Tetraena mongolica is restrictedly distributed in the western Ordos plateau of China and has been listed as a national protected plant. We generated 13 chloroplast (cp) genomes of T. mongolica  and conducted a series of comparative analyses of six Zygophyllaceae cp genomes. T. mongolica cp genome exhibited a quadripartite structure with drastically reduced inverted repeats  and undergone the loss of a suit of ndh genes and a copy of rRNAs. Furthermore, all the T. mongolica populations were divided into two genetic groups based on complete cp phylogenomics. In addition, notably variable genome size, gene order and structural changes had been observed among the six Zygophyllaceae cp genomes. Overall, our findings provide insights into the cp genome evolution mode and intraspecific relationships of T. mongolica, and provide a molecular basis for scientific conservation of this endangered plant.A comprehensive understanding of genetic background for rare species will provide an important theoretical basis for the future species management, monitoring and conservation.  Dramatic global climate change and human activities have triggered the loss of biodiversity and the sixth mass extinction . It is nTetraena mongolica Maxim, a super-xerophilous shrub, is a monotypic genus of Zygophyllaceae  genome, which can provide more phylogenetic signals compared with a limited number of markers and reflect the evolutionary history of a single parental lineage.Previous molecular studies based on a limited number of markers have provided insights into the genetic diversity, genetic structure and phylogenetic relationships of ongolica . HoweverPaphiopedilum, Passiflora, Picea, Sequencing technical feasibility has made cp genome a workhorse of plant phylogeny and genome evolution e.g., . Given tZygophyllaceae comprising 13 accessions of T. mongolica and one each of Zygophyllum mucronatum, Zygophyllum xanthoxylon. We intend to 1) explore the cp genome evolution mode of T. mongolica by comparing with all genera for which reliable cp genomes have been published in Zygophyllaceae; and 2) estimate the intraspecific relationships of T. mongolica with different data partitions.In this study, we generated 15 cp genomes representing three species of T. mongolica, Z. mucronatum, and Z. xanthoxylon. Detailed information of sampling is provided in T. mongolica. Currently, the best-supported taxonomy of Z. mucronatum and Z. xanthoxylon is that they belong to genus Zygophyllum , Z. xanthoxylon (MZ427318) and Zygophyllum fabago (MK341052) were used as reference genome in cp genome assembly of T. mongolica, Z. xanthoxylon and Z. mucronatum, respectively. Annotation of the cp genomes was performed in GENEIOUS v.11  coupled with manual adjustments.Total genomic DNA was extracted for the 15 accessions from silica-dried leaf material following the modified CTAB method . SequencT. mongolica was assessed. Sequences were aligned by MAFFT with the default settings  is the ratio of the observed frequency of a codon to the expected frequency and is a good indicator of codon usage bias .To avoid sampling bias, protein-coding regions with the length greater than 300 bp were extracted for the analysis . Finallyage bias . RSCU vaGA v 5.0 . The heaZygophyllaceae cp genomes were analyzed, respectively. Firstly, REPuter  within six  REPuter  was used REPuter  with def REPuter . The minT. mongolica, the following datasets: 1) complete cp genome sequences (CCS); 2) noncoding region sequences (NCS); and 3) protein-coding region sequences (PCS) were analyzed. In these inferences, L. tridentata (MK726018) and G. angustifolium (MK726011) were treated as outgroups. For Zygophyllaceae, we used Krameria bicolor (MK726015) and Krameria lanceolata (MK726016) as outgroups. Datasets:1) complete cp genome sequences (CCS); and 2) SSC + single IR sequences (SIS) were conducted for phylogenetic analysis. Sequence alignment was performed using MAFFT  and Bayesian inference (BI) methods, which were conducted using RAxML v7.2.8  and MrBang MAFFT  with theng MAFFT  based onT. mongolica was performed by PopArt v 1.7 (A TCS network of intraspecific relationships of rt v 1.7  using thrt v 1.7 .T. mongolica lineages were estimated using BEAST v1.8.0 (http://beast.community/figtree). Considering the ambiguity of Zygophyllaceae fossil record  to 106,230 bp (MX23) (trnL (UAG) end to the junction of IRa/SSC boundary of HNHCQ39 and the other accessions were 555 bp and 553 bp, respectively (To assess intraspecific variation within approach . T. mongp (MX23) . In the ectively . The GC ectively .T. mongolica composed of 104 genes, including 67 protein-coding genes, 33 tRNA genes and four rRNA genes  across the complete cp genomes of total populations was relatively low (Pi = 0.00032). In contrast, non-coding regions showed the highest sequence diversity (Pi = 0.00054) and protein-coding genes exhibited extremely low sequence diversity (Pi = 0.00012) .T. mongolica cp genome, a total of six species of Zygophyllaceae were analyzed, including T. terrestris (subfam. Tribuloideae), L. tridentata (subfam. Larreoideae), G. angustifolium (subfam. Larreoideae), T. mongolica (subfam. Zygophylloideae), Z. mucronatum (subfam. Zygophylloideae) and Z. xanthoxylon (subfam. Zygophylloideae), which covered all genera for which reliable cp genomes have been published in Zygophyllaceae. Due to the highly conserved cp genomes, we used WD01 to represent T. mongolica for the subsequent analyses.To investigate the evolution mode of Z. mucronatum) to 158,184 bp (T. terrestris) (T. mongolica) to 35.81% (T. terrestris).The complete cp genome sequences of six Zygophyllaceae species were 105,251 bp (restris) . All of Zygophyllaceae exhibited two patterns of gene order and content: the cp genomes of subfam. Zygophylloideae possessed basically identical genes, and the remaining three cp genomes shared similar coding genes. The specific details of gene loss were shown in the G. angustifolium) in IRs, which attributed to the gene loss  and the transfer of genes from IRs to SSC region . Although most IR boundary shifts are small  among land plants  to 24,733 (T. terrestris) codons. The codon usage and RSCU values of six Zygophyllaceae cp genomes were summarized in T. terrestris genes than in the remaining species genes, however, UUA was always the most frequently occurring codon  relative to the other codons encoding Leu in six species. Codons AUG and UGG showed no bias (RSCU = 1), as Methionine (Met) and Tryptophan (Trp) were encoded only by AUG and UGG, respectively. Concurrently, 30 codons were translation-preferred (RSCU>1) and characterized with a bias in favor of purine (A/U) at the third codon position (except UUG).To avoid sampling bias, protein-coding regions with the length greater than 300 bp were analyzed , resultiT. terrestris contained the largest number of repeats (50) while Z. xanthoxylon had the fewest (25). We found that 96.9% of tandem repeats had the repeat motif less than five and only three of the 226 repeats had larger repeat unit (>100 bp). We detected 121 dispersed repeats, and the majority were forward repeats (52.07%), followed by palindromic repeats (37.2%). In contrast, reverse repeats and complement repeats were found with much lower frequency or even absent in some species. Moreover, there were 115 (95.0%) smaller dispersed repeats (<50 bp) while Z. mucronatum and Z. xanthoxylon had four and two larger dispersed repeats (>100 bp), respectively. Among the 452 SSRs, mononucleotide repeats accounted for the highest proportion (84.7%) and all consisted of A/T repeats. Penta- and hexanucleotide repeats were absent in all species.A total of 226 tandem repeats, 121 dispersed repeats and 452 SSRs were identified in the six cp genomes  and the T. mongolica (T. mongolica populations were mainly divided into two groups: the northern group (WJRGC35 and northern populations) and the southern group (the remaining populations).Three datasets  were used to reconstruct the intraspecific relationships of ongolica . The topongolica . With thongolica . OverallZygophyllaceae were performed based on CCS dataset . The split between the mainly northern group and southern group was ca. 0.225 Ma. The onset of diversification of the mainly northern group and southern group occurred at 0.097 Ma (node B) and 0.082 Ma (node C), respectively.The BEAST-derived ages of n points  were larn points . Thus, wMonotropa and Hypopitys, suggesting that speciation may be occurring , the remaining species in Zygophyllaceae had smaller cp genomes (c. 106–135 kb) compared with most land plants (an average size of 151 kb) (Z. xanthoxylon) and up to 25,842 bp (T. terrestris), accounted for Zygophyllaceae cp genome size variation overall . The phenomenon that IR has undergone expansion and contraction is nearly omnipresent while significant IR boundary shifts are relatively rare , contributing to their smaller IR size . Overall GC content is typically in the range of 30–40% (average 37%) in most cp genomes of land plants  legumes, some species of Geraniaceae;  151 kb) . Unlike  151 kb) , variatiely rare . In all d plants , and theZygophyllaceae cp genomes differed greatly, from 104 in subfam. Zygophylloideae, to 130 in T. terrestris. Especially, gene numbers varied from 12 (subfam. Zygophylloideae) to 35 (G. angustifolium) located in IRs. Despite there were different numbers of protein-coding genes (67–84), the codon usage preference of each amino acid showed highly similar in these Zygophyllaceae cp genomes, even more broadly across land plants, as previous research results  occurred in subfam. Zygophylloideae, which had also been detected and verified by PCR product sequencing (ndhA–ndhK) constitute ndh family and encode subunits of the NDH complex that is involved in photosystem I cyclic electron flow and chlororespiration , an arid and semi-arid plant, possesses all 11 ndh genes in cp genome, possibly indicating the different evolutionary history among the subfamilies of Zygophyllaceae  are often present in the IRa and IRb respectively. In subfam. ce versa , and thece versa .Passiflora  with significantly improved support values, which is generally consistent with previous result inferred from cpDNA marker , which may provide opportunities for the long-distance dispersal of seeds from other populations. Despite two groups were well supported, the phylogenetic relationships within group were recovered with low to medium support in all datasets, which may be largely attributed to the low genetic differentiation among the populations rather than the insufficient phylogenetic signals. Such a similar result was also observed in previous study, which inferred from a total of 38,097 SNPs  rather than two genera (Sarcozygium and Zygophyllum). Such a taxonomy was also suggested in most previous studies (e.g., Although representatives of Morkillioideae and Seetzenioideae were absent, the remaining three of the five subfamilies of  studies . To somees e.g., .


