The loss of cholinergic neurotransmission in Alzheimer's disease (AD) patients' brain is accompanied by a reduced concentration of Acetylcholine (ACh) within synaptic clefts. Thus, the use of acetylcholinesterase inhibitors (AChEIs) to block the cholinergic degradation of ACh is a promising approach for AD treatment. In the present study, a series of 2-chloro-3-hydrazinopyrazine derivatives (CHP1-5) were designed, synthesized, and biologically evaluated as potential multifunctional anti-AD agents.1H and 13C NMR, and elemental analyses. Further, the intended compounds were assessed in vitro for their AChE inhibitory and neuroprotective effects. Furthermore, DPPH, FRAP and ABTS assays were utilized to determine their antioxidant activity. The statistical analysis was performed using one-way ANOVA.In addition, the chemical structures and purity of the synthesized compounds were elucidated through using IR, 50 values of 3.76 and 4.2 µM compared to the donepezil (0.53 µM), respectively. The study examined the effect and molecular mechanism of CHP4 on the Ab1–42-induced cytotoxicity in differentiated PC12 cells. At concentrations of 0–100 μM, CHP4 was non-toxic in PC12. Additionally, Ab1–42 significantly stimulated tau hyperphosphorylation and induced differentiated PC12 cell death. Further, CHP4 resulted in diminishing the Ab1–42-induced toxicity in PC12 cell significantly. CHP4 at 30 μM concentration significantly increased the Ab1–42-induced HSP70 expression and decreased tau hyperphosphorylation.Based on the results, CHP4 and CHP5 exhibited strong AChE inhibitory effects with the ICAccording to the results of our studies CHP4 can be considered as safe and efficient AChEI and employed as a potential multifunctional anti-AD agent.The online version contains supplementary material available at 10.1186/s13065-022-00799-w. AD is considered as one of the most causes of dementia and neurodegenerative disease among senile individuals, which may occur in 60% of cases. About 50 million individuals have dementia worldwide, to which approximately 10 million new cases are added every year. The total number of dementia individuals is expected to reach 82 and 152 million in 2030 and 2050, respectively –6. FurthN-methylenebenzenamine and 4-(methyleneamino) benzoic acid derivatives comprising of N-methylenebenzenamine nucleus  [Oxidative stress is detected as one of the causing factors in aging, which contributes to the development of multiple neurodegenerative disease such as AD, and plays an important role in destroying neuronal cells. Therefore, this process is considered as one of the primary incidents in AD. Accordingly, the effective protection of neuronal cells against oxidative stress damage can potentially prevent AD . In AD, n-metal) . Pyrazinn-metal) . So far,n-metal) , 23. Pyrn-metal) . The preChemical materials, 2-chloro-3-hydrazinopyrazine; chromene; methoxy benzaldehyde; 3-hydroxybenzaldehyde; 2-hydroxybenzaldehyde; 4-chlorobenzaldehyde; glacial acetic acid and TLC-RP 18 (Silica gel 60) were purchased from Sigma-Aldrich.n-hexane–ethyl acetate solvent system was used as an eluent. Thin layer chromatography  was employed for determining the substrate purity and monitoring reaction. Further, an electrothermal melting point device was applied for specifying all melting points (mp). The visible spectra and FTIR ones in KBr pellets were respectively measured on a Pharmacia Biotech spectrophotometer and a Shimadzu 8400 FT-IR spectrophotometer. Furthermore, a FT-NMR (400 MHz) Brucker apparatus was used for recording 1H and 13C NMR spectra. Chemical shifts were expressed in δ ppm using TMS as an internal standard and coupling constants (J) were given in Hz.The 2-chloro-3-hydrazinopyrazine and formyl derivatives utilized to synthesize Schiff-bases and other chemicals were obtained from Sigma-Aldrich and Merck. Additionally, double-distilled water was consumed throughout the experiment and 2O. Finally, the structure of the obtained Schiff-bases was confirmed by using FT-IR and 1H and 13C NMR spectroscopy.The pyrazine-based Schiff-bases were prepared through the condensation reaction of 2-chloro-3-hydrazinopyrazine (0.5 mmol) with various formyl derivatives (0.5 mmol) in ethanol (15 mL) in high yield. As a typical procedure, 0.5 mmol 2-chloro-3-hydrazinopyrazine was added to a mixture of a formyl derivative (0.5 mmol), absolute ethanol (15 mL), and glacial acetic acid  in a 50-mL round-bottom flask and heated to reflux with stirring for 7 h. The reaction progress was monitored through TLC using n-hexane: ethyl acetate (6:4) as solvent. After completing the reaction, the reaction mixture was cooled to room temperature, and the precipitate was collected by using vacuum filtration and washed with deionized water several times for affording the pyrazine-based Schiff-bases. Then, the products were isolated through recrystallization from DMF/Hmax (DMF): 365 nm. FT-IR : 610 (C–Cl), 1170 (C–N), 1242 (C–O), 1511 (C=C), 1647 (C=N), 3248 (N–H). 1H NMR ): 3.86 , 6.94 ,7.73 ,7.81 , 8.07 , 8.21, 8.58 . 13C NMR ): 55.39, 109.94, 114.20, 126.30, 129.04, 133.31, 141.52, 146.01, 147.52, 161.33.Yellow solid, mp: 196–198 °C, yield: 87%, molecular weight (Mw): 262.69 g/mol, λmax (DMF): 365 nm.Yellow solid, mp: 265–268 °C, yield: 85%, Mw: 314.76 g/mol, λ−1): 719 (C–Cl), 1170 (C–N), 1207 (C–O), 1518 (C=C), 1645 (C=N), 1665 (C=O), 3256 (N–H). 1H NMR ): 2.51 , 7.46 ,7.56 , 7.88 , 8.09 , 8.23 , 8.41 , 8.74 , 8.77 . 13C NMR ): 21.04, 118.30, 125.36, 125.4, 133.8, 134.1, 134.3, 135.50, 135.54, 136.05, 136.11, 138.51, 141.37, 153.98.FT-IR : 365 nm.Yellow solid, mp: 210–215 °C, yield: 89%, Mw: 248.67 g/mol, λ−1): 689 (C–Cl), 1170 (C–N), (C–O), 1666 (C=N), 3291 (O–H), 3420 (N–H). 1H NMR ): 6.80 , 7.08 , 7.24 , 7.85 , 8.23 , 8.43 , 9.59 , 10.77 . 13C NMR ): 112.76, 117.35, 118.87, 130.32, 132.91, 133.79, 136.52, 141.97, 146.31, 148.37, 158.11.FT-IR : 386 nm.Brown solid, mp: 140–144 °C, Mw: 248.67 g/mol, λ−1): 747 (C–Cl), 1053 (C–N), 1168 (C–O), 1666 (C=N), 3287 (O–H), 3420 (N–H). 1H NMR ): 6.93 , 7.29 , 7.46 , 7.89 , 8.27 , 8.70 , 11.2 , 11.55 . 13C NMR ): 116.89, 119.32, 119.74, 130.09, 131.20, 132.98, 134.20, 142.08, 147.36, 147.86, 157.69.FT-IR : 390 nm.Brown solid, mp: 201–204 °C, yield: 92%, Mw: 267.11 g/mol, λ−1): 707 (C–Cl), 1168 (C–O), 1487 (C=C), 1647 (C=N), 3207 (N–H). 1H NMR ): 7.52 , 7.73 , 7.87 , 8.26 , 8.50 , 10.92 . 13C NMR ): 128.73, 129.37, 133.01, 134.10, 134.23, 134.29, 142.01, 144.70, 148.29.FT-IR  from Electrophorus electricus was utilized in the study . Further3+-TPTZ) to ferrous in the presence of prepared compounds. Finally, a standard curve was drawn by considering the different concentrations (10–1000 µM) of ferrous sulfate.The radical scavenging activity of prepared compounds was assessed through using ABTS decolorization assay and method reported by Re et al.  with fewRattus norvegicus pheochromocytoma PC12 cells  were cultured in the Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Gibco) at 37 °C with 5% CO2. In order to examine the role of CHP4, PC12 cells were treated with the different concentrations (10–100 µM) of CHP4 for 24 and 48 h. In addition, Ab1-42 peptides were dissolved in distilled water and primarily incubated at 37 °C for 7 d before using for constructing pre-aggregation. Further, PC12 cells were incubated with 100 µM aggregated Ab1-42 for 24 h to make a cellular AD model in vitro. Furthermore, 50 µL of DMSO and 10 µL of 0.5 mg/mL MTT stock solution were added to each well containing about 1 mL of medium and the mixture was incubated for 4 h. Then, the plates were agitated on a plate shaker for 30 min and optical density was read at 570 nm by using an ELISA reader (Biotech). A slightly-modified MTT method of Jamalzadeh et al. [h et al.  was emplg for 10 min, the supernatants were transferred to new tubes. Protein concentration was detected through using the Bradford assay [To this end, the cells were seeded in the 6-well plate and treated with 20 and 30 µM CHP4 for 24 h. Additionally, they were washed with cold PBS, followed by lysing with lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, protease and phosphatase inhibitor cocktails) for 30 min on ice. After centrifuging lysates at 4 °C in 12,000rd assay . FurtherGeometry optimization of all five compounds was performed at DFT level theory with B3LYP hybrid exchange–correlation energy functional method and 6-31G++  basis by Gaussian 09 software . From thhttp://www.chemaxon.com). All docking calculations were performed by considering the protein and ligand as inflexible and flexible, respectively. Furthermore, donepezil (DPZ) was docked into 6O4W as a positive control ligand to validate the docking protocol. Finally, the Vina output file was entered into ADT for analyzing the docking results, and the hydrophobic interactions of AChE-ligand complexes, as well as bond lengths were examined.AutoDock Vina software program was applied for docking CHP4 into hAChE protein . In addiFor the purpose of identification of conserved sequences of AChE from an evolutionary aspect, ConSurf software was used with HMMER homolog search algorithm, MAFFT-L-INS-i method for alignments and Bayesian method for calculations .Each concentration was assayed in triplicates (n = 3) and repeated in three independent experiments. The values were expressed as mean ± SD and one-way ANOVA was utilized to determine the significant differences from controls statistically.13C and 1H NMR spectroscopic data . The preliminary results in Fig. 50 = 3.76 µM). In fact, the activity of the synthesized imine derivatives depends on the nature of the substituents attached to formyl group.All of obtained imine derivatives (CHP1-5) were evaluated for their inhibitory activities toward AChE through using an in vitro assay based on the reported protocol in comparison with donepezil as a standard drug  of compounds and their inhibition was assessed. The compounds were tested to determine by decolorizing the ABTS radical and assessed as a quenching of the absorbance at 734 nm. Based on the results, CHP1 exhibited high scavenging activity (48%)  complex to the TPTZ-Fe (II), the results of which are displayed in Fig. 2+ exhibited significant antioxidant properties in all synthesized compounds. Finally, the highest and least antioxidant effect was respectively obtained in CHP4 and CHP5.The ABTSration 1–0 μM of cration 1–0 μM of cration 1–0 μM of cration 1–0 μM of cIn order to examine the neuroprotective effects of CHP4 against the Ab1-42-induced toxicity, PC12 cells were treated with CHP4 in the presence or absence of 100 µM Ab1-42 for 24 h. Then, cell viability was determined through using MTT assay. As depicted in Fig. In the pathological conditions, tau can be more phosphorylated than the normal phosphorylation, which is AD hallmarks . Based o6 of other compounds. Additionally, while CHP1, CHP2 and CHP4 have their least electron-rich regions at their N–H of hydrazine group, for CHP3 and CHP4 it is related to the phenolic hydrogen.First, the optimized structural properties of all compounds, which can be found in supplementary information, were obtained at DFT level of theory. The colored scheme of MEP visualization is presented in Fig. First, it was predicted that all compounds are capable of passing from BBB and their diagrams are presented in supplementary information. Lipinski’s rule of five was employed as the basis of investigating the drug