L. brownii (Lb) and its variety, L. brownii var. giganteum (Lbg), during in vitro bulblet initiation were obtained. A total of 53,576 and 65,050 high-quality non-redundant full-length transcripts of Lbg and Lb were generated, respectively. Morphological observation showed that Lbg possessed a stronger capacity to generate bulblets in vitro than Lb, and 1 mg L−1 2,4-dichlorophenoxyacetic acid  significantly increased bulblet regeneration rate in two lilies. Screening of differentially expressed transcripts (DETs) between different stages and Mfuzz analysis showed 0 DAT to 1 DAT was the crucial stage with the most complex transcriptional change, with carbohydrate metabolism pathway was significantly enriched. In addition, 6,218 and 8,965 DETs were screened between the 2,4-D-treated group and the control group in Lbg and Lb, respectively. 2,4-D application had evident effects on the expression of genes involved in auxin signaling pathway, such as TIRs, ARFs, Aux/IAAs, GH3s and SAURs. Then, we compared the expression profiles of crucial genes of carbohydrate metabolism between different stages and different treatments. SUSs, SUTs, TPSs, AGPLs, GBSSs and SSs showed significant responses during bulblet initiation. The expression of CWINs, SUTs and SWEETs were significantly upregulated by 2,4-D in two lilies. In addition, 2,4-D increased the expression of starch degradation genes (AMYs and BAMs) and inhibited starch synthesis genes . SBEs were significantly upregulated in Lbg but not in Lb. Significant co-expression was showed between genes involved in carbohydrate metabolism and auxin signaling, together with transcription factors such as bHLHs, MYBs, ERFs and C3Hs. This study indicates the coordinate regulation of bulblet initiation by carbohydrate metabolism and auxin signaling, serving as a basis for further studies on the molecular mechanism of bulblet initiation in lily and other bulbous flowers.Bulblet initiation, including adventitious bud initiation and bulblet formation, is a crucial process for lily and other bulbous flowers that are commercially propagated by vegetative means. Here, by a hybrid strategy combining Pacific Biosciences (PacBio) full-length sequencing and Illumina RNA sequencing (RNA-seq), high-quality transcripts of  Similar AGPases . Sucrose AGPases . A clearorbonne’ . Similar Lycoris . The aboLycoris, endogenous IAA content showed an increase and then a decrease during bulblet initiation and development, which were consistent with the expression patterns of genes involved in IAA synthesis and signal transduction   sequencing technology of the PacBio system has offered a new third-generation sequencing platform, which possesses advantages such as long read lengths (length > 10 kb), high consensus accuracy and a low degree of bias  technology has been widely used in the study of the process of bulblet formation in multiple bulbous plants, including Lilium , Lycoris(Lycoris  and sweebatatas) . In Lililopment. . However of bias , which ichnology .in vitro bulblet initiation process of Lilium brownii (Lb) and Lilium brownii var. giganteum (Lbg), a variant of Lb  and then cultured for 14 days on basal Murashige and Skoog (MS) medium , China. ) medium  containi2.2Lb and Lbg were observed under a stereomicroscope . The regeneration rate and propagation efficiency of bulblets were calculated as follows: Regeneration rate = number of scales that produced adventitious buds/total number of scales; Propagation efficiency = total number of produced buds/total number of scales. Representative data were supported by three biological replicates, each containing 120 repeats. Transverse sections of scales at the proximal end where adventitious buds initiated were stained with periodic acid-Schiff (PAS) and Naphthol Yellow S as previously described , stage of wounding response and early regeneration competence (1 DAT), stage of adventitious bud initiation (8 DAT) and stage of adventitious bud swelling and bulblet formation (14 DAT). The entire scales used for bulblet induction at 0 DAT, 1 DAT, 8 DAT and 14 DAT were sampled, frozen in liquid nitrogen and stored at -80°C for total RNA extraction. Sampling was performed with three biological replicates for each stage.The bulblet initiation process of 2.4Lb and Lbg cultured on 2,4-D-free  and 2,4-D-containing  medium was extracted using an EASYspin Plus Complex RNA Kit  according to the manufacturer’s instructions. Total RNA from each sample was equally mixed to generate a pool and then synthesized to first-strand cDNA using Clontech SMARTer PCR cDNA Synthesis Kit . Large-scale PCR was performed using the BluePippin™ Size Selection System . The SMRTbell template libraries were constructed and then sequenced on the PacBio Sequel platform. The following methods for generating full-length reference transcripts referenced Total RNA of scale samples at four stages of Total RNA of scale samples was sequenced with the Illumina Xten 4000 platform . Quality control (QC) for each Illumina transcriptome was performed by fastp (v0.19.7) with default parameters . Clean r2.5http://eggnog-mapper.embl.de/) with an e-value of ≤ 1e-5 and identity of ≥ 60% with Viridiplantae (green plants) selected as the taxonomic scope  . Transcr2.7Lbg and six DETs of Lb were selected for expression analysis using quantitative real-time PCR (qRT-PCR). Total RNA (1 μg) of each sample was reverse transcribed by the PrimeScript™ RT reagent Kit with gDNA Eraser . The diluted (1:30) cDNA was used as the template for qRT-PCR analysis. Gene-specific primers were designed by the NCBI Primer-BLAST tool  following the manufacturer’s protocol. Three replicates were included in each assay. Sucrose, fructose, and glucose contents were measured by high-performance liquid chromatography (HPLC)  equipped with Refractive Index (RI) Detector 2414 (Waters) according to the previous method in 2.9−1. The tubes were centrifuged at 12 000 rpm for 10 min at 4°C. The resulting supernatant was dried by blowing under N2. The residue was resuspended in 0.5 mL of 70% (v/v) methanol and centrifuged, and the supernatants were then analyzed in a triple quadrupole mass spectrometer .IAA extraction and quantification were performed using a previously described method with slight modifications . Briefly2.10Lb and Lbg were performed using Pearson’s two-tailed tests and visualized by Cytoscape v3.7.1  was used to compare differences among different indices or treatments via SPSS 26.0 . Correlation analyses between gene expression data in e v3.7.1 .33.1Lbg and Lb  than those in Lb  (Lbg possesses a stronger capacity to generate bulblets in vitro than Lb.Throughout the bulblet initiation and development process (0 DAT to 49 DAT), the regeneration rate and propagation efficiency were calculated. It is showed that visible adventitious buds occurred at 8 DAT, and the number of newly formed bulblets was significantly increased until 14 DAT and then tended to plateau until 25 DAT or later in both 3.2Lbg were first obtained with 53,576 nonredundant transcripts of an average length of 3,108 bp, and 80.79% of the Lbg clean reads obtained by Illumina RNA-Seq mapped to the Lbg reference transcripts  showed that there were obvious differences of expression patterns among different stages under the same treatment conditions, except for 8 DAT and 14 DAT in Lbg  and six transcripts of Lb  were randomly selected for qRT-PCR to validate the differential expression by RNA-Seq. The results showed that the differential expression levels of these selected transcripts by qRT-PCR were highly consistent with those obtained by RNA-Seq  0 DAT (Group 1), 8 DAT vs 1 DAT (Group 2) and 14 DAT vs 8 DAT (Group 3). In total, 11,964 and 18,834 stage-specific DETs were screened in Lbg and Lb, respectively. The transcripts were divided into eight and ten clusters in Lbg and Lb, respectively ” (map00020), which is involved in carbohydrate metabolism, was upregulated in all the four candidate clusters  and “glycosaminoglycan degradation” (map00531) enriched in cluster 7 (Lbg), and “cell cycle” (map04110), “cellular senescence” (map04218) and “necroptosis” (map04217) enriched in cluster 8 (Lb), indicating that this stage could be associated with cell growth and death (Lbg) and cluster 2 (Lb) were increased at 14 DAT, and transcripts in cluster 2 (Lb) were mainly enriched in “biosynthesis of other secondary metabolites” and “xenobiotics biodegradation and metabolism” classes, which might play important functional roles in the bulblet swelling stage  in 2,4-D-treated group than in the control group in both Lbg and in Lb  and 8,965  DETs between 2,4-D-treated group and the control group at 1 DAT, 8 DAT and 14 DAT in Lbg and Lb, respectively  were significantly downregulated (p < 0.001) at 1 DAT in both Lbg and Lb  were significantly enhanced (p < 0.001) by 2,4-D treatment (p < 0.001) the expression of two Tryptophan Aminotransferase (TAA) and Tryptophan Aminotransferase Related (TAR) (Isoform 49388 and 40427) in Lb, which could promote the endogenous synthesis of auxin (p < 0.05) under 2,4-D treatment than that of the control group at 8 DAT and 14 DAT in Lb  were significantly (p < 0.001) upregulated at 8 DAT in the control group, and 15 ARFs in Lbg and two ARFs in Lb were significantly (p < 0.001) upregulated at 8 DAT with 2,4-D treatment, following the downregulation of TIR1s (p < 0.001) encoding some GH3 (five in Lbg and seven in Lb), Small Auxin Upregulated RNA (SAUR) (two in Lbg and seven in Lb) and Indole-3-acetic Acid Inducible (Aux/IAA) (14 in Lbg and eight in Lb) in the 2,4-D-treated group than in the control group at 1 DAT or 8 DAT further support the above idea (Lbg and three in Lb) after 1 DAT (Three Auxin Response Factors (ARFs) of 3.6Lbg and 17 in Lb) were significantly upregulated (p < 0.001) at 1 DAT or 8 DAT (Lbg (Isoform 29531) were upregulated at 1 DAT and then downregulated, and the expression level of four CWINs  were significantly increased (p < 0.001) in the 2,4-D-treated group  in the 2,4-D treated group than that in the control group in both Lbg and Lb  and five TPSs in Lb  were significantly upregulated (p < 0.001) at 1 DAT, 8 DAT or 14 DAT (Lbg and two in Lb) were significantly upregulated (p < 0.001) at 1 DAT, among them, the upregulation of one SUT in Lbg (Isoform 31091) and two SUTs (Isoform 36404 and 36445) in Lb was significantly enhanced (p < 0.001) by 2,4-D  and one in Lb (Isoform 48829) without 2,4-D during bulblet initiation, but these SWEETs significantly upregulated (p < 0.001) at 8 DAT with 2,4-D application  at 1 DAT without 2,4-D, but some of these upregulations were inhibited (Isoform 33352 in Lbg), attenuated  or delayed  in the 2,4-D-treated group  and β-Amylases (BAMs) , which were involved in starch degradation, were significantly upregulated (p < 0.001) by 2,4-D  by 2,4-D at 1 DAT, 8 DAT and 14 DAT  (p < 0.001) in Lbg  but not in Lb , Granule-Bound Starch Synthase (GBSS), and Starch Synthase (SS) were significantly upregulated  between these candidate TFs and DETs involved in auxin signaling and carbohydrate metabolism was conducted. In 44.1Tissue culture is a main asexual reproduction method for many bulbous crops and has a significant advantage in promoting regeneration efficiency and shortening the breeding and propagation cycle . AdventiLbg and Lb were obtained through PacBio Iso-seq together with Illumina short-read sequencing during in vitro bulblet initiation. To date, Illumina sequencing technology has been widely applied to explore the process of bulblet formation and development in lily . The N50 of transcripts of Lbg and Lb was 5,422 bp and 5,199 bp, respectively, and the