1a/2b . Thus, iSIC1a/2b , 43, 49.SIC1a/2b , 43, 49.2+-activated K+ channel  activated by intracellular Ca2+. It contributes to controlling the cell membrane potential [Phosphoinositide 3-kinases (PI3K) are members of the PI3K/Akt/MTOR pathway, which is often mutated in cancer cells. PI3K is also known to be activated by hypoxia  and is lotential  and has otential , 42, hepotential , 33, andotential , lung adotential , and panotential .e). While we found no evidence for the involvement of ASICs or KCa3.1 in enhanced migration, blockade of PI3K abrogated enhanced migration at acidic pHe, identifying PI3K as a novel mediator of the more aggressive migration at acidic pHe.Here, we established a 2.5D migration assay and show much more aggressive migration of GSCs at an acidic extracellular pH . Culturing the spheres under normoxic or hypoxic (< 3% O2) conditions did not affect migration . We found that ASIC1 was most strongly expressed and that ASIC3 and ASIC4 were expressed at two- to tenfold lower levels  in ASIC1a knockout cells, likely due to the knock-out inducing a frameshift in the second exon of ASIC1a, thereby impairing the binding of the qRT-PCR probe at the exon junction of exon 2 to exon 3.We assessed the expression of p = 0.65 and p = 0.14, respectively) , tubulin beta 3 (TUBB3), and galactocerebrosidase (GALC), which are markers of astrocytes, neurons, and oligodendrocytes, respectively. While nestin expression was high, the expression of TUBB3 and, in particular, of GFAP and GALC was much lower. There was a tendency for increased expression of nestin and of GFAP at pH 6.6, but this change was not statistically significant (ly) Fig. .e, we assessed migration of GSCs at acidic pH 6.6 in the presence of the potent ASIC1a inhibitor psalmotoxin 1  or the potent ASIC3 inhibitor APETx2 (500 nM). Neither inhibitor had an effect on migration, irrespective of pH  or the pan-PI3K inhibitor wortmannin (1 µM) to the spheres 30 min before the start of the migration assay; we performed these assays with spheres maintained at pH 7.4 or at pH 6.6. While TRAM-34 did not affect migration under any conditions, wortmannin strongly reduced migration, but only when outgrowth was tested at pHe 6.6 . This suggests that PI3K plays an essential role in the acute induction of migration by acidic pHe and that the priming of migration or selection of aggressively migrating GSCs by acidic pH is PI3K-independent and relies on a different mechanism, at least when tested at pH 7.4. The finding that wortmannin strongly reduced migration at pHe 6.6 of cell maintained at acidic pH below the level of migration of these cells at pHe 7.4 is not entirely consistent with this interpretation and suggests a complex interplay of different mechanisms in acid-dependent migration of GSCs.We then considered other targets affecting the pH-dependent migration of GSCs, and turned to Kigration , 16, 42 igration , 21, 34.ived GBM , whereasived GBM . To asse6.6 Fig. . In conte than the abrupt changes used in our experiments. In addition, serum-free conditions may not accurately represent the micro-environment of tumours in vivo. Nevertheless, we believe that our assay provides an important addition to earlier migration assays assessing the role of ASICs in tumour cell migration, which used serum-cultured glioma cell lines, which do not well represent the parental tumour [We note that the 2.5D sphere migration assay can only partially reproduce migration of tumour cells in situ. Cancer cells in situ are likely to be confronted with slower changes in pHl tumour , 30, in Our findings using patient-derived R54 GSCs and a 2.5D migration assay do not confirm previous publications reporting that ASIC1, ASIC2, or ASIC3 play a role in pH- or hypoxia-dependent migration in CNS , 43, 49,Ca3.1 has been reported in numerous studies [Ca3.1 is a marker for the mesenchymal subtype of GSCs [Ca3.1 plays a role in acid-induced migration of mesenchymal subtype GSCs.Likewise, a role for K studies , 42, 48  studies , while i of GSCs . It wille: activation of PI3K is crucial for triggering fatty acid synthesis in liver cancer cells by an acidic TME [In contrast, our findings reveal a novel and prominent role for PI3K in acid-induced migration of R54 GSCs. Because PI3K is an