Four ethyl methanesulfonate mutagenized populations (4032 mutant families) have been screened for the presence of induced mutations in targeted genes. The mutation types and effects have been characterized for a total of 138 soybean genes involved in soybean seed composition, disease resistance, and many other quality traits. To test the efficiency of TbyS+ in complex genomes, we used soybeans as a model with a focus on three desaturase gene families, GmSACPD, GmFAD2, and GmFAD3, that are involved in the soybean fatty acid biosynthesis pathway. We successfully isolated mutants from all the six gene family members. Unsurprisingly, most of the characterized mutants showed significant changes either in their stearic, oleic, or linolenic acids. By using TbyS+, we discovered novel sources of soybean oil traits, including high saturated and monosaturated fatty acids in addition to low polyunsaturated fatty acid contents. This technology provides an unprecedented platform for highly effective screening of polyploid mutant populations and functional gene analysis. The obtained soybean mutants from this study can be used in subsequent soybean breeding programs for improved oil composition traits.Reverse genetic approaches have been widely applied to study gene function in crop species; however, these techniques, including gel-based TILLING, present low efficiency to characterize genes in soybeans due to genome complexity, gene duplication, and the presence of multiple gene family members that share high homology in their DNA sequence. Chemical mutagenesis emerges as a genetically modified-free strategy to produce large-scale soybean mutants for economically important traits improvement. The current study uses an optimized high-throughput TILLING by target capture sequencing technology, or TILLING-by-Sequencing Glycine max (L.) Merr.] is the world’s largest oilseed crop and provides about 53% of vegetable oil in the U.S. . Ol. Ol13]. G111000) ,17. The an seeds . The assulations ,20. A coan seeds ,22,23. San seeds ,25.GmFAD3 genes have been previously identified as GmFAD3A (Glyma.14g194300), GmFAD3B (Glyma.02g227200), GmFAD3C (Glyma.18g062000), and GmFAD3D (Glyma.11g174100) [GmFAD3A have been reported as a source that reduces linolenic acid content [GmFAD3C gene have also been identified and resulted in low linolenic acid content in soybean seeds [GmFAD3 genes [GmFAD2s and GmFAD3s mutations, non-transgenic soybean lines with low linolenic acid content (<3%) and extremely high oleic acid content (>80%) have been achieved [The microsomal omega-3 fatty acid desaturase (FAD3) converts linoleic acid to linolenic acid in the fatty acid biosynthetic pathway. Polyunsaturated fatty acids, including linoleic acid  and α-linolenic acid , typically constitute 60% of conventional soybean oil . High pog174100) . A serie content ,30. Rein content  isolatedan seeds ,33. SoybD3 genes ,35. By sachieved ,37.Targeting Induced Local Lesions INGenomes), was used to identify induced mutations from a mutagenized population. It typically entails chemical mutagenesis and a high-throughput mutation screening method. Ethyl methanesulfonate (EMS) is regarded as the most commonly used chemical mutagen to randomly create point mutations in plant genomes [TILLING bySequencing (TbyS), several recent studies have been reported to improve the efficiency of mutation detection from polyploid species, including durum wheat , rice (diploid), soybean , crambe (hexaploid), and peanut  [As an efficient reverse genetic method in functional genomics; TILLING  ,44,45,46raploid) ,48.TILLING-by-Sequencing+ (TbyS+) technology and its application in identifying induced mutations over 138 genes, including members of three key soybean fatty acid desaturase gene families. A comprehensive analysis of the three fatty acid desaturase gene families was conducted including gene evolution, gene duplication, gene structure, and expression profile. An extra-large number of EMS-induced mutagenized soybean populations have been developed (4032 M2 families) as a diverse genetic resource for economically important traits. The soybean mutants carrying mutations at the six fatty acid biosynthetic genes were phenotyped for their fatty acid profiles. The identified new alleles could be used in soybean breeding programs for improved oil composition traits.Here, we report the use of +. Young leaves were collected from independent M2 plant, and then genomic DNA were extracted and quantified. A total of 42 plates comprising 4032 DNA samples were pooled into three plates  using a bidimensional-arraying strategy. From 168 vertical pools in P1 and P2, each pool contained 24 DNA samples, whereas 48 DNA samples were pooled into each of the 84 horizontal pools in P3, for high-throughput mutation screening  from Pooled sequencing data (CRISP) for SNP discovery. The original Variant Call Format (VCF) files were filtered through SnpSift and stats of SNPs for each gene were visualized using Integrative Genomics Viewer (IGV) . For eac+ were summarized for each of the 138 soybean genes and organized under each of the 20 soybean chromosomes  program at Ensembl Plants . The majGmFAD2-1B is the only gene whose number of another type of mutation is larger than the number of either G to A or C to T (GmFAD2-1B (1), GmFAD3B (3), and GmFAD3C (1) (About 79.6% of SNP mutations are the EMS-type mutations (G/C to A/T) while the other types of mutations account for 20.4% of the total base changes. The mutation density for the five fatty acid desaturase genes is estimated as 1/212 kb . GmFAD2-r C to T . Within r C to T . SeveralAD3C (1) .+, we selected a total of 12 previously identified mutants in GmSACPD-C [GmFAD2-1A/1B [+, including four GmSACPD-C mutants , and three GmFAD2-1A mutants  [+ technology provides a high success rate for true mutation detection, 4/5 (80%) for GmSACPD-C and 3/4 (75%) for GmFAD2-1A.To test the performance of TbySmSACPD-C , and GmFD2-1A/1B  genes asn F1274)   were at the first exon and the other four  were located at the second exon  and GmFAD2-1B  (FAD2-1A mutants showed increased oleic acid content (25.0–34.5%), in which the F258 mutant contained the highest level of oleic acid (34.5%). The oleic acid contents of five FAD2-1B mutants ranged between 23.0% and 27.8%  . Four FAnd 27.8% .GmFAD3 mutants ranged from 4.5% to 5.8% compared to Forrest wild type (7.2%). Among them, one GmFAD3A mutant (F1012) presented the lowest linolenic acid content (4.5%), and two GmFAD3B mutants (F475 and F728) contained 4.6% linolenic acid content  and another two carried nonsense mutations (W247* at GmFAD3B and W267* at GmFAD3C) . The lin content .A. thaliana), three legume species , two dicot species (L. usitatissimum and O. europaea), and two monocot species (E. guineensis and O. sativa). Three mutations at the SACPD-C were located at highly conserved residue positions  and one at A less conserved residue position, G261 , F2146 (SACPD-CR108W), F186 (SACPD-CG185E), and F1202 (SACPD-CG244R) belonged to conserved residues group. Out of the four mutations identified at the GmFAD2-1A, three mutations  were found at conserved residues , while only one mutation (F258) was found at non-conserved residues (FAD2-1AK168Q)  and two conserved residues (FAD2-1BH113P in F966 and FAD2-1BP262S in F720) at the GmFAD2-1B were identified (Unlike the F1320 (SACPD-C1AK168Q) . Three nentified .GmFAD3-A/B/C were located in highly conserved residue positions except two (WX247 and EX333) , F180 (FAD3AG277D), F1012 (FAD3AL279F), and F1428 (FAD3AD360N). Besides the F560 (FAD3BW247*) nonsense mutant, the other three GmFAD3B mutations, F475 (FAD3BP154L), F728 (FAD3BG282D), and F461 (FAD3BH306Y), were located at conserved residues. Two conserved residues (FAD3CA38T in F953 and FAD3CG128E in F846) and one non-conserved residue (FAD3CQ333P in F1739) were identified from three GmFAD3C mutations  . All fouutations .GmSACPD), omega-6 fatty acid desaturase (GmFAD2), and omega-3 fatty acid desaturase . Previous studies described three actively transcribed members of the GmSACPD gene family and seven members of GmFAD2 gene family in soybean [O. europaea) clustered apart from GmFAD2 (L. usitatissimum), which is well known to accumulate ALA, clustered in the same sub-clade with the soybean microsomal omega-3 desaturases  with an average of 1134 bp. The average CDS length of the five GmFAD2 members was 1152 bp while it was limited to only 651 bp and 882 bp for the other two members, GmFAD2-2A and GmFAD2-2E, respectively. The average CDS length of the four microsomal GmFAD3 was 1140 bp, which is 221 bp shorter than that of the plastidial GmFAD7 and GmFAD8 (1361 bp) (The coding sequence (CDS) lengths of the four 1361 bp) . Regardi1361 bp) .GmSACPD-A/B subfamily possessed three exons while the GmSACPD-C/D subfamily carry two exons. The overall gene length of GmSACPD-A/B was also larger than that of GmSACPD-C/D due to their extended intron length (GmFAD2-1 gene family carried two exons with similar gene lengths (1164 bp)  . The gen7/GmFAD8 .GmFAD2-1 subfamily presented remarkably high expression levels in the late stages of seed development, leaf, flower, and pod. For the omega-3 desaturase gene family, except GmFAD3D, the rest of the GmFAD3 members showed fairly high expression in seeds, while the transcripts of two GmFAD8 genes were abundant in leaf. GmFAD3B and GmFAD3C were also highly expressed in leaf and pod. In contrast, two GmFAD7 genes were expressed at drastically low levels in all tissues except pod shells. A low expression level of all eight omega-3 desaturase genes was detected in root and nodule , Chr02 (GmSACPD-B), Chr14 (GmSACPD-C), and Chr13 (GmSACPD-D). Six chromosomes contained the seven GmFAD2 gene family members, including GmFAD2-1A on Chr10, GmFAD2-1B on Chr20, GmFAD2-2A and GmFAD2-2B on Chr19, GmFAD2-2C on Chr03, GmFAD2-2D on Chr09, and GmFAD2-2E on Chr15. Similarly, eight soybean omega-3 desaturase genes were distributed on seven chromosomes, in which GmFAD3A was located on Chr14, GmFAD3B on Chr02, GmFAD3C and GmFAD7-1 on Chr18, GmFAD3D on Chr11, GmFAD7-2 on Chr07, GmFAD8-1 on Chr01, and GmFAD8-2 on Chr03  , including eight segmental and one-tandem duplication blocks (FAD2-2B) . The ratGmSACPD-C, GmFAD2-1A/1B, and GmFAD3A/B/C alleles, the goal of developing soybean with high stearic acid, high oleic acid and low polyunsaturated fatty acid contents can be achieved under the same genetic background in a timely manner.Soybean is a leading oilseed that is grown worldwide. Improving seed oil composition traits is at the core of soybean breeding. However, traditional breeding is labor-consuming, and therefore, it hindered the rapid development of new soybean germplasm into the market. Currently, the major source of improved oil/fatty acid composition traits was mainly produced by genetically modified crops. Such products must comply with the restrictive regulations and cause solicitude from consumers. To avoid health risks and meet nutritional needs, numerous efforts have been made to produce soybeans with elevated oleic acid and low polyunsaturated fatty acid contents ,37. In t+ technology maximized the multiplexing in the mutant library without sacrificing the accuracy of detecting mutations [In the past decade, there were tremendous efforts to develop functional analysis tools in soybean. RNAi and virus-induced gene silencing have been extensively used to study soybean