Schinus molle is a medicinal plant used as an anti-inflammatory and for rheumatic pain in the traditional medicine of Peru. On the other hand, Aedes aegypti is the main vector of several tropical diseases and the transmitter of yellow fever, chikungunya, malaria, dengue, and Zika virus. In this study, the aim was to investigate the antioxidant activity in vitro and the insecticidal activity in silico, in the presence of the mosquito juvenile hormone-binding protein (mJHBP) from Aedes aegypti, of the essential oil from S. molle leaves. The volatile phytochemicals were analyzed by gas chromatography-mass spectrometry (GC-MS), and the profile antioxidants were examined by DPPH, ABTS, and FRAP assays. The evaluation in silico was carried out on mJHBP (PDB: 5V13) with an insecticidal approach. The results revealed that EO presented as the main volatile components to alpha-phellandrene (32.68%), D-limonene (12.59%), and beta-phellandrene (12.24%). The antioxidant activity showed values for DPPH = 11.42 ± 0.08 μmol ET/g, ABTS = 134.88 ± 4.37 μmol ET/g, and FRAP = 65.16 ± 1.46 μmol ET/g. Regarding the insecticidal approach in silico, alpha-muurolene and gamma-cadinene had the best biding energy on mJHBP , followed by beta-cadinene . Additionally, the volatile components did not reveal antioxidant activity, and its potential insecticidal effect would be acting on mJHBP from A. aegypti. Schinus molle L., internationally commonly known as peruvian pepper, false pepper, American pepper, or pink pepper, is a perennial tree belonging to the Anacardiaceae family, which is native to subtropical regions of South and Central America [S. molle (leaves and fruits) exhibits biological activities such as antibacterial (gram-positive and gram-negative bacteria), antifungal, insecticidal, repellent, and cytotoxic activities [Essential oils (EOs) are a class of natural products characterized by their volatile components, especially terpenes and sesquiterpenes . EOs are America . In Peru America . The esstivities . Locallytivities .Aedes aegypti [Regarding insecticidal activity, several synthetized chemicals are used as repellents, vector control, or insecticides, but during the last year, some of them have been retired due to their carcinogenic, genotoxic, or harmful-to-the-environment effects by bioaccumulation . The app aegypti .A. aegypti, which consist of two modified odorant-binding protein domains with its respective ligand in the N-terminal domain named JH-III [Recently, a group of salivary D7 proteins known as mosquito juvenile hormone-binding protein (mJHBP) has been identified in d JH-III . This hod JH-III . In reced JH-III .S. molle EO was evaluated in several species such as Trogoderma granarium, Tribolium castaneum [Sitophilus oryzae [Haematobia irritans [Ctenocephalides felis felis [Gonipterus platensis [A. aegypti. Thus, we investigated the potential insecticidal effect in silico of the volatile components of S. molle EO to find any responsible molecule as a JH analogue, which might combat the presence of mosquitoes in tropical regions and reduce the prevalence of diseases transmitted by them. The aim in this study was (1) to determine the total volatile component of the EO from S. molle leaves by gas chromatography-mass spectrometry (GC-MS), (2) to evaluate the antioxidant activity using the DPPH, ABTS, and FRAP methods, and (3) to determine the insecticidal activity using a virtual screening of the EO from S. molle leaves on the mosquito juvenile hormone-binding protein from Aedes aegypti.Although the insecticidal activity of astaneum , Sitophis oryzae , Haematoirritans , Ctenoceis felis , and Gonlatensis , currentAll the solvents , of analytical grade (99.5%), were purchased from Merck . 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ), and Trolox were purchased from Sigma-Aldrich .Schinus molle (leaves) cultivated in Tinguiña, Ica Region, Peru (406 m.a.s.l.) in December 2020 was received. Leaves were cleaned and peeled to be incorporated in a Clevenger equipment and to obtain essential oil by hydrodistillation for 2 h [2SO4 was added to eliminate any water drops. Finally, the EO was stored in a sealed amber vial until further use.A quantity of 4700 g of  for 2 h . The essμL of EO was mixed with 1.0 mL of dichloromethane. Next, 1.0 μL of the working solution was injected into the equipment in splitless mode (split: 20 : 1). The EO was run on a J&W 122-1545.67659 DB-5 ms column, (60 m × 250 μm × 0.25 μm) . The working conditions were as follows: the temperature program was 40°C, starting with increments of 5°C/min up to 180°C, followed by increases of 2.5°C/min up to 200°C for 5 min, and finally 10°C/min up to 300°C, remaining for 3 min. The helium flow rate was at 1 mL/min. Volatile components' identification was based on a comparison of relative retention indices (RIs) and mass spectra data with the NIST20 library data and the published literature [Volatile chemicals were determined with a GC-MS system . Then, 20 μL of EO (10 mg/mL) was mixed with 2850 μL of a methanolic solution of DPPH (20 mg/L) with an absorbance adjusted to 1.1 ± 0.02 nm. After mixing, it was incubated in the dark for 30 minutes, and the absorbance reading was carried out at 515 nm. The standard curve was elaborated with Trolox at concentrations of 0 to 800 μmol/mL. The Trolox equivalent antioxidant capacity (TEAC) was expressed as μmol TE/g of essential oil [A 150 tial oil . To calcμL of the EO  was mixed with 2850 μL of ABTS radical and incubated for 7 min, the same procedure was used with Trolox standard ranging between 0 and 400 μmol/mL. The absorbance was read to 0.7 ± 0.02 at a wavelength of 734 nm. The Trolox equivalent antioxidant capacity (TEAC) was expressed as μmol TE/g of essential oil [50), a linear regression method was used based on the different concentrations of the EO.To carry out the antioxidant activity using the ABTS radical, a solution was prepared using the mixing of 10 mL of ABTS (4.06 mg/mL) and 10 mL of potassium persulfate (0.7 mg/mL), both reacted for 16 hours. Then, 150 tial oil . To calcμL of EO (1 mg/mL) was mixed with 2850 μL of FRAP reagent and reacted for 4 min at room temperature. The absorbance reading was carried out at 593 nm. A standard curve was prepared with Trolox (50-800 μM). The results were expressed as μmol equivalents of Trolox per gram of essential oil (μmol TE/g EO) [50), the linear regression method was used based on the different concentrations of the EO.In the determination of FRAP of EO, a reagent battery was made using 25 mL of acetate buffer pH 3.6; 2.5 mL of 20 mM TPTZ was dissolved in 40 mM HCl and 2.5 mL of 20 mM ferric chloride hexahydrate. The mixing of this reagents constituted the FRAP solution which reacted with the EO at different concentrations as well as Trolox standard. 