mean length of transcripts of Lbg and Lb were 3,108 bp and 2,965 bp, respectively  upregulated the expression level of LrSS1, LrSS2, and LrGBSS1 genes, which could enhance carbohydrate accumulation in the bulblets, thus promoted their development  showed a rapid decline during bulblet initiation in the efficient bulblet regeneration system  were downregulated, while key enzymes involved in starch degradation (AMY and BAM) were upregulated compared to the control group (Starch accounts for approximately 70% of the dry weight of lily bulbs . Starch elopment . Similar and SBE . In the ormation . Moreoven system . Here, wLilium, SUSs and INVs were both highly expressed in the mother scales and bulblets during bulblet emergence and swelling (SUSs and INVs were greatly upregulated accompanied by a decrease in sucrose content in mother scales during bulblet initiation in Lycoris (Lbg and 17 SUSs in Lb were upregulated at 1 DAT or 8 DAT, and one CWIN in Lbg and two CWINs in Lb were upregulated at 1 DAT and then downregulated (SUS and CWIN often presented an opposite expression pattern during bulblet initiation, and this change was considered to be a possible sign of the transition from bulblet initiation to development (CWINs were highly expressed during the early bulblet initiation stage and produced glucose, which might act as sugar signaling rather than carbon resources (Lycoris, the more highly LsCWIN2 was expressed, the more bulblets were produced (Lbg and Lb were significantly increased by 2,4-D, showing a possible role of CWINs in increasing bulblet regeneration rate. Recent study showed that LbgCWIN1 significantly upregulated endogenous starch was degraded during in vitro bulblet initiation in lily (CWIN can be selected as a candidate gene subsequent function verification.Soluble sugars in mother scales were transported into the region where bulblets were initiated to supply the follow-up bulblet development . Sucroseswelling . Similar Lycoris . In thiselopment . In partesources . In Lycoproduced . Here, t in lily , indicatLbg and two SUTs in Lb was significantly enhanced by 2,4-D at 1 DAT (Lbg and one SWEET had no significant expression change in Lb without 2,4-D during bulblet initiation, but significantly upregulated at 8 DAT and 14 DAT with 2,4-D application (in vitro bulblet initiation mainly through promoting sucrose unloading from the SE/CC to the sink cells, and SWEETs and SUTs can be considered as good candidates for future functional studies.Interestingly, the application of 2,4-D in the medium for bulblet induction had significant effects on the expression of genes involved in carbohydrate metabolism, especially SUTs and SWEETs . The upr4.4Lbg, three SWEETs, two AMYs, one BAM and one SUS were co-expressed with one AUX1, six Aux/IAAs, five ARFs and five GH3s, and ten SUSs, two INHs, one CIN and three AGPLs were co-expressed with two PINs (in vitro bulblet initiation in lily. A recent study showed that bHLH, bZIP, WRKY, TCP, MYB, YABBY, NAC, C2H2 were identified to be involved in bulbil induction of ‘Sorbonne’ lily by coexpression analysis (Lycoris, coexpression analysis revealed that transcripts encoding WOX14, MYB117, and ULT1 coexpressed with LsCWIN2, and transcripts encoding ERFs were coexpressed with LsSUS4 during bulb vegetative production (in vitro bulblet initiation. Among them, several TFs might be involved in the regulation the expression of key enzymes in carbohydrate metabolism during in vitro bulblet initiation in both Lbg and Lb, for example, C3Hs and bHLHs were co-expressed with AMYs and SWEETs, and some MYBs and bHLHs were co-expressed with SUSs in both Lbg and in Lb (Although carbohydrate metabolism and the auxin signaling pathway have been respectively demonstrated to participate in bulblet initiation, their cooperative function during this process has not yet been reported. Here, we found that in analysis . In Lycooduction . In the The data presented in the study are deposited in the GenBank repository, accession number PRJNA933000.LZ, YW and ZMR designed the experiment. LZ, CG, YCX, YL and XX carried out the experiment. CG and LC analyzed the data. CG drafted the manuscript. ZR, YW and YPX revised the manuscript. All authors contributed to the article and approved the submitted version.


Salivary duct carcinoma with rhabdoid features (SDC-RF) is a rare form of salivary gland neoplasm that was recently described. We report a case of SDC-RF of the parotid gland with loss of E-cadherin and decreased β-catenin expression in a 73-year-old male who presented with right facial/neck swelling and intermittent pain. Morphologically, the tumor presented with a discohesive infiltrate of isolated and cords of pleomorphic round cells containing moderate amount of eosinophilic to fine-vacuolated cytoplasm and hyperchromatic nuclei infiltrating through fibroadipose tissue and salivary parenchyma. Immunophenotypically, the tumor was positive for Cytokeratins Oscar and 7, GATA3, GCDFP, HER2, and an androgen receptor but negative for CK20, S100, p40, Melan A, CDX2, TTF1, ER, SATB2, DOG1, synaptophysin, and chromogranin. Due to its diffuse infiltrating pattern, involvement of the parapharyngeal space, supraclavicular fat pad, dermis, and skin without a defined surgical target, the tumor was deemed unresectable. Anti-HER2 therapy (Herceptin and Pertuzumab) was utilized. At the last follow-up, the patient is alive, with complete locoregional control and brain metastases. An electronic search was performed in the following registries for papers published up to June 2023: PubMed, Embase, and Web of Science. For the database searches, the keywords searched were “salivary gland”, “salivary duct carcinoma”, and “salivary duct carcinoma with rhabdoid features”. Our review of the literature identified 30 cases of SDC-RF that reveal there is a predilection for males (83%), parotid gland (72%), and patients older than the 6th decade of life (83%). Immunophenotypically, all SDC-RF cases except one were positive for AR and GCDFP (97%), 81% were positive for HER2, and loss or decreased expression of E-cadherin in 93% of cases. In conclusion, we described a rare case of SDF-RF of the parotid gland with no E-cadherin expression, decreased β-catenin expression, and its immunophenotypic profile.  CDH1 mutations [Salivary duct carcinoma (SDC) is a relatively uncommon high-grade malignancy of the salivary gland that may arise de novo or observed in the carcinomatous component of carcinoma ex-pleomorphic adenoma. SDC was originally described as resembling high-grade ductal carcinoma of the breast with cribriform morphology, roman bridge formation, and prominent central necrosis ,2. Howevutations ,13. A reutations . In thisA 73-year-old male with a significant history of tobacco smoking (100 pack years) presented to the Otolaryngology clinic of University of Tennessee Health Science Center with right facial/neck swelling and intermittent pain of 2 months duration. The patient stated the symptoms had gotten worse over time. There was no facial twitch or weakness. Palpation of the right parotid gland revealed firmness to the tail and inferior aspects of the gland. The patient’s medical history was positive for hypertension, chronic kidney disease, chronic pancreatitis, diabetes mellitus, and pulmonary emphysema. Imaging studies revealed an ill-defined increased density and enhancement of the right parotid gland involving the superficial and deep portions of the gland. There was a persistent loss of delineation within the posterior parotid gland and the adjacent posterior belly of the digastric muscle. An impression of inflammatory changes versus an infiltrating parotid neoplasm was rendered. The patient was prescribed Augmentin 875 mg BID for 10 days to help rule out an infectious/inflammatory process. Two weeks later, the patient returned for a follow-up review with no improvement of symptoms. The initial post-contrast CT scan of the neck demonstrated a nodular and infiltrative lesion centered in the right parotid gland. There was significant surrounding fat stranding and fat plane effacement involving the adjacent neck spaces and structures. Both the right sternocleidomastoid and posterior belly digastric muscles had ill-defined margins with the lesion. Additionally, there was thickening of the fascial plane and overlying skin . A folloAn ultrasound-guided core needle biopsy was performed. Histopathologically, the biopsy showed a diffuse infiltrate of discohesive pleomorphic round cells singly and in single cell files within the parotid gland and surrounding adipose tissue. The stroma ranged from being fibrotic to histologically indiscernible. Typical rhabdoid morphology was not identified. While nuclei were eccentrically placed giving a plasmacytoid appearance, the cytoplasm had a fine, foamy appearance instead of dense eosinophilia of true rhabdoid cells A–F. An eThe patient’s proposed care was discussed at the Methodist University Hospital Multidisciplinary Head and Neck Tumor Board. The imaging studies were reviewed, including PET–CT images which showed ipsilateral nodal metastases but no distant metastases. Given the patient’s comorbid conditions and involvement of the parapharyngeal space, supraclavicular fat pad, dermis, and skin without a clearly defined surgical target, the tumor was deemed unresectable. The recommendation of the tumor board was for induction systemic treatment with anti-HER2 therapy (Herceptin and Pertuzumab). At the last follow-up, the patient was alive, with complete locoregional control and brain metastases . An electronic search was performed in the following registries for papers published up to June 2023: PubMed, Embase, and Web of Science. For the database searches, the keywords searched were “salivary gland”, “salivary duct carcinoma”, and “salivary duct carcinoma with rhabdoid features”.th decade of life  [CDH1 (gene that encodes E-cadherin) have been reported in up to 72–86% of SDC-RF cases [SDC-RF is a rare form of salivary gland neoplasm with only 30 cases reported in the literature ,9,10,11.e 25/30, 3% [8,9,1RF cases . GATA3 iRF cases . Our casE-cadherin is a transmembrane protein that forms homotypic cell–cell adhesion structures called adherens junctions. The intracellular domain binds to catenins, of which β-catenin interacts with the actin cytoskeleton and plays a pivotal role in intracellular signaling promoting cell proliferation and survival . Loss ofCDH1 alteration, mostly mutations and a recently identified CDH1::CORO7 fusion [PIK3CA, HRAS, TP53, PTEN, and AKT1 [PIK3CA, HRAS, and NRAS have been reported in classic SDC [PLAG1  in pleomorphic adenoma (PA), carcinoma ex-PA, and myoepithelial carcinoma [HMGA2  in PA and carcinoma ex-PA [MYB/MYBL1::NFIB in adenoid cystic carcinoma [MAML2::CRTC1/3 in mucoepidermoid carcinoma [PRKD1/2/3  in polymorphous adenocarcinoma/cribriform adenocarcinoma of the salivary gland [ETV6  in secretory carcinoma [SS18 (fusion partners MEF2C and ZBTB7A) in microsecretory adenocarcinoma [TRIM27/33 and NCOA4) in intraductal carcinoma [HTN3-MSANTD3 and NR4A3 upregulation in acinic cell carcinoma [AKT1 mutations in epithelia–myoepithelial carcinoma, intraductal papillary mucinous neoplasm, and salivary mucinous adenocarcinoma [CTNNB1 mutations in basal cell adenoma and basal cell adenocarcinoma [BRAF mutations in sialadenoma papilliferum [Molecularly, SDC-RF appears to be defined by 7 fusion ,11. Othessic SDC ,20. Manyarcinoma ,23,24,25ma ex-PA ,27; MYB/arcinoma ,29; MAMLarcinoma ; PRKD1/2ry gland ,34,35,36arcinoma ,38,39; Sarcinoma ; RET , alive with complete locoregional control and distant brain metastases .


