Veillonella spp. and Prevotella spp. in the salivary microbiomes of IBD patients. Noteworthy, Prevotella salivae emerged as a distinct species significantly associated with IBD. P. salivae is an under-recognized pathogen that was found to play a role in both oral and systemic diseases. In this study, we delve deeper into the salivary microbiomes of both IBD patients and healthy controls. Employing diverse cultivation techniques and real-time quantitative polymerase chain reactions (RT-qPCR), we gauged the prevalence and abundance of Veillonella spp., Prevotella spp., and P. salivae. Our isolation efforts yielded 407 and 168 strains of Veillonella spp., as well as 173 and 90 strains of Prevotella spp., from the saliva samples of IBD patients and healthy controls, respectively. Veillonella-vancomycin agar emerged as the discerning choice for optimal Veillonella spp. cultivation, while Schaedler kanamycin-vancomycin agar proved to be the most suitable medium for cultivating Prevotella spp. strains. Comparing our RT-qPCR findings to the previous 16S rRNA amplicon sequencing data, the results corroborated the higher abundance of Veillonella spp., Prevotella spp., and P. salivae in the saliva of IBD patients compared to healthy controls. However, it’s worth noting that in contrast to RT-qPCR, the 16S rRNA amplicon sequencing data revealed greater absolute abundance of all three bacterial groups in both IBD patients and controls.The global prevalence of inflammatory bowel disease (IBD) is on the rise, prompting significant attention from researchers worldwide. IBD entails chronic inflammatory disorders of the intestinal tract, characterized by alternating flares and remissions. Through high-throughput sequencing, numerous studies have unveiled a potential microbial signature for IBD patients showing intestinal enrichment of oral-associated bacteria. Simultaneously, the oral microbiome can be perturbed by intestinal inflammation. Our prior investigation, based on 16S rRNA amplicon sequencing, underscored elevated abundance of  Veillonella spp. and Prevotella spp. bacteria than that of healthy controls  of periodontal and endodontic infections  being remarkably raised in their saliva samples. P. salivae, despite being an often overlooked pathogen, has demonstrated its involvement in various conditions, including periodontitis  Veillonella agar base +/- vancomycin M416 , previously utilized for the selective isolation of Veillonella species  light visualization, a technique employed to identify distinct colonies exhibiting a characteristic brick-red fluorescence, setting them apart from other pigmented genera. The isolated strains underwent identification using the Matrix-assisted LASER Desorption/Ionization-Time of Flight device . Subsequently, all saliva samples were subjected to dilution with Brain Heart Infusion (BHI) broth, maintaining a ratio of 1:4. To further enhance the proliferation of Gram-negative anaerobic P. salivae bacteria, while restraining the growth of Gram-positive counterparts, the diluted saliva samples were once again streaked onto Schaedler KV agar plates and incubated at 37°C under anaerobic conditions.In the context of identifying the presence of  species , 2) Bloo species , 3) Bruclla spp. , 4) GC–Llla spp. , 5) non-lla spp. , and 6)  salivae . To achiP. salivae, a distinctive reliance on the presence of Prevotella melaninogenica came to light, prompting the exploration of co-culture techniques. Utilizing Schaedler KV agar plates, five discrete strains of P. melaninogenica  were concurrently streaked. Subsequent to this, six strains of P. salivae  were horizontally streaked in six parallel lines perpendicular to the streaks of P. melaninogenica. Importantly, all chosen P. salivae strains had been isolated from the very same samples as the P. melaninogenica strains.During the endeavor to isolate P. melaninogenica and P. salivae was meticulously monitored, with particular focus on the intersections of the vertical and horizontal streaks. Furthermore, a pairwise and triplet arrangement of the same strains was replicated on separate Schaedler KV agar plates. Following the attainment of substantial growth, the utilization of MALDI-TOF assisted in the meticulous analysis of colony identity, especially in cases of uncertainty, such as those arising in the intersection regions.These plates were maintained within an anaerobic environment at 37°C, and they underwent three separate checks over a span of 2 to 7 days. The growth of both Veillonella spp. strains and 28 Prevotella spp. strains, which had been successfully isolated multiple times, were chosen for further evaluation. Specifically, a single representative isolate for each species was selected from each patient or healthy control. The biomass of each isolated strain was carefully gathered using sterile 10 μl inoculation loops and subsequently re-suspended within 1.5 ml Eppendorf tubes, with each tube containing 1 ml of 0.9% sodium chloride solution. The tubes were centrifuged at 8,000 rpm for 1 minute and the supernatant was discarded. For molecular based identification, DNA extraction was performed from all isolated Veillonella spp., Prevotella spp., and P. salivae strains using the QIAamp® DNA Mini Kit  according to the manufacturer’s instructions. DNA was then frozen and stored at -72°C. Subsequently, amplification of the 16S rRNA gene (V1-V9) was performed using the universal forward primer pF1: 5’ AGAGTTTGATCCTGGCTCAG and the universal reverse primer pR1: 5’ GGCTACCTTGTTACGACTT  and OTU14 sequences which were detected in the previous study  denaturation at 95° C for 15 seconds, followed by 40 cycles for P.s primers and 35 cycles for all other primers  , all applied on Schaedler KV agar plates.It’s important to note that despite the frequent detection of  (OTU14) , its isoP. salivae manifested varying colors and sizes. On Schaedler KV agar medium, three different sizes were discernible: large, medium, and small  and Prevotella spp. (25 isolates), BLAST results concurred with MALDI-TOF identifications  RT-qPCR: This method employed specific PCR primers, as previously detailed. ii) Comparative analysis: The 16S rRNA amplicon sequencing outcomes from the earlier study (The assessment of absolute abundance for the three target groups—er study  served aVeillonella genus in IBD patients (P<0.001) and controls (P<0.01) was significantly elevated when assessed using the 16S rRNA amplicon sequencing technique, compared to RT-qPCR  of isolated Veillonella, Prevotella, and P. salivae strains. Additionally, we employed RT-qPCRs to quantitatively assess the Veillonella spp., Prevotella spp., and P. salivae groups. To provide context, our findings will be interpreted in relation to the microbiome data from the same samples, as previously published  or to cultivate various fastidious oral bacteria, as indicated by previous studies (Fusobacterium nucleatum culture filtrate, which facilitated the growth of Prevotella sp. HOT-376. Additionally, it has been demonstrated that Anaerolineae HOT-439, Bacteroidetes HOT-365 and Bacteroidaceae HOT-272 exhibit a reliance on one or both of the helper bacteria Cutibacterium acnes and F. nucleatum. Notably, C. acnes and P. intermedia played crucial roles as helpers for promoting the growth of Tannerella sp. HOT-286 (P. salivae is a bacterium with a delicate constitution, exhibiting intolerance to oxygen, and necessitating a span of five to seven days for its optimal growth on agar plates. Intriguingly, in our investigations, P. salivae was frequently observed in close adjacency to P. melaninogenica colonies, exhibiting a phenomenon akin to the satellite concept, wherein P. salivae seemed to orbit P. melaninogenica. This observation hints at a potential dependence of P. salivae on the presence of P. melaninogenica, as visually represented in P. melaninogenica might act as a nurturing influence for P. salivae, fostering its growth until it reaches a certain colony size and/or quantity. An unprecedented observation that emerged from these experiments was the phenomenon where, after achieving a certain level of growth, P. salivae appeared to hinder the growth of P. melaninogenica (Prevotella species, it is highly advisable to conduct further investigations using a more extensive strain collection.The technique of cross-streaking an agar plate with a helper bacterium has been previously employed to successfully isolate  studies . An illu HOT-286 . Based onogenica Figure 2V. nakazawae and P. vespertina were absent in the Bruker database but present in NCBI BLAST, leading to discrepancies.In clinical laboratories, microorganisms are increasingly being identified using MALDI-TOF MS, a technique that records protein mass spectra from whole cell or intracellular content and compares them to known database references . AccuratBoth MALDI-TOF and traditional bacterial identification methods, which are based on phenotypic traits, are generally less precise than identification through genotypic approaches . AnalysiP. salivae observed in both IBD patients and controls when assessed through RT-qPCR, compared to the number of colonies/strains isolated via cultivation. Notably, the quantification of Prevotella spp. in general exhibited substantial discrepancies in P10, P12, P13, and P21 samples, reflecting the intrinsic challenges associated with cultivating and isolating Prevotella spp. from these particular saliva samples. However, it’s important to acknowledge that quantifying bacterial load via RT-qPCR can lead to divergent profiles (Addressing the challenges posed by culture-based approaches, RT-qPCR has emerged as a valuable tool for enhancing diagnostic sensitivity and lowering the limit of detection, particularly for fastidious organisms . This heprofiles .The application of RT-qPCR does come with certain limitations. It necessitates the pre-specification of amplification targets, and the selection of an external reference organism introduces a potential bias. To mitigate this, various common microbiological taxa can serve as reference organisms for the standard curve . NonetheVeillonella species was 72% with RT-qPCR, as opposed to 81.5% with 16S rRNA amplicon sequencing. Similarly, for Prevotella species, the RT-qPCR primer coverage was 68.5%, while the 16S rRNA amplicon sequencing primer coverage was 90.4%. This divergence is also evident in the case of P. salivae, where the broader coverage of the 16S rRNA amplicon sequencing primer contributes to a greater absolute abundance measurement (Prior research has illuminated the impact of primer coverage on accurate microbial diversity assessment . Primer surement Table 1.surement . Howeversurement .In conclusion, absolute quantification data is heavily influenced by primer choice, necessitating caution when comparing abundance data across studies unless identical primer sets are employed . MoreoveWhile this study provides valuable insights, few limitations need to be acknowledged. The relatively small sample size restricts definitive conclusions regarding significance. Furthermore, factors impacting the microbiome, such as diet, smoking, oral inflammation, and oral hygiene, were not controlled for prior to sample collection, even though they are important parameters .Veillonella spp., Prevotella spp., and P. salivae in IBD patients compared to healthy controls. Additionally, the study sheds light on the optimal cultivation conditions for P. salivae and the potential role of helper strains in its growth. The implications of helper-dependency, whether self or reliant on P. melaninogenica, warrant further exploration to unravel the clinical significance of P. salivae. Ultimately, our findings highlight the significance of primer coverage, as demonstrated by the broader scope of the 16S rRNA amplicon sequencing primer, leading to a more comprehensive assessment of absolute abundance.Despite these limitations, our findings highlight the higher prevalence of MH: Data curation, Investigation, Visualization, Writing – original draft, Writing – review and editing. GC: Writing – review and editing, Funding acquisition. MA: Funding acquisition, Writing – review and editing, Conceptualization, Data curation, Investigation, Methodology, Project administration, Software, Supervision, Validation, Visualization.