genes. However, gene silencing technology lacked precision due to the similarity between the soybean gene family members. Its application also largely depends on tissue and time specificity expression. Therefore, there is a strong need to develop a new gene functional analysis tool. Coupled with chemical mutagenesis, the high-throughput screening techniques have increasingly progressed to meet the needs of functional genomic studies in major crops. Here, we presented a target capture method facilitated by NGS to efficiently identify population-wide induced mutations in genes controlling economically important traits. In this study, the size of screening mutant populations (4032 M2 families) and the number of targeted genes (>138) were far more than previous studies ,45,51. Mutations . Insteadfreebayes, a haplotype-based variant detector, failed to call mutations in our initial SNP calling test [Furthermore, right after DNA library preparation, the use of capture-seq-enrichment facilitated by target recovery technology before moving to the NGS considerably increases the designed probes specificity and the number of true positive mutants obtained. This step eliminated the issues related to gene families, the gene with high copy number and similarities within the soybean genome, and provided a robust solution to isolate mutants in complex and duplicated genomes. Using parallel commands, all raw sequencing data were automatically processed before SNP calling. Due to the complexity of multiplexing, ing test . After switching to CRISP, we were able to detect SNPs and small InDels from sequencing pooled samples using the Illumina platform. Based on the quality score (QUAL) and Alt Allele Counts (AC), the filtering retained up to 92.3% of novel mutations but removed the majority of spurious ones when applying such thresholds (QUAL ≥ 700 and AC ≥ 4). Although the individual mutant line was not revealed directly after SNP calling, the number of lines with the targeted mutation would be confined to only two candidates, and the mutant can be eventually determined by Sanger sequencing, which is still the most reliable way of mutation validation ,43. Our GmSACPD-C, GmFAD2-1A, GmFAD2-1B, GmFAD3A, GmFAD3B, and GmFAD3C, respectively. Unlike other mutagen agents (like sodium azide or fast neutron), EMS mutagenesis does not generate large deletions. At the M2 generation, most of the mutations are heterozygous. Therefore, the plants are advanced to the M3 generation, where, 25% revert to the wild type, 50% are heterozygous, and 25% are homozygous for the desired mutation. The M3 mutants are sanger sequenced to validate the homozygous mutations for further phenotyping.Ethyl methanesulfonate (EMS) is the most commonly used chemical mutagen to randomly create point mutations in plant genomes ,39. Most+ is justified by confirming our published mutations in genes controlling the fatty acid composition and disease resistance traits [+, a variety of novel missense/nonsense mutations in fatty acid desaturase genes within the fatty acid biosynthesis pathway have been identified. Besides the five mutations that were previously identified in the first exon of GmSACPD-C [GmFAD2-1B as well as six novel missense mutations in GmFAD2-1A. From 18 newly identified GmFAD3 mutations, one nonsense mutation each was detected at GmFAD3B and GmFAD3C  have shown a greater impact on stearic acid content (GmFAD2-1A mutation in non-conserved residue (K168Q) presented a higher oleic acid content than other three GmFAD2-1A mutations in conserved residues.The novel identified mutations within the six fatty acid desaturase genes at the fatty acid biosynthesis pathway present novel sources to improve soybean oil composition traits. Within four  content ; Table 2 content . InteresGmFAD2-1B mutations , olive (high oleic acid content), and flax (high polyunsaturated fatty acids level) . A largeunctions . One SACathogens . The gen soybean . FAD2 gene family, FAD2 from four legume species showed a close evolutionary relationship besides other five plant species. FAD2 from monocot species are more likely to be the ancestors for the rest of FAD2 . Although soybean microsomal and plastidial omega-3 desaturases were grouped in different sub-clades, they presented a highly conserved gene structure and catalytic residues in their structural motifs  was disrupted with tungsten carbide beads in a 96-well plate using TissueLyser System (QIAGEN), and DNA was extracted using DNeasy 96 Plant Kit . The quantity of DNA was estimated using Synergy 2 Multi-Mode Microplate Reader . The concentration of each DNA sample was normalized to 100 ng/uL. All DNA samples in the 96-well plates were stored at −20 °C for long-term purposes.Genomic DNA samples from 96-well plates were pooled using bidimensional-arraying strategy to increase the screening throughput. In vertical pools, DNA arrayed from each of two plates were placed vertically in one column of P1 or P2 plates. Each well in the P1 and P2 plates contains 24 samples from the original DNA plates. In horizontal pools, DNA arrayed from the same row of each six plates were pooled in one row of P3 plate, and 48 samples were contained in each well of the P3 plate. The soybean-targeted capture design was developed on the capture-seq platform at Rapid Genomics  . The cusThe FASTQ raw reads were subjected to quality control using FastQC v0.11.9, while trimming and filtering of low-quality reads were performed using Trimmomatic V0.39 . The cleGmSACPD-C, GmFAD2-1A, GmFAD2-1B, GmFAD3A, GmFAD3B, and GmFAD3C using Primer3 [https://www.genewiz.com/). Putative mutations were identified by alignment of sampled sequences to the reference genome using Unipro UGENE [Sanger sequencing was used to confirm all identified mutations at the three fatty acid desaturase gene family members . PCR(Pol Primer3 . The PCRro UGENE .v/v) methanolic HCl, incubated at 80 °C for 10 min, and cooled for 7 min. 7.5 mL of 6% (w/v) potassium carbonate and 2 mL of hexane were added to each tube and centrifuged at 1200× g for 5 min. The upper layers were transferred to vials, from which the individual fatty acid contents were determined as a percentage of the total fatty acid content in soybean seed by gas chromatography. A Shimadzu GC-2010  gas chromatograph, fitted with a flame ionization detector, was equipped with a 60-m SP-2560 fused silica capillary famewax column (0.25 mm i.d. × 0.25 μm film thickness) . The oven temperature was set at 190 °C for 2 min, then increased 5 °C/min to 250 °C and maintained for 5 min. The injector and detector temperatures were 255 °C. Standard fatty acids  were run first to create a calibration reference.Five major fatty acid contents were measured from individual seeds of mutant lines according to the two-step methylation procedure . At leasGlycine max, Wm82.a2.v1) at Phytozome (v12.1) (https://phytozome.jgi.doe.gov). The fatty acid desaturases from other plant species, including Arabidopsis thaliana, Phaseolus vulgaris (v2.1), Medicago truncatula (Mt4.0v1), Linum usitatissimum (v1.0), Oryza sativa (v7_JGI), Olea europaea var. sylvestris (v1.0), and Lotus japonicas genome assembly build 3.0 (http://www.kazusa.or.jp/lotus/) were identified by BLASTP searches using the soybean fatty acid desaturase protein sequences as queries against the corresponding references of [The nucleotide and amino acid sequences of putative soybean fatty acid desaturases were retrieved from the soybean reference genome . An unrooted phylogenetic gene tree was then constructed by maximum likelihood (ML) method in MEGA X using the Jones–Taylor–Thornton Gamma Distributed (JTT+G) model with a bootstrap analysis of 1000 replicates ,81.https://www.soybase.org/soyseq/). The results were used to generate heatmap and hierarchical clustering using an expression-based heat map [The genomic and coding sequences of soybean fatty acid desaturases retrieved from Phytozome v12.1 were aligned to generate the gene exon-intron structure diagram using the Gene Structure Display Server . The norheat map .http://chibba.agtec.uga.edu/duplication/) Nonsynonymous (Ka) versus synonymous substitution (Ks) rates were calculated based on their values retrieved from PGDD. Given the Ks values and a rate of 6.1 × 10−9 substitutions per site per year, the divergence time (T) was equal to Ks/(2 × 6.1 × 10−9) × 10−6 million years ago (Mya) for each gene pair [The locations of the soybean fatty acid desaturase genes and their corresponding chromosomes were drawn based on the soybean genome annotation a2.v1 on SoyBase. Syntenic analysis were performed using soybean fatty acid desaturases as locus identifiers in the plant genome duplication database (PGDD)  (http://ene pair .https://www.ncbi.nlm.nih.gov/cdd).Multiple sequence alignments were performed using MUSCLE, in which the fatty acid desaturase mutations were overlaid on the amino acid sequence of Forrest wild type. Catalytic residues in conserved motifs of soybean fatty acid desaturases were identified from NCBI Conserved Domain Database (The protein sequence of Forrest cv. GmSACPD-C was subjected to modeling with Deepview and Swiss Model Workspace software, as shown previously . Mutatio+, a versatile extension of the conventional TbyS and a high throughput technology coupled with bioinformatic tools to identify population-wide mutations, proved to be the method of choice to study gene function in soybean. TbyS+ allows for a fast identification of causal mutations based on target capture sequencing enrichment of selected genes, including promoters, exons, introns or any other regions of interest within a variety of sequenced genomes. This technology can be used efficiently to target and distinguish between gene family members that share in general high similarities and also genes with high copy numbers. We were able to discriminate mutations between members of each fatty acid desaturase gene family using capture sequencing enrichment and target recovery technology prior to next-generation sequencing. Meanwhile, large number of novel mutants in gene of interest were uncovered and became immediately available for soybean community.As the mainstream of gene functional analysis, complementation by gene transformation is still time-consuming and a costly process despite the efforts by the scientific community to establish the services-oriented transformation center. Recently, gene editing using CRISPR has emerged as a promising technology for functional gene analysis in soybeans; however, it is still very limited in its use and requires highly trained laboratories and focus only on few genes. Therefore, there is a strong need for new technologies combining high throughput screening methods while keeping lower cost for gene functional characterization. In this study, TbyS


Scientific Reports 10.1038/s41598-020-80730-2, published online 08 January 2021Correction to: This Article contains a typographical error in the Acknowledgements section.“This research was supported by Leading University Project for International Cooperation through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (MOE) (NRF-2019R1F1A1063170); and partially supported by NRF-2019R1A2C1006972.”should read:“This research was supported by Leading University Project for International Cooperation through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (MOE) (NRF-2019R1A2C2011563); and partially supported by NRF-2019R1A2C1006972.”


