Corynespora cassiicola, as a necrotrophic phytopathogenic ascomycetous fungus, can infect hundreds of species of plants and rarely causes human diseases. This pathogen infects cucumber species and causes cucumber target spot, which has recently caused large cucumber yield losses in China. Genome sequence and spore germination-associated transcriptome analysis will contribute to the understanding of the molecular mechanism of pathogenicity and spore germination of C. cassiicola.C. cassiicola isolate HGCC with high virulence. Although conspecific, HGCC exhibited distinct genome sequence differences from a rubber tree-sampled isolate (CCP) and a human-sampled isolate (UM591). The proportion of secreted proteins was 7.2% in HGCC. A total of 28.9% (4232) of HGCC genes, 29.5% (4298) of CCP genes and 28.6% (4214) of UM591 genes were highly homologous to experimentally proven virulence-associated genes, respectively, which were not significantly different (P = 0.866) from the average (29.7%) of 10 other phytopathogenic fungi. Thousands of putative virulence-associated genes in various pathways or families were identified in C. cassiicola. Second, a global view of the transcriptome of C. cassiicola spores during germination was evaluated using RNA sequencing (RNA-Seq). A total of 3288 differentially expressed genes (DEGs) were identified. The majority of KEGG-annotated DEGs were involved in metabolism, genetic information processing, cellular processes, the organismal system, human diseases and environmental information processing.First, we reported the draft genome sequences of the cucumber-sampled C. cassiicola in cucumbers and the understanding of molecular and cellular processes during spore germination.These results facilitate the exploration of the molecular pathogenic mechanism of  C. cassiicola (BerK & Curt) Wei has recently caused tremendous cucumber yield losses in China [C. cassiicola, as a necrotrophic parasitic fungus, can infect more than 500 plant species, including tomato, eggplant, tobacco, rubber, cotton, soybean and balsam pear, in addition to cucumber and cause plant spot diseases [C. cassiicola is an opportunistic pathogen in humans and causes fungal keratitis under suitable conditions [C. cassiicola against plants should be well studied to effectively control diseases caused by this pathogen.Cucumber target spot caused by in China . More imdiseases , which hnditions  and subcnditions . TherefoC. cassiicola has mainly focused on biological characteristics, pathogenicity differentiation, cloning of virulence-associated genes, etc. It was reported that C. cassiicola sampled from cucumber could grow at 10–35 °C, and its optimum growth temperature was approximately 30 °C [C. cassiicola spores germinated from one end or both ends at 25–30 °C with > 90% relative humidity, and the germination rate in drops of water was the highest. C. cassiicola was able to invade cucumber leaves primarily through direct contact or via stomata [C. cassiicola showed pathogenic and genetic variation among isolates sampled in different host plants, proving that the intraspecific strains of C. cassiicola showed high host specialization [C. cassiicola isolates from perilla, cucumber, tomato, aubergine and sweet pepper in Japan were divided into seven pathogenicity groups (PG1-PG7) [C. cassiicola, and contains six different cassiicolin isoforms, Cas1, Cas2, Cas3, Cas4, Cas5 and Cas6, in different C. cassiicola isolates sampled from various hosts and geographical origins [Cas1 gene were the most aggressive to rubber trees. Additionally, some isolates with no Cas gene also generated moderate symptoms on rubber tree leaves, showing that uncharacterized effectors existed in C. cassiicola [For the past few years, research on the pathogenic mechanism of  stomata . C. casslization . Sixty-fPG1-PG7) . Cassiic origins . The aggC. cassiicola requires multiple pathogenic factors, such as cutinase, cell wall-degrading enzymes [2+ and cAMP signal pathways etc. [C. cassiicola have rarely been cloned and functionally characterized except for two MAPK genes CCk1 [CMP1 [Cas [C. cassiicola. Thus, a great number of virulence-associated genes remain to be identified, cloned and functionally characterized.Similar to other filamentous fungal pathogens,  enzymes  and cytoays etc. . To datenes CCk1  and CMP1k1 [CMP1 , and theMP1 [Cas , 17, whiC. cassiicola thus far.The fungal conidium involved in reproduction is the main type of fungal asexual spore, and is the main form of inoculation and infection . RestingC. cassiicola be globally analyzed. Although the genome sequencing of two C. cassiicola isolates, CCP from a rubber tree and UM591 from the contact lens of a patient with keratomycosis, was completed [C. cassiicola isolate (HGCC) with high virulence to cucumber, and comparatively analyzed its genome with two other C. cassiicola isolates (CCP from a rubber tree and UM591 from a human) and other phytopathogenic ascomycete fungi in multiple families or pathways of virulence-associated genes in this study. Second, we studied the relative transcriptional levels of genes during the spore germination of C. cassiicola HGCC using RNA-Seq. These research results provide a great deal of information revealing the molecular mechanism of pathogenicity and spore germination of C. cassiicola.Genome sequencing is a high throughput means to identify functional genes combined with homologous matches against functional databases or conserved domain searches , 23. Furompleted , 25, it C. cassiicola sampled from cucumber. By being subcultured on potato dextrose agar (PDA) plates at 25 °C in the dark, HGCC produced a layer of fluffy aerial mycelium that was whitish gray when young and dark green when old  from the average number  of 10 other phytopathogenic fungi, including Curvularia lunata, Bipolaris maydis, Cercospora zeae-maydis, Parastagonospora nodorum, Setospaeria turcica, Pyrenophora tritici-repentis, Magnaporthe oryzae, Aspergillus flavus, Fusarium graminearum and Botrytis cinerea  using an Illumina HiSeq X 10 system. A total of 54,580,316 high-quality reads were assembled into 1032 scaffolds (N50: 500 kb) with a genome size of 42.7 Mb, which was slightly less than that of CCP (44.8 Mb) (JGI: 1019537) and greater than that of UM591 (41.4 Mb) (GenBank: JAQF00000000.1) (Table C. lunata (57.4%), B. maydis (57.1%), S. turcica (57.0%), P. nodorum (57.8%), P. tritici-repentis (56.8%), C. zeae-maydis (47.0%), A. flavus (47.5%), B. cinerea (47.2%), F. graminearum (46.0%) and M. oryzae (44.9%). More than 70% of HGCC genes had a > 90.0% amino acid sequence identity with CCP (75.5%) and UM591 (72.4%), which was far more than the 55% between two C. lunata isolates, CX-3 from maize and m118 from sorghum [C. cassiicola isolates had distinct differences in their genomes, although HGCC had a high amino acid sequence identity with both CCP and UM591.The HGCC genome had a 91.6 and 90.5% amino acid sequence identity with CCP and UM591, respectively, and a 44.9–57.8% identity with other phytopathogenic fungi, such as  sorghum . A totalnerea 47.%, F. granerea 47.%, F. granerea 47.%, F. graC. cassiicola , the major facilitator superfamily (MFS) (P = 0.043), cytochrome P450 enzymes (CYPs) (P = 0.011), G protein-coupled receptors (GPCRs) (P = 0.028), Pth11-like G protein-coupled receptors (P = 0.011), protein kinases (P = 0.011), proteases (P = 0.011), cysteine proteases (P = 0.017), metallo proteases (P = 0.022), serine proteases (P = 0.011), GHs (P = 0.011), pectate lyases (P = 0.011), pectinesterases (P = 0.044), cellulases (P = 0.045), glucosidases (P = 0.010) and secondary metabolite backbone genes (P = 0.042) between C. cassiicola and the 10 other phytopathogenic fungi, and the number of proteins in the each family in the former was more than that in the latter, suggesting that C. cassiicola had family expansions in these families. These protein families were expected to play important roles in fungal survival in various adverse environments.A total of 11,111 gene-encoding proteins were classified into 3892 conserved protein families in HGCC by Pfam matches with profile hidden Markov models, which was close to the 3847 families containing 11,187 proteins in CCP and the 3860 families containing 11,110 proteins in UM591. Glycoside hydrolase (GH), pectate lyase, cutinase and cellulose were important pathogenic factors that were highly abundant in C. cassiicola and other phytopathogenic fungi were conducted against the pathogen-host interaction (PHI) database at a E-value of 1e-5. A total of 28.9% (4232) of HGCC genes, 29.5% (4298) of CCP genes and 28.6% (4214) of UM591 genes were matched with PHI database, respectively. There was no significant difference (P = 0.866) in the percentage of PHI-associated genes between the three C. cassiicola and the 10 other phytopathogenic fungi (average: 29.7%)  from the average (113) of the 10 other phytopathogenic fungi , C  and PTH11  classes in C. cassiicola were significantly different (P < 0.05) from the average  of the other phytopathogenic fungi, respectively. More than 80% of Pth11-like GPCRs were identified as PHI-associated genes in HGCC (53/62), CCP (50/61), UM591 (48/57)), and the 10 other phytopathogenic fungi (average: 32/37), respectively. The G protein alpha subunit is an important component of the heterotrimeric G protein complex [Pseudocercospora fijiensis  and two G protein alpha subunits of Stemphylium lycopersici , respectively.GPCRs transduce external environmental signals by way of heterotrimeric G proteins into secondary messengers to regulate gene expression and the subsequent cellular response . These pgenicity , and its average 13 of the complex  and can  complex , 31. In 2+ signaling are part of major signaling pathways and are associated with virulence in phytopathogenic fungi, which are controlled by a series of protein kinases [Saccharomyces cerevisiae, FUS3/KSS1, Mpk1 and Hog1, have been well studied and are highly conserved in other fungi [2+ pathway-associated genes in S. cerevisiae, 48, 12 and 23 screened genes in HGCC were highly homologous with MAPK, cAMP and Ca2+ signal pathway genes of S. cerevisiae, respectively  from 116 to 140 in other phytopathogenic fungi  in the numbers of protein kinases in the Other and STE classes between C. cassiicola and the other phytopathogenic fungi, respectively. The STE class  and MAPK family  in the CMGC class were involved in the MAPK pathway. The PKA family  of the AGC class was involved in the cAMP pathway. The CLK family  and the RCK family  of the CMGC class, the CAMK class , and the PKC family  in the AGC class were related to the Ca2+ pathway. Interestingly, almost all protein kinases were PHI-associated genes in HGCC (150/154), CCP (154/156), UM591 (148/151), and the 10 other phytopathogenic fungi (average: 