Background: Transcription factors in Foxo signaling pathway influence hepatocellular carcinoma metastasis through epithelial mesenchymal transition-related pathways. Prognostic factors in the Foxo signaling pathway are feasible for HCC prognosis and therapeutic management.Methods: Based on the differentially expressed genes and Foxo signaling pathway genes in HCC, the ConsensusClusterPlus package was conducted to identify Foxo signaling pathway-related molecular subtypes in HCC. Based on the DEGs in the FMSs, the optimal prognostic factors in HCC were screened by cox and least absolute shrinkage and selection operator (LASSO) cox analysis to form the Foxo prognosis score (FPS). The prognostic predictive effectiveness of FPS was assessed by Kaplan Meier (K-M) analysis and Receiver Operating Characteristic (ROC) analysis. Additionally, tumor microenvironment (TME) score, tumor mutation burden (TMB) and treatment sensitivity differences in FMSs and FPS groups were also evaluated.Results: There were low, medium and high Foxo signaling pathway activity molecular subtypes in HCC named FMS 1, FMS 2 and FMS 3, respectively. FMS 1 with lowest Foxo signaling pathway activity presented an excellent survival advantage, while FMS 3 with highest Foxo signaling pathway activity exhibited an inhibitory TME status. According to FPS grouping, low FPS exhibited favorable survival, low TMB and anti-tumor activity. Patients in the low FPS group were mostly in the early stage of cancer. Moreover, we found that patients with high and low FPS exhibited different sensitivity to chemotherapy, and patients with low FPS were more sensitive to immunotherapy.Conclusion: We revealed a novel molecular subtype and prognostic tool based on Foxo signaling pathway signature, which could potentially provide a direction for accurate and effective assessment of potential personalized treatment options and prognostic management for HCC patients. Hepatocellular carcinoma (HCC) is the most prevalent pathological type of liver cancer, with 90% of liver cancer cases being HCC . AccordiThe transcription factors in the Foxo signaling pathway, FOXO1 and FOXO3, contributed critical proteins to the development of malignant progression of HCC . In HCC,In this study, we integrated high-throughput sequencing data of HCC from multiple databases  to conduct a comprehensive theoretical analysis. The intention of this study was aimed to establish molecular subtypes of Foxo signaling pathway activity in HCC, followed by an attempt to construct the prognostic scoring system for HCC based on the specialized genes in the subtypes. To our knowledge, this is the first time that Foxo signaling pathway was studied in the prognosis of liver cancer.https://portal.gdc.cancer.gov/) database. This study was conducted using the HCC samples cohort in TCGA as the training set. To validate the results of this investigation, three additional datasets were accessed as validation sets. RNA-Seq data for HCC sequencing researches were accessed from HCCDB (http://lifeome.net/database/hccdb/home.html) (project name: ICGC-LIRI-JP) and GENE EXPRESSION OMNIBUS (GEO) database (https://www.ncbi.nlm.nih.gov/gds) (registration number: GSE14520 (https://www.genome.jp/kegg/) , respecp/kegg/) .Clinical data and RNA-Seq data of samples in TCGA-LIHC, ICGC-LIRI-JP, GSE14520 and GSE76427 were imported into the sangerbox database  for the p < 0.05). Overlapping genes in prognosis-related genes in DEGs and Foxo signaling pathway were extracted, and the consistency clustering analysis was performed based on the expression matrix of overlapping genes. The ConsensusClusterPlus R package (k = 2–10. Foxo Molecular subtypes (FMS) in HCC were determined according to the consistency matrix and consistency cumulative distribution function for each k-value range.In TCGA-LIHC, the limma package  was perf package  was perfThe limma package was used to perform differential analysis to identify DEGs in each FMS according to the gene expression matrix in the different FMSs. The DEGs were imported into the STRING database  to constBased on the gene expression matrix in MCODE 1, the coxph function in the survival package  was perfThe FPS of samples in TCGA-LIHC was calculated according to the FPS evaluation equation. Samples with FPS>0 was defined as high FPS group and samples with FPS<0 was defined as low FPS group. The survival package was performed to perform Kaplan-Meier (K-M) survival analysis and graphed K-M survival curves. The timeROC package  was perfThe somatic mutation data corresponding to HCC samples in TCGA-LIHC were also sourced from the TCGA database. The Maftools package (version: 2.8.05)  was deplFor HCC samples in TCGA-LIHC, multiple tumor microenvironment (TME) infiltration algorithms were conducted to assess the immune score or infiltration abundance of infiltrating immune cells in these samples. The CIBERSORT algorithm  was deveThe h.all.v7.5.1.symbols.gmt gene set from MSigDB database was imported from the GSEA website for single sample gene set enrichment analysis (ssGSEA) via the GSVA package. The Hmisc package  was emplhttp://tide.dfci.harvard.edu/login/) (https://www.cancerrxgene.org/) database. pRRophetic package   website. package  was deveAge, Gender, Stage, Grade, and FPS information of HCC samples in TCGA-LIHC were extracted and univariate COX and multivariate COX analyses were performed to identify pivotal independent prognostic parameters in HCC for Nomogram construction. Meanwhile, the calibration curves were performed to assess the prognostic predictive value of Nomogram based on the 1-year, 3-year, and 5-year survival rates of HCC predicted by Nomogram versus the actual survival rates of recorded HCC. Additionally, Decision curve (DCA) was graphed for assessing the clinical predictive value of FPS, Nomogram based on its information.http://sangerbox.com/home.html) also assisted in data processing. p value < 0.05 was treated as statistically significant.All statistical analyses were performed using R software (version 4.0.3). Moreover, Sangerbox (p < 0.05)  . Subsequ < 0.05) . 19-gene < 0.05) . Finally < 0.05) .k = 3, the clustering of HCC patients exhibited excellent consistency with low interference between samples . When  samples . Therefo samples . In TCGA samples . In TCGA samples .According to CIBERSORT results, B cells naïve, T cells CD4 memory resting, Monocytes, Macrophages M1, Mast cells resting expressed high infiltration abundance in FMS 1; T cells regulatory (Tregs), Macrophages M0 expressed high infiltration abundance in FMS 3 . DependiTo explore the prognostic differences and TME activity differences, differential analysis was performed in FMS 1-3 to construct PPI networks to explore the protein regulatory network differences among FMSs. Firstly, differential analysis was performed. 576 DEGs were presented in FMS 1, consisting of 188 upregulated expression DEGs and 388 downregulated expression DEGs; 2 upregulated expression DEGs were presented in FMS 2; 1,048 DEGs were presented in FMS 3, consisting of 658 upregulated expression DEGs and 390 downregulated expression DEGs. 1,108 overlapping DEGs were presented in FMSs, and these genes were included in STRING to construct PPI network, and 717 genes were included in PPI network with confidence score >0.7 . AccordiIn TCGA-LIHC, 92 prognosis-related genes were identified by univariate COX analysis, we found that these genes were all Risk genes. According to the trajectory diagram of the change of independent variable coefficients in LASSO COX analysis, the interference of similar genes in the model was minimized at lambda = 0.0172, and the model fit was minimized at this time, when 16 genes were included in the model . FinallyIn TCGA-LUAD, the clinicopathological statistical information, 9-gene expression level, and FPS distribution of HCC samples were demonstrated in To observe the connection between FPS and biological functions, the spearman correlation between ssGSEA scores and FPS was evaluated for each pathway in the h.all.v7.5.1.symbols.gmt gene set. We found that FPS exhibited remarkably negative correlations with metabolism-related pathways and remarkably positive correlations with cell cycle-related pathways . FurtherThe immune cell infiltration characteristics in HCC samples in FPS grouping were then characterized. According to the CIBERSORT results, T cells regulatory (Tregs), Macrophages M0 exhibited higher infiltration abundance in the high FPS; B cells naïve, T cells CD4 memory resting, Mast cells resting exhibited higher infiltration abundance in the low FPS group . FPS exhUnivariate COX and multivariate COX analyses were performed in TCGA-LIHC by integrating information on Age, Gender, Stage, Grade, and FPS. We identified FPS and Stage as independent clinical prognostic factors for HCC . TherefoHCC remains a major cause of cancer death progressing rapidly . Inhibit+ T cells being depleted or their function being suppressed  family, plays an important role in the deacetylation of FOXO and regulation of autophagy in cells . Based oNotwithstanding original molecular subtype and prognostic tool based on Foxo signaling pathway signature was developed for HCC patients, some limitations need to be solved for future applying. First of all, more clinical statistics are necessary to calibrate the model. Secondly, 9 gens selected for FPS riskscore signature requires more studies on mechanism to confirm their roles as biomarkers for HCC prognosis.Overall, we defined novel molecular subtypes in HCC in accordance with genes in the Foxo signaling pathway, providing new research perspectives in the study of HCC tumor heterogeneity. In this study, we also constructed the HCC prognostic scoring system for Foxo signaling pathway activity characteristics based on DEGs in FMSs, which demonstrated excellent robustness in assessing HCC prognosis, immune activity, and mutational status. Encouragingly, the FPS also exhibited excellent and promising outcome in guiding HCC drug selection. The clinical value of FPS was not further validated in a large sample of clinical cases. Further prospective trials are needed to investigate the prognostic value and therapeutic guidance of FPS in the future.