Objective: Few real-world studies have shown clear association between interleukin (IL)-17 inhibitors and inflammatory bowel disease (IBD) onset. This study investigated the reporting prevalence and evaluated the clinical features and management of IL-17 inhibitor-related IBD events.Methods: We used the US FDA Adverse Event Reporting System database and retrieved data, from 2015 to 2022, on IL-17 inhibitors to identify gastrointestinal inflammatory events and conduct disproportionality analyses by estimating the reporting odds ratios (RORs) and corresponding 95% confidence intervals (CIs). Furthermore, case reports and case series, from 2015 to 30 November 2022, on IBD induced by IL-17 inhibitors were collected for retrospective analysis.Results: A total of 388 cases of primary suspected IL-17 inhibitor-associated gastrointestinal events were reported (268 IBD and 120 colitis), including 348 cases involving secukinumab (SEC), 36 cases involving ixekizumab (IXE), and 4 cases involving brodalumab (BRO). Statistically significant reporting rates of total IBD events were observed for SEC and IXE , whereas BRO did not trigger a safety signal. Twenty-nine studies, which included 34 cases, showed evidence of IBD, following SEC (79.4%) and IXE (20.6%) treatment. The median age was 42 years; typical initial symptoms included diarrhea (90.9%), abdominal pain (57.6%), bloody diarrhea (51.5%), and fever (36.4%). The median time to onset of IBD symptoms was 2.9 months. Some cases were accompanied by elevated white blood cell (WBC) count (87.5%), erythrocyte sedimentation rate , C-reactive protein , and fecal calprotectin . Cessation of IL-17 inhibitors plus treatment with corticosteroids and TNF antagonists, as either monotherapy or in combination, could lead to complete clinical remission. The median time to remission after IL-17 inhibitor discontinuation was 4 weeks.Conclusion: IL-17 inhibitor treatment is associated with exacerbation and new onset of IBD and colitis. Obtaining a detailed patient history before initiation of treatment and monitoring gastrointestinal symptoms and intestinal inflammatory biomarkers during IL-17 inhibitor treatment is important for safe use of these drugs. Interleukin (IL)-17 is a significant proinflammatory cytokine and a therapeutic target for rheumatological diseases as blocking IL-17 reduces disease activity and improves patient quality of life . The antTo date, the pathophysiological mechanisms underlying these paradoxical effects are not well-defined, there is limited real-world data demonstrating the association between IL-17 inhibitor therapy and the onset of IBD, and there are no clear guidelines for the management of patients who experience these adverse events. We aimed to assess differences in safety signals using the FDA Adverse Event Reporting System (FAERS) database, and describe characteristics, management, and prognosis of IBD flare or new onset in patients after anti-IL-17 drug therapy in a real-life setting, which provides valuable information about emerging gastrointestinal safety issues related to IL-17 inhibitors.In this study, we conducted a retrospective, disproportionality, pharmacovigilance analysis. Data from 2015 Quarter 1 (Q1) to 2022 Q3 were retrieved from the publicly available FAERS database in FDA website to evaluate the risk of gastrointestinal inflammatory conditions with the use of different IL-7 inhibitor therapies.We assessed the use of IL-17 inhibitors in a large-scale population. Study drugs were IL-17 inhibitors on the market: SEC, IXE, and BRO. To identify IL-17 inhibitor-related records, both brand names and generic names were used. Furthermore, IBD and colitis case reports in FAERS were coded using the preferred term (PT) according to the Medical Dictionary for Regulatory Activities Terminology (MedDRA). Drugs were assigned a role  by the person reporting the adverse drug reaction (ADR). ADRs were categorized into two main groups: 1) “IBD,” including reported diagnoses of ulcerative colitis (UC), Crohn’s disease (CD), and undifferentiated IBD (uIBD); 2) “colitis,” including reported diagnoses of colitis , proctitis, and enteritis. The effects of disproportionality analysis were evaluated using the established pharmacovigilance index reporting odds ratio (ROR), which were calculated as (a*d)/(b*c) . In our A comprehensive search of multiple electronic databases, including PubMed, Embase, Wanfang, China National Knowledge Infrastructure (CNKI), and China Biology Medicine disc (CBMdisc), from January 2015 to September 2022, regarding IL-17 inhibitor-induced IBD was conducted, with no language restrictions. The search terms were “secukinumab or Cosentyx”, “Ixekizumab or Taltz,” “Brodalumab or Siliq,” “Ulcerative colitis,” “Crohn’s disease,” and “Inflammatory bowel disease”. Case reports and case series were included, and reviews, mechanistic studies, animal studies, and articles for which the full text was not available were excluded. Two reviewers searched the literature independently and examined the relevant studies for further assessment of inclusion/exclusion criteria and to identify clinical characteristics; collected data included the region of patient location, age, sex, Indication of IL-17 inhibitors, medical history, time to onset, clinical manifestations, laboratory tests, histopathological examinations, treatment, and prognosis of IL-17 inhibitor-induced IBD. The time to onset of target IBD was defined as the time from the start date of IL-17 inhibitor administration to the onset of the associated IBD.Up to 2022 Q3, the numbers of reported adverse event (ADE) cases in the FAERS database related to SEC, IXE, and BRO were 44,688, 24,406, and 1796, respectively. From screening individual IL-17 inhibitor-related gastrointestinal inflammatory case reports, a total of 388 cases of primary suspect IL-17 inhibitor-associated inflammatory bowel disease and colitis were reported, of which 348 were SEC, 36 were IXE, and 4 were BRO. These cases included 268 IBD diagnoses and 120 colitis diagnoses . The maiFrom the entire database, the disproportionality analyses identified three unique preferred terms (PTs) for IBD and 12 PTs for colitis. Globally, all cases of IBD after SEC and IXE treatment accounted for 1.36% and 0.66% of all reported cases, respectively, and there was a statistically significant reporting rate of total IBD events observed among patients treated with SEC and IXE , whereas this proportion was lower for BRO  . All colA total of 29 studies were identified in the published literature, including 25 case reports  and 4 caAmong the 34 cases included, IBD classification was reported in 28 cases, with 13 cases (46.4%) identified as UC, 10 cases (35.7%) as CD, and 5 cases (17.9%) as unclassified IBD. Symptoms were reported for 33 patients; the most common initial symptoms were diarrhea , followed by abdominal pain , bloody diarrhea , and fever . Other accompanying symptoms included weight loss , chills , and nausea . One patient underwent colonoscopy for colorectal cancer screening, which revealed asymptomatic Crohn’s disease .The laboratory results for IL-17 inhibitor-induced IBD cases are summarized in Computed tomography (CT) results were reported for 10 patients. Typical imaging features were bowel wall thickening , pancolitis , and colon perforation ; two patients showed normal CT results.Colonoscopy results were reported for 28 patients, with deep ulcerations found in 13 patients (46.4%) and superficial ulcerations found in 6 patients (21.4%); eight patients (28.5%) showed active inflammation, four patients (14.3%) had friable mucosa, and four patients (14.3%) had loss of the normal vascular pattern. Intestinal mucosal biopsies were performed in 25 patients, and the characteristics of histopathological examination presented mainly as chronic inflammation , cryptitis , crypt abscesses , and granulomatous lesions .A total of 31 patients discontinued IL-17 inhibitor therapy after the onset of IBD. Among the 34 included patients, 19 (61.3%) underwent monotherapy for IBD; corticosteroid therapy was administered in 10 cases (32.3%), TNF antagonist therapy in 6 cases (19.4%), ustekinumab therapy in 2 cases (6.4%), and mesalazine therapy in 1 case (3.2%). A total of 11 patients (35.5%) received combination therapy, and one patient did not receive any treatment. Six patients failed in treatment with corticosteroids alone or in combination with mesalazine; they obtained significant improvement when treatment was changed to infliximab alone or in combination with corticosteroids . All patvia VigiBase in 2021, no IBD was detected among patients in the BRO group, and the number of IBD cases associated with SEC treatment was five times that observed in the IXE treatment group (83.3% vs. 16.2%)  and IXE  can trigger IBD, while BRO did not trigger safety signals. We have also provided practical information about IBD and colitis types, time to symptom onset, and outcomes of treatment with IL-17 inhibitors, which had not been the focus of previous studies. A total of 388 case reports of primary suspect IL-17 inhibitor-related gastrointestinal inflammatory conditions were identified in the FAERS database, which included 268 IBD diagnoses and 120 colitis diagnoses. We detected four cases associated with BRO treatment, whereas the number of IBD cases that occurred after SEC treatment was 10 times greater than that observed after IXE therapy (89.7% vs. 9.3%). In a post-marketing study . 16.2%) , which i. 16.2%) . These dTo date, studies based on pooled data from clinical studies, observational studies, retrospective studies, and meta-analyses have not provided substantial detail regarding the clinical features of IBD induced by IL-17 inhibitors . Our stuIn the descriptive analysis, we observed that 91.2% of the cases were new-onset IBD, and about half of the new-onset cases were detected within 3 months of starting anti-IL-17 therapy. A study by At present, there is no clinical guidance for the management of patients with disease flare or new-onset IBD after IL-17 inhibitor therapy. Our study suggests that alternative treatments to improve gastrointestinal symptoms and treatment of the underlying pathology have included mainly corticosteroids and TNF antagonists, as either monotherapy or combination therapy; mesalazine and other biological agents (such as ustekinumab) were shown to be effective as well. Corticosteroids were used in 66.7% of cases in our study, and the type, dose, and duration in each case depended on the improvement of symptoms. Discontinuation of IL-17 inhibitors resulted in substantial improvement of symptoms in all patients, except for two who experienced relapse after 2 or 3 months of remission. Generally, the prognosis of IBD related to IL-17 inhibitors was excellent, and we observed that most patients (66.7%) recovered within 4 weeks. However, we still recommend that these patients be followed up for a longer period to monitor for potential recurrence.via prompt evaluation, cessation of IL-17 inhibitors, and initiation of alternative treatment  can generally lead to clinical remission of IBD. Further prospective studies are needed to evaluate and confirm risk factors for development of IBD associated with IL-17 inhibitor therapy.Our retrospective analysis demonstrated that use of IL-17 inhibitors is associated with exacerbation or new-onset of IBD and colitis within 6 months of therapy. Rheumatologists and dermatologists should be on alert for gastrointestinal symptoms, such as diarrhea, bloody diarrhea, abdominal pain, and fever, along with elevated WBC count, ESR, C-reactive protein, and fecal calprotectin levels during IL-17 inhibitor therapy. It is essential to complete a comprehensive patient history prior to the initiation of treatment, include appropriate workup to monitor intestinal inflammatory markers, such as fecal calprotectin, and perform colonoscopy during treatment to help identify early symptoms and prevent severe complications. Early detection of adverse events


