Introduction: Prostate cancer is the second leading cause of cancer death among men in the United States. Castration-Resistant Prostate Cancer (CRPC) often develops resistance to androgen deprivation therapy. Resistance in CRPC is often driven by AR variants and glucocorticoid receptor (GR). Thus, drugs that target both could be vital in overcoming resistance.Methods: Utilizing the STAR Drug Discovery Platform, three hundred medicinal plant extracts were examined across 25 signaling pathways to identify potential drug candidates. Effects of the botanical drug YIV-818-A, derived from optimized water extracts of Rubia cordifolia (R.C.), on Dihydrotestosterone (DHT) or Dexamethasone (DEX) induced luciferase activity were assessed in 22RV1 cells harboring the ARE luciferase reporter. Furthermore, the key active compounds in YIV-818-A were identified through activity guided purification. The inhibitory effects of YIV-818-A, RA-V, and RA-VII on AR and GR activities, their impact on AR target genes, and their roles in modifying epigenetic status were investigated. Finally, the synergistic effects of these compounds with established CRPC drugs were evaluated both in vitro and in vivo.Results: YIV-818-A was found to effectively inhibit DHT or DEX induced luciferase activity in 22RV1 cells. Deoxybouvardin (RA-V) was identified as the key active compound responsible for inhibiting AR and GR activities. Both YIV-818-A and RA-V, along with RA-VII, effectively downregulated AR and AR-V proteins through inhibiting protein synthesis, impacted the expression of AR target genes, and modified the epigenetic status by reducing levels of Bromodomain and Extra-Terminal proteins (Brd2/Brd4) and H3K27Ac. Furthermore, these compounds exhibited synergistic effects with apalutamide, darolutamide, or enzalutamide, and suppressed AR mediated luciferase activity of 22RV1 cells. Co-administration of YIV-818-A and enzalutamide led to a significant reduction of 22RV1 tumor growth in vivo. Different sources of R.C. had variable levels of RA-V, correlating with their potency in AR inhibition.Discussion: YIV-818-A, RA-V, and RA-VII show considerable promise in addressing drug resistance in CRPC by targeting both AR protein and GR function, along with modulation of vital epigenetic markers. Given the established safety profile of YIV-818-A, these findings suggest its potential as a chemopreventive agent and a robust anti-prostate cancer drug. Prostate cancer is the second leading cause of cancer death among men in the United States. According to the National Cancer Institute over 3 million men in the United States are living with prostate cancer, with 288,300 additional cases and 34,700 deaths expected in 2023. Androgen receptor (AR) signaling plays a key role in development of Benign prostatic hyperplasia (BPH) and prostate cancer . TherefoMany mechanisms for CRPC are proposed and some are responsible for abiraterone resistance and enzalutamide resistance. Resistance mechanisms include altering the metabolic enzymes expression favorable for DHT synthesis, AR gene amplification , AR mutaRubia cordifolia (R.C.), was discovered as a novel drug candidate. YIV-818-A, Deoxybouvardin (RA-V), or RA-VII were found to have the ability to effectively target both the androgen receptor and glucocorticoid receptor by down-regulating the androgen receptor protein, impairing the glucocorticoid receptor function, and reducing Brd2/Brd4 levels and H3K27Ac levels, which play a crucial role in AR and GR function. Additionally, the compounds showed synergistic effects with apalutamide, darolutamide, and enzalutamide in inhibiting androgen receptor activity and suppressing the growth of 22RV1 cells. Co-administration of YIV-818-A and enzalutamide also resulted in a marked reduction of 22RV1 tumor growth in vivo. In conclusion, YIV-818-A, RA-V, or RA-VII hold promise for overcoming current drug resistance and could be used for the treatment of androgen-dependent disease and castration-resistant prostate cancer (CRPC).Creating a multi-targeted medication that can simultaneously hinder both the androgen receptor variants and glucocorticoid receptor action holds the potential to overcome drug resistance, extend the duration of treatment efficacy, and ultimately enhance the therapeutic outcomes for prostate cancer patients. Through our STAR  Drug Discovery Platform, we studied the effects of three hundred medicinal plant extracts across 25 signaling pathways to identify a drug candidate to target the androgen signaling pathway. YIV-818-A, optimized water extracts of Rubia Cordifolia  water extract spray dried powder) 100 mg/mL was extracted with 1 mL HPLC grade water and heated at 80°C for 30 min. The herbal extract was centrifuged at 12,000 rpm in desktop centrifuge at room temperature for 5 min. The supernatant was transferred into a 2 mL tube and used as a 100 mg/mL. RA-V, RA-VII and RA-XI standard compounds were purchased from Chemfaces.com and were dissolved in DMSO.YIV-818-A (+m/z = 757.3549 (positive mode) was dissolved in CDCl3 and subjected to NMR analysis  for chemical structure determination.25 g of YIV-818-A was extracted in 100 mL HPLC grade water at 80°C for 30 min. The extract of YIV-818-A was then centrifuged at 10,000 rpm for 10 min and the supernatant was passed through a solid phase column . The column was washed sequentially with 50 mL 10% EtOH and 50 mL 30% EtOH and finally 30 mL 50% EtOH was used to elute the fraction containing. The 50% EtOH fraction was vacuum dried and re-dissolved in 3 mL 50% EtOH before passing through a second solid phase column . The second column was then washed with 30 mL 0.1% formic acid, 30 mL 10:90 [(Methanol:Acetonitrile 88:12): (0.1% formic acid)], 30 mL 30:70 [(Methanol:Acetonitrile 88:12): (0.1% formic acid)], and 30 mL 45:55 [(Methanol:Acetonitrile 88:12): (0.1% formic acid)]. A fraction was collected by eluting with 30 mL 60:40 [(Methanol:Acetonitrile 88:12): (0.1% formic acid)] and was vacuum dried. Next, the dried material was dissolved and treated with 0.2 N NaOH in a 50% methanol solution (5 mL) for 10 min. The NaOH treated fraction was passed through a C18 column and eluted with AM60 and AM75 as procedure used in the second column. AM75 fraction was then vacuum dried. The dried AM75 fraction was re-dissolved in 3 mL 50% EtOH and then passed through a C18 column (2.5 cm diameter x 50 cm length packed with Discovery DSC18 resin). The C18 column was washed with a gradient of acetonitrile:water from 10% to 80% . All collected fractions were then vacuum dried and dissolved in EtOH for the AR luciferase assay and Western blotting. The fractions which successfully inhibited the AR activity and downregulate the AR and CycD1 protein of 22RV1 cells were subjected to UHPLC Q-Exactive Orbitrap MS for analysis . The fraction with the purified active compound with [M+H]m/z 120–1,000. The key parameters were as follows: spray voltage, 3.5 kV (+); the sheath gas flow rate, 35 arb; aux gas flow rate, 10 arb; capillary temperature, 320°C; heater temperature, 350°C; resolution, 70,000. Data acquisition and processing were carried out with the Xcalibur version 4.2.A Dionex Ultimate 3000 UHPLC and the Q-Exactive Focus Orbitrap MS was connected via an electrospray ionization (ESI) source. The fractions were performed on a Thermo Scientifific Hypersil GOLD™ aQ  at the flow rate of 0.3 mL/min. The mobile phases consisted of 0.1% formic acid aqueous solution (solvent A) and acetonitrile (solvent B) with the following gradient program: 0–2 min, 95%–90% A; 2–5 min, 90%–80% A; 5–10 min, 80%–75% A; 10–12 min, 75%–45% A; 12–20 min, 45%–20% A; 20–25 min, 20%–5% A; 25–26 min, 5%–95% A; 26–30 min, 95% A. The sample injection volume was 2 µL. The key mass spectrometer was running at positive mode with the mass range at 2 incubator.22RV1 and LNCaP prostate cancer cells were grown in corning T75 cell culture flasks in RPMI1640 media supplemented with 5% FBS, 50 μg/mL Kanamycin in a 37°C, 5% COThe 22RV1, LNCaP and PC3 cell lines were stably transfected with a PGL4.2 luciferase reporter . This reporter contains a sequence (3xgtaattgcagaacagcaagtgctagctctc) representing the wild type of Androgen Response Element (ARE) derived from the Prostate-Specific Antigen (PSA) promoter. As a control, cells were also transfected with the PGL4.2 luciferase reporter that does not include the PSA promoter. This transfection was accomplished using Lipofectamine 3000 . Stable clones were selected and maintained with puromycin (0.5 μg/mL). DHT (25 nM) and DEX (50 nM) was used to stimulate androgen receptor activity for a 24-hour period. The pcDNA5 plasmid , carrying the full open reading frame of GR, was transfected into LNCaP cells, and hygromycin (50 μg/mL) was employed to select and maintain stable clones.4 reporter cells per well of 96-well plate were seeded in RPMI1640 media supplemented with 5% dialysis activated carbon treated FBS, 50 μg/mL Kanamycin in a 37°C, 5% CO2 incubator for 48 h. Cells were treated with herbal extracts at 30, 100, 300, and 600 μg/mL for 24 h in a 37°C-CO2 incubator. DHT (25 nM) was used to stimulate androgen receptor activity. Dexamethasone (50 nM) was used to stimulate glucocorticoid receptor activity. Cells were lysed using luciferase lysis buffer after which luciferase buffer with luciferin was added to generate luminescence. Luminescence was recorded using a luminescence microplate reader. The IC50 is the concentration of treatment agent required to reduce the measured luminescence to half of its maximal value. IC50 was determined by the median-effect equation of the Chou-Talalay method . Cell lysates were sonicated for 10 s to shear DNA. Cell extracts were then electrophoresed through 10% SDS-polyacrylamide gels and transferred to 0.2 µm nitrocellulose membranes  with a Miniprotein II transferring apparatus (Bio-Rad). The non-specific interaction of membrane was blocked with 5% non-fat milk and probed in TBS-T buffer  containing. Monoclonal