Periodontal diseases are pathological processes resulting from infections and inflammation affecting the periodontium or the tissue surrounding and supporting the teeth. Pathogenic bacteria living in complex biofilms initiate and perpetuate this disease in susceptible hosts. In some cases, broad-spectrum antibiotic therapy has been a treatment of choice to control bacterial infection. However, increasing antibiotic resistance among periodontal pathogens has become a significant challenge when treating periodontal diseases. Thanks to the improved understanding of the pathogenesis of periodontal disease, which involves the host immune response, and the importance of the human microbiome, the primary goal of periodontal therapy has shifted, in recent years, to the restoration of homeostasis in oral microbiota and its harmonious balance with the host periodontal tissues. This shift in therapeutic goals and the drug resistance challenge call for alternative approaches to antibiotic therapy that indiscriminately eliminate harmful or beneficial bacteria. In this review, we summarize the recent advancement of alternative methods and new compounds that offer promising potential for the treatment and prevention of periodontal disease. Agents that target biofilm formation, bacterial quorum-sensing systems and other virulence factors have been reviewed. New and exciting microbiome approaches, such as oral microbiota replacement therapy and probiotic therapy for periodontal disease, are also discussed. Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola. These bacteria are known as the red complex as they are found together in periodontal pockets where extensive periodontal tissue damage is evident . To. ToP. gingivalis .P. gingivalis possess two antigenically distinct fimbriae subunits, FimA and Mfa1. FimA is found in short fimbriae, and Mfa1 is found in long fimbriae [P. gingivalis in a germ-free rat model [P. gingivalis and interfer with biofilm formation. The study also showed that treating these peptides reduces P. gingivalis adhesion to Streptococcus gordonii in a dual-species model [Fimbriae are proteinaceous appendages that protrude from the bacterial outer membrane that helps microbes attach to host cell surfaces and other microbial surfaces. fimbriae . Immunizat model . In a rees model .A. actinomycetemcomitans, associated with periodontal disease, produces leukotoxin (LtxA) during colonization in the host to escape the host immune response [A. actinomycetemcomitans, which kills leukocytes by recognizing cholesterol and the β2 strands of lymphocyte function-associated antigen-1 (LFA-1) integrin [A. actinomycetemcomitans calls for developing alternative treatment methods [Toxins play an essential role in bacterial pathogenesis. The Gram-negative bacteria response . Anotherresponse . LtxA isintegrin . Mechani methods . It has  methods . Studies methods , 55. Cat methods . At the  methods .Proteases, including gingipains, aminopeptidase IV, and collagenases, play a role in bacterial adhesion, degradation of host proteins (i.e. immunoglobulins), modulating inflammatory responses, and damaging host tissues . Thus, rMicrobial proteases play a crucial role in the progression of periodontal disease, directly damaging host tissues or indirectly activating a myriad of host proteases and inactivating protease inhibitors in the host . ExtraceP. gingivalis are called “trypsin-like enzymes” as they cut after the arginine or lysine residue at the C-terminal of polypeptides [P. gingivalis [Members of cysteine proteinases in peptides . These epeptides . Gingipangivalis .P. gingivalis before infection by a Kgp-specific inhibitor (A71561) designed by Curtis et al. decreased the P. gingivalis virulence significantly. In contrast, the Rgp-specific inhibitor leupeptin did not influence the pathogenicity [In a mouse model, treatment of itor A715 designedgenicity . rgpA and kgp, in P. gingivalis when combined with polyphenols synergistically [rgpA and kgp proteases and show anti-inflammatory responses [P. gingivalis by decreasing the activity of gingipains in vivo model [It has been reported that a compound in rhubarb roots named rhein can cause the downregulation of both genes,stically . Anotheresponses . Recent vo model .rgpA and kgp protease genes at sub-MICs but also inhibits the hydrolytic activity of gingipains in a dose-dependent manner [rgpA and kgp activities in P. gingivalis at a concentration of one µg/ml [Eucalyptus globules inhibited both Arg- and Lys-specific gingipains in P. gingivalis in a dose-dependent manner [The primary component of black tea, theaflavin, has dual effects on gene expression and the catalytic activity of these proteases. Theaflavin not only downregulates the expression of t manner , 67. Frane µg/ml . Macrocat manner .Dental biofilm formation in the supragingival and subgingival areas is required to develop dental caries and periodontitis . BacteriArtocarpus lakoocha, has been shown to have antioxidant properties [A. actinomycetemcomitans [Oxyresveratol or trans-2,4,3′,5′-tetrahydroxystilbene, the main bioactive element of tropical tree operties . This cocomitans .Kaempferia pandurate, has antibiofilm and bactericidal activities [P. gingivalis [Panduratin A, a natural compound found in tivities . Epigalltivities . This cotivities . Studiesngivalis .Cranberry extract containing bioactive compounds such as Licocalchone A and Proanthocyanidins also shows promising benefits against gingivitis by reducing inflammation, biofilm formation and inhibiting proteolytic activity of periodontal pathogens , 80. PomThe effect of the bioactive compounds of plant origin on the growth and biofilm formation of periodontal pathogens and their potential mechanisms of action are summarized in Table P. gingivalis and A. actinomycetemcomitans isolated from patients with dental biofilm-associated diseases [P. gingivalis uses signalling molecules for interspecies interaction to promote a microbial dysbiosis which leads to periodontal disease [P. intermedia, and F. nucleatum, as well as in Eikenella corrodens, initiates the formation of biofilms in the oral cavity through LuxS-dependent pathway [A. actinomycetemcomitans can also form mature biofilm through autoinducer-2 dependent quorum sensing manner [Aliivibrio fischeri, has not been reported in periodontal pathogens [Bacterial communication plays a vital role in establishing a host-infection process successfully . Communidiseases . P. ging disease . Thus, t disease . A study pathway . A. actig manner . AHL-medathogens .Delisea pulchra, a marine alga produces various components of vanilla extract, garlic, halogenated furanones and vanilla extract. These compounds have quorum quenching properties that help block the quorum sensing singling in periodontal pathogens [A. actinomycetemcomitans [Developing a drug that could inhibit quorum sensing has been gaining attention in recent years. Quorum quenching, commonly known as inhibiting the signalling pathway of quorum sensing, can be an attractive solution for targeting periodontal bacteria by destroying bacterial communication, a critical aspect of biofilm formation and virulence factor expression , 90. Plaathogens –94. It hcomitans .P. gingivalis, although they do not affect the growth of P. gingivalis [Similar functional properties are shared by ribose binding protein and autoinducer-2; ribose binding protein acts as an antagonist on the receptors of autoinducer-2 . Brominangivalis . Studiesngivalis . Thus, aRecently, immunomodulatory therapy that regulates the immune response has received much attention. Immunomodulatory therapy is a promising option for treating periodontal diseases . ImmunomThe immune microenvironment of periodontitis-related tissue is critically essential for periodontal disease. Increased infiltration of leukocytes and the release of inflammatory molecules can resolve inflammation effectively. However, excessive amounts of this inflammatory response can cause serious harm to the periodontal tissues and the alveolar bone. Modulation of this microenvironment can not only supplement traditional treatments for periodontal disease but also may promote periodontal regeneration . DiffereAmong the leukocytes, macrophages play a significant role in various stages of periodontitis . NeutropF. nucleatum by upregulation of the antioxidant pathway [Drug therapy targeting immune response is promising for treating periodontal diseases. Resveratrol is a natural polyphenol and can inhibit NF-kB activation in macrophages, and it shows immunomodulatory effects on  pathway , 107. Me pathway . Metform pathway . Catechi pathway . Gliclaz pathway . Moreove pathway .Recent studies have shown that curcumin can release pro-inflammatory molecules, which is effective against periodontal pathogens . CMC2.24A. actinomycetemcomitans, which involves the downregulation of TNF-α and IL-1β cytokines [P. gingivalis. Other drug therapy includes carnosic acid, glyburide, and bismuth drugs, which show their immunomodulation by infiltrating inflammatory cells and reducing pro-inflammatory cytokines such as IL-6, IL-1β and TNF-α [Trans-cinnamic aldehyde is also used as a drug for treating ytokines . Kava-20nd TNF-α , 117.Immuno-related gene therapy is another novel approach targeting host immunity. It has been shown that downregulation of T-cell immune response cDNA7 (TIRC7) leads to a lower number of T-cells in gingival tissue, which affects the periodontal disease . PlasmidStem cell therapy has significant potential to impact periodontal health. Different mesenchymal stem cells can modulate the microenvironment coupled with the inflammatory response of leukocytes and cytokines associated with periodontal disease. These stem cells are being explored as a promising therapeutic intervention for periodontal disease. Periodontal ligament stem cells (PDLSCs) can interact with immune cells by modulating their activities and thus work as a potential therapeutic approach for treating periodontal diseases . PDLSc cLow dose antibiotics are another group of agents that can modulate host immune response, potentially contributing to the periodontal disease progression or resolution. Doxycycline at a low dose, i.e. at sub-inhibitory concentrations, inhibits matrix metalloproteinase‐8 (MMP-8), which is present abundantly in periodontitis . A pilotTable In contrast to the traditional view of individual pathogens being responsible for disease onset, the new perspective of periodontal disease deems that the transition from health to disease status is attributed to a shift in the global balance of the microbial flora . Periodontal disease is considered a result of disrupted microbial homeostasis and microbe-mediated disruption of host homeostasis. Such a perspective has opened new paths for periodontal disease treatment, including restoring the homeostasis of the microbiota associated with periodontal health.P. intermedia or P. gingivalis, which was high in the diseased patient sample [Bifidobacterium, Streptococcus, and Actinomyces. Commercially available dietary probiotics have been shown to have more substantial inhibitory properties against periodontal bacteria [Recent studies have suggested probiotic therapy for treating periodontal diseases in the oral cavity , 144. Prt sample . These pbacteria . A succebacteria .Lactobacillus fermentum and Lactobacillus gasseri were the most common species in healthy subjects. In contrast, Lactobacillus plantarum was the most prevalent species in patients with periodontitis [Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus rhamnosus and Lactobacillus paracasei, which can be used as a probiotics for maintaining oral health [Lactobacillus reuteri could be as efficacious as probiotics for reducing dental-biofilm induced by systemic or local risk factors [Bifidobacterium animalis significantly impacted inflammatory parameters of gingiva and bacterial plaque [A study comparing the prevalence of oral Lactobacilli in healthy subjects and patients with periodontitis (chronic) showed that dontitis . In addil health . A repor factors . Studiesl plaque .Clostridium difficile infections. Also, it has shown efficacy for treating one class of inflammatory bowel diseases known as Crohn’s disease. [Microbiota replacement therapy is a new disease treatment/prevention measure in which microbiota from healthy donors is transplanted to diseased patients. Replacement therapies have been successfully used to treat disease. , 154. Midisease. . Naturaldisease. , 156. Redisease. . With thAlthough anti-virulence therapeutic strategies are relatively new in preventing and treating periodontal disease, novel drugs targeting the critical virulence factors of periodontal pathogens have already been identified, which serve as a promising alternative in the era of antibiotic resistance. A growing library of novel targets will open the door to rapidly identifying anti-virulence drugs.Among the approaches that target specific virulence factors to treat periodontal diseases, protease inhibitors against gingipains have been tested in vivo. Although receptor-based peptide inhibitors against LtxA activity and peptides interfering with fimbrial biogenesis show significant results, animal models' potency must be tested to validate their effectiveness.The formation of biofilm by oral pathogens makes these pathogens challenging to treat with antibiotics. Compounds with bioactive in quorum quenching and downregulating adherence factors in microbes can be used to prevent or clear up microbial biofilms. Further studies are required to test the quorum quenching strategies to combat biofilm formation by periodontal pathogens.Plant-derived natural bioactive compounds impart general inhibitory effects on many oral pathogens. The growing emergence and spread of antimicrobial resistance call for renewed interests in plant-based compounds such as polyphenols, which have few side effects to the host. Antimicrobial activity and anti-inflammatory effects of these natural compounds make them an attractive remedy for periodontal diseases. However, the effective doses of these natural antimicrobial compounds need to be determined and their antimicrobial mechanisms assessed before they can be used in conjunction with traditional treatment approaches.Immunomodulatory therapy and microbiome-based therapies show exciting promises in treating periodontal diseases. Still, further research is needed to improve their efficacy and to understand their long-term effects.In summary, significant progress has been made in our understanding of periodontal disease and its treatment strategies in recent years. We can expect novel approaches will become available for periodontal disease prevention and treatment in the near future.