150 The molecules isolated from phytochemicals of essential oil were docked against the mosquito juvenile hormone-binding protein (PDB id: 5V13). In order to validate the docking, the crystal structures were docked with the native ligand pyriproxifen and JH3 bound to X-ray structures of mosquito juvenile hormone-binding protein. In addition to the former for comparison, better validation of docking at the binding cavity of mosquito juvenile hormone-binding protein was determined surrounding the 3 Å distance from the bound ligand JH3 using the DoGSiteScorer server. The binding cavity residues comprised mainly TYR33, LEU37, TRP50, AL51, TRP53, TYR64, SER69, TYR129, TYR133, ILE140, PHE269, TRP278, and ALA281. Protein and ligand preparations were performed using AutoDock Tools (v. 1.5.6) . Gasteiger charges were added to the ligand molecules prior to converting to PDBQT format. The online server DoGSiteScorer and the information about the binding site residues of the native ligand were used to construct the grid box. The grid box of dimensions 25 × 25 × 14 Å for the mosquito juvenile hormone-binding protein with 0.375 Å grid spacing was constructed using AutoGrid 4.2. Semiflexible docking was performed keeping the receptor molecule rigid and ligands flexible. AutoDock 4.2 using the Lamarckian genetic algorithm (LGA) scoring function with number of GA runs = 100, population size = 500, and maximum number of evaluations = 25,000,000 was used to develop the molecular docking of all volatile components of EO. Then, the RMSD clustering maps were obtained by a reclustering command with a clustering tolerance of 0.25 Å, 0.5 Å, and 1 Å, respectively, in order to obtain the best cluster having the lowest energy score with a high number of populations .α-phellandrene  and α-muurolene  with the mosquito juvenile hormone-binding protein (mJHBP) using the Desmond 2020.1 from Schrödinger, LLC. The reproducibility was completed using three replicates for each MD run. The OPLS-2005 force field and explicit solvent model with the SPC water molecules were used in this system. To neutralize and simulate the physiological conditions, the charge Na+ ions and 0.15 M NaCl were added, respectively. The system was equilibrated using constant-temperature and constant-volume ensemble (NVT) for 100 ps. This was followed by a short run equilibration and minimization using constant-temperature and constant-pressure ensemble (NPT) for 12 ps and was set up using the Nose-Hoover chain coupling scheme with temperature 27°C, the relaxation time of 1.0 ps, pressure 1 bar, and a time of 2 fs. The Martyna-Tuckerman-Klein chain coupling scheme barostat method was used for pressure control using a relaxation time of 2 ps. The particle mesh Ewald method was used for calculating long-range electrostatic interactions with a radius of 9 Å for the coulomb interactions. Bonded forces were calculated using a RESPA integrator with a time step of 2 fs for each trajectory. The root mean square deviation (RMSD), radius of gyration (Rg), root mean square fluctuation (RMSF), and solvent accessible surface area (SAS area) were calculated to monitor the stability of the MD simulation.This evaluation was carried on the dock complexes for S. molle, the IC50 values were estimated by linear regression statistics. A Spearman's Rho coefficient was calculated to establish a correlation between the evaluated concentrations and the antioxidant response. P values less than 0.01 were considered statistically significant. GraphPad Prism program version 6.0  was used to carry out the statistical analysis.In the analysis of the antioxidant profile of essential oil from S. molle essential oil were analyzed by GC-MS and are presented in 15H26O and a molecular weight of 222.37 g/mol. The analysis identified alpha-phellandrene as the main volatile chemical with 32.68%, followed by D-limonene (12.59%) and beta-phellandrene (12.24%). According to The obtained EO of fresh leaves showed a light-yellow color, a density of 0.873 ± 0.02 g/mL at 20°C, and an extraction yield of 0.73% during 2 hours of distillation. The profiles of volatile constituents of β-phellandrene (15.7%) and α-phellandrene (12.1%) were the main components of the EO from S. molle leaves cultivated in Tepetitla de Lardizábal, Mexico [S. molle EO obtained of dried leaves from Seropédica, Rio de Janeiro, Brazil, epi-α-cadinol (22.85%) was the major component [S. molle from Caxias do Sul, Brazil, alpha-pinene (60.04 ± 0.07%) was the most abundant constituent [α-cadinol with 21.0 ± 1.1%, followed by myrcene (16.7 ± 1.1) and sabinene (14.5 ± 0.8) [S. molle from North Cyprus showed values of alpha-phellandrene, limonene, and beta-phellandrene equivalent to 31.5%, 10.1%, and 9.9%, respectively [S. molle from Sonora, Mexico, revealed 60.8% of D-limonene (17.8%), o-cymene (16.4%), β-phellandrene (12.6%), δ-cadinene (7.5%), and caryophyllene (6.5%) [In the phytochemical analysis, our results differed from those of other investigations and could be explained by different types of factor such as type of extraction, temperature conditions, storage conditions, edaphic conditions, plant part extracted, ecosystem, environmental, harvesting time, and the presence of environmental pollution . In this, Mexico . In an aomponent . Additiostituent . In Navi5 ± 0.8) . S. mollectively . On the e (6.5%) . HoweverS. molle EO exhibited a low antioxidant activity, as is shown in P = 0.0004). Other reports have shown different values; according to Eryigit et al., EO showed a TEAC (ABTS) = 4.7 ± 1.2 mM Trolox of S. molle grown in Turkey [α and β-phellandrene, α-pinene, sabinene, limonene, β-myrcene, and others [50 against DPPH was 41.84 μg/mL, which was higher compared to the findings of the EO from southeast Portugal [S. molle from Egypt showed an IC50 at DPPH of 172.45 μg/mL and a high percentage of oxygenated sesquiterpene (17.73%) [S. molle EO could be acted by electron transfer to reduce the free radicals.n Turkey . EO fromd others . In thisPortugal . In anot(17.73%) . AccordiS. molle EO leaves with mJHBP from A. aegypti. Out of 32 specific compounds found abundantly in chromatography, gamma-cadinene and alpha-muurolene displayed a lower binding energy (ΔG) of -9.7 kcal/mol and predicted inhibitory concentration (Ki) of 0.134 μM , compared to alpha-muurolene, the total components might be synergizing the insecticidal effect.The virtual screening of the 32 chemical components, additionally JH3 and the pyriproxyfen (synthetic insecticide), was carried out in order to understand the interaction profile of various volatile compounds present in 0.134 μM . AlthougG > −9 kcal/mol) of some volatile components was evidenced in those interactions with the Tyr133, Trp53, Tyr33, and Phe144 residues as shown in Supplementary Materials Figures The principal residues of mJHBP, TYR133, TRP53, TYR33, and PHE144 were involved in Pi-Sigma bond formation with alpha-muurolene . We obseα-phellandrene and mJHBP+α-muurolene complexes. Each simulation of 100 ns displayed stable conformation comparing the root mean square deviation (RMSD) values. The Cα backbone of mJHBP bound to α-phellandrene exhibited a deviation of 0.5 Å (α-muurolene exhibited a deviation of 1.0 Å (α-phellandrene and α-muurolene-bound mJHBP are quite stable in complex due to the higher affinity of the ligand. The plots for root mean square fluctuations (mMSF) displayed a significant spike in fluctuation (3.5 Å) at amino acid residue 220 in mJHBP, while