likeness properties of compounds. According to this rule, an ideal drug candidate must not have more than 5 hydrogen bond donors, more than ten hydrogen bond acceptors, a molecular mass less than 500 Da and logP more than 5 . As has Following the end of docking process, the best conformation was selected and the scoring functions indicated that conformations were the best complement to the AChE. Additionally, AutoDock 4.2 software was applied for all of the docking calculations . The effectiveness of CHP4 may be related to the electron withdrawing property of the phenyl ring [N-phenyl ring improved AChE inhibitory effect slightly [Designing, synthesizing, and producing the molecules valuable as human therapeutic agents are considered one of the principal purpose of pharmaceutical and organic chemistry. Pyrazines possess various types of biological activity, which represents a range of their pharmacological activities such as antibacterial, antimycobacterial, antiviral, antifungal, anti-diabetic, anti-cancer, analgesic, hypnotic, diuretic , and antnyl ring . Computaslightly . Furtherslightly .50 = 0.026 mM) in the DPPH assay due to its higher capacity to donate electron compared to the others (CHP2 > CHP3 > CHP4 > CHP1 > CHP5). In the ABTS assay, CHP1 performed better than the others due to the presence of electron-withdrawing and electron-donating group, methoxy group, with 48% inhibition (CHP1 > CHP4 > CHP3 > CHP5 > CHP2). Furthermore, FRAP assay is based on the ability of antioxidants to reduce Fe3+ to Fe2+. The highest ability for Fe3+ reduction was observed in CHP4 (3.016 per 1 mM) (CHP4 > CHP2 > CHP5 > CHP3 > CHP1). The ortho position of a hydroxyl group on benzoic ring exhibited excellent antioxidant activity, while the meta position led to weak effect [In the AD patients, the use of antioxidant therapies for the disease is necessary to reduce pathogenic symptoms . The resk effect . Based ok effect , CHP4 waAD is a common neurodegenerative diseases, the hallmark pathologic characteristics of Aβ plaques, tau hyperphosphorylation, and neuronal cell death . HSP70 aIn addition, the effects of CHP4 on the expression of Tau-p and HSP70 were assessed using western blot analysis, which demonstrates that the exposure of CHP4 resulted in decreasing the Ab1-42-induced phosphorylation of tau and increased HSP70 expression. According to Lu et al., HSP700 plays cytoprotective roles in AD, blocks Aβ self-assembly, moderates caspase-dependent and caspase-independent apoptotic pathways and reduces it in neuron cells, it also directly prevents tau aggregation and enhance Aβ clearance by upregulates the expression of insulin degrading enzyme and transforming growth factor beta . Some stIn summary, five compounds (CHP1–CHP5) were designed, synthesized, and evaluated. In the following, all synthesized compounds were analyzed and identified by using IR, 1H and 13C NMR. Novel compounds have been assayed for assessing the inhibitory effects on AChE, which CHP4 was effective (IC50 = 3.76 µM). According to the results of DPPH, ABTS and FRAP assays, CHP4 was chosen for subsequent the study. We have shown that CHP4 has a protective effect on PC12 neuronal cells against the toxicity of Ab1-42 peptide. In supplemental studies of western blot analysis, CHP4 reduce the expression of Tau-p as an important cause to AD On the other hand, a decrease in HSP70 expression was also seen, which could be a sign of a diminish in the pathological symptoms of this disease.The multifunctional properties  highlight CHP4 as a promising candidate for further studies on the development of novel drugs against AD. However, more extensive research should be conducted to assess the exact mechanism of effectiveness, as well as dose and potential in medical applications.Additional file 1:Figures S1-S20. 2D Structure image and 1H-NMR, 13C-NMR, FT-IR spectra of compounds CHP1, CHP2, CHP3, CHP4 and CHP5. Figures S21-S23. Original photos of western bot of CHP4. Tables S1-S5. Optimized geometric properties of CHP1 to CHP5. Tables S6-S7. Vina docking results. Figures S24-S28. Diagram of the capability of the synthesized compounds in passing from BBB. Figures S29-S30. Protein and ligand interaction, ribbon model of the dimeric structure (S29), and binding pocket (S30) of AChE.


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the resulting coronavirus disease 2019 (COVID-19) have a substantial burden on health-care systems around the world. This is a randomized parallel controlled trial for assessment of the immunogenicity and safety of an inactivated SARS-CoV-2 vaccine, aiming to determine an appropriate vaccination interval of the vaccine for high-risk occupational population.In an ongoing randomized, parallel, controlled phase IV trial between January and May 2021 in Taiyuan City, Shanxi Province, China, we randomly assigned the airport ground staff and public security officers aged 18 to 59 years to receive two doses of inactivated SARS-CoV-2 vaccine at 14 days, 21 days, or 28 days. The serum neutralizing antibody to live SARS-CoV-2 was performed at baseline and 28 days after immunization. Long-term data are being collected. The primary immunogenicity endpoints were neutralization antibody seroconversion and geometric mean titer (GMT) at 28 days after the second dose. Analysis of variance (ANOVA), chi-square, and logistic regression analysis were used for data analysis.CI: 88.4–108.4) in the 0–14 group, which was significantly lower compared with 134.4 (95% CI: 123.1–145.7) in the 0–21 group (P < 0.001 vs 0–14 group) and 145.5 (95% CI: 131.3–159.6) in the 0–28 group (P < 0.001 vs 0–14 group), resulting in the seroconversion rates to neutralizing antibodies (GMT ≥ 16) of 100.0% for all three groups, respectively. The intention-to-treat (ITT) analysis yielded similar results. All reported adverse reactions were mild.A total of 809 participants underwent randomization and received two doses of injections: 270, 270, 269 in the 0–14, 0–21, and 0–28 vaccination group, respectively. By day 28 after the second injection, SARS-CoV-2 neutralizing antibody of GMT was 98.4 (95% Both a two-dose of inactivated SARS-CoV-2 vaccine at 0–21 days and 0–28 days regimens significantly improved SARS-CoV-2 neutralizing antibody level compared to the 0–14 days regimen in high-risk occupational population, with seroconversion rates of 100.0%.www.chictr.org.cn.Chinese Clinical Trial Registry, ChiCTR2100041705, ChiCTR2100041706. Registered 1 January 2021, The online version contains supplementary material available at 10.1186/s40249-021-00924-2. The ongoing pandemic of coronavirus disease 2019 (COVID-19) induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to an unprecedented global public health crisis. Globally, as of 26 October 2021, more than 243 million cases of SARS-CoV-2 infection and more than 4.9 million deaths have been reported , influenza vaccination history, smoking, drinking, and chronic diseases were collected via questionnaire investigation.After each dose was vaccinated, the participants were observed for any immediate reaction for 30 min, and local and systemic adverse reactions were collected. Participants were required to record the local adverse events and systemic adverse events on diary cards within 7 days of each injection. Any other unsolicited symptoms were also recorded during a 28-day follow-up period after each injection by spontaneous report from the participants combined with the regular visit. The solicited adverse reactions included local reactions  and systematic reactions .Oropharyngeal/nasal swabs were collected for detecting SARS-CoV-2 nucleic acid from all subjects by using reverse transcriptase-polymerase chain reaction (RT-PCR) test to determine whether subjects have occurred SARS-COV-2 infection before the first, second dose of vaccine vaccination, and 28 days after the whole course of vaccination, respectively. Blood samples were taken from participants for serology tests before the first injection and on day 28 after the second injection. The neutralizing antibody to live SARS-CoV-2 [strain 19nCoV-CDC-Tan-Strain 05 (QD01)] were quantified using a micro cytopathogenic effect assay at baseline and 28 days after immunization. A positive antibody response (seroconversion) was defined as post-injection titer of at least 1:16 if the baseline titer was below 1:4, or at least a fourfold increase in post-injection titer from baseline if the baseline titer was at least 1:4 . We defiThe primary immunogenic endpoints were the seroconversion rates [geometric mean titer (GMT) ≥ 16] and GMT of neutralizing antibody to live SARS-CoV-2 at day 28 after the last dose. Secondary immunogenic endpoints were the positive rates  28 days after the whole course of vaccination, respectively. The primary endpoint for safety was the occurrence of adverse reactions within 7 days after the first and second vaccinations. Adverse events within 28 days after the first and the second vaccinations across the three groups were analyzed as secondary safety endpoints. Figure P < 0.05.The study sample size of 360 participants provided 84.4% power to detect a difference of 5% (85% vs 80%) of responders in the 0–21 and 0–28 vaccination groups compared with the 0–14 group, respectively in airport ground staff and public security officers. Data were recorded using EpiData version 3.1 , and analyses were performed using SAS version 9.3 . Analysis of variance (ANOVA) was used to analyze continuous data, and the chi-square or Fisher’s exact test was used for categorical data. We assessed immunogenic endpoints by the intention-to-treat (ITT) analysis  and per-protocol (PP) analysis . Multinomial logistic regression analysis and unconditional logistic regression model were used to determine the influencing factors of SARS-CoV-2 neutralizing antibody immunization. The safety analysis was performed on data from all subjects who received vaccination after randomization. The level of statistical significance for all analyses was P > 0.05; Table Between January and May 2021, 810 participants were screened, and 809 were enrolled . Of the 809 participants who were enrolled, 405 participants were the public security officers and 404 participants were the airport ground staff; with 270, 270, and 269 participants in the group 0–14, 0–21, 0–28 vaccination cohort. All enrolled participants received the first injection and completed the two-dose vaccination schedule. A total of 256, 247 and 241 patients in the 0–14, 0–21, and 0–28 groups, respectively, completed the follow-up 28 days after the whole course of vaccination  was noted in the 0–14 group, which was significantly lower compared with 134.4 (95% CI: 123.1–145.7) in the 0–21 group (P < 0.001 vs 0–14 group) and 145.5 (95% CI: 131.3–159.6) in the 0–28 group (P < 0.001 vs 0–14 group). The ITT analysis showed similar results that the GMT were between 93.5 and 129.8 in the three groups.By day 28 after the second injection, the seroconversion rates of neutralizing antibody (GMT ≥ 16) were all 100.0% in the 0–14, 0–21, and 0–28 groups. SARS-CoV-2 neutralizing antibody with a GMT of 98.4 (95% P < 0.001) and 95.9%  in the 0–14, 0–21, and 0–28 groups, respectively. The positive rates of neutralizing antibody GMT ≥ 64, 128, and 256 were 60.9% (156/256), 31.6% (81/256) and 9.0% (23/256) in the 0–14 group, 84.6% (209/247), 55.9% (138/247) and 15.0% (37/247) in the 0–21 group, 80.9% (195/241), 