Bacteria and Archaea) from surface (0–30 m) down to deep waters  in summer ice-covered and ice-free regions of the Fram Strait, the main gateway for Atlantic inflow into the Arctic Ocean. Cell abundances of the bacterioplankton communities in surface waters varied from 105 cells mL–1 in ice-covered regions to 106 cells mL–1 in the ice-free regions. Observations suggest that these were overall driven by variations in phytoplankton bloom conditions across the Strait. The bacterial groups Bacteroidetes and Gammaproteobacteria showed several-fold higher cell abundances under late phytoplankton bloom conditions of the ice-free regions. Other taxonomic groups, such as the Rhodobacteraceae, revealed a distinct association of cell abundances with the surface Atlantic waters. With increasing depth (>500 m), the total cell abundances of the bacterioplankton communities decreased by up to two orders of magnitude, while largely unknown taxonomic groups  maintained constant cell abundances throughout the entire water column (ca. 103 cells mL–1). This suggests that these enigmatic groups may occupy a specific ecological niche in the entire water column. Our results provide the first quantitative spatial variations assessment of bacterioplankton in the summer ice-covered and ice-free Arctic water column, and suggest that further shift toward ice-free Arctic summers with longer phytoplankton blooms can lead to major changes in the associated standing stock of the bacterioplankton communities.The Arctic is impacted by climate warming faster than any other oceanic region on Earth. Assessing the baseline of microbial communities in this rapidly changing ecosystem is vital for understanding the implications of ocean warming and sea ice retreat on ecosystem functioning. Using CARD-FISH and semi-automated counting, we quantified 14 ecologically relevant taxonomic groups of bacterioplankton ( Atmospheric and oceanic warming has a substantial impact on the Arctic Ocean already today . The strSea ice conditions have a strong impact on the seasonal ecological dynamics in Fram Strait and the whole Arctic Ocean , affectiLong-term summer observations in the region, conducted in the framework of the Long-Term Ecological Research site HAUSGARTEN, revealed strong ecological variations associated with the Atlantic Meridional Overturning Circulation . WarmingIn Situ Hybridization  primers selection , as wellUsing CARD-FISH and semi-automated cell counting, we quantified bacterial and archaeal cell abundances of 12 taxonomic groups selected based on a 16S rRNA gene survey of water column microbial communities during the same summer expedition in the Fram Strait  Supplem. SamplesBased on the known hydrography of the Strait  and the a concentrations, suggested a late stage of the phytoplankton bloom across the Strait (a maximum 10–28 m depth), revealed that the communities at the time of sampling in the ice-covered EG and the ice-margin N stations had a higher abundance of diatoms, in contrast to the ice-free HG stations that had a higher abundance of Phaeocystis spp., . The communities were dominated by bacterial cells that comprised 8–11 × 105 cells mL–1 in the HG and N stations, and 2 × 105 cells mL–1 in the EG stations (Bacteroidetes (2.1 × 105 cells mL–1) in the HG and N stations, followed by Gammaproteobacteria (from 1.6 to 2.1 × 105 cells mL–1) and Verrucomicrobia (from 1.6 to 2.1 × 105 cells mL–1), with a several-fold higher cell abundance, compared to the EG stations (where together they comprised between 0.1 and 0.3 × 105 cells mL–1)  . These tls mL–1) . Previouls mL–1) . Our micstations . Hence, Bacteroidetes, Gammaproteobacteria, and Verrucomicrobia were consistent with in 16S rRNA gene observations of size-fractionated bacterioplankton communities  conducted during the same expedition (>75%) (Alteromonadaceae) showed two- to threefold lower relative abundance in the molecular study (Bacteroidetes) may have altered their representation in the PCR-based 16S rRNA gene dataset  . Howeverar study . These dar study , which s dataset , and thua concentration and consumed inorganic nutrients) across the Strait. We did not find significant correlations between these physical and biogeochemical parameters, which suggest to some extent their independent effect on the bacterioplankton communities , showed significant positive correlations to water temperature  showed strong positive correlation with statistical significance to salinity  and the different phytoplankton bloom conditions  and inorganic phosphate (PO4) was below 16 (3) and phosphate (ΔPO4) revealed a very strong positive correlation with statistical significance , used by diatoms, did not show a significant correlation to ΔPO4 and ΔNO3. This further supports the impact of different phytoplankton populations across the Strait  revealed weaker relationships with cell abundances of different taxonomic groups  were higher in some stations with more advanced phytoplankton bloom conditions. However, as we have only limited observations of phytoplankton for this study, we cannot test previous hypotheses of direct associations between the abundances of specific phytoplankton groups and bacterioplankton taxa  and up to 8% was covered by the Archaea-specific probe , while the coverage by the Archaea-specific probe significantly increased up to 17% of DAPI-stained cells . Members of the domain Bacteria dominated the communities throughout the entire water column, with highest cell abundances in surface waters (105–106 cells mL–1), and significantly lower 104 cells mL–1 at depth . Archaeal cells had an overall lower abundance than bacterial cells by an order of magnitude throughout the entire water column, ranging from 104 cells mL–1 at surface down to 103 cells mL–1 in bathypelagic waters . Compared to the stronger decline in bacterial cell numbers, this pattern mirrors the known global trend of relative archaeal enrichment in epipelagic waters , SAR324 (class Deltaproteobacteria), and SAR406 (phylum Marinimicrobia)  reaching up to 15% of the sequences in mesopelagic waters (>200 m) , where they may realize an ecological niche , in both ice-free and ice-covered regions of the Strait, decreased by two orders of magnitude in their cell abundances at meso- and bathypelagic depths  have a rather homogeneous hydrography , and areicrobia) . There w(>200 m) . Howeveral niche . We obsells mL–1 , reflectlls mL–1 . Bacteriep basin . Previouep basin . The celep basin . Very liep basin , or, in ep basin . Their hep basin , and thuBacteria , where they comprised half of the total archaeal community . Surface water bacterioplankton communities were dominated by Gammaproteobacteria, Bacteroidetes, and Verrucomicrobia, which corresponded with biogeochemical conditions in the ongoing seasonal phytoplankton bloom. This suggests that currently predicted longer seasonal phytoplankton blooms, as well as the increasing Atlantic influence on the Arctic Ocean , may have a strong impact on the composition and biogeographical distribution of certain bacterioplankton taxonomic groups in the surface Arctic waters.Using state-of-the-art semi-automatic microscopy cell counting, we quantified the absolute cell abundance of 12 key taxonomic groups in summer bacterioplankton communities of both ice-free and ice-covered regions of the Fram Strait. We found that in surface waters some taxonomic groups were associated with the distinct water masses of the Strait . With depth, some taxonomic groups, such as the SAR202 clade, maintained similar abundances throughout the entire water column , where other taxa decline by several-fold. The observation of a homogenous abundance further supports the previously established hypothesis that through high functional plasticity these taxonomic groups are realizing various ecological niches throughout the entire water column.Altogether, our quantitative data on cell abundances of ecologically relevant taxonomic bacterioplankton groups provide insights into factors structuring pelagic bacterioplankton communities from surface to the deep waters of the Arctic Ocean, and add to a baseline to better assess future changes in a rapidly warming region.Sampling was carried out during the RV Polarstern expedition PS99.2 to the Long-Term Ecological Research (LTER) site HAUSGARTEN in Fram Strait . Sampling was carried out with 12 L Niskin bottles mounted on a CTD rosette (Sea-Bird Electronics Inc. SBE 911 plus probe) equipped with temperature and conductivity sensors, a pressure sensor, altimeter, and a chlorophyll fluorometer. In ice-covered regions the samples were collected through holes in the ice kept open by the research vessel. On board, the samples were fixed with formalin in a final concentration of 2% for 10–12 h, then filtered onto 0.2 μm polycarbonate Nucleopore Track-Etched filters , and stored at −20°C for further analysis.a concentration  for 1 h at 37°C. Filters for enumerating Archaea and Thaumarchaeota were treated for an additional 30 min in 36 U mL–1 achromopeptidase  and 15 μg mL–1 proteinase K at 37°C. Subsequently, endogenous peroxidases were inactivated by submerging the filter pieces in 0.15% H2O2 in methanol for 30 min before rinsing in Milli-Q water and dehydration in 96% ethanol. Then, the filters were covered in hybridization buffer and a probe concentration of 0.2 ng μL–1. Hybridization was performed at 46°C for 2.5 h, followed by washing in pre-warmed washing buffer at 48°C for 10 min, and 15 min in 1x PBS. Signal amplification was carried out for 45 min at 46°C with amplification buffer containing either tyramide-bound Alexa 488 (1 μg/mL) or Alexa 594 (0.33 μg mL–1). Afterward, the cells were counterstained in 1 μg/mL DAPI  for 10 min at 46°C. After rinsing with Milli-Q water and 96% ethanol, the filter pieces were embedded in a 4:1 mix of Citifluor  and Vectashield , and stored overnight at −20°C for later microscopy evaluation.We quantified absolute cell abundances of 12 key bacterioplankton groups , membersc waters . The selc waters . CARD-FIc waters , using hThe filters were evaluated microscopically under a Zeiss Axio Imager.Z2 stand , equipped with a multipurpose fully automated microscope imaging system (MPISYS), a Colibri LED light source illumination system, and a multi-filter set 62HE . Pictures were taken via a cooled charged-coupled-device (CCD) camera  with a 63 × oil objective, a numerical aperture of 1.4, and a pixel size of 0.1016 μm/pixel, coupled to the AxioVision SE64 Rel.4.9.1 software  as described by Cell