Artabotrys hexapetalus (synonyms: A.uncinatus and odoratissimusA. ) is known as yingzhao in Chinese. Extracts of the plant have long been used in Asian folk medicine to treat various symptoms and diseases, including fevers, microbial infections, ulcers, hepatic disorders and other health problems. In particular, extracts from the roots and fruits of the plant are used for treating malaria. Numerous bioactive natural products have been isolated from the plant, mainly aporphine  and benzylisoquinoline  alkaloids, terpenoids (artaboterpenoids), flavonoids (artabotrysides), butanolides  and a small series of endoperoxides known as yingzhaosu A-to-D. These natural products confer antioxidant, anti-inflammatory and antiproliferative properties to the plant extracts. The lead compound yingzhaosu A displays marked activities against the malaria parasites Plasmodium falciparum and P. berghei. Total syntheses have been developed to access yingzhaosu compounds and analogues, such as the potent compound C14-epi-yingzhaosu A and simpler molecules with a dioxane unit. The mechanism of action of yingzhaosu A points to an iron(II)-induced degradation leading to the formation of two alkylating species, an unsaturated ketone and a cyclohexyl radical, which can then react with vital parasitic proteins. A bioreductive activation of yingzhaosu A endoperoxide can also occur with the heme iron complex. The mechanism of action of yingzhaosu endoperoxides is discussed, to promote further chemical and pharmacological studies of these neglected, but highly interesting bioactive compounds. Yingzhaosu A/C represent useful templates for designing novel antimalarial drugs.The medicinal plant  Despite new treatment modalities, malaria remains a major public health threat worldwide, causing more than 400,000 deaths per year, predominantly in children in sub-Saharan Africa and tropical regions . RecentlPlasmodium falciparum mild malaria generally relies on combination therapy, including artemisinin (ART) and/or chloroquine, whereas intravenous artesunate is often preferred in cases of severe malaria, at least in developed countries [®) is also approved to treat uncomplicated malaria [Artemisia family (qinghao in Chinese) have been extensively studied for their pharmacological properties and phytochemical content.The first-line treatment of ountries . The com malaria ,6. ART a malaria . Herbs oArtabotrys unciatus (L.) Meer.) has been considerably less investigated. Moreover, the main phytochemical compounds found in yingzhao, known as yingzhaosu, have been essentially neglected, at least from a pharmacological viewpoint  Meer. (Annonaceae), from which yingzhaosu compounds have been isolated, as discussed above  Bhandari, which is the accepted botanical name [A. odoratissimus R.Brown.  Bhand. growing at the Fairchild Tropical Garden in Miami (US) have been used to study plant architecture during growth [The Chinese medicine yingzhao is usually associated with the plant name ed above . Howevercal name . Other sR.Brown. . This plg growth . It can g growth .Cananga odorata (ylang-ylang) used in perfumery. A. hexapetalus is a woody scandent climbing shrub. The leaves are oblong to broadly lanceolate in shape and the flowers are fragrant, with yellow petals . There is also mention of the use of the plant to treat fever, diarrhea, dysentery, cuts, sprains, ulcers, and asthma.A. hexapetalus extracts. These can range from antibacterial and antifungal effects to hepato-protective and anti-ulcer activities (A. odoratissimus R.Br. (synonym) has shown a broad-spectrum activity against 14 different storage fungi, and interestingly, it was reported to arrest aflatoxin B1 secretion by a toxic strain of Aspergillus flavus [A. hexapetalus also revealed antifungal effects [Recently, Kousalya and Doss  have invtivities . In genes flavus . Flower  effects .A. hexapetalus extracts [Alcoholic leave extracts display cytoprotective effects in vitro and in vivo. An ethanolic extract orally administered (100–200 mg/k for seven days) to mice with drug-induced liver injury was found to reduce the oxidative damages at least at the biochemical level and to alleviate the sign of cellular degeneration and necrosis. The extract was well tolerated in mice . The obsextracts ,37,38.A. hexapetalus have been found to reduce sperm count and mobility in rats, reducing the diameter of seminiferous tubules. The extract lowered the testosterone level and significantly reduced fertility in rats [A. odoratissimus Roxb. (synonym). In this case, the extracts  were found to disturb the oestrus cycle and reduce implantation and thus fertility [A. odoratissimus Roxb. in female rats. The extracts altered the level of cholesterol and steroidal hormones (estradiol and progesterone) and caused polycystic ovaries in rats [A. odoratissimus plant extracts is known for several decades [The hydroalcoholic leave extracts of  in rats . The obsertility . Antifer in rats ,34. In I decades ,42 and r decades .A. hexapetalus refers to antiparasitic effects. For a long time, yingzhao (roots and fruits) has been used to combat parasites such as Plasmodium falciparum and Leishmania donovani. A petroleum ether extract of A. hexapetalus was shown to moderately reduce the growth of the promastigote forms of cultured L. donovani. The effect was attributed to the presence of flavonoids such as quercetin and apigenin [A. hexapetalus display activities against Plasmodium and Leishmania, but not against the African earthworm Eudrilus eugeniae (African nightcrawler) [A. hexapetalus. Solid data have been reported with other species, such as A. crassifolius [The main bioactivity of apigenin . Notwithapigenin ,44. Hydrcrawler) ,28. It ssifolius , but notA. odoratissimus fruit extract against MIA PaCa-2 pancreatic cancer cells. The organic (ethyl acetate) extract reduced cell growth, revealed an antioxidant effect, and induced apoptotic cell death associated with DNA damage [A. uncinatus (synonym), including cytotoxic oxoaporphines and other alkaloids endowed with cytotoxic effects such as atherospermidine and squamolone  of artabonatine A has been revised in 2018, based on the chemical synthesis of the two diastereomeric isomers of (−)-artabonatine A (anti-isomer was found to function as a potent inhibitor of serotonergic 5-HT2C receptor (Ki = 1.6 μM) whereas the syn-isomer inhibited dopamine transporter (Ki = 3.8 μM) [Annona cherimola Mill (cherimoya), which is also a tropical species of Annonaceae [Pseuduvaria trimera and the compound was shown to exhibit mild cytotoxic properties toward cancer cells in vitro [A. hexapetalus. Hex-A proved to be more cytotoxic toward cancer cells than Hex-B. Its antiproliferative action was comparable to that of the reference anticancer drug cisplatin [The oxazoloaporphine alkaloids artabonatines A-F have been isolated from fresh unripe fruits of ncinatus ,47. Theyncinatus . The strnatine A . Their c 3.8 μM) . These cnonaceae . Anotherin vitro . Howeverisplatin .A. uncinatus, such as liriodenine, anonaine, norushinsunine, asimilobine and stepharine [50 = 23.2 μg/mL against P. falciparum) [Annona species and contribute to the antiparasitic properties of the plants [Known alkaloids have been isolated from epharine . Anonainciparum) . It can ciparum) . Alkaloie plants .A. uncinatus is interesting to underline because it has revealed marked cytotoxic properties, inhibiting the growth of HepG2 liver cancer cells (IC50 = 6.1 μg/mL) [Artabotrys species, such as A. uncinatus and A. crassifolius [A. uncinatus has been named artacinatine [A. spinosus together with artacinatine C [A. hongkongensis [In the case of the rare butanolide, the alkaloid uncinine from 1 μg/mL) . This or1 μg/mL) , but itssifolius ,58. Thissifolius . A relatcinatine . The dercinatine . Artacinnatine C , and froongensis .A. uncinatus, including (i) the alkaloid asimilobine which has antibacterial properties, (ii) the catecholic berberine alkaloid artavenustine, and (iii) a variety of classical sterol derivatives such as β-sitosterol and stigmasterol [A. odoratissimus (synonym) [Lan and coworkers have identified more than 30 compounds from masterol . They almasterol . β-Sitossynonym) ,66.R)-artabotriol has also been identified, together with artabotrycinol [50 values in the range 1.38−8.19 μM [A. hexapetalus, including various yingzhaosu derivatives endowed with antiviral effects. This is the case of the derivative -yingzhaosu C which revealed a noticeable antiviral effect against Coxsackie virus B3 (TC50 = 23.11 μM) [Lignans and flavonoids have been identified as well, including the dibenzylbutyrolactone lignans (iso)americanin, the flavonol glycoside artabotrysides A and B, and diverse flavonoids (taxifolin) and flavonoid glycosides such as quercetin/kaempferol/luetolin glycosides ,67. Artatrycinol . Artabottrycinol . The two−8.19 μM . This or3.11 μM) .Artabotrys species [A. uncinatus is quebrachitol, a cyclic polyol (or cyclitol) which displays antidiabetic properties [Various volatile compounds have been identified from the flowers of the plant, including the abundant sesquiterpene called β-gurjunene  as a maj species ,75. They species . Anotheroperties . Other boperties , but in A. uncinatus (synonym) during storage in the shade for two months, as mentioned later in a report [The first two compounds in the series, yingzhaosu A and B , were isa report . They ara report ,82. YingR-(−)-carvone, a common monoterpene found in many plants [R-(−)-carvone is known for its hypolipidemic, cytoprotective and sedative effects [S-(−)-limonene, providing thus information about the configuration at positions C-4 and C-8 [The first total synthesis of yingzhaosu A was presented in 1991, starting from the precursor y plants . R-(−)-c effects ,85,86,87 effects . Subsequ and C-8 . Then, t and C-8 , as well and C-8 . Over th and C-8 .S-(−)-limonene, with an overall yield of 7.3% [2 and autoxidation [50 = 36.6 and 0.57 μg/mL for yingzhaosu A and C(14)epi-yingzhaosu A, respectively). The epimer exhibited a higher antiplasmodial activity than the parent product . The epimer was much more potent than the parent product in vivo against the chloroquine-sensitive P. berghei NY strain (ED50 = 250 and 90 mg/Kg for yingzhaosu A and C(14)epi-yingzhaosu A, respectively). However, the epimer remained much less active than the reference sodium artesunate in the same in vivo test (ED50 = 4.2 mg/Kg) [A remarkable effort led to the synthesis of yingzhaosu A in only 8 steps starting also from  of 7.3%   . This maThe synthesis of cyclic peroxide has attracted considerable interest in the chemistry field for different reasons  ,97. The P. falciparum (EC50 = 100 nM) but still less active than artemisinin (EC50 = 7.8 nM). Nevertheless, this compound presented a good cell selectivity, being weakly cytotoxic toward FM3A mouse mammary carcinoma cells (IC50 = 33 μM) [P. falciparum (EC50 = 13 nM) with an efficacy comparable to that of