We show that adjustment of materials textural propertiesand therefore metal single-atom coordination mode enables ligand-to-metalcharge transfer (LMCT) or ligand-to-metal-to-ligand charge transfer(LMLCT), properties that were long speculated in single-atom catalysisbut never observed. We employ the developed materials in the degradationof organic pollutants under irradiation with visible light. Kineticinvestigations under flow conditions show that single atoms of Agand Cu decrease the number of toxic organic fragmentation productswhile leading to a higher selectivity toward full degradation. Theresults correlate with the selected mode of charge transfer in thedesigned photocatalysts and provide a new understanding of how thelocal environment of a single-atom catalyst affects the surface structureand reactivity. The concepts can be exploited further to rationallydesign and optimize other single-atom materials.Because of theirpeculiar nitrogen-rich structure, carbon nitridesare convenient polydentate ligands for designing single atom-dispersedphotocatalysts. However, the relation between catalysts’ texturalproperties and their photophysical–photocatalytic propertiesis rarely elaborated. Herein, we report the preparation and characterizationof a series of single-atom heterogeneous catalysts featuring highlydispersed Ag and Cu species on mesoporous graphitic C The inherent nitrogen-lined porescan accommodate the metal single atoms in a stable manner16via “strong metal–supportinteraction” (SMSI),18 and this can enhancethe photocatalytic performance. Carbon nitrides and Ni salts havebeen elegantly employed in photoredox catalysis.22 At the same time, poly(heptazineimides) possessing alkali, alkaline-earth abundant, and transitionmetal cations have been extensively studied in organic photoredoxcatalysis and H2 evolution.29 Despite these efforts, structure–property–performancerelationship in single-atom photocatalysis is rarely elaborated. Itis also unclear which properties make single-atom catalysts special.Deposition of stable single atoms on nitrogen-framedpores of carbonand carbon nitrides is perhaps the most successful strategy existingtoday to prepare SACs.3N4 polymers witha metal salt, followed by metal reduction and/or thermal treatment.30 However, this often leads to an undesirabledecrease in the total surface area of the final composite as wellas potential metal clustering. An alternative approach to homogeneouslyintroduce metals into the C3N4 framework comprisesthe addition of a metal salt during the synthesis of carbon nitride.To avoid demixing, it has been shown that it is just crucial to selecta suitable miscible and reactive metal precursor that would undergoco-condensation with the C3N4 precursors toensure the homogeneity of the final composite.30Typically, metal deposition is performed post-synthetically byimpregnation of prepared C32 Among those, the removalof leftover pharmaceutical contaminants from water remains as oneof the most challenging chemical transformations.34 By using light and a photocatalyst, it is potentially possible tophoto-oxidize organic pollutants to CO2 and H2O. However, during degradation, stable product fragments that aremore toxic than the initial substrate are frequently formed, limitingthe industrial and societal exploitation of this technology.33 For example, in the TiO2/carbon dot-mediatedphotocatalytic degradation of Gemfibrozil , the contaminant easilydegrades, but benzene derivatives are formed and released in the “purified”water, creating acute toxicity.36 Catalysts showing anincreased selectivity toward CO2 have not been reportedso far, although this would enable a form of carbon mining from wastewater.38From a practical standpoint, SACs can exhibit verysharp productselectivities, just as their homogeneous analogues. This makes themparticularly suitable to overcome low selectivity patterns in manychemical transformations.3N4) enables control and observationof ligand–metal charge transfer. In particular, using a seriesof spectroscopic techniques, we reveal that due to different coordinationenvironments of metal single atoms on the surface of mpg-C3N4 or in the bulk of the material, ligand-to-metal chargetransfer (LMCT) or ligand-to-metal-to-ligand charge transfer (LMLCT)is operative. The selected mode of charge transfer determines theactivity of the single-atom catalyst and, for the specific degradationof Gemfibrozil, it drives the selectivity toward enhanced formationof CO2. The results correlate with the selected mode ofcharge transfer in the designed single-atom materials and open newavenues for the understanding and rational design of single-atom photocatalysts.In this work, we demonstrate that photocatalyst design involvingatomically dispersed metal species at mesoporous graphitic carbonnitride (mpg-C3 (≥99%), CuCl2·2H2O (≥99%), cyanamide (99%), LudoxHS40 (40 wt % suspension in water), and NH4HF2 (95%) were purchased from Sigma-Aldrich. The preparation of metaltricyanomethanides was performed according to the procedure detailedbelow.Sodium tricyanomethanide (98%)was purchased from IoLiTec. AgNO3  in water (10 mL) was addedin one portion to a stirred solution of sodium tricyanomethanide  in water (10 mL). The mixture was maintained under stirringin the dark for 3 h. A white solid was separated by centrifugationat 4000 rpm, washed with water (3 × 10 mL), and dried in vacuum. Yield: 1.92 g, 97%.A solutionof AgNO2·2H2O  in water(10 mL) was added in one portion to a stirred solution of sodium tricyanomethanide in water (10 mL). The mixture was maintained understirring in the dark for 3 h. A brown solid was separated by centrifugationat 4000 rpm, washed with water (3 × 10 mL), and dried in vacuum. Yield: 0.95 g, 39%.A solutionof CuCl3N4 and Cu@mpg-C3N4 was performedmodifying an established procedure.30 Cyanamide(3.0 g), metal tricyanomethanide, and a 40% aqueous dispersion of12 nm SiO2 particles (Ludox HS40) (7.5 g) were mixed andheated at 70 °C under stirring for 16 h until water was completelyevaporated. The resulting mixture was heated at a rate of 2.2 °Cmin–1 over 4 h to reach a temperatureof 550 °C. The temperature was kept constant for another 4 h.The resulting brown-yellow powder was briefly ground and treated withan NH4HF2 solution (12 g in 50 mL of water)for 24 h to remove the silica template. The suspension was centrifugedand the precipitate was washed three times with distilled water andonce with ethanol. Finally, the product was dried at 60 °C undervacuum overnight. For Ag1@mpg-C3N4, Ag2@mpg-C3N4, Cu1@mpg-C3N4, and Cu2@mpg-C3N4, 30 mg (0.15 mmol) and 105 mg (0.53 mmol) ofsilver(I) tricyanomethanide and 37 mg (0.15 mmol) and 131 mg (0.53mmol) of copper(II) tricyanomethanide were added, respectively. Themass values of products obtained for Ag1@mpg-C3N4, Ag2@mpg-C3N4, Cu1@mpg-C3N4 and Cu2@mpg-C3N4 were 1.64, 1.35, 1.78,and 1.61 g, respectively.The synthesis of Ag@mpg-C3N4 was similar with that of Ag@mpg-C3N4 and Cu@mpg-C3N4 except that nometal tricyanomethanide was added.The mass of the product obtained was 1.48 g.The procedure of preparing mpg-Cp/p0 < 0.3 using the QuadraWin 5.05software package. The pore size distribution was obtained by applyingthe quenched solid density functional theory (QSDFT) model for N2 adsorbed on carbon with a cylindrical pore shape at 77 K.The optical absorbance spectra were measured on a Shimadzu UV 2600spectrophotometer equipped with an integrating sphere. The photoluminescence(PL) spectra were recorded and the quantum yield (QY) was measuredusing an FP-8300 fluorescence spectrometer. The excitation wavelengthwas set to 365 nm. The time-resolved PL spectra were obtained on afluorescence lifetime spectrometer  equippedwith a PDL 800-D picosecond pulsed diode laser drive. The decay curveswere fitted using a nonlinear method with a multicomponent decay lawgiven by the general formula I (t) = a1 exp(−t/τ1) + a2 exp(−t/τ2) + a1 exp(−t/τ3). The electron paramagnetic resonance(EPR) study was conducted on a Bruker EMXnano benchtop X-Band EPRspectrometer. The following settings were used: center field, 3200G; sweep width, 3000 G; receiver gain, 40 dB; modulation amplitude,1.000 G; number of scans, 1; microwave attenuation, 25 dB (0.3162mW); room temperature. The Mott–Schottky measurements werecarried out with the Arbin electrochemical testing station (ArbinInstrument) in a standard three-electrode quartz cell. The workingelectrode was prepared as follows: 2 mg of sample was suspended in0.2 mL of deionized water containing 0.02 mL of 5 wt % Nafion D-520dispersion, and the mixture was then dispersed by ultrasonicationand spread onto an fluorine-doped tin oxide (FTO) glass. After beingdried naturally, the FTO glass was heated at 120 °C for 1 h.The prepared thin film was employed as a working electrode, with aplatinum plate as a counter electrode and Ag/AgCl as a reference electrode(3 M KCl). A 0.5 M Na2SO4 aqueous solution wasused as an electrolyte (pH = 8.2). The measurement was carried outupon a frequency of 10 kHz in a potential range from −1.0 to0.4 V vs Ag/AgCl. The measured potentials vs Ag/AgCl were converted to the reversible hydrogen electrode(RHE) scale according to the Nernst equation ERHE = EAg/AgCl + E0Ag/AgCl + 0.059 pH, where ERHE is the converted potential vs RHE, E0Ag/AgCl = 0.1976 at 25 °C,and EAg/AgCl is the experimentally measuredpotential against the Ag/AgCl reference. X-ray photoelectron spectroscopy(XPS) measurements were carried out with an X-ray gun Mg Kαradiation (1254.6 eV) using the CISSY end-station under ultra-highvacuum (UHV) at 1.5 × 10–8 Pa, equipped witha SPECS XR 50 and Combined Lens Analyzer Module (CLAM). The bindingenergy scale and Fermi level were calibrated using a gold film. TheXPS quantitative analysis was performed through CasaXPS software usingLorentzian–Gaussian functions and Shirley background deletionin the photoemission spectra. The ultraviolet photoelectron spectroscopy(UPS) spectra were acquired with a He I (21.2 eV) radiation source.The detector was a combined lens with an analyzer module thermoVG(TLAM).Scanning electron microscopy(SEM) and energy-dispersive X-ray (EDX) images were obtained on aJSM-7500F (JEOL) at an accelerating voltage of 3 kV. EDX investigationswere conducted using a Link ISIS-300 system  equipped with a Si(Li) detector and an energy resolution of133 eV. Transmission electron microscopy (TEM) studies were performedusing a double Cs-corrected JEOL JEM-ARM200F (S)TEM operated at 80kV equipped with a cold field emission gun. Powder X-ray diffraction(XRD) was performed on a Bruker D8 Advance diffractometer equippedwith a scintillation counter detector with Cu Kα radiation (λ= 0.15418 nm). Elemental analysis was accomplished by combustion analysisusing a Vario Micro device. An inductively coupled plasma opticalemission spectroscopy (ICP-OES) study was performed using an Optima8000 ICP-OES spectrometer (PerkinElmer). Nitrogen adsorption–desorptionmeasurements were performed after degassing the samples at 150 °Cfor 20 h using a Quantachrome Quadrasorb SI-MP porosimeter at 77 K.The specific surface areas were calculated by applying the Brunauer–Emmett–Teller(BET) model to adsorption isotherms for 0.05 < –1. The concentration, conversion, and selectivity were calculatedby a peak area method. To determine the concentration of organic moietyin water, product solutions were also measured by elemental CHN analysis.The photocatalytic performance ofthe prepared samples was studied using a solution of Gemfibrozil (10mg) in deionized water (100 mL). This corresponds to a Gemfibrozilconcentration of 100 ppm. All experiments were carried out using visiblelight (450 nm) irradiation. For the continuous-flow reactions, thewater sample containing the pharmaceutical contaminant was pumpedusing a peristaltic pump through a custom-made photocatalytic reactorfeaturing a transparent column with a thin layer of catalyst . The LEDs extended verticallyalong each side of the reactor and illuminated the central manifoldwhere the packed catalyst layer was present. The experimental conditions were varied to exploretheir effect on the pollutant degradation. For the case of batch experiments,Gemfibrozil (10 mg) and the catalyst  were placedin deionized water (100 mL) and the suspension was placed into a round-bottomflask and irradiated using LED lamps placed along each side of theflask. The product solutions were analyzed by a high-performance liquidchromatograph (Waters 1525 Binary HPLC pump). The stationary phaseconsisted of a Purospher Star RP-18 column . The eluent phase was a mixture of water and methanolwith a gradient concentration at a flow rate of 1.0 mL min3N4 with a low surface area(below 10 m2 g–1), resulting in limitedactivity in heterogeneous catalytic reactions.39 Therefore, in this work, a “hard”templating synthetic route using SiO2 nanoparticles hasbeen chosen as an effective technique to obtain an ordered mesoporousstructure. We have prepared, characterized, and evaluated a seriesof photocatalysts based on mpg-C3N4, containingisolated Ag and Cu single atoms on it. The introduction of silverand copper ions into a C3N4 network has beenaccomplished by co-condensation of the respective tricyanomethanidesalts with cyanamide in the presence of 12 nm SiO2 nanoparticlesas a hard template at 550 °C. During this process, the matrixnucleates and grows, embedding into the template. Removal of the templateby treatment with (NH4)HF2 gives the mesoporousstructure of the material with a high surface area, i.e., the template replica. The resultant mesostructured Ag/Cu mpg-C3N4 with ordered porosity is expected to providesufficient surface active sites and enhance