intracellular enzyme, it must have been activated either by a non-ASIC proton-sensor on the surface of R54 cells or by a drop in intracellular pH that often accompanies extracellular acidification. A few previous studies already reported an activation of PI3K by acidic pHidic TME , for actidic TME , and foridic TME . The preCa3.1 in pH-dependent migration of GBM cells. However, wortmannin reduced migration induced by acute extracellular acidosis suggesting that PI3K plays a crucial role in pH-dependent migration of GBM cells. The mechanism by which acidic pHe activates PI3K to induce migration remains unclear and needs to be elucidated in the future.In this study, using a 2.5D sphere migration assay and patient-derived R54 GSCs, we could not confirm a role for ASIC1, ASIC2, ASIC3, or K+ primary patient-derived GBM stem cell (GSC) line that grows as a heterogeneous tumour sphere [3, supplemented with 2% Neuropan 27 (PAN-Biotech), 1% of 200 mM L-Glutamine , 20 ng/ml recombinant human epidermal growth factor , and 20 ng/ml recombinant human fibroblast growth factor . They were regularly confirmed to be mycoplasma-negative via PCR. Medium with a pH of 6.6 was prepared with powdered medium not containing NaHCO3, which was added at a concentration of 0.4002 g/l, and also contained 15 mM HEPES, additionally to the supplements listed above. Cells were split with 0.05% Trypsin–EDTA solution (Thermo Fisher Scientific) into single cells once per week for regular cell maintenance .The cell line R54  is an IDr sphere , 35. R54n = 3 independent cell batches. The Rotor-Gene Q software  was then used for analysis. Statistical analysis was performed with ΔCt values using an unpaired two-sided Student’s t test, comparing R54 cells at pH 7.4 and pH 6.6, as well as R54 cells and R54 ASIC1 KO cells at pH 7.4. ΔCt values are normalized logarithmic expression values and as such expected to be normally distributed [R54 GSCs were incubated in pH 7.4 or pH 6.6 for 3 days. RNA was then isolated from GSCs using the NucleoSpin RNA isolation kit , and concentration and quality were measured with the NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific). RNA was reverse transcribed into cDNA with the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific), according to the manufacturers’ instruction. Quantitative real-time PCR (qRT-PCR) was conducted using 20 ng of cDNA template per reaction, as well as the Luna mastermix (NEB) in combination with FAM-MGB labelled hydrolysis (TaqMan™) probes from Thermo Fisher Scientific, namely: ASIC1a (Hs00952802), ASIC1b , ASIC2 (Hs00153756), ASIC3 (Hs00245092), ASIC4 (Hs00539823), nestin (Hs00707120), GFAP (Hs00909233), TUBB3 (Hs00801390), and GALC (Hs00164660), with GAPDH as a housekeeping gene (Hs02758991). Reactions containing 5 µl Luna NEB mastermix, 1 µl cDNA, 1 µl TaqMan probe, and 3 µl sterile water were pipetted into 4-Strip 0.1 ml Tubes  and transferred to the Rotor-Gene Q thermocycler  for real-time measurements. Reactions were performed in technical triplicates for each biological replicate, with technical duplicate negative controls for each TaqMan probe. The qRT-PCR programme was conducted with an initial denaturation step , 40 cycles of denaturation , annealing , and elongation , followed by a final elongation step . Experiments were repeated with RNA isolated from at least tributed .2. The plates were then incubated overnight at 4 °C. GSCs were grown for 7 or 14 days into spheres; for some experiments, spheres were grown for 14 days in experimental pH conditions. The spheres were then gently spun down (400 g for 1.5 min) and resuspended in medium with different pH, with and without pharmacological inhibitors. Medium of ASIC2a over-expressing GSCs also contained 4-hydroxytamoxifen (4-HT). For 4-HT induction, the spheres had already been preincubated for 3 days with 4-HT at a concentration of 100 nM. Afterwards, a bystander blinded the experiment. The laminin was aspirated, the wells were washed once with PBS, and then the spheres were gently added into the wells, at a concentration that was empirically determined previously. The 24-well plate was then incubated at 37 °C, 5% CO2 for 5–6 h. Afterwards, the plates were carefully placed under the microscope and pictures taken with the IC