rabbit anti-AR (1:5000), was used to detect the androgen receptor (Abcam #133273), Glucocorticoid Receptor (D8H2) XP® Rabbit mAb #3660, ERα Antibody (F-10): sc-8002, BRD4 (E2A7X) Rabbit mAb #13440, Brd2 (D89B4) Rabbit mAb #5848, PARP (46D11) Rabbit mAb #9532, Acetyl-Histone H3 (Lys27) (D5E4) XP® Rabbit mAb #8173 and a monoclonal actin antibody diluted 1:2500  was used to detect β-actin as the internal control to normalize protein loading. The membranes were then incubated with horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG . Enhanced chemiluminescence reagents  were used to visualize the immunoreactive bands and the densities of protein bands were scanned analyzed using ImageJ software from the NIH.RNA was extracted from herb treated cells using the Roche High Pure RNA isolation kit. cDNA was then generated from RNA samples using a Bio-rad iScript Advanced cDNA synthesis kit for RT-qPCR. qPCR was performed using human AR, KLK2, PSA, CycD1 and β-actin primers  and iTaq6 cells in 100 μL Matrigel, BD Biosciences) were transplanted subcutaneously into 8-week-old female NCR-nude mice . Body weight, tumor size, and mortality of the mice were monitored daily. After 10–14 days, mice with tumor sizes of 180 mm3 were selected. Tumor volume was examined by using the formula length × width2 × π/6. Each group consisted of five mice. YIV-818-A was administered orally for 7 days  and/or enzalutamide was administered orally for 7 days . In the control groups, mice were administered water orally. All animal experiments were carried out in accordance with the relevant guidelines and regulations approved Yale University Institutional Animal Care and Use Committee (IACUC) protocol. Animal experimental protocols were approved by Yale University Institutional Animal Care and Use Committee (IACUC). Animal studies were carried out in compliance with the ARRIVE guidelines.22RV1 cells /KLK3 Human ELISA Kit (Thermofisher Scietific) was used to detect PSA protein of plasma collected from animal using PST tube with heparin (BD biosciences) according to the manual provided in the kit.p < 0.05.Two-way ANOVA analysis (GraphPad Prism 9) was employed to discern differences between two data curves. Additionally, t-tests (Microsoft Excel) were conducted to ascertain statistical significance between group comparisons. Differences were considered statistically significant at +m/z = 757.3549 that was detected using high resolution LC-MS  C40H49N6O9 is 757.3555 which is closest to our detected mass (m/z = 757.3549) of the purified compound. RA-V was purchased from a commercial source and was subjected to LCMS. Both chemicals of F11 and RA-V had some retention time with [M+H]+m/z = 757.3549  had same chemical shifts with Deoxybouvardin (RA-V) . The eluted fractions (F9 to F15) from the final C18 column had a relatively strong inhibitory effect on AR driven luciferase activity and could also downregulate AR/AR-V (AR splice variants) protein of 22RV1 . Fractioon LC-MS . By marc757.3549 . They al779.3364 . In addin (RA-V)  as previn (RA-V) . Please 50 observed in LNCaP cells. This finding is consistent with previous studies  , thereby promising to substantially attenuate, if not completely nullify, the drug resistance manifested in 22RV1 cells.YIV-818A demonstrated enhanced inhibitory action in 22RV1 cells in the presence of DHT, suggesting a potential heightened susceptibility of wild type AR to YIV-818A in these cells . Notablyariants) . The abonfluence . Even thAdditionally, it has been reported that around 30% of enzalutamide-resistant patients exhibit overexpression of the glucocorticoid receptor (GR), which can substitute for AR function and activate downstream targets of the androgen receptor. In our study, we observed that enzalutamide was unable to inhibit GR-mediated luciferase activity in the AR luciferase reporter assay using 22RV1 cells that express GR . In contPSA and KLK2 of 22RV1 cells by using qRT-PCR  and might be beneficial in enzalutamide-resistant cases with GR overexpression.Based on the LC-MS analysis, it was determined that the water extract of YIV-818-A at 250 μg/mL contained approximately 75 ng/mL of RA-V. As a result, we utilized a concentration range of YIV-818-A spanning from 28 μg/mL to 250 μg/mL, and a concentration range of RA-V from 8.3 ng/mL to 75 ng/mL, to treat 22RV1 cells with or without DHT for a duration of 24 h . These sde novo protein synthesis  subfamily of bromodomain proteins plays a crucial role in AR and GR-dependent enhancer activation, leading to the transcription of target genes regulated by these receptors . By recoThese findings suggest that YIV-818-A, RA-V, or RA-VII might reduce the binding of Brd proteins to promoter/enhancer regions of AR and GR target genes, thereby impacting their transcription. Additionally, the downregulation of Brd proteins and H3K27Ac could represent one mechanism of action through which YIV-818-A, RA-V, or RA-VII inhibit GR transcriptional activity. These results also imply that YIV-818-A, RA-V, or RA-VII are not pan-inhibitors for histone acetyltransferases (HAT), but rather may selectively inhibit the transcription of a subset of genes that are dependent on H3K27Ac.Given that YIV-818-A, RA-V, or RA-VII exhibit distinct mechanisms of action in inhibiting AR compared to the FDA-approved androgen inhibitors, enzalutamide, apalutamide, and darolutamide used in prostate cancer treatments, we speculated that YIV-818-A, RA-V, or RA-VII might have the potential to synergistically enhance the therapeutic effects of enzalutamide, apalutamide, or darolutamide.To assess drug interactions between YIV-818-A, RA-V, or RA-VII and the current drugs, we performed an isobologram analysis based on the combination index. Our results demonstrated that combinations of YIV-818-A, RA-V, or RA-VII with enzalutamide, apalutamide, or darolutamide exhibited synergistic effects  in inhibiting DHT-driven AR activity in both 22RV1 and LNCaP cells . Therefoin vivo. The compelling in vitro evidence of synergistic AR activity inhibition by YIV-818-A and enzalutamide led us to explore their combined efficacy in an in vivo setting. We proceeded to validate this hypothesis using a 22RV1 xenograft model in nude mice.YIV-818-A enhanced enzalutamide action against 22RV1 tumor growth p = 0.018). On the other hand, enzalutamide  was not found to substantially decrease the growth of 22RV1 tumors in the nude mice (p < 0.0001) was observed when YIV-818-A and enzalutamide were co-administered (p < 0.007)  resulted in a significant deceleration of tumor growth (ude mice . Howevernistered . It is inistered . In a denistered . Further< 0.007) .in vitro findings and indicate that a combined therapeutic approach with YIV-818-A and enzalutamide could offer an enhanced strategy to inhibit the growth of prostate cancer.These results robustly align with our Rubia cordifolia (R.C.) and YIV-818-A water extracts were used to treat 22RV1-AR-luciferase reporter cells in present of DHT for 24 h. Results indicated that different batches of Rubia cordifolia (R.C.) had different potency in inhibiting DHT triggered AR activity (Rubia cordifolia (R.C.) had some degree of correlation to the AR inhibition (%) (R = 0.819) (Rubia cordifolia (R.C.) could be used to predict its potency of AR inhibition. An increased content of RA-V in the batches of Rubia cordifolia (R.C.) increased the likelihood of stronger AR activity inhibition.27 different batches of activity . The RA-= 0.819) . TherefoEnzalutamide, apalutamide, and darolutamide are second-generation androgen receptor (AR) blockers approved for both non-metastatic and metastatic castration-resistant prostate cancer (CRPC) . These dIn this study, we present YIV-818-A and its active component, RA-V, as novel compounds capable of inhibiting both AR and GR activity. Similarly, RA-VII, a bicyclic hexapeptide analog of RA-V, exhibits comparable activity. Our Western blotting results reveal that YIV-818-A, RA-V, or RA-VII can reduce AR protein levels and diminish mRNA expression of AR target genes such as PSA and KLK2. Previous research demonstrated that SVC112, an analog of RA-VII, selectively reduces Cyclin D1 (CycD1) protein levels by obstructing protein translation in Head and Neck Squamous Carcinoma . FurtherInterestingly, the inhibition of glucocorticoid receptor (GR) activity by YIV-818-A, RA-V, or RA-VII does not appear to directly correspond with protein downregulation. This led us to hypothesize that these compounds might be influencing the cofactors associated with the GR protein. Supporting this premise, our findings revealed that the levels of bromodomain proteins (Brd) and histone 3 lysine 27 acetylation (H3K27ac) were attenuated by YIV-818, RA-V, and RA-VII. Considering the pivotal role of Brd and H3K27ac in AR and GR-dependent enhancer activation and target gene transcription , this miin vivo . By virtue of their ability to downregulate AR-V (AR splice variants) and hinder the GR activity of CRPC cells, the combination of these compounds with enzalutamide, apalutamide, or darolutamide might considerably decrease the chance of drug resistance development. Moreover, a potential reduction in drug dosage could possibly mitigate adverse effects experienced by patients. Therefore, YIV-818-A, RA-V, or RA-VII represent a promising approach to augment the therapeutic impact of enzalutamide, apalutamide, and darolutamide.Notably, YIV-818-A, RA-V, or RA-VII have all exhibited a synergistic effect with enzalutamide, apalutamide, or darolutamide in the inhibition of AR activity, both in cell culture and Furthermore, palbociclib, abemaciclib, and ribociclib, which are CDK4/6 inhibitors, are approved for the treatment of metastatic CRPC . Cyclin Rubia cordifolia (R.C.) (At least twenty-four distinct bicyclic hexapeptides have been discovered and isolated from a (R.C.) . Existina (R.C.) . Moreovea (R.C.) .Our study underlines the varying potency of different bicyclic hexapeptides, such