51.5% (124/241) and 17.4% (42/241) in the 0–28 group, respectively , 96.4% .In the 0–14 group, the proportion of neutralizing antibody was higher at the GMT 32–63  and 64–127 , while the proportion of neutralizing antibody in the 0–21 and 0–28 groups was higher at the GMT of 64–127  and 128–255 , respectively , and it was 4.4-fold higher than that in 0–14 vaccination group at GMT ≥ 128 (95% CI: 2.8–7.1). Participants in the 0–28 group had 2.0-fold odds when GMT was 64–127 (95% CI 1.2–3.3) than that in the 0–14 group and had 3.3-fold odds of GMT ≥ 128 (95% CI: 2.1–5.2)  and the results showed that only the vaccination regimen was associated with the antibody response. After adjusting for age, gender, BMI, marital status, education level, influenza vaccination history, smoking, drinking, and chronic diseases, participants who received 0–21 vaccination regimen was 2.5 times higher than the 0–14 vaccination group when GMT was 64–127 (95% CI: 1.9–8.8) and 3.6 (95% CI: 1.7–7.6) times more likely to be positive (GMT ≥ 32) than those in 0–14 groups, respectively; and the participants in 0–21 and 0–28 groups showed higher positive rates than those in 0–14 group (GMT ≥ 64) . The similar results were found at GMT ≥ 128 or 256 , 4.8% (13/270), and 3.7% (10/269) in the 0–14, 0–21 and 0–28 vaccination cohort group.P > 0.05). None of the subjects reported serious adverse reactions or became SARS-CoV-2 infected during the follow-up period (Table Solicited adverse reactions were reported by 8 (3.0%) in the 0–14 vaccination cohort group, 11 (4.1%) in the 0–21 vaccination cohort group, and 7 (2.6%) in the 0–28 vaccination cohort group within 7 days after injection. No significant differences were found in the occurrence of solicited and unsolicited adverse reactions among the three groups. Pain, swelling, pruritus, diarrhea and fatigue within 7 days after vaccination were reported by 4 1.5%), 2 (0.7%), 1 (0.4%), 0 (0.0%), and 1 (0.4%) subject in the 0–14 group, 7 (2.6%), 0 (0.0%), 2 (0.7%), 1 (0.4%), and 1 (0.4%) subject in the 0–21 group, and 2 (0.7%), 2 (0.7%), 0 (0.0%), 1 (0.4%), and 2 (0.7%) subject in the 0–28 vaccination group, respectively. Rash, cough and headache within 28 days after vaccination were reported by 1 (0.4%), 1 (0.4%), and 1 (0.4%) subject in the 0–14 group, 1 (0.4%), 1 (0.4%), and 0 (0.0%) subject in the 0–21 group, and 1 (0.4%), 1 (0.4%), and 1 (0.4%) subject in the 0–28 group, respectively. The reported adverse reactions did not differ significantly among the three study groups  being 100% in the three groups. The current clinical trials have also assessed the immunogenicity of SARS-CoV-2 vaccines, and have seen comparable results. The existing inactivated virus vaccines have shown significant immune responses (79–100%) and neutralizing antibody titers 18.9–282.7) [ being 10.9–282.7 In our research, the SARS-CoV-2 neutralizing antibody titers of the 0–28, 0–21 groups were significantly greater than that of 0–14 group. Xia et al. , 13 in bThe incidence of adverse reactions in the 0–14, 0–21 and 0–28 groups were similarly low. Moreover, we did not find severe adverse reaction, with the most common symptom being injection-site pain, indicating no safety concerns. The overall incidence of adverse events after vaccination was 3.7–4.8% in our vaccine-treated groups, which is noticeably lower than that of other SARS-CoV-2 vaccine platform candidates such as viral-vectored vaccines, DNA or RNA vaccines –30. The As the COVID-19 pandemic continues to spread, multiple SARS-COV-2 variants have emerged. Of these, the Delta variant has currently become the dominant strain of SARS-CoV-2, causing public concern around the world . One stuIn addition, our study had several limitations. First, since the majority of public security officers are male, there may be insufficient representation of the population. And we only reported immune response data for the high-risk occupational population aged 18 to 59 years. Further studies are required to assess the immunogenicity of inactivated SARS-CoV-2 vaccine in various populations, including general population, older people, children and adolescents. Second, data on long-term immunogenicity is not yet available, and the ongoing trial will provide more information. Third, cellular immunity and immune memory were not measured in the current study which need to be further studied.In summary, a two-dose of inactivated SARS-CoV-2 vaccine at 0–21 days and 0–28 days regimens significantly improved SARS-CoV-2 neutralizing antibody level compared to the 0–14 days regimen in high-risk occupational population, with seroconversion rates of 100.0%, which to some extent provided a basis for optimizing the immunization strategy of inactivated vaccine against COVID-19 among high-risk occupational population. The results were interim and the long-term immunogenicity and actual protection needs further study.Additional file 1: Supplement 1. Demographic and Behavioral Characteristics of High-risk Occupational Population 28 Days after the Whole Course of Vaccination. Supplement 2. SARS-CoV-2 Neutralizing Antibody Immunization 28 Days after the Whole Course of Vaccination Stratified by Age and Gender. b. SARS-CoV-2 neutralizing antibody immunization by gender. Supplement 3. GMT of SARS-CoV-2 Neutralizing Antibody 28 Days after the Whole Course of Vaccination Stratified by Age and Gender. Supplement 4. Distribution of SARS-CoV-2 Neutralizing Antibody 28 Days after the Whole Course of Vaccination Stratified by Age and Gender


Q.Clear is a Bayesian penalized likelihood (BPL) reconstruction algorithm that presents improvements in signal-to-noise ratio (SNR) in clinical positron emission tomography (PET) scans. Brain studies in research require a reconstruction that provides a good spatial resolution and accentuates contrast features however, filtered back-projection (FBP) reconstruction is not available on GE SIGNA PET-Magnetic Resonance (PET-MR) and studies have been reconstructed with an ordered subset expectation maximization (OSEM) algorithm. This study aims to propose a strategy to approximate brain PET quantitative outcomes obtained from images reconstructed with Q.Clear versus traditional FBP and OSEM.β values and FBP obtained on the PET-CT were determined.Contrast recovery and background variability were investigated with the National Electrical Manufacturers Association (NEMA) Image Quality (IQ) phantom. Resolution, axial uniformity and SNR were investigated using the Hoffman phantom. Both phantoms were scanned on a Siemens Biograph 6 TruePoint PET-Computed Tomography (CT) and a General Electric SIGNA PET-MR, for FBP, OSEM and Q.Clear. Differences between the metrics obtained with Q.Clear with different β values presented the best results, whereas for SNR Q.Clear with higher β gave the best results. The uniformity results are greatly impacted by the β value, where β < 600 can yield worse uniformity results compared with the FBP reconstruction.For in plane and axial resolution, Q.Clear with low β values, Q.Clear can provide similar results to the ones obtained with traditional FBP reconstruction, suggesting it can be used for quantitative brain PET kinetic modelling studies.This study shows that Q.Clear improves contrast recovery and provides better resolution and SNR, in comparison to OSEM, on the PET-MR. When using low The online version contains supplementary material available at 10.1186/s40658-021-00386-3. Positron emission tomography (PET) is an imaging technique that allows for non-invasive quantitative measurement of biological processes in vivo. Image reconstruction methods can broadly be divided into analytical and iterative algorithms. Whereas analytical reconstruction algorithms  assume continuous data and introduce a discrete character to it a posteriori, iterative reconstruction algorithms  assume discretely sampled data. Although iterative reconstruction algorithms are routinely used in the clinical setting, where image quality and lesion contrast are of great importance, analytical reconstruction algorithms are still used in research for accurate PET data quantification via kinetic modelling .β BCPP-EF  solution to the phantom, ensuring that the hot spheres contained a concentration four times that of the background (22.4 kBq/mL versus 5.6 kBq/mL) [3  and NEMA NU 2–2007 full-width half maximum at 1 cm from centre of 4.1 mm transverse and 4.7 mm axial [3 and NEMA NU 2–2007 full-width half maximum at 1 cm from centre of 4.05 mm transverse and 6.08 mm axial [The National Electrical Manufacturers Association (NEMA) Image Quality (IQ) phantom was prepared by adding [ kBq/mL) . The twomm axial ) and oncmm axial ). In bot18F]BCPP-EF, or 34.4 MBq of [11C]SA4503, or 36.4 MBq of [11C]UCB-J in water and then filling the phantom, ensuring the removal of large air bubbles. The 18F phantom was scanned for 40 min in the PET-CT scanner, reconstructed with a matrix of 256 × 256 and for 40 min in the PET-MR scanner, reconstructed with a matrix of 384 × 384, in order to keep the voxel size as similar as possible across all PET datasets. The matrix size on z-direction for Hoffman scans acquired in the PET-MR is 89, for Hoffman scans acquired in the PET-CT is 109, for NEMA IQ acquired in the PET-MR is 89 and for NEMA IQ acquired in the PET-CT is 111. The voxel size for the Hoffman scans acquired in the PET-MR is 1 × 1 × 2.78 mm3, for the Hoffman scans acquired in the PET-CT is 1.02 × 1.02 × 2.03 mm3, for the NEMA IQ acquired in PET-MR is 4.69 × 4.69 × 2.78 mm3 and for the NEMA IQ acquired in the PET-CT is 5.35 × 5.35 × 5 mm3. Due to the short half-life of 11C, the Hoffman phantom was filled with [11C]SA4503 solution and scanned in the PET-MR and subsequently filled with [11C]UCB-J solution and scanned in the PET-CT. The duration of the acquisition and acquisition parameters were the same as for the 18F phantom and the data was acquired in listmode for both the 11C and 18F phantoms.The Hoffman phantom was prepared by mixing 29.6 MBq of [β value (up to 1000) and the size of the FBP and OSEM filter kernel for two different isotopes and brain phantoms in a variety of outcome measures , a wide range of filter from 5 to 15 mm was used in this study. Attenuation correction on the PET-CT was performed with a low-dose attenuation correction CT scan performed prior to the PET acquisition . Attenuation correction on the PET-MR was performed with a GE CT-based template of the respective phantoms. All images acquired in the PET-CT and in the PET-MR have been reconstructed with random and scatter correction. These protocols were designed based on centre benchmark during this single-centre project and based on previous literature as detailed above.Each NEMA and Hoffman phantom scans acquired on the PET-CT scanner was reconstructed 6 times and each NEMA and Hoffman phantoms acquired on the PET-MR scanner was reconstructed 13 times, as can be observed in Table The NEMA phantom scans were analysed using a customised Interactive Data Language (IDL ®) program according to NEMA standards , 22. CirThe percentage contrast for each hot sphere was calculated according to Eq. CH is the average of the counts found in the ROI for a hot sphere, CB is the average of the background counts in the background ROI for the same sphere, aH and aB the activity concentration in the hot sphere and in the background, respectively [where ectively .The Percentage contrast for each cold sphere was calculated according to Eq. CC represents the average of counts in the ROI for a cold sphere and CB represents the average of the 60 background ROI counts for the same sphere size [where ere size .For the background variability, the standard deviation of the background ROI counts for each sphere size was calculated according to Eq. k equals the 60 background ROI counts and the background variability was calculated according to Eq. where 3) and calculating the percentage standard deviation according to Eq. The Hoffman phantom data were analysed using the VivoQuant® software version 3.5 patch 2  . The resCP is the average counts in the VOI and σp the standard deviation.Where The signal-to-noise ratio (SNR) was determined by drawing a VOI in the right putamen and a VOI in the background “white matter” region of the Hoffman phantom (devoid of radioactivity) and it was calculated according to Eq. CP is the average counts in the VOI for the putamen, CW is the average counts in the VOI placed in a uniform area in the background and σW the standard deviation in the background [Where ckground .