Chronic spontaneous urticaria (CSU) is a histamine-mediated inflammatory skin disease, and second-generation non-sedating H1-antihistamines (nsAH) at licensed doses have long been the first-line therapy in CSU. However, about 50% of patients are resistant to nsAH, and the precise pathogenesis remains largely unknown but seems to be associated with low-level systemic or intestinal inflammation. We aim to determine the fecal microbial composition and clarify its correlation with the clinical profiles og CSU with nsAH resistance.R language software.A total of 25 CSU patients with or 19 CSU patients without nsAH resistance and 19 healthy controls (HC) were enrolled in this study. The intestinal microbiome was detected by 16S rRNA sequencing. The data were analyzed using P <0.05 for all variables). Compared to patients with nsAH-responsiveness, the abundance of fecal genera Prevotella, Megamonas, and Escherichia were significantly increased, while that of Blautia, Alistipes, Anaerostipes, and Lachnospira were remarkably reduced in nsAH-resistant patients . Finally, systemic not intestinal inflammation degree was positively correlated with genera Escherichia, while negatively with genera Blautia, Dorea, Lactobacillus, Eubacterium_hallii_group, and Roseburia. CSU without nsAH resistance and HC individuals showed almost unchanged genera bacterium.Significantly higher urticarial activity score for 7 days, stool calprotectin, erythrocyte sedimentation rate, serum C-reactive protein, and interleukin-6, but much lower alpha-diversity and evenness of fecal bacterial community were observed in CSU patients with nsAH resistance than in those without (Among CSU patients, pro-inflammation phenotype relating to enteric dysbacteriosis features nsAH resistance in CSU patients. The results provide clues for future microbial-based or anti-inflammatory therapies on nsAH resistant CSU. Whether a spontaneous or inducible subtype, chronic urticaria is characterized by recurrent episodes of pruritic wheals with or without associated angioedema persisting longer than 6 weeks . GloballCSU is generally believed to be a histamine-mediated allergic reaction, and second-generation non-sedating H1-antihistamines (nsAH) at licensed doses have long been the first-line therapy in CSU. Clinically, resistant CSU (RCSU) is defined by the patients who are unresponsive to any nsAH at approved dosage after 2 to 4 weeks of treatment, and RCSU accounts for 40 to 60% of patients receiving the standard treatment . The mecTogether with other colleagues, we previously proved that inflammation abnormalities mediated by the imbalance of Th1/Th2/Th17 cytokines might contribute to CSU pathogenesis, and thus, understanding of the possible sources of inflammation in real-world settings is critical in enabling measures to improve symptom control and minimize disease burden among CSU patients . Inflamm2LEN/WAO/EDF guidelines . By 2 weeks of follow-up, a total of 25 RCSU patients aged ≥18 years (RCSU group) and 19 age- and sex-matched CSU patients with an adequate response to levocetirizine (CSU group) were enrolled, and meanwhile, 19 age- and sex-matched healthy controls (HC group) were recruited. The diagnosis of CSU was made by board-certified dermatologists following the criteria proposed by the new EAACI/GAidelines . A CSU pExclusion criteria included: (a) pregnant or lactating women, (b) patients with previous or current allergic and autoimmune diseases, systemic diseases (such as inflammatory bowel diseases and metabolic diseases), or skin diseases other than urticaria , (c) patient with a history of gastrointestinal surgery, (d) smokers with smoking index >400, (e) drinkers with daily ethanol intake ≥40 g for men (20 g for women) in the past five years or ≥80 g in past two weeks, and (f) use of systemic antibiotics, retinoids, corticosteroids or any other immunosuppressive therapy in the recent 6 months. The study was approved by the Medical Ethics Committee of the Third Affiliated Hospital of Chongqing Medical University, and written informed consent was obtained from all the participants at the time of recruitment.2LEN/WAO/EDF guideline , medical history, previous or current medications, smoking habits, and alcohol intake. After 2 weeks of treatment, fasting venous blood collected from each patient was used to determine erythrocyte sedimentation rate (ESR) and white blood cells, and the serum was used to quantify protein levels of total IgE , C-reactive protein , and interleukin (IL)-6  by enzyme-linked immunosorbent assay (ELISA) following the instructions of the manufacturer from commercially-available kits. Autologous serum skin testing (ASST) was performed in all patients as previously described, and morphologically positive reactions  are assessed as auto-allergic. The urticarial activity score for 7 days (UAS7) at the time of patient recruitment and UAS at sample collecting were assessed for each patient according to the EAACI/GAuideline .® Flash Master Mix , 10 μM primers, and 100 ng of template DNA. The amplification conditions were 98°C for 30 s (1 cycle), followed by 98°C for 10 s, 56°C for 5 s, 72°C for 5 s (30 cycles), and 72°C for 1 min (1 cycle). Amplicons were then recovered from 2% agarose gels and further purified using an AxyPrep DNA Gel Extraction Kit  and quantified by QuantiFluor-ST  according to corresponding protocols. According to the instruction, purified amplicons were pooled in equimolar and paired-end sequenced (2 × 300) on an Illumina MiSeq platform . The raw reads were deposited into the NCBI Sequence Read Archive (SRA) database (Accession Number: PRJNA809140).After 2 weeks of treatment with levocetirizine, approximately 5 g of fresh stool samples from each patient were collected in a sterile plastic cup and stored at −80°C immediately. The fresh stool was used to detect fecal calprotectin protein by a corresponding ELISA kit . A QIAamp DNA stool mini kit  was used to extract bacterial DNA from 200 ± 20 mg of feces according to the instructions of the manufacturer and a NanoDrop ND-1000 Spectrophotometer (Nucliber) to confirm the purity of extracted DNA (A260/280 ratio of 1.8); all extracted DNA samples were stored at −80°C until further analysis. To analyze bacterial colonies in feces, the amplification of the V3–V4 region of the 16S rRNA gene was performed using polymerase chain reaction (PCR) with bacterial universal primers 341F (5’-CCTACGGGNGGCWGCAG-3’) and 806R (5’-GGACTACHVGGGTATCTAAT-3’). All PCR reactions were performed in triplicate using a 50-μl mixture containing 1× Phantahttp://drive5.com/uparse/). The taxonomy of each 16S rRNA gene sequence was analyzed using the RDP Classifier Algorithm (http://rdp.cme.msu.edu/) against the Silva (SSU128) 16S rRNA database, with a confidence threshold of 70%. QIIME and R packages (v3.2.0) from the free online Majorbio I-Sanger Cloud Platform (www.i-sanger.com) were applied to analyze sequencing data. Briefly, alpha diversity indices (Shannon index and Simpson index), evenness indices (Simpson even and Shannon even indices), and richness estimators  at OTU level were calculated using Mothur v.1.30.2. At the OTU level, partial least squares-discriminant analysis (PLS-DA) was applied to show the distance rank among the three groups. Non-metric multidimensional scaling (NMDS) was used to assess similarities in the microbial composition among three groups (beta diversity) using the R package, and stress <0.2 is generally expressed by the two-dimensional dot pattern of NMDS. Statistically, significant differences in the relative abundances of taxa were determined by Wilcoxon rank-sum test. Linear discriminant analysis (LDA) was used for assessing taxa responsible for the differences between groups. The gene function profiles of gut bacteria were predicted with a Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis. Partial Pearson’s correlation controlling for age, sex, and BMI was calculated between the differential abundance at the genus level and clinical characteristics in the VGAM package. In addition, the continuous and categorical variables were presented as mean (M) ± standard deviation (SD) and percentage (%), respectively. The chi-square test or Student’s t-test was conducted to compare the demographic information and clinical characteristics of the two groups. A P-value <0.05 (two sides) was accepted as the cutoff for statistical significance, and a false discovery rate (FDR) was considered for the correction of the P-value.Paired-end raw reads with overlap were merged to tags, and tags were clustered to operational taxonomic units (OTUs) at 97% sequence similarity using UPARSE . Compared with the CSU group, the RCSU group showed a remarkably longer course of disease , significantly higher value of UAS7 , UAS at sample collecting , ESR , the serum protein level of CRP , and IL-6 , and gut calprotectin . The state of ASST and the total serum IgE did not differ in the two groups .The demographic and clinical characteristics of the three groups are summarized in We obtained 3,163,836 , 2,447,932 , and 2,524,720  raw sequences from 25 fecal samples in the RCSU, 19 in the CSU, and 19 in the HC groups, respectively. After quality filtering and trimming, 1,491,943  valid sequences with a mean length of 419 bp, 926,357  with that 415 bp and 930,462  with that 416 bp were screened in the three groups, respectively. The reads from the three cohorts involved 1,344 OTUs, namely, 19 Phylum, 28 Classes, 68 Orders, 139 Families, 357 Genera, and 721 Species. The coverage estimator of the value of good was 99.99%. Clear asymptotes observed in refraction curve analyses with Shannon or Sobs index at OTU level indicates a near-complete sampling of the communities . In comparison with the CSU group, Simpson estimator and index values for Shannon, Shannoneven, or Simpsoneven in the RCSU group were remarkably higher and lower, respectively , which implies decreased alpha-diversity and evenness of fecal bacterial community in the RCSU patients. No significant difference in the alpha-diversity, richness, and evenness of the fecal bacterial community were observed between the CSU