artemisinin and a very high cellular selectivity [50 = 3 and 10 nM, respectively) [Various cyclic peroxides structurally close to yingzhaosu A have been described, such as compound 14b , which w= 33 μM) . Other pectivity ,26. Othectively) . These cOne particular compound derived from yingzhaosu A has been developed, the synthetic endoperoxide arteflene (Ro 42-1611) with a 1,2-dioxane unit . This coA better knowledge of the molecular target and pathways involved in the antiplasmodial action of yingzhaosu A would help the design of analogues, but thus far, no study has been specifically devoted to elucidating the mechanism of action of the natural product. However, two important elements point toward the implication of iron and iron complexes in the bioactivity process. On the one hand, it is known that yingzhaosu A can be activated by an iron(II) compound in a Fenton-type reaction . YingzhaP. falciparum. They showed that the compound was attacked by heme to break the endoperoxide bond, thus generating sterically hindered tertiary oxygen-centered radicals. The yingzhaosu A molecule was broken into two parts after a rearrangement to remove the side chain, as represented in On the other hand, a recent study has pointed out the rearrangement and cleavage of yingzhaosu A in the presence of iron-bound heme. The authors built a heme-activatable probe based on the structure of yingzhaosu A to identify novel inhibitors of The role of the iron complex in the mechanism of action of endoperoxide drugs has been largely debated ,119. TheArtabotrys hexapetalus (L.f.) Bhandari, or yingzhao in Chinese, has been known for decades and is extensively used in traditional medicine in Asia for the treatment of malaria and associated fevers. The plant is well known, but the various synonyms can result in confusion and complexity. The accepted name A. hexapetalus should be used primarily, not the synonyms, such as. A. uncinatus and A. odoratissimus in scientific communications. The plant has a large medicinal potential, well recognized in Asia, in particular for the treatment of parasitic diseases [The medicinal plant diseases .The chemical diversity of bioactive natural products identified from the plant is large. It is a rich source of secondary metabolites with diverse chemical classes , as is frequently the case with medicinal species. The presence of many aporphine alkaloids is remarkable, notably in the oxazoloaporphine series . These products could be useful to combat various metabolic diseases, such as type 2 diabetes mellitus, endothelial dysfunction, hypertension and cardiovascular diseases . There aThe high potency of artemisinin and the recognized clinical efficacy of artemisinin combination therapy, which is recommended by the World Health Organization, contribute to promoting the design and development of novel compounds with a cyclic endoperoxide core. A huge diversity of compounds has been synthesized, including second and third generations of artemisinin derivatives , trioxolanes, tri- and tetra-oxanes, and a variety of non-artemisinin-derived synthetic endoperoxide-containing molecules ,132,133.


Chlamydomonas reinhardtii, Arabidopsis thaliana, Oryza sativa, and Zea mays as well as the psychrophilic green alga Chlamydomonas sp. UWO241, and was proven to be a practical and accurate tool for gene duplication analyses. The web tool is free to use at http://hsdfinder.com. Documentation and tutorials can be found via the GitHub: https://github.com/zx0223winner/HSDFinder.Gene duplication is an important evolutionary mechanism capable of providing new genetic material for adaptive and nonadaptive evolution. However, bioinformatics tools for identifying duplicate genes are often limited to the detection of paralogs in multiple species or to specific types of gene duplicates, such as retrocopies. Here, we present a user-friendly, BLAST-based web tool, called HSDFinder, which can identify, annotate, categorize, and visualize highly similar duplicate genes (HSDs) in eukaryotic nuclear genomes. HSDFinder includes an online heatmap plotting option, allowing users to compare HSDs among different species and visualize the results in different Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway functional categories. The external software requirements are BLAST, InterProScan, and KEGG. The utility of HSDFinder was tested on various model eukaryotic species, including  Gene duplication is a near-ubiquitous phenomenon throughout the eukaryotic tree of life . SometimSaccharomyces cerevisiae and Schizosaccharomyces pombe provided evidence for the role of gene duplication in organismal adaptation .There are some previously developed tools and databases for studying gene duplication. The Duplicated Gene Database (DGD), for instance, collected the co-localized and duplicate genes from nine species but has not updated any new species since 2012 . In the Chlamydomonas sp. UWO241 harbours hundreds of HSDs, which might be aiding its survival in the cold via gene dosage  arose more recently than more diverged paralogs . From a functional perspective, HSDs/HS-paralogs probably encode proteins that carry out the same function and thus are more likely to have a role in gene dosage as compared to more divergent duplicates/paralogs.This software is also designed with a user-friendly interface for parsing BLAST all-against-all protein sequence similarity searches via homology assessment metrics ; it integrates structural information, including Pfam domains and InterPro annotations, in order to better annotate gene duplicates; it displays the duplicates to be categorized over KEGG pathway schematics; and it offers an online publication-ready heatmap plotting option for visualizing the duplicates across species.http://hsdfinder.com) or through a local environment (Linux and Python 3) after downloading the software package from GitHub (https://github.com/zx0223winner/HSDFinder). But to run it locally, the pre-installed Python (preferably Python 3) and Linux  environments are required. Usually, a minimum specification requirement is a machine with two cores and 4 GB of random-access memory (RAM), which should allow HSDs to be identified and visualized within a few minutes. The tested data and external software resources, including links, are listed in the key resources table (https://github.com/zx0223winner/HSDFinder.HSDFinder can be run on the Apache server through an online web interface designed using HTML and Python scripts (es table . The docThe software implementation is written in Python 3. There are three groups of custom scripts and platforms: 1) HSDFinder.py, operation.py, and pfam.py filter, which annotate the duplicates from BLAST all-against-all protein similarity search results and protein annotation databases ; 2) HSD_to_KEGG.py categorizes the duplicates under KEGG pathway functional categories; and 3) Django (3.1.5), a Python-based web platform used to maintain the web server as well as pandas (1.2.2), the software library used for manipulating the data. The full HSDFinder source code can be found in the GitHub repository. Necessary input files include the 12-column BLAST all-against-all protein similarity search output in tab-delimited file and the 13-column InterProScan  search oBefore running HSDFinder, two tab-delimited files created by external tools are needed . The firResults section), these default thresholds can capture a large number of HSDs. For poorly curated genomes, potential bottlenecks include an increase in the number of hypothetical proteins among predicted HSDs. But since the similarity of duplicated genes within and among genomes can vary significantly, the thresholds can be adjusted  to acquire more possible HSD candidates  and ≥90% pairwise amino acid identities. Choosing such a strict cut-off might rule out other genuine duplicates from the list. But based on our past experience with green algal genomes  and valindidates . Similarndidates , gene condidates . This isA. thaliana, O. sativa, D. melanogaster were 61.7, 15.5 and 50.0, respectively, while the largest HSDs/Mb value among selected green algae was 2.5 in Chlamydomonas eustigma. This might reflect the diploid nature of the plant and animal genomes, which can yield more gene duplicates via whole-genome duplication events as compared to their haploid green algal counterparts. This can be observed from the results of 3-group HSDs and at least 4-group HSDs in diploid species, which still retain large numbers of HSDs  and ≥ 4-group: 995  in A. thaliana) compared to the haploid algal species  and ≥ 4-group: 27  in Chlamydomonas sp. ICE-L)  . Note, HChlamydomonas sp. ICE-L (0.013) and the acidophilic species C. eustigma (0.020) (C. eustigma: 8/16), histone functional domains   and  (0.020) . The idea: 4/13) . AlthougA. thaliana, O. sativa, Z. mays, D. melanogaster, M. musculus, and R. norvegicus. The statistics of HSD candidates in each species via different thresholds are summarized in A. thaliana genome as an example, an all-against-all protein sequence similarity search using BLASTP  was filtered via the following criteria: from 10 to 100 amino acid length differences and from ≥60% to ≥90% amino acid pairwise identities  . But we introduced a simple equation to roughly evaluate the performance of each metric. For example, in  example , the pervegicus) . Thus, fA. thaliana, Z. mays, and O. sativa  vs 21,622 for A. thaliana  vs 21,461   genome has released ten annotation versions over the past decade.To validate the performance of these parameters in HSDFinder, we compared the number of duplicated genes predicted by HSDFinder to other previously used methods for . sativa . Our detthaliana ; 34,581 thaliana  for Z. ms 21,461  for O. sdentity)  and BLASdentity) . This laDupGen_finder (http://hsdfinder.com/database/).HSDFinder can identify duplicated genes when the duplicates satisfy the assigned criteria: near-identical protein lengths (within 10 amino acids of each other) and ≥90% pairwise amino acid identities. However, it does not rule out another widespread method for duplication detection based on Best BLAST Mutual Hits (BBMH) . Unlike n_finder , our sofn_finder . In our n_finder , the defWith the decreasing cost of biological analyses , biologists are dealing with larger amounts of data, and many bioinformatics software analysis suites require considerable knowledge of computer scripting and microprogramming. HSDFinder is designed to fill the demand for custom-made scripts to move from one analysis step to another. It can analyze duplicated genes from genome sequences by integrating the results from InterProScan and KEGG. HSDFinder aims to become a useful platform for the identification and comprehensive analysis of HSDs in eukaryotic genomes. In the future, the software will be improved by taking into account more scientific discoveries in the field of gene duplication, particularly substitution rate analyses and expression levels.