β values.Differences in contrast, background variability, resolution, uniformity and SNR were calculated relative to the FBP reconstruction with 5 mm FWHM Gaussian filter, the standard FBP reconstruction for the department. Bland-Altman plots were used to investigate the quantitative differences between the FBP with 5 mm FWHM Gaussian filter (obtained in the PET-CT) and the TOF-OSEM with 4 iterations, 8 subsets and 5 mm filter (obtained in the PET-MR) versus Q.Clear with different GraphPad Prism version 8.1.0 for Windows  was used for statistical analysis and graphical representation .β values) from the PET-MR provided consistently higher percentage contrast compared to OSEM reconstructions on the PET-CT and the PET-MR, as well as the FBP on the PET-CT. For all reconstruction methods, the percentage contrast was highest for large diameter spheres of the NEMA phantom and reduced with sphere size  to 1.89% (39 mm sphere). The same trend was observed for the standard deviation (0.58 to 0.53 %) and median (2.28 to 1.61%), while the coefficients of variation were relatively stable at 23.8%, 18.6%, 15.1%, 19.0%, 20.1% and 28.4% for the 10, 13, 17, 22, 30, and 39 mm sphere, respectively. The lowest quantitative difference was found for Q.Clear with β100 (0.32) when comparing with FBP with a 5-mm kernel and for Q.Clear with β1000 (0.11) when TOF-OSEM with 4 iterations, 8 subsets and 5 mm kernel   of 16.5 mm observed for the Hoffman phantom, was with 18F in the PET-MR, for OSEM 4 iterations, 16 subsets and a 15 mm filter  was for PET-MR OSEM 4 iterations, 16 subsets and 15 mm filter; while the smallest difference (0.0 mm) was for PET-MR OSEM 4 iterations, 16 subsets and 5 mm filter together with Q.Clear β value of 1000.Images of the Hoffman phantom filled with the z)  of 16.5 mm was observed for FBP with a 15-mm filter  of 6.5 mm was for Q.Clear reconstruction with β of 100. Relative to FBP with a 5-mm filter, the largest difference was for FBP with a 15-mm filter (− 7.5 mm), while the smallest was for the Q.Clear with β800 or 900 (0.0 mm).The highest FWHM   was for PET-MR OSEM 4 iterations, 16 subsets, 5 mm filter.The Q.Clear with filter 8.%  was for FBP with 10 mm filter.For SNR, the largest value (84.8) was for Q.Clear with 000 Fig. . The poo11C solutions and reconstructed with different methods are presented in Fig. x,y) was 16.5 mm for PET-MR OSEM reconstruction with 4 iterations, 16 subsets and 15 mm filter  was for PET-MR OSEM 4 iterations, 16 subsets and 15 mm filter, while the smallest difference (0.0 mm) was for PET-CT OSEM 4 iterations, 16 subsets and 5 mm filter together with PET-MR OSEM 4 iterations, 16 subsets and 5 mm filter and Q.Clear with β800 and 900.Images of the Hoffman phantom filled with the z) was 16.5 mm for FBP and 15 mm filter , while the smallest was for Q.Clear with β400 (0.0 mm).The highest FWHM   was for Q.Clear with β of 700.The Q.Clear reconstruction with 8%) Fig. . Relativβ of 1000  was for FBP with 10 mm filter.For SNR, the highest value (65.3) was for Q.Clear with 000 Fig. . The pooThis study investigated the performance of the Q.Clear reconstruction algorithm using a PET-MR against OSEM (PET-MR and PET-CT) and FBP (PET-CT) algorithms on general use and brain phantom data. Different isotopes were used to characterise noise, uniformity, SNR and quantitative bias outcomes, and the Hoffman brain phantom was also selected to simulate radioisotope distribution in the grey and white matter of the brain.18F or 11C. This data is in accordance with Conti et al.’s [+ emitters and scanned up until 200 million net true counts were obtained. In their study, the 18F and 11C images presented very similar radial profiles.Carbon-11 and Fluorine-18 tracers are used in clinical and research PET not only because of their short half-life but also due to the short-range of the positrons in tissue . Our stuet al.’s  findingsβ value increases, the contrast recovery and background variability decrease. Using the same phantom filled with 18F-FDG and a GE Discovery 690 PET/CT scanner, Teoh et al. also found that when Q.Clear β values increased, the contrast recovery and background variability decreased [Our NEMA phantom data demonstrate that as the Q.Clear ecreased . Furtherecreased .β > 200 [18F-filled phantom in the GE Signa PET-MR and reconstructed the acquired dataset with TOF-OSEM with 4 iterations and 28 subsets. The team obtained a background variability of 6.1% for the sphere with 10 mm decreasing with the increase in sphere diameter to 2.7% for the 37 mm sphere [18F-filled NEMA phantom on a GE Discovery IQ PET-CT scanner. They found that as β values increased, the background variability and the contrast recovery coefficients decreased [The background variability results are higher for Q.Clear than for OSEM when reconstructing data on the PET-MR. This is in contrast with Teoh et al.’s findings in the PET-CT scanner as in the study mentioned above the group reported OSEM background variability results higher or equal to the background variability results obtained with Q.Clear with β > 200 . This maβ > 200 . The FBPm sphere . Reynés-ecreased .x,y) and FWHM(z) results show that the Q.Clear reconstructions with different β values on the PET-MR are more closely related to the FBP reconstruction, with a 5-mm kernel, rather than the FBP reconstructions with the 10 mm and 15 mm kernel in the PET-CT. The FWHM results obtained for the Q.Clear reconstructions in the PET-MR are lower although still related to the results obtained for the FBP reconstruction with 5 mm filter in the PET-CT. The FWHM(z) results obtained for the Q.Clear reconstructions with β < 400 are considerably lower than the ones obtained for the FBP and OSEM reconstructions performed in the PET-CT. These metrics indicate an improvement in the in plane and axial resolution with this algorithm. This is consistent with the data obtained by Rogasch et al., who scanned a NEMA phantom during 30 min in a GE Discover MI PET-CT system and reconstructed the data with TOF-OSEM 4 iterations, 16 subsets and 2 mm filter, TOF-OSEM 2 iterations, 17 subsets and 2 mm filter, TOF-OSEM 2 iterarions, 8 subsets and 6.4 mm filter, Q.Clear β150, Q.Clear β300 and Q.Clear β450 [The FWHM and FWHM(z), whereas Q.Clear with higher β levels improves uniformity and SNR. The findings in our study which was conducted in a GE Signa PET-MR scanner are consistent with those obtained by Reynés-Llompart et al. on a GE Discovery IQ PET-CT scanner. The team conducted a clinical evaluation of torso and brain acquisition and found that, after subjective quality assessment, β values between 300 and 400 are recommended for reconstructing torso acquisitions and β values between 100 and 200 are recommended for brain acquisitions [Overall, Q.Clear with lower isitions .β value of 100 will provide the best results based on our novel data with the Hoffman phantom, albeit with lower SNR compared with β value of 1000 and equivalent values to FBP.Q.Clear improves contrast recovery on the PET-MR in comparison to OSEM. Moreover, Q.Clear also provides better in plane, axial resolution and signal-to-noise; however, its effect on image uniformity requires further investigations. For brain PET studies, in which spatial resolution is paramount, the Q.Clear reconstruction with Additional file 1: Supplementary file 1. Fig. 1 Bland-Altman plots assessing agreement between FBP with 5mm and Q.Clear with different β values, for the contrast recovery data obtained from the NEMA phantom. FBP 5mm and Q.Clear β100 (A); FBP 5mm and Q.Clear β200 (B); FBP 5mm and Q.Clear β300 (C); FBP 5mm and Q.Clear β400 (D); FBP 5mm and Q.Clear β500 (E). Fig. 2 Bland-Altman plots assessing agreement between FBP with 5mm and Q.Clear with different β values, for the contrast recovery data obtained from the NEMA phantom. FBP 5mm and Q.Clear β600 (F); FBP 5mm and Q.Clear β700 (G); FBP 5mm and Q.Clear β800 (H); FBP 5mm and Q.Clear β900 (I); FBP 5mm and Q.Clear β1000 (J).Additional file 2: Supplementary file 2. Fig. 1 Bland-Altman plots assessing agreement between TOF-OSEM 4iteration, 8subsets with 5mm (4i8s5mm) and Q.Clear with different β values, for the contrast recovery data obtained from the NEMA phantom. TOF-OSEM 4i8s5mm and Q.Clear β100 (A); TOF-OSEM 4i8s5mm and Q.Clear β200 (B); TOF-OSEM 4i8s5mm and Q.Clear β300 (C); TOF-OSEM 4i8s5mm and Q.Clear β400 (D); TOF-OSEM 4i8s5mm and Q.Clear β500 (E). Fig. 2 Bland-Altman plots assessing agreement between TOF-OSEM 4iteration, 8subsets with 5mm (4i8s5mm) and Q.Clear with different β values, for the contrast recovery data obtained from the NEMA phantom. TOF-OSEM 4i8s5mm and Q.Clear β600 (F); TOF-OSEM 4i8s5mm and Q.Clear β700 (G); TOF-OSEM 4i8s5mm and Q.Clear β800 (H); TOF-OSEM 4i8s5mm and Q.Clear β900 (I); TOF-OSEM 4i8s5mm and Q.Clear β1000 (J).Additional file 3: Supplementary file 3. Fig. 1 Bland-Altman plots assessing agreement between FBP with 5mm and Q.Clear with different β values, for the background variability data obtained from the NEMA phantom. FBP 5mm and Q.Clear β100 (A); FBP 5mm and Q.Clear β200 (B); FBP 5mm and Q.Clear β300 (C); FBP 5mm and Q.Clear β400 (D); FBP 5mm and Q.Clear β500 (E). Fig. 2 Bland-Altman plots assessing agreement between FBP with 5mm and Q.Clear with different β values, for the background variability data obtained from the NEMA phantom. FBP 5mm and Q.Clear β600 (F); FBP 5mm and Q.Clear β700 (G); FBP 5mm and Q.Clear β800 (H); FBP 5mm and Q.Clear β900 (I); FBP 5mm and Q.Clear β1000 (J).Additional file 4: Supplementary file 4. Fig. 1 Bland-Altman plots assessing agreement between TOF-OSEM 4iteration, 8subsets with 5mm (4i8s5mm) and Q.Clear with different β values, for the background variability data obtained from the NEMA phantom. TOF-OSEM 4i8s5mm and Q.Clear β100 (A); TOF-OSEM 4i8s5mm and Q.Clear β200 (B); TOF-OSEM 4i8s5mm and Q.Clear β300 (C); TOF-OSEM 4i8s5mm and Q.Clear β400 (D); TOF-OSEM 4i8s5mm and Q.Clear β500 (E). Fig. 2 Bland-Altman plots assessing agreement between TOF-OSEM 4iteration, 8subsets with 5mm (4i8s5mm)and Q.Clear with different β values, for the background variability data obtained from the NEMA phantom. TOF-OSEM 4i8s5mm and Q.Clear β600 (F); TOF-OSEM 4i8s5mm and Q.Clear β700 (G); TOF-OSEM 4i8s5mm and Q.Clear β800 (H); TOF-OSEM 4i8s5mm and Q.Clear β900 (I); TOF-OSEM 4i8s5mm and Q.Clear β1000 (J).