and the HC groups . At the OTU level, distinguished cohorts were visualized by PLS-DA, and beta-diversity analyzed using the NMDS analysis based on unweighted UniFrac metrics also showed significant distinct clusters among the three cohorts , Bacteroides (16.72%), Megamonas (10.98%), Faecalibacterium (5.55%), Lachnoclostridium (3.13%), Agathobacter (2.34%), Blautia (2.07%), and Megasphaera (1.61%), and that in the CSU group were assigned to Bacteroides (26.16%), Prevotella (15.11%), Faecalibacterium (8.28%), Blautia (4.04%), Agathobacter (4.32%), Megamonas (3.38%), Bifidobacterium (2.76%), and Alistipes(1.41%), and that in the HC group were Bacteroides (28.68%), Prevotella (12.82%), Faecalibacterium (7.40%), Agathobacter (4.41%), Blautia (3.62%), Megamonas (3.34%), Succinivibrio (2.84%), and Roseburia (1.67%).There was 208 shared bacterium in three groups on Genus level, with 23, 6, and 36 unique ones in the RCSU, the CSU, and the HC groups, respectively. More than 90% of fecal bacteria were Phyla P >0.05 for all variables). Differences in the relative abundances of the top 30 genera composition of the intestinal microflora in the three groups were further analyzed using the Wilcox rank-sum test. Results showed that the RCSU group displayed a significant increase in Prevotella , Megamonas , and Escherichia , while members of the Blautia , Alistipes , Anaerostipes , and Lachnospira  were relatively decreased compared to the CSU group At the phylum level, no significant differences in the relative abundance of the bacterium were detected among the three groups , whereas the CSU group was enriched with genera Agathobacter, Blautia, Acetanaerobacterium, Alistipes, and Eubacterium_hallii_group, etc.  and serum IL-6 , moderate with disease duration  and serum CRP , and weak with UAS7 . In addition, Prevotella is also positively associated with ESR . Contrastly, ESR , UAS7 , serum IL-6 , and CRP  all were negative with that of genera Blautia. Although not strong, negative associations between the systemic inflammation indicators and Dorea, Lactobacillus, Eubacterium_hallii_group, and Roseburia were shown . However, no correlation was found between the content of gut calprotectin and the observed genera.Finally, analyses of the relationship between observed genera in all the patients and clinical characteristics were conducted. Variance inflation factor (VIF) of clinical indicator more than 10 was considered to influence subsequent correlation analysis as the existence of multicollinearity between clinical factors. By inspection, VIF of disease duration, UAS at sample collecting, UAS7, ASST, serum IgE, ESR, IL-6, CRP, and calprotectin was 7.29, 3.07, 5.81, 2.11, 1.48, 6.11, 1.54, 6.10, and 1.86, respectively. As the correlation Heatmap graph shows  such as atopic dermatitis, psoriasis, and acne vulgaris . Gut micAkkermansia muciniphila, Faecalibacterium prausnitzii, Clostridium leptum, Lactobacillus, and Bifidobacterium in fecal samples from patients with chronic urticaria versus the healthy measured by real-time polymerase chain reaction were firstly reported in the team of Nabizadeh  expression and FcϵRI -mediated mast cell degranulation  the causal relationships among specific enterobacteria, systemic inflammation, and nsAH resistance in CSU failed to be adequately explained as with all case-controlled clinical studies, 2) assessments of intestinal flora, and systemic inflammation before and after nsAH treatment are further required to determine the effect of poor treatment response on related changes, 3) within-subject verification of related biomarkers and replication procedures in larger study populations from multicenter are lacking, and assessment of systemic inflammation in the healthy control group in parallel is deficient, 4) nsAHs other than levocetirizine such as ebastine and olopatadine were not included in this study. However, the present study indicates that pro-inflammation phenotype relating to enteric dysbacteriosis features nsAH resistance in CSU patients, which provides clues for future microbial-based or anti-inflammatory therapies on nsAH resistant CSU.The data presented in the study are deposited in the NCBI Sequence Read Archive (SRA) database, accession number: PRJNA809140.The studies involving human participants were reviewed and approved by the Medical Ethics Committee of the Third Affiliated Hospital of Chongqing Medical University. The patients/participants provided their written informed consent to participate in this study.FH and BC designed the study. YS wrote the manuscript. ZY and XY collected serum samples. YS and KD carried out the experiments and analyzed the data. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This work was funded by grants from the National Natural Science Foundation of China (82003337), the China Postdoctoral Science Foundation (2020M683268), the Chongqing Natural Science Foundation (cstc2020jcyj-bshX0023), the Chongqing Excellence Programme (cstc2021ycjh-bgzxm0291), the Postdoctoral Foundation of Chongqing Medical University (R9001) and the Funding for Key Disciplines of Third Affiliated Hospital of Chongqing Medical University (ZK201902).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.


Tff-deficient mice, which have completely different phenotypes. In particular, TFF2 is secreted by myeloid cells  and lymphocytes , where it modulates immune reactions triggering inflammation. As a new concept, in addition to lectin-triggered activation, a hypothetical lectin-triggered inhibition of glycosylated transmembrane receptors by TFF peptides is discussed. Thus, TFFs are promising players in the field of glycoimmunology, such as galectins and C-type lectins.Trefoil factor family peptides , together with mucins, are typical exocrine products of mucous epithelia. Here, they act as a gastric tumor suppressor (TFF1) or they play different roles in mucosal innate immune defense . Minute amounts are also secreted as endocrine, e.g., by the immune and central nervous systems. As a hallmark, TFF peptides have different lectin activities, best characterized for TFF2, but also TFF1. Pathologically, ectopic expression occurs during inflammation and in various tumors. In this review, the role of TFF peptides during inflammation is discussed on two levels. On the one hand, the expression of TFF1-3 is regulated by inflammatory signals in different ways (upstream links). On the other hand, TFF peptides influence inflammatory processes (downstream links). The latter are recognized best in various  I-V, CysII-IV, and CysIII-VI . In contrast, TFF2 contains two TFF domains and two additional cysteine residues, the latter connecting the C- and N-terminal via a disulfide bridge  peptides comprise TFF1, TFF2, and TFF3 (reviews: ). They sysIII-VI . Both TFe bridge . There aThe major amounts of TFF peptides are secreted from mucous epithelia, where they are released together with mucins in an exocrine manner ,11,12,13Furthermore, minute amounts of TFF peptides undergo endocrine secretion. Typical examples are lymphoid organs and tissues  [Helicobacter pylori lipopolysaccharide as well as the carbohydrate moiety of the mucin MUC6 [Gastric TFF1 occurs mainly as a monomer with a highly exposed free thiol group at Cysr humans , mice [9olog xP1 . Such anolog xP1 . Similarolog xP1 , TFF1 obxP1 [VII . Furtherroximity . Thus, CROS/RNS) ,9,37,38.ROS/RNS) ,37,41. FROS/RNS) ,37,42. Fcin MUC6 ,44. An α→4Galβ1→R moiety of the mucin MUC6 [Tff2-deficient (Tff2KO) mice, TFF2 is expected to play a role in the innate immune defense of the gastric mucosa [H. pylori growth [TFF2 is a lectin recognizing specifically the O-linked GlcNAcα1cin MUC6 ,35,36,45cin MUC6 ,47. The cin MUC6  and physcin MUC6 ,49. The c mucosa ,49. Of ni growth .In the intestine as well as saliva, TFF3 mainly occurs as a disulfide-linked hetero-dimer with FCGBP ,51. FCGBSoon after their discovery, ectopic expression of TFF peptides was detected in pathological conditions, particularly during chronic inflammation, such as gastro-esophageal reflux disease, Barrett esophagus, gastric and duodenal ulcers, diverticulitis, inflammatory bowel disease, pancreatitis, hepatholithiasis, cholecystitis, salpingitis, and inflammatory nasal polypi  in different metaplasias ,63, as w+ T lymphocytes , each secreting signature cytokines and expressing a lineage-specifying transcription factor [An acute inflammation is a defense mechanism of the immune system driven primarily by myeloid cells . Macrophages are phagocytic cells of the innate immune system and they have remarkable plasticity. They have two states of polarized activation: classically activated (M1) and alternatively activated (M2) phenotypes. The latter is subdivided at least into three subtypes  . The actn factor ,71,72. Tn factor . Furthern factor ,75. An in factor ,77. Thusn factor ,79.Numerous studies addressed the following question: which signals trigger the expression of TFF peptides during inflammation, and are there differences between the three TFF genes? Further questions asked were as follows: (i) is a loss of TFF peptides linked to inflammation, and (ii) is the expression of cytokines regulated by TFF peptides? The data concerning the multiple links of TFF peptides and inflammation are rather complex, partly seemingly controversial and often based upon single observations in a variety of very specialized systems. In order to get a glimpse on this complex interplay, the topic is discussed on two different levels : (i) comIn contrast to previous reviews describing the situation in healthy mucous epithelia , herMultiple reports indicate a complex regulation of TFF gene expression. Typical regulatory signals include estrogen, pro- and anti-inflammatory cytokines, transforming growth factor α (TGFα), fibroblast growth factors (FGFs), gastrin, TFF peptides (interregulation), prostaglandins, arachidonic acid, indomethacin, aspirin, omeprazole, butyrate, hydrogen peroxide, osmotic stress, hypoxia, X-ray irradiation, and pathogens  release interleukin (IL)-8 [H. pylori infection; this shifts the immune response toward a Th1-axis (pro-inflammatory) [An infection