Spiroplasma ixodetis infection in an immunocompetent patient and an immunocompromised patient who had frequent tick exposure. Fever, thrombocytopenia, and increased liver aminotransferase levels raised the suspicion of anaplasmosis, but 16S rRNA PCR and Sanger sequencing yielded a diagnosis of spiroplasmosis. Both patients recovered after doxycycline treatment.We report 2 cases of  Borrelia spp., tick-borne encephalitis virus, Babesia spp., Rickettsia spp., Neoehrlichia mikurensis, Anaplasma phagocytophilum). Spiroplasma ixodetis was initially described as a cause of neonatal cataract and uveitis  in July 2021 because of a 3-day history of fever (temperature up to 39°C) and mild headache. She reported frequent tick exposure in southeastern Sweden but no history of opportunistic infections or immunosuppressive diseases or treatments that would have compromised immune defenses. She was admitted because of clinical suspicion of anaplasmosis.A. phagocytophilum (N. mikurensis (S. ixodetis that had 99.72% sequence homology with a reference strain of S. ixodetis (GenBank accession no. MN166761) (S. ixodetis sequence has been deposited in GenBank (accession no. OL636349).Blood tests showed thrombocytopenia and increased levels of C-reactive protein (CRP) and alanine aminotransferase (ALT) . Real-tikurensis  identifiN166761) . The S. A. phagocytophilum was observed . However, the result was disregarded because of the negative A. phagocytophilum PCR result at admission.A slight increase in IgG convalescent-phase titer against The fever decreased promptly when oral doxycycline treatment (100 mg 2×/d) was initiated. The patient was discharged, and treatment was continued for a total of 10 days. Upon follow-up, the patient had recovered and had no remaining laboratory result abnormalities . Total sCase-patient 2 was a 76-year-old man who had insulin-dependent type 2 diabetes and Crohn’s disease who had been given infliximab maintenance therapy. He was on a prednisolone taper after an exacerbation of his inflammatory bowel disease. The patient sought care at the emergency department of Visby County Hospital  in October 2021 for a 2-week history of spiking fevers and fatigue. He reported multiple tick bites throughout summer and had been given penicillin V for erythema migrans. No other focal signs or symptoms were reported.At admission, blood tests showed pancytopenia with predominant thrombocytopenia and increased CRP and ALT levels . EmpiricN. mikurensis and A. phagocytophilum by PCR and unbiased bacterial 16S rRNA sequencing. Analysis identified S. ixodetis in serum that had 99.86% sequence homology with a reference strain of S. ixodetis. The patient sequence has been deposited in GenBank (accession no. OL636350) , which resulted in return of liver and kidney functions to reference values within 1 week and improved clinical condition . The patL636350) . The patS. ixodetis infection that were presumably acquired by tick bites in southeastern Sweden. This organism has not been reported in Ix. ricinus ticks from Sweden, but A. phagocytophilum, N. mikurensis, Rickettsia spp., and Babesia spp. are endemic tickborne microorganisms that may cause febrile illness. However, thrombocytopenia and increased levels of liver enzymes rarely occur in neoehrlichiosis . The route of transmission was unclear except for the S. apis case, for which a hornet sting was the plausible route of transmission. In contrast, the S. ixodetis patients we describe were most likely infected via tick bites acquired in the coastal areas of southeastern Sweden, including the islands of Öland and Gotland.In reports on systemic S. ixodetis infection. Previous case reports of human Spiroplasma infection have been associated with an immunocompromised state, either in the form of immature eyes of newborns or conditions requiring immunosuppressive treatment. We report a systemic Spiroplasma infection in an apparently immunocompetent person. However, immunosenescence of the aged immune system might have compromised innate or acquired immune defenses (Our study suggests an association between tick exposure and human S. ixodetis infection and acute febrile illness after tick exposure. Treatment with doxycycline was successful. This finding shows the clinical utility of unbiased 16S rRNA analysis for correct diagnosis and treatment, as well as its potential for identifying novel pathogens in the febrile host. We are developing a Spiroplasma-specific PCR that might increase sensitivity of detection. S. ixodetis is an emerging pathogen that should be considered in patients with febrile illness after tick exposure.In conclusion, we report 2 case-patients who had Spiroplasma ixodetis infections in immunocompetent and immunosuppressed patients after tick exposure, Sweden.Additional information on


Legionella, including the most prevalent Legionella pneumophila, has been treated primarily with antibiotics. Environmental water and soil are the reservoirs for L. pneumophila. Studying antimicrobial susceptibility using a large number of isolates from various environmental sources and regions could provide an unbiased result. In the present study, antimicrobial susceptibility of 1464 environmental L. pneumophila isolates that were derived from various environmental water and soil sources of 12 cities in China to rifampin (RIF), erythromycin (ERY), clarithromycin (CLA), azithromycin (AZI), ciprofloxacin (CIP), moxifloxacin (MOX), levofloxacin (LEV), and doxycycline (DOX) was investigated, and minimum inhibitory concentration (MIC) data were obtained. We show that regarding macrolides, ERY was least active (MIC90 = 0.5 mg/L), while CLA was most active (MIC90 = 0.063 mg/L). A total of three fluoroquinolones have similar MICs on L. pneumophila. Among these antimicrobials, RIF was the most active agent, while DOX was the most inactive one. We observed different susceptibility profiles between serogroup 1 (sg1) and sg2-15 or between water and soil isolates from different regions. The ECOFFs were ERY and AZI (0.5 mg/L), RIF (0.002 mg/L), CIP, CLA and MOX (0.125 mg/L), LEV (0.063 mg/), and DOX (32 mg/L). Overall, two fluoroquinolone-resistant environmental isolates (0.14%) were first documented based on the wild-type MIC distribution. Not all azithromycin-resistant isolates  harbored the lpeAB efflux pump. The MICs of the ERY and CLA on the lpeAB + isolates were not elevated. These results suggested that the lpeAB efflux pump might be only responsible for AZI resistance, and undiscovered AZI-specific resistant mechanisms exist in L. pneumophila. Based on the big MIC data obtained in the present study, the same defense strategies, particularly against both CLA and RIF, may exist in L. pneumophila. The results determined in our study will guide further research on antimicrobial resistance mechanisms of L. pneumophila and could be used as a reference for setting clinical breakpoints and discovering antimicrobial-resistant isolates in the clinic, contributing to the antibiotic choice in the treatment of LD.Legionnaires’ disease (LD), caused by  Legionella pneumophila (L. pneumophila) is the most common causative agent of LD. It is a Gram-negative, facultative intracellular opportunistic pathogen of humans and is responsible for both sporadic and epidemic community-acquired and hospital-acquired cases of LD. Legionella pneumophila is ubiquitously present in aquatic environments, including moist soil and water systems, where aquatic protozoa act as its natural hosts  appears to be responsible for over 84% of diagnosed cases and 80–90% of community-acquired LD , which was first identified in 1977, manifests as atypical pneumonia and can evoke severe disease and death . Legioneal hosts . The co-cause LD . Aquatic persons . Direct uired LD . Almost nfection .L. pneumophila, due to their intracellular permeability  of the gyrA gene of L. pneumophila  distribution and ECOFFs were a noteworthy issue  , early aeability . Howeverreported . Some AZumophila . These fumophila , which iumophila . In the umophila ; howeverumophila . Meanwhihy issue . In addiL. pneumophila isolates to investigate the in vitro activity of RIF, ERY, CLA, AZI, CIP, MOX, LEV, and DOX against L. pneumophila derived from diverse environments , which could represent the most common aquatic environmental reservoirs of L. pneumophila in China. Antimicrobial susceptibility profiles of these isolates belonging to different serogroups (sgs) or from different environmental sources and regions were investigated. Furthermore, we aimed to determine an unbiased tentative ECOFF for these antibiotics against L. pneumophila since EUCAST provides widely accepted ECOFFs for these antimicrobial agents that are used for the treatment of LD.In the present study, we compiled a large (∼1500 isolates) and comprehensive collection of Legionella bacterial isolates from environmental sources were used.Ethical approval was not required as only L. pneumophila isolates (including 329 sg1 and 1135 sg2-15 or 1079 water and 385 soil isolates) derived from 138 samples, which were obtained from 12 cities in seven provinces of China between April 2019 and January 2021, were investigated in this study. These isolates were from various aquatic sources, including water sources of rivers, sea, lakes, ponds, small streams, and grass puddles, and soil sources of the garden and potting soils , LEV , and DOX . The testing concentration range is 0.016–32.0 mg/L for ERY and DOX; 0.004–8.0 mg/L for CIP, CLA, AZI, MOX, and LEV; and 0.0000625–0.125 mg/L for RIF based on the data obtained from the reference strain ATCC33152. The Legionella bacteria were inoculated on buffered charcoal yeast extract agar supplemented with 1g/L α-ketoglutarate, 0.25 g/L L-cysteine, and 0.4 g/L ferric pyrophosphates (BCYEα) and then incubated at 37 °C with 5% v/v CO2 atmosphere with a higher humidity for 72-96 h before the antibiotic susceptibility test (AST).A total of 1464 ng soils . The samus study . The L. Legionella species  in a 1.5-mL tube. A final Legionella bacterial concentration of ∼105/mL in each well was obtained by dilution. A volume of 20 μL