125/129), suggesting that protein kinases played key roles in pathogenic processes mainly mediated by the three pathogenic signal pathways.MAPK, cAMP and Ca kinases . Three Mer fungi . Based oP = 0.492) from the average (10) of the other phytopathogenic fungi  phosphorelay signaling is usually used by fungi, bacteria, plants and slime molds to sense and adapt to their environment . Fungal  average 0 of the P = 0.051) from the average (11) in the 10 other phytopathogenic fungi. There was significant difference (P = 0.011) in the number of pectate lyases, a type of pectinase, between C. cassiicola  and the other phytopathogenic fungi (average: 12). However, pectinesterase, another type of pectinase, was less abundant than pectate lyase in C. cassiicola  and the other phytopathogenic fungi (0–5). Fourteen, 17 and 16 cellulases were identified in HGCC, CCP and UM591, respectively, which were significantly different (P = 0.045) from the average (12) in the other phytopathogenic fungi. The hemicellulase glucanase , glucosidase  and xylanase  were abundant in C. cassiicola, of which only glucosidase showed significant difference (P = 0.010) in number between C. cassiicola and the other phytopathogenic fungi (average: 9). More than half of cutinases and xylanases and almost all of pectate lyases, pectinesterases, glucanases and glucosidases were PHI-associated genes in these phytopathogenic fungi.To successfully invade the host plant, phytopathogenic fungi are expected to produce and secrete multiple extracellular degrading enzymes, such as cutinase for the degradation of the cuticle , cell waC. cassiicola genomes , which were significantly different (P = 0.011) from the average value (521) of the other phytopathogenic fungi  from the average of the other phytopathogenic fungi   from the average (20) of the other phytopathogenic fungi  from the average (194) in the other phytopathogenic fungi. Many GH families exhibited gene expansion in C. cassiicola when compared to the other phytopathogenic fungi, including the GH1, GH2, GH3, GH7, GH12, GH20, GH27, GH35, GH43, GH53, GH61, GH72, GH88, GH106 and GH107 families; however, the GH30 and GH 65 families exhibited gene constriction. More than half of GHs were PHI-associated genes in HGCC (141/262), CCP (138/262), UM591 (141/265), and the other phytopathogenic fungi (average: 100/194). Interestingly, almost all GHs of the GH3, GH7, GH10, GH17, GH18, GH20, GH28, GH31, GH61 and GH72 families were PHI-associated genes in C. cassiicola and the 10 other phytopathogenic fungi. It was reported that the GH6, GH7, GH45 and GH61 cellulases and GH10 xlyanases were absent in insect pathogenic fungi but present in phytopathogenic fungi [GH catalyzes the hydrolysis of glycosidic bonds in complex sugars , playingic fungi . The GH C. cassiicola contained a large number of transporters , which were significantly different (P = 0.043) from the average (548) of the other phytopathogenic fungi  and ATP-binding cassette (ABC) superfamily  superfamily were the two largest superfamilies of transporters. The former is capable of transporting small solutes in response to chemiosmotic ion gradients, and the latter transports small molecules and macromolecules under ATP hydrolysis [P = 0.043) in the number of MFS transporters between C. cassiicola and the other phytopathogenic fungi (average: 160). However, there was no significant difference (P = 0.127) in the number of ABC transporters between C. cassiicola and the other phytopathogenic fungi (average: 43). Notably, almost all MFS transporters and all ABC transporters were PHI-associated genes in these phytopathogenic fungi, showing that MFS and ABC transporters played key roles in fungal pathogenicity.drolysis , 42. The+ antiporter (DHA) subfamilies (DHA1 and DHA2), drug transporters of the MFS superfamily, can secrete toxic compounds into the outer environment [P = 0.042) in the number of DHA1 transporters between C. cassiicola and the other phytopathogenic fungi (average: 32). Interestingly, all drug transporters were PHI-associated genes except for DHA2 (4/5).In phytopathogenic fungi, drug transporters of ABC and MFS superfamilies can secrete endogenous fungal virulence factors such as toxins and protect pathogens against exogenous plant defense compounds such as phytoalexins , 43. Twoironment . The mulironment . There wilies DHA and DHA2C. cassiicola HGCC (218), CCP (214) and UM591 (209) genomes and classified into 112 families; there was significant difference (P = 0.011) in the number of CYPs between C. cassiicola and the other phytopathogenic fungi (average: 122)  in the number of CYP65s between C. cassiicola  and the other phytopathogenic fungi (average: 11). Additionally, CYP505 was scarce in C. cassiicola  and the other phytopathogenic fungi (average: 3).CYPs are proteins of a superfamily, are ubiquitous in all biological kingdoms, and contain heme as a cofactor and are therefore hemoproteins. Fungal CYPs play key roles in various metabolic processes, such as housekeeping biochemical reactions, detoxification of chemicals and adaptation to adverse surroundings . A largela HGCC 28, CCP  in the number of backbone genes between C. cassiicola  and the other phytopathogenic fungi (average: 42)  were significantly different (P = 0.011) from that of the other phytopathogenic fungi (average: 17).Melanin and mycotoxin are important virulence factors in phytopathogenic fungi . To dataC. cassiicola PKSs and role-known PKSs in other pathogenic fungi  were specific to CCP when compared to HGCC. The second cluster contained nonreducing PKSs with KS and AT domains at a minimum and did not include dehydratase (DH), enoyl reductase (ER) and ketoreductase (KR) domains; this cluster contained 10 PKSs of C. cassiicola and 9 known PKSs related to melanin biosynthesis in other fungal pathogens such as Aspergillus fumigatus Alb1p, Ceratocystis resinifera PKS1, Colletotrichum lagenarium PKS1, Chaetomium globosum PKS-1, Ascochyta rabie PKS1, B. maydis PKS18, S. turcica StPKS, Bipolaris oryzae PKS1, and A. alternata ALM1. It was difficult to identify which reducing PKSs were involved in mycotoxin biosynthesis in C. cassiicola due to the complicated evolutionary relationship for the KS domain of the reducing PKSs in C. cassiicola and known mycotoxin-related PKSs in other fungi. Nevertheless, the nonreducing PKS HGCC_7666 of C. cassiicola had the closest evolutionary relationship with known melanin-associated nonreducing PKSs, suggesting that HGCC_7666 was probably the backbone gene for melanin synthesis in C. cassiicola.To analyze the relationship between the PKS domain and its function, phylogenetic analysis for the ketoacyl CoA synthase (KS) domain of PKS was performed among ngi Fig. . These PAvr genes, trigger or suppress effector-triggered immunity (ETI) mediated by a gene-for-gene system in PHI [Avr-encoded proteins (AvrLm4–7) that were putative effector proteins  can be secreted directly into host plant cells to function in host recognition or colonization  and the m in PHI . Fifty-nLysin motif (LysM)-containing effectors are ubiquitous in phytopathogenic fungi and are secreted out of the cell to suppress the immune response of the host , 58. ComC. cassiicola, 6 h- and 12 h-germination times were selected for RNA-Seq based transcriptome analysis. After 12 h at 25 °C in sterile ddH2O, the majority of spores germinated at two ends, and only a few germinated at one end , suggesting that samples in the same treatment group were repeatable and that the RNA-Seq data were reliable. A total of 66–73% of C. cassiicola filtered reads per library mapped to the gene predictions of C. cassiicola. A total of 3288 genes were differentially expressed, of which 1552 and 1736 were up-regulated and down-regulated, respectively, during spore germination of C. cassiicola with ungerminated spores as the reference  and pathway. A total of 2600 DEGs 79.08%) were annotated by gene ontology (GO). GO term analysis for 9.08% wer2 = 0.7434). The expression patterns of 56 DEGs were confirmed by qRT-PCR, and 10 were not , and catalase [nheC, cytK and hblC during Bacillus thuringiensis spore germination [C. cassiicola in this study. A total of 3288 genes were found to be differentially expressed, which was similar to the 3026 DEGs observed during spore germination of P. expansum [C. cassiicola and P. expansum that the majority of spore germination-associated DEGs were enriched in cellular processese, localization, metabolic processes, regulation of biological processes, single-organism processes, binding, and catalytic activity. Similar pathway classification results for DEGs were also found between C. cassiicola and P. expansum, and the majority of KEGG-annotated DEGs were classified in metabolism, genetic information processing, environmental information processing, cellular processes, and human diseases. This phenomenon showed that RNA-Seq data for spore germination were reliable and probably provided valuable information on the molecular mechanism of C. cassiicola germination.Gene expression profiles can provide insight into the molecular and cellular processes of spore germination at either the transcriptional level or at the proteome level. Zhou et al. found that a total of 3026 genes were differentially expressed during spore germination of ocessing . Liu et  RNA-Seq . Joise Hcatalase . Bassi emination . These rexpansum . It was C. cassiicola spore germination. Similar results were found in the spore germination process of Penicillium expansum [C. cassiicola spore germination, and most of these DEGs were up-regulated, which was nearly opposite to Zhou et al.’s results that all DEGs involved in energy metabolism were down-regulated both in the transcriptional and protein levels [Carbohydrate metabolism involves the various biochemical processes responsible for the formation, breakdown and interconversion of carbohydrates, which serve as short-term fuel for organisms . Therefoexpansum . Energy n levels .C. cassiicola from cucumber was presented, and thousands of genome-wide virulence-associated genes were mined by homologous searches against multiple functional databases and conserved domain searches. A total of 3288 genes were differentially expressed during the spore germination of C. cassiicola. Most of the KEGG annotated DEGs were involved in metabolism, genetic information processing, cellular processes, organismal system, human diseases and environmental information processing. These results not only facilitate the understanding of the molecular pathogenic mechanism of C. cassiicola in cucumber and the molecular and cellular processes during the spore germination, but also lay the foundation for disease control.In conclusion, the genome sequence of C. cassiicola (Berk. & M.A. Curtis) strain HGCC, which we isolated from infected cucumber