of the stomach with ividuals . In a monfection . The down (IL)-8 , which immatory) . From thmmatory) . Of notemmatory) . Furthermmatory) .H. pylori infection is probably not that simple. For example, TFF1 expression (together with IL-8 expression) is strongly induced in vitro in the gastric adenocarcinoma cell line AGS after H. pylori infection [H. pylori via ERK signaling [H. pylori-induced gastric inflammation in vivo and in vitro [However, the situation concerning TFF1 expression during nfection ,90. Hereignaling ,91. Furtin vitro ,92.Toxoplasma gondii infection [In sharp contrast to the down-regulation of TFF1 in gastric inflammation, TFF1 expression is ectopically induced in different organs in chronic inflammatory diseases ,93,94 asnfection ,98. The nfection . Furthernfection . These wnfection . Furthernfection . In a munfection ,96.gp130757F and gp130ΔSTAT, respectively) for IL-6/IL-11 revealed that IL-6 can also potently positively regulate the expression of TFF peptides [gp130757F mutants, SHP2-Ras-ERK signaling is blocked, the Tff1 level is decreased, and antral adenomas and carcinomas are developed [Tff1KO . Thus, rcinomas ,103,104.Minute amounts of TFF1 are also synthesized in the brain, for example, in astrocytes . In the T. gondii infection (two models) [TFF1 (but not TFF2 or TFF3) is also up-regulated in the immune system, e.g., in the murine spleen after  models) ,98. Here models) , which c models) . Further models) .H. pylori infection [In contrast to TFF1, TFF2 was only transiently reduced in the human stomach after nfection . TFF2 isnfection , as wellnfection  and allenfection . For exanfection . Using tnfection . In MKN4nfection ; other tnfection .Aspergillus fumigatus) [Furthermore, TFF2 was strongly induced in the lung in murine asthma models by the Th2 cytokines, IL-4 and IL-13 . TFF2 inmigatus) . The Th2migatus) . TFF2 wamigatus) . Thus, TMinute amounts of TFF2 are also expressed in the immune system, such as the thymus, bone marrow, spleen (memory T cells), lymph nodes, and peritoneal macrophages ,16,17,18Nippostrongylus brasiliensis [Bifidobacterium dentium [Giardia muris and Citrobacter rodentium [Citrobacter rodentium alone reduces TFF3 expression and RagKO (T and B cell-deficient) mice did not exhibit this reduction [The Th2 cytokines IL-4 and IL-13 up-regulate TFF3 expression in vitro via the transcription factor STAT6 . Of speciliensis , Bifidob dentium , and co-odentium . The latodentium . In conteduction ,117. ThuIn contrast, the Th1 cytokine TNF-α inhibits TFF3 expression via NF-κB . TFF3 regp130ΔSTAT) of the IL-6/IL-11 receptor had a reduced Tff3 level and impaired intestinal wound healing [Tff3KO mice [On the other hand, mice with a mutated gp130 signal-transducing chain  by commensal bacteria . This isMinor amounts of TFF3 are expressed in lymphatic organs such as the thymus and bone marrow as well as the spleen (memory T cells) and lymph nodes ,15,123. In the rodent and human brain, minute amounts of TFF3 are expressed mainly in neurons and also in the choroid plexus, but not in astrocytes or resting microglial cells ,126,127.Tff-deficient mice) and by gain-of-function studies . Of special note, there is a remarkably limited number of convincing reports describing significant effects of TFF peptides  in gain-of-function studies; there are specific and sensitive readouts missing, which would allow direct functional measurements. This might be a further indication that the major functions of TFF peptides probably do not rely on simple ligation to high-affinity transmembrane receptors and triggering signaling cascades.Generally, the role of TFF peptides in influencing inflammatory processes can be investigated by loss-of-function models -induced colitis [Interestingly, at 5 months, the villi of the small intestinal mucosa were enlarged (hyperplasia) in ry cells . A role ntestine . However colitis .Tff1-CreERT2 and Tff1-Cre mice showed that Tff1 is also expressed in long-lived stem and progenitor cells of the gastric antrum, which finally re-populate the entire antral units [Troy+ progenitor cells at their base [Lgr5+ progenitor cells are found, which probably originate from +Cckbr progenitor cells at the +4 position [Tff1-CreERT2 and Tff1-Cre mice would explain why inflammation and carcinogenesis in Tff1KO mice are restricted to the antrum, as Tff1 is expressed possibly already in Lgr5+  progenitor cells, but not in fundic Troy+ progenitor cells [Cckbr and the transcription factor Mist, specifically in the gastric antrum of Tff1KO mice [Lineage tracing studies using transgenic al units ,138. In al units ,138. Thial units ). The clal units . Fundic eir base , whereasposition ,143. Thu1KO mice .Finally, the question arises on the precise molecular function of TFF1 and how a loss of TFF1 triggers gastric inflammation and carcinogenesis. Currently, at least four hypothetical models (or a combination of these) are plausible.Tff1KO mice, the unfolded protein response (UPR) is activated [r of 60k) in the human stomach; X might be a disulfide isomerase of the endoplasmic reticulum (ER) related to ERp57 [Pdia3  is significantly up-regulated in the gastric fundus and antrum of Tff1KO mice [First, TFF1 could be an intracellular chaperone, as, in ctivated ,105. Thito ERp57 ,37. ERp5to ERp57 . Of note1KO mice . This mo1KO mice .VII residue [VII of TFF1 cures Tff1KO mice from developing adenomas and carcinomas.Second, TFF1 was postulated to act as a scavenger for extracellular ROS/RNS due to its exposed and probably highly nucleophilic Cys residue ,9,37,38. residue . In part→R moiety or a similar structure. This unusual sugar moiety is characteristic of the mucin MUC6 from frog to human and is essential for binding the lectin TFF2 [N-acetylglucosaminyltransferase (α4GnT) [A4gntKO mice have a very similar phenotype to Tff1KO mice [Tff2KO mice have a completely different phenotype to A4gntKO mice.Third, TFF1 could serve as an extracellular lectin, recognizing a yet not identified glycoprotein with a terminal GlcNAcα1tin TFF2 ,36. The  (α4GnT) . Remarka1KO mice . Recentl1KO mice ,44. Thus1KO mice , and may1KO mice . HoweverTff1KO mice, only (particularly in the antrum) [Fourth, a 37k-entity of Gkn2, probably a Gkn2 homodimer, was recently detected in  antrum) ; the usuTff2KO mice did not show obvious gastrointestinal abnormalities [Tff2KO mice, the degree of gastric ulceration after administration of the COX1/2 inhibitor indomethacin was significantly increased [Tff2KO mice also exhibited accelerated progression of gastritis to dysplasia in the gastric antrum after infection with H. pylori [H. felis-induced gastritis, with enhanced gastric inflammation [malities . Howeverncreased  and the ncreased . Tff2KO . pylori  and thesammation . All theammation ,8,20.Tff2KO mice were hyperresponsive to IL-1β stimulation concerning the secretion of IL-6 [Lactococcus lactis strain, which had a therapeutic effect even in chronic colitis in Il10KO mice [Tff2KO mice exhibited a more severe response to and a delayed recovery from DSS-induced colitis [Tff2KO mice, colonic Tff3 expression is also strongly reduced, which could be the cause of this phenotype [TFF2 expression is not limited to the gastrointestinal tract but is also present in macrophages and lymphocytes. Remarkably, peritoneal macrophages from  of IL-6 . Thus, T of IL-6 . A simil0KO mice . In cont colitis ,17. A coTff2KO mice were orally infected with T. gondii [Tff2KO mice showed an increased baseline level of IL-12/23p40 when compared with the wild type, but they did not develop the typical intestinal immunopathology [Tff2KO mice, the baseline production of IFN-γ was not different, but the expansion of IFN-γ-producing Th1 cells was greatly induced after T. gondii infection [T. gondii infection. Currently, the precise molecular mechanism of how TFF2 inhibits TLR-driven IL-12 expression is not known. A receptor blocking mechanism may be involved, as discussed in In another animal model of intestinal inflammation, . gondii . In wildathology . Generalathology . This inathology . IL-12 infection . Taken tT. gondii [Yersinia enterocolitica resulted in a lethal outcome in Tff2KO mice, but not in wild type mice [Tff2KO mice, the reduced amount of macrophages allowed Y. enterocolitica to cross the epithelial barrier of the ileum [Tff3 synthesis in Tff2KO mice [In contrast to infection with . gondii , oral inype mice . In Tff2he ileum . Current2KO mice  may alsoTff2KO rats were orally infected with E. coli, which led to bacteremia, in contrast to the wild type [Tff2 expression reaches a peak and drops sharply thereafter [Tff2KO rats is in agreement with a function of TFF2 for the barrier integrity of the neonatal rat intestine.In another set-up, nine day-old (P9) ild type . At thisereafter . Thus, tTff2KO mice had an increased number of MDSCs and exhibited a greater number of tumors in an azoxymethane/DSS model of inflammatory colorectal carcinogenesis [In a further study, TFF2 from splenic memory T cells suppressed the expansion of splenic myeloid-derived suppressor cells (MDSC) via CXCR4 . The numogenesis .Tff2 (Cd11cCreTff2flox mice) [Nippostrongylus brasiliensis, the lung pathology was exacerbated in these mice and the proliferative expansion of epithelial alveolar type 2 cells was reduced [Wnt4 and Wnt16. Thus, myeloid-derived TFF2 also drives macrophages to accelerate epithelial regeneration after lung injury [The inhibitory effect of TFF2 on macrophages was also demonstrated by a myeloid-specific deletion of ox mice) . After i reduced . The latg injury .N. brasiliensis, TFF2 expression increased first in the lung (early stage) and then in the intestine (late stage); this is a prerequisite for the induction of IL-33 production, a Th2-promoting cytokine, in lung epithelial cells, alveolar macrophages, and inflammatory dendritic cells [Tff2KO mice, the IL-33 levels are only slightly increased [After the infection of mice with ic cells . Thus, increased . Of specncreased , which increased ,27.Tff2KO mice, IL-33 expression is significantly reduced at least in the gastric fundus [H. pylori infection also changed IL-33 expression