Hypericum (Hypericaceae) are recognized as an abundant source of natural products with interesting chemical structures and intriguing biological activities. In the course of our continuing study on constituents of Hypericum plants, aiming at searching natural product-based lead compounds for therapeutic agents, we have isolated more than 100 new characteristic metabolites classified as prenylated acylphloroglucinols, meroterpenes, ketides, dibenzo-1,4-dioxane derivatives, and xanthones including prenylated xanthones, phenylxanthones, and xanthonolignoids from 11 Hypericum plants and one Triadenum plant collected in Japan, China, and Uzbekistan or cultivated in Japan. This review summarizes their chemical structures and biological activities.Plants belonging to the genus  Hypericum plants of the family Hypericaceae, consisting of over 500 perennial herbs or shrubs subdivided into 30 sections, are mainly distributed in temperate area -xanthone (15), 1,3,5-trihydroxy-6,7-[2′-(1-hydroxy-1-methylethyl)-dihydrofurano]-xanthone (16), and 1,3,5-trihydroxy-6-O-prenylxanthone (17) were isolated from the aerial parts of H. ascyron  and B (19)  is a unique conjugate of 19 and a sesquiterpene, eremophil-9,11(13)-dien-8β,12-olide, possessing an unprecedented heptacyclic ring system. The structures of 18 and 19 were assigned by detailed spectroscopic analyses, including application of a modified Mosher’s method to a derivative of 19. An evaluation of biological activity of 18 and 19 is ongoing.xanthone , 1,3,5-txanthone , and 1,3Hypericum monogynum  (Biyouyanagi in Japanese), an evergreen shrub originated in China, is cultivated as an ornamental plant in Japan. Its stems and leaves have been used for the treatment of female disorders in Japan. In contrast, the roots of this plant have been used to treat various disorders, such as rheumatism, snakebite, and furuncle, in China. Chemical constituents of the roots, stems, and leaves of H. monogynum cultivated in Tokushima prefecture were separately and thoroughly investigated to isolate new characteristic metabolites. Chipericumins A–D (20–23) are spirocyclic PAPs isolated from the roots  and B (21) have a unique 5/6/6/5 tetracyclic ring system. Chinesins I and II , were also isolated from the roots of H. monogynum. Among others, biyoulactones A–C (24–26) are novel pentacyclic meroterpenes possessing bi- and tricyclic γ-lactone moieties connected through a C–C single bond , 22H. moa et al.  Fig. 4)H. monogy9 and 30 –26. Xie d 30 , of, ofH. mo) and B 3 had a noH. monogyum D + 15.4 }. Assignments of the absolute configuration for the major enantiomer of 39 as well as the ratio of enantiomers (88:12) were elucidated by analyzing their MTPA ester derivatives -decane core highly substituted by prenyl groups, whose absolute configuration was also deduced on the basis of ECD calculation. Hypatulin A (44) exhibited a moderate antimicrobial activity against B. subtilis octane-2,4,8-trione core with a characteristic C4 alkyl moiety  and B (48) are type B PAPs, whereas takaneone C (49) is the first example of type A PAP with a bicyclo[3.2.1]octane-2,4,8-trione core. Takaneols A (50) and B (51) are PAPs with a dihydrofuran moiety fused to the phloroglucinol moiety -octane-2,4,8-trione core  exhibited antimicrobial activites against Escherichia coli (MIC 4.0 μg/mL) and S. aureus (MIC 8.0 μg/mL) [81) Fig.  51]. We. WeHyperogirin A . Furtherogirin A , 54. YezHokkaido . Yezo’otins A–C 7–81  .Fig. 10THypericum erecturm is a perennial herb widely distributed in east Asia. This plant is called “Otogirisou” in Japanese and a representative species of Hypericum plants seen in Japan. The aerial parts of H. erectum have been used as a traditional remedy to heal wounds, burn wounds, bruises, swelling, and rheumatism. Interestingly, the aerial parts of H. erectum were also used for treating disorders of birds. We, however, had an interest in the root constituents of H. erectum, and investigated them to isolated PAPs named erecricins A–E (87–91) and adotogirin (92)  are PAPs possessing a chromane or a chromene skeleton. Adotogirin (92), a simple acylphloroglucinol with an O-geranyl moiety, displayed antimicrobial activities against MRSA {MIC range 0.5–4.0 μg/mL for seven strains (MIC50 1.0 μg/mL)}, methicillin-sensitive Staphylococcus aureus (MSSA) (MICs 1.0 μg/mL for five strains), and B. subtilis (MIC 2.0 μg/mL), while 87–91 did not show any antimicrobial activities [92) Fig.  56]. Er. ErHyperHypericum plants are cultivated as ornamental plants because of their beautiful yellow flowers that bloom in early summer. H. frondosum ‘Sunburst’ is a cultivar with larger flowers, and the investigation on the aerial parts of this plant cultivated at the botanical garden of Tokushima University gave four new ketides, frondhyperins A–D (93–96)  had novel chemical structures comprising short ketide and phenylketide moieties in common. The absolute configuration of 94 was assigned by ECD calculation, while those of 93 and 95 were revealed by their chemical correlation of 94. Frondhyperin D (96) was shown to be a racemate. It is noteworthy that PAPs, common constituents of Hypericum plants, were not found in this plant material in our study, although frondhyperin B (94) was isolated as a major constituent  [Some woody 96) Fig.  57]. Fr. FrHyperHypericum elodeoides and H. scabrum are medicinally used perennial herbs grown in central to west regions of China and in central Asia, respectively. H. elodeoides has been used for the treatment of diarrhea and snake bite in China. Chromatographic separations of the extract from the aerial parts of H. elodeoides collected in Yunnan province, China furnished two PAPs, hypelodins A (97) and B (98)  is a bicyclic PAP with three prenyl groups and one 4-methyl-1,3-pentadiene moiety, while hypelodin B (98) has a cage-like structure with a 6/6/5/7/6/5 hexacyclic ring system. Recently, Park et al. isolated hyperlodin B (98) from H. ascyron and reported its inhibitory activity against human neutrophil elastase [98) Fig.  58]. Hy. HyHyperelastase .Fig. 13TH. scabrum is one of the most popular medicinal herbs in Uzbekistan to treat numerous disorders, such as liver, gall bladder, intestinal, and heart diseases, rheumatism, and cystitis. Investigation on constituents of the aerial parts of H. scabrum collected at Chimgan, Uzbekistan showed this plant to be a rich source of polycyclic PAPs with a benzoyl group as their acyl moieties. Hyperibone K (99) is the first example of type B PAP possessing a “diamond-like” adamantane skeleton  was assigned based on the enantioselective total synthesis of an enantiomer of 99 by Porco, Jr. et al. [100) is a polycyclic PAP with bicyclo[3.3.1]nonane-2,4,9-trione core  was also achieved by Plietker et al. [99) and L (100) against human cancer cell lines (A549 and MCF-7) [99 were reported by Gu et al. [101)  Fig.  on interTriadenum is a sister genus of Hypericum consisting of six species. T. japonicum, a perennial herb bearing small pale pink flowers in contrast with yellow flowers of Hypericum plants, grows in marshy places in the eastern Asia and coastal area of eastern Russia. Our phytochemical investigation on the aerial parts of T. japonicum collected at Hokkaido resulted in the isolation of six new PAPs, (−)-nemorosonol (102) and trijapins A–E (103–107) [102 was assigned by NMR analysis and TDDFT ECD calculation. Interestingly, 102 was an enantiomer of (+)-nemorosonol previously isolated from Clusia nemorosa (Clusiaceae) [103–105) were assigned as analogues of (−)-nemorosonol (102) with an additional tetrahydrofuran ring, whereas trijapin D (100) was shown to be a PAP with an endperoxy moiety. (−)-Nemorosonol (102) exhibited antimicrobial activities against A. niger (IC50 16 μg/mL), T. mentagrophytes (IC50 8 μg/mL), C. albicans (IC50 32 μg/mL), E. coli (MIC 8 μg/mL), S. aureus (MIC 16 μg/mL), B. subtilis (MIC 16 μg/mL), and M. luteus (MIC 32 μg/mL), while trijapin D (106) showed an antimicrobial activity against C. albicans (IC50 8 μg/mL) [103–107) . The strsiaceae) . Trijapi8 μg/mL)   isolated from H. monogynum, hypatulins A (44) and B (45) isolated from H. patulum, and yezo’otogirins A–C (79–81) isolated from H. yezoense were meroterpenes structurally and biosynthetically related to PAPs, while plausible biosynthetic pathway of the PAPs was summarized in previous reviews [18) isolated from H. ascyron} or a spirolactone derivative {biyouyanagins A (29) and B (30) isolated from H. monogynum}. Simple meroterpenes {yojironins A (77) and B (78) isolated from H. yojiroanum and yezo’otogirins D (85) and E (86) isolated from H. yezoense} and ketides {frondhyperins A–D (93–96) isolated from a cultivar H. frondosum ‘Sunburst’} were also biogenetically interesting compounds. Thus, Hypericum plants are an attractive source of various characteristic metabolites, and therefore a systematic biological evaluation of our compounds isolated from Hypericum plants is in progress.This review summarized the chemical structures of 107 characteristic metabolites isolated from 11 nt years . Interesnes A–E 2–28 isola) and B 4 isolated


Xenopus laevis oocytes. Thus, membrane cholesterol regulates trabecular transduction of mechanical information, with TRPV4 channels mainly located outside the cholesterol-enriched membrane domains. Moreover, the biomechanical milieu itself shapes the lipid content of TM membranes. Diet, cholesterol metabolism, and mechanical stress might modulate the conventional outflow pathway and intraocular pressure in glaucoma and diabetes in part by modulating TM mechanosensing.Despite the association of cholesterol with debilitating pressure-related diseases such as glaucoma, heart disease, and diabetes, its role in mechanotransduction is not well understood. We investigated the relationship between mechanical strain, free membrane cholesterol, actin cytoskeleton, and the stretch-activated transient receptor potential vanilloid isoform 4 (TRPV4) channel in human trabecular meshwork (TM) cells. Physiological levels of cyclic stretch resulted in time-dependent decreases in membrane cholesterol/phosphatidylcholine ratio and upregulation of stress fibers. Depleting free membrane cholesterol with m-β-cyclodextrin (MβCD) augmented TRPV4 activation by the agonist GSK1016790A, swelling and strain, with the effects reversed by cholesterol supplementation. MβCD increased membrane expression of TRPV4, caveolin-1, and flotillin. TRPV4 did not colocalize or interact with caveolae or lipid rafts, apart from a truncated ∼75 kDa variant partially precipitated by a caveolin-1 antibody. MβCD induced currents in TRPV4-expressing   Conversion of sensory information into electrical and chemical signals in eukaryotic cells is modulated by unesterified cholesterol, a planar 27-carbon polycyclic molecule that constitutes ∼20% of the total mass of membrane lipids , 2. Its Vertebrates maintain cholesterol levels within a narrow range, with deficits or oversupply often harmful for health , 6. DefeWe recently identified the mechanosensitive transient receptor potential vanilloid 4 (TRPV4) channel as a principal regulator of mechanically induced signaling in mouse and human TM , 26, 27.2. The phenotype was periodically validated by profiling for markers, including Aqp1, Timp3, Myoc, MGP, Acta2 (α-smooth muscle actin [αSMA]), and dexamethasone-induced upregulation of myocilin expression. These data are shown in our previous characterizations of the cell line  as described previously , 27, 33 ell line , 26, 33.Xenopus laevis oocyte experiments were performed according to the guidelines of the Danish Veterinary and Food Administration (Ministry of Environment and Food) and approved by the animal facility at the Faculty of Health and Medical Sciences, University of Copenhagen. The experiments conform to the principles and regulations described  and cholesterol were obtained from Sigma or VWR. GSK101 (1 mM) stock aliquots were prepared in dimethyl sulfoxide and subsequently diluted into working saline concentrations . Chemical reagents for biochemical experiments—methanol, isopropanol, Methyl-β-cyclodextrin  was dissolved in TMCM and used at 10 mM, the concentration that removes 80–90% of free membrane cholesterol , 38. CelThe swelling studies