leaves in Shanghai in 2010, was highly virulent to cucumber; thus, it was used for genome sequencing. The isolate was cultured on PDA medium in test tubes at 25 °C for 7 d and then maintained at 4 °C. The HGCC isolate was subcultured on PDA plates at 25 °C in the dark for 10 d, and the mycelium was then collected by peeling it off using a glass slide and ground in liquid nitrogen. Genomic DNA was extracted from finely ground material using the CTAB method [B method . DNA qua2O was added to each plate and spores were peeled off using sterile writing brushes and filtered with three layers of sterile gauze to prepare the spore suspension. Spores were collected through centrifugation at 4602×g for 5 min. Spores were resuspended and adjusted to 105 spores/mL with sterile ddH2O. The spore suspension was incubated at 25 °C in the dark for 6 h and 12 h to perform the spore germination test. Equal volumes of the germinated spore suspensions incubated for 6 h or 12 h were mixed. The germinated spores in the mixed suspension were collected by centrifugation at 4602×g for 5 min. The germinated spores and ungerminated spores were ground for 1 min using a Geno/Grinder 2010  after being frozen rapidly with liquid nitrogen for the extraction of total RNA. Total RNA was extracted using the TaKaRa MiniBEST universal RNA extraction kit following the manufacturer’s recommended method and stored at − 80 °C. The test was conducted in biological triplicate.Spore production tests were conducted on PDA plates. After being activated on PDA plates at 25 °C, the HGCC strain was subcultured on PDA plates at 25 °C for 10 d with 12 h of light and 12 h of dark per day. Five milliliters of sterile ddHTotal RNA was quantified and assessed for purity using a Nanodrop instrument, and the integrity was evaluated using an Agilent 2100 instrument. RNA samples returning an RNA integrity number (RIN) greater than 6.3 were considered acceptable for sequencing. A cDNA library was made from each qualified RNA sample. The libraries were sequenced using an Illumina HiSeq X 10 instrument at the Novogene Bioinformatics Institute, Beijing.The whole genome of HGCC was de novo sequenced using an Illumina HiSeq X Ten in 2 × 149 bp paired-end mode with a 400 bp insert sizes of the library at Personalbio . High-quality data were obtained from raw data by removing adapter contamination with AdapterRemoval (version 2) , collectC. cassiicola genome sequences were predicted using Augustus software with the annotated gene information of B. cinerea [C. cassiicola was performed by sequence alignment against the Pfam database with profile hidden Markov models (http://xfam.org/) using hmmer 3.1b2 (http://www.hmmer.org/).Gene structures of  cinerea . Putativ cinerea , SignalP cinerea , TMHMM S cinerea , and Big cinerea . Putativ cinerea . ProteinC. cassiicola HGCC protein sequences against the PHI database  with a cutoff E-value of 1e-5. MAPK pathway associated genes of C. cassiicola were screened by local BLASTP against S. cerevisiae MAPK pathway-associated genes with a cutoff E-value of 1e-5 that were confirmed by experiments. GPCRs were identified by local BLASTP against the GPCRDB database (http://gpcrdb.org/) with the best hits and further confirmed by searching seven transmembrane helices with TMHMM Server v. 2.0. Kinases, proteases and transporters were identified by local BLASTP against the KinBase database with a cutoff E-value of 1e-10, the MEROPS peptidase database (https://www.ebi.ac.uk/merops/) with a cutoff E-value of 1e-20 [E-value of 1e-40, respectively [http://drnelson.uthsc.edu/CytochromeP450.html) and CAZy database (http://www.cazy.org/), respectively, with a cutoff E-value of 1e-10.Potential virulence-associated genes were identified by local BLASTP searches of of 1e-20 , and theof 1e-20  with a cectively . CYPs anhttp://smurf.jcvi.org/index.php) [C. cassiicola PKSs and other functionally known PKSs in other fungi were submitted to the SBSPKS database (http://www.nii.ac.in/~pksdb/sbspks/master.html) to modulate and analyze conserved domains. Phylogenetic analysis of PKSs was performed by aligning KS domain sequences and creating a maximum likelihood tree using MEGA 7.0 with Jones-Taylor-Thornton (JTT) model. These functionally known PKS-related toxin biosynthesis pathways included A. ochraceus AoLC35–12 (GenBank: AAT92023) for ochratoxin production, A. alternate ACTTS3 (GenBank: BAJ14522) for ACT-toxin production, B. maydis PKS1 (GenBank: AAB08104) and PKS2 (GenBank: AAR90257) for T-toxin production, G. zeae PKS4 (GenBank: ABB90283) for zearalenones production and G. moniliformis Fum1p (GenBank: AAD43562) for fumonisin production. Melanin-associated PKSs included A. fumigatus Alb1p (GenBank: ACJ13039), Ceratocystis resinifera PKS1 (GenBank: AAO60166), C. lagenarium PKS1 (GenBank: BAA18956), C. globosum PKS-1 (GenBank: AFP82905), A. rabie PKS1 (GenBank: ACS74449), B. maydis PKS18 (GenBank: AAR90272), S. turcica StPKS (GenBank: AEE68981), B. oryzae PKS1 (GenBank: BAD22832), and A. alternate ALM1 (GenBank: BAK64048).Secondary metabolism backbone genes such as PKS, NRPS and NRPS-PKS were identified in the SMURF system (dex.php) . C. cassC. cassiicola HGCC and the other two C. cassiicola isolates (UM591 and CCP) or other phytopathogenic fungi, the same pipeline was used for the gene prediction and annotation of the genome for all species. Accession numbers of the genomes are JAQF00000000.1 (GenBanK) for C. cassiicola UM591, GCA_003016335.1 (GenBanK) for C. cassiicola CCP, GCA_000743335.1 (GenBanK) for C. lunata, GCA_000338975.1 (GenBanK) for B. maydis, 2,761,201,826 (JGI) for C. zeae-maydis, GCA_002267025.1 (GenBanK) for P. nodorum, GCA_000359705.1 (GenBanK) for S. turcica, GCA_000149985.1 (GenBanK) for P. tritici-repentis, GCA_000002495.2 (GenBanK) for M. oryzae, GCA_009017415.1 (GenBanK) for A. flavus, GCA_000240135.3 (GenBanK) for F. graminearum, and GCA_000143535.4 (GenBanK) for B. cinerea. To construct an interspecific phylogenetic tree among these fungi, 2831 orthologous protein sequences were screened by local reciprocal Blast search with a cutoff of E-value 1e-20. These orthologous proteins were aligned using Clustal W 2.1 [To compare the gene annotation between C. cassiicola  compared to 10 other phytopathogenic fungi, including C. lunata, B. maydis, C. zeae-maydis, P. nodorum, S. turcica, P. tritici-repentis, M. oryzae, A. flavus, F. graminearum, and B. cinerea. The cutoff of significance was set at P < 0.05.Statistical analysis was conducted by a non-parametric test (Mann-Whitney U test) of SSPS v18.0 using the gene amount of each gene family in C. cassiicola RNA-Seq libraries were mapped on the predicted CDS of C. cassiicola using Bowtie2 with default settings [The quality of the raw reads generated from RNA-Seq was checked with FastQC . The clesettings . The numThe counts of RNA-Seq reads over transcripts were used to calculate the fold change of gene expression using DESeq2 . DEGs weGapdh gene was used as the internal control. The first strand of cDNA was synthesized from total RNA of germinated and ungerminated spores using EasyScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR  . The qRT-PCR test was conducted with a QuanStudio 6 Flex instrument . Twenty microliters of reaction mixture contained 2 μL diluted cDNA (1–100 ng), 0.4 μL of each primer (10 μM), 7.2 μL of nuclease-free water and 10 μL of 2 × TransStart® Top Green qPCR SuperMix . The reaction program was follows: one cycle at 94 °C for 30 s and 40 cycles at 94 °C for 5 s, 60 °C for 15 s and 72 °C for 10 s. The specificities of the amplification products were tested through a melting-curve analysis between 60 °C and 95 °C after each PCR reaction. The relative gene expression data were analyzed using the 2-ΔΔCt method [Sixty-six DEGs involved in the cell growth and death of cellular processes were selected for the confirmation of the RNA-Seq data using qRT-PCR . bP value (highlighted in bold for comparisons showing significant differences) is calculated by a non-parametric test (Mann-Whitney U test) of SSPS v18.0 using the gene amount of each family in C. cassiicola  compared to the 10 other plant pathogenic fungi (grey column). The cutoff of significance is set at P < 0.05 (highlighted). c PHI: pathogen-host interaction. d Percentage: The percentage of PHI-associated gene in protein-coding genes. Table S2 The number of genes for selected gene families in C. cassiicola and other ascomycetes. a PHI: pathogen-host interaction. b Average is calculated using the gene/PHI-associated gene amount of each family in the 10 other plant pathogenic fungi (grey column). cP value (highlighted in bold for comparisons showing significant differences) is calculated by a non-parametric test (Mann-Whitney U test) of SSPS v18.0 using the gene amount of each family in C. cassiicola  compared to the 10 other plant pathogenic fungi (grey column). The cutoff of significance is set at P < 0.05 (highlighted). Fungal species: HGCC, C. cassiicola HGCC; CCP, C. cassiicola CCP; UM591, C. cassiicola UM591; CL, C. lunata; BM, B. maydis; CZM, C. zeae-maydis; PN, P. nodorum; ST, S. turcica; PTR, P. tritici-repentis; MO, M. oryzae; AF, A. flavus; FG, F. graminearum; BC, B. cinerea. Table S3 G protein-coupled receptors in different fungal genomes. a PHI: pathogen-host interaction. b Average is calculated using the gene/PHI-associated gene amount of each family in the 10 other plant pathogenic fungi (grey column). cP value (highlighted in bold for comparisons showing significant differences) is calculated by a non-parametric test (Mann-Whitney U test) of SSPS v18.0 using the gene amount of each family in C. cassiicola  compared to the 10 other plant pathogenic fungi (grey column). The cutoff of significance is set at P < 0.05 (highlighted). Fungal species: HGCC, C. cassiicola HGCC; CCP, C. cassiicola CCP; UM591, C. cassiicola UM591; CL, C. lunata; BM, B. maydis; CZM, C. zeae-maydis; PN, P. nodorum; ST, S. turcica; PTR, P. tritici-repentis; MO, M. oryzae; AF, A. flavus; FG, F. graminearum; BC, B. cinerea. Table S4 Gene-encoding proteins for the MAPK signaling pathway in C. cassiicola HGCC. The colour of the protein names indicates the degree of conservation between S. cerevisiae and C. cassiicola HGCC proteins based on Blastp. Blue, e-value <1e-5 and > 1e-20; Green, e-value <1e-100 and > 1e-20; Red, e-value <1e-100. Table S5 Gene-encoding proteins for the cAMP signaling pathway in C. cassiicola HGCC. Table S6 Gene-encoding proteins for the Ca2+ signaling pathway in C. cassiicola HGCC. Table S7 The number of protein kinases in different fungal genomes. a PHI: pathogen-host interaction. b Average is calculated using the gene/PHI-associated gene amount of each family in the 10 other plant pathogenic fungi (grey column). cP value (highlighted in bold for comparisons