as RA-V, RA-VII, or RA-XI, in inhibiting AR and GR. It is intriguing to note how a seemingly minor change, such as substituting glutamic acid at the 2nd position of the hexapeptide (as in RA-XI) instead of alanine (as in RA-V or RA-VII), can significantly reduce the anti-AR and anti-GR activity of RA-XI. This could be attributed to the bulky carboxylic acid group of glutamic acid potentially impeding its binding to the target molecule. On the other hand, the presence of a CH3 group on the tyrosine at position 6 in RA-VII appears to enhance its anti-AR and anti-GR activity compared to RA-V, which has an H group at the same position. The region spanning the 2nd to 6th positions could dictate the hexapeptide’s orientation towards its target, but this hypothesis warrants further investigation. A comprehensive study exploring the structure-activity relationship of this class of compounds in relation to AR should be pursued further. Such efforts could pave the way for the identification of more potent compounds with high selectivity towards AR, GR, or certain epigenetic states.In addition to AR, AR-V (AR splice variants), and GR, prostate cancer progression and drug resistance involve a myriad of alternative mechanisms that have been extensively studied . NotablyRubia cordifolia (R.C) has a long-standing history of safe usage in Asia as a dietary supplement designed to improve health. Rubia cordifolia (R.C.) has been recognized for its diverse biological properties, including promoting coagulation, modulating the immune system, providing anti-inflammatory and neuroprotective effects, acting as an antioxidant, and exhibiting anti-tumor properties (Rubia cordifolia (R.C.) may inhibit the growth of prostate cancer by disrupting AR and GR signaling. Thus, Rubia cordifolia (R.C.) emerges as a potentially powerful therapeutic agent for prostate cancer treatment or as a chemopreventive measure. This promising prospect is deserving of further investigation and consideration in future research endeavors.operties . HoweverRubia cordifolia (R.C.) across different batches. Interestingly, the amount of RA-V present in Rubia cordifolia (R.C.) showed a strong correlation with AR inhibition across different batches. This suggests that RA-V could serve as a chemical marker to select high-potency Rubia cordifolia (R.C.) batches for development in the treatment or prevention of prostate cancer.We identified RA-V, a component of YIV-818-A, as the agent responsible for inhibiting AR and GR. However, we observed considerable variation in the quantity of RA-V present in Rubia cordifolia (R.C.), could serve as a chemical marker for quality control in the preparation of this herbal supplement. Given the longstanding safe usage of Rubia cordifolia (R.C.) in Asia as a dietary supplement for health improvement, YIV-818-A could potentially be developed as a chemoprevention agent and/or anti-cancer drug for the treatment of prostate cancer.In summary, YIV-818-A, RA-V, or RA-VII show potential in overcoming the drug resistance encountered in castration-resistant prostate cancer (CRPC) cells caused by AR variants and GR. This is achieved by down-regulating AR protein, inhibiting GR function, and influencing epigenetic regulation. Agents such as YIV-818-A, RA-V, and RA-VII could potentially enhance the therapeutic effect of FDA-approved androgen inhibitors—enzalutamide, apalutamide, or darolutamide—against castration-resistant prostate cancer (CRPC). Moreover, RA-V, found in


RB1, TP53, and PTEN expression and MYCN and AURKA amplification appear to be key drivers for NEPC differentiation. Epigenetic modifications also play an important role in the transition to a neuroendocrine phenotype. DNA methylation of specific gene promoters can regulate lineage commitment and differentiation. Histone methylation can suppress AR expression and promote neuroendocrine-specific gene expression. Emerging data suggest that EZH2 is a key regulator of this epigenetic rewiring. Several mechanisms drive AR-dependent castration resistance, notably AR splice variant expression, expression of the adrenal-permissive 3βHSD1 allele, and glucocorticoid receptor expression. Aberrant epigenetic regulation also promotes radioresistance by altering the expression of DNA repair- and cell cycle-related genes. Novel therapies are currently being developed to target these diverse genetic, epigenetic, and hormonal mechanisms promoting lineage plasticity-driven NEPC.Androgen deprivation therapy is a cornerstone of treatment for advanced prostate cancer, and the development of castrate-resistant prostate cancer (CRPC) is the primary cause of prostate cancer-related mortality. While CRPC typically develops through a gain in androgen receptor (AR) signaling, a subset of CRPC will lose reliance on the AR. This process involves genetic, epigenetic, and hormonal changes that promote cellular plasticity, leading to AR-indifferent disease, with neuroendocrine prostate cancer (NEPC) being the quintessential example. NEPC is enriched following treatment with second-generation anti-androgens and exhibits resistance to endocrine therapy. Loss of  The traUnique to PCa is its reliance on the AR to regulate tumor progression via transcriptional regulation of AR targeted genes  and the 22.1For decades, androgens have been implicated in driving PCa development and progression through their interactions with the AR, a ligand-dependent nuclear transcription factor . With th2.2RB1, TP53, and PTEN are individually among the most common genetic alterations in PCa. However, the concomitant loss of more than one of these critical factors is enriched in the NE subtype , PTEN and RB1 loss (DKO), and PTEN, RB1, and TP53 loss (TKO) has demonstrated lower AR expression and activation of NEPC programming in the DKO and TKO groups. Notably, the DKO and TKO tumors exhibited increased expression of SOX2, a key downstream effector for NEPC lineage plasticity  program gene sets  family of proteins, especially FOXA1 and FOXA2, have been implicated in NEPC development. The FOXA family is a group of pioneer transcription factors that can access condensed chromatin, facilitating the local binding of other regulatory factors . FOXA1 enversion , with FOprograms . FOXA1 iprograms . On the programs , 52. Howprograms . FOXA2 iprograms , 54, and in NEPC , a recep in NEPC , 56. Inh in NEPC . Neverth3Epigenetic modifications  are critical regulators of transcriptional states, are known drivers of carcinogenesis, and are tightly associated with NEPC lineage plasticity. Despite similarities in their genomic profiles, NEPC and prostate adenocarcinoma have markedly distinct epigenetic landscapes , 37, 57.3.1De novo DNA methylation is catalyzed by the DNA methyltransferases (DNMT) DNMT3a and DNMT3b, while maintenance DNA methylation is catalyzed by DNMT1 . by DNMT1 . Inverseby DNMT1 . Global by DNMT1 . DNA hypby DNMT1 . Site-spby DNMT1 , 62. DNAby DNMT1 , 64. Addcurrence –68. As sthylated .AR promoter, driven by PTEN, RB1, and TP53 loss, can partially explain the inactivation of canonical AR signaling in NEPC  and is deacetylated by histone deacetylases (HDACs). Similarly, histone methylation is catalyzed by histone methyltransferases (KMTs) and reversed by histone demethylases (KDMs). Histone acetylation is commonly associated with transcriptional activation, resulting from alterations in histone net charge. This leads to an open chromatin conformation that is more accessible to transcription factor binding and the regulation of gene transcription. Unlike histone acetylation, methylation does not exclusively lead to translational activation but is instead associated with either gene activation or repression.3.2.1de novo DNA methylation via a scaffolding mechanism mediated by the long ncRNA HOTAIR , a key transcription factor involved in Wnt/-catenin signaling. Elevated Wnt signaling is a common feature in NEPC, and TCF4 inhibition has been shown to preclude the transdifferentiation of adenocarcinoma to NEPC, following ADT . FurtherIn addition to histone methylation, histone demethylation also plays a significant role in regulating NEPC. LSD1 is known to demethylate H3K4 and lysine residues on several non-histone proteins, including, TP53, E2F1, DNMT1, and HIF-1. Notably, LSD1 is an important regulator of AR transcriptional activity, suppressing the transcription of AR target genes . Indirec3.2.2Recent studies have shown that the use of modern ARPIs can result in widespread changes in chromatin structure and contribute to lineage plasticity. Histone acetylation and deacetylation are two factors that affect chromatin accessibility –92. NotaCDH1 loss, for example, has been shown to promote neuronal differentiation and enzalutamide resistance , which originate from adrenal precursor steroids such as dehydroepiandrosterone (DHEA) , 115. SeThe upregulation of glucocorticoid receptors (GR) is one AR-independent mechanism known to mediate ADT resistance in PCa. GRs are a part of the nuclear receptor superfamily of transcription factors . Like AR4.2Radiotherapy is a cornerstone of curative PCa treatment, and its combination with ADT has been shown to significantly increase patients’ overall survival . Neverth4.2.1in vitro and in vivo. AR expression was also correlated with survival in vitro and time to tumor progression in vivo. Finally, AR pathway upregulation was found in nearly 20% of patients following RT  repair genes. Treatment with second-generation anti-androgens was shown to downregulate DNA repair genes in a CRPC model. Moreover, primary PCa was observed to displays a wide spectrum of AR transcriptional output that correlates with the expression of a set of DNA repair genes. The addition of androgens to ionizing radiation (IR) treated PCa cells enhanced DNA damage repair and treatment with anti-androgens increased DNA damage and decreased clonogenic survival . Similarowing RT . Taken tGiven the association between AR signaling