To evaluate the prevalence of aortic regurgitation (AR) and associations between the individual aortic root components and AR severity in the general population. The study included the first 10,000 participants of the population-based Hamburg City Health Study (HCHS) of whom 8259 subjects, aged 62.23 ± 8.46 years , enrolled 2016–2018, provided echocardiographic data. 69 subjects with bicuspid valves and 23 subjects with moderate/severe aortic stenosis were excluded. Aortic root dimensions were measured using state-of-the-art cardiac ultrasound, including the aortic annulus, sinus of Valsalva, sinotubular junction (STJ), and ascending aorta, in diastole and systole. The distribution of AR was: 932 (11.4%) mild, 208 (2.5%) moderate, and 20 (0.24%) severe. Patients with moderate or severe AR were predominantly male at advanced age who had hypertension, coronary artery disease, atrial fibrillation, and renal dysfunction. Increasing AR severity correlated with higher absolute and indexed aortic root diameters . In binary logistic regression analysis, all aortic root components showed significant associations with moderate/severe AR. Mid-systolic STJ showed the strongest association with moderate/severe AR . AR was prevalent in 14.2%, of whom 2.8% showed moderate/severe AR. All assessed aortic root diameters correlated with the prevalence and severity of AR. STJ diameter had the strongest association with moderate/severe AR possibly reflecting the pathophysiological impact of an increasingly dilated STJ in the context of an ageing aorta.The online version contains supplementary material available at 10.1007/s10554-021-02337-6. The aortic root is a complex and dynamic entity, varying in size proportionally to height, weight, and age. The interplay of its four different parts, the aortic annulus (AoAn), sinus of Valsalva (SoV), sinotubular junction (STJ), and the proximal ascending aorta (AscAo), is crucial for aortic valve (AV) competence. Along with a dramatic decrease of rheumatic heart disease in the Western world, aortic root dilatation evolved into the predominant cause of aortic regurgitation (AR) –4. HowevHence, first we aimed at providing up-to-date data on the prevalence of AR as assessed by state-of-the-art TTE in the general population. Second, we investigated the correlation between AR severity and each individual part of the aortic root measured systematically in end-diastole and mid-systole.www.hchs.hamburg) who underwent TTE. As previously described, the HCHS is a single-center, prospective, long-term, population-based cohort study [The study population derived from a sample of 10,000 consecutive, at random selected participants from the Hamburg City Health Study  at the baseline visit on dedicated ultrasound machines  following standard operating procedures at the HCHS Epidemiological Study Center Hamburg-Eppendorf, Hamburg, Germany . All TTEFor aortic root assessment, a zoomed transthoracic parasternal long axis view was recorded at breath hold . SystemaAV morphology  and potential degenerative changes were assessed in the parasternal long axis, AV short axis and apical views. Aortic stenosis and regurgitation were assessed according to current ESC guidelines . An in-dχ2) for categorical variables. BSA was calculated using the DuBois formula. Aortic root measurements were divided by BSA to obtain indexed measurements. Correlation of these indexed measurements with AR severity as well as the ratio of sinotubular junction and aortic annulus were presented as boxplots using ANOVA to draw multiple group comparisons.Continuous variables are given as mean ± standard deviation (SD). Categorical variables are given as absolute numbers and percentage of participants. Multiple group comparisons of different severities of AR were assessed by analysis of variance (ANOVA) for continuous variables and chi-squared test . Therefore, we constructed separate models for all eight aortic root measurements. Age, sex, BSA, hypertension, and diabetes were pre-defined as relevant confounders and controlled for in all aortic root models. Results of logistic regression were corrected post-hoc using the Holm method and are depicted by odds ratios (OR) and corresponding 95% confidence intervals (CI). Differences were considered statistically significant at a two-sided p-value level of 0.05 after post-hoc correction. All statistical analyses were performed using R (version 3.6.2). A list of the used packages and versions can be found in the appendix.The analyzed study population included 8167 HCHS subjects of the first 10,000 HCHS participants, with 4191 female (51.3%) and a mean age of 62.23 ± 8.46 years (range 45–74 years) . Systolic were greater than diastolic measurements of the aortic root. Both the absolute and indexed diameters of aortic root components showed the lowest values in the no AR group and the highest values in the severe AR group Table ; Fig. 2.The present study analyzes standardized, state-of-the-art echocardiographic aortic root measurements in association with the severity of aortic regurgitation in a large sample of the population-based HCHS. Major findings include that AR is common in the general population and that the size of all parts of the aortic root are associated with moderate/severe AR. Of the different aortic root parts, MS STJ size showed the strongest correlation with moderate/severe AR.The overall prevalence of AR in the population-based HCHS cohort, aged 45 to 74 years, was 14.2%, while the rate of moderate/severe AR was 2.8%. Subjects with moderate/severe AS or bicuspid valves were excluded from our analysis. Previously published studies on AR prevalence at the populational level reported very heterogeneous results –17. AR pIn the present study, all mid-systolic and end-diastolic aortic root diameters were larger with increasing severity and prevalence of AR. A significant correlation between aortic root size and AR has been previously demonstrated by several research groups , 16, 20.Several previous studies focused on the definition of cut-off values of aortic root size which would be associated with AR occurrence . Roman eThe results of this study are influenced by the subjects included. Our study cohort represents the first 10.000 subjects of the HCHS. Most of the subjects were middle-aged and free of symptoms of cardiovascular disease. Therefore, the translation into other populations and utilization in the clinical setting is limited. Only a small proportion of the overall cohort showed severe AR. Although our study is derived from a large sample, most subjects showed mild AR, which led to an underrepresentation of the moderate/severe group. The degree of association of the different aortic root components with AR severity could vary considerably according to patients’ baseline characteristics. The presented regression analysis was adjusted for all detectable confounding variables. Nevertheless, we cannot exclude that other relevant interacting variables were not considered. The pathogenetic mechanism by which STJ diameter is associated with AR was not addressed. Whether AR itself induced the aorta to dilate or aortic root dilatation led to regurgitation was not answered. Most importantly, our study was not designed to investigate the causality of AR, which is not possible due to cross-sectional study design, but to evaluate the correlation between aortic root diameters and AR severity.In a large population-based sample, AR prevalence was 14.2%, with 2.8% being moderate/severe AR. Higher aortic root diameters at all levels were significantly associated with the prevalence and severity of AR. Of those, mid-systolic STJ had the strongest association with moderate/severe AR, reflecting its pathophysiological role in the context of an ageing aorta. Clinicians should closely monitor aortic root dilatation in the context of AR severity. Special focus should be paid to STJ dilatation as it showed the strongest association with a moderate/severe AR. Further validation in a broader, prospective population-based sample to evaluate the prognostic role of dilated aortic root diameters in the context of AR is needed.Supplementary file1 (DOCX 141 kb)Below is the link to the electronic supplementary material.


Substance P (SP) and toll‐like receptors (TLRs) contribute to airway disease, particularly during viral infection. We recently demonstrated that SP can act as an initial response to viral stimuli in the upper airway by upregulating TLRs in the nasal epithelia (the SP‐TLR axis). Patients with allergic rhinitis (AR) suffer from prolonged airway infections. The aim of the present study was to examine if patients with AR exhibit a disturbance in the SP‐TLR axis.Human nasal biopsies and human nasal epithelial cells (HNEC) from healthy volunteers and patients with AR were cultured in the presence of SP. Epithelial expression of TLR4, neutral endopeptidase (NEP) and neurokinin 1 (NK1) were evaluated with flow cytometry and/or quantitative polymerase chain reaction after 30 min to 24 h. The effect of SP on nasal lipopolysaccharide‐induced interleukin‐8 (IL‐8) release was investigated.SP stimulation of tissue from healthy volunteers resulted in a transient increase of the TLR4 expression, whereas stimulation of AR patient‐derived material led to a delayed and prolonged upregulation of TLR4. NEP expression in HNEC was lower in AR than healthy controls whereas NK1 receptor expression was increased. SP pretreatment increased TLR4‐dependent IL‐8 expression in healthy controls, but not in AR.SP‐induced regulation of TLR4 in the human nasal mucosa is disturbed in AR. An altered SP‐mediated innate immune response may contribute to the dysfunctional and often prolonged responses to infection in AR. It is a well‐founded clinical observation that patients with AR often are more prone to develop infectious airway disease than nonallergic individuals. This includes sinusitis, as well as otitis media with effusion. The close association of nasal allergies with these conditions has been supported by extensive epidemiological evidence.Substance P (SP) is known to be locally induced upon nasal allergen challengeThe action of SP is known to be controlled by a number of mechanisms. First, SP is broken down by a variety of peptidases, most notably neutral endopeptidase (NEP).As AR is associated with alterations in the expression of SP, the present study was designed to compare elements in SP‐TLR axis in nasal biopsies and epithelial cells derived from patients with AR with corresponding material from healthy individuals.22.1The study was approved by the local Ethical Committee, Stockholm, Sweden (number 2014/299 and 2016/826). All participants gave their written, informed consent. All procedures were conducted according to the principles expressed in the Declaration of Helsinki.2.2The study included 30 nonsmoking, non‐astmatic, patients with moderate to severe birch and/or grass pollen‐induced seasonal AR, and 35 healthy volunteers. Human nasal epithelial cells (HNEC) were isolated from 24 healthy subjects and 22 AR patients; nasal biopsies were isolated from 11 healthy subjects and 8 AR patients. All samples were acquired outside the pollen season. Patient characteristics are listed in Table The diagnosis of birch or grass pollen‐induced AR was based on a positive history of seasonal AR as well as a positive ImmunoCap Rapid (Asthma/Rhinitis Adult) test . Patients who did not meet both criteria were excluded from the study. The ImmunoCap Rapid (Asthma/Rhinitis Adult) assesses the presence of circulating specific immunoglobulin E against 10 common airborne allergens, including pollen , house dust mites (HDM), mould and common animal allergens . Exclusion criteria included a history of upper airway infection within 2 weeks prior to visit, treatment