Dietary phytochemicals are particularly attractive for chemoprevention and are able to modulate several signal transduction pathways linked with cancer. Olive oil, a major component of the Mediterranean diet, is an abundant source of phenolic compounds. Olive oil production is associated with the generation of a waste material, termed ‘olive mill wastewater’ (OMWW) that have been reported to contain water-soluble polyphenols. Prostate cancer (PCa) is considered as an ideal cancer type for chemopreventive approaches, due to its wide incidence but relatively long latency period and progression time. Here, we investigated activities associated with potential preventive properties of a polyphenol-rich olive mill wastewater extract, OMWW (A009), on three in vitro models of PCa. A009 was able to inhibit PCa cell proliferation, adhesion, migration, and invasion. Molecularly, we found that A009 targeted NF-κB and reduced pro-angiogenic growth factor, VEGF, CXCL8, and CXCL12 production. IL-6/STAT3 axis was also regulated by the extract. A009 shows promising properties, and purified hydroxytyrosol (HyT), the major polyphenol component of A009, was also active but not always as effective as A009. Finally, our results support the idea of repositioning a food waste-derived material for nutraceutical employment, with environmental and industrial cost management benefits. Adherence to a Mediterranean dietary pattern has generally been associated with a decreased risk of developing most non-communicable diseases, including cardiovascular, neurodegenerative pathologies, and cancer ,2,3,4. Tp-coumaric, o-coumaric, protocatechuic, synaptic, p-hydroxybenzoic, and gallic acid) and phenolic alcohols, principally represented by 3,4-dihydroxyphenylethanol  and (p-hydroxyphenyl)ethanol (p-HPEA). Current data strongly support the fact that the beneficial effects of olive oil consumption are also due to its minor bioactive components [Among other beneficial foods, olive oil is considered a reservoir of chemopreventive molecules . Extra vmponents .Large volumes of wastewater  are generated during olive oil production, particularly during the malaxation process . OMWDespite recent advances in diagnosis and treatment, prostate cancer (PCa) remains one of the leading causes of cancer-related deaths in men worldwide . CurrentNF-κB activation and many other signals associated with inflammation are known to contribute to prostate cancer development [Inflammatory responses are involved in cancer initiation and metastasis ,26,27. Celopment ,32,33,34elopment . Several NF-κB and IL-6/STAT3.Here, we focused on the properties of the A009 and the equivalent HyT concentration by investigating the capabilities to block PCa cell aggressiveness in vitro, and analyzed the cellular mechanisms involved, in a potential chemopreventive approach. We showed that the tested extracts inhibit cytokines involved in angiogenesis by reducing the release of pro-angiogenic factors: VEGF, CXCL8, and CXCL12. Furthermore, A009 blocked the pro-inflammatory pathwaysTo investigate the anti-proliferative effects of A009 we employed three PCa cell lines: hormone-sensitive (LNCaP) and human metastatic hormone-resistant PCa cells . Cells were treated with various dilutions (ranging from 1:50 to 1:5000) of A009 and compared with HyT alone, at the same concentrations as within the A009 extracts ,19. Two We observed moderate induction of apoptosis by A009 on PC-3 and DU-145 cells A,B whileWe also investigated the effects of A009 on cell cycle, in DU-145 and LNCaP PCa cell lines, following 24 and 48 h of treatment. We observed a trend of decreased ability to undergo the S-phase in DU-145 and LNCaP cells treated with A009, and in LNCaP cells at 24 and 48 h following treatment however, no statistical significance was found . EtOH diWe investigated the adhesion capabilities of PC-3, DU-145, and LNCaP human PCa cells, which were pre-treated for 24 h with A009 or HyT . A009 significantly prevented PC-3, DU-145, and LNCaP adhesion A. A compMigration and invasiveness of tumor cells are crucial steps for the development of malignancies and cancer progression ,37. The We investigated whether the A009 extracts were effective in limiting the release of pro-angiogenic factors in PC-3, DU-145, and LnCaP PCa cell lines. FACS analysis showed decreased production of VEGF, CXCL8, and CXCL12 by the three PCa cell lines, exposed to A009 at 1:500 and 1:250 dilutions, following 6 h of treatment A,B. HyT We validated our FACS data on VEGF and CXCL8, by evaluating the release of the same cytokines by BIOPLEX assay, described in the Materials and Methods section. Secreted products were collected from PC-3, DU-145, and LNCaP cells, following 24 h exposure to two different lots of A009 (L3 and L4), at 1:250 dilution. We observed a statistically significant reduction in VEGF and CXCL8 release by the three PCa cell lines exposed to the A009, confirming the FACS data. Angiogenin (ANG) was modulated only in LNCaP cells, following A009 treatment C.Given the antioxidant and anti-inflammatory properties, we explored the ability of A009 to target NF-κB on PCa cells. We found that PC-3, DU-145, and LNCaP PCa cell lines all exhibited reduction of NF-κB activity, following treatment with A009 for 24 h, normalizing to both β-actin , and prostaglandins, favor PCa initiation and progression ,40,41,42 β-actin A and NF-It has been shown that NF-κB controls the expression of numerous genes, including that of interleukin 6 (IL-6) ,44,45,46Prostate cancer represents the second most common cancer in men and the fifth leading cause of cancer-related deaths worldwide ,53. The A growing amount of data show that polyphenols can prevent prostate cancer development and invasiveness . HyT hasTumor cells need to