Serous ovarian cancer is the most common and primary death type in ovarian cancer. In recent studies, tumor microenvironment and tumor immune infiltration significantly affect the prognosis of ovarian cancer. This study analyzed the four gene expression types of ovarian cancer in TCGA database to extract differentially expressed genes and verify the prognostic significance. Meanwhile, functional enrichment and protein interaction network analysis exposed that these genes were related to immune response and immune infiltration. Subsequently, we proved these prognostic genes in an independent data set from the GEO database. Finally, multivariate cox regression analysis revealed the prognostic significance of TAP1 and CXCL13. The genetic alteration and interaction network of these two genes were shown. Then, we established a nomogram model related to the two genes and clinical risk factors. This model performed well in Calibration plot and Decision Curve Analysis. In conclusion, we have obtained a list of genes related to the immune microenvironment with a better prognosis for serous ovarian cancer, and based on this, we have tried to establish a clinical prognosis model. In female, ovarian cancer was the third most common cancer and the second leading cause of cancer-related deaths in 2018, as 295,414 people being diagnosed and 184,799 deaths being reported globally . Ovarianp = 0.016; and HR 0.78 [95%CI = 0.44–1.40], p = 0.41, respectively) (Gene expression profiling has been widely used in ovarian cancer to identify distinct molecular subtypes. The Cancer Genome Atlas (TCGA) project provides gene expression data and clinical and survival information . Ovarianctively) . These tctively) .Different gene expression profiles not only provide valuable clues regarding the molecular subtypes , but alsThe role of tumor microenvironment in predicting clinical outcome and efficacy was gradually recognized. Researchers were trying to find reliable immune-related prognostic genes in ovarian cancer, including TAP1  and CXCLThis study aims to screen the immune-related genes of serous ovarian cancer from different perspective based on the differences in the prognosis and immune scores among four molecular types. Moreover, we establish a new risk classification system based on two immune-related prognostic genes to predict overall survival, and propose potential prognostic markers and therapeutic targets for advanced ovarian cancers.https://tcga-data.nci.nih.gov/tcga/) on June 7, 2020. In TCGA data portal, we also downloaded gene expression subtype (n = 308) and clinical data such as age, pathological stage, grade, and survival information. The level of stromal cells and the infiltration level of immune cells in OV tissues were viewed by scores from the ESTIMATE website (https://bioinformatics.mdanderson.org/estimate/index.html). The gene expression subgroups were used as the test group, while all TCGA data of OV were used as the internal verification group for the survival prognosis of the selected genes. GSE32062 data set(27) from the GPL6480 platform was downloaded for external verification.Ovarian serous cystadenocarcinoma gene expression data by AffyU133a array was obtained from TCGA dataset  and Heatmaps were used to visualize the DEGs. The heatmaps and clustering were based on an open-source web tool Morpheus (https://software.broadinstitute.org/morpheus).Package limma  was perfhttps://www.oncomine.org/) is a database for tumor-related gene research from GEO, TCGA, and published literature. The transcription levels of up-regulated genes with prognostic significance were compared with those of normal control samples. The statistical method used for comparison was Student’s t-test.Oncomine (http://gepia.cancer-pku.cn/).This tool was developed by http://metascape.org), which provided comprehensive gene list annotation and analysis resources. Gene Ontology (GO) process and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were conducted in functional enrichment analysis. Only terms with p-value < 0.01, minimum overlap of 3, and enrichment factor of >1.5 were considered significant. To understand the underlying interaction, Metascape was also employed to construct protein network analysis. Molecular Complex Detection (MCODE) algorithm was applied to identify densely connected network components. The results of enrichment analysis with prognostic genes were shown by a bubble diagram using an online platform (http://www.bioinformatics.com.cn).Metascape is a web-based portal .The prognostic significance of differentially expressed genes was assessed by univariate analysis. + T cells, CD8+ T cells, neutrophils, macrophages, and dendritic cells .t-test was used to compare the difference between the tumor tissues and normal ones.The detailed protocols were well established and described in our previous studies . In briehttps://www.cbioportal.org/) stores DNA copy number data, mRNA and microRNA expression data, and other useful information. Samples with mRNA data (RNA Seq V2) of 585 ovarian cancer data were obtained from the cBioPortal to further analyze the genetic alterations and Co-expression of TAP1 and CXCL13. The first 100 co-expressed genes of CXCL13 and TAP1 were obtained from the cBioPortal database. The protein-protein interaction network of shared genes was retrieved from the STRING database (high confident: 0.7) (https://string-db.org/). The network was further constructed by Cytoscape software.The cBioPortal website (http://sangerbox.com/). The regression coefficients in the multivariate Cox regression model were used to generate a nomogram. The performance of the nomogram was evaluated using the Calibration plot, C index, and Decision Curve Analysis. All statistical analyses were performed using R . “rms” package, “survival” package, “survivalROC” package and “pheatmap” package were installed and used.Cox proportional hazards regression model was used for multivariate analysis of prognostic factors. The heatmap of the relationship between risk classification and gene expression was drawn on the sangerbox website (p = 0.092), grade (p = 0.861), new event type in the course of the disease (p = 0.141), cancer status at enrollment (p = 0.335), anatomic site (p = 0.577), and initial pathologic diagnosis method (p = 0.402), but were related to age (p = 0.002) and OS (p = 0.035)  cases of immunoreactive subtype, 85 (27.60%) cases of proliferative subtype, 69 (22.40%) cases of mesenchymal subtype, and 70 (22.73%) cases of differentiated subtype. Gene expression types were not statistically significant with pathological stage (= 0.035) . It has ESTIMATE was used to compare the infiltration of immune cells of the four types. Based on the ESTIMATE algorithm, stromal scores ranged from -1593.24 to 1837.43, and immune scores were distributed between −1400.6 and 2774.16. Among the immune scores of ovarian cancers, the average score of immunoreactive type was the highest, followed by mesenchymal and differentiation ones, and proliferative type had the lowest score . On the p-value = 0.00), differentiation type , and proliferative type , Kaplan-Meier survival curves showed that the immunoreactive type had the best prognosis   . Subsequ= 0.008) .In this section, we identified the DEGs between the immunoreactive subtype and other subtypes to reveal the correlation of survival differences. First, the difference between gene expression subtypes was analyzed by package limma. Then, we applied heatmaps to show the differential gene expression matrix. Hierarchical clustering was used to cluster different subtypes, and we measured them by the Euclidean distance method. Compared with the proliferative group, 607 genes were upregulated, and 356 genes were down-regulated in the immunoreactive group. 138 genes were upregulated, and 412 genes were down-regulated when compared with the mesenchymal group; 276 genes were upregulated, and 72 genes were down-regulated when compared with differentiation type. Upregulated and downregulated genes in each comparison group were visualized using heatmaps  and volcTo analyze the potential function of DEGs between the immunoreactive group and other groups, we performed functional enrichment analysis and protein-protein interactions. Gene ontology (GO) terms showed the analysis results through Biological Processes, Cellular Components, and Molecular Functions. In the immunoreactive vs. proliferative groups and immunoreactive vs. differentiated groups, functional concentration mainly focused on immune response, immune regulation, and defense response. While immunoreactive vs. mesenchymal subtypes primarily concentrated on the regulation of chemotaxis and cytokine and the formation of extracellular matrix, blood vessels, and other tissue structures .KEGG analysis concentrated on infection, cancer, chemokine signaling pathway, cytokine action, phagocytosis, and the inflammatory response when compared immunoreactive to proliferative types. Immunoreactive vs. differentiated groups were focused on infection, cancer, and inflammatory response. Immunoreactive vs. mesenchymal groups were concentrated in infection, cytokines, and inflammation .Metascap uses BioGrid, InWeb_IM, OmniPath databases to analyze interactions between proteins. Enrichment analysis is a collection of physically related proteins. If the network contains 3 and 500 proteins, the MCODE algorithm is used to identify the network components. p < 0.05), including CXCL11, TAP1, CXCL13, STAT1, CD38, UBD, ISG20, LAMP3, CXCL9, PSMB9, GBP1, USP18, HLA-DOB, WARS   . To furt < 0.05) . The gen < 0.05) . We extr < 0.05) .+ T cell, CD4+ T cell, macrophage, neutrophil, and dendritic cell, though ISG20 and UBD were negatively correlated with macrophage infiltration . Among them, six immune cells infiltration were significantly associated with the expression of STAT1, CXCL11, CXCL13, PSMB9, and GBP1, but no macrophage cell infiltration was found statistically significant  for the remaining six genes: CD38, CXCL9, TAP1, WARS, ISG20, and UBD  . Further, we set up a risk score formula based on the two genes, risk score = TAP1*(−1.2281) + CXCL13 *(−0.8237).To further evaluate the prognostic value of these screened genes, we used Cox proportional-hazards model to evaluate the impact of these gene expression on the prognosis. This analysis yielded a risk score. Based on this score, the OV samples in TCGA were divided into two parts: the high-risk group and the low-risk group . The dot0.00011) , while Thttps://www.proteinatlas.org/)  database (as.org/) . Meanwhimal ones . On the mal ones . The Peamal ones . Subsequmal ones .We developed a model to assess survival in advanced ovarian cancer based on the risk score. We considered some clinicopathological factors such as age, stage, and grade in advanced ovarian cancer patients (stage III–IV) . Then, wAs a potential treatment option, tumor immunotherapy has made great progress in recent years . Due to p = 0.0264; immunoreactive vs. mesenchymal, p = 0.0009; immunoreactive vs. differentiated, p = 0.0123), which agrees with the results reported by an Australian study   .BRAC1/2 mutation status and outstanding response to immunotherapy . These genes with prognostic significance mainly focused on immune response, inflammatory response, and protein metabolism. Protein-protein interaction analysis showed that chemokine family members CXCL9, CXCL11, and CXCL13, which are important mediators of leukocyte migration to inflammatory sites (Herein, DEGs shared by the three OV groups were revealed ry sites , formed ry sites .+ and CD8+ TILs are closely related to improved PFS, disease-free survival, and OS (+ TILs also have prognostic value in ovarian cancer (+ B cells could improve patient survival (+ TILs that will promote tumor cell lysis (Finally, all 14 genes identified herein were found to have prognostic value, as confirmed by internal validation. Using an independent GEO cohort of 260 high-grade serous ovarian cancer samples, 12 genes showed significant correlations between gene expression and overall survival. TIMER showed that these validated genes were positively correlated with TILs and inversely proportional to tumor purity. Similar results were reported by , and OS . Other rn cancer . Moreovesurvival , as B cell lysis .Among the herein validated genes, TAP1 and CXCL13 were identified as positive risk factors through the Cox proportional-hazards model. TAP1 encodes a transporter that is responsible for presenting tumor antigens in major histocompatibility complex I or human leukocyte antigen complexes . Hence, As the frequently altered neighbor genes of TAP1 and CXCL13, the immune-related genes CTLA-4, IFNG, and PRF1 were found to have the higher interconnected nodes according to the protein-protein interaction network. CTLA-4 has served as targets of immunotherapy by immune checkpoint pathways , whereasHerein, the Tumor-Node-Metastasis staging system was used to evaluate the survival outcome of patients; however, patients with the same anatomical distribution were classified into the same stage without considering other variables, such as genetic differences or histology . NomograIn general, this study highlights the differences in immune scores and survival prognosis between the immunoreactive and other gene expression types in serous ovarian cancers. In particular, the common differential genes with prognostic significance were determined, and their correlation with immune infiltrating cells was described. Finally, a nomogram model for advanced ovarian cancer based on TAP1 and CXCL13 was established for the first time. Although the accuracy of this nomogram model was verified. Its predictive effect still needs to be further investigated in the basic experiment and large-scale multicenter clinical trials.