and radioresistance in localized hormone sensitive disease, it follows that castrate-resistance is similarly tied to radiation response. A possible pathway leading to radioresistance in CRPC is the emergence of AR-Vs. One study has shown that AR-Vs can promote non-homologous end joining (NHEJ) repair when canonical AR signaling is blocked by ARPI . Similar4.2.2GPX3, MGMT, and ASC , DNA dependent protein kinases, -H2AX, breast cancer gene 1/2 (BRCA 1/2), PARP-1, and RAD51, are important in DDR induction . Defecti and ASC . Moreovemutation . The metowing RT .CDKs), CDKN1A (p21Cip1), CDKN1B (p27Kip1), and CDKN2A (p16Ink4A), reprimo (RPRM), stratifin (SFN), and CDK1 are all frequently hypermethylated and play various roles in tumor radioresistance  are one of the best characterized Ras effector groups, and their activation leads to the induction of the PI3K/Akt/mTOR pathway that affects cell growth, cell cycle, and cell survival . Uncontr in-vivo , 163. DN in-vivo , 165. Mo in-vivo .5With lineage plasticity driven CRPC posing a challenge for clinical management, novel therapeutic options targeting NEPC are needed. Several clinical trials focusing on agents that target NEPC have been reported, with one of the earliest Phase 2 trials testing the AURKA inhibitor alisertib. Investigators tested alisertib in NEPC patients as AURKA appears to be important in stabilizing MYCN . It shouIn addition to NEPC targeted therapies, drugs that target the NEPC epigenome and delay or reverse NE differentiation are promising. EZH2 is the most well studied epigenetic factor that is dysregulated in NEPC , 77. PreBET bromodomain proteins also offer a potential route for epigenetic targeting of NEPC. Preclinically, treatment with BRD4 inhibitors, either in monotherapy or in combination with ARPIs, have exhibited anti-tumor properties in PCa –172. EarOther potential avenues for epigenetic targeting are LSD1 and the DNMTs. LSD1 is a KDM that is overexpressed in androgen-independent PCa and modulates FOXA1-dependent, AR-associated lineage plasticity and stem cell-associated gene expression , 175. SeDrugs that inhibit lineage plasticity by targeting the epigenome may also be used as radiosensitizers in the future. For instance, one pre-clinical study showed that EZH2 inhibition dramatically enhanced CRPC cells to genotoxic stress, demonstrating the potential utility in EZH2 inhibitors in sensitizing cancers that overexpress EZH2-activated DDR genes to genotoxic agents . MoreoveDespite the potential of new epigenetic and NE-targeting therapies in NEPC treatment, their development and clinical deployment has proven challenging. One major hurdle for many novel epigenetic therapies is their lack of specificity, often affecting a wide range of transcriptional networks leading to undesired off-target results and side-effects . Neverth6Prostate cancer encompasses a range of natural histories, spanning from indolent disease to treatment-refractory CRPC/NEPC. Therapy-induced NEPC is marked by lineage plasticity, loss of luminal markers, and acquisition of NE features, such as SYP and CHGA. While genetic mutations, such as loss of PTEN, TP53, and RB1, and MYCN amplification have been implicated in this lineage transition, they are insufficient to induce the transdifferentiation of adenocarcinoma to NEPC. Instead, increasing evidence points to an aberrant epigenome as a major driver of lineage plasticity. Hyper-methylation of the AR promoter may partially explain the loss of canonical AR signaling observed in NEPC. Similarly, abnormal function of master epigenetic regulators, such as EZH2, promote NE lineage programs. Epigenetic dysregulation can also affect PCa radioresistance by altering the expression of genes involved in DDR and cell cycle. NEPC’s reliance on epigenetic machinery has provided a new avenue for treating a disease that, so far, has been nearly incurable. Epigenome-targeting drugs have shown promise for treating NEPC in preclinical and early-clinical studies, and similar mechanisms are beginning to be exploited to re-sensitize tumors to RT. Despite their potential, numerous challenges remain before these epigenetic therapies can be successfully implemented in modern treatment regimens for NEPC. In summary, understanding the mechanisms that promote lineage plasticity, from tumorigenesis to the emergence of treatment resistance, is imperative in contemporary prostate cancer research and represents a critical step in the discovery of new targets and more effective therapies to prevent and treat lethal PCa.JI and SGa did the literature review and wrote the initial draft, made the figure, and worked on the revisions. AM constructed the tables. RL wrote the abstract. JN, CEW, CW, and SGu provided valuable suggestions for the manuscript. OM reviewed the final version and approved the final version for submission. All authors contributed to the article and approved the submitted version.


Yi Fei Qing Hua Granules (YQG) is a traditional Chinese herbal medicine with the effects of inhibiting the proliferation of lung cancer cells. Luteolin is one of the active compounds of YQG. Luteolin is a common flavonoid extracted from natural herbs and it can promote cancer cells apoptosis has been reported. However, the underlying molecular mechanism and effects of luteolin on human lung cancer needs to be validated.Molecular docking, network pharmacology methods and quantitative structure–activity relationship (QSAR) model were used to identify the active components of YQG and their possible mechanisms of action. Western blot analysis was used to measure AR expression in A549 cells. Cell migration assays were used to detect A549 cells proliferation transfected by AR plasmid and AR mutation plasmid, respectively.TCMSP search results revealed that there are 182 active compounds in YQG, which correspond to 232 target genes. Sixty-one genes were overlapping genes in the 2 datasets of TCMSP and GeneCards. Through bioinformatics tagging of these overlapping genes, a total of 1,951 GO functional tagging analysis and 133 KEGG pathways were obtained. Through molecular docking technology and QSAR model verification, the multi-target active compound luteolin was screened out as one of the active components of YQG for in vitro verification. Androgen receptor (AR) was the hub protein with the highest docking score of luteolin. Western blot showed that luteolin could inhibit AR protein expression in lung cancer cell line A549. After the phosphorylation site of AR protein 877 was inactivated, the ability of luteolin to inhibit the proliferation of lung cancer cells was weakened. Luteolin significantly inhibited the growth of A549 xenogeneic tumors at day 25 and 28 and inhibited the expression of AR.In this study, we have explored luteolin as one of the active components of YQG, and may inhibit the proliferation and migration of A549 cells by decreasing the expression of AR and the regulation of phosphorylation at AR-binding sites. Lung cancer is the leading cause of cancer death worldwide and is highly prevalent in China . Most paOne of the Chinese medicines for the clinical treatment of lung cancer, Yi Fei Qing Hua granules (YQG), has such an effect. YQG is mainly composed of Astragalus, Codonopsis, Glehnia littoralis, Ophiopogon, Agrimonia, Fistula, Patriniaceae, Oldenlandia diffusa, Fritillaria, Aster, Platycodon, bitter almond, and licorice. Pharmacological studies have found that YQG demonstrates anti-cancer, immunoregulation, and tumor angiogenesis–inhibitory effects, and clinical observations and experimental studies have shown that its prescription has a certain effect on the treatment of NSCLC . HoweverAndrogen Receptor (AR) is a ligand-dependent transcription factor which belongs to the steroid hormone nuclear receptors superfamily. AR is up-regulation in the prostate cancers, and is a valuable therapeutic target in prostate cancer , 12. StuWhether NSCLC is a sex hormone-related tumor is still controversial, the cancer genome atlas (TCGA) has identified androgen receptor (AR) to be mutated, deleted and amplified across human lung squamous cell carcinoma. Expression of AR is critical for early lung development .In the present study, we found that lung cancer A549 cells could express androgen receptor (AR). We used network pharmacology, computer molecular docking, and bioinformatics technology to study the effective ingredients and mechanism of Yi Fei Qing Hua Granules (YQG) inhibited lung cancer AR expression and proliferation. This study was validated in combination with in vitro experiments to provide a scientific basis for the mechanism of the anti-cancer effect of YQG.https://tcmsp-e.com/). The screening conditions were oral bioavailability (OB) of at least 30% and drug-like (DL) degree of at least 0.18. In addition, UniProt was used to verify the gene symbols of the genes screened in TCMSP and to construct a dataset of Chinese medicine chemical components and targets.Lung cancer-related genes were collected from GeneCards. The chemical constituents and target genes of 13 Chinese herbal medicines in YQG were screened using the TCM System Pharmacology Database (TCMSP) (R software (version 3.6.3) was used to calculate the overlapping genes between lung cancer-related genes and known YQG lung cancer treatment target genes to construct a protein–protein interaction (PPI) network. The STRING database (version 11.0) was then used to analyze the interaction of target genes in the network. To ensure the robustness of the results, when the interaction score was greater than or equal to 0.4, the network type was selected as a physical subnetwork, and the Cytoscape software program (version 3.7.1) was used for analysis. The top ten proteins with three-dimensional structure with the strongest interaction between proteins were selected as the main target proteins (hub proteins).We downloaded the corresponding three-dimensional structure diagram of the active compounds