Multiple approaches are applied to obtain inorganic, organic and composite nanomaterials with different functionality. In the present work, self-assembling nanocomplexes (NCs) were prepared on the basis of enzymes and polypeptides followed by the investigation of the influence of low-molecular weight biologically active compounds on the properties of the NCs. For that, the initially possible formation of catalytically active self-assembling NCs of four hydrolytic enzymes with nine effectors was screened via molecular modeling. It allowed the selection of two enzymes (hexahistidine-tagged organophosphorus hydrolase and penicillin acylase) and two compounds (emodin and naringenin) having biological activity. Further, such NCs based on surface-modified enzymes were characterized by a batch of physical and biochemical methods. At least three NCs containing emodin and enzyme (His N-acyl homoserine lactones and pheromone-like peptides are among the most well-known QS inductors of Gram-negative and Gram-positive bacteria, respectively. In the case of Quorum Quenching (QQ) of Gram-negative bacterial cells, the action of enzymes hydrolyzing the N-acyl homoserine lactones appeared very effective, and the combinations of the QQ-enzymes with antibiotics significantly improves the efficiency of the latter   as described previously [Hiseviously . Furthereviously . Enzyme eviously  with an 6-OPH or penicillin acylase (Sigma-Aldrich) in PBS buffer (pH 7.4) was gently mixed with a water solution of PEG-PLE50 , PLE50 (Alamanda Polymers) or Gelofusine®  in an equimolar ratio and filtered through a 0.2-μm Chromafil® Xtra PES filter ; 10 μg/mL naringenin or 1 μg/mL emodin (Sigma-Aldrich) in EtOH were added to the nanocomplexes at dosage 1 vol.% before filtering.To obtain enzyme NCs with polypeptides, a previously published method ,27 was uThe hydrodynamic size of the NCs was determined as described previously  by PTA uiOH, piranha solution (H2SO4:H2O2 = 2:1) and water. After drying at 120 °C for 1 h, samples were immersed into a 0.25 vol.% APTMS in hexane (0.5 mL per 2 replicas) and gently shaken at 30 °C for 30 min. Then, the liquid phase was rapidly removed and 0.5 mL of CHCl3 was added and further shaken at 30 °C for 10 min. After that, the liquid was discarded and samples were washed two times with two portions of 2 mL water, with intermediate shaking at 30 °C for 30 min. Samples were dried at 90 °C for 30 min and stored at room temperature in closed vessels. A 5 μL sample of as-prepared enzyme NCs was applied per wafer in duplicate and exposed at room temperature for 5 min. After that samples were washed two times in 2 mL water, spin-dried and stored under vacuum before analysis.Silicon wafers for AFM were functionalized by 3-aminopropyltrimethoxysilane  according to the known protocol  with minhttp://gwyddion.net/, accessed on 15 December 2022) [2) were discarded also. Finally, the data were exported to OriginPro  and envelopes were approximated under the assumption of Gaussian distribution .The tapping mode of SmartSPM-1000  was used for obtaining the topography profiles. The diamond single crystal cantilevers AFM Probe ART™ D300  used in this study had a typical spring constant of 40 N/m and a rounded tip of 5–10 nm radius. The spring constant of each cantilever was determined using a technique based on measuring the change in the resonant frequency of the fundamental mode of vibration [er 2022) . A 50% tBacillus subtilis B-522  and Gram-negative bacterial cells of Escherichia coli DH5α . Briefly, 20 µL of (3 ± 1) × 106 cells/mL in a 0.9% NaCl was loaded onto BC samples and exposed for 24 h at 25 °C. Then, samples were immersed in 1 mL of DMSO and gently stirred for 1 h. The residual ATP concentration in DMSO extract was determined using a standard luciferin–luciferase ATP reagent  according to the method [A 10 μL sample of 10 μg/mL naringenin or 1 μg/mL emodin (Sigma-Aldrich) in EtOH diluted 1–1000 times was loaded per BC sample (1 × 1 cm) and dried for 72 h at +8 °C under sterile conditions. The antibacterial activity was determined by the previously published procedure ,42 with e method  using a 6-OPH in PBS buffer, or a mixture of 4 μL penicillin acylase with 1.5 μL His6-OPH was applied to the same samples and dried for 24 h at 8 °C under sterile conditions. Thus, while omitting antibiotics, QS effectors, or enzymes, various compositions were prepared. All samples were simultaneously investigated towards B. subtilis and E. coli cells as described above.A 10 μL sample of 10 mg/mL colistin or polymyxin B was applied per BC sample (1 × 1 cm) and dried for 24 h at 8 °C under sterile conditions. Then, 10 μL of 1 μg/mL naringenin or 0.1 μg/mL emodin in EtOH was loaded per the same samples and dried for 72 h at 8 °C under sterile conditions. A further 4 μL of 3 mg/mL penicillin acylase in PBS buffer (pH 7.4), or 5 μL of 0.15 mg/mL HisE.coli cells was used. A 5 μL sample of 2.4 mg/mL penicillin acylase and 5 μL of 0.5 mg/mL His6-OPH in PBS buffer were mixed with 5 μL of 0.2 μg/mL emodin in H2O. Then, 10 μL of PBS buffer or 10 mg/mL polymyxin B was added. These mixtures were introduced to a 25 μL suspension of (3 ± 1) × 105 cells/mL in a 0.9% NaCl. As a negative control, 25 μL of PBS buffer was used. All samples were gently shaken for 3 h at 25 °C. After that, a 5 μL of the sample was applied on an APS-modified silicon wafer and dried in the air at room temperature (ca. 30 min). Before SEM analysis, samples were freeze-dried , sputtered with gold and studied at various magnifications with a Supra 40-30-87 microscope .To prepare samples for SEM analysis, a mature culture of Pseudomonas aeruginosa (PDB 4M1J), carboxypeptidase A from Bos taurus (PDB 1YME) and protease (stearolysin or thermolysin) from Geobacillus stearothermophilus (PDB 6GHX) were obtained from the Protein Data Bank. The structure of His6-OPH was prepared previously [Crystallographic structures of acylase PvdQ from eviously  and basehttp://www.poissonboltzmann.org/, accessed on 15 December 2022) with a PARSE force field and default settings [https://ccsb.scripps.edu/mgltools/, accessed on 15 December 2022) [Previously published procedures ,27 were settings . Then, ter 2022) .Structures of QS effectors were drawn using ChemBioDraw software  and then minimized using ChemBio3D with force field MM2. Further, the structures in the PDB format were converted to the PDBQT format using AutoDockTools with atomic charges calculated with the Gasteiger–Marsili method.http://vina.scripps.edu/, accessed on 15 December 2022) [get_area” function of PyMOL Molecular Graphics System . Statistical analysis was performed using SigmaPlot , and the data are presented as means ± standard deviation (±SD) unless otherwise stated.Enzyme–ligand complexes were simulated using AutoDock Vina   on a desThus, self-assembling NCs of QQ enzymes with QS effectors were designed, prepared and characterized in the work. Such NCs can be rationally preselected to minimize the negative influence on each other and were obtained via simple pooling of several components. The method can be promising to prepare enzyme NCs with different functionality.


It has been demonstrated that reactive astrocytes can be polarized into pro-inflammatory A1 phenotype or anti-inflammatory A2 phenotype under neurotoxic and neurodegenerative conditions. Microglia have been suggested to play a critical role in astrocyte phenotype polarization by releasing pro- and anti-inflammatory mediators. In this study, we examined whether trimethyltin (TMT) insult can induce astrocyte polarization in the dentate gyrus of mice, and whether protein kinase Cδ (PKCδ) plays a role in TMT-induced astrocyte phenotype polarization.Male C57BL/6 N mice received TMT , and temporal changes in the mRNA expression of A1 and A2 phenotype markers were evaluated in the hippocampus. In addition, temporal and spatial changes in the protein expression of C3, S100A10, Iba-1, and p-PKCδ were examined in the dentate gyrus. Rottlerin  was administered 3–5 days after TMT treatment, and the expression of A1 and A2 transcripts, p-PKCδ, Iba-1, C3, S100A10, and C1q was evaluated 6 days after TMT treatment.TMT treatment significantly increased the mRNA expression of A1 and A2 phenotype markers, and the increased expression of A1 markers remained longer than that of A2 markers. The immunoreactivity of the representative A1 phenotype marker, C3 and A2 phenotype marker, S100A10 peaked 6 days after TMT insult in the dentate gyrus. While C3 was expressed evenly throughout the dentate gyrus, S100A10 was highly expressed in the hilus and inner molecular layer. In addition, TMT insult induced microglial p-PKCδ expression. Treatment with rottlerin, a PKCδ inhibitor, decreased Iba-1 and C3 expression, but did not affect S100A10 expression, suggesting that PKCδ inhibition attenuates microglial activation and A1 astrocyte phenotype polarization. Consistently, rottlerin significantly reduced the expression of C1q and tumor necrosis factor-α (TNFα), which has been suggested to be released by activated microglia and induce A1 astrocyte polarization.We demonstrated the temporal and spatial profiles of astrocyte polarization after TMT insult in the dentate gyrus of mice. Taken together, our results suggest that PKCδ plays a role in inducing A1 astrocyte polarization by promoting microglial activation and consequently increasing the expression of pro-inflammatory mediators after TMT insult.The online version contains supplementary material available at 10.1186/s12974-022-02507-w. Astrocytes are the most abundant glial cell type in the brain, and play diverse physiological roles in maintaining brain homeostasis through regulation of ion and neurotransmitter concentrations, tissue pH, neurogenesis, synaptogenesis, and blood–brain barrier integrity , 2. Astr Protein kinase Cδ (PKCδ) is a novel PKC isozyme , and has. They were adapted to these conditions for 2 weeks prior to the experiment. In the present study, male mice were used to avoid the influence of the estrus cycle of female mice, because previous studies have suggested that estrogen or the estrus cycle could affect astroglial and microglial activation, and consequently neuroinflammation  and 42.3% of S100A10-positive cells were co-localized with C3 [tM for S100A10 = 0.423 ± 0.0123 (n = 4)]. The findings indicate significant co-localization of C3 and S100A10 expression induced by TMT treatment in the dentate gyrus of mice , and this increase in Iba-1 expression was more pronounced at 6 and 10 days after TMT , suggesting that the temporal pattern of microglial activation is consistent with that of astroglial activation and phenotypic alteration, particularly A1 phenotype polarization, in the dentate gyrus of mice after TMT treatment , and became more evident at 6 and 10 days after TMT treatment   Fig. B. Doublene) Fig. C.P < 0.01 vs. vehicle + TMT), and rottlerin-mediated attenuation of p-PKCδ expression appeared to be more pronounced in the molecular layer than in the granular cell layer of dentate gyrus treated with TMT , suggesting that PKCδ inhibition attenuates TMT-induced Iba-1 expression in the dentate gyrus. Similar to p-PKCδ expression, the effect of rottlerin appeared to be more prominent in the molecular layer than in the granular cell layer in TMT-treated mice  by rottlerin in the dentate gyrus of TMT-treated mice, suggesting that rottlerin attenuates microglial proliferation or migration in response to TMT insult  and cell body size/cell size ratio . Significant increases were observed in the number of branches , number of junctions , average branch length , and summed branch length  after rottlerin administration in the dentate gyrus of TMT-treated mice   Fig. .Fig. 7EfP < 0.01 vs. vehicle + TMT)  Fig. . These rP < 0.05 vs. saline), and became more pronounced at 6 and 10 days after TMT treatment    6 days after TMT treatment  Fig. A. In Wesent Fig. B, suggesP < 0.01 vs. vehicle + TMT)  Fig. , suggestIn the present study, we characterized the temporal and spatial profiles of astrocyte polarization after TMT insult