Orbicella annularis and O. franksi, two young species with contrasting vertical distribution in the Caribbean. When transplanted from deep to shallow, O. franksi experienced fast photoacclimation and low mortality, and maintained a consistent bacterial community. By contrast, O. annularis experienced high mortality and limited photoacclimation when transplanted from shallow to deep. The photophysiological collapse of O. annularis in the deep environment was associated with an increased microbiome variability and reduction of some bacterial taxa. Differences in the symbiotic algal community were more pronounced between coral species than between depths. Our study suggests that these sibling species are adapted to distinctive light environments partially driven by the algae photoacclimation capacity and the microbiome robustness, highlighting the importance of niche specialization in symbiotic corals for the maintenance of species diversity. Our findings have implications for the management of these threatened Caribbean corals and the effectiveness of coral reef restoration efforts.Metazoans host complex communities of microorganisms that include dinoflagellates, fungi, bacteria, archaea and viruses. Interactions among members of these complex assemblages allow hosts to adjust their physiology and metabolism to cope with environmental variation and occupy different habitats. Here, using reciprocal transplantation across depths, we studied adaptive divergence in the corals  MicrobiWhile the influence of different species of symbiotic algae on the ecophysiology of reef-building corals has been studied, the effect of other coral-associated microorganisms is less well known, especially across depth-segregated species ,10. HoweOrbicella species complex, dominant in Caribbean reefs, was regarded as one species with ecotypic variation, but recent research revealed three species partially segregated by depth   . ΔF/Fm′ ris n = 3 and O. f species , leadingv. 3.6.1 .. 2.4. 2.4.1O. annularis and O. franksi is in part due to their microbial communities. Tissue samples were collected at the end of the transplant experiment using 1/8″ metal corers by divers wearing nitrile gloves and were immediately deposited in whirl pack bags. Once returned to the boat, each sample was gently washed with filter-sterile (0.2 µm) seawater, deposited in a sterile cryovial and immediately preserved in liquid nitrogen. We extracted DNA from coral tissue samples using the MoBio Powersoil DNA Isolation Kit (MoBio Laboratories). Two-stage amplicon PCR was performed on the V4 region of the 16S small subunit prokaryotic rRNA gene [We quantified coral-associated microbiome communities in coral transplants to test if adaptive divergence between RNA gene ,28  package . Primer . 2.4.2O. annularis and O. franksi) and species-specific models (i.e. O. annularis or O. franksi) were tested. Heatmaps of OTU abundances were created using the phyloseq::plot_heatmap function [t-tests (1000 Monte Carlo permutations) with Bonferroni correction. To test for significant differences in OTU abundances across host species and depths, we employed negative binomial modelling using DESeq2 [O. annularis and O. franksi) and species-specific models (i.e. O. annularis or O. franksi) were tested. p-values for the significance of contrasts were generated based on Wald statistics, and false discovery rates were calculated using the Benjamini–Hochberg procedure.To quantify differences among treatments, we used weighted UniFrac (wUniFrac) dissimilarity matrices using OTU-level relative abundances. Significant differences in bacterial assemblages were assessed by permutational multivariate analysis of variance (PERMANOVA) with wUniFrac distances and the explanatory variables host species and depth (i.e. vegan::adonis) . Both ovfunction . Within-g DESeq2 ,38. Both. 2.5. 2.5.1To quantify differences in dinoflagellate communities across species and depths, we used a two-stage amplicon PCR on the same DNA that was extracted and used for the 16S amplification. We amplified the internal transcribed spacer 2 (ITS2) rRNA marker gene to characterize microalgal taxa within the family Symbiodiniaceae . Once th. 2.5.2To compare dinoflagellate communities across samples, we constructed Bray–Curtis and Jaccard dissimilarity matrices using absolute abundances. Significant differences in bacterial communities between sample types were assessed by PERMANOVA with Bray–Curtis and Jaccard distances and explanatory variables including host species, season and depth using the adonis function from the vegan package . We test. 3. 3.1Kd near the transplant sites was 0.40 m−1, indicating that corals from the shallow (3.5 m) and deep (9.5 m) sites receive, respectively, approximately 25% and approximately 2% sea surface irradiance. Across the vertical distribution range of each species (a), it is estimated that the light intensity varies between 18% and 62% sea surface irradiance for O. annularis and between 5% and 33% for O. franksi. Relative light levels recorded by data loggers indicated that the light exposure was nearly five times more variable in shallow water than in deep water. Daily temperatures were significantly higher in the shallow site  than in the deep site . However, based on the scaling quotient of temperature (Q10) of Orbicella spp. [The  species a, it is lla spp. , it is e. 3.2O. annularis S-D (Δdepth = 6 m) resulted in 26% mortality (Fisher exact test: p = 0.003) and was significantly higher than that of O. franksi colonies transplanted D-D . Orbicella franksi therefore has an advantage of 26% over O. annularis in deep habitats. By contrast, O. franksi when transplanted D-S did remarkably well with only 2% mortality (Fisher exact test: p = 0.63). Mortality of the two species was not significantly different , suggesting that O. franksi in shallow areas has no perceivable short-term (less than six months) disadvantage relative to O. annularis (Fisher exact test: p = 0.60).Transplantation of . 3.3O. annularis exhibited a significant increase in Fv/Fm when transplanted S-D (0.622 ± 0.034) relative to corals transplanted S-S (0.541 ± 0.007) . On the contrary, symbionts of O. franksi transplanted D-S experienced a reduction in Fv/Fm (0.470 ± 0.052) relative to D-D transplants . Transplantation of O. annularis S-D induced a significant reduction in Qm (0.008 ± 0.076), relative to S-S transplantation  (a), while O. franksi exhibited a significant increase in Qm (0.226 ± 0.156) when transplanted D-S, relative to D-D transplants  (b).Symbionts of  < 0.01) a, while  < 0.01) b.FigureQm on coral colonies along the vertical distribution of each species ranged from 0.099 to 0.673 in O. annularis and from 0.020 to 0.492 in O. franksi (c). We found a significant species by depth interaction , indicating that the slope of the regression model describing the relationship between Qm and depth was significantly different between species, being more than twice as pronounced in O. annularis  than in O. franksi . The linear regression of Qm with depth indicated that the potential depth limit described by the bioenergetics of the coral–algae symbiosis  is 5.5 m for O. annularis and 7.8 m for O. franksi (c), which nearly coincide with the observed lower limit of distribution of both species in the study area , but not host species (p = 0.12) or depth by species interaction .After quality control, sequencing resulted in a total of 577 930 microbial reads  partitioned across 14 274 unique OTUs. Overall, coral-associated prokaryote communities were significantly structured according to depth . OTUs enriched in shallow-water coral microbiomes are from the bacterial orders Acidimicrobiales (1 OTU), Alteromonadales (1), Kiloniellales (2), Lactobacillales (1), Neisseriales (1), Oceanospirillales (3) and Synechococcales (1). The mean logO. annularis  and O. franksi fragments (0.582 ± 0.008) (padj = 0.358). By contrast, microbiome variability differed significantly between depths, being greatest in O. annularis transplanted S-D  and significantly higher than O. annularis transplanted S-S  or O. franksi transplanted D-S  (b). The larger microbiome variability in O. annularis transplanted deep is consistent with higher mortality and limited photoacclimation potential.Microbiome variability did not differ significantly between species with = 0.006) b. The la. 3.5O. annularis were significantly different from those of O. franksi regardless of the depth to which they were transplanted . Symbiodiniaceae genotypes belonging to the genera Symbiodinium (ITS2 type A3) and Cladocopium  occurred in both coral species, although Cladocopium genotypes were more abundant in O. franksi. Genotypes from the genus Breviolum (B1 and B1/B1t) were detected in high abundance in O. annularis, and in many colonies from the shallow site (40% of them) were the only dominant symbiont. Only one O. franksi colony transplanted D-S hosted a Breviolum (B1) population. Genotypes belonging to the genus Durusdinium  were detected only in O. franksi transplanted D-D .Algal communities of nted D-D . Neither. 4O. annularis and O. franksi have diverged physiologically and occupy distinct light environments in part due to the variation in their associated microbiotas . Following transplantation to deep habitats, O. annularis experiences a limited photoacclimation potential and disruption of the photosynthetic performance of its algal symbionts, consistent with increased mortality and significant microbiome community shifts with increased variability. By contrast, O. franksi maintained a robust physiological performance, a resilient microbiome composition with no significant community shifts or increased variability, and low mortality at both depths. Our study suggests that O. annularis is adapted to shallow environments characterized by a higher and more variable temperature and light regimes, while O. franksi is physiologically able to live in both shallow and deep habitats. The niches of these sibling species have diverged, and a large component of the niche separation seems to be related to variations in the photoacclimation capabilities and the microbial community of each species. The absence of O. franksi in shallow areas may be related to other ecological aspects not considered in our experiment, such as slow growth in an area of intense space competition, a restricted morphological plasticity for regulating the light capture (see below) and/or disadvantages at the larval or recruitment stages in shallow waters.Our study demonstrates that despite being genetically close  , which is notably higher than in clear-water sites . The vel Toro 0.0 m−1, whn Belize ) and refn Belize . This ven Belize ,46 and sth rates .O. annularis experiences an almost complete loss of photosynthetic activity when transplanted deep. Orbicella annularis fragments photoacclimate to low-light conditions by increasing the light energy conversion efficiency (i.e. increase in Fv/Fm) [Qm reflect a trivial photosynthetic contribution of O. annularis symbionts to the host metabolism due to light-limited photosynthesis [O. annularis in the deep environment supports the insufficient autotrophic contribution to the host metabolism. Photoacclimation of O. franksi fragments transplanted to the shallow environment resulted in an increased