Klebsiella pneumoniae (CRKP) is a serious public health issue. The study aimed to identify the antimicrobial resistance and accessory genes, the clonal relatedness, and the evolutionary dynamics of selected CRKP isolates recovered in an adult and pediatric intensive care unit of a tertiary hospital in Greece. Methods: Twenty-four CRKP isolates recovered during 2018–2022 were included in the study. Next-generation sequencing was performed using the Ion Torrent PGM Platform. The identification of the plasmid content, MLST, and antimicrobial resistance genes, as well as the comparison of multiple genome alignments and the identification of core genome single-nucleotide polymorphism sites, were performed using various bioinformatics software. Results: The isolates belonged to eight sequence types: 11, 15, 30, 35, 39, 307, 323, and 512. A variety of carbapenemases  and resistance genes were detected. CRKP strains shared visually common genomic regions with the reference strain (NTUH-K2044). ST15, ST323, ST39, and ST11 CRKP isolates presented on average 17, 6, 16, and 866 recombined SNPs, respectively. All isolates belonging to ST15, ST323, and ST39 were classified into distinct phylogenetic branches, while ST11 isolates were assigned to a two-subclade branch. For large CRKP sets, the phylogeny seems to change approximately every seven SNPs. Conclusions: The current study provides insight into the genetic characterization of CRKP isolates in the ICUs of a tertiary hospital. Our results indicate clonal dispersion of ST15, ST323, and ST39 and highly diverged ST11 isolates.Background: Carbapenem-resistant  Klebsiella pneumoniae is recognized as an important Gram-negative opportunistic pathogen that causes community- and hospital-acquired infections globally [K. pneumoniae has become a major public health problem, causing a significant increase in morbidity and mortality worldwide [K. pneumoniae (CRKP) has generally been considered an increasingly serious issue for clinical practice due to the limitation of therapeutic options [globally . It can globally ,3,4. Theorldwide . The spr options ,7,8. Gre options ,11,12,13 options .K. pneumoniae carbapenemase (KPC) being the most prevalent, followed by Verona integron-encoded metallo-β-lactamase (VIM), New Delhi metallo-β-lactamase (NDM), and OXA-48 carbapenemases [OmpK35, OmpK36, and OmpK37, is considered a secondary mechanism in the emergence of CRKP [The rapid dissemination of CRKP is closely related to the antimicrobial resistance genes carried by plasmids and transferable genetic elements ,16,17,18enemases ,21,22. T of CRKP ,24,25. T of CRKP .K. pneumoniae strains, as well as for the surveillance of virulence genes [The application of next-generation sequencing (NGS) has become a powerful tool for obtaining whole genome sequences (WGS), resulting in genetic characterization of the strains and a better understanding of the genomic diversity, providing fast and in-depth information about bacterial pathogenicity . WGS anace genes . The aimblaKPC showed susceptibility to ceftazidime-avibactam (CAZ-AVI). The phenotypic resistance rates to antimicrobials of CRKP isolates are shown in the heatmap in All CRKP isolates were resistant to β-lactam antibiotics . Nineteen isolates (79.2%) were resistant to at least one aminoglycoside, 23 isolates (95.8%) showed resistance to quinolones, eight isolates (33.3%) were resistant to fosfomycin and tigecycline, and 21 (87.5%) to cotrimoxazole. Four out of five isolates that carried the class A β-lactamase The CRKP isolates belonged to eight different STs: ST11 , ST39 , ST323 , ST15 , ST307 , ST30 , ST512 , and ST35 .The resistance genes and the plasmid content per isolate are detailed in mrk cluster (type 3 fimbriae); siderophores, as yersiniabactin genes  were present in 17 isolates; and genes encoding aerobactin siderophore (iut) were identified in five isolates . In addition, the kfu gene  was detected in four isolates. The virulence factors detected included fimbrial genes, as 23/24 isolates possessed the acr, mar, sox, rob, ram, sdiA, fis, and envR) or the OqxAB efflux pump (oqx and rar) were present in all isolates . It was found that isolates belonging to the same ST shared many identical regions with the reference strain. CRKP isolates C4112 and C746 of the ST11, D2452, and D6184 of the ST39, Z866 of the ST323, and B2562 of the ST15 displayed visually the highest similarity with the reference strain throughout their whole genome .The length of the multiple core genome alignments of all CRKP isolates was 1,892,845 base pairs. The core genome length corresponds to about 36.1% of the reference genome length. Nearly 1.9 million columns in the core genome alignment  showed identical nucleotides for all isolates, meaning that 1.5% of the genome is polymorphic. A phylogenetic tree including the cgSNP-based phylogenetic analysis of all isolates, along with the reference strain, is seen in When performing all possible pairwise analyses among ST15 isolates, an average of 17 recombined SNPs (range 5–26) were detected; the SNP diversity along the core genome was very low, that is, 0–3 cgSNPs per kilobase. ST323 CRKP strains presented an average of six recombined SNPs (range 5–6), and the SNP diversity was extremely low, that is, 0-2 cgSNPs per kilobase. ST39 CRKP strains displayed an average of 16 recombined SNPs (range 4–43), with the SNP diversity being low, that is, 0–5 cgSNPs per kilobase. Finally, ST11 CRKP strains showed an average of 866 recombined SNPs (range 1–1652), with the SNP diversity being clearly higher, that is, 0–66 cgSNPs per kilobase.−4) (−3) (−3), for instance, A5051 and C2482  G, the im−4) A and com−4) C. For so−4) (−3) G, the fr−4) (−3) B, until nd C2482 C,G. The nd C2482 D–F.n = 2, and stretches were normally around 7–12 consecutive SNPs and very rarely longer than 15–20 consecutive SNPs .ven SNPs . In addiThe present study included 24 CRKP isolates collected from June 2018 to July 2022 from 18 patients hospitalized in the ICU  and six patients hospitalized in the PICU  of a tertiary university-affiliated general hospital in Greece. The median age of the adult patients was 57.5 years (range 30–88), while the median age of the children was 6.2 years (range 0.25–17). The selection of the isolates was based on the type of carbapenemase(s) previously detected in order to include all possible combinations, as they were pre-screened as previously described . Eight iThe study was approved by the Ethics Committee of Aristotle’s University Medical Faculty .CRKP isolates were identified through the automated system VITEK 2  using the Gram-negative identification card (GN ID). Antimicrobial susceptibility testing (AST) of isolates was performed using the AST318 and XN10 cards, while the use of minimum inhibitory concentration (MIC) test strips  was applied to confirm susceptibility to ceftazidime-avibactam (CAZ-AVI). Identification and susceptibility cards were interpreted according to the manufacturer’s instructions. The Clinical and Laboratory Standards Institute (CLSI) susceptibility breakpoints were applied for the interpretation of results . The breAll CRKP isolates were previously screened for carbapenemase production using a lateral flow immunochromatographic assay (LFIA) . The results were confirmed by multiplex PCR in a single reaction following a modified protocol that was previously described ,31.Bacterial DNA was extracted using the DNA extraction kit . The DNA concentration (sample starting concentration between 10–100 ng/μL) was measured using the Qubit double-strand DNA HS assay kit . NGS was performed using the Ion Torrent PGM Platform (Life Technologies Corporation). All procedures regarding purification, ligation, barcoding, library preparation, emulsion PCR, and enrichment were performed following the manufacturer’s instructions. PCR products were loaded on an Ion-316™ chip. The Ion PGM Hi-Q View (200) chemistry was applied using the Ion PGM Hi-Q view sequencing kit (A25592). K. pneumoniae NTUH-K2044 strain (GenBank accession number NC_012731) was used as a reference sequence, which is an invasive and hypervirulent K. pneumoniae strain with the hypervirulent plasmid pK2044 [The software Geneious Prime version 2021.2.1 and BLAST Ring Image Generator (BRIG) were used to produce genome assemblies and annotation data ,33. K. pd pK2044 .https://bigsdb.pasteur.fr/cgi-bin/bigsdb/bigsdb.pl?db=pubmlst_klebsiella_seqdef (accessed on 20 October 2022).The sequence types (ST), plasmid types, and antimicrobial resistance genes were identified through the Center for Genomic Epidemiology website using the MLST web server and the related online databases, PlasmidFinder version 2.0 and Resfinder version 4.1, respectively ,36,37. Thttps://realphy.unibas.ch/realphy/ (accessed on 15 November 2022) was used to identify relevant SNP sites for core genome phylogenetic analysis [K. pneumoniae NTUH-K2044 sequence was used as a reference for the multiple alignments and the visual comparison of the whole genomes [T that a randomly chosen position in the core genome alignment has been overwritten by recombination in its history was calculated, that is, the time since the genetic ancestors of the position in the alignment diverged from a common ancestor.The BRIG software was used to visually compare multiple genome alignments using a default minimum threshold of 50% . The refanalysis . Default genomes . The pro genomes . Pairwis genomes . The cal genomes . In addi genomes . MoreoveblaNDM-1, and two of them (8.3%) transferred concurrently blaNDM-1 and blaOXA-48. Six out of 10 NDM-producers (60.0%) were assigned to ST11, which is common in NDM-positive CRKP strains and is globally distributed, especially in Asia and particularly in China, but also in Europe [K. pneumoniae ST11 clonal strain occurred during 2011–2012 [K. pneumoniae ST11 clinical isolate co-producing NDM-1 and OXA-48 carbapenemases was reported in 2016 [In the current study, the WGSs of 24 CRKP isolates were analyzed. It was found that they belonged to eight different STs, with ST11 and ST39 being the most prevalent. Ten out of 24 isolates (41.6%) carried n Europe ,44. In G in 2016 . NDM-1-p in 2016 . However in 2016 .blaKPC-2, while one of them (14.3%) harbored blaKPC-33 (a KPC-2 variant). CRKP strains belonging to ST39 have been detected only in sporadic cases so far, like ST39 carrying blaKPC-2 [blaVIM-1 [blaKPC-2 and blaVIM-1 [blaNDM-1, blaKPC-2, and blaOXA-48 in ST39 CRKP isolates [ST39 was also the most prevalent type, encompassing seven isolates (29.1%); two of them carried blaKPC-2  or blaVIblaVIM-1 . ST39 isblaVIM-1 . The preblaVIM-1 . Recentlisolates . Three Cisolates ,51. Anotisolates . It is oisolates ,54.armA, rmtB). In addition, 12 of these isolates carried at least one gene of the three AME classes . A disagreement between phenotype and genotype has been detected in five isolates (20.8%) . A disagreement of approximately 10% between genotype and phenotype for aminoglycosides for the VITEK 2 automated system has been reported previously [Twenty of the 24 isolates harbored at least two genes encoding aminoglycoside-modifying enzymes (AMEs), and six of them carried one 16S rRNA methylase gene -Ib-cr gene  has been identified in 16 isolates, as previously described [Fluoroquinolone resistance was observed in seven isolates (29.2%), which carried the plasmid-encoded isolates . The mulisolates , while tescribed .fosA was detected in all isolates, but 16 of them (66.7%) were phenotypically susceptible, evidenced by an increase in the MIC, as previously described [sul or dfrA) were found in all isolates except one. The Inc-type plasmids detected in all isolates and the Col-type plasmids detected in 15 isolates have been reported in other studies [For fosfomycin resistance, the gene escribed . Resista studies ,59.mrk cluster, characteristic of type 3 fimbriae, which promotes bacterial adhesion to both abiotic and biotic surfaces [K. pneumoniae [In terms of adhesive structures, almost all isolates possessed the surfaces . Regardisurfaces , while aeumoniae . The acqeumoniae .NGS allows for more accurate analyses regarding the clonal relatedness of strains compared to conventional typing methods . SeveralIn pairwise comparisons, isolates of ST15, ST323, and ST39 presented an average of 17, 6, and 16 recombined SNPs, respectively, implying the clonal relatedness of these isolates in accordance with their ST classification. Similarly, Miro et al. report the clonal relatedness of OXA-48-producing ST405 CRKP strains further studied with core genome MLST, showing between 6 and 17 cgSNPs . MoreoveOn the contrary, ST11 isolates showed an average of 866 cgSNPs, indicating the polyclonality of these isolates. This indication is further strengthened by the variability in their content of carbapenemase and antimicrobial resistance genes  and by tE. coli isolates, and they disclosed a similar C/S ratio [In addition, the current study highlighted novel ways in which these cgSNPs could be used to quantify phylogenetic structures and the role of recombination in genome evolution. It was shown that there are pairs of closely related isolates and that most of their DNA has been clonally inherited; however, most of the changes are derived from recombination events. When focusing on the core genome alignment, it has been shown that, until the current genetic state of CRKP strains, each genomic locus has been overwritten thousands of times by recombination in the history of their evolution. Sakoparnig et al. reached the same conclusions when focusing on /S ratio . Moreove/S ratio . Several/S ratio ,68. To tOne of the basic challenges among WGS-based typing methods is the definition of a cut-off value for the identification of the clonal relatedness between isolates; however, this remains doubtful, as it seems to be not only species-dependent but also population-dependent ,67,69. TIn conclusion, the current study provides insight into the genetic characterization of CRKP isolates circulating during a 4-year period in the ICUs of a Greek tertiary hospital. Phylogenetic analysis showed that some of them were epidemiologically related. Given that NGS has become more affordable, its use in hospital settings is extremely helpful, as the obtained data could guide infection control and prevention strategies.