of each antibiotic solution was then added to microtiter wells in 2-fold decreasing concentrations. The final volume of each well was 100 μL, containing 104Legionella bacteria. A blank well consisting of 100 μL of the BYE broth was used as a negative control. The plates were sealed and incubated for 48 h without agitation at 37°C before determining the MIC by observing the presence or absence of turbidity. The MIC was defined as the lowest concentration of antibiotics yielding complete inhibition of visible growth. Each isolate was tested at least two times to verify the results.Minimum inhibitory concentrations (MICs) were determined by using the broth microdilution (BMD) test using buffered yeast extract broth supplemented with 1g/L α-ketoglutarate, 0.25 g/L L-cysteine, and 0.4 g/L ferric pyrophosphates (BYE) on 96-well microtiter plates as it can provide unbiased MICs due to the absence of charcoal and it was the internationally agreed gold standard for  species . In brie50 and MIC90, respectively. The ECOFF is used to separate bacterial populations based on MIC distributions and is defined as the highest MIC value of isolates that are not known to have resistance  and rplV (protein L22) (lpeA and lpeB (designated as lpeAB) is mainly the cause of AZI resistance in L. pneumophila , lpeA_R: GGTGTCTTCGTCGAGCA; lpeB_F: CA TCCTGTAATCACCATCATC, lpeB_R: CAACGGAAGCA ACACCTTG. These primers could not only detect the presence of lpeAB but also define if the two genes were correctly arranged in spatial.More and more antibiotic-resistant isolates are found in clinical isolates . These iisolates . L. pneuumophila . Many maein L22) . Howeverumophila . Therefot-test or one-way ANOVA. For categorical variables, we calculated the frequencies and percentages of isolates in each category by chi-square or Fisher’s exact tests. Non-parametric Spearman’s correlation analysis was used to analyze the relationship between different antibiotics against L. pneumophila isolates. Statistical analyses were performed using SPSS 25.0 (IBM software). A principal component analysis (PCA) for MICs of the eight antibiotics was performed using Origin 2021b (OriginLab). P < 0.05 was considered statistically significant. Simpson’s diversity index was used to measure the homogeneity of antibiotic susceptibility of the isolates using the formula: D = 1-Σ(Ni/N)2.Continuous variables (log MICs) were compared using Student’s in vitro susceptibility profiles of the 1464 L. pneumophila isolates against the eight antibiotics, as judged by MIC distributions, are shown in L. pneumophila isolates, almost all tested ones were inhibited by low concentrations of RIF, fluoroquinolones, and macrolides. ERY yielded MICs ranging between 0.031 and 0.5 mg/L. Similar results were found in CIP (0.008–0.5 mg/L), which had the same upper limit but a lower limit as compared with ERY. By contrast, AZI produced a higher upper limit of MIC than CIP (0.008–1 mg/L). This result was following the average MICs of each antibiotic against L. pneumophila (L. pneumophila sg 1 ATCC 33152 (two repetitions) showed higher sensitivities  to all the eight antibiotics, including RIF (0.0005 mg/L), ERY (0.125 mg/L), CLA (0.016 mg/L), AZI (0.25 mg/L), CIP (0.063 mg/L), MOX (0.031 mg/L), LEV (0.031 mg/L), and DOX (8 mg/L). MIC diversity among the isolates was highest in ERY, while was lowest in MOX (0.70 vs. 0.23), indicating more mechanisms might be existed in confronting ERY for L. pneumophila, and vice versa. Previous studies using the BMD method to perform susceptibility testing showed variability in the MIC50, MIC90, and MIC ranges  is the major cause of decreased RIF susceptibility in many bacteria  have been observed to play a role in regulating RIF susceptibility in another intracellular bacterium Mycobacterium tuberculosis  and could be defined as resistant isolates. These results were roughly consistent with our results obtained by the ECOFFinder, although we did not find an obvious bimodal distribution of MIC on the 1464 isolates. L. pneumophila  from a pneumonia patient, probably due to the gyrA mutation during antimicrobial therapy. Fluoroquinolone-resistant strains from environmental sources were not documented yet, and our findings revealed the hidden isolates in the environments and emphasized the need for environmental surveillance and susceptibility testing of L. pneumophila isolates. Given that the two isolates were resistant to all fluoroquinolones  based on the WT distribution of MIC, resistance-associated mutations in the QRDR of DNA gyrase and topoisomerase IV coding genes may exist and requires further research. The two studies that utilized limited numbers (122 and 149 strains) of L. pneumophila composed of environmental and clinical strains from Norway or China showed that frequencies of AZI-resistant ones were both about 17% (21/122 and 25/149), and environmental strains displayed similar frequencies of resistant ones with the clinical strains (L. pneumophila were found with an MIC ≥ 0.5 mg/L (BMD method)  than ever thought.The primary MIC distribution of the eight antimicrobials against e E-test . They she E-test  for DOX .’s data , 3  . In addi method) . HoweverL. pneumophila against AZI, a multiplex PCR was conducted not only to detect the efflux pump coding genes lpeA and lpeB but also to detect the genetic arrangement of the two genes because the arrangement may have a potential impact on the gene expression  harbored lpeAB efflux. For the 33 AZI-resistant isolates that had an MIC of 0.5 mg/L, 31 (93.94%) harbored lpeAB efflux. All the lepAB-positive (lpeAB +) isolates had the right spatial arrangement of two genes. Conversely, none of the AZI-sensitive isolates (MIC < 0.5 mg/L) harbored the lpeAB efflux according to the PCR for 273 representative isolates , but an elevated MIC of CLA in lpeAB- isolates was observed  were distinct between isolates from different environmental sources or regions in China. Natas et al. and Jia et al. both showed that all AZI-resistant L. pneumophila isolates harbored an lpeAB efflux pump; however, we did find 2 AZI-resistant isolates without lpeAB. These isolates also showed similar MICs of other two macrolides, as well as other types of antibiotics, when compared with those lpeAB- AZI-sensitive isolates  resistance and 46 isolates with AZI resistance were determined in the present study. Genomic research, multiplex PCR assay, and statistical analysis confirmed the correction of lepAB efflux with AZI-resistance in L. pneumophila. However, we found this mechanism was only restricted to AZI but no other macrolides . Furthermore, some undefined AZI-specific resistance mechanisms that are not the same as other macrolides  in L. pneumophila might exist. With the statistical analysis using our big data, we also proposed the existence of the shared defense strategies, particularly against both RIF and CLA in L. pneumophila, which deserves further research.A large number of The original contributions presented in this study are included in the article/Legionella bacteria. J-LY, HS, XZ, and MY verified the data. All authors contributed to the article and approved the submitted version.X-YZ designed the study, made data interpretation, and revised the manuscript. J-LY carried out the experiments. X-YZ and J-LY analyzed the data and wrote the manuscript. HS collected the environmental samples for the isolation of


Staphylococcus capitis is primarily described as a human skin commensal but is now emergent as an opportunistic pathogen isolated from the bloodstream and prosthetic joint infections, and neonatal intensive care unit (NICU)-associated sepsis. We used comparative genomic analyses of S. capitis to provide new insights into commensal scalp isolates from varying skin states , and to expand our current knowledge of the species populations . A highly recombinogenic population structure was revealed, with genomes including the presence of a range of previously described staphylococcal virulence factors, cell wall-associated proteins, and two-component systems. Genomic differences between the two described S. capitis subspecies were explored, which revealed the determinants associated exclusively with each subspecies. The subspecies ureolyticus was distinguished from subspecies capitis based on the differences in antimicrobial resistance genes, β-lactam resistance genes, and β-class phenol soluble modulins and gene clusters linked to biofilm formation and survival on skin. This study will aid further research into the classification of S. capitis and virulence-linked phylogroups to monitor the spread and evolution of S. capitis. Staphylococcus capitis was first isolated from healthy human skin in 1975 and classified as a coagulase-negative Staphylococcus species (CoNS) , incorporating a further 127 publicly available sequences, with the aim to expand knowledge of S. capitis population structure, the genotypic definition of subspecies, and identify factors that are likely to contribute to virulence, competition, and colonization.In the study presented here, we whole-genome sequenced 59 S. capitis isolates of scalp skin  collected in the UK in 2017. Scalp samples were obtained using the method of Williamson and Kligman , 0.2% (w/v) lithium chloride , 0.5 % (v/v) glycerol , 1.0% (v/v) sodium azide (Sigma Aldrich), and 3.0% (v/v) sterile egg yolk emulsion (Lab M), pH 7.2].Whole-genome sequencing (WGS) was performed on 59  Kligman . The colS. capitis subspecies [ATCC 49325 (S. capitis subsp. ureolyticus) and ATCC 27840 (S. capitis subsp. capitis)] (https://www.ncbi.nlm.nih.gov/genbank/), Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra), or European Nucleotide Archive (https://www.ebi.ac.uk/ena), and accession numbers are listed in S. capitis genomes were downloaded. All publicly available S. capitis genome reads were also downloaded and subject to quality control. This study included WGS reads from Illumina sequencing only, with more than 10X coverage. All publicly available datasets were then phylogenetically reconstructed (as described below), and Treemmer v0.3 was used to reduce redundancies within the public dataset, leaving 120 genomes to be included in further analyses ] . Also inAll isolates selectively obtained in this study, together with seven further strains comprising culture collection type strains that were included within a formally defined taxonomy (included in this study: NCTC 11045 and DSM 6717) and skin isolates, were grown in 10 mL of BHI broth (Lab M) overnight by shaking at 37 °C. Subsequently, 1 mL of each overnight culture was centrifuged for 1 min at 5,000 rpm and resuspended in 180 μl of lysis buffer ; the cells of each clone were extracted to obtain high-quality genomic DNA using the QIAGEN DNeasy Blood and Tissue Kit and eluted in 10 mM Tris-HCl (pH 8.5).