fraction of photo-inactivated PSII reaction centres and capacity for thermal dissipation of excessive light energy absorbed [O. annularis, the estimated Qm in O. franksi does not indicate the occurrence of chronic photoinhibition in the shallow environment nor light-limitation in the deep environment, suggesting that O. franksi can maintain a more robust physiological performance across depths. The photoacclimation responses of both species in the transplant experiment were consistent with the rates of change in Qm across their vertical distribution range, which collectively suggest that the symbiotic algae of O. annularis are more sensitive to changes in light intensity with depth than symbionts of O. franksi.Our findings indicate that n Fv/Fm) ,49. Howeynthesis , suggestabsorbed ,49. But Orbicella species complex is unusual among other scleractinians in that they can simultaneously host multi-species communities of symbiotic dinoflagellates, whose composition follows environmental gradients of irradiance within colonies and across depths [O. annularis in shallow habitats correlates with its faster vertical growth among Orbicella species and with symbiotic partnerships with particular photoacclimation potential [Orbicella franksi, on the other hand, produce plate-like colonies to maximize light capture in deep environments. When transplanted to shallow well-lit environments, despite a potential for successful photoacclimation as indicated by our results, the plate-like morphology limits the capacity to regulate the internal light climate and allows very slow vertical growth. This slow growth makes O. franksi a poor competitor, probably explaining why this species is rare in shallow areas. Additionally, disadvantages at the larval or recruitment stage [Orbicella species, as seems to happen in other anthozoans [Colony morphology can help modulate the light capture and photosynthetic energy acquisition along the vertical distribution range of corals ,50. The s depths ,18. The otential . Its mornt stage  may keepthozoans .Orbicella species is their heterotrophic feeding capacity. It has been indicated that corals from deep environments increase their metabolic reliance on heterotrophy to compensate for reduced photosynthesis [O. franksi in deep environments might be related to an increased heterotrophic feeding capacity relative to its sibling species. Unevenly distributed corallites that give the bumpy appearance to O. franksi colonies with larger corallites [O. franksi grow near the lowest growth potential determined by light availability in deep environments [Orbicella species is conflicting and inconclusive.Another aspect that may influence the vertical distribution of ynthesis ,54. The rallites  may confrallites . Howeverronments , suggestronments . Overall. 4.2O. annularis and O. franksi. Despite the higher and more variable temperature and light intensity in shallow areas, Durusdinium trenchii was not detected in O. annularis colonies. This symbiont is common among Orbicella and other coral species growing under harsh environmental conditions [O. franksi colonies from the deep environment. The increased abundance of D. trenchii in O. franksi may be related to the run-off impacts in the water column (e.g. sedimentation and nutrient enrichment), a reduction in light penetration and the mechanisms by which the coral–algae symbiosis interact with these environmental conditions [Breviolum genotypes in O. annularis and Cladocopium genotypes in O. franksi, both in the shallow and deep transplant sites, is consistent with previous reports [Cladocopium spp. or Breviolum spp. uniquely associated with each Orbicella species like in other depth-segregated anthozoans  [Species-specific associations with algal symbionts with contrasting photoacclimation capabilities may be a key axis of differentiation between nditions ,58. Surpnditions . The pre reports ,60 and m reports ,62. It iis spp.) ,63.. 4.3Several Endozoicomonas OTUs were significantly enriched in shallow habitats. Endozoicimonaceae are diverse gammaproteobacterial symbionts of numerous marine hosts at varying depths and with a wide global distribution . MembersMembers of the family Alteromonadaceae and the order Acidimicrobiales were also enriched in shallow areas. Alteromonadaceae belong to a diverse group of heterotrophic gammaproteobacteria known to associate with marine hosts and nutrient-rich environments. Members of this group tolerate relatively high temperatures and have been used in coral probiotic studies as coral-associated bacteria capable of scavenging free radicals  and therAlloiococcus and Synechococcus. Alloiococcus belongs to the group of gram-positive lactic acid bacteria, which are recognized for producing bacterial growth inhibitors that function to deter invading bacteria in their hosts [Synechococcus is a photoautotrophic cyanobacterium found in surface waters harbouring abundant light. Both corals and their symbiotic algae are known to actively feed on Synechococcus [Synechococcus cells may increase bleaching recovery and coral health [Finally, corals in shallow areas were also enriched for ir hosts . Synechohococcus ,71 whichhococcus . As a fol health .et al. [Seriatopora hysterix, not host genotype or Symbiodiniaceae strain. Our results do not contradict this finding but suggest that responsiveness to environmental conditions can differ significantly even among very closely related coral taxa.There is a continuing debate as to the relative role of coral host versus environment in shaping coral microbiomes. This study demonstrates that the responsiveness of coral microbiomes to environmental conditions differs significantly even among very closely related coral species . These differences in microbiome shifts may be related to the resilience of the coral host and its associated algal community to a particular habitat. Pantos et al.  found th. 4.4O. annularis when transplanted to low-light environments. We suggest that to enhance survivorship during restoration, the particular light environment of source populations should be similar to the transplant sites. In this study, due to the high vertical attenuation of light (Kd = 0.40 m−1), a 6 m increase in depth resulted in an order of magnitude reduction in irradiance and increased mortality of O. annularis by 26%. In a clear-water site (e.g. Kd = 0.06 m−1), this response would be expected to occur with an increase in depth of approximately 40 m. Given the expensive nature of coral restoration, equating the light environment of donor and transplant sites will probably increase yield and decrease costs. Minimally, our approach can be used to estimate the maximum theoretical depth for each species in a given location with certain water optical quality, thereby providing guidance when choosing the location and depth for coral transplantation.A key finding in our study with implications for coral restoration is the increased mortality of O. annularis, may be adapted to an environment with strong changes in light, temperature and salinity. A potential, and relatively unexplored, outcome from ongoing environmental change and disturbances on reef corals, is the instability of associated microbiomes across the vertical distribution range of species. Our study suggests that the microbiome of shallow-water species respond strongly to environmental change, with potential detrimental effects on the corals' survival. The instability in the coral microbiomes of shallow-water corals will potentially increase as a result of climate change, contributing to the ongoing coral decline in these reef areas [The second aspect of our findings is related to microbiome composition in different habitats across reefs. Our study suggests that the microbiome of shallow-water specialists, like ef areas ,74.O. annularis, can be extremely vulnerable to these changes as their physiology/morphology is specialized for high-light habitats. As the degradation of water optical properties in coral reefs continue, shallow-water specialists, which are typically major reef-building species, will probably become rare, shifting the structural and functional integrity of reefs.Lastly, subtle differences in the water optical conditions can result in changes in the underwater light environment and the vertical distribution of coral species. Most coral reefs around the globe are currently threatened by the direct effects of sediments, pollutants and nutrients associated with coastal development and terrestrial run-off . These c. 5O. annularis and O. franksi, are adapted to distinctive light environments along depth gradients. The limited photoacclimation potential and less robust microbiome community restricts O. annularis to shallow, high-light environments. Orbicella franksi is more versatile, but other ecological aspects such as slow growth in areas of intense space competition restrict the species to deep environments. These contrasting responses associated with the microbial communities highlight the importance of niche specialization in symbiotic corals for the maintenance of species diversity. Our study has implications for coral reef restoration efforts, providing guidance when choosing the location, depth and light environment for coral transplantation.Our study suggests that the sibling coral species,


Acinetobacter baumannii is a leading cause of nosocomial infections that severely threaten public health. The formidable adaptability and resistance of this opportunistic pathogen have hampered the development of antimicrobial therapies which consequently leads to very limited treatment options. We mapped the global prevalence of multidrug-resistant A. baumannii and showed that carbapenem-resistant A. baumannii is widespread throughout Asia and the Americas. Moreover, when antimicrobial resistance rates of Acinetobacter spp. exceed a threshold level, the proportion of A. baumannii isolates from clinical samples surges. Therefore, vaccines represent a realistic alternative strategy to tackle this pathogen. Research into anti-A. baumannii vaccines have enhanced in the past decade and multiple antigens have been investigated preclinically with varying results. This review summarises the current knowledge of virulence factors relating to A. baumannii–host interactions and its implication in vaccine design, with a view to understanding the current state of A. baumannii vaccine development and the direction of future efforts. Acinetobacter baumannii is an opportunistic gram-negative coccobacillus that demonstrates exquisite survival under various environmental conditions and intrinsic resistance to routinely prescribed antibiotics [A. baumannii strains are now prevalent worldwide, with the highest percentage of carbapenem resistance of over 90% being reported in the Mediterranean region, imposing serious burdens on healthcare systems. The vast majority of A. baumannii isolates arise in medical institutions and are closely associated with nosocomial infections, particularly in patients receiving intensive care and in immunocompromised individuals. Ventilator-associated pneumonia (VAP), central line-associated bloodstream infections (BSI), urinal tract infections (UTI), and meningitis are the most common clinical manifestations [A. baumannii as a critical priority, for which, new