were conducted as reported previously , 41. ExtLipids were extracted using Folch method . Total lCell lysis was performed in lysis buffer containing 2% octylglucoside, 150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 0.1% Triton X-100, and protease inhibitor cocktail (Roche). Lysates were cleared by centrifugation, and protein concentrations determined using a BCA assay (Thermo Fisher Scientific). Immunoprecipitation of Cav-1 was performed using Dynabeads™ Protein G Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, Cav-1 primary antibody (10 ng/μl) was conjugated to Dynabeads magnetic beads at room temperature. Then, an equal amount of protein from each sample was incubated with the beads-antibody conjugates for 15 min at room temperature, with gentle agitation. The beads were removed from solution by DynaMag™-2 magnet (Thermo Fisher Scientific), washed in wash buffer, and resuspended in lysis buffer. The original cell lysates, immunoprecipitates, and unbound fractions (flow-through) were boiled in Laemmli buffer, separated by reducing SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% BSA for 1 h, and probed with Cav-1  and TRPV4 primary antibodies (Alomone Lab). Primary antibodies were detected using Clean-Blot® IP detection reagent (Thermo Fisher Scientific) conjugated to HRP.2, 10 mM NaCl, 0.0005 mg/ml, 2 mM sodium vanadate, 20 mM sodium fluoride, 0.5 mM DTT, and 1 mM PMSF) on ice and centrifuged at 15,000 g for 30 min at 4°C to separate cytosolic proteins from intracellular and plasma membranes. The pellet was resuspended in 0.5 M Na2CO3, transferred to a 5%/35%/45% sucrose (in Na2CO3) flotation gradient and spun at 36,000 rpm for 18 h using a preparative ultracentrifuge model XL-90 . Fractions obtained from the sucrose gradient were diluted in hypotonic buffer and spun at 15,000 g for 30 min at 4°C. Pellets (25 μl) were resuspended in RIPA buffer and 2× Laemmli buffer. About 30 μl of each sample was loaded in 10% SDS-PAGE and transferred to PVDF membranes for 1 h at 220 mA. Nonspecific binding was blocked with 5% nonfat milk and 2% BSA. The samples were incubated overnight at 4°C with TRPV4 , flotillin , Cav-1 , and α-SMA  antibodies, followed by anti-mouse  or anti-rabbit  HRP-conjugated secondary antibodies. The blotted proteins were developed with an enhanced chemiluminescence kit (Thermo Fisher Scientific).Samples were homogenized in hypotonic homogenization buffer  rinse, PBS containing 5% FBS and 0.3% Triton X-100 blocking solution was applied for 20 min. Filamentous actin (F-actin) was labeled with AlexaFluor 488 phalloidin . Primary antibodies  were diluted in antibody solution (2% BSA and 0.2% Triton X-100 in PBS) and applied overnight at 4 tissues , 44. Aft tissues , 0.005% Binary → Convert to mask with white background and automatic threshold level. Immunoreactive puncta (number/cell area) with the segmented area were counted with the Analyze particles plug-in. Minimum (3 pixel2) and maximum (30 pixel2) pixel area sizes were defined to exclude regions outside the regions of interest (ROIs), calculate the particle number/cell area, and determine the relative puncta numbers. Individual particle sizes for each cell were averaged and normalized.Images were acquired using identical parameters , resulting in very similar signal-to-noise ratios across datasets. ImageJ  was used to extract and quantify the mean intensities and particle analysis of immunoreactive signals, with ∼40–50 cells per slide averaged across at least three independent experiments. The fluorescence intensity of F-actin was measured in arbitrary units using the area integrated intensity measurement tool of ImageJ with background compensation. Data were plotted as the signal as averaged and normalized fluorescence intensity (in percent) per cell area compared with the control. In particle analysis, color images were converted to black and white using 2PO4 1.5, MgCl2 (6H20) 1.5, CaCl2 2, glucose 10, Hepes hemisodium salt 10, pyruvic acid 1, lactic acid 1, l-glutamine 0.5, glutathione 0.5, sodium ascorbate 0.3, with pH 7.4, and osmolarity at 300 ± 10 mOsm, delivered through a gravity-fed 8-reservoir system (Warner Instruments) that converged toward a manifold tube inserted into the experimental chamber. Epifluorescence was detected with Photometrics Delta or Prime BSI cameras. Ratiometric Ca2+ imaging was performed on ROIs that marked a central somatic region and were typically binned at 3 × 3 (R/R (peak F340/F380 ratio − baseline/baseline) was used to quantify the amplitude of Ca2+ signals. Data acquisition and F340/F380 ratio calculations were performed by NIS Elements 3.22 software .Calcium responses in TM cells were tracked following published protocols , 46, 47.at 3 × 3 , 46. Bac2+]i was monitored using a plate reader (Turner Biosystems). Cells were seeded onto noncoated 96-well plates and loaded with 2 μM of Fluo-4 AM for 30–45 min at 37°C. Fluorescence was measured at an excitation of 490 nm and an emission of 520 nm, with intervals of 90 s and 6–10 measurements per experiment. About 490/520 nm ratios were normalized to the control untreated samples. Baseline measurements were recorded in control wells at the same time without addition of agonist or hypotonic stimuli (HTS).The intracellular calcium concentration TM (P < 0.0001)   A, B (∼10< 0.001) A, B.P < 0.05; P < 0.05)  C. A simi < 0.05) A. These P < 0.0001) increases in [Ca2+]i (P < 0.001), with the facilitatory effect inhibited by cholesterol supplementation   and augmentation by cholesterol removal  (P < 0.05).Originally identified as a regulator of cellular swelling , 54, TRP [Ca2+]i A, B. Dep< 0.001) A, B. Spe; N = 3) B  (P < 0.0001) and reduced by cholesterol supplementation to 0.38 ± 0.05    A, B. The 0.0001) A, B. Sim+ puncta A, B. Acc; N = 2) C, D.P < 0.001) (P < 0.01) (P < 0.0001) while reducing filipin fluorescence to 0.24 ± 0.03 . MβCD treatment of stretch-exposed cells did not further affect filipin fluorescence  (P < 0.05) (Changes in membrane cholesterol content have been shown to influence cell surface tension and actomyosin assembly . To test< 0.001) A, C. Int < 0.01) A, B, sug; N = 3) A, B, whe < 0.05) C. These P < 0.05) (P < 0.05)   A, B, D. < 0.05) C, D.Fig.Xenopus laevis expression system that has been used in previous studies of cholesterol depletion  by cholesterol depletion  ultracentrifugation, and fractions were analyzed by Western blot. Flotillin-1, Cav-1, and α-SMA antibodies were used as markers for noncaveolar lipid, caveolar lipid, and cytosolic fractions, respectively. The 48 kDa noncaveolar lipid raft marker flotillin-1 labeled fractions 3–6. The caveolar marker Cav-1 (∼22 kDa) predominantly labeled fractions 3–6, with weak signal present in fractions 1 and 2, whereas α-SMA (∼42 kDa) was confined to the supernatant/cytosol fraction. The TRPV4 protein mainly partitioned into the flotillin-free fraction 2, which showed a weak Cav-1 signal A. Thus, TRPV4 in cultured endothelial cells coimmunoprecipitates with Cav-1 , 32. To Raft/caveolar localization of TRPV4 was examined further with immunochemistry. TRPV4-ir did not colocalize with Cav-1 D, therebi) physiological levels of mechanical stretch regulate the TM membrane C/PC ratio, ii) the majority of TRPV4 protein is excluded from raft/caveolar regions, with the possible exception of a small proportion of a truncated splice variant that interacts with Cav-1; iii) membrane cholesterol negatively modulates TRPV4 activation by agonists and mechanical stimuli; iv) TM F-actin expression is cholesterol and stretch dependent; and v) cholesterol tonically suppresses TRPV4 activity in a nonmammalian expression system. These findings implicate cholesterol-TRPV4 interactions in dynamic and use-dependent regulation of cellular mechanosensing.In this study, we demonstrate that the mechanical milieu regulates the lipid composition of the membrane and that TRPV4, a nonselective cation channel that mediates a wide range of physical and chemical inputs, is highly sensitive to free membrane cholesterol levels. The key findings are In contrast to the extensive body of knowledge about how lipid composition affects the structural and mechanical properties of biological membranes, much less is known about the relationship between mechanical stress and membrane lipid content. Our finding that cyclic strain lowers the membrane C/PC ratio suggests that biophysical and modulatory properties of biological membranes reflect the biomechanical environment. The sensitivity of stretch-induced reorganization of the lipid content to HC067047 further identifies TRPV4 activation as an obligatory step that couples mechanical strain to lipid signaling. Our second conclusion is that changes in the lipid content may profoundly modulate function of embedded mechanoregulated proteins such as TRPV4. This could be relevant in blinding diseases such as glaucoma, which has been associated with elevated C/PC ratios in TM cells .2+ signals induced by the TRPV4 agonist GSK1016790A, cyclic strain, and cell swelling. Given that MβCD similarly facilitates transmembrane currents in TRPV4-expressing, but not control, X. laevis oocytes, we propose that the channel is directly and negatively regulated by membrane cholesterol. Supplementation with exogenous MβCD:cholesterol obviated the facilitation induced by the cyclodextrin alone without affecting peak amplitudes of agonist-evoked and mechanically evoked Ca2+ signals. This may reflect saturation of control membranes with free cholesterol, which constitutes approximately a third of the total membrane lipid mass (Free cholesterol is a central constituent of caveolae and lipid rafts, which serve as regulatory portals for cellular cholesterol homeostasis , 7, 72. pid mass , 38. A spid mass . The ∼4.pid mass  suggestspid mass , 74, Kv1pid mass , Kir (39pid mass , and TRPpid mass  channelspid mass  and endopid mass .suppresses TRPV4 activation in glia (R616Q inverted cholesterol recognition/interaction amino acid consensus motif mutation was associated with gain of function for TRPV4 (Lipids regulate the gating of ion channels through direct interactions within membrane microdomains and indirectly by shaping the biophysical properties of the membrane. An increase in membrane tension, caused by cholesterol removal, might facilitate opening of stretch-activated channels , 80 yet  in glia  and endo in glia . Consist in glia , TRPM3 ( in glia , TRPC3 ( in glia , and vol in glia  channels in glia , 85, 86, in glia . Alloste in glia , 88, 89.or TRPV4 . Cholestor TRPV4 , whereasor TRPV4 , 93 whilor TRPV4 .2+ signals. Differential effects of simvastatin versus MβCD on stress fiber formation and cell contractility have been reported in endothelial cells and fibroblasts (Mechanosensing often involves interactions between the cell membrane and the cytoskeleton. Our finding that MβCD treatment dissolves lipid rafts and stimulates actin polymerization in TM cells accords with its effects on osteoblasts, fibroblasts, endothelial cells, and myocytes , 94, 95.roblasts .2+-binding proteins.The outflow of aqueous humor is fine tuned by arrays of mechanosensitive molecules that include lipid rafts, caveolae, focal complexes, changes in gene expression, ECM and TRPV4, Piezo1, and TREK-1 channels , 103. TRIn summary, our study suggests that TM mechanotransduction is integrated with cholesterol homeostasis. The interdependence between membrane tension, cholesterol content, TRPV4 signaling, and calcium homeostasis suggests that mechanical sensing is a highly dynamic and integrated process that could influence regulation of the conventional outflow pathway. Cholesterol may protect the TM from hypertension-induced injury by downregulating the sensitivity to mechanical stress, but this adaptive function could be compromised by cholesterol dysregulation in glaucoma, diabetic retinopathy, Niemann-Pick disease, and/or macular degeneration.The data described are contained within the article.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