in the dentate gyrus of mice. We also found that the expression of p-PKCδ, an important mediator of neuroinflammation, was induced in microglial cells concomitant with TMT excitotoxicity. Late- and post-ictal treatment with rottlerin, a PKCδ inhibitor, significantly attenuated microglial activation and decreased the expression of C1q and TNFα, and consequently significantly attenuated A1 astrocyte polarization induced by TMT. Thus, our results suggest that PKCδ is involved in A1 astrocyte polarization through the promotion of microglial activation and subsequent release of pro-inflammatory mediators under excitotoxic conditions.It has long been demonstrated that astrocytes become reactive in response to various brain injuries and neurodegenerative changes. Reactive astrocytes form a functional barrier in neuropathological conditions, limiting neuroinflammation to the injured area, preventing their spread to adjacent brain tissues, and promoting tissue repair –5. In thRecently, it has been reported that reactive astrocytes can be polarized into the pro-inflammatory A1 phenotype or the anti-inflammatory A2 phenotype in various neuropathological conditions , 13–15. Immunohistochemical analysis showed that the expression of both C3 and S100A10 peaked at 6 days after TMT treatment in the dentate gyrus of mice. C3 expression remained increased at 10 days, but S100A10 expression significantly decreased at this time point, suggesting that A1 astrocyte polarization lasts longer than A2 astrocyte polarization in this model. Interestingly, C3 mRNA expression started to significantly increase from 1 day after TMT treatment; however, C3 protein expression, as examined by immunohistochemistry, did not significantly increase in astrocytes until 2 days after TMT treatment. Instead, C3 expression appeared to slightly increase in the microvessels 2 days after TMT treatment, which was in line with previous findings showing that C3 expression can be increased by ischemic or inflammatory stimuli in the epithelial cells of brain microvessels –73. ThusC3 and EMP1 genes in their in situ hybridization study. Recently, it has been shown that A1 and A2 phenotypes can be mutually converted rather than permanently fixed in vitro and in vivo [Previous studies have suggested that C3 and S100A10 are specific and exclusive markers of A1 and A2 astrocytes, respectively , 49. Int in vivo –77. One  in vivo  reportedcsf1r gene knockout has been reported to attenuate A1 phenotype polarization induced by kainate excitotoxicity [Activated microglia have been demonstrated to be important for inducing A1 astrocyte polarization through the release of pro-inflammatory factors , 11, 48.toxicity  or lipoptoxicity . In conttoxicity , suggesttoxicity , 79–81, In the present study, Iba-1 expression was examined to evaluate microglial activation. However, Iba-1 is expressed in monocytes/macrophages as well as in microglia. It has been suggested that peripheral monocytes/macrophages can infiltrate into the brain parenchyma when the blood–brain barrier is disrupted following brain injury and neuroinflammation , 83. InfPKCδ has been demonstrated to play an important role in neuroinflammation , 91, 92.The present study found that rottlerin treatment decreased the mRNA expression of A1 astrocyte markers, but not A2 astrocyte markers, induced by TMT in the dentate gyrus of mice. Consistently, rottlerin significantly decreased C3-immunoreactivity, but did not significantly affect S100A10-immunoreactivity after TMT insult. Among the pro-inflammatory factors released by activated microglia, C1q, TNFα, and IL-1α have been shown to be critical for the specific induction of astroglial A1 phenotype polarization upon inflammatory stimulus . HoweverIt has been suggested that A1 phenotype astrocytes release pro-inflammatory and neurotoxic factors, such as TNFα, IL-1β, nitric oxide, and reactive oxygen species , 10, andOur study demonstrated the temporal and spatial profiles of astrocyte polarization after TMT-induced excitotoxicity in the dentate gyrus of mice. In addition, it was found that TMT induced microglial p-PKCδ expression, and that the PKCδ inhibitor rottlerin suppressed the neurotoxic A1 polarization of astrocytes by inhibiting microglial activation and consequent expression of pro-inflammatory factors, including C1q and TNFα. Therefore, these results suggest that PKCδ inhibition might be an important target for suppressing A1 astrocyte polarization and delayed neuronal damage under excitotoxic conditions.Additional file 1. Additional Information and Additional Tables S1–S5.Additional file 2. Additional Figures S1–S5.


Discontinuities in the vibrational frequenciesobserved in Raman spectra measured between ambient pressure and ∼5.5GPa show that both polymorphs undergo phase transitions, the αphase at 0.8 GPa and the β phase at 2.1 GPa. The structuralsignatures of the transitions, which signal the onset of compressionof initially more rigid intermolecular contacts, were identified fromthe trends in the occupied and unoccupied volumes of the unit cellwith pressure and in the case of the β phase by deviations froman ideal model of compression defined by Birch–Murnaghan equationsof state.The effect of pressureon the α and β polymorphsofa derivative of Blatter’s radical, 3-phenyl-1-(pyrid-2-yl)-1,4-dihydrobenzo[ e]triazin-4-yl at pressures of 0.8 and 2.1 GPa.Subtle, second order phase transitionsoccur in the αand β polymorphs of the Blatter radical, 3-phenyl-1-(pyrid-2-yl)-1,4-dihydrobenzo[ Raman spectroscopy and an analysis of intermolecularinteractions suggest subtle phase transitions in both, at ∼2and ∼1.5 GPa, respectively.5Although studies of molecularmaterials at high-pressure have becomequite common, there are relatively few structural reports in whichthe responses of different polymorphs of the same compound are compared.e]triazin-4-yl6  and its derivativesare air- and moisture-stable, open-shell species with potential applicationsas photodetectors,8 emissive materials for OLEDs,9 pH sensors,10 liquidcrystalline photoconductors,14 and electroactive building blocks of polymers inpurely organic batteries.15 The large,delocalized SOMO surface of the benzotriazinyl core in Blatter radicalshas many potential sites for intermolecular interactions in crystalstructures which, tentatively, can increase the propensity towardpolymorphism.16 The aim of the presentcontribution is to compare the effect of pressure on two polymorphsof a derivative of Blatter’s radical, 3-phenyl-1-(pyrid-2-yl)-1,4-dihydrobenzo[e]triazin-4-yl (1), , 1. The po22.1e]triazin-4-yl (1) was prepared accordingto the procedure in ref -1,4-dihydrobenzo,23 and refinements athigher pressure started from the atomic coordinates of the precedingpressure point. Refinement was by full-matrix least-squares on |F|2 .25 Intramolecular bond distancesand angles in all refinements against data collected at high pressurewere restrained to those observed at ambient pressure. Where possible,non-hydrogen atoms were refined with anisotropic displacement parameterssubject to enhanced rigid-bond restraints.26 Crystals of 1β diffracted more weakly than thoseof 1α, leading to poorer data quality statisticsand lower precision in the refined parameters; in some cases it wasnecessary to mix isotropic and anisotropic refinement for differentatoms in the same structure. Nevertheless, the data do establish consistenttrends in volumes and contact distances with pressure. Hydrogen atomswere placed in calculated positions and constrained to ride on theirparent atoms. Crystal and refinement data for both 1α and 1β are listed in Data were collected asin ref  and proc2.2–1) equipped with a gratingwith 1800 grooves/mm and a charge-coupled device (CCD) cooled withliquid nitrogen. An argon laser operating at a wavelength of 488 nmwas used for the excitation of the samples. The laser beam was focusedon the sample surface using a microscope (OLYMPUS) with a long-workingdistance 50× lens with a numerical aperture of 0.75, forminga spot of approximately 4 μm on the surface of the sample. Highpressure was applied to the samples using a diamond anvil cell (DAC).To avoid sample decomposition under laser irradiation, MeOH/EtOH wasreplaced as a pressure-transmitting medium by nujol. Nujol is a mineraloil (CAS number 8012–95–1), which provides quasi-hydrostaticconditions to 7–10 GPa,27 confirmedin our measurements by the symmetrical ruby emission bands observedat all pressures. Pressures were measured using the ruby fluorescencemethod.20 The maximum pressures reachedwere 5.36 GPa for 1α and 5.43 GPa for 1β. The deconvolution of the recorded spectra into a set of Lorentzianline profiles was performed using the Fityk software.28Raman spectrawere acquired on a LabRAM HR (Jobin-Yvon HORIBA) spectrometer, whichis an 800 mm focal length Czerny-Turner type spectrograph  -1,4-dihydrobenzo39 and other molecularsolids such as C6Br6 (K0 = 9.07 GPa), anthracene (7.5 GPa), and hexamethylbenzene(7.2 GPa).40 Animations showing the pathof compression of each polymorph are available in the 1α and 1β).The variation of unit cell volume with pressure is shown for bothpolymorphs in a axis compressingby 13% for polymorph 1α and b axisby 17% for 1β. The changes in the b and c directions are similar for 1α at 6% and 5%, respectively, while the a axis changesby 8% and the c axis by 4% in 1β. In polymorph 1β the β angle decreasesby 1.5%, causing sin β to increase over the pressure series and double bonds (1530 to 1660 cm–1). Elsewhere, bands between 300 and 1100 cm–1 are at similar wavenumbers despite minor relative intensity changes.Since polymorphs 1β) and 5.43 GPa (1β) from those seen in the diffractionexperiments  is probably areflection of the differing hydrostatic properties of the pressure-transmittingmedia, nujol and 4:1 methanol–ethanol in the two experiments.The vibrational spectra are recovered upon releasing the pressure and another where these forms can be fingerprinted(∼1600 cm–1). The same effects are seen inother spectral regions.A more detailed analysis of the Raman spectra isenabled by deconvolutionof the bands into a set of Lorentzian profiles. As the pressure dependenceof the Raman spectra is quite subtle, the analysis will focus on oneregion where there is little difference between the polymorphs .Although an acceptable fit to asingle equation ofstate acrossthe full pressure range can also be obtained for the network volumeof  0.8 GPa 6a. The n1β, which consists of two separate linearregions .A similar, but much clearer, discontinuity occursin the networkvolume of regions 6babove 1α at 0.8GPa and in 1β above 2.1 GPa are related to a switchin which the mechanism of compression shifts toward the network ofintermolecular interactions. The discontinuity in compressibilityrather than the unit cell volume itself implies that both transitionsare second order.The discontinuities suggest, in agreement with theRaman data,that the phase transitions occurring in 3.51α at ambientpressure is 268.2(2) Å3 and at 5.76 GPa is 267.6(3)Å3. The molecular volume for 1β at ambient pressure is 267.8(2) Å3 and at 7.42 GPais 267.6(3) Å3. The small magnitude of these differencesindicates that most of the reduction in the network volume in bothpolymorphs is due to intermolecular compression.The molecular volume for polymorph 39 a first order phase transitionat 5.34 GPa was driven by the onset of rotation of the N-phenyl rings.The ring substituents in polymorphs 1α and 1β rotate in a similar fashion with increasing pressure,but the rotation is continuous. The N-Pyr torsion in 1α decreases from 39.0(3)° at ambient to 32.1(7)° at 4.40GPa and then increases to 36(3)° at 5.76 GPa. In 1β, the angle changes from 37.9(5)° to 28.7(7)° from ambientto 7.42 GPa, with no discontinuity. The C-Ph torsion angle changesfrom −13.8(3)° to −16(4)° between ambientand 5.76 GPa in 1α and from −1.6(2)°to −1.5(4)° in 1β. Plots of each torsionangle with pressure are available in the In Blatter’sradical itself,41 The hinge angle  shows distinct and oppositereactions to pressure increase, 1α exhibits