interact with stromal components and recruit inflammatory and endothelial cells to fuel nutrients and disseminate to distant organs. This requires the secretion of pro-inflammatory and pro-angiogenic factors acting both at tissue and systemic levels ,69,70. WWe performed further biochemical analysis to identify molecular mechanisms associated with the functional properties exhibited by our A009 extracts.cellular transformation, prostate cancer growth, lymph node metastases, resistance to chemotherapy, and disease outcome [. The NF-κB family of transcription factors have been shown to be important and implicated in prostate cancer [. Due to the many links between inflammatory signals and the malignant development of prostate cancer, targeting NF-κB has been suggested as a promising therapeutic option [. We found that our A009 extracts were able to reduce, in vitro, NF-κB activation in the PCa cell lines following 24 h of treatment , ≥98% in purity, was purchased from Cayman Chemicals . HyT was dissolved in ethanol (EtOH) and added to the medium, while EtOH vehicle alone was also used as a control. Two different lots of A009 (L3 and L4) were used. When not specified, experiments showing only one A009 extract were performed using the L4 lot.2. The RO permeate, representing ultrapure water, was discarded. Finally, the RO concentrate, with a volume concentration ratio (VCR) of 3.6, constituted the A009. Phenolic composition of A009 was determined using high-performance liquid chromatography (HPLC) analysis. Quantification revealed that the major phenolic component of A009 is HyT, along with several other phenolic compounds  and used to obtain the phenol-rich purified extract A009 . The experiments were performed using two different batches of A009 , 100 U/mL penicillin, and 100 µg/mL . LNCaP was grown on culture dishes coated with 0.01% polylysine solution (Sigma-Aldrich). All cell lines were maintained at 37 °C in 5% CO2.All experiments were performed using three cell lines of prostate cancer, human metastatic hormone-sensitive LNCaP and hormone-resistant cells PC-3, and DU-145. All prostate cancer cell lines were purchased from ATCC and grown in RPMI 1640 medium  supplemented with 10% fetal bovine serum , 2 mM 3 cells/well) were seeded into a 96-well plate. Following cell adhesion, fresh complete medium with decreasing dilutions  of A009 or HyT were added for 24 to 96 h. EtOH dissolved in the RPMI complete medium was used as control vehicle. MTT reagent 5 (mg/mL) was added to cell cultures, which was then replaced with 100 μL of DMSO, and the amount of solubilized formazan was quantified at 570 nm in a SpectraMax M2 .Cell proliferation was assessed by MTT -2,5-diphenyltetrazolium bromide; Sigma Aldrich, Milan, Italy). PC-3, DU-145, and LNCaP cancer cells  and HyT with the same concentrations for 24 and 48 h. Control cells receive complete medium alone. Apoptosis was detected by PI (Sigma Aldrich) and anti-annexin V-FITC  staining, followed by flow cytometry analysis, using a BD FACSCantoII flow cytometer. Flow data were analyzed with the FACSDiva 6.1.2 software .PC-3, DU-145, and LNCaP cells were treated with two concentrations of A009  and HyT with the same concentrations as in the two batches of A009, for 24 and 48 h. Control cells received complete medium alone (negative control) or vincristine . At each time point, cells were detached and fixed in cold 70% ethanol. DNA was stained with 100 μg/mL DAPI 2 µg/mL (Sigma-Aldrich) in 1× PBS solution. Stained nuclei were analyzed for V450 fluorescence signal using a FACSCanto flow cytometer. G0/G1, S, G2/M, and apoptotic phase of the cell cycle were determined using the FACSDiva 6.1.2  and FlowLogic software .3 cells were seeded on a 4-well chamber slide coated with fibronectin (2 µg/mL). Following 90 min of incubation, cells were washed, fixed with 4% paraformaldehyde (PFA), and then stained with DAPI (Sigma Aldrich). Assays were performed in triplicates. Cells within 5 blind fields per well were randomly counted to determine the number of adherent cells resulting from each treatment, using a Zeiss Microscope associated with a Nikon camera. Experiments were performed in triplicate, three times.Cell adhesion assay was performed as previously described . PC-3, D4), treated with A009 and HyT for 24 h, were washed with PBS, resuspended in serum-free RPMI medium, and placed in the upper compartment of the Boyden chamber. RPMI complete medium was added in the lower compartment to induce migration and invasion, and in the related in vitro assays. Polycarbonate filters, of 10 µm pore size, pre-coated with matrigel  for the chemoinvasion assay or with collagen IV  for the chemotaxis assay, were used as the interface between two Boyden chamber compartments. Following 6 h (chemotaxis) or 24 h (chemoinvasion) of incubation at 37 °C in 5% CO2, the filters were recovered, and cells on the upper surface were mechanically removed with a cotton swab and migrated or invaded cells on the lower filter surface were fixed with absolute ethanol and stained with DAPI. Migrated/invaded cells for each treatment were counted in a double-blind manner in 5 random fields using a Zeiss Microscope associated with a Nikon camera . Experiments were performed in triplicate.Migration and invasion assays were performed using a modified Boyden chamber, as previously described ,84. PC-3Cells were lysed in RIPA buffer containing protease and phosphatase inhibitor cocktails (Roche Diagnostics GmbH). Proteins (30 µg) were loaded on NuPAGE Novex 4%–12% Bis-Tris Gel (Life Technologies) and transferred to a PVDF membrane Amersham Hybond . Membranes were incubated overnight at 4 °C with the primary antibodies: anti-NF-κB p65, anti-p-STAT3, anti-IL-6 . The secondary antibody peroxidase-linked