showing significant differences) is calculated by a non-parametric test (Mann-Whitney U test) of SSPS v18.0 using the gene amount of each family in C. cassiicola  compared to the 10 other plant pathogenic fungi (grey column). The cutoff of significance is set at P < 0.05 (highlighted). Fungal species: HGCC, C. cassiicola HGCC; CCP, C. cassiicola CCP; UM591, C. cassiicola UM591; CL, C. lunata; BM, B. maydis; CZM, C. zeae-maydis; PN, P. nodorum; ST, S. turcica; PTR, P. tritici-repentis; MO, M. oryzae; AF, A. flavus; FG, F. graminearum; BC, B. cinerea. Table S8 Protease genes classified by the MEROPS family in different fungal genomes. a PHI: pathogen-host interaction. b Average is calculated using the gene/PHI-associated gene amount of each family in the 10 other plant pathogenic fungi (grey column). cP value (highlighted in bold for comparisons showing significant differences) is calculated by a non-parametric test (Mann-Whitney U test) of SSPS v18.0 using the gene amount of each family in C. cassiicola  compared to the 10 other plant pathogenic fungi (grey column). The cutoff of significance is set at P < 0.05 (highlighted). Fungal species: HGCC, C. cassiicola HGCC; CCP, C. cassiicola CCP; UM591, C. cassiicola UM591; CL, C. lunata; BM, B. maydis; CZM, C. zeae-maydis; PN, P. nodorum; ST, S. turcica; PTR, P. tritici-repentis; MO, M. oryzae; AF, A. flavus; FG, F. graminearum; BC, B. cinerea. Table S9 Glycoside hydrolase in C. cassiicola and other phytopathogenic ascomycetes. a PHI: pathogen-host interaction. b Average is calculated using the gene/PHI-associated gene amount of each family in the 10 other plant pathogenic fungi (grey column). cP value (highlighted in bold for comparisons showing significant differences) is calculated by a non-parametric test (Mann-Whitney U test) of SSPS v18.0 using the gene amount of each family in C. cassiicola  compared to the 10 other plant pathogenic fungi (grey column). The cutoff of significance is set at P < 0.05 (highlighted). Fungal species: HGCC, C. cassiicola HGCC; CCP, C. cassiicola CCP; UM591, C. cassiicola UM591; CL, C. lunata; BM, B. maydis; CZM, C. zeae-maydis; PN, P. nodorum; ST, S. turcica; PTR, P. tritici-repentis; MO, M. oryzae; AF, A. flavus; FG, F. graminearum; BC, B. cinerea. Table S10 Transporters in C. cassiicola and other phytopathogenic ascomycetes. a PHI: pathogen-host interaction. b Average is calculated using the gene/PHI-associated gene amount of each family in the 10 other plant pathogenic fungi (grey column). cP value (highlighted in bold for comparisons showing significant differences) is calculated by a non-parametric test (Mann-Whitney U test) of SSPS v18.0 using the gene amount of each family in C. cassiicola  compared to the 10 other plant pathogenic fungi (grey column). The cutoff of significance is set at P < 0.05 (highlighted). Fungal species: HGCC, C. cassiicola HGCC; CCP, C. cassiicola CCP; UM591, C. cassiicola UM591; CL, C. lunata; BM, B. maydis; CZM, C. zeae-maydis; PN, P. nodorum; ST, S. turcica; PTR, P. tritici-repentis; MO, M. oryzae; AF, A. flavus; FG, F. graminearum; BC, B. cinerea. Table S11 Drug transporters in C. cassiicola and other phytopathogenic ascomycetes. a PHI: pathogen-host interaction. b Average is calculated using the gene/PHI-associated gene amount of each family in the 10 other plant pathogenic fungi (grey column). cP value (highlighted in bold for comparisons showing significant differences) is calculated by a non-parametric test (Mann-Whitney U test) of SSPS v18.0 using the gene amount of each family in C. cassiicola  compared to the 10 other plant pathogenic fungi (grey column). The cutoff of significance is set at P < 0.05 (highlighted). Fungal species: HGCC, C. cassiicola HGCC; CCP, C. cassiicola CCP; UM591, C. cassiicola UM591; CL, C. lunata; BM, B. maydis; CZM, C. zeae-maydis; PN, P. nodorum; ST, S. turcica; PTR, P. tritici-repentis; MO, M. oryzae; AF, A. flavus; FG, F. graminearum; BC, B. cinerea. Table S12 Cytochrome P450 genes classified by the CYP family in different fungal genomes. a PHI: pathogen-host interaction. b Average is calculated using the gene/PHI-associated gene amount of each family in the 10 other plant pathogenic fungi (grey column). cP value (highlighted in bold for comparisons showing significant differences) is calculated by a non-parametric test (Mann-Whitney U test) of SSPS v18.0 using the gene amount of each family in C. cassiicola  compared to the 10 other plant pathogenic fungi (grey column). The cutoff of significance is set at P < 0.05 (highlighted). Fungal species: HGCC, C. cassiicola HGCC; CCP, C. cassiicola CCP; UM591, C. cassiicola UM591; CL, C. lunata; BM, B. maydis; CZM, C. zeae-maydis; PN, P. nodorum; ST, S. turcica; PTR, P. tritici-repentis; MO, M. oryzae; AF, A. flavus; FG, F. graminearum; BC, B. cinerea. Table S13 Numbers of backbone genes for the biosynthesis of secondary metabolites in different pathogenic fungi. a PHI: pathogen-host interaction. b Average is calculated using the gene/PHI-associated gene amount of each family in the 10 other plant pathogenic fungi (grey column). cP value (highlighted in bold for comparisons showing significant differences) is calculated by a non-parametric test (Mann-Whitney U test) of SSPS v18.0 using the gene amount of each family in C. cassiicola  compared to the 10 other plant pathogenic fungi (grey column). The cutoff of significance is set at P < 0.05 (highlighted). Fungal species: HGCC, C. cassiicola HGCC; CCP, C. cassiicola CCP; UM591, C. cassiicola UM591; CL, C. lunata; BM, B. maydis; CZM, C. zeae-maydis; PN, P. nodorum; ST, S. turcica; PTR, P. tritici-repentis; MO, M. oryzae; AF, A. flavus; FG, F. graminearum; BC, B. cinerea. Table S14 Putative small, cysteine-rich proteins (SCRPs) in C. cassiicola HGCC.Additional file 2: Table S15 Sequencing yield from individual libraries per sample. a CC_0_1, b CC_0_2 and c CC_0_3 are triplicate ungerminated spore samples. d CC_6_12_1, e CC_6_12_2, and f CC_6_12_3 are triplicate mixed 6 h- and 12 h-germinated spore samples. Table S16 3288 DEGs during spore germination identified by RNA-Seq. a CC_6_12: mixed 6 h- and 12 h-germinated spore samples; b CC_0: ungerminated spore samples. Table S17 KEGG pathway enrichment analysis of DEGs. Table S18 qRT-PCR for selected DEGs involved in cell growth and death. * The express pattern of DEGs from RNA-Seq was not validated by qRT-PCR.Additional file 3: Fig. S1 Pearson correlation between samples in gene expression levels. CC_0_1, CC_0_2 and CC_0_3 are triplicate ungerminated spore samples. CC_6_12_1, CC_6_12_2, and CC_6_12_3 are triplicate mixed 6 h- and 12 h-germinated spore samples. R2 is the square of the Pearson correlation coefficient.


The inhibitory activities of AChE and BChE were evaluated in vitro by Ellman method. The results show that some compounds have good inhibitory activity against AChE and BChE. Among them, compound 8i showed the strongest inhibitory effect on both AChE (eeAChE IC50 = 0.39 μM) and BChE (eqBChE IC50 = 0.28 μM). Enzyme inhibition kinetics and molecular modeling studies have shown that compound 8i bind simultaneously to the peripheral anionic site (PAS) and the catalytic sites (CAS) of AChE and BChE. In addition, the cytotoxicity of compound 8i is lower than that of Tacrine, indicating its potential safety as anti-Alzheimer’s disease (anti-AD) agents. In summary, these data suggest that compound 8i is a promising multipotent agent for the treatment of AD.A series of novel compounds  According to the report of the International Alzheimer’s Association in the last three years, there are more than 36 million AD patients in the world. With the acceleration of the aging process of the population, the amount of AD incidence is increasing year by year, and the number of AD patients in the world will exceed 130 million by 2050 . AD is tAD was initially characterized by memory loss, cognitive dysfunction, inability to take care of themselves in daily life, and subsequent exacerbations of mental and behavioral abnormalities ,8. AD isAD is a complex neurodegenerative syndrome. Due to the complicated pathogenesis of AD, its etiology is not completely clear. The therapeutic drugs used in the clinical and research stages can only delay the course of AD, but there are no drugs that cure or delay the course of AD ,12. A laPrevious studies have identified three pathological features of AD: decreased levels of AChE in the neurotransmitter matrix, deposition of Aβ, and hyperphosphorylation of tau protein. Scientists have been trying to find the etiology and treatment strategy of AD through further study of the above three pathological characteristics ,20. At pThere are two types of ChEs in the central nervous system, namely AChE and BChE. AChE and BChE are important targets for the development of anti-AD drugs. The physiological function of AChE has been preliminarily understood, but the understanding of BChE is less. Studies have shown that ACh activity in some brain regions of patients with mild to severe AD decreases to 10%–15% of the normal value, and AChEIs have significant effects on patients with mild to moderate AD and can repair their cognitive impairment and other symptoms .When AD develops to the middle and late stage, AChE activity decreases, whereas BChE activity increases, and BChE acts as a metabolic compensation for AChE, partially compensating for the role of AChE in hydrolyzing ACh ,23,24. TWith the study of AChE crystal structure, it was found that the ligand binding pocket of AChE in general is a long and narrow channel extending from the surface to the interior . The chaG801-0274 AChE (eeAChE IC50 = 2.05 μM), BChE (eqBChE IC50 = 0.03 μM) can inhibit ChEs. In this paper, we used it as the lead compound for structural modification   triazole (11f) > 1H-Benzo[d]imidazole (6d) > 1H-Indazole (6c) > 1H-Indole (6a); its activity on BChE are 2-Oxoindoline (8i) > 1H-Benzo[d]imidazole (6d) > 1H-Indole (6a) > 1H-Benzo[d]  triazole (11f). Among them, compound 8i (50 = 0.39 μM) and BChE (eqBChE IC50 = 0.28 μM), these results indicated that compound 8i was a potent dual inhibitor against AChE and BChE. It was speculated that 2-Oxoindoline is the key structure for inhibiting two ChEs and is not selective for both ChEs. According to the molecular docking results, for eeAChE, 2-Oxoindoline of compound 8i bound with Trp286 via π-π stacking interaction; for huAChE, 2-Oxoindoline of compound 8i bound with Trp286 and Tyr341 via π-π stacking interaction, and for huBChE, 2-Oxoindoline of compound 8i bound with Phe329 via π-π stacking interaction. These interactions increase the inhibitory activity by enhancing the binding affinity.First, we synthesized compounds pound 8i  showed t3, 4-CF3), compared with compound 6d, when R is 4-CF3 (6e), 4-OCH3 (11d), 2-Cl (11e), the ChEs inhibitory activity decreased. In particular, compound 11d showed little inhibitory activity on BChE at 100 μM. In general, with the same Y, when R is H, the compounds have the highest inhibitory activity against ChEs; when Y is substituted by 2-Cl or 4-CF3, the inhibitory activity of the compounds to ChEs were weakened; and when Y is substituted by 4-OCH3, the compounds have the worst inhibitory activity against AChEs. We speculate that enhancing the electron-withdrawing effect or the donor effect on the aromatic ring is not conducive to improving the performance of the analog, and the appropriate space may facilitate the analog to enter the CAS pocket of ChEs.Then, we evaluated the effect of R on ChEs activity. We modified R with different substituent  in the previous compounds was replaced by Tosyl moiety. We synthesized compounds 10s–v. Except for compound 10u  has low inhibitory activity against ChEs, the compounds 10s ), 10t , 10v  all can maintain ChEs inhibitory activity at micromolar levels, indicating that the p-Toluenesulfonamide moiety is responsible for maintaining the inhibitory activity of ChEs. On one hand, we speculate that methyl occupies the pocket of the active site and interacts with amino acid residues to increase inhibitory activity. On the other hand, Sulfonamide moiety is very important for maintaining ChEs inhibitory activity, which may be related to the bond angle between Sulfonamide and molecules + 348.2070, found 348.2070.N-((1-(2-Chlorobenzyl)piperidin-4-yl)methyl)-1H-Indole-5-carboxamide (6b). 