with local or systemic corticosteroids within 2 months before the visit, and history of chronic rhinosinusitis with or without presence of nasal polyposis.Five AR patients tested positive for allergy towards common animals allergens and one AR patient tested positive for allergy towards HDM. Two AR patients had comorbid allergic asthma.2.32. Cells from passages 2–5 were used, and all were positive for EpCAM (>90%), an epithelial‐specific adhesion molecule.HNECs were isolated by nasal brushing as described previously.2.42.Human nasal biopsies (HNBs) were isolated as described previously.2.5HNEC were seeded onto 24‐well culture plates  in 1 ml complete KSFM and incubated overnight. Cells were subsequently stimulated with 30 µg/ml R837 (InvivoGen) for 15, 30, 60 or 240 min or 30 nM SP (Sigma‐Aldrich) for 30, 60 or 240 min. Cell culture supernatants were collected and stored at −80°C until further use. Cells were assessed for expression of TLR4, NEP or NK1R with flow cytometry or mRNA expression of TLR4 with quantitative polymerase chain reaction (qPCR).2.6Biopsies were dissected into four equal pieces and placed into 48‐well culture plates in 500 μl complete KSFM, where they were allowed to equilibrate for 30 min prior to stimulation. Biopsies were subsequently stimulated with 30 µg/ml R837 for 15, 30, 60 or 240 min or 30 nM SP for 30, 60 or 240 min. Cell culture supernatants were collected and stored at −80°C until further use. Biopsies were placed through a 100 μm cell strainer  into complete KSFM. The cells were washed and centrifuged, after which the supernatant was aspirated and discarded. Cells were subsequently assessed for expression of TLR4, TLR7, NEP and NK1R with flow cytometry.2.7For detection of EpCAM, TLR4, TLR7, NEP and NK1R, epithelial cells and biopsies were stained with the following antibodies: EpCAM‐fluoroscein‐isothiocyanate , TLR4‐AlexaFluor700 , TLR7‐phycoerythrin , CD10‐PE‐Cy7  and NK1R‐allophycocyanin . Cells were stained with aqua LIVE/DEAD® Fixable dead stain (Thermo Fisher Scientific) to detect dead cells; Fc Block (BD) was used to block nonspecific binding. Fluorescence minus one controls were used to assess background fluorescence levels. The IntraPrep Permeabilisation Reagent kit  was used to detect intracellular TLRs. Cells were analysed on an LSR Fortessa Flow Cytomter (BD Biosciences). Events in the range of 5000–30,000 were recorded. Data were analysed with FlowJo Analysis Software (TreeStar).2.818 primers (Thermo Fisher Scientific) on a xPeltier Thermal Cykler 200 (MJ Research).Epithelial cells were lysed with RLT Buffer (Qiagen) supplemented with 40 mM dithiothreitol. Total RNA was extracted using the RNeasy Micro Kit Plus (Qiagen); quantity and quality of RNA was determined by spectrophotometer using the wavelength absorption ratios 260/280 and 260/230. Four hundred nanograms RNA was reverse transcribed into cDNA using the Omniscript Reverse Transcription Kit (Qiagen) with Oligo (dT)2.9actb, gapdh) and target genes (tlr4) were acquired from Sigma‐Aldrich and are detailed in Table cxcl8 (Hs00174103_m1) were acquired from Applied Biosystems. Analysis of Ct values was performed using the ΔΔCt method.Real‐time multiplex qPCR was performed using a dual‐labelled probe technique with the Quantifast Multiplex PCR + R kit (Qiagen) on a Stratagene Mx3000P Cycler (Agilent Technologies). All reactions were run with the following program: activation at 95°C for 5 min, followed by 40 cycles of two‐step cycling at 95°C for 45 s and 60°C for 45 s. Primers and probes for reference genes , according to the manufacturer's instructions.2.11SEM. In all experiments, n is equal to the number of subjects. Analysis was performed using a two‐way analysis of variance, followed by a Fisher's posttest. A p value of 0.05 or less was considered statistically significant.Data were analysed with GraphPad Prism software (Version 6.0). Results are expressed as mean ± 33.1, HNBs and HNECs from healthy volunteers and patients with AR were stimulated with R837. SP release was unaffected by the allergic versus non‐allergic status, neither in the HNB specimens (p = 0.9), nor in the HNEC material . A similar increase was seen in AR samples, but with a later TLR4 peak . TLR4 increases were significantly higher in HNB from AR patients at 240 min (p = 0.029) and in HNEC at 60 min (p = 0.046), as compared to respective material from healthy volunteers. Also, at the mRNA level, after 5–24 h, the TLR4 upregulation appeared earlier and was higher in HNECs from healthy individuals than in material from patients with AR , and significantly elevated above values in AR patients (p = 0.032).As no differences in SP release between healthy and AR patients were evident, the second arm of the two‐step SP‐TLR axis was investigated. HNBs and HNEC were stimulated with SP and TLR4 expression was assessed. Representative histograms of TLR4 expression in unstimulated HNB and cultured HNEC are presented in Figures k Figure , demonstR Figure ; at 4 ho3.3p = 0.7745). However, when only epithelial cells of the same biopsies were analysed, the NEP levels appeared to be lower in specimens from allergic patients . This difference was more marked, reaching significance (p = 0.0003) when the NEP analysis was repeated on the HNEC   Figure . SP stim3.4p = 0.038). In HNEC, SP induced a trend towards an increase in the NK1R expression in cells from both healthy and allergic patients. However, the increases among the controls appeared earlier than in material from allergic volunteers  on the surface of epithelial cells.s Figure .3.5TLR4 is responsible for inducing early inflammatory responses against bacterial lipoproteins, such as lipopolysaccharide (LPS). In order to determine whether altered expression of TLR4 following SP stimulation resulted in altered responses to bacterial exposure, isolated HNEC were stimulated with SP for 30 or 60 min and then subsequently exposed to LPS. When the levels of IL‐8 mRNA were assessed 3 h after the LPS exposure, increases were evident in the control, nonallergic material, but failed to appear in cells from patients with AR , leading to large degree of variability in the results. A larger sample size would likely result in greater statistical stability of the data. Second, results from the study were based on in vitro stimulations; in vivo stimulations would be of interest to further confirm the differences in SP‐TLR axis in atopic and nonatopic individuals. Finally, only a subset of factors involved in the regulation of SP responses, namely NEP and NK1R, were investigated in this study. It is likely that other neuropeptides and inflammatory mediators are involved in this response. Further work on these mediators would offer a more complete picture of the SP‐TLR axis and would more intricately explain the differences seen in AR patients.The present study demonstrates that in healthy individuals, SP functions as a regulator of innate immunity, particularly in upregulation TLR4 and thus priming LPS‐induced inflammation. In patients with AR this SP‐TLR axis appears to be impaired, with a heightened and prolonged upregulation of TLR4, associated with the lack of the expected increased response to LPS. This compromised ability for SP to regulate immunity may, at least in part, contribute to the more severe and prolonged infection‐induced diseases often seen in conjunction with airway allergy.Olivia Larsson, Susanna Kumlien Georén, Claus Bachert and Lars Olaf Cardell designed the experiments and interpreted the results. Ola Sunnergren recruited patients and took all biopsies. Olivia Larsson performed the experiments, analysed the data and wrote the paper. All authors read and approved the final manuscript.Supporting Information S1Click here for additional data file.Supporting Information S2Click here for additional data file.Supporting Information S3Click here for additional data file.


Prostate cancer is one of the most frequently diagnosed cancers in men and is the second leading cause of cancer death in developed countries. Current therapeutics that target the androgen receptor (AR) are only transiently effective. Anti-AR therapy-resistant tumors often emerge with vast cellular and molecular alterations and present themselves at the clinic in more deadly forms including metastatic castration-resistant prostate cancer (mCRPC) or neuroendocrine prostate cancer (NEPC). One emerging strategy in effective treatment of the advanced forms of prostate cancer is to target drivers other than AR. The present study shows that the nuclear receptor RORγ and the serine/threonine kinase PBK form a regulatory loop in hyperactive AR signaling. It also demonstrates that orally administered, small-molecule antagonists/inverse agonists of RORγ are effective in blocking the growth of the mCRPC tumors. Our findings provide a rationale for therapeutic targeting of RORγ alone or in combination with PBK inhibitors for the advanced forms of prostate cancer.Metastatic castration-resistant prostate cancer (mCRPC) is a highly aggressive disease with few therapeutic options. Hyperactive androgen receptor (AR) signaling plays a key role in CRPC progression. Previously, we identified RAR-related orphan receptor gamma (RORγ) as a novel key driver of AR gene overexpression and increased AR signaling. We report here that several RORγ antagonists/inverse agonists including XY018 and compound 31 were orally effective in potent inhibition of the growth of tumor models including patient-derived xenograft (PDX) tumors. RORγ controls the expression of multiple aggressive-tumor gene programs including those of epithelial-mesenchymal transition (EMT) and invasion. We found that PDZ binding kinase (PBK), a serine/threonine kinase, is a downstream target of RORγ that exerts the cellular effects. Alterations of RORγ expression or function significantly downregulated the mRNA and protein level of PBK. Our further analyses demonstrated that elevated PBK associates with and stabilizes RORγ and AR proteins, thus constituting novel, interlocked feed-forward loops in hyperactive AR and RORγ signaling. Indeed, dual inhibition of RORγ and PBK synergistically inhibited the expression and function of RORγ, AR, and AR-V7, and the growth and survival of CRPC cells. Therefore, our study provided a promising, new strategy for treatment of advanced forms of prostate cancer. The hyperactivity of androgen receptor (AR), a member of the nuclear receptor (NR) family of transcription factors, is the major driver of prostate cancer (PCa) progression to metastatic castration-resistant PCa or mCRPC. Mechanisms contributing to AR hyperactivity in the tumors include AR gene overexpression, amplification, mutations, and aberrant alternative splicing that generates constitutively active AR variants, such as AR-V7, that lack the ligand binding domain (LBD) ,2,3. OthCurrently, therapeutics suppressing AR activity are the mainstay of systemic therapy for advanced PCa. Widely used AR antagonists such as bicalutamide and enzalutamide targeting the AR LBD exhibit initial favorable response in most patients. However, resistance to those drugs inevitably develops and the disease relapses through tumor genetic and epigenetic changes, including the generation of AR variants such as AR-V7 ,3,10,11.In our pursuit of developing new therapeutics for advanced PCa, we identified retinoic acid receptor-related orphan receptor-γ (RORγ), another member of the NR family , as one In this study, we demonstrate that via oral dosing several RORγ antagonists/inverse agonists including compound 31 (cmpd 31 hereafter) are highly effective in inhibition of the growth of patient-derived xenograft (PDX) tumors. We found that RORγ controls the expression of multiple aggressive-tumor gene programs including epithelial-mesenchymal transition (EMT) genes such as PTTG and PBK. Interestingly, PBK associates with and stabilizes RORγ protein, thus constituting novel, interconnected feedforward loops in AR hyperactivity and a vulnerability point in effective therapeutic targeting for mCRPC.50 of 1.51 μM) than XY018 or GSK805   . Our colctively) . Similarctively) E. While ctively) E. Furthectively) F. In furctively) . TogetheThe remarkable activity of cmpd 31 in inhibition of CRPC cell growth prompted us to examine its anti-tumor potency. As shown in To examine the mechanism of the RORγ antagonists in inhibition of CRPC, we performed RNA-seq analysis with C4-2B cells treated with the antagonists. As shown in The pro-oncogenic serine/threonine kinase PBK was recently shown being overexpressed in metastatic PCa tumors and regulated by AR . Given tTo further explore the functional interactions between PBK and RORγ, we treated C4-2B cells with increasing doses of the PBK inhibitor (PBKi) and measured the expression of AR, RORγ, and PBK. Interestingly, we found that the protein levels of RORγ, AR, and PBK were significantly decreased by the PBKi in a dose-dependent manner A whereasOur identification of RORγ-PBK as a novel regulatory axis prompted us to examine the consequences of simultaneous targeting of the two proteins. Indeed, treatment with PBKi at 0.4 μM was sufficient to sensitize C4-2B cells to RORγ antagonist cmpd 31 as suggested by an approximately 4-fold decrease (from 0.156 to 0.039 μM) of cmpd 31 concentrations needed in suppression of C4-2B colony formation to close to 140 colonies . SimilarHyperactive AR signaling due to AR gene alterations and AR variants is a well- known major driver of PCa progression to mCRPCs ,30. We pIn our attempt of revealing the major gene programs perturbed by the RORγ antagonists, we identified several major EMT regulators such as PBK, FN1, NEK2, and PTTG1 that are strongly inhibited by the antagonists. Indeed, the serine/threonine kinase PBK was of particular interest to us. Recently, several studies showed that PBK is overexpressed in multiple cancer types including prostate cancer ,32,33,342 at 37 °C. siRNAs were purchased from Dharmacon . The siRNA sequences targeting RORγ gene RORC were CGAGGATGAGATTGCCCTCTA for siRORC-1, and CACCTCACAAATTGAAGTGAT for siRORC-2. siRNA sequence CAGTCGCGTTTGCGACTGG was used as non-targeting control. Transfection of siRNAs was performed with OptiMEM  and Dharmafect1 , following the manufacturer’s instructions.C4-2B cells were from UroCor Inc. 22Rv1 cells were from ATCC. They were cultured in RPMI 1640  containing 10% Fetal Bovine Serum (FBS) . To mimic the castration-resistant prostate cancer (CRPC) condition, C4-2B cells were cultured in phenol red-free RPMI 1640 (Thermo Fisher Scientific) supplemented with 9% charcoal-dextran-stripped (cds) FBS, plus 1% FBS for experiment. All cells were grown in a humidified incubator with 5% COSR2211 was purchased from TOCRIS . XY018, Cmpd 31 and GSK805 were synthesized and purified to >98–99% purity by WuXi AppTec. PBK/TOPK inhibitor HI-TOPK-032 was from Sigma . Other chemicals were purchased from Thermo Fisher Scientific.5 per well and transfected with siRNA or treated with compounds as indicated. Total cell number were counted with a Coulter counter. For colony formation assay, 500 cells were seeded in a well of 6-well plate and cultured for 14 days. Then cell colonies were stained with Fix/Stain buffer  for 20 min at room temperature (RT) and washed with H2O five times and air-dried overnight. Cell colonies were then scanned and counted.For cell viability, cells were seeded to 96-well plates at 1500/well density. Indicated concentrations of compounds were added to cells after 24 h. After 4 days of incubation, cell viability was measured using Cell-Titer GLO reagents  per manufacturer instructions. For cell growth assay, cells were seeded in 6-well plates at a density of 2 × 10−ΔΔCT method was used to obtain the relative quantifications. Experiments were repeated three times. The primers are listed in Cells were lysed with RIPA lysis buffer . Protein concentrations were measured and adjusted using DC™ Protein Assay Kit I . Then proteins were separated by 10% SDS-PAGE and transferred onto PVDF membranes, which were then blocked by 5% fat-free milk. Membranes were incubated with indicated primary antibodies at 4 °C overnight and then subjected to second antibody (1:2000) incubation at RT for 1 h. Antibody-recognized proteins were visualized using ChemiDocTM MP imaging system (Bio-Rad) after incubation with HRP substrate . Antibodies used in this study are described in 2O2 and then 10% goat serum were used for eliminating non-specific binding. Subsequently, tissue sections were incubated overnight at 4 °C with anti-Ki-67 monoclonal antibody  or anti-cleaved-Caspase-3 monoclonal antibody , at 1:1000 and 1:500 dilutions, respectively. Then the slides were washed and incubated with biotin-conjugated secondary antibodies for 30 min followed by incubation with ABC reagents in the Vectastain Elite ABC kits  and counterstaining with hematoxylin, as previously described [Briefly, dissected tumor tissues were fixed in 10% neutral buffered formalin overnight and then embedded in paraffin and cut into 4μm-thick sections. The sections were deparaffinized with xylene and rehydrated with gradient alcohol before antigen retrieval was performed in 0.01 M citrate buffer at just below boiling temperature for 10 min. Endogenous peroxidase activity was quenched with 3% Hescribed . Images 5) were then resuspended in RPMI medium containing 0.1% FBS and placed into the upper chamber of Transwell inserts with 8 μm pores  for migration assays or 2 mg/mL Matrigel-coated Transwell inserts for invasion assays. The bottom wells contained 500 µL 10% FBS-RPMI as a chemo-attractant. After 24 h (for migration assay) or 48 h (for invasion assay), cells on the upper surfaces of the inserts were removed using a cotton swab. Migrated cells on the lower surface of the membrane were fixed in 4% formaldehyde and stained with 0.1% crystal violet for 15 min. Cells were counted under microscopy for ten random fields.For Transwell assays, C4-2B cells were pretreated with siRNAs, compounds or RORγ-V5 lentivirus for 48 h and then detached into single-cell suspensions. Cells (1 × 106 per 100 mm dish) were co-transfected with pLX304-V5-RORγ or lentiCRISPR V2 and packaging plasmids psPAX2 and pMD2.G using lipofectamine 2000 overnight. Then medium was replaced with fresh medium. After 48 h, virus-containing supernatants were harvested and stored in −80 °C freezers before use. Cells (2 × 105) in 6-well were infected with the lentiviruses for expressing V5-RORγ or empty vector by incubation with 1 mL of the supernatant supplemented with 10 ng polybrene for 6 h. For co-IP assay, magnetic beads were precoated with 4 μg indicated antibodies at 4 °C overnight. Cells were lysed with lysis buffer  for 30 min at 4 °C. Then the lysates were centrifuged at 15,000× g for 1 min to collect nuclei pellets that were then incubated in extraction buffer  for 15 min to extract nuclear proteins. After protein concentrations were adjusted, 1% nuclear extracts were kept as input. The remaining extracts were divided into two equal groups: one group was incubated with anti-V5 antibody-coated magnetic beads and the other was incubated with IgG-coated magnetic beads. After incubation at 4 °C overnight, the beads were washed using washing buffer  for 5 times and eluted in 2 × SDS protein loading buffer . Then the eluted proteins were subjected to Western blotting. For ubiquitin pull down assay, cells were lysed with NP40 lysis buffer  for 30 min at 4 °C. Then cell lysates were centrifuged at 12,000× g for 10 min to collect supernatants. Equal amounts of supernatants were incubated with anti-V5 Ab-coated or IgG coated magnetic beads. The subsequent steps were the same as Co-IP assay.Human RORγ cDNA was cloned into pLX304-V5 plasmid as previously described . sgRNA s3, the mice were randomized and then treated orally (p.o.) with 100 µL of either vehicle or RORγ antagonist XY018, Cmpd 31 or GSK805 ) for five times per week. Tumor growth was monitored by calipers, and volume was calculated with the equation V = 0.5 × (length × width2). Mice carrying C4-2B tumors were established and treated in the same way as with LuCaP 35CR model. Mouse body weight during the study was also monitored. At the end of the studies, mice were euthanized, and tumors were dissected, weighed, and processed for further analyses.All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of California, Davis. Four-weeks-old male NOD-SCID mice  were castrated and two weeks later subcutaneously implanted with LuCaP 35CR  tumor tihttps://cran.r-project.org/web/packages/pheatmap/index.html, access date 4 January 2019). GSEA was conducted using the Java desktop software , as described previously [C4-2B cells were treated with RORγ antagonists XY018 (5 µM), Cmpd 31 (5 µM), GSK805 (5 µM), or vehicle for 48 h before RNA isolation. RNA-seq libraries from 1 µg total RNA were prepared and validated as previously described  was subseviously . Genes weviously , and epieviously  were useCell culture–based experiments were performed at least three times, with assay points triplicated, as indicated. The data are presented as mean ± SD from three independent experiments. Statistical analysis was performed using one-way ANOVA for qRT-PCR analysis, tumor xenografts, quantitative analysis of IHC data, wound healing, and transwell assay. Statistical significances were considered when were less than 0.05.In conclusion, our study revealed RORγ-controlled aggressive gene programs and the interlocked feedforward loops as a new mechanism in hyperactive AR signaling in mCRPC, and further demonstrated the therapeutic efficacy of ROR-γ antagonists when orally administered in a clinically relevant PDX model. Thus, our study highlights the potential value of the RORγ antagonists for further development as attractive therapeutics for mCRPC.


ABSTRACT IMPACT: This work will accelerate the translation of post stroke rehabilitation devices from the research lab to clinic use. OBJECTIVES/GOALS: Rehabilitation device efficacy alone does not lead to adoption into clinical practice. The objective of this work was to increase understanding of the landscape for clinical adoption of post-stroke physical rehabilitation devices. METHODS/STUDY POPULATION: We conducted interviews with 107 stakeholders including patients who have had strokes, rehab directors, and physical/occupational therapists to understand their viewpoints for adopting new rehabilitation devices. To contribute to previous literature, interviews were analyzed qualitatively using direct content analysis to provide more specific details about the most appropriate adoption settings, specific roles for stakeholders, and drivers for all stakeholders involved in the adoption process. RESULTS/ANTICIPATED RESULTS: Unique to this work, care settings in which therapy goals are best aligned for restorative devices were found to be outpatient rehabilitation, followed by inpatient rehabilitation. Therapists are the major influencers for adoption because they typically introduce new rehabilitation devices to patients for both clinic and home use. We also learned therapists’ utilization rate of a rehabilitation device influences a rehabilitation director’s decision to acquire the device for facility use. Additionally, device setup in <7 minutes will allow for increased use without reducing therapist productivity. DISCUSSION/SIGNIFICANCE OF FINDINGS: Rehabilitation device development should consider the best settings to first introduce the device, roles of each stakeholder, and drivers that influence each stakeholder to accelerate successful adoption of the developed device.