anti-rabbit IgG or anti-mouse IgG  was diluted at 1:3000 for 1 h at room temperature. Specific protein bands were detected using Pierce ECL Western Blotting Substrate (ThermoFisher Scientific). Protein expressions were normalized to beta-actin at 1:5000 (Abcam). Western blot data were analyzed using ImageJ software to determine the optical density (OD) of the bands.The production of intracellular cytokines was performed by flow cytometry. PC-3, DU-145, and LNCaP PCa cells were treated for 6 h with 1:500 and 1:250 A009 or HyT, in complete RPMI medium supplemented with 10 ng/mL phorbol 12-myristate 13-acetate , 500 ng/mL ionomycin (Sigma-Aldrich), and protein transport inhibitor brefeldin A (Golgi Plug) ). Following 6 h of stimulation, cells were harvested and treated with Cytofix/Cytoperm fixation and permeabilization kit (BD). Cells were stained with phycoerythrin (PE)-conjugated anti-human VEGF (R&D Systems), PE-conjugated anti-human CXCL-8 (R&D Systems), and PE-conjugated anti-human CXCL12 (BD). Negative controls included directly labeled isotype-matched irrelevant mAbs (BD). The expression of specific cytokines and angiogenic growth factors were determined by flow cytometry, using a BD FACSCantoII analyzer.Cytokine profiling was investigated by secretome analysis. Following 24 h of treatment of the PCa cells, supernatants were collected, centrifuged to remove residual dead cells and debris, and concentrated using Concentricon devices , with a 3 kDa membrane pore cut-off, to obtain concentrated supernatants. Cytokine release profiling was performed using the Bio-Plex Pro Assays  in a customized cytokine panel.The statistical significance between multiple datasets was determined by one-way ANOVA using GraphPad Prism 7 and 8. Data are expressed as means ± SEM or SD.Our results demonstrate that purified extract A009 from the waste material OMWW exert promising preventive activities, in vitro, on human prostate cancer cells. We demonstrated that our A009 extracts target NF-κB and the IL-6/STAT3 axis in vitro, providing a rationale for the potential use of a water-soluble olive polyphenol-rich nutraceutical product in a potential chemopreventive approach.


Amylin, , a 37-amino acid peptide hormone, is released in response to nutrients, including glucose, lipids or amino acids. Amylin is co-stored and co-secreted with insulin by pancreatic islet β-cells. Amylin inhibits food intake, delays gastric emptying, and decreases blood glucose levels, leading to the reduction of body weight. Therefore, amylin as well as insulin play important roles in controlling the level of blood glucose. However, human amylin aggregates and human amylin oligomers cause membrane disruption, endoplasmic reticulum (ER) stress and mitochondrial damage. Since cytotoxicity of human amylin oligomers to pancreatic islet β-cells can lead to diabetes, the protection of pancreatic islet β cells from cytotoxic amylin is crucial. Human amylin oligomers also inhibit autophagy, although autophagy can function to remove amylin aggregates and damaged organelles. Small molecules, including β-sheet breaker peptides, chemical chaperones, and foldamers, inhibit and disaggregate amyloid formed by human amylin, suggesting the possible use of these small molecules in the treatment of diabetes. In this review, we summarize recent findings regarding the role and cytotoxicity of amylin and the protection of pancreatic islet β-cells from cytotoxicity of amylin. Amylin, also known as islet amyloid polypeptide (IAPP), is a 37-amino acid peptide hormone. Amylin is released in response to nutrients, including glucose, lipids, or amino acid (arginine) ,2. Pancr1, AMY2 and AMY3 receptors. AMY1 receptor consists of CTR and RAMP1, AMY2 receptor consists of CTR and RAMP2 and AMY3 receptor consists of CTR and RAMP3  family, which includes CT, αcalcitonin gene-related peptide (αCGRP), βCGRP, adrenomedullin (AM), and AM2 . The members of this family comprise a disulfide bond at the N-terminus and an amidated C-terminus. CT receptor (CTR) and CTR-like receptor (CLR) are receptors for the members of CT family. CTR has splice variants with tissue-specific expression patterns ,11,12,13nd RAMP3 .d-ribose prevents the formation of aggregates, and d-ribose-glycated insulin is highly cytotoxic. However, aggregated insulin is not cytotoxic [Numerous proteins, including human amylin, aggregate and form amyloids. The formation of amyloids is associated with the induction of cytotoxicity, leading to diseases such as type 2 diabetes (T2D), Creutzfeldt–Jakob disease, Alzheimer’s disease, Parkinson’s disease and Huntington’s disease . Howeverytotoxic . W7FW14Fytotoxic . On the ytotoxic .Amylin inhibits food intake and delays gastric emptying, leading to the reduction of blood glucose levels and body weight. Moreover, pramlintide, an amylin analogue, also reduces body weight in humans . TherefoThe area postrema (AP), located in the caudal hindbrain, is considered as one of the major sites of amylin action, and ablation of the AP leads to a reduction of the inhibitory effect of amylin on food intake . In addiAmylin functions to slow gastric emptying in rodents ,37,38. FA recent study reported that in the VMH, amylin enhances leptin signaling via the induction of interleukin-6 (IL-6) and the expression of the mRNA of Lepr-b , which iAmylin exerts effects on pancreatic islet β-cells as well as the CNS. Knockout of amylin results in an increase in glucose-induced insulin secretion from pancreatic islet β-cells  while phAmyloid aggregates composed of amylin are found in pancreatic islet β-cells of patients with T2D ,55. The Secretory granules are acidic (approximately pH 5.5) . The uncHuman amylin induces cell membrane permeabilization and perturbation, endoplasmic