1H-Indole-5-carboxylic acid , CDI , (1-(2-Chlorobenzyl) piperidin-4-yl)methanamine , THF (15 mL) White solid, m.p.: 88–89 °C, yield: 66%, 1H NMR  δ 8.08 , 7.58 , 7.49 , 7.41–7.35 , 7.30–7.24 , 6.51 , 3.78 , 3.05 , 2.31 , 1.77 , 1.45–1.33 . 13C NMR  δ 170.60, 138.15, 134.64, 131.71, 129.37, 129.10, 127.70, 126.78, 125.91, 125.17, 120.18, 119.95, 110.67, 102.17, 58.57, 53.06, 44.79, 35.56, 28.93. HRMS (ESI): calcd. For C22H24ClN3O [M + H]+ 382.1681, found 382.1703.N-((1-Benzylpiperidin-4-yl) methyl)-1H-indazole-5-carboxamide (6c). 1H-Indazole-5-carboxylic acid , CDI , (1-Benzylpiperidin-4-yl)methanamine , THF (15 mL) White solid, m.p.: 111–112 °C, yield: 56%, 1H NMR  δ 13.30 , 8.48 , 8.35 , 8.20 , 7.87 , 7.57 , 7.33–7.27 , 7.24 , 3.45 , 3.18 , 2.81 , 1.92 , 1.67 , 1.59 , 1.22 . 13C NMR  δ 167.07, 141.33, 138.83, 135.11, 129.24, 128.56, 127.67, 127.62, 127.30, 125.79, 122.79, 120.90, 110.16, 62.78, 53.38, 45.31, 36.17, 30.23. HRMS (ESI): calcd. For C21H24N4O [M + H]+ 349.2023, found 349.2019.N-((1-Benzylpiperidin-4-yl)methyl)-1H-benzo[d]imidazole-5-carboxamide (6d). 1H-Benzo[d]imidazole-5-carboxylic acid , CDI , (1-Benzylpiperidin-4-yl)methanamine , THF (15 mL) Yellow oil, yield: 72%, 1H NMR  δ 8.53 , 8.27 , 7.78 , 7.70–7.61 , 7.28 , 7.21 , 7.04 , 3.39 , 3.19 , 2.76 , 1.85 , 1.65 , 1.20 . 13C NMR  δ 167.40, 144.24, 139.08, 135.64, 129.16, 128.51, 127.18, 121.88, 115.66, 114.85, 62.93, 53.43, 53.35, 45.39, 36.25, 30.32. HRMS (ESI): calcd. For C21H24N4O [M + H]+ 349.2023, found 349.2023.N-((1-(4-(Trifluoromethyl)benzyl)piperidin-4-yl)methyl)-1H-benzo[d]imidazole-5-carboxamide (6e). 1H-Benzo[d]imidazole-5-carboxylic acid , CDI , (1-(4-(Trifluoromethyl)benzyl)piperidin-4-yl)methanamine , THF (15 mL) White solid, m.p.: 92–93 °C, yield: 77%, 1H NMR  δ 8.29–8.27 , 8.12 , 7.87–7.77 , 7.62 , 7.56 , 7.39 , 3.66 , 3.30 , 3.27 , 2.90 , 2.15–2.06 , 1.79–1.63 , 1.44–1.24 . 13C NMR  δ 169.23, 141.32, 134.81, 131.92, 130.75, 130.34, 128.00, 127.37, 127.30, 125.41, 122.55, 120.67, 114.13, 113.58, 109.69, 58.94, 57.96, 53.34, 53.01, 44.93, 44.83, 35.64, 35.52, 29.26, 29.06. HRMS (ESI): calcd. For C22H23F3N4O [M + H]+ 417.1897, found 417.1895.N-((1-(4-Methoxybenzyl)piperidin-4-yl)methyl)-1H-benzo[d]triazole-5-carboxamide (6f). 1H-Benzo[d]triazole-5-carboxylic acid , CDI , (1-(4-Methoxybenzyl)piperidin-4-yl)methanamine , THF (15 mL) Yellow oil, yield: 63%, 1H NMR  δ 8.53 , 8.32 , 8.16 , 7.75 , 7.61 , 7.32 , 6.91 , 3.74 , 3.69 , 3.18 , 2.97 , 2.26 , 1.73 , 1.68 , 1.34 . 13C NMR  δ 167.31, 159.33, 144.22, 131.57, 128.92, 121.94, 114.16, 60.78, 55.52, 52.34, 44.84, 35.34, 28.86. HRMS (ESI): calcd. For C21H25N5O2 [M + H]+ 380.2081, found 380.2077.N-((1-(2-Chlorobenzyl)piperidin-4-yl)methyl)-1H-benzo[d]triazole-5-carboxamide (6g). 1H-Benzo[d]triazole-5-carboxylic acid , CDI , (1-(2-Chlorobenzyl)piperidin-4-yl)methanamine , THF (15 mL) White solid, m.p.: 77–79 °C, yield: 57%, 1H NMR  δ 8.63 , 8.44 , 7.87 , 7.47 , 7.39 , 7.30 , 7.27–7.25 , 7.03 , 3.52 , 3.21 , 2.82 , 2.00 , 1.68 , 1.61 , 1.23 . 13C NMR  δ 166.50, 140.30, 139.38, 138.70, 131.96, 129.27, 128.56, 127.32, 125.46, 115.61, 114.32, 62.73, 53.33, 45.41, 36.08, 30.16. HRMS (ESI): calcd. For C20H22ClN5O [M + H]+ 384.1586, found 384.1584.N-((1-(4-(Trifluoromethyl)benzyl)piperidin-4-yl)methyl)-1H-benzo[d]triazole-5-carboxamide (6h). 1H-Benzo[d]triazole-5-carboxylic acid , CDI , (1-(4-(Trifluoromethyl)benzyl)piperidin-4-yl)methanamine , THF (15 mL) White solid, m.p.: 90–91 °C, yield: 73%, 1H NMR  δ 8.38 , 7.92–7.90 , 7.88 , 7.81 , 7.68 , 7.61 , 7.51–7.42 , 3.89 , 3.33 , 3.13–3.01 , 2.47–2.31 , 1.90–1.68 , 1.52–1.32 . 13C NMR  δ 166.50, 140.30, 139.38, 138.70, 131.96, 129.27, 128.56, 127.32, 125.46, 115.61, 114.32, 62.73, 53.33, 45.41, 36.08, 30.16. HRMS (ESI): calcd. For C21H22F3N5O [M + H]+ 418.1849, found 418.1850.7) , PyBOP (1.2 equiv.) and DIPEA (1.5 equiv.) were added to DMF and stirred at room temperature for 20 min. Then, intermediates (4a–d) (1.0 equiv.) was added and stirred at room temperature for 4 h. After completion of the reaction, the reaction mixture was quenched with saturated NaCl solution (25 mL). The aqueous phase was extracted with DCM (25 × 3 mL). The DCM layer was combined and washed with brine solution (25 × 3 mL). The organic layer was dried over anhydrous Na2SO4 and the solvent was removed under reduced pressure. After concentration, the crude product was purified by silica gel column chromatograph using a methanol in DCM gradient (DCM: methanol= 60:1–5:1) yielded compounds 8i–l.Intermediates (N-((1-Benzylpiperidin-4-yl)methyl)-2-Oxoindoline-5-carboxamide (8i). 2-Oxoindoline-5-carboxylic acid , (1-Benzylpiperidin-4-yl)methanamine , PyBOP , DIPEA, DMF (6 mL). White solid, m.p.:155–156 °C, yield: 56%, 1H NMR  δ 7.73–7.68 , 7.53–7.42 , 6.91 , 4.26 , 3.44 , 3.31 , 2.98 , 1.97 , 1.54 . 13C NMR  δ 178.52, 168.90, 146.69, 130.99, 129.80, 129.40, 128.99, 127.92, 127.60, 125.95, 123.42, 123.40, 108.97, 60.21, 51.86, 43.82, 42.11, 34.01, 26.82. HRMS (ESI): calcd. For C22H25N3O2 [M + H]+ 364.2020, found 364.2032.N-((1-(2-Chlorobenzyl)piperidin-4-yl)methyl)-2-Oxoindoline-5-carboxamide (8j). 2-Oxoindoline-5-carboxylic acid , (1-(2-Chlorobenzyl)piperidin-4-yl)methanamine , PyBOP , DIPEA, DMF (6 mL). White solid, m.p.: 151–152 °C, yield: 35%, 1H NMR  δ 7.74–7.69 , 7.50 , 7.39 , 7.28 , 6.93 , 3.71 , 3.27 , 3.00 , 2.21 , 1.76 , 1.69 , 1.37 . 13C NMR  δ 168.83, 146.63, 135.02, 132.40, 130.20, 129.68, 128.07, 127.58, 127.18, 125.93, 123.41, 108.96, 57.94, 52.76, 44.36, 34.83, 28.01. HRMS (ESI): calcd. For C22H24ClN3O2 [M + H]+ 398.1630, found 398.1652.N-((1-(4-Methoxybenzyl)piperidin-4-yl)methyl)-2-Oxoindoline-5-carboxamide (8k). 2-Oxoindoline-5-carboxylic acid , (1-(4-Methoxybenzyl)piperidin-4-yl)methanamine , PyBOP , DIPEA, DMF (6 mL). White solid, m.p.: 158–159 °C, yield: 43%, 1H NMR  δ 7.74–7.68 , 7.40 , 6.98 , 6.91 , 4.19 , 3.79 , 3.43 , 3.31 , 2.95 , 1.97 , 1.60–1.45 , 1.26 . 13C NMR  δ 178.54, 168.87, 161.10, 146.66, 132.51, 127.95, 127.62, 125.93, 123.46, 121.09, 114.21, 108.98, 54.55, 51.51, 43.78, 34.03, 27.22, 26.85. HRMS (ESI): calcd. For C23H27N3O3 [M + H]+ 394.2125, found 394.2131.2-Oxo-N-((1-(4-(trifluoromethyl)benzyl)piperidin-4-yl)methyl)indoline-5-carboxamide (8l). 2-Oxoindoline-5-carboxylic acid , (1-(4-(Trifluoromethyl)benzyl)piperidin-4-yl)methanamine , PyBOP , DIPEA, DMF (6 mL). White solid, m.p.: 113–115 °C, yield: 40%, 1H NMR  δ 7.79 , 7.73–7.69 , 7.62 , 7.56 , 7.44–7.32 , 6.95–6.90 , 3.60 , 3.24 , 2.91–2.77 , 2.09–1.93 , 1.78–1.61 , 1.40–1.21 . 13C NMR ) δ 168.70, 146.44, 131.73, 130.35, 130.30, 128.23, 127.47, 127.30, 126.79, 125.81, 125.26, 123.34, 108.90, 59.79, 59.79, 58.24, 53.43, 53.14, 45.07, 36.04, 36.04, 36.00, 36.00, 29.82, 29.76. HRMS (ESI): calcd. For C23H24F3N3O2 [M + H]+ 432.1893, found 432.1886.4a–d)  in DCM (5 mL) was added Et3N (1.8 equiv.). The mixture was cooled to 0 °C, TsCl (1.3 equiv.) in DCM (5 mL) was added dropwise, and the reaction mixture was stirred for an additional 1 h at 0 °C. After completion of the reaction, the reaction mixture was dissolved in 15 mL saturated sodium bicarbonate (NaHCO3) and extracted with DCM (25 × 3 mL). Organic phases were combined and washed with saturated NaCl solution (25 × 3 mL). The organic layer was dried over anhydrous Na2SO4 and the solvent was removed under reduced pressure. After concentration, the crude product was purified by silica gel column chromatograph using a methanol in dichloromethane gradient (dichloromethane: methanol = 60:1–5:1) yielded compounds 10s–v.To a solution of (1-Benzylpiperidin-4-yl) methanamine derivatives (N-((1-Benzylpiperidin-4-yl)methyl)-4-methylbenzenesulfonamide (10s). (1-Benzylpiperidin-4-yl) methanamine , TsCl , Et3N , DCM (9 mL) White solid, m.p.:78–79 °C, yield: 80%, 1H NMR  δ 7.68 , 7.36–7.24 , 3.60 , 2.93 , 2.67 , 2.39 , 2.09 , 1.68 , 1.44 , 1.28–1.13 . 13C NMR  δ 143.22, 137.71, 136.09, 129.69, 129.36, 128.05, 127.39, 126.67, 62.59, 52.72, 35.66, 28.79, 20.11. HRMS (ESI): calcd. For C20H26N2O2S [M + H]+ 359.1788, found 359.1802.N-((1-(2-Chlorobenzyl)piperidin-4-yl)methyl)-4-methylbenzenesulfonamide (10t). (1-(4-Chlorobenzyl)piperidin-4-yl)methanamine , TsCl , Et3N , DCM (9 mL) White solid, m.p.: 111–112 °C, yield: 73%, 1H NMR  δ 7.69 , 7.44 , 7.37–7.31 , 7.23 , 3.62 , 2.89 , 2.67 , 2.39 , 2.06 , 1.65 , 1.37 , 1.20 , 1.15 . 13C NMR  δ 143.42, 137.49, 135.59, 133.53, 131.79, 130.10, 129.48, 127.74, 127.35, 126.68, 126.68, 20.13. HRMS (ESI): calcd. For C20H25ClN2O2S [M + H]+ 393.1398, found 393.1397.N-((1-(4-Methoxybenzyl)piperidin-4-yl)methyl)-4-methylbenzenesulfonamide (10u). (1-(4-Methoxybenzyl)piperidin-4-yl)methanamine , TsCl , Et3N , DCM (9 mL) White solid, m.p.: 76–77 °C, yield: 87%, 1H NMR  δ 7.61 , 7.25 , 6.85 , 3.80 , 3.70 , 3.10 , 2.63 , 2.44 , 2.32 , 2.05 , 1.73 , 1.52 , 1.27–1.19 . 13C NMR  δ 160.41, 143.33, 137.60, 131.82, 129.42, 126.66, 113.87, 60.67, 54.47, 51.88, 34.72, 27.47, 20.10. HRMS (ESI): calcd. For C21H28N2O3S [M + H]+ 389.1893, found 389.1894.4-Methyl-N-((1-(4-(trifluoromethyl)benzyl)piperidin-4-yl)methyl)benzenesulfonamide (10v). (1-(4-(Trifluoromethyl)benzyl)piperidin-4-yl)methanamine , TsCl , Et3N , DCM (9 mL) White solid, m.p.: 87–88 °C, yield: 88%, 1H NMR  δ 7.76 , 7.71–7.66 , 7.61 , 7.57–7.51 , 7.40–7.31 , 3.58 , 2.79 , 2.67 , 2.40 , 1.97 , 1.69–1.59 , 1.40 , 1.15 . 