the rest of the residues fluctuated less during the entire 100 ns simulation  from 19.6 Å to 19.4 Å, and the lowering of the Rg indicates the compactness of the complex  studies were carried out in order to determine the stability and convergence of mJHBP+of 0.5 Å , while mof 1.0 Å . RMSD pl Figures  indicati complex . On the nd state . The ove Figures . The int Figures .S. molle leaves presented 34 volatile constituents determined by gas chromatography-mass spectrometry (GC-MS). Alpha-phellandrene was the major component, which represented 32.68% of the total composition; furthermore, beta-phellandrene and D-limonene were the following more abundant metabolites with 12.24% and 12.59%, respectively. Additionally, the antioxidant activity was evaluated in order to determine the antioxidant profile of EO using the three methods of DPPH, ABTS, and FRAP. According to the results, the EO showed better affinity and a good effect on FRAP assay and IC50 equivalent to 1.50 ± 0.02 mg/mL. However, this study revealed that S. molle EO is not a good antioxidant. Regarding the insecticidal activity in silico based on a virtual screening on mosquito juvenile hormone-binding protein, several volatile compounds were active against this target such as alpha-muurolene and gamma-cadinene, being similar to pyriproxyfen, which is a synthetic insecticide analogue of JH. The molecular dynamics carried out for alpha muurolene (the best result in molecular docking) and alpha-phellandrene (the most abundant molecule) were very stable during 100 ns of evaluation. In the future, S. molle EO might be used as bioinsecticide on A aegypti, but an in vitro and in vivo assay has to be evaluated to validate our findings.The essential oil of


Recent evidence suggests that dexmedetomidine might also directly inhibit hyperpolarization-activated cyclic-nucleotide gated (HCN) channels. We analyzed the effects of dexmedetomidine on native HCN channel function in thalamocortical relay neurons of the ventrobasal complex of the thalamus from mice, performing whole-cell patch-clamp recordings. Over a clinically relevant range of concentrations (1–10 µM), the effects of dexmedetomidine were modest. At a concentration of 10 µM, dexmedetomidine significantly reduced maximal Ih amplitude , yet changes to the half-maximal activation potential V1/2 occurred exclusively in the presence of the very high concentration of 100 µM . Coincidentally, only the very high concentration of 100 µM induced a significant deceleration of the fast component of the HCN activation time course . With the exception of significantly increasing the membrane input resistance (starting at 10 µM), dexmedetomidine did not affect biophysical membrane properties and HCN channel-mediated parameters of neuronal excitability. Hence, the sedative qualities of dexmedetomidine and its effect on the thalamocortical network are not decisively shaped by direct inhibition of HCN channel function.Dexmedetomidine is a selective α By decreasing noradrenaline release in a dose-dependent manner  mV, n = 11, p > 0.999; Δ 10 µM DEX: +0.8 [−1.9–+1.5] mV, n = 10, p > 0.999; and Δ 100 µM DEX: −0.8 [−2.6–+0.2] mV, n = 10, p > 0.342). In between concentration groups, there were no significant differences with regard to baseline conditions . A total of 40 µM of HCN channel blocker ZD7288, which has a reported half maximal inhibitory concentration (IC50) of 15 µM [control: −60.9 [−63.2–−58.5] mV]; +40 µM ZD7288: −65.5 [−74.0–−61.0] mV; and +10 µm DEX: −63.5 [−72.1–−61.0] mV, n = 5). See Passive and active membrane biophysics were derived from current–voltage relationship recordings. Under baseline recordings, TC neurons displayed a median resting membrane potential of −58.2 (IQR: −59.7–−56.9) mV, n = 31. For all three DEX concentration groups, we did not detect a significant change in the resting membrane potential after 30 min and 45 min of DEX application, see of 15 µM , inducedΔ 1 µM DEX: +24.0 [−10.0–+37.2] MΩ, n = 11, p = 0.602; Δ 10 µM DEX: +27.9 [+9.6–+89.0] MΩ, n = 10, p = 0.011; and Δ 100 µM DEX: +95.3 [+73.2–+120.5] MΩ, n = 10, p < 0.001). A total of 40 µM ZD7288 also resulted in an increase in the input resistance in all cells recorded, which again persisted after applying 10 µM DEX  p = 0.037 and at −60 pA: +40 [+3.2–+64.9] MΩ, n = 10, p = 0.084).Beginning at a concentration of 10 µM DEX, we observed a significant increase in the membrane input resistance, indicative of decreased neuronal leakiness. Under control conditions, the median input resistance was at 343.4 (252.3–365.6) MΩ, n = 31. The effects of DEX are presented as an absolute difference from the baseline recordings , only the high concentration of 100 µM DEX persistently induced a significant hyperpolarization in the whole range between −90 and +350 pA, whereas 10 µM DEX only induced significant changes during strongly hyperpolarizing currents, see Δ 1 µM DEX: −0.6 [−2.6–−0.2] mV, n = 11, p = 0.583; Δ 10 µM DEX: −0.4 [−2.4–+0.9] mV, n = 10, p = 0.850; and Δ 100 µM DEX: +1.3 [+1.1–+5.4] mV, n = 10, p = 0.013). In contrast, HCN channel blocker ZD7288 shifted the threshold potential toward hyperpolarization. The co-application of ZD7288 and 10 µM DEX did not reverse the observed hyperpolarization , see Similarly, intrinsic firing properties of TC neurons were only significantly affected by a very high concentration of 100 µM DEX. Under the baseline conditions, the threshold potential for action potential firing was determined at −39.2 (−40.5–−37.6) mV. 100 µM DEX shifted this threshold toward depolarization, with no significant effects occurring at lower concentrations , see We further analyzed the effect of DEX on the maximum frequency of tonic action potential firing. The frequency was derived from the current injection of +190 pA, as we did not observe a further increase in maximum frequency beyond this value. Median tonic action potential firing at the baseline conditions occurred at 40.2 (36.3–44.9) Hz, n = 31. Again, significant changes were exclusively observable in the presence of the high DEX concentration, with a decrease in tonic action potential frequency  and an increase in the input resistance , whereas there were no significant changes regarding the action potential threshold (p = 0.578) as well as tonic action potential firing (p = 0.094).All experiments were conducted in the presence of 150 µM Bah, as well as modalities of HCN channel function. Since the extent of HCN channel activation correlates with increasing hyperpolarization , n = 11, p > 0.999; 10 µM DEX: 0.90 [0.80–1.00], n = 10, p = 0.293; 100 µM DEX: 0.57 [0.42–0.58], n = 10, p < 0.001; and 40 µM ZD7288: 0.05 [0.03–0.14], n = 5), see h current amplitude became significant beyond membrane potentials of −83 mV.We analyzed the effects of DEX on the HCN channel-mediated, mixed cation current Irization , the Ih tail activation curves fitted with a Boltzmann function are shown in 1/2) of −86.6 (−85.2–−88.0) mV for TC relay neurons under baseline conditions. Only in the presence of the high concentration of 100 µM DEX was the V1/2 shifted toward hyperpolarization in accordance with a reduced