Panax ginseng, a historical herbal plant used in East Asia, have been used as possible options for curing inflammatory diseases. All of the ginsenosides tested target different steps of the NF-κB signaling pathway, ameliorating the symptoms of severe illnesses. Moreover, ginsenosides inhibit the NF-κB-mediated activation of cancer metastasis and immune resistance, significantly attenuating the expression of MMPs, Snail, Slug, TWIST1, and PD-L1. This review introduces current studies on the therapeutic efficacy of ginsenosides in alleviating NF-κB responses and emphasizes the critical role of ginsenosides in severe inflammatory diseases as well as cancers.Nuclear factor kappa B (NF-κB) signaling pathways progress inflammation and immune cell differentiation in the host immune response; however, the uncontrollable stimulation of NF-κB signaling is responsible for several inflammatory illnesses regardless of whether the conditions are acute or chronic. Innate immune cells, such as macrophages, microglia, and Kupffer cells, secrete pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-1β, via the activation of NF-κB subunits, which may lead to the damage of normal cells, including neurons, cardiomyocytes, hepatocytes, and alveolar cells. This results in the occurrence of neurodegenerative disorders, cardiac infarction, or liver injury, which may eventually lead to systemic inflammation or cancer. Recently, ginsenosides from  Inflammation is a pre-emptive defense mechanism against infections to protect the host’s body ; howeverNuclear factor kappa B (NF-κB) was first discovered by Ranjan Sen and David Baltimore in 1986 ,8. ThereiNOS), and pro-inflammatory cytokines, such as interleukin (IL)-1, IL-6, and tumor necrosis factor-alpha (TNF-α) . Mo. Mo42]. Panax ginseng is known to have various health benefits, which are applied widely, such as improving psychologic function or reducing stress and anxiety. Twenty days of 40 μM/kg of ginsenoside Rh2 administration recovered spatial and non-spatial memory in a sleep-deprivation-induced ICR mouse model by suppressing oxidative stress and antioxidant activity . In. In66]. P. ginseng mainly targets NF-κB activation. The P. ginseng saponin fraction with a rich amount of ginsenosides blocks the phosphorylation of IKKα/β, downregulating IκBα degradation and eventually inhibiting the translocation of NF-κB subunits [Inflammation is initiated by various molecular pathways. Mitogen-activated protein kinase (MAPK), which triggers the phosphorylation-induced activation of AP-1 subunits, activates pro-inflammatory signals that enhance T-cell differentiation and B-cell class switching . The Jansubunits . Interessubunits . The mecsubunits .In the central nervous system (CNS), microglia play a key role in innate immunity. Microglia can be either polarized into M1 microglia, which secrete pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, or M2 microglia, which are responsible for neurogenesis and remyelination . MicroglStroke is the second most fatal disease following cancer. About 13 million new incidents of stroke are reported annually . SurprisiNOS, COX-2, IL-1β, IL-6, and TNF-α by suppressing p65 activity, eventually attenuating stress-induced inflammation. Rg1 also recovered the cuprizone (CPZ)-induced populational increase in microglia [Ginsenosides not only recover brain damage, but they also cause behavioral improvements. For instance, 20(S)-PPD attenuated depression and improved the sucrose preference of Sprague Dawley rats with chronic unpredictable mild stress (CUMS)  in additicroglia . Ionizedicroglia . The oraTo sum up, ginsenosides modified pro-inflammatory microglial activities by modulating the NF-κB signaling pathway; therefore, they can be used as a possible option for neural disorders.According to the studies from the American Heart Association in 2013, patients with heart failure in the United States were projected to be 3% of the population by 2030 . CurrentJin et al. used human umbilical vascular endothelial cells (HUVECs) to examine the therapeutic potential of ginsenoside Rh1 on LPS-induced inflammation . LPS treGinsenoside Rg1 and Rg3 both reduced NLRP3 inflammasome activity in cardiomyocytes ,95. Rg1 The above evidence suggests that various ginsenosides could protect endothelial cells, cardiomyocytes, and heart myoblasts from inflammatory responses stimulated by the NF-κB signaling pathway, attenuating heart failure.The National Vital Statistics Report showed that about 4.5 million adults in the United States had chronic liver disease in 2017 . GloballAccording to bioinformatics analyses, ginseng is known to have 31 bioactive ingredients that show anti-ALD properties . Among tIn NAFLD patients, excessive fat accumulation in the liver by a high-fat or high-cholesterol diet puts a strain on liver function ,108,109.20(S)-Rh1 inhibited the phosphorylation of p65, inactivating the downstream NLRP3 inflammasome activation in the liver tissue of C57BL/6 mice induced by a high-fat diet accompanied with a streptozotocin (STZ) injection . InteresLin et al. discovered the fact that Rb2 ameliorates fat accumulation and induces a decease in body weight in C57BL/6 mice fed a high-fat diet . The daiThe above studies demonstrate that ginsenosides can be applied to chronic liver diseases or metabolic disorders by reversing Kupffer cell polarization and ameliorating lipotoxicity via targeting the NF-κB signaling pathway.Pulmonary inflammation, affecting more than 510 million patients globally, is a major cause of respiratory disorders such as acute respiratory distress syndrome (ARDS), asthma, and COPD . PulmonaCigarette smoke is the primary cause of COPD. According to Li et al., when primary airway basal cells from healthy donors are exposed to cigarette smoke, the activation of the NF-κB signaling pathway is induced ; howeverGinsenoside Rb1 deactivates the NLRP3 inflammasome, which participates in IL-1β maturation via caspase-1 activation . IL-1β iA daily oral injection of 30 mg/kg of Rg1 also alleviates acute pulmonary inflammation (ALI) induced via the intratracheal administration of LPS . Rg1 ameIn summary, ginsenosides can also be applied as drugs against inflammatory pulmonary diseases since ginsenosides can successfully ameliorate immune cell infiltration to the alveolar space, lung edema, the thickening of the alveolar wall, and alveolar cell injury by regulating the NF-κB signaling pathway.Kidney inflammation, which accounts for 40% of kidney disease, affects kidney function and its ability to filter blood as well as excrete waste through urine. Diabetic nephropathy (DN) is one of the leading causes of chronic kidney disease, especially in developed countries . In the The levels of TNF-α and IL-1β were found to be regulated by 20(R)-ginsenoside Rg3 in a DN mouse model. To induce DN, a high-fat diet (HFD) was continuously provided and STZ (100 mg/kg) was injected. NF-κB was overexpressed in the HFD/STZ group compared to the normal group, whereas reduced NF-κB expression was observed in the 20(R)-Rg3 treated group. Moreover, the increased expression of p-NF-κB, p-IκBα, and p-IKKβ was downregulated by 20(R)-Rg3 at the protein level . This stThe protective effect of ginsenoside Rg5 has been significantly shown in HFD/STZ-induced diabetic mice. The expression of IL-6, TNF-α, and IL-18, the main inflammatory cytokines, are downregulated in renal tissues and sera following high doses of Rg5. When NLRP3 is activated an inflammasome complex is formed, which produces and secretes inflammatory cytokines—IL-1β. A Western blot analysis found that elevated levels of NLRP3, pro-IL-1β, and IL-1β were reduced in the kidneys of the DN group by Rg5. In addition, Rg5 significantly ameliorates the level of NF-κB p65 expression, which illustrates the fact that Rg5 plays a protective role in DN kidneys by modulating the NF-κB pathway .Using C57BL/6 mice with contrast-induced nephropathy (CIN), researchers have demonstrated that ginsenoside Rb1 (GRb1), a key active component of ginseng, dose-dependently suppresses the expression of IL-6, IL-1β, and TNF-α. Moreover, high mobility group box 1 (HMGB1), an extracellular inflammatory cytokine involved in transcriptional regulation , is knowOverall, these results imply that these ginsenosides can be applied in diabetic kidney injury by blocking the secretion of inflammatory cytokines in addition to alleviation of oxidative stress and apoptosis via mediation of the NF-κB signaling pathway.Colitis refers to inflammation of the colon, as well as a type of digestive disorder. A range of causes can attack the lining of the colon, including dietary factors, infections, and autoimmune disorders . EvidencLactobacillus fermentum induces the production of anti-inflammatory cytokines such as IL-10 [Recently, gut microbiota are some of the hot keywords related to intestinal health. The gut microbiota refer to the numerous bacteria living in the human gut, forming a complex ecosystem. Gut bacteria regulate a host’s immune system by modulating epithelial mucosa and inflammatory cytokine production . For insas IL-10 . On the as IL-10 . Thus, tB. Furthermore, microbial dysbiosis was ameliorated by Rk3 via its suppressive effect on pro-inflammatory bacteria, such as Bacteroidete.Chen et al. examined the role of Rk3 in obesity-induced colitis . Rk3 dimB in LPS-induced human intestinal epithelial LS174T cells. This effect was also investigated at an in vivo level in a UC model induced by dextran sulfate sodium (DSS). The expression of IL-1β, IL-6, COX-2, TNF-α, and ICAM was repressed by Rc. Specifically, the researchers focused on the function of the farnesoid x receptor (FXR), a nuclear receptor activated by bile acids, in the inflammatory response of the intestine [−/− mice. Their results indicate that Rc suppressed the inflammatory response and recovered damage to intestinal barriers [Ginsenoside Rc (Rc) inhibits the expression of IL-1β, TNF-α, and IL-6, as well as the nuclear translocation of NF-κntestine . The mRNbarriers .Lactobacillus and Ruminococcus, was increased while the population of pro-inflammatory bacteria, such as Alloprevotella, was decreased [In acute radiation proctitis (ARP), the effect of ginsenoside Rg3 (GRg3) was investigated in an ARP rat model. Rg3 plays an active role in anti-inflammation dose-dependently by restricting the expression of TLR4, MyD88, NF-κB p65, IL-10, and IL-1β at the mRNA and protein levels. This indicates that GRg3 regulates NF-κB activation via MyD88-dependent TLR4. In addition, imbalances in the composition of gut microbiota were improved after treatment with GRg3, insofar that the diminished population of immunomodulatory microbiota, such as ecreased .In sum, ginsenosides have a potential therapeutic effect on colitis by regulating as well as protecting the intestinal mucosa and altering the composition of microbiota.Bone is a living growing tissue that plays an important role in supporting the body and protecting internal organs. If abnormal bone function occurs, bone fractures can be induced through decreased bone microstructure and bone strength. Activated T and B cells promote bone loss by secreting cytokines, including TNF-α, RANKL, and IL-17A . Common In OP, fractures occur easily due to weakened bone strength, which mainly occurs in the spine and wrist bones but may affect the whole body. When the amount of bone removed and decomposed without the proper formation of new bones becomes excessive, a lack of bone mass ensues . OP can OA is a degenerative joint disease that impacts cartilage, which is the protective tissue that covers the