antibiotics are urgently needed [A. baumannii occur, it is difficult to thoroughly eradicate from the environment due to its remarkable resistance to disinfectants and its capacity to rapidly develop tolerance to these antibacterial agents, contributing to prolonged colonisation and transmission [A. baumannii infections. Compared with other nosocomial pathogens such as Pseudomonas aeruginosa, Staphylococcus aureus, and Clostridium difficile, the search for vaccines against A. baumannii is in its infancy [A. baumannii.ibiotics . Multidrstations . Unfortustations ,4,5,6. Iy needed . In addismission . TherefoAcinetobacter currently comprises more than 60 validly named species [Moraxellaceae, within the order Pseudomonadales, and the class Gammaproteobacteria since 1991 [A. baumannii (strain ATCC 17978) was first sequenced in 2007 by Smith et al. [A. baumannii is phenotypically similar to, and phylogenetically close to, several other Acinetobacter species  complex consists of six valid species: A. baumannii, A. calcoaceticus (formerly Acinetobacter genomic species 1), A. nosocomialis (formerly Acinetobacter genomic species 13TU) [A. pittii (formerly Acinetobacter genomic species 3) [A. seifertii (formerly Acinetobacter genomic species ‘close to 13TU’) [A. lactucae  [A. calcoaceticus is non-pathogenic, the other five members are all clinically relevant and are frequently identified in a broad sense as A. baumannii, or A. baumannii group, in clinical microbial identification. The dominant species among the clinically isolated A. baumannii group vary with region [A. baumannii is the most virulent [The genus  species . It has nce 1991 . The whoh et al. . However species . The A. o 13TU’) , and theoorniae) ,15,16,17h region ,19, but Acinetobacter spp. are ubiquitous organisms and the first isolate, described as Micrococcus calcoaceticus back in 1911, was from soil [A. baumannii, however, are still poorly defined as it is almost exclusively isolated from hospital environments and communities involving close contact [A. baumannii was rarely detected on human skin , indicating that this is not the natural habitat of A. baumannii, despite Acinetobacter spp. being associated with human skin flora [A. baumannii in the human body louse has been reported in many countries worldwide [A. baumannii strain SDF which is responsible for community-acquired infections was first isolated from the interior of body lice collected on homeless people in France [rom soil . Their wrom soil . The nat contact . A. baumin flora . The preorldwide ,28,29. Fn France .A. baumannii in environmental soil and waterbody samples has frequently been reported [Acinetobacter spp., with A. baumannii representing 14.7% (n = 34) [A. baumannii strain from acid paleosol in Croatia [A. baumannii strains were from sewage or urban streams, and hence it is challenging to define whether they are native residents or contaminants from other A. baumannii positive fomites [A. baumannii colonisation has also been observed in livestock animals, mostly from cattle and poultry [A. baumannii isolates from livestock avian in Germany to the lineages spread in hospitals worldwide and found there were several strains closely related to human clinical isolates indicating that livestock avian may serve as a powerful vector for the spread of A. baumannii [A. baumannii barely existed in the choana and rectums of wild songbirds and gulls captured in Germany and Poland  [Identification of traces of reported ,35,36,37n = 34) . More re Croatia . Most wa . More r poultry ,41,42,43 poultry , but the = 1051) . More cuA. baumannii is of major concern. It is classified as an ESKAPE pathogen, an acronym referring to Enterococcus faecium, S. aureus, Klebsiella pneumoniae, A. baumannii, P. aeruginosa, and Enterobacter spp., all of which are recognised nosocomial bacteria with high potential to exhibit MDR and virulence [A. baumannii strains are frequently reported to demonstrate resistance to the most routinely prescribed antibiotics in varying degrees, including carbapenems such as imipenem and meropenem. Carbapenem is one of the β-lactam antibiotics with the broadest spectrum and highest efficiency. Resistance to carbapenem is considered to be a marker for extensively resistant bacteria because it involves a broad range of co-resistance to unrelated antibiotic classes [A. baumannii strains documented over the past decade  surveillance annual reports, and in the case of those without applicable surveillance data, published figures from the most recent research or reviews were used. It is apparent that carbapenem-resistant A. baumannii is now problematic across Asia and the Americas, except in Japan and Canada. Moreover, Oceania, Western Europe, the Nordic region, and part of central Europe have the lowest R% (<10%), however, in areas surrounding the Mediterranean, including southern Europe, the Middle East, North Africa, up to 90% of A. baumannii clinical isolates are resistant to carbapenems. Details for central Africa and island countries are limited.The antimicrobial resistance of irulence . Clinica classes . We havet decade . The parA. baumannii infections, MDR often compels the adoption of last-resort antibiotics such as colistin when essentially no other options are available. However, the increasing quantity of recent cases reporting the emergence of pan-drug-resistant (PDR) strains tolerant to last-resort therapies has dimmed their potential [In the treatment of otential ,50,51,52A. baumannii has been long been recognised as a major opportunistic pathogen causing nosocomial infections, or healthcare-associated infections, and manifests as a broad range of infections, including respiratory infections, BSIs, UTIs, and meningitis. [A. baumannii infections [A. baumannii infection was over 50% [A. baumannii infections was 17.9%, of which infections occurring at normally sterile sites had much higher mortality of 41.3% compared with UTIs (8.3%) [A. baumannii is one of the leading causes of VAP worldwide, especially in Asia, Latin America, and the Middle East [A. baumannii caused VAP has been reported to be as high as 84.3% [A. baumannii bacteraemia ranges from 37% to 52% in the U.S. [A. baumannii meningitis is comparably low, it is an increasing threat in post-neurosurgical patients, with a mortality rate approaching 70% [A. baumannii skin and soft tissue infections in trauma  victims following natural disasters or wars have also been recorded, such as those during the Iraq conflicts, the Syrian war, the Wenchuan earthquake, the Marmara earthquake, and the Indian Ocean tsunami [A. baumannii [A. baumannii were reported worldwide [A. baumannii infections developed VAP and 85% (17/20) of them had co-infections with SARS-CoV-2 [A. baumannii infections experienced a two-fold higher mortality rate relative to COVID-19 patients without A. baumannii infections. These reports suggested that A. baumannii co-infection may exacerbate the development and prognosis of COVID-19 and hinder the clinical diagnosis and treatment [ingitis. . Patientfections . Previouover 50% , althougdle East . The ICUhing 70% . A. baum tsunami ,56,57,58orldwide ,60,61. Areatment .A. baumannii infections are rarely reported but comparably more often in some tropical or sub-tropical areas in the Asia–Pacific such as northern Australia and Taiwan [A. baumannii strains involved in community-acquired infection are more susceptible to antibiotics. Farrugia et al. sequenced the whole genome of a community strain D1279779 obtained from Darwin, Australia, and found that the antibiotic resistance island AbaR, associated with nosocomial strains, was absent in that strain [A. baumannii infections are characterised by an acute fulminant course involving septic shock, respiratory failure, severe sepsis, pneumonia, and the mortality can be quite high, i.e., over 60% [Community-acquired d Taiwan ,64,65,66d Taiwan . Unlike t strain . Despiteover 60% ,68.A. baumannii in clinical sites have often been reported [Acinetobacter spp. isolates identified among all the clinical samples of monitored AMR species . The parameter F% was compared to R% by region . Curve estimation (0.0304×R% (adjusted R2 = 0.736). This exponential model suggests that when antibiotic resistance rates are low in a region, the clinical isolation rates of Acinetobacter spp. generally remain relatively stable, but once the R% exceeds a certain level, the proportion of A. baumannii isolates from clinical samples surges, a stark warning that there might be considerably more A. baumannii associated infections and a higher risk of nosocomial outbreaks in the future. In this case, with an R% of less than 10%, the detecting frequency F% can be as low as <1%; whereas F% increases by ten-fold if the R% is more than 90%. However, due to the small sample size (n = 41) and high variance, the relationships between the prevalence of A. baumannii outbreaks and its antibiotic resistance should be further validated with more relevant data. No evidence of gender or age difference in incidence has been reported so far.Since the 1990s, outbreaks of reported , particureported ,71, and y region  using thy region . There itimation  shows thA. baumannii among all clinical isolated, aerobic, and facultative gram-negative pathogens have been reported, ranging from 0.7% in North America to 4.6% in the Middle East [A. baumannii. Historically, A. baumannii was also known as ‘Iraqibacter’ because it caused outbreaks of MDR strains in the US military hospitals based in Iraq, Kuwait, and Afghanistan during the Iraq War [A. baumannii have been reported [A. baumannii was the most prevalent organism recovered (22%) during the study period of 2003–2008 [A. baumannii has spread from the US military system to the civilian settings.Regional differences in the overall proportion of dle East . The MidIraq War ,73. ThesIraq War ,55, and reported ,51,74, rreported . Moreovereported ,77. For 003–2008 . Subsequ003–2008 . In cont003–2008 , suggestA. baumannii infectious sites. High levels of interleukin 2 (IL-2), IL-4, IL-17, tumour necrosis factor α (TNF-α) and interferon γ (IFN-γ), as well as a lower CD4+/CD8+ ratio, have been reported [A. baumannii is strain-dependent and involves complicated mechanisms of pathogenicity which are only beginning to be elucidated. Many efficacious vaccines against other pathogens have been developed based on virulence factors, consequently, A. baumannii virulence factors are worth reviewing.Severe tissue damage with infiltration of large numbers of inflammatory cells is frequently observed at reported . The virA. baumannii than non-respiratory epithelial cells [A. baumannii pneumonia. Outer membrane proteins (OMP), including OmpA , are the most well-studied A. baumannii adhesins [A. baumannii with a molecular weight of 38 kDa and plays an essential role in adhesion and invasion. Competitive binding studies with recombinant A. baumannii OmpA attenuated both cell binding and invasion [A. baumannii attaches with moderate affinity to epithelial cells, secreting OmpA into the cells, which leads to bacterial uptake via actin rearrangement and membrane reorganisation of host cells, known as a zipper-like mechanism [A. baumannii OmpA (UniProt ID: Q6RYW5) has no significant homology with E. coli (K12) OmpA  but shares 46% and 41% identities