Sonchus alliance, a spectacular example of adaptive radiation with six genera and approximately 31 species, is found exclusively on three Macaronesian Islands  in the Atlantic Ocean. Four of the Sonchus taxa are restricted to Madeira, including shrubs and small trees at higher elevations (S. fruticosus and S. pinnatus), and caudex perennials in the lower coastal areas (S. ustulatus subsp. maderensis and S. ustulatus subsp. ustulatus). The Madeiran Sonchus stemmed from a single colonization event that originated from the Canaries < 3 million years ago. However, the plastome evolution and species relationships remains insufficiently explored. We therefore assembled and characterized the plastomes of four Sonchus taxa from Madeira and conducted a phylogenomic analysis. We found highly conserved plastome sequences among the taxa, further supporting a single and recent origin. We also found highly conserved plastomes among the cosmopolitan weedy Sonchus, Macaronesian Sonchus in the Atlantic, and Juan Fernández Islands Dendroseris in the Pacific. Furthermore, we identified four mutation hotspot regions  and simple sequence repeat motifs. This study strongly supports the monophyly of Madeiran Sonchus. However, its relationship with the remaining woody Sonchus alliance from the Canary Islands requires further investigation.The woody  Drosophila   in Australia, and Sonchus novae-zelandiae (Hook.f.) Garn.-Jones  and S. grandifolius Kirk  in New Zealand, as well as the woody Sonchus alliance in the Macaronesian Islands. The subgenus Dendrosonchus, also known as the woody Sonchus alliance, consists of approximately 31 species and is found in Macaronesia, mainly in the Canary Islands, Madeira archipelagos, and Cape Verde. However, S. pinnatifidus is found on both the Canary Islands and Morocco. They are endemic to the Canary Islands with the exception of five species, among which S. daltonii is endemic to the Cape Verde, and the remaining four species are endemic to the Madeira archipelagos . S.. S.Sonchhipelago . SonchusSonchus and related genera in the subtribe Hyoseridinae, we characterized several herbaceous and woody Sonchus species [Sonchus alliance have been characterized with special emphasis on species from the Canaries [Sonchus based on cpDNA is also lacking. Therefore, this study aimed to: (1) determine the complete plastomes of four Madeira-endemic Sonchus taxa; (2) compare their gene content, order, and any changes in organization; (3) conduct comparative genomic analyses to identify highly variable hotspot regions and chloroplast simple sequence repeat (cpSSR) markers; and (4) elucidate the phylogenetic relationships among the four Madeira Sonchus taxa and assess their relationships with the Canary Islands congeneric taxa.The advent of next generation sequencing (NGS) allowed for the rapid assembly and characterization of whole plastid genome sequences in numerous land plant lineages. These plastome resources have proven useful in resolving difficult and obscure phylogenetic relationships and developing efficient plastid markers for DNA barcoding and phylogeographic and phylogenetic studies . In an o species –22. In pCanaries ,21. HoweSonchus taxa were collected freshly from the island . Sonchus fruticosus’s DNA was extracted using the CTAB method [One the 2002 expedition to Madeira, four endemic ation in ). The toB method . The conde novo genomic assembler Velvet 1.2.10 [S. pinnatus, we performed PCR confirmation for three ambiguous regions  to assemble the circular plastid genome. PCR gap filling was performed using the Inclone™ Taq DNA polymerase kit , with a final volume of 50 μL. The PCR product was purified using the Inclone™ Gel & PCR Purification Kit  and sequenced at Macrogen Corporation .An Illumina paired-end (PE) genomic library was constructed and sequenced using the Illumina HiSeq platform, according to the standard Illumina PE protocol in the TruSeq Nano DNA Sample Preparation Guide. The sequence reads were assembled using the t 1.2.10 . In the http://mbio-serv2.mbioekol.lu.se/ARAGORN/; [http://lowelab.ucsc.edu/tRNAscan-SE/; [S. canariensis (NC042381), S. acaulis (NC042382), S. webbii (NC042383), and S. leptocephalus (MN334533) using Geneious Prime 2020.0.3 . Each circular genome map was visualized using OGDRAW v. 1.3.1  were annotated using CPGAVAS2 , and tRNRAGORN/; ) and tRNcan-SE/; ). RNAmmecan-SE/;  was usedmpg.de/; ) and MPISonchus species and analyzed the DNA polymorphisms using DnaSP v. 6 [Pi) with a 100 bp window length and a 25 bp step size. We executed the LAGAN alignment mode using the mVISTA program  to aftware/; ), and gehttp://pgrc.ipk-gatersleben.de/misa/) was used to detect simple sequence repeat (SSR) motifs with repeat units ranging from one to six nucleotides [MIcroSAtellite identification software . Thirty-five of the total CDSs were analyzed as 52 plastid genes were not supported by the PREP-Cp. We calculated the Ka/Ks ratio of each pair in Madeira Sonchus using DnaSP v. 6 [The codon usage bias was calculated using MEGA-X . We compnl.edu/; ), with daSP v. 6  to evaluSonchus species, we selected a total of 18 complete chloroplast genomes of the subtribe Hyoseridinae (formerly known as Sonchinae). Reichardia ligulata (MN893255), which diverged early in Hyoseridinae, was used as the outgroup. We selected herbaceous Sonchus spp., including S. boulosii (NC042244), S. arvensis (NC054161), S. oleraceus (MK371006), S. asper (MK371015), and S. brachyotus (MT850047). Moreover, we included five previously sequenced the woody Sonchus alliance taxa from the Canary Islands, namely S. bupleuroides (MK371017), S. webbii (NC042383), S. leptocephalus (MN334533), S. acaulis (MK033507), and S. canariensis (MK033506). Finally, we included seven Dendroseris species, which are endemic to the Juan Fernández Islands in the Pacific Ocean and belong to the Hyoseridinae, namely D. berteroana (NC051923), D. pinnata (NC051926), D. marginata (NC051925), D. litoralis (NC051922), D. macrantha (NC051924), D. micrantha (NC051921), and D. pruinata (NC051920). We aligned the chloroplast genomes using MAFFT v. 7 with default settings and edited them manually using Geneious software. A maximum-likelihood (ML) tree and a Bayesian tree were inferred using IQ-TREE v. 1. 6.12 [To determine the phylogenetic position and relationships of four Madeiran raceus MK1006, S. bii NC0423, S. lep 1. 6.12  implemen 1. 6.12 . The defS. fruticosus and S. pinnatus), 152,411 bp (S. ustulatus subsp. ustulatus) to 152,426 bp (S. ustulatus subsp. maderensis) (S. pinnatus) to 2770 (S. fruticosus). The sizes of the LSC, SSC, and IR were nearly identical among the four plastomes  contained a single intron and three genes  contained two introns  was slightly longer than the others (471 bp).Each Madeira chloroplast genome was composed of 131 CDSs, 87 genes, 37 tRNAs, and six rRNAs . The toterensis) , which alastomes . In partositions . Seven g introns . We foun Islands . The MadSonchus plastomes and found a similar total number of SSRs  to 22,776  among the four Madeira Sonchus taxa, with consistent patterns of frequently used codons among them  based on the protein-coding genes revealed an average codon usage ranging from 22,774  to thymine (T) transitions. The most frequent transitions were conversions from proline (P) to leucine (L).We also predicted RNA editing sites in the Madeira e of 0.8 . These eSonchus alliances on the Macaronesian Islands  using DnaSP  in the LSC, and one protein-coding (ycf1) and one intergenic region (ndhF-ψycf1) in the SSC  when only Madeiran species plastomes were considered  region, followed by the SSC (23.2%) and IR (1.7%) regions.The mVISTA plots, with lastomes . We founlastomes . Approxiand ycf1 . The mosSonchus alliance in the Macaronesian Islands (100% BS and 1.0 posterior probability (PP)) . The two subspecies of S. ustulatus, namely subsp. ustulatus and subsp. maderensis, are not in a monophyletic clade. Rather, subsp. ustulatus shares a more recent common ancestor with S. pinnatus and S. fruticosus than with conspecific subsp. maderensis (The topologies of the ML tree and the Bayesian inference (BI) tree were identical, and both strongly suggested the monophyly of the woody ty (PP)) . The MP derensis .Sonchus. The Madeira Sonchus species  had slightly larger plastomes than the weedy Sonchus  and the Canary Islands Sonchus species . However, the gene content and organization were highly conserved among the groups [Sonchus alliance from Madeira and the Canaries had slightly larger plastomes than the continental herbaceous congeneric species. In addition, very similar plastome sizes , gene content, and organization was observed between the Atlantic Ocean diploid Sonchus endemics and the Pacific Ocean Juan Fernández Islands tetraploid Dendroseris, which belongs to the same subtribe Hyoseridinae as Sonchus [We found highly conserved plastomes based on their size, gene content, order, and organization between the four Madeira e groups ,21. The Sonchus  as the weedy eraceus; ) and the webbii; ), slight species ,21. We fdroseris . Howevernsistent . The cod8 sites; ) and mos4 sites; ). In gen studies –48. Two  Sonchus  and Maca Islands ,14,49.Sonchus alliance in the Macaronesian Islands. Although the current study was based on a limited number of species, it is most likely that the woody Sonchus alliance from the Macaronesian Islands was derived from a common ancestor, as previously reported [Sonchus was strongly supported in every ML, BI and MP analysis based on the complete plastome sequences. The monophyly of the Madeira clade and interspecific relationships were weakly supported based on the nrDNA ITS  and few chloroplast regions . One novel finding of this study is that the two conspecific subspecies of S. ustulatus are not closely related. Rather, S. ustulatus subsp. ustulatus shares a more recent common ancestor with S. pinnatus and S. fruticosus than with conspecific S. ustulatus subsp. maderensis. The previous ITS tree weakly suggested that two subspecies from coastal areas, without forming monophyly, represent basal lineages that first diverged within Madeira [The phylogenetic tree shows the monophyletic clade of the woody reported ,14,41. USonchus suggests that two currently recognized subspecies of S. ustulatus may represent distinct taxonomic entities, warranting recognition at the species level. Sonchus ustulatus subsp. maderensis was proposed by Aldridge [Sonchus ustulatus subsp. maderensis occurs in moist rocky areas on the northern coast of Madeira (and rarely in Porto Santo and Desertas). In contrast, S. ustulatus subsp. ustulatus occurs in dry rocky and sunny areas on the southern coast of Madeira. These taxa are small, herbaceous perennials with acaulous or short, subwoody stems that can reach up to 30 cm in height. In contrast, S. pinnatus and S. fruticose are perennial shrubs that reach heights of up to 2 m and 4 m, respectively. Therefore, given the species distribution and chloroplast phylogenomic tree topology, it is highly likely that the common ancestor of the Madeiran Sonchus was somewhat similar to S. ustulatus. These were likely herbaceous perennials that first colonized the northern coastal areas of Madeira and diversified into laurel forests and moist ravines in the interior of Madeira, becoming tall shrubs. The species-level recognition of the two subspecies is further supported by a recent report of a new species, S. parathalassius J.G.Costa ex R.Jardim & M.Seq., from Porto Santo and the taxonomic recognition of S. latifolia (= S. ustulauts subsp. maderensis) [The plastome-based phylogenetic analysis of Madeira Aldridge , based oerensis) . OverallSonchus was fully and strongly established