Measure programme . For experiments conducted under hypoxic conditions, the 24-well plates were incubated for 5–6 h in an Incucyte Live-Cell Analysis System , kindly provided by Dr. Jochen Maurer, at 37 °C, 5% CO2, < 3% O2. Pictures were taken from the Incucyte System with the built-in analysis system. Diameter of spheres and number of migrating cells were counted by hand and positions of cells marked, using ImageJ . Tumourspheres with a high background of freely migrating cells were disregarded, and only cells that could be clearly assigned to a tumoursphere were counted. Furthermore, only spheres with a diameter > 50 µm were quantified. After counting, the experiment was unblinded. The effect of ASICs on migration was tested using PcTx1 , APETx2 , or MitTx (Smartox Biotech) and the effect of KCNN4 and PI3K by TRAM-34  and wortmannin , respectively.We used a 2.5D sphere migration assay (SMA) that relies on the attachment of spheres to laminin-coated plates and the subsequent migration of individual GSCs out of the spheres. The day before the experiment, 24-well plates (Thermo Fisher Scientific) were coated with 180 µl laminin from mouse Engelbreth-Holm-Swarm  sarcoma (20 ng/µl), resulting in a final laminin density of 1–2 µg per cmA two-vector inducible system was used to generate ASIC2 overexpressing R54 cells. The cells were transduced first with a lentiviral vector pF GEV16 Super PGKHygro, which expresses a Gal4 DNA binding domain fused to a mutant oestrogen receptor and GEV16. Secondly, the cells were transduced with a lentiviral vector pF 5 × UAS W SV40 Puro ASIC2, which expresses ASIC2 in a Gal4-dependent manner. To generate lentiviral supernatants, 293 T cells were transfected with 3 mg pMD2.G, 5 mg pMDlg/pRRE, and 2.5 mg pRSV-Rev of the lentiviral packaging vectors  together2PO4, 4.8 Na2HPO4, 5 glucose, 2.38 MgCl2, 1 EGTA, and 0.726 Ca2+-gluconate. The pH was adjusted to pH 7.2 using KOH and HCl. The cells were recorded in a perfused bath system at RT with an extracellular solution containing (in mM) 115 NaCl, 0.4 KH2PO4, 1.6 K2HPO4, 5 Glucose, 1 MgCl2, 25 Na+-gluconate, 3 Ca2+-gluconate, and 5 HEPES/MES. HEPES was used as a buffer for the standard bath solution of pH 7.3, and MES was used as a buffer for the stimulating bath solutions of pH 6 and 5. The pH-values were adjusted using NaOH and HCl. Each stimulation lasted 10 s. The cells were stimulated at least once with pH 6 before being activated with pH 5. Between stimulations, the cells were perfused with standard bath solution pH 7.3 for at least 60 s. To test the effect of PcTx1, 50 nM PcTx1 was added to the pH 6 and the pH 7.3 solution. It was pre-applied for 2 min. For analysis, the software ClampFit was used. Only fast activating (time to peak < 1.5 s) and transient currents with amplitudes > 10 pA were considered as ASIC-currents.R54 GSCs overexpressing ASIC2 by lentiviral transduction were induced with 100 nM 4-HT 3 days before splitting. After splitting with 0.05% Trypsin/EDTA (Thermo Fisher Scientific), single cells were seeded on coverslips coated with poly-D-lysine and analysed by patch-clamp during the following 48 h. An Axopatch 200B amplifier and Digidata 1440A digitizer  controlled by Clampex (Version 10.6) were used for current recordings. The holding potential was − 70 mV. Micropipettes with a resistance of 4–6 MΩ were filled with an intracellular solution containing (in mM) 95 K-gluconate, 30 KCl, 1.2 NaHt test in Prism 8 (Version 8.4.3), with significance threshold set to p ≤ 0.05.Statistical analyses of qRT-PCR results were performed using two-sided padj < 0.05.For sphere migration assays (SMAs), the experimenter was blinded for the condition. We performed SMAs with at least three independent biological replicates with a variable number of technical replicates. To avoid pseudoreplication due to clustering of technical replicates from one biological replicate  and to ap < 0.05. Normal distribution of the data for one-way ANOVA was confirmed via Anderson–Darling, D’Agostino-Pearson omnibus normality, Shapiro–Wilk normality and Kolmogorov–Smirnov normality tests in Prism 8 (Version 8.4.3), with significance threshold set to p < 0.05.Sphere diameters were analysed with one-way ANOVA in Prism 8 (Version 8.4.3), with significance set at