open-channel probability , see Furthermore, we evaluated whether DEX affects the voltage-dependent activation of HCN channels and open channel probability via a tail current analysis. The respective I2+ did not significantly change Ih max  nor V1/2 .In comparison to native recordings, the addition of 150 µM Bah activation, which was evaluated by fitting the Ih current recorded at −133 mV to a biexponential function, see fast and τslow change considerably, depending on the voltage step analyzed, as well as the duration of the current recording. Due to the limited effects observed with regard to Ih amplitude, we chose to apply the biexponential fitting to the maximum voltage step at −133 mV. The median value of τfast at −133 mV was 231 ms , while τslow was 1143 ms  under control conditions. Only the fast component of HCN activation kinetics was subjected to a significant deceleration, while the slow component remained, overall, unaffected. Likewise, the observed deceleration was only significant at a high concentration of 100 µM DEX , see A similar pattern of only modest effects at high DEX concentrations was seen with regard to the time course of I2+. In a subset of experiments, we evaluated the impact of Ba2+ on native current-clamp recordings. As shown in 2+ unmasks an otherwise discreet voltage sag  and significantly accelerates the median rebound burst delay . In comparison, HCN channel blocker ZD7288 completely attenuates the inwardly directed voltage rectification. Further, in two out of five cells, we were not able to induce bursting activity in the presence of ZD7288, even when increasing the current injection to −500 pA or compensating for the ZD7288-induced membrane hyperpolarization. In comparison, the effects of DEX were negligible, see Lastly, we evaluated whether DEX affects modalities of neuronal excitability in TC relay neurons generally associated with HCN channel function. Therefore, we performed additional whole-cell current-clamp recordings. Upon injection of a hyperpolarizing current , TC neurons display an anomalous rectification of the membrane potential, termed “voltage sag”, followed by a low-threshold, calcium burst of action potential firing. Upon burst cessation, the neurons show an afterdepolarization. Both afterdepolarization and voltage sag are largely mediated by HCN channels , see Figh amplitude, half-maximal activation potential, as well as a deceleration of the fast activation kinetics time constant, did not affect the voltage sag , see The median voltage sag amplitude displayed by TC neurons under baseline conditions was 86.4 (70.7–92.9) mV (n = 31). Interestingly, even the high concentration of 100 µM DEX, which was sufficient to elicit a significant change in IΔ 100 µM DEX: n = 10, p = 0.003; lower concentrations not shown), see Δ 100 µM DEX: n = 10, p = 0.002, see Δ 100 µM DEX: −140.3 [−247.1–−72.8] ms, n = 10, p = 0.016), see Significant changes with regard to rebound delay, the number of action potentials during rebound spike activation, as well as median burst duration exclusively occurred at the high concentration of 100 µM DEX. During control conditions, the median delay of rebound burst-spiking upon hyperpolarization was 29.0 ms , with a significant prolongation of 6.5 ms (5.1–8.9) ms  of DEX required for sedation and analgesia in rats was determined to be at 1 µM [h amplitude already within 5 min after application [h and HCN channel function was characterized as non-recoverable even after a prolonged washout [An obvious concern with respect to in vitro studies relates to the question of whether the applied concentrations of a pharmacological agent are indeed clinically relevant. A recent study  states t at 1 µM . Some so at 1 µM . However at 1 µM . Therefo at 1 µM . In vitr at 1 µM ,32, 0.1– at 1 µM , and 10  at 1 µM ). We, thlication ,48. Our 2-adrenoceptor agonist, has been shown to directly inhibit HCN2 and HCN4  channels in transfected HEK293 cells and block Ih in isolated sinoatrial node pacemaker cells. Comparable to the HCN blocking agent ZD7288, clonidine also shifted V1/2 toward more hyperpolarized potentials [h amplitude and slowed its rate of activation independently from cAMP via the activation of protein kinase C [h in further neuronal cell types, including rat trigeminal root ganglion neurons [The goal of this study was to investigate whether DEX can directly inhibit HCN channel function. One indication of this assumption was based on the observation that clonidine, another αtentials . In dopakinase C . Further neurons , rat hyp neurons , and rat neurons .3). DEX induced a concentration-dependent reduction in the amplitude of Ih (IC50 of 1.21 µM) and a substantial hyperpolarizing shift in the voltage-dependent activation course of Ih (−10 mV). Further, DEX attenuated Ih in pheochromocytoma PC12 cells and slowed its activation time course. Endocrine and neuroendocrine cells, including GH3 cells, intrinsically express isoforms HCN2 and HCN3 [h amplitude irreversibly and the V1/2 was shifted toward hyperpolarization by −8 mV This effect was only marginally dependent on intracellular cAMP. Rat PVN neurons have been shown to express mRNA of HCN1, HCN2, and HCN3, but not HCN4 [So far, our knowledge about the effects of DEX on HCN channels is still limited. There is indirect evidence from a study using an in vivo model of peripheral nerve blockade in rats . Here, aand HCN3 . Lastly,and HCN3 . DEX rednot HCN4 . 2+ [ir and certain potassium K2P channels [2+. In contrast with the reported decrease in input resistance in the study by Shirasaka et al. [A-receptors [h and increases input resistance [Furthermore, DEX dose-dependently hyperpolarized PVN neurons and decreased input resistance, but this hyperpolarization was blocked by pretreatment with 0.3 mM Ba2+ . Therefochannels . This coa et al. , we obsea et al. . Howevera et al. . Many aneceptors ,30,55. Tsistance  while elsistance . Howeverh are known to be strongly sensitive to changes in experimental conditions, including the ionic composition of extra- and intra-cellular solutions, temperature, pH, and patch-clamp configuration [h currents are present throughout the mammalian nervous system, but the distribution of specific HCN isoforms and their combinations display a more heterogeneous pattern [h conductance in TC relay neurons is largely dependent on HCN2 [As a matter of principle, recordings of HCN-mediated Iguration . In contguration . Howeverguration ,30. Instguration ,32,47,48guration  and diffguration ,59,60. Tguration . HCN cha pattern . TC rela pattern ,62 and,  on HCN2  and also on HCN2 . Importa on HCN2 ,65,66. W on HCN2 . As the  on HCN2 . As in p on HCN2 ,30, we u1/2 values for HCN2 and HCN4 range between −70 and −100 mV [1/2 10 to 25 mV toward depolarization [1/2 of −86.6 mV, as well as a median τslow of 1143 ms. Together with the existing research evidence, our data strongly indicate that the recorded conductance stems from HCN2 and HCN4, whereas the other studies cited include recordings of HCN1 and HCN2, HCN1, HCN2 and HCN3, and HCN2 andHCN3. It is further noteworthy that the DEX-induced inhibition of Ih in slice