Furthermore, the molecular docking results showed that fangchinoline bound with AChE residues in the peripheral anionic site, and cyclanoline bound with AChE residues in the peripheral anionic site, anionic site, and catalytic site. In parallel, cyclanoline bound with butyrylcholinesterase (BChE) residues in the anionic site, catalytic site, and aromatic site. The results support that fangchinoline and cyclanoline, alkaloids derived from STR, could account for the anti-AChE function of STR. Thus, STR extract or its alkaloids may potentially be developed as a therapeutic strategy for Alzheimer’s patients.Stephaniae tetrandrae radix (STR) is a commonly used traditional Chinese medicine in alleviating edema by inducing diuresis. In the clinic, STR extracts or its components are widely used in the treatment of edema, dysuria, and rheumatism for the regulation of water metabolism. Furthermore, STR has been used in treating emotional problems for years by combining with other Chinese herbs. However, the material basis and mechanism of STR on the nervous system have not been revealed. Here, the main components of STR extracts with different extracting solvents were identified, including three major alkaloids, i.e., cyclanoline, fangchinoline, and tetrandrine. The cholinesterase inhibitory activity of STR extracts and its alkaloids was determined using the Ellman assay. Both cyclanoline and fangchinoline showed acetylcholinesterase (AChE) inhibitory activity, demonstrating noncompetitive enzyme inhibition. In contrast, tetrandrine did not show enzymatic inhibition. The synergism of STR alkaloids with huperzine A or donepezil was calculated by the median-effect principle. The drug combination of fangchinoline–huperzine A or donepezil synergistically inhibited AChE, having a combination index (CI) < 1 at  Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by memory loss, cognitive impairment, and behavioral disturbances ; the patCoptis chinensis Franch.), Lycopodii herba (the whole herb of Lycopodium japonicum Thunb.), and Zingiberis rhizoma recens , have shown good potency in treating rheumatic disease for their properties in eliminating dampness or dispelling rheumatism [Stephania tetrandra S. Moore) is a common TCM herb in treating edema, dysuria, and rheumatism. The main components of STR are alkaloids, i.e., tetrandrine and fangchinoline, which show beneficial effects on brain disease by regulating the synthesis, expression, and metabolism of neurotransmitters [Recent studies suggested that inflammatory response plays an essential role in the development of AD ,14. In ieumatism ,18,19. Ieumatism ,21. Stepsmitters ,23,24,25n-butanol, and water for extraction of STR based on polarity in n-butanol extract of STR. Furthermore, the amounts of individual alkaloids in STR crude drug were determined, and the linear relationships (R2 of each component > 0.999) were calibrated from the calibration curve  were tested for AChE inhibitory activity using the Ellman assay. As shown in n-butanol, and water) of STR were prepared and tested for AChE and BChE activities. As shown in 50) of 11.82 ± 2.25 μg/mL, while it showed no inhibitory activity on BChE. The n-butanol extract showed a good dose–effect relationship with AChE and BChE inhibition with IC50 values of 69.13 ± 4.41 and 108.35 ± 5.16 μg/mL, respectively. However, the water extract showed no AChE inhibitory activity and a weak inhibitory activity on BChE at high concentration. The chloroform extract of STR mainly contained bisbenzylisoquinoline alkaloids, including fangchinoline and tetrandrine, while the n-butanol extract of STR mainly contained cyclanoline. The water extract contained much lower levels of these alkaloids. Therefore, it was speculated that the above alkaloids were closely related to the potency of STR in terms of cholinesterase inhibition.The different polar extracts  of STR were further tested. As shown in 50 of 2.58 ± 0.28 μM, while it demonstrated no inhibitory activity on BChE. In parallel, its structural analogue tetrandrine exhibited no inhibitory activity on AChE at the maximal dissolvable concentration (data was not shown), as well as no or little inhibitory activity on BChE. However, cyclanoline exhibited weak AChE and BChE inhibitory activities with IC50 values of 105.6 ± 2.29 and 246.65 ± 5.46 μM, respectively. Galanthamine served as a positive control, showing inhibitory activity on AChE and BChE with IC50 values of 2.46 ± 0.17 and 49.82 ± 2.50 μM, respectively. In addition, the kinetics of AChE inhibition by fangchinoline and cyclanoline were studied. Serial concentrations of fangchinoline (0.5–5 μM) and cyclanoline (30–120 μM) were added to AChE lysate containing a range of acetylthiocholine iodide . As determined from the Lineweaver–Burk plots, the iK values were estimated as 1.79 mM for fangchinoline and 72.30 mM for cyclanoline  of 28.93 ± 1.41 nM and 0.68 ± 0.08 nM, respectively, which were much lower than when using their single drug alone. The synergistic effects of fangchinoline and huperzine A or donepezil on AChE inhibition were further evaluated by the median-effect principle. As shown from the aF–combination index (CI) plot in aF = 0.5, the CI values of the fangchinoline and huperzine A or donepezil combination  were 0.80 and 0.72, respectively, suggesting a synergistic effect. However, the CI value of the fangchinoline and huperzine A combination increased with increasing dose or aF value, which indicated that the synergy decreased as a function of the dose. When aF > 0.7, the CI value of fangchinoline and huperzine A was close to or greater than one, indicating that the inhibitory effect of their combination might be additive or antagonistic. In parallel, the synergistic effect of fangchinoline and donepezil on AChE slowly decreased with increasing dose, while the CI value of the combination was less than one at different aF values.Considering the compatibility theory of drugs, fangchinoline and the represented compound huperzine A or donepezil were tested for their synergistic effect on AChE inhibition. Fangchinoline, huperzine A, and donepezil inhibited AChE in dose-dependent manners, having ICectively A. The coectively B. In addThe active site of AChE is a deep and narrow gorge . The binAs for cyclanoline, there were similar binding targets with the peripheral anionic site B, i.e., It was previously shown that BChE shares ~55% sequence homology with AChE , and theiK value of fangchinoline was much lower than that of cyclanoline. Through molecular docking analysis, the binding modes of fangchinoline, tetrandrine, and cyclanoline with AChE or BChE were found to be different  by reflux with 0%, 25%, 50%, 75%, and 100% ethanol volume fractions. The two filtrates were combined, concentrated to a sticky state, and dried at low temperature.Fangchinoline , tetrandrine , and galanthamine  were purchased from Chengdu Ruifensi Biotechnology ; huperzine A  and donepezil  were purchased from Chengdu DeSiTe Biotechnology . STR is the root of n-butanol with a volume ratio of 1:1. The chloroform, n-butanol, and remaining water solutions were concentrated to a sticky state and dried at low temperature as above. The STR crude drug solution was prepared according to the Chinese Pharmacopoeia .In order to prepare different polar extracts of STR, appropriate amounts of STR herb were extracted with 75% ethanol, according to the operation described above. The concentrated solution was extracted three times using chloroform with a volume ratio of 1:1. The chloroform solution was combined, and the remaining solution was subjected to extraction three times using n-butanol, and water were weighed and dissolved to 0.80, 1.50, and 1.80 mg/mL, respectively. The dissolved extracts were filtered using a 0.22 μm Millipore filter, and the filtrate was subsequently collected for HPLC determination. The standards of fangchinoline, tetrandrine, and cyclanoline were weighed accurately and dissolved to a stock solution of 1 mg/mL using MeOH. The mixed standard solutions of fangchinoline, tetrandrine, and cyclanoline  were prepared by mixing different volumes of the stock solution. The mixed stock solutions were diluted to a series of working standards with MeOH for HPLC determination. The HPLC chromatographic analysis was performed using a Waters 2695 HPLC (Waters Technology (Shanghai) Co., Ltd., Shanghai, China) equipped with an ultraviolet–visible light (UV-Vis) photodiode array detector. The sample separation was achieved on an Innoval C18 column  with a flow rate of 1.0 mL/min at 25 °C. The mobile phase of STR was composed of MeCN (A) and water  using a gradient elution of 10–30% A at 0–20 min and 30–55% A at 20–25 min. The detection was performed at 280 nm. The injection volume was set at 10 µL.STR extracts  were weighed accurately and dissolved in 50% MeOH at a concentration of 2 mg/mL. Different polar extractives of STR with solvents of chloroform, n-butanol, and water were weighed accurately and dissolved to a stock solution with concentrations of 30, 60, and 90 mg/mL using DMSO, respectively. The different concentrations of STR extracts were prepared for the evaluation of cholinesterase inhibitory activity by diluting the initial stock concentrations. The standards of cyclanoline, fangchinoline, tetrandrine, galanthamine, huperzine A, and donepezil were weighed accurately and dissolved to stock solutions with concentrations of 100, 50, 10, 50, 25, and 25 mM using DMSO, respectively. Different concentrations of these alkaloids were prepared for the evaluation of cholinesterase inhibitory activity by diluting initial stock concentrations. The combinations of fangchinoline