light multireflection,to enable efficient photocatalytic reactions. The final compositionand textural properties of the reference and single-atom mesoporousgraphitic carbon nitrides are shown in 3N4 molecular formula for the synthesized organic part of the material,the metal content in Ag@mpg-C3N4, i.e., 0.31–0.32 wt %, suggests that statistically one silver atomis surrounded by ca. 100 heptazine units. In thecase of Cu@mpg-C3N4, the concentration of metalsingle atoms is higher, and for Cu1@mpg-C3N4, it is one atom per 70 heptazine units, while for Cu2@mpg-C3N4, it is one atom per 20 units. The percentageof metal transferred from the tricyanomethanide precursor into thematerial is higher for Cu (>79%) compared to Ag (7–32%).Inparticular, it is quantitative for Cu1@mpg-C3N4 (Table S2). Independent from the presenceof isolated metal species on it, the mesoporous structure of the preparedmaterials is confirmed  peak.41 Single metal atoms disturb the local structure of the C3N4 network as illustrated by an intensity decrease andslight broadening of the stacking diffraction peak at 27° 2θ,suggesting an increased distortion of the stacking arrangement ofthe carbon nitride layers upon an increase in the number of Ag andCu precursors.The chemical structure of the finalCpatterns 1a. All mFigures S2 and S3). Quantitativeanalysis from the XPS spectra reveals that the C/N atomic ratio ofthe samples is ca. 0.66 (Table S3), in line with the total C/N ratio. In the XPS C 1s spectra,a peak at 284.0 eV 42 is assigned to electron-rich, e.g., carbide-like, carbon (Ccarb).30 The peak at 287.4 eV is assigned to carbon bound to terminal NH2 groups (C–NH2). The peak at 288.7 eV isassigned to N—C=N moieties in heptazine rings.41 The N 1s peak can be deconvoluted into threepeaks centered at 398.8, 399.5, and 401.0 eV, corresponding to pyridinicN (C=N—C), tertiary nitrogen (N–(C)3), and amino functional groups (C–NH2 and C–NH–C),respectively.43 In general, introductionof either Ag or Cu single atoms leads to the shift of the Ccarb peak by 0.5–0.8 eV to higher energies,44 indicating that metal single atoms bind selectively toelectron-rich sites in the carbon nitride and induce redistributionof electron density in the material.X-ray photoelectron spectroscopy (XPS) wasperformed followed by fitting with Lorentzian–Gaussian curvesand analysis  to that for C 1s, correcting for different cross sectionsfor radiation. Elsewhere, it has been reported that Cu single atomsdeposited at carbon nitride47 and graphene49 carry partial positive charge. Based on integrated areas of thepeaks, in Ag2@mpg-C3N4, we estimate that 0.61e is transferred to silver  and Ag(0),respectively 1b. Similo silver 1b, whileo copper 1c, which3N4 and is represented by particles witha diameter of ca. 20–200 nm as seen from theSEM images . TEM images showthe presence of abundant mesopores, which originate from the removalof the template . These havean average pore size of ca. 5–15 nm, whichis in line with the results of N2 physisorption studies,showing type IV isotherms and type H3 hysteresis loops . The energy-dispersive X-ray spectraof all materials retrieved from the collected SEM and TEM maps confirmthe presence of all relevant chemical elements . High-angle annular dark-field scanningtransmission electron microscopy (HAADF-STEM) investigation performedon an aberration-corrected microscope showed the presence of silverand copper elements in an atomically dispersed form throughout thesamples  absorption spectroscopy, which originates from π–π*transitions . This effect is the most pronounced for Cu2@mpg-C3N4 and is apparently due to the highest Cu content, 1.66 wt%, in the studied series of materials. Second, light absorption inthe range of 500–800 nm is enhanced due to the introductionof metal ions, which facilitate LMCT. Charge transfer between carbonnitride and metal atoms is further confirmed by quenching of mpg-C3N4 fluorescence (3N4 to 0.59% for Ag2@mpg-C3N4 and0.36% for Cu2@mpg-C3N4 (Table S4). For comparative studies, we have selected materialsmodified with Cu because of similar textural properties, i.e., surface area and specific pore volume −1.52 The results of steady-state fluorescence quenchingare supported by the time-resolved emission spectra (TRES) (Table S5). Thus, photogenerated charge carriersin Cu2@mpg-C3N4 have a ca. 5times shorter lifetime compared to the reference mpg-C3N4, namely, 172 ± 3 ps versus 778 ± 18 ps. Thefluorescence quenching rate constant kq was calculated to be (2.63 ± 0.06) × 109 (wt%)−1 s–1. Despite the fact thatexcitons in the reference mpg-C3N4 possess alreadyvery short lifetime compared, for example, to solutions of molecularphotocatalysts,53 introduction of Ag andCu is still able to efficiently quench fluorescence, because singleatoms are within the subnanometer distance from the fluorophore, whichfacilitates their interaction, while diffusion as a potential rate-limitingstep is eliminated.54 Taking into accountlow IQE for the set of the synthesized materials and TRES data, weconclude that excitons in mpg-C3N4 reach surfacestates in less than 0.8 ns, while those in Cu2@mpg-C3N4 reach surface states in less than 0.2 ns. In the notationadopted in the semiconductor community, metal single atoms are consideredas unoccupied defect states located close to the conduction band (CB)edge . Materialswith nearly identical textural properties, Ag2@mpg-C3N4, Cu1@mpg-C3N4, and Cu2@mpg-C3N4 . For Cu@mpg-C3N4 along with the quenching of the narrow signal,a broad peak covering the full spectral window evolves (57Electron paramagnetic resonance(EPR) spectroscopy was employedto probe the unpaired electrons in the catalysts 3f. The rbackbone S6. Matermpg-C3N4 3c. At thntensity 3b and lo evolves 3f. This 59 Such a charge transfer process in Ag1@mpg-C3N4 occurs on the subpicosecond scale,60 whileenhanced electron mobility is registered as the amplified EPR signal(Table S6), enhanced fluorescence . Introduction of metal single atoms results inthe shift of the VB by 0.04–0.45 eV to more positive numberscompared to pristine mpg-C3N4  of mpg-C3N4 was determined tobe −1.05 V vs RHE, while for mpg-C3N4 modified with Ag and Cu single atoms, the corresponding UFB values are shifted to more positive values, i.e., ca. −0.81 V and −0.46V vs RHE, respectively  or when the metal has low empty orbitalsbut, to our knowledge, was never experimentally observed over single-atomcatalysts.To investigate the influence of metal singleatoms on the alignmentof the energy levels, we determined the valence band (VB) energiesusing ultraviolet photoelectron spectroscopy (UPS) S13. Intr61 This compound is hard to degrade using state-of-the-arttechnologies62 and, when this happens,toxic organic moieties that are stable and soluble in water are formed.36 Our catalytic tests have been conducted under continuous-flow conditions.As shown in rt) in the photoreactor until it reaches a plateauwhen rt > 25 min. Such a trend is common and hasbeen reported elsewhere.64 In fact, a heterogeneousphotocatalytic reaction involves adsorption of reactants from a fluidphase onto a solid surface, surface reaction of the adsorbed species,and desorption of products into the fluid phase. Clearly, substancesother than the reactants, including impurities, reaction intermediates,or (by)products, can also be adsorbed on the catalyst surface andinhibit the reaction due to partial occupation of the catalyst activesites, resulting in a decrease in the degradation rate. It is importantto remark, however, that this effect is more pronounced over pristinempg-C3N4 and less over the single-atom catalysts.Using the materials described above, we havestudied the photocatalytic removal of Gemfibrozil from deionized water.Gemfibrozil is a major contaminant present in water samples and isamong the emerging pollutants listed on the 3rd Watch List under theWater Framework Directive of the European Union.3N4 >Cu1@mpg-C3N4 > Ag2@mpg-C3N4 > Ag1@mpg-C3N4 > mpg-C3N4. This effect does not change after fixing the residencetime andvarying the temperature or the pressure  or using a slurry batch-type photoreactor . Also, the degradation takes placeonly in the presence of light. Finally, a stable photocatalytic performanceis observed for all materials .The order of reactivity is Cu2@mpg-Cc = c0 exp(−kt), where c represents the concentration of Gemfibrozil over time, and c0 is its initial concentration in water. Thisequation represents the best practice to simulate the photoreactorbehavior, considering that an in-depth analysis of Gemfibrozil degradationthrough ab initio simulations is not yet available.In particular, the equation corresponds to a pseudo-first-order concentrationdecay, which is well applicable since the curves of ln(c/c0) versus retention time (rt) show a nearly linear relationship . From this equation, the rate constant (k) andestimated pollutant half-life (t1/2) havebeen determined (Table S7) to provide valuablekinetic insights. By comparing the rate constants with values availablein the literature (Table S8), it is possibleto appreciate the efficiency of the single-atom materials. The estimatedhalf-life for the contaminant degradation is in line with this resultand further demonstrates that Cu2@mpg-C3N4 requiresonly 4 min to halve the contaminant concentration.Based on the data in 3N4 (τ = 172 ps), which correlates with thehighest activity in the degradation of Gemfibrozil. The nature ofmetal single atoms has a clear influence on the performance of thephotocatalyst. Upon photoexcitation of Cu@mpg-C3N4, LMCT promotes the reduction of Cu(II) to Cu(I)/Cu(0) mononuclearcomplexes. The latter activates oxygen via SET and therefore acceleratesthe degradation of the pollutant.67 A similar process inAg@mpg-C3N4 leads to the more stable Ag(0) species(EAg(I)/Ag(0) = +0.8 V vs SHE). Therefore, metal single atoms not only adjust the photophysicalproperties of the carbon nitride semiconductor but also are efficientlyinvolved in the process of Gemfibrozil degradation.The photocatalyticperformance can be correlated with the above-discussedphotophysical properties of the materials influenced by the introductionof single atoms 3c. Thus,3N4 and Cu@mpg-C3N4 comparedto the reference mpg-C3N4. Ag1@mpg-C3N4 clearly stands out as the most selective catalyst towardCO2, followed by Ag2@mpg-C3N4. Despitethe fact that Cu2@mpg-C3N4 showed the highestactivity in the removal of Gemfibrozil, its selectivity toward CO2 is moderate, suggesting the existence of a follow-up cascade, i.e., the drug molecule undergoes transformation quickly,but calcination is a slower process. Note that the catalysts werecompared at a similar degree of conversion and under kinetic conditions(10%). These results point that single-atom photocatalysts can reducethe amount of toxic species of benzene during the pollutant degradation.Although Ag1@mpg-C3N4 and Ag2@mpg-C3N4 have nearly the same Ag content, their textural propertiesare different (3N4 facilitate electron transportthat is beneficial to promote the multistep pathway of Gemfibrozildegradation to CO2.To unravelthe product selectivity formed over the different photocatalysts,we have determined the organic content in the product solutions viaCHN analysis 4b. Organifferent 1. As ded3N4. Using this seriesof catalysts, we have shown that Ag and Cu single metal species candecrease the extent of aromatic byproducts in the degradation of emergingwater contaminants and promote the selective formation of CO2. By combining advanced characterization methods, we show that suchresults can be linked to ligand–metal charge transfer in thedesigned materials, as confirmed by DRUV–vis, TRES, fluorescencequantum efficiency, and EPR measurements. Specifically, textural propertiesof mpg-C3N4 modified with metal single atomsinfluence the mode and type of charge transfer in the studied materials.Overall, this work not only shows the incorporation and use of Cuand Ag atoms on carbon nitride for photocatalytic water decontaminationbut also opens fundamental directions in the design of selective single-atomphotocatalysts, showing how fine-tuning the band structure of thehybrid semiconductor nanomaterials can lead to improved photocatalyticdata.Wehave prepared, characterized, and evaluated a series of single-atomheterogeneous catalysts featuring highly dispersed metal species onmesoporous graphitic C