with probable lipoprotein YiaD and YfiB, respectively. Considering the query covers are relatively low (31% for both), the results should be interpreted cautiously. Similarly, Omp33–36  is another outer membrane virulence factor involved in host–cell attachment. It is also cytotoxic, inducing apoptosis by activation of caspases and modulation of autophagy, with the consequent accumulation of sequestosome 1 and autophagosome LC3B-II [A. baumannii mutant deficient in the omp33 gene resulted in a significant reduction in adherence, invasion, and cytotoxicity of human lung epithelial cells, compared to the wild type [A. baumannii adhesins include the biofilm-associated protein (Bap), which increases the membrane hydrophobicity [Acinetobacter trimeric autotransporter (Ata). Ata mediates adherence through binding to type IV collagen in the extracellular matrix  of host cells [Studies have demonstrated that the respiratory epithelial cells are more easily invaded by al cells , which ladhesins ,83,84,85invasion . A. baumechanism . The intechanism  and the  LC3B-II . An isogild type ,87. Othehobicity ; the Fhahobicity ; and thest cells ,91,92. IA. baumannii is capable of producing different adhesins and pilus assembly systems in response to changes in environmental conditions, such as the BlsA mediated, photoregulated type I pilus assembly system PrpABCD [A. baumannii strain with enhanced adherence and motility [A. baumannii bound via ChoP to the platelet-activating factor receptor (PAFR) on human lung cells, resulting in activation of signalling involving G protein, clathrin, β-arrestins, proteins involved in the direction of the vacuolar movement, and intracellular calcium, which eventually led to invasion [A. baumannii to various glycosphingolipids on thin-layer chromatograms [Recently, non-OMP adhesins and invasins have been identified. Both the response regulator, BfmR, and the sensor kinase, BfmS, in the BfmRS two-component system have been reported to play a role in cell adherence ,94. Seve PrpABCD . Eijkelk PrpABCD . Interesmotility . Thioredmotility . In contmotility . Phosphomotility ,101,102.motility . A. bauminvasion . Host ceatograms . Collectatograms .A. baumannii, particularly lipooligosaccharides (LOS), contributes to its cytotoxicity towards host cells. LOS is the major component on the outer leaflet of the outer membrane [A. baumannii. Erridge et al. showed that A. baumannii LOS stimulates TNFs and IL-8 secretion from macrophages [A. baumannii LOS.The envelope of membrane  and controphages . Severalrophages ,109,110,A. baumannii GGT and showed A. baumannii strains with higher extracellular GGT activity resulted in more severe tissue damage by inducing increased inflammation and oxidation activities with elevated phenoloxidase, lysozyme, lactate dehydrogenase, and lipid peroxidation observed [As previously mentioned, several envelope proteins including OmpA, Omp33–36, and Ata, also contribute to cytotoxicity. Bacterial γ-glutamyl transferase enzyme (GGT), secreted via the type II secretion system, was also reported to cause cell damage directly by caspase activation and apoptosis, depletion of ATP and subsequent necrosis, and cell-cycle arrest. A high level of serum GGT was associated with oxidative stress and the exacerbation of chronic obstructive pulmonary disease (COPD) ,112. Verobserved .A. baumannii based on its identification within pathogenicity islands (PAI) after whole-genome sequencing, but its specific roles have not been studied to date [A. baumannii may utilise a type VI secretion system to kill competing bacteria [There are additional virulence factors whose roles and molecular interactions are not fully understood, but their absence has resulted in impaired virulence or lethality. For example, Smith et al. reported a type IV secretion system may contribute to the pathogenesis of  to date . Furtherbacteria .A. baumannii pathogenesis, serum persistence also affects the infection process indirectly. Capsular exopolysaccharide (CPS) is an envelope component mediating the persistence of many invasive bacteria, enabling organisms to survive better under unfavourable conditions, e.g., desiccation. It also effectively protects pathogens from phagocytosis and humoral immune attack and inhibits the activation of the alternative complement pathway [A. baumannii express the K locus gene cluster, responsible for the biosynthesis and export of CPS [ptk and epsA, that were predicted to encode a putative protein tyrosine kinase (PTK) and a putative polysaccharide export OMP (EpsA), respectively, and the ptk and epsA mutants showed significantly decreased survival in soft tissue infection sites [pglC and pglL mutants showed impaired survival in mouse septicaemia models [In addition to adhesion, invasion, and cytotoxicity contributing to  pathway . Many clt of CPS ,116. Howon sites . Similara models ,119, sugA. baumannii serum resistance, conferring a competitive advantage in vivo, as mutants lacking LpsB, a highly conserved glycosyltransferase involved in LOS synthesis, showed reduced resistance to human serum [A. baumannii by human serum and impaired its ability to penetrate the endothelial monolayers [A. baumannii from H2O2, low pH, and 2,4-DNP, and the uspA mutant formed fewer colonies in sepsis or pneumonia mouse models [A. baumannii to confer resistance to and degrade H2O2 is also largely dependent on the catalases KatE and KatG, which may attenuate the production of reactive oxygen species (ROS) by phagocytic cells of the innate immune system [LOS may be important for an serum . Metal aan serum ,122,123.nolayers . Universe models ,126,127.e system . Finallye system .vaccineA. baumannii worldwide and increasingly frequent reports of emerging PDR strains, more effective control implications are urgently needed to combat this imminent threat. Thus, novel antibiotics and anti-A. baumannii vaccines are the two main routes being followed, with the latter being especially important in view of the likelihood of the development of resistance to new antibiotics. A. baumannii has an intracellular lifestyle, invading host cells via a zipper-like mechanism [A. baumannii vaccines should induce acquired cellular immunity with long-term memory [A. baumannii vaccines for targeted populations.With the extensive spread of MDR echanism  and therm memory , while tm memory ,137,138,A. baumannii rarely causes diseases in healthy people, nevertheless, protection is needed for vulnerable individuals, particularly those residing in, or frequently travelling to, areas with a high prevalence or historic outbreaks of A. baumannii. These susceptible cohorts are the main target population of A. baumannii vaccines and were nominally identified and characterised into three groups  challenge for a period of four weeks post-challenge. Moreover, immunisation-induced antisera provided fully effective protection in passive immunisation experiments, highlighting that this attenuated live vaccine induced robust humoral responses despite minimal splenic cell-mediated immunity [Given that a considerable proportion of the individuals susceptible to  adhesin . Mice imimmunity .A. baumannii was a formalin-killed ATCC 19606 whole-cell vaccine administered with aluminium phosphate adjuvant. Mice intramuscularly (i.m.) immunised with this vaccine were effectively protected from a lethal sepsis challenge raised by heterologous clinical strains, including one PDR strain, with seven-day survival rates between 85%–100%. Significant increases in the serum levels of immunoglobulin G1 (IgG1), IgG2a, and IgM were detected, suggesting a mixed Th1/Th2-mediated response was stimulated [A. baumannii strain LAC-4 successfully elicited both mucosal and humoral immune responses in mice with significant levels of IgA, IgG1, and IgG2a in bronchoalveolar lavage (BAL) fluid being achieved. All immunised mice were completely protected from lethal i.n. or i.p. challenges raised by the LAC-4 strain for up to 10 days [Several inactivated whole-cell vaccine candidates formulated either with or without adjuvants were also evaluated ,141,142.imulated . InteresA. baumannii and their molecular interactions continue to be identified in the past decade, their immunogenicity and their potential as subunit vaccines have been more extensively investigated  across clinical isolates [A. baumannii OmpA would be less likely to cause mutual interference with other OmpA-containing commensal bacteria, minimising the potential side effects as a vaccine. Several studies have presented positive results demonstrating active immunisation with OmpA-based vaccines could confer prophylactic benefits against A. baumannii . Intranasally immunised rOmpA conjugated to cholera toxin, showed 15-day survival rates from 40% to 100% following i.p. challenges compared with 100% mortality in two days in the control groups [Probably the most understood and promising subunit candidate for isolates ,145, whiaumannii . Intranal groups . A studyl groups  suggestil groups .A. baumannii colonised patients and showed no cross-reaction with sera from other infections, [A. baumannii with ≥98% identity [A. baumannii and its co-administration with OmpA may confer even greater protection; however, data from active immunisation studies are currently insufficient. In silico simulation also predicted peptides constructed by exposed epitopes of Omp33–36 could trigger antibody responses with higher avidity [A. baumannii-induced pneumonia by engendering anti-adhesive and robust opsonic activities [A. baumannii infections [6 × LD50 (1013 CFU), a five-day survival rate of 60% was obtained [A. baumannii, BauA, and BfnH, proved only moderately effective against lethal sepsis challenges with survival rates of 40% and 44.5%, respectively [Besides OmpA, several other virulence factors have also been tested as prospective vaccine candidates. Omp33–36 was specifically recognised by IgG, IgM, and IgA from ections, , highligidentity . Omp33–3 avidity . Alternativities , making tivities  as a monfections ,153 but obtained . It shouectively .vaccineA. baumannii pathogenesis and overlooked as virulence factors may also have a potential impact as vaccines. These immunogens are likely to be safer even when a high dose is given [A. baumannii porin involved in iron uptake and colistin binding which is conserved across 804 strains with more than 91% identity in protein sequence [Burkholderia pseudomallei OmpW homolog afforded extended protection (81 days) against a lethal melioidosis challenge [A. baumannii strains and with almost negligible homology to human proteins. Again, a recombinant Trx–Omp22 fusion protein showed 100% protection of immunised mice for seven days after lethal challenge at high doses, with lower doses showing protection of a third of the mice [A. baumannii to a higher level than those immunised with either of the two proteins individually [Additionally, some OMPs which are less relevant to is given  and haveis given . Purifieis given . The caris given . OmpW issequence . We havehallenge . Immunishallenge . Later, the mice . Althougvidually ,161 demoA. baumannii cells or recombinant A. baumannii proteins produced by E. coli customarily require a detoxification step reducing the content of LOS to a minimal level to weaken the endotoxin activity, ensuring