and huperzine A or donepezil at a concentration ratio of 50:1 or 1000:1, respectively, were individually prepared by mixing appropriate volumes of fangchinoline and huperzine A or donepezil stock solutions. After that, the stock solutions of fangchinoline and huperzine A or donepezil were diluted to six concentrations  using DMSO.STR extracts  were weighed accurately and dissolved to a stock solution with a concentration of 40 mg/mL using dimethyl sulfoxide (DMSO). Different polar extracts of STR with chloroform, w/v; containing 1 mM ethylenediaminetetraacetic acid (EDTA) and ethylene glycol tetraacetic acid (EGTA), 0.5% triton X-100, 150 mM NaCl, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid , 50 μg/mL benzamidine HCL, 10 μg/mL aprotinin, leupeptin, and pepstatin) for the AChE activity assay. The AChE assay medium consisted of brain lysate (5 mg/mL) with testing drugs, 80 mM Na2HPO4 (pH 7.4), 0.1 mM tetraisopropylpyro-phosphoramide (iso-OMPA), 0.5 mM DTNB (5′-dithiobis (2-nitrobenzenoic acid)), and 0.625 mM ATCh. The mixture of brain lysate, Na2HPO4 buffer, and iso-OMPA solution was incubated at 37 °C for 10 min, and then the solutions of DNTB and ATCh were added. After re-incubating at 37 °C for 30 min, AChE activity was tested by determining the absorbance at 405 nm.The extracts and alkaloids of STR were tested for cholinesterase activity using a 96-well microtiter plate  with a final volume of 200 μL, according to the Ellman method ,32. The v/v, diluted with precooled low-salt lysis buffer) was prepared for the BChE activity assay. The BChE assay medium consisted of the same composition as the AChE assay medium, except that the brain lysate and iso-OMPA were replaced with diluted serum solution and 0.1 μM BW284c51. The mixture of diluted serum, Na2HPO4 buffer, and BW284c51 solution was incubated at 37 °C for 10 min, and then the solutions of DNTB and ATCh were added. After re-incubating at 37 °C for 30 min, the BChE activity was tested by determining the absorbance at 405 nm. The concentration of DMSO in the reaction system of AChE/BChE assay was controlled at 0.5%. The background contrast without brain lysate or serum was set to eliminate the color interference at 405 nm. The AChE/BChE inhibitory activity of drugs was calculated as a percentage of the control absorbance value, AChE/BChE inhibition (%) = (1 − absorbance with inhibitor/absorbance without inhibitor) × 100%. Galanthamine was used as a positive control. The protein concentrations were measured using the Bio-Rad Protein Assay kit .Mouse serum (1: 50 mK), maximum velocity (maxV), and inhibitory constant (iK) were used to evaluate the kinetics of fangchinoline and cyclanoline. Lineweaver–Burk plots were then plotted using initial velocities, from which the mK and maxV values were calculated . The slopes of reciprocal plots were then plotted against the doses of fangchinoline and cyclanoline, and iK values were determined as the absolute value of the x-axis intercept. Donepezil was used as a positive control. All experiments were performed in triplicate.The Michaelis constant (aF/uF = (D/mD)m) was used to calculate the dose of fangchinoline, huperzine A, and donepezil contributing to AChE-specific inhibition according to the dose–effect curve. For comparison, the median-effect equation was transformed to a linear equation (log(aF/uF) = mlogD − mlogmD) by taking the logarithm of both sides, where D is the drug dose, mD represents the dose at 50% potency, aF is the AChE inhibition expressed as a decimal fraction, uF represents the unaffected fraction (uF = 1 − aF), and m is the coefficient of the median-effect equation (slope of the linear equation). The linearity curves and equations of fangchinoline, huperzine A, and donepezil were constructed by plotting mlogD versus log(aF/uF) at each dose effect. The mD of fangchinoline, huperzine A, and donepezil was calculated according to their equations. The combination index (CI) was used to evaluate the synergistic effect of fangchinoline and huperzine A or donepezil according to the Chou–Talalay equation (CI = D1/Dx1+ D2/Dx2), where D1 and D2 are the doses of the single drug required to produce aF in their combination, and Dx1 and Dx2 are the doses of the single drug producing the same effect alone. The values of D1, D2, Dx1, and Dx2 at different doses  were similarly calculated according to the linear equation (log(aF/uF) = mlogD − mlogmD), and then the CI values of fangchinoline and huperzine A or donepezil were calculated. CI values <1, =1, and >1 indicated synergistic, additive, and antagonistic effects, respectively.The synergistic effect between fangchinoline and huperzine A or donepezil was calculated by the median-effect principle ,33,34. TTo reveal the underlying binding targets of STR active components with AChE or BChE, a molecular docking study was used to analyze the interactions of fangchinoline, tetrandrine, and cyclanoline with the related residues of AChE or BChE. The three-dimensional (3D) structures of AChE (PDB code: 4EY7) and BChE (PDB code: 4BDS) were obtained from the Protein Data Bank ,36, and Statistical tests were done with DPS 18.10 software , using the bioassay module . Data weiK value of fangchinoline was much lower than that of cyclanoline. This result was also supported by the molecular docking simulation. In addition, the combination of fangchinoline and huperzine A or donepezil showed a good synergistic effect on AChE inhibition.The chemical composition of natural plants and herbs represents the effective material basis of TCM. Here, we proposed that alkaloids are the main active ingredients of STR in AChE and BChE inhibition. The enzyme activity results showed that the AChE inhibitory activities of STR extracts increased in accordance with the alkaloid amount. Bisbenzylisoquinoline and protoberberine alkaloids were the two main types of alkaloids contributing to the AChE/BChE inhibitory activity of STR, whereby the bisbenzylisoquinoline alkaloids inhibited AChE and the water-soluble protoberberine alkaloids inhibited both AChE and BChE activity. Fangchinoline was the representative bisbenzylisoquinoline alkaloid accounting for AChE inhibition, while its structural analogue, tetrandrine, did not have AChE inhibitory activity. Cyclanoline was the main proberberine alkaloid, and its inhibitory activity of AChE was comparable to that of BChE. Furthermore, both fangchinoline and cyclanoline exhibited noncompetitive AChE inhibition. The


Pseudomonas protegens EMM-1 based on phylogenetic analysis of 16S rDNA, rpoB, rpoD, and gyrB gene sequences. We uncovered genes involved in the production of antimicrobial compounds like 2,4-diacetylphloroglucinol , pyoluteorin, and lectin-like bacteriocins. These antimicrobial compounds are also produced by other fluorescent pseudomonads alike P. protegens. Double-layer agar assay showed that P. protegens EMM-1 inhibited the growth of several multidrug-resistant (MDR) bacteria, especially clinical isolates of the genera Klebsiella and β-hemolytic Streptococcus. This strain also displayed inhibitory effects against diverse fungi, such as Aspergillus, Botrytis, and Fusarium. Besides, a crude extract of inhibitory substances secreted into agar was obtained after the cold-leaching process, and physicochemical characterization was performed. The partially purified inhibitory substances produced by P. protegens EMM-1 inhibited the growth of Streptococcus sp. and Microbacterium sp., but no inhibitory effect was noted for other bacterial or fungal strains. The molecular weight determined after ultrafiltration was between 3 and 10 kDa. The inhibitory activity was thermally stable up to 60°C (but completely lost at 100°C), and the inhibitory activity remained active in a wide pH range (from 3 to 9). After treatment with a protease from Bacillus licheniformis, the inhibitory activity was decreased by 90%, suggesting the presence of proteic natural compounds. All these findings suggested that P. protegens EMM-1 is a potential source of antimicrobials to be used against pathogens for humans and plants.The bacterial strain, EMM-1, was isolated from the rhizosphere of red maize (“Rojo Criollo”) and identified as  Pseudomonas protegens represents a new bacterial group that stands out for its ability to produce diverse inhibitory substances, including pyoluteorin (Plt), pyrrolnitrin (Prn), 2,4-diacetylphloroglucinol , and hydrogen cyanide [P. protegens Pf-5, CHA0, and Cab57 for contributing to plant protection [P. protegens produces LlpA bacteriocins [Pseudomonas. These bacteriocins likely provide P. protegens with competitive advantages in the rhizosphere [Microorganisms have developed diverse strategies to survive and compete for the resources of their habitat, and one of them is the production of inhibitory substances . Pseudom cyanide , 7. Someotection . Besideseriocins , capablein vivo experiments has led the consideration of their clinical use to treat different bacterial infections. For example, amyloliquecidine (produced by Bacillus amyloliquefaciens) and pyocin SD2 (from P. aeruginosa) are effective in treating skin and lung infections in animal models, respectively [Although the commercial use of inhibitory substances, such as bacteriocins, is limited, they have been considered as promising weapons against diverse pathogens because they have been used for years as natural food preservatives . At the ectively , 12.Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, P. aeruginosa, and Enterobacter spp. [S. aureus isolates are resistant to methicillin. The frequency of resistance of Klebsiella strains to β-lactam and carbapenem antibiotics is 56 and 4.6%, respectively. K. pneumoniae is resistant to third-generation cephalosporins and carbapenems [Mycobacterium tuberculosis. Approximately 1.5 million people die per year due to the increase of multidrug-resistant (MDR) strains. In 2015, 10.4 million new cases of tuberculosis were registered worldwide, and 580,000 cases corresponded to MDR tuberculosis [Antimicrobial resistance (AMR) is an emerging problem that conditions the greater use of broad-spectrum antibiotics, causing negative consequences in the health, environment, and agriculture sectors . Moreoveter spp. . In Mexibapenems . Anotherrculosis . In 2016P. syringae and Xanthomonas campestris [Antimicrobial resistance has also affected other areas, such as agriculture, and the indiscriminate use of agrochemicals has affected human health . For exampestris . The incmpestris .Currently, the study of inhibitory substances produced by antagonistic bacteria is gaining importance in developing alternative solutions for food preservation and probiotic therapeutics as well as antibacterial agents against MDR pathogens or to control fungal diseases in crop plants . In thisP. protegens EMM-1, a strain isolated from the “Rojo Criollo” maize rhizosphere. The molecular identification was performed by amplification, sequencing, and phylogenetic analysis of the 16S rDNA gene and three housekeeping genes . Amplification of genes related to the production of antibiotics in P. protegens species was also included.In this work, we report the identification of P. protegens EMM-1 inhibited several Gram-positive and Gram-negative bacteria  as well as some important fungi. Additionally, the physicochemical characterization of partially purified inhibitory substances produced by P. protegens EMM-1 in the solid medium was performed. These findings suggested a promising use of P. protegens EMM-1 as a source of antimicrobials with the potential to be used against human or plant pathogens.Our results demonstrated that Enterobacter sp. UAPS03001 [Pseudomonas sp. EMM-1 based on microscopic observation of Gram-negative bacilli, after Gram staining, and amplification of the 16S rDNA gene sequence (GenBank accession number: MN959751.1).The bacterial strain EMM-1 was isolated in 2008 from the rhizosphere of the autochthonous red maize (named “Rojo Criollo”) at the Ecology and Survival of Microorganisms Group from the Benemérita Universidad Autónoma de Puebla at Puebla, Mexico. “Rojo Criollo” maize was extracted from rhizospheric soil  . SeveralAPS03001  and otheAPS03001  and subsTo study the range of inhibition of EMM-1 strain, the inhibitory activity was tested against several bacterial and fungal strains . We seleMicrobial cultures were grown at 30°C and preserved in 50% glycerol at -80°C to provide stable inoculums during the study. Growth conditions and culture media used for each microorganism are specified in rpoB, rpoD, and gyrB) were amplified by PCR using the primers listed in the For bacterial identification, genomic DNA from the EMM-1 strain was extracted using the Promega Wizard® Genomic DNA Purification Kit  according to the manufacturer’s instructions. The 16S rDNA gene and three housekeeping genes  with the following amplification conditions: 1) amplification of 16S rDNA consisted of denaturation at 95°C for 5 min followed by 30 cycles of denaturation at 95°C for 30 sec, annealing at 52°C for 40 sec, and extension at 72°C for 1 min with a final extension at 72°C for 5 min; 2) amplification of nding Tm  for 45 sPCR amplifications were verified by agarose gel electrophoresis, and the amplified genes were purified using the QIAquick Gel Extraction Kit  according to the manufacturer’s instructions. The amplified genes were sequenced by Unidad de Síntesis y Secuenciación de ADN . Nucleotide sequences were analyzed using BlastN  against P. fluorescens group retrieved from GenBank. Sequence alignment was performed using the Clustal W program  [rpoB, rpoD, and gyrB. This analysis was carried out with the same strains used for 16S rDNA gene phylogeny, using nucleotide sequences available from GenBank, and following the same methodology.Phylogenetic analysis of partial 16S rDNA gene sequences was carried out using EMM-1 strain and 24 strains belonging to the Ireland)  and corrIreland) . A neighIreland)  tree wasIreland) , and conP. protegens synthesize the antimicrobial metabolites, 2,4-DAPG, Plt, and LlpA which require the phlD, plt, and llpA genes, respectively [P. protegens CHA0 .ectively . TherefophlD consisted of denaturation at 94°C for 2 min followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 62°C for 45 sec, and extension at 72°C for 1 min with a final extension at 72°C for 10 min. Amplification conditions for plt were the same as for phlD with exception of extension time (2.5 min). Amplification of llpA consisted of denaturation at 95°C for 2 min followed by 30 cycles of denaturation at 95°C for 30 sec, annealing at 60°C for 45 sec, and extension at 72°C for 1 min with a final extension at 72°C for 5 min.Amplification of PCR amplifications were verified by agarose gel electrophoresis, and the amplified genes were purified using the QIAquick Gel Extraction Kit  according to the manufacturer’s instructions. The amplified genes were sequenced by Unidad de Síntesis y Secuenciación de ADN . Nucleotide sequences were analyzed using BlastN  against P. protegens EMM-1 nucleotide sequences determined herein were deposited in the GenBank database under the following accession numbers: MN959751.1 (16S rDNA), MT799749 (rpoB-EMM1), MT798860 (rpoD-EMM1), MT798861 (gyrB-EMM1), MT798862 (phlD-EMM1), MT798863 (pltM-EMM1), MT798859 (bacteriocin-EMM1). The sequence of the 16S rDNA gene from Microbacterium sp. strain UAPS01-201 was deposited in GenBank with the accession number MT095120.1.The ability of EMM-1 strain to inhibit the growth of microorganisms listed in 6−108 CFU/mL. Microbial quantification was performed by the drop-plate method [For the double-layer agar method, EMM-1 was first grown in LB broth and incubated at 30°C for 24 h with reciprocal shaking (180 rpm). A 20 μL drop of the EMM-1 culture was placed in the middle of glass Petri plates containing previously poured LB agar. Plates were incubated for 48 h at 30°C. After incubation, EMM-1 colonies were removed with a sterile glass slide, and the plates were exposed to chloroform vapor for 1.5 h and left semiopen for 20 min to allow evaporation of residual chloroform. Plates were covered with 10 mL of soft agar (8 g/L) inoculated with a microbial suspension of each indicator strain, independently, and adjusted to a final concentration of 10e method . For micFinally, covered plates were incubated at 30°C for 24 h (for bacteria) and 120 h (for fungi). The presence of an inhibition halo was considered indicative of the production of inhibitory substances. All assays were performed in triplicate.In order to isolate the inhibitory substances produced by EMM-1, we performed the cold-leaching methodology . BrieflyThe antimicrobial activity of the crude extract was determined by the agar-well diffusion assay according to De Giani et al.  with som3PO4, NaH2PO4, and Na2HPO4 to obtain pH values ranging from 3 to 9. To test the stability of inhibitory compounds, at different temperatures, 500 μL of the crude extract was placed in Eppendorf tubes and incubated at different temperatures (ranging from -20°C to 100°C) for 80 min. Additionally, to determine the effect of proteolytic enzymes, 6 mL of the crude extract was treated with 1 mL of a B. licheniformis protease solution  at a concentration of 2.4 U/g at 30°C for 2 h. After each treatment, 300 μL of the samples were assayed for the inhibitory activity (AU/mL) against a sensitive strain (Streptococcus sp. SP10).To determine the approximate size of the inhibitory substances, 10 mL of the crude extract was centrifuged using Millipore tubes with nitrocellulose membranes of different pore sizes  at 5000 rpm/10 min/6°C. The activity of each fraction was tested by the double-layer agar assay. For the determination of pH stability, 500 μL of the crude extract was mixed with sterile 0.01 M phosphate buffers in equal parts (1:1) and incubated for 2 h at 30°C before activity assay. Phosphate buffers were prepared using the appropriate pairs of HPseudomonas species. The most closely related sequence belonged to P. protegens CHA0T (99.77% identity). Moreover,16S rDNA sequences of 24 Pseudomonas strains belonging to the P. fluorescens group were selected from the NCBI database to elaborate the phylogenetic tree  useful in the identification of Pseudomonas species were amplified for accurate identification of the EMM-1 strain. Amplification by PCR yielded amplicons of 381 bp (rpoB), 631 bp (rpoD), and 470 bp (gyrB)  . After a strains . Phylogetic tree .P. protegens EMM-1 carries genes involved in the production of antimicrobials, we amplified and sequenced some genes related to the production of 2,4-DAPG, pyoluteorin, and Putidacin L1. Nucleotide sequences of 681 bp (phlD), 840 bp (llpA), and 216 bp (pltM) were obtained. After BlastN analysis, sequence similarity resulted in > 98% identity with sequences from P. protegens strains  showed a clear inhibition halo in the middle of the plate . Microscopic observation of Gram-positive cocci instead of Gram-negative P. protegens bacilli confirmed that the growth belonged to Streptococcus (data not shown).The inhibitory activity of he plate . As a rehibition . Gram stStreptococcus sp. SP10 (64.6 ± 6.11 mm), Microbacterium sp. UAPS01-201 (48.3 ± 2.88 mm), Klebsiella sp. KP12 (36.6 ± 0.57 mm) and P. tropica MTo-293 (43.6 ± 0.57 mm) (The best inhibitory effect was observed for the following bacteria: 0.57 mm) .Aspergillus sp. and Botrytis sp. were considered the most sensitive with inhibition halos of 39 ± 0.00 mm and 38.8 ± 2.93 mm, respectively  are used . In the activity . For thiar assay . Althoug 0.57 mm  and rematococcus  instead tococcus . MicrobaTo estimate the molecular size of the inhibitory substances contained in the crude extract, ultrafiltration with 50, 30, 10 and 3 kDa nitrocellulose membranes was performed, and the inhibitory activity of each fraction was evaluated using the double-layer agar assay. The fractions retained in the 50, 30, and 10 kDa membranes did not show inhibitory activity, but molecules retained in the 3 kDa filter displayed inhibitory activity, indicating an estimated molecular size between 3 to 10 kDa.B. licheniformis decreased the inhibitory activity of the crude extract by 90%  antimicrobial compounds. This bacterium has been studied for its biocontrol properties because it inhibits several phytopathogenic fungi. However, little is known about the antibacterial activity of P. protegens against bacterial clinical isolates [munities . The orgompounds , 42. P. isolates .P. protegens species based on the phylogenetic analysis of 16S rDNA, rpoB, rpoD, and gyrB gene sequences (phlD) and pyoluteorin (pltM)  in P. protegens has been well documented. LlpAs are 31-kDa proteins composed of two monocot mannose-binding lectin domains [P. protegens EMM-1 carries a gene involved in the synthesis of a lectin-like bacteriocin of the Putidacin L1 family from P. protegens CHA0  . Therefont fungi , but no  domains . Here, wens CHA0 . UnderstP. protegens against bacterial strains such as Pseudomonas putida, Pseudomonas syringae, Ralstonia solanacearum, Enterobacter sp., and Bacillus subtilis has been demonstrated [P. protegens against MDR clinical isolates has not been reported. In this study, the inhibitory potential of P. protegens EMM-1 against clinical isolates was revealed. P. protegens EMM-1 inhibited Gram-positive and Gram-negative bacteria, including MDR opportunistic pathogens, through the production of inhibitory substances Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S1 Raw imagerpoB; 4. rpoD; 5. gyrB; 6. phlD; 7. plt; 8. llpA. The PCR products were electrophoresed on a 1% agarose gel (50 min/90 volts) and visualized under UV light in a Spectroline Ultraviolet Transilluminator, using GelRed® Nucleic Acid Gel Stain . Nonspecific bands are observed in lanes 2, 3, and 8. The photography was captured with a Huawei P9 Lite VNS-L53 camera and 1–8 lanes were selected to generate the Lane 1: 1 kb Plus DNA Ladder ; 2. 16S rDNA; 3. (PDF)Click here for additional data file.


TSC2 or PIK3CA mutations obviate the requirement for PIM kinase and circumvent the inhibition of S6 phosphorylation by abemaciclib. Combination with a PI3K inhibitor restored suppression of S6 phosphorylation and synergized to curtail cell growth. By combining abemaciclib with a PI3K inhibitor, three pathways  to mTOR activation are neutralized, suggesting a potential combination strategy for the treatment of PIK3CA-mutant ER+ breast cancer.Aberrant activation of mitogenic signaling pathways in cancer promotes growth and proliferation of cells by activating mTOR and S6 phosphorylation, and D-cyclin kinases and Rb phosphorylation, respectively. Correspondingly, inhibition of phosphorylation of both Rb and S6 is required for robust anti-tumor efficacy of drugs that inhibit cell signaling. The best-established mechanism of mTOR activation in cancer is via PI3K/Akt signaling, but mTOR activity can also be stimulated by CDK4 and PIM kinases. In this study, we show that the CDK4/6 inhibitor abemaciclib inhibits PIM kinase and S6 phosphorylation in cancer cells and concurrent inhibition of PIM, CDK4, and CDK6 suppresses both S6 and Rb phosphorylation.  Cyclin-dependent kinases (CDK) 4 and 6, which phosphorylate the retinoblastoma tumor suppressor protein Rb to promote G1 to S transition, are commonly dysregulated in cancer . D-cycliin vitro assays [The CDK4/6 inhibitors abemaciclib, palbociclib, and ribociclib have emerged as important new treatment options for HR+, HER2- advanced breast cancer in combination with endocrine therapy and are under investigation for additional indications –19. Seveo assays –27, but,o assays .PIK3CA and TSC2. Additionally, we evaluate the potential utility of concurrent treatment with abemaciclib and the PI3K inhibitor BYL719  in PIK3CA mutant breast cancer. Our results suggest that abemaciclib can inhibit the mTOR pathway independently of its effects on Rb and support combining abemaciclib with PI3K/mTOR pathway inhibitors to fully suppress phosphorylation of S6 via multiple inputs.Here we show that abemaciclib can suppress the kinase activity of the oncoprotein PIM, and that, similar to PIM inhibitors, abemaciclib inhibits S6 phosphorylation in cells with wild-type In vivo, S6 phosphorylation was inhibited by abemaciclib, but not by palbociclib, in A549 xenograft tumors, while reduced Rb phosphorylation was evident following treatment with either drug  and MDA-MB-175 breast cancer cells . Inhibitors of DYRK1B  or CDK9pound 33 , did notA structural comparison of how abemaciclib and palbociclib could bind in the ATP pocket of the PIM1 isoform allowed us to understand their different activities. We modeled abemaciclib binding to the PIM1 catalytic site. Overlaying palbociclib on this model and inspection of the catalytic Lys67 , gatekeeper (Leu120) and DFG domain showed multiple clashes which would prevent binding . Abemaciclib, by contrast, is seen to (i) fill the ATP pocket under the p-loop, (ii) make hydrophobic contact with Val52, and (iii) make a favorable interaction with Lys67 explaining its potency against PIM kinase.50 68 v 539 nM), despite similar inhibition of phospho-Rb (50 76 v 68 nM), and the combination was not synergistic . The potency improvement conferred by addition of the PIM inhibitor to palbociclib is accompanied by inhibition of S6 phosphorylation to a degree comparable to abemaciclib monotherapy .Several mechanisms have been postulated to underlie the control of mTOR signaling by PIM kinases, including phosphorylation and inactivation of mTOR suppressors PRAS40 and tuberin (TSC2) , 35. PRAn DMS-53 , or knocn DMS-53 , preventIn addition to phosphorylation of TSC2, PIM was also reported to phosphorylate glycogen synthase kinase 3β (GSK3β) at Ser9, resulting in its inactivation . As GSK3PIK3CA and/or HER2 alterations would compensate for PIM. Inhibition of S6 and p70S6K phosphorylation by abemaciclib was substantially reduced in PIK3CA mut/HER2+ MDA-MB-453 and PIK3CA mut T-47D, as compared to PIK3CA WT MDA-MB-175 . Combination treatment resulted in synergistic inhibition of cell growth in twelve cell lines  [Interpretation of ± 42 nM) , this ac± 42 nM) . Inhibit± 42 nM) . In the ± 42 nM) , and thup < 0.001), and RB1 depletion conferred resistance to abemaciclib, suggesting that the predominant anti-tumor activity is driven by CDK4/6 inhibition [50 of abemaciclib was at least 5× lower than palbociclib. Intriguingly, several of these, including MDA-MB-157 and HCC1143, are from triple negative breast cancer, which has been reported to depend on PIM kinase [Though much work on PIM kinases has focused on their roles in hematological malignancies, with clinical testing of PIM inhibitors as monotherapy in multiple myeloma, acute myeloid leukemia, malignant lymphoma, and non-Hodgkin’s lymphoma , they hahibition . Additiohibition , there wM kinase , 52. FurM kinase , in partM kinase , 55. WheM kinase  remains PIM and CDK4/6 have each been implicated in resistance to PI3K inhibition , while PSeveral beneficial combinations of CDK4/6 inhibitors with compounds targeting PI3K/Akt/mTOR signaling have been reported in the preclinical setting , 61–65. A549, BT-20, BT-474, BT-549, CAMA-1, DMS-53, DU4475, HCC38, HCC70, HCC202, HCC1143, HCC1187, HCC1395, HCC1569, HCC1806, HCC1937, HCC1954, HCC2218, HCT-116, Hs578T, Jeko-1, MCF-7, MDA-MB-134-VI, MDA-MB-157, MDA-MB-175-VII, MDA-MB-231, MDA-MB-361, MDA-MB-415, MDA-MB-436, MDA-MB-453, MDA-MB-468, MiaPaCa2, SK-BR-3, NCI-H441, SK-MEL-28, T-47D, U2OS, and ZR-75-1 were obtained from the American Type Culture Collection (ATCC) and cultured according to vendor recommendations. EFM-19 were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ) and SNU-886 were from the Korean Cell Line Bank (KCLB).Abemaciclib, palbociclib, ribociclib, PIM447, BYL719, LY2090314, everolimus, DYRK1Bi AZ cpd 33 , dinacicCells were treated as indicated and western blot analysis was performed as previously described . The pS6® Luminescent Cell Viability Assay , according to manufacturer’s instructions, or PI staining following treatment for two cell doubling times (2DT). PI staining was performed as previously described [Cells were plated in 96-well plates and allowed to attach overnight prior to treatment with increasing concentrations of the indicated compounds. Cell growth was assessed by CellTiter-Gloescribed . Responsescribed  or Blissescribed .6) were suspended in a 1:1 mixture of HBSS and Matrigel (BD Biosciences) and injected subcutaneously into the rear flank of female CB17SCID mice (Envigo). Mice were subsequently randomized into treatment groups (n = 5/group) based on tumor volume and body weight when mean tumor volume reached 200–250 mm3. A single dose (50 mg/kg) of abemaciclib or palbociclib was administered by oral gavage (PO) with tumors collected 2, 4, 8, or 24 h post-treatment. In vivo doses were selected in part based on previous pharmacokinetic data (data not shown), with 50 mg/kg palbociclib in mice corresponding to ~3× clinical exposures at 125 mg QD dose. Abemaciclib was formulated in 1% hydroxyethyl cellulose  in 25 mM phosphate buffer, pH 2, while palbociclib was formulated in 5% N-methyl-2-pyrrolidone , 0.05% antifoam in 25 mM phosphate buffer, pH 2. Vehicle 1% HEC in 25 mM phosphate buffer served as control. Tumors were homogenized and lysates were analyzed by western blot as described above.All animal studies were performed in accordance with American Association for Laboratory Animal Care institutional guidelines, and all protocols were approved by the Eli Lilly and Company Animal Care and Use Committee. A549 cells (5 × 10CDK4 (L-003238-00-0005), CDK6 (L-003240-00-0005), PIM1 (L-003923-00-0005), PIM2 (L-005359-00-0005), PIM3 (L-032287-00-0005), TSC2 (L-003029-00-0005), or non-targeting control (D-001810-10-05) SMARTpool siRNA (Dharmacon) using DharmaFECT1 transfection reagent . Following 48 h transfection, cells were treated as indicated and/or lysed and analyzed by western blot.Cells plated in 6-well plates were transfected with 25 nM RB1- or TSC2-specific sgRNAs, respectively, and subjected to puromycin selection . RB1, 5′CGGTGCCGGGGGTTCCGCGG 3′; TSC2, 5′ CTGTCGCACCATCAACGTCA 3′; 5′ CTGCAACTACCACGCTGCT 3′ (Sangon Biotech). For RB1, pooled KO cells were additionally subjected to selection with 2 µM palbociclib and RB1 KO was confirmed by western blot. For TSC2, single clones were isolated and TSC2 KO was validated by Sanger sequencing.DMS-53 and A549 cells were transfected with a plasmid containing Cas9 and Preparation of the in-house PIM1 protein-ligand complex x-ray, the glide docking model and the superposition of palbociclib on top of abemaciclib were performed using the available Protein Preparation Wizard, Glide and manual atom pairs superposition panels of Maestro version 12.0.12 (Schrodinger Suites Release 2019-2).An in-house PIM1 protein-ligand complex crystal structure where the ligand was structurally similar to abemaciclib (benzimidazole ring and fluorine atoms and their positions were conserved) was prepared. Waters were removed and not considered. A glide grid centered in the ligand and with defined two constrains to Glu121 (aromatic hydrogen bond) and Lys67 (H-bond) was built. This grid was used to dock abemaciclib 3D structure   using XPPostmenopausal early stage  HR+, HER2- breast cancer patients were randomized to one of three treatment arms: abemaciclib monotherapy , anastrozole monotherapy , or abemaciclib in combination with anastrozole for two weeks, followed by an additional fourteen weeks of treatment with combination abemaciclib and anastrozole. Biopsies were obtained at baseline, following two weeks of initial therapy, and following the additional fourteen weeks of combination therapy. The study was conducted in compliance with the principles of good clinical practice, applicable laws and regulations, and the Declaration of Helsinki. The protocol was approved by each institution’s review board, and written informed consent was collected from patients before enrollment. This study is registered with ClinicalTrials. gov NCT02441946). Extracted RNA from FFPE biopsy samples was analyzed by Illumina Truseq RNA exome RNA sequencing (RNAseq) and GSEA as previously described [946. Extr


Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of children and adolescents. The fusion-positive (FP)-RMS variant expressing chimeric oncoproteins such as PAX3-FOXO1 and PAX7-FOXO1 is at high risk. The fusion negative subgroup, FN-RMS, has a good prognosis when non-metastatic. Despite a multimodal therapeutic approach, FP-RMS and metastatic FN-RMS often show a dismal prognosis with 5-year survival of less than 30%. Therefore, novel targets need to be discovered to develop therapies that halt tumor progression, reducing long-term side effects in young patients. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that regulates focal contacts at the cellular edges. It plays a role in cell motility, survival, and proliferation in response to integrin and growth factor receptors’ activation. FAK is often dysregulated in cancer, being upregulated and/or overactivated in several adult and pediatric tumor types. In RMS, both in vitro and preclinical studies point to a role of FAK in tumor cell motility/invasion and proliferation, which is inhibited by FAK inhibitors. In this review, we summarize the data on FAK expression and modulation in RMS. Moreover, we give an overview of the approaches to inhibit FAK in both preclinical and clinical cancer settings. Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood, accounting for about 50% of all the soft tissue sarcomas and 8% of all pediatric cancers . RMS celwww.clinicaltrials.gov) (follow-up is still ongoing). Five-year disease-free survival (DFS) and overall survival (OS) resulted both higher than those in the group without maintenance therapy  prompting the EpSSG to incorporate this approach in the new standard of care for these types of patients [The first-line standard of care for high-risk RMS includes vincristine and dactinomycin combined with an alkylating agent which is cyclophosphamide for COG (VAC) and ifosfamide for EpSSG (IVA) ,14. Recepatients .Of note, patients with bone or bone marrow metastases or metastases in more than 3 sites show a 5-year survival rate <10% (reviewed in ). TherefPTK2 gene, mainly localized to cellular focal contacts at the cellular edges, which plays a critical role in adhesion-dependent cell motility, survival, and proliferation in response to integrin and RTK signaling [Focal adhesion kinase (FAK) is a 125 kDa non-receptor tyrosine kinase encoded by the ignaling . Overallignaling ,31. Anoiignaling . While iignaling ,34. FAK In this review, we summarize the insights into the involvement of FAK in RMS pathogenesis. We also discuss the perspectives and challenge of potential clinical applications of FAK inhibitors in RMS.FAK is not only a sensor of environmental rigidity, but it is also involved in an intricate network of intramolecular interactions existing among the microenvironment, the adhesion receptor complexes, and the nucleus coordinating signals through the focal adhesion multiprotein complex . FunctioThe structure of FAK consists of multiple domains, including the N-terminal 4.1, ezrin, radixin, moesin homology domain FERM, a central catalytic tyrosine kinase domain important for its activity, and a C-terminal region containing a focal-adhesion targeting (FAT) domain and a proline-rich region  Figure . The FERThe activation of FAK is under the control of different phosphorylation events. An important autoregulatory mechanism occurs through an interaction between the FERM and kinase domains to maintain an autoinhibited state. A main interaction is formed between the FERM F2 lobe and the kinase C-lobe. In addition, an indirect contact is formed in autoinhibited FAK between the FERM-F1 lobe and the kinase N-lobe. This connection includes the Y397 autophosphorylation site, important for the activation of the protein . FERM haOnce Y397 autophosphorylation occurs, this site is converted into a high affinity-binding site for proteins containing SH2 and SH3 domains, among which is the Src kinase. In this way, Src interacts via its SH2 domain with pY397 and via the SH3 domain with a PxxP motif located between the FERM and the kinase domain. These interactions contribute to Src-dependent activation, triggering phosphorylation of several FAK tyrosines, including Y576 and Y577 in the activation loop of the kinase domain, which induces full catalytic activity of the protein . FurtherOtherwise, FAK plays its roles via non-canonical kinase-independent scaffold functions. Lim et al. found that FAK was also functional into the nucleus . Indeed,However, the molecular mechanisms are still unclear and further studies on the roles of nuclear FAK are needed .During muscle development, FAK is involved in several processes including differentiation and migration. Crosstalk between myoblasts and ECM, in particular laminin, regulates myogenic processes by stabilizing multinucleated structures that will form muscle fibers . TherefAltogether, these data suggest that FAK plays fundamental roles in skeletal muscle participating, in addition to myogenesis/regeneration, in the control of energy metabolism also via PI3K signaling.Cas were unmodified by IGF1, the growth factor caused robust phosphorylation of the three proteins, which was prevented by pretreatment with rapamycin which was ineffective in cells expressing mTORrr [The levels of FAK expression cannot be used as a prognostic indicator in RMS since FAK and Y379 phosphorylated FAK are both also expressed in normal skeletal muscle. The expression of FAK in RMS has been evaluated in a recent study showing that, in a small cohort of pediatric RMSs including both alveolar and embryonal histotypes, FAK was expressed and phosphorylated with no difference between the two subtypes . Functiog mTORrr . IGF1-deg mTORrr . ConsideCell motility can be regulated by FAK as a downstream effector of the MET oncogene , which i® layer and cell migration was detected after PF-573228 treatment at different concentrations. The effects of FAK inhibition in vivo were evaluated using Y15, a small molecule that inhibits FAK by decreasing its protein levels [A subsequent study directly involved FAK in cell survival, invasion, and migration of RMS cells, regardless their fusion gene status . As a man levels . Y15 tren levels . When trn levels . This woThe Y397 phosphorylated FAK was detected in the FN-RMS cell line RD18, a subclone of RD cells , by massInterestingly, the alkylating agent Temozolomide has been shown to impair RH30 FP-RMS cells’ survival and, at the same time, to activate autophagy, the inhibition of which synergizes with the drug to induce tumor cell death . ThroughRecently, Fanzani et al. demonstrated that caveolin-1, when overexpressed in FN-RMS cells in vitro, promotes migration and invasion of tumor cells, a phenomenon blocked by ERK inhibition . CaveoliThe multiple roles of FAK make this protein crucial for oncogenesis and, in agreement, FAK is overexpressed in most types of cancer ,82. The Since FAK has several interactors, a huge effort is in place to discover protein–protein interaction (PPI) inhibitors of FAK as a strategy to avoid issues of selectivity related to competitive inhibitors of the ATP-binding site. Among the FERM interaction inhibitors, roslin 2 is a compound that, blocking the p53-binding site in the F1 lobe of FERM, reduces FAK levels and reactivates p53 in colon cancer cells, synergizing with conventional chemotherapeutics .With the same principle, the small molecule M13 blocked interactions between the FERM domain of FAK and MDM2, leading to an increase in p53 levels and a decrease of tumor cells’ survival .The compound INT2-31 targets the FAK/IGF1-R interaction and has been used in preclinical research on melanoma, showing the ability to block cell proliferation inducing apoptosis in vitro and decreasing tumor growth in vivo by inhibition, at least in part, of AKT signaling .An in silico screening to target the Y397 site identified two small molecules, Y11 and Y15, which inhibit FAK autophosphorylation, reducing the levels of the protein, thus targeting the FAK binding to SH2-containing proteins ,94. Y11 One of the therapeutic advances in which FAK inhibition can be exploited is chemoresistance, often developed by some tumors following the first-line interventions. Several preclinical studies highlighted the anti-tumor effects of an anti-FAK approach in combination with conventional or targeted therapeutics promoting tumor remission in xenografts models (reviewed in ). As an What emerged from the preliminary data obtained from those trials is that FAK inhibitors could be potential cytostatic drugs, capable of keeping the disease stable for more than 12 weeks (reviewed in ). These Future studies should identify the best combinations using FAK inhibitors and small molecules against signaling cross-talking with FAK that could have potentiality in RMS patients. They could be those affecting key pathways involved in the developmental signals that are fundamental for the survival, motility, and stemness of RMS cells. Among these, inhibitors of IGF1-R, as already mentioned, as well as CDK4/6 or PARP inhibitors could be investigated in a preclinical RMS setting. Moreover, a combination strategy could allow for the reduction of the doses for each compound that in therapy with a single agent are higher, thus lowering the side effects often related to targeted therapies.However, more studies are needed to understand how FAK regulates tumor survival and the exchanges with the tumor microenvironment in cancer and, in particular, in RMS. In addition, the nuclear role of FAK in gene expression should be better clarified. Preclinical models of pediatric cancers can help in elucidating the role of FAK and its signaling in tumor progression and in validating FAK inhibition in combinatorial therapeutic strategies.


A receptor-mediated currents in epilepsy, raising the question of the source of the inhibitory transmitter. We used the unilateral intracortical kainate mouse model of temporal lobe epilepsy (TLE) with hippocampal sclerosis (HS) to answer this question. In our model we observed profound loss of interneurons in the sclerotic hippocampal CA1 region and dentate gyrus already 5 days after epilepsy induction. Consistent with the literature, the absence of interneurons caused no reduction of tonic inhibition of CA1 pyramidal neurons. In dentate granule cells the inhibitory currents were even increased in epileptic tissue. Intriguingly, immunostaining of brain sections from epileptic mice with antibodies against GABA revealed strong and progressive accumulation of the neurotransmitter in reactive astrocytes. Pharmacological inhibition of the astrocytic GABA transporter GAT3 did not affect tonic inhibition in the sclerotic hippocampus, suggesting that this transporter is not responsible for astrocytic GABA accumulation or release. Immunostaining further indicated that both decarboxylation of glutamate and putrescine degradation accounted for the increased GABA levels in reactive astrocytes. Together, our data provide evidence that the preserved tonic inhibitory currents in the epileptic brain are mediated by GABA overproduction and release from astrocytes. A deeper understanding of the underlying mechanisms may lead to new strategies for antiepileptic drug therapy.An imbalance of excitation and inhibition has been associated with the pathophysiology of epilepsy. Loss of GABAergic interneurons and/or synaptic inhibition has been shown in various epilepsy models and in human epilepsy. Despite this loss, several studies reported preserved or increased tonic GABA A receptor-mediated currents are preserved in the sclerotic hippocampal CA1 region and increased in the dentate gyrus.- Despite massive loss of interneurons, tonic GABA- Reactive astrocytes in the sclerotic mouse hippocampus display pronounced GABA accumulation.- Both decarboxylation of glutamate and putrescine degradation may underlie astrocytic GABA accumulation.A receptors (GABAARs) (AR-mediated currents (often termed “tonic inhibition”) are preserved or even increased in focal epilepsy (Epilepsy is a disorder of the brain characterized by recurrent unprovoked seizures that affects 1–2% of the population worldwide . The maiGABAARs) . In lineepilepsy , 20, 21.To gain further insight into this highly relevant topic, in the present study we used immunohistochemical and electrophysiological methods to unravel the relationship between the extent of interneuronal loss, tonic inhibition and astrocytic GABA content in the hippocampal CA1 region and dentate gyrus during the early chronic phase of epileptogenesis in the unilateral intracortical kainate mouse model of TLE-HS. The results further support the hypothesis that the preserved tonic inhibition in TLE-HS is mediated by ambient GABA released from reactive astrocytes.ad libitum). Male FVB  or transgenic mice with human GFAP (hGFAP) promoter-controlled expression of EGFP . Data acortical kainate injection model reliably mimics key morphological and functional features of chronic human TLE-HS . The number of contralateral GABAergic interneurons was not different from sham injected controls , has been documented in human epilepsy and in several different experimental models of the disease, suggesting that it is critically involved in epileptogenesis. We have previously shown that the unilateral intran TLE-HS . Howevern TLE-HS . Since ocontrols .Together, these findings are consistent with other work demonstrating loss of interneurons in epilepsy, and indicate that this pathological process represents a very early event during epileptogenesis.B receptor antagonist CGP52432. Tonic current amplitude was calculated as the difference in holding current before and after bicuculline (20 μM) application. Interestingly, tonic inhibition on the ipsi- vs. contralateral sides in kainate injected mice and vs. untreated control animals were not different in CA1 pyramidal neurons .The preserved or aberrantly increased tonic GABAnditions , 32–34. nditions . Contralnditions . GFAP imnditions . We founIn the DG of sham injected animals, 88% of total  GABA was found in neurons and merely 12% in astrocytes. Intriguingly, after kainate injection the astrocytic contribution increased to 67% at 5 dpi and reached 90% at 14 dpi . In the These data show that during epileptogenesis the loss of GABAergic neurons goes along with a pronounced increase in astrocytic GABA content. Release of GABA from astrocytes might thus mediate tonic inhibition in the epileptic hippocampus.AR current as an indirect read-out of extracellular GABA levels. On the contralateral side, the blocker caused the expected increase in tonic current amplitudes in both CA1 and DG . Ipsilaterally, however, SNAP-5114 had no effect , GABA may have excitatory effects in epilepsy , but not affect expression and/or activation of the release machinery. It is also conceivable that astrocytic GABA release occurs only in the absence of GABA uptake. Clearly, the functional significance of astrocytic GABA on the contralateral side remains to be established and requires further studies.Since the latent period of epileptogenesis in our model lasts about 5 days , the incAR currents are preserved in CA1 pyramidal neurons and increased in dentate granule cells of the sclerotic hippocampus in a chronic TLE model. Furthermore, we gained evidence that GABA overproduction and release from reactive astrocytes represents the main source of ambient GABA responsible for inhibition under this condition. As human and rodent astrocytes display many similar functional properties (In this study we show that despite massive interneuron loss, tonic GABAoperties , it is rThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by LANUV 84-02.04.2012.A212, 84-02-04.2015.A393.JM designed experiments, acquired, analyzed, and interpreted data. AT and LH acquired, analyzed, and interpreted data. HM analyzed and interpreted data. CS and PB designed and supervised experiments and wrote the manuscript. All authors revised the work critically and approved the manuscript.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.


Cell Death & DiseaseCorrection to: 10.1038/s41419-020-2703-x published online 30 June 2020The original version of this Article contained an error in the author affiliations.Affiliation 1 incorrectly read ‘Department of Orthopedics, Yangzhou University Affiliated Hospital, No. 368, Hanjiang middle Rd, 225000 Yangzhou, People’s Republic of China’, whereas it should have read ‘Department of Orthopedics, Affiliated Hospital of Yangzhou University, Yangzhou University, No. 368, Hanjiang Middle Road, 225000 Yangzhou, People’s Republic of China’.This has now been corrected in both the PDF and HTML versions of the Article.


Inflammasomes are protein complexes which are important in several inflammatory diseases. Inflammasomes form part of the innate immune system that triggers the activation of inflammatory cytokines interleukin (IL)-1β and IL-18. The inflammasome most studied in sterile inflammation and non-communicable disease is the NLRP3 inflammasome. Upon activation by diverse pathogen or disease associated signals, NLRP3 nucleates the oligomerization of an adaptor protein ASC forming a platform (the inflammasome) for the recruitment and activation of the protease caspase-1. Active caspase-1 catalyzes the processing and release of IL-1β and IL-18, and via cleavage of the pore forming protein gasdermin D can drive pyroptotic cell death. This review focuses on the structural basis and mechanism for NLRP3 inflammasome signaling in the context of drug design, providing chemical structures, activities, and clinical potential of direct inflammasome inhibitors. A cryo-EM structure of NLRP3 bound to NEK7 protein provides structural insight and aids in the discovery of novel NLRP3 inhibitors utilizing ligand-based or structure-based approaches. Since our 2015 review titled “Inhibiting the Inflammasome: A Chemical Perspective” ,4,5,6. TInflammation is generally regarded as a protective mechanism in case of infection or injury by the removal of toxins by immune cells. Non-sterile inflammation occurs from either parasites, fungi, viruses or bacteria. Sterile inflammation results from injury, foreign bodies, or hypersensitivity. Under normal conditions, immune cells are found circulating in the blood. However, upon recognition of toxins by host cells, immune cells, and their components are recruited to remove the damaging agents and promote tissue repair. Receptors that recognize pathogen-associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs) are located on innate immune cells and are termed pattern recognition receptors (PRRs). NOD-Like receptors (NLRs) are cytosolic PRRs detecting both PAMP and DAMP signals. Some NLRs form an inflammatory complex called an inflammasome . This reThe activation of the canonical NLRP3 inflammasome pathway requires two stages. The first is the priming stage which involves myeloid differentiation primary response 88 (MYD88), and Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling responsible for the expression of NLRP3 and pro-IL-1β . Under nNLRP3 is composed of the nucleotide binding oligomerization domain , a pyrin domain (PYD) at the amino terminal and a leucine-rich repeat (LRR) at the carboxy terminal. The NACHT domain (ATPase domain) of NLRP3 is composed of the nucleotide binding domain (NBD), helical domains HD1 and HD2 and a winged helix domain (WHD) in between HD1 and HD2 a 7]..7].The cryo-EM structure of human NLRP3 bound to an artificially engineered dimer of NIMA (never in mitosis gene a)-related kinase 7 (NEK7)  has been reported b [7]. ThNACHT include Walker A and Walker B , IN, IN29],  of 2 µM  [29]. INof 10 µM  30]. IN. IN50 ofin INF58  , is present downstream of the Walker A motif. ATP hydrolysis requires a glutamate residue, which together with aspartate are involved in the formation of co-ordinate bonds with magnesium ions. Aspartate, the first acidic residue in the NLRP3 Walker B site, was found to be within 5Å of ATP-Mg2+ .50 of 7.5 nM in BMDMs  a is a poin BMDMs  33]. MC. MC50 ofin BMDMs b 34]. F. F50 of in BMDMs c, again Tapia-Abellán and coworkers  used BioIn summary, these mechanism of action studies show that MCC950 is proposed to bind non-covalently with high affinity close to the Walker B site on NLRP3. This prevents a structural conformational change of the ATP-binding site keeping the NLRP3 inflammasome in an inactive state, preventing hydrolysis of ATP needed for NLRP3 assembly ,35,36. ASeveral companies, including Inflazome , IFM The1) or 2-pyrazine (2) groups  when tested in the whole blood assay when compared with MCC950 [Nodthera has been granted patents on NLRP3 inhibitors covering carbamoyl derivatives . NodTher) groups , with ICMC cells . These th MCC950 ,44.Inflazome, a biotech company whose vision is to develop potent, best-in-class, small molecule NLRP3 modulators for the treatment of a broad range of inflammatory diseases, has been granted US and Europe patents for its potent and selective lead NLRP3 inflammasome inhibitors: Somalix  is in phase 2 clinical trials and inzomelid (CNS penetrant) has completed phase 1 ,45,46.50 values of <100 nM [3) being the most active with a low nM IC50 value in THP1 cells . Perricone and coworkers used this homology model in a Molecular Dynamics (MD) study with MCC950  upregulation . A virtud 3.5 µM , althougD = 3.41 × 10−6), blocking interaction of ASC with NLRP3 resulting in inhibition of inflammasome activation. Mutation studies and the BMDM gouty arthritis mouse model activated with MSU were used to further confirm the anti-inflammatory effect of β-carotene [The crystal structure of NLRP3 pyrin domain (PDB: 3QF2)  has widecarotene .50 of 10–15 µM [It is interesting to note that tranilast , a drug 10–15 µM . Tranila10–15 µM . Inhibit50 of 7.5 nM, acts directly at or near the Walker B site, whereas CY09 is thought to act directly through covalent modification of the Walker A binding site. The cryo-EM structure of human NLRP3 bound to NEK7 has provided insights on the mechanism of signaling and assembly of the NLRP3 inflammasome, with molecular docking in the ADP-binding site, molecular dynamic simulations, pharmacophore-based screening and virtual screening supporting the discovery of potent NLRP3 inhibitors with appropriate PK properties.The NLRP3 inflammasome has been the focus of drug discovery research due to its involvement in several inflammatory diseases. Inhibitors either have an indirect effect or a direct effect on NLRP3 and in this review there is a focus on those with a direct effect. MCC950, a potent inflammasome inhibitor with an ICA number of pharmaceutical companies have drug discovery programs to identify novel NLRP3 inflammasome inhibitors, with several compounds currently undergoing clinical trials. The outcome of these trials will of course have a major impact on the future direction of NLRP3 inflammasome research in the pharmaceutical industry. For example, inhibition of the inflammasome comes with a risk of infection, therefore formulations designed for localized or targeted use may be beneficial. In addition, recent studies have shown that post-translational modification of the inflammasome is essential ,59, for Although this review focuses only on direct inhibition of the NLRP3 inflammasome, there is significant interest in the inhibition of upstream processes, which again may lessen the risk of infection. For example, our research group has recently shown that chloride channels are important regulators of the inflammasome, with novel urea-based compounds showing promising indirect inhibition of the NLRP3 inflammasome ,61. What


The landscape scenes were pre-selected based on their contemplative landscape score (CLS) to represent different levels of aggregation of contemplative features within each view. Participants went to each of the sites in a random order to passively view the scenes, while their electroencephalography (EEG) signal was being recorded concurrently. Frontal alpha asymmetry (FAA) values, commonly associated with the approach-related motivation and positive emotions, were extracted. The preliminary results show trends for the main effect of site on FAA, suggestive of stronger FAA in park compared to the control site, akin to more positive mood. There was also a trend for the interaction between the site and scene, which suggests that even within the individual sites, there is variability depending on the specific scene. Adjusting for environmental covariate strengthened these effects, these interim findings are promising in supporting the study hypothesis and suggest that exposure to urban green spaces may be linked to mental health outcomes.A growing body of evidence from observational and experimental studies shows the associations between exposure to urban green spaces (UGSs) and mental health outcomes. Little is known about which specific features of UGS that might be the most beneficial. In addition, there is potential in utilizing objective physiological markers of mental health, such as assessing brain activity, but the subject requires further investigation. This paper presents the preliminary findings from an on-going within-subject experiment where adult participants ( Environmental exposures are a sum of all sensory stimuli we receive along the life cycle, closely intertwined with our mental health and well-being (MH&WB) ,2. As viUrbanized areas are characterized by the concentration of built elements and infrastructure, crowdedness, noise and pollution. Visually, the urbanized environment is highly transformed from its natural manifestation. Potential negative effects of the urban visual exposures on MH&WB have been already noted by Sir Fredrick Law Olmsted, the front runner of American sanitary reform in early XIX century, known as the Father of Landscape Architecture: “A man’s eyes cannot be as much occupied as they are in large cities by artificial things…without harmful effect, first on his mental and nervous system and ultimately on his entire constitutional organization” . Recent Therefore, urban green spaces (UGSs) play a major role in mitigating the negative effects of urban environment exposures on MH&WB. There is an established consensus among researchers that contact with natural environments has beneficial influence on MH&WB of people ,14,15,16FAA is one of the most studied brainwave patterns that is an objective measure of current mood. It is associated with an increased alpha power in the right frontal lobe when compared to the left. As in brain science more alpha power indicates less activation, FAA can be also referred to as decreased activation of the right frontal lobe when compared to the left. According to the approach-withdrawal hypothesis  FAA is aPrevious studies on the role of UGS on MH&WB have several limitations—including the lack of reliable physiological biomarkers and tools that can be deployed for an outdoor environment. For example, a relatively inexpensive and highly portable electroencephalography (EEG) apparatus, Emotiv EPOC has been used previously but its reliability is questionable due to high noise-sensitivity and generally poor performance . Furthern = 100. The inclusion criteria were: age between 21 and 75 years old, right-handed, no serious visual impairment, no pacemaker or recent otologic surgery and willing to commit to the full study period of three outdoor sessions. Participants were reimbursed with S$50 worth vouchers after completion of the sessions. One participant did not complete the experiment, without providing a reason, and therefore was not included in the analyzed sample. The study was granted NUS Ethical Committee’s approval (ref#: S-18-352).This is an on-going study and the results presented here are based on a sub-sample of 22 healthy volunteers  from Singapore. Participants were of Chinese (73%), Indian (5%) and other (22%) ethnicities. The full sample on completion will comprise The selected sites are two UGS in Singapore (an urban park and a neighborhood green space), and one busy urban street (control) characterized by negligible greenery. Geographically, Singapore is located near the equator and has tropical climate with abundant rainfall , high and uniform temperatures and humidity all year round . Meteorological variables do not show large month to month variability and diurnal variations are strongly influenced by solar radiation .Within each site, participants were exposed to three different landscape scenes with different landscape quality values and features within the view. Each of the scenes was previously annotated by four landscape architecture experts using the contemplative landscape model (CLM) and a mean was used to score each scene . CLM is In order to control for depressive symptoms we utilized Beck Depression Inventory II (BDI), a 21-item self-reported multiple choice inventory, widely used as an indicator of severity of depression (81% of sensitivity and 92% specificity) .Data was collected between March and May 2019, during morning or late afternoon hours of the working week. Experimental sessions were scheduled individually, during one session there was one site with three scenes visited (order randomized ). ParticThe EEG signal was processed offline using the Brain Analyzer 2 software . The raw signal had a 500 Hz sampling rate, with 2000 µS sampling interval, and was filtered with the high cutoff 40 Hz and 50 Hz notch filter. Channels were referenced according to the 10–20 international system and the visual inspection was performed to evaluate the signal quality, noisy or missing channels. Topographic interpolation of noisy or lost channels was performed where necessary, and ocular movements were corrected using the Independent Component Analysis (ICA) ocular correction function. Later, the 60 s long segments for both baseline and viewing conditions were extracted and non-complex fast Fourier transformation was performed on both segments. Further, the frequencies from two segments were averaged, and the alpha power (8–13 Hz) values for each electrode was extracted. The exported viewing data was then corrected for the baseline (viewing-baseline), and alpha power values for the left and right frontal regions were computed  ,22. Posit-test was used to compare the before and after POMS scores. Analyses were conducted in IBM SPSS v.23  and the alpha level of 0.05 was used as an indicator of statistical significance.A repeated measures general linear model (3 × 3) was used to test the main effect of site and the interaction between the site and the view on FAA. Unadjusted post-hoc comparisons were conducted to investigate specific group differences. In addition, to examine whether the landscape score (CLS) predicts FAA independently of the experimental condition, we used a linear regression model with bootstrapped 95% confidence intervals (CI), with 1000 samples and bias corrected accelerated CIs. For this purpose, CLS from the control condition were dummy coded and assigned a score of 1, while the raw CLS from the experimental conditions remained unchanged. As external conditions may impact the quality of exposure, data were re-analyzed after adjusting for Thom’s discomfort index (TDI), which is a good summary measure of the external conditions . Due to Sample characteristics, including selected environmental metrics, are described in p < 0.05), so multivariate tests (Pillai’s trace) were reported alongside the within-subject effects, with degrees of freedom corrected using Greenhouse–Geisser estimates of sphericity.Sphericity assumption was violated for the main effect and the interaction (p = 0.078), which was attenuated in the within-subjects tests  = 1.37, p = 0.26). After adjusting for TDI, the multivariate tests showed a significant main effect of site  = 5.14, p = 0.024), which was slightly attenuated in the within-subjects tests .The multivariate effects showed a non-significant trend for the main effect of site  = 3.04, p = 0.61) or Site 3 (p = 0.20), but Site 2 was significantly different from Site 3 (p = 0.023).The highest mean FAA were recorded in Site 1 (urban park), and the lowest in Site 3 (urban street) and these are depicted in p = 0.088), which was not present in the within-subjects tests  = 0.36, p = 0.64). These trends were further attenuated after adjusting for TDI in multivariate tests  = 1.84, p = 0.20) and though within-subjects comparisons showed significant interaction between the site and the view . The mean FAA across the sites and the views are summarized in The multivariate tests showed a non-significant trend for the interaction between the site and the view  = 2.57, p = 0.16), and adjusting for TDI did not influence that association .Independently of the site, there was a non-significant weak positive association between the CLS and FAA  and Site 2 (>0.05). However, the total mood disturbance seemed to increase from before to after the exposure in the control group .In addition, there were no POMS changes before and after the exposure in Site 1  when exposed to the busy street environment in high-density downtown with negligible greenery. Importantly, there was a trend for a non-significant interaction between the site and the view type, suggesting that even within the individual sites, there may be a heterogeneous response to different views and some scenes may induce more or less FAA response than others b. This fThese preliminary findings support previous research findings suggesting that quality of green exposure can play a more important role than the quantity or accessibility of UGS for the MH&WB outcomes ,42 and bFuture research will examine the differences in brain oscillations between indoor and outdoor exposure to contemplative and non-contemplative landscapes, including in the sample patients with diagnosed depression. We will also run the dose-response analysis, which will evaluate the optimal exposure time for the best MH&WB outcomes.The strength of this study was a well-controlled experimental design with multiple conditions and objective physiological measures. The main limitation of the study was the small sample size at this preliminary stage, as the data collection was still ongoing. There also was a possibility of bias regarding the outlying FAA values, noticeable through the values of standard deviation varying between Site 1 and Site 2 and 3. However, the bias risk was mitigated by a within-subjects design, as each participant went through the same data collecting procedure in three different environments and to remain conservative all FAA values were adjusted for baseline.On completion, the results of this study will provide a basis for prevention and interventions that target mental health outcomes and will be used as guidelines for designing UGS that are optimal for mental health promotion.