To examine the interaction of working memory (WM) type with emotional interference in trait anxiety, event-related potentials were measured in a combined WM and emotional task. Participants completed a delayed matching-to-sample task of WM, and emotional pictures were presented during the maintenance interval. The results indicated that negative affect interfered with spatial WM; task-related changes in amplitude were observed in the late positive potential (LPP) and slow waves in both the high and low anxiety groups. We also found an interaction among WM type, emotion, and trait anxiety such that participants with high levels of trait anxiety showed an opposite neural response to verbal and spatial WM tasks compared with individuals with low trait anxiety during the sustained brain activity involved in processing negative or neutral pictures in the delay phase. Our results increase our understanding of the influence of emotions on recognition and the vulnerability of those with trait anxiety to emotional stimuli. Early components of LPP have been interpreted as responses to attentional capture, recognition, and stimulus evaluation   = −68.43, P < 0.001] and more highly arousing  than were the neutral pictures.The emotional stimuli were 120 pictures consisting of 60 aversive pictures and 60 neutral pictures, which were selected from the International Affective Picture System  for 750 ms, a white “+” appeared for 1,000 ms. Finally, a small letter appeared at 1 of the 12 points corresponding to the clock . ParticiParticipants completed a short training session consisted of 12 stimuli, followed by two formal sessions. Only one kind of emotional picture appeared in one session. Sessions were separated by a 5-min interval. In order to avoid emotional disturbance, the neutral session appeared before the aversive session. Each session was composed of two blocks, one verbal and one spatial, appearing in random order. Each block included 60 trials, resulting in a total of 240 trials, which appeared completely randomized. There was a brief rest when participants finished 30 trials.Electroencephalograms (EEGs) were recorded from 64 scalp electrodes located in standard 10/20 electrode positions embedded in an elastic cap recording device (NeuroScan version 4.3 system). All electrodes were referenced to the M2 (right mastoid) and then re-referenced offline to the average of M1 (left mastoid) and M2. EEGs were recorded with a 0.01–100-Hz bandpass filter and 1,000-Hz sampling rate. Electrode impedances were always kept below 5 kΩ. Vertical electro-oculogram (EOG) recording electrodes were positioned above and below the left eye, and horizontal EOG recording electrodes were positioned at the outer canthi of both eyes. Each epoch was filtered with a 24-Hz low-pass filter. Trials with various artifacts were rejected using a criterion of ±100 μV. The ERPs were averaged for trials with correct responses.The EEG was segmented for each trial beginning 100 ms before to 1,000 ms after the picture onset. Based on previous studies  were entered into a 2 × 2 × 2 mixed analysis of variance (ANOVA), with task type  and valence  as within-subject factors and group (LA/HA) as between-subject factor. Besides, all electrophysiological data were analyzed by repeated-measures ANOVA also including electrode (FC/CP/P/PO) and laterality (left/midline/right) as within-subject factors. Greenhouse–Geisser adjustments to the degrees of freedom were performed.F = 17.3, p < 0.001, η2 = 0.31]; the accuracy (ACC) under the negative-emotion condition (91.5 ± 0.6%) was higher than that under the neutral-emotion condition (86 ± 1.5%). The task type × emotion interaction was significant, F = 4.74, p < 0.05, η2 = 0.11. A further simple-effect test reflected that verbal WM (85.58 ± 0.7%) was significantly more accurate than was spatial WM (82.34 ± 0.9%) under the negative-emotion condition . Additionally, the accuracy of verbal WM was higher under the neutral-emotion condition (94 ± 0.7%) than it was under the negative-emotion condition (85.6 ± 2.3%). ACC values for each condition in each group are shown in ANOVA results revealed a main effect of emotion ; the RTs for spatial WM tasks (786.13 ± 15 ms) were longer than those for verbal WM tasks (714.11 ± 14.83 ms). The main effect of emotion was significant , with the RTs under the negative-emotion condition (774.81 ± 16.25 ms) being longer than those under the neutral-emotion condition (725.43 ± 13.15 ms). RT values for each condition in each group are shown in ANOVA results revealed a main effect of task type  and electrode , with the amplitudes of early LPPs higher under the negative-emotion (5.17 ± 0.64 μV) than the neutral-emotion (0.5 ± 0.41 μV), and the amplitude of the parietal–occipital electrodes highest (6.43 ± 0.72 μV) and that of the frontal–central electrodes lowest (−3.74 ± 0.53 μV). The main effect of laterality was significant, F = 66.85, p < 0.001, η2 = 0.63. According to the amplitude from large to small: left side (3.93 ± 0.52 μV) > right side (3.72 ± 0.47 μV) > middle line position (0.86 ± 0.55 μV).The ANOVA conducted on early LPP  showed aF = 6.23, p < 0.05, η2 = 0.14, was followed by simple effect analysis for verbal WM and spatial WM, and then neutral-emotion and negative-emotion, respectively. The negative pictures elicited larger early LPPs compared to the neutral pictures in both the verbal [(5.41 ± 0.69) vs. (0.26 ± 0.46) μV] and the spatial [(4.93 ± 0.64) vs. (0.74 ± 0.42) μV] tasks.The task type × emotion interaction, F = 14.03, p < 0.001, η2 = 0.27. The simple effect of group for neutral emotion × spatial WM interaction was significant, and the HA group showed larger early LPP (1.65 ± 0.59 μV) amplitude than did the LA group (−0.17 ± 0.61 μV). The simple effect of task type was significant for the HA group under the neutral  and negative-emotion . When the HA group viewed neutral pictures during the maintenance phase, the pictures elicited higher early LPP amplitude during spatial WM task (1.65 ± 0.59 μV) than the verbal WM task (0.45 ± 0.64 μV). In contrast, after the HA group viewed negative pictures, the pictures elicited higher early LPP amplitude during the verbal WM task (6.01 ± 0.95 μV) than the spatial WM task (4.8 ± 0.89 μV). No significant differences were found for these other group comparisons.The task type × emotion × group interaction was significant, F = 149.6, p < 0.001, η2 = 0.8, the negative emotion elicited a higher amplitude of the late LPP (4.34 ± 0.64 μV) compared to the neutral emotion (−1.69 ± 0.38 μV).Regarding late LPP amplitude , analysiF = 6.45, p < 0.05, η2 = 0.14. The interaction effect was examined by comparing the effect of group for each combination of task type and emotional condition. Under both of the two WM task conditions, the late LPP elicited under the negative-emotion was larger than the neutral-emotion in the HA group in comparison to the LA group. The simple main effect of emotion was significant for the HA group under the verbal  and spatial WM tasks . During verbal WM task, the negative pictures elicited higher late LPP amplitude (4.99 ± 0.95 μV) than the neutral pictures (−1.87 ± 0.64 μV). Similarly, during spatial WM task, the negative pictures elicited higher late LPP amplitude (3.84 ± 0.92 μV) than the neutral pictures (−1.37 ± 0.53 μV). No significant differences were found for these other group comparisons.The task type × emotion × group interaction was significant, F = 37.53, p < 0.001, η2 = 0.66. According to the amplitude from large to small: left side (1.88 ± 0.47 μV) > right side (1.71 ± 0.44 μV) > middle line position (0.38 ± 0.52 μV).The main effect of laterality was significant, F = 4.51, p < 0.05, η2 = 0.19. Further simple effect test showed that the simple main effect of laterality was significant in verbal  and spatial  in the LA group. In the low anxiety group, the amplitude of LPP in the left side of the brain was the largest both in verbal (2.03 ± 0.76 μV) and spatial (1.61 ± 0.44 μV) tasks. In the HA group, The simple main effect of laterality was significant both in the verbal  and spatial WM tasks in the HA group. In the HA group, the LPP amplitude in the left side of the brain (2.31 ± 0.74 μV) was the largest in verbal tasks, similar to the low anxiety group. In contrast to the LA group, the LPP amplitude in the right side of the brain was the largest (1.71 ± 0.62 μV) in spatial tasks in the HA group.The interaction effect of task type × laterality × group was significant, F = 178.23, p < 0.001, η2 = 0.82], with slow-wave amplitude under the negative emotion (4.67 ± 0.31 μV) higher than the neutral emotion (1.89 ± 0.32 μV). The main effect of laterality was also significant , and the amplitude of the left lateral electrodes was the highest (2.56 ± 0.42 μV).The ANOVA conducted on slow wave showed a main effect of emotional state  between the amplitudes elicited by the interference pictures during the verbal and spatial tasks in the HA group over only the parietal–occipital electrodes in comparison to the LA group, and the interference pictures elicited higher slow-wave amplitudes in the spatial (1.57 ± 0.59 μV) than in the verbal tasks (0.45 ± 0.66 μV).We found a marginally significant task type × electrode × group interaction effect, F = 3.55, p < 0.05, η2 = 0.08. The comparisons of the effects of emotional state for each task type revealed that negative emotion elicited higher amplitudes than neutral emotion in both the verbal and spatial tasks   and the higher early LPP amplitude during spatial WM task compared to the verbal WM task in the HA group.In conclusion, our findings demonstrate that negative emotional distractors have differential effects on neural attentional processing for individuals with high vs. low trait anxiety, suggestive of possible compensatory/resilience mechanisms at work to allow comparative task scores in individuals with high trait anxiety. Our results suggest that such mechanisms have important effects on the performance of cognitive tasks and that the kind of effect depends on the type of the task , on the level of trait anxiety (higher levels of trait anxiety are associated with more interference), and on the type of emotional interference . Our results contribute to our understanding of the influence of emotions on recognition performance and trait anxiety, which are associated with increased susceptibility to emotional disruptions.954147041@qq.com.The datasets presented in this article are not readily available because the data also forms part of the ongoing study. Requests to access the datasets should be directed to Huifang yang, The studies involving human participants were reviewed and approved by the Ethics Committee of Lingnan Normal University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from all participants for the publication of any potentially identifiable data included in this article.HY: experiment design. JL: data collection. XZ: major revision of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