in this study, but that of the closely related or progenitor Sonchus species from the Canary Islands remains unclear. Based on the limited Canary Islands Sonchus samples in this study, the plastome tree suggested that S. acaulis and S. canariensis are sisters to the Madeira clade, suggesting a possible origin from Tenerife and Gran Canaria. We gained insufficient insight into this using previous cpDNA phylogeny because of a lack of resolution within subg. Dendrosonchus and genus Taeckholmia [Sonchus as a sister to the clade of some Canary Islands Sonchus from various islands, including the older central islands  and relatively younger western islands . It makes the identification of the ancestral island and species of origin of Madeira Sonchus uncertain. Nevertheless, it was suggested that the origin of Madeira Sonchus happened relatively recently , despite the geological age (ca. 5 million years ago) of the Madeira archipelago [Sonchus alliances in the Macaronesian Islands, is required to further determine the geographical origin of the Madeira Sonchus species.The monophyly of the Madeira ckholmia . The morhipelago . An exteSonchus species, enabling the comparison of their genomes to those of the congeneric cosmopolitan Sonchus, the woody Canary Islands endemic Sonchus from the Atlantic Ocean, and the Juan Fernández Islands endemic Dendroseris in the Pacific Ocean. Although the four Madeira endemic Sonchus species are highly morphologically and ecologically differentiated, we found nearly identical and highly conserved chloroplast genomes, supporting a single and recent origin. We identified four mutation hotspots, namely trnK-rps16, petN-psbM, ndhF-Ψycf1, and ycf, and 70–71 cpSSRs for population genetics, phylogeography, and phylogenetic investigation. We also characterized the codon usage bias and RNA editing sites among the four Madeira taxa and compared them with those of the Canary Islands Sonchus and the Juan Fernández Islands Dendroseris. The plastome-based phylogenetic tree strongly supported the monophyly of the Madeira lineage for the first time, but its relationship with the Canary Islands congenerics requires further study based on genome-wide nuclear and chloroplast genomes. The phylogenomic tree also suggests that the herbaceous perennial, S. ustulatus subsp. maderensis, which occurs on the northern coast of Madeira, was the first to diverge within the Madeira archipelago. Lastly, S. ustulatus subsp. ustulatus, which are herbaceous perennials occurring on the southern coast of Madeira, are more closely related to the clade of perennial shrubs, S. pinnatus and S. fruticose, than to conspecific S. ustulatus subsp. maderensis. This relationship corroborates the species-level recognition of S. ustulatus subsp. maderensis as a distinct species, S. latifolia. Further genome-wide investigations may clarify the relationship among Sonchus species within Madeira.This is the first study to report the complete chloroplast genome of four Madeira S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


Withania somnifera L. and its safety applications in CRC management. Important databases were rigorously searched for relevant literature, and only 82 full-text publications matched the inclusion requirements from a massive collection of 10,002 titles and abstracts. W. somnifera L. contains a high concentration of active plant-based compounds. The pharmacological activity of the plant from our study has been demonstrated to exert antiproliferation, upregulation of apoptosis, decrease in oxidative stress, downregulation of cyclooxygenase-2 (COX-2), induction of targeted cytotoxic effects on cancerous cells, and exertion of both antiangiogenesis and antimigratory effects. We advise further research before recommending W. somnifera L. for clinical use to identify the optimal concentrations required to elicit beneficial effects in CRC management in humans, singly or in combination.Research into tumorigenic pathways can aid in the development of more efficient cancer therapies and provide insight into the physiological regulatory mechanisms employed by rapidly proliferating cancer cells. Due to the severe side effects of cancer chemotherapeutic medications, plant chemicals and their analogues are now explored more frequently for the treatment and prevention of colorectal cancer (CRC), opening the stage for new phytotherapeutic strategies that are considered effective and safe substitutes. Our study aimed to evaluate the medicinal properties of  Withania somnifera (L.) (W. somnifera (L.)) belongs to the kingdom Plantae (plants), subkingdom Tracheophytes (vascular plants), division Angiospermae, class Eudicots, clade Asterids, order Solanales, family Solanaceae, subfamily Solanoideae, tribe Physaleae, genus Withania, and species somnifera, according to the biological classification system [W. somnifera L. is a Solanaceae plant commonly called “ashwagandha” in Hindi/Sanskrit and is a small shrub growing in abundance in the subtropics. The leaves and roots are used in Ayurveda, the Indian traditional medicine [n system ,2,3. W. medicine . The aermedicine . It is amedicine . Flowersmedicine , and aremedicine .W. somnifera Dunal and W. somnifera Kaul have been identified as two subspecies [Withania species W. somnifera and W. coagulans are both widely farmed [W. somnifera cultivars utilized in Sri Lanka, although the Indian cultivar is better suited for drug research due to its starchy character, whereas the local species has fibrous roots that are challenging to powder [Withania somnifera L. [Additionally, bspecies . In Indiy farmed . There ao powder . The leao powder . It is go powder . The trao powder . The Ayuifera L. .Cancer is the second-largest cause of death, trailing only cardiovascular disease. Colorectal cancer is now the third-biggest cause of cancer-related deaths in Western nations . Cancer Understanding tumorigenic pathways can help researchers create more effective cancer treatments and gain insight into the metabolic control mechanisms used by swiftly reproducing malignant cells . Even thThe extracellular matrix, which includes the stromal and immune cells it surrounds and the chemical compounds and signaling substances in the intercellular space, constitute the tumor microenvironment (TME) . A TME mDue to the severe side effects of these chemotherapeutic medications, plant chemicals and their analogues are now being used more frequently in both the treatment and the prevention of CRC . ChemothW. somnifera (L.) uses has not been fully understood and established [Withania somnifera L. and its safety application in CRC management. This updated review presents the plant as a viable alternative with favorable benefits in CRC therapeutic management. As it is, however, there is little information available on this significant plant species, particularly in in vivo and in vitro CRC experimental trials. Therefore, this is the first comprehensive review evaluating the proactive potential for W. somnifera (L.) in the treatment of CRC in humans.Despite its lengthy history of therapeutic uses, its medicinal benefit has garnered less attention. The technique that ablished . Our stuW. somnifera species of plants against CRC. To identify primary studies examining the effects of W. somnifera on CRC carcinogenesis, relevant literature was comprehensively searched in the Web of Science, Ovid, BMC, Springer, Elsevier, MDPI, and MEDLINE databases. Google Scholar was useful in acquiring more articles. This exercise was conducted in adherence to PRISMA guidelines [This study used an in-depth systematic review technique to assemble data on botanical descriptions, ethnomedical uses, plant-based compounds, and pharmacological activities of idelines  but withidelines ,29. Withania somnifera L. efficacious in colon cancer management?” “Are active compounds of Withania somnifera L. safe for human therapeutic application?” After setting up the review questions, the authors individually examined only English research titles and abstracts from primary sources [To make the search criteria easier, review questions were created. Two specific questions were set up: “Are the active compounds of  sources ,31. Rese sources ,31. OnlyWithania somnifera.A systematic search criterion was created to ensure that the screening method was effective. The MeSH terms “growth”, “proliferation”, “development”, “anti-cancer”, “bioactivity”, “phytochemicals”, “physiological activity”, “pharmacological activities”, and “multiplication” were combined with either “colorectal”, “colon cancer”, “adenocarcinomas”, “polyps”, “colorectal tumor”, “colon tumor”, keywords, or their synonyms. These terms were ultimately combined with the plant species under review, Withania’s biochemistry has been thoroughly investigated, and several distinct metabolites have been identified and their biological activities described , interleukin (IL)-10, and glutathione (GSH) levels are all lowered by aqueous root extracts of W. somnifera L. (0.05–0.4 mg/mL), which also regulate peripheral blood mononuclear cell (PBMC) and leukemic THP-1 cell viability [Through gene silencing and pathway analysis, Widolo et al.  reportediability .Withaferin A suppressed IL-6, COX-2, TNF-α, and prosurvival biomarkers  in APC Min/+ and dextran sodium sulfate/azoxymethane models and prevented CRC in both models. The treatment of withaferin A reduced cancer proliferation in a transgenic adenomatous polyposis coli (APCMin/+) mice model . W. somnifera L. extract on HT-29 colon cells was investigated. The investigators found that methanolic extract of W. somnifera L. root had an antiproliferative effect due to increased ROS generation and mitochondrial dysfunction [In the CRC cell lines SW-480, SW-620, and HCT-116, withaferin A suppressed Notch-1 and inhibited the Akt/NF-B/Bcl-2 pathway. In colon cancer cells, withaferin A inhibited the rapamycin signaling factors p4E BP1 and pS6K and stimulated c-Jun-NH2-kinase-mediated apoptosis . It has W. somnifera (L.) extract has been demonstrated to improve immunological dysfunction and CRC rehabilitation [Alnuqaydan et al.  investiglitation . The immlitation . The dowlitation ,58,59 anlitation  in CRC. litation ,53,54,60When treating oncologic disorders, the impact of anticancer medications on healthy cells is a major source of worry. Researchers have undertaken very little research to fully grasp this significant problem. Withanolides have been investigated for their potential cytotoxicity toward normal cells as well as their powerful anticancer properties , includiW. somnifera (L.) did not exhibit any toxicity at a concentration of 2000 mg/kg, according to a specific acute and subacute toxicity investigation [W. somnifera L. roots. The W. somnifera L. root extract did not have any maternal or fetal toxicity in mice, according to an investigation on gestational developmental adverse effects [A combination of withanolide A and withaferin A offers multimodal activities against CRC cell lines. These chemical components are reported to trigger the cytotoxicity of cells in various cancerous cell lines . The protigation ,63. Anottigation  reported effects . W. somnifera L. extract’s antiproliferative efficacy additionally demonstrated lower cytotoxicity in normal cells as compared with DU145 prostate cancer cells [W. somnifera L. extract on normal Vero cells compared with the human MDA-MB-231 breast carcinoma cell line [According to research by Baig et al. , withanoer cells . Similarell line . Withafeell line . Withania and its numerous species have also been examined for their harmful effects [Withania somnifera L. roots were shown to be effective and safe. It has been demonstrated