biphasically—an acute phase with increased IL-33 followed by suppression in the chronic phase [The TFF2-IL-33 axis has also been described in the stomach, where IL-33 is synthesized in a subpopulation of surface mucous cells, probably in precursors of surface mucous cells . In the c fundus . Furtheric phase . Chronicic phase . Taken tic phase .Tff2KO mice have a compromised immune system [Taken together, TFF2 has a function in the normal stomach as a constituent of the gastric mucus barrier , which is a first line defense against microbial infections  is also synthesized in these epithelia. Another interesting goal would also be the generation of FcgbpKO mice and to determine their phenotype in the DSS colitis model. Furthermore, the binding of TFF3 to DMBT1gp340, a pattern recognition receptor with a function in mucosal innate immunity, could play a protective role here [The murine colonic mucus consists of two layers: a firmly adherent inner layer, and a loose outer layer. Normally, the inner layer is devoid of bacteria ,166. Aftsociated  and is esociated . It woulole here ,25,170.Of note, the expression of pro-inflammatory cytokines in cultured microglial cells was reduced by TFF3 . This poT. gondii infection, the expression of the inflammasome constituent Nlrp12 was significantly reduced in Tff3KO mice when compared with wild type mice [An immunomodulatory role of TFF3 is also in line with the observation that in the murine spleen after ype mice .Tff-deficient mice have completely different phenotypes, but all are related to inflammatory processes, either directly or after various mucosal challenges. The following picture concerning the multiple and different functions of TFF peptides has emerged , or in a variety of mucous epithelia (TFF3). Here, they play a role as a gastric tumor suppressor (TFF1) or they are involved in the mucosal innate immune defense as integral parts of the mucus barrier  ,20. As a→4Galβ1→R moiety of the mucin MUC6, a lectin-triggered activation of a glycosylated transmembrane protein seems reasonable [Under pathological conditions, e.g., after mucosal injury or infection, TFF2, in particular, is secreted in an endocrine fashion by myeloid cells  and lymphocytes . Here, at least TFF2 is a modulator of immune reactions triggering inflammatory processes. On the one hand, TFF2 induces the synthesis and release of the nuclear alarmin IL-33, at least in mucous epithelia, which leads to a Th2 immune response. There are multiple indications that certain TFF2 effects are mediated by activating ligation to CXCR4 and/or a plethora of other glycosylated transmembrane proteins. Unfortunately, the details are not known currently. As TFF2 is a lectin recognizing, at least, the O-linked GlcNAcα1asonable . On the Tff1KO mice [In contrast to a proposed lectin-triggered activation of receptors , TFF pep1KO mice . The preTff2KO mice to IL-1β stimulation [Accordingly, TFF1 is a natural antagonist of the IL-6 receptor system and comparable with the action of tocilizumab, a humanized anti-IL-6 receptor antibody, which is used in the clinics for treating rheumatoid arthritis, cytokine release syndrome and even COVID-19 . A similmulation  might beIn the future, TFF peptides might be used to specifically block a series of glycosylated receptors playing mayor roles in inflammatory processes. As glycosylation patterns are relatively cell-specific, the use of TFF peptides could be selective for specific cells. Furthermore, TFF peptides recognize different carbohydrate moieties. This combination might allow interesting future clinical applications for TFF peptides and might open new therapeutic strategies, e.g., as anti-inflammatory agents. Thus, it is now a promising goal to test systematically receptors for their binding of TFF peptides, e.g., in vitro. When using different cell lines, the knowledge of the specific glycosylation pattern is of particular interest. The use of different cell lines might explain contrary past results. As a prerequisite for such studies, the carbohydrate specificities of TFF peptides have to be elucidated in detail. Thus far, it is clear that the lectin characteristics of the three TFF peptides are different, but partly related. GlcNAc seems to be a common moiety recognized at least by TFF1 and TFF2 . FurtherTff2KO mice  to reduce oral mucositis, which is a side effect of radio- and chemotherapy . For the


Ahr actsas a xenosensor and modulates the transcription of biotransformationgenes in vertebrates, such as cytochrome P450 1A (cyp1a). Atlantic cod (Gadus morhua) possessestwo Ahr proteins, Ahr1a and Ahr2a, which diverge in their primarystructure, tissue-specific expression, ligand affinities, and transactivationprofiles. Here, a luciferase reporter gene assay was used to assessthe sensitivity of the Atlantic cod Ahrs to 31 polycyclic aromaticcompounds (PACs), including two- to five-ring native PAHs, a sulfur-containingheterocyclic PAC, as well as several methylated, methoxylated, andhydroxylated congeners. Notably, most parent compounds, includingnaphthalene, phenanthrene, and partly, chrysene, did not act as agonistsfor the Ahrs, while hydroxylated and/or alkylated versions of thesePAHs were potent agonists. Importantly, the greater potencies of substitutedPAH derivatives and their ubiquitous occurrence in nature emphasizethat more knowledge on the toxicity of these environmentally and toxicologicallyrelevant compounds is imperative.Polycyclic aromatichydrocarbons (PAHs) are among the most toxicand bioavailable components found in petroleum and represent a highrisk to aquatic organisms. The aryl hydrocarbon receptor (Ahr) isa ligand-activated transcription factor that mediates the toxicityof 2,3,7,8-tetrachlorodibenzo- SubstitutedPAH congeners act as strong aryl hydrocarbonreceptor agonists, emphasizing the need to consider these compoundsin environmental monitoring and risk assessment of aquatic environments. PACs include polycyclic aromatic hydrocarbons (PAHs) andalso nitrogen-, oxygen-, or sulfur-containing heterocyclic aromaticcompounds (NSO-PACs), as well as compounds with heteroatoms containingfunctional groups .4 However, somePAHs (with Kow > 6) tend to bioaccumulatein fish. Nevertheless, these PAHs have relatively short half-livesdue to efficient metabolism and excretion and thus do not biomagnify.8 In vertebrates, such as fish, birds, and mammals,hepatic cytochrome P450 monooxygenase enzyme systems are mostly responsiblefor this rapid metabolism. Due to the carcinogenic and mutagenic propertiesof PAH metabolites, PAHs can cause adverse effects in aquatic organismsand potentially to humans through fish and shellfish consumption.15 For these reasons, they have been regarded as high priority compoundsfor environmental pollution monitoring, and a priority list of 16PAHs (PAH-16) was made by the US Environmental Protection Agency (EPA).16 Today, PAH-16 are routinely analyzed in environmentalmonitoring programs and risk assessments of PAH-polluted sites.In general, PACs have low water solubility and are mainly foundassociated with suspended particulate matter in water and do eventuallyaccumulate in sediments.17 Substituted PAHs have been shown to contributeto the toxicity of both pyrolytic and petrolytic PAH mixtures in theearly life stages of rainbow trout (Oncorhynchus mykiss), Japanese medaka (Oryzias latipes), and zebrafish (Danio rerio).22 Hydroxylation of alkylated phenanthrenes has also been shown toenhance early life stage toxicity in Japanese medaka,23 further emphasizing the importance of considering the contributionof substituted PAHs in mediating toxicity in fishes and other aquaticorganisms. Furthermore, as most PAHs are subject to metabolic activationby cytochrome P450s and epoxide hydrolases, epoxide and diol metabolitesare formed in vivo in vertebrates.25 The major PAH oxidation products formedin fish are trans-dihydrodiols, including -1,2-dihydrochrysene-1,2-diol, -1,2-dihydrophenantrene-1,2-diol,and -1,2-dihydronaphthalene-1,2-diol,which are some of the trans-dihydrodiols derivedfrom chrysene, phenanthrene, and naphthalene, respectively.27 In addition to being formed duringbiotransformation, hydroxylated PAHs may also be produced during incompletecombustion of, e.g., fire wood.28 Notably, the toxic potential of alkylated and oxygenatedPACs has received less attention compared to the 16 PAHs prioritizedfor environmental monitoring.1 As the PAH-16only encompass parent PAHs, the importance of expanding our knowledgeof the toxicities of heterocycles and alkyl derivatives and includesuch compounds in a more extensive panel of PACs for environmentalmonitoring has been proposed.29In addition to the unsubstituted parent compounds, substitutedPAHs, such as alkylated PAH derivatives, can be found in the environment,and these have been reported to be more toxic than their unsubstitutedcongeners.33 AHR is a ligand-activated transcriptionfactor and a member of the basic helix–loop–helix PER-ARNT-SIM(bHLH-PAS) superfamily, which has been widely studied because of itsimportant role in mediating cellular responses to halogenated aromatichydrocarbons. 