However, the high toxicity associated with MoS2 has limited its biological applications for in vivo photothermal therapy and drug delivery systems. Herein, we have developed cationic hydroxyethyl cellulose (JR400) surface-modified MoS2 nanoparticles (NPs) that are responsive to near-infrared (NIR) laser irradiation as a transdermal drug delivery system (TDDS). Herein, we confirmed the preparation of hexagonal phase MoS2 with robust surface modification with JR400. The flower-like morphology of the NPs had an average diameter of 355 ± 69.3 nm limiting the absorption of the NPs through the stratum corneum. With the ability to efficiently load 90.4 ± 0.3% of the model drug atenolol (ATE), where 1 g of JR400-MoS2 NPs was able to load 3.6 g ATE, we assayed the controlled release capacity in vitro skin penetration studies. These JR400-MoS2 NPs showed further enhancement under NIR stimulation, with a 2.3-fold increase in ATE skin penetration. Furthermore, we verified in vivo that these JR400-MoS2 NPs do not cause skin irritation suggesting that they are promising new TDDS candidates for small molecule drugs.Molybdenum disulfide (MoS TDDSs avoid the first-pass effect and improve patient compliance because they are applied topically  flower-like MoS2 nanoparticles (NPs) produced via a simple hydrothermal approach  were further characterized using attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, X-ray diffraction (XRD), and transmission electron microscopy (TEM). We chose to load the J400-MoS2 NPs with the model drug atenolol (ATE), a β1-adrenergic receptor blocking agent prescribed for hypertension. The drug load efficacy and photothermal conversion effect were evaluated, and the controlled release capacity of JR400-MoS2 NPs was demonstrated in in vitro drug release and skin penetration assays.Herein, we aimed to overcome the toxicity of MoS2.2.1.Ammonium molybdate tetrahydrate and thiourea were obtained from Shanghai Macklin Biochemical Co. Ltd. . Cationic hydroxyethyl cellulose  was purchased from Shandong Usolf Chemical Technology Co. Ltd. . Atenolol  was purchased from Rhawn Chemical Technology Co. Ltd. . HPLC grade methanol and phosphoric acid were used throughout. All other chemicals were reagent grade and obtained commercially. All chemicals were used as received without any further purification. Ultrapure water was purified by a Milli-Q system (18.2 MΩ).2.2.2 NPs .Afterward, 1.0 g of the obtained flower-like MoS2.3.2 NPs was collected using the KBr pellets method with ATR-FTIR spectroscopy . The crystalline structure of JR400-MoS2 NPs was studied using Rigaku SmartLab X-Ray Diffraction with Cu Kα radiation. The microstructure of the samples was investigated using TEM , which was operated at 200 kV. The particle size and zeta potential were measured using dynamic light scattering . The colloidal stability was analyzed by zeta potential and sedimentation volume ratio. The sedimentation volume ratio was determined by the ratio of the height after (Hu) and before (H0) the sedimentation.Spectrum of JR400-MoS2.4.2 NPs were suspended in water at varying concentrations from 0.1 to 1.0 mg/mL for irradiation with an 808-nm laser (0.5 W/cm2) to measure the photothermal effects. The laser power density was also varied between 0.2 and 1.0 W/cm2. The thermal stability of the JR400-MoS2 NPs at 0.5 mg/mL was determined by NIR-stimulation (0.5 W/cm2) for 5 min over three on-off cycles. The temperature of the solution was measured by a magnetic stirrer equipped with a temperature probe.JR400-MoS2.5.2 NPs by mixing different concentrations of the drug with 0.5 mg/mL JR400-MoS2 NPs in 5 mL of phosphate-buffered saline (PBS) (pH = 7.0). The mixture was stirred for 2 h and incubated at 32 °C for 24 h. The free drug was removed by centrifugation at 5000 r/min for 10 min, and the JR400-MoS2-ATE NPs were washed with ultrapure water three times. The JR400-MoS2-ATE NPs were dried at 60 °C for 24 h under vacuum and stored at room temperature. The drug loading was calculated by the concentration of free drug in the supernatant, as analyzed by HPLC. Drug loading efficiency and drug loading percent were calculated using the following equations:ATE was loaded onto the surface of JR400-MoS2.6.2.6.1.2-ATE into 50 mL PBS. In order to form a uniform suspension, the mixture was stirred for 1 h, followed by sonication for 15 min. The suspension was added into the donor chamber of a two-chamber diffusion cell immediately after preparation.A donor solution was prepared by adding 25 mg JR400-MoS2.6.2.2-ATE solution was added into the donor chamber, and an equal volume of PBS (pH 7.4) containing 15% PEG400 (PBS-PEG400) was added into the receptor chamber. Both chambers were continuously stirred with a magnetic stirrer at 600 rpm and kept at 32 °C. 2.0 mL from receptor chamber was collected at 1, 2, 3, 4, 5, 6, 7, 8, 12, 24, 28, 32, 36, and 48 h, and then replaced with the same volume of fresh PBS-PEG400. The donor solution was irradiated with laser (0.5 W/cm2) for 5 min after sampling.Drug release was assayed using a two-chamber diffusion cell separated by a 0.22-μm cellulose microporous membrane. 4.0 mL of JR400-MoS2.6.3.Male Wistar rats  were supplied by Liaoning Changsheng . Full-thickness skin was prepared as follows: (1) the rat was anesthetized with urethane , and the abdominal hair was shaved off. (2) Abdominal skin was excised after the rat was sacrificed. (3) The adhering subcutaneous tissues were carefully removed. All the procedures were performed in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of Shenyang Medical College.The two-chamber diffusion cell was used for the permeation experiments. The skin was mounted between the two chambers with SC facing the donor solution and epidermis facing the receptor solution. Sample collection and drug concentration measurements were performed in the same manner as drug release study, except that sample collection was conducted at 2, 4, 6, 8, 12, 24, 28, 32, 36, and 48 h with NIR-stimulation after every sampling. The cumulative drug skin penetration amount was calculated using the drug concentration in the receptor solution, taking the effect of sampling into account.2.6.4.The drug concentration was determined by Hitachi HPLC , which consisted of a Pump L-2130, AutoSampler L-2200, UV-detector L-2420, and C18 reversed-phase column . The mobile phase for the ATE was a mixture of methanol, water, and phosphoric acid solution . The column temperature was 25 °C, the flow rate was set at 0.7 mL/min, and the free drug was detected at a wavelength of 275 nm.2.7.®  was used to measure the biocompatibility of JR400-MoS2 NPs by measuring the erythema index (EI) of the skin. Rabbits were used to assess potential skin irritation caused by JR400-MoS2 NPs compared with a 10% (w/v) aqueous solution of sodium dodecyl sulfate (SDS) as the positive control. The dorsal skin of the rabbits was shaved and divided into three separate sections, each with an area of 2.5 cm × 2.5 cm. The initial EI values of the chosen sections were measured as a baseline value (EI0) before topical application of 500 μL of PBS, JR400-MoS2 NPs, or 10% SDS in their respective sections. The application sites were covered with double-layer gauze in order to prevent perturbation. After 8 h, the excess solutions were removed, and the predefined sections were gently cleaned with cotton wool swabs. The EIt was measured at set intervals and ΔEI was calculated by subtracting EI0 from EIt. This experiment was performed in quadruplicate.A Mexameter2.8.p< .05.Results are reported as the mean ± SD. The data were subjected to analysis of variance (ANOVA) using the SPSS 16.0 software . Significance levels are reported for 3.3.1.2 NPs were synthesized using a previously reported hydrothermal process using ammonium molybdate tetrahydrate and thiourea precursors to produce polyacrylamide (PAM) modified MoS2 NPs , (100), (103), (105), and (110) diffraction planes, respectively (2 (JCPDS no. 37-1492) , TEM, and high-resolution TEM (HRTEM)  plane was observed in the enlarged HRTEM image  . The SEMnosheets . The strnosheets , consistEM image , where t2 NPs had an average diameter of 355 ± 69.3 nm (PDI = 0.259), as obtained by DLS, similar to the size obtained by HRTEM image  drug release from the matrix and (2) percutaneous absorption. JR400-MoS3.6.in vivo skin erythema measurement was used to monitor irritation caused by the JR400-MoS2 NPs. As shown in 2 NPs suspended in water did not increase ΔEI, indicating high biocompatibility.Visual observation of erythema is widely used to evaluate the skin irritant potential of substances. However, this method has been criticized as imprecise and subjective. A noninvasive 4.2 NPs were synthesized as a TDDS for ATE, a β1-adrenergic receptor blocking agent prescribed for hypertension. The ultra-high specific surface area of the JR400-functionalized MoS2 NPs allowed for efficient binding of ATE with 90% loading efficiency. NIR stimulation enhanced drug release and facilitated drug skin penetration. The application of JR400-MoS2 NPs as a TDDS shows the potential to mitigate issues associated with the toxicity of MoS2 use in vivo and allows for the controlled release of ATE. To our knowledge, this is the first demonstration of a functional MoS2-based TDDS. The promising results showed herein warrant further studies to evaluate the in vivo application of JR400-MoS2 NPs based TDDSs.JR400-functionalized MoS


An accurate, reliable, and specific method was developed for the quantitative determination of fumonisins B1, B2, B3, and their hydrolyzed metabolites, HFB1, HFB2, and HFB3, in broiler chicken feed and excreta using ultra-performance liquid chromatography combined with tandem mass spectrometry (UPLC-MS/MS). The samples were extracted and diluted for the determination of parent fumonisins. Another portion of the extracted samples was alkaline-hydrolyzed and cleaned using a strong anionic exchange adsorbent (MAX) for the determination of hydrolyzed fumonisins. Chromatographic separation was performed on a CORTECS C18 column  using 0.2% formic acid aqueous solution and methanol with 0.2% formic acid as the mobile phase under gradient elution. The six fumonisins, FB1, FB2, FB3, HFB1, HFB2, and HFB3, were analyzed by tandem mass spectrometry using multiple-reaction monitoring (MRM) mode. The six fumonisins showed good linearity, with relative coefficients of r > 0.99. The limits of quantitation (LOQs) were 160 μg/kg. At the low, medium, and high spiked levels, the recovery of fumonisins in chicken feed and excreta ranged from 82.6 to 115.8%, with a precision (RSD) of 3.9–18.9%. This method was successfully applied to investigate the migration and transformation of fumonisins in broiler chickens. Fusarium and Aspergillus species. Among these analogues, fumonisin Bs (FBs) are the most widely distributed in animal feed and human food through contamination of grain crops -FB1, [13C]-HFB1, and [13C]-pHFB1a+b as isotope internal standards ,13,14. HC-FB1, -FB1, [13C34]-FB2, and [13C34]-FB3 standard solutions with certified concentrations were also purchased from Biopure .Acetonitrile, methanol, and formic acid were of LC/MS grade . All other chemicals were of analytical grade or better. Deionized water (18.2 MΩ cm) was collected from a Milli-Q system . Oasis MAX 3cc 60 mg cartridges and Oasis HLB 3cc 60 mg cartridges were purchased from Waters . Standard solutions of mixed FB1, FB2, and FB3 with certified concentrations of each analyte were purchased from Biopure . [All samples were obtained from a broiler chicken two-week fumonisin exposure study. Twelve 7-day-old male Ross 308 broiler chickens were given an FB-contaminated diet for two weeks. The FB-incurred feed was prepared, and the levels of 6 analytes were measured. The remaining feed was weighed every 24 h. Thus, the consumption of feed for 12 chickens could be calculated. Every 24 h, excreta samples of the 12 broiler chickens were pooled, weighed, and homogenized. All samples were stored at −20 °C until further analysis. The animal experiment was approved by the Ethics Committee of Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (project identification code: SV-20200906-Y06) on 6 September 2020.v/v/v). Samples were then shaken for 30 min and ultrasonicated for another 30 min. After centrifugation, 50 µL of supernatant (15 min at 8000 rpm) was combined and vortexed with 950 µL of distilled water. Then, a 50 μL aliquot of the resulting solution was mixed with 10 μL 13C-FBs IS working solution and diluted with 850 µL of 0.2% FA in methanol/water (1:9) prior to LC-MS/MS injection for FBs analysis. A total of 5 g of feed sample or excreta sample was homogenized in 20 mL of ACN/water/formic acid  prior to LC-MS/MS injection for HFBs analysis.Another 50 µL aliquot was added with 10 μL of LC–MS/MS analysis was performed on an ultra-high-performance liquid chromatography system  coupled to a tandem mass spectrometer  in multiple-reaction monitoring (MRM) mode with an electrospray ionization source in positive mode (ESI+). Chromatographic separation was achieved on a CORTEX C18 column . The temperatures of the column oven and autosampler tray were set at 40 °C and 10 °C, respectively. The flow rate was 0.4 mL/min. Mobile phase A consisted of 0.2% FA in water, whereas mobile phase B consisted of 0.2% FA in methanol. Gradient elution was performed: 0 min , 0–6 min (linear gradient to 90% B), 6–8 min , 8–8.1 min (linear gradient to 90% A), 8.1–10.0 min . The following mass spectrometry parameters were used: capillary voltage: 2.5 kV; desolvation temperature: 500 °C; desolvation gas flow: 800 L/h; collision gas: 0.15 mL/min. The MRM transitions that were monitored for analytes are shown in