to be nontoxic and effective clinically for human health and wellness, from its use in antiquity to its present applications [ effects . Accordiications .Apoptosis is a mechanism that has undergone little change throughout evolution and is crucial for maintaining tissue homeostasis . The proThe mitochondrial caspase pathway’s starter, caspase-9, is a crucial mediator in the control of apoptosis. These enzymes (caspases) are part of a cascade that is initiated by proapoptotic commands, which leads to the cleavage of an assortment of peptides and the disintegration of the cell. The comprehension of caspase programming is essential for strategically regulating apoptosis for therapeutic benefit ,72. The W. somnifera L. roots could stimulate mitochondria-mediated apoptosis in triple-negative breast cancer cells (MDA-MB-231) at an IC50 dose of 92 g/mL. Reported also [It has been suggested that the activity of withanolides is mediated via the control of nuclear factor kappa B (NF-kB) expression because NF-kB regulates numerous genes that affect cell division, malignancy, cancer metastases, and inflammation. This suggests that withanolides prevent NF-kB activation and NF-kB-regulated gene expression, which could help clarify why they can increase apoptosis while preventing invasion . Accordited also  were casted also . W. somnifera L. ethanolic extract is cytotoxic (99.7 g/mL) on cancerous cells, induces apoptosis, and inhibits angiogenesis and cell migration [W. somnifera L. has been shown to be due to the increased autophagy induction and apoptotic effects of the plant. Withaferin A causes apoptosis and Mad2 and Cdc20, crucial components of the spindle checkpoint complex, which are degraded by proteasomes. By restoring correct anaphase initiation and maintaining a greater number of viable cells, further overexpression of Mad2 partially reverses the harmful effect of withaferin A. It is hypothesized that withaferin A kills cancer cells by delaying the mitotic exit and then causing chromosome instability [W. somnifera L. compositions have highly promising anticancer properties in both in vitro and in vivo applications, there are currently no authorized therapeutic candidates. For the discovery of novel anticancer pharmaceuticals, conducting clinical trials with W. somnifera L. phytochemicals/formulations is urgently necessary.The association between caspase-9 and CRC is still poorly understood to date. Studying its association with clinicopathological characteristics and longevity may provide insightful data for predicting survival and choosing additional treatment strategies. igration . The postability . AlthougAngiogenesis is essential for the survival, development, and metastasis of cancer cells. Sajida  demonstrW. somnifera L. extract has been implicated with significantly reducing cell migration, thus demonstrating its antimigratory ability [Withania somnifera L. decreased the levels of the migration-promoting proteins hnRNP-K, VEGF, and MMP, making them potential alternative therapies for the management of metastatic cancer [Additionally, withaferin A demonstrated antiangiogenic efficacy in the mouse model with reduced p-ERK and p-Akt levels. By reducing the expression of MMP in HeLa and PC3 cells, this molecule also prevented the invasion of cancer cells . At lowe ability . It has c cancer . It has further been elucidated that withanone and withaferin A in combination had powerful antimigratory and antiangiogenic effects in vitro and were specifically lethal to cancer cells. These actions were corroborated by molecular studies of marker proteins, such as MMPs, which are essential for the invasion and metastasis of cancer ,62,82. TWithania somnifera L.’s pharmacological activity has been shown in our study to unquestionably exert antiproliferation, upregulation of apoptosis, decrease in oxidative stress, downregulation of cyclooxygenase-2 (COX-2), induction of targeted cytotoxic effects on cancerous cells, and exertion of both antiangiogenesis and antimigratory effects. Numerous macromolecules found in W. somnifera L. suggest the value of using this plant as a possible anti-CRC agent with the guarantee of phytotherapeutic benefits. W. somnifera L. and its constituent parts kill cancer cells via at least five separate routes, including p53 signaling, GM-CSF signaling, death receptor signaling, apoptosis signaling, and the G2/M DNA damage regulation pathway, as has been reported. However, due to the lack of established quantities and concentration measurements, we advise further research before recommending W. somnifera L. for clinical use to identify the minimal and optimal concentrations/levels required to elicit beneficial effects in the management of CRC in humans, either alone or in combination. Evidence adduced herein demonstrates this plant as an efficacious and safe substitute for human medicinal developments. Finally, it may also be investigated as prospective raw materials for the formulation of conventional CRC pharmaceuticals.


IntroductionHelicobacter pylori (H. pylori) is a bacteria causing chronic stomach infections, influenced by various factors, including host traits and bacterial virulence. It uses both urease-dependent and independent mechanisms to survive acidic gastric environments. Management entails diagnosis, treatment, and eradication verification. Combining drugs is key to overcoming resistance and ensuring bacteria elimination, thus preventing recurrence and complications. H. Pylori eradication mitigates gastric cancer risk and alleviates symptoms. Racial disparities persist despite declining H. pylori and gastric cancer incidence in the United States (US). African Americans (AA) have higher gastric cancer risks than non-Hispanic Whites. Addressing these disparities is crucial to protect high-risk populations.MethodsThis study retrospectively compiled H. pylori infection data from 2009 to 2022, categorized by race. Propensity score matching balanced initial group characteristics before analysis. Chi-squared and odds ratio tests were used on the cohort, with Kaplan Meier and Log Rank methods evaluating disease clearance in ethnic groups. Data were extracted from the Sunrise Electronic Medical Record software, including patient demographics, health details, and treatment specifics. Patients aged 18-65 with H. pylori infection at Nassau University Medical Center, who followed their treatment, were selected. Data were processed using Statistical Package for the Social Sciences (SPSS) and RStudio software.ResultsThe study initially included 10,040 H. pylori-diagnosed patients, with 9,288 meeting the study's criteria after attrition. Predominantly female (64.7%), the cohort was racially diverse. A longer disease clearance time was noted among Hispanics (p=0.044). Binomial logistic regression analysis identified influential factors like high school graduation rates, poverty level income, and language proficiency on disease clearance. An odds ratio analysis further emphasized language barriers  and education status  as primary covariates impacting disease clearance, underlining the role of socio-economic factors and language proficiency in health outcomes.ConclusionThe study highlights racial disparities in H. pylori clearance rates, particularly among Hispanics, necessitating culturally sensitive interventions. It advocates for improved diagnostics, increased healthcare access, and social determinants of health-focused initiatives. It identifies socio-economic status and language proficiency as key factors impacting health outcomes, calling for actions to bridge these disparities. Addressing these differences can decrease healthcare inequalities and economic burden, improving overall health outcomes and reducing costs associated with H. pylori clearance. Helicobacter pylori (H. pylori) is a gram-negative, helical-shaped bacterial pathogen known to cause chronic infections in the human gastric epithelium . The facThis paper was accepted for presentation at Digestive Disease Week (DDW) conference 2023 in Chicago.Study designInformation related to H. pylori infection was retrospectively compiled from June 2009 to June 2022, with patients sorted by their race. Prior to the result analysis, the initial characteristics of each group were balanced via propensity score matching, ensuring a fair comparison. A chi-squared test and odds ratio analysis were conducted across the entire cohort. The Kaplan Meier survival analysis and Log Rank (Mantel-Cox) model were used to study disease clearance among ethnic groups. Moreover, this study aids in unearthing potential inequities in healthcare accessibility or differing health outcomes across varied racial and ethnic groups.Data sourceData were collected retrospectively using the Sunrise Electronic Medical Record software . The data collected included detailed information such as age, race, gender, socio-economic status, education level, length of treatment, medication, alcohol abuse, liver disorders, upper GI bleeding, and comorbidities. This information was obtained using Current Procedural Terminology (CPT) 10 codes, including K92.2, K29.71, I86.4, I10, B96.81, 0W3P8ZZ, 0W3P7ZZ, D64.9, K70.3, K74.4, K74.5, K25.9, E08, E78.5, and K21.9.Inclusion and exclusion criteriaThe study selected all patients between the ages of 18 and 65 who were diagnosed with H. pylori infection at Nassau University Medical Center in East Meadow, New York, between 2009 and 2022. However, patients who did not maintain their follow-up appointments and those who did not complete their prescribed antibiotic therapy were omitted from the study. This exclusion likely guaranteed that any unidentified or unmeasured comorbidities that could impact patient outcomes did not skew the study results.Data analysisThe data were processed using the Statistical Package for Social Sciences (SPSS) for Windows  and RStudio software . Categorical data were represented by the number of patients (n), whereas continuous data were displayed as the mean value ± the standard deviation; the p-value for the study was <0.05.The research initially involved 10,040 patients diagnosed with H pylori. However, due to attrition during the follow-up period, the final group comprised 9,288 patients who fulfilled the study's criteria. The patient cohort was predominantly female, accounting for 64.7% , as shown in Table A binomial logistic regression analysis was carried out to determine influential factors affecting the disease clearance process. The results highlighted that certain variables, namely the percentage of high school graduates p=0.012), the percentage of income below the poverty level (p=0.023), and language proficiency (p=0.011), exhibited a significant impact across all demographic groups. Subsequently, an odds ratio analysis was conducted to pinpoint primary covariates that could contribute to an extended time for disease clearance, as shown in Table , the perThe principal covariates identified were language barriers  and education status . These factors were revealed to substantially shape the health outcomes for patients battling H pylori. This finding underscores the considerable influence of socio-economic factors and language proficiency on the health outcomes of individuals affected by this condition.Racial disparities in Helicobacter pylori infectionRecent studies have shed light on the global extent of H.pylori infection, revealing a staggering number of around 4.4 billion affected individuals . Focusing on the United States, a considerable variation can be observed in the prevalence of H. pylori infection across diverse racial and ethnic populations. It has been reported that the infection rate ranges from 18.4% to 