ends of bones in a joint. This disease can affect any joint, but mostly affects the knees and is prevalent globally in those older than 60 years old . ChronicThe therapeutic effects of Rb1 are shown in both OP and OA. First, in a dexamethasone (DEX)-induced rat model of OP, the NF-κB p65 protein expression was inhibited. According to recent studies, an aryl hydrocarbon receptor (AHR), related to immune responses, triggers the growth and differentiation of osteoblasts, which leads to the recovery of bone injury. This study demonstrated that, in OP rats, AHR elevated the level of proline and arginine-rich end leucine-rich repeat protein (PRELP), known as a factor involved in the inhibition of osteoclastogenesis . SubsequRb1 was also examined in a rabbit knee OA model. The increased expression of TNF-α, caspase-3, and BAX was reversed in the Rb1-treated group. Rb1 also diminished PGE2 production and MMP expression by targeting p-Akt/Akt, p-P65/NF-κB, and p-P38/P38 proteins in a dose-dependent manner, which was confirmed through macroscopic images that showed recovered cartilage injuries. These results indicate that GRb1 can restore an impaired articular cartilage area by downregulating the level of MMPs through the suppression of apoptosis and the NF-κB signaling pathway .P. gingivalis LPS were shrunk by Rb3 under non-cytotoxic conditions. Rb3 also attenuated p38 MAPK, AKT, and NF-κB activation in human periodontal ligament cells (HPLCs). In particular, Rb3 significantly reduced TLR2 mRNA expression. This tendency was demonstrated in an experimental periodontitis rat model. Osteoclast genesis and bone loss were diminished after Rb3 treatment. These data support the protective effect of Rb3 against alveolar bone resorption with the deactivation of the immune response [Lastly, the impact of Rb3 on alveolar bone resorption was explored by Sun et al. Enhanced IL-8, IL-6, and IL-1β levels induced by response .In conclusion, these ginsenosides can be considered therapeutic agents of bone disorders by differentiating osteoblasts and through the downregulation of NF-κB signaling pathways.Cancer is a major public health concern with a high mortality rate. Approximately two million new cases of cancer and 0.6 million deaths are reported annually in the United States . AlthougThe invasion and migration of cancer cells result in tumor metastasis, making treatment with surgical resection difficult. Malignant tumor cells lose cell–cell adhesion capacity and detach from the primary tumor mass . The disOncogenic receptors, especially receptor tyrosine kinases such as epidermal growth factor (EGFR), proliferate the activation of protein kinase B (Akt) . Akt actThere are thousands of studies on the anticancer capacity of ginsenosides ,170,171.Recent studies on Rh1, Rk1, and Rg5 have shown that ginsenosides can block the translocation of NF-κB subunits, which reduces the NF-κB-mediated expression of MMPs ,173. SinThe therapeutic activity of Rk1 was also evaluated in an A549 xenograft mouse model . Rk1 treGinsenoside Rh2 is well known for its anticancer properties . Nearly P. ginseng has been studied for thousands of years; however, research on its modulatory effect on the NF-κB signaling pathway does not have a long history. The first paper reported, published in 2001, deals with P. ginseng’s antitumor activity, derived from its inhibitory effects on the NF-κB and MAPK signaling pathways examined in human leukemia cells [P. ginseng, ameliorated p65 phosphorylation, reducing liver inflammation in a mouse NASH model [The therapeutic efficacy of ia cells . RecentlSH model . MeanwhiSH model .P. ginseng. This review presents recent studies that have demonstrated the anti-inflammatory and anticancer activities of ginsenosides, especially in targeting the NF-κB pathway (summarized in In this review, we narrowed down the effects of each single ginsenoside rich in The effects of ginsenosides on chronic diseases are quite promising, since small doses of ginsenosides showed significant therapeutic effects. As we can see in Panax ginseng that exert anti-inflammatory, antimicrobial, antidiabetic, and anticancer activities. The global ginseng market size is estimated to be USD 2084 million, and the development of extraction techniques allows for the mass production of ginsenosides, such as Rg1, Re, Rh2, and Rg3, in tens of kilograms [Ginsenosides are bioactive compounds abundant in ilograms ; howeverilograms . Moreoveilograms . Ginsenoilograms . The reailograms ,187. Proilograms ,189; forilograms .In conclusion, based on this review, we hope that more researchers understand the specific mechanisms of the therapeutic efficacy of ginsenosides, and that more clinical trials are conducted to design commercialized drugs with ginsenosides as active compounds.


RAMP1, CRIP1, CORT, CHST13, and DDX60L) in OS patients that can be further explored for therapeutic applications.Osteosarcoma (OS), which accounts for about 35% of bone malignancy, shows aggressive progression in adults, adolescents, and children. Neoadjuvant chemotherapy provides an improvement in survival for OS patients; however, a molecular level understanding of the disease mechanisms is needed. We utilized publicly available multiomics data from the TARGET database, to perform survival analyses, differential expression analyses, mutational analyses, and subtyping using integrative clustering. Our results have identified several prognostic biomarkers  for mRNA, lncRNA, and miRNA expression; DNA methylation; and mutation data from the TARGET dataset revealed two distinct molecular subtypes, each with a distinctive gene expression profile. Between the two subtypes, three upregulated genes, POP4, HEY1, CERKL, and seven downregulated genes, CEACAM1, ABLIM1, LTBP2, ISLR, LRRC32, PTPRF, and GPX3, associated with OS metastasis were found to be differentially regulated. Thus, the molecular subtyping results provide a strong basis for classification of OS patients that could be used to develop better prognostic treatment strategies.Osteosarcoma (OS) is a common bone malignancy in children and adolescents. Although histological subtyping followed by improved OS treatment regimens have helped achieve favorable outcomes, a lack of understanding of the molecular subtypes remains a challenge to characterize its genetic heterogeneity and subsequently to identify diagnostic and prognostic biomarkers for developing effective treatments. In the present study, global analysis of DNA methylation, and mRNA and miRNA gene expression in OS patient samples were correlated with their clinical characteristics. The mucin family of genes,  Osteosarcoma (OS), a highly aggressive bone marrow malignancy that originates in mesenchymal tissue, accounts for about 35% of bone cancer cases worldwide. Other bone tumors include chondrosarcomas 25%), and Ewing sarcomas 16%) [6% [1,2,35%, and EFor various cancer types, including OS, genetic heterogeneity due to genomic instability poses challenges to the efficacy of current treatment strategies ,11,12, dhttps://dcc.icgc.org/, accessed on 1 December 2021), The Cancer Genome Atlas (TCGA), and Gene Expression Omnibus (GEO) for cancer sub-classification studies on the genomic, transcriptomic, and other clinical data.In parallel, a variety of statistical and machine learning approaches such as Support Vector Machines (SVMs) and Deep Neural Networks (DNN) have been developed for molecular characterization and subclassification of cancers by assessing multiomics data ,26. ThesIn OS, the common subclassification method relies upon the characteristics of the dominant matrix present in the tumor. Conventionally, tumors occurring in fibrous tissue, bone, and cartilage are referred to as fibroblastic, osteoblastic, and chondroblastic OS, respectively ,29. ThesUnlike traditional histology-driven classifications, molecular subtyping of OS requires the identification of gene-based biomarkers and effective classifiers that can distinguish subtypes based on the molecular data. Prognostic biomarkers provide information about the patients’ overall cancer outcome and help to identify an appropriate therapeutic approach to cure or manage the disease. There is no systematic genome-wide analysis of these prognosis factors in OS ,33,34,35Multiomic data: We downloaded gene expression, DNA methylation, miRNA expression, and clinical data for 85 common OS patients from Therapeutically Applicable Research To Generate Effective Treatments  using a Bioconductor tool, TCGAbiolinks  [GA data) . We systProtein coding and lncRNA data: TCGAbiolinks was used to query and download the HTSeq–FPKM (Fragments Per Kilobase of Exon per Million) gene expression count data from the TARGET-OS project. Genes having more than 20% missing values across patients were not considered further for analysis. Additionally, only those genes with CPM (count per million) values > 1 for more than 10 % samples were retained. Genes were annotated as protein-coding or lncRNA using gencode reference annotation v22. The normalized count data were gene median centered, and the top 2500 protein-coding and 500 lncRNA based on median absolute deviation (MAD) were retained for further clustering analysis.miRNA data: Normalized miRNA data were downloaded from NCI TARGET’s web portal. The dataset was first filtered by removing duplicated miRNA expression data. Further, clustering was performed using nonnegative matrix factorization (NMF) by retaining 190 (25%) of the most variable miRNAs.Methylation array data: The raw IDAT files from the methylation study  [p array)  were dowp array)  tool in p array) . This wap array) . CpG proMutation data: Mutation data from 123 OS patients was downloaded as a tab-delimited Mutation Annotation Format (MAF) file along with the scores, all-lesions, amplification, and deletion gene files generated by the Genepattern GISTIC module [n = 1107) having a mutation in more than 20% of the samples were retained in the final matrix for clustering analysis.C module . These dC module . Genes hClustering analysis: NMF based clustering was performed using nrun = 500 for the number of clusters, k = 2 to 10 for miRNA, mutation, protein-coding, lncRNA, and methylation data for the same set of patients across all datasets by using R package NMF. The preferred clustering result was determined by using the observed cophenetic correlation coefficients between clusters and the average silhouette width of consensus cluster members.Cluster-Of-Clusters Analysis (COCA): The R package, COCA [ge, COCA , an inteDifferential gene expression analysis: Differential gene expression (DGE) analysis between the samples belonging to the two molecular subtypes obtained from the COCA was performed using the Bioconductor tool, DESeq2. The Benjamini-Hochberg (BH) adjusted p-value cut-off of 0.01, and an absolute log2Fold Change of 1 was used to obtain the list of differentially expressed genes. The heatmap was generated using tidyheatmap package in R.Pathway analysis: All differentially expressed genes  between Subtypes 1 and 2 were used as input into QIAGEN IPA  for pathway enrichment analysis.Survival analysis: We performed survival analyses for protein-coding, lncRNA, and miRNA genes using the log-rank test and Cox-regression analysis  by constructing high and low expression groups, using the median expression of genes as the cut-off value. For gene expression analysis, we used HTSeq–FPKM gene expression count data of OS patients. The R tool, “survival” was used for survival analysis, and the Kaplan–Meier survival curve plots were generated for all analyses. In addition, a