Directional migration of corneal epithelial cells is essential for healing of corneal wounds, which is a robust response mediated by biochemical and bioelectrical cues. Naturally occurring electric fields at corneal wounds provide a powerful guidance cue for directional cell migration, as does extracellular ATP. Our recent large-scale siRNA library screening identified a role for purinergic signaling in the electric field-guided migration  of human corneal epithelial (hTCEpi) cells.We examined the effect of extracellular ATP on galvanotaxis of hTCEpi cells. Galvanotactic cell migration was recorded by video microscopy, and directedness and migration speed was calculated. The role of purinergic receptors in galvanotaxis regulation was evaluated by pharmacological inhibition or knocking down of P2X and P2Y receptors.Addition of ATP enhanced galvanotaxis, and most remarkably sensitized galvanotaxis response to very low level of electric fields in the physiological range (10–30 mV/mm). The stimulatory effect of extracellular ATP was diminished by apyrase treatment. Importantly, cells stimulated with extracellular ATP migrated with significantly increased directedness and speed, which were diminished by knocking down or pharmacological inhibition of P2X and P2Y receptors. Inhibition of pannexin-1 (ATP permeable channel) significantly impaired galvanotaxis. Moreover, pharmacological inhibition of ecto-ATPase enhanced galvanotaxis.Extracellular ATP and physiological electric fields synergistically enhanced the galvanotaxis response of hTCEpi cells. hTCEpi cells are likely to secrete ATP actively, and purinergic signaling is down-regulated by ecto-ATPases. Both P2X and P2Y receptors coordinately play a role for galvanotaxis of hTCEpi cells. Many types of cells in our body can sense weak extracellular electric fields (EFs) and migrate toward the cathode or the anode, termed galvanotaxis/electrotaxis –4. Galva+ and Ca2+ to flow through the pore. The entry of Na+ via P2X leads to the depolarization of the plasma membrane, and the activation of various subsequent intracellular processes. P2Y is a G-protein coupled receptor that activates intracellular signaling cascades to regulate various cellular functions [4, one type of P2Y receptor, is activated by UTP, but is antagonized by ATP in human cells [Adenosine-5’-triphosphate (ATP) is well known as an energy source in cells. Every cell in our body continuously generates ATP molecules from various saccharides (mainly glucose) and triglycerides –8, and Aunctions , 25. P2Yunctions , 26. Intan cells . These pan cells –31. In aan cells , 33.We recently performed large-scale siRNA library screening to identify important genes for galvanotaxis regulation and found that knocking down several genes impaired or enhanced galvanotaxis of corneal epithelial cells , 35. Amo2+ (60 μM), EpiLife defined growth supplement (EDGS), penicillin/streptomycin, and Lipofectamine 2000 reagent were purchased from Thermo Fisher Scientific . On-target plus siRNA was purchased from GE healthcare . FNC coating mixture was purchased from Athena ES . Suramin hexasodium salt (an inhibitor of P2X and P2Y receptors), PPADS tetrasodium salt (an inhibitor of P2X and P2Y receptors), TNP-ATP triethylammonium salt (an inhibitor of P2X receptor), NF 157 (an inhibitor of P2Y11 and P2X1), ARL 67156 (an inhibitor of ecto-ATPases), and 10Panx (an inhibitor of Pannexin-1 channel) were purchased from Tocris Bioscience . Adenosine-5’-(γ-thio)-triphosphate tetralithium salt , Brilliant blue G , and apyrase were purchased from Sigma-Aldrich .EpiLife culture medium with Ca2+ supplemented with EDGS and penicillin/streptomycin at 37 °C with air containing 5% carbon dioxide, as described previously [4 cells/well in 12-well plate 1 day before transfection. siRNA was transfected by using Lipofectamine 2000 reagent according to the manufacturer’s protocol. At 2 days after transfection, the cells were trypsinized and seeded onto galvanotaxis chamber using FNC coating mixture and subjected to migration experiments described below.Telomerase immortalized human corneal epithelial cells (hTCEpi cells) were generously provided by Dr. James Jester , and were grown in EpiLife with 60 μM Caeviously . For siR3)2, 1.3 mM MgSO4 and 4.6 mM Tris base, pH 7.4) to pooled medium on either side of the galvanotaxis chamber. Cells were exposed to 0–100 mV/mm direct current EF for 1 h. Cell behavior was observed with a Carl Zeiss Observer Z1 inverted microscope with a Photometrics QuantEM EMCCD camera  and MetaMorph NX software , and serial time-lapse images were captured. Cell migration was analyzed to determine directedness (cos θ) and track speed by using ImageJ software  with MTrackJ and Chemotaxis tool plugins. Briefly, trajectories of cells were pooled to make composite graphs. The directedness of migration was assessed as cos θ, where θ is the angle between the EF vector and a straight line connecting start and end positions of a cell. A cell moving directly to cathode would have a directedness of 1; a cell moving directly to the anode would have a directedness of −1. A value close to 0 represents random cell movement. Speed is the total length travelled by the cell divided by time.EF-induced cell migration experiments were performed as described previously . Brieflyt-test were used for statistical analysis as appropriate and a p value less than 0.05 was considered as statistically significant.All data are represented as means ± SEM. We used Statcel4 plugin  with Microsoft Excel for statistical analysis. ANOVA, Tukey’s HSD and the Student’s + channel Kir4.2 (encoded by the KCNJ15 gene) plays a crucial role in sensing weak extracellular EFs in concert with intracellular small molecules (polyamines) to facilitate galvanotaxis [We recently performed a large-scale siRNA library screen to identify important molecules for galvanotaxis regulation using human corneal epithelial (hTCEpi) cells. We found that an inwardly rectifying Kanotaxis . During Recent studies have revealed that many types of cells in our body secrete ATP, and secreted extracellular ATP regulates various cellular functions via binding with P2 receptors –17. This11 and P2X1, also significantly attenuated the stimulatory effect of ATP. However, another P2X inhibitor TNP-ATP had little effect on galvanotaxis of hTCEpi cells . Suramin and PPADS, both non-specific inhibitors of several P2X and P2Y receptors, abolished the effect of extracellular ATP application. BBG, an inhibitor of several P2X receptors, and NF 157, an inhibitor of P2Ypi cells  and D.Of note, in addition to ATP, a non-hydrolysable analogue of ATP, ATPγS, also significantly enhanced galvanotaxis of hTCEpi cells  and F. I10Panx and evaluated its effect on galvanotaxis in hTCEpi cells. As shown in 10Panx slightly but significantly lowered directedness (cos θ) at the EF strength of 30 mV/mm. This result suggests that hTCEpi cells actively secrete ATP through pannexin-1 channel.Pannexin channels act as the ATP secreting channels in mammalian cells . SecreteExtracellular ATP is catabolized by a variety of extracellular ATPases and nucleotidases. These ATPases and nucleotidases, termed ecto-ATPases and ectonucleotidases, are variably expressed on the cell surface of various cell types –47. It h5, P2X6, and P2X7) and four P2Y receptors  expressed in these cells.In purinergic signaling, extracellular ATP binds with its receptor molecules on the plasma membrane, purinergic P2X and P2Y receptors. As mentioned above, P2X receptors are ligand-gated cation channels. P2Y receptors are G-protein coupled receptor (GPCR). Seven P2X receptor-encoding genes , and ten P2Y receptor-encoding genes  have been identified in the human genome. Four P2X receptor-encoding genes  and four P2Y receptor-encoding genes  are expressed in hTCEpi cells, at least at the mRNA level . We knocked down all P2 receptors expressed in hTCEpi cells individually by siRNA. We transfected siRNA into hTCEpi cells using Lipofectamine 2000. At 2 days after transfection, galvanotaxis of those cells were evaluated at the EF strength of 30 mV/mm in the presence or absence of ATP. As shown in +/K+ ATPase), and wound size and depth [In the present study, we demonstrated that extracellular ATP significantly lowered threshold EF strength for the galvanotaxis response in hTCEpi cells via purinergic receptors. Recent studies have revealed that purinergic signaling is one of key factors in corneal wound healing . For exand depth . Wound-gnd depth . Our res11 and P2X1). The effect of extracellular ATP application was attenuated by treatment with suramin, PPADS, BBG, and NF 157, but not with TNP-ATP. Those inhibitors have different specificity against P2X or P2Y receptors, or both P2X and P2Y receptors. For example, suramin inhibits several P2X and P2Y receptors , whereas TNP-ATP inhibits several P2X receptors . As mentioned above, four P2X receptors-encoding genes (P2RX4 to P2RX7) and four P2Y receptor-encoding genes  are expressed in hTCEpi cells . To evaluate which P2 receptors are functionally expressed and contribute to galvanotaxis in hTCEpi cells, we knocked down those four P2X-encoding genes and four P2Y-encoding genes individually by using siRNA. As shown in 5, P2X6, and P2X7) and four P2Y receptors  are functionally expressed and contribute to galvanotaxis of hTCEpi cells. Consistent with the results of inhibitor experiments shown in 4 seems to play a small role in the galvanotaxis reaction of hTCEpi cells, since knocking down of P2RX4 and treatment with TNP-ATP, an inhibitor of P2X1, P2X3, and P2X4, had little effect on galvanotaxis. As mentioned above, multiple P2X and P2Y receptors are expressed in hTCEpi cells. Therefore, knocking down of one receptor may cause compensatory action by other receptor(s). In the present study, knocking down of some receptors (except for P2X4) significantly impaired galvanotaxis, but the effect was not so strong as that of some inhibitors such as PPADS and suramin, which inhibit multiple receptors, suggesting possible compensatory effects. Knocking down of one receptor, for example P2X5, may up-regulate P2X6/P2X7 expression and/or activity, and up-regulated P2X6/P2X7 may compensate the loss of P2X5. This might be the reason why the inhibitory effect by knocking down of several receptors was not so strong as with acute drug treatment, despite their knocking down significantly impairing galvanotaxis.Importantly, the effect of extracellular ATP application was diminished by treatment with various inhibitors of P2 receptors. We used suramin (a non-specific inhibitor of both P2X and P2Y receptors), PPADS (a non-specific inhibitor of both P2X and P2Y receptors), TNP-ATP (an inhibitor of P2X receptors), BBG (an inhibitor of P2X receptors), and NF 157  have been shown to play pivotal roles in galvanotaxis regulation [3) is generated upon PLC activation, and IP3 triggers Ca2+ release from endoplasmic reticulum. Both DAG and Ca2+ (released from endoplasmic reticulum) activate the MEK-ERK pathway to enhance cell migration [P2X receptors are ligand-gated non-selective cation channels that have permeability toward cations such as Namotility . Moreovenockdown . Furthernockdown . It is wnockdown . Castellnockdown . Phosphagulation , 52. In gulation . Those dgulation –56. For igration . Elucida4 [4, P2X5, P2X6, and P2X7) and four P2Ys  are expressed, and all of those P2 receptors except P2X4 play a role in galvanotaxis regulation , such as ADP, UTP, UDP, and UDP-glucose , 26. Int4 . In hTCEgulation . Among tthan ATP . In the 10Panx, an inhibitor of the pannexin-1 channel, significantly impaired galvanotaxis of hTCEpi cells. On the other hand, application of ARL 67156, an inhibitor of ecto-ATPases, significantly enhanced galvanotaxis. These results suggest that hTCEpi cells secrete ATP via a pannexin-1 ATP permeable channel, and this “autocrine/paracrine purinergic signaling” is negatively regulated by ecto-ATPases. Active ATP secretion and autocrine/paracrine ATP signaling is thought to be important for the maintenance of homeostasis in various tissues, including epithelia. Three pannexin channel-encoding genes have been identified in the human genome, namely Panx1, Panx2, and Panx3. Pannexin-1 is encoded by the Panx1 gene and expressed ubiquitously. Pannexin channels can be activated by various factors, such as mechanical stimulation, caspase cleavage, cytosolic Ca2+, extracellular ATP and K+, and membrane depolarization [Application of rization –64. Exporization . In the rization –47. ATP rization . Adenosirization . SimilarWe found that extracellular ATP and physiological EF synergistically induced the galvanotaxis response of corneal epithelial cells. Corneal epithelial cells are likely to secrete ATP actively, and purinergic signaling is down-regulated by ecto-ATPases. Both P2X and P2Y receptors coordinately play a role for galvanotaxis of hTCEpi cells. Our proposed model is depicted in