anangle increase by 5.98°, while in 1β it decreasesby 2.13°  isalso the most compressiblein polymorph  D and F 8. The laM0) of each interactionwas calculated using third order Birch–Murnaghan EoSs fittedto the centroid distances from the entire pressure series for 1α .The already soft interactions A and D compress ideally, while forF the deviation from ideality is relatively modest. The phase transitionin 1β is thus associated with a general softeningof the more incompressible contacts, rather than a step change inany one contact.Attempts to fit the data for 1α occursat only 0.8 GPa, and as shown in 1β. Asa result, all interactions in polymorph 1α appearto conform to ideal behavior across the entire pressure range studied.The phase transition in polymorph 4e]triazin-4-yl polymorphs, 1α and 1β, have been investigated up to 5.76 and 7.42 GPa,respectively. The strongest contacts in both polymorphs are π-stackinginteractions, but the crystallographic axes along which they are distributedexperience the most compression at elevated pressure. Perpendicularto this direction, the compression is governed by the distributionof interstitial voids.The crystal structuresof the 3-phenyl-1-(pyrid-2-yl)-1,4-dihydrobenzo[As is the case with other molecular structures,the path of compressionin both polymorphs involves reduction of the void space. This is veryclearly illustrated by the difference in the bulk moduli of the voidvolume, in the region of 2 GPa for both phases, compared to the occupied“network” volume, ∼100 GPa in both cases, similarto the total bulk modulus of a first-row transition metal.1β above 2.1 GPa. Analysisof the intermolecular contact distances shows that this change isassociated with a shift in the mechanism of compression toward theinitially less compressible intermolecular contacts. Specific contactscould be identified by their deviations from an ideal model of compressionexpressed in a linear third order Birch–Murnaghan equationof state. The term “linear” here refers to the variationof distance with pressure instead of volume, rather than indictingthat the equation of state follows a straight line.Althoughno first order phase transitions occur for either polymorph,there is a very clear discontinuity in the vibrational frequenciesand network volume of 1α. TheRaman data show a very clear anomaly at 0.8 GPa, but correspondingdiscontinuity in the network volume is very slight and, in all candor,was not detected until the Raman data were available. There are noobvious deviations from ideality in the compression of the intermolecularcontacts.The situationis more ambiguous for 1 show subtle, second order,isosymmetric phase transitions. The results obtained here demonstratethe great sensitivity of Raman spectroscopy to such transitions andsuggest that the trends in the occupied and unoccupied volumes ofthe crystal structure together with deviations from ideal behaviorin the intermolecular contacts can both help identify their structuralsignatures. These results mirror similar results obtained for thearomatic hydrocarbon naphthalene, where trends in occupied volumewith pressure revealed38 the structuraleffects of a phase transition which had been debated in the literaturesince Bridgman’s first investigation in 1938,42 reconciling crystallographic and spectroscopic data. Thepresent study also demonstrates the limitations of this approach ifa transition occurs at relatively low pressure before a clear volumetrend or equation of state can be established.High-pressure phase transitions are far from uncommonin molecularmaterials, but unless there is a change in space group or a discontinuityin the unit cell dimensions or other structural features, it can bedifficult to discern a clear structural difference between the twophases. Both polymorphs of


An electrochemical-DNA (E-DNA) sensor was constructed by using DNA metallization to produce an electrochemical signal reporter in situ and hybridization chain reaction (HCR) as signal amplification strategy. The cyclic voltammetry (CV) technique was used to characterize the electrochemical solid-state Ag/AgCl process. Moreover, the enzyme cleavage technique was introduced to reduce background signals and further improve recognition accuracy. On the basis of these techniques, the as-prepared E-DNA sensor exhibited superior sensing performance for trace ctDNA analysis with a detection range of 0.5 fM to 10 pM and a detection limit of 7 aM. The proposed E-DNA sensor also displayed excellent selectivity, satisfied repeatability and stability, and had good recovery, all of which supports its potential applications for future clinical sample analysis. Liquid biopsy has long been accepted as an advanced technique to obtain testing samples from body fluids because of its convenience and low invasiveness ,2,3; it Electrochemical DNA (E-DNA) sensors exhibit natural advantages for ctDNA detection due to their convenient construction, low cost, time saving, and high specificity. In addition, these sensors are easy to miniaturize into portable instruments for point-of-care testing ,18,19. IIn this study, taking KRAS gene as a ctDNA model, an E-DNA sensor was constructed for ctDNA sensing by fully integrating the advantages of DNA metallization ,27, HCR,3Fe(CN)6, K4Fe(CN)6, and N,N,N′,N′-Tetramethylethylenediamine (TEMED) were obtained from Shanghai Macklin Biochemical Co., Ltd. HNO3, AgNO3 and HCl were obtained from Shanghai Lingfeng Chemical Reagents Co., Ltd. Orthoboric acid was prepared from Sigma-Aldrich (Shanghai) Trading Co, Ltd. MCH was obtained from Shanghai Yien Chemical Technology Co., Ltd. NaNO3, Mg(NO3)2·6 H2O and H2SO4 were obtained from Sinopharm Chemical Reagent Co., Ltd. NaCO3 was obtained from Shanghai Runjie Chemical Reagent Co., Ltd.Acrylamide, N,N′-Methylenebisacrylamide, Tris Base, and NaCl were obtained from Shanghai Aladdin Bio-Chem Technology Co., Ltd. Ammonium persulfate, HCHO, KAll the sequences used in this study were synthesized and offered by Sangon Biotech Co., Ltd.  . Exo Ι wThe measurements of cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were performed on an Autolab PGSTAT 302 N electrochemical station . A polyacrylamide gel electrophoresis experiment was used on a DYCZ-25 D electrophoresis apparatus  and imaged on a Tanon 2500 BR Multifunctional gel imaging analyzer . The determination of the nucleic acids’ concentration was conducted with a UV-1900 spectrophotometer . Chronocoulometric experiments were conducted at a CHI 660 E electrochemical workstation .2SO4, 0.01 M KCl/0.1 M H2SO4, and 0.05 M H2SO4).An Au electrode was polished by 0.3 μm and 0.05 μm aluminum oxide suspension and cleaned via muti-step electrochemical cyclic voltammetry cleaning steps of four different solutions 2·6H2O and 100 mM NaNO3 in 20 mM HEPES (pH 7.4). The HCR solution contains 0.5 µM initiator solution and 1.5 µM hairpin H1 and H2 mixture.First, the Au electrode was dropped by a 6 μL capture probe (0.05 μM) and incubated for 1.5 h at room temperature. Then, 6 µL MCH (1 mM) was dripped onto the Au electrode and incubated for 20 min to fill the blank area and prevent unspecific absorption. In the following step, 6 µL target solution was dropped on the electrode for 1 h. Then the electrode was rinsed with ultrapure water. Furthermore, the modified electrode was incubated with Exo Ι (0.1 U/mL) for 10 min at 37 °C to cleavage the capture probes, which were not hybridized to duplex with the target. Then the electrode was again rinsed with ultrapure water. 6 µL initiator solution (0.5 µM) was put on the electrode for 1 h to leave the trigger domain to trigger subsequent aggregation between hairpins H1 and H2. After the electrode was rinsed again with ultrapure water, hairpins H1 and H2 were annealed in their own tubes by heating to 95 °C for 5 min, then slowly cooled down to room temperature. Afterward, we mixed them to 1.5 µM by DNA hybridization buffer for further use. Finally, the mixed solution (1.5 µM) was applied to the electrode incubation for 1.5 h to generate HCR. Then the electrode was rinsed once more with ultrapure water to proceed with the later operation. The DNA hybridization buffer and HCR buffer contain 50 mM Mg(NO3[Fe(CN)6] containing 1 M KCl, at a 0.1 V/s of scan rate and ranging from −0.2 V to 0.7 V. Electrochemical impedance spectroscopy (EIS) was conducted in 10 mM K3[Fe(CN)6]/K4[Fe(CN)6] containing 1 M KCl. The parameters for the EIS were set as 0.01 V of the amplitude and 0.01 Hz to 1 × 105 Hz of the frequency range. The Chronocoulometric experiment was operated in 10 mM Tris-HCl (pH 7.4) containing 50 μM RuHeX. The three-electrode system was applied in the electrochemical measurements used, including Au electrode as the work electrode, Ag/AgCl (3 M KCl) as the reference electrode, and platinum wire as the auxiliary electrode. Cyclic voltammetry (CV) was carried out in 10 mM K3 solution (100 µM) for 1 h and the electrode was then rinsed with ultrapure water. Subsequently, the electrode was soaked in the freshly prepared NaBH4 (10 mM) for 10 min to reduce the Ag ions to Ag on the DNA strand. Then the electrode was rinsed with ultrapure water. The electrode was placed in 0.1 M NaCl containing 0.1 M NaNO3 as electrolyte and measured ranging from −0.2 to 0.4 V.For the DNA metallization, the modified electrode was immersed in AgNO3, 0.2% AgNO3, developer) and scanned in a gel imaging analyzer.5 μL HCR product with 1 μL loading buffer was analyzed in 1 × TBE buffer by 15% PAGE , 10% Ammonium persulfate (APS), TEMED). The conditions of PAGE were 100 V of voltage and 40 min of time. Gel electrophoresis was stained using the silver-stained method . The results showed that lanes 1, 3, 4, 5, and 6 corresponded to the five components  participating in HCR reaction, respectively , while t+ is reduced to a metal cluster in situ. The implementation of this method is restricted to a negatively charged ribose phosphate backbone, which possesses excellent selectivity and avoids interference caused by nonspecific signals, thus improving accuracy. 6]3−/4− at the electrode was altered and the current was increased, as shown in 6]3−/4− (the new peak appeared in curve b). Additionally, the decrease in the EIS Ret value (from over 2000 Ω to about 70 Ω) can be observed clearly  is one of the key indicators to evaluate the sensing performance of the established sensor. Although DNA metallization is an excellent technique to amplify electrochemical signals, it is often subject to the factor of excessive background signals. Exo Ι can digest a capture probe from 3′ to 5′ direction, while the cutting products of the capture probe would eventually be removed from the surface by a buffer. From The extended duplex DNA via HCR reaction created more sites for DNA metallization, which amplified the signal. Under the same target concentration condition (1 nM), the current without HCR amplification was about 1.2 µA while the current with HCR treatment was about 4.9 µA a. When t2) was 0.992. The linear correlation curve was described as y = 1.0399 x + 0.5314. According to the calculation basis (3σ rule), the final LOD was calculated to be as low as 7 aM, which was lower than in previous studies, indicating that the proposed sensor exhibited excellent sensitivity  and the RSD (3.8–6.5%) were achieved , exhibitIn summary, an EC-HCR sensor was proposed by using target recognition–induced in situ generation of silver nanoparticles as electrochemical probes for ctDNA KRAS gene detection, while HCR was used to amplify the output signal, and enzyme digestion technique was applied to suppress the background signal. Silver metallization can be used to indicate DNA composition like traditional electrophoresis, providing a specific-target nucleic acid–dependent electrochemical signal. On the basis of the above design, the as-prepared electrochemical DNA sensors exhibited wider linear detection range and lower detection limit. They also displayed outstanding selectivity, repeatability, and stability. This recovery experiment has huge potential for the future analysis of clinical samples.