vaccine safety, and critically ensuring that the immunological response is mediated by the protein antigen being evaluated. LOS is also not a popular target for vaccine development due to the short-lasting B cell memory it induces; however, mice vaccinated with LOS alone were protected from a lethal challenge [A. baumannii mutant deficient in LOS production showed significantly reduced protection (40%) compared with those derived from the wild-type cells, and administration of exogenous LOS restored the survival levels [A. baumannii can acquire resistance to colistin and collateral sensitivity to azithromycin, rifampicin, and vancomycin through the complete loss of LOS, suggesting that LOS-based vaccines may deliver weakened protection against some problematic LOS-deficient A. baumannii MDR or PDR strains [A rather controversial and neglected antigen for subunit vaccine candidates is LOS . OMPs exhallenge . LOS is l levels . Noticea strains . DespiteA. baumannii strains. To date, single recombinant protein vaccines have performed poorly in providing protection against bacteria that mediate pathogenesis via multiple virulence mechanisms. A. baumannii expresses several virulence factors that allow successful evasion of innate immune defence [A. baumannii are insufficiently studied and most of them are outer member complex (OMC) or outer membrane vesicle (OMV) based.Unfortunately, despite encouraging results in pre-clinical studies, single subunit antigens are unlikely to elicit comprehensive protection in humans against heterologous  defence , contribA. baumannii reference strain ATCC 19606 effectively elicited both humoral and cellular responses in vaccinated mice and induced 80% to 100% protection against sepsis following challenge with heterologous A. baumannii isolates. Notably, the antisera elicited with this OMC vaccine also cleared established A. baumannii infections (70–100% recovery rate) [OMPs extracted from ry rate) . However®, an OMV-based product [A. baumannii reference strain ATCC 19606 proved highly effective in immunising mice, and 90% of the immunised mice were protected against heterologous strains. Subsequent MALDI–TOF MS analysis identified six proteins with high immunogenicity in the OMVs, including previously mentioned, OmpA, OmpW, Omp33–36, and CarO, as well as two other putative proteins [E. coli-derived OMVs packaging fused proteins of exogenous A. baumannii Omp22 and a pore-forming haemolytic protein ClyA, which protected over 60% and 100% of mice against a sepsis challenge, without any additional adjuvants. It is worth noting that the carrier control groups administered unmodified E. coli OMVs showed 35–63.6% survival indicating that E. coli-derived OMVs elicited cross-immunity to A. baumannii infection [Despite this, the use of naturally secreted outer membrane vesicles (OMV) as vaccine technology platforms has gained renewed interest in recent years particularly since the approval of meningococcal group B vaccine, Bexsero product . Crucial product ,170. Natproteins  which wenfection .A. baumannii. Very recently, DNA vaccines delivering A. baumannii OmpA gene were reported to induce a mixed humoral and cellular response protecting lethal bacterial challenges in murine pneumonia models [A. baumannii OmpA and proteoglycan associated lipoprotein (PAL) genes administered intramuscularly resulted in the protection of >80% of immunised mice to lethal pulmonary challenge with four heterologous strains, while immunisation with an OmpA encoding vector elicited 50% survival in response to challenge. A high level of humoral responses and mixed Th1/Th2/Th17 cellular responses, and reduced bacterial loads were reported with reduced inflammatory cytokines and inflammatory cell infiltration in the BAL [ompA vaccine utilising a eukaryotic expression vector pBudCE4.1 protected 60% of the immunised mice against pulmonary infection for up to 15 days post-challenge, eliciting moderate Il-2, IL-4, Il-12, and IFN-γ responses [Despite nucleic acid vaccines have gained increasing interest as vaccine platforms in recent years, there are barely any studies on nucleic acid vaccines against a models ,164. Rec the BAL . ImmunisA. baumannii has lagged behind that for nosocomial pathogens such as C. difficile, P. aeruginosa, and S. aureus [A. baumannii vaccines.Despite the aforementioned efforts, the progress of vaccines against . aureus , and to A. baumannii vaccines are still in their infancy, with most research efforts looking into conventional approaches and platforms, e.g., subunit and killed vaccines. Novel antigen carriers and advanced delivery systems, such as nucleic acid vaccines, virus vectors, conjugated carriers, and co-delivery of multiple antigens are rarely explored. Nevertheless, as the mRNA and DNA vaccine technologies have been acknowledged throughout the world during the COVID-19 vaccine race, it is foreseeable that burgeoning attention may be paid to nucleic acid vaccines against A. baumannii and other pathogens in the coming future. In addition, proteomic approaches and reverse vaccinology exploit modern bioinformatics, allowing the systematic in silico evaluation and selection of putative immunogens for vaccine candidates, which may have a wider application for future antigen selection and vaccine design against A. baumannii infections.Current investigations on anti-A. baumannii should demonstrate its protective benefits in immunised animals with a significantly improved survival rate, reduced bacterial burdens within organs, and the suppressed accumulation of inflammatory cytokines and chemokines in sera. Furthermore, the resulting anti-sera are expected to contain sufficient antibody titre for effective passive immunisation and even for antibody therapy treating established infections. Therefore, most studies have laid emphasis on evaluating post-immunisation or post-infection humoral immune responses, while cell-mediated immunity remains largely unstudied. However, as an invasive pathogen, it is essential that A. baumannii vaccines elicit dominant cellular immune responses and prime sustained T memory cells for long-term protection. In this context, though most studies reported robust vaccine-induced humoral or Th2-mediated immune responses, their performance in stimulating cellular immunity was not specified. Besides, the intervals between the last immunisation and bacterial challenge were only one week in some studies, making the evaluation of the endurance of the protective response even less reliable. It is hence important for the A. baumannii vaccine research community to standardise the evaluation protocols, such as the animal models, intervals between immunisation and challenges, challenge strains, doses, and routes, so that the comparison in the efficacy of the experimental vaccines between different laboratories is more rigorous [In pre-clinical trials, a promising vaccine candidate against rigorous .A. baumannii vaccine studies have evaluated the protection against not only homologous strain challenges but also challenges raised by other clinical isolates to demonstrate the broad protection offered by the vaccine candidates. However, as the protection rates reported in these studies varied considerably with strain, immunisation, and/or challenging dose, their protective effects require more comprehensive evaluations covering a broader range of heterologous strains in diverse challenging scenarios, especially for those involving highly virulent strains and massive exposure dose. Strain-dependent diversity in the selected antigens may also increase the challenges in developing a vaccine providing broad or multivalent protection, as even in a specific bacterial strain, the amino acid sequences of some antigen proteins, e.g., OmpA, may vary among multiple subclasses [A. baumannii has an exceptional ability to acquire foreign DNA and generate new variants as the whole genome sequencing of ATCC 179878 found a significant fraction of open reading frames (ORF) (17.2%) in 28 putative alien islands [It is encouraging to note that many bclasses ,171. At  islands , and sucA. baumannii arose following outbreaks in the US army during the Iraq conflicts, and since then is becoming a global concern. Currently available treatment options for MDR A. baumannii-caused infections are quite limited where colistin is often reported as the only antibiotic delivering therapeutic effects, making the pathogen extremely difficult to treat. Massive research efforts have been devoted to its antibiotic resistance mechanisms and epidemiology but so far, our understanding of its pathology is comparably limited. In this context, vaccines against A. baumannii infections, are urgently needed and will be of great value in the prevention of infection and control of this canny pathogen. The past decade has witnessed the very first efforts in A. baumannii vaccine development, however, none have entered clinical trials, which is clear evidence of the challenges involved and the need for further progress in understanding the complex A. baumannii–host relationships at each stage of the infection process. Most A. baumannii vaccine candidates developed to date explored conventional protein-based technologies, inducing opsonophagocytic antibody-mediated killing and/or antibody-mediated toxin inhibition. However, a polarised Th2 response may not be sufficient for effective and long-term protection, considering that A. baumannii is an invasive pathogen; consequently, a mixed Th1/Th2 orTh1/Th17 response may be more beneficial. Investigations of novel platforms, as well as adjuvant development, could offer the possibility of optimising immune mechanisms and targeting multiple antigens, which will eventually deliver vaccines with broad coverage and clinical efficacy. In addition, the COVID vaccine and associated technology acceleration could change the landscape of future vaccine research and development. With the licensing of the first mRNA vaccines, nucleic acid vaccines against A. baumannii and other invasive pathogens are expected to generate more interest in the near future. There is some urgency, however, in order to tackle the global spread of MDR A. baumannii, and vaccine development needs to accelerate in order to keep up with the pace of spread.The increasing attention to