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) has been established as the most potent exogenous agonist forvarious AHR orthologs. AHR-dependent toxicities are both species-and tissue-specific and can cause a wide spectrum of effects, includingteratogenicity, immuno-, hepato-, cardio- and dermal toxicity, modulationof cell growth, proliferation and differentiation, endocrine disruption,and tumor promotion.33 Accordingly, severaladverse outcome pathways involving AHR and/or activation of AHR asthe molecular initiating event have been described and are currentlybeing developed .To abide to different nomenclature rules for mammalian and fish proteins,35 abbreviated protein names have two different formats in scientificpublications. Thus, AHR or Ahr is used when referring to mammalianor fish aryl hydrocarbon receptor orthologs, respectively. Ligand-activatedAHR heterodimerizes with the aryl hydrocarbon receptor nuclear translocator(ARNT) and specifically binds to xenobiotic response elements (XREs)upstream of the AHR target genes, modulating the transcription ofa battery of genes encoding enzymes involved in the biotransformationof xenobiotics, including CYP1A.Thetoxicity of PACs has, to a large extent, been attributed tothe activation of the aryl hydrocarbon receptor (AHR) and the subsequentalteration of its target gene expression.36 Recently, two Ahrproteins were identified and functionally characterized in Atlanticcod, i.e., Ahr1a and Ahr2a. The Ahr paralogs differin both tissue-specific and spatiotemporal gene expression, ligand-bindingaffinities, and transactivation activities, suggesting that Ahr1aand Ahr2a have acquired different functional roles in Atlantic codthrough a process of subfunction partitioning.38 Moreover, current data suggests that Ahr2a is the main subtype involvedin mediating responses to xenobiotics, while Ahr1a appears to be importantin the development of the eye in cod embryos and larvae. However,the high sensitivity of Ahr1a to different ligands, including benzo[a]pyrene, suggests that Ahr1a activity can be modulatedby pollutants.Atlantic cod is a culturally,ecologically, and economically importantteleost species residing in the Barents Sea, the North Atlantic Ocean,and the Baltic Sea.in vitro, and their sensitivities and efficacies were compared. Among the31 PACs, seven were unsubstituted and represented congeners previouslydetected in Atlantic cod liver and bile, as well as PACs that aremajor constituents of crude oil, such as naphthalene, phenanthrene,fluorene, and chrysene.39 Moreover, substituted versionsof chrysene and phenanthrene are abundant constituents of petrogenicsubstances present in marine environments, and an extensive libraryof alkylated phenanthrenes and substituted chrysenes, including dimethylatedphenanthrenes as well as methylated, methoxylated, and hydroxylatedchrysene congeners, has therefore been assessed in this study. Finally, trans-diols of naphthalene and phenanthrene, which are consideredthe most prominent biotransformation products of these compounds commonlyfound in fish bile, are included in these analyses, and to our knowledge,this is the first time these metabolites have been evaluated as agonistsfor Ahr.40 Importantly, although in vitro Ahr activation not necessarily correlates to adverseoutcomes in organisms, reporter gene assays as applied in this studymay still expand our understanding of PAC-mediated toxicities. Suchdata could also prove important for future risk assessment and furtherreveal functional differences between receptors and receptor subtypesand potentially divergences in species susceptibility to PAC exposure.39As the AHR/Ahr pathway plays a central role inPAH-mediated toxicity,it is necessary to obtain knowledge of the sensitivities of Atlanticcod Ahrs to the wide array of PACs present in the environment. Inthis study, a total of 31 compounds were tested for their abilityto transactivate the Atlantic cod Ahr1a and Ahr2a p-dioxin (TCDD) was purchased from LGC Standards .The PAHs chrysene (CHR), 5-methylchrysene (5-MC), phenanthrene (PHE),naphthalene (NAP), pyrene (PYR), benzo[a]pyrene (BAP),fluorene (FLU), -1,2-dihydronaphthalene-1,2-diol, and dibenzothiophene(DBT) were purchased from Merck KGaA . The synthesisof -1,2-dihydrophenanthrene-1,2-diol,11 1-,2-,3-,4-methoxychrysene  and chrysene-1-ol,-2-ol,-3-ol,-4-ol ,41 1-,2-,3-,4-,6-methylchrysene,42 3-ethylphenanthrene(3-EP), and 3-propylphenanthrene (3-PP)43 is described elsewhere. 1,7-, 2,3-, and 2,7-dimethylphenanthrene were prepared as described by Böhmeet al.,44 and the preparation of 2,3-dimethoxychrysene is described in the Supporting Information.2,3,7,8-tetrachlorodibenzo-l-glutamate, 1 mM sodium pyruvate,and 100 U/mL penicillin–streptomycin , and cultivated at 37 °C with 5% CO2 for24 h. Cells were transiently co-transfected with a eukaryotic expressionplasmid (pcDNA3.1/Zeo(+), 31 ng/well), pcDNA3.1/Zeo(+)-based plasmidsencoding gmAhr1a or gmAhr2a (3 ng/well), gmArnt1 (6 ng/well),37 a luciferase reporter plasmid containing fourDREs , and a β-galactosidase normalizationplasmid ,46 using a MirusTransIT LT-1 transfection reagent according to the recommendationsof the supplier. COS-7 cells were seeded and cultivated in DMEM (4500mg/L glucose), supplemented with 10% FBS, 1 mM sodium pyruvate, and4 mM l-glutamine. In the exposure medium, FBS was substitutedwith 10% charcoal-stripped FBS . To createthe exposure media with the highest test concentration, compoundssolved in dimethyl sulfoxide (DMSO) were diluted 1:200 in exposuremedium, resulting in exposure media with 1× test compound and0.5% DMSO. The highest concentration exposure media were seriallydiluted five times  in exposuremedium supplemented with 0.5% DMSO. Following transfection, cellswere incubated in exposure media containing PACs or solvent control(0.5% DMSO) for 24 h. Reporter gene assays were repeated at leastthree times and with three technical replicates per exposure. TCDD(30 pM to 100 nM) was used as a known agonist in each experiment.Absorbance and luminescence measurements were performed on an EnSpireMultimode plate reader . While no wavelengthfiltering was used to measure firefly luciferase activity by luminescence,absorbance measurements to quantify β-galactosidase activitywere performed at 420 nm wavelength. The viability of exposed cellswas evaluated using the resazurin reduction assay as previously described.48COS-7 simian kidney cells were seeded onto96 well plates (10 000 cells/well) in Dulbecco’s modifiedEagle’s medium (DMEM) with phenol red, supplemented with 10%fetal bovine serum (FBS), 4 mM 50) and maximum efficacies (Emax). EC50 values were only determinedfor compounds that produced a sigmoidal concentration–responsecurve. Relative effect potencies 25 (REP25) were determinedby dividing the EC25 of TCDD by the concentration of PACnecessary to produce a response equal to 25% of the response of TCDDwith gmAhrs, essentially as described by Villeneuve et al. and Lamet al.50 The D’Agostino–Pearson (Ahractivation data) or Kolmogorov–Smirnov (viability data) normalitytests were used to confirm the normal distribution of the data. One-wayanalysis of variance (ANOVA) and Dunnett′s test were used tocompare responses at different concentrations of compounds and solventcontrol mediated via the same Ahr, in addition to comparing metabolicactivity in PAC-exposed cells and cells exposed to solvent controlin the resazurin reduction assay. Welch′s t-test was used tocompare the maximum Ahr-mediated response and potency (Emax and EC50) between Ahr1a or Ahr2a producedby the same test compound (Prism v7).Recorded luminescencewas normalized for variation in transfection efficiencies using β-galactosidaseenzyme activity. The difference in normalized light units measuredin lysates from exposed cells to solvent-exposed cells was calculatedand expressed relative to the maximum response in TCDD-exposed cells.Response curves were prepared by nonlinear regression analyses inPrism v7 and used for the determination of half-maximal effectiveconcentration values .Luciferasereporter gene assays were used to assess the abilityof 31 PACs to transactivate the Atlantic cod Ahr1a and Ahr2a hthalene 1. The trSupporting Tables S1–S5). Eleven PACs  did not activate either Ahr1a or Ahr2a. Receptor efficacies(Emax) and potencies (EC50 andREP25) are visualized in 37 while chrysene was shown hereto solely activate Ahr1a . Furthermore, six monomethylated chrysenes, fourchrysenols, four methoxychrysenes, and 2,3-DMOC also activated thetwo Ahrs (Supporting Tables S2–S4).Twenty-one and 20 PACs were found to activate Ahr1aand Ahr2a,respectively  produced higher efficacies (superefficacies) than TCDDwith Ahr1a , while 3-MC, 5-MC, 6-MC, 2-COH, 4-COH, 1-MOC, and 2-MOC were agonistswith efficacies similar to that of TCDD. In contrast, CHR, 1-MC, 3-COH,3-MOC, 4-MOC, and 2,3-DMOC produced lower efficacies than TCDD viaAhr1a (Supporting Table S6). Similarly,for responses via Ahr2a, two of the tested compounds (2-MC and 2-MOC)elicited superefficacies that were 127 and 115% of the TCDD maximuminduction, while five of the tested compounds  produced similar efficacies (Supporting Table S6). Ten PACs  demonstrated lower efficacies in comparison to TCDD(Supporting Table S6). REP25 valueswere calculated to express relative potencies of the PACs to TCDDwith the two cod Ahrs, and as expected, none of the PACs were morepotent agonists than TCDD for either Ahr1a or Ahr2a (25 of 0.97 (TCDD = 1.0) with Ahr2a. With thissubtype, five compounds  demonstratedREP25 values of 0.17–0.97, whereas with Ahr1a, only2-MC and 2-MOC were in the same range .The resazurin reduction assay was used to monitor potential effectson the metabolic activities in COS-7 cells after PAC exposure. Whilethe majority of the compounds did not affect cell viability, a slightreduction in metabolic activities in cells exposed to the highestconcentrations used of 5-MC, 6-MC, 1-COH, 3-COD, 3-MOC, 4-MOC, and2,3-DMP was observed  activity in waterborneexposures of juvenile rainbow trout.64 However,the related 3-ethylphenanthrene compound was not able to transactivatethe Ahrs in the current study. Notably, 3-PP transactivated both Ahr1aand Ahr2a as a weak agonist, supporting that some alkylated phenanthrenescan activate the Ahr-signaling pathway in fish.Environmentalexposures to PAHs most often involve mixtures ratherthan single compounds, and dependent on their sources, the contentof substituted and unsubstituted PAHs varies to a large extent.66 CHR has previously been found in mollusks, crustaceans, and fishand appears to be diluted in the marine food webs.69 CHR and itsderivatives have been shown to activate rat and human AHR in vitro(Poeciliopsis lucida) hepatoma cells,49 and in vivo studies have indicated thatCHR can activate both Ahr1 and Ahr2 in zebrafish.74 Unsubstituted CHR, in the current study, was found to actsolely as an agonist of Ahr1a. However, each of the assessed monomethylatedchrysenes was found to activate both Ahr1a and Ahr2a. Furthermore,all of the monomethylated chrysenes that were assessed produced increasedefficacies in comparison to unsubstituted CHR. The high activitiesof