enumeration was performed with the software Automated Cell Measuring and Enumeration Tool . Total bacterioplankton cells were determined as the total amount of DAPI-stained cells. Counts for each taxonomic group included only cells that were simultaneously stained by DAPI and the taxa-specific FISH probe.Following  the nutrwww.r-project.org) in RStudio (v1.3.1056), i.e., statistical tests for normality, ANOVA and Kruskal–Wallis. Post hoc Wilcoxon test and Pearson’s rank correlation coefficient were conducted with the R package “rstatix” (v0.6.0)  ((v0.6.0) . Plots w(v0.6.0)  and “tid(v0.6.0) .The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.MC-M, EF, and VS-C designed and conducted the study, and wrote the manuscript with guidance from AB. MC-M performed the hybridizations, cell counting, data and statistical analysis with guidance from VS-C  and EF . All authors critically revised the manuscript and gave their approval of the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Salvia miltiorrhiza that have demonstrated neuroprotective effects against dopaminergic neurotoxins and the inhibition of MAO-A. Since MAO-B inhibition is considered an effective therapeutic strategy for PD, we tested the inhibitory activities of three abundant tanshinone congeners against recombinant human MAO (hMAO) isoenzymes through in vitro experiments. In our study, tanshinone I (1) exhibited the highest potency against hMAO-A, followed by tanshinone IIA and cryptotanshinone, with an IC50 less than 10 µM. They also suppressed hMAO-B activity, with an IC50 below 25 µM. Although tanshinones are known to inhibit hMAO-A, their enzyme inhibition mechanism and binding sites have yet to be investigated. Enzyme kinetics and molecular docking studies have revealed the mode of inhibition and interactions of tanshinones during enzyme inhibition. Proteochemometric modeling predicted mAChRs as possible pharmacological targets of 1, and in vitro functional assays confirmed the selective M4 antagonist nature of 1 (56.1% ± 2.40% inhibition of control agonist response at 100 µM). These findings indicate that 1 is a potential therapeutic molecule for managing the motor dysfunction and depression associated with PD.Monoamine oxidases (MAOs) and muscarinic acetylcholine receptors (mAChRs) are considered important therapeutic targets for Parkinson’s disease (PD). Lipophilic tanshinones are major phytoconstituents in the dried roots of  Although dopaminergic therapies offer considerable benefits, l-dopa-induced dyskinesia (LID) and motor fluctuation and l-dopa-resistant or nondopaminergic motor features, as well as nonmotor complaints, demand the use of nondopaminergic treatment options. For example, the N-methyl-d-aspartate (NMDA) receptor antagonist amantadine is used clinically for LID during PD treatment. Other nondopaminergic neurotransmitters and neuromodulatory systems, such as glutamatergic, serotonergic, cholinergic, histaminergic, cannabinoid, and non-adrenergic, influence the basal ganglia circuit and are also the targets of ongoing studies to enhance the treatment of PD and alleviate the side effects of DA replacement therapy [Parkinson’s disease (PD) is the second-most prevalent age-dependent neurodegenerative disease (NDD) and is characterized by the progressive degeneration of dopaminergic and nondopaminergic systems in the substantia nigra, the striatal deficiency of dopamine (DA), and the intracellular aggregation of α-synuclein. It affects nearly 2% to 3% of the elderly population aged over 65 years and presents as motor dysfunctions such as tremor, rigidity, bradykinesia, and postural instability. Besides motor symptoms, nonmotor features such as cognitive impairment, depression, dysautonomia, and other social and behavioral abnormalities exacerbate the disease condition ,2,3. Cur therapy ,4.Monoamine oxidases  are the mitochondria-bound flavoenzymes responsible for the catalytic degradation of biogenic amines, including monoaminergic neurotransmitters, dietary amines, and xenobiotics in the brain and peripheral tissues. In the human brain, MAOs occur in two isoenzyme forms, MAO-A and MAO-B, which are approximately 72% identical in amino acid sequence makeup but vary in substrate preferences, inhibitor specificities, cell and tissue distribution, and immunological properties. MAO-A selectively deaminates serotonin 5-HT), whereas MAO-B preferentially degrades benzylamine and 2-phenylethylamine. DA, noradrenaline, and adrenaline are the common substrates for both isoforms of MAOs. Thus, the inhibitors of MAOs are considered as prophylactic and therapeutic agents for NDDs such as PD, Alzheimer’s disease (AD), schizophrenia, anxiety, and depression. These conditions are characterized by elevated levels and activities of MAOs, which lead to decreased levels of neurotransmitters, increased oxidative stress resulting from the oxidoreductase activity of the enzymes, and the subsequent degeneration of neurons -HT, wher,7.1, M3, and M5, which activate phospholipase C and regulate intracellular calcium mobilization, and Gi/o-coupled M2 and M4, which inhibit adenylyl cyclase, causing a reduction in the cAMP levels [1, M2, and M4 are the predominant mAChRs in the human brain. Quantitative analyses such as immunohistochemical and radioligand-binding studies, which were performed to determine the mAChR distribution, showed that the M1, M2, and M4 receptors are the highly expressed muscarinic receptor subtypes in the frontal, temporal, parietal, and occipital cortices. The M1 and M4 receptors are highly localized in the hippocampus and the basal ganglia, while the M2, M3, and M5 receptors predominate in the thalamus, peripheral vasculature, and cerebrovasculature, respectively [Acetylcholine (ACh) is a prime neurotransmitter in the striatum released by innervated cholinergic interneurons. Cholinergic transmission is proposed to play a critical role in regulating the local circuits of the striatal complex and the modulation of presynaptic DA release ,9. ACh aP levels ,11. M1, ectively ,13. Concectively .Salvia miltiorrhiza, known as Dansen in traditional Chinese medicine (TCM), have been extensively used as a traditional medicine for various diseases, such as coronary heart diseases, cerebrovascular diseases, AD, PD, renal deficiency, cancer, hepatocirrhosis, and bone loss, either as a single herb or in combination with other herbal medicines. Salvia miltiorrhiza is a perennial plant widely distributed in China, Japan, and Korea [Salvia constitute a high amount of phenolic acids, flavonoids, terpenes, and tanshinones [The dried roots of nd Korea ,16. Compshinones . The majshinones . Likewisshinones .S. miltiorrhiza have demonstrated the potential activities of its constituents in NDDs, including AD and PD. Phytoconstituents from this species exhibit the inhibition of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-secretase (BACE1), which are implicated in the pathogenesis of AD [1) showed a remarkable suppression of the proinflammatory M1 factors—namely, nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 expressed in activated microglia. Further, it enhanced the motor function in vivo and protected against MPTP-induced neurodegeneration [2), in multiple experiments, prevented the loss of dopaminergic neurons via different molecular mechanisms [Salvia phytochemicals can act as anti-Parkinson agents. Selegiline and rasagiline are the two most well-known MAO-B inhibitors that display the protection of dopaminergic neurons from cell death induced by dopaminergic neurotoxins ; ischemia; excitotoxins; and other insults [S. miltiorrhiza: 1, 2, cryptotanshinone (3), and dihydrotanshinone I (Dtan I) for recombinant human MAO-A (hMAO-A) inhibition and found Dtan I as the most active compound, with an IC50 value of 23 µM, followed by 1, 3, and 2, with IC50 values of 80, 84, and >400 µM, respectively [1, 2, and 3 isolated from the roots of S. miltiorrhiza. We examined the enzyme inhibition mode and intermolecular interactions engaged in enzyme inhibition through kinetic experiments and computational docking to elucidate the mechanism of enzyme inhibition. Many natural and synthetic compounds can target more than one locus of insult during pathogenesis, and such multifunctional agents have emerged as master keys or magic bullets in the treatment of multifactorial diseases [Recent pharmacological studies focusing on the neuroprotective roles of is of AD ,20,21. Tneration . Tanshinchanisms ,24,25. Tchanisms . Overall insults . Previouectively . Howeverdiseases . Since i1), tanshinone IIA (2), and cryptotanshinone (3) of >98% purity were obtained from the dried roots of Salvia miltiorrhiza, as described in our published report [1, 2, and 3 are shown in l-deprenyl·HCl, clorgyline·HCl, 3-isobutyl-1-methylxanthine (IBMX), and acetylcholine chloride (ACh) were purchased from Sigma Aldrich . Dulbecco’s modified Eagle’s medium (DMEM), Hank’s balanced salt solution (HBSS), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were bought from Invitrogen . The remaining reagents and chemicals were purchased from commercial suppliers.Tanshinone I . The experimental procedures for this experiment were as described earlier [l-deprenyl/clorgyline.HCl was added to a 12.5-µL aliquot of beetle luciferin derivative substrate  in each well of a 96-well plate. An enzyme solution (25 µL) was then added to the test samples to initiate the reaction. After an hour of incubation at 25 °C, a reconstituted luciferin detection reagent (50 µL) was added to each well to stop the reaction and produce a luminescent signal. The final mixture was incubated for an additional 20 min at 25 °C. Then, the luminescence was recorded on a FilterMax F5 Multi-Mode microplate reader . The amount of luminescence was directly proportional to the residual activity of the hMAO isoenzymes.The inhibitory activities on hMAO-A and hMAO-B by three tanshinones:  earlier ,31. Brie1, 2, and 3 using varying concentrations of the hMAO substrate (40–160 µM for the hMAO-A kinetics and 4–16 µM for the hMAO-B kinetics). Kinetic experiments were carried out as described by Seong et al. [Ki) were obtained from secondary plots analyzed using SigmaPlot 12.0 software .Enzyme inhibition kinetics was performed with tanshinones g et al.  and Paudg et al. . The dif1, 2, and 3. The experimental model for determining the targets was based on a machine learning prototype with a Parzen Rosenblatt window covering 55,079 compounds against 99 human proteins. Representations of