reticulum (ER) stress, mitochondrial damage, and β-cell death ,86,87,88Human amylin oligomers are considered to be more toxic than fibrils and amyloid deposits, and oligomers may intracellularly form in pancreatic islet β-cells ,85,94. HHuman amylin aggregates in pancreatic islet β-cells induce ER stress, mitochondrial damage, and membrane disruption, leading to β-cell death and diabetes ,86,100. Reportedly, amylin aggregates could be disaggregated by small molecules, such as β-sheet breaker peptides, chemical chaperones and foldamers . β-sheetAmylin is co-stored and co-secreted with insulin and functions to control blood glucose levels by inhibiting food intake and slowing gastric emptying. However, human amylin aggregates in pancreatic islet β-cells and aggregates of human amylin induce cell death, leading to T2D. Human amylin oligomers are considered to be the most toxic form of amylin aggregates. These can cross the plasma membrane and appear to act similarly to prions. Therefore, there is the possibility that T2D could be a transmissible disease via transfer of amylin oligomers between individuals. Thus, the inhibition and disaggregation of amylin aggregates might be a viable therapeutic strategy against T2D. Further investigation of the mechanisms of amylin aggregate formation and their cytotoxic modes of action is crucial.


Objectives: To unveil the role of SIRT1 in limiting oxidative stress in psoriasis and to further discuss the therapeutic prospects of salidroside in psoriasis.Methods: Literature from 2002 to 2019 was searched with “psoriasis”, “oxidative stress”, “SIRT1”, “salidroside” as the key words. Then, Oxidative stress in psoriasis and the role of SIRT1 were summarized and the potential role of salidroside in the disease was speculated.Results: Oxidative stress might contribute to the pathogenesis of psoriasis. High levels of ROS produced during oxidative stress lead to the release of inflammatory mediators, that, in turn, induce angiogenesis and excessive proliferation of keratinocytes. SIRT1 is a member of the sirtuin family, of which the activation lead to the inhibition of such oxidative stress signaling pathways MAPK, NF-κB, and STAT3, down-regulation of inflammatory factors, suppression of inflammation and keratinocyte hyperproliferation, and inhibition of angiogenesis. Salidroside, the main ingredient of Rhodiola, is known to exert antioxidant roles, which has been attributed to SIRT1 activation.Conclusion: Salidroside might inhibit oxidative stress singling pathways via SIRT1 activation, and could be as an ideal candidate for management of psoriasis. It is characterized by erythema, plaques, and scales, and is sometimes accompanied by itching . The hisseverity ,4.Salidroside – the main ingredient of Rhodiola – has anti-aging and antioxidant properties. Recent reports have emphasized its protective effects on the nervous and cardiovascular systems, and in the treatment of tumors and inflammatory diseases . Salidro2−), hydroxyl radical (HO−), hydrogen peroxide (H2O2), and lipid radicals, which are the byproducts of cellular metabolism [Oxidative stress is generated due to an imbalance between levels of reactive oxygen species (ROS) and antioxidants . ROS maitabolism . Some entabolism .Oxidative stress is an essential factor that induces and aggravates psoriasis, which is known to be induced by a variety of factors – alcohol consumption, smoking, infection, drugs, obesity, cell metabolism, immune response, and pathological state . Previou−, and inducible nitric oxide synthase (iNOS), and decrease in the levels of SOD, CAT, and GSH-px [γ, IL-2, TNF-α, and TNF-β, produced by Th11; IL-17, IL-22, IL-23, and TNF-α, by Th17; and antimicrobial peptide (AMP), TNF-α, IL-6, IL-8, polymorphonuclears (PMN), and VGEF, by activated keratinocytes [α, IL-8, and IL-17 [α induce endothelial dysfunction, which can consequently contribute to autoimmune and cardiovascular diseases [Generation of ROS is a crucial step in the induction of oxidative stress in psoriasis. ROS generally act as second messengers during this process and lead to an increase in the levels of malondialdehyde (MDA), NO, HOd GSH-px ,9,13. Eld GSH-px ,14. Thisinocytes ,15. Thesinocytes . Angiogend IL-17 ,17. As rdiseases ,19.+-dependent histone deacetylase that mediates the antioxidative stress in psoriasis [Sirtuins are a family of proteins that are involved in multiple cellular functions related to metabolism, cell cycle, aging, inflammation, apoptosis, cell proliferation, and DNA repair . Among tsoriasis ,24. It wsoriasis  and inhisoriasis . Moreovesoriasis . Additiosoriasis . Recentlsoriasis . Accordisoriasis .α, IL-6, and MCP-1ex. The involvement of the MAPK/AP-1 pathway in oxidative stress has been extensively investigated [α responsive genes and that of genes involved in keratinocyte differentiation were significantly downregulated [MAPKs are a family of serine-threonine protein kinases that are involved in signal transduction, cell differentiation, proliferation, apoptosis, and immune response. Members of this family include – extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and the p38 MAPKs ,30. AP-1stigated . It has stigated , and JNKstigated ,40. Studstigated . When SRegulated .2O2, imported via AQP3, participates in the activation of the NF-κB signaling pathway in keratinocytes and is involved in the pathogenesis of psoriasis [α, thereby enhancing the downstream inflammatory response [α [α, and PPARα; concomitantly, NF-κB also downregulated SIRT1 expression and signaling via ROS, IFN-γ, and PARP-1 under oxidative stress conditions [Nuclear factor-κB (NF-κB) is an essential inflammatory mediator in the pathogenesis of psoriasis; increased expression of NF-κB has been demonstrated in psoriatic lesions . ROS cansoriasis . Alteredsoriasis . Furtherresponse ,46. SIRTresponse ; overexpponse [α . It has nditions .Besides MAPK/AP-1 and NF-κB pathways, STAT3 also plays an essential role in psoriasis. STAT3 has been implicated in the regulation of fundamental biological processes such as cell proliferation, differentiation, oncogenesis, survival, and apoptosis . In restIn summary, activation of SIRT1 can counteract the damage caused by oxidative stress by inhibiting the MAPK, NF-κB, and STAT3 pathways , leadingα [2O2 [Recently, a detailed review of the pharmacological effects of salidroside in various diseases has demonstrated its anti-aging, anti-inflammatory, antioxidation, anti-cancer, and liver protection properties . It is aα . Previouα . Xiang-jα . Studiesα [2O2 . Besidesα [2O2 .