Saccharomyces cerevisiae mutants overexpressing Candida albicans ABC pump Cdr1 variants resistant to fluconazole efflux inhibition by milbemycin α25 (8 mutants), enniatin B (8), or beauvericin (4). The 20 mutations were in just 9 residues at the centers of transmembrane segment 1 (TMS1) (6 mutations), TMS4 (4), TMS5 (4), TMS8 (1), and TMS11 (2) and in A713P (3), a previously reported FK506-resistant “hot spot 1” mutation in extracellular loop 3. Six Cdr1-G521S/C/V/R (TMS1) variants were resistant to all four inhibitors, four Cdr1-M639I (TMS4) variants were resistant to milbemycin α25 and enniatin B, and two Cdr1-V668I/D (TMS5) variants were resistant to enniatin B and beauvericin. The eight milbemycin α25-resistant mutants were altered in four amino acids as follows: G521R, M639I, A713P, and T1355N (TMS11). These four Cdr1 variants responded differently to various types of inhibitors, and each exhibited altered substrate specificity and kinetic properties. The data infer an entry gate function for Cdr1-G521 and a role for Cdr1-A713 in the constitutively high Cdr1 ATPase activity. Cdr1-M639I and -T1355N possibly cause inhibitor resistance by altering TMS contacts near the substrate/inhibitor-binding pocket. Models for the interactions of substrates and different types of inhibitors with Cdr1 at various stages of the transport cycle are presented.Overexpression of ATP-binding cassette (ABC) transporters is a major cause of drug resistance in fungal pathogens. Milbemycins, enniatin B, beauvericin, and FK506 are promising leads for broad-spectrum fungal multidrug efflux pump inhibitors. The characterization of naturally generated inhibitor-resistant mutants is a powerful tool to elucidate structure-activity relationships in ABC transporters. We isolated 20  Candida albicans protects cells from a wide range of structurally unrelated cytotoxic agents, including azole antifungals  transporter superfamily . Overexpifungals . ABC traifungals , 4. Eukaifungals –7. Sevenfungals –, 7. Planfungals ––11 typicungals –– of the AC. albicans Cdr1 and Saccharomyces cerevisiae Pdr5  of Cdr1/Pdr5, of which about half were in “hot spot 1” in, or proximal to, A713/A723 in the extracellular loop 3 (EL3) of the ED and T540/T550 and S542/T552 in EL1  in growth inhibitory zones around discs containing milbemycin α25 on yeast extract-peptone-dextrose (YPD) agar plates with high, but sub-MICs of FLC (CDR1 open reading frame (ORF) that caused 4 different amino acid substitutions  (Table S1). The four milbemycin α25 resistance mutations were in the large extracellular loop 3  and near the centers of TMS1 (G521R), TMS4 (M639I), and TMS11 (T1355N) (S. cerevisiae strains used in this study is provided in s of FLC . Of 10 m(T1355N)  and D. PFLC) compared with wt Cdr1 , for which the MIC was 16-fold reduced in both mutants, but this finding may be an artifact due to its low water solubility. Significant changes in substrate specificities, however, were observed for A713P. The mutant was modestly (2- to 4-fold reduced MICs) affected in the transport of FLC, voriconazole (VRC), cerulenin (CER), cycloheximide (CHX), and latrunculin A (LaA); it had an unchanged MIC for rhodamine 6G (R6G) and monensin (MON), but it was severely (8- to 32-fold reduced MICs) affected in the transport of clotrimazole (CLT), miconazole (MCZ), ketoconazole (KTC), PSC, itraconazole (ITC), and nigericin (NIG) (2 = 0.63) of larger compounds by Cdr1, a trend that was reversed completely (R2 = 0.51) for G521R  and diverse chemistries  were determined for AD/pABC3, AD/CDR1, and the four milbemycin α25-resistant mutants . The MICs were determined to be an indirect measure of the ability of Cdr1 to efflux the substrates. As expected, all mutants effluxed FLC to some degree (as they were isolated on FLC-containing media), although A713P and T1355N had 2-fold and G521R had 4-fold reduced minimum growth inhibitory concentrations of FLC (MIC wt Cdr1 . S. cerein (NIG) . G521R ein (NIG) . The onlin (NIG) . A graphor G521R . The 8-for G521R  were refS. cerevisiae AD/CDR1  values for FLC efflux inhibition in whole cells (FLC (1 mg/L) of AD/pABC3. The concentration required to inhibit FLC transport  of AD/CDR1 by 50%  (50 of 0.08 μM) followed by enniatin B (0.16 μM), beauvericin (0.31 μM), and FK506 (2.5 μM). G521R was 20 to 100 times more resistant to all four inhibitors   varied shibitors . M639I ahibitors . Strong hibitors  with frahibitors . Even thhibitors  for thishibitors . A713P whibitors . The othhibitors .50 values for the inhibition of the ATPase activities by milbemycin α25, FK506, enniatin B, and beauvericin were calculated from the data in 50 values for the inhibition of FLC efflux. For Cdr1, G521R, M639I, and T1355N, there was a good correlation between the IC50 values for FLC efflux inhibition and the IC50 values for the inhibition of the ATPase activities by FK506, enniatin B, and beauvericin  . There was no such correlation, however, for milbemycin α25 , enniatin B (0.18 μM), and beauvericin (0.24 μM) were comparable to the IC50 values for the inhibition of FLC efflux by whole cells , M639I (75 times), and A713P (>75 times) than those in T1355N (15 times) or G521R (3.5 times) , the presence of FLC impaired this interaction in T1355N so that the FLC efflux pump function of T1355N remained equally susceptible to beauvericin and was even ∼4 times more resistant to enniatin B than wt Cdr1  of the FLC efflux pump function and the ATPase activity of M639I and the FK506 susceptibilities of T1355N  . These dF mutant . The preF mutant . Even th wt Cdr1 . The res wt Cdr1 . Its ATP wt Cdr1  to D. Ye wt Cdr1 . There wf T1355N .Unlike most ABC proteins, the ATPase activity of PDR transporters is oligomycin sensitive, and its oligomycin sensitivity is used to determine PDR transporter-specific ATPase activities in crude plasma membrane preparations . Vanadat34–mK (0.62 mM), Vmax (303 nmol/min/mg), and kcat (8.6 s−1) values of the ATPase activity of wt Cdr1  and T1355N (0.58 mM) were comparable to those of wt Cdr1 but the Vmax values of M639I (233 nmol/min/mg) and T1355N (189 nmol/min/mg) were reduced by ∼20% and ∼40%, respectively (mK (0.35 mM), Vmax (101 nmol/min/mg), and kcat (2.9 s−1) values for A713P were ∼40%, ∼70%, and ∼70% lower, respectively, than those for wt Cdr1, and the kcat/mK ratio (8.3 s−1 mM−1), an indicator of catalytic efficiency, was also decreased to ∼60% of wt Cdr1 (13.9 s−1 mM−1) (mK (0.92 mM), Vmax (497 nmol/min/mg), and kcat (14.1 s−1) values of G521R were ∼50% to 65% higher than those in wt Cdr1, although the overall catalytic efficiency  was similar to that of wt Cdr1 because the reduced affinity of ATP for the NBD  was compensated for by an increased turnover rate   . This re−1 mM−1) . In cont4.1 s−1) .mK and Vmax values for the Cdr1 variants in the absence and presence of the different inhibitor concentrations were calculated  were also observed for the inhibition of the ATPase activities of G521R, M639I, and T1355N by FK506, enniatin B, beauvericin, and oligomycin (mK increases and Vmax decreases) for FK506 (mK increases and Vmax constant) for enniatin B (mK and Vmax increase) (50 of wt Cdr1) the ATPase activity of A713P. A further increase of the enniatin B and beauvericin concentrations did, however, begin to inhibit the ATPase activity of A713P (see mK of the A713P ATPase activity more than 2-fold (mK of wt Cdr1 (0.62 mM)  , FK506 aiKs) for FK506, enniatin B, beauvericin, oligomycin, and vanadate of the five Cdr1 variants were determined using Dixon plots  and KTC (∼100 μM) were 1.8 and 8 times higher, respectively, than those for wt Cdr1, and A713P was resistant to the remaining four substrates  . The ATPase activity of G521R was practically resistant to CLT, KTC, MON, and NIG; it was more sensitive to ITC than wt Cdr1; and its R6G susceptibility remained unchanged from wt Cdr1 . The altered substrate susceptibilities of the ATPase activities of G521R and A713P were independent of whether these compounds were effluxed by these mutant pumps or not. For example, G521R did not efflux CLT and effluxed ITC poorly (Table S2); yet, its ATPase activity was resistant to CLT and more sensitive to ITC than wt Cdr1. Although A713P effluxed MON as efficiently as wt Cdr1 (Table S2) and was unable to efflux NIG, its ATPase activity was insensitive to both compounds .Most smaller substrates, such as FLC, had no effect on the ATPase activity of Cdr1 (data not shown). Six larger substrates  inhibited the ATPase activity of wt Cdr1; however none inhibited the ATPase activity by more than 75% . Only CLT, KTC, and R6G inhibited the ATPase activity of wt Cdr1 by more than 50%. The ICBecause the ATPase activities of the four milbemycin α25-resistant mutants responded in a similar fashion to milbemycin α25, enniatin B, and beauvericin, we expected to find Cdr1 efflux pump mutations in similar regions, and possibly identical residues, in a search for enniatin B- and beauvericin-resistant Cdr1 efflux pump mutants. Eight enniatin B- and four beauvericin-resistant efflux pump mutants were isolated . As expeThe high frequency of multiple independent milbemycin α25-resistant , enniatin B-resistant , and beauvericin-resistant  isolates with mutations in the same residues see  indicateCandida krusei multigene family ABC1, ABC11, and ABC12, for example, evolved specialized transport functions optimized for the transport of either smaller (ABC12) or larger (ABC11) compounds, with ABC1 having the broadest substrate specificity but apparently at the expense of efflux efficiency of particular compounds   and moreompounds .The Cdr1 mutations causing milbemycin α25, enniatin B, and beauvericin resistance were near the center of the TMDs and were mostly different from the previously identified extracellular hot spot 1 and the TMS8-9-10 hot spot 2 regions that enabled cotransport of FK506 with FLC or CHX  and wereBased on the findings presented in this study, the following models were constructed to explain (i) the ATPase kinetics of wt Cdr1, G521R, and A713P; (ii) how these variants transport substrates; and (iii) how the efflux pump function of wt Cdr1 and the four milbemycin α25-resistant Cdr1 variants are inhibited by various types of efflux pump inhibitors.mK (step 1), increased Vmax (step 2), and an ∼20-fold increased vanadate dissociation constant iK (Km and Vmax—the distance between the two NBDs in G521R is greater in step 1 and step 2 than for wt Cdr1 (mK in step 1 and decreased Vmax in step 2) (iK for vanadate)   and ATP anadate)  at CNBD2 by ∼70% ; Fig. 7Ces lower  or higheModels accounting for the transport properties of wt Cdr1, G521R, and A713P are presented in Models for the interactions of efflux pump inhibitors with wt Cdr1, G521R, and A713P are presented in At concentrations that inhibited wt Cdr1, the access of inhibitors to the substrate binding pocket of G521R was prevented and FLC efflux occurred unobstructed . Much hiModels explaining the complex interactions of the efflux pump inhibitors with A713P are presented in mK (0.35 mM) and Vmax (101 nmol/min/mg) values of the A713P ATPase activity. Two enniatin B and beauvericin molecules separate the NBDs further than in wt Cdr1, thus increasing the mK to 0.81 mM and 0.79 mM  value of the A713P ATPase activity but, unlike with the other inhibitors, two FK506 molecules partially (∼25%)   inhibit y (∼25%) . Interesy (∼25%)  and D. Py (∼25%) .RC21v3 binds with similar affinities to the extracellular domain in the inward-open conformation of wt Cdr1 and G521R . The draThe oligomycin sensitivities of the ATPase activities were least affected by any of the Cdr1 mutations , suggestM639I and T1355N mutations caused only subtle changes to the substrate transport efficiencies and the inhibitor susceptibilities of wt Cdr1. T1355 was previously noted as an important residue involved in substrate transport and inhibitor binding, both in Cdr1  and Pdr5ab initio model of the extracellular domain of Cdr1  of these typically large, circular compounds. Two options are available, however, for FLC transport to become resistant to inhibition by weak substrates, such as FK506. Option 1 is to prevent entry (G521 mutations), and option 2 is to enhance cotransport  of FLC with FK506. A detailed characterization of the milbemycin α25-, enniatin B-, and beauvericin-resistant mutants provided important clues about how efflux pump substrates and inhibitors interact with Cdr1. We discovered a gate function for G521 and propose that A713 contributes to a critical contact point at the top of the transporter involved in the constitutively high, basal, noninducible ATPase activity of Cdr1. Interestingly, the action of some inhibitors was influenced by the presence of FLC in the substrate binding cavity of Cdr1. Some inhibitor/FLC combinations  enhanced, while others  reduced, the inhibitory potential of the broad-spectrum efflux pump inhibitors. The rather complex and combinatorial interactions have important implications for the design and development of novel efflux pump inhibitors. Our results indicate that an ideal efflux pump inhibitor should not be an efflux pump substrate; rather, it should have broad-spectrum activity against a range of efflux pumps, and it should not be compromised by the presence of other antifungal efflux pump substrates.d-octapeptide Candia albicans Cdr1-specific inhibitor . FK506 was kindly provided by Astellas Pharma Inc. . FLC was purchased from LKT Laboratories Inc. . Clotrimazole (CLT), miconazole (MCZ), ketoconazole (KTC), itraconazole (ITC), voriconazole (VRC), posaconazole (PSC), nigericin (NIG), monensin (MON), latrunculin A (LaA), cerulenin (CER), rhodamine 6G (R6G), beauvericin, and amphotericin B (AMB) were obtained from Sigma-Aldrich Japan Inc. . Enniatin B was purchased from Alexis Biochemicals . Cycloheximide (CHX) was purchased from Nacalai Tesque Inc. . RC21v3 was our in-house-developed nhibitor .S. cerevisiae strains used in this study are listed in S. cerevisiae strain used to overexpress Cdr1 is based on AD1-8u-  (PDR5 locus of AD1-8u- as described previously (- was grown in buffered CSM-URA medium supplemented with 0.02% (wt/vol) uridine. AD1-8u- . Strainss 10261)  at the Peviously . Yeast ceviously . Strain - and its derivative strains do not grow in RPMI medium used in the CLSI method. The MIC for an antifungal was defined as the lowest concentration of the drug that inhibited growth yield by at least 90%.The susceptibility of yeast to antifungal agents was determined using a modification of the CLSI M27-A3 broth microdilution reference method  as descr6 mid-logarithmic-phase yeast cells suspended in 5 mL of melted (50°C) top-agarose medium (YPD plus 0.6% [wt/vol] agarose supplemented with FLC at 0.25× MIC). Filter discs containing 5 μg milbemycin α25, 10 μg FK506, 0.2 μg enniatin B, or 0.5 μg beauvericin were placed onto the solidified top agarose, and the plates were incubated at 30°C for 48 h.Compounds were tested for inhibition of FLC transport by yeast strains overexpressing Cdr1 or Cdr1 mutations. Petri dishes containing FLC at 0.25× MIC in solidified YPD were seeded with 1 × 10FLC at various concentrations of a test compound in order to identify potential synergistic effects between the test compound and FLC. The assay was carried out in CSM medium (pH 7.0) as described previously , and small-scale plasma membrane preparations were used to determine the kinetic properties of the ATPase activities of Cdr1 variants. Harvesting 40 optical density units  of cells at mid-exponential growth phase and using a small-scale plasma membrane isolation protocol optimized for 40 ODU of cells   , 58. LarAll data are contained within the manuscript and the supplemental information.