the compound molecular characterizations were obtained by extended connectivity fingerprints (ECFP_4) generated using jCompoundMapper. The chemical similarity was calculated using the Aitchison and Aitken kernel function. Protein sequences were aligned using the MUSCLE algorithm created by the bui3d package; comparisons between two protein sequences were made, and the similarities were calculated. Internal and external validation were done for the prediction model, evaluating the sensitivity, specificity, Matthews correlation coefficient, and area under the curve. The detailed PCM was described in an earlier report [PCM was used to predict the potential protein targets of r report .1R, hM2R, hM3R, hM4R, and hM5R). The functional assay provided the readouts based on the measurements of calcium ion mobilization (for the Gαq/11-coupled receptors) and cAMP level (for the Gαi/o-coupled receptors).The cellular and nuclear receptor functional assays were carried out at Eurofins Cerep  following the in-house protocols . The experimental methods for the assays were as described in the previous reports ,35,36 an2 receptors were suspended in a medium containing HBSS buffer complemented with 20-mM HEPES buffer (pH 7.4) and 500-µM IBMX, whereas the stable human M4 receptor cloned CHO cells were distributed in HBSS buffer with 20-mM HEPES/NaOH (pH 7.4), 70-mM NaCl, 5.33-mM KCl, 1.25-mM CaCl2, 0.5-mM MgCl2, 0.41-mM MgSO4, 0.441-mM KH2PO4, 0.3-mM Na2HPO4, 0.1% glucose, and 500-µM IBMX. These cell suspensions were distributed into their respective assay plates at a density of 104 cells/well and incubated for 5 min at 25°C with/without the test compound or standard. After that, NKH 477 was added to make the final concentrations of either 5 µM (for M2R) or 1 µM (for M4R) and incubated for 10 min at 37 °C. The cells were then lysed, and the fluorescence acceptor (D2-labeled CAMP) and fluorescence donor (anti-CAMP antibody labeled with europium cryptate) were dispensed into the cell plate. The resulting mixture was incubated for the next 1 h at RT. The HTRF reading was subsequently taken using a PerkinElmer Envision microplate reader  at an excitation intensity of 337 nm and emission intensities of 620 and 665 nm. The cAMP level was calculated as the ratio of the signal measured at 665 nm to that measured at 620 nm. The agonist and antagonist activities were illustrated as a percentage (%) stimulation of the control agonist response and as a % inhibition of the control agonist response. The agonist effect was determined as a % of the control response to 3-µM and 1-µM acetylcholine for M2R and M4R, respectively. Likewise, the antagonist behavior was represented as a % of the inhibition of the control response to 0.3-µM and 100-nM acetylcholine for M2R and M4R, respectively. The reference agonist for the assay was acetylcholine chloride, and the reference antagonists were methoctramine (for M2R) and PD 102807 (for M4R).Stable CHO cells expressing the transfected cDNA of human M1 on M1R, M3R, and M5R. Stable human M1R and M3R cloned CHO cells were suspended in DMEM buffer complemented with and without 0.1% delipidated fetal calf serum and dispensed in a microplate at a density of 3 × 104 cells/well and 2.5 × 104 cells/well, respectively. A fluorescent probe  blended with probenecid and 20-M HEPES (pH 7.4) was applied to each well of cells and equilibrated at 37 °C for 60 min, followed by an additional 15 min at 22 °C. The assay plate was placed in a microplate reader , and the test solution, reference agonist, or HBSS buffer (control) were added to the plate, and ultimately, the fluorescence was measured.The calcium ion influx was evaluated fluorometrically to determine the functional activity of 5R-transfected RBL cells were distributed in HBSS buffer containing 20-mM HEPES (pH 7.4) and dispensed into the assay plate at a concentration of 2.0 × 104 cells/well. The fluorescent probe  was blended with probenecid in the buffer and added to each well and left to equilibrate for 60 min at 30 °C. The assay plate was placed in a microplate reader , and the test solution, reference agonist, or HBSS buffer (control) were added. The fluorescence was then measured.Likewise, hM1R, M3R, and M5R, respectively. Likewise, the antagonist behavior was measured as a % inhibition of the control response to 3-nM, 100-nM, and 10-nM acetylcholine for M1R, M3R, and M5R, respectively. The reference antagonist drugs for M1R, M3R, and M5R were pirenzepine, 4-DAMP, and atropine sulfate, respectively.The agonist activity was determined as the % of the control response to 100-nM, 1-µM, and 624-nM acetylcholine for M1, 2, and 3 to the X-ray crystallographic structures of hMAO-A and hMAO-B acquired from the RCSB Protein Data Bank (PBD) with IDs 2BXR and 2BYB, respectively [4 receptor bound to tiotropium with a RCSB PDB ID 5DSG was used for studying the interactions between 1 and the M4 receptor. The source of the 3D chemical structures of 1, 2, and 3 was the PubChem Compound database (NCBI) with CIDs 114917, 164676, and 160254. Similarly, the crystal structures of harmine, deprenyl, acetylcholine, and tiotropium were also derived from NCBI under CIDs 5280953, 5195, 187, and 5487427, respectively. For docking hMAOs, the water and ligand molecules were eliminated using Discovery Studio , but the cofactor FAD was retained. AutoDockTool (ADT) was used for adding Kollman charges and polar hydrogens to the cleaned proteins. The 3D structures of the compounds were generated using MarvinSketch . The Gasteiger charges and number of rotatable bonds were computed automatically for the ligands using ADT. The coordinates of the active sites of the proteins were generated using AutoGrid. The number of generic algorithm runs was set to ten, and the other docking parameters were set as the default using AutoDock. The ligand–protein complexes with the least binding energies from their respective populated clusters were selected for the docking analysis. The interactions in the complexes were visualized using Discovery Studio.Autodock4.2 software was used for the molecular docking of compounds ectively . The cry), drug-likeness, and lead-likeness of 1 were predicted by SwissADME [The pharmacokinetic behaviors, such as human intestinal absorption (HIA), Caco-2 permeability, the blood–brain barrier (BBB) and CNS permeability, and the toxicity profile, were predicted using a web-based pkCSM application , whereaswissADME .1, 2, and 3, obtained from the roots of S. miltiorrhiza, were investigated through a chemiluminescent in vitro assay using a MAO-Glo kit. All three tanshinone congeners 1, 2, and 3 showed considerable activity against both hMAO-A and hMAO-B .The recombinant human monoamine oxidase (hMAO-A and hMAO-B) inhibition potential of the tanshinones d hMAO-B . Compoun1, 2, and 3 are potent MAO inhibitors, we performed an enzyme kinetics study to identify the enzyme inhibition modes and Ki values. Lineweaver-Burk plots (1/V vs. 1/S) and secondary plots (Kmapp/Vmaxapp and 1/Vmaxapp vs. the inhibitor concentration) were used to determine the kinetic parameters  was docked with the reference standard harmine, and hMAO-B (2BYB) was docked with the reference hMAO-B inhibitor deprenyl, to validate the docking results. To comprehend the specific binding sites and interactions at the orthosteric and/or allosteric sites of the enzymes responsible for the potent inhibitory actions of compounds 1, 2, and 3 occupied the active site of hMAO-A and interacted with the catalytic binding site with low binding energies of –10.06, –9.91, and –10.07 kcal/mol, respectively. The 1-methylbenzofuran ring of 1 was aligned toward the catalytic aromatic cage comprising Tyr407, Tyr444, and the flavin adenine dinucleotide (FAD) cofactor of hMAO-A and interacted with these residues and FAD through strong hydrophobic bonds, such as π-π stacking, π-sigma, and π-alkyl bonds. The O–atom at C11 further associated with the FAD through a polar H-bond. The tyrosine residues surrounding FAD, Tyr444, and Tyr407 were considered recognition residues crucial for stable substrate/inhibitor binding through π-π interactions between the aromatic rings, while Ile335 and Phe208 of the active site were responsible for substrate selectivity [1 further linked with the substrate-specific residues Ile335 and Phe208 with aromatic π-sigma and π-π stacking interactions. The other active site amino acid residues in the ligand–enzyme complex formation were Ile180, Ile35, and Leu337. Compounds 2 and 3, which differed in the 1,2-ene group in their benzofuran ring structures, faced towards the important catalytic site constituting FAD, Tyr407, and Tyr444 through their 6,6-dimethylnaphthalene moiety. Interactions with these catalytic residues, together with Phe352 and Tyr69, occurred via two methyl substituents, while the 1-methylbenzofuran-10,11-dione moiety participated in interactions with many active site residues of hMAO-A, such as Ile335, Ile325, Leu337, Ile 180, Cys323, Phe208, Val210, and others, as shown in As shown in ectivity ,42. The 1, 2, and 3 could be positioned in the allosteric binding sites of the hMAO-A enzyme, with similar orientations and binding energies of –7.87, –7.3, and –8.67 kcal/mol, respectively. Interactions with amino acid residues such as His488, Phe112, Trp128, Tyr121, and Tyr124 through hydrophobic bonds were common in these three compounds  and Arg127 ; electrostatic bonding with Asp123 (by the o-quinone moiety) and Glu483 (via 6-methylbenzene ring); and hydrophobic π-π stacking, π-π T-shaped interaction, and alkyl and π-alkyl bonding to Gly194, Ile477, Arg120, and Thr479.Even though the tanshinones ne group a,b. hMAO2 also exhibited interactions with both the catalytic and allosteric site residues of hMAO-B with binding energies of −10.29 and −7.98 kcal/mol, respectively. As with 1, the compound could anchor with Cys172 via a H-bond to the 11-O group and with Leu199 and Leu171 via hydrophobic interactions. From the binding pose analysis of 2 at the catalytic site, it was observed that the 6,6-dimethylcyclohexane ring projected outside the entrance cavity and interacted with the allosteric site residues Pro104, Phe103, Trp119, and Leu164  and at what target site the compound binds. The results of the molecular docking study, including the interacting residues and the binding energies, are presented in 1 occupies the orthosteric binding site of M4R and interacts with the amino acid residues of transmembranes (TM) 3, 5, and 7. Compound 1 binds with the catalytic