and the proteins related to the hub genes from PubChem and the Protein Data Bank, respectively, and then used Discovery Studio (DS) 2019 software program  for molecular docking and absorption, distribution, metabolism, and excretion (ADME) prediction.https://www.ebi.ac.uk/chembl/) database to obtain the biological activity data of drug molecules that act on hub proteins as a training data set for modeling. The SMILES sequences of the top 10 multi-target active ingredients were obtained from the PubChem (https://pubchem.ncbi.nlm.nih.gov/) database. Descriptor calculation and feature selection: Use the PaDEL-Descriptor library in python to calculate the molecular descriptors of compounds. The input to the PaDEL-Descriptor software is the SMILES sequence of the molecule, and then features with low variance are removed using the python sklearn library feature selection method Variance Threshold. Model selection: Use the LazyRegressor function in python’s lazypredict library for automatic training, compare 41 machine learning models, and use the Adjusted R-squared, R-squared, and root mean square error (RMSE), time taken four criteria to evaluate the effect of the model, select the model with the best evaluation results to establish the QSAR model of the effective Chinese medicine monomer in the treatment of lung cancer. Use the four indicators of explained_variance_score, mean_absolute_error (MAE), mean_squared_error (MSE), and R-squared value (r2_score) provided by the sklearn library metrics to judge the reliability of the regression model. Finally, the descriptors of TCM monomer molecules are input into the model to predict the biological activity value.Using the method of machine learning, the hub proteins and the top 10 multi-target active components screened were analyzed based on QSAR. Download data: Use the ChEMBL .The human lung cancer cell lines PC9, A549, and H460 were provided by the Clinical Research Center of Jiading District Central Hospital. They were cultured in Dulbecco’s Modified Eagle Medium (DMEM)  containing 10% fetal bovine serum (Gibco) at 37 °C, with 5% CO4 cells/well), incubated alone at 37 °C for 24 h, then incubated with different doses of luteolin in a 5% CO2 incubator at 37 °C for 48 h. Luteolin (80 µM) was applied to A549 cells after transfection of AR wild-type and mutant-type plasmids for 24 h. Next, 10 µL of CCK-8 was added to each well, followed by incubation for one hour. The optical density value was measured at 450 nm using a microplate reader . Each experiment was repeated three times.The cell counting kit 8  colorimetric method was used to detect the effect of luteolin  on the proliferation of A549, PC9, and H460 cells and the effect of luteolin on A549 cells after mutation of androgen receptor phosphorylation sites at the binding site. The cells were inoculated in a 96-well plate (1 × 105 cells/well), and a scratch test was performed after 24 h of attachment, where we added 80 μM luteolin and took pictures of scratches at 0, 24, and 48 h, respectively. Each experiment was repeated three times. Luteolin (80 μM) was applied to untransfected A549 cells (Control), A549 cells transfected with empty vector plasmids (NC), AR wild-type (AR-WT) and mutant-type plasmids (AR-MU) for 24 h, and scratch pictures were taken at 0, 24, and 48 h. The cell migration rate was calculated using Image J , and the GraphPad Prism software program was used for statistics and graphing.A549 cells were inoculated in a six-well plate  cytotoxicity detection kit was purchased from Beyotime Biotechnology . The 2 × 10A549 cells were seeded on a 6-well culture plate for 12 h, treated with different concentrations of luteolin for 48 h and collected. The cells were resuspended with PBS (4 °C) and centrifuged at 2000 rpm for 5–10 min more, and then washed. According to the instructions for the production of the Annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V‐FITC/PI) Apoptosis Kit , the cells were suspended with 500 μL of binding buffer, and 5 μL of Annexin V-FITC and PI were added and incubated at room temperature and in the dark for 15 min. Cell apoptosis was detected by flow cytometer (BD FACS Canto II).AR wild-type plasmid was constructed by primers F and R to amplify the target gene. The primer sequences are listed in Table AR mutant expression plasmids (AR-MU) were constructed using the point mutation PCR method. Using primers F and R-MUT to amplify the region fragments, and primers F-MUT and R to amplify the region fragments, both DNA fragments were mixed and used as templates for PCR amplification, and primers F and R are used to amplify the fragments of the target gene. The PCR amplification conditions were the same as with the AR wild-type plasmid.5 cells/well). Then, we mixed the empty vector, wild-type (WT) AR, and the mutant AR plasmids with lipofectamine 2000 (Thermo Fisher Scientific) at a ratio of 5 μg to 8 μL, and transferred them into different wells of the six-well plate. Next, 2 mL of serum-free and dual-antibody-free DMEM was added to each well. After 4 h, the medium with serum and non-double antibody was replaced. Twenty-four hours after transfection, transfection was observed under a fluorescence microscope .A549 cells were inoculated in a six-well plate (3.5 × 105/well) cells were seeded in a six-well plate overnight. After the cells adhered, DMEM complete medium containing luteolin 40 μM or 80 μM was added to three wells of the six-well plate, and treated for 48 h. Cells were collected after rinsing and scraping with PBS. Cells were lysed on ice with lysis buffer . The concentration of protein was quantified using a BCA kit . An equivalent amount of protein was loaded on 10% SDS-PAGE gels and then transferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat milk for an hour, and then incubated with primary antibodies (1:1000) at 4 ℃ overnight. The antibodies were as follows: GAPDH , AR . On the next day, the membranes were washed three times (5 min each time) with TBST solution, and incubated with secondary antibody (1:5000) for 2 h. After three washes with TBST (10 min each time), images were acquired using a gel imaging system .A549 . In the control group, 10 μl/g 10% DMSO/corn oil (vehicle) was given through intragastric administration. Mice in the experimental groups were given 150 mg/kg luteolin oral gavage, 1 mg/kg 5α-Dihydrotestosterone  (0.2 mg/ml) subcutaneous injection, and 150 mg/kg luteolin combined with 1 mg/kg DHT subcutaneous injection). All treatments were observed every 2 days for 28 days. The weight of the experimental animals was measured about once a week after inoculation until before grouping, and the tumor volume was measured once a week when the tumor was visible. After grouping, the weight of experimental animals and the tumor volume of experimental animals were measured twice a week. The mice were euthanized 1 days after the last dose and tumors were removed and photographed. Tumors were fixed in 4% paraformaldehyde for 12 h, embedded in paraffin, and cut into 5-μm sections. The sections were assigned for hematoxylin–eosin (HE) stain and immunohistochemistry (IHC) assay and examined by light microscopy. For IHC, anti-AR  antibodies were used.All animal studies were approved by the Ethics Committee of Shanghai University of Medicine and Health Sciences . Male Balb/c nude mice 6–7 weeks of age were purchased from Daoke Medical Technology Co., LTD . 1 × 10P < 0.05indicated that the difference was statistically significant.All of the experiments were repeated three times. Results are expressed as mean and standard deviation (SD) values, and all of the statistical comparisons were evaluated using one-way or two-way analysis of variance. Significant differences between means were measured using Duncan’s multiple range test. Statistical analysis was performed using GraphPad Prism software (version 8.0.2) and R-studio (R version 3.6.3). STAT3, EGFR, ESR1, JUN, CCND1, STAT1, AKT1, AR, MYC, and VEGFA) were selected as hub genes. Since CAV1 has no three-dimensional protein structure, DS was unable to conduct molecular docking, so it was temporarily excluded . As seen from the line chart , and the results showed that there was no significant difference in the release of luteolin at different concentrations   .FCM analysis was used to further verify the pro-apoptotic effect of luteolin on A549 cells. It can be seen from Fig. We used the DS proteomic analysis software to calculate the three-dimensional structure diagram of luteolin and AR binding as well as the sites of their stable interaction Fig. A and B. Western Blot showed that AR protein expression was different after treating A549 cells with different concentrations of luteolin for 48 h: When luteolin was 80 μM, AR protein expression was significantly decreased Fig. A and B.FP < 0.037) and 28 (P < 0.046) of administration, and compared with the DHT group, luteolin group also showed significant differences at day 25 (P < 0.046) and 28 (P < 0.035) of administration . Since LibDock Score in molecular docking is a parameter for comprehensive evaluation of energy, geometry and chemical environment, and the significance of AR in lung cancer needs to be further confirmed.T877A and membrane receptors for androgens. The results found all six flavonoids assumed an alternative orientation in ART877A orienting closer to helix12 (H12), and resembling the pose of known antagonists [Some studies have pointed out that luteolin has anti-tumor activity for many cancers . In lungagonists , 27. In agonists . InactivIn order to further validate these findings, we firstly tested the effects of luteolin on the proliferation, migration and apoptosis of human lung cancer cells in vitro. The results showed that 80 μM luteolin significantly reduced proliferation and migration in A549 cells, however, the LDH test showed that its toxicity was weak. Flow cytometry for A549 cells showed no significant increase in apoptosis or necrosis