+ [In addition, salidroside could act as an activator of SIRT1 to exert its antioxidant effects. Chun-Yang Wang et al. proved that salidroside increased SOD activity and GSH levels and reduced the phosphorylation of MAPK by activating SIRT1; they also found that it protects SH-SY5Y cells from apoptosis and oxidative stress induced by MPP+ . Similar+ . It has + . Meanwhi+ . Further+ .However, the antioxidative mechanism of salidroside in psoriasis has not yet been reported. In our review, we highlight the role of SIRT1 in reducing oxidative stress in psoriasis. The activation of SIRT1 could effectively inhibit MAPK, NF-κB, and STAT3 phosphorylation, thereby decreasing the expression of downstream genes and secretion of inflammatory cytokines, eventually blocking the development of psoriasis. Salidroside, the activator of SIRT1, may cure psoriasis resistant to oxidative stress damage by inhibiting the MAPK, NF-κB, and STAT3 pathways; however, this property of salidroside needs further research.This review, overall, indicates that psoriasis is a common dermatitis associated with oxidative stress mediated by MAPK, NF-κB, and STAT3 signaling cascade. Salidroside, the activator of SIRT1, may cure psoriasis by blocking these three targets. Thus, salidroside, alone or in combination with other compounds, could be an ideal candidate for the management. However, to date, no research on salidroside as a therapeutic option to treat psoriasis has been reported. Hence, further investigation is required to validate the effects of salidroside on psoriasis pathophysiology and further verify the effect of SIRT1 on MAPK, NF-κB, and STAT3 pathways in the skin.


H2AFJ) more expressed in responsive tumors, including PTCHD1 and genes involved in abnormal cytotoxic T cell physiology, TOX, LY75, and SH2D1A. The following four pairs of markers could correctly classify all tumor samples according to response: PTCHD1/PDXDC2P, LOC100506731/NEURL4, SH2D1A/ENST00000478672, and TOX/H2AFJ. Gene sets correlated with tumor downstaging (FDR < 0.01) were mainly involved in immune response or lymphocyte activation, including CD47, LCK, NCK1, CD24, CD3E, ZAP70, FOXP3, and CD74, among others. In locally advanced breast cancer, stromal cells may present specific features of immune response that may be associated with chemotherapy response.Breast cancer stromal compartment, may influence responsiveness to chemotherapy. Our aim was to detect a stromal cell signature  associated with response to neoadjuvant chemotherapy (neoCT) in locally advanced breast cancer (LABC). The tumor samples were collected from 44 patients with LABC (29 estrogen receptor (ER) positive and 15 ER negative) before the start of any treatment. Neoadjuvant chemotherapy consisted of doxorubicin and cyclophosphamide, followed by paclitaxel. Response was defined as downstaging to maximum ypT1a-b/ypN0. The stromal cells, mainly composed of fibroblast and immune cells, were microdissected from fresh frozen tumor samples and gene expression profile was determined using Agilent SurePrint G3 Human Gene Expression microarrays. Expression levels were compared using MeV (MultiExperiment Viewer) software, applying SAM . To classify samples according to tumor response, the order of median based on confidence statements (MedOr) was used, and to identify gene sets correlated with the phenotype downstaging, gene set enrichment analysis (GSEA). Nine patients presented disease downstaging. Eleven sequences (FDR 17) were differentially expressed, all of which (except  Neoadjuvant chemotherapy (neoCT) has a central role in the management of locally advanced breast cancer and a number of different response predictive signatures have been identified, however, to the present moment, none of them is used in clinical practice ,2. In coBreast cancer behavior is a reflection of an interactive signaling between the malignant epithelial compartment and the surrounding microenvironment, composed of stromal cells, including carcinoma-associated fibroblasts, mesenchymal stem cells, tumor-associated macrophages, endothelial cells, pericytes, adipocytes, and lymphocytes, as well as extracellular matrix components . The intThere is evidence that stromal cells of normal tissues behave quite distinctly from stromal cells of tumoral tissues. Normal stroma may inhibit cell proliferation, in contrast with tumor stroma that may support tumor development and progression, through the induction of cancer cells proliferation, migration, and invasion, as well as the activation of angiogenesis ,4,5. TheHence, our aim was to evaluate whether a stromal cell transcriptional signature might be associated with response to neoadjuvant anthracycline and taxane, in locally advanced breast cancer, using a direct approach of stromal cell selection.ClinicalTrials.gov (Identifier NCT00820690). A written informed consent was signed by all participants.This protocol was approved by the Institutional Ethics Committee . This study was registered