Staphylococcus (Staph)  was evaluated in milk samples collected from: (1) lactating cows with SCM (n = 504), and (2) cows in the post-partum period (PP) . Rapid identification of Gram-positive bacteria in chromogenic media was performed by visual inspection of colony colors after 24 h of incubation at 37°C. Bacterial identification by MALDI-TOF mass spectrometry was considered the reference methodology for calculating: Acc, Se, Sp, PPV, NPV, and Cohen's Kappa coefficient of agreement (k). The chromogenic media GP showed high Acc for Strep. agalactiae/dysgalactiae identification in both samples of SCM  and of cows in PP . Similar results were observed for Strep. uberis/Enterococcus spp. identification  in SCM samples and Se: 100%; Sp: 99.6% and Acc: 99.6% in samples of PP cows using the GP media. However, the GP chromogenic media showed low Se (25.0% in SCM samples and 50.0% in samples of cows in PP) for Staph. aureus identification, despite Sp and Acc were high (Sp: 98.3% and Acc: 95.4% in SCM and Sp samples: 99.4% and Acc: 98.9% in PP cow samples). Staph culture media showed high Acc for Staph. aureus identification , although the low prevalence of Staph. epidermidis and Staph. saprophyticus limit inferences about the performance of identifying these pathogens in Staph media. In conclusion, despite the limitation of the GP media for identification of Staph. aureus, GP, and Staph chromogenic media obtained satisfactory diagnostic performance results for the rapid identification of the main Gram-positive pathogens associated with SCM.The present study aimed to evaluate the diagnostic performance specificity (Sp), sensitivity (Se), positive predictive value (PPV), negative predictive value (NPV), and accuracy (Acc) of two chromogenic culture media for rapid identification of Gram-positive bacteria causing subclinical mastitis (SCM) in dairy cows. For this, the performance of chromogenic culture media Gram-positive (GP) and  Mastitis is a disease that causes major losses in dairy production . The mosThe occurrence of SCM in the dairy herd leads to direct as well as indirect economic losses, such as the decrease in milk production, which reduces the profitability of farms , 7. ThesThe use of microbiological culture associated with biochemical tests is the standard method for identifying mastitis-causing agents. However, laboratory microbiological culture is not widely used in dairy herds across different countries . The maiChromogenic culture media are alternatives for rapid microbiological identification, as they make it possible to presumptively differentiate bacterial species and/or groups according to colony color, reducing the need for biochemical tests . These cStaph. aureus in CM milk samples. Therefore, the use of chromogenic culture media on the dairy farm routine could be an alternative for the rapid identification of SCM. However, no previous study evaluated the effectiveness of chromogenic media in milk samples from cows with SCM, and for the monitoring of cows in post-partum (PP); which is a period of the high risk of new IMI and the manifestation of IMI not cured during the dry period can manifest , respectively, present high Acc, Se, and Sp for rapid identification of the main SCM causing agents during lactation and in the PP period compared to standard laboratory microbiological identification methods such as mass spectrometry.Thus, the present study hypothesizes that the chromogenic media Mastitis GP and Staph. , which are selective for Gram-positive (GP) and The present study was carried out under the ethical principles of animal experimentation and followed the rules established by the National Council for the Control of Animal Experimentation (CONCEA). The experimental protocol adopted was approved by the Ethics Committee on the Use of Animals (CEUA) of the Faculty of Veterinary Medicine and Zootechnics of the University of São Paulo under registration No. 4579250719.n = 5) and Minas Gerais (n = 1), Brazil. Dairy herds were selected based on non-probabilistic convenience sampling . Herds had an average of 125 (± 60) lactating cows housed in compost-bedded pack barns, with average milk production of 27.5 (± 2.5) liters/cow/day. Also, all herds performed individual monthly SCC analysis of all lactating cows. Before the beginning of the study, milking personnel and/or those responsible for the udder health of each herd were trained for procedures of sampling collecting collection, and clinical mastitis diagnosis.The milk samples were obtained from six dairy herds located in the states of São Paulo (n = 226) or individual samples (n = 315) of mammary quarter collected for microbiological culture. In our study, cows with CM, or cows that present concomitant diseases other than mastitis and/or treated with antimicrobials (systemic or intramammary) in <14 days of sample collection, were excluded.Five hundred and four lactating cows with SCM  had composite milk samples (pool of the four mammary quarters) collected for microbiological identification of mastitis-causing pathogens. Additionally, 541 cows in the PP period  had composite milk samples , for identification of Gram-positive bacteria and Streptococcus agalactiae/dysgalactiae; (b) dark blue/metallic blue—Streptococcus uberis/Enterococcus spp.; (c) pink/mauve—Staphylococcus aureus  colorless/pinky colony—Staphylococcus epidermidis; (c) turquoise blue—Staphylococcus saprophyticus. In both culture media, colonies showing other colors not described in the manufacturer's recommendations were considered as “other microorganisms.”The GP culture media identification results were interpreted according to the following colony colors: (a) turquoise blue—s aureus . AdditioStrep. uberis/Enterococcus spp. or Strep. agalactiae/dysgalactiae when they presented a growth of 3 or more colonies with the color pattern defined for the species. For Staph. aureus, the growth of only 1 colony was considered the threshold for the identification of the agent. In the Staph culture media, the growth of ≥ 1 colony was sufficient for the identification of Staph. aureus, Staph. epidermidis, and Staph. saprophyticus. After the visual inspection of morphological characteristics, all the isolates that showed positive growth in blood agar and the chromogenic culture media were submitted to microbiological identification by Matrix Associated Laser Desorption-Ionization—Time of Flight (MALDI-TOF MS), as previously described  blood agar supplemented with 5% bovine blood; b) GP chromogenic media and c) Staph chromogenic media. After inoculation, the plates were incubated at 37°C for 24 h, and then they were subjected to visual inspection to evaluate the microbiological growth. Samples that presented ≥ 3 microorganisms with different characteristics in composite milk samples, or individual mammary quarter, were considered contaminated . Besidesescribed . ThroughThe visual identification of mastitis-causing agents by GP and Staph chromogenic media was compared to the gold standard method (MALDI-TOF MS) to estimate performance indicators. Samples that showed contamination in one of the two results (blood agar and/or chromogenic media) were excluded in the final analysis.The performance indicators of Acc, Se, Sp, Positive Predictive Value (PPV), and Negative Predictive Value (NPV) of the chromogenic media were calculated based on the following results: True Positive , True Negative , False Positive , False Negative  .The SENSPAC option of PROC FREQ; SAS version 9.4  was used to calculate Se, Sp, PPV, and NPV. Acc was also calculated by PROC FREQ, considering the TP and TN results. The results of Acc, Se, Sp, PPV, and NPV for the microbiological identification of chromogenic culture media were classified as low (results <60%), intermediate (between 60 and 80%), and high (> 80%) .The results of Cohen's Kappa coefficient of agreement (k) were calculated by PROC FREQ of SAS version 9.4 . Almost perfect agreement was considered when results of the coefficient between 0.81 and 1.00; substantial agreement (results from 0.61 to 0.80); moderate agreement (results from 0.41 to 0.60); fair agreement (results between 0.21 and 0.40), slight agreement (results between 0.00 and 0.20), and values ≤ 0.00 poor agreement  were isolated from the three culture media , considering that the microorganisms isolated in each media are not necessarily the same for the same sample. No contamination was observed in SCM samples evaluated.n = 5; four composite and one of the mammary quarter) contamination was observed, and these milk samples were not evaluated in diagnostic performance analysis.A total of 541 milk samples (226 composite and 315 from mammary quarter) from post-partum dairy cows were evaluated during the 04/12/2019–07/25/2019 periods. A total of 36 different species of microorganisms  were isolated considering the growth results for the three culture media evaluated. In 0.9% milk samples (Staphylococcus (NAS) was the microorganism group that had the largest isolation frequency . Staph. chromogenes was the most frequently isolated pathogen in both SCM  and PP  of the SCM milk samples and 30.5% (165/541) of PP milk samples had microbiological growth. Of these, mixed culture was observed in 3.8% (19/504) SCM and in 2.4% (13/541) PP milk samples. Gram-positive bacteria had higher isolation frequency  and non-aureus Staph. chromogenes were the most frequent pathogen isolated in GP media  of SCM and 13.3% (72/541) of PP milk samples had microbiological growth. Mixed culture was observed in 5.8% (29/504) of SCM and 1.5% (8/541) of PP milk samples. Also, 4.8% (24/504) of SCM and 0.6% (3/541) of PP milk samples had microbiological isolation in GP chromogenic media and had no growth in blood agar. Staph. chromogenes was the most isolated pathogen , followed by Staph. aureus . Strep. uberis was isolated in Staph media in 0.4% (2/504) of SCM milk samples. Colonies of Strep. uberis identified in the Staph media and the respective milk sample were re-inoculated in Staph media plates and no microbiological isolation was observed.In Staph chromogenic media, 32.74% (165/504) of SCM and 5.5% (30/541) of PP milk samples had microbiological isolation. Of these, mixed culture was observed in 2.4% (12/504) of SCM and 0.6% (3/541) of PP milk samples. Also, 5.6% (28/504) of SCM and 0.7% (4/541) of PP milk samples had no growth in blood agar and had microbiological isolate in Staph chromogenic media. Strep. uberis/Enterococcus spp.) to 95.6%  for SCM milk samples, and from 98.9% (Staph. aureus) to 99.3% (Strep. uberis/Enterococcus spp.) for PP milk samples. Additionally, Se of GP media varied according to the group of pathogens from 25.0% (Staph. aureus) to 90.5% (Strep. uberis/Enterococcus spp.) in SCM milk samples, and from 50% (Staph. aureus) to 100%  for PP milk samples. Gram-positive culture media Sp for SCM ranged from 92.5% (Strep. uberis/Enterococcus spp.) to 98.7% (Staph. aureus), and in PP milk samples ranged from 99.1%  to 99.6% (Strep. uberis/Enterococcus spp.).Accuracy of GP chromogenic media identification varied according to the pathogen group from 92.3% (Staph. aureus) to 70.4%  in SCM milk samples and 50.0% (Staph. aureus) to 71.4% (Strep. uberis/Enterococcus spp.) in PP milk samples. Also, NPV results range from 97.0% (Staph. aureus) to 98.9%  in SCM milk samples, and range from 99.4% (Staph. aureus) to 100%  in PP milk samples.Positive predictive value results of GP media identification varied according to the pathogen group from 38.5% (Staph. aureus) to 0.76  in SCM milk samples and range from 0.45 (Staph. aureus) to 0.72 (Strep. uberis/Enterococcus spp.) in PP milk samples. Indicators of diagnostic performance for negative results of SCM in GP chromogenic media were: Acc = 82.1%, Se = 88.7%, Sp = 77.3%, PPV = 74.1%, NPV = 90.4% and k = 0.60. Similarly, for PP negative milk samples, Acc was 83.4%, Se = 99.2%, Sp = 