site residues Tyr439 and Tyr433 via π-σ bonds, similar to the cognate agonist ACh. Interactions with Ala200 and Ala203 occur via π-alkyl bonds, similar to the cognate inverse agonist tiotropium. Additionally, compound 1 interacts with the active site residues Tyr113 via π-π stacking and Cys442 via π-alkyl and alkyl bonds. Unlike the reference ligands ACh and tiotropium, H-bonds and electrostatic interactions were not observed in Compound 1 and the M4R complex. Aromatic residues Tyr113 (TM3), Tyr416 (TM6), Tyr439 (TM7), and Tyr443 (TM7) have been linked to modulating the dissociation of antagonists from the orthosteric binding site of M4R. The mutations of these residues were found to reduce the binding affinity significantly. Moreover, the interactions of tiotropium with Asp112, Ser85, Tryp108, Tyr439, and Tyr443 were found specifically in M4R [4R complex.A computational study was used to investigate how the compound interacts with the My in M4R , suggest1 by SwissADME showed a drug-likeness but no lead-likeness due to the violation of a condition where XLOGP3 should be less than 3.5 [1. The pkCSM showed no toxicity on hepatocytes but is likely to be positive in the Ames test  is composed of S. miltiorrhiza, Panax notoginseng, and Dryobalanops aromatica [S. miltiorrhiza, such as anti-Alzheimer’s, through the inhibition of cholinesterase [Herbal medicines have been used in TCM for numerous ailments in China and other Asian countries for thousands of years. The roots of romatica . The phyromatica ,49. The esterase , β-secreesterase , and amyesterase ; the attesterase ; and theesterase ,25, haveS. miltiorrhiza, supplemented with enzyme kinetics and molecular docking studies. Our research found that the test compounds were more active against hMAO-A than hMAO-B, consistent with a previous report [1 showed the most potent and selective hMAO-A inhibition, with an IC50 value of 2.62 ± 0.52 µM and Ki value of 1.69 ± 0.19 µM. It showed a mixed-mode of enzyme inhibition, corroborating with catalytic and allosteric site binding in the docking studies. These results signify that 1 can bind to both free and substrate-bound hMAO-A enzymes. Compounds 2 and 3 also exhibited a strong mixed-mode of hMAO-A inhibition, with IC50 values of 6.08 ± 0.06 and 8.70 ± 0.06 µM and Ki values of 0.72 ± 0.13 and 4.99 ± 0.34 µM, respectively. The potency of the hMAO-A inhibition obtained in our work differs from that of a previously published article [Our study investigated the hMAO isoenzyme inhibition potential of three abundant tanshinones of s report . Among t article , because1, 2, and 3 show selectivity toward hMAO-A, the ability of the compounds to suppress hMAO-B cannot be neglected, because they displayed enzyme inhibition at concentrations below 25 µM. MAO-B accounts for 80% of the total MAO activity and major dopamine oxidation in the striatum of the human brain compared to MAO-A. Moreover, MAO-B increases with aging and neurodegenerative diseases such as PD and AD, even though the age-related decline of many neurons and related neurotransmitters and enzymes occurs [1, 2, and 3 were selective and notable inhibitors of hMAO-A, with moderate action against hMAO-B. The inhibition potential of these compounds was more significant for BChE than for AChE, suggesting their selectivity toward BChE [1 was portrayed as the most efficacious for inhibiting BACE1 [Although tanshinones s occurs ,53,54,55ard BChE . Among tng BACE1  and Aβ ang BACE1 .1 was necessary for the strong π-π interactions with the critical catalytic site residues of hMAO-A. The 6,6-dimethyl substitution oriented the molecules in such a way that the methyl groups interacted with the Tyr444, Tyr407, and FAD600, preventing the aromatic sandwich interaction between the compounds and the enzyme, which is important for potent enzyme inhibition. Likewise, docking with hMAO-B showed that the structural features of 1 allowed it to interact with catalytic substrate-binding sites comprising FAD600, Tyr398, and Tyr 435, along with the second cavity residues, through polar and nonpolar bonding. In accord with the kinetic study, the inhibition mechanism established by the computational study showed catalytic and allosteric inhibition for 1 at the hMAO-B binding sites. The noncompetitive hMAO-B inhibitor 2, on the other hand, was found to bind to both the catalytic and allosteric site residues to a similar degree. Similarly, 3 was engaged in the active site of hMAO-B interacting with most of the known second cavity loop residues and one substrate residue, Tyr398, of hMAO-B, which complied with its competitive binding mode.The in silico study revealed that the 1-methylbenzofuran ring with a single methyl substitution at C6 found in 1. The cell and nuclear receptor-based functional assay showed 1 to be a selective M4 antagonist. The in silico study revealed that the compound occupies the orthosteric binding site of the M4 receptor through hydrophobic interactions with active site residues, which were previously described by Thal et al. (2016) [Besides selective hMAO-A inhibition, tanshinones can target other proteins and enzymes. Among the high NR score targets, mAChRs have been linked to nondopaminergic motor and nonmotor symptoms of PD, and the only compound presumed to modulate these receptors through the PCM was . (2016) .1 revealed a poor solubility and low permeability (Caco-2 permeability was only 1.40 cm/s). As predicted for the CNS and BBB permeability, a recent in vivo pharmacokinetic and tissue distribution study using rats showed the distribution of 1 into the brain, with the highest amount reaching the liver, kidney, and lungs after oral administration. It was also observed that combinations of tanshinones had greater bioavailability  than single tanshinones administered orally, except for 1 and Dtan I, which showed similar pharmacokinetic values compared to that of the tanshinone mixture. Among tanshinones 1, 2, 3, and Dtan I, 1 had the most extended half-life [1 to protect from hepatotoxicity was reported by Park et al. (2009), which conformed to the predicted non-hepatotoxic property [1 to be Ames-positive, indicating its mutagenic ability, its anticancer effects, together with its neuroprotective and anti-inflammatory effects, have been verified experimentally [Although several studies have demonstrated the pharmacological benefits of tanshinones, the pharmacokinetics study showed a low bioavailability due to their low solubility and permeability. The in silico prediction of the pharmacokinetic characteristics of alf-life . The abiproperty ,57. Thoumentally ,50,58,59Neuropathological investigations in the autopsied brain of patients with PD have indicated that the disease manifests as dopaminergic neuronal degeneration in the ventrolateral substantia nigra in the early stage, which progresses to the midbrain and other regions of the brain in the later stages, along with increased levels of MAO-A and MAO-B ,54. Sinc4 is the major mAChR subtype in the striatum, where it is enriched in the D1 DA receptor-expressing spiny projection neurons (SPNs) comprising the basal ganglia direct pathway (D1-SPNs) but not in D2 expressing indirect pathway SPNs. This is a critical pathway for motor activation. Studies using M4 deletion from D1-SPNs have found genetically modified mice to be hyperlocomotive, with elevated baseline DA, and more sensitive to dopaminergic stimulants than their littermate controls [4 muscarinic receptors regulate the dynamic balance between cholinergic and dopaminergic systems. In the M4 knockout mouse model, it was observed that the loss of M4 receptors enhanced the DA basal levels and induced dopaminergic hyperexcitability in response to psychostimulants, suggesting that M4 receptors are crucial muscarinic autoreceptors that regulate DA neurotransmission and dopaminergic activity [4R antagonists could improve a parkinsonian motor disability by relieving the M4-mediated inhibition of DA elevation and hyperexcitation of the dopaminergic system in the striatum. Mcontrols . Tzavaraactivity . Thus, sS. miltiorrhiza in AD, tanshinone congeners appreciably attenuated the scopolamine-induced cognitive impairments and reversed learning and memory dysfunctions induced by scopolamine and diazepam. These observations indicated that the memory-enhancing effect of tanshinones might be associated with the cholinergic signaling activation or the inhibition of cholinesterases that led to increased acetylcholine levels [1, 2, and 3 are known, and the nAChR antagonist effects of the lipophilic extract of S. miltiorrhiza have been reported, whether the cognitive improvement by these tanshinone congeners is directly linked to cholinergic signaling remains unknown [2 and M4 receptors are the most relevant targets for 1, and the in vitro experiment determined the M4 selective antagonist property of 1, which might rule out the role of tanshinones in the stimulation of muscarinic receptors for learning and memory. Kim et al. (2009) found that the activation of the ERK/CREB signaling pathway by 1 in the hippocampus reversed GABAA receptor agonist- and NMDA receptor antagonist-induced cognitive dysfunctions [In former investigations that focused on the implications of diterpenoids from e levels . Althoug unknown ,21,62,63unctions .1–M4), designing a drug to act selectively at one of the mAChRs is challenging. Since M4 represents a crucial target for PD and selective M4 antagonists could produce therapeutic benefits, avoiding unwanted cholinergic side effects, the discovery of safe and novel M4-selective antagonists is vital. To date, a few M4-selective antagonists are known, and most clinically available antimuscarinic agents are either nonselective or only partially selective [1 from S. miltiorrhiza is a fully selective antagonist of M4. The investigation into the monoamine oxidase inhibitory activity of the compounds led to the acknowledgment that 1 is a potent and selective hMAO-A inhibitor with a mixed-mode of enzyme inhibition. The other tanshinones, 2 and 3, also showed significant and selective hMAO-A inhibition with moderate activity against hMAO-B. Among the three tanshinones, 1 exhibited the most desirable pharmacological activities, along with the reported abilities to inhibit cholinesterases, BACE1, and Aβ aggregation, suggesting that 1 may have a promising role in neurodegenerative diseases. Overall, our present in vitro study revealed compound 1 as a potent hMAO-A/B inhibitor and a selective M4R antagonist. These bioactivities suggest the potential therapeutic benefits of 1 in alleviating DA deficiency, motor symptoms, and depression in NDDs. However, further in vivo investigations are important to establish 1 as a potential M4R antagonist and a MAO inhibitor to treat motor dysfunctions and depression.Due to the extreme similarity of the conserved residues that comprise the orthosteric sites of muscarinic receptors (Melective . Our pre