Saccharomyces cerevisiae and further restrict the development of industrial bioethanol production. Transcription factors are regarded as targets for constructing robust S. cerevisiae by genetic engineering. The tolerance-related transcription factors have been successively reported, while their regulatory mechanisms are not clear. In this study, we revealed the regulation mechanisms of Haa1p and Tye7p that had outstanding contributions to the improvement of the fermentation performance and multiple inhibitor tolerance of S. cerevisiae.Various inhibitors coexist in the hydrolysate derived from lignocellulosic biomass. They inhibit the performance of HAA1-overexpressing strain s6H3 (H), and the TYE7-overexpressing strain s6T3 (T). The expression of the pathways related to carbohydrate, amino acid, transcription, translation, cofactors, and vitamins metabolism was enhanced in the strains s6H3 and s6T3. Compared to C_H vs. C_S group, the unique DEGs in AFur_H vs. AFur_S group were further involved in oxidative phosphorylation, purine metabolism, vitamin B6 metabolism, and spliceosome under the regulation of Haa1p. A similar pattern appeared under the regulation of Tye7p, and the unique DEGs in AFur_T vs. AFur_S group were also involved in riboflavin metabolism and spliceosome. The most significant difference between the regulations of Haa1p and Tye7p was the intracellular energy supply. Haa1p preferred to enhance oxidative phosphorylation, while Tye7p tended to upregulate glycolysis/gluconeogenesis.Comparative transcriptomic analyses were applied to reveal the regulatory mechanisms of Haa1p and Tye7p under mixed sugar fermentation conditions with mixed inhibitors [acetic acid and furfural (AFur)] or without inhibitor (C) using the original strain s6 (S), the HAA1 or TYE7. The positive perturbations of energy and amino acid metabolism were beneficial to the improvement of the fermentation performance of the strain. Furthermore, strengthening of key cofactor metabolism, and transcriptional and translational regulation were helpful in improving the strain tolerance. This work provides a novel and comprehensive understanding of the regulation mechanisms of Haa1p and Tye7p in S. cerevisiae.Global gene expressions could be rewired with the overexpression of The online version contains supplementary material available at 10.1186/s12934-022-01822-4. Saccharomyces cerevisiae has superior ethanol fermentation performance, but its xylose metabolism and stress tolerance are two key bottlenecks restricting the development of lignocellulosic bioethanol [S. cerevisiae with xylose metabolic ability, which improves the utilization of lignocellulose biomass [S. cerevisiae, can not only improve the conversion efficiency of the fermentable sugars to ethanol but also reduce the stringent requirements in the biomass pretreatment process [Bioethanol production using lignocellulosic biomass as feedstock could ease the pressure on fossil energy consumption and further advance carbon neutrality plans . Saccharoethanol , 3. The  biomass . However biomass –7. There process .S. cerevisiae, ethanol production is still not ideal when using lignocellulosic biomass as feedstock due to the heterogeneous composition and the multifarious inhibitors in pretreated slurries [Various inhibitors  are inevitably released in hydrolysate with the dissolution of the fermentable sugars in the pretreatment process , 10. Theslurries , 17.S. cerevisiae overexpressing TF of SFP1 or ACE2 could be increased by 300–400% when fermenting in a synthetic medium with acetic acid and furfural [HAA1 [MSN2/4 [TYE7 [YAP1 [S. cerevisiae.Transcription factors (TFs) can involve in the regulation of genes, which is a feasible target for constructing robust strains using genetic engineering. The ethanol productivity of furfural . Besidesal [HAA1 , 18, MSN [MSN2/4 , TYE7 [5E7 [YAP1 , 21, etcS. cerevisiae. At present, those revealed regulatory mechanisms of the key TFs are limited to the conditions with single sugar and single inhibitor [S. cerevisiae are different among various fermentable sugars  under the conditions with/without inhibitor [S. cerevisiae is more sensitive to inhibitors in the xylose fermentation stage than that in the glucose fermentation stage when mixed sugars are fermented [Understanding the regulatory mechanism of TF is very important for improving the robustness of nhibitor –24. Whilnhibitor , 25–27. ermented , 7. ConsHAA1 and TYE7 in the parental strain s6 using the CRISPR/Cas9 gene engineering method [TPO2, TPO3, YRO2, etc.) [HAA1 is involved in cellular copper/iron ion homeostasis [In our previous study, two tolerant strains s6H3 and s6T3 were constructed by respectively overexpressing g method , 28. Mixg method , 18. Haa2, etc.) . Some tr2, etc.) , 23. GO eostasis , and poseostasis , 31. HowENO1 and ENO2 (enolase), TDH , PGK1 (phosphoglycerate kinase), PGM1 (phosphoglycerate mutase), PYK1 (pyruvate kinase), and TPI1 (triosephosphate isomerase) [Tye7p, a transcriptional activator, contributes to glycolytic genes activation, such as omerase) , 33. Resomerase) , 35. HowS. cerevisiae.In the present study, the regulatory mechanisms of Haa1p and Tye7p under the conditions with/without the mixed acetic acid and furfural were studied by comparative transcriptomics analysis using the strains s6, s6H3, and s6T3 when fermenting mixed glucose and xylose , 28. TheCompared with the parent strain s6, the strains s6H3 and s6T3 had much better fermentation performance in 10% YPDX  medium with/without mixed acetic acid (2.4 g/L) and furfural (1.9 g/L)  Haa1p and Tye7p in enhancing the fermentation performance and inhibitor tolerance of the strains, we designed comparative transcriptomic experiments by focusing on mixed glucose and xylose fermentation with or without mixed acetic acid and furfural. The fermentation process with mixed sugars as carbon sources could be divided into the glucose fermentation stage and the xylose fermentation stage. The effect of glucose repression lasted after glucose depletion. The transcriptional response of the glucose stage caused disturbance to the subsequent xylose stage, resulting in specific transcriptomic profiles . ConsideS. cerevisiae S288C after quality control for the raw data , and the results were consistent with the results of the transcriptome analysis, suggesting that the transcriptomic results were reliable , s6H3 (H), and s6T3 (T) were C_S, C_H, and C_T under the condition without inhibitor (C), and AFur_S, AFur_H, and AFur_T under the condition with mixed acetic acid and furfural (AFur), respectively. The transcriptome data were aligned with HAA1 and TYE7 in strains s6, s6H3, and s6T3 under the conditions with/without mixed acetic acid and furfural were shown in Table HAA1 and TYE7 were significantly increased under the initiation of UBI4 promoter (PUBI4), which has little fluctuation under mixed acetic acid and furfural stress. These results indicated that the expressions of HAA1 and TYE7 were upregulated by PUBI4. To reveal the regulation mechanisms of Haa1p and Tye7p, the differences in genome expression profiles of the groups C_H vs. C_S, AFur_H vs. AFur_S, C_T vs. C_S, and AFur_T vs. AFur_S were analyzed based on differentially expressed genes (DEGs) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment. The DEGs were filtered with a threshold of false discovery rate (FDR) < 0.05 and a fold change (Sample B/Sample A) ≥ 1.5. The KEGG enrichment analysis was accorded to the KEGG database, and the enriched pathways were filtered with a threshold of enrichment ratio (E) ≥ 0.1 and/or P < 0.05.The fragments per kilobase of exon per million reads mapped (FPKM) values of HAA1, especially in the presence of inhibitors. The distribution of the DEGs in these two groups were shown in Fig. The numbers of DEGs were 234 and 629 in C_H vs. C_S and AFur_H vs. AFur_S groups, respectively, in which 154 (234) and 517 (629) were upregulated. This result suggested that the transcription process was significantly affected by the overexpression of P < 0.05 from those pathways that only meet E ≥ 0.1, two radar maps were drawn to reveal the regulation mechanism of Haa1p in improving the sugar consumption performance and inhibitor tolerance . There were 17 specific pathways when pathways from these two groups were combined into a whole. Only starch and sucrose metabolism was concurrently enriched in these two groups. In C_H vs. C_S group, the enriched pathways were mainly involved in carbohydrate metabolism , amino acid metabolism , lipid metabolism (fatty acid biosynthesis), cofactors and vitamins metabolism (nicotinate and nicotinamide metabolism), and cell growth and death (necroptosis). While carbohydrate metabolism , amino acid metabolism (tryptophan metabolism), energy metabolism (oxidative phosphorylation), cofactors and vitamins metabolism (vitamin B6 metabolism), nucleotide metabolism (purine metabolism), and transcription (spliceosome) were significantly enriched in AFur_H vs. AFur_S group.These DEGs were used for KEGG enrichment analysis. In AFur_H vs. AFur_S group, the number of the enriched pathways (E ≥ 0.1) was much larger than that in C_H vs. C_S group Fig. . Screenence Fig. A. 11 andTo explore new regulation perspectives of Haa1p in the conditions with or without inhibitors, the KEGG enrichment analysis of the DEGs in the different regions of the Venn plot was discussed in detail Fig. . The shaHAA1, the overexpression of TYE7 seemed to have a temperate influence on the transcription regulation in the presence of inhibitors. The proportion of the shared DEGs (86) was 38.22% and 33.33% of the total DEGs in C_T vs. C_S and AFur_T vs. AFur_S groups, respectively  and 200 (258) were upregulated. Compared with the overexpression of P < 0.05 were further screened to draw the radar map. As shown in Figs. KEGG enrichment analysis showed that the number of the enriched pathways (E ≥ 0.1) in AFur_T vs. AFur_S group was larger than that in C_T vs. C_S group Fig. . The patThe shared DEGs  were mainly involved in biotin metabolism, suggesting this pathway should occupy key loci in Tye7p regulation Fig. ; Table 2Combined with the results of the KEGG enrichment analysis for the total DEGs and the regional DEGs, the key role of central carbon metabolism and amino acid metabolism in improving strain performance was demonstrated Figs. ; Table 2The DEGs regulated by Haa1p and Tye7p were mixed with some potential TFs. 13, 20, 8, and 5 potential TFs were picked up from the DEGs in C_H vs. C_S, AFur_H vs. AFur_S, C_T vs. C_S, and AFur_T vs. AFur_S groups, respectively  was overexpressed, positive perturbations were activated at the cellular level. The regulatory mechanisms of Haa1p and Tye7p were visually presented based on those key DEGs and KEGG pathways  was “1 + 1 < 2” rather than “1 + 1 > 2” , XYL2 (xylitol dehydrogenase) from Scheffersomyces stipitis as well as XKS1 (xylulokinase) from S. cerevisiae) and pIWBGL1 (contains BGL1 (β-glucosidase) from Aspergillus aculeatus) in the industrial flocculating yeast strain KF-7 [HAA1 alone (UBI4P-HAA1-HAA1T), overexpressing TYE7 alone (UBI4P-TYE7-TYE7T), and co-overexpressing HAA1 and TYE7 , respectively, in the parental strain s6 in our previous work [us study . Strain ain KF-7 . Strainsous work .g, 2 min), and transferred into 100 mL of 10% YPDX medium  in a 300-mL conical flask. Flasks were incubated in a thermostat water bath (35 °C). The broth in flasks was stirred (200 rpm) using a magnetic stirring system. If necessary, acetic acid (2.4 g/L) and furfural (1.9 g/L) were added to the sterilized medium. The batch fermentation method was described previously [The strains s6, s6H3, and s6T3 were activated at 30 ℃ on the 2% YPD-agar plate . After 24 h, a loopful of cells was transferred into a 500-mL conical flask with 100 mL of 5% YPD medium , and cultivated aerobically for 16 h  in a shaker. Fresh cells (0.5 g dry cell weight (DCW)) were collected by centrifugation (8000×www.majorbio.com) after sequencing in Shanghai Majorbio Bio-pharm Technology Co., Ltd.Cells used for RNA extraction were collected at 7 h from the control , and mixed acetic acid (2.4 g/L) and furfural (1.9 g/L) (AFur) groups. The methods for extracting and measuring total RNA and RNA-seq were performed as previously described . Three iADY2, ATO2, BTN2, ENO1, ENO2, and HSP30, with varied transcript abundance, were chosen to quantify the relative expression levels  were used to search for genes that have been experimentally shown to be regulated by the TFs from documented associations in the YEASTRACT database. The analysis was conducted as previously described [Quantified gene expression results used FPKM (fragments per kilobase of exon per million reads mapped) as a unit. The gene filtered with a threshold of false discovery rate (FDR) < 0.05 and a fold change (Sample B/Sample A) ≥ 1.5 were considered as differentially expressed genes (DEGs). The KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway terms with a escribed .The concentrations of glucose, xylose, and ethanol were determined as previously described . GlucoseAdditional file 1: Fig. S1. The glucose (A and D), xylose (B and E), and ethanol (C and F) concentration curves of strains s6, s6H3, and s6T3 underthe condition without inhibitor  and the condition with mixed acetic acid and furfural . Black squaresrepresent strain s6; red circles represent strain s6H3; green upwards triangles represent strain s6T3 (Ref. 5). Fig. S2. Evaluation of inhibitor tolerance of strains by batch fermentation using 10% YPDX medium (A), 10% YPDX medium containing mixed acetic acid and furfural (2.4+1.9 g/L) (B),and pretreated corn stover slurry (Ref. 5) (C). Black squares represent strain s6; red circles represent strain s6H3; blue upwards triangles represent strain s6T3, green stars represent strain s6H3T10. Fig. S3. The cluster graph of expression pattern of DEGs in each group. C_S_1, C_S_2, and C_S_3 represent the three biological replicates of strain s6 in the control (C) group, Afur_S_1, Afur_S_2, and Afur_S_3 represent the three biological replicates of strain s6 in mixed acetic acid and furfural (Afur) group; C_H_1, C_H_2, and C_H_3 represent the three biological replicates of strain s6H3 in control (C) group, Afur_H_1, Afur_H_2, and Afur_H_3 represent the three biological replicates of strain s6H3 in mixed acetic acid and furfural (Afur) group; C_T_1, C_T_2, and C_T_3 represent the three biological replicates of strain s6T3 in control (C) group, Afur_T_1, Afur_T_2, and Afur_T_3 represent the three biological replicates of strain s6T3 in mixed acetic acid and furfural (Afur) group. Fig. S4. Validation of transcriptome data by RT-qPCR. The changed fold means the ratio of the expression level of a specific gene in the experimental group to that in the control group.The ACT1 expression level was used as a reference in RT-qPCR. Fig. S5. Venn diagrams of the enriched pathways when overexpressed Haa1p and Tye7p, respectively. The black font represents the enriched pathways, and the red font represents the classification of each pathway. Table S1. The results of transcriptome data alignment with S. cerevisiae S288C. Table S2. The differently expressed TFs in C_H vs. C_S (234), C_T vs. C_S (225), AFur_H vs. AFur_S (629), and AFur_T vs. AFur_S (258) groups. Table S4. The differently expressed genes involved in the key KEGG pathway in C_H vs. C_S, AFur_H vs.AFur_S, C_T vs. C_S, and AFur_T vs. AFur_S groups. Table S5. The primers used for RT-qPCR.Additional file 2: Table S3. The genes regulated by the potential TFs in C_H vs. C_S (13), C_T vs. C_S (20), AFur_H vs. AFur_S (8), and AFur_T vs. AFur_S (5) groups. The light yellow highlighted values are calculated by DEGs/genes (the number of the DEGs regulated by the TFs to the number of the genes regulated by the TFs) or DEGs/all the DEGs . The green highlighted genes are the DEGs that regulated by the TFs.


A dinickel catalyst promotes the [2 + 2 + 1]-cycloaddition of two aldehyde equivalents and a vinylidene. The resulting methylenedioxolane products can be deprotected in one pot under acidic conditions to reveal α-hydroxy ketones. This method provides convenient access to unsymmetrical alkyl-substituted α-hydroxy ketones, which are challenging to synthesize selectively using cross-benzoin reactions. Mechanistic studies are consistent with an initial migratory insertion of the aldehyde into a dinickel bridging vinylidene. Insertion of the second aldehyde followed by C–O reductive elimination furnishes the cycloadduct. Under dilute conditions, an enone side product is generated due to a competing β-hydride elimination from the proposed metallacyclic intermediate. A DFT model consistent with the concentration-dependent formation of the methylenedioxolane and enone is presented. A dinickel catalyst promotes reductive [2 + 2 + 1]-cycloaddition reactions using readily available 1,1-dichloroalkenes as vinylidene precursors. The observation that Zn can be replaced with Cp2Co indicates that the reaction does not require the formation of an organozinc intermediate.Reaction optimization studies were carried out using aldehyde 2 (2.0 equiv.) and 1,1-dichloroalkene 3 (1.0 equiv.) as model substrates. Under conditions similar to those previously employed in [2 + 1]-cycloaddition reactions, dinickel catalyst 7 affords methylenedioxolane 4 in 89% yield as an 8 : 1 mixture of diastereomers and with exclusively c alkene , entry 12O was critical for reaction efficiency. With DMA alone, the yield of 4 decreased to 47% (entry 4). Both the identity of the reductant and the solvent appeared to have a significant effect on d.r. but did not impact the high Z selectivity at the exocyclic alkene. Under standard conditions, a premetallated dinickel catalyst was used (7). However, the active catalyst could also be assembled in situ by stirring free i-PrNDI (8) and Ni(dme)Cl2 over Zn, and there was no significant change in reaction outcome (entry 5). Increasing (entry 6) or decreasing (entry 7) the steric profile of the NDI ligand decreased the yield of 4. Finally, Ni(dme)Cl2 alone or other mononickel catalysts bearing imine and/or pyridine donors analogous to those found in 7 proved to be ineffective in the reaction .During reaction development, we found that the inclusion of a nonpolar cosolvent such as Etanti/syn diastereomers.Following the [2 + 2 + 1]-cycloaddition, methylenedioxolane 4 was deprotected using TFA to form α-hydroxy ketone 5. This two-step sequence can be carried out in a single pot without the need to isolate the intermediate dioxolane (62% yield over the two steps). Alternatively, hydrogenation of methylenedioxolane 4 followed by Lewis acid-catalyzed deprotection yields diol 6 as a 3 : 1 ratio of 15 The benzoin reaction, which involves the Umpolung coupling of two aldehydes via an acyl anion intermediate, is one of the most common C–C coupling strategies used to form α-hydroxy ketones. In certain cases, it is possible to carry out selective cross-benzoin reactions by exploiting differences in the steric or electronic properties of the two partners.16 However, in cases where the two aldehydes are relatively unbiased, catalytic cross-coupling is generally not feasible. In these cases, a pregenerated acyl anion equivalent, such as a dithiane17 or a cyanohydrin,18 is required. The drawbacks of this approach are the need for additional synthetic steps and the use of a strong base  to generate the requisite anion.α-Hydroxy ketones are found in many biologically active natural products .15 The bTo demonstrate the synthetic utility of this α-hydroxy ketone synthesis, we explored the substrate scope of the one-pot [2 + 2 + 1]-cycloaddition and deprotection sequence . The rea19 Phenylacetaldehyde is an effective substrate, forming (±)-sattabacin (38) in 73% yield. Although free phenols are not tolerated, (±)-4-hydroxysattabacin (39) could also be synthesized in 41% yield by utilizing a TMS protecting group. The TMS group is conveniently removed under the same TFA conditions used to deprotect the dioxolane.As a synthetic application of this method, two acyloin-containing natural products were prepared.i-PrNDI)Ni2Cl (44), were carried out at two different concentrations. A mixture of 42 and 43 was formed at higher concentrations, but dioxolane formation was suppressed at lower concentrations.There is evidence supporting a metallacyclic intermediate in the [2 + 2 + 1]-cycloaddition similar to that proposed in the dinickel-catalyzed vinylidene–alkene cyclization reaction . When thd1 was used, the enone product was labelled exclusively at the α-carbon (>95% deuterium incorporation), confirming the fate of the hydrogen undergoing β-hydride elimination  level of theory, which had previously produced stationary points that corresponded closely to X-ray structures. Single point energy calculations were carried out with an empirical dispersion correction and an SMD model.20 In order to compare DFT methods, single point energy calculations were also carried out at the M06L/def2-TZVP level of theory , which is the aldehyde adduct of the putative Ni2(μ-vinylidene) intermediate. There are four possible migratory insertion transition states, differing in the orientation of the vinylidene and aldehyde substituents. The most favorable pathway has a barrier of 10.1 kcal mol−1 and generates Ni2 metallacycle 47a . Reductive elimination transition states from metallacycle 50 are prohibitively high in energy. However, dissociation of the ether oxygen and association of the C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>C π-bond to one of the Ni atoms would form to an isomeric metallacycle (51) that is similar in energy . Metallacycle 51 has an elongated Ni–Ni distance of 2.9 Å but a notably shortened distance between the C and O undergoing reductive elimination (2.5 Å vs. 2.7 Å in 50). Accordingly, C–O reductive elimination from 51 has a barrier of only 9.9 kcal mol−1, and this step yields the product adduct 53. Key to this overall process is the ability of the dinuclear active site to form and break weak Ni–Ni interactions in order to traverse different intermediates in the catalytic cycle.Binding of the second aldehyde to form 48 is exothermic by 3.8 kcal molE/Z selectivity of the reaction should be determined in this step. Consistent with the high Z selectivity