26.2% in non-Hispanic white populations . In starAnother point of interest is the comparative prevalence ratios of H. pylori infection across different racial groups. Specifically, the infection ratio for Blacks compared to non-Hispanic whites has been observed to vary from 1.3 to 5.4 [Racial disparities in Helicobacter pylori clearanceResearch indicates that African Americans show lower H. pylori clearance rates compared to other ethnic and racial groups. For example, a US study reported 74% clearance in Caucasians and 68% in Hispanics, compared to only 47% in African Americans post a 7-day treatment. The reasons behind these disparities remain unclear, possibly due to genetic factors or varied host responses to the infection .Treatment failure indicated by non-serologic-based testing includes unsuccessful H. pylori eradication . Around Poor adherence and antibiotic resistance are two major risk factors for eradication failure . Other rPossible solutions to address racial disparities in Helicobacter pylori clearanceAdvancement in Screening and Identification ProceduresInfections caused by Helicobacter pylori are linked to almost half of the cases of gastric body or antrum tumors . The evoCurrently, there are multiple diagnostic procedures for H. pylori infection, ranging from less invasive to invasive testing . The mosConcentrated Interventions to Address Health's Social Determinants & Expanding Access to Healthcare and Treatment OptionsSocial determinants of health are conditions in the environments where individuals are born, live, learn, work, play, worship, and age, influencing healthcare, quality of life, and health risks over a lifetime . These sSocio-economic status (SES) plays a vital role in patient access to healthcare . In the Consequences of racial disparities in Helicobacter pylori clearancePatients with H. pylori infection have a heightened risk of developing gastric adenocarcinoma, barring tumors of the Gastro Esophageal junction. The odds ratio is 3.6 in general, but it surges to 18 in women and 9 in blacks, indicating a particularly pronounced risk in these groups . If not An effective response to this medical challenge is the eradication of H. pylori, which has been found to lower the rates of gastric cancer and its recurrence . EradicaThe study's retrospective design may introduce biases and limit causal inferences. Using data from electronic medical records could result in incomplete or inaccurate information, potentially affecting the analysis. Although propensity score matching was employed to balance group characteristics, unmeasured confounding variables may still influence the results. Additionally, the study's focus on a single medical center may restrict the generalizability of findings to other populations or healthcare settings. The study's observational nature precludes establishing a cause-and-effect relationship between race, socioeconomic factors, language proficiency, and H. pylori clearance.Furthermore, excluding patients who did not follow their treatment plan may introduce selection bias. Despite efforts to control for influential factors, other unexplored variables may impact disease clearance rates among different racial groups. Therefore, a cautious interpretation of the study's findings is necessary, and further research is warranted to comprehensively address racial disparities in H. pylori clearance and improve health outcomes.This study underscores the racial disparities in H. pylori clearance across diverse demographic groups. The results revealed a prolonged disease clearance time, particularly among Hispanics, emphasizing the need for culturally competent interventions. This necessitates improvements in diagnostic procedures, broader access to healthcare, and robust interventions addressing the social determinants of health. Crucially, socio-economic factors and language proficiency were found to significantly impact health outcomes in H. pylori-infected patients, warranting initiatives to bridge racial, ethnic, and financial disparities. Addressing these disparities is not only essential for reducing healthcare inequalities but also holds significant implications for the economic burden on the healthcare system. Therefore, a concerted effort toward understanding and addressing these racial disparities can improve health outcomes and reduce the overall economic cost associated with H. pylori clearance.


Entomotherapy, the use of insects for medicinal purposes, has been practised for centuries in many countries around the world. More than 2100 edible insect species are eaten by humans, but little is known about the possibility of using these insects as a promising alternative to traditional pharmaceuticals for treating diseases. This review offers a fundamental understanding of the therapeutic applications of insects and how they might be used in medicine. In this review, 235 insect species from 15 orders are reported to be used as medicine. Hymenoptera contains the largest medicinal insect species, followed by Coleoptera, Orthoptera, Lepidoptera, and Blattodea. Scientists have examined and validated the potential uses of insects along with their products and by-products in treating various diseases, and records show that they are primarily used to treat digestive and skin disorders. Insects are known to be rich sources of bioactive compounds, explaining their therapeutic features such as anti-inflammatory, antimicrobial, antiviral, and so on. Challenges associated with the consumption of insects (entomophagy) and their therapeutic uses include regulation barriers and consumer acceptance. Moreover, the overexploitation of medicinal insects in their natural habitat has led to a population crisis, thus necessitating the investigation and development of their mass-rearing procedure. Lastly, this review suggests potential directions for developing insects used in medicine and offers advice for scientists interested in entomotherapy. In future, entomotherapy may become a sustainable and cost-effective solution for treating various ailments and has the potential to revolutionize modern medicine. InsecChartergellus communis wasp venom in two different breast cancer cell lines (HR+ and triple-negative) showed encouraging results by killing just cancer cells while leaving healthy cells alone [Entomotherapy has been practised in many countries around the world. According to Wigglesworth , many pels alone . Blisterls alone . Accordils alone , these bIn this review, we also discussed many insect species used for medicinal purposes, at which stages these species are utilized, and the impact of these insects on human health. We examine if entomotherapy is met with the same opposition as entomophagy. The earlier ideas of gathering insects, the requirement for industrial manufacturing to create significant amounts of insect-based medication, and what insect mass production would entail are discussed. Our review suggests potential directions for developing insects used in medicine and offers advice for scientists interested in entomotherapy.2Bombyx mori L.), date back to at least 3000 years in China. At the same time, honeybees (Apis mellifera L.) were first recorded during the Xizho Dynasty (about 1100–771 B.C.). Tao Hongjing's “Mingyi Bielu”  expanded “Shennong Bencaojing” to include information on nine additional species of medicinal insects. In his book “Compendium of Materia Medica” (1587), Li Shizhen listed seventy-three different insects used for medical purposes. As a result, 105 bug species were included in the supplementary volume to the “Compendium of Materia Medica” by Zhao Xuemin . According to Robert James, who quoted the Dioscorides, “grasshoppers in a suffumigation relieve under a dysury (difficult micturition), especially as is incident to the female sex”. When insects are bruised and mixed with sugar, they are used to treat ulcers and also serve as dewormers [Kerria lacca Kerr.) has been used as medicines since the 3rd century [Insects in medicine have a long history of application in many societies worldwide by different tribes. Medicinal uses of insects, such as silkworms , Cordyceps sinensis infected Thitarodes armoricanus Oberthür, and Cordyceps hawkesii infected Agrotis ipsilon (Hufnagel).Among those 235 species, 151 were documented with the specific stage or product  used. Adult stage (90 species) was the most documented stage, followed by larvae/nymphs (60), pupae/cocoon (13), and eggs (7). The usage of adults and larvae/nymph are distributed widely among different orders . On the contrary, the usage of eggs  and pupae/cocoon  are limited in two orders, respectively. Besides, fungus infected larvae are only documented in three species in Lepidoptera, which are the Other than insects per se, byproducts from 31 species were documented. Byproducts from species in Hymenoptera are the most documented , for example, bee wax, honey, royal jelly, bee pollen, bee comb, and bee venom from the family Apidae, bee comb from the family Vespidae, and nest from the family Formicidae .3.2https://apps.who.int/classifications/apps/icd/icd10training/). Modern research  that studied medicinal functions with species family documented in the above five summarized literatures [The international classification of diseases system ICD10  is used eratures ,63,65,66The focus on health effects of insects in modern medicine has changed significantly compared to folk medicine. Insects that used to fight against infectious and parasitic diseases (ICD A00-B99) counted ∼36% of the total research, followed by insects promote wound healing (counted ∼17%) and anti-neoplasms . Heatmap analysis a–c showeThe choice of insects in disease research seems to be heavily impacted by the documentary of folk medicine and the market availability. Below is a detailed description of functions mentioned in modern research based on the ICD system with mechanisms  and ingr3.2.1Bacillus sp., Staphylococcus sp., and Proteus sp.), digestive system infection , urinary tract infection , and other infections . Besides, at least 13 species of fungus    of the research focused on antibacterial effects. At least 30 species of bacteria were determined to be inhibited by insect derivatives , includiida sp.) , five viida sp.) , and tenntortus)  were dettidae sp . and the[B. mori .Table 6B3.2.2Eupolyphaga sinensis  (Blattodea: Corydiidae) is another well-known traditional Chinese anti-tumor medicine, from which the extracted polysaccharide [At least 15 types of anticancer activities were documented, including breast cancer, liver cancer, colorectal cancer, lung cancer, ovarian cancer, colon cancer, pancreatic cancer, esophageal cancer, cervical cancer, tongue cancer, bladder cancer, leukemia, murine melanoma, and two types of ascites cancer. Insect-derived ingredients can inhibit tumor cell adhesion , restraiccharide  and a prccharide  have beeccharide  and blowccharide  were det3.2.3Not much research can be classified in this group besides nutritional anemia. Insects are rich in nutrients and have been proved to be effective diet supplements . For exa3.2.4B. mori [p-coumaric from A. mellifera propolis [Anti-hyperlipidemic effects works through energy metabolism balancing [E. sinensis [Vespa basalis Smith (Hymenoptera: Vespidae) [Gryllus bimaculatus De Geer (Orthoptera: Gryllidae) [Bombus ignitus (Smith) (Hymenoptera: Apidae) [The anti-hyperglycemic effect works through advanced glycation end products (AGEs) inhibition , α-glucoB. mori  and epicpropolis  have shoalancing , AMPK/mTalancing , and choalancing , and thealancing . Peptidealancing  and AR-9sinensis  and Mastespidae)  and glycyllidae)  and Bomb Apidae)  have bee3.2.5Polybia