The percutaneous iliosacral screw is a common procedure for treating pelvic posterior ring instability. Traditional X-ray fluoroscopy screw placement has the advantages of decreased bleeding and trauma, but it also has some drawbacks, such as increased radiation exposure and screw dislocation. The purpose of this study was to establish a safe, effective, and quick approach for putting iliosacral screws for the treatment of unstable posterior pelvic ring damage utilizing simulated screws based on preoperative computed tomography (CT) planning.n = 20) and preoperative planning (n = 21). Pelvic radiographs , as well as normal CT scans of the pelvis, were all taken postoperatively to confirm the screw position. After that, the screw insertion time, the radiation exposure time, and the screw misplacement rate (as assessed by postoperative CT) were all examined. Screw position grading was evaluated by Smith grading.From February 2019 to June 2020, we retrospectively assessed 41 patients with posterior pelvic ring instability who were treated with percutaneous iliosacral screws in our institution, and randomly separated them into two groups: conventional surgery (P < 0.05).In the conventional surgery group, 26 screws were inserted in 20 patients, with each screw insertion taking 23.15 ± 4.19 min and 1.02 ± 0.17 min to expose to radiation. Eight of the 26 screws were misplaced (30.8%). In the preoperative planning group, 24 screws were inserted in 21 patients, with each screw taking 19.57 ± 4.05 min to implant and 0.67 ± 0.09 min to expose to radiation. One of 24 screws was misplaced (4.2%). Screw insertion time, radiation exposure time, and screw dislocation rate were all significantly reduced when preoperative planning aided iliosacral screw placement (Preoperative CT simulation of iliosacral screws for placement planning, screw trajectory, and intraoperative screw placement is a safe way for reducing surgical time, radiation exposure, and ensuring accurate screw placement. Pelvic fractures caused by high-energy injuries are becoming increasingly common as the social economy increase . The purThe usual approach of iliosacral screw placement is to use standard lateral radiographs of the sacrum to establish the insertion point during the procedure and then adjust the direction of the guidewire based on the pelvic inlet and outlet views , 5, 8. TPreoperative high-quality images are now easier to obtain because updated instruments, and proper preoperative planning can minimize operation time and raise surgeon confidence by evaluating preoperative 3D reconstruction . We usedThis study was approved by the ethics committee of Bengal Medical College (Number: BYYFY-2019KY03). From February 2019 to June 2020, a total of 124 patients with pelvic fractures were treated in our hospital. The following were the inclusion criteria: 1. Age > 14 years; 2. Injury posterior pelvic ring instability (Tile B or C injury); 3. Sacral fracture: Denis zone 1 or zone 2; 4. Pelvic fractures may be minimized by preoperative or intraoperative traction. Exclusion criteria include: 1. Age < 14 years; 2. 0pen pelvic fracture; 3. Having a skin infection in the incision location. 4. Surgical incision and investigation are indicated when vascular and nerve damage is present. 41 patients with pelvic fractures who satisfied the inclusion criteria were divided into two groups at random: the traditional surgery group had 20 patients, whereas the preoperative planning group had 21.According to the patient's ante-posterior, inlet and outlet X-rays, and a preoperative thin CT scan (0.625 mm thickness), the fracture type was diagnosed. The surgical procedure was determined based on the type of fracture.All of the patients were supine for the procedure, which was carried out by the same skilled surgeon. The physician determined and utilized standard intraoperative lateral, inlet, and outlet radiographs of the pelvis. Simultaneously, a skilled radiologist operates the same C-arm machine and adjusts it to seek the optimal X-ray image.According to the standards of screw position published by Smith et al. , the scrGender, age, fracture type, mechanism of injury, time of screw placement (from the insertion of the guidewire into the skin to the completion of the screw placement), time of radiation exposure during screw placement, and postoperative screw position were all recorded.After admission, the patient underwent a routine plain pelvic CT  scan, and CT images of 0.625 mm thickness were obtained. CT images were imported into Mimics software  to obtain the patient's pelvic standard lateral views, and inlet and outlet views Fig. a, b, c. The iliosacral screw trajectory was generated using the patient's preoperative CT three-dimensional model. The patient's body and the operating table were adjusted so that the patient's body was parallel to the floor at the center of the operating table. After disinfection, the c-arm angle was adjusted to obtain standard lateral and inlet and outlet radiographs of the sacrum. The ground was marked and the rotation angle of the c-arm was recorded. The guiding needle tip was adjusted according to the pre-planned screw insertion location on standard lateral radiographs of the sacrum. The guide needle was gently hammered into the bone using a bone hammer once it had been adjusted to the proper insertion site. At this stage, the inlet and outlet views, as well as the guidewire pointing at the inlet and outlet views, were changed under the preoperative planning. To prevent the guidewire from altering the track, the bone hammer was gradually hammered into the region according to this angle, and fluoroscopy was conducted again after the cortex was penetrated. After the needle was placed along the preoperative trajectory, standard lateral and inlet and outlet radiographs of the sacrum were obtained again. After measuring the required length of the screw with a needle tester, a screw  of proper length was inserted Fig. .Fig. 2PrThe patient lay supine on a fluoroscopy carbon fiber operating table while the same surgeon used a C-arm machine to execute standard screw placement. The guidewire (diameter: 2.5 mm) was first put in a safe screw placement region after obtaining standard lateral imaging of the sacrum. The guidewire was then carefully hammered into the bone with a bone hammer to prevent the position of the guidewire from being lost. Adjust the guide wire's orientation at the inlet and outlet views, then hammer into it in this direction after it's found in the osseous suitable safe path at the inlet and outlet views. Measure the guide wire's length and insert it in the proper length Fig. .Anterior–posterior pelvic radiographs, inlet and outlet radiographs, and a postoperative CT scan were taken three days after surgery to determine screw placement and fracture reduction. The quadriceps femoris muscle in both lower limbs was intensively trained on the second day after surgery. One week after a type B fracture, the patient was able to sit up in bed. 2 weeks after surgery: partial weight-bearing; 3 weeks after surgery: full weight-bearing Sit up in bed two weeks after a type C fracture operation; partial weight-bearing four weeks after surgery; full weight-bearing six weeks after surgery. Patients returned to the hospital 1.5, 3, 6, and 12 months following surgery for follow-up.p-value < 0.05.Following data collection, t-test and Chi-square tests were performed to compare the traditional surgery group to the preoperative planning group in terms of screw placement time, radiation exposure time, and precision. For categorical data, descriptive statistics are reported as ratios, and for continuous variables, mean plus standard deviation. Fisher's exact test was used to generate p values whenever the anticipated numbers of cell entries were less than 5. SPSS statistics software version 22.0  was used for all statistical analyses. Statistical significance was defined as There were no statistically significant differences between the two groups in demographic or preoperative characteristics Table . There wP < 0.05) as compared to the traditional surgery group. Each screw in the traditional surgery group received 1.02 ± 0.17 min of radiation exposures. Each screw in the preoperative planning group received 0.67 ± 0.09 min of radiation exposure, which was statistically significant difference (P < 0.001).The results for these two groups are shown in Table Eighteen screws (69.2%) were totally inserted in the bone channel in the conventional surgery group, six screws (23.1%) were close to the sacral foramen cortex but did not extend into the sacral foramen, and two screw (7.7%) penetrated the sacral foramen. 23 screws (95.8%) were totally inserted in the bone channel in the preoperative planning group, one screw 4.2%) was close to the cortex of the sacral foramen, and no screws entered the sacral foramen (Fig. .2% was cThe majority of orthopedic surgeons utilize percutaneous iliosacral screw fixation as a standard surgical procedure , 13, 18.Ebraheim et al.  proposedStudies have demonstrated that intraoperative CT fluoroscopy navigation and computer 2D/3D navigation can guide screw placement with greater accuracy, but it has not been widely adopted due to the high technical requirements and high overall costs , 26–29. Clinicians face a significant challenge in treating unstable pelvic fractures. First, high-energy injury patients frequently have many injuries throughout the body, and poor blood perfusion caused by large blood loss frequently exacerbates tissue and organ harm . Second,Preoperative planning assists and guides intraoperative placement of iliosacral screws for the treatment of unstable posterior pelvic ring injuries, simplifying the surgical procedure and permitting surgeons to insert iliosacral screws more safely. It can significantly reduce radiation exposure, shorten operation time, and improve precision when compared to standard surgical approaches. Preoperative planning opens up a novel option for percutaneous iliosacral screw fixation that warrants further investigation.