observed experimentally, the migratory insertion transition state that would lead to the E product is 2.0 kcal mol−1 higher in energy. When this transition state was modeled with i-Pr groups on the catalyst instead of the Me truncation, the difference in energy increased to 3.1 kcal mol−1 (see ESIAccording to the calculated mechanism, the migratory insertion of the first aldehyde is rate-limiting and irreversible. Thus, the −1, making it significantly less favorable than the pathways leading to the methylenedioxolane or to the enone.To model the enone formation under dilute reaction conditions, we searched for β-hydride elimination transition states from intermediate 47. Isomer 47a cannot undergo β–hydride elimination, because the β–H is positioned away from Ni. However, isomer 47b, where the Me and H substituents of the aldehyde are swapped, is poised to undergo a nearly barrierless β-hydride elimination. Finally, we also examined the C–O reductive elimination from 47a, which would form a hypothetical methylene epoxide product. This step has a calculated barrier of 21.4 kcal molIn summary, a dinickel catalyst promotes [2 + 2 + 1]-cycloadditions of two aldehyde equivalents and a vinylidene to form methylenedioxolane products. Unlike the Pauson–Khand reaction, these cycloadditions involve an initial coupling of a vinylidene, which is the C1 component, and an aldehyde to form a metallacyclic intermediate. By avoiding competing reductive elimination and β-hydride elimination processes, it is possible to intercept this intermediate with a second aldehyde and achieve a net three-component cycloaddition. In principle, this mechanism should be compatible with other classes of 2π-systems, and future studies will focus on expanding the scope of partners that can be used.Conceptualization, C. U. and A. E. K.; methodology, C. U. and A. E. K.; investigation, A. E. K., M. L., and M. I. B.; writing – original draft, C. U. and A. E. K.; writing – reviewing and editing, C. U., A. E. K., and M. L.; supervision, C. U.; project administration, C. U. and A. E. K.; funding acquisition, C. U.There are no conflicts to declare.SC-013-D2SC02696A-s001


Moreover, GAA also suppressed H2O2 induced major matrix degrading proteases  associated with NP degradation. Additionally, we found NP protective ability of GAA by up-regulating extra cellular matrix anabolic factors like type II collagen (Col II) and aggrecan in NP cells. Furthermore, we also demonstrated that GAA suppressed the activation of TLR4/NLRP3 in H2O2-stimulated NP cells. Thus, our results demonstrate that GAA inhibited the H2O2 induced apoptosis, oxidative stress, and inflammatory responses through the depression of TLR4/NLRP3 signaling axis. GAA possess NP protective properties and may be of value in suppressing the pathogenesis of IVDD.As a multifactorial disease, intervertebral disc degeneration (IVDD) causes many spinal-related diseases, which causes disability in the workforce and heavy social costs all over the world. Recently, Ganoderic Acid A (GAA) has been reported to play many pharmacological effects. However, its effect on IVDD remains unclear. In the present study, our study determined that GAA significantly inhibited H  Ganoderic Acid A alleviates the degeneration of intervertebral discGanoderic Acid A suppresses the activation of TLR4/NLRP3 signal pathwayGanoderic Acid A suppresses nucleus pulposus cells apoptosis.Intervertebral disc degeneration (IVDD) is a disease that develops over age worldwide . The preOxidative stress is also considered to be another important factor in the development of IVDD . Targeti2O2-induced IVDD in nucleus pulposus (NP) cells via modulating the TLR4/NLRP3 signaling pathway and to demonstrate the protective effect of GAA on IVDD. It is significant for looking for novel therapeutic drug in the treatment of IVDD.Ganoderic Acid A (GAA), a triterpene extracted from the fungus Ganoderma lucidum ), has an2.First, NP tissues were macroscopically separated from the lumbar intervertebral disc of 4-week-old male Sprague-Dawley rats. And then cut the tissues into small pieces, washed with phosphate buffered salin (PBS) and incubated with 0.25% trypsin solution (containing 0.2% mg/ml type II collagenase) for 12 h at 37°C . NP cell2O2 for 24 h and the viability of the cells in order to determine the appropriate treatment concentration. Subsequently, 10 µl of CCK-8 reagent was added to the cells and incubated for 4 h according to the manufacturer’s protocol. The cell viability was detected at 450 nm using a microplate reader  [Cell counting kit (CCK)-8 assays  were performed to detect the viability of NP cells according to the manufacturer’s instructions. NP cells were treated with 0, 1, 2, 4, 8, 16, 32, or 64 μM GAA or 0, 25, 50, 100, 200, 400, or 800 μM HVT, USA) .2 at 37°C and then pretreated with GAA for 1 h, and then treated with 400 μM H2O2 for 24 h. Next, treated NP cells were collected, washed three times with cold PBS, resuspended in 100 μl 1× binding buffer (1 × 105 cells) with 5 μL Annexin V-FITC and 5 μL propidium iodide (PI), and incubated for 15 min in the dark at room temperature. Flow cytometry was applied to detect cell apoptosis [The NP cells were cultured in an incubator of 5% COpoptosis .TM Fluorometric TUNEL System  according to the manufacturer’s protocols to detect the apoptotic cells.NP cells were fixed and then indicated with 0.1% Triton X-100. NP cells were stained with DeadEndNP cell supernatant was obtained by centrifugation at 4,000 g for 5 min at 4°C. And the SOD and MDA were detected using kits and performed according to the manufacturer’s protocols . For SODDetermination of MDA was based upon the lipid peroxidation MDA Assay Kit . Cells were lysed and reacted with thiobarbituric acid. Absorbance of the supernatant was measured spectrophotometrically at 532 nm.Determination of GSH is based on the reaction of DTNB (5´5-dithiobis-(2-nitrobenzoic acid)) with GSH and yield a yellow colored chromophore; 5-thio-nitrobenzoic acid with a maximum absorbance at 412 nm.GPX catalyzes the reduction of hydroperoxides by utilizing GSH as a reluctant. Determination of GPX activity was carried out according to the method of Chiu et al . The act−ΔΔCt method was applied to analyze the relative expression levels.Total RNA was extracted from NP cells using Trizol reagent  and the purity of the extracted RNA was determined using the NanoDrop 1000 spectrophotometer . Then, the mRNA levels were detected using quantitative real-time reverse transcriptase PCR analyses with SYBR Premix Ex Taq . The primers were listed in The cells were extracted with radio-immunoprecipitation assay (RIPA) lysis buffer supplemented with Protease Inhibitor Cocktail, and total protein was obtained for subsequent analysis. The concentration of protein was determined using bicinchoninic acid (BCA) method, while protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane for labeling. After blocking with 5% nonfat milk for 1 hour, the membranes were incubated with primary antibodies against Bax , Caspase-3 , Bcl-2 , IL-6 , IL-1β , TNF-α , TLR2 , TLR4 , NLRP3 , Caspase-1  and β-actin  at 4°C overnight. The membranes were washed and incubated with the secondary antibodies at room temperature for 2 h. An enhanced chemiluminescence detection system  was finally used to determine the emission of the membrane. The western blot results were analyzed by Image J .Data were analyzed by GraphPad Prism Version 7.0 software. All values are expressed as the mean ± standard deviation (SD). Differences between the groups were analyzed by Student’s t test and analysis of variance (ANOVA). P-value <0.05 was considered as statistical difference.2O2 treatment. GAA was determined to be mainly mediated TLR4/ NLRP3 signaling activity to process its protective effect on IVDD.Our results demonstrated that GAA could inhibit NP cell apoptosis and inflammation mediators and oxidative stress mediators which was induced by HInitially, to investigate the cytotoxicity of GAA on NP cells, our findings indicated that pretreatment with GAA did no cell cytotoxicity when the concentrations below 16 μM for either 24 ) or 48 h2O2  1 h prior to H2O2.Moreover, we detected the cell viability treated with different doses of H2O2 ). There 2O2 (16.35%). Interestingly, pretreatment with GAA significantly suppressed the apoptosis rate induced by H2O2. Consistent with those results, pretreatment with GAA also could significantly inhibit the apoptosis-related protein expression (Bax and caspase-3), and promote the levels of anti-apoptotic protein (Bcl-2), which were induced by H2O2 treatment (2O2 in a dose dependent manner (As shown in reatment ). In addt manner ).Figure2O2-treated NP cells by qPCR (Inflammation is known as a key mechanism of IVDD pathogenesis . In this by qPCR ) and wes by qPCR ) analysi2O2 treatment notably inhibited the activity of glutathione (GSH), superoxide dismutase (SOD) and glutathione peroxidase (GPX). However, those were restored by GAA  in NP c2O2 ). Thus, 2O2-induced NP cell apoptosis, release of inflammatory cytokines, oxidative stress mediators, and major matrix-degrading proteases associated with NP degradation. Moreover, we reported the protective effect of GAA on the NP cells caused by the up-regulation of ECM anabolic factors via regulating the TLR4/NLRP3 signal pathway, which indicated that GAA may be a valuable therapeutic drug on IVDD.In this study, we demonstrated that GAA inhibited H2O2-induced mRNA expression of proteases and ECM marker components in NP cells. H2O2 stimulation led to a significant up-regulation of MMP3, MMP13, ADAMTS4 and ADAMTS5 mRNAs, whereas pretreatment with GAA significantly inhibited the expression of all of these enzymes. In contrast, H2O2 stimulation significantly down-regulated the mRNA expression of collagen II and aggrecan in NP cells, whereas pretreatment with GAA significantly enhanced their mRNA expression. ECM is a transcription factor that has biological effects in inflammation and oxidative stress. Research has shown the participation of H2O2 was used to induce the IVD model in NP cells [2O2 for 24 h as well as the oxidative stress and inflammation. Interestingly, GAA treatment suppressed the apoptosis and inhibited the release of inflammation factors and oxidative stress in NP cell.Recently, ECM degradation has been reported to play the important role in IVDD progression by matrix-degrading proteases ,25. HencNP cells . Consist2O2-induced NP cells; however, GAA administration could significantly inhibit TLR4/NLRP3 signaling.Recently, activation of the TLR4/NLRP3 pathway has been reported to cause the up-regulation of inflammation and oxidative stress related genes . TherefoIn this study, we determined that GAA processed its protective effect on IVDD by inhibiting TLR4/ NLRP3 signaling. This finding may provide a potential therapeutic drug for IVDD. However, the use of cell experiment puts some limitations on the transferability of the results to the human situation. Future studies will benefit from exploring a possible dose-dependent protective effect of GAA on IVDD of animal and human.Click here for additional data file.