44.5%, PPV = 81.5%, NPV = 95.8%, and k = 0.50.Kappa coefficient of agreement values varied according to the pathogen group from 0.27 (Staph. epidermidis (n = 1 in SCM and n = 4 in PP milk samples) and Staph. saprophyticus (n = 1 in SCM and n = 0 in PP milk samples), it was not possible to evaluate the diagnostic performance of Staph chromogenic media for microbiological identification of these pathogens. However, Staph. aureus identification by Staph media in SCM milk samples had Acc = 98.0%, Se = 80.0%; Sp = 98.8%; PPV = 72.7% and NPV = 99.2%. In addition, for Staph. aureus identification in PP milk samples, Staph media had results of Acc = 99.6%; Se = 66.7%; Sp = 100%; PPV = 100%, NPV = 99.6%, and k was 0.71 for SCM and 0.80 for PP milk samples  were similar to those obtained for the identification of Streptococcus spp. from the Minnesota Tri-Plate triplet   indicate that it is possible to obtain identification results reliable for these pathogens from PP samples.In chromogenic media GP, it was observed high Se and Sp for identification of ctively) . Howeverctively)  and MacCStrep. agalactiae/dysgalactiae and Strep. uberis/Enterococcus spp. groups, both in SCM samples  and for PP samples . These results were inferior to those obtained by Ganda et al.  which would match the results obtained by Accumast and Minnesota plate.The positive predictive values were classified as low/moderate for the a et al.   and for PP samples (NPV = 100% and NPV = 99.6). The results obtained were superior to those of the Minnesota Easy Culture System biplane for Streptococcus spp., and similar to those obtained by the Minnesota Easy Culture System Tri-Plate  and PP samples (Se = 50%). This low Se can be attributed to an inconsistency in the color pattern generated for the diversity of Staph. aureus isolates that were evaluated in this study. Different shades of pink were observed, not necessarily consistent with the characteristic color associated with Staph. aureus identification according to the manufacturer's recommendations, which caused misidentification and high numbers of FN results. Due to the high amount of FN results, both in SCM samples  and in PP samples (FN = 50% 3/6), PPV was also compromised . Similar results for Se and PPV were obtained for Staph. aureus in SCM samples by the rapid microbiological identification method VetoRapid kit  and moderate to high for PP samples . The PPV result was intermediate for SCM samples (PPV = 72.7) and the NPV result was high (NPV = 99.2). However, PPV would be higher (PPV = 90.9%) if we included samples with the isolation of Staph. aureus in Staph culture media (correctly identified by the chromogenic media), but without growth on blood agar were considered TP. These results are similar to those obtained by Accumast , which has not been described in Minnesota Easy Culture System II and Accumast. However, due to the low isolation frequency of Staph. saprophyticus and Staph. epidermidis, it was not possible to determine the diagnostic performance in these species.For the chromogenic media Staph, the results of Se and Sp for Accumast  and MinnAccumast . The abiure risk . The resk coefficient, was substantial for the Strep. agalactiae/dysgalactiae (k = 0.76) and Strep uberis/Enterococcus spp. (k = 0.70) groups for SCM samples. In PP samples, we also observed substantial agreement for the groups of pathogens mentioned above . The agreement observed in the present study was higher than that obtained for Streptococcus spp. by the bi-plate (k < 0.6) and similar to the tri-plate (k between 0.73 and 0.85) Minnesota Easy Culture System (k = 0.91)  . The abindicated .k result obtained indicates that GP chromogenic media had a fair agreement for the rapid identification of Staph. aureus (k = 0.30), which corroborates the unsatisfactory results of Se (25.0%), and PPV (38.5%). For PP samples, the agreement was moderate (k = 0.49). The agreement results were lower than those obtained by Accumast (k = 0.93)  (k = 0.33)   and by tctively)  but were = 0.33) . The lowStaph. aureus for both SCM samples (k = 0.73) and PP samples (k = 0.79). Our results were slightly lower than the almost perfect agreement obtained by Accumast (k = 0.93) (k coefficient of the Staph media was higher than that obtained by the Minnesota Easy Culture System Bi-Plate and Tri-Plate (both k < 0.7)    in CM sak < 0.7) , CHROMagctively) , indicatn = 86). The results obtained for the GP chromogenic media were superior to those described by Ferreira et al.  and moderate Sp (77.3%) results were obtained in SCM samples, indicating the assertiveness of the media for negative results. In the PP samples, GP chromogenic media obtained high Se for negative results (99.2%) but presented low Sp (44.5%). The low Sp may be associated with the high amount of FP . Also, two samples of SCM had isolation of Strep. uberis, a pathogen that should have growth inhibited in the Staph chromogenic media, which is selective for Staphylococcus spp. However, in both cases, when these colonies were transferred to a new plate of the Staph chromogenic media, there was no microbial isolation, and the same occurred when a new inoculation of the milk sample was performed.The Se of samples without growth in the Staph chromogenic media was also high in SCM (Se = 91.4%), while the Sp was moderate (Sp = 77%). As observed in the GP chromogenic media, for PP samples, Sp was low (Sp = 20.5%) and Se was high (Se = 99%). The results indicate assertiveness in the diagnosis of negative samples for microbiological isolation, but little Sp in this identification, which again may be partially associated with the occurrence of high numbers of FP results, especially in PP samples , which were considered FP results. These results may suggest that the selectivity of the chromogenic media could reduce the competition between microorganisms, thus allowing the growth of non-isolated microorganisms in blood agar . The ability of GP chromogenic culture media to differentiate between the groups Strep. uberis/Enterococcus spp. from Strep. agalactiae/dysgalactiae would be helpful for immediate SCM control, such as the segregation of suspected positive cows for Strep. agalactiae. Additionally, the identification of Strep. agalactiae/dysgalactiae positive cows using the GP media is an indication of the need to screen the whole herd for identification of Strep. agalactiae cows based on laboratory microbiological identification.In general, regardless of the type of sample (SCM or PP), with the only exception of Staph. aureus, which is possibly related to the inconsistency of the color pattern associated with the identification of this pathogen. The performance of the Staph chromogenic media was generally satisfactory for Staph. aureus, the main pathogen to which the media proposed to identify, what indicates the potential of the Staph media to be used in on-farm culture systems for specific identification of Staph. aureus. The identification of Staph. aureus positive cows would be helpful for control measures, such as the segregation and/or culling of infected cows.In this study, GP media presented limitations in the identification of Strep. uberis and Strep. agalactiae/dysgalactiae in GP media, as well as it possible to differentiate between Staph. aureus; Staph. epidermidis and Staph. saprophyticus, when using Staph media.Finally, the tested chromogenic media obtained diagnostic performance similar to other methods of rapid microbiological identification used in on-farm culture systems, with the advantage of allowing between Staph. aureus identification using GP media. The possibility of rapid microbiological identification (from 18 to 24 h), the high values obtained for the diagnostic performance in most groups of pathogens, and the ability to differentiate between some species of Staphylococcus spp. and Streptococcus spp. genus suggests that GP and Staph media are adequate alternatives for using rapid identification of Gram-positive pathogens causing SCM.The results of diagnostic performance of GP and Staph chromogenic media were satisfactory in identifying the main pathogens associated with mastitis in milk samples from cows in PP and SCM samples during lactation, except for The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by National Council for the Control of Animal Experimentation (CONCEA) Ethics Committee on the Use of Animals (CEUA) of the Faculty of Veterinary Medicine and Zootechnics of the University of São Paulo. Written informed consent was obtained from the owners for the participation of their animals in this study.BGa and MS took part in designing the experiment. BGa, CF, GF, BGr, and MS performed the experiment, data analysis, and wrote the paper. All authors participated in reading, provided a critical review, and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


No difference was observed in pedometry and quality of life. Adherence was above 63%. Patient satisfaction was high. Tele-rehabilitation with VAPA appears to be useful in patients with IPF. Exercise capacity was better at follow up at 3 and 6 months compared with the control group. There was no change in quality of life or pedometry. Adherence and patient satisfaction were all high.We investigated the usefulness and effectiveness of tele-rehabilitation on exercise capacity in patients with idiopathic pulmonary fibrosis (IPF). A randomized study was carried out, including stable patients with IPF for 3 months of tele-rehabilitation: video and chat consultations with a physiotherapist and workout sessions with a virtual physiotherapist agent (VAPA). Moreover, 6-min walk test distance (6MWTD), forced vital capacity (FVC), diffusion capacity for carbon monoxide (DLCO), 7 days pedometry, Saint George Respiratory Questionnaire for interstitial lung disease, The King’s Brief Interstitial Lung Disease Questionnaire, and General Anxiety Disorder 7 Questionnaire were tested before and after 3 months of tele-rehabilitation, as well as after 3 and 6 months follow-up. Patient satisfaction and adherence were also measured for tele-rehabilitation with VAPA. Twenty-nine patients aged 70.9 ± 8.6 years, male 72.4%, FVC% 83.5 ± 17.7, DLCO% 50.6 ± 13.0, 6MWTD 468.4 ± 14.8 were included. Fifteen patients were randomized to tele-rehabilitation with VAPA and 14 to the control group. Differences in 6MWTD between groups were at baseline (+10 m ( Idiopathic pulmonary fibrosis (IPF) is a rare progressive fibrotic lung disease with a poor prognosis . The adjIn patients with IPF, pulmonary rehabilitation (PR) can improve dyspnea, quality of life, and functional exercise capacity in both the short and long term . RehabilTele-rehabilitation is flexible, allowing patients to exercise whenever they find it suitable, and it can reach patients across long distances as well as increase disease awareness . In otheOur aim was to assess adherence, usefulness, and patient satisfaction of tele-rehabilitation, and to compare the effect of tele-rehabilitation to usual care without rehabilitation in patients with IPF. A new platform, the virtual autonomous physiotherapist agent (VAPA), was used to deliver the tele-rehabilitation.www.randomization.com, (accessed on 20 September 2021)). All subjects were randomized into one block (reproducible using seed 26058).The study was a single-center, prospective, randomized clinical trial. Randomization was undertaken electronically at randomization.com  scan, lung function tests, bronchoscopy, and lung biopsies, if available . InclusiPatients were randomized 1:1 to a 12-week tele-rehabilitation program on top of usual care or to a control group receiving only usual care (without rehabilitation). After 12 weeks of rehabilitation, patients in the intervention arm were offered daily use of VAPA during follow-up, but without physiotherapist guidance. End parameters were measured at baseline, after the end of the