TPSs) that are the key enzymes in terpenoids biosynthesis. Orchidacea, containing more than 800 genera and 28,000 species, is one of the largest and most diverse families of flowering plants, and is widely distributed. Here, the diversification of the TPSs evolution in Orchidaceae is revealed. A characterization and phylogeny of TPSs from four different species with whole genome sequences is available. Phylogenetic analysis of orchid TPSs indicates these genes are divided into TPS-a, -b, -e/f, and g subfamilies, and their duplicated copies are increased in derived orchid species compared to that in the early divergence orchid, A. shenzhenica. The large increase of both TPS-a and TPS-b copies can probably be attributed to the pro-duction of different volatile compounds for attracting pollinators or generating chemical defenses in derived orchid lineages; while the duplications of TPS-g and TPS-e/f copies occurred in a species-dependent manner.Terpenoids are the largest class of plant secondary metabolites and are one of the major emitted volatile compounds released to the atmosphere. They have functions of attracting pollinators or defense function, insecticidal properties, and are even used as pharmaceutical agents. Because of the importance of terpenoids, an increasing number of plants are required to investigate the function and evolution of terpene synthases ( Accordind TPS-h . TPS-a, ndorffii .TPSs has three conserved motifs on C- and N-terminal regions. The conserved motif of N-terminal domain is R(R)X8W  and the C-terminal domain contains two highly conserved aspartate-rich motifs. One of them is the DDXXD motif, which is involved in the coordination of divalent ion(s), water molecules, and the stabilization of the active site [8W’ motif is also essential for monoterpene cyclization, while some MTPSs do not have it [Oryza sativa L. cv. Nipponbare and Hinohikari) [Glycine max cv. ‘Bagao’), which has a signal peptide and is believed to be functional in plastid [The full length of plant ive site ,18,19. Tive site ,21. Mostive site , sesquitive site . They al have it . These cohikari) ; nerol s plastid ; and FaN plastid .TPSs in the same subfamilies are similar in sequence and have similar functions. Based on the protein sequence, angiosperm STPSs and DTPSs belong to TPS-a subfamily and monoterpene synthases belong to TPS-b subfamily. Subfamilies in TPS-c and e/f have enzyme activities of DTPSs; Gymnosperm-specific TPS-d subfamily owns the enzyme activities for MTPSs, STPSs, and DTPSs. TPS-g encodes MTPSs, STPSs, and DTPSs that produce mainly acyclic terpenoids. TPS-h is Selaginella moellendorffii-specific subfamily and putative encodes DTPSs [TPS are usually further modified by cytochromes p450 (CYPs) to generate diverse structures [es DTPSs ,27. Recees DTPSs . Moreovees DTPSs . Some TPes DTPSs , others es DTPSs ,31. Moleructures .Orchids show extraordinary morphological, structural, and physiological characteristics unique in the plant kingdom . ContainCycnoches densiflorum and C. dianae [Bulbophyllum vinaceum [e-carvone oxide for Catasetum integerrimum [p-dimethoxybenzene for Cycnoches ventricosum and Mormodes lineata [Notylia barkeri [e-ocimene and linalool for Gongora galeata [Orchis mascula and Orchis pauciflora [Dendrobium sinense [Calanthe sylvatica [Cyclopogon elatus [E)-β-ocimene and (E)-epoxyocimene for Catasetum cernuum and Gongora bufonia [Cymbidium goeringii [Floral VOCs are characterized into several orchids, including α- and β-pinene for . dianae ; phenylpvinaceum ; α-pinengerrimum ; p-dimet lineata ; β-bisab barkeri ; e-ocime galeata ; monoteruciflora ; (Z)-11- sinense ; terpenoylvatica  and Cycln elatus ; (E)-β-o bufonia ; and faroeringii .Phalaenopsis spp. is very popular worldwide for its spectacular flower morphology and colors. Most Phalaenopsis orchids are scentless but some do emit scent VOCs [P. amboinensis, P. bellina, P. javanica, P. lueddemanniana, P. schilleriana, P. stuartiana, P. venosa, and P. violace [P. bellina and P. violacea are two scented orchids that are very popular in breeding scented cultivars. P. bellina emits mainly monoterpenoids, including citronellol, geraniol, linalool, myrcene, nerol, and ocimene [P. violacea emits monoterpenoids accompanied with a phenylpropanoid, cinnamyl alcohol [P. schilleriana contain monoterpenoids as well, including citronellol, nerol, and neryl acetate [TPSs.ent VOCs . The sce violace . P. bell ocimene ,48, whil alcohol . The VOC acetate . BecauseTPSs in Orchidaceae. TPSs from different orchid species are reported to explore the evolutionary history and the evolution diversification of Orchidaceae TPSs.In the present review, we summarized the recent progress in the understanding of the biosynthesis and biological function of terpenoids, and the latest advances in research on the evolution and functional diversification of 10-C20 diphosphates go through cyclization and rearrangement to produce the basic carbon skeletons for terpenoids catalyzed by TPS [TPS family consists of enzymes that use GDP to form cyclic and acylic monoterpenes (C10), FDP for sesquiterpene (C15), and GGDP for diterpene (C20) [30 and C40. The final step converts terpenes into different skeletons by oxidation, reduction, isomerization, conjugation, and other transformation [TPSs are the key enzymes in terpenoid biosynthesis.There are two compartmentalized terpenoid biosynthesis pathways, the mevalonic acid (MVA) pathway that occurs in the cytosol, and the methylerythritol phosphate (MEP) pathway that occurs in plastids to produce isopentenyl diphosphate (IPP) and its allylic isomer-dimethylallyl diphosphate (DMAPP) converted by isopentenyl diphosphate isomerase (IDI)  50,51,5,551,52. d by TPS . The TPSne (C20) . Moreoveormation . TPSs arA. shenzhenica [Vanilla planifolia [D. catenatum [P. equestris [https://www.ncbi.nlm.nih.gov/ (accessed on 6 January 2021).) and OrchidBase database [http://orchidbase.itps.ncku.edu.tw/est/home2012.aspx (accessed on 9 August 2020).). Second, A. ashenzhenica is the most original orchid, and P. equestris is the first whole genome sequenced orchid. V. planifolia produces vanillin and is important in the food industry, and D. catenatum is a medicinal orchid and produces important secondary metabolites for pharmaceutical purpose. We isolated the TPS genes of Orchidaceae through KAAS (http://www.genome.jp/tools/kaas/ (accessed on 21 February 2017).) annotation and BLASTp from the whole genome sequences of four orchids. Each full-length TPS is characterized by two conserved domains with Pfam [TPS genes were identified from the whole genome sequences of A. shenzhenica, V. planifolia, D. catenatum, and P. equestris, respectively. In addition, P. aphrodite with white, scentless flowers and P. bellina scented flowers are native species. Their floral transcriptomes are available in Orchidstra and OrchidBase transcriptome database, respectively. 17 TPS genes in P. aphrodite and 11 TPS genes in P. bellina were identified from the transcriptome database. The TPS genes were denoted with numbers Ash-, KAG-, Dca-, Peq-, PATC-, and PbTPS- identified from A. shenzhenica, V. planifolia, D. catenatum, P. equestris, P. aphrodite, and P. bellina, respectively.We chose the whole genome sequences of four orchids, including nzhenica  in Apostanifolia  in Vanilatenatum  and P. equestris  in Epidedatabase  . So, we further investigated TPS evolution in Orchidaceae and provided insight into TPSs at the genome level. In this review, the encoded amino acid sequences of identified orchid TPS genes were aligned with those from Arabidopsis and Abies grandis, and those from Selaginella moellendorffii were used as outgroups  TPSs have more members in TPS-b subfamily than in TPS-a subfamily. Within TPS-b subfamily, these orchid TPSs form distinct clades separated from those of Arabidopsis (dicot) TPSs . In particular, the number of duplicated orchid TPS-a and TPS-b copies increased in V. planifolia and D. catenatum  TPSs . Taken tatenatum . These dTPS-e/f subfamily has increased copy numbers compared to that from A. thaliana  of angiosperm [TPS-d and TPS-h are gymnosperm and Selaginella moellendorffii specific, respectively [TPSs were grouped in these subfamilies, in accordance with previous conclusions by Chen et.al, and Trapp et.al. [Our phylogenetic analysis also reveals that the orchid thaliana . Orchid anifolia , whereasgiosperm . TPS-d aectively . Our anap et.al. ,62.https://meme-suite.org/meme/tools/meme (accessed on 19 March 2021).) (TPSs (R(R)X8W, EDXXD, RXR, DDXXD, and NSE/DTE) were elucidated X8W, except PATC144727, Peq011664, Dca017107, and PATC155674 in TPS-a subfamily (TPS-b subfamily that encodes MTPSs contains the highly conserved R(R)X8W motif. All TPSs in Arabidopsis TPS-b subfamily contain conserved R(R)X8W motif, except AtTPS02 X8W motif of orchid TPSs is divergent in TPS-b subfamily.Motifs of identified orchid TPS proteins were predicted using MEME software ( 2021).) A, and fiucidated B. The TPt plants ,16,63. Ireaction , or in sreaction . Compareubfamily A. In con AtTPS02 B. Howeve8W motif B. MotifsA. shenzhenica has clear evidence of whole-genome duplication that is shared by all orchids [TPS in A. shenzhenica are among the fewest and are worthwhile for further investigation. For Phalaenopsis orchids, paralogs of TPS genes could be identified from each species, implying the duplications were attributed to their common ancestor, and some persisted or lost in current species  in three parallel clades of the phylogenetic tree   in eight parallel clades, indicating the TPS-b gene copy duplications could be traced back to the common ancestor of Phalaenopsis species   and CPS. MTPSs and STPSs are evolved from ancestral DTPS through duplication and then sub- or neo-functionalization during evolution . A. shen orchids . Yet, th species . For exaTPS-e/f subfamilies are mainly detected in angiosperm and conifers DTPSs of primary metabolism  [TPS-e/f subfamilies comprise orthologous genes without R(R)X8W  ,67. Orch R(R)X8W C, which TPS-g subfamily is closely related to the TPS-b but lacks the N-terminal “R(R)X8W” motif and encodes MTPSs, STPSs, and DTPSs that produce mainly acyclic terpenoids [TPS-g subfamily in Arabidopsis (AtTPS14) lacks both “R(R)X8W” and “RXR” motifs. However, although TPSs of V. planifolia in TPS-g subfamily  class [Dendrobium is believed to be responsible for its medical property [TPS-a TPSs was detected in D. catenatumas as compared to that of other orchid species, which is responsible for sesquiterpene biosynthesis  emission in P. bellina [P. bellina. TPS-e/f enzymes have diverse functions, including linalool synthase, geranyllinalool synthase, and farnesene synthase in kiwifruit [TPSs in the TPS-e/f subfamily are thought to be dicot-specific because so far no TPS-e/f activity has been reported in monocots. However, the number of TPS in TPS-e/f expands from 1 in Apostasia to 4 in Phalaenopsis  class , and manproperty . Concomiynthesis . The inclinators . PbTPSs ubfamily . Previou bellina . This suiwifruit ,74. TPSsaenopsis , suggestTPSs in each subfamily evolved from the early divergence orchid species, such as A. shenzhenica and/or V. planifolia. The large expansion of TPS copies in orchid groups such as V. planifolia, D. catenatum, and Phalaenopsis species might be due to high flexibility for adaptation