at Inclusion criteria were women with locally advanced breast cancer and clinical conditions to receive treatment with doxorubicin, cyclophosphamide, and paclitaxel. Exclusion criteria were patients diagnosed with inflammatory breast cancer or previous treatment for breast cancer. All tumor samples were collected before the start of neoadjuvant chemotherapy.2 and cyclophosphamide 600 mg/m2, every 21 days, followed by 4 cycles of paclitaxel 174 mg/m2 every 21 days (or 80 mg/m2 weekly for 12 weeks). Response was defined as pathological complete response (PCR) or downstaging to maximum ypT1a-b/ypN0, after chemotherapy.Neoadjuvant chemotherapy followed the hospital treatment protocol, consisting of 4 cycles of doxorubicin 60 mg/mFresh frozen tumor fragments collected before any treatment were cut in 10 µm slices. Intratumoral stroma, mainly comprising fibroblasts, sometimes accompanied by immune cells was microdissected using CapSure HS LCM  in a Pix Cell II Arcturus Laser Capture Microdissection (Thermo Fisher Scientific) (LCM) . Tumor nPlus2-round (Thermo Technologies) and Low Input Quick Amp kit  protocols and labelled with CY5 or CY3, respectively. Samples were analyzed on a NanoDrop spectrometer (Thermo Fisher Scientific) and yield values varied between 0.79 and 5.93 µg of cRNA and specific activity between 6.36 and 10.75 pMol Cy3 or Cy5 per µg cRNA. Competitive hybridization was performed in the SurePrint G38×60K slides (Agilent Technologies) and fluorescence intensities from scanned image files in an Agilent Bundle Model B Microarray Scanner System (Agilent Technologies) were preprocessed with Agilent Feature Extraction software (v10.7.1) and normalized using GeneSpring GX12.1 software (Agilent Technologies). A detailed description of methods is provided in Both total RNA extracted from samples and Universal Reference RNA  were linearly amplified, using two-round RiboAmp HSComparisons of expression levels were performed using MeV  software applying SAM . Analysis was done using all sequences (without filtering). Unsupervised hierarchical clustering based on Euclidean distance and average linkage was used to verify association patterns. The reliability of the clustering was assessed by bootstrap technique using MeV. Raw data from microarray was deposited at the Gene Expression Omnibus (GEO).Classification of stromal samples according to response was also analyzed using the order of medians, based on confidence statements ,13. Thisp value cut off ≤ 0.05 (https://toppgene.cchmc.org/enrichment.jsp) [Gene list enrichment analysis and candidate gene prioritization, based on functional annotations and protein interactions network, was performed using Toppgene suite, with FDR correction ent.jsp) .http://software.broadinstitute.org/gsea/index.jsp). In this pairwise comparison, all genes are ranked based on signal-to-noise ratio. Then, the alternative hypothesis, that rank ordering of distinct pathway members is associated with a specific phenotype, is tested [Gene set enrichment analysis (GSEA) was used to identify whether predefined gene sets might associate with gene expression differences between phenotypes .The ROC plotter, an online transcriptome-level validation tool for predictive biomarkers, was used to investigate the potential association of 10 highlighted genes with pathological complete response to any chemotherapy .Forty-four patients diagnosed with locally advanced breast cancer, between July 2008 and January 2012, at the Hospital de Câncer de Barretos, Barretos, SP, Brazil, were included. Patients’ median age was 43 years (21–64 y). All patients presented stage III disease and mean tumor dimension prechemotherapy was 7.0 cm (± 2.0) and postchemotherapy was 4.2 cm (± 3.4). All patients, except for three, were diagnosed with invasive ductal carcinoma and among tumors, 29 were classified as estrogen receptor (ER) positive and 15 were classified as ER negative . All patTo characterize the microdissected samples, we selected the top 50 highly expressed genes among all samples and performed gene list enrichment analysis  . These gACTA2 (smooth muscle α actin) and FAP , that are expressed by myofibroblasts and by reactive stromal fibroblasts of epithelial cancers (https://www.ncbi.nlm.nih.gov/gene/2191) [We then selected some classical fibroblast, lymphocyte, and epithelial markers to evaluate their expression, which is shown on ne/2191) , respectESR1, GATA3, NAT1, TFF3), and 19 more expressed in ER negative samples  would classify the microdissected stromal cells. Using SAM test (FDR zero), 51 sequences were differentially expressed, 32 more expressed in stromal cells from ER positive , in contrast with AR vs. ESR1, AR vs. PGR, AR vs. ESR2, ESR1 vs. ESR2, and PGR vs. ESR2, which were not correlated .Additionally, we further explored the relative gene expression of other important hormonal receptors in breast cancer, such as estrogen receptor beta (ESR2), progesterone receptor (PGR) and androgen receptor (AR), concomitantly with estrogen receptor alpha (ESR1) ,19. In srrelated . Furtherhttp://toppgene.cchmc.org/enrichment.jsp).Then, we evaluated whether information on the age of the patients, categorized as ≤40 years (young adults) and >40 years, would classify the microdissected stromal cells. A differential gene expression profile, according to the patients´ ages, included 30 sequences  , all of H2AFJ) more expressed in responsive tumors, from patients who presented tumor downstaging  were more expressed in responsive samples. In addition, ingenuity pathway analysis, IPA, revealed a network enriched in the gene list “cancer” . Using SAM test (FDR 17), 11 sequences (including nine genes) were differentially expressed, all of which ”,  results . HoweverOur next step was to use GSEA