paulista Ihering (Hymenoptera: Vespidae) venom was determined to be the active ingredients again anxiety, which may work with the B2-receptors and B1-receptors [B. mori cocoon was determined to have anti-depression effect, which may be due to its antioxidant and estrogenic properties [The bradykinin-related peptide isolated from eceptors . The siloperties .3.2.6The mechanisms of insect neuroprotective effects include antioxidation , anti-in3.2.7E. sinensis [B. mori [p-coumaric) from A. mellifera, and N-acetyldopamine dimers from Oxya chinensis sinuosa Mishchenko (Orthoptera: Acrididae) [Anti-thrombosis works through plasminogen activation and fibrinogen (a major determinant of plasma and blood viscosity) hydrolyzation , FXa inhsinensis , sericin[B. mori , polypherididae) .3.2.8Bombyx batryticatus  and cicada periostracum ) were well known against respiratory disease in traditional Chinese medicine and have been recommended as potential medicines fighting against SARS-CoV-2 [RS-CoV-2 . They heRS-CoV-2 , TRPA1/TRS-CoV-2 , and GATRS-CoV-2  regulati3.2.9B. mori [E. sinensis [Musca domestica L. (Diptera: Muscidae) [P. americana [P. americana gained a lot of attention in digestive system protection, from which the oligosaccharides [P. americana has been developed into a commercial medicine named Kangfuxin solution in China [The inflammation reduction  and antiB. mori , E. sinesinensis , Musca duscidae) , and P. mericana  have beecharides  and an acharides  have beein China .3.2.10B. mori have been used in skin protection for a long history. The cocoon sericin [Besides the antibacterial function described in group A00-B99, the antioxidation and anti-inflammation effects of insects help to reduce psoriasis , dermati sericin , freeze- sericin , and eve sericin  were det3.2.11G. bimaculatus was determined to be an effective ingredient, which produced a significant anti-edema effect [Insects used in rheumatism and arthritis are well-known in traditional medical systems. The anti-inflammation  and antia effect .3.2.12Lucilia sericata (Meigen) is the most used insect in maggot therapy. The excretions and secretions from maggot larvae have shown outstanding effects in wound healing, from which mainly proteins/enzymes , disinfection  and wound healing  is an edible insect with kidney-detoxifying and antiinflammatory properties [The Chinese black ant  to treat inflammatory illnesses by administering a sterile bee sting or injecting a prepared B.V. solution ,257. TheNagaland  a-i and rovinces  j-r [28,o et al.  showed tmericana , to relihe world . In manyhe world . Coughs,he world ,28.Fig. 4.1Eating insects, or “entomophagy,” has evoked a wide range of feelings in people. Many psychological hurdles must be overcome before it can become mainstream because it is commonly held that neophobia and revulsion are the primary psychological factors that people use to reject entomophagy. With insects in medicine many illnesses have been treated with insects and insect extracts in folk medicine . However4.2Acceptance is still a barrier to the widespread use of insects as a medicinal resource for treating diseases and illnesses, especially in developed countries where most people view insects with distaste . However55.1A review of medical use of insects can at least date back to 2000 years ago when the book Sheng Nong's Herbal Classic described multiple medicinal applications of insects . Till noB. mori adult used in medicine should be the newly emerged male with wings and legs removed [Mylabris sp. Beetles is the first month of autumn among the thorns of specific plants  [Rhynchophorus palmarum (L.) (Coleoptera: Curculionidae) larvae collected from the native palm tree Mauritia flexuosa (L.f.) (Arecaceae: Arecaceae) is with the best healing properties [Insects for medicinal purposes differ from insects as food and feed for nutritional purposes in their exact medical effects requirements. Since the nutrients of insects vary even within species , princip removed . Cicadid removed . The besora sp.) . Rhynchooperties .Due to the uncertainty  of wide harvesting, some insects have been domesticated for their commercial value. Insect domestication has at least 7000 years history , for exaC. sinensis is widely used in commercial sale with similar medical function, which has been an effective substitute of wide collection of infected ghost months, Hepialus armoricanus (Oberthür)  [Alternatively, if the effective ingredients have been identified, producing the specific ingredients rather than the insects would be a promising option. For example, the fermentation extract of mycelia from cultivated of ialidae) .5.2The need for insect-based medicine is increasing . The emeC. sinensis infected ghost moth larva is an important anti-cancer Chinese medicine resource. However, the geographic distribution is confined, which is only available in soil of Qinghai-Tibet Plateau with 3500–5000 altitudes [Omphisa fuscidentalis Hampson (Lepidoptera: Crambidae). The traditional harvesting activity usually cut down the entire bamboo clumps which is destructive [The increased need for medicinal insects has led to overexploitation resulting in severe insect population crisis and habitat damage. For example, the ltitudes . Overexpltitudes . Anothertructive .Locusta migratoria (L.) (Orthoptera: Acrididae) is only available in November and December while the Termite is only available from November to next April [Besides, the variation across season is a major challenge in commercialization of wild harvested insects. For example, in Republic of Congo, the migratory locust, xt April . Referrixt April , geograpxt April , types oxt April , and thext April .Mylabris sp. (Coleoptera: Meloidae), which used as an anti-cancer resource, was once determined reaching ∼45 mg/kg resulting in carcinogenic risk [Bacillus sp. and Staphylococcus sp.) associated with foodborne disease have been determined in raw edible grasshoppers, Ruspolia differens (Serville) (Orthoptera: Terrigoniidae), in Uganda [More importantly, wild harvested insects have huge safety concerns. Heavy metal is a big concern nowadays due to civilization pollution. For example, the copper level of the wide harvested nic risk . Pathogen Uganda . Besidesn Uganda .5.3Musca macellaria Fabricius  [Medicinal insects are only small parts of the beneficial use of insects. Besides bees and silkworms, the mass production of sterile screwworm, horidae)  on artifhoridae) . The edihoridae) . Accordihoridae)  after thhoridae) .Though the nutrient and environmental requirements differ by species. There are following issues need to be considered before setting up an insect farm . To set 5.3.1Now most of the edible insects are obtained by trading and a few are wide-collected and reproduced indoor, which the generic diversity is generally uncleared while the concept of insect breeding for food and feed is new . Domesti5.3.2The standard quality and continuous supply of feed is essential for insect mass rearing. Depending on the type of insect, the range of feed sources availability varies. For oligophagous like silkworms, the mass production of mulberries is required traditionally. In order to facilitate the sericulture, artificial diets for silkworms have been particularly developed . Under t5.3.3The location, mass and energy/heat balances , modelli5.3.4P. americana [Traditionally, most of the medicinal insects were sun dried and then boiled or fried before consumption . Open anmericana .5.3.5Lipid oxidation can generate toxic products, which are correlated with inflammatory diseases, cancer, atherosclerosis, and aging . Oxidati5.3.6A. domesticus densovirus (AdDNV) has caused mass mortality in cricket farms [Mass production pushed insects to growth and develop at a high density, which provides optimal conditions for insect disease transmission . For exaet farms . Hygieneet farms . Up to det farms .5.3.7While the above aspects are more related to technical issues, other challenges  need to be overcome by the whole community . For example, to avoid food-borne disease contamination, insect farms should follow some hygiene and sanitation protocol. A detail guide on hygiene practices of insect farming can be found on the website of international platform of insects for food and feed . The gui6Insects have been widely used as medicinal resources in many parts of the world since ancient times. Insects can be used alone or combination with medicinal plants in the treatment of diseases . The proIn effect, captive farming of edible insects can offer feed and food for animals and humans, respectively, and provide resources for the pharmaceutical industry to discover drugs for various health-related complications. Specifically, among others, future research should provide proper identification of these medicinal insects using molecular tools and conduct further investigation to verify and assess the viability of utilizing insects in the drug discovery process. Limited information exists on the reservations about entomotherapy as there are about entomophagy. Moreover, a clear understanding of the therapeutic use of insects and insect-derived products between Western countries and other regions worldwide requires further investigations. There is also a need to assess consumer opinions/consumer science on entomotherapy. Progress has been towards using insects for medicinal purposes. However, knowledge about their side effects is generally lacking. The relationship between disease treatment using insects and insect-derived products and allergenic effects in patients should be considered in future studies. While a clinical evaluation of medicinal plants’ efficacy and safety have been considered by several researchers, such information on medicinal uses insects is poorly documented. Future works should also investigate knowledge, perception, and willingness to apply and pay for entomotherapy. In addition, implications of using insects and insect-derived products on insect biodiversity conservation, the use of insects for the treatment of animal diseases and the contribution of insects to drug discovery may offer a new direction and solution to emerging diseases. Furthermore, among insects used for therapeutic purposes, some are pests responsible for diseases in plants and humans and others play a role in biological control as predators and pollinators of crops. In view for environment and biodiversity conservation, there is a need to select few samples of these insects in investigations necessary to ecological balance.S.A.S. – Conceptualization, Validation, Formal Analysis, Resources, Writing - Original Draft, Writing - Review and Editing, Visualization, Data Curation, Project administration, Supervision. C.L. – Writing - Original Draft. O.F.A. – Writing - Original Draft. I.F. – Conceptualization, Writing - Review and Editing. M.A.H. – Formal Analysis, Validation. J.A.M.P. - Review and Editing. A.B. - Data Curation. A.G. - Data Curation. J.S.C. - Review and Editing.This research was funded by FCT-Fundação para a Ciência e a Tecnologia through the CQM Base Fund - UIDB/00674/2020, and Programmatic Fund - UIDP/00674/2020, by Interreg MAC 2014–2020 Cooperacion territorial through AD4MAC project (MAC2/1.1 b/350), and by ARDITI-Agência Regional para o Desenvolvimento da Investigação Tecnologia e Inovação, through the project M1420-01-0145-FEDER-000005 - Centro de Química da Madeira - CQM+ (Madeira 14–20 Program) and the Post-Doctoral fellowship given to JAMP (Project M1420–09–5369-FSE-000001). The authors also acknowledge FCT and Madeira 14–2020 program to the Portuguese Mass Spectrometry Network (RNEM) through PROEQUIPRAM program, M14-20 M1420-01-0145-FEDER-000008).The authors declare no conflict of interest.Not applicable.