and evolution through natural selection.Together, our analyses suggest that orchid TPS genes was detected in orchids. Orchid TPS gene clusters diverged with tandem or segmental duplications  in different scaffolds in the TPS-g subfamily . The percentages of clustered TPS genes were 66.7%, 81.5%, 57.1%, and 53.3% for A. shenzhenica, V. planifolia, D. catenatum, and P. equestris, respectively, while that was 40.6% in Arabidopsis thaliana  were increased compared with those in A. shenzhenica, the total number was not linked to the increased genome size.The functional cluster phenomenon of ications . Tandem ubfamily C. TPS geubfamily A,B. We iscaffold A–D. In aectively . Each clquestris . TPS genthaliana . The cluectively . The cluectively . InteresArabidopsis related to triterpene biosynthesis pathway [TPS gene families in plants has been described and often results in tandem repeats, as an important driver for the expansion [TPS genes have already been reported in several plant species, such as Arabidopsis thaliana [Vitis vinifera [Solanum lycopersicum [Eucalpyus grandis [TPS genes in E. grandis are up to 20 genes [Solanum species, the gene duplications and divergence give rise to TPS gene clusters for terpene biosynthesis [V. vinifera TPSs are present on chromosome 18 [Arabidopsis TPS genes are reported with the phenomenon of several gene clusters [TPS members, including Os08g07080, Os08g07100, and Os08g07120, is observed in Asian rice Oryza sativa and also appears in various rice species including O. glaberrima, O. rufipogon, O. nivara, O. barthii, and O. punctata. [TPSs indicate the functions in insect-damaged plants [TPS genes and their catalytic activities for emission patterns of volatile terpenes is induced by insect damage and is largely consistent [TPSs with other biosynthesis-related genes was also found to form unexpected connection with time passed. For instance, the evolution of TPS/CYP pairs is different in monocot and dicot [TPS/CYP pairs duplicate with ancestral TPS/CYP pairs as templates to be evolved in dicots, but the evolutionary mechanism of monocot shows that the genome rearrangement of TPS and CYP occurred independently [Solanum spp., TPS forms functional clusters with cis-prenyl transferase [In plants, gene clusters were often observed for metabolic pathways, such as gene clusters found in oat and  pathway . Local dxpansion ,76. The xpansion . The funvinifera , Solanumpersicum , Eucalpy grandis , and ric grandis ,80. Geno20 genes . In seveynthesis . A denseosome 18 . Arabidond dicot . TPS/CYPendently . In Solansferase . Both taTPS genes formed genomic clusters, and the clusters increased in V. planifolia and D. catenatum. Combining the results from phylogenetic analysis and functional gene clusters, orchid TPSs may be expanded by tandem or segmental duplications. Interestingly, the genome duplication events occurred all the way along the evolution from Apostasioideae to Vanilloideae and Epidendroideae; the TPS clusters and copy numbers increased in orchid lineages, such as the early divergence A. shenzhenica. The large expansion of orchid TPS copies in V. planifolia, and D. catenatum species might have high flexibility in secondary biosynthesis through natural selection.Together, the orchid TPS is from duplication and loss of TPS genes. In Orchidaceae, we discover that the duplication event of TPS occurred among all TPS subfamilies. TPs-a, TPS-b, and TPS-e/f subfamilies went through gene duplication, while TPS-g duplicated from Apostaceae to Vaniloideae, and then lost from Vaniloideae to Epidendroideae. The driving force of TPS evolution in each subfamily may be different. For example, in TPS-a and TPS-b, the necessity of generating volatile compounds for the interaction of orchids with their pollinators, producing chemical defenses and being responsive to environmental stress, may be the major reason for their rapid evolution. On the other hand, the duplications of TPS-g and TPS-e/f copies were mainly species dependent and the reason remains to be uncovered.The basic evolution of


Significance: The majority of the drugs which target membrane-embedded protease γ-secretase show an unusual biphasic activation–inhibition dose-response in cells, model animals, and humans. Semagacestat and avagacestat are two biphasic drugs that can facilitate cognitive decline in patients with Alzheimer’s disease. Initial mechanistic studies showed that the biphasic drugs, and pathogenic mutations, can produce the same type of changes in γ-secretase activity. Results: DAPT, semagacestat LY-411,575, and avagacestat are four drugs that show different binding constants, and a biphasic activation–inhibition dose-response for amyloid-β-40 products in SH-SY5 cells. Multiscale molecular dynamics studies have shown that all four drugs bind to the most mobile parts in the presenilin structure, at different ends of the 29 Å long active site tunnel. The biphasic dose-response assays are a result of the modulation of γ-secretase activity by the concurrent binding of multiple drug molecules at each end of the active site tunnel. The drugs activate γ-secretase by facilitating the opening of the active site tunnel, when the rate-limiting step is the tunnel opening, and the formation of the enzyme–substrate complex. The drugs inhibit γ-secretase as uncompetitive inhibitors by binding next to the substrate, to dynamic enzyme structures which regulate processive catalysis. The drugs can modulate the production of different amyloid-β catalytic intermediates by penetration into the active site tunnel, to different depths, with different flexibility and different binding affinity. Conclusions: Biphasic drugs and pathogenic mutations can affect the same dynamic protein structures that control processive catalysis. Successful drug-design strategies must incorporate transient changes in the γ-secretase structure in the development of specific modulators of its catalytic activity. Alzheimer’s disease is a slowly-progressing neurodegenerative disorder with a fatal outcome . AlzheimThe majority of the potential drugs for Alzheimer’s disease have targeted the metabolism of the C-terminal part of amyloid precursor protein (C99-APP) ,4,5,6. TThe activation and inhibition of γ-secretase activity which can be observed in the biphasic dose-response to drugs are good examples of the complex enzymatic mechanisms that were not adequately recognized in the drug-development studies ,13,14,15The great majority of the drug-screening and optimization studies used an unphysiological high saturation of γ-secretase with its substrate, and a limited concentration range for the drugs. Such an approach can be a good time-and money-saving strategy in the early screening process ,16,18,19The mechanistic studies of the biphasic dose-response drugs are also important because these compounds can produce the same type of changes in γ-secretase activity as the disease-causing Familial Alzheimer’s disease (FAD) mutations ,25. FAD Here, we present combined structure and activity studies of γ-secretase in order to describe the biphasic dose-response in the presence of the four best-known drugs. Semagacestat and avagacestat were chosen as two biphasic drugs which can facilitate cognitive decline in patients in clinical trials ,16,28,31We measured the biphasic dose-response in Aβ 1-40 production in SH-SY5 cells . One batThe Aβ 1-40 production in the SH-SY5 cells treated with DAPT, semagacestat, LY-411, 575, and avagacestat show biphasic activation–inhibition dose-response curves with clear differences between the drugs in their activation and inhibition constants, and Hill's coefficients . Thus, tWe find that activation and inhibition curves always overlap; the compounds that show low EC50 values for activation also show low IC50 for inhibition . These rDAPT, semagacestat, and LY-411,575 show quantitative differences in their biphasic dose-response curves , and a vAvagacestat and LY-411,575 have similar EC50 and IC50, and notable differences in the Hill's coefficients . AvagaceThe flexible parts in the γ-secretase structure can be described using residue-based coarse-grained protocols  37]. We. We37]. open and closed structures [Substrate binding and Aβ catalytic intermediates lead to an increase in the width and mobility in the presenilin structure at its cytosolic end A 38]. T. T38]. Tructures .The highest mobility is observed with Aβ 43 and Aβ 46 catalytic intermediates A–C. ThesWhen Aβ substrates of different lengths are bound to γ-secretase, the active site tunnel is predominately closed at its cytosolic end . The acthttps://www.alzforum.org/mutations accessed on 2 April 2021). The active site aspartates are part of helix structures, and show low mobility deep in the protein interior; however, they are directly adjacent to the high mobility sites (Root mean square fluctuation (RMSF) graphs ,41 showety sites C 40,41],41https:Possible binding sites for different drugs can be identified by searching Connolly surface using molecular docking studies . Theril 2021 ,11,13.The possibility that several drug molecules can bind to γ-secretase simultaneously was suggested in some of the earlier studies . MultiplWe have combined molecular docking studies  and moleWe found that the drugs bound at the different ends of the active site tunnel could mimic conformational changes in the enzyme structure that drive the release of tripeptide byproducts and processive catalysis , the druThe binding site at the cytosolic end of the presenilin structure is the most dynamic . In the The second binding site, at the membrane end of the active site tunnel, can bind only one molecule with each of the four drugs  Figure . In the The third binding site is in the hydrophobic gap between the Aph1 subunit and the TM8 in presenilin 1. Any analysis at this site has to be taken with some reservations, since that part of the structure lacks 73 amino acids on presenilin 1 ,27. NeveThe structures of γ-secretase in complex with different Aβ catalytic intermediates were used for the analysis of the modulation of Aβ production by biphasic drugs ,9,13,27 open and closed structures of γ-secretase . FADil 2021,  and the il 2021, . Biphasiil 2021, ,22,26,27The insights from mechanistic studies of biphasic dose-response can help to achieve reproducible results and sustained progress in future drug-development efforts ,23,27,32The majority of preclinical drug development studies did not pay attention to the extent of γ-secretase saturation with its substrate, and most often used a saturating substrate. The assays with saturating substrate are the fastest and the cheapest, but also the least relevant to cell physiology ,9,10,16.The biphasic dose-response curves can give a false impression that the drugs can be used at precise doses to act only as activators or only as inhibitors of γ-secretase  10,15,1,115,16. In conclusion, numerous and expensive failures of different drug-design strategies are not evidence that γ-secretase is not a good therapeutic target, but are rather evidence that ‘fast-and-cheap’ protocols have overlooked the key features of this uniquely complex protease. The drug-screening strategies must follow fundamental protocols for the analysis of enzyme activity ,19,24 inDrugs were purchased from Calbiochem: DAPT -l-alanyl]-S-phenylglycine t-butyl