to identify gene sets that were correlated with the phenotype downstaging or with the phenotype non-downstaging. Considering gene expression derived from all 44 tumor samples, GSEA revealed that 117 gene sets correlated with tumor downstaging (FDR < 0.01), including seven KEGG gene sets, comprehending two immune system-related gene sets , as well as 15 GO biological process gene sets among which, at least 10, involved with immune response, including five implicated in induction or activation of this process: positive regulation of lymphocyte activation, positive regulation of immune system process, positive regulation of T cell activation, T cell activation, and lymphocyte activation . In addiAs many gene sets associated with tumor downstaging were related with immune response, we evaluated expression of some classical T cell markers individually (using the Mann–Whitney test) in this group of microdissected samples. CD8, CD3E, and CD247 (CD3Z) were all more expressed in DS samples, whereas CD4, CD3D, and CD3G were not differentially expressed between DS and NDS. In addition, markers of an immunosuppressive microenvironment, such as FOXP3, CD274 (PDL1), and CTLA4 were also more expressed in responsive tumors . HoweverIn addition, we evaluated whether the stromal mRNA expression of hormone receptors might be predictive of tumor response. Nonetheless, ESR1, ESR2, PGR, and AR were not differentially expressed in DS vs. NDS samples .We also used GSEA to identify gene sets that correlated with tumor response specifically in ER positive tumors, as well as in ER negative tumors, separately.CD74, CD8A, CD8B, and nine molecules of HLA complex, among others) and four GO gene sets , including one related with immune response, i.e., regulation of T cell activation .In ER positive tumors, the GSEA revealed 214 gene sets (FDR < 0.25) that correlated with tumor downstaging . Using aIn ER negative samples, the GSEA identified 998 gene sets, using FDR < 0.25, that were correlated with tumor downstaging. Using a more stringent FDR cut off (<0.05), five KEGG gene sets were correlated with tumor downstaging, including two related ith immune response, i.e., antigen processing and presentation and T cell receptor signaling pathway, as well as at least 12 GO gene sets  related with the immune process, including two with FDR < 0.01, which were adaptive immune response and positive regulation of T cell activation .In ER positive samples that did not present tumor downstaging, 49 gene sets were found in the GSEA analysis (FDR < 0.25) including one KEGG gene set, i.e., ECM receptor interaction ; eight GO gene sets;  cell adhesion; cell recognition; embryonic development; (cellular component) cortical cytoskeleton; cell projection part; cell surface; cytosolic part; and (molecular function) oxidoreductase activity .KRT6A, KRT1, KRT19, KRT18, and KRT31); synaptic vesicle; intermediate filament; and (molecular function) neuropeptide receptor activity  including two KEGG pathways, related with arginine and proline metabolism, as well as the following four GO gene sets (FDR < 0.1): (cellular component) intermediate filament cytoskeleton , COL1A2, MMP2, TGFB3, SPARC and DCN (decorin), as well as a relatively low expression of another group of genes, including SNAI2 and THBS2 , however, could not cluster the samples, according to disease outcome  [HSD17B2, which codes for an enzyme involved in metabolizing androgens and estrogen to less active metabolites, further indicating that the estrogen pathway is not critical in these tumors.In our study, genes, such as t cells) ,21,22. Tt cells) , as wellIn accordance with previous reports in prostate cancer fibroblasts , we obseWe could identify a panel of differentially expressed genes between young (less than 41 years) and older patients. This was rather expected because differences in expression levels of dermal fibroblast from young and old human beings had already been described . In addiPTCHD1, LY75, SH2D1A, and TOX were more expressed, while H2AFJ was less expressed. More interestingly, a group of four pairs of genes were sufficient to classify samples according to response which included: PTCHD1/PDXDC2P, LOC100506731/NEURL4, SH2D1A/ENST00000478672, and TOX/H2AFJ with a high confidence.In stromal cells from responsive tumors, genes such as H2AFJ encodes a member of the histone H2A super family, that may be overexpressed in breast cancer samples through gene amplification and considered a putative breast cancer oncogene [H2AFJ overexpression observed in non-responsive breast cancer samples, in colorectal cancer cell lines, H2AFJ was described as a mediator of chemoradiation resistance  [PTCHD1 codes for a patched-related protein, structurally similar to the Hedgehog (Hh) receptors PTCH1 and PTCH2. PTCHD1 exhibits biochemical activity in Hh-dependent processes similar to the inhibitory effect of PTCH1 and PTCH2 on Gli-dependent transcription and may be expressed in mammary glands [oncogene . Consist., 2019) . PTCHD1 y glands . PTCH1 dy glands . This meSH2D1A and TOX were previously shown to be upregulated in germinal center T helper cells as compared with other CD4+ T cell subsets [LY75 (CD205) is expressed at relatively high levels on myeloid blood dendritic cells and monocytes and plays a role in endocytic uptake of antigen and presentation to lymphocytes via MHC class II molecules [ subsets . However subsets . In turnolecules ,34. The CD24, that modulates B cell activation responses, CD3D and CD3E, which are part of the T-cell receptor/CD3 complex, that couples antigen recognition to several intracellular signal-transduction pathways; CD7, found on thymocytes and mature T cells and playing an essential role in T cell interactions; CD40LGT, expressed on the surface of T