13C NMR  δ 143.14, 137.67, 133.70, 131.73, 130.36, 130.33, 130.03, 129.28, 127.36, 126.84, 126.60, 125.27, 113.19, 59.65, 58.14, 53.26, 52.97, 48.19, 48.15, 48.12, 48.06, 47.95, 47.89, 47.78, 47.72, 47.61, 47.44, 47.32, 47.27, 47.10, 35.94, 35.86, 29.45, 20.03. HRMS (ESI): calcd. For C21H25F3N2O2S [M + H]+ 427.1662, found 427.1660.11  and substituted (2-romoethyl)benzene derivatives (12a–b)  were dissolved in 20 mL acetone. Then, anhydrous K2CO3  and catalytic amount KI were added. The reaction mixture was refluxed for 4 h. After completion of the reaction, acetone was concentrated, and the residue was dissolved in water (60 mL) and extracted with ethyl acetate (60 × 3 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuum. The obtained oil was used in further synthesis without purification yielded compounds 13a–b (Yields were 67% and 72%). Then, 4 mol/L KOH (2.5 equiv.) was added to the solution of compounds 13a–b in C2H5OH: H2O = 5:1(6 mL). The reaction mixture was stirred at room temperature for 7 h. After completion of the reaction, the reaction mixture was evaporated to dryness after neutralization with dilute hydrochloric acid solution. Poured into ethyl acetate to deposit the solid, after cooling off, the mixture was filtered and washed with cold ethyl acetate to give compounds 14a–d.Ethyl 2-piperidin-4-ylacetate 14a–b) (1.2 equiv.), PyBOP (1.2 equiv.) and DIPEA (1.5 equiv.) were added to 6 mL DMF and stirred at room temperature for 20 min. Then, intermediates (15a–b) (1.0 equiv.) was added and stirred at room temperature for 4 h. After completion of the reaction, the reaction mixture was quenched with saturated NaCl solution. The aqueous phase was extracted with DCM. The DCM layer was combined and washed with brine solution. The organic layer was dried over anhydrous Na2SO4 and the solvent was removed under reduced pressure. After concentration, the crude product was purified by silica gel column chromatograph using a methanol in dichloromethane gradient (DCM:methanol = 60:1–5:1) yielded compounds 16a–d.Finally, intermediates (N-(1H-Indol-5-yl)-2-(1-phenethylpiperidin-4-yl)acetamide (16a). 2-(1-Phenethylpiperidin-4-yl) acetic acid , 1H-Indol-5-amine , PyBOP , DIPEA , DMF (6 mL). Yellow solid, m.p.: 165–167 °C, yield: 80.70%, 1H NMR  δ 7.74 , 7.28 , 7.20 , 7.15 , 6.37 , 3.60 , 3.26 , 3.08–3.01 , 2.39 , 2.21–2.13 , 2.04 , 1.74–1.60 , 1.27 . 13C NMR  δ 170.66, 136.38, 133.81, 129.79, 128.59, 128.42, 127.97, 126.86, 125.29, 115.83, 115.76, 112.47, 110.75, 101.07, 72.24, 70.09, 60.80, 57.69, 53.42, 52.37, 41.90, 31.68, 31.23, 29.38, 28.89, 22.34, 13.05. HRMS (ESI): calcd. For C23H27N3O [M + H]+ 362.2227, found 362.2242.2-(1-(2-Chlorophenethyl)piperidin-4-yl)-N-(1H-indol-5-yl)acetamide (16b). 2-(1-(2-Chlorophenethyl)piperidin-4-yl)acetic acid , 1H-Indol-5-amine , PyBOP , DIPEA, DMF (6 mL). Yellow solid, m.p.: 172–173 °C, yield: 30.41%, 1H NMR  δ 7.75 , 7.39–7.29 , 7.28–7.20 , 7.19 , 7.16 , 6.37 , 3.45 , 3.15–3.09 , 3.08–3.01 , 2.79 , 2.36 , 2.10 , 2.00–1.92 , 1.60 . 13C NMR  δ 171.22, 135.56, 133.78, 133.57, 130.84, 129.85, 129.30, 128.28, 127.96, 127.17, 125.21, 115.80, 112.48, 110.73, 101.06, 56.91, 52.68, 42.55, 32.24, 29.95, 29.32, 29.01. HRMS (ESI): calcd. For C23H26ClN3O [M + H]+ 396.1837, found 396.1838.N-(1H-Benzo[d]imidazol-5-yl)-2-(1-phenethylpiperidin-4-yl)acetamide (16c). 2-(1-phenethylpiperidin-4-yl)acetic acid , 1H-Benzo[d]imidazol-5-amine , PyBOP , DIPEA, DMF (6 mL). Red solid, m.p.: 167–168 °C, yield: 45.92%, 1H NMR  δ 8.09 , 8.03 , 7.52 , 7.29–7.12 , 3.07 , 2.84–2.78 , 2.64–2.58 , 2.32 , 2.17 , 1.93 , 1.81 , 1.42 . 13C NMR  δ 171.84, 141.67, 139.67, 133.93, 128.33, 128.21, 128.20, 125.93, 116.32, 60.33, 53.19, 47.89, 43.30, 33.27, 32.51, 31.15. HRMS (ESI): calcd. For C22H26N4O [M + H]+ 363.2179, found 363.2197.N-(1H-Benzo[d]imidazol-5-yl)-2-(1-(2-chlorophenethyl)piperidin-4-yl)acetamide (16d). 2-(1-(2-Chlorophenethyl)piperidin-4-yl)acetic acid , 1H-Benzo[d]imidazol-5-amine , PyBOP , DIPEA, DMF (6 mL). Red solid, m.p.: 176–177 °C, yield: 40.33%, 1H NMR  δ 7.95 , 7.70 , 7.66 , 7.48 , 7.35 , 7.24 , 7.19 , 6.90 , 3.62 , 3.57 , 3.37 , 2.95 , 2.10 , 1.75 , 1.66 , 1.60 , 1.39 . 13C NMR  δ 171.90, 141.65, 137.34, 134.98, 133.95, 133.58, 130.73, 129.20, 127.73, 126.97, 121.27, 116.28, 58.31, 58.27, 53.17, 53.06, 50.62, 43.39, 40.15, 33.39, 32.59, 31.29, 31.15, 30.24. HRMS (ESI): calcd. For C22H25ClN4O [M + H]+ 397.1790, found 397.1797.The inhibitory activity of the target compound against AChE (from Electrophorus electricus (eeAChE), Sigma-Aldrich, Munich, Germany) and horse serum BChE  were measured by Ellman’s method . In 96-w50) value was calculated according to the inhibition curve and the data were shown in the layout of mean ± SEM by GraphPad Prism 6.0.Changes in absorbance were measured at 412 nm by using microplate reader . The measurement of each concentration for each compound was detected in triplicate. GraphPad Prism 6.0  was used for data processing. The inhibition curve was fitted by plotting the logarithm of the concentration of the tested compounds with the percentage of enzyme activity (reference to 100%). The  concentrations of 90, 150, 226, 452 and 904 μM. The concentration of compound 8i. The three protein structures are pretreated  by the “prepare protein” module in DS to provide the structures suitable for docking. The “prepare ligand” module in DS is used to test the structural preparation of the compound. The native ligand in the crystal structure was used to define the binding site. The binding site was defined as the site sphere ((in 10 Å radius) around the original ligand in the co-crystal structures. The docking program CDOCKER encoded in DS 2016 was applied to identify the potential binding of compound 8i to eeAChE, huAChE, and huBChE. Other CDOCKER parameters were set to default values. Compound 8i was chosen for molecular modeling as the most active compounds in the series  , huAChE e series . Compoun2. To carry out the experiment, cells (6 × 103 cells/well) were seeded in 96-well plate in complete medium. After 24 h, the culture medium was removed and the cells were exposed to increasing concentrations of compounds 6b, 6c, 6d, 8i, 10s, 10v or Tacrine  in DMEM for further 24 h. Cell survival was measured by 3--2,5-Diphenyltetrazolium Bromide (MTT) assay [Pheochromocytoma-derived cell line (PC12 cells) were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS at 37 °C in a humidified atmosphere containing 5% CO6a–h, 8i–1, 10s–v, and 16a–d were synthesized and evaluated, together with the known analogs 11a–f, for their inhibitory activities towards AChE and BChE. The results show that most of the compounds have AChE and/or BChE inhibitory activity. Compound 8i showed the strongest inhibitory effect on both AChE (eeAChE IC50 = 0.39 μM) and BChE (eqBChE IC50 = 0.28 μM). Compared with compound G801-0274, compound 8i has comparable inhibitory activity against two ChEs, so that it can exert an anti-ChEs effect in a balanced manner. Kinetic studies indicated a mixed-type inhibition of compound 8i, including competitive inhibition and non-competitive inhibition. Subsequently, molecular docking was performed to evaluate the interaction mechanism between compound 8i and enzymes. Enzyme inhibition kinetics and molecular modeling studies have shown that compound 8i bind simultaneously to the PAS and the CAS of AChE and BChE. Therefore, compounds 8i may be promising scaffold for treatment, and further modifications have been made to obtain novel AChE and BChE dual-target inhibitors.In this paper, a series of novel compounds


Haloterrigena turkmenica and Haloterrigena salina and has been denominated Haloterrigena sp. strain SGH1. Strain SGH1 grew at 20–40°C (optimum 37°C), at salinities between 15 and 30% (w/v) NaCl (optimum 25%) and growth was improved by addition of 50 mM KCl and 0.5% w/v casamino acids. Growth was severely restricted at salinities below 15% NaCl and cell lysis is avoided at a minimal 10% NaCl. Maximal concentrations of magnesium chloride and sodium or magnesium perchlorates that supported SGH1 growth were 0.5 and 0.15M, respectively. Haloterrigena sp. strain SGH1 accumulates bacterioruberin (BR), a C50 xanthophyll, as the major carotenoid. Total carotenoids in strain SGH1 amounted to nearly 400 μg BR per gram of dry biomass. Nearly 80% of total carotenoids accumulated as geometric isomers of BR: all-trans-BR (50%), 5-cis-BR (15%), 9-cis-BR (10%), 13-cis-BR (4%); other carotenoids were dehydrated derivatives of BR. Carotenogenesis in SGH1 was a reversible and salt-dependent process; transferring BR-rich cells grown in 25% (w/v) NaCl to 15% (w/v) NaCl medium resulted in depigmentation, and BR content was recovered after transference and growth of unpigmented cells to high salinity medium. Methanol extracts and purified BR isomers showed an 8–9-fold higher antioxidant activity than Trolox or β-carotene. Both, plasma membrane integrity and mitochondrial membrane potential measurements under acute 18-h assays showed that purified BR isomers were non-toxic to cultured human THP-1 cells.An extreme halophilic archaeon, strain SGH1, is a novel microorganism isolated from endolithic microbial communities colonizing halites at Salar Grande, Atacama Desert, in northern Chile. Our study provides structural, biochemical, genomic, and physiological information on this new isolate living at the edge of the physical and chemical extremes at the Atacama Desert. SGH1 is a Gram-negative, red-pigmented, non-motile unicellular coccoid organism. Under the transmission electron microscope, strain SGH1 showed an abundant electro-dense material surrounding electron-lucent globular structures resembling gas vacuoles. Strain SGH1 showed a 16S rRNA gene sequence with a close phylogenetic relationship to the extreme halophilic archaea  The Atacama Desert is considered to be the driest and oldest dryland on Earth . The micLithobiontic colonization can be considered as one of the most successful strategies for microbial survival under the extreme desiccation and high solar radiation on the Atacama Desert. Lithic habitats provide one of the most efficient niches to sustain microbial life by acting as a filter to solar and ultraviolet radiations, as a liquid water harvesting device from fogs and dew events, by salt deliquescence and capillary condensation . In AtacHaloterrigena sp. strain SGH1 and it is an extreme halophilic archaeon with a high content of the carotenoid bacterioruberin . Collection and further work were carried out aseptically with all materials previously sterilized.Salar Grande, with an approximate surface of 500 km3, 50 mM KCl, with NaCl at concentrations between 0 and 30% w/v (medium Z8-NK). Compatible organic solutes  were also added to the Z8-NK medium at 10 mM final concentration. Cultures were incubated at 30°C, in