NSP3) is an oncogenic molecule that has been concomitantly implicated in the progression of coronavirus infection. However, its oncological role in lung cancer and whether it plays a role in modulating the tumor immune microenvironment is not properly understood. In the present in silico study, we demonstrated that NSP3 (SH2D3C) is associated with advanced stage and poor prognoses of lung cancer cohorts. Genetic alterations of NSP3 (SH2D3C) co-occurred inversely with Epidermal Growth Factor Receptor (EGFR) alterations and elicited its pathological role via modulation of various components of the immune and inflammatory pathways in lung cancer. Our correlation analysis suggested that NSP3 (SH2D3C) promotes tumor immune evasion via dysfunctional T-cell phenotypes and T-cell exclusion mechanisms in lung cancer patients. NSP3 (SH2D3C) demonstrated a high predictive value and association with therapy resistance in lung cancer, hence serving as an attractive target for therapy exploration. We evaluated the in silico drug-likeness and NSP3 (SH2D3C) target efficacy of six organosulfur small molecules from Allium sativum using a molecular docking study. We found that the six organosulfur compounds demonstrated selective cytotoxic potential against cancer cell lines and good predictions for ADMET properties, drug-likeness, and safety profile. E-ajoene, alliin, diallyl sulfide, 2-vinyl-4H-1,3-dithiin, allicin, and S-allyl-cysteine docked well into the NSP3 (SH2D3C)-binding cavity with binding affinities ranging from –4.3~–6.70 Ă and random forest (RF) scores ranging from 4.31~5.26 pKd. However, S-allyl-cysteine interaction with NSP3 (SH2D3C) is unfavorable and hence less susceptible to NSP3 ligandability. In conclusion, our study revealed that NSP3 is an important onco-immunological biomarker encompassing the tumor microenvironment, disease staging and prognosis in lung cancer and could serve as an attractive target for cancer therapy. The organosulfur compounds from A. sativum have molecular properties to efficiently interact with the binding site of NSP3 and are currently under vigorous preclinical study in our laboratory.The multi-domain non-structural protein 3 ( Lung cancer is the second most commonly diagnosed cancer, with an estimated 2.2 million new cases 11.4%) worldwide, and remains the leading cause of cancer deaths, with an estimated 1.8 million deaths (18%) in 2020 1.4% worl,4. HistoA tumor-driven immune imbalance plays an important role in tumor initiation and progression. The tumor microenvironment (TME) is a complex system that encompasses various stroma cell types, immune cell types, and other extracellular components . InteracNSP3) is a multi-domain, multifunctional protein that is an essential component of the replication/transcription complex (RTC), responsible for the synthesis and processing of RNA, and interference with the innate immune system of host cells [NSP3 has been implicated in cancer progression and metastasis [Non-structural protein 3  is an aromatic herbaceous annual spice with various biological activities [NSP3 expression as well as genetic and epigenetic alterations on the TME with respect to tumor immune evasion mechanisms and prognoses in lung cancer cohorts. In addition, we reported the drug-likeness, pharmacokinetics (PKs), toxicity, and potential therapeutic properties of some sulfur-containing compounds from A. sativum. Lastly, we also demonstrated the potential of the compounds to regulate the activity of NSP3 using molecular docking studies of receptor−ligand interactions.In drug discovery pipelines, natural products, particularly medicinal plants, serve as an important source of natural therapeutic agents for treating several diseases ,26,27,28tivities ,31,32,33tivities ,39,40,41tivities ,43,44. Ihttp://timer.cistrome.org/ accessed on 23 July 2021) [http://gepia.cancer-pku.cn/ accessed on 23 July 2021) [NSP3 (SH2D3C) in tumor vs. normal tissues from various cancer types of The Cancer Genome Atlas (TCGA) database. In addition, we used the TNMplot server [NSP3 (SH2D3C) between the pathological free , primary cancer, and metastatic lung cancer tissues.We used the Tumor IMmune Estimation Resource (TIMER2.0) algorithm (ly 2021) , and thely 2021)  for the t server  to conduNSP3, the expression level and survival information of lung cancer cohorts from GEO, EGA, and TCGA were integrated via PostgreSQL server. We split the patient samples into high and low expression levels based on the quantile expressions levels of the gene. The survival differences between the two groups was visualized using the Kaplan−Meier survival analysis  [p value are calculated.To analyze the prognostic value of st 2021) . The hazhttp://www.cbioportal.org/ accessed on 20 August 2021) [NSP3 genetic alterations in lung cancer cohorts. The survival differences between cohorts with and without alterations in NSP3 (SH2D3C) genome was also analyzed through the server and visualized using the Kaplan−Meier plots. In addition, we analyzed the gene mutation co-occurrence patterns between NSP3 (SH2D3C) and other genes in TCGA lung cancer cohorts. Alteration co-occurrence was considered significant only at p < 0.05 and q < 0.05.We explore the cancer genomic data set through the cBioPortal tool (st 2021) ,50 to anhttp://timer.comp-genomics.org/ accessed on 5 August 2021) to comprehensively analyze correlations between NSP3 (SH2D3C) expressions and tumor infiltration levels of six immune cell types  T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells (DCs)) and immunosuppressive cells (cancer-associated fibroblasts (CAFs), T reg cells, and tumor-associated macrophages (TAMs)) in TCGA cohorts of lung cancer. We used the Tumor Immune Dysfunction and Exclusion (TIDE) (http://tide.dfci.harvard.edu accessed on 3 August 2021) tools [NSP3 (SH2D3C) with cytotoxic T lymphocyte (CTL) infiltration, dysfunctional T-cell phenotypes, and T-cell exclusion phenotypes in lung cancer cohorts.We explore the immune module of the TIMER algorithm (1) tools  to analyNSP3 was constructed and analyzed via the search tool for retrieval of interacting genes/proteins (STRING) server  [NSP3 were loaded to the single protein modules of the server and analyzed for both predictive and known interactions in Homo sapiens under the confidence search of 0.70 and at p < 0.05. In addition, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and gene ontology (GO) of the biological process enrichment of the network was downloaded via the server and was further confirmed using the Enrichr platform, an online gene set enrichment analysis (GSEA) server [http://www.ehbio.com/ImageGP/ accessed on 27 August 2021) platform. Analysis and visualization of the gene−gene interaction (GGI) network of the NSP3 was performed through the GeneMANIA platform, a real-time multiple association network integration algorithm for predicting gene functions [NSP3 was analyzed at the confident levels of 0.4 by using the OPENTARGET platform , a bio-web algorithm that integrates genetic, omic, and chemical data to identify the involvement of genes in diseases and aid systematic drug target identification and prioritization [The protein−protein (PPI) interaction network of ly 2021) . The off) server ,54. The unctions . The gentization .SH2D3 knockdown (shSH2D3C) in association with the expression of a number gene. A total of 58 samples are classified into two groups by the median value of shSH2D3C and target genes. Results were presented in the form of predictivity and descriptivity. The predictivity is defined as the fold change (FC) of shSH2D3C efficacy between samples of high and low expression of the gene. Descriptivity is defined as the FC of gene expression between samples of high and low shSH2D3C efficacy.We evaluated the efficacy of A. sativum  using the SwissADME algorithm [https://www.cbligand.org/BBB/ accessed on 21 July 2021), which operates based on the support vector machine (SVM) and LiCABEDS algorithms to analyze the BBB permeation ability of the compounds [We analyzed the drug-likeness, pharmacokinetics, and medicinal chemistry of six organosulfur small molecules from lgorithm . We usedompounds . We evalompounds  created 50) of the compounds for the different routes of administrations  in rats using the GUSAR software for quantitative structure-activity relationship (QSAR)/quantitative structure-property relationship (QSPR) modeling [4 chemical structures with data on acute rat toxicity represented by the LD50 values (log10 (mmol/kg)). In addition, we also evaluated the ecotoxicity properties of the compounds using the bioaccumulation factor Log10(BCF), Daphnia magna 50% lethal concentration (LC50) -Log10(mol/L), Fathead Minnow LC50 Log10(mmol/L), and Tetrahymena pyriformis 50% inhibitory growth concentration (IGC50) -log10(mol/L) as indicators.We predicted the 50% lethal dose (LDmodeling . The GUSNSP3 (PDB: 6W6Y) were downloaded from the Protein Data Bank (PDB) (https://www.rcsb.org/ accessed on 15 August 2021) in PDB file format and subsequently converted into the Auto Dock PDBQT format using AutoDock Vina  [A. sativum  were drawn using the Avogadro molecular builder and visualization tool ver. 1.XX and subsequently converted to PDB format using the PyMOL tool and then to PDBQT format using AutoDock Vina. The receptor was prepared by pre-docking removal of water (H20) molecules and the addition of hydrogen atoms (polar only) and Kolman charges [The crystal structures of CA, USA) . Three-d charges ,63. Mole charges ,62,64,65 charges .NSP3 expression and infiltration of immunosuppressive cells. A KM curve was employed to visualize survival differences between lung cancer cohorts with high and those with low expression levels of NSP3 (SH2D3C). The statistical significance of the differential expression of NSP3 between lung cancer tumor and adjacent normal tissue was evaluated using the Wilcoxon test. Statistical significance was indicated by * p < 0.05; ** p < 0.01; *** p < 0.001.Spearman’s rank correlations were used to assess correlations between NSP3 (SH2D3C) between tumor and adjacent normal tissue revealed that NSP3 (SH2D3C) has deregulatory expression in tumor samples of TCGA cancer types (NSP3 (SH2D3C) is expressed at a lower level in lung cancer tumors than the adjacent normal tissue (NSP3 (SH2D3C) expression levels than the primary tumor (NSP3 (SH2D3C) between lung cancer tissue and disease-free tissue. Interestingly, we found that lung cancer tumors exhibited higher intensity of NSP3 (SH2D3C) than the pathological free tissue (NSP3 (SH2D3C) exhibited shorter survival duration than cohorts with low mRNA expression levels (NSP3 (SH2D3C) is associated with the advanced stage and