recordings of rat hypothalamic PVN neurons was almost completely antagonized by the application of yohimbine, an α2-adrenoceptor antagonist. This suggests that the inhibition of Ih was reliant on an intact α2-adrenoceptor mechanism [V −100 mV  and, in rization . In thisechanism . h plays a crucial role in the generation, amplification, and stable maintenance of rhythmic oscillations within the TC network [2+ current IT [2A-adrenergic receptor subtype [2-adrenergic receptors are ascribed to this specific subtype [2B-adrenergic receptor subtype [2A-adrenergic receptors in DEX-mediated sedation.The non-inactivating, inwardly rectifying current I network  and furt network . The intrrent IT , promoterrent IT , and witrrent IT . EEG recrrent IT ,4. Interrrent IT . Both EErrent IT . This isrrent IT . As DEX rrent IT , TC inte subtype ,14,73. M subtype . Interes subtype . Therefoh might be regulated by circulating estradiol in a cyclical manner [Naturally, the findings of this study should be interpreted in light of its limitations. Firstly, all recordings in this study were performed at room temperature. Albeit not uncommon ,32,47,48l manner , though l manner ,67. HoweUltimately, our data demonstrate that DEX, in a clinically relevant concentration range, does not significantly inhibit the native HCN channel function of TC neurons in the thalamic VB complex ex vivo, and parameters of neuronal excitability remain largely unaffected by its application. In comparison with anesthetic agents such as propofol, xenon, halothane, and sevoflurane, which substantially inhibit native HCN channel function in TC relay neurons in vitro ,27,29,302PO4, 25.0 D-glucose, 25.0 NaHCO3, 6.0 MgCl2, and 0.5 CaCl2. During the cutting procedure, aCSF was continuously aerated with carbogen  to ensure oxygenation and a stable pH of approximately 7.4. Slices containing the region of interest (3–4 per brain) were then transferred into a storage chamber containing a carbogen-saturated standard aCSF  and incubated for a minimum of 30 min at 34 °C in a warm water bath, followed by an additional 30 min of recovery time at room temperature.Animal handling was reviewed and approved by the competent veterinary office . We used female C57Bl/6N mice (P28–P35), which were put in deep isoflurane anesthesia (as indicated by loss of righting reflex) before surgical brain extraction. Brain slices with a 350 µm-thickness were prepared according to a well-established protocol , which a2, 10.0 HEPES, 0.5 EGTA, 2.0 K2-ATP, 0.3 Na2-GTP, and pH: 7.25. As an electrophysiological defining feature, TC relay neurons exhibit an Ih-dependent inward rectification of the membrane potential following injection of hyperpolarizing currents, termed voltage sag [Individual slices were transferred into the recording chamber of the patch-clamp setup and mechanically fixated with a custom-made grid. During experiments, slices were continuously perfused with carbogen-saturated standard aCSF at a flow rate of 5–8 mL/min. Upon visual allocation of the thalamic VB , individual TC relay neurons were identified with infrared videomicroscopy . We established whole-cell recordings of single cells with fire-polished, borosilicate glass pipettes . Pipettes were fabricated with open tip resistances between 4 and 6 MΩ, using a Flaming/Brown micropipette puller  and filled with an intracellular solution with the following composition (in mM): 130.0 K-D-gluconate, 5.0 NaCl, 2.0 MgCltage sag , which wh currents were recorded in the voltage-clamp mode, according to a protocol previously described [h currents of TC neurons were gradually activated by applying hyperpolarizing voltage steps . As the stability of whole-cell recordings tends to deteriorate at highly hyperpolarized membrane potentials and factoring in the concurrent acceleration of Ih activation kinetics through hyperpolarization, we opted for incrementally reducing the pulse length of each voltage step by 500 ms, resulting in a 2.0 s pulse duration at −133 mV [h appears to reliably reach its maximum at membrane potentials beyond −110 mV [h current amplitude was derived from the −133 mV voltage step and used for normalization. Each voltage step also included a tail step of −103 mV before returning to the baseline of −43 mV. Respective tail current amplitudes (Itail) were normalized to the tail current acquired from the −133 mV voltage step and were used to assess the voltage dependency of steady-state activation. For this purpose, the equation (I–Imin)/(Imax–Imin) was applied, with Imax representing the tail current amplitude derived from the voltage step of −133 mV to −103 mV and Imin derived from the voltage step from −43 mV to −103 mV. These data fit well with a Boltzmann distribution, which is usually applied to estimate steady-state activation. The voltage-dependent time course of Ih activation was approximated by fitting a biexponential function to the currents evoked during the hyperpolarizing step of −133 mV, yielding fast (τfast) and slow (τslow) time constants of Ih activation, as previously described for TC relay neurons [h currents evoked at strongly hyperpolarized membrane potentials, while a monoexponential fit approach yields more robust results at more depolarized membrane potentials [All patch-clamp recordings were conducted in the whole-cell configuration with a discontinuous single-electrode voltage-clamp (dSEVC) amplifier . Though series resistance is effectively compensated by applying the dSEVC technique with high switching frequencies (60–80 kHz and a 25% duty cycle), it was continuously monitored. Iescribed ,30. Star −133 mV ,41. Ih a −110 mV . Therefotentials . As we dFurther current-clamp recordings were recorded from the same TC neurons. After injection of a hyperpolarizing current of −350 pA for a duration of 500 ms at the resting membrane potential, TC neurons display an inwardly directed rectification of the membrane potential, termed voltage sag. We defined the voltage sag as the difference between the maximum voltage deflection and the steady-state potential. Subsequently, we measured the time between the start of the membrane repolarization at the end of the hyperpolarizing current injection and the peak of the first action potential during rebound burst-firing (rebound burst delay). This current-clamp protocol was repeated eight times for each recording and later analyzed as a graphically averaged composite trace with Igor 5 . The impact of DEX on active and passive electrical membrane properties, comprising resting membrane potential, membrane input resistance, the current–voltage relationship, the threshold value of action potential firing, and the tonic action potential frequency were derived from membrane voltage changes induced by successive increases of current injections  at the resting membrane potential. For current-clamp recordings, we only adjusted the resting membrane to control (pre-drug application) levels when significant changes in the resting membrane potential were observed.2+), which was added to the aCSF circulation. Briefly summarized, Ba2+ inhibits the potentially interfering effects on inwardly rectifying