cells, regulating B cell function by engaging CD40 on the B cell surface; CD74 that associates with class II major histocompatibility complex (MHC) and is an important chaperone that regulates antigen presentation for immune response; CD79B, the B lymphocyte antigen receptor, a multimeric complex that includes the antigen-specific component, surface immunoglobulin (Ig), necessary for expression and function of the B-cell antigen receptor; CD83, involved in the regulation of antigen presentation; CD96 that plays a role in the adhesive interactions of activated T and NK cells during the late phase of the immune response, as well as function in antigen presentation.In addition, various immune response gene sets were positively correlated with tumor downstaging. These gene sets comprehended genes such as In an exploratory analysis, we individually analyzed the expression of some classical markers of tumor immune response. We observed a higher expression of CD8, CD3E, and CD247 (CD4Z) in responsive tumors. In contrast, a significant correlation between pCR and higher prechemotherapy infiltration by CD3, CD4, and CD20 has been previously described .q-values). In accordance, it has been previously shown, in a cohort of triple negative tumors and HER2 positive tumors, that an increased percentage of stromal T infiltrating lymphocytes, as well as expression of immune activating and immunossupressive genes, are positively correlated to response to neoadjuvant chemotherapy [CD74, CD8A, and CD8B, and various genes of the HLA complex, as well as GO biological process gene set such as “regulation of T cell activation”, comprehending ZAP70, CD24, CD3E, LCK, IL21, among others.Interestingly, gene sets enriched in immune-related genes were more strongly correlated with tumor downstaging in ER negative tumors , we could not predict the clinical outcome for patients bearing ER negative tumors in the present series . It is iStrengths of our study include the specific evaluation of microdissected stromal cells from patients with locally advanced breast cancer, who underwent the same neoadjuvant chemotherapy regimen and the identification of pairs of genes that might classify tumors according to response to neoadjuvant chemotherapy. Limitations include a relatively small number of patients and the intensive labor associated with tumor microdissection, that complicates the use in the clinical practice.In summary, tumor stromal cells, which are non-malignant cells, represent an interesting option to evaluate response to neoadjuvant chemotherapy. In locally advanced breast cancer, stromal cells may present specific features of immune response that may be associated with chemotherapy response.


Arabidopsis and rice that have physical barriers between their photosynthetic organs and soils, the photosynthetic organs of duckweeds face directly to their nutrient suppliers (waters), therefore, their responses to salinity may be distinct. In this research, we found that the duckweed Spirodela polyrhiza L. accumulated high content of sodium and reduced potassium and calcium contents in large amounts under salt stress. Fresh weight, Rubisco and AGPase activities, and starch content were significantly decreaseded in the first day but recovered gradually in the following days and accumulated more starch than control from Day 3 to Day 5 when treated with 100 mM and 150 mM NaCl. A total of 2156 differentially expressed genes were identified. Overall, the genes related to ethylene metabolism, major CHO degradation, lipid degradation, N-metabolism, secondary metabolism of flavonoids, and abiotic stress were significantly increased, while those involved in cell cycle and organization, cell wall, mitochondrial electron transport of ATP synthesis, light reaction of photosynthesis, auxin metabolism, and tetrapyrrole synthesis were greatly inhibited. Moreover, salt stress also significantly influenced the expression of transcription factors that are mainly involved in abiotic stress and cell differentiation. However, most of the osmosensing calcium antiporters (OSCA) and the potassium inward channels were downregulated, Na+/H+ antiporters (SOS1 and NHX) and a Na+/Ca2+ exchanger were slightly upregulated, but most of them did not respond significantly to salt stress. These results indicated that the ion homeostasis was strongly disturbed. Finally, the shared and distinct regulatory networks of salt stress responses between duckweeds and other plants were intensively discussed. Taken together, these findings provide novel insights into the underlying mechanisms of salt stress response in duckweeds, and can be served as a useful foundation for salt tolerance improvement of duckweeds for the application in salinity conditions.Duckweeds are a family of freshwater angiosperms with morphology reduced to fronds and propagation by vegetative budding. Unlike other angiosperm plants such as  Spirodela, Landoltia, Lemna, Wolffiella, and Wolffia with a total of 37 species [Arabidopsis and rice). This may explain why duckweeds are highly sensitive to salt stress, a major known factor that causes cellular damage and affects plant growth and development. To date, many efforts have been made to characterize the performances of diverse duckweed species in response to salt. In Spirodela polyrhiza L., NaCl treatment significantly reduced photosynthetic pigment accumulation, inhibited plant growth, and simultaneously enhanced malondialdehyde (MDA) and hydrogen peroxide (H2O2) contents in plant tissues [Lemna minor L., salt stress strongly reduced the removal capacity of nitrogen (N) and phosphorus (P) from water. Moreover, higher salt stress with longer cultivation period exerted more injury to duckweed and greater inhibition of N and P removal [Lemna gