Lysiphyllum strychnifolium (Craib) A. Schmitz, a traditional Thai medicinal plant, is mainly composed of polyphenols and flavonoids and exhibits several pharmacological activities, including antioxidant, anticancer, antimicrobial, and antidiabetic activities. However, the mechanism by which pure compounds from L. strychnifolium inhibit glucose catalysis in the small intestine and their effect on the glucose transporter remain unknown.O-𝛼-L-rhamnopyranoside (compound 1) and 3,5,7,3’,5’-pentahydroxy-flavanonol-3-O-𝛼-L-rhamnopyranoside (compound 2) on the inhibition of α-amylase and α-glucosidase, as well as glucose transporters, such as sodium-glucose cotransporter 1 (SGLT1), glucose transporter 2 (GLUT2), and glucose transporter 5 (GLUT5), using Caco-2 cells as a model of human intestinal epithelial cells. Additionally, the binding affinity and interaction patterns of compounds against two receptor proteins (SGLT1 and GLUT2) were determined for the first time utilizing a molecular docking approach.The objectives of this research were to examine the effect of 3,5,7-trihydroxychromone-3-50 values of 3.32 ± 0.30 µg/mL and 2.86 ± 0.10 µg/mL, respectively) in addition to a moderate inhibitory effect for compound 2 (IC50 = 10.15 ± 0.53 µg/mL). Interestingly, compounds 1 and 2 significantly inhibited α-glucosidase and exhibited better inhibition than that of acarbose, with IC50 values of 5.35 ± 1.66 µg/mL, 510.15 ± 1.46 µg/mL, and 736.93 ± 7.02 µg/mL, respectively. Additionally, α-glucosidase activity in the supernatant of the Caco-2 cell monolayer was observed. In comparison to acarbose, compounds 1 and 2 inhibited α-glucosidase activity more effectively in Caco-2 cells without cytotoxicity at a concentration of 62.5 µg/mL. Furthermore, the glucose uptake pathways mediated by SGLT1, GLUT2, and GLUT5- were downregulated in Caco-2 cells treated with compounds 1 and 2. Additionally, molecular modeling studies revealed that compounds 1 and 2 presented high binding activity with SGLT1 and GLUT2.In the α-amylase inhibition assay, a concentration-dependent inhibitory response was observed against the enzyme. The results indicated that compound 1 inhibited α-amylase activity in a manner similar to that of acarbose (which exhibit ICL. strychnifolium extracts. Our findings indicated that compounds 1 and 2 reduced glucose uptake in Caco-2 cells by decreasing the expression of glucose transporter genes and inhibiting the binding sites of SGLT1 and GLUT2. Therefore, compounds 1 and 2 may be used as functional foods in dietary therapy for postprandial hyperglycemia modulation of type 2 diabetes.In summary, our present study was the first to perform molecular docking with compounds present in  Diabetes mellitus (DM) is a chronic metabolic disorder caused by insufficient insulin secretion, impaired insulin action, or a combination of the two, resulting in hyperglycemia. DM is among the most prevalent diseases in the twenty-first century, mainly due to a variety of lifestyle problems . The chrCarbohydrates are converted into glucose and enter the bloodstream in the small intestine. Alpha-amylase enzyme (α-amylase) and alpha-glucosidase (α-glucosidase) enzymes play an important role in catalyzing the hydrolysis of starch to glucose. Glucose cannot diffuse freely across the hydrophobic area of the lipid layer of the cell membrane and can only be absorbed and utilized in the small intestine via glucose transporters . NumerouL. strychnifolium is a reddish climbing plant that is found in montane tropical rainforests and mixed deciduous forests. The leaves, vines, and roots of L. strychnifolium have an astringent, sweet, and cold flavor. Additionally, L. strychnifolium, a traditional Thai medicinal plant, is widely used for detoxification purposes [L. strychnifolium are polyphenols and flavonoids, and L. strychnifolium exhibits several pharmacological activities, including antioxidant, anticancer, antibacterial, antiviral, and antidiabetic activities [L. strychnifolium contains 3,5,7-trihydroxychromone-3-O-α-L-rhamnopyranoside and 3,5,7,3’,5’-pentahydroxy-flavanonol-3-O-𝛼-L-rhamnopyranoside, which inhibit α-glucosidase activity [L. strychnifolium extracts inhibit α-amylase activity and α-glucosidase in the small intestine and affects glucose transporters remains unknown.Managing type-2 diabetes by conventional therapy involves inhibiting the degradation of dietary starch by glucosidases such as α-amylase and α-glucosidase . Recentlpurposes . The maitivities –11. The activity . HoweverO-α-L-rhamnopyranoside (compound 1) and 3,5,7,3’,5’-pentahydroxy-flavanonol-3-O-𝛼-L-rhamnopyranoside (compound 2), that were isolated from L. strychnifolium for in vitro studies of α-amylase activity and α-glucosidase activity. Moreover, the effect of these compounds on glucose transporters in human intestinal epithelial Caco-2 cells was investigated. Additionally, the cytotoxicity and mRNA expression of genes involved in glucose uptake  were determined using the MTT assay and RT‒PCR, respectively. Furthermore, we explored the compound expected to be responsible for the antidiabetic activity by performing molecular docking with the identified compounds toward the glucose transporter.Herein, we emphasized the antidiabetic effects of pure compounds, 3,5,7-trihydroxychromone-3-Culture medium DMEM (high glucose), DMEM (without glucose), fetal bovine serum, penicillin‒streptomycin and phosphate buffer solution were purchased from Invitrogen Corporation . 3--2,5-diphenyl tetrazolium bromide (MTT), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2’-azinobis (3-ethylbenzothiaz oline-6-sulfonic acid) diammonium salt (ABTS), α-amylase, α-glucosidase, maltose and acarbose were purchased from Sigma‒Aldrich Corporation . Analytical grade chemicals and reagents were utilized in the studies.L. strychnifolium stems were collected in 2020 at the Suan Ya Thai Thongnoppakhun herbal garden in Chonburi Province. A voucher specimen (SKP 072021901) is currently being stored at the Herbarium of the Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Thailand.L. strychnifolium (4.5 kg) was extracted twice at room temperature with EtOH (28 L). Under reduced pressure, a 993.5 g EtOH extract was obtained and maintained at 4 °C. The EtOH extract (993.5 g) was sequentially partitioned with hexane, dichloromethane, ethyl acetate, and water. The water and chloroform fractions that exhibited good activity in enzyme assays were selected to further isolate bioactive compounds. The 40 g water fraction was chromatographed in water, water/methanol, and methanol to yield 6 fractions (F1-F6). Fraction F6 (6.2 g) was separated by silica gel column chromatography using 10% methanol in ethyl acetate. Subfraction F6/3a (3.24 g) was purified by column chromatography on silica gel using 20% methanol in chloroform. Subfraction F6/2b was purified by silica gel column chromatography using 20% methanol in chloroform to extract 3,5,7-trihydroxychromone-3-O-𝛼-L-rhamnopyranoside (compound 1) and 3,5,7,3’,5’-pentahydroxy-flavanonol-3-O-𝛼-L-rhamnopyranoside (compound 2). Pure chemicals (compounds 1 and 2) were isolated by chromatography, and their spectroscopic data were compared to earlier reports [ reports , 12.50).The method by Kumar, Sanjiv et al.  was adop50).The method by Kumar, Sanjiv et al.  was adop50).The method by Poovitha S and Parani M  was adop2CO3 reagent was added. The absorbance of the reaction mixture was measured at 405 nm. The percent inhibition of all samples was calculated using the following equation and was expressed as the 50% inhibitory concentration (IC50).The method by Poovitha S and Parani M  was adop2. For the viability test, the Caco-2 cells were seeded on 96-well plates, and 24-well Transwell plates were used to determine α-glucosidase inhibition. The cells differentiated after 21 days and exhibited intestinal microvillus membrane hydrolases [Caco-2 cells were obtained from the American Type Culture Collection (ATCC). The cells were cultured in DMEM with 10% FBS and 1% penicillin–streptomycin solution and were maintained at 37 °C in a humidified environment of 95% air and 5% COdrolases . Fresh mCaco-2 cells were obtained from the American Type Culture Collection (ATCC). The cells were grown in complete medium  and plated at a density of 10,000 cells per well in 96-well plates for 21 days. Then, the cells were treated with extracts at concentrations ranging from 0 to 500 g/ml for another 24 h. DMSO (0.1%) was added to the control group. The MTT test was used to measure cell viability after treatment. In a 96-well plate, 100 µl of MTT at a concentration of 0.5 mg/ml was applied to each well. The MTT solution was withdrawn after 3 h of incubation at 37 °C, and formazan crystals were dissolved in 100 µl of DMSO. At 570 nm, the purple color that was generated was detected spectrophotometrically (with a reference OD of 690) . Prism 6Caco-2 cells were seeded in 24-transwell plates at a density of 10,000 cells per well for 21 days. The cells were washed in 1X PBS after the culture media was removed. The upper chamber was then filled with compounds/acarbose and 28 mM maltose in glucose-free media (300 µl) for 24 h overnight. At the same time, the lower chamber was filled with glucose-free medium (500 µl). Then, 50 µl of culture media from the upper chamber was taken and measured by the glucose oxidase method . The comRNA was extracted from Caco-2 cells using an RNA extraction kit . Reverse transcription was performed with SuperScript III Reverse Transcriptionase . cDNA was synthesized from 2 µg of total RNA according to the manufacturer’s instructions. Then, the cDNA was maintained at − 20°C for further analysis by real-time PCR. The specific primers used in the experiment were as follows: SGLT1, 5’-TGGCATCAACGCTGTCTTCT-3’ (forward) and 3’-AGCCAATGGTGGCATACACA-5’ (reverse); GLUT2, 5’-CTGCCGCTGAGAAGATTAGAC-3’ (forward) and 3’-CAGGTCTTGTGTGAGTGTGG-5’ (reverse); GLUT5, 5’- TCCATTTGGAGGGTTTATCG-3’ (forward) and 3’-AACAGCAAGGCCCCCTTTT-5’ (reverse). All conditions were performed in triplicate with 3 independent experiments. The real-time qPCR mixture (10 µL) comprised 1 µL of DNA polymerase, template DNA (25 ng), 1 µL of DEPC, and 1.2 µL of forward and reverse primers. Thermal cycling  consisted of , 3,5,7,3’,5’-pentahydroxy-flavanonol-3-O-𝛼-L-rhamnopyranoside (compound 2), acarbose, and the glucose transporter membrane (SGLT1 and GLUT2). The crystalized structures of SGLT1 and GLUT2 were downloaded from the RCSB protein data bank as PDB IDs 2XQ2 and 4ZWB, respectively [in silico experiments. The binding poses, interactions, and binding affinities of acarbose, compounds and SGLT1 and GLUT2 were determined by using the built-in AutoDock Vina 1.1 module of LigandScout 4.4 advanced. The native ligand was redocked into its binding site to validate the methods. The redocking RMSD values were 0.60 and 0.00 Å, respectively.Molecular docking was performed to illustrate the molecular interaction between the two major compounds derived from ectively , 19. Allectively . The binp value of less than 0.05.The results are presented as the means ± SEMs and were analyzed by using GraphPad Prism 5.0 (GraphPad Software). Statistical comparisons were determined by one-way ANOVA, followed by two-tailed Student’s t test. Statistical significance was defined as a 50 (the concentration that causes 50% inhibition) values of 10.26 ± 0.20 µM and 14.40 ± 0.10 µM, respectively, when compared to that of ascorbic acid (25.10 ± 0.19 µM). In addition, the EC50 values of compounds 1 and 2 were 5.22 ± 0.20 µM and 5.29 ± 0.12 µM, respectively, as determined by the DPPH assay. All compounds demonstrated significantly higher antioxidant activity than that of positive controls.Two methods were used to evaluate the antioxidant activities, including the DPPH radical scavenging activity and ABTS radical scavenging activity. These assays offer a redox-functioning proton ion for unstable free radicals and play a critical role in stabilizing detrimental free radicals in the human body. The scavenging capacity of the compounds is indicated by the degree of reduction in absorbance measurement. Thus, we examined the free radical scavenging activity of compound 1 and compound 2 in comparison to ascorbic acid (positive control), as shown in Table 50 values of 7.37 ± 0.30 µM and 4.43 ± 0.10 µM, respectively. In addition, compound 2 showed less efficient inhibition of α-amylase activity than that of acarbose, with IC50 values of 29.82 ± 0.53 µM  in the human small intestine Table A-F. The Type 2 DM is a metabolic disorder characterized by prolonged periods of elevated blood glucose (hyperglycemia), which results in insulin resistance in peripheral tissues or impaired insulin production by pancreatic B cells , 3. ContO-𝛼-L-rhamnopyranoside (compound 1) and 3,5,7,3’,5’-pentahydroxy-flavanonol-3-O-𝛼-L-rhamnopyranoside (compound 2) on α-amylase inhibition and α-glucosidase inhibitory activity. We considered that compounds 1 and 2 are involved in the degradation of polysaccharides into disaccharides, in which compound 1 exhibited strong inhibition of both α-amylase and α-glucosidase. Comparison of the half maximal inhibitory concentration (IC50) values revealed that the α-amylase inhibitory activity of compound 1 (IC50 3.32 µg/mL) was similar to that of acarbose (IC50 2.86 µg/mL). In addition, the IC50 values of compound 1 (IC50 5.35 µg/mL) inhibited α-glucosidase activity by approximately 133% compared with that of acarbose (IC50 736.93 µg/mL). In comparison to previous studies, Ficus racemosa Linn. fruit ethanolic extract showed an inhibitory effect of α-amylase with an IC50 value of 7.44 µg/mL [50 values of Ficus carica L. in the inhibition of α-amylase and α-glucosidase were 315.89 ± 3.83 µg/mL and 255.57 ± 36.46 µg/mL, respectively [Here, we examined the effects of 3,5,7-trihydroxychromone-3-44 µg/mL . The IC5ectively .α-Glucosidase degrades disaccharides into monosaccharides, such as glucose, in the small intestine prior to absorption. According to cell lines, Caco-2 cells express the same morphological characteristics and most of the functional properties of terminally differentiated small intestinal enterocytes and express brush-border enzymes, such as α-glucosidase , 21, 29.in silico results is necessary to ensure that the identification of efficacy is accurate, as there is a possibility of false positive and negative results, which is one of the limitations of the study.Glucose uptake in the small intestine is most notably mediated by sodium-dependent glucose transporter 1 (SGLT1), epithelium glucose transporter 2 (GLUT2), and epithelial glucose transporter 5 (GLUT5) . Thus, tOxidative stress plays a role in the pathogenesis of type 2 diabetes and its complications. Increased levels of free radical and oxidative stress are associated with lipid peroxidation, nonenzymatic glycation of proteins, and glucose oxidation, all of which contribute to the development of diabetes and its complications, including coronary artery disease, nephropathy, retinopathy, and neuropathy . We founO-α-L-rhamnopyranoside and 3,5,7,3’,5’-pentahydroxy-flavanonol-3-O-𝛼-L-rhamnopyranoside derived from L. strychnifolium have antioxidant and antidiabetic effects, as evidenced by α-amylase inhibition activities, α-glucosidase inhibition activities, and decreased glucose absorption in enterocytes of the small intestine by suppressing the gene expression of glucose transporters and inhibiting the binding sites of SGLT1 and GLUT2 (Fig. O-α-L-rhamnopyranoside and 3,5,7,3’,5’-pentahydroxy-flavanonol-3-O-𝛼-L-rhamnopyranoside may be used as functional foods in dietary therapy for postprandial hyperglycemia modulation of type 2 diabetes without the side effects associated with acarbose treatments.Our findings demonstrate that 3,5,7-trihydroxychromone-3-UT2 Fig. . Therefo


Latilactobacillus sakei, a frequently used meat starter, remains scarce.Lactic acid bacteria (LAB) are used as starters in a wide variety of food fermentations. While the number of reports of phages infecting other LAB steadily increased over the years, information about phage associated with Latilactobacillus sakei genomes revealed the presence of 26 intact, eleven questionable and 52 incomplete prophage sequences across all analysed genomes with a range of one to five predicted prophage sequences per strain. Screening 24 sakei strains for inducible prophages by utilising UV light or mitomycin C, we identified seven lysogenic strains showing lysis after induction during subsequent growth monitoring.In this study, a predictive genomic analysis of 43 Latilactobacillus sakei TMW 1.46, we demonstrate that simultaneous induction of multiple prophages is possible.Electron microscopic analysis revealed fully assembled virions in the purified lysates of four samples, thus confirming successful prophage induction. All virions featured icosahedral, isomeric heads and long, most likely non-contractile tails indicating siphoviruses. By performing phylogenetic analyses with various marker genes as well as full prophage sequences, we displayed a remarkably high diversity of prophages, that share a similar gene module organisation and six different chromosomal integration sites were identified. By sequencing viral DNA purified from lysates of Latilactobacillus sakei, we also demonstrated their potential to impact growth of their host after induction, as well as forming seemingly fully assembled virions.With this work, we not only provide data about the incidence of prophages harboured by the meat starter The online version contains supplementary material available at 10.1186/s12866-022-02675-y. After being patented by Jensen et al. in 1940  and 1.46_P2_LYS_REV [5′-CCGCACATACACAGTCCGATAC-3′] were used (expected amplificate length: 2.3 kb). Successful DNA amplification was checked by sequencing of the amplified DNA products via Illumina (Eurofins Genomics) after purification of the PCR reactions using the E.Z.N.A.® Cycle Pure Kit (Omega Bio-Tek) according to manufacturer’s instructions.L. sakei TMW 1.46 lysate was used, which was sterile filtered using Filtropur S filters with 0.45 μm pore size (SARSTEDT AG & Co. KG) 24 h after UV light induction and was digested with DNase I. For DNase I digestion 16 μl lysate, 2 μl of solubilised DNase I (stock solution: 1500 Kunitz units in 550 μl RNase-free water) from the RNase-Free DNase Set (QIAGEN) and 2 μl of 10x DNase I Reaction Buffer (Invitrogen) were incubated for 10 minutes at 37 °C, followed by DNase I inactivation at 75 °C for 10 minutes.For the viral DNA detection via PCR, 1 μl of L. sakei TMW 1.46 (expected amplificate length: 127 bp). Viral DNA of TMW 1.46 P2 was amplified with the primers 1.46_P2_TER_FWD [5′-GAAATGGGAACAGATGGGGC-3′] and 1.46_P2_TER_REV [5′-GGCAAATTGATGGCTTAAATGG-3′] (expected amplificate length: 125 bp). Complete digestion of L. sakei TMW 1.46 genomic DNA in the lysates was indicated by a negative result using the primers 616 V [5′-AGAGTTTGATYMTGGCTCAG-3′] and 609R [5′-ACTACYVGGGTATCTAAKCC-3′] for partial amplification of the 16 s rDNA genes [The primers 1.46_P1_INT_FWD [5′-GCTACAATTCAACCGCTTGGG-3′] and 1.46_P1_INT_REV [5′-CAAAGCATTATCCGCAGAC-3′] were used for the detection of TMW 1.46 P1 DNA in the DNase I digested, post induced lysate of NA genes  (expectewww.snapgene.com) was used to colourise prophage genes according to their predicted function. Easyfig 2.2.5 [CLC Main Workbench version 8.1.4 (Qiagen) for BLAST, (whole genome) alignments, phylogenetic trees and bootstrap analyses. SnapGene Viewer version 5.2.3: SnapGene software  over the aligned nucleotides (both in percent) using the web tool JSpeciesWS . PairwisLatilactobacillus sakei genomes, the free web application “Phage search tool enhanced release” (PHASTER) by [uniprot.org; BLASTp; E-Threshold: 10; Matrix: Auto; Filtering: None; Gapped: yes; Hits: 250) and replaced where reasonable. For the prophage sequence visualisation and allocation of gene module colours the program SnapGene Viewer  was used. The colour coding in this study was based on the colour coding of previous works with Levilactobacillus brevis phages [For the detection, evaluation of completeness  and a first annotation of prophage sequences in analysed STER) by , 12 was s phages . The phas phages .The following blast settings were used for the prophage genome comparison with Easyfig 2.2.5 : Min. leAdditional file 1. Additional File A1Additional file 2. Additional File A2Additional file 3. Additional File A3Additional file 4 Fig. S1 PCR-mediated DNA amplification, verifying circularization of both prophages (TMW 1.46 P1 and TMW 1.46 P2) within a non-induced cryo stock of L. sakei TMW 1.46, visualised by agarose gel electrophoresis. “1” + “7”: GeneRuler 1 kb DNA Ladder (Thermo Scientific). “2”: TMW 1.46 P1 circularization. “3”: Negative control for “2” (water instead of a washed cryo culture). “4”: Empty. “5”: TMW 1.46 P2 circularization. “6”: Negative control for “5” (water instead of a washed cryo culture).Additional file 5 Fig. S2 PCR-mediated DNA amplification, verifying the presence of virions originating from both prophages (TMW 1.46 P1 and TMW 1.46 P2) in post UV light induced, sterile filtered and DNase I digested lysates of L. sakei TMW 1.46, visualised by agarose gel electrophoresis. “1” + “11”: GeneRuler 1 kb DNA Ladder (Thermo Scientific). “2”: Post UV light induced, DNase I treated lysate amplificated with TMW 1.46 P1 marker. “3”: Washed L. s. TMW 1.46 cryo stock amplificated with TMW 1.46 P1 marker. “4”: Negative control for “2” + “3” (water instead of a washed cryo culture). “5”: Post UV light induced, DNase I treated lysate amplificated with TMW 1.46 P2 marker. “6”: Washed L. s. TMW 1.46 cryo stock amplificated with TMW 1.46 P2 marker. “7”: Negative control for “5” + “6” (water instead of a washed cryo culture). “8”: Post UV light induced, DNase I treated lysate amplificated with 16 s rDNA marker. “9”: Washed L. s. TMW 1.46 cryo stock amplificated with 16 s rDNA marker. “10”: Negative control for “8” + “9”  gene of as intact predicted prophages in L. sakei strains. Bootstrap values are based on a Jukes-Cantor model (1000 replicates). The phage terminase (large subunit) genes of phages infecting other lactobacilli were included as outgroups.Additional file 7 Fig. S4 Neighbor joining tree of the phage tape measure protein (TMP) gene of as intact predicted prophages in L. sakei strains. Bootstrap values are based on a Jukes-Cantor model (996 replicates). The TMP genes of phages infecting other lactobacilli were included as outgroups.Additional file 8 Table S1 Examples of genes/putative tasks of annotated proteins within the different phage gene modules and their respective color-coding used in the genome comparison.


The Cu atom is coordinated in a distorted tetrahedral geometry by two N atoms from the 2,2′-biquinoline ligands and the two μ2-bridging iodide ligands. The molecules are in contact via π–π-stacking interactions. Hirshfeld surface analysis showed that the most important contributions to the intermolecular interactions are H⋯H (39.7%), H⋯I/I⋯H (17.8%), C⋯H/H⋯C (17.5%), C⋯C (16.5%), N⋯C/C⋯N (3.9%) and N⋯H/H⋯N (3.5%).The molecular and crystal structures of the title compound, [Cu The Cu1—I1 and Cu1i—I1 distances  are 2.5734 (2) and 2.6487 (2) Å, which are close to the distances in similar compounds  and 0.026 (2) Å. The angle between the quinolines described by rings 1/2 -2,2′-biquinoline-di-copper(I)]. We only found data for compounds with substituted quinoline rings in position-4 with carboxylate fragments. All compounds crystallize in the triclinic space group Pet al., 2010n-hexyl carboxylate groups are attached to the quinoline rings at position 4. In YIJFAA, YIJFEE, and YIJFII  to 1.3919 (blue) a.u., as illustrated in Fig. 3a). Red spots on the surface correspond to C⋯C and I⋯H interactions. The presence of π-stacking interactions is confirmed by the characteristic red and blue triangles on the shape-index surface [Fig. 3b)]. Fingerprint plots of the most important non-covalent interactions for the title compound are shown in Fig. 46.36H24Cu2I2N4. Calculated (%): C, 48.61; H, 2.64; N, 6.19.The title compound was prepared by refluxing CuI with one equivalent of 2,2′-biquinoline in ethanol for 24 h. The compound precipitates as a purple solid in 87% yield. Found (%): C, 48.39; H, 2.71; N, 6.27. forC7.Uiso(H) = 1.2Ueq(C)].Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989023000634/dj2055sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989023000634/dj2055Isup2.hklStructure factors: contains datablock(s) I. DOI: 2237760CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


All of them were obtained as racemic mixtures, chiral HPLC was used to produce their (+)- and (−)-antipodes. The structures of these new compounds, including their absolute configurations, were elucidated by spectroscopic and computational methods. This isolation provides new insight into the chemical profiling of F. sinkiangensis resins beyond the well-investigated structure types such as sesquiterpene coumarins and disulfides. Compounds 2a and 3a were found to significantly inhibit the invasion and migration of triple-negative breast cancer (TNBC) cell lines via CCK-8 assay. On the other hand, the wound-healing assay also demonstrated that compounds 4a and 4b could promote the proliferation of human umbilical vein endothelial cells (HUVECs). Notably, the promoting effects of 4a and 4b were observed as more significant versus a positive control using basic fibroblast growth factor (bFGF).Ferulasinkins A–D ( Ferula, belonging to the Apiaceae family, includes more than 150 species that are widespread throughout the Mediterranean area and Central Asia, such as Turkey, Iran, Pakistan, and northwest China 20D −19.3 ; CD (MeOH) Δε203 −16.08, Δε219 −0.87, Δε233 −2.43; 1a}; {[α]20D +26.0 ; CD (MeOH) Δε200 +20.86, Δε216 +1.03, Δε223 +2.17; 1b}; HR-ESI-MS ; 1H- and 13C-NMR data, see Ferulasinkin A (2): colorless gum; UV (MeOH) λmax (logε) 202 (3.93), 217 (3.01), 230 (3.13), 280 (2.69) nm; {[α]20D −19.3 ; CD (MeOH) Δε209 +2.90, Δε221 −0.02, Δε233 –0.83; 2a}; {[α]20D +23.3 ; CD (MeOH) Δε209 −5.94, Δε221 +0.17, Δε237 +0.58; 2b}; HR-ESI-MS ; 1H- and 13C-NMR data, see Ferulasinkin B (3): colorless gum; UV (MeOH) λmax (logε) 202 (4.18), 216 (3.21), 231 (3.35), 282 (2.89) nm; {[α]20D −21.0 ; CD (MeOH) Δε204 −15.68, Δε219 −1.60, Δε232 −2.82; 3a}; {[α]20D +19.3 ; CD (MeOH) Δε200 +6.85, Δε218 +0.54, Δε233 +0.87; 3b}; HR-ESI-MS ; 1H- and 13C-NMR data, see Ferulasinkin C (4): colorless gum; UV (MeOH) λmax (logε) 201 (3.76), 215 (2.76), 231 (2.86), 280 (2.41) nm; {[α]20D −6.0 ; CD (MeOH) Δε208 +0.85, Δε225 +0.27, Δε238 +0.57; 4a}; {[α]20D +8.6 ; CD (MeOH) Δε209 −5.41, Δε223 −1.57, Δε234 −2.70; 4b}; HR-ESI-MS ; 1H- and 13C-NMR data see Ferulasinkin D  level in a Gaussian 09 program  [The CONFLEX searches based on molecular mechanics with the Molecular Merck force field (MMFF) were performed for compounds CT, USA) . Finallyv/v) fetal bovine serum (FBS) with 100 U/mL penicillin and incubated at 37 °C in a 5% CO2, 95% humidified atmosphere. Cellular metabolic activity was measured with a Cell Count Kit-8 (CCK-8)  assay kit after a 40 μM compound or DMSO treatment. The cells were seeded in 96-well plates at a density of 3–5 × 103 cells/well for 1 day and then incubated in 2% FBS media with or without compounds for 48 h. Subsequently, 10 μL of 5 mg/mL CCK-8 solution was added to each well and incubated at 37 °C for an additional 4 h. The absorbance of each well was determined at 450 nm with a microplate reader . The confluent TNBC cells in 96 cultured plates were scratched vertically by using Scratch Assay Starter Kit  and incubated with compounds (40 μM) and DMSO as vehicles in DMEM containing 0.5% FBS for 48 h. Images of the wounded region were analyzed by CYTATION1  after scratches occurred. Cell migration of this region was monitored at 0 h, 24 h, and 48 h , respectively. The migration width of T24 (width at T0 − width at T24) and T48 (width at T0 − width at T48) were recorded.The human TNBC cell lines HCC1806 and MDA-MB231  were cultured in 10 cm dishes and maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10%  and DMSO as vehicles in ECM containing 0.5% FBS for 24 h. Images of the wounded region were analyzed by CYTATION1 after scratches occurred. Cell migration of this region was monitored at 0 h, 24 h, and 48 h , respectively. The migration width of T24 (width at T0 − width at T24) and T48 (width at T0 − width at T48) were recorded.HUVECs  were cultured in 10 cm dishes and maintained in endothelial cell medium (ECM)  containing 5% FBS, 1% penicillin, 1% streptomycin, and 1% endothelial cell growth supplement (ECGS) , and incubated at 37 °C in a 5% COt-test and a one-way ANOVA test. When * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001, differences were considered significant.All biological experiments in this study were carried out in triplicate. The data are means ±SEM. Graphpad Prism 7  was used to conduct statistical analyses, which included a Student’s F. sinkiangensis. Their respective enantiomers were obtained via chiral HPLC separation. Compounds 2a and 3a could significantly inhibit the invasion and migration of TNBC cell lines. Additionally, compounds 4a and 4b could promote cell proliferation of HUVECs, showing their wound-healing effects. These findings help us to gain a deep insight into the chemical profiling and biological properties of F. sinkiangensis resins.In summary, four new norlignans, ferulasinkins A–D, were isolated as racemic mixtures from the resins of


Bacillus safensis, designated as BsXyn10. The xylanase gene (bsxyn10) was cloned from Bacillus safensis and expressed in Escherichia coli. The reduced molecular mass of BsXyn10 was 48 kDa upon SDS-PAGE. Bsxyn10 was optimally active at pH 7.0 and 60 °C, stable over a broad range of pH (5.0–8.0), and also revealed tolerance toward different modulators . The enzyme was active toward various xylans with no activity on the glucose-based polysaccharides. KM, vmax, and kcat for oat spelt xylan hydrolysis were found to be 1.96 g·L−1, 58.6 μmole·min−1·(mg protein)−1, and 49 s−1, respectively. Thermodynamic parameters for oat spelt xylan hydrolysis at 60 °C were ΔS* = −61.9 J·mol−1·K−1, ΔH* = 37.0 kJ·mol−1 and ΔG* = 57.6 kJ·mol−1. BsXyn10 retained high levels of activity at temperatures up to 60 °C. The thermodynamic parameters  for the thermal deactivation of BsXyn10 at a temperature range of 40–80 °C were: 192.5 ≤ ΔH*D ≤ 192.8 kJ·mol−1, 262.1 ≤ ΔS*D ≤ 265.8 J·mol−1·K−1, and 99.9 ≤ ΔG*D ≤ 109.6 kJ·mol−1. The BsXyn10-treated oat spelt xylan manifested the catalytic release of xylooligosaccharides of 2–6 DP, suggesting that BsXyn10 represents a promising candidate biocatalyst appropriate for several biotechnological applications.Xylanases have a broad range of applications in agro-industrial processes. In this study, we report on the discovery and characterization of a new thermotolerant GH10 xylanase from  Hemicelluloses, the second most abundant biopolymer group in nature after cellulose, are mainly composed of xylan, an amorphous structural polysaccharide that encompasses a xylose backbone, decorated with a source-dependent variety of side-chain substitutions . The exaDue to its high heterogeneity, complete xylan degradation is performed by the synergistic action of different xylanolytic enzymes, which are cumulatively referred to as xylanases. The xylanase group consists of endo-1,4-β-D-xylanases EC 3.2.1.8), β-D-xylosidases EC3.2.1.37), α-glucuronidases (EC 3.2.1.139) acetylxylan esterases (EC 3.1.1.72), α-L-arabinofuranosidases EC3.2.1.55), p-coumaric esterases (3.1.1.B10), and ferulic acid esterases (EC 3.1.1.73) involved in the depolymerization of xylan into simple pentoses, xylooligosaccharides, and side-chain groups  was detected. This result matched our original taxonomic analysis, which assigned the 16S sequencing reads of the isolate to the genus Bacillus. BlastP analysis against the UniProtKB/SwissProt database revealed that the closest characterized homolog of BsXyn10 is an endo-1,4-β-xylanase from Acetivibrio thermocellus ATCC 27,405 [NCBI Reference Sequence: A3DH97.1]  with a 29% sequence identity (query coverage 59%) [B. safensis [PDB entry number: 7D88_A] [Following the identification (through 16S rRNA sequencing) of our strain as h BlastP  against 3DH97.1]  with a s 7D88_A] . Like BsIn order to study the biochemical properties of the new xylanase, BsXyn10 was recombinantly produced and purified to homogeneity, as confirmed by the single band on the SDS-PAGE, b, by appThe molecular mass of BsXyn10 is in the range of molecular masses reported for GH10 xylanases of different origin .Bacillus sp. N16-5  vs. time  and decimal reduction time  are important parameters in industrial applications because the higher its value, the higher the enzyme thermostability. The t1/2 and D values for BsXyn10 over the temperature range tested are presented in 1/2 and D values of 5.3 h and 17.4 h, respectively, at 50 °C, while at 60 °C, the values were 25 and 83 min.As shown in d vs. 1/T (Arrhenius plot) at 195.4 kJ·mol−1  [T. naphthophila RKU- 10Τ (516.3 kJ·mol−1) [Cohnella sp. A01 (232.8 kJ·mol−1) [In general, the thermal inactivation of enzymes is a two-step process: Native enzyme (N) → Unfolded enzyme (U) → Inactive enzyme (I). Upon exposure of the native enzyme at elevated temperatures, an unstable intermediate (U) is formed. Thermal inactivation energy n E(a)d) ,39. The kJ·mol−1 . This vaJ·mol−1) , much loJ·mol−1) .D) is a measure of the number of non-covalent linkages broken in forming a transition state for enzyme inactivation while entropy change (ΔS*D) indicates the enzyme disorder due to disruptions of the enzyme structure [The thermal inactivation of enzymes is also accompanied by the disruption of non-covalent linkages (hydrophobic interactions). Enthalpy change  were fairly stable when the temperature increased, as also reported for the B. halodurans TSEV1 GH10 xylanase (ΔH*D = 251.7 kJ·mole−1) at a temperature range of 65–80 °C [D values (230.07–229.86 kJ·mole−1) at a temperature range of 55–80 °C was recorded for XynB-A01 [2 moiety from a hydrophobic bond is approximately 5.4 kJ·mole−1, and thus the formation of the transition state leading to inactivation of the BSXyn10 implies the disruption, as an average, of 35.7 non-covalent bonds [The thermodynamic parameters of BsXyn10 were calculated in the temperature range of 40–80 °C and presented in 65–80 °C  while a nt bonds .Positive ΔS* values at each temperature clarified that no noteworthy aggregation processes occurred; a similar trend has previously been reported ,41.D) includes both the entropic and enthalpic contributions and thus is considered as a more accurate and reliable variable to assess the enzyme’s stability. The value of ΔG*D for BsXyn10 ranged from 109.6–99.9 kJ·mole−1 at 40–80 °C  at a temperature range of 55–80 °C [B. halodurans TSEV1 GH10 xylanase seemed less thermodynamically stable compared to BsXyn10 as ΔG*D decreased from 98.8 to 21.95 kJ·mole−1 when the temperature increased from 65 to 70 °C, respectively [The Gibbs free energy of inactivation (ΔG*40–80 °C . The low55–80 °C . The B. ectively .The main xylooligosaccharides produced after 24 h of oat spelt xylan hydrolysis by BsXyn10 were xylotriose (X3 approximately 32%), followed by xylotetraose . Xylobiose (X2) and xylopentaose (X5) accounted for 18 and 12%, respectively, while smaller amounts of xylose  and xylohexaose  were detected . An almoF. johnsoniae, which produced X2-X6 xylooligosaccharides and a low amount of xylose [BsXyn10 exhibited the same hydrolytic pattern as Xyn10A of f xylose  , but no xylose was detected [According to Collins et al. , lower Ddetected ,38 , a novel thermotolerant xylanolytic enzyme with great potential for various industrial biotransformations due to its high specific activity and significant thermal tolerance, as corroborated by the thermodynamic and kinetic analysis of the enzyme. The hydrolytic efficacy of BsXyn10 on different xylan types is consistent with the broad substrate specificity of the GH10 family xylanase. BsXyn10 produces X2–X6 xylooligosaccharides and a low amount of xylose. The production of XOs of similar DP at moderate temperatures indicated the suitability of BsXyn10 in the bioprocesses, preferably performed with less (heat) energy input. Overall, this study illustrates the discovery of a novel member of the GH10 family of xylanases where BsXyn10 has emerged as a promising candidate for use in various applications as it features characteristics favorable for different industrial setups.


N-oxide-κO)zinc(II); dibromidobis(quinoline N-oxide-κO)zinc(II) and diiodidobis(quinoline N-oxide-κO)zinc(II) are presented.The structures of the three related compounds dichloridobis(quinoline  N-oxide (QNO) in methanol yields compounds as ZnX2(QNO)2, where X = Cl (I), Br (II) and I (III), namely, dichloridobis(quinoline N-oxide-κO)zinc(II), [ZnCl2(C9H7NO)2], dibromidobis(quinoline N-oxide-κO)zinc(II), [ZnBr2(C9H7NO)2], and diiodidobis(quinoline N-oxide-κO)zinc(II) [ZnI2(C9H7NO)2]. In all three complexes, Zn cations are coordinated by two QNO ligands bound through the oxygen atoms and two halide atoms, with X—Zn—X bond angles ca 20° wider than the O—Zn—O, giving rise to a distorted tetrahedral geometry. Crystals of (II) and (III) are isostructural and both show pairwise π-stacking of QNO ligands and weak C—H⋯X hydrogen bonds, while (I) packs differently, with a shorter C—H⋯Cl bond and without π-stacking.The reaction of one equivalent of zinc(II) halide with two equivalents of quinoline  The latter has the distances H⋯Cl = 2.53 Å  and C⋯Cl = 3.416 (9) Å within the previously observed range but shorter than the average values of 2.64 and 3.66 Å, respectively  and (III), the red spots correspond to C—H⋯X interactions, viz. C18—H18⋯X1, C5—H5⋯X1, C16—H16⋯X2, and C9—H9⋯X2, which can be also regarded as weak hydrogen bonds  (X = Br) are 2.85, 2.88, 2.88 and 2.89 Å, respectively, while in (III) (X = I) they are 3.03, 3.12, 3.03 and 2.96 Å, respectively.For  < (II) < (III), together with the size of the halogen atoms and hence their share of the molecular surface . Interestingly, π-stacking in the structures of (II) and (III) gives only a modest increase of C⋯C contacts compared to (I), probably because it is counterbalanced by an overall decrease of carbon atoms’ share of the surface (21.4 > 19.5 > 18.3%). No halogen⋯halogen contacts are observed in any of the three structures.Analysis of the two-dimensional fingerprint plots Table 2 indicate5.et al., 2016N-oxides and halogen ligands bound to zinc returned 21 unique entries, the majority (15) of which contain pyridine N-oxide and its derivatives. Of these, the most closely related are pyridine N-oxide complexes, dichlorobis(pyridine N-oxide)zinc(II) zinc(II) zinc(II) zinc(II) zinc(II) , for which only unit-cell parameters were determined zinc(II) zinc(II)  and ZnBr2·0.86H2O (FW = 240.69 g mol−1).The water content of QNO and ZnBrI) was synthesized by dissolving 0.0986 g of QNO·0.28H2O  in 33 mL of methanol to which 0.0440 g of ZnCl2  were added at 295 K. The solution was covered with parafilm then allowed to sit; X-ray quality crystals were grown by slow evaporation at 295 K. Yield, 0.0822 g (60.2%). Selected IR bands : 3107 (w), 3083 (w), 3057 (w), 1579 (m), 1513 (m), 1447 (m), 1402 (s), 1269 (s), 1227 (m), 1203 (s), 1179 (m), 1144 (m), 1089 (s), 1050 (m), 883 (s), 800 (s), 768 (s), 723 (m), 584 (m), 559 (m), 542 (m).The title compounds were all synthesized in a similar manner. Compound (II) was synthesized by dissolving 0.0983 g of QNO·0.28H2O (0.654 mmol), in 40 mL of methanol to which 0.0778 g of ZnBr2·0.86H2O  were added at 295 K. The solution was covered with parafilm then allowed to sit; X-ray quality crystals were grown by slow evaporation at 295 K. Yield, 0.0866 g (46.7%). Selected IR bands : 3106 (w), 3075 (w), 3061 (w), 3016 (w), 1580 (m), 1510 (s), 1455 (m), 1270 (s), 1227 (m), 1214 (s), 1204 (s), 1173 (m), 1138 (m), 1086 (s), 1048 (m), 877 (m), 800 (s), 767 (s), 720 (s), 581 (m), 563 (m), 500 (m).Compound (III) was synthesized by dissolving 0.0517 g of QNO·0.28H2O (0.352 mmol) in approximately 36 mL of methanol to which 0.0524 g of ZnI2  were added at 295 K. The solution was covered with parafilm then allowed to sit; X-ray quality crystals were grown by slow evaporation at 295 K. Yield, 0.0910 g (52.3%). Selected IR Bands : 3100 (w), 3090 (w), 2076 (w), 3059 (w), 3027 (w),1580 (s), 1507 (s), 1382 (s), 1267 (m), 1225 (m), 1207 (s), 1169 (m), 1141 (m), 1044 (m), 880 (s), 807 (s), 769 (s), 720 (m), 580 (m), 562 (m), 499 (m).Compound  is absent in all of the metal complexes  = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989022005953/zv2014sup1.cifCrystal structure: contains datablock(s) I, II, III. DOI: 10.1107/S2056989022005953/zv2014Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989022005953/zv2014IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989022005953/zv2014IIIsup4.hklStructure factors: contains datablock(s) III. DOI: 2176715, 2176714, 2176713CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


NH3 volatilization from fertilizers and wastes (v-NH3) has long been assumed to be the primary NH3 source, but the contribution of combustion-related NH3  remains unconstrained. Here, we collated nitrogen isotopes of atmospheric NH3 and NH4+ and established a robust method to differentiate v-NH3 and c-NH3. We found that the relative contribution of the c-NH3 in the total NH3 emissions reached up to 40 ± 21% (6.6 ± 3.4 Tg N yr−1), 49 ± 16% (2.8 ± 0.9 Tg N yr−1), and 44 ± 19% (2.8 ± 1.3 Tg N yr−1) in East Asia, North America, and Europe, respectively, though its fractions and amounts in these regions generally decreased over the past decades. Given its importance, c-NH3 emission should be considered in making emission inventories, dispersion modeling, mitigation strategies, budgeting deposition fluxes, and evaluating the ecological effects of atmospheric NH3 loading.Atmospheric ammonia (NH By integrating nitrogen isotope systematics of ammonia emissions and transformations in the atmosphere, this study quantified the combustion-related ammonia emission and uncovered its importance for mitigating strategies of ammonia pollution. The uptake of NH3 within clouds and rain and onto atmospheric aerosols causes the formation of ammonium (NH4+) within atmospheric particulates and precipitation 3. Over the last century, urbanization and industrial and agricultural intensification have greatly increased NH3 production and have led to a continuous increase in the NH3 emission amounts and deposition fluxes8. Globally, the NH3 emission has increased from 20.6 Tg N yr−1 in 1860 to 58.2 Tg N yr−1 in 1993 and may double to 118.0 Tg N yr−1 by 20504. Since the 1950s, East Asia, North America, and Europe have been three regions of high NH3 emissions10, with total emission amounts of 10.7 ± 0.4, 3.7 ± 0.2, and 3.5 ± 0.3 Tg N yr−1 during 2000–2015, respectively, according to emission inventory data  and NHx deposition   in the above three regions also remain high. In human-disturbed areas, excessive NH3 has promoted secondary aerosol production and air pollution4. Elevated NHx concentrations and deposition have caused negative impacts on ecosystem structure and function  and huge economic loss17.Ammonia  (denoted as v-NH3)19. The dissolved NH3 volatilizes from liquid-phase substrates containing NH4+ at favorable pH, temperature, and pressure conditions20. The other is NH3 emission from combustion-related sources  (denoted as c-NH3)24. NH3 would be released from industrial coal combustion, heavy-duty and light-duty diesel vehicles equipped with selective catalytic or non-catalytic reduction systems because of excessive urea/NH3 used for the catalytic degradation of nitrogen oxides (NOx)26. It is also produced via steam reforming from hydrocarbons27 and or catalytic reaction of nitric oxide with molecular hydrogen28 in light and medium-dust gasoline vehicles equipped with three-way catalytic converters, relating to the catalyst temperatures and air-to-fuel ratios29. The biomass N, typically as amides (R-(C = O)-NH-R’) and amines (R-NH2), can produce NH3 under poor mixing conditions during biomass burning30. So far, there have been direct observations on emission factors of various v-NH3 sources to budget the v-NH3 emission amount 23, biomass burning (8.2 Tg N yr−1)31, and other combustion sources (6.3 Tg N yr−1)10 has reached up to 15.9 Tg N yr−1, which accounts for 30% of the global NH3 emission (54.3 Tg N yr−1). Additionally, isotopic evidence demonstrated that c-NH3 had reached 29–62% in NH3 of the ambient atmosphere34 and 45–90% of the NH4+ deposition in cities of China and the USA36. All the above evidence suggests that the relative importance and amount of regional c-NH3 emissions are still open questions and should be re-evaluated.However, evidence from laboratory simulations, in-situ observations, satellite observations, and emission inventories points to an underestimation of c-NHδ15N, δ15N = (15N/14N)sample/(15N/14N)standard −1, where atmospheric N2 is used as the standard) of primary v-NH3 and c-NH3 emission sources , NH4+ in atmospheric particulates (p-NH4+) and precipitation (w-NH4+) in East Asia, North America, and Europe  to a-NH3, p-NH4+, or w-NH4+, respectively , we established a set of isotopic methods to calculate relative contributions between v-NH3 and c-NH3 in above study regions  average −18.9 ± 4.2‰, significantly lower than c-NH3   integrate their δ15N signatures and fractional contributions   during the unidirectional reaction of NH3 → p-NH4+ or equilibrium isotope fractionations (εeq) during reversible reactions of NH3 ↔ p-NH4+  are generally higher than δ15Na-NH3, which is generally negative  of δ15Na-NH3, δ15Np-NH4+, or δ15Nw-NH4+ to the corresponding δ15Ni-NH3 . In this work, we developed a new set of methods to constrain 15∆a-NH3, 15∆p-NH4+, and 15∆w-NH4+, and then reconstruct δ15Ni-NH3 (detailed in Methods).Practically, site-based red Fig. . However3 in Eq. , δ15N inive Fig. . The maiCa-NH3, Cp-NH4+, δ15Na-NH3, δ15Np-NH4+, and δ15Nw-NH4+ at six sites )   reductions on mean annual fp-NH4+  than in Europe (−7.7 ± 6.3‰) and East Asia (−8.0 ± 6.0‰)  Fig. . Becauseδ15Ni-NH3 decreased significantly between 1972 and 2018 in North America (p < 0.01) and decreased slightly from 2001 to 2018 in East Asia (p = 0.13) and 1974–2017 in Europe (p = 0.39)  in Eq. .Fc-NH3 averages 40 ± 21% in East Asia, 49 ± 16% in North America, and 44 ± 19% in Europe . One possible explanation is that the study regions are hotspots of globally high c-NH3 emissions24. Supportively, the total consumption of fossil fuels in these regions accounts for more than 60% of the global energy consumption  in emission inventories  and total NH3 emissions (detailed in Methods), which average 6.6 ± 3.4 Tg N yr−1 and 16.6 ± 3.5 Tg N yr−1 in East Asia, 2.8 ± 0.9 Tg N yr−1 and 5.8 ± 1.1 Tg N yr−1 in North America, and 2.8 ± 1.3 Tg N yr−1 and 7.0 ± 1.6 Tg N yr−1 in Europe, respectively  over the past decades in North America  and also Av-NH3 to Ac-NH3 (p < 0.05) generally from lower than 1.0 to higher than 1.0   and also Av-NH3 to Ac-NH3 over the past decades   and non-urban (65 sites) or between agricultural (32 sites) and non-agricultural sites (33 sites)  have comparable emission strengths with c-NH3 sources (mainly from fossil fuel combustion) in urban areas. Human excreta contributed 11.4% to the total NH3 emissions in Shanghai urban of eastern China49. The v-NH3 from urban green space contributed up to 60% to ambient NH3 in Qingdao in northern China50. The urban NH3 concentrations influenced by the v-NH3 from urban waste containers, sewage systems, humans, and open markets were 2.5 times higher than that of traffic-influenced urban areas in Spain51. Ambient NH3 concentrations in the Beijing urban peaked when fertilizer was intensively applied on the North China Plain52. Secondly, the c-NH3 emission  is undoubtedly as significant as the v-NH3 (mainly fertilizer application and live stocks) in non-urban areas. For example, the c-NH3 from biomass burning control seasonal variations of surface NH3 concentrations in major disturbed regions of the Northern Hemisphere, which is even stronger in the Southern Hemisphere with frequent wildfires  for sites in Beijing urban53, while the c-NH3 accounted for 33% (25–69%) in the total NH4+ deposition of the cropland 370 km far from the Beijing urban11. Collectively, site-based δ15Na-NH3 and δ15NNH4+ represent mixing signatures and can be feasibly used to evaluate regional c-NH3 and v-NH3 emissions.In this study, the non-urban ultural 3 sites anultural 3 sites an3 emissions have been considerably underestimated in regions with significant anthropogenic sources. In the process, the new estimates of c-NH3 emissions have brought measured NHx deposition data into closer agreement with emissions. Our conclusion about the underestimation of c-NH3 emission has important implications for control measures since the current efforts have been focused almost exclusively on agricultural sources. Although the marginal abatement cost of NH3 emissions is only 10% of the global NOx emission, with 162 billion US dollars net benefit, the reduction can effectively improve air pollution and its negative impact55. For example, measures to reduce c-NH3 emissions should be considered in the methods for mitigating v-NH3 emissions by improved farm management practices with N use reductions, deep machine placement of fertilizer, enhanced-efficiency fertilizer use, and enhanced manure management in the agriculture sector57. The revelation of high c-NH3 emissions also implies that the potential, costs, and impacts of NH3 emissions reduction need to be re-assessed. Steps to reduce c-NH3 emissions may alleviate the pressure of reducing agricultural NH3 emissions and achieve ‘win-win’ outcomes for agricultural production and food supply, human and environmental health56.This study demonstrates that c-NHδ15N for all NH3 emission sources in each region. It is even more challenging to conduct simultaneous observations on Ca-NH3, Cp-NH4+, δ15Na-NH3, δ15Np-NH4+, and δ15Nw-NH4+ among landscapes. As a result, a limited number of data is available in terms of observation sites and their spatial distribution in the three regions, and the corresponding data comparison among the three regions is preliminary in the current stage. Moreover, δ15N observations focus largely on the middle- and low-latitudes and low altitudes of the Northern Hemisphere but rare on the high mountains and high latitudes of the Northern Hemisphere and the Southern Hemisphere. More observations on the concentration and δ15N parameters of NH3 and NH4+ in both source emissions and deposition are necessary to improve the isotope source apportionment, especially in remote areas. Finally, despite the updated regional c-NH3 and total NH3 emissions, future in-depth cooperation among different observational and modeling methods should be encouraged.The uncertainty of this study lies in the technical difficulty in measuring δ15N data of major NH3 emission sources , coal combustion (cc-NH3), and biomass burning (bb-NH3)  in total c-NH3 emissions (15.9 Tg N yr−1) are 8%, 40%, and 52%, respectively  and waste materials (wm-NH3) account for 56% and 44% of the total v-NH3 emission, respectively10. Similarly, we calculated the δ15Nv-NH3 by Eq. (For v-NHδ15Nc-NH3 and δ15Nv-NH3 is propagated errors estimated using the Monte Carlo method (MCM). Briefly, we ran 10000 trials for the MCM in the software of Microsoft Excel-Add-In and calibrated the SD to match the corresponding true values.The standard deviation (SD) of 3’, ‘ammonium/NH4+’, ‘rainfall’, ‘rain’, ‘rain water’, ‘precipitation’, ‘aerosol’, and ‘particulate’. The databases include the Web of Science (http://isiknowledge.com), Google Scholar (http://scholar.google.com.hk), and Baidu Scholar (http://xueshu.baidu.com). By December 2020, there are 18 publications on δ15Na-NH3 , 857 measurements of δ15Np-NH4+ at 33 sites , and 1540 measurements of δ15Nw-NH4+ at 80 sites   N2 as an end product using the Elemental Analyzer combustion method or 2) N2O as an end product using the bromate oxidation and azide or hydroxylamine reduction, or using the persulfate oxidation and denitrifier method58, but such difference would not change the spatiotemporal patterns of δ15Ni-NH3  in the same databases as described above. There are 107 publications published by July 2021  values  in each region  and fp-NH4+ = Cp-NH4+ / (Ca-NH3 + Cp-NH4+).Based on simultaneous observations of seasonal ion (Eq. ).7\documδ15Ni-NH3 and δ15Na-NH3 , betweeH4+, Eq. ), betweeH4+, Eq. ) for thefp-NH4+ values in Supplementary Fig. 15∆a-NH3, 15∆p-NH4+, and 15∆w-NH4+ in each region  and in North America 61 , δ15Ni-NH3(p-NH4+), or δ15Ni-NH3(w-NH4+); Eq. ; Eq.  − 13, reδ15Ni-NH3(a-NH3) and δ15Ni-NH3(p-NH4+), δ15Ni-NH3(a-NH3) and δ15Ni-NH3(w-NH4+), δ15Ni-NH3(p-NH4+) and δ15Ni-NH3(w-NH4+) are negligible, averaging 0.4 ± 2.3‰, −0.2 ± 1.3‰, and −0.4 ± 2.5‰, respectively  into the source contributions. In each run, the percentage data (n = 10000) output from the SIAR model was used to calculate the mean ± SD values of corresponding Fv-NH3 and Fc-NH3 at each site, then site-based mean ± SD values of Fv-NH3 and Fc-NH3  were  were Fv-data n = 000 outpuite Fig. .3 emission (Av-NH3), we calculated corresponding amounts of total NH3 emissions  and theNH3, Eq. ).14\docuAv-NH3 and Fv-NH3 were used to calculate regional mean ± SD of Atotal and Ac-NH3  and Origin 2016 statistical package  for Windows were used for data analyses in this study. The Tukey honest significant difference (Tukey HSD) and the least significant difference (LSD) tests of the one-way analysis of variance (ANOVA) were used to identify significant differences in H4+ Fig. , East AsH4+ Fig. , major sSupplementary Information


Following publication of the original article , the autThe incorrect Fig. 5 is:Fig. 5 Expression of Lr-pgrn-s1 after lamprey skin and spinal cord injury. A Expression of Lr-pgrn-s1 in lamprey skin lesions 1d and 2d. B Expression of Lr-pgrn-s1 in lamprey spinal cord injury at 6 h, 1d and 3d by qRT-PCR. C Detection of tissue localization of Lr-PGRN-S1 in skin injury 1d by immunohistochemistry. Take pictures at 40× magnification. D Immunohistochemistry of Lr-pgrn-s1 was knocked down 6 h after lamprey spinal cord injury. Results under 20× and 40× microscopy in Sham, NC  and Lr-PGRN-S1 groups. E The qRT-PCR analysis of genes in MAPK, Notch, Wnt and TGF-β signaling pathways. The statistical differences between experimental groups were detected by the Student’s t test. All data were presented as the means ± SDs based on three independent samples with three replicates per sample. ns non-significant, *P < 0.05, **P < 0.001, ***P < 0.0001.The correct Fig.


Consistently, Aβ induced the senescence-associated mitochondrial dysfunctions such as repression of ATP production, oxygen consumption rate (OCR), and mitochondrial membrane potential and enhancement of ROS production. Furthermore, Aβ was found to significantly suppress mRNA expression and protein level of Sirtuin-1 (SIRT1), a key regulator of senescence, and inhibit mRNA expression and translocation of NRF2, a critical transcription factor in inflammatory responses, leading to impairment of phagocytosis. Rescue of SIRT1, as expected, could counteract the pathological effects of Aβ. In summary, our findings revealed that Aβ accelerates human microglial senescence mainly through its suppression of the SIRT1/NRF2 pathway and suggested that genetic and pharmaceutical rescue of SIRT1 may provide a potential alternative treatment.Microglia play important roles in maintenance of brain homeostasis, while due to some pathological stimuli in aging-related neurodegenerative diseases including Alzheimer's disease, they are malfunctioning. Here, we demonstrated that amyloid- It inescence –5. Micronescence , 7. Comprophages . Cell serophages . Cell serophages , 10, 11.rophages  telomererophages , 14, androphages . During rophages , 15, charophages –18. Aβ, nescence . Howeverβ and tau proteins in AD patients [β deposition and toxicity and improved AD pathology based on several earlier in vitro and in vivo studies [Sirtuin-1 (SIRT1) is a NAD-dependent deacetylase that participates in the regulation of cell senescence, metabolism, inflammation, and mitochondrial function , 20. Undpatients –28. SIRT studies –31. Furt studies –34. Therβ to induce cellular senescence in human microglial cells. After Aβ stimulation, we found the senescence-related mitochondrial functions were exacerbated significantly. We also detected that Aβ induction affected phagocytosis and ROS production of microglia and downregulated the SIRT1/NRF2 pathway. Overexpression of SIRT1 or using SIRT1 activator such as aspirin can counteract Aβ-induced cellular senescence. In summary, our results suggest that the SIRT1/NRF2 pathway is a therapeutic target for AD-related cellular senescence.Here, we used A2/95% air at 37°C.Human microglial cells HMC3 were obtained from ATCC (#CRL0314). Cells were cultured in MEM with 10% FBS and 1% penicillin-streptomycin in a humidified incubator with 5% COβ peptides (Chinese peptide) were prepared according to the protocols described previously [β peptides were dissolved in HFIP  to a final concentration of 1 mM, the HFIP-treated Aβ peptides were resolved in DMSO and then diluted to a concentration of 100 μM with DMEM/F12 phenol-red free medium and incubated at 4°C for 24 h. After centrifugation at 12,000 g for 10 min, the supernatant with soluble Aβ was added to cultures. Aβ42-1 peptides  were used as a negative control. Aβ42-1 peptides were prepared using the same protocol. The concentration of Aβ42-1 was 10 μM. In this paper, Aβ presented Aβ1-42.The Aeviously , 35. In β-galactosidase  activity was performed using the SA-β-gal staining kit , according to the manufacturer's instructions. In brief, cells were plated in the density of 40,000 cells per well into a 24-well plate. After 24 h seeding, cells were treated with Aβ for 72 h, then the cells were fixed with 4% formaldehyde in PBS for 15 min, and the fixed cells were stained with SA-β-Gal staining solution at 37°C for 15 h. The percentage of positively stained cells were calculated based on three replicates.Senescence-associated β for 72 h; then, the cells were extracted by TRI Reagent  to obtain total RNA according to the manufacturer's instructions. cDNA was synthesized using cDNA Synthesis kit  and qPCR analysis was done with power SYBR Green PCR master mix . Primers used were as follows: PAI-1 (forward: 5′-ACCGCAACGTGGTTTTCTCA-3′ and reverse: 5′-TTGAATCCCATAGCTGCTTGAAT-3′), p21 (forward: 5′-CGAAGTCAGTTCCTTGTGGAG-3′ and reverse: 5′-AGTCGTGGTCTTTG GGAGTC-3′), CCNA1 (forward: 5′-GAAATTGTGCCTTGCCTGAGTG-3′ and reverse: 5′-TCTGATATGGAGGTGAAGTTCTGGA-3′), CCND1 (forward: 5′-ATGTTCGTGGCCTCTA AGATGA-3′ and reverse: 5′-CAGGTTCCACTTGAGCTTGTTC-3′), SIRT1 (forward: 5′-TAG CCTTGTCAGATAAGGAAGGA-3′ and reverse: 5′-ACAGCTTCACAGTCAACTTTGT-3′), SIRT5 (forward: 5′-GCCATAGCCGAGTGTGAGAC-3′ and reverse: 5′-CAACTCCACAAGA GGTACATCG -3′), NRF2 (forward: 5′-TCAGCGACGGAAAGAG TATGA-3′ and reverse: 5′- CCACTGGTTTCTGACTGGATGT-3′), TNFα (forward: 5′-CCTCTCTCTAATCAGCCCTCT G-3′ and reverse: 5′-GAGGACCTGGGAGTAGATGAG-3′), IL1β (forward: 5′-ATGATGGCT TATTACAGTGGCAA-3′ and reverse: 5′-GTCGGAGATTCGTAGCTGGA-3′), IL6 (forward: 5′-ACTCACCTCTTCAGAACGAATTG-3′ and reverse: 5′-CCATCTTTGGAAGGTTCAGGT TG-3′), and HPRT (forward: 5′-CCTGGCGTCGTGATTAGTGAT-3′ and reverse: 5′-AGACGTTC AGTCCTGTCCATAA-3′). The reaction parameters were as follows: 95°C for 10 min; 95°C for 30 s, 40 cycle; 60°C for 30 s; and 72°C for 30 s. An additional cycle was performed for evaluation of primer's dissociation curve: 95°C for 1 min, 60°C for 30 s, and 95°C for 30 s. 2ΔΔCT– was used to analyze expression of genes. The gene levels were to HPRT endogenous control.HMC3 cells were stimulated with Aμg samples were loaded and separated on 10% or 12% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% milk in TBST for 1 h, we used TBST to dilute primary and secondary antibodies. Membranes were incubated with primary antibody overnight at 4°C, washed in TBST, and incubated with HRP-conjugated secondary antibody for 60 min. The proteins of interest were performed using an ECL western blot detection kit (Bio-Rad). ImageJ software was used to evaluate the densitometry. Actin or proliferating cell nuclear antigen (PCNA) was used as loading control. Antibodies used were as follows: p53 , PAI-1 , p21 , SIRT1 , SIRT5 , NRF2 , PCNA , and Actin .Western blotting was performed as described previously . BrieflyμM Aβ for 72 h. JC-1 kit  was used to detect the MMP level of cells according to the manufacturer's instructions. In brief, cells were loaded with JC-1 staining solution for 30 min at 37°C and then washed with staining buffer for twice. The cells were captured using Zeiss confocal laser scanning microscope (Zeiss 880 Airyscan). The fluorescence intensity was measured at 490/530 nm (green) for monomers and 525/590 nm for aggregates (red) using the BioTek SynergyNEO (BioTek), and the ratio of red/green fluorescence intensity was presented as MMP.HMC3 cells were plated at 9,000 cells per well in black-walled 96-wells plates and cultured overnight, then stimulated with 10 μM Aβ for 72 h and followed by staining with 10 μM DCFH-DA in PBS for 30 min at 37°C. PBS was used to wash the cells for three times. Then, the cells were detected using Zeiss confocal laser scanning microscope (Zeiss 880 Airyscan). Lastly, fluorescence was measured at 485 nm excitation/538 nm emission using a BioTek SynergyNEO , and the fluorescence signal was normalized to the Hoechst.DCFH-DA  was used to assess intracellular ROS levels. Briefly, HMC3 cells were seeded into black-walled 96-well plate at 9,000 cells/well density. Cells were stimulated with 10 μM Aβ for 72 h. Then, the cells were washed twice and maintained in XF assay medium. OCR was measured under basal condition and also after the injection of oligomycin (1 μM), FCCP (1 μM), rotenone (1 μM), and antimycin A (1 μM). After baseline measurements, oligomycin (1 μM), FCCP (1 μM), rotenone (1 μM), and antimycin A (1 μM) was injected sequentially. Data were analyzed using Seahorse XF24 Wave software, and the results were normalized to cell number.Oxygen consumption rate (OCR) was measured using a Seahorse XF24 analyzer (Seahorse Bioscience) according to the manufacturer's guidance. Briefly, 9,000 cells were plated on the XF24 cell culture microplate and cultured with 10 β for 72 h. After that, the cells were washed with PBS and the medium were changed to FBS-free DMEM alone at 37°C for 6 h. The fluorescent latex beads  were preopsonised in 50% FBS and PBS, and the beads were loaded to the cells at concentrations of 20 beads per cell and incubated at 37°C for 3 h. After Aβ uptake, the cells were processed for immunofluorescence using Zeiss confocal laser scanning microscope (Zeiss 880 Airyscan). Hoechst was used to stain the nuclei. Lastly, the fluorescence intensity was also detected using the BioTek SynergyNEO  at 485 nm excitation/538 nm emission.HMC3 cells were plated into 96-well plates at 9,000 cells per well and cultured overnight and then treated with Aβ for indicated time. Nuclear and Cytoplasmic Protein Extraction Kit  was used in this experiment according to the manufacturer's instructions. In brief, 80 μl Buffer A was added to cells for 10 min, next 5 μl Buffer B was added, then the cells were centrifugated at 12,000 g for 5 min, and the supernatant was the cytoplasmic protein. The precipitation was resolved with 25 μl Buffer C for 30 min and centrifuged at 16,000 g for 10 min at 4°C to obtain the nuclear fraction.HMC3 cells were cultured in 6 cm plates, grew for 24 h, and then were treated with Aμg plasmid per well into 6 cm dish. SIRT1 plasmid or FUGW plasmid was transfected using ViaFect reagent  according to manufacturer's instructions. After transfection for 24 h, cells were stimulated by Aβ for 72 h. The knockdown of SIRT1 was performed by the transfection with specific siRNA  using ViaFect reagent. The cloning primers used were as follows: SIRT1 (forward: 5′-TGGGCTGCAGGTCGACTCTAGAATGGCAGATGAAGCAGCTCTC-3′ and reverse: 5′-TTG ATATCGAATTCTAGACTATGATTTGTTTGATGGATAGTTCATGTCT-3′). The siRNA primers were as follows: siSIRT1-1 (forward: 5′-CACCUGAGUUGGA UGAUAUTT-3′ and reverse: 5′-AUAUCAUCCAACUCAGGUGTT-3′) and siSIRT1-2 (forward: 5′-GUCUGUUUCAUG UGGAAUATT-3′and reverse: 5′-UAUUCCACAUGAAACAGACTT-3′).SIRT1 cDNA was made from pCMV-SIRT1-t1-Flag  via PCR amplification. SIRT1 cDNA was cloned into the FUGW vector using Seamless Cloning Kit  and confirmed by DNA sequencing. HMC3 cells were plated into 24-well or 6 cm dish at appropriate intensity and cultured overnight. We transfected 150 ng plasmid per well into 24-well or 1.5 n ≥ 3 independent experiments; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001, analyzed by one-way ANOVA followed by Bonferroni's test.GraphPad Prism 7.0 was used to draw graphs and perform data analysis. The data are presented as mean ± SEM, μM Aβ for different times and evaluated the gene expression of senescence. Compared with the Aβ42-1 treatment control group, cells treated with Aβ displayed significantly higher expression of senescence-associated genes  assay to confirm the senescence phenotype of HMC3 cells. As shown in Figures β-gal-positive cells is observed in cell culture treated with 10 μM Aβ. We also evaluated SASP markers such as TNF-α, IL-1β, and IL-6 by qPCR  in HMC3 after treatment with oligomycin (ATP synthase inhibitor), FCCP (H+ ionophore), or rotenone and antimycin A (electron-transport chain inhibitor). These results revealed that Aβ treatment significantly diminished the maximal respiratory capacity of mitochondria and ATP production in microglia compared with vehicle. Aβ treatment also reduced basal respiration and spare capacity OCR, but no statistically significant change was observed . In this study, we used JC-1 probe to evaluate MMP in HMC3 cells. Red fluorescence and green fluorescence characterized high and low mitochondrial membrane permeability, respectively, and the ratio could signify the change of MMP. Cells treated with Aβ increased green fluorescence intensity  [β treatment affected nuclear translocation of NRF2. The results indicated that Aβ reduced the mRNA expression of NRF2 (β inhibited NRF2 nuclear translocation (N-Nrf2) in a time-dependent manner  , 33. NRF of NRF2 . Moreove Figures , NRF2 le Figures . In conc Figures  and B. Tβ treatment, we next tested whether overexpression of SIRT1 could rescue Aβ defects including senescence, mitochondrial disability, and microglial dysfunctions. Here, we transfected HMC3 cells with SIRT1 plasmid or FUGW plasmid. As shown in Figures β markedly downregulated SIRT1 expression and upregulated senescence genes including PAI-1 and p21, but SIRT1 overexpression almost counteracted the influence of Aβ-induced senescence in HMC3 cells (Figures β-gal signal in the cells relative to the vehicle controls (Figures β impaired mitochondrial membrane potential (β-induced ROS production was significantly rescued by SIRT1 overexpression (β phagocytosis (β-mediated cell senescence, mitochondrial dysfunctions, and microglial state.Since the protein level of SIRT1 has been reduced with A Figures . Further Figures . We next Figures . Interesotential . Similarpression . Moreoveocytosis . Togetheβ stimulation (Figures μM aspirin markedly increased SIRT1 level. To assess the effect of aspirin on senescent microglia cells, SA-β-gal activity was detected and there was a significant reduction in the number of SA-β-gal-positive cells in aspirin-treated cells (Figures μM aspirin significantly inhibited ROS production (In this paper, we wanted to find some drugs which could relieve microglial cellular senescence. Surprisingly, treatment of aspirin in HMC3 was found to rescue cellular senescence after A Figures . Aspirin Figures . Meanwhi Figures . We also Figures . Lastly,oduction  and incroduction . In summβ [β, IL-6, IL-12, and TNF-α and generate nitrogen species and reactive oxygen. However, protective microglia produce anti-inflammatory cytokines such as IL-10 and TGF-β and growth factors. The dynamic changes of toxic/protective phenotypes are critically associated with AD. Endogenous stimuli including Aβ and tau may persistently activate proinflammatory responses and finally aggravate progression of neurodegenerative disease [β could induce microglial senescence. Srinivasan et al. revealed Alzheimer's patient microglia exhibited enhanced aging [β aggravate senescence-associated mitochondrial dysfunctions and impair microglial functions. Interestingly, we revealed that the SIRT1/NRF2 pathway is partly reduced by Aβ stimulation. Notably, overexpression of SIRT1 or use SIRT1 activator such as aspirin may rescue Aβ defects.AD is a neurodegenerative disease mainly characterized by the progressive aggregation of Aβ , 51. Theβ , 53. Recβ , 55. Tox disease . The exp disease . In the ed aging . We alsoβ stimulation in our work. Previous studies reported that a metabolic switch from mitochondrial OXPHOS to anaerobic glycolysis in Aβ-treated primary mouse microglia is associated with microglia phagocytosis [β treatment significantly reduced OCR levels in human microglia cells and impaired the capacity of microglia phagocytosis. Thus, therapies targeting basic mitochondrial processes hold great promise.Increasing evidences point out that mitochondrial dysfunction is one of the hallmarks of aging , 58, attocytosis , 42. Herβ and tau proteins than in normal aging individuals [β stimulation in human microglia cells. At the same time, SIRT1-associated NRF2 nuclear translocation was also reduced after Aβ treatment. Aspirin is one of the most widely used treatments for cardiovascular disease. Aspirin has obviously anti-inflammatory function. We found that aspirin could increase SIRT1 production and alleviate human microglial senescence. Aspirin has been reported to reduce amyloid plaque in a mouse model of AD [Sirtuins are class III histone deacylases possessing outstanding properties in preventing diseases and reversing some aspects of aging –64. SIRTividuals , 67. Herel of AD . Howeverel of AD . There a


A) receptors, the primary inhibitory ion channels in the mammalian brain. Moreover, among ten EMC subunits, EMC3 and EMC6 have the most prominent effect, and overexpression of EMC3 or EMC6 is sufficient to restore the function of epilepsy-associated GABAA receptor variants. In addition, EMC3 and EMC6 demonstrate endogenous interactions with major neuroreceptors, which depends on their transmembrane domains, suggesting a general role of the EMC in the biogenesis of neuroreceptors.The endoplasmic reticulum membrane complex (EMC) plays a critical role in the biogenesis of tail-anchored proteins and a subset of multi-pass membrane proteins in the endoplasmic reticulum (ER). However, because of nearly exclusive expression of neurotransmitter-gated ion channels in the central nervous system (CNS), the role of the EMC in their biogenesis is not well understood. In this study, we demonstrated that the EMC positively regulates the surface trafficking and thus function of endogenous γ-aminobutyric acid type A (GABA  •A receptorsEMC3 and EMC6 positively regulate the function of endogenous GABA•The EMC interacts with major endogenous neuroreceptors•A receptorsThe EMC transmembrane domains contribute to the interaction with GABA•A receptor variantsOverexpressing the EMC restores the function of GABA Biological sciences; Molecular biology; Neuroscience; Molecular neuroscience; Cell biology Acquiring the correct transmembrane topology is essential for the function of membrane proteins, which consist of about 30% of the eukaryotic proteome. The endoplasmic reticulum membrane complex (EMC) plays a critical role in the insertion of membrane proteins into the lipid bilayer of the endoplasmic reticulum (ER) . The EMCC. elegans, and rhodopsins and transient receptor potential (TRP) channels in Drosophila   . In addiubunits) , which ieriments . To furteriments C–1L.A receptor protein levels. The most substantial reduction of the total α1 protein levels was observed with the depletion of EMC3 . EMC3 is the only EMC subunit that exhibits homology to Oxa1 family proteins, which are known membrane protein insertases  that are critical for its assembly and activity, EMC1, EMC3 and EMC6 positively regulated α1 protein levels D and 1G, p<0.01, E and EMCsertases , and EMC elegans .A receptor biogenesis in the ER membrane .Among the five peripheral subunits of the EMC  that are not essential for the complex’s stability or assembly, EMC4 and EMC10 positively regulated α1 protein levels F and 1L,A receptors  to 375 and 573 pA, corresponding to 55 and 32% reduction respectively  of functional GABAA receptors are abundantly expressed in the plasma membrane in the cortex  digestion assay. The human α1 subunit of GABAA receptors has two N-linked glycosylation sites at N38 and N138. Endo H resistant bands represent glycoproteins, which have correctly folded in the ER, trafficked to the Golgi and fully modified with the N-linked complex glycans, thus becoming resistant to Endo H. On the other hand, acting on glycoproteins remaining in ER, Endo H may remove the high mannose structure after the asparaginyl-N-acetyl-D-glucosamine on the α1 subunits of GABAA receptors. We carried out EMC3 or EMC6 siRNA transfection in HEK293T cells expressing α1β2γ2 GABAA receptors; 48 h after transfection, proteins were extracted, and subjected to Endo H digestion and western blot analysis  in transmembrane helix 1 (TM1) and Arg180 (R180) in transmembrane helix 3 (TM3)  D, and th 3 (TM3) . For thaectively E. TherefHomo sapiens, M musculus, and Saccharomyces cerevisiae , α1(G251D), and α1(P260L) (A receptors was observed in HEK293T cells stably expressing α1(D219N)β2γ2  . We carr19N)β2γ2 A, α1(G2519N)β2γ2 B and α1(19N)β2γ2 C, to 26019N)β2γ2 A, α1(G2519N)β2γ2 B and α1(19N)β2γ2 C. Moreoveceptors D. These A receptors are also influenced by several kinetic parameters, such as activation, desensitization, and deactivation time scales. In addition, GABAA receptors on the cell surface could have binary forms (with α1 and β2 subunits) in addition to triheteromeric forms ; these two forms have different single channel conductance. Since the cell surface α1 subunits could have different opening probability and single channel conductance, their quantity is not strictly proportional to the observed macroscopic peak current amplitude. Such a discrepancy was also reported in GABAA receptors containing ρ1 variants , numerous subtypes of GABAA receptors containing the combination of subunits can form either in the synaptic sites to trigger fast, transient phasic inhibition or in the extrasynaptic sites to generate persistent tonic inhibition  of GABAA receptors , γ2(R82Q), or γ2(R177G) by increasing the protein levels of pro-folding chaperones, such as BiP, and the interactions between these chaperones and GABAA variants; importantly, ATF6 activators are more selective for folding defective variants compared to wild type receptors .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Ting-Wei Mu  were obtained from ATCC. GT1-7 cells  were obtained from Millipore. Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) with 10% heat-inactivated fetal bovine serum (FBS) and 1% Penicillin-Streptomycin at 37°C in 5% COA receptor α1 (Uniprot no. P14867-1), β2 , γ2  subunits, and pCMV6 Entry Vector plasmid (pCMV6-EV) were obtained from OriGene. The human FLAG-tagged EMC3 plasmid was purchased from GenScript . The human FLAG-tagged EMC5 plasmid  and FLAG-tagged EMC6 plasmid  were obtained from OriGene. The mutations GABRA1-D219N, GABRA1-G251D, GABRA1-P260L, EMC3-R31A, EMC3-R180A, EMC6-D27A, and EMC6-N22A were constructed using QuikChange II site-directed mutagenesis Kit . All cDNA sequences were confirmed by DNA sequencing.The pCMV6 plasmids containing human GABAThe following small interfering RNA (siRNA) duplexes were obtained from Dharmacon: EMC1.1 (J-059370-09-0005), EMC1.2 (J-059370-10-0005), EMC2.1 (J-049743-09-0005), EMC2.2 (J-049743-10-0005), EMC3.1 (J-056059-09-0005), EMC3.2 (J-056059-11-0005), EMC4.1 (J-046351-09-0005), EMC4.2 (J-046351-10-0005), EMC5.1 (J-041149-09-0005) EMC5.2 (J-041149-11-0005), EMC6.1 (J-047425-10-0005), EMC6.2 (J-047425-12-0005), EMC7.1 (J-051219-09-0005), EMC7.2 (J-051219-11-0005), EMC8.1 (J-046488-09-0005), EMC8.2 (J-046488-10-0005), EMC9.1 (J-046998-09-0005), EMC9.2 (J-046998-10-0005), EMC10.1 (J-041588-11-0005), EMC10.2 (J-041588-12-0005), human EMC3.1 (J-010715-17-0005), human EMC3.2 (J-010715-18-0005), human EMC6.1 (J-014711-19-0005), human EMC6.2 (J-014711-20-0005), and Non-Targeting (NT) siRNA (D-001810-01-20), which was used as a negative control. The designation of EMCn.1 and EMCn.2 (n = 1 to 10) indicates two distinct siRNA sequences against each EMC subunit.The rabbit polyclonal EMC1 antibody , rabbit polyclonal EMC3 antibody , rabbit polyclonal EMC4 antibody , rabbit polyclonal EMC9 antibody , and rabbit polyclonal EMC10 antibody  were from Abcepta. The rabbit polyclonal EMC2 antibody  and mouse monoclonal EMC8 antibody  were obtained from Proteintech. The rabbit polyclonal EMC5 antibody , rabbit polyclonal EMC6 antibody , and rabbit polyclonal EMC7 antibody  were from Pierce.A α1 subunit antibody (clone BD24)  and mouse monoclonal anti-GABAA β2/β3 antibody  were obtained from Millipore. The rabbit monoclonal anti-GABAA α1 subunit antibody  and rabbit polyclonal anti-GABAA γ2 antibody  were obtained from Synaptic systems. The rabbit polyclonal anti-GABAA α1 antibody came from R&D systems . The mouse monoclonal anti-β-actin antibody  and mouse monoclonal FLAG antibody  came from Sigma Aldrich. The fluorescent anti-β-actin antibody Rhodamine came from Biorad . The rabbit polyclonal anti-calnexin  and rat polyclonal anti-Grp94  antibodies were purchased from Enzo Life Sciences. The rabbit polyclonal anti-VCP  antibody was obtained from Abgent. The rabbit polyclonal anti-SEC61α antibody was obtained from Proteintech . The rabbit polyclonal anti-Grp78 antibody , rabbit monoclonal anti-NR1 antibody , rabbit monoclonal anti-NR2A antibody , rabbit monoclonal anti-NR2B antibody , and rabbit polyclonal anti-nAChR α7 antibody , and sodium potassium ATPase antibody  were purchased from Abcam.The mouse monoclonal anti-GABAThe secondary antibodies used from Invitrogen included: HRP-conjugated goat-anti-rabbit , goat-anti-mouse , and goat-anti-rat , and Alexa Fluor 594-conjugated goat-anti-rabbit  and goat-anti-mouse .2. Cells were grown in 6-well plates or 10-cm dishes and allowed to reach ∼70% confluency before transient transfection using TransIT-2020 , or siRNA treatment (50 nM) using the HiPerfect Transfection Reagent  according to the manufacturer’s instruction. A second siRNA transfection was performed 24 h after the first siRNA treatment to increase knockdown efficiency. Forty-eight hours after transfection, cells were harvested for further analysis.HEK293T cells  were obtained from ATCC, and GT1-7 cells  were obtained from Millipore. Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM)  with 10% heat-inactivated fetal bovine serum  and 1% Penicillin-Streptomycin  at 37°C in 5% COStable cell lines for α1β2γ2, α1(D219N)β2γ2, α1(G251D)β2γ2, and α1(P260L)β2γ2 were generated using the G-418 selection method. Briefly, cells were transfected with α1:β2:γ2 (1:1:1), α1(D219N):β2:γ2 (1:1:1), α1(G251D):β2:γ2 (1:1:1) or α1(P260L):β2:γ2 (1:1:1) plasmids, selected in DMEM supplemented with 0.8 mg/mL G418 (Enzo Life Sciences) for 10 days, and then maintained in DMEM supplemented with 0.4 mg/mL G418. G-418 resistant cells were used for experiments.g, 4°C for 10 min to obtain the supernatant as total proteins. The protein concentration was measured according to Thermo Fisher MicroBCA kit protocol. Cell lysates were loaded with Laemmli sample buffer  with β-mercaptoethanol (1:10 v/v) and separated through SDS-PAGE gel.To harvest total proteins, cells were washed with Dulbecco’s phosphate-buffered saline (DPBS) . Trypsin (0.05%)  was added to lift the cells, and DMEM was added to harvest cells from the dish and pipette into a centrifuge tube. Cells were spun down for 3 min at 1000 rpm and then DMEM was removed while avoiding the pellet. Cell pellets were washed with DPBS and centrifuged again at 1000 rpm for 3 min. The DPBS was removed, and pellets were stored on ice during transport to a −80°C freezer. Cells were lysed with lysis buffer  ) supplemented with complete protease inhibitor cocktail (Roche). Cells were vortexed for 30 s followed by ultrasonication for 30 s for three times. Then they were centrifuged at 15,000 × Before proceeding to western blot, gels were made ranging from 8% to 20% resolving gel depending on the size of the protein with 4% stacking gel on top. To run SDS-PAGE gel electrophoresis, gels were place in a holding voltage cassette and submerged in the running buffer, which contained the following: 25 mM Tris , 192 mM Glycine , and 0.1% (w/v) of sodium dodecyl sulfate . Protein ladder was added . Gels were run at 10 min at 100 volts until the samples passed the stacking gel and were uniformly aligned. For the remaining time of 45 min to 1 h, gels were run at 150 V. After running samples to sufficient molecular weight, gels were transferred at 100 V for 1 h to a nitrocellulose membrane. The transfer buffer contained the following: 25 mM Tris , 192 mM Glycine , 20% (v/v) of methanol . After the transfer, membranes were washed briefly in TBS-T, which contained the following: 20 mM Tris , 150 mM NaCl , pH 7.6, and 0.1% (v/v) Tween20 . They were further incubated in 5% non-fat milk powder  in TBS-T for 30 min to 2 h. Following the blocking step, the membranes were incubated in 1% milk with the primary antibody added starting at 1:1000 dilution and adjusted accordingly on subsequent runs. The following day, the membrane was washed 3 times with TBS-T for 10 min each and incubated with their secondary antibody  for 1 h. This was followed by 3 more washed with TBS-T. Afterward gels were exposed with Pico PLUS  or Femto  SuperSignal West chemiluminescent substrates from Thermo Scientific for three minutes. After using different exposure times to get optimal images, results were analyzed to quantify band intensity using ImageJ software from the NIH.Cell lysates (500 μg) were pre-cleared with 30 μl of protein A/G plus-agarose beads  and 1 μg of normal mouse IgG antibody  for 1 h at 4°C to remove nonspecific binding proteins. The pre-cleared cell lysates were incubated with 2.0 μg of mouse anti-α1 antibody for 1 h at 4°C, and then with 30 μl of protein A/G plus agarose beads overnight at 4°C. For FLAG-tagged proteins, the pre-cleared cell lysates were incubated with 30 μl of anti-FLAG M2 magnetic beads  overnight at 4°C. IgG serves as negative control. The beads were collected by centrifugation at 8000 × g for 30 s or using a magnet separator (Promega), and washed three times with lysis buffer. The complex was eluted by incubation with 30 μl of Laemmli sample loading buffer in the presence of β-mercaptoethanol. The immuno-purified eluents were separated through SDS-PAGE gel, and western blot analysis was performed.http://n2t.net/addgene:12260; RRID:Addgene_12260) and pMD2.G  were a gift from Didier Trono. Media was changed after 8 h; after 52 additional h, media was harvested and passed through 0.45 μm filter  to collect the lentivirus. Furthermore, the lentivirus were concentrated using Lenti-X concentrator  and quantified with the qPCR lentivirus titration kit  according to the manufacturer’s instruction, and saved to −80°C for neuron cells transduction.Lentivirus were generated from transiently transfected HEK293T cells and collected after 60 h from the media. Briefly, HEK293T cells were grown in 10-cm dishes and allowed to reach ∼70% confluency before transient transfection using TransIT-2020 (Mirus), according to the manufacturer’s instruction. The following plasmid (6 μg) was added to 10-cm dishes: EMC3-set of four siRNA lentivectors , or EMC6-set of four siRNA lentivectors , or scrambled siRNA lentivector  as the control. Additionally, to form the lentivirus, the following packaging and envelop plasmids were added to all of the 10-cm dishes as well: psPAX2 (6 μg) and pMD2.G (0.75 μg). psPAX2  and NbActiv1 media . Prior to plating cells, 10-cm dishes or coverslips  in a 24-well plate were coated with 4 mL per plate or 500 μL per well of 50 μg/ml poly-D-lysine  at 4°C overnight. The next day PDL was removed and the plates were washed with sterile distilled water two times. Then they were coated with the same volumes of 5 μg/mL Laminin  overnight at 4°C. The plates were then put into a 37°C cell incubator while preparing the neurons for 1 h and Laminin was removed immediately before plating neurons.Neurons were extracted according to instructions from BrainBits. Briefly, connective tissues of cortex were digested with 2 mL of 2 mg/mL of papain  at 30°C water bath for 15 min, gently swirling every 5 min. Papain was then removed without disturbing the tissue at the bottom of the tube. The HEB media was then added to the tissue tube. The salinized Pasteur pipette was then used to triturate 30 times very slowly and carefully to not add air bubbles with the tip in the middle of the tube. The undispersed pieces were allowed to settle for one minute. The supernatant with dispersed cells were then transferred to a sterile 15 mL tube. This was spun down at 200 × g for one minute and the supernatant was discarded. Afterwards 1-2 mL of NbActiv1 was added while carefully avoiding air bubbles. Neurons were plated at 1 million per 10-cm dish or 25,000 per well of 24-well plate. The neuronal culture media contained the following: 100 mL Neurobasal A , 2 mL B27 , 0.25 mL GlutaMAX  and 1 mL Penicillin-Streptomycin . Additionally, cytosine β-D-arabinofuranoside hydrochloride (Ara-C hydrochloride)  was added from day-in-vitro (DIV) 3 at a final concentration of 2 μM each time as half of the media was changed every three days. At DIV 6, lentivirus transduction was carried out to the neurons as indicated; the multiplicity-of-infection (MOI) was at 10, that is, the ratio of lentivirus count to neuron cells count in each well. At DIV 12, neurons were subjected to protein extraction for co-IP or immunofluorescence staining for confocal microscopy.C57BL/6J mice (Jackson laboratory) at 8–10 weeks were sacrificed, and the cortex was isolated and stored at −80°C. On the day of experiments, the cortex was thawed on ice and homogenized in homogenization buffer  using a plastic micro tissue homogenizer, and then sheared by passing 10 times through a 21G needle. Homogenates were centrifuged at 800 g for 10 min at 4°C and supernatants were collected. The pellet was re-homogenized in additional homogenization buffer and centrifuged at 800 × g for 10 min at 4°C, and the supernatant was collected again. The supernatants were combined and rotated at 4°C for 2 h, and then centrifuged at 18,000 × g for 30 min at 4°C. The resulting supernatant was collected as mouse brain homogenate, and its protein concentration was determined by a MicroBCA assay. The animal studies followed the guidelines of the Institutional Animal Care and Use Committees (IACUC) at Case Western Reserve University.A receptors, primary neurons that were cultured on coverslips, were fixed with 2% paraformaldehyde in DPBS, blocked with goat serum for 0.5 h at room temperature, and labeled with 100 μL of appropriate anti-α1 , β2/3 , or γ2  antibodies (1:200) for 1 h without detergent permeabilization. Afterwards, they were incubated at room temperature with 500 μL (1:400) of Alexa 594-conjugated goat anti-rabbit antibody , or Alexa 594-conjugated goat anti-mouse antibody  for 1 h. Afterwards, cells were permeabilized with saponin (0.2%) for 5 min and incubated with DAPI (1 μg/mL) for 3 min to stain the nucleus. The coverslips were then mounted and sealed. For confocal immunofluorescence microscopy, an Olympus IX-81 Fluoview FV1000 confocal laser scanning system was used. A 60× objective was used to collect images using FV10-ASW software. Quantification of the fluorescence intensity was achieved using the ImageJ software from the NIH.To label cell surface GABA2 and 1 mM MgCl2 (PBS+ CM) for 30minat 4°C to label surface membrane proteins. In order to quench the reaction, the cells were incubated with 1.5 mL of 50 mM glycine in ice-cold DPBS-CM twice for 5 min at 4°C. They were then washed twice with DPBS. The sulfhydryl groups were then blocked by incubating the cells with 5 nM N-ethylmaleimide (NEM) in DPBS-CM for 15 min at room temperature and then the liquid was removed. Cells were scraped off plates and solubilized overnight at 4°C in lysis buffer  supplemented with Roche complete protease inhibitor cocktail and 5 mM NEM. The next day the lysates were centrifuged at 16,000 × g for 10 min at 4°C to pellet cellular debris. The supernatant was saved, and the protein concentration was measured with a MicroBCA assay. Biotinylated surface proteins were affinity-purified from the above supernatant by incubating for 2hat 4°C with 50 μL of immobilized neutravidin-conjugated agarose bead slurry . The samples were then subjected to centrifugation . The beads were washed three times with buffer  and three times further without Triton X-100. Surface proteins were eluted from beads by incubating for 30minat room temperature with 80 μL of LSB ⁄ Urea buffer (2x Laemmli sample buffer (LSB) with 100 mM DTT and 6 M Urea, pH 6.8) for SDS-PAGE and Western blotting analysis.To perform biotinylation assay, 6-cm dishes were coated in a 300-fold dilution of poly-L-lysine  at 2 mL per plate for one hour at 37°C. Plates were rinsed twice with DPBS and allowed to air dry. The cells were plated on the coated dishes and incubated for 48 h post-transfection. The intact cells were washed one time with ice-cold DPBS and incubated with the membrane impermeable biotinylation reagent Sulfo-NHS SS-Biotin  in PBS containing 0.1 mM CaClCycloheximide-chase assay to evaluate protein degradation in cells was carried out according to published procedures . BrieflyN-acetyl-D-glucosamine in the N-linked glycans incorporated on the α1 subunit in the ER, total cell lysates were digested with Endo H enzyme  with G5 reaction buffer at 37°C for 1h. The Peptide-N-Glycosidase F (PNGase F) enzyme  treated samples served as a control for unglycosylated α1 subunits. Treated samples were then subjected to Western blot analysis.To remove asparaginyl-2 , 1 mM CaCl2 , 10 mM glucose , 10 mM HEPES . The intracellular solution (ICS) contained the following: 153 mM KCl , 1 mM MgCl2 , 5 mM EGTA , 10 mM HEPES . Briefly, cells were grown to 50 to 70 percent confluence on 10-cm dishes. Then 3 mL Accutase  was added and the cells were incubated for 3 min at 37°C until the cells were floating as observed under microscope with minimal clumps. We then pelleted cells with centrifugation for 1 min at 200 × g, removed supernatant and resuspended cells in serum free medium HEK293 SFM II , supplemented with 25 mM HEPES  and 1% penicillin streptomycin . Cells were put on gentle shaking at room temperature for 0.5 to 1 h. Mercury 16 plates were prepared according to manufacture suggestions. Whole-cell GABA-induced currents were recorded at a holding potential of −60 mV, at 100 μM or 1 mM GABA concentration as indicated. The signals were acquired and analyzed by Fluxion Data Analyzer.Whole-cell currents were recorded 48 h post transfection of GT1-7 or HEK293T cells. Automated patch clamping was performed on the Ionflux Mercury 16 instrument . The extracellular solution (ECS) contained the following: 142 mM NaCl, 8 mM KCl , 6 mM MgClt-test if two groups were compared and one-way ANOVA followed by post-hoc Tukey test if more than two groups were compared. A p< 0.05 was considered statistically significant. ∗, p< 0.05; ∗∗, p< 0.01.All data are presented as mean ± SEM. Statistical significance was evaluated using Student’s