poor prognosis of lung cancer and thus could serve as a prognostic biomarker of lung cancer progression and follow-up.Our differential expression profile of er types A. Howevel tissue B. On a cry tumor C. Our sury tumor D. Furthee tissue E. Moreovn levels F. CollecNSP3 (SH2D3C) in 566 lung adenocarcinoma cases  and found that 2.1% of the cohorts harbored genetically altered NSP3 (SH2D3C) (NSP3 (SH2D3C) in lung adenocarcinoma cohorts (NSP3 (SH2D3C) mutation in the database, 83.33% were missense (NSP3 (SH2D3C) are associated with shorter overall survival (NSP3 (SH2D3C) alterations with other genetic alterations and found that NSP3 (SH2D3C) alteration is inversely associated with Epidermal Growth Factor Receptor (EGFR) alterations in lung cancer, i.e., lung cancer patients with NSP3 (SH2D3C) alteration are devoid of EGFR alterations  A. Howeve cohorts B. Specifmissense B, C and missense C. Furthesurvival D, diseassurvival E, and prsurvival F of the erations G.NSP3 gene interacts with a number of oncogenic, immune, and inflammatory genes including AR, BCAR1, BCAR3, CAV1, CSRNP1, EFS, EGFR, EPHB2, GIMAP6, ICAM2, IL7R, KIT, MET, MYO1G, NAALADL1, NEDD9, PTK2B, RGL4, SEPTIN1, and TNNT , EIF4G1 (7), PABPC1 (7), BCAR1 (5), CAPRIN1 (5), EIF4A1 (5), and EIF4A2 (5) (VEGFR) signaling pathway, immune response-activating cell surface receptor signaling, Fc receptor signaling pathway, cytoplasmic translational initiation, viral process, transmembrane receptor protein tyrosine kinase signaling pathway, post-transcriptional regulation of gene expression, regulation of substrate adhesion-dependent cell spreading, I-kappaB kinase/NF-kappaB signaling, and Fc-gamma receptor signaling pathway involved in phagocytosis  B. To anaocytosis C, while ocytosis D. CollecNSP3 (SH2D3C) expression with tumor infiltration by immune cells and found that NSP3 (SH2D3C) expression showed strong correlations with the infiltration of six immune cell types, including the B cells , CD8+ T cells , CD4+ T cells , M1-macrophages , neutrophils , and DCs  (NSP3 (SH2D3C) expression was inversely associated with CTL levels in lung cancer patients (NSP3 (SH2D3C) in cohorts with higher CTL levels exhibited a shorter survival period, while a lower expression profile of NSP3 (SH2D3C) in cohorts with higher CTL levels exhibited a longer survival period, suggesting a correlation with dysfunctional T-cell phenotypes (NSP3 (SH2D3C) expressions with infiltration levels of immunosuppressive cells and found that NSP3 (SH2D3C) expression was positively correlated with tumor infiltration of CAFs , T reg cells , and M2 macrophages  in LUAD and LUSC, respectively (NSP3 (SH2D3C) promotes tumor immune evasion via dysfunctional T-cell phenotypes and T-cell exclusion mechanisms in lung cancer patients.We analyzed the relationship of × 10–10) A. Interepatients B. In addenotypes C. Furtheectively D. AltogeNSP3 (SH2D3C) in lung cancer (NSP3 (SH2D3C) is hypermethylated in tumors of lung cancer patients compared to the normal tissue (NSP3 (SH2D3C) exhibited shorter survival duration than cohorts with low methylation levels (NSP3 (SH2D3C) revealed that cohorts with high levels of NSP3 (SH2D3C) exhibited dysfunctional T-cell phenotypes and poor prognosis (NSP3 (SH2D3C) are a risk factor for LUAD and LUSC cohorts (NSP3 (SH2D3C) exhibited shorter overall survival duration than the cohort with low CNA (NSP3 (SH2D3C) including deep deletion, arm-level deletion, arm-level gain, and high amplification also mediated the infiltrations of the tumor-immune cells in both LUAD and LUSC (NSP3 (SH2D3C) are associated with infiltration of immune cells and poorer prognosis of lung cancer cohorts.We evaluated the effect of CNA and methylation of g cancer A–F, and l tissue D. In addn levels E. Analysrognosis F. Nevert cohorts A. Analys low CNA B. In addand LUSC C. CollecSH2D3C and the sensitivity of NSCLC cell lines to various clinical drugs. Our results revealed that high expression level of SH2D3C is associated with the resistance of NSCLC cell lines to doxorubicin, mitoxantrone, GSK1070916, obatoclax mesylate, alisertib, NSC319726, mitomycin-C, bleomycin, etoposide, pelitinib, and BX-912 (SH2D3 knockdown (shSH2D3C) in association with the expression of a number of target genes. Interestingly, we found that higher expression levels of the target genes are associated with the low efficacy of shSH2D3C in NSCLC cell lines  of allicin against leukemia cell line (HL-60), and strong activity of E-ajoene on the liver (pa = 0.881) and breast (pa = 0.878) cancer cell lines. Alliin was identified to be active against lung, liver, and pancreatic cancer cells (pa = 0.51~0.671). 2-vinyl-4H-1,3-dithiin demonstrated in silico cytotoxic activity against leukemia and brain cancer cell lines (pa = 0.518~0.810), while S-allyl-cysteine demonstrated activity against the lung and pancreatic cancer cell lines (pa = 0.519~0.739). Collectively, this study revealed that the six organosulfur compounds in garlic evaluated in this study exhibited selective cytotoxic activity on cancer cell lines and are not cytotoxic to normal human cell lines  binding cavity with binding affinities ranging –4.3~–6.70 Ă and random forest (RF) ranging 4.31~5.26 pKd (NSP3 (SH2D3C) is unfavorable  is differentially expressed in different cancer types and thus could serve different prognostic purposes in different cancer types. As such, different therapeutic approaches must be considered when targeting NSP3 (SH2D3C) in different cancer types. Controversial roles of proteins in different cancer types have been reported in previous studies; for instance, EPHB4 was described as an oncogene in some cancers and as a tumor suppressor in other cancers [NSP3 signaling in cancer, characterized by multi-domain and multifunctional properties. Notwithstanding, NSP3 was proposed to be a promising therapeutic target in both cancer and COVID-19. Specifically, our differential expression and survival analyses strongly suggested that NSP3 (SH2D3C) is a potential biomarker of advanced stages of lung cancer.The impacts of cancer incidences and mortality are increasing at a global level. An estimated 19.3 million new cancer cases and almost 10.0 million cancer deaths occurred in 2020 ,67. Our  cancers ,69,70. TSH2D3C. Consequently, GO and KEGG pathway enrichment analyses were conducted to identify functional processes and pathways that may be mediated by these genes. Interestingly, our results suggested that NSP3-associated networks were involved in several pathways and biological processes associated with bacterial and viral infections, immune and inflammatory diseases, and cell proliferation and lung cancer development.Genes with similar expression patterns share similar functions. Therefore, gene functional enrichment analysis is important in an in silico approach and has been widely used in the analysis of gene-related pathological processes ,72. Our NSP3 could regulate lung cancer tumor evasion of immune cells via both T-cell exclusion and induction of dysfunctional T-cell phenotypes. In addition, both genetic alterations and gene methylation of NSP3 were also found to regulate dysfunctional T-cell phenotypes and are associated with the early death of lung cancer patients. Altogether, our findings strongly suggest that NSP3 is an important onco-immunological biomarker encompassing the TME, disease staging, and prognosis in lung cancer, and can serve as an attractive target for cancer therapies. However, subsequent development of NSP3 as a reliable therapeutic target in lung cancer requires preclinical and clinical validation.Tumor immune cell infiltrations are indicators of host immune responses to cancer cells ,74. SeveA. sativum are good drug-like candidates and exhibited selective in silico cytotoxic activities against lung, brain, liver, and pancreatic cancers with no cytotoxic effects on normal cell lines. The compounds are BBB-penetrant and therefore will be very useful in treating glioblastomas and other diseases associated with the central nervous system (CNS). Evaluation of toxicity of natural products and active compounds is an important factor in the development of drugs for therapeutic applications [50 levels in rats. The overall analysis, however, revealed that the oral route was the safest route for administering these compounds; this route should be well tolerated when used for acute administration and is currently being employed for in vivo evaluations of these compounds against lung cancer and glioblastomas in our laboratory.Drug-likeness is a complex balance of molecular properties and structural features which determine whether a molecule is like known drugs ,81. Inteications ,83,84. OAllium sativum have the molecular properties to interact efficiently with the binding site of NSP3 and are currently under rigorous experimental validation in our laboratory. E-ajoene, alliin, diallyl sulfide, 2-vinyl-4H-1,3-dithiin, and allicin docked well into the binding cavity of NSP3 (SH2D3C). Hydrogen bonds and other noncovalent interactions, such as hydrophobic and ionic interactions and van der Waals forces, play important roles in stabilizing ligand−protein complexes [NSP3 predominantly involved hydrogen bonds, van der Waals forces, π-alkyl, and various hydrophobic interactions. The van der Waals forces created around the compound’s backbone with the several amino acids create a strong cohesive environment, which further stabilized the complexes [NSP3 (SH2D3C) is unfavorable and hence less susceptible to NSP3 ligandability. Altogether, our molecular docking experiments suggested that the organosulfur compounds from A. sativum have the molecular properties to interact efficiently with the binding site of NSP3. This interaction is under rigorous experimental validation in in vitro as well as in vivo models of GBM and lung cancer.Molecular docking has become an increasingly valuable tool for depicting the possible interaction between a small-molecule drug candidate and a protein target during the early stage of drug development ,86,87. Oomplexes . We founomplexes . HoweverNSP3 is an important onco-immunological biomarker encompassing the tumor microenvironment, disease staging, and prognoses in lung cancer and could serve as an attractive target for cancer therapies. Our molecular docking experiments suggested that the organosulfur compounds from Allium sativum, including alliin, allicin, E-ajoene, Z-ajoene, 2-vinyl-4H-1,3-dithiin, diallyl sulfide, and allyl methyl sulfide, have molecular properties allowing them to efficiently interact with the binding site of NSP3 and are currently under rigorous experimental validation in our laboratory.In conclusion, our in silico study suggested that