log-rank or Mantel–Haenszel test was conducted to assess differences between survival curves of the two groups and to calculate the p-values. A hazard ratio (HR) > 1 indicates that patients in the high expression group had low survival, and HR < 1 suggested high or better survival.Correlation analysis: Pearson correlation analysis between gene expression for the protein-coding genes was performed using the R cor.test function. The association was considered significant at a p-value < 0.05.In the present work, we have utilized the OS omics data of five distinct datatypes which include miRNA, mRNA, lncRNA, methylation, and mutation data, along with the survival data from public resources including the TARGET database and the PubMed repository. Initially, three data types  along with the survival information were used to understand the associations of these biomolecules to the survival of OS patients. The later part of the study mainly focused on COCA-based clustering of 85 patients using multi-level omics information including miRNA, mRNA, lncRNA, methylation, and mutation, resulting in the identification of two OS subtypes. Results from each analysis are described in the following sections.PABPC3, HRNR, KMT2C, and PRSS3 in more than 65% of the samples (MUC6 (80%), MUC12 (76%), and MUC4 (76%). An oncoplot of members of the MUC family genes with their mutation frequencies is shown in For mutational signature analysis, we read MAF files using maftool, which provided frequencies of six classes of the mutation types  in each sample. The contribution of each mutational signature was analyzed across the samples. Our analyses showed that the commonly mutated genes include  samples . A misseTEAD2, WWC1, AJUBA, CSNK1D, and LLGL2 in hippo signaling, IGF1R, RAC1, ERBB4, GRB2, and NF1 in RTK-RAS pathway, RFNG, LFNG, MAML3, APH1A, and NOTCH2 in Notch pathway, and WNT7B, LZTR1, DKK1, APC, and FZD6 in Wnt pathway. In the TP53 pathway, genes including CHEK2, and ATM mostly showed missense mutations (blue color), while the TP53 gene had multiple mutations including missense, Amplification (Amp), Deletion (Del), and Frameshift. NOTCH signaling pathways mostly showed Amp mutation as presented in brick color. Similarly, the genes LLGL2, CSNK1D, and AJUBA in the HIPPO pathway showed Amp mutation; while other genes, WWC1 and TEAD2 in the same pathway, were deleted. Cell cycle pathways include genes RB1 which showed multiple mutation types, whereas, other genes CCNE1 and CDK4 showed mainly amplification. Multiple types of mutation were identified in RB1 in Cell Cycle and TP53 in TP53 pathways, NAML3 in NOTCH pathways, MGA in MYC pathways, and NF1 in RTK-RAS pathways. In general, amplifications were most prevalent across pathways. The fraction of affected genes in the most enriched pathways included 94% in Hippo, 86% in Wnt, 84% in RTK-RAS, and 84% in Notch signaling pathways  using protein-coding HTSeq-FPKM gene expression identified 430 genes associated with OS prognosis . The genes with HR more than 4.5 include RAMP1, TAC4, MT1A, CRIP1, NHEJ1, TIMM23B, COL5A2, UFC1, and CORT. The higher expression of these genes indicate low survival of OS patients. In contrast, higher expression of genes such as METTL20, MYH10, PDE1B, CCDC42, and many more which got HR < 1 refers to better survival rates. In each category, survival plots for the top three potential genes are provided in p-value of all 430 genes are provided in p-value < 0.001) related to the survival of OS patients, are shown in The log-Rank test and cox-regression analysis (p < 0.05) in our analysis. As shown in p < 0.05. Only a few genes such as SOST and WNT5A in WNT signaling pathways, MAPK1 and PEBP1 in RTK-RAS pathways, and PIK3R3 and RPS6 in PI3K pathways, showed negative expression pattern within the pathways. In our analyses, none of the genes in the NRF2 and TGFβ signaling pathways were significant in survival and are, therefore, not shown in ATM in TP53 pathway, CDKN2A in Cell cycle, FAT1 and FAT3 in HIPPO signaling, MXl1 in MYC pathway, DTX3, DTX2, JAG2, HES4, and HES5 in NOTCH signaling showed HR values greater than 1 indicating their association with low survival in OS patients. Other genes such as LRP5, SOST, WNT1, RPS6 in different pathways also have HR value more than 1 with possible association with survival  or therapeutic targets , and aresurvival B. In conp-value < 0.01) or 142 lncRNAs  are correlated with survival. ELFN1-AS1 (HR > 1) is among the top 3 lncRNAs identified  in our study, which has been recognized as an important signature to predicting OS patient survival in a previous study [p-value < 0.05. Higher expression of these lncRNAs are associated with better survival of the patients; hence, they serve as potential biomarkers for OS prognosis. Survival plots of the top lncRNAs with HR > 1 and HR < 1 are provided in the Survival analysis in relation to lncRNAs showed that 36 lncRNAs (us study . Similarp < 0.05) or 31 miRNAs (p < 0.01), which showed association with the survival , resulting in three clusters each for protein-coding, lncRNA, miRNA, and methylation data, and four clusters for the mutation data .A matrix of clusters was generated and used as input for COCA, which resulted in two major integrated molecular clusters/subtypes of OS, consisting of 44 and 41 patients in Subtype-1 and Subtype-2, respectively. The matrix of clusters was plotted using plotMOC function. 2Fold Change > 1.5 and adjusted p-value < 0.05  using IPA identified WNT signaling and Axonal Guidance signaling pathways, which are related to the OS pathogenesis [Differential Gene Expression analysis was performed between the two molecular subtypes obtained from COCA using the Bioconductor package, DESeq2 . A totale < 0.05 A. Among e < 0.05 B clearlye < 0.05 B. Howeveogenesis ,51,52 , MUC12 (76% of the patients), and MUC4 (76% of the patients). Previously, Tirabosco et al. have reported MUC4 as the immunoreactive gene in fibromyxoid and sclerosing tumors in bone [MUC12 and MUC4 were identified as top-altered genes in a cohort of 21 OS patients [MUC15 which promote cell proliferation, migration, and invasion [Mutation analysis identified significant mutations in the Mucin (MUC) family of genes, notably  in bone . Similarpatients . Howeverinvasion , mutatedKMT2C, which is mutated in 67% of all OS samples, involved in OS carcinogenesis and progression [KMT2C, which can have histone modification-related activity in OS [Further, the gene gression . Similarty in OS . Furtherty in OS ,63,64.We also observed enrichment of pathway members that contained recurrent or known alterations that are likely to be cancer drivers or therapeutic targets in the Hippo, RTK-RAS, Notch, and Wnt signaling pathways  Figure . The HipRAMP1): a protein of calcium signaling receptor complex; Tachykinin Precursor 4 (TAC4), Metallothionein 1A (MT1A), CRIP1, and CORT. Our analysis showed that these genes are associated with low survival of OS patients based on their HR values  (CHST13 (HR: 2.4) is associated with low survival, while DDX60L (HR 0.37), METTL20 (HR 0.14), MYH10 (HR 0.16), and PDE1B (HR 0.15) are associated with higher survival outcomes in OS patients  . Similarpatients . These greceptor ,70. Knoc sarcoma .TAC4 and MT1A expression. Although the role of TAC4 in metastasis and prognosis of OS is unknown, other bioinformatic studies have shown a possible link [MT1A expression levels were used as one of the genes to develop a machine learning-based prognostic risk model in OS patients, suggesting that this gene is associated with OS survival [A lower survival rate in patients was related to high ble link . MT1A exsurvival .CRIP1 and CORT was associated with lower survival (p < 0.001). CRIP1 has been previously shown to be over-expressed in pre-therapeutic OS samples [CORT is an endogenous cyclic neuropeptide known to regulate the growth and metastasis of lung and thyroid cancer [CORT gene has also been associated with high-risk group OS patients [From our study, higher expression of  samples . CORT isd cancer ,75. The patients . In addi2Fold Change > 1.5 and adjusted p-value < 0.05), including genes associated with Wnt signaling, calcium signaling, and PI3K-Akt signaling pathways, were highly enriched and found related to OS pathogenesis.Our global clustering strategy built upon the multiomics landscape has identified two main subtypes , which opens up the potential for developing effective treatments. Differential expression and pathway enrichment analyses suggest that both subtypes had different deregulated pathways associated with OS pathology. We observed over 250 differentially expressed genes between two subtypes  were found to be highly mutated in our study. miRNAs: hsa-miR-190, hsa-miR-452, hsa-miR-142, hsa-miR-122, and hsa-miR-200, and lncRNAs: ELFN1-AS1, lnc01060, RP11-283C24.1, RP11-283C24.1, CTD-2078B5.2, RP11-78O7.2, and RP11-446N19.1 were also found to be significantly associated with the survival of OS patients; and therefore, interpreted as potential biomarkers that can to be further validated with experimental studies. Our detailed clustering approach using COCA has identified two main OS subtypes, which are significantly different at the molecular level. These findings offer a strong basis to understand the genetic heterogeneity of OS and develop next generation prognostic biomarkers for effective treatment of OS patients.This study has identified potential prognostic genes including


The rise of antimicrobial resistance has created an urgent need for antibiotic-alternative strategies for disease control and prevention. Host defense peptides (HDPs), which have both antimicrobial and immunomodulatory properties, are an important component of the innate immune system. A host-directed approach to stimulate the synthesis of endogenous HDPs has emerged as a promising solution to treat infections with a minimum risk for developing antimicrobial resistance. Among a diverse group of compounds that have been identified as inducers of HDP synthesis are polyphenols, which are naturally occurring secondary metabolites of plants characterized by the presence of multiple phenol units. In addition to their well-known antioxidant and anti-inflammatory activities, a variety of polyphenols have been shown to stimulate HDP synthesis across animal species. This review summarizes both the in vitro and in vivo evidence of polyphenols regulating HDP synthesis. The mechanisms by which polyphenols induce HDP gene expression are also discussed. Natural polyphenols warrant further investigation as potential antibiotic alternatives for the control and prevention of infectious diseases. Antimicrobial resistance has emerged as a major world health concern over past decades . ExcessiAlthough synthetic and recombinant HDPs have shown clinical promise  and are The innate immune system, consisting of physical, chemical, and cellular barriers, serves as the host’s first line of defense against a wide variety of insults. As an important chemical barrier of the innate immune system, HDPs are small peptides (approximately 15–45 amino acids) found in virtually all organisms including mammals, birds, reptiles, amphibians, plants, and fungi ,4,11.Two major HDP families in vertebrate animals are cathelicidins and defensins ,4. CatheHDPs are active against both Gram-positive and Gram-negative bacteria, fungi, parasites, and enveloped viruses, as well as cancer cells ,4. SeverIn addition to direct antimicrobial activity, HDPs possess immunomodulatory activities ,12 Figu. HDPs arAlthough they are capable of eliciting proinflammatory and immune responses to facilitate the clearance of pathogens, HDPs mediate largely anti-inflammatory responses during inflammation and infection by inhibiting the expression of proinflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α), while stimulating the production of anti-inflammatory cytokines such as IL-10 ,26 Figu. FurtherPsuedomonas aeruginosa rhamnolipids act as a PAMP to activate transcription factors, such as nuclear factor κB (NF-κB) and activator protein-1 (AP-1), causing induction of HBD2 in keratinocytes [As an important component of innate defense, certain HDPs such as human α-defensins and HBD1 are constitutively produced, while other HDPs like HBD2, HBD3, and HBD4 are induced upon infection and injury ,28. Pathinocytes ,30. Proiinocytes .3, zinc, β-glucan, fructan, and lactose are also capable of inducing HDP synthesis [Lactobacillus strains have the ability to upregulate β-defensin synthesis as well [Additionally, a diverse group of small-molecule compounds have been shown to induce expression of HDPs ,8,9,10.  