three-month tele-rehabilitation period (3 months from baseline), and again after three and six months of follow-up (6- and 9 months from baseline respectively).Tele-rehabilitation was delivered using a virtual autonomous physiotherapist agent (VAPA), a platform developed in a European consortium  and two university hospitals  and funded by the Eurostars program . The telThe content of the tele-rehabilitation program is shown in The primary endpoint was exercise capacity measured as 6MWTD in meters (m) from baseline to 3 months (end of tele-rehabilitation) . SecondaThere are no available studies of tele-rehabilitation in IPF, and previous studies used other QoL measures. Thus, there are no reliable results on which to base a sample size calculation. Our study was planned as a pilot study to estimate the effect of a tele-rehabilitation solution, including 24 patients divided into two groups with 12 in each group. A drop-out rate of 25% was expected, thus we aimed to recruit 30 patients in total.Changes in endpoints after tele-rehabilitation and follow-up were compared between groups using the unpaired t-test for parametric data and Mann–Whitney rank-sum test non-parametric data using 95% confidence intervals. Changes from baseline to after both tele-rehabilitation and follow-up were analyzed within groups using the paired t-test for parametric data and the Wilcoxon signed-rank test for non-parametric data. Data results are expressed as means ± standard deviation The analysis was run in the R program and IBM SPSS Statistics version 25. All results were analyzed using the intention-to-treat approach .p-value 0.03); all other baseline parameters were not statistically significant different between groups. Baseline demographics are displayed in Ninety-one patients with IPF were screened for participation and twenty-nine patients were included. Fifteen patients were randomized to tele-rehabilitation with VAPA and 14 patients to the control group . Mean agEligible patients declining participation suffered from more advanced lung disease . Excludep = 0.03) and at 6 months from baseline , but not at 9 months from baseline .Tele-rehabilitation with VAPA resulted in sustained 6MWTD of 465.64 ± 122.75 at baseline compared with 469.91 ± 115.63 after 3 months, 469.10 ± 135.88 at 6 months, and 447.67 ± 132.97 at 9 month, while controls experienced a decline from baseline at 456.00 ± 51.73 to 420.80 ± 70.16 after 3 months, 422.6 ± 76 after 6 months, and 389.6 ± 84.8 at 9 months . StatistNo statistically significant differences between groups were observed regarding exercise activity as measured by pedometry, QoL, and pulmonary function test (PFT) . No adveNo participants in the control group reported any form of training during the study. In the tele-rehabilitation with VAPA group, five and three patients reported continuous training with the tele-rehabilitation with VAPA program, but without interaction with a therapist after 6 and 9 months, respectively.As shown in Additional results from this study regarding total group baseline data, results on all variables in relation to pulmonary function, physical performance and quality of life in control and Tele-rehabilitation with VAPA groups and comparison between groups can be found in the Supplementary File S1.Our aim was to assess adherence, usefulness, and patient satisfaction of tele-rehabilitation, as well as to compare the effect of tele-rehabilitation to usual care without rehabilitation in patients with IPF. This new platform, the virtual autonomous physiotherapist agent (VAPA), appears to be useful. We found significant differences between groups after 3- and 6 months from baseline. No difference was observed in pedometry and quality of life. In addition, the study shows high adherence and patient satisfaction. This study is the first to evaluate the usefulness of a new platform, VAPA, for tele-rehabilitation in patients with IPF as an alternative to standard care. Tele-rehabilitation with VAPA sustained exercise capacity and showed high patient adherence and satisfaction.According to a recent review, rehabilitation in patients with IPF resulted in improved exercise capacity, with a mean difference of 6MWTD of 40.07 m compared with controls . Our resIn our study, training with tele-rehabilitation seemed to somewhat stabilize physical capacity, while the control group continuously deteriorated in 6MWTD as time advanced . PulmonaHospital-based pulmonary rehabilitation programs combine aerobic exercise, respiratory muscle training, oxygen therapy, nutritional intervention, education, and self-management approaches, among others. In tele-rehabilitation with VAPA, we digitalized the content of the rehabilitation program as much as possible, but still included aerobic exercise and respiratory muscle training with nutritional intervention, education, and self-management approaches by creating e-learning packages with direct messages and “know how” on how to cope with disease symptoms. We were not able to show improved QoL, in accordance with most other studies ,32,35, bRegarding the 7 days pedometry and 7 dVMCPM, we were not able to detect any changes between groups, but we observed a trend, though statistically insignificant, towards better performance in the tele-rehabilitation with VAPA group. The low number of patients and the fact that patients started the study in different seasons of the year may partly explain this. For instance, a patient enrolled during the summer may potentially have walked less at follow-up in the winter.Considering the different tele-rehabilitation platforms that are available ,10,11,12In virtual reality, the avatar is a representation of the patient in the virtual world. The avatar interacts with the virtual agent, a computer-based entity who guides the user to perform the intended tasks. An avatar can, for instance, be a graphical representation of a patient’s body shape, or a sound representing the pulse of the patient, whereas an agent can be presented by a human-like graphical representation or just a voice. Importantly, the avatar, as a representation of the patient, is followed continuously by the agent, giving the agent the opportunity to know where the patient is in real time when performing a certain task. Such a capability triggers behavioral changes in the agent who follows specific rules programmed in a decision support system. Rules are based on avatars’ behavioral changes.In the VAPA set-up aimed at patients with cardio-pulmonary diseases, the best parameter to follow is the heart rate. Here, the heart rate creates a numerical avatar in the VAPA agent virtual world and triggers the agent’s guidance according to whether the patient’s heart rate is above or below a certain pre-specified threshold during exercise performance.Video platforms such as the one used in the studies by Hansen and Rayce ,38,39 alHaving to attend training at scheduled appointments virtually at home can also affect patients’ partners who need to change their way of living as they may need to be available in the case of problems with the ongoing video meeting . MoreoveIn our study, patient adherence was moderate to high and higher than the 20–50% reported in previous studies ,35, and Our study has several limitations. The low number of participants and the drop-out rate may have influenced the results. However, this is not uncommon in patients with severe and advanced disease and not different from what is seen in rehabilitation programs for patients with other chronic respiratory diseases ,42. Our Tele-rehabilitation with VAPA appears to be useful for patients with IPF and the results are sustained exercise capacity at 3- and 6 months from baseline, while reduced exercise capacity was observed in the control group. No change in QoL or 7 days pedometry was observed. Tele-rehabilitation had a relatively high adherence as well as high patient satisfaction and safety. Tele-rehabilitation with VAPA thus seems to be a promising alternative rehabilitation strategy in IPF and a useful technology to increase IPF positive behavioral change towards a more physically active lifestyle.


Dear Editor,Frontal Fibrosing Alopecia (FFA) is primary lymphocytic scarring alopecia with an unclear etiopathogenesis. Hormonal, immune-mediated, genetic, and environmental hypotheses have been proposed; one of which relates to increased sunscreen use.A case-control study with thirty-six Hispanic females; eighteen cases and eighteen sex-and age-matched controls, was designed. The protocol was approved by the local ethics committee of the University Hospital “Dr. José Eleuterio González” in Monterrey, Mexico. A questionnaire inquiring about hygiene products and sunscreens, including time of use, was formulated. Patients and controls were patch tested using allergens from allergEAZE’s Standard, Cosmetic, and Photopatch series. Readings were performed 48 and 96 hours after application. Reactions were measured by the North American Contact Dermatitis Group criteria.The mean age of FFA patients was 59.1 and 56.5 years for controls. Sixteen cases were postmenopausal. Ten subjects referred minimally one symptom (mostly pruritus). Seven cases had at least one autoimmune-mediated disease, being lichen planus pigments the most common. Nine patients had a family history of autoimmunity.Hair loss outside of the scalp was noted in fifteen cases. All had eyebrow, twelve limb, and ten eyelash involvements. Eight individuals presented facial papules.p = 0.035). Makeup foundation was used by fourteen patients in each group. FFA cases used more facial moisturizers compared to controls, without statistical significance.Fifteen patients had positive reactions to at least one allergen against ten controls, no significant difference was found. The most common allergens in FFA were iodopropynyl butylcarbamate and propolis . We revi2) nanoparticles in hair shafts of FFA patients and asymptomatic controls; no positive reactions in eight FFA patients patch tested to five titanium types were found. It is unclear if TiO2 plays a role in FFA pathogenesis.Aldoori et al. suggested environmental factors associated to FFA; with a significant difference in sunscreen use compared to controls. Forty patients were patch tested; more positive reactions to linalool hydroperoxide and Balsam of Peru were found in subjects with FFA versus general patch tested subjects.Our study included sex-and age-matched healthy controls, while most of the previous publications have a general patch test population. Limitations include small sample size, recall bias for sunscreen use, lack of genetic studies, and generalizability due to racial/ethnic homogeneity.In conclusion, FFA’s etiopathogenesis remains unknown. Increased sunscreen use and a greater prevalence of contact allergy was demonstrated in our FFA patients. Raising the question of whether allergic/immunologic responses in genetically predisposed patients could be triggered by exogenous substances. Our statistically non-significant greater prevalence of contact allergy and heterogeneity of results in previous studies endorses the need for further research to confirm this association.None declared.Sonia Sofia Ocampo-Garza: Conception and design of the study; acquisition of data; analysis and interpretation; drafting the article; final approval of the version submitted.Maira Elizabeth Herz-Ruelas: Conception and design of the study; analysis and interpretation; revising the article; final approval of the version submitted.Sonia Chavez-Alvarez: Conception and design of the study; revising the article; final approval of the version submitted.David Marcelo de la Fuente-Rodriguez: Acquisition of data; final approval of the version submitted.Jorge Ocampo-Candiani: Conception and design of the study; analysis and interpretation; revising the article; final approval of the version submitted.None declared.


Triple-negative breast cancer (TNBC) represents a heterogeneous group of breast cancers that lack estrogen receptor (ER), progesterone receptor (PR), and human epidermal factor 2 (HER2) amplifications. This triple negativity represents a challenge in choosing the right treatment, as without the aforementioned biomarkers there are no efficient therapeutic targets. Nevertheless, some triple-negative breast cancers express androgen receptor (AR), which could be used as a novel therapeutic target in such subgroup of breast cancers. In our review, we aimed to identify clinical features and proposed potential therapeutic approaches of this specific subgroup—AR-positive triple-negative breast cancer. Our findings contributed to a better understanding of the current problematics regarding AR-positive TNBC. This review summarizes the recent findings of a vast array of studies conducted on androgen receptor-positive triple-negative breast cancer (AR-positive TNBC) to provide a better understanding of this specific breast cancer subgroup. AR expression is correlated with higher age, lower histological grade, lower proliferation index Ki-67, spiculated masses, and calcifications on mammography. Studies investigating the correlation between AR expression and lymph node metastasis are highly discordant. In addition, results regarding prognosis are highly contradictory. AR antagonists are a promising novel therapeutic approach in AR-positive TNBC. However, AR signaling pathways should be more investigated in order to understand the influence of AR expression on TNBC more thoroughly.  Triple-negative breast cancer (TNBC) represents a heterogeneous group of breast cancers that are defined by absence of molecular markers. They lack estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) amplifications . ExpressWhen AR is inactive, it is bound on heat shock proteins (HSP). Most commonly in carcinogenesis, HSP70 impacts cell survival and could be a potential target for therapy . They stNevertheless, the role of AR in breast cancer remains unclear and therefore it is cardinal that its function is thoroughly researched. Furthermore, the interactions between the AR and androgens are complex and they differ between different sex, age, tissue type, and hormonal status . AR posiThe aim of our review is to focus on TNBCs that express AR and illustrate the current problematics in their diagnosis, prognosis, associations with clinicopathological features, and treatment. The studies investigating associations between clinicopathological features and AR expression are highly discordant. Age at diagnosis was greater among the LAR subtype  and AR-pInterestingly, a recent study  found siAmong all TNBC subtypes, LAR is the most highly differentiated tumor . It was Ki-67 was lower among the LAR subtype than in the other subtypes ,44,45. SThere was no correlation found between AR and body mass index (BMI), but there was a strong association between AR negativity and diabetes . As thisBreast cancer can metastasize to almost any organ. Since differentiation of carcinomas of unknown primary origin can be a challenge for a clinician, it is of great importance that novel biomarkers for diagnosis of primary breast cancer are found .SOX10 plays a role in mediated mammary epithelial cell growth in vitro and it labels benign myoepithelial cells of the breast, as well as myoepithelial cell-derived neoplasms of the salivary glands. Therefore, it is suggested that it could be attributable to a basal-like or myoepithelial cell-like phenotype of these neoplasms, and it could be a potential biomarker for TNBCs. In relation to the AR, the incidence of AR expression is lower in SOX10-positive TNBC than in SOX10-negative TNBC [SOX10 and AR expression are inversely correlated. Taken together, AR is expressed in many breast carcinomas, but cannot be used as an independent biomarker, whereas SOX10 could be [SOX10 and AR, it could still be used for diagnosis of AR-negative TNBC, the so-called quadruple-negative breast cancer (QNBC). Furthermore, SOX10 in conjunction with GATA3, could be used as additional diagnostic marker of metastasis of unknown origin [SOX10 is useful because its expression is stable between primary and metastatic lesions and exhibits the highest level of concordance in comparison with GATA3 and AR [ive TNBC ,50. In on origin ,50. Addi3 and AR . GATA3 is a zinc finger transcription factor, and it is a key regulator of cellular differentiation and lineage specification and has different roles in the human body. In mammary gland, it is responsible for differentiation of luminal cells and low GATA3 expression correlates with worse breast cancer prognosis [GATA3 expression was strongly correlated with AR positivity, suggesting a pathway linking GATA3 and AR signaling [GATA3 and AR was stronger in lobular carcinomas, suggesting that the pathways linking them are more active in tumors with lobular differentiation. Furthermore, GATA3 expression was negatively associated with nuclear grade, which suggests that it is a marker of better differentiation [rognosis . GATA3 eignaling ,53. SpecGATA3 staining is proved useful, especially in differentiation between breast and urothelial carcinoma, as they share overlapping morphology [GATA3 and AR correlation can be used in GATA3-positive tumors of unknown origin to distinguish metastatic GATA3-positive carcinoma of breast origin from urothelial origin [The significance of rphology . (DAVIS)l origin .GATA3 expression [On the contrary, the expression of AR was not associated with pression , but theSOX10 and GATA3 are useful in distinguishing between primary tumor and metastatic breast cancer [Taken together, both t cancer , the forRecent scientific research is emerging to identify novel prognostic biomarkers for targeted analysis and therapy of TNBCs . PD1 is FOXA1 is a key determinant of ER function and endocrine response. There is a significant association between AR and FOXA1 status ,58. In oER-beta receptor is the other form of ER and it has been, unlike ER-alfa receptor, detected in TNBC patients , more frThe growth pattern of a tumour also contributes to identifying appropriate therapeutic regimen for TNBC patients along with the pathological characteristics. Even though the differences are not so significant between TNBC and non-TNBC patients, it is believed that there are differences among different histological subtypes . StudiesDue to its distinct growth pattern, high rate of missed malignant diagnoses between different TNBC subtypes was observed. The lowest rate of misdiagnosis in mammography was observed among the LAR subtype . However, it was found that AR negativity was associated with higher density on mammography among clinically detected tumors, whereas there was no association found among screening-detected cases .The influence of AR expression on prognosis is still being controversial, as the role of AR signaling in TNBC tumor cells is still not well understood.It was found that prognosis highly depends on the ER status. AR expression is associated with a better prognosis in patients that have ER-positive tumors, whereas ER-negative tumors do not display such a pattern . It was AR positivity is associated with greater mortality in TNBC patients . FurtherOn the contrary, Thike et al. reported that women with AR-positive TNBC had significantly improved DFS and better OS. Additionally, these tumors have a decreased likelihood of recurrence, whereas AR-negative tumors are associated with development of recurrences and prone to develop metastases ,39,40,66AR immunohistochemistry (IHC) expression was statistically associated with better survival, even though it had a statistical association with LNM metastases . Lymph nAdditionally, it was demonstrated that AR-positivity was significantly correlated with better survival in specific high-risk subgroups: young, premenopausal, large tumor size, more node involvement (4+), high stage, high grade, positive vascular invasion, positive p53, negative CK5/6, and higher Ki-67. Thus, it was suggested that expression of AR reveals tumors with better biological behavior in high-risk patients . Some stDistant-metastasis-free survival did not vary between different TNBC subtypes, whereas relapse-free survival (RFS) was increased in the LAR subtype, which suggests that the recurrence was due to different mechanisms and not connected to AR . AR was demonstrated to be an independent prognostic marker for both DFS and OS . On the A high discordance rate is present in biomarker expression between primary and metastatic lesions ,24, as wSince the role of AR in involvement of breast cancer is unclear, it has been contentious whether AR agonists or antagonists should be used in the treatment. It was discovered that AR agonists can inhibit proliferation of AR-positive breast cancer, except in ER-negative and HER2-positive breast cancer . Hence, In multiple non-LAR breast cancer subtypes, relatively low AR expression depended on AR for proliferation, migration, and invasion . FurtherBicalutamide, a first-generation AR antagonist, was shown to induce cell apoptosis  and inhiEnzalutamide, a second-generation AR antagonist, which is a competitive inhibitor that prevents AR nuclear localization , was shoMore than that, AR inhibition with enzalutamide was shown to be an inductor of radiation sensitivity in AR-positive TNBC cell lines, proposing AR inhibition as an effective radiosensitization strategy . Additio2+-activated K+ channel KCa1.1. plays an important role in the promotion of breast cancer cell proliferation and metastasis. It was also found to be an androgen-responsive gene in AR-positive breast cancer. Anti-androgens also affect KCa1.1. in AR-positive breast cancer cells and its down-regulation may contribute at least in part to the anti-proliferative and anti-metastatic effects of androgen receptor inhibitors. The mechanism behind anti-androgen actions in the inhibition of KCa1.1. activity is thought to be the enhancement of KCa1.1. protein degradation [The large-conductance Caradation .Abiraterone inhibits CYP17A1—an enzyme involved in androgen biosynthesis. It leads to reduced AR signaling, as there is a decreased amount of androgens required to stimulate the signaling cascade . It was The phosphatidylinositol 3-kinase (PI3K) signaling pathway is vital for cell growth and survival , and PIKRecently, a lot of interest has been put into investigation of TNBC, as it represents a highly heterogeneous and challenging disease. Our review shows that age at diagnosis is higher in AR subtype breast cancer and AR-positive TNBC . Despite enormous amount of knowledge gathered on TNBC to date, the data regarding prognosis are still contradictory. Research has shown that AR could be a promising therapeutic target. However, AR signaling pathways are not fully understood and more complicated than thought, as AR also interplays with other signaling pathways, especially PIK3CA and MAPK pathways. In conclusion, we recommend that more personalized approach is taken in treatment of TNBC.