after treatment with 80 μM luteolin alone. When 80 μM luteolin was used in combination with DDP (10 μg/mL), its effect on promoting apoptosis was better than that of DDP alone. Some studies believe that luteolin exerts pro-apoptotic effect on A549 lung adenocarcinoma cells , 30. HowNext, we used a plasmid construction approach to mutate AR (Thr-Ala877) so that this site exhibits allosteric inactivation and loss of phosphorylation before transfecting it into the A549 cell line. The results of CCK-8 and cell scratch assay showed that the proliferation and migration of A549 cells were inhibited to a certain extent by the treatment of 80 μM luteolin. The inhibitory effect of luteolin on the proliferation and migration of A549 transfected with AR wild plasmid and mutant plasmid was weaker than that of A549 transfected with un-transfected or empty vector. In particular, the inhibitory effect of luteolin was weaker after site mutation. The results of a western blot showed that AR expression was significantly inhibited by treatment with 80 μM luteolin. The transfection experiments revealed three possible problems: firstly, AR plays a role in promoting tumor proliferation and migration in A549. Secondly, luteolin may inhibit cell proliferation and migration mainly by binding to the phosphorylation site Thr (877) of the AR. Thirdly, luteolin could suppress the expression of AR protein and indirectly inhibit the proliferation of A549, which could explain the weak inhibitory effect of 80 μM luteolin on the proliferation of A549 even in AR mutant plasmid transfected A549.In vivo experiment, luteolin administration was used to verify its anti-tumor effect in A549 xenograft mice. Although not all mouse transplanted tumors were effectively inhibited in this study, it was still observed that the anti-tumor effect of luteolin group began to appear at 25 days and 28 days compared with the control group and the DHT group. Additionally, immunohistochemical staining for AR expression in sections of mice transplanted tumors showed AR expression was decreased in luteolin group compared with other groups, which further validate the results of in vitro experiments.Some studies have found that AR may play an important role in lung cancer biology, however, the role of AR signals in the progression of sex-unrelated cancers presents a complex situation and evidence remains contradictory –35. In tIn summary, we applied silico prediction to study the material basis and mechanism of YQG against lung cancer, and confirmed that luteolin can inhibit the proliferation and migration of lung cancer cells in vitro validation, and the mechanism of luteolin's action on lung cancer cells may be related to its docking at the phosphorylation site of THR 877 of the AR protein. This study also has some limitations. In this study, we selected the active compound with the highest score for docking with hub proteins for research through computer molecular docking, and we did not rule out the inhibitory effect of other active compounds on lung cancer cells. In addition, more studies are needed to further demonstrate if different AR-downstreams are modulated and if the AR-inhibition, in A549 or in the in vivo experiments induces the same effect induced by luteolin treatment, etc. More studies will be necessary in the future to further disentangle these questions.This study preliminarily explored the mechanism of Chinese herbal compounds inhibiting lung cancer proliferation. These results seem to indicate that some flavonoids in Chinese herbal medicine inhibiting lung cancer are closely related to AR of lung cancer. Therefore, it seems that this direction of research is worthy of attention. They also provide some new directions for lung cancer therapy , 31–33


Simple sequence repeats (SSRs), also known as microsatellites, are typically comprised of 1–6 nucleotide units repeated in tandem patterns Ellegren . Due to https://github.com/aaranyue/SSR2Marker.Here, we report a novel pipeline, SSR2Marker, to specifically explore the candidate polymorphic SSR markers between any two given sequences at a large scale. It enables users to identify both monomorphic and dimorphic SSR markers for different purposes. Meanwhile, detailed information, including SSR motifs, primer pairs, amplified fragments, sequence sizes, length polymorphisms and statistics calculations, is also provided to facilitate subsequent genetic analyses and marker-assisted breeding. The source codes, examples and a complete manual of the SSR2Marker pipeline are freely available at The SSR2Marker pipeline is designed as a local program consisting of the six main steps as follows Fig. .Fig. 1S(i). Identification of SSR motifs: The required input data could be two DNA sequences from whatever source derived. SSR motifs as well as their corresponding information, such as SSR length, repeat class and number, are internally executed using MISA with slightly modified parameters . Comparison of repeat classes: By default, the basic nucleotide unit of SSR motif starts from the first nucleotide of the identified SSR marker . As shown, frame shift and reverse complementary might cause diversity of the appearance of nucleotide units, which should be standardized for further processing. Here, a pre-prepared dictionary is employed to classify all possible nucleotide units into their representative classes, e.g., the AAC class = AAC, ACA, CAA, GTT, TGT and TTG. Only the SSR motifs within the same class are retained for further analysis.E-value <= 1e-5, Identity > = 80). In consideration of inversion sequences, the defined upstream sequences are compared against not only themselves but also the downstream sequences, and vice versa. The desired SSR markers should simultaneously contain the alignments of upstream and downstream sequences from both FASTA files.(iii). Alignment of SSR-flanking regions: After obtaining the same SSR class, the flanking regions on both sides (upstream and downstream) of each SSR motif are extracted at a given length value (default setting is 200 bp). To reduce the influence of alignment biases that might be caused by SSR motifs, only the flanking regions are retrieved and aligned by using the BLAST program ((iv). Selection of identical fragments: Even the alignments possess high homologies, there could be still numerous SNPs and/or gaps existed in the SSR-flanking regions, which could affect the ability of the PCR primers to anneal and eventually reduce the across-species transferability. Therefore, only the identical DNA fragments within the upstream and downstream sequences are retained with the assistance of MAFFT . Design of common primers: Among each SSR-flanking region, the longest identical DNA fragments of both sides are separately extracted and then jointly used as reference for the design of PCR primers using Primer3 . Classification of length polymorphism: Due to the high rates of DNA replication errors within SSR motifs, both the size of SSR repeats and the length of SSR-flanking regions show extensive intraspecies and interspecies variations. With the common primers in hand, it is easy to extract PCR amplicons from both of the input sequences. Based on the difference of amplicons in length, these SSR motifs can be classified into monomorphic (with length difference = 0) or dimorphic (with length difference > 0) markers.Collectively, all these steps are implemented in the SSR2Marker pipeline written in Perl script. Only two separate files in FASTA format are required for the identification of SSR markers. When running, users can rapidly obtain the accurate and comprehensive information stored in several output files, such as monomorphism and dimorphism of SSR markers, length and location of SSR motifs, class and unit of SSR repeats, a maximum of five primer pairs with detailed parameters, amplified fragments by each pair of primers, and statistical results for every procedure during this pipeline.Actinidia chinensis and Actinidia eriantha , only a single command line ‘perl SSR2Marker.pl -f1 Hongyang.fasta -f2 White.fasta’ is needed to start running the SSR2Marker pipeline on a Linux operating system . This task took approximately 2.4 h on a Linux server, with 4 Gb memory and 2.50 GHz Dual-Core CPU. As a result, 1561 monomorphic and 13,733 dimorphic SSR markers are identified. Among these dimorphisms, only 2475 (~ 18.02%) are caused by the length variations of SSR motifs between A. chinensis and A. eriantha. On the contrary, the majority of dimorphic markers  are resulted from length variations of SSR-flanking regions, which have often been routinely overlooked by most algorithms or simply considered as special cases.For illustration purposes, we present a case study on the economically important kiwifruit cultivar ‘Jinyan’, which was bred from the interspecific hybridization between We present SSR2Marker, a user-friendly pipeline for the identification of SSR markers and the design of primer pairs. By using the identical SSR-flanking regions as references, the designed primers are completely applicable to both of the two given sequences, exhibiting satisfactory performance in transferability rate and marker quantity. Meanwhile, SSR2Marker considers the polymorphic status of SSR markers based on sequence length differences of both SSR motifs and SSR-flanking regions, which have dramatically expanded the number of informative SSR markers. As shown in the case study of kiwifruit, 11,258 dimorphic markers are generated from the length variations in the SSR-flanking regions, accounting for ~ 81.98%. Furthermore, this pipeline is qualified for the detection of taxon-specific markers . Taken together, SSR2Marker can help researchers to easily obtain plentiful SSR markers that will undoubtedly accelerate genetic studies on a wide range of organisms.Additional file 1: Supplementary Table 1. The major limitations of the currently available tools or databases developed for SSR marker analysis.