a rotary incubator (Zichen ZWWY-100B) at 120 rpm, under constant illumination with white fluorescent light (34 μmoles m–2 s–1). After 2–3 weeks, growth was observed in cultures growing in Z8-NK containing 25% NaCl and 10 mM L-proline, as an evident increase in turbidity. Aliquots were transferred to 3% agar plates prepared in Z8-NK plus 25% NaCl and 10 mM L-proline and incubated until colonies were observed. Light microscopy observations, before or after Gram staining, confirmed the presence of only one type of microorganism in the isolated colonies. The isolated microorganism was denominated strain SGH1. SGH1 growth improvement was obtained after L-proline was replaced by 0.5% w/v casamino acids. This new medium, Z8-MOD at 25% NaCl, was used for maintenance and biomass production. Strain SGH1 was conserved using glycerol 25% at −80°C.Halites samples were broken open to expose the endolithic colonization which can be observed as a greenish line that runs parallel at few mm below the rock surface . Using aAliquots of liquid cultures and agar colonies were fixed 3 h at 5°C with 3% glutaraldehyde (EMS grade) in 0.1 mM cacodylate buffer. Samples were washed several times by centrifugation and pelleted in fresh sterile agar. Pellets were post-fixed with 1% (w/v) osmium tetroxide for 5 h at room temperature. Dehydration included a step with 70% ethanol with 2% uranyl acetate to increase contrast of intracellular components. Samples were infiltrate with LR-White resin and polymerized using gelatin capsules at 60°C for 24 h. An Ultracut E ultramicrotome (Reichert-Jung) was used for preparation of ultrathin 80 nm sections that were placed on copper grids and contrasted with lead citrate. Grids were examined at 80 kV with a Leo 910  transmission electron microscope equipped with a Gatan BioScan 792 camera .660: 0.1) was cultured in 25 mL of liquid medium Z8-MOD at different final salinities from 0 to 30% w/v NaCl. Once the optimal salinity was determined, the SGH1 cultures were grown at various pH (from 6 to 10) and temperatures (from 20 to 50°C) in order to define optimal growth conditions. SGH1 growth was followed as increments in turbidity at 660 nm and doubling time were computed at the exponential phase of growth. Tolerance of strain SGH1 to MgCl2 x 6H2O (50 to 500 mM), to sodium or magnesium perchlorate (50 to 300 mM) was conducted in independent growth experiments, in triplicates, in medium Z8-MOD. The specific growth rate for each condition was computed after 96 h of growth.Strain SGH1  and finally stored in amber bottles at 4°C.Strain SGH1 grown in Z8-MOD medium was harvested at the early stationary phase of growth by centrifugation . Cells were washed with fresh growth medium at a final concentration of 10% (w/v) NaCl and, cells were recovered by centrifugation . The cell pellet was lyophilized and the dried biomass was exhaustively extracted with 100% methanol (HPLC grade) at 4°C, under dim light, in order to obtain a fraction enriched in total carotenoids. The methanolic extracts were clarified by centrifugation  and the uncolored cell residues were discarded. The methanolic extract was dried by N–1 cm–1) (Absorption spectrum (350–600 nm) of carotenoid extracts were obtained in a Shimadzu spectrophotometer UV-1601 and absorbance values at 494 nm were used to compute total SGH1 carotenoids concentration in SGH1 methanol extracts, using the following equation and the extinction coefficient for BR in methanol  :2 and dissolved in 100% methanol. Each recovered HPLC fraction (10 μL) was injected in a Thermo Scientific Dionex Ultimate-3000 UHPLC system with a C18 column ; the mobile phase for elution was a methanol/acetonitrile/water gradient  for 2 min, 30:70:0 (v/v) for 10 min, 50:50:0 (v/v) for 15 min, and 90:10:0 (v/v) for 20 min, at 25°C, at a flow rate of 1.0 mL per min. Data analysis was managed with the Xcalibur 2.3 software (Thermo Fisher Scientific). Absorbance spectra were conducted at 494 nm. The UHPLC chromatographer was coupled to a high-resolution mass spectrometer Q Exactive (Thermo Fisher Scientific) controlled by the Q Exactive 2.0 SP 2 software (Thermo Fisher Scientific). Carotenoid ionization was conducted with an Electrospray Ionization Source at 400°C and 2,500 V, under nitrogen (Genus NM32LA Peak Scientific) to generate ionized fragments. The scanning of positively ionized fragments was conducted at 100 to 1,000 m/z. Caffeine, N-butylamine, buspirone, sodium dodecyl sulfate, and taurocholate served as standards (Sigma-Aldrich). The scanning of positively ionized fragments was conducted at 100 to 1,000 m/z.ethanol extracts (50 μL) were injected to a Shimatzu Hitachi CL-20A HPLC system containing a C18 reverse phase column  as stationary phase. For elution, the mobile phase used was methanol/acetonitrile/water  at a flow rate of 1.0 mL/min during 12 min. The collection of fractions was conducted after programming the HPLC system software to start and finish at specific retention times to avoid contamination among the collected peaks. The more abundant HPLC fractions were recovered, dried under NHaloterrigena sp. SGH1 was conducted by data analyses of Raman spectroscopy, chromatographic migrations, UV-Vis spectra, spectral fine structure and MS fragmentation patterns  using a 25% w/v NaCl solution. Next, the samples were dried at room temperature for 24 h in darkness. Micro-Raman analyses of upper region of the dried samples were performed on a multichannel Raman DXL Thermo Fischer spectrometer coupled with a Peltier-cooled (−50°C) CCD detector and equipped with 10x objective. The grating with 900 lines/mm was used. Excitation was provided by the 523 nm Argon laser. Spectrograph slit aperture was 25 μm. To achieve enhanced signal-to-noise ratios, 44 scans were accumulated, each of 2.70 s exposure time with a laser power of 8.0 mW at the samples. The background exposures were 512. Spectra were recorded with a spectral resolution of 1.9 cmThree assays were employed to evaluate antioxidative activity of the methanolic extracts and fractions purified by HPLC chromatography.2O2 in the presence of ferrous ions and, oxidative damage to DNA can be observed by a differential electrophoretic mobility of relaxed and supercoiled molecules (2O2 plus 15 μL 100 μM FeCl2). Oxidized DNA decreases its electrophoretic mobility when compared with intact non-oxidized supercoiled DNA. Protection of plasmid DNA to oxidation was evaluated with 5 μL of Trolox (50 μg/μL), 5 μL of β-carotene (50 μg/μL) or 5 μL of SGH1 extract . The assays were incubated at 37°C during 30 min and stopped by adding 1.0 μL of 5 mM EDTA. Electrophoresis of intact or damaged plasmid DNA was carried out at room temperature on 0.8% agarose gels for 40 min at 80 mV. The gels were stained with ethidium bromide, dried and bands densitometry was estimated by ImageJ, an open source image processing program.1This assay involves the formation of hydroxyl radicals from Holecules . The ass*+. This mix was diluted with 70% ethanol until the solution reached an absorbance of 0.7 ± 0.02 at 734 nm. The assay included 1.98 mL of the ABTS*+ solution plus 0.02 mL of a carotenoid solution and it was incubated during 7 min at room temperature, in darkness. A decrease in absorbance at 734 nm was used to compute the antioxidant activity of the carotenoid samples, expressed as percentage of inhibition of the oxidation of ABTS*+. The antioxidant activity of SGH1 carotenoids was finally expressed as TEAC  and IC50  (A solution of 7 mM ABTS dissolved in water was mixed with 2.45 mM potassium persulfate in a 1:1 (v/v) ratio during 16 h to generate the radical ABTS ABTS*+) . Each in+3  which was prepared with 10 mM TPTZ, 20 mM FeCl3 x 6 H2O and 0.3M acetate buffer, pH 3.6, at a ratio 1:1:10 (v/v). The assay was conducted during 30 min at room temperature, in darkness. Absorbance at 593 nm was computed to determine the antioxidant activity of carotenoids with respect to Trolox (Albiochem) (The reducing power of SGH1 carotenoids was done mixing SGH1 carotenoid samples (100 μL) with a freshly prepared solution containing TPTZ-Febiochem) . The ant® 30-2001) derived from the blood of a 1-year-old boy with acute monocytic leukemia  containing 10% fetal bovine serum (Gibco Cat. 11875085) at 37°C, in an atmosphere of 5% CO2 in air. HP-1 cells were selected for these studies since cytotoxicity and other cellular responses can be easily and rapidly conducted by flow cytometry; also, they are non-adherent easily cultured cells and, as bloodstream circulating cells, they are a reasonable experimental model for exposure to high concentrations of novel compounds that could eventually be used in biomedical applications (THP-1 cell line (ATCCleukemia  was usedications .5 cells/mL) were starved for 24 h in FluoroBrite DMEM medium (Gibco Cat. A18967-01), free of fetal bovine serum and glutathione. Next, cells were incubated with 0.5% (v/v) methanol , methanol extract (500 μg/mL), purified Fractions I, II, III, IV, or V (500 μg/mL), 0.5% (v/v) ethanol  or Antimycin A (30 μM), as final concentrations in the assays. Eighteen hours after treatment, cells were incubated with 2.4 μM PI during 15 min. PI positive cells were measured by flow cytometry using an excitation laser beam at 488 and 585 nm detector. Results were expressed as percentage of viable cells. The assays were done in four independent experiments.Cell viability was evaluated by dye exclusion using a membrane impermeant compound. Propidium iodide  is a membrane impermeant fluorogenic dye generally excluded from viable cells . PI bindTM Cat. 564697) is a cationic, lipophilic and fluorescent dye that is readily sequestered by active mitochondria of healthy cells, but not within mitochondria that have lost its ΔΨm (5 cells/mL) were starved for 24 h in FluoroBrite DMEM medium (Gibco Cat. A18967-01), free of fetal bovine serum and glutathione. Next, cells were incubated with 0.5% (v/v) methanol , methanol extract (500 μg/mL), purified Fractions I, II, III, IV, or V (500 μg/mL), 0.5% (v/v) ethanol  or Antimycin A (30 μM), as final concentrations in the assays. Eighteen hours after treatment, the cells were incubated with 0.8 μM MTRed during 15 min and MTRed positive cells were quantified by flow cytometry using an excitation laser beam at 640 nm and emission detector at 660 nm. Based on the MTRed intensities of the cell, they were distributed in three different populations nominated as THP-1 cells with low, middle or high ΔΨm. Results were expressed as percentage of cells distributed in each ΔΨm level. The assays were done in four independent experiments.Cell viability was also evaluated by measurements of the mitochondrial membrane potential (ΔΨm), which can be lost in cells undergoing apoptosis or necrosis . MitoSta its ΔΨm . CellulaMN410435) against the sequences of different groups