Monocytes play an important role in innate immunity. Some epidemiological evidence indicates an association between peripheral blood monocytes and ulcerative colitis (UC). The association between peripheral blood monocytes and mucosal healing (MH), however, remains unclear. We evaluated this issue in patients with UC.Study subjects consisted of 272 Japanese patients with UC. Monocyte counts were taken in the morning after overnight fasting. Monocyte count was divided into tertiles based on the distribution of values among all study subjects. Information on clinical remission was obtained from medical records. MH was assessed using the Mayo endoscopic subscore.3. Rates of clinical remission, MH, and complete MH were 61.0%, 66.2%, and 27.9%, respectively. High monocyte count was significantly inversely associated with clinical remission, MH, and complete MH . Patients were also classified according to C-reactive protein (CRP) levels; in the low CRP group (<0.1 mg/dL), high monocyte count was independently inversely associated with complete MH but not with clinical remission or MH . In the high CRP group, there was no association between monocyte count and clinical outcomes.The mean monocyte count was 360.1 ± 155.3/mmOur findings suggest that peripheral blood monocyte count can be used as a serum supplemental marker for MH in UC patients with low CRP levels. The clinical course of ulcerative colitis (UC) is characterized by repeated relapses and remissions . MucosalPeripheral blood leukocyte subtypes including monocytes can be monitored at clinical visits; such data are routinely available. Dysregulation of mucosal innate immunity seems to be a key factor in the pathogenesis of UC , and monIn general, serological biomarkers for intestinal inflammation have lower sensitivity and less specificity in patients with UC compared with those in other patients . We haveStudy subjects in the baseline survey consisted of 387 Japanese patients with UC who visited the Department of Gastroenterology and Metabology at the Ehime University Graduate School of Medicine or several affiliated hospitals in Ehime Prefecture. All patients who were able to consent to the study and respond to the self-administered questionnaire were considered as candidates for inclusion. The final analysis sample in this study consisted of 272 patients because 115 patients were excluded because of incomplete data. All patients had been diagnosed with UC based on radiological, endoscopic, histological, and clinical criteria. This study was conducted in accordance with the Declaration of Helsinki, and the study protocol was approved by the institutional review board of the Ehime University Graduate School of Medicine (#1505011). Trained staff obtained written informed consent from all enrolled patients. This study cohort was registered during the period 2015–2019.Information on CRP, peripheral blood monocyte count, endoscopic findings, disease extent, clinical remission, and the use of medication for UC was obtained from medical records. Other variables were reported in a self-administered paper-and-pencil questionnaire. Blood samples were taken in the morning after overnight fasting. A well-trained laboratory medical technologist measured each patient's monocyte count using a flow cytometer. Height was measured to the nearest millimeter using a stadiometer with the patient standing completely erect. Weight was measured in light clothing. Body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared. Blood examination was performed when a colonoscopy was booked or when the colonoscopy examination was performed, and up to 2 months may have passed between the blood examination and the colonoscopy.Clinical remission was defined as no rectal bleeding and stool frequency < 3 times per day.MH was assessed using the Mayo endoscopic subscore (MES) . In this3 (reference category); (ii) moderate monocyte count, 281–391/mm3; and (iii) high monocyte count, ≥ 391/mm3. CRP values were classified as either low (<0.1 mg/dL) or high (≥0.1 mg/dL). Crude odds ratios (ORs) and their 95% confidence intervals (CIs) for clinical remission, MH, and complete MH in relation to peripheral blood monocyte count were estimated using logistic regression analysis. Multiple logistic regression analyses were used to adjust for potential confounding factors. Age, sex, BMI, disease extent (proctitis/nonproctitis), prednisolone use, and antitumor necrosis factor (TNF)-α monoclonal antibody preparation use were selected a priori as potential confounding factors. All statistical analyses were mainly performed using SAS software package, version 9.4 . A receiver operating characteristic (ROC) curve was generated, and the area under the curve was calculated to measure the utility of monocyte count in indicating MH. JMP 14.2 (SAS Institute) was used to analyze the ROC curve and to identify the cutoff value, specificity, and sensitivity of peripheral blood monocyte count. All probability values for statistical tests were 2-tailed, and P < 0.05 was considered statistically significant.Monocyte count was divided into tertiles according to the distribution of this value among all study subjects as follows: (i) low monocyte count, < 281/mm3. Peripheral blood monocyte count was significantly higher in patients treated with prednisolone than in patients not treated with prednisolone  but did not differ between patients treated with and without any other medications.Table P for trend = 0.036, MH: adjusted OR 0.45 [95% CI: 0.23–0.89], P for trend = 0.023, and complete MH: adjusted OR 0.48 [95% CI: 0.23–0.97], P for trend = 0.021).Table P for trend = 0.027), but not with clinical remission or MH. In the high CRP group (CRP ≥ 0.1 mg/dL), there was no association between peripheral blood monocyte count and clinical outcomes. In the low CRP group, the ROC curve of the peripheral blood monocyte count for identifying complete MH had an area under the curve of 0.614  found that peripheral blood monocyte count did not significantly differ between active-stage and inactive-stage IBD, regardless of ctive UC . The disAlthough we have shown a link between peripheral blood monocyte count and MH in patients with UC, the underlying mechanism linking peripheral blood monocyte count and MH remains unclear. Persistent activation of monocytes and dysregulation of innate immune responses are involved in the development of UC . ProinflIL-17 also plays a pathological role in UC : IL-17A This study has some limitations. First, because a cross-sectional design was used, we cannot confirm a causal relationship between peripheral blood monocyte count and MH. Second, it is likely that most of the patients in this cohort had been receiving long-term treatment before the study. The long duration of treatment might have affected peripheral blood monocyte counts and endoscopic activity. Third, the possibility of other concurrent collagen diseases that might elevate CRP such as primary sclerosis cholangitis and rheumatoid arthritis was not excluded, and such diseases may have confounded the results. Fourth, CRP was measured using different kits in different patients, hospitals, and periods. In Japan, however, the measurement methods for common laboratory data, including CRP, are standardized, and all common laboratory data in this cohort are quality controlled using standard substances . FinallyIn conclusion, peripheral blood monocyte count may be significantly inversely associated with clinical remission, partial MH, and MH in Japanese patients with UC. In UC patients with low CRP, peripheral blood monocyte count might be significantly inversely associated with only MH. In UC patients with low CRP, therefore, peripheral blood monocyte count might serve as a supplemental blood marker for MH.Guarantor of the article: Shinya Furukawa, MD, PhD.Specific author contributions: Study conception and design: S.F., E.T., Y.I., and Y.H.: Material preparation and data collection: S.F., S.Y., K.S., K.T., Y.H., K.M., T.N., S.S., N.S., H.M., K.O., A.H., H.T., Y.Y., E.T., and Y.I.: Data analysis: S.F. Interpretation of data: S.F., T.M. and Y.I.: First draft writing: S.F. All authors revised and edited drafts and read and approved the final manuscript.Financial support: None to report.Potential competing interests: None to report.✓ The number of patients with ulcerative colitis (UC) is increasing.✓ Mucosal healing (MH) is the therapeutic goal for UC.✓ Monocytes play an important role in innate immunity.✓ Some studies have shown an association between peripheral blood monocyte count and disease activity in patients with UC.✓ The association between peripheral blood monocyte count and MH remains unclear.✓ Peripheral blood monocyte count is significantly inversely associated with clinical remission, MH, and complete MH in Japanese patients with UC.✓ In patients with low C-reactive protein, peripheral blood monocyte count might be significantly inversely associated with only MH but not clinical remission.✓ In patients with high C-reactive protein, no association between peripheral blood monocyte count and clinical outcomes was found.


Dendrobium nobile, belonging to Dendrobium, the second largest genus in Orchidaceae, has high ornamental, medicinal, and ecological value. D. nobile is the source of many popular horticultural varieties. Among the Dendrobium species, D. nobile has the highest amount of dendrobine, which is regarded as one of the criteria for evaluating medicinal quality. Due to lack of data and analysis at the genomic level, the biosynthesis pathways of dendrobine and other related medicinal ingredients in D. nobile are unknown. In this paper, we report a chromosome-scale reference genome of D. nobile to facilitate the investigation of its genomic characteristics for comparison with other Dendrobium species. The assembled genome size of D. nobile was 1.19 Gb. Of the sequences, 99.45% were anchored to 19 chromosomes. Furthermore, we identified differences in gene number and gene expression patterns compared with two other Dendrobium species by integrating whole-genome sequencing and transcriptomic analysis . Differences in the TPS and CYP450 gene families were also found among orchid species. All the above differences might contribute to the species-specific medicinal ingredient biosynthesis pathways. The metabolic pathway-related analysis will provide further insight into orchid responses to the environment. Additionally, the reference genome will provide important insights for further molecular elucidation of the medicinal active ingredients of Dendrobium and enhance the understanding of orchid evolution.Orchids constitute approximately 10% of flowering plant species. However, only about 10 orchid genomes have been published. Metabolites are the main way through which orchids respond to their environment.  