potassium channels (Kir) and members of the two-pore domain acid-sensitive potassium channels (K2P), as well as facilitates improved analysis by significantly enhancing voltage sag amplitude [The liquid junction potential was corrected online, as estimated based on the ionic compositions of intra- and extra-cellular solutions and with help of the Liquid Junction Potential Calculator tool . As previously elaborated on  with a sampling frequency of 9 kHz, and stored on a hard drive . For data acquisition, we used Pulse version 8.5 . Data from individual cells were only included in the analysis if they did not meet one of the following exclusion criteria: (1) no GΩ-seal resistance formation upon establishment of cell-attached configuration, (2) an initial resting membrane potential below −50 mV, (3) an unstable series resistance or holding current (>20% change), and (4) no stable baseline conditions after 15 min, as assessed by the experimenter. Analysis of electrophysiological recordings was performed offline with Igor 5 . Action potentials were evaluated using Mini Analysis .p-values were adjusted for multiplicity and differences were considered statistically significant if p < 0.05, as graphically indicated by asterisks.For statistical evaluation of the data obtained and the creation of graphs, we used GraphPad Prism 7.03 . Sample sizes for different concentration groups were chosen based on previous experience. As formally testing for normal distribution inherently poses a challenge in case of limited sample sizes , we opte


This study investigates the inhibitory effect of ATR on A549 lung cancer cells. Cell viability was detected by the Cell Counting Kit-8 assay, and results showed that ATR could significantly inhibit the proliferation of A549 cells. Apoptosis was detected by Annexin V-FITC/PI staining, and apoptosis rate and mitochondrial membrane potential were detected by flow cytometry. Results showed that the effect of ATR on the apoptosis of A549 cells was negatively correlated with the change in mitochondrial membrane potential. Western blot analysis showed that ATR regulated apoptosis induced by mitogen-activated protein kinase, signal transducer and activator of transcription 3, and nuclear factor kappa B signaling pathways. Analyses of reactive oxygen species (ROS), cell cycle, and cell migration showed that ATR induced intracellular ROS accumulation as an initiation signal to induce cell cycle arrest regulated by the AKT signaling pathway and cell migration inhibition regulated by the Wnt signaling pathway. Results showed that ATR can inhibit cell proliferation, induce cell apoptosis, induce cell cycle arrest, and inhibit the migration of A549 cells ( Lung cancer is a malignant tumor that originates from the bronchial mucosa, gland, or alveolar epithelium . Lung caInducing the apoptosis of cancer cells is an effective way to treat cancer, and many signaling pathways are involved in this . A largeThe breakdown of adhesion between cells promotes the migration and invasion of cancer cells . The actAtractylodes lancea, and its hypoglycemic, anti-inflammatory, and gastric empty-promoting effects were widely reported [Natural drugs are popular in all fields of research and development because of their high efficiency and low toxicity ,23,24. Areported ,26,27. Sreported . In addireported . ATR alsreported ,31; howeThis study investigated the mechanism by which ATR inhibits growth and migration, and induces the apoptosis of A549 lung cancer cells. The mechanism of action of ATR in A549 cells, and the relationship between ROS accumulations induced by ATR in A549 cells was discussed.50) values of the four lung cancer cells were 37.92 ± 1.59, 76.88 ± 2.21, 63.27 ± 1.48, and 61.05 ± 1.66 µM  [2− is decomposed by the superoxide dismutase of mitochondria into hydrogen peroxide, which eventually generates hydroxyl radical (•OH), and these are the main components of ROS [During electron transfer in the mitochondrial respiratory chain, most electrons are transferred to On (•O2−) . •O2− iss of ROS ,44,45. Rs of ROS . TherefoEach stage of the cell cycle is tightly regulated, and checkpoints exist to detect potential DNA damage and allow for repair before the cell divides . If the Cell migration is involved in embryonic development, wound healing, inflammatory response, and cancer metastasis . Some stIn conclusion, ATR induces the apoptosis, G2/M phase arrest, and migration inhibition of A549 cells through ROS accumulation . TherefoATR (purity > 98%)  or 5-FU  was dissolved in dimethyl sulfoxide (DMSO)  and stored at −20 °C. The final concentration of DMSO was less than 0.5%, which could rule out the cytotoxic effect of the solvent on cells. The chemical concentration was diluted with cell culture medium before use.2.Lung cancer cells  and normal human cells  were used in the study. Lung-cancer and IMR-90 cells were purchased from American Type Culture Collection . GES-1 and 293T cells were purchased from Sage Biotechnology Co. . THLE-2 cells were purchased from Otwo Biotech . All cells were grown in Dulbecco’s Modified Eagles Medium (DMEM) . The cell culture medium was supplemented with 10% fetal bovine serum (FBS)  and 1% penicillin/streptomycin . Cells were grown in an incubator at 37 °C and 5% CO4 cells/well) were cultured in 96-well plates for 24 h. Cells were treated with ATR and 5-FU at 20, 40, 60, 80, and 100 µM. A total of 10 μL CCK-8 was added to each well and incubated for 3 h in the dark. Absorbance was measured at 450 nm using a multifunctional microplate reader . IC50 values were calculated using GraphPad Prism software. In addition, all cells were treated with IC50 values of ATR and 5-FU for 6, 12, 18, 24, and 30 h to analyze the cell survival rate.Cell viability was detected using Cell Counting Kit-8 (CCK-8) . Briefly, all cells (1 × 105 cells/well) were cultured in 6-well plates for 24 h. Cells were treated with 38 µM  ATR and 38 µM 5-FU for 3, 6, 12, and 24 h. Collected cells were evenly mixed with 200 µL 1× binding buffer, 3 µL Annexin V-FITC, and 5 µL PI, and incubated for 15 min in the dark. Cell morphology was observed by fluorescence microscopy . In addition, ATR-treated A549 cells were mixed with 100 µL 1× binding buffer, followed by the addition of 4 µL Annexin V-FITC and 3 µL PI and incubation for 5 min away from light. The rate of cell apoptosis was detected by flow cytometry .The apoptosis of A549 cells was detected using the Annexin V-FITC/PI Apoptosis Kit . Briefly, A549 cells (1 × 105 cells/well) were cultured in 3.5 cm cell culture dishes for 24 h. Cells were treated with 38 µM ATR for 3, 6, 12, and 24 h. Collected cells were stained with 1 mL JC-1 staining solution for 30 min, followed by the addition of 1 mL 1× JC-1 binding buffer and 300 µL phosphate-buffered saline (PBS). MMP was analyzed using flow cytometry.The MMP of A549 cells was detected using the JC-1 MMP Detection Kit . Briefly, A549 cells (1 × 105 cells/well) were cultured in 6 cm cell culture dishes for 24 h. Cells were treated with 38 µM ATR for 3, 6, 12, and 24 h. Collected cells were centrifuged at 500× g for 3 min, followed by the addition of 200 