−1) was submitted for Illumina DNA sequencing by MicrobesNG  using Nextera XT library protocol on a HiSeq platform, generating 250 bp paired-end reads . The resulting datasets are available from the SRA under BioProject number PRJEB47273. Adapters and low-quality bases were trimmed with Trimmomatic v???  and MultiQC v1.0  agarose gels (at 90 mV for 40 min with a 1 kb ladder). For sequencing, the extracted DNA in a final volume of 60 μl  were mapped to the reference genome S. capitis AYP1020, which was isolated from human blood  . To examine the phenotypic basis for the separation of the two subspecies within the phylogenetic reconstruction . The API Staph-Ident Strip system was used according to the manufacturer's instructions.To measure the extent of truction , 3, the Pan-genome analysis of all 186 isolates was performed using the Panaroo v1.1.2 software package with default parameters, MAFFT alignment, and a core gene threshold of 90%  > 1 were considered to be overrepresented in the subspecies cluster.To identify the genes enriched in https://github.com/tseemann/abricate) with NCBI Bacterial Antimicrobial Resistance Reference Gene Database, with a default minimum DNA percentage identity of 80% , S. epidermidis RP62a (Genbank assembly accession: GCA_000011925.1), and S. aureus MW2 (Genbank assembly accession: GCA_00307695.1) and pan-genome data from various studies  indices were used to quantify the genetic relatedness of the two nd Kloos .Written informed consent was obtained from all enrolled participants. The study protocol was reviewed and approved by an Independent Ethics Committee and operated at Unilever Port Sunlight, United Kingdom. The study was conducted in compliance with the Declaration of Helsinki and its subsequent revisions.Staphylococcus capitis sequence reads  were assembled into draft genomes with an average of 85 contigs. The mean size of the assembled genomes ranged from 2.2 to 2.6 Mb. Each genome had a mean 2,335  predicted protein sequences (CDSs) with a meanGC content of 32.7%, similar to the S. capitis reference genome AYP1020 . The pan-genome was further divided into 2,034 (45.4%) core genes  and 2,437 (54.5%) accessory genes (shared by some genomes)  . Gene acgenomes) . S. capiS. capitis subspecies revealed that most annotated genes in the accessory genomes of both S. capitis ssp. capitis (53 %) and ssp. ureolyticus (57 %) were poorly characterized, which was also true of the core genome (ssp. capitis 41 % and ssp. ureolyticus 38 %). The finding could indicate the presence of novel gene clusters in the S. capitis genome and a low level of curation. The most abundant categories in the core genomes of both subspecies were those linked to essential gene functions, such as transcription (ssp. capitis 7 % and ssp. ureolyticus 7 %). In contrast, the accessory genomes were enriched with gene clusters associated with replication, recombination, and repair (ssp. capitis 9 % and ssp. ureolyticus 9 %), as observed in the pan-genome analysis of each subspecies , while commensal isolates were associated with the S. capitis ssp. capitis clade (59/82). Of note, 22 clinical isolates are interspersed across the dominant subclade within the S. capitis ssp. capitis clade and 23 commensal isolates are interspersed across the two S. capitis ssp. ureolyticus subclades ; however, this is likely due to the reference genome itself being clustered in this group. Recombination was inferred across large regions of the chromosome, predominantly within the first ~750 kb and the last ~1 Mb of the genome . The recS. capitis genomes analyzed, 48% (5% S. capitis ssp. capitis and 44% S. capitis ssp. ureolyticus) were classified as MDR, carrying genetic determinants conferring resistance against three or more classes of drugs  compared to 169 found in commensal isolates (n = 81).The CoNS that colonize human skin are generally considered to be non-pathogenic species specialized for healthy human skin and mucosal surfaces, but they are now emerging as important opportunistic pathogens Otto, . Antimicof drugs . MDR wasS. capitis. PSMs are a novel toxin family that have antimicrobial activity  contributing to the virulence potential of O'Neill, . A totalO'Neill, , with ge O'Neill . Similaris clade . Specifiis clade .rd and/or tryptophan at the 20th position are putatively associated with providing antibacterial activity of β-class family peptides  proteins curated with potential virulence roles; biofilm-associated proteins IcaRADBC; capsule biosynthesis proteins CapDACB; surface adhesins AtlE, Pls, Aap, FnbpA, SesA, SesB, SesC, SesG, Ebp, and Bap; and MSCRAMMs SdrX, SdrZL, SdrH, SdrF, and SdrG. Of these CWA proteins, 11 were encoded in S. capitis AYP1020. Across the 186 S. capitis genomes analyzed, sesA, sesB, sesC, sesG, icaRADBC, fbnpA, capDACB, and atlE were found in all isolates. MSCRAMM genes sdrX and sdrZL were found in >95 % of genomes investigated. Contrastingly, the surface adhesin gene pls was found in <10 % of S. capitis genomes and was absent from ssp. capitis (S. capitis AYP1020 genome (determined in this study as S. capitis ssp. ureolyticus) but present in other CoNS species, such as S. epidermidis RP62a  and S. saprophyticus (11 TCS) with numbers determined by Rapun-Araiz et al. (The 16 two-component systems (TCS) described in z et al.  to be inz et al. .S. capitis ssp. capitis and S. capitis ssp. ureolyticus based upon the original descriptions of Bannerman and Kloos . A total of 22 isolates sampled in this study that were spread across the phylogenetic tree (11 assigned to the S. capitis ssp. capitis clade and 11 assigned to the S. capitis ssp. ureolyticus clade), as well as the type and culture collection stains, were tested for classifying the phenotypic traits. Among the 11 isolates belonging to the S. capitis ssp. ureolyticus clade, only 4 (33%) tested urease-positive/maltose-positive, and 10 (91%) S. capitis ssp. capitis isolates tested urease-negative/maltose-negative, indicating an unreliable phenotype of urease activity and maltose fermentation as a subspecies definition  between isolates determined here as S. capitis ssp. ureolyticus and S. capitis ssp. capitis revealed that genomes from the two subspecies shared little genetic differences with 96% ANI. Pan-genomic comparative analysis also revealed limited gene content variation of 1% between the two subspecies. To help identify genes that could be used to discriminate between the subspecies and serve as diagnostic markers for rapid identification by PCR in future studies, we identified significantly overrepresented genes of each subspecies. This approach identified a total of 38 gene clusters found across all S. capitis ssp. capitis genomes and 13 across all S. capitis ssp. ureolyticus genomes , another version sharing blastp identity of 22% was only present in S. capitis ssp. capitis isolates (0%).Since the original phenotypic trait descriptors for  genomes , 3. Upon genomes . An examS. capitis subspecies, we applied Scoary to identify genes that are overrepresented in S. capitis ssp. capitis and S. capitis ssp. ureolyticus. A total of 1,086 predicted gene clusters were found to differ significantly (p < 0.05) between the subspecies, although no significant differences were found between the assigned functional COG categories (S. capitis ssp. capitis isolates include those for the arginine catabolic mobile element (ACME), encoding arginine deiminase activity found in various species of Staphylococcus. Most S. capitis isolates in this study contain a Type V ACME gene cluster; however, different conserved versions are found in each subspecies. ACME types are currently characterized by the presence and absence of the opp3 operon, encoding an arginine deaminase pathway, the arc operon, an oligopeptide permease ABC transporter, and kdp operon, encoding a potassium transporter  that encodes enzymes to counteract low pH  element  can be found below: The studies involving human participants were reviewed and approved by an Independent Ethics Committee and operated at Unilever Port Sunlight, United Kingdom. The patients/participants provided their written informed consent to participate in this study.CC and MH conceived and designed this study and drafted the manuscript. CC performed all the data analysis and interpretation for the results under the scientific guidance of RB and MH. All authors contributed to the editing and approved the final manuscript.This work was funded by a Biotechnology and Biological Sciences Research Council Doctoral Training Partnership CASE studentship BB/M011186/1 awarded to MH, with support from Unilever PLC.