Arterial resection (AR) during pancreatectomy for curative R0 resection of pancreatic ductal adenocarcinoma (PDAC) remains a controversial procedure with high morbidity.To investigate the feasibility and oncological outcomes of pancreatectomy combined with AR at a high-volume center for pancreatic surgery.We retrospectively analyzed our experience in PDAC patients, who underwent pancreatic resection with AR and/or venous resection (VR) between 2007 and 2021.P = 0.11). The final histopathological findings of PDAC patients were similar, including M stage, regional lymph node metastases, and R0 margin resection. The mortality of the entire cohort was 6.2% (16/259), with a tendency to increase mortality in the AR cohort, yet without statistical significance . Although 19 (82.6%) patients had PDAC in the final histopathology, only 6 were confirmed to have infiltrated arteria. The microscopic distribution of PDAC in these infiltrated arterial walls on hematoxylin-eosin staining was classified into 3 patterns. Strikingly, the perivascular nerves frequently exhibited perineural invasion.In total 259 PDAC patients with borderline resectable (n = 138) or locally advanced (n = 121) PDAC underwent vascular resection during tumor resection. From these, 23 patients had AR . However, 12 out of 23 patients (52.2%) underwent simultaneous VR including 1 case with intraoperative arterial injury. In comparison, 11 patients (47.8%) underwent AR only including 3 intraoperative arterial injury patients. Although the operation time and bleeding rate of patients with AR were respectively longer and higher than in VR, no significant difference was detected in postoperative complications between VR and AR  during pancreatectomy for curative R0 resection of pancreatic ductal adenocarcinoma (PDAC) remains a controversial procedure. Here, we report the outcome of patients who underwent AR over the past 14 years in our institution and propose a novel classification for arterial wall invasion in PDAC, which may guide future decision-making during AR in pancreatic surgery. Owing to the implementation of thorough preoperative workup, improved perioperative care, new multimodal treatment regimens, and improved technical approaches during surgery,7 pancreatic surgery combined with VR in high-volume centers (HVCs) has remarkably improved the oncological outcomes. However, pancreatectomy combined with AR remains controversial.8 Nearly 50% of patients with PDAC are diagnosed at an advanced stage,9 which is defined as tumor encasement of the celiac artery (CA), common hepatic artery, or superior mesenteric artery. R0 resection is an independent prognostic factor for overall survival in PDAC,10 and is the only chance for long-term survival for patients with locally advanced (LA) PDAC.11 As such, pancreatic surgeons are continuously striving for new strategies to increase the chances of R0 resection.13 Attaining an R0 status frequently requires vascular resection,14 including, in some cases, also an AR. However, arterial infiltration has been classically regarded as a contraindication to surgery, which can be ascribed to the high morbidity and mortality associated with AR and/or reconstruction.Surgical resection is the cardinal effective therapy for patients with resectable pancreatic ductal adenocarcinoma (PDAC) and without major infiltration of important visceral arteries.20 The frequent occurrence of venous thrombosis in PDAC patients is ascribed to early and excessive activation of platelets and procoagulant factors.22 In patients receiving AR and/or arterial reconstruction, the intraoperative blood loss aggressively stimulates the stress responses and feedback mechanisms in the body, thereby further promoting prothrombin activation and platelet activation to compensate for blood loss.22 The hemodynamic variations provide a condition for platelet aggregation, which is caused by the hypercoagulable state of blood in advanced cancers,23 temporary blood flow blockage during microvascular anastomosis, or the blood eddy currents in vascular stumps. Moreover, anastomotic sutures will create attachment points for platelet aggregation. Notably, visceral thrombosis, predominantly portal vein thrombosis,24 usually occurs in PDAC and has been identified as a prognostic factor for short-term survival.25 Moreover, the expansion of portal vein thrombosis into the liver can cause intrahepatic thrombosis, leading to impaired liver function and even liver failure.28Ostensibly, PDAC, especially pancreatic head cancer, can frequently and directly infiltrate the superior mesenteric vein and portal vein (PV) by local tumor extension. Intrinsically, PDAC is a malignancy associated with venous thrombosis, which is linked with 17%−36% morbidity.The present study assessed the perioperative morbidity and mortality, postoperative complications, and the long-term oncologic outcome in borderline resectable (BR)/LA PDAC patients who received AR and/or VR during pancreatectomy in an HVC for pancreatic surgery.The retrospective analysis of the perioperative and oncological outcome of our resected PDAC patients was approved by the Ethics Committee of the Technical University of Munich (nr. 2022-407-S-NP)., neoadjuvant/adjuvant treatment, survival, and vascular  resection (including the cause and type). All the relevant histopathological findings were obtained from the pathology reports.We retrospectively reviewed our electronic database of 138 BR and 121 LA PDAC patients who underwent pancreatic resection at Klinikum rechts der Isar, Technical University of Munich, Germany, between 2007 and 2021. The following clinicopathologic data were collected to analyze prospectively: baseline characteristics , surgical procedures , operative time, postoperative complications [Clavien–Dindo] and intraoperative bleeding classification, mortality, histopathological findings . Regional lymph node metastases were identified in 180 patients with (75%) VR only,4 (50%) with AR only, and 9 (81.8%) with AR and VR. The distribution of T1/T2 and T3/T4 were also comparable between patients with VR AR only, AR and VR . R0 resection was achieved in 21.6% (56/259) of all patients . We detected a tendency toward increased mortality in the AR group, yet without statistical significance . The patients mainly died due to postoperative hemorrhage in the AR cohort. The postoperative bleeding rate after AR  was significantly higher than that after VR (7.9) . Surgery-related complications including pancreatic fistula, biliary fistula, and intestinal fistula showed no significance between VR and AR cohorts  underwent AR only, and 11 patients (57.9%) underwent simultaneous VR. No difference was present with regard to tumor grade or M stage among PDAC patients with VR .1% underIn addition to the 19 PDAC patients who underwent AR, there were 4 cases with intraoperative arterial injury, which compelled us to perform AR only in 1 patient, and simultaneously AR and VR in 3 patients. In fact, these patients suffered from a recurrence of gastric cancer infiltrating the pancreas, chronic pancreatitis, bile duct cancer, or pancreatic neuroendocrine tumor. The types of AR in our all AR were celiac trunk resection (n = 5), hepatic artery resection (n = 16), and other AR types (n = 2). Furthermore, only in 6 (1 patient underwent AR only and 5 patients underwent AR and VR) out of 19 cases with PDAC, the resected artery was microscopically infiltrated (Table P = 0.11). The mortality rate of the entire cohort was 6.2% (16/259). In the AR cohort, 4 deaths due to surgical complications were reported . There were 4 cases of liver complications, namely, liver abscess, hepatic encephalitis, thrombosis, and sepsis. The overall survival in AR presented likewise poor, all 4 non-PDAC-patients died due to complications respectively within 30 days, after 3, 5, and 12 months; in the 19 PDAC-AR cohort, 4 patients died due to complications, whereas 9 died after a median survival of 14.8 months, and only 2 patients had a recent survival of 48.8 and 65 months, but both of them developed tumor recurrence.Although the complication rate in the AR cohort was higher than that in the VR cohort, no significant difference was detected between VR and AR  at rates as high as 17.4% (4/23). These complications undoubtedly reduced the quality of life and increased psychological trauma in patients undergoing pancreatic surgery for PDAC. In these regards alone, AR can inevitably be perilous, even in high-volume centers for pancreatic surgery.PDAC is commonly diagnosed as a LA disease with local invasion or distant metastasis, and its 5-year survival is only 20%–27% even after receiving neoadjuvant chemotherapy followed by radical surgery.46 Similar to our findings, these studies reported a longer operation time and higher bleeding amount due to AR when compared with standard surgery without vascular resection.46 In our cohort, 4 out of 19 PDAC patients with AR died due to complications, and 9 died in follow-up with a median survival of 14.8 months. The remaining 6 still alive PDAC patients, respectively had a survival of 65, 49, 12, 13, 15, and 6 months . Heretofore, the published results on the resection and reconstruction of major peripancreatic arteries have been conflicting.44 In our study, in the final pathological findings, a true arterial tumor infiltration was found in only 6 out of the 19 patients with AR. As such, surgeons might tend to overestimate arterial infiltration in PDAC, which justifies the performance of periarterial divestment in selected cases.Other centers reported that combining AR is feasible and safe in PDAC patients for attaining a longer recurrence-free and overall survival, and the postoperative morbidity and mortality did not significantly increase compared with palliative surgery.47 and is identified as an independent prognostic factor.48 Therefore, we need to better understand the extent to which surgeons should and can resect or, at least, divest arteries during PDAC surgery for attaining the best balance between radicality and prognostic benefit, while avoiding extensive traumas.The main limitation of our study is its low sample size of AR, and the ultimate benefit status of AR should be constantly scrutinized and updated. Considering the prognostic relevance of PDAC, R0 resection involving AR in PDAC is still of importance for improving overall survival. AR in pancreatectomy increases the feasibility of R0 resection, which remains the only option to improve long-term survivalThe hazards coupled to AR in pancreatic surgery is an unavoidable topic even in HVCs, although AR can evolve as a key surgical approach in the future, just like VR evolved over the past decade. The poor perioperative and oncologic outcomes of BR/LA PDAC patients with AR lead to the dismal current situation. However, with an appropriate learning curve, AR can be offered to selected patients for R0 resection with acceptable postoperative outcomes.