μL cytoplasmic extraction reagent and incubation on ice for 10 min. The cell solution was centrifuged at 15,000× g for 10 min, and the supernatant was cytoplasmic protein. Then, 80 μL nuclear protein extraction reagent was added to the cell precipitate, followed by centrifugation at 14,000× g for 10 min; the supernatant was nuclear protein.Nuclear protein was extracted from A549 cells using nuclear protein extraction kits . Briefly, A549 cells (6 × 105 cells/well) were cultured in 6 cm cell culture dishes for 24 h. Cells were treated with 38 µM ATR for 3, 6, 12, and 24 h. Proteins were separated on 12% SDS-PAGE electrophoresis gels and electrotransferred to nitrocellulose membranes. After adding 5% skim milk to avoid nonspecific binding, the membrane was incubated with primary antibodies  at 4 °C overnight. The primary antibodies used were Bad (25 kDa), Bcl-2 (26 kDa), cyto-c (15 kDa), cle-caspase-3 (17 kDa), cle-PARP (89 kDa), α-tubulin (55 kDa), p-ERK (42 kDa), ERK (42 kDa), p-JNK (46 kDa), JNK (46 kDa), p-p38 (38 kDa), p38 (38 kDa), p-STAT3 (91/86 kDa), STAT3 (91/86 kDa), NF-κB-p65 (65 kDa), IκBα (35–41 kDa), p-IκBα (41 kDa), Lamin B1 (67 kDa), p-AKT (56 kDa), AKT (56 kDa), CDK1/2 (33 kDa), Cyclin B (60 kDa), p21 (21 kDa), p27 (27 kDa), Wnt-3a (65 kDa), p-GSK-3β (47 kDa), GSK-3β (47 kDa), E-cadherin (120 kDa), N-cadherin (130 kDa) and β-catenin (92 kDa). Next, the membrane was incubated with secondary antibodies  for 2 h. Lastly, proteins were detected with enhanced chemiluminescence . Images were exposed with a multifunction imager  and analyzed with ImageJ software.Protein expression was detected by Western blot analysis. Briefly, A549 cells (6 × 105 cells/well) were cultured in 3.5 cm cell culture dishes for 24 h and treated with 38 µM ATR for 3, 6, 12, and 24 h. Collected cells were stained with 10 µL 2’,7’-dichlorofluorescein diacetate (DCFH-DA) and incubated for 30 min in the dark, followed by the addition of 300 µL PBS. ROS accumulation was detected by flow cytometry. In addition, A549 cells had been pretreated with NAC (Beyotime Institute Biotechnology) before being treated with ATR and tested again.The ROS levels of A549 cells were detected using ROS detection kits . Briefly, A549 cells (1 × 105 cells/well) were cultured in 3.5 cm cell culture dishes for 24 h. Cells were treated with 38 µM ATR for 3, 6, 12, and 24 h. The collected cells were fixed in 70% alcohol at 4 °C overnight. Then 100 µL RNase was added and incubated for 30 min at 37 °C, followed by the addition of 400 µL PI and staining at 4 °C for 30 min in the dark. The number of cells at different stages was detected by flow cytometry. Then the A549 cells were pretreated with NAC before being treated with ATR and tested again.The cell cycle of A549 cells was detected using DNA quantification kits . Briefly, A549 cells (1 × 105 cells/well) were cultured in 6-well plates for 24 h and then vertically scratched. After 2 h culture in serum-free DMEM, cells were treated with 38 µM ATR for 3, 6, 12, and 24 h. Cell migration was observed by fluorescence microscope. In addition, A549 cells were pretreated with NAC before being treated with ATR and tested again. Cell migration was detected by Transwell assay. Briefly, a serum-free suspension of A549 cells (1 × 105 cells/well) treated with 38 µM ATR was cultured in the upper chamber. DMEM containing 20% FBS was added to the lower chamber. A549 cells were stained with 1% crystal violet solution, and cell migration was observed at 3, 6, 12, and 24 h.Cell migration was detected by wound-healing assay. Briefly, A549 cells (1 × 1050, and SPSS 21.0 software was used for Tukey’s post hoc test. p < 0.05 was considered to be statistically significant.The experiment was repeated three times, and data are expressed as mean ± standard deviation. GraphPad Prism software was used to calculate IC


Nannochloropsis salina that heterologously expressed sysr1, which encodes ADH in the cyanobacterium Synechocystis sp. PCC 6906. To drive sysr1 expression, the heat shock protein 70 (HSP70) promoter isolated from N. salina was used, as its transcript levels were significantly increased under either cold or heat stress growth conditions. When subjected to cold stress, transgenic N. salina cells were more cold-tolerant than wild-type cells, showing less ROS production but increased activity of antioxidant enzymes such as superoxide dismutase, ascorbate peroxidase, and catalase. Thus, we suggest that reinforcement of alcohol metabolism could be a target for genetic manipulation to endow algae with cold temperature stress tolerance.Temperature is an important regulator of growth in algae and other photosynthetic organisms. Temperatures above or below the optimal growth temperature could cause oxidative stress to algae through accumulation of oxidizing compounds such as reactive oxygen species (ROS). Thus, algal temperature stress tolerance could be attained by enhancing oxidative stress resistance. In plants, alcohol dehydrogenase (ADH) has been implicated in cold stress tolerance, eliciting a signal for the synthesis of antioxidant enzymes that counteract oxidative damage associated with several abiotic stresses. Little is known whether temperature stress could be alleviated by ADH in algae. Here, we generated transgenic lines of the unicellular oleaginous alga  Temperature is a key regulator of the growth and development of living organisms such as algae and plants. Each organism has its own optimal temperature range; temperatures outside of this range are detrimental to growth and development . Accumul6f complex, resulting in photoinhibition of photosynthetic apparatus, including photosystem II and I , members of the dehydrogenase enzyme superfamily, are widely distributed across all living organisms . These ebidopsis , rice  was cultured in modified f/2N medium , FeCl3·6H2O (3.15 g/L), CoCl2·6H2O (10 mg/L), ZnSO4·7H2O (22 mg/L), MnCl2·4H2O (180 mg/L), CuSO4·5H2O (9.8 mg/L), and Na2MoO4·2H2O (6.3 mg/L)], and 2.5 mL/L vitamin stock , as well as 10 mM Tris-HCl to maintain pH 7.6. The cells were maintained in ambient air at 25°C under a light intensity of 120 μmol/m2/s.N medium  containiN. salina heterologously expressing an ADH were generated using a cyanobacterial sysr1 that encodes ADH from Synechocystis sp. PCC 6906, as sysr1 has been successfully used to generate oxidative-stress-resistant Arabidopsis , washed three times with fresh 375 mM sorbitol, and then resuspended in 375 mM sorbitol to a concentration of 5 × 109 cells/mL. One hundred microliters of concentrated cells  and 5 μg of XbaI-linearized pNsSysr1 vector were mixed in 2-mm cuvettes  prior to transformation. Then, electroporation was performed with the prepared cells and DNA mixtures using an ECM 850 square wave electroporation system . The following conditions: 50 pulses, 100-μs pulses, 500-ms intervals, and a high-voltage field strength of 12,000 V cm−1. After electroporation, the cells were resuspended in 10 mL f/2N media and incubated for 1 day at 25°C in the dark. Cells were centrifuged at 1,000 x g for 5 min and then plated onto f/2N agar medium containing Zeocin (2.5 