Programmed cell death 1 (PD-1) mRNA expression significantly increased, and either PD-1 or PD-1 plus CTLA4 (cytotoxic T-lymphocyte associated protein 4) inhibitors showed unsatisfactory efficacy in high-ferrscore patients. Our study demonstrates the implication of FRGs in prognosis prediction and evaluation of immunotherapy efficacy in LIHC patients.Ferroptosis is a type of iron-dependent programmed cell death. Ferroptosis inducers have been shown to have a great potential for cancer therapy. We aimed to generate a risk scoring model based on ferroptosis-related genes (FRGs) and validate its predictive performances in overall survival (OS) prediction and immunotherapy efficacy evaluation in liver hepatocellular carcinoma (LIHC). Differential and Univariate Cox regression analyses were applied to analyze RNA-seq data of LIHC samples from TCGA and GEO databases to identify prognosis-related ferroptosis genes. Patients were assigned to three clusters  based on the cluster analysis of prognostic ferroptosis genes. The principal component analysis (PCA) was performed to build a risk scoring model based on differentially expressed FRGs. Survival analysis revealed that Ferrcluster B LIHC patients had a lower OS rate alongside more severe immune cell infiltration versus Ferrcluster A and C patients; moreover, the LIHC patients in high-ferrscore group had significantly lower survival than the low-ferrscore group. Compared to low-ferrscore patients,  LIHC, the most common primary malignant tumor of liver, is one of the most threatening malignancies to human life, with the rising morbidity and the growing mortality , 2. AccoFerroptosis refers to a novel cell death mode that is determined by iron and reactive oxygen species (ROS) , 6. In gPD-1, PD-L1 and CTLA4) in LIHC patients, and then the prognosis, tumor microenvironment and application prospect of the immunotherapy in LIHC patients were analyzed and predicted.Tumor microenvironment (TME) refers to the growth and survival of a tumor or cancer stem cell, which consists of the surrounding immune cells, blood vessels, extracellular matrix, lymphocytes, etc . Cells iACSL1, ACSL5, SLC39A14, SLA39A8, MAP1LC3B, ATG5, ALOX15, TP53, CYBB and SAT2 exceeded that of copy number increase   . The exp tissues .TP53 could act as a prognostic biomarker of LIHC by univariate COX analysis (P=0. 016). The overall survival rate of patients with Ferrcluster A and C reached over that of Ferrcluster B. According to the results of the heat map, most ferroptosis genes were slightly expressed in Ferrcluster A and highly expressed in Ferrcluster B. Moreover, there were more patients with advanced stage and death in Ferrcluster B, which further explained why the overall survival rate of s Ferrcluster B was lower than that of Ferrcluster A and C , and the Ferrcluster C was significantly lower than the other two types of B cells and T cells. , and the overall survival rate of Genecluster A and Genecluster C reached over that of Genecluster B  (P < 0.001)  . The sur< 0.001) , 4D. Forr. cells . Accordir. cells , 4G.P < 0.001). The low mutation load group achieved the better survival than the high mutation load group (P < 0.001) , primarily the missense mutation  . Moreovemutation , 5D.P < 0.05)    , 6B, andre group , 6D. Acc < 0.05) , 6F. The > 0.05) .+T cells, classic tumor kill T cells, inhibit cystine uptake by tumor cells by down-regulating SLC3A2 and SLC7A11, as an attempt to enhance Ferroptosis-specific lipid peroxidation in tumor cells. After the anti-PD-L1 treatment with an immune checkpoint inhibitor, the ferroptosis-specific lipid peroxidation levels were significantly elevated. However, blockage of ferroptosis results in a significant decrease in the sensitivity of tumor cells to immunotherapy, showing that ferroptosis can increase the anti-tumor efficacy of the immune system [Ferroptosis refers to an iron-dependent, non-apoptotic form of cell death. It can occur in numerous organ systems, which is associated with considerable diseases  . As the e system . In LIHCe system . AccordiTP53 achieved the maximal mutation rate according to the results. TP53 plays a dual role in tumor ferroptosis. Existing studies reported that TP53 can inhibit SLC7A11 to enhance ferroptosis. In colorectal cancer, the activity of DPP4 is blocked by transcription-independent method, so as to inhibit Ferroptosis [TP53 in LIHC remains to be unclear. Subsequently, TCGA and GEO databases are combined. According to the expression level of ferroptosis genes, LIHC samples fell to the Ferrcluster A-C. TME immunocyte infiltration analysis indicated the significantly different characteristics of infiltrated immunocytes in the three types. The occurrence and development of cancer were affected by TME infiltration and associated with the host immune system [In the present study, the somatic mutation rates, CNV and TMB of 40 ferroptosis genes in LIHC was initially analyzed. In addition, roptosis , 25. Howe system . Thus, Te system .As revealed from existing studies, according to the infiltration of immune cells, the immune microenvironment generally falls to three types, i.e., the immune rejection, the immunoinflammatory type as well as the immune desert type. The mentioned phenotypes prevent immune response from removing tumor cells through their own unique mechanism. In the present study, according to the difference of immune cell infiltration among the three types, Ferrcluster A may be immuno-inflammatory, which is characterized by infiltration of immune cell subtypes. The main reason is the increase of TIL. Ferrcluster B may be immune rejection. Despite the abundant infiltration of immune cells, the aggregated immune cells increased at the edge of the infiltration, and no effective infiltration was achieved. Ferrcluster C may be immune desert type, and the main reason is the lack of activation and initiation of T cells . As suggSubsequently, the potential biological behaviors among the three genotypes were analyzed by screening the differential gene of the three genotypes. the Univariate COX analysis was conducted to screen the prognosis-related differential genes, and the gene subtype analysis was conducted. The survival analysis reported the differences among the three gene types. Given the individual heterogeneity of ferroptosis genes and the heterogeneity and complexity of LIHC patients, a ferroptosis scoring model (using the ferrscore as a quantitative indicator) was built to quantitatively analyze individual LIHC patients. Consistent with the ferrcluster survival analysis, the overall survival rate of the Genecluster B was lower than that of the Genecluster A and C as well. Furthermore, the higher the ferrscore, the worse the survival would be. As the immune rejection type, the Ferrcluster B and the Genecluster B achieved the maximal scores, while as the immune inflammation type, the Ferrcluster A and the Genecluster A had the minimal scores, suggesting that based on the proposed ferroptosis scoring model, it has high clinical practicability, can analyze the type of TME infiltration in a single LIHC patient, and can also play a role in predicting the prognosis of LIHC patients.TP53 was 50%, complying with the result achieved in existing studies. On the whole, TP53 mutations were missense mutations [CTNNB1 (27%). As reported from existing studies, CTNNB1 mutations occur in nearly 19%-26% of LIHC patients, capable of activating Wnt-β-catenia signaling pathway and promoting tumor progression [PD-1/PD-L1. Lastly, the efficacy of the combination therapy of PD-1 and CTLA4 in LIHC patients was assessed. Nevertheless, there are still many shortcomings in this study, including the relatively uneven distribution of sample size after cluster typing, and the prognosis of patients with high- and low-ferrscore groups still needs internal verification. In addition, the predictive efficacy of ferroptosis score in clinical immunotherapy of LIHC patients still needs to be further evaluated and verified.In addition, this study revealed somatic mutations in the high and low ferrscore groups. In the high ferrscore group, the mutation rate of utations , 31. In gression . It has gression . The pregression . With thIn brief, based on the ferroptosis scoring model built in this study, the infiltration of the TME immune cells in LIHC patients could be comprehensively assessed to determine the different immunophenotypes of LIHC patients, as well as the correlation between the ferrscore and clinicopathological characteristics of LIHC patients. Such a model can accurately predict the survival rate of LIHC patients, and it critical to assessing the sensitivity of patients to the PD-1/PD-L1 inhibitors and the efficacy of combination therapy of the PD-1 and the CTLA4 on LIHC patients. This study could present more insights into the clinical immunotherapy of LIHC and formulate novel strategies to develop LIHC drugs.https://portal.gdc.cancer.gov/) database. The R language (version 4.0.2) and the “Bioconductor limma” package were adopted to classify the transcriptome data, and transform the RNA sequencing data  to the TPM value (transcripts per kilobasemillion). With “heptocelluar carcinoma survival” as the keyword, the probe matrix file and the platform annotation file of LIHC were downloaded from GEO  database, and the matrix file should involve the survival time and survival status of the patients. GSE76427 contained 116 patients with LIHC, and the clinical data of the patients were complete and qualified to be included in the subsequent analysis here. The LIHC somatic mutation data originated from TCGA. Furthermore, the data of LIHC Copy Number Variation (CNV) originated from the UCSC xena (https://xena.ucsc.edu/) database.The transcriptome data and clinical data of LIHC originated from TCGA GDC . The GSVA analysis was conducted by complying with the “GSEABase” and “GSVA” R package [P value < 0.01 showed the statistically significant difference. The results are illustrated in the heat map.To verify whether there are functional and pathway differences in the three subtypes of LIHC, the “c2. cp. kegg. v7.4. symbols.gmt” gene set file was downloaded from the MsiDB database  [The SSGSEA  algorithm was adopted to assess the infiltration content of immune cells in different subtypes of LIHC . The immT cells) . The SSGT cells) .P value < 0.01. With the classical Bayesian method of R language limma package, the differential genes in the three types of LIHC were analyzed. The GO and KEGG enrichment analysis were conducted to examine the main functions and pathways involved by the differential genes [P < 0.05 indicated the association with prognosis. The consensus clustering algorithm was adopted to define the number of gene clusters as well as their stability, Kaplan-Meier survival analysis was conducted to determine the survival differences among different subtypes.Based on the expressions of 40 ferroptosis genes, the filter condition was set as regulated al genes . Given tThe principal component analysis (PCA) method was conducted to build the ferroptosis scoring model, and principal component 1 and principal component 2 were selected as signature scores. Ferroptosis score (ferrscore) = is gene) . The feris gene) . The “Suis gene) . The allP < 0.05, and the correlation graph of ferrscore and immune cells was drawn. “ggboxplot” R package was used to analyze whether there were differences in ferrscore between ferrcluster and genecluster, and P value < 0.05 means that the difference showed statistical significance. According to the surv-cutpoint command of “suivminer” R package, the LIHC samples were classified as the high and low mutation load groups. Kaplan-Meier was adopted to analyze the survival difference between high and low mutation loads. The TMB and ferrScore groups were combined, and the joint survival curve was drawn. Based on the “maftools” R package, the gene mutation situation in the high and low ferrscore groups was analyzed and the oncoplot command was used to visualize the data and generate the waterfall plot [The “corrplot” R package was adopted, the filter condition was set as all plot .https://tcia.at) database, and the difference between ferroptosis score and immunotherapy score was examined, as an attempt to analyze the efficacy of the PD1 and the CTLA4 and their combination treatment in patients with high and low ferrscore groups.The clinical application scope of ferrscore model was first assessed, so as to analyze whether ferrscore is capable of distinguishing different clinical stages of LIHC, the survival status of the LIHC, as well as whether the ferroptosis gene can successfully predict the response of LIHC patients to immune checkpoint inhibitors. The boxplot of the PD-1/PD-L1 expression in the high and low ferrscore groups was drawn. The immune score file of LIHC originated from the TCIA . The one-way ANOVA and the Kruskal-Wallis test were performed to compare the differences among three or more groups. The Kaplan-Meier method was adopted to plot the prognosis curve, and the logarithmic rank test was performed to determine the significance of the difference. The Spearman test was performed to conduct the correlation analysis. All statistical Supplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Table 5Supplementary Table 6Supplementary Table 7