of Haloarchaea Class that were retrieved from NCBI. Multiple sequence alignment was created by ClustalW in MEGA 7  or t-test .Statistical data analysis was done by ANOVA with Bonferroni correction  against NCBI non-redundant nucleotide collection showed a 99% of identity to Haloterrigena turkmenica  NaCl at 45°C . Strain 2 x 6 H2O). SGH1 cells were also able to grow at 150 mM sodium or magnesium perchlorate with a decrease in its specific growth rate close to 40% (The effect of magnesium chloride (50 to 500 mM) and sodium or magnesium perchlorate (50 to 300 mM) on SGH1 aerobic growth was considered as a measure of tolerance of strain SGH1 for these salts, and results are shown in e to 40% . Higher SGH1 accumulated nearly 400 μg of BR per gram of dry biomass at late exponential phase of growth at 25–30% NaCl and 25–35°C . Strain Haloterrigena sp. SGH1 was based on the data analyses of their chromatographic migrations, UV-Vis spectra, spectral fine structure and MS fragmentation patterns (–1) that were in close proximity with those informed for extracts from other haloarchaea  were used to discriminate among geometric isomers and anhydrous derivatives of BR in strain Haloterrigena sp. SGH1 (absorbance ratios%III/II and%DII/II)  .Haloterrigena sp. strain SGH1 cells contain six C-50 molecules related to the BR family: four geometric isomers  and two dehydrated derivatives  and, one the metabolic intermediate . Also, all-trans-BR was the major BR isomer accounting for nearly 65% of all BR-type carotenoids in SGH1. Fractions FI and FII are enriched in all-trans-BR and 5-cis-BR, respectively, and 9-cis-BR is the major carotenoid in FIII. Carotenoid composition plus UHPLC-MS and spectrophotometric data are summarized in Mass spectrometry provided the corresponding profiles for positively ionized fragments and their m/z for each major subfraction as shown in Haloterrigena sp. SGH1. First, the protective role of SGH1 extracts against oxidative damage of DNA was tested using plasmid pUC19. Incubation of plasmid DNA with the Fenton’s reagent rendered a relaxation of the original supercoiled DNA with a decreased electrophoretic mobility  to H. turkmenica 4kT  NaCl  NaCl , which iHaloterrigena sp. strain SGH1 is a reversible, salt-dependent metabolic process and the maximum carotenoid content was obtained in cells grown at 25°C and 25% NaCl. Comparatively, a lower carotenoid content has been registered for other halophilic archaea; e.g., 45 μg g–1 of dry biomass in Halococcus morrhuae  in a hyperpigmented, genetically modified Haloferax volcanii strain HVLON3 is higher than in other haloarchaea. The red-pigmented strain SGH1 accumulates BR when growing at high salinity (25% NaCl) but the BR content decreased nearly 20-fold after the SGH1 cells are transferred and adapted to grow at low salinity (15% NaCl). Depigmentation is a reversible process in which the BR biosynthesis resumed after the unpigmented cells are transferred and grown at high salinity, reaching the maximum BR content observed in SGH1 cells adapted to grow at high salinity. Thus, BR content correlates reversibly with the salinity of the growth medium, an adaptive process not previously reported in other Haloterrigena species. Several studies on the reversible effect of salinity on carotenoid content are available; for example, (i) two extreme halophilic archaea, Halobacterium cutirubrum and Halobacterium mediterranei, showed that their C40 and C50 carotenoids content was also salt-modulated in an opposite fashion, that is, carotenoids in H. mediterranei decreased severely at high salinity, while high carotenoid content was observed when H. cutirubrum were grown at 20–35% salinity  to high salinity (20% NaCl) showed a twofold increment on the expression and a threefold increase in specific activity of HMG-CoA reductase, an enzyme involved in isoprenoide biosynthesis leading to carotenoids in haloarchaea, but a 1.5-fold decrease in carotenoid content  Kuechanism  and,  and the three major Raman displacement signals  observed in the methanolic extracts , two dehydrated derivatives  and one metabolic intermediate (cis-mono-anhydrous-BR). Among them, all-trans-BR was the most abundant carotenoid accounting for 65% of total carotenoids in strain SGH1 and all geometric isomers of BR represented nearly 80% of total carotenoids in SGH1. The presence of minor carotenoids  was not investigated. In addition, the use of a C18 column in the HPLC system showed limitations in the separation of fractions; for example, -cis-BR. In a similar way, the major signal in Fig. FIII.2 has a RT of 11.71 min, and it was identified as 9-cis-BR in Methanolic extracts of isolate SGH1 were chromatographically separated into 17 fractions. Those fractions with the highest relative abundance were selected for further analyses. The identification of the carotenoids was based on their UV-Vis spectra, spectral fine structure and MS fragmentation patterns. We found that BR molecules from 50 carotenoids have been reported in other halophilic archaea, as expected for studies carried out with different genera and species. Abundant C50 carotenoids and minor contents of C30, C40, and C51 carotenoids were found in H. turkmenica DMS-5511 , five different cis-isomers of BR and 10 different dehydrated isomer derivatives of BR, and all-trans-BR (Different CDMS-5511 . Extracttrans-BR . The pretrans BR isomers have a linear arrangement of its doble bonds but cis-BR isomers are bended molecules (cis-isomers have higher absorbance than trans-isomers at the cis-peak (388 nm) which becomes more intense as the cis double bond gets closer to the center of the molecules  NaCl at a maximal concentration of 150 mM magnesium or sodium perchlorate, with a 40% drop in its specific growth rate, suggesting the existence and expression of SGH1 genes involved in perchlorate tolerance.Lithobiontic microbial consortia thriving within halite pinnacles from Salar Grande are in a ‘foggy environment’ . These eoarchaea . The halte salts . Similard medium . EvidencHaloterrigena sp. strain SGH1 to magnesium was investigated by adding increasing concentrations of MgCl2 until it was equivalent to a 2,500-fold increment from 0.2 mM MgCl2, the concentration in the growth medium. A drop of 25% on its growth rate was observed when isolate SGH1 was grown in 25% (w/v) NaCl plus 0.5M magnesium chloride. The magnesium concentration within the endolithic colonization zone in halites from Salar Grande is 0.4 mg kg–1 nearly 10 times lower than the concentration in the growth medium. Thus, isolate SGH1 is tolerant to chaotropic agents such as high magnesium concentrations during growth in a medium at high salinity (25% NaCl). This agrees with growth at a maximal concentration of 0.42M MgCl2 and 2.76M NaCl, of halophilic isolates from a MgCl2-dominated hypersaline lake at the Mediterranean Sea  were similar or higher than their original methanolic extracts, depending upon de antioxidant assay used. This is the first report of antioxidant activity of purified isomers of BR. Our results are in clear correspondence with previous reports using extracts of halophilic archaea from various origins; for example, TEAC values for carotenoid extracts from two halophilic archaea, H. morrhuae and Halobacterium salinarium, were 5.1–5.3, with IC50 of 0.8 μg/mL  in comparison with control cells (80% viability). Then, BR isomers and derivatives are not toxic to cultured human cells. The addition of these antioxidant and non-toxic molecules improved the viability of starved THP-1 cells for at least 42 h (24 h of starving plus 18 h of treatment).We report here the effect of BR isomers and derivatives on human THP-1 monocytes. HP-1 cells are bloodstream-circulating, non-adherent cells and a good model to be exposed to high concentrations of novel compounds that eventually may be used in biomedical applications . All celHaloterrigena sp. strain SGH1 decreased the mitochondrial membrane potential of THP-1 cells at a non-toxic level. The mechanism for this effect requires more investigation and we propose that the activation of mitochondrial uncoupling proteins may be involved. The existence of a feedback loop between ROS and the inner mitochondrial membrane proton leak has been proposed . A parallel increase in the percentage of middle ΔΨm-cells was observed, and Fractions II and Fraction III were most effective . Neither the methanolic extract nor all the purified fractions increased the percentage of cells showing low-ΔΨm, in contrast to the effect of 30 μM Antimycin A (46% increase), which is used as a positive control for a partial disruption of ΔΨm by inhibiting Complex III at the mitochondrial electron transport chains. In consequence, BR molecules from proposed : ROS wouproposed . Since pproposed , we propArthrobacter agilis 50Cyt, containing all-trans-BR, was shown to be not phototoxic to cultured murine fibroblasts; however, the glycosylated BR molecules were highly phototoxic and cytotoxic to these cells  showed maximal BR content, but growth at low salinity (15%) rendered unpigmented cells. Growth of strain SGH1 at 25% (w/v) NaCl, was limited by magnesium chloride and sodium or magnesium perchlorate at maximal tolerable concentrations of 500 and 150 mM, respectively; thus, isolate SGH1 can be considered a new experimental model in which to study genes and enzymes associated to aerobic/anaerobic perchlorate metabolism and its applications on biological removal of perchlorates from nitrate-rich fertilizers and natural waters. Also, this is the first report that shows that the antioxidant activity of purified isomers of BR is higher than Trolox, beta-carotene, and astaxanthin. In addition, we report the first evaluation of BR toxicity on THP-1 cells concluding that purified BR isomers are not toxic to cultured human cells and open the opportunity for new applications in pharmaceutical, food and medical products.Lithobiontic microbial consortia in Atacama halites form biofilms with a surprisingly complex diversity that includes unculturable members of the three domains of life and viruses. MN410435.Nucleotide sequence used in this work can be found in NCBI accession number NF conducted extraction, purification and identification of carotenoids and evaluated their biochemical properties. SH isolated and purifies strain SGH1. NF, MV, and AG implemented and evaluated the antioxidant activity of extracts and purified fractions. LZ, FF, and BP conducted cytotoxic assays. CS-A applied HPLC to extracts and collected fractions. CA, JW, and VS-E conducted TEM and JW the Raman studies. SH, AG, CV, and JA conducted genomic studies. RB-G analyzed results and reviewed critically the manuscript. BG-S conceived and designed the studies and wrote the manuscript. All authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.