Dexmedetomidine has controversial influence on cardiac electrophysiology. The aim of this study was to explore the effects of dexmedetomidine on perioperative cardiac electrophysiology in patients undergoing general anesthesia.1, D2, D3 receiving dexmedetomidine 1, 1, 0.5 μg/kg over 10 min and 1, 0.5, 0.5 μg/kg/h continuous infusion respectively, and control group (group C) receiving normal saline. Twelve-lead electrocardiograms were recorded at the time before dexmedetomidine/normal saline infusion (T1), loading dose finish (T2), surgery ending (T6), 1 h (T7) after entering PACU, 24 h (T8), 48 h (T9), 72 h (T10) and 1 month (T11) postoperatively. Cardiac circulation efficiency (CCE) were also recorded.Eighty-one patients were randomly divided into four groups: groups D2 in groups D1 and D2 while decreased at T7 and T8 in group D3 (P < 0.05), iCEB were decreased at T8 (P < 0.05). Compared with group D1, QTc at T2, T6, T7, T9 and T10 and iCEB at T8 were decreased, and CCE at T2-T4 were increased in group D3 significantly (P < 0.05). Compared with group D2, QTc at T2 and iCEB at T8 were decreased and CCE at T2 and T3 were increased in group D3 significantly (P < 0.05).Compared with group C, QTc were significantly increased at TDexmedetomidine at a loading dose of 0.5 μg/kg and a maintenance dose of 0.5 μg/kg/h can maintain stability of cardiac electrophysiology during perioperative period and has no significant adverse effects on CCE.ClinicalTrials.gov NCT04577430 (Date of registration: 06/10/2020). The loading dose of dexmedetomidine was infused intravenously at a constant speed with an infusion pump for 10 min.The patients were assigned to four groups using a computer-generated number table randomly: dexmedetomidine loaded with 1 μg/kg and maintained with 1 μg/kg/h (group DPO2) after entering the operating theater. Venous access was established in a wrist cephalic vein with a 20-G intravenous cannula . Ringer’s solution was transfused at a rate of 10–15 ml/kg/h. For those with normal Allen’s test results, the left radial artery was punctured under ultrasound guidance after local anesthesia to monitor the invasive blood pressure (IBP). IBP and cardiac function monitoring equipment  was connected to the arterial line. Dexmedetomidine or normal saline loading dose was initiated after 5 min’s rest. In each group, the loading dose was infused intravenously for 10 min before induction of anesthesia, and then the maintenance dose was initiated. An anesthetist took charge of the preparation of dexmedetomidine and saline, which were covered with a towel when on the infusion pump. Another anesthetist who was blinded to the drugs and group allocations was responsible for data collection and intraoperative management. The patients and surgeons were both blinded to group allocations. All research data were recorded at 8.00 a.m. - 12.00 a.m.No patient received preoperative medication. All patients were monitored routinely with electrocardiogram (ECG), non-invasive blood pressure (NBP) and pulse oxygen saturation . General anesthesia was maintained with intravenous propofol 4–12 mg/kg/h, remifentanil 5–15 μg/kg/h, and dexmedetomidine/normal saline with the corresponding doses in each group. Muscle relaxation was maintained with intermittent intravenous injection of rocuronium. The depth of anesthesia was monitored with Narcotrend monitoring , the stage of which was maintained between D2 to E1 (sedation index 20 – 46) in all groups. Propofol and remifentanil were infused until surgery ending, and dexmedetomidine/saline and rocuronium stopped infusion at about 30 min before the end of surgery. Propofol and remifentanil doses were adjusted according to anesthesia depth from the Narcotrend monitoring. If mean arterial pressure (MAP) decreased > 20% of baseline value, ephedrine or phenylephrine was used. If HR decreased > 20% of basic value, atropine was injected. Cases were excluded if vasoactive drugs or atropine was used. Patients were called their names every 1 min until eyes opening after surgery finish. The tracheal tube was removed when the patient met extubation indications. All subjects were then transferred to the post-anesthesia care unit (PACU).Total intravenous general anesthesia was induced with midazolam 0.05 mg/kg, propofol 1.0–2.0 mg/kg, sufentanil 0.2–0.4 μg/kg and rocuronium 0.6 mg/kg. After sufficient muscle relaxation, the tracheal tube was inserted and fixed and then mechanical ventilation was performed. The ventilator parameters were set at inhaled oxygen concentration 60%, tidal volume 6–8 ml/kg, respiratory rate 12–14 times/min, inspiration/expiration ratio 1:2, positive end-expiratory pressure 3 cmH1), dexmedetomidine loading dose finish (T2), surgery beginning (T3), 30 min after surgery beginning (T4), 1 h after surgery beginning (T5), surgery ending (T6) and 1 h after transferring to PACU (T7). The twelve-lead ECG was measured at T1, T2, T6, T7, 24 h (T8), 48 h (T9), 72 h (T10) and 1 month (T11) postoperatively using the digital electrocardiograph  with ECG paper speeding at 25 mm/s and the gain at 10 mm/mv. Measurements and analyzation of the ECG were manipulated by an electrophysiologist who was blinded to group allocations. Each ECG in leads II and V5 was measured for 3 complete P-QRS-T cycles, and the averages were recorded respectively. The PR in lead II interval was measured, and the intervals of QRS, QT, QTc and Tp-e were measured in lead V5. The QT interval was measured from the start of QRS complex to the T wave ending, and the T wave ending was the intersection of the descending branch tangent and the baseline. If U waves appeared, the T wave ending was regard as the nadir of the curve between T and U waves. QTc was calculated based on the Bazett formula [1, T2, T5, T6 and T7, and changes in potassium (K+) and ionized calcium (iCa2+) levels were recorded. The surgery time, duration of anesthesia  and waking time from surgery were recorded, respectively. The dosages of propofol and remifentanil, total fluid intake from entering the operating theater to PACU and use of vasoactive drugs were recorded. The occurrence of arrhythmia during perioperative period was also recorded.Cardiac circulation efficiency (CCE), maximum pressure gradient (dp/dt), HR, MAP, and systemic vascular resistance (SVR) were recorded at the following time points: just before dexmedetomidine infusion  was applied to sample size calculation in this research. QTc was the main research indicator, showing a mean ± standard deviation (SD) of 1.0 ± 12.2 ms after dexmedetomidine loading dose used in our pilot study. As a result, at least 15 patients should be included in every group to reach a power of 80% (β = 0.2) with two-tailed significance level of less than 0.05. An additional 20% was added to make up loss by protocol violations, 18 patients were recruited per group to minimize the impact of missing data consequently.χ2) test or Fisher's exact test. Bonferroni correction was used for group differences between baseline and each time point. Two-sided P values of less than 0.05 were considered to be significant.SPSS 23.0  was used for complete statistical analysis. Data with normal distributions were expressed as means ± SD. One-way analysis of variance (ANOVA) was used for comparisons between groups (variance homogeneity), and Welch’s ANOVA was used for variance nonhomogeneity. Repeated measurement ANOVA was used for repeated measurements data in groups, and categorical data were compared by chi-square (P > 0.05). No significant differences were observed in clinical characteristics including surgery time, duration of anesthesia, waking time from surgery, total fluid intake from the operating theater to the PACU or patients with hypertension   (Table + and iCa2+ concentrations existed in any group (P > 0.05)  were available for the analysis finally. Figure 1. Compared with T1, the PR intervals were prolonged significantly at T2, T6 and T7 in group D1 , at T2 in group D2 (P < 0.001), and prolonged at T2, T6, and T7  but shortened significantly at T10 (P = 0.008) in group D3. Compared with T2, the PR intervals were shortened significantly at T8-11 in groups D1 and D2  and at T9-11 in group D3 .PR intervals in all groups were normal at T2 in groups D1 and D2 (P < 0.001). Compared with groups D1 and D2, the PR intervals were shortened significantly at T2 in group D3 .Compared with group C, the PR intervals were prolonged significantly at TP > 0.05).There were no significant differences in QRS intervals within and among the four groups , at T2 and T6 in group D2 , and at T6-8 in group C (P < 0.01), while shortened significantly at T2 and T10 in group D3 . Compared with T2, QTc decreased significantly at T10 and T11 in group D1 , at T9-11 in group D2 , while increased significantly at T6 in group D3 (P = 0.001) and at T6-8 in group C .QTc values in all groups were normal and no differences existed at T1 and D2 were significantly prolonged at T2 , while shortened significantly at T7 and T8 in group D3 . QTc in group D3 was shortened significantly at T2, T6, T7, T9 and T10 compared with group D1 , and shortened significantly at T2 compared with group D2 (P < 0.001).Compared with group C, QTc in groups D1 and T2. There were no statistical differences among the four groups both at T1 and T2 (P > 0.05).Tp-e values in all groups were normal and no differences existed at T8 in group D1 was prolonged significantly (P = 0.007) while shortened significantly in group D3 (P = 0.032). Tp-e in group D3 was shortened significantly at T7-9 compared with group D1  and at T8 and T9 compared with group D2 .Compared with group C, Tp-e at T1 among the four groups were comparable. Compared with T1, iCEB at T2, T6-8 in groups D1 and D2 , at T6 in group D3 (P < 0.001), and at T6 and T7 in group C  were significantly prolonged. Compared with T2, iCEB at T10 in groups D1 and D2 were significantly shortened , at T6 was prolonged and at T8 was shortened significantly in group D3 , and at T6 and T7 in group C was significantly shortened .iCEB values at T8 in group D3 was significantly shortened compared with groups C, D1 and D2  . Compared with group C, CCE decreased significantly at T2 in groups D1 and D2 , and at T3 in group D1 (P = 0.005). Compared with group D1, CCE increased significantly in group D3 at T2-4 . Compared with group D2, CCE increased significantly in group D3 at T2 and T3  , and at T3-6 in group C . Compared with T2, dp/dt decreased significantly at T4-7 in group D1 (P < 0.001) and at T4-6 in groups D2, D3 and C . Compared with group C, dp/dt decreased significantly in group D1 at T7 and in D2 at T4 and T7 . Compared with groups D1 and D2, dp/dt increased significantly in group D3 at T7  , and at T3-7 in group C . Compared with T2, HR decreased significantly at T4 in group D1 (P = 0.014), and at T3-6 in group C (P < 0.001). Compared with group C, HR decreased significantly at T2 in each dexmedetomidine group , and at T4 in group D1 (P = 0.013)  and decreased at T6 (P = 0.024) significantly, increased at T2 (P < 0.001) and decreased at T5-6 in group D2  significantly, decreased significantly at T2, T5 and T6 in group D3 , and decreased significantly at T5 in group C (P = 0.006). Compared with T2, MAP in groups D1 at T5-7, D2 at T4-7, D3 at T6, and C at T5 decreased significantly . Compared with group C, MAP in group D1 increased significantly at T2-5 , in group D2 increased significantly at T2 (P = 0.047). Compared with groups D1, MAP in group D3 decreased significantly at T2 and T3 . Compared with groups D2, MAP in group D3 decreased significantly at T2 (P = 0.033) . Compared with T2, SVR in groups D1 and D2 was significantly lower at T6 and T7 . Compared with group C, SVR was significantly increased at T2, T4 and T5 in group D1 , and at T2 in group D2 (P = 0.047). Compared with group D1, SVR was significantly lower at T2-5 in group D3 . Compared with group D2, SVR in group D3 was significantly lower at T2 and T3 . No arrhythmia occurred during perioperative period in the four groups  channel subtype α—Nav1.5 specifically expressed in cardiac tissues in a dose-dependent manner [+ channel continuous current (INap) peak values under stress state, thus reduces effects on myocardial repolarization dispersion and the occurrence rates of malignant arrhythmia. Previous studies reported that dexmedetomidine could prolong the action potential duration of cardiomyocytes, reduce the incidence of early afterdepolarization, and decrease the autonomy of cardiomyocytes.Dexmedetomidine contains the imidazole ring, which has