the methylated chrysenes are in line with previous studies, where1-MC and 5-MC have been shown to contribute significantly to the totalTEQ value of PAH-contaminated environmental samples.66 When expressing the toxic potential of CHR and its derivativesrelative to the toxicity of BAP , Richter-Brockmannand Achten noted that 1-MC and 5-MC had an agonistic potential of10 and 100 times higher than CHR .66 While our data showed that the six methylchryseneswere moderate to strong agonists of the Ahrs, we did not observe anevident correlation between efficacies and potencies of chrysene andmethylated chrysenes and the previously reported TEF values for thesecompounds. However, these discrepancies may be ascribed to species-specificdifferences in ligand recognition and binding affinities between Atlanticcod Ahrs and human AHR. 1–6 MCs have also previously been demonstratedto activate rat AHR in the H4IIE-luc reporter geneassay, and REP25 values were calculated for these compounds.73 As REPx values are based on internal relative comparisonsof potency  to that of a reference compound with an assay-specific receptor, comparing REP valuesacross studies and receptors is problematic. Both assay conditions,reporter system, and the sensitivity of the species studied will influencethe baseline EC50. The EC50 for rat AHR withTCDD in the H4IIE-luc reporter assay is, in mostcases, reported to be in the 8–18 pM range,76 whereas in our assay, we determined a TCDD EC50 of 1.7and 6.2 nM with cod Ahr1a and Ahr2a, respectively, in the same range,as reported by Aranguren-Abadía et al.37 Taking this into account, REPs are still useful in comparing therelative transactivating potency of compounds across studies, species,and receptors. It is, for example, interesting to note that with the1–6 MC series, Machala et al. found 3-MC to give the highestREP values, followed by 6-MC, 4-MC, 2-MC, 5-MC, and 1-MC. In our study,with cod Ahr2a, we found 2-MC to be the most potent, followed by 3-MC,5-MC, 1-MC, 4-MC, and 6-MC. The REPs observed for MCs with cod Ahr2aalso covered a larger range (150-fold) compared to that for rat AHR(40-fold). These differences in REPs between compounds may point tointeresting differences in the structural features of the ligand-bindingpocket of cod Ahrs versus rat AHR.CHR and methylchrysenesoriginate from both pyrogenic and petrogenicsources, and methylchrysenes can also be formed from chrysene by bioalkylation. in vitro−73 and t50Furthermore, we also demonstratedthat intermediates formed inthe synthesis of chrysenols, such as mono- and dimethoxylated chrysene,produced high efficacies and were potent Atlantic cod Ahr agonists.This is also in accordance with a previous report, where 2-MOC hasbeen shown to be a stronger agonist of rat AHR than chrysene.77 and PAH-mediated activation ofAhr induces the expressionof phase 1 and phase 2 biotransformation enzymes important for theirelimination.78trans-dihydrodiolmetabolites formed by CYP-mediated oxygenation are major hepatic oxidationproducts of PAHs and are excreted in the bile of bony fishes.80 The most abundant trans-dihydrodiols identified in Atlantic cod exposed to crude oil are R,R-1,2-DHP and R,R-1,2-DHN that are derived from phenanthrene and naphthalene,respectively.26 Noteworthy, we found that the trans-dihydrodiols of naphthalene and phenanthrene can actas agonists for the Ahrs, which was in contrast to their parent compoundsthat did not activate either Ahr1a or Ahr2a. To our knowledge, thisis the first time that the biotransformation products of two- andthree-ring compounds have been shown to act as Ahr agonists in a vertebrateorganism. This observation emphasizes the promiscuity of the Atlanticcod Ahr ligand-binding pockets, which accommodate the recognitionand binding of PAHs ranging from two- to at least five-ring structures.Intriguingly, activation of the Ahr-signaling pathway by the nonactivenaphthalene and phenanthrene may thus occur after in vivo exposure, while their Ahr-activating properties should probablybe ascribed to the trans-dihydrodiol metabolitesformed after CYP-mediated hydroxylation of the two mother compounds.Fish have a high capacity to metabolizePAHs,Scophthalmusmaximus) that were exposed to various PAH mixtures,77 while 4-COH and 6-COH were found to constitute6–9% of the metabolites detected in liver microsomes preparedfrom rainbow trout exposed to CHR.81 Notably,it has been reported that the exposure of zebrafish embryos to 2-COHand 6-COH caused circulatory, cardiac, and ocular effects, in contrastto their parent compound, which did not induce any toxicities.82 All of the chrysenols assessed in our study,including 1-COH, 2-COH, 3-COH, and 4-COH, were stronger agonists ofboth Ahr subtypes than unsubstituted chrysene. While it is temptingto explain the observed differences in toxicity of CHR and chrysenolsin zebrafish by differences in potential to activate Ahr, this isnot straightforward due to the number of possible Ahr-dependent andAhr-independent mechanisms that could be involved.74 However, our findings demonstrated that several chrysenolcongeners can act as potent Ahr agonists and may potentially causetoxic effects in Atlantic cod via activation of the Ahr-signalingpathway.Chrysenols, such as 1-COH, 4-COH, and 6-COH, are other examplesof PAH metabolites that have been detected in fish. Chrysene-1-olwas detected in the bile of juvenile turbot . Interestingly, their TCDD activationprofiles also differ, even though the amino acids known to bind andcoordinate TCDD are conserved between the two Ahrs,37 suggesting that the observed differences must be attributedto features located elsewhere in these proteins. In a similar vein,the observed differences in affinities to TCDD of Ahr variants fromdifferent populations of Atlantic tomcod (Microgadustomcod) from Hudson River  could not be ascribedto differences in the LBD but rather to other structural differencesthat affect the stability of the protein and result in lesser affinityof TCDD.83Many of the PACs assessed in this study produced differentactivationprofiles for Ahr1a and Ahr2a. In accordance with our previous findingsfor β-naphthoflavone and PCB-126,85 While adverse effects on Atlantic cod have been demonstrated aftercrude oil and produced water exposure,88 limited informationexists for this species regarding the toxicity mediated by the individualchemical constituents, such as unsubstituted and substituted PACs.However, it was shown in juvenile Atlantic haddock (Melanogrammus aeglefinus), which is another Gadiformspecies, that among 12 injected unsubstituted heavy PAHs, includingBAP, benz[a]anthracene, dibenzanthracene, and CHR, produced high levels ofDNA adducts in the liver.89 Furthermore,it has also been shown that intramuscular injections of NAP, CHR,and their corresponding dihydrodiol metabolites R,R-1,2-DHN and -1,2-dihydrochrysene-1,2-diol result in the formation of PAH-proteinadducts in the Atlantic cod plasma proteome and possibly a triggeredimmune response.90 Importantly, it wasrecently demonstrated that BAP activated the Ahr-signaling pathwayin early life stages of Atlantic cod.38Ahr2a expression was induced inthe cardiovascular system in both cod embryos and larvae, indicatingcardiotoxicity responses in an Ahr-dependent mode of action. Thisis similar to observations of BAP-mediated activation of Ahr2 in zebrafishembryos, which produced cardiotoxic effects via induction of cyp1a expression.91 Thus, as several PACs are demonstrated in this study to act as Ahr2aagonists with potencies and efficacies in the same range as BAP, including2-MC, 3-MC, 2-MOC, and 4-MOC, it is not unlikely that exposure tosuch PAHs during early life stages of cod produces cardiotoxic effects.In contrast to ahr2, ahr1a transcriptswere solely detected in the eye of cod embryos and larvae, and itsexpression was unaffected by BAP exposure, supporting that Ahr2a isthe major subtype involved in mediating PAH-induced responses duringearly life stages.38 Thus, although Ahr1awas shown here to be sensitive toward a wide array of substitutedand unsubstituted PACs in vitro, it appears thatAhr1a does not have a prominent role in producing adverse effectsof PAH exposure during early development. However, it cannot be excludedthat activation of Ahr1a may produce adverse effects in Atlantic codvia modulation of yet undescribed Ahr1a-regulated pathways.Molecular mechanisms andphysiological effects of PAH exposurehave been shown to differ among individual PAH/PAC congeners. Hence,three modes of action have previously been described in teleost species,including Ahr-independent, Ahr-dependent, and Cyp1a metabolism-dependent.In conclusion, we have shown that substituted PAHs, including methylated,mono- and dihydroxylated and methoxylated PACs, are strong agonistsof the Atlantic cod Ahrs. The substituted PAHs are also more potentand produce higher efficacies in comparison to their unsubstitutedparent compounds. Ahr1a and Ahr2a were mostly activated by the samePACs, but Ahr2a was, in general, the most sensitive receptor, displayingthe highest potencies of the compounds. Importantly, our results stronglysupport that substituted PACs may contribute significantly to thebiological effects of PAHs in the environment, and their contributionshould be considered when assessing the risk and hazards of PACs.Usually, assessments of the risk and hazard, as well as monitoringof PACs, are based solely on the quantification of the 16 priorityPAHs. However, as substituted PACs may contribute significantly toAhr-mediated toxicity of environmental samples, it becomes apparentthat measurement of the 16 priority PAHs is insufficient for predictingPAH-induced toxicity in aquatic environments. Complementing chemicalanalyses with reporter gene assays as used in this study could significantlyaid the risk assessment of environmental samples. Such assays canintegrate individual potencies and mixture interactions of compoundsthat act via a common mode of action. As toxicological data on substitutedand heterocyclic PACs is still limited, further studies are necessaryto elucidate their mode of action and their joint potencies in mixtures.