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Salvia miltiorrhiza, CUL genes have not been characterized and its role in plant development, stress response and secondary metabolite synthesis have not been studied. In this study, genome-wide analyses were performed to identify and to predict the structure and function of CUL of S. miltiorrhiza. Eight CUL genes were identified from the genome of S. miltiorrhiza. The CUL genes were clustered into four subgroups according to phylogenetic relationships. The CUL domain was highly conserved across the family of CUL genes. Analysis of cis-acting elements suggested that CUL genes might play important roles in a variety of biological processes, including abscission reaction acid (ABA) processing. To investigate this hypothesis, we treated hairy roots of S. miltiorrhiza with ABA. The expression of CUL genes varied obviously after ABA treatment. Co-expression network results indicated that three CUL genes might be involved in the biosynthesis of phenolic acid or tanshinone. In summary, the mining of the CUL genes in the whole genome of S. miltiorrhiza contribute novel information to the understanding of the CUL genes and its functional roles in plant secondary metabolites, growth and development.CULLIN (CUL) proteins are E3 ubiquitin ligases that are involved in a wide variety of biological processes as well as in response to stress in plants. In   •Salvia miltiorrhiza.Eight CUL ubiquitin ligase genes were identified from the genome of •CUL genes were clustered into four subgroups according to phylogenetic relationships.The •CUL genes varied obviously after ABA treatment.The expression of seven •CUL genes might be involved in the phenolic acid or tanshinone biosynthesis.Three  Then, the seed file of the CULLIN domain (PF00888) was retrieved from the Pfam database. The HMMER program was used to identify the potential CUL genes in S. miltiorrhiza (CUL genes obtained from the result of HMMsearch were further submitted to SMART website (http://smart.embl-heidelberg.de/) to determine completeness of CUL conserved domain (CUL gene candidates (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/)  .2.2A. thaliana and S. miltiorrhiza were collected. In final, the total of 14 CUL protein sequences were downloaded. Sequence alignment was performed using MEGA 6.0 software with the ClustalW function (https://itol.embl.de/) (CUL protein sequences from function . The phymbl.de/) .2.3S. miltiorrhiza CUL genes, the GSDS (Gene Structure display System) tool was utilized (http://gsds.cbi.pku.edu.cn/)   of the edu.cn/) . The novte.org/) . For a tsoftware .2.4CUL genes was specified as putative promoter sequences. Eight promoter sequences of CUL genes were retrieved using Tbtools  (A length of 3000-bp in the upstream of initiation codon of the  Tbtools . The cise online . Accordiart.com) .2.5S. miltiorrhiza plants were cultivated on Murashige and Skoog (MS) media at 25 °C under a 16 h light/8 h dark photoperiod (Agrobacterium rhizogenes strain C58C1 (pRi A4) cultures were used to infect the sterile stems and/or leaves of S. miltiorrhiza to generate hairy roots . Then, Cytoscape software is used to visualize the above results  according to protocols. Double stranded cDNAs were separated on agarose gel, and were recovered for the RNA-seq . Construeviously . Using teviously . Based o results .2.6SmActin gene was used as an internal control. The primer pairs for qRT-PCR are summarized in −ΔΔCt method was introduced to perform the quantification of gene expression  and hairy roots grown for 40 days were treated with 50 μM ABA for RNA isolation, and was converted into cDNA using a plant RNA prep pure kit  . cDNA ofpression . Each ge33.1CUL genes in the S. miltiorrhiza genome. The HMM (Hidden Markov Model) profile of the CUL domain was obtained from the Pfam database. The HMMER tools were employed to convert the multiple sequence alignment into the position-specific scoring system, then to conduct large-scale sequence analysis. In final, we obtained eight putative sequences using HMMER with default parameters and a significant P-value of 0.01. We have analyzed the molecular weight, number of amino acids, gene length, pI, instability index, grand average of hydropathy, introns, class, and subcellular localization of all the CUL genes , Arabidopsis (6 members)  . AccordiCUL genes in each group were counted. In S. miltiorrhiza, it was found that Groups I–IV contained 4, 2, 1, 1 CUL genes, respectively. In Arabidopsis, Groups I to IV contained 2, 2, 1, 1 CUL genes, respectively. Comparison of S. miltiorrhiza and Arabidopsis, the increased gene number in group I implies the presence of gene expansion in S. miltiorrhiza.In order to evaluate the degree of gene expansion or loss during evolution, the 3.3CUL genes, we compared the full-length cDNA sequences of all genes with the corresponding genomic DNA. By comparing the number and location of exons and introns , salicylic acid (SA), gibberellin (GA), auxin and methyl jasmonate (MeJA) are very important in most of the CUL genes , which indicates that most of the CUL genes in S. miltiorrhiza may be particularly sensitive to ABA treatment. In addition, 7 SmCUL genes  are confirmed to have MeJA responsive elements, and 6 SmCUL genes  have cis-acting elements related to drought, indicating that these genes may have special resistance under drought stress. It is worth noting that the MYB transcription factor (TF) binding elements exists in the six SmCUL genes , indicating that the six SmCUL genes may be regulated by MYB genes in response to drought stress. The promoter elements are clustered and represented by a word cloud image. As shown in cis-acting elements including light responsive element (ATTAAT), abscisic acid (ABA) responsive element (ACGTG), MeJA responsive element (TGACG and CGTCA), low temperature responsive element (CCGAAA), MYB drought-induced binding site (CAACTG), auxin responsive element (AACGAC), salicylic acid responsive element (CCATCTTTTT), gibberellin responsive element (CCTTTTG and TCTGTTG) and stress responsive element (ATTCTCTAAC), are abundant in the promoters of CUL ubiquitin ligase genes  were examined to explore the function of the gene participating in plant growth, development and secondary metabolism in S. miltiorrhiza  exhibited the highest expression level in the vegetative tissue of stem. Whereas, SmCUL3A got the highest expression level in root, which is used as the medicinal harvesting tissue in traditional chinese medicine. The differential expression of CUL genes in various tissues indicated the diverse function in plant secondary metabolite synthesis, growth and development in S. miltiorrhiza.The expression profiles of the eight iorrhiza . The res3.7CUL genes with phenolic acids and tanshinone biosynthetic genes was constructed, and the result indicated that 3 out of 8 CUL genes  showed a negative correlation with phenolic acids biosynthetic genes (CUL gene (SmCUL2) showed a positive correlation with CPS gene in tanshinone biosynthetic pathway with Pearson correlation coefficient(r) > 0.8 and P-value <0.05 as a cutoff , tyrosineamino transferase (TAT) .The co-eic genes . Co-expra cutoff . Overall4CUL ubiquitin ligase genes widely exist in plants and have been validated to participate in diverse biological functions  (unctions . Due to diseases . Genome-nia (11) .Arabidopsis, 6 out of 11 CUL genes have complete C-terminal or N-terminal sequence, and the other five CUL genes cannot be translated normally because they do not have complete N-terminal and C-terminal sequence. Based on the results of evolutionary tree clustering, eight CUL genes in S. miltiorrhiza were named according to the names of CUL genes in A. thaliana , were grouped into four groups , Cul-BTB (Group II), Cul-DDB1 (Group III) and Cul-APC/C (Group IV). CUL ubiquitin ligases can form multisubunit enzymes with complex structures . The inte family .The dive species . In the  introns . It was utations . The rescis-acting elements within promoters, it indicates that the CUL gene family is involved in stress-related mechanisms, hormone regulation, growth and development  promoters all contained ABA responsive elements, among of which SmCUL1 contains the largest number of ABA responsive elements reaching to six. The result indicates that the CUL gene may play an essential role in the ABA signal transduction process in S. miltiorrhiza. In A. thaliana, AtCUL3 was validated to interact with AtHB6 to respond to ABA induction  were identified in S. miltiorrhiza, suggesting that CUL genes might be regulated by related MYB genes mediating the drought stress signaling (CUL genes in S. miltiorrhiza. These results indicated that CUL genes might participate in diverse biological processes during growth and development in S. miltiorrhiza (Through analyzing elopment A (Belda-elopment . In our  regions B. In parnduction . Meanwhinduction . Herein,ignaling , and thiiorrhiza .CULgenes were thought to participate in various abiotic stress and hormone induction (CUL genes (CUL genes responding to ABA treatment. Based on RNA-sequencing databases, it revealed that except to SmAPC2, the other seven CUL genes could be induced by ABA treatment (CUL genes exposed to ABA treatment were consistent with the transcriptome database (cis-elements in the CUL gene family are associated with hormone regulation not only contain ABA, but also include SA, GA and other auxins (The nduction . Due to UL genes , it pushreatment . Our quadatabase b In factr auxins .CUL gene family in S. miltiorrhiza based on the whole genome, transcriptome dataset and qRT-PCR expression analysis. Our research is the first systematic and comprehensive analysis of the CUL genes family in S. miltiorrhiza, and provides a valuable information for further elucidating the molecular mechanism of CUL genes responding to ABA induction. It may also help us to recognize the diverse biological functions of CUL genes in other species.In conclusion, we have characterized the Xiankui Gao: Writing – original draft, Drafting the manuscript, Validation, Methodology, Formal analysis, Funding acquisition, of, Data curation. Xiujuan Li: Writing – original draft, Drafting the manuscript, Formal analysis, Funding acquisition, of, Data curation. Chengan Chen: Formal analysis. Can Wang: Formal analysis. Yuqi Fu: Resources, Investigation. ZiZhen Zheng: Resources, Investigation. Min Shi: Writing – review & editing, Supervision. Xiaolong Hao: Writing – review & editing. Limei Zhao: Methodology. Minghua Qiu: Writing – review & editing. Guoyin Kai: Conceptualization, and design of study, Acquisition of data, Revising the manuscript. Wei Zhou: Writing – original draft, Drafting the manuscript, Formal analysis, Conceptualization, and design of study, Acquisition of data, Revising the manuscript, Approval of the version of the manuscript to be published.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.