as well . Recent  as well ,39,40,41Polyphenols are secondary metabolites produced abundantly in fruits, vegetables, tea, coffee, wine, and nuts as a mechanism of defense against pathogen invasion . PolyphePolyphenols are well-known natural antioxidants and anti-inflammatory agents ,43,44. TPolyphenols are also known to have anticancer activities and improve cardiovascular, metabolic, and neuronal health as supported by multiple epidemiological studies ,50,51. MA number of polyphenols have been shown with the capacity to enhance HDP expression at mRNA and/or protein levels both in vitro and in vivo across a number of animal species including humans, mice, pigs, chickens, fishes, and insects .HAMP) by 4-fold in human HepG2 hepatocytes and enhanced HAMP mRNA expression by over 600-fold in the liver of rats when injected intraperitoneally [LEAP2) in the liver [Bombyx mori, a silkworm, in response to dietary supplementation of quercetin [AvBD) genes in chicken HTC macrophage cells, further showing a strong synergy with butyrate [AvBD genes in the chicken ileum [Quercetin is capable of inducing the synthesis of HDPs in multiple animal species. Quercetin augmented the mRNA expression of hepcidin antimicrobial peptide  expression in SW620 colon epithelial cells [CAMP expression by approximately 100-fold in A549 cells, a human lung epithelial cell line [CAMP expression, but decreased DEFB1 expression in human HaCaT keratinocytes [DEFB1, DEFB2, DEFB3, and CAMP mRNA expression in human epidermal keratinocytes [CAMP, DEFB2, S100A7 (psoriasin), S100A8 , and S100A9  in human NHEK epidermal keratinocytes in response to stimulation with proinflammatory cytokines TNF-α, IL-17A, or IL-22 [Flavone induced human cathelicidin  and PBD3 in endometrial epithelial cells [PBD2 and PBD3 was further enhanced in porcine endometrial epithelial cells treated with polyinosinic:polycytidylic acid (Poly (I:C)) [Astragalus membranaceus, triggered the expression of multiple porcine HDP genes, PBD2, PEP2C, and PG1-5, in 3D4/31 alveolar macrophages [PBD2, PBD3, and PEP2C expression in porcine IPEC-J2 cells and also enhanced PBD3 expression in jejunal explants [Genistein, a soy isoflavone, upregulated ell line  and alsoinocytes . Similaral cells  and incral cells . Genistey (I:C)) , suggestrophages . Moreoveexplants .DEFB3 gene abolished the antiviral effect of EGCG, indicating that EGCG-induced upregulation of HBD3 synthesis is essential for the antiviral activity of EGCG [AvBD9 mRNA expression by 50-fold in chicken peripheral blood mononuclear cells (PBMCs) and further synergized with butyrate to induce AvBD9 expression by over 3500-fold [PBD1 and PBD2 mRNA and protein expression in a dose-dependent manner [Epigallocatechin gallate (EGCG), a flavanol of green tea, and theaflavins, flavanols of black tea, significantly enhanced mRNA expression and protein secretion of HBD1 and HBD2 in human gingival epithelial cells and oral epithelial cells, respectively ,70. EGCG of EGCG . Additio500-fold . In porct manner . Moreovet manner .Defb2 mRNA expression by 2-fold in the ileum compared to rats on a low-fat diet [REG3G) to levels similar to rats on a low-fat diet [S100A7 and S100A15 (koebnerisin), together with many other proinflammatory cytokines, in inflamed psoriatic keratinocytes [Anthocyanins such as cyanidin 3-O-glucoside increased total β-defensin protein levels in the intestinal mucosa of mice in an ovalbumin-induced food allergy model . A bluebfat diet . Mice fefat diet . Howeverinocytes .Defb3 in mice by nearly 10-fold and reduced the colonization of Candida albicans in the oral cavity [Galleria mellonella [AvBD9 mRNA expression by 100-fold in chicken PBMCs and further synergized with sodium butyrate to induce AvBD9 mRNA expression over 1600-fold [Ellagic acid, a hydroxybenzoic acid, induced both mRNA and protein expression levels of HBD2 in primary human gingival epithelial cells . Oral adl cavity . In the llonella . Anacard600-fold .CAMP mRNA and protein expression in human U937 monocytes, further showing a strong synergy with 1,25-dihydroxyvitamin D3 [CAMP mRNA and protein expression in both cultured human keratinocytes and mouse skin and further blocked invasion of Staphylococcus aureus into mouse skin [Defa3, Defa5, and Defa20 in the ileum of mice [HAMP mRNA expression in liver cells by 4-fold as well as an 8-fold mRNA increase in the liver of rats [AvBD9 mRNA expression and also synergized with sodium butyrate to induce AvBD9 expression in chicken PBMCs [Streptococcus pneumonia-induced expression of DEFB2 in human A549 lung epithelial cells [Pseudomonas aeruginosa or nontypeable Haemophilus influenzae [PBD3 and PG1-5 in porcine 3D4/31 alveolar macrophages [PBD3, PEP2C, and PG1-5 mRNA expression in porcine IPEC-J2 intestinal epithelial cells and porcine jejunal explants [Resveratrol, a well-known stilbenoid of grapes, has been found to regulate HDP expression both in vitro and in vivo across animal species. For example, resveratrol induced tamin D3 . Resverause skin . Similar of mice . Resvera of rats . Additioen PBMCs . While ral cells , the oppfluenzae ,85. Pterrophages . Isorhapexplants .CAMP mRNA in human U937 monocytes and HT-29 intestinal epithelial cells [DEFB1, HAMP, and LEAP2 in the liver and blood and also improved the survival of grass carp infected with Aeromonas hydrophilla [DEFB1, HAMP, LEAP2A, and LEAP2B mRNA expression in the intestine and exacerbate A. hydrophila infection in grass carp [HAMP in HepG2 cells [DEFB3 mRNA expression by 30-fold in human colonic epithelial cells [PBD3 expression in porcine IPEC-J2 cells [PBD3 in porcine IPEC-J2 intestinal epithelial cells, 3D4/31 alveolar macrophages, and jejunal explants [Several other classes of polyphenols have also been shown to regulate HDP expression. For example, curcumin, a curcuminoid isolated from turmeric, was found to cause a 3-fold induction of al cells . Dietaryrophilla . Howeverass carp ,89. NariG2 cells . Chalconal cells . More thJ2 cells . Of thosexplants .p-coumaric, gallic acid, vanilic acid, and trans-cinnamic acid [REG3B and 2-fold increase in REG3G mRNA expression in the ileum of mice [Defb1 and Defb2, as well as improved barrier junction in mice [Several studies have also revealed the benefits of polyphenol-rich foods, rather than an individual polyphenol, on HDP regulation. For instance, supplementation of pinto beans, which contain high amounts of phenolic compounds such as kaempferol, mic acid , caused  of mice . Dietary in mice .The mechanisms by which structurally diverse polyphenols regulate HDP expression are beginning to be revealed. The MAPK signaling pathway, the sphingosine-1-phopshate (S1P) pathway, the Kelch-like ECH-associated protein 1- nuclear factor erythroid 2-related factor 2 (KEAP1-NRF2) pathway, and epigenetics are involved in the regulation of certain HDP genes. In live animals, polyphenol-mediated alternations of intestinal bacterial metabolites are likely to contribute to enhanced HDP synthesis as well.DEFB1 and DEFB2 expression is diminished in the presence of ERK/12 and p38 MAPK inhibitors in human gingival epithelial cells [DEFB3 induction [PBD2 expression by EGCG is significantly reduced in porcine IPEC-J2 cells upon inhibition of p38 MAPK, but not ERK1/2 or JNK [The MAPK signaling pathway is essential for a variety of cellular processes such as stress response and cell proliferation and differentiation . The MAPal cells . Similarnduction . Inducti2 or JNK . While t2 or JNK ,71, poly2 or JNK ,62,71.CAMP gene expression [CAMP gene expression [CAMP gene expression, SIP generated by SPHK2 appears to be a negative regulator of CAMP expression [SPHK2 mRNA expression [SPHK1 mRNA expression [S1P is produced intracellularly from sphingosine on the cell membrane, acting as a signaling molecule involved in many cellular processes such as cell growth and cell trafficking . Genistepression . Resverapression . Two sphpression . While Spression . It is npression , and respression .HAMP gene promotor by inhibiting KEAP1 to allow increased binding of NRF2 to the antioxidant response element (ARE) site of target gene promoters [KEAP1 is a repressor of NRF2, a major transcription factor responsible for transcription of many genes with antioxidant properties . Thereforomoters  , which leads to a condensed chromatin structure associated frequently with diminished gene transcription . Methylacription ,109. 5-Apression . Polyphepression ,112, sugpression ,101. Hispression . Dependipression . Inhibitpression . Howeverpression , a commopression ,66.Allobaculum, Roseburia, Butyricicoccus, Bifidobacterium, and Anaerotruncus along with increasing SCFA production in mice [SLC5a8, a butyrate transporter, was observed in the colon of mice supplemented with polyphenol-rich extracts of cinnamon bark and grape pomace [Defb2 and SCFA receptor, Gpr43, in mice [REG3G and DEFB expression in intestinal epithelial cells [Most dietary polyphenol intake is unabsorbed in the small intestine, thus the gut microbiota is critically important for biotransformation and metabolism of these products . Polyphe in mice ,82, whice pomace . Supplem in mice . GPR43 ial cells . TherefoThe use of HDPs as antibiotic alternatives to infectious disease control and prevention is promising due to the beneficial antimicrobial and immunomodulatory properties of these peptides ,4,11. ExNatural polyphenols are known to be antioxidant, anti-inflammatory, anticancer, cardio-protective, and neuro-protective. Current research further highlights the ability of polyphenols to boost HDP synthesis and innate defense, suggesting its potential for anti-infective application. Further studies on the in vivo efficacy of these polyphenols in different animal disease models are required to demonstrate the utility of these natural compounds as effective antibiotic alternatives. The MAPK signaling pathway, the S1P pathway, and the KEAP1-NRF2 pathway have been shown to be involved in polyphenol-mediated induction of certain HDP genes. In addition, epigenetic modifications such as histone acetylation, histone methylation, and DNA methylation by polyphenols play a critical role in HDP induction as well. Moreover, bacterial metabolites, SCFAs in particular, contribute to HDP gene induction in vivo upon polyphenol consumption. Further investigations on the mechanisms of action of these polyphenols are warranted to fully realize their potential in antimicrobial therapy.Although polyphenols demonstrate many alluring health benefits, their use in human medicine has been limited due to the low oral bioavailability of many polyphenols . Polyphe