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Fusobacterium nucleatum is a prevalent periodontal pathogen and is associated with many systemic diseases. Our knowledge of the genomic characteristics and pathogenic effectors of different F. nucleatum strains is limited. In this study, we completed the whole genome assembly of the 4 F. nucleatum strains and carried out a comprehensive pangenomic study of 30 strains with their complete genome sequences. Phylogenetic analysis revealed that the F. nucleatum strains are mainly divided into 4 subspecies, while 1 of the sequenced strains was classified into a new subspecies. Gene composition analysis revealed that a total of 517 “core/soft-core genes” with housekeeping functions widely distributed in almost all the strains. Each subspecies had a unique gene cluster shared by strains within the subspecies. Analysis of the virulence factors revealed that many virulence factors were widely distributed across all the strains, with some present in multiple copies. Some virulence genes showed no consistent occurrence rule at the subspecies level and were specifically distributed in certain strains. The genomic islands mainly revealed strain-specific characteristics instead of subspecies level consistency, while CRISPR types and secondary metabolite biosynthetic gene clusters were identically distributed in F. nucleatum strains from the same subspecies. The variation in amino acid sites in the adhesion protein FadA did not affect the monomer and dimer 3D structures, but it may affect the binding surface and the stability of binding to host receptors. This study provides a basis for the pathogenic study of F. nucleatum at the subspecies and strain levels.IMPORTANCE We used F. nucleatum as an example to analyze the genomic characteristics of oral pathogens at the species, subspecies, and strain levels and elucidate the similarities and differences in functional genes and virulence factors among different subspecies/strains of the same oral pathogen. We believe that the unique biological characteristics of each subspecies/strain can be attributed to the differences in functional gene clusters or the presence/absence of certain virulence genes. This study showed that F. nucleatum strains from the same subspecies had similar functional gene compositions, CRISPR types, and secondary metabolite biosynthetic gene clusters, while pathogenic genes, such as virulence genes, antibiotic resistance genes, and GIs, had more strain level specificity. The findings of this study suggest that, for microbial pathogenicity studies, we should carefully consider the subspecies/strains being used, as different strains may vary greatly. Fusobacterium nucleatum is a Gram-negative, spindle, non-spore-forming anaerobic bacterium belonging to the phylum Fusobacterium and family Bacteroidaceae , and animalis  than subsp. nucleatum and vincentii  , and animalis. Three of the sequenced strains, named FNV, FNA, and FNP, were classified into subsp. vincentii, animalis, and polymorphum, respectively. However, 1 new strain (FNU) could not be classified into any of these subspecies. Furthermore, we selected Fusobacterium periodonticum (F. periodonticum) and Fusobacterium hwasookii (F. hwasookii) as outgroups to construct a phylogenetic tree with F. nucleatum . The results showed that sequence similarity between F. hwasookii strains and F. nucleatum subsp. polymorphum was much higher than those between different F. nucleatum subspecies.We obtained 4 strains of Methods) . We consF. nucleatum strains in the NCBI database (table S1). The phylogenetic tree showed the evolutionary interrelations and classification of the four strains, and FNU formed an independent evolutionary branch with the strain 13_08_02 . Next, 30 F. nucleatum strains with complete genome information (26 strains from the NCBI database and the 4 strains that we sequenced) were used to carry out phylogenetic analysis and an average nucleotide identity (ANI) analysis. The results showed that FNU was in a new branch , 42 soft-core genes (28 <= strains < 29), 3,954 shell genes (4 <= strains < 28) and 8,277 cloud genes (strains < 4) were identified across the 30 strains . The results showed that the relationship between subsp. animalis 4_8 and subsp. polymorphum ChDC_F306 might be closer. There are shared plasmid elements. We carried out gene annotation and gene similarity analysis on all plasmids using the OrthoFinder (version 2.2.6) software and found that multiple genes were shared in the plasmids of different strains (Table S8 plasmid). We found that there were no virulence genes and AMR-associated genes contained in any of these plasmids.Among the plasmids, there was no subspecies level specificity for the presence and number of plasmids and the separation locations among the 30 strains. For instance, 7 plasmids . We consanimalis-unique genes were associated with membrane composition and transmembrane transporter-related terms, such as integral component of the plasma membrane and transmembrane transporter activity  and phosphoenolpyruvate-dependent sugar phosphotransferase system  was used to define virulence factors (VFs) in 30 strains. Globally, 107 VFs from 11 virulence categories were detected in the genome of F. nucleatum   . VFs asse copies . In addicdiA and exlA were present in subsp. animalis 4_8 and FNU; cap8P only resided in subsp. vincentii KCOM 1231; wbfU could be detected in only subsp. polymorphum FNP; and ctpV, which is associated with nutritional/metabolic factors, was present in only subsp. animalis 4_8  was used to identify antibiotic resistance genes (ARGs) among  strains . The OXAffh and YadA had identical distribution patterns, while SecA was mainly detected in subsp. polymorphum.In contrast to VFs and ARGs, the secretion system-related genes showed no specificity at the subspecies or strain level  and D. TF. nucleatum strains. nucleatum 25586 and animalis KCOM 1325 had only 3.We used IslandViewer based on the IslandPick, IslandPath-DIMOB, SIGI-HMM and Islander databases to reveal the characteristics of genomic islands (GIs) among animalis and polymorphum contained genes encoding a recombinant protein, the murein hydrolase activator EnvC, a NAD kinase, the DNA repair protein RecN and the tyrosine recombinase XerD. CRISPR-associated endoribonuclease/proteins were identified in vincentii FNV, animalis KCOM 1279, and animalis THCT7A2. Type II secretion system proteins were present in vincentii 3_1_27, and vincentii KCOM_1231, etc. Toxin zeta was found in vincentii 3_1_27, animalis 4_8, animalis 7_1, animalis KCOM_1325, etc. Antitoxin HigA/YwqK was found in vincentii KCOM 2931, vincentii KCOM_1231, etc. PTS system component was identified in vincentii FNV, vincentii KCOM 2931, and vincentii THCT14A3, etc.Functional annotation showed that GIs from different subspecies/strains carried genes with different functions. Specifically, GIs in subsp. F. nucleatum strains (Table S7). The results showed that strains from the same subspecies had identical CRISPR types  is an evolving and adaptive immune defense element that widely distributes in bacteria and archaea. We used CRISPRCasFinder and CRISPRDetect to classify CRISPR sequences among the 30 PR types . For exaspecific .F. nucleatum strains were annotated using the antiSMASH database. As shown in F. nucleatum was synthesized by nonribosomal peptide synthetase (NRPS), which is a biosynthetic enzyme that produces various nonribosomal peptides and can be widely used in clinical applications, for example, as antibiotics, antitumor inhibitors, and immune-suppressants  biosynthetic gene clusters in ressants , 40. Theymorphum . From sufamilies .F. nucleatum strains  surface was analyzed using PDBePISA. e energy  and I. T surface F. nucleatum is a well-known pathogen associated with periodontitis and multiple systemic diseases, but knowledge of the genomic characteristics of different F. nucleatum strains is limited. In this study, we performed a pangenomic analysis of 30 F. nucleatum strains, providing a fundamental basis for the pathogenesis research and clinical treatment. The phylogenetic tree obtained for multiple protein units in this study was consistent with the results of previous studies. Similar to W1481  solid medium  and cultured in anaerobic incubators  at 37 degrees C (37°C anaerobic incubators) for 72 h. Then, single colonies were selected and placed in BHI liquid medium for proliferation to the logarithmic growth stage. The cultures were then centrifuged at 4500 rpm for 5 min to collect the bacterial cell, which were re-suspended in BHI liquid medium for subculture. The purity of the bacteria was determined by PCR and 16S rRNA sequencing. The bacterial cell culture methods were performed as described previously.We obtained 4 strains of vincentii FNV, FNU, subsp. animalis FNA, and subsp. polymorphum FNP) were sequenced using the PacBio Sequel platform and Illumina NovaSeq PE150 at the Beijing Novogene Bioinformatics Technology Co., Ltd. The whole genome sequences of the four strains were deposited as a BioProject (PRJNA932162) in GenBank under the accession numbers CP117525 (FNV), CP117526 (FNU), CP117797 (FNA), and CP117825 (FNP).Genomic DNA was extracted by the SDS method . The detF. nucleatum strains available in the NCBI database were downloaded. The genomic information of F. nucleatum is summarized in Table S1. We downloaded the genome sequences of F. periodonticum and F. hwasookii from the NCBI, whose detailed information was summarized in Table S1. Then, all the downloaded genomic nucleic acid sequences were annotated by Prokka software (version 1.12)   with defF. nucleatum strain. Protein family analysis was performed by OrthoFinder software (version 2.2.6)   with defhttp://tree.bio.ed.ac.uk/software/figtree) was used to visualize the best ML tree result and analyze the phylogenetic relationships among different F. nucleatum strains and subspecies.The MAFFT software (version 7.407)  was usedF. nucleatum, an ANI analysis was performed using the FastANI software , 'soft core' (95% <= strains < 99%), 'shell' (15% <= strains < 95%) and 'cloud' (0% <= strains < 15%) groups.Based on the gff files obtained from the Prokka annotation results, we used Roary software  to perfohttp://jvenn.toulouse.inra.fr/app/example.html) to identify subspecies-specific gene families from the pangenomic analysis results. The representative sequence of each gene family was extracted by the Perl program and further annotated by KOBAS software (version 3.0.3) with the parameter -s: ko. Then, the R package clusterProfiler (version 4.4.4) was applied for GO and KEGG enrichment analysis based on the KOBAS annotation results of KOBAS. The statistically significant results (P < 0.05) of core gene families and subspecies-unique gene families were visualized in bar plots.Next, we used the jveen tool  , and these genes are gradually lost  was used to perform multiple sequence alignments of FadA sequences, and the results were visualized using ESPript 3 (http://espript.ibcp.fr). Based on the multiple sequence alignment results, we selected a representative FadA sequence in each subspecies. AlphaFold2  molecular dynamics simulation method was selected. It has been reported that CG molecular simulation can be used to accurately predict the strength of protein-protein interaction, while achieving a 500-times acceleration compared with all-atom molecular simulation . We usedPRJNA932162) in GenBank under the accession numbers CP117525 (FNV), CP117526 (FNU), CP117797 (FNA), and CP117825 (FNP).The whole genome sequences of the four strains were deposited as a BioProject (