To compare clinical outcomes between patients for whom their participation in inpatient rehabilitation was and was not impacted by dementia through matching patients reporting dementia (dementia group) with those not reporting dementia (non‐dementia group).Prospectively collected data held by the Australasian Rehabilitation Outcome Centre (AROC) were analyzed for patients aged 65 years or older receiving inpatient rehabilitation in public hospitals in Australia following a hip fracture and discharged between July 1, 2014, and June 30, 2019. Patients reported as having dementia impacting their rehabilitation program were matched to patients not reporting dementia based on age, admission motor Functional Independence Measure (FIM) score, and accommodation prior to rehabilitation. The matched cohorts were compared in relation to clinical outcomes  following participation in hospital‐based rehabilitation using univariate analysis.P < 0.001) and their median length of stay was 2 days shorter than those without dementia . Relative change in FIM score and FIM efficiency (per week) were lower in the dementia group . Discharge destination between the two groups was statistically different, with 35.7% of patients with dementia being discharged to residential aged care facilities (RACFs) compared to 21.7% of those without dementia (P < 0.001). More patients with dementia had carers in their private residence in the post‐rehabilitation phase, 82.2% vs. 57.6% (P < 0.001).Patients with dementia had significantly lower cognitive FIM scores on commencing rehabilitation (17.6 and 26.9, respectively, Patients with dementia who sustain a fractured hip benefit from inpatient rehabilitation, although their clinical outcomes are not as good as those without dementia. FIM change and FIM efficiency were lower in the dementia group. Length of stay in the hospital for patients with dementia was shorter due to earlier recognition for the need for placement in either an RACF or at home with carer support. The need for placement in an RACF or carer support in a private residence was significantly greater in the dementia group. Patients with dementia who sustain a fractured hip benefit from inpatient rehabilitation though their clinical outcomes are not as good as those without dementia. FIM change and FIM efficiency were lower in the dementia group. Length of stay in hospital for dementia patients was shorter due to earlier recognition for the need for placement in either a RACF or at home with carer support. It affects memory, thinking, orientation, comprehension, calculation, learning capacity, language, and judgment.Hip fractures contribute to both mortality and morbidity in the elderly. Dementia is associated with a hip fracture hospitalization rate 2.5 times that of adults without dementia.The changing demographic of dementia worldwide will continue to impact on hip fracture rehabilitation. Fragility fractures are relatively common in the elderly, particularly among those with dementia. Studies exploring the predictors of poor clinical outcomes and level of function on discharge for those who underwent rehabilitation in subacute settings are limited. A recent Cochrane review of hip fracture management by Smith et al suggested that models of enhanced rehabilitation and care used in the included trials show benefits over usual care for preventing delirium and reducing the length of stay (LOS) for people with dementia. However, the level of evidence of these results is low as data were available from only seven trials with a total of 555 participants.2There were two objectives of this study. First, we described the patient profiles on admission to inpatient rehabilitation for those who did and did not report dementia impacting their rehabilitation. Second, we compared patient profiles and clinical outcomes for patients who reported dementia impacting on their participation in rehabilitation (dementia group) to a matched cohort of those who did not report dementia (non‐dementia group).33.1This study used prospectively collected episodes of inpatient rehabilitation routinely submitted to the Australasian Rehabilitation Outcomes Centre (AROC) for benchmarking. The AROC is the national rehabilitation medicine integrated outcomes center of Australia and New Zealand. It is a joint initiative of the Australian rehabilitation sector 4Patients receiving inpatient rehabilitation following a fractured neck of femur, who were aged 65 years or older and were treated in an Australian public hospital between July 1, 2014, and June 30, 2019, were included in this study.4.1The AROC dataset includes comorbidities, defined as pre‐existing illness/impairments, not part of the principal presenting condition, impacting the rehabilitation process. Dementia is one such comorbidity.4.2On admission only: age, sex, and date of fracture.On admission and discharge: accommodation, carer status, services received, and functional status.The AROC data collection includes:4.34.3.1An inpatient rehabilitation episode of care is the date that the patient’s care is transferred to a rehabilitation physician or physician with interest in rehabilitation, and it is recorded in the medical record that the rehabilitation team has commenced the rehabilitation program/provision of care.4.3.2The Functional Independence Measure (FIM) instrument is a primary indicator of patient disability with FIM being used to track changes in functional ability of a patient during an episode of hospital rehabilitation care.4.4To create the matched cohort, patients with dementia (dementia group) were matched by age, motor functional status on admission, and residence at the time of fracture with patients not reporting dementia (non‐dementia group). Four passes were required to obtain the full matched cohort Table , with sut test was used with continuous variables. A P value < 0.05 was regarded as statistically significant.Univariate analyses were used to describe group differences; the chi‐square test was used with categorical variables, and the The Australian National Subacute and Non Acute Patient (AN‐SNAP) is a case‐mix classification that categorizes rehabilitation, palliative care, geriatric evaluation and management, psychogeriatric care, and non‐acute care. The rehabilitation branch classifies patients based on impairment, level of function on admission, and age.55.1During the study period 20,905 episodes of care were admitted to inpatient rehabilitation units following a fractured neck of femur. Of these, 1901 episodes reported dementia impacted participation in rehabilitation. Table vs. 70.1% in the non‐dementia group . Most of the admitted patients were aged between 80 and 94 years, accounting for 74.6% and 60.6%, respectively, in the dementia and non‐dementia groups. Both groups had a similar proportion of extremely old people aged 95 years or older (5.9% in the dementia group and 3.9% in the non‐dementia group). Of the 20,905 patients in this study, 69.2% were women in both groups. A large proportion of the patients were from private residences at the time of admission, 80.5% in the dementia group and 90.2% in the non‐dementia group. Compared to those without dementia, patients with dementia admitted for hip fracture rehabilitation had a higher likelihood of residing in residential aged care facilities . At the time of admission, 71% of patients with dementia had carer assistance compared with 40% for non‐dementia patients. Service requirements were similar between the dementia and non‐dementia groups for domestic assistance , with increased requirements for both personal care  and meal preparation  in the dementia group.Most patients were admitted to the rehabilitation unit within 2 weeks since the initial injury with 67.3% in the dementia group (n = 1279/1901) Patients with dementia had lower motor FIM scores (mean 33.9 and median 32) on admission compared to those without dementia (mean 46.0 and median 47). Similarly, patients with dementia had significantly lower cognitive FIM scores (mean 17.6 and median 18) than those without dementia (mean 28.0 and median 30).5.2P = 0.074; Table P < 0.001).After controlling for age, motor function, and prior accommodation by matching, there was no difference between the two groups in the timing of the rehabilitation unit admission since the initial insult (P < 0.001).In comparing clinical outcomes Table , this stP < 0.001). Patients reporting dementia were statistically more likely to be discharged to an RACF . Allied health care and social support increased in both groups, being greater for the non‐dementia group. Personal care support increased about 20% in both groups, whereas support for meals declined. A higher proportion of patients with dementia (5.8% on admission and 21.4% on discharge) needed case management support in the post‐rehabilitation phase than those without dementia (5.7% on admission and 16.2% on discharge).Both groups showed increased needs for services post‐rehabilitation of almost 30% compared to admission  scores being significantly lower.P < 0.001) in the matched cohort, as they are more likely to go to an RACF or home with a carer support. This is consistent with recent study by Rasu et al that demonstrated that patients with dementia and without a known diagnosis of osteoporosis had a 5% shorter LOS (P = 0.04) when hospitalized for hip fracture.We noted that patients who had dementia on admission had a shorter inpatient rehabilitation stay in the hospital; the median LOS was 2 days less compared to those without dementia  = 1.19, 95% confidence interval (CI) : 1.09–1.30], 90‐day entry into permanent care , and increased likelihood of activities of daily living limitations .P < 0.001). Consulting teams should carefully design goal‐orientated rehabilitation programs suitable for people with dementia to ensure optimum clinical outcomes following hip fracture. Dewing and Dijk's review stresses the importance of recognizing negative consequences that can be associated with inpatient and post discharge management of those with dementia due to health care personnel having insufficient understanding and the requirements associated with dementia.Health care outcomes for people living with dementia are dependent on the availability of appropriate services, which vary by region and country, being more likely to result in more inequitable outcomes for those with dementia when compared with outcomes for people without cognitive impairments.6.1This study is a large retrospective analysis of rehabilitation outcomes following hip fracture which examines the impact of underlying dementia in determining outcome by comparison with a matched non‐dementia cohort. Use of AROC data allowed for analysis of an extremely large data set as it incorporated all public hospital admissions within Australia, the period of analysis extending over 5 years (2014–2019).A limitation of the study was the inability to delineate the severity of dementia and therefore its impact on rehabilitation outcomes. Non‐dementia group could have undiagnosed cognitive impairment but did not impact on rehabilitation at the time of admission and therefore not recorded. Although FIM scoring has both motor and cognitive components, FIM scoring is heavily weighted for motor functioning.7With the rising incidence of dementia and hip fractures associated with a globally aging population, providing optimal inpatient rehabilitation and post‐discharge support services for this vulnerable group is essential to maximize functional gains and reduce long‐term health costs. Despite the increased discharge to an RACF for the dementia group, we demonstrated that improvements in both motor and cognitive function are likely to have a beneficial effect on long‐term management. By identifying functional and cognitive limitations and assistance prior to admission, strategic points of intervention across the trajectory of recovery can be introduced thereby improving clinical outcomes. Future research is needed to delineate the impact of severity of dementia on rehabilitation outcome as the disease progresses.Y.L. was responsible for initial drafting and revising the article. J.W. contributed to idea generation and revision of the article. T.A. was responsible for data analysis and revision of article.The authors received no financial support for the research, authorship, and/or publication of this article.The authors declared no potential conflict of interest.Ethics approval for this study (NRR_ AROC_ 2020_04) was granted according to the AROC ethics approval from the University of Wollongong Human Research Ethics Committee .Waiver of consent was obtained from the Australasian Rehabilitation Outcomes Centre (AROC).