T, CPCC 205215, and CPCC 205251 were isolated from different biological soil crust samples collected from Tengger Desert, China. The 16S rRNA gene sequence comparison of these three strains showed they had almost identical 16S rRNA genes, which were closely related to members of the family Geodermatophilaceae, with the highest similarities of 96.3–97.3% to the species of Modestobacter. In the phylogenetic tree based on 16S rRNA gene sequences, these isolates clustered into a subclade next to the branch containing the species of Modestobacter lapidis and Modestobacter multiseptatus, within the lineage of the genus Modestobacter. The comparative genomic characteristics  and the phenotypic properties  of these isolates readily supported to affiliate them to the genus Modestobacter as a single separate species. For which, we proposed that the isolates CPCC 205119T, CPCC 205215, and CPCC 205251 represent a novel species of the genus Modestobacter as Modestobacter deserti sp. nov. CPCC 205119T (=I12A-02624=NBRC 113528T=KCTC 49201T) is the type strain. The genome of strain CPCC 205119T consisted of one chromosome  containing 4,424 coding genes, 48 tRNA genes, five rRNA genes, three other ncRNA genes, and 101 pseudogenes, with G+C content of 74.7%. The whole-genome sequences analysis indicated that this species contained alkaline phosphatase genes (phoA/phoD), phosphate transport-related genes , trehalose-phosphate synthase gene (otsA), trehalose 6-phosphate phosphatase gene (otsB) and other encoding genes for the properties that help the microorganisms to adapt to harsh environmental conditions prevalent in deserts. Strains of this species could solubilize tricalcium phosphate [Ca3(PO4)2] and phytin, assimilate pyrophosphate, thiophosphate, dithiophosphate, phosphoenol pyruvate, 2-deoxy-d-glucose-6-phosphate, and cysteamine-S-phosphate.Three Gram-stain-positive, aerobic, motile actinobacterial strains designated as CPCC 205119 Geodermatophilaceae  members were isolated from harsh environments, including desert soils , katA , and uvrACD , phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylmethylethanolamine (PME), phosphatidylinositol (PI), and phosphatidylinositol mannosides (PIM). The major respiratory quinone is MK-9(H4), and MK-8(H4), MK-9(H6), MK-9(H2), and MK-10(H4) may be present. Major fatty acids are iso-C16:0 and iso-C15:0. The DNA G+C content ranges from 68 to 74.1%.Water scarcity, violent temperature fluctuation, strong ultraviolet radiation, and low-substrate supply are the key abiotic stress factors for life in desert environments. Microbiological soil crusts, the primary stage of biological soil crusts, contribute greatly in stabilization of the sandy surface, soil formation, and carbon and nitrogen assimilation, which provide foundation for other organisms to survive. The “National Desert Ecological Reserve” (NDER) located in Shapotou desert region, on the south east edge of the Tengger Desert, south to the Yellow River, in the northwest of China is the first Chinese NDER. This NDER acts as a model for studying the development of biological soil crusts. Previous study  found thhilaceae  were ubirt soils , sandstort soils , stone srt soils , as wellModestobacter cultures from Shapotou NDER, and study the properties of Modestobacter members inhabiting in the biological soil crusts in desert niches. The detailed phenotypic and genotypic properties resulted from these strains could be helpful to systematically demonstrate their ecological adaptation mechanism and their ecological function. As a result, strains CPCC 205119T, CPCC 205215, and CPCC 205251 were isolated from biological soil crusts collected from Tengger Desert, showing phosphate-solubilizing activity. Based on the phenotypic and genotypic data, a novel species Modestobacter deserti sp. nov. is proposed, with the isolate CPCC 205119T as the type strain.The primary goal of the present research was to collect and identify the The moss-dominated soil crusts samples designated IMB12100 and IMB12108 were collected from Cuiliu ditch  and Yiwan spring , respectively, and the cyanobacteria-dominated soil crusts sample CCL12125 was from the North-road experimental area , in the middle of Shapotou NDER . Different samples were sealed in sterilized envelopes following collection and taken to the laboratory within 1week of collection. All samples were immediately processed for the isolation of microorganisms after arriving at the laboratory, and the remaining samples were maintained at −80°C.−3 dilution was dropped into isolation agar Petri dishes and spread evenly on the surface. The isolation medium of PYG  and agar (1.5% w/v) was prepared and autoclaved separately, to avoid high H2O2 concentration in media prepared by autoclaving agar and phosphate buffer together (v/v) of compound trace salts solution , 1% (v/v) of compound vitamin mixture  and cycloheximide to the final concentration of 50mgL−1. Vitamin mixture and cycloheximide were all filter-sterilized.Dilution plating method was employed for the isolation of microorganisms from the sand samples. Approximately 0.2–0.3ml of the 10together , and thev/v) at −80°C.The Petri dishes spread with soil suspension were incubated at 28°C for 3weeks and distinct colonies were streaked into newly prepared PYG agar dishes to purify the cultures. The purified isolates were maintained on PYG slants at 4°C and also as glycerol suspensions , Tryptic soy agar , Reasoner’s 2A agar , nutrition agar (NA), and PYG agar. The temperature range for growth was tested at 4, 10, 15, 20, 28, 30, 32, 35, 37, and 40°C using ISP 2 broth for cultivation 2weeks. The pH range  for growth was observed in ISP 2 broth using the buffer system described by w/v; at 1% intervals)].Growth characteristics of the strains were tested using ISP 2 , GYM (gLw/v) and then checked using light microscopy. The cellular morphology of the test strains was studied using transmission electron microscopy (JEOL JEM-1010) after 6-day incubation on GYM semi-solid slant agar. Before cells were mounted on formvar-coated copper grids (Electron Microscopy Science), they were negatively stained using 2% (w/v) uranyl acetate for 15s.Colonies characteristics and pigments production were observed and recorded after 3-day incubation on GYM agarM. lapidis DSM 100206T and M. multiseptatus DSM 44406T obtained from DSMZ were carried out some assays in parallel.The reference strains of v/v) H2O2. The abilities of strains to produce H2S and indole, hydrolysis of cellulose, gelatin, and starch were examined according to previously described procedures . Other metabolic characters were determined by API 50CH and API ZYM test kits (bioMerieux) according to the manufacturer’s instructions. Results were evaluated after incubation at 28°C for 72–168h. The oxidase activity was detected using API oxidase reagent (bioMeriéux) according to the manufacturer’s instructions. The catalase activity was determined by observation of bubble production in 3% , and the collected biomass re-suspended in small aliquots of sterilized distilled water. Cell suspension, adjusted to approximately 1×106CFU/ml, was inoculated into the phosphate-solubilizing medium (gL−1) . For the control, the bacterial cell inoculum was replaced with sterile water. Culture broth was centrifuged  on the 2nd day of incubation, and the amount of available phosphorus in the supernatant determined colorimetrically using standard protocol as described previously . Cultures were centrifuged  sulfuric acid, 2.87mol; ammonium molybdate, 8.1mmol; antimonyl potassium tartrate, 1.5mmol; ascorbic acid, 85.2mmol (added into the solution just before use).Concentration of the available phosphorus in the culture media was calculated based on the following equation:X represents available phosphorus in the culture media; P, available phosphorus calculated for a reaction mixture; V1, total volume of reaction mixture; V2 volume of culture supernatant added in the reaction mixture; K, the dilution ratio used for measuring the absorbance.where Biomass for chemotaxonomic studies of the strains was obtained by cultivation in flasks on a rotary shaker (180rpm) using GYM broth at 28°C for 4days except that cellular fatty acid extraction and analysis were conducted using the cultures harvested from Tryptic soy broth (TSB). The diagnostic isomers of diaminopimelic acid in the whole cell hydrolysates  of these strains were subjected to thin-layer chromatography on cellulose plates using the solvent system of The whole-genome sequencing was implemented using an Illumina HiSeq 4000 system  at the Beijing Genomics Institute . Genomic DNA was sheared randomly to construct three read libraries with length of 300bp by a Bioruptor ultrasonicator  and physico-chemical methods. The paired-end fragment libraries were sequenced according to the Illumina HiSeq 4000 system’s protocol. Raw reads of low quality from paired-end sequencing (those with consecutive bases covered by fewer than five reads) were discarded. The sequenced reads were assembled using SOAPdenovo v1.05 software.Gene prediction was performed on the genome assembly by glimmer3Geodermatophilaceae. The protein sequence of each genome annotated by RAST was used for downstream analysis such as stress-responding genes retrieval, pan-genome analysis, and calculation of AAI and POCP values. For pathway analysis, the predicted proteins sequences were uploaded to KEGG Automatic Annotation Server with “for prokaryotes” and “bidirectional best hit” options. The website of UniProtRapid Annotation using Subsystem Technology RAST;  was applModestobacter. ANI Calculator , dDDH , AAI (average amino identity), and POCP (percentage of conserved proteins) were calculated for the definition and classification of the novel species of the genus lculator ,9 Genomelculator ,10 Averalculator ,11 and PModestobacter members. The protein sequences of strains CPCC 205119T, CPCC 205215, and CPCC 205251 were annotated by RAST 2.0. The protein sequences of other Modestobacter strains were accessed from NCBI. Pan-genome analysis was performed by BPGA 1.3 using default settings  based on the frequency it occurs in the three genomes. Different CV reflects the distribution frequency of the homologous gene in these three strains. The larger CV indicates that the gene is more widely distributed in the strains of the species M. deserti sp. nov. and the more conserved the gene family is in the species M. deserti. In the pan-genome of the three strains, if the CVs of some homologous families were 3, these homologous families were considered as the part of core genome; those homologous families with the CV of 2 or 1 were regarded as accessory genes or unique genes, respectively. The core, accessory, unique and exclusively absent genes were retrieved using USEARCH clustering tool. BPGA performed the evolutionary analysis based on concatenated core gene alignments and binary (presence/absence) pan-matrix. Gene matrix was calculated using similarity or dissimilarity in contribution of genes to orthologous gene clusters. For core genome based phylogenetic tree, BPGA first extracted the protein sequences  from 20 random orthologous gene clusters to generate core genome phylogeny tree. BPGA automated multiple sequences alignments using MUSCLE. All alignments were concatenated and a neighbor-joining phylogenetic tree was constructed.Bacterial Pan-genome Analysis (BPGA) pipeline was applied for analysis of the genomic diversity of the settings , and thepipeline . A totalT and CPCC 205215 were isolated from moss-dominated soil crust samples IMB12100 and IMB12108, respectively, and strain CPCC 205251 was acquired from cyanobacteria-dominated soil crust sample CCL12125, collected from Shapotou NDER in Tengger Desert, China.Strains designated as CPCC 205119T , CPCC 205215 , and CPCC 205251  were obtained. Pairwise alignment indicated that these three newly isolated strains contained almost identical 16S rRNA genes. BLAST search results showed that they exhibited the highest similarities with the validly described species of the genus Modestobacter, i.e., M. lapidis DSM 100206T (97.3%), M. multiseptatus DSM 44406T (97.3%), and other type strains of the genus Modestobacter (<97.0%). These similarity values