ester). Semagacestat, LY-450,139 2S)-2-Hydroxy-3-methyl-N-amino]ethyl]-butanamide; LY-411,575, N2-[(2S)-2--2-hydroxyethanoyl]-N1-azepin-7-yl]-L-alaninamide; Avagacestat, (2R)-2-[N-[(4-Chlorophenyl)sulfonyl]-N-[[2-fluoro-4-phenyl]methyl]amino]-5,5,5-trifluoropentanamide, (R)-2-benzyl)phenylsulfonamido)-5,5,5-trifluoropentanamide, BMS-708163.v/v). The DMSO vehicle represents 0 nM drugs. The cells were incubated in 6-well plates, with the drugs at the given concentrations, for between 12–18 h. We paid maximal attention to measure all four drugs under identical conditions. The same batch of cells was used in parallel for the measurements with all four drugs. Identical conditions were used for the incubation of the drugs with the cells, sample harvesting, and measurements of Aβ 1-40.SH-SY5 cells were purchased from ATCC as passage 11, and were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum. The measurements of the biphasic response in the presence of the drugs, and the corresponding data analysis were described in detail in our earlier studies . BrieflyThe assays closely followed the manufacturer’s instructions. Briefly, sandwich ELISA kits for the quantitative detection of human Aβ 1-40 peptides in a flexible 96-well format were purchased from Millipore . The assay had a linear response in the range from 6 to 125 pM of Aβ 1–40. The Aβ 1–40 samples from the cell cultures were used immediately after their collection, following the manufacturer suggestion and our earlier reported experimental experiences . The welThe binding sites for semagacestat, avagacestat, LY-411,575, and DAPT were calculated following the standard protocols ,67. BrieCoarse-grained molecular dynamics calculations with the γ-secretase structure within the lipid bilayer were prepared using CHARMM-GUI 32 Martini Bilayer Maker 33 ,68. The γ-Secretase structures in complex with different drugs from the molecular docking studies ,72 were The all-atom simulations of γ-secretase in the lipid bilayer were prepared using CHARMM-GUI Membrane Builder 35–37 . OPM proThe validities of the presenilin structures in each of the molecular dynamic simulations were analyzed by comparing the active site structures with the mechanistic studies of the active site of aspartic protease , and by The ligand parameterization was prepared using ACPYPE tools  or the CS(x) represents the measured activity at inhibitor concentration x. PA is the physiological Aβ 1–40 production activity at inhibitor concentration zero. MA is the calculated maximal activity caused by activation if there is no competing inhibition. The MA values roughly correlate with the capacity of drugs to induce an increase in the enzyme–substrate affinity. MI is the maximal inhibition. EC50 and IC50 represent the activation and inhibition constants, respectively, while p and q represent the corresponding Hill’s coefficients.Molecular imaging and analyses of the molecular properties used VMD 1.9.3 and UCSF Chimera 1.14 ,78. The


Diminished synaptic gain—the sensitivity of postsynaptic responses to neural inputs—may be a fundamental synaptic pathology in schizophrenia. Evidence for this is indirect, however. Furthermore, it is unclear whether pyramidal cells or interneurons (or both) are affected, or how these deficits relate to symptoms.n = 108), their relatives (n = 57), and control subjects (n = 107) underwent 3 electroencephalography (EEG) paradigms—resting, mismatch negativity, and 40-Hz auditory steady-state response—and resting functional magnetic resonance imaging. Dynamic causal modeling was used to quantify synaptic connectivity in cortical microcircuits.People with schizophrenia diagnoses (PScz) , reduced mismatch negativity, and reduced 40-Hz power. Across all 4 paradigms, characteristic PScz data features were all best explained by models with greater self-inhibition (decreased synaptic gain) in pyramidal cells. Furthermore, disinhibition in auditory areas predicted abnormal auditory perception (and positive symptoms) in PScz in 3 paradigms.First, characteristic EEG changes in PScz in 3 classic paradigms are all attributable to the same underlying parameter change: greater self-inhibition in pyramidal cells. Second, psychotic symptoms in PScz relate to disinhibition in neural circuits. These findings are more commensurate with the hypothesis that in PScz, a primary loss of synaptic gain on pyramidal cells is then compensated by interneuron downregulation (rather than the converse). They further suggest that psychotic symptoms relate to this secondary downregulation. SEE COMMENTARY ON PAGE 167Reduced excitatory synaptic gain  is believed to be a primary deficit in schizophrenia ,2. This Various mechanisms may reduce synaptic gain in schizophrenia: the most important is probably hypofunction of NMDA receptors (NMDARs) and their postsynaptic signaling cascade ,2. EvideAn alternative way to investigate synaptic gain is by using electroencephalography (EEG) paradigms such as the mismatch negativity (MMN), an auditory oddball paradigm , and audNeural mass models of noninvasive data can be parameterized in terms of synaptic gain and these parameters estimated, for example, using dynamic causal modeling (DCM) , furnishTo date, DCM studies of PScz have used modest sample sizes and single paradigms but have found reasonably consistent results, e.g., cortical disinhibition in EEG , 19, 20 n = 107) recruited from outpatient clinics, first-degree relatives (n = 57), and control subjects (n = 108) recruited from media advertisements, who each underwent rsEEG, MMN, 40-Hz ASSR, and rsfMRI paradigms and recorded symptoms and other measures. PScz and Con were well matched in terms of age , sex  and smoking status . PScz had mean Brief Psychiatric Rating Scale scores of 14.4 out of 49 for positive symptoms and 7.3 out of 28 for negative symptoms  parameters, with the exception of rsEEG, where characteristic group responses were modeled. We interpret greater self-inhibition of pyramidal cells as an effective loss of pyramidal synaptic gain. Given known pathophysiology in PScz, NMDAR hypofunction seems the most likely explanation for loss of pyramidal gain, but other explanations are possible . The mean adjusted power spectra within the Con (n = 98) and PScz (n = 95) groups are shown in F3,573 = 6.59, p < .001) but not of eyes × group  or of frequency × eyes × group . We therefore averaged the power in each frequency band across eyes open and closed conditions and performed Wilcoxon rank sum tests (as some distributions were skewed), Bonferroni-corrected for 4 frequency bands , decreased β , and increased γ , but unchanged α .We first examined rsEEG power spectra by subtracting the 1/f gradient, noting that gradients did not differ between groups with eyes open or closed  or PScz . In the averaged deviant and standard waveforms   and in Rel  , as did Rel, although this was only significant at an uncorrected peak threshold   , which w = 3.83) . Likewis = 3.02) .p = .89  than in PScz  and Rel   .p > .95) and right (R) IFG (p > .99)  of PScz on the microcircuit parameters  (p > .99) (p > .95). Rel did not show effects of p > .95 in either analysis.We then used PEB to ask which parameters best explained group differences in MMN: self-inhibition within areas or connections between areas. The reduced mismatch amplitude in PScz was best explained by increased self-inhibition in deviant—relative to standard—trials in left (L) inferior frontal gyrus (IFG) (p > .99) . Includip < .95) , left unp > .99)—within the Broca area—but increased self-inhibition in R IFG (p > .95) in the mismatch contrast .In PScz, the auditory perceptual abnormalities state measure was associated with disinhibition in L IFG , and Rel = 39.9 Hz . Adjusted time-frequency plots are shown in p < .05; t tests at each frequency and time point are circled on the middle and right plots, for Con vs. PScz and Con vs. Rel in black and PScz vs. Rel in white; this many differences are unlikely due to chance—Con vs. PScz and Con vs. Rel both p < .001, PScz vs. Rel p = .006, permutation tests). Maximum ASSR γ power correlated with MMN amplitude in PScz  but not in Con  or Rel .We next considered induced responses during auditory steady-state stimulation. Group-averaged 40-Hz ASSR are shown in w ≤ 4 Hz (see R2 = 0.53)  —was (50 −12 4), hence bilateral sources at (±50 −12 4) were used as priors for reconstruction of virtual electrode data: the DCM comprised these bilateral sources and their thalamic drive . Empiric4 Hz see ; this wi4 Hz see . Model f = 0.53) . Group d p > .1) .p > .95) (p > .99) (p > .99) (We performed group comparisons with PEB using schizophrenia genetic risk (PScz + Rel > Con) and diagnosis (PScz > Rel) as explanatory variables ,19, instp > .95) , and redp > .99) , left. Tp > .99) , right.p > .99) and reduced ii-sp connectivity (p > .99), and greater self-inhibition in L A1 (p > .99) .In PScz, the auditory perceptual abnormalities trait measure related to a disinhibited sp-ii-sp circuit, i.e., increased sp-ii (p > .99) . The audR2s were >0.7 in all groups, with no group differences   based on MMN source locations see : bilaterp > .05) .p > .99 and p > .95, respectively) (p > .95). These effects did not survive addition of chlorpromazine dose equivalents (L IFG self-inhibition fell to p > .75). However, Rel > Con showed the same increase in self-inhibition in bilateral IFG (both p > .95) . These differences were not detected using conventional functional connectivity analyses, which cannot assess self-inhibition, or analyses of regional variance  . These ep > .95) . This grp > .99) (p > .95) and L A1 and superior temporal gyrus (STG) (both p > .99) and STG-A1 backward connectivity bilaterally (both p > .99)  , left. Cp > .99) , right.p > .99 except L IFG and R A1, both p > .95) and stronger forward connections everywhere except R Al-STG  (p > .99)  were associated with decreased self-inhibition everywhere except R STG  , left. Bp > .99) , right.Note that many rsfMRI results were lost if global signal regression was not performed .In summary, we found clear evidence for increased self-inhibition (evidence of reduced synaptic gain) in PScz  in all dDCM of EEG and fMRI produced two key cross-paradigm findings. First, well-established effects in rsEEG , MMN 1111, and 4Second, abnormal auditory percepts in PScz were associated with decreased self-inhibition in auditory areas selectively across 3 paradigms . This isIn rsEEG, increased θ power in PScz is a well-established finding ,30. A U-Decreased mismatch amplitude in PScz [and especially in chronic PScz ] is wellIn the 40-Hz ASSR, PScz showed decreased γ power and peak frequency, and Rel showed decreased power . A previous rsfMRI DCM analysis in PScz found disinhibition in the anterior cingulate cortex , rather p > .95 despite Rel’s similar data features to PScz. In 40-Hz ASSR, pyramidal self-inhibition was reduced in Rel  in cortical glutamate  in PScz, especially in frontal areas, but disinhibition—in auditory areas in particular—correlated with auditory perceptual abnormalities. Psychotic symptoms may therefore be caused by interneuronal downregulation that restores cortical excitation/inhibition balance in PScz. These complex processes may explain why successful glutamatergic treatments for PScz are elusive, and why such treatments may have narrow therapeutic windows  or depen