Dendrobium nobile is a medicinal species belonging to Dendrobium, the second largest genus in the Orchidaceae. With high amounts of medicinal active ingredients, D. nobile is also one of the five Dendrobium species recorded in the Chinese Pharmacopoeia (2020 Edition). Polysaccharides and alkaloids are the main medicinal components of Dendrobium. They are mainly stored in the stem and have strong antioxidant, neuroprotective, antidiabetic, antihypertensive, and immunomodulatory activities . Dendrobine, a sesquiterpenoid alkaloid, is the signature bioactive component and is regarded as one of the criteria for evaluating the quality of D. nobile. The biosynthesis of these medicinal ingredients varies with factors including the tissues sampled, species, and genetics (Dendrobium is an important research topic.Jinchai Shihu (金钗石斛) genetics . ElucidaDendrobium contains active polysaccharides  plants were collected. The species was identified by comparison with the D. nobile specimen deposited in the herbarium of the National Orchid Conservation Center of China. Green young leaves were frozen for short reads sequencing and PacBio sequencing. Leaf buds from the plants were fixed as described in The wild D. nobile plants using a modified cetyltrimethylammonium bromide (CTAB) protocol, and then was shorn by Covaris ultrasonicator. DNA fragments between 300bp and 400bp were then selected by Agencourt AMPure XP-Medium kit for library construction and finally sequencing was performed on the MGISEQ-2000 platform. The raw data was generated from constructed paired-end libraries (PE150), and the clean data was obtained after data filtering, which was carried out by SOAPnuke v1.6.5 software (https://github.com/BGI-flexlab/SOAPnuke)  , with thD. nobile using the RNAprep Pure Plant Kit and genomic DNA contamination was removed using RNase-Free DNase I (both from Tiangen), respectively. The integrity of RNA was evaluated on a 1.0% agarose gel stained with ethidium bromide (EB), and its quality and quantity were assessed using a Qubit2.0 Fluorometer and an Agilent 2,100 Bioanalyzer (Agilent Technologies). As the RNA integrity number (RIN) was greater than 7.0 for all samples, they were used in cDNA library construction and Illumina sequencing. The cDNA library was constructed using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB) and 3 μ g RNA per sample, following the manufacturer’s recommendations. The PCR products obtained were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2,100 system. Library preparations were sequenced on the Illumina Novaseq6000 sequencer, generating 150-bp paired-end reads.For assisting gene annotation and gene expression analysis, total RNA was extracted from tender leaves, stems, and roots of three different individuals in the same growing stage of Before genome assembly, genome size and heterozygosity were estimated by Jellyfish v.2.1.4  and Genohttp://www.repeatmasker.org) were used to identify repetitive sequences based on the RepBase v21.12 database (http://www.girinst.org/repbase). For de novo prediction, a repetitive sequence database was constructed through RepeatModeler (http://www.repeatmasker.org/RepeatModeler/) and LTR_FINDER v1.06 (http://tlife.fudan.edu.cn/ltr_finder/). RepeatMasker software was then used to predict the repeat sequences. Tandem Repeats Finder v4.09 (http://tandem.bu.edu/trf/trf.html) was used to find tandem repeats in the genome. The repeat-masked genome assembly was used for annotating high-quality protein-coding genes through an integration of homology-based, de novo, and transcriptome-based predictions. For homology-based prediction, protein sequences from seven species  were used to align the D. nobile genome sequences through Genewise v2.4.1 (https://www.ebi.ac.uk/Tools/psa/genewise/). Then, 5,000 complete genes from the homology-based prediction method were used to produce a training model using the Augustus  and SNAP (http://homepage.mac.com/iankorf/) software. The RNA sequencing data of D. nobile were mapped to the genome sequences through Hisat v2.1.0 and StringTie v1.3.4d , TrEMBL (http://www.uniprot.org/), Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.genome.jp/kegg/), InterPro (https://www.ebi.ac.uk/interpro/), Nr (non-redundant database), KOG (http://lowelab.ucsc.edu/tRNAscan-SE/) was used to annotate the tRNA sequences. BLASTN was used to search for rRNA. The miRNA and snRNA sequences were predicted by the INFERNAL  software.Functional annotation of the predicted gene models was carried out by Blast v2.2.26  softwareInterPro  (https:/se), KOG  (clusterse), KOG  databasese), KOG   were used for orthologous gene family identification and clustering. BLASTP was used to calculate the similarities between sequence pairs with a cut-off e value of 1e−5. Finally, OrthoMCL v.2.0.9 (The protein sequences of  v.2.0.9  with defhttp://www.drive5.com/muscle/). Then, a super-alignment matrix generated from all the alignment results was used for the construction of the phylogenetic tree. Finally, RAxML  was used to estimate the divergence time. The following constraints were used for time calibrations: i) the B. distachyon and O. sativa divergence time [40.0–54.0 million years (Ma)]  (PAML MCMCTREE (rs (Ma)] ; ii) the20.0 Ma) ; iii) th20.0 Ma) ; and iv)20.0 Ma) .Ks distribution was used to infer whole-genome duplication (WGD) events in D. nobile based on paralogous gene pairs and the divergence between species based on orthologues. MCscanX v1.5.2  was used to find the collinear regions between D. nobile and D. chrysotoxum, D. nobile and V. planifolia, and D. nobile and C. sinense.For further analysis of the features of collinear regions, MCscan (http://sourceforge.net/projects/cafehahnlab/) was used to determine the expansion and contraction of orthologous gene families combined with divergence times.Based on the gene families clustered by OrthoMCL , we filtCMS genes, PF01264.22 for CS genes, PF13292.7 for DXS genes, PF02401.19 for HDR genes, PF04551.15 for HDS genes, PF08540.11 and PF01154.18 for HMGS genes, PF00288.27 for PMK genes, PF00275.21 for SHKG genes, PF01202.23 for SK genes, and PF03088.17 for STR genes) were downloaded from Pfam (pfam.xfam.org/), and the query sequences for the BLASTP methods were mostly from homologous genes of Arabidopsis, except for the SKDH genes from Pisum sativum , and two profiles were used to carry out HMM searches against the protein database for six species . These sequences were then manually checked, and the sequences with at least one of these domains were retained. The retained amino acid sequences were aligned using MAFFT (pfam.xfam.org/). HMM searches were used to obtain the homologs in the protein database for seven species . The next steps were the same as those for TPS gene family identification, except for the rice genome. The identified CYP450 genes in A. thaliana and O. sativa in this study were further confirmed with the Arabidopsis Cytochrome P450 database (http://www.p450.kvl.dk/At_cyps/family.shtml) and rice (The HMM profiles for PF01397 (Terpene_synth) and PF03936 (Terpene_synth_C) were downloaded from Pfam  predicted genes were functionally annotated . Among tD. nobile, D. catenatum, and D. chrysotoxum were sisters to P. equestris, forming an Epidenroideae clade. G. elata and A. shenzhenica were located at the base of the Orchidaceae branches (D. nobile and P. equestris was 41.7 (29.2–54.9) Mya (D. chrysotoxum appeared at 11.9 (6.5–18.1) Mya, and the divergence time between D. nobile and D. catenatum was 5.6 (2.9–8.6) Mya. Then, the expansion and contraction of orthologous gene families were determined. According to the results  Mya . Finally results , 110 genst genes . This maD. nobile, the Ks distribution pattern of paralogous genes in collinear regions was analyzed. There were two peaks, and the Ks values were 0.8 and 1.5  occurred before the divergence between D. nobile and A. shenzhenica, suggesting that the WGD events were shared among all extant orchids . The second recent WGD event in D. nobile (Ks = 1.5) was shared with most monocots  genes, two PGM genes, three SPS genes, four SUS genes, two UGP genes, and five UGE genes in D. nobile. Based on the phylogenetic analysis, these gene families could be further divided into several branches. The number of genes in different branches among these species varied greatly, especially those in the NI, PGM, and UGE gene families , suggesting a Dendrobium-specific gene  and three other species  , Dnobile17G01430.1 (DHS), Dnobile08G00887.1 (DHQS), and Dnobile02G00904.1 (DHD-SKDH) (Dnobile08G00649.1 (HMGS) and Dnobile01G00682.1 (MVD) genes in the MVA pathway also had higher expression in stems , Guchui-Maker69398 (DXR), Guchui-Maker111118 (MCS), Guchui-Maker68853 (HDS), and Guchui-Maker109159 (HDR). For STR genes, Dnobile14G00298.1 (STR12/13) was mainly expressed in stems (Guchui-Maker98640 (STR9) was mainly expressed in stems, and Guchui-Maker96976 (STR10) was mainly expressed in leaves  biosynthesis.To determine the genetic variation of the alkaloid biosynthesis pathway, all key genes (20 genes or gene families) upstream of the alkaloid biosynthesis pathway were identified among three haliana) ). Most wHD-SKDH) . The Dnoin stems , such asin stems , while on leaves . Dendrob. nobile  and is rD. nobile. TPSs catalyze the biosynthesis of sesquiterpenes (C15) using farnesyl diphosphate (FPP) as a substrate  and TPS21 genes are involved in dendrobine biosynthesis , two other orchid species (P. equestris and A. shenzhenica), A. thaliana, and rice. There were 123, 170, 228, 257, and 210 CYP450 gene members in A. shenzhenica, P. equestris, D. nobile, D. chrysotoxum, and D. catenatum, respectively. The number of genes in Dendrobium species was much higher than that in P. equestris and A. shenzhenica, mainly because of tandem gene duplication (Cytochrome P450 monooxygenases (CYP450s) are integral components in terpenoid and alkaloid pathways . Biosyntlication .CYP720 in orchids were identified  have had their complete genome sequenced. The chromosome-level genome assembly of D. nobile should be helpful for further functional research as the model of dendrobine biosynthesis in Dendrobium. As one of the most important medicinal plants in traditional Chinese medicine, D. nobile is mainly composed of dendrobine and polysaccharides with pharmacological activity. This study contributed to improving the understanding of the metabolism of these medicinal components.To date, three D. nobile genome is 1.35%, meaning that it is difficult to assemble  interconverts glucose-6-phosphate and glucose-1-phosphate and is a key enzyme in central metabolism  is a plant enzyme that plays vital roles in sucrose production across various plant species and is involved in photosynthesis  and TPS21 genes are involved in dendrobine biosynthesis , the higher number of TPS-a genes in D. nobile and their expression mainly in the stem may be important factors involved in the production of more dendrobine compared to other Dendrobium species . Furthermore, the opposite gene distribution in TPS-b-II and TPS-b-III among the three Dendrobium species may contribute to the differences in the amounts and constitution of alkaloids between D. nobile and D. chrysotoxum in various tissues, as the TPS-b subfamily encodes monoTPSs . Phylogenetic analysis of the D. nobile gene family involved in the polysaccharide synthesis pathway showed that the gene number variation and Dendrobium-specific genes may be related to the fleshy stems with abundant polysaccharides. The analysis results of the TPS and CYP450 gene families suggest that there are more TPS-a genes in D. nobile, and the opposite distribution pattern in TPS-b-II and TPS-b-III among Dendrobium species may contribute to the species-specific alkaloid biosynthesis pathways. The differences in CYP85 clans among Dendrobium species may also play important roles in alkaloid biosynthesis and floral development. The analysis of D. nobile revealed the mechanism through which the fleshy stem produces abundant polysaccharides and alkaloids, as well as the floral development regulation, which is critical for industrial development. This was the first study to fully analyze the characteristics of genes in the biosynthesis pathways of polysaccharides and alkaloids in Dendrobium. The results of this study provide a high-quality genome of Dendrobium and important insights into the molecular elucidation of medicinal active ingredients, molecular breeding, and orchid responses to the environment, enhancing the understanding of orchid evolution.Although All data from this study were submitted to the NCBI database under Bioproject ID: PRJNA725550. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession JAGYWB000000000. The version described in this paper is version JAGYWB010000000.