agonistic effects on αeceptors , inhibiteceptors . It inhit manner  to decre1 and D2 were significantly prolonged than these in groups D3 and C when dexmedetomidine loading dose finish, and no significant difference existed between groups D3 and C. It indicated that the incidence of AV conduction block induced by dexmedetomidine might be related to its dose, that is, higher dose might produce higher incidence of AV conduction block. PR intervals recovered to normal levels at 48 h, 72 h and 1 month postoperatively in each dexmedetomidine group.PR interval reflects AV conduction. In this study, the PR intervals in each dexmedetomidine group were significantly prolonged after loading dose, indicating that dexmedetomidine had inhibitory effects on AV conduction, which was consistent with the conclusion of Ergul et al.’s study . The PR 1 and D2 were significantly longer than the baseline values, while in group D3 shortened significantly after dexmedetomidine loading dose injection, suggesting that dexmedetomidine loading dose 1 μg/kg injected in 10 min could prolong ventricular repolarization duration and interfere with cardiac conduction system significantly, which was not conducive to cardiac electrophysiological stability. QTc in groups D1 and D2 was significantly prolonged than those in groups D3 and C when dexmedetomidine loading dose finish. Meanwhile, QTc in group D3 was significantly shorter than those in groups D1 and C at surgery ending, 1 h in PACU, 24 h and 48 h postoperatively. It indicated that dexmedetomidine in group D3 could reduce myocardial electrophysiological heterogeneity [Abnormal prolongation of QTc usually indicates increased sensitivity to arrhythmia. In this study, the QTc intervals in groups Dogeneity , suggestogeneity , 23, 24.1 was significantly prolonged, while in group D3 shortened significantly, indicating an increased risk of desynchronization of myocardial cells repolarization existed in group D1, and suggesting that the effects of dexmedetomidine on ventricular transmembrane repolarization might be related to its dose. It has been reported that the Tp-e interval may be prolonged in response to sympathetic hyperactivity [3 compared with group C in our study might be related to the balanced vagal efference and sympathetic activity originating from the lower dose of dexmedetomidine [Tp-e is an indicator of the synchronization of myocardial cells repolarization and can be used as a predictor of torsades de pointes. Matthias et al.  found thactivity . The shotomidine .1 and D2 increased significantly at dexmedetomidine loading dose finish, surgery ending and 24 h postoperatively compared with baseline values. Although iCEB in group D3 increased significantly compared with the baseline at surgery ending, it returned to the baseline at 24 h postoperatively and was significantly lower than groups D1, D2 and C, suggesting that higher dose of dexmedetomidine might not be beneficial to cardiac electrophysiological stability and lower dose of dexmedetomidine in D3 was more conducive to keep cardiac electrophysiological balance during perioperative period. Studies have shown [iCEB is an index that can reflect cardiac electrophysiological balance quickly and intuitively, which predicts the risk of arrhythmias, including torsades de pointes (TdP) and non-TdP ventricular tachycardia/ventricular fibrillation , 29. Theve shown  that abn1 and D2, SVR and MAP increased significantly compared to baseline values and were higher than groups C and D3 when dexmedetomidine loading doses completed, suggesting that dexmedetomidine at higher dose of 1 μg/kg infused for 10 min could stimulate the α2B receptors on vascular smooth muscle cells and contract resistance blood vessels [1 and D2 were significantly lower than basic values, which indicated that the cardiac ejection function was influenced to a certain extent.In groups D vessels –32. Mean1 and D2 after dexmedetomidine loading dose were significantly lower than those in groups C and D3, indicating that dexmedetomidine loading dose 1 μg/kg might produce inhibitory effect on cardiac function. The reason might be related to the significantly increased SVR and cardiac afterload [3 was significantly higher than those in groups D1 and D2 at 1 h in PACU and had no significant difference compared with that in group C, illustrating that lower dose of dexmedetomidine at 0.5 μg/kg/h significantly improve the cardiac prognosis, which was consistent with the previous studies [3 had no significant adverse effects on myocardial function during perioperative period.CCE is the cardiac circulation efficiency, which reflects the work efficiency of the heart for maintaining the circulation dynamic balance. The higher the CCE, the greater the efficiency, which is negatively correlated with the sudden death rate . In thisfterload , producifterload . Dp/dt i studies –38. HencCa-L of cardiomyocytes significantly at a concentration of 10 ng/ml and above, that is, time of the ion channel inactivation was prolonged. In this study, HR in each dexmedetomidine group decreased significantly than the basic values and that in group C when the loading dose was completed. The reason might be related to the changes of Ca2+ ion channel inactivation [+ and iCa2+were comparable with standard values to avoid adverse effects on QTc, PR and QRS intervals [In vitro studies , 39, dextivation . Since ctivation . Furtherntervals .Our study still has certain limitations. Considering that sevoflurane might prolong QTc intervals , this stIn summary, dexmedetomidine at a loading dose of 0.5 μg/kg and a maintenance dose of 0.5 μg/kg/h could maintain the stability of cardiac electrophysiology during perioperative period and has no significant adverse effects on CCE.


In this work, the substitution of Ca2+ cation in Sr5Ti4O13 sintered ceramics was chosen to enhance the structural, optical, and dielectric properties of the product. It was found that the Ca2+ content has significant effects on enhancing the dielectric properties as compared to Mn and glass additions. It was observed that the relative density, band gap energy, and dielectric loss (tangent loss) increase while relative permittivity decreases along with Ca2+ content. High relative density (96.7%), low porosity, and high band gap energy (2.241 eV) values were obtained in (Sr1−xCax)5Ti4O13, 0.0 ≤ x ≤ 0.06 sintered ceramics. These results will play a key role in the application of dielectric resonators.Low loss Ruddlesden–Popper (RP) series, i.e., (Sr Lately, the RP series ceramics have been the subject of numerous scientific studies, with results showing their interesting dielectric properties , resulting consecutive perovskite pieces due to shifting by direction 1 ÷ 2 [111], w.r.t the unit cell of RP series. The known RP structure has closely alike lattice parameters i.e., (a = b = 0.385 to 0.389 nm) but c = 2.812 nm for n = 4 [2+ contents as shown in 2+ and Ca2+ cations as shown in n+1TinO3n+1 (n = 4) sintered ceramic. 2+ contents of Srn+1TinO3n+1 (n = 4) sintered ceramics. It has been noted that the relative density increases with the Ca2+ content, which further modified the optical and dielectric properties. The highest values of relative density is (96.7%) of Srn+1TinO3n+1 (n = 4) sintered ceramics was observed at x = 0.06 content.or n = 4 ,30,31,321−xCax)5Ti4O13, 0.00 ≤ x ≤ 0.06 sintered ceramics. The variation of relative densities and grain size of all the samples has been investigated. The SEM micrographs of (Sr5Ti4O13) green pellets with doping of Mn or glasses at different sintering temperature were studied by many scientific researchers [5Ti4O13) sintered ceramic [2+ concentration has been observed to increase the porosity and grain size of all samples in (Sr5Ti4O13) sintered ceramic. These factors will affect the structure, optical, and dielectric properties of the base product. In order to improve these properties, numerous studies have examined the synthesis settings used to create various dopants in the base product [earchers . It has  ceramic ,28,29,30 product ,32.1-xCax)5Ti4O13, 0 ≤ x ≤ 0.06 sintered ceramic. FTIR spectrometer plays a key role to characterize the vibrational stretching and un-stretching mode of the base sample synthesized by chemical reaction route [−1, and 3200.0 cm−1. This mode of vibration is produced by the absorption of vapors during synthesis process. Only asymmetric mode at wave number (3700.0 per cm) was recorded in the base product which shown carboxylates family [on route ,34. The s family . In this1−xCax)5Ti4O13, 0.00 ≤ x ≤ 0.06 sintered ceramics. Many of the researchers reported that the Sr5Ti4O13 base sample was found to be transparent for white light [5Ti4O13, is translucent for visible light. The band structure and electronic transition were characterized using photon energy [α), which was calculated using Equation (1) [gE = bandgap energy, A = constant of proportionality, and hv = photon energy.te light . It is vn energy . The eletion (1) .(1)α=Ah−1). The band gap energy of all the samples was calculated using the Tauc plots method. It was reported that the values of band gap energy increase with Ca2+ content.The coefficient of absorption will be defined how distant light of specific wavelength can be penetrated into material before being absorbed. When light absorbed poorly by material have low coefficient of absorption looks to be thin at specific wavelength. The unit of absorption coefficient is (cm1−xCax)5Ti4O13, 0.00 ≤ x ≤ 0.06 sintered ceramics. The emission line spectrum will be produced by the recombination of holes and electron charge carriers. Using the equation (E = hc/λ), where E = optical excitation energy, h = Plank’s constant (~6.63 × 10−34 Js) c = speed of light (3 × 108 m/s) and λ is the emission wavelength, we can find the value of excitation energy of all the samples.Emission at photoluminescence peak of the samples has been noted at the range of ~400–550 nm. Multiple photonic processes such as PL have certain common uses, and PL is a multiple photonic process that has some typical applications, i.e., (i) determination of band gape energy, (ii) material quality, as well as (iii) molecular structure and crystallinity, reported by many researchers ,39. It h1−xCax)5Ti4O13, 0.0 ≤ x ≤ 0.06 sintered ceramics using the vector network analyzer, as shown in r) and tangent loss (tanδ) were observed for the contents (at x = 0.0 and 0.02), which shows the transition of ferroelectric to Para electric phases. The same behavior was recorded in the values of ‘εr’ and ‘tanδ’ for Ba5-xSrxDyTi3V7O30 (0 ≤ x ≤ 5) sintered ceramics at temperatures of 430 °C, 350 °C, 325 °C, 85 °C, and 42 °C, respectively [r (~1400) was observed for (Sr1−xCax)5Ti4O13, (composition with x = 0.02) at 100 Hz frequency, and found to decrease with increasing operating frequency, which may be due to the interfacial polarizations. Moreover, the value of εr decreased with increasing Ca2+ contents, which is due to the difference of ionic polarizebilities of Ca2+ (3.16 Å3) and Sr2+ (4.24 Å3) [The low frequency dielectric properties of all the synthesized samples sintered at 1200 °C for 3 h in air were better due to their high relative densities. The variation of relative permittivity (εr) and tangent loss (tanδ) with varying temperature was measured at 100 Hz–1 MHz for  ,45. It h4.24 Å3) . The low4.24 Å3) . General4.24 Å3) .1−xCax)5Ti4O13, 0.0 ≤ x ≤ 0.06 sintered ceramics is shown in 2+contents.The complex impedance spectroscopy mechanism is generally used to investigate the structural properties and bonding of the various types of materials, comprising the ferroelectric, ionic insulator, and linked ceramics under different experimental conditions . The var1−xCax)5Ti4O13, 0.0 ≤ x ≤ 0.06 sintered ceramics was synthesized by conventional solid state method. The structural, microstructural, optical, and dielectric properties of all the samples have been investigated. The XRD patterns revealed the tetragonal phase with space group (I4/mmm). The SEM image revealed that the grain size and porosity increase with increasing Ca2+ contents, which was due to the difference of ionic radii. The results of UV spectroscopy declared that the bandgap energy increases from 1.68 eV to 2.24 eV along with increasing Ca2+ concentrations. The good values of dielectric properties  in the frequency range from 100 Hz to 1 MHz was observed. It has been observed that the magnitude of Z″ increases with Z′ and Ca2+ contents. The overall findings are suitable for the application of dielectric devices.The solid solutions of (Sr