were all lower than 98.65%, which was proposed by Kim et al. as the threshold for differentiating two genic species according to the 16S rRNA gene similarity  used to classify bacterial strains of the same species , while lower than the threshold for bacterial species delineation  of the genus Modestobacter. The calculated G+C contents of these strains were in the range of 74.6–74.7%.Assembly sequences for the draft genomes of CPCC 205119aterials . In the SM 44402 . The dDD species ; the ANI 95–96%; ; the AAIn ~ 90%; ; the POCion 70%; ; Table 1ion 70%; . TherefoT, CPCC 205215, and CPCC 205251 contained 4,480, 4,443, and 4,523 genes, respectively , pyrophosphatase coding gene (ppa), and phytase coding gene , phosphate transport-related genes , various metal resistance genes (czcD), DNA inducible enzyme gene (dnaG), thioredoxin reductase gene (trx), and mitomycin free radical oxidase gene  than that in Antarctica’s Valley (M. multisptatus DSM 44402).The whole genome of strains CPCC 205119ectively . The str results . The heane phyC; , phosphane mcrA;  were preM. deserti, which were divided into 4,592 homologous families by cluster analysis. Histograms were constructed according to different CVs  and the number of genomes (n) was obtained by fitting, as follows:The relationship between the pan-genome size and the number of genomes of the species, and the relationship between the number of core genes and the number of genomes were deduced  by usingfcore) and the number of genomes (n) was obtained by fitting, as follows:Meanwhile, the functional relationship between the number of core genes  were involved in carbohydrate metabolism, amino acid metabolism (14.3%), some other elementary metabolism , energy metabolism (6.8%), metabolism of cofactors and vitamins (5.6%), lipid metabolism (5.3%), nucleotide metabolism (5.3%), and membrane transport (5.1%). Accessory and unique genes appeared to be enriched in membrane transport, carbohydrate metabolism, as well as metabolism of cofactors and vitamins. Among the accessory genes (41), the major portion of genes seemed related to membrane transport (19.5%), metabolism of cofactors and vitamins (14.6%), carbohydrate metabolism (12.2%), amino acid metabolism (9.8%), some other elementary metabolism , signal transduction (9.8%), signal transduction (9.8%). Unique genes (4) seemed to be mainly enriched in carbohydrate metabolism (75%), and carbon metabolism , BPGA could map 2,508 (54.6%) to KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, i.e., core genes , accessory genes  and unique genes . Having filtered some KEGG pathway related to eukaryotes, we obtained an overview on the metabolic pathway corresponding to the gene(s) in the pan-genome of the species ism 25%; .2A, NA, and PYG agar after incubation for 72h at 28°C. No diffusible pigments were observed on any tested media. Cells of strains CPCC 205119T, CPCC 205215, and CPCC 205251 were aerobic, Gram-staining-positive, non-spore-forming, cocci (diameter of 0.5–0.8μm) and/or short rods (0.5–0.6μm in width and 0.8–1.1μm in length), motile with polar flagella. Bud-like structure was observed for some cells  NaCl. The optimum growth occurred at 28–32°C, pH 6.0–8.0, with the absence of NaCl. The catalase, oxidase and phosphate solubilizing activity of these strains were positive. None of these strains can hydrolyze gelatin, CM-cellulose (carboxymethyl cellulose), urea and starch. Nitrate was not reduced and H2S was not produced.Orange to red colored colonies with a maximum diameter of 1.1mm were opaque, convex and irregular circle on ISP 2, GYM, TSA, Rme cells . These tDetailed physiological and biochemical characteristics of these strains from Biolog GEN III, PM3B, and PM4A Microplates, and API 50CH and API ZYM test kits are given in the description of the species and in the supplementary materials as T, CPCC 205215, and CPCC 205251 could solubilize tricalcium phosphate [Ca3(PO4)2] and phytin with a detection of additional available phosphorus of (4.72±0.34) mg/L and (36.78±1.75) mg/L, respectively.In the liquid culture assay, strains CPCC 205119meso-diaminopimelic (meso-DAP) in the cell wall. Whole-cell hydrolysates yielded arabinose, glucose and ribose. The cellular polar lipids contained DPG, PE, PG, PI and PIM, PME as well as small amounts of unidentified phospholipid (UPL) and aminophospholipid   with minor amounts of MK-8(H4) (17.7–21.2%); The cellular fatty acids were C18:1ω9c (18.9–29.7%), iso-C16:0 (11.2–20.9%), C16:0 (6.7–11.0%), C17:1ω8c (4.2–11.8%), C16:1ω7c (6.6–9.0%), iso-C15:0 (6.1–8.0%), C18:0 .2S, indole and melanin, hydrolysis of gelatin, starch, and cellulose are negative. Cells are positive for acid phosphatase, alkaline phosphatase, cystine arylamidase, esterase lipase (C8), esterase (C4), leucine arylamidase, lipase (C14), naphthol-AS-B1-phosphohydrolase, trypsin, valine arylamidase, α-glucosidase, β-glucosidase, β-galactosidase, and weekly positive for α-chymotrypsin, α-galactosidase in API ZYM strip. Acetic acid, bromo-succinic acid, d-cellobiose, Dextrin, d-fructose, d-galactose, d-malic acid, d-maltose, d-mannose, d-salicin, d-trehalose, d-turanose, gelatin, glycerol, l-aspartic acid, l-glutamic acid, l-malic acid, propionic acid, sucrose, Tween 40, α-d-glucose, α-d-lactose, and α-keto-butyric acid can be utilized as sole carbon sources in Biolog GEN III Micro-Plates. Acid is produced from esculin ferric citrate and potassium 5-ketogluconate. Cell wall contains alanine, glutamic acid, glycine and meso-diaminopimelic as diagnostic amino acids. The whole-cell hydrolysis contains arabinose, galactose, ribose and traces of glucose. The cellular polar lipid system includes DPG, PE, PG, PI, and PIM, as well as small amount of APL. The predominant respiratory quinone is MK-9(H4), with minor of MK-8(H4). The major fatty acids are C18:1ω9c, iso-C16:0, C16:0, iso-C15:0, and C16:1ω7c. The G+C content of the genomic DNA is 74.7%. The type strain is CPCC 205119T (=I12A-02624T=NBRC 113528T=KCTC 49201T), isolated from the moss-dominated soil crust from the Shapotou NDER located in Tengger Desert situated at the Ningxia Hui Autonomous Region, the north of China. The assembly sequences for the draft genome of CPCC 205119T has been deposited in GenBank with the accession number JAAGWK000000000.Cells are aerobic, Gram-staining-positive, cocci- to rod-shaped, motile, and non-sporing actinobacterium. The colonies are irregular, opaque, orange-red on most tested media. Grows at 10–37°C and pH 6.0–11.0, with optima at 28–30°C and pH 7.0–8.0, respectively. Grows in NaCl at concentrations up to not more than 3%. The activity of oxidase and catalase is positive. The production of HM. deserti sp. nov. accommodating three isolates from the desert microbiological soil crusts. The detailed phenotypic properties illustrated the abilities of these strains to adapt to environmental stress.Based on the genomic information, we summarized genetic characteristics of the novel species M. deserti, 37 gene clusters (1.6%) were mapped to bacterial chemotaxis  and flagellar assembly (ko02040) pathway which was related to cell motility . These gene clusters, including chemotaxis family related genes cheA, cheB, cheR, cheW, cheY, mcp, motA, motB, rbsB, tar, and tap, flagellar basal-body rod protein coding genes flgB and flgC, flagellar basal-body rod modification protein coding gene flgD, flagellar hook protein coding genes flgE and flgL, flagellar biosynthesis protein coding genes flhA, flhB, fliP, fliQ, and fliR, RNA polymerase sigma factor for flagellar operon coding gene fliA, flagellin coding gene fliC, flagellar hook-basal body complex protein coding gene fliE, flagellar M-ring protein coding gene fliF, flagellar motor switch protein coding genes fliG and fliY, flagellum-specific ATP synthase gene fliI, flagellar motor switch protein coding gene fliM. These genes might encode some factors and proteins to help the cells to adapt to harsh environment by sensing chemical gradients in their habitats and then move toward more favorable conditions. The interaction between above factors and proteins caused a change in behavior, such as in direction or speed of rotation of flagella , PhoC (coded by phoC gene), and the phospholipase PhoD  acquisition and assimilation are of fundamental importance in cell physiology because P is a kind of required nutrient in many of the metabolic and energy-producing pathways in bacteria. The phosphorus sources include organic phosphorus and inorganic phosphorus. Bacteria transport inorganic phosphate by high affinity phosphate transport system PstSCAB coded by the B operon , PhnCDE d-glucose 6-phosphate, and cysteamine-S-phosphate, especially, they could solubilize tricalcium phosphate [Ca3(PO4)2] and phytin.The phenotypic assays showed that all the three strains could assimilate pyrophosphate, thiophosphate, dithiophosphate, phosphoenol pyruvate, 2-deoxy-via the KEGG database, 2-deoxy-d-glucose-6-phosphate (C06369), dithiophosphate, phosphoenol pyruvate (C00074), and pyrophosphate (C00013) could be predicted from these three strains’ metabolic pathway. In the RAST annotation results (phyC) was retrieved from all the three genomes. It could be proposed that phytase could catalyze the hydrolysis of inorganic orthophosphate from phytin  and phosphate transport-related genes , as well as inorganic pyrophosphatase coding gene (ppa), phytase coding gene (phyC), phosphate starvation-inducible protein coding gene (phoH), and a polyphosphate kinase coding gene (ppk) were retrieved from all these three genomes , one copy of aglG gene (α-glucoside transport system permease protein AglG coding gene), one copy of otsA gene , one copy of otsB gene , two copies of treS genes , one copy of treY gene , and one copy of treZ gene . The proteins coded by these genes reported to be involved in the pathway of trehalose uptake and biosynthesis, which is part of starch and sucrose metabolism (ko00500), and these proteins were supposed to respond to the osmotic stress  were mapped to replication and repair (KO09124) contained DNA replication (ko03030), base excision repair (ko03410), nucleotide excision repair (ko03420), mismatch repair (ko03430), homologous recombination (ko03440), and non-homologous end-joining (ko03450) pathway, such as uvrA (UvrABC system protein A coding gene), uvrB (UvrABC system protein B coding gene), uvrC (UvrABC system protein C coding gene), uvrD (DNA helicase II coding gene), recA (protein RecA coding gene), recB (RecBCD enzyme subunit RecB coding gene), recC (RecBCD enzyme subunit RecC coding gene), recD (RecBCD enzyme subunit RecD coding gene), recF (DNA replication and repair protein RecF coding gene), recG (ATP-dependent DNA helicase RecG coding gene), recO (DNA repair protein RecO coding gene), recQ (ATP-dependent DNA helicase RecQ coding gene), and recR (recombination protein RecR coding gene). Thirty-seven gene clusters (1.5%) were mapped to carbon fixation pathways in prokaryotes (ko00720), such as coxS , coxM (carbon monoxide dehydrogenase medium chain coding gene), and coxL (carbon monoxide dehydrogenase large chain coding gene). In the core genome, katG gene  was retrieved, and the protein coded by this gene could protect strains against toxic reactive oxygen species (ROS) including hydrogen peroxide as well as organic peroxides. The gene csrA was also retrieved from the core genome, this key regulator could bind to mRNA to regulate the gene expression, thereby, shift from rapid growth to stress survival, or might help the strain to survive in low carbon habitats by activating peptide uptake , glycerol, hexosephosphates, amino acids, carboxylic acids, fatty acids, and phosphate esters  can be found in the article/Z-MJ, B-HZ, and H-MS carried out the experiments and prepared the manuscript. TZ and L-YY collected the samples. Y-QZ designed the research and analyzed the data. All authors contributed to the article and approved the submitted version.This research was supported by National Natural Science Foundation of China (32170021 and 31670010), Beijing Natural Science Foundation (5212018), the National Infrastructure of Microbial Resources (NIMR-2021-3), and CAMS Innovation Fund for Medical Sciences .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.