μg/mL).Transgenic lines of bidopsis . Gene syeviously . Cells g7 cells/mL) in liquid or agar solid media at either 4°C for 7 days or 37°C for 3 days (for liquid medium) or 0.5 days (for solid medium) under white light (120 μmol/m2/s), and supplied with high (2%) CO2. Then, temperature-stressed cultures were transferred to a growth chamber at 25°C with high CO2. For liquid cultures, absorbance (750 nm) of cells after 3 days of incubation was measured using a spectrophotometer . Specific growth rates (µ) were calculated as follows: µ = ln(x2) - In(x1)/t2-t1, where µ is the specific growth rate and x1 and x2 represent the number of cells at time 1(t1) and time 2(t2), respectively. For the spot growth test, cells (7 × 104 –7 × 106) were inoculated onto the f/2N agar plates were transferred to temperature treatment conditions. Cell images were taken 14 days after incubation at 25°C. Light intensity and CO2 concentration during and after temperature treatments were kept the same.Cold- and high-temperature stress was imposed by incubating cells  , according to the manufacturer’s instructions. The qRT-PCR was performed using the CFX96 qPCR system (Bio-Rad) and TOPreal™ qPCR 2X PreMIX . The ΔCt = Target Ct mean – actin gene Ct,ΔΔCt = Sample ΔCt mean - Reference ΔCt mean,(-ΔΔCt) = fold difference.2^8 cells) after temperature stress treatments. ADH activity was determined colorimetrically  by quantifying the amount of NADH produced using an Alcohol Dehydrogenase Activity Colorimetric Assay Kit (Sigma-Aldrich). ALDH activity was determined colorimetrically  by a coupled enzyme assay in which acetaldehyde was oxidized by ALDH generating NADH using an Aldehyde Dehydrogenase Activity Colorimetric Assay Kit (Sigma-Aldrich). An aliquot of cell extract (50 µL) was added to the assay medium before the assay and incubated for 1 h at 37°C or 25°C.The activities of ADH and ALDH were measured in cells from wild-type and transgenic lines .Multiple alignments with other known ADH proteins were performed using Clustal Omega. Phylogenetic analyses of putative sysr1-expressing transgenic cells using a standard protocol . A 0.3-kb PCR fragment corresponding to the C-terminal region of sysr1 was used as a probe. Hybridization with 32P-labeled probes was performed in accordance with manufacturer’s instructions. The hybridization signals were detected using a Bio-Imaging Analyzer BAS-1800II . For Northern blotting, total RNAs were isolated using Nucleozol , according to the manufacturer’s instructions. Total RNA (15 μg) was separated on a 1.2% agarose gel and then blotted onto a nylon membrane. The 0.3-kb sysr1 fragment was used as a probe. The primer sequences used for the PCR probe in Southern and Northern blots are listed in the Genomic DNAs were extracted from wild-type and protocol . An aliq7 cells/mL in 2 mL of f/2N medium supplemented with 5 µM 2′,7′-dichlorofluorescein-diacetate (DCF-DA). Cells were then incubated in the dark at 25°C for 1 h, with gentle shaking. The fluorescence of the DCF-DA-treated cells was measured with a spectrofluorometer  at room temperature, with an excitation wavelength of 485 nm and an emission band between 500 and 600 nm. To determine relative ROS production, fluorescence intensity at 520 nm (F520) was used.Accumulation of ROS was assessed using a modified version of the method described by 9 cells) suspended in 1.5 mL of cold extraction buffer containing 50 mM Tris–HCl (pH 7.8), 1 mM EDTA, 1 mM MgCl2, and 1% (w/w) polyvinylpyrrolidone were disrupted by sonication on ice . For the APX assay, this buffer also contained 1 mM ascorbate. After sonication, crude extract was centrifuged at 14,000 x g at 4°C for 10 min, and the supernatant was collected. Protein concentration was calculated using the Bradford method, using bovine serum albumin as a standard  . The rea2O2, and 10 μL of enzyme extract. The decrease in absorbance at 240 nm was measured for up to 2 min, and CAT activity was calculated using the extinction coefficient of 0.036 mM−1 cm−1 , 10 mM HM−1 cm−1 .2O2, and 10 μL of the enzyme extract. APX activity was calculated from the change in absorbance at 290 nm due to ascorbate oxidation, and determined using an extinction coefficient of 2.8 mM−1 cm−1  contained 50 mM sodium phosphate buffer (pH 7.0), 0.5 mM ascorbate, 0.1 mM HM−1 cm−1 .The gene sequences used in this study can be found in the NCBI database library under the following accession numbers: OP168497 (NsADH1), OP168498 (NsADH2), OP168499 (NsADH3), OP168500 (NsALDH1), OP168501 (NsALDH2), OP168502 (NsHSF), OP168503 (NsHSP70), OP168504 (NsHSP90), OP168505 (NsHSP100), OP168506 (NsAPX), OP168507 (NsSOD), OP168508 (NsCAT), and OP168509 (NsACT1).p < 0.05 was considered statistically significant. Different letters indicate a significant difference.Statistical analysis was conducted using one-way ANOVA. Data are expressed as the mean ± SE, and N. salina cells. To set up temperatures below or above optimal growth, N. salina cells were grown under room (25°C), low (4°C), and high (37°C) temperature growth conditions. Cells were cultured for 6 days under continuous white light illumination (120 μmol/m2/s) with 2% CO2. A high CO2 concentration and continuous illumination were maintained throughout temperature stress treatments to avoid disturbance of photosynthetic electron transport due to limited CO2. Thus, under such conditions, responses of N. salina to temperature changes are highly related to the effects of temperature on light-driven photosynthetic electron transport activities. Consistent with previous reports  cells  in l) cells . Thus, AN. salina cells with robust heterologous expression of ADH were more tolerant to temperature stress than wild-type N. salina cells, sysr1-expressing N. salina transgenic lines were generated. Rather than using the actual N. salina ADH gene as a homologous alternative, we searched for a heterologous ADH gene for transgenic construction  promoter of N. salina rather than a constitutive promoter such as actin, so that the gene would be induced mostly under temperature-stress conditions. Among HSP and heat shock transcription factor (HSF) promoters, the HSP70 promoter was chosen rather than other heat shock transcription factors (HSF) or HSP90 and HSP100, as in contrast to these, transcript levels of HSP70 increased by ~1.9-fold under both temperature-stress conditions  were measured in N. salina wild-type and transgenic lines. At normal temperatures, expression of the genes encoding these enzymes was slightly higher in the transgenic lines than in the wild-type, whereas under cold stress, levels were significantly greater in the transgenic lines than in the wild-type. These increases were 1.4–1.6-fold for APX, 1.6–2.0-fold for SOD, and 1.8–3.6-fold for CAT  and two ALDH (ALDH1 and ALDH2) genes in the genome, their transcript levels and enzyme activities were downregulated during temperature stress  can be found in the article/W-JJ designed the research. J-ML, SJ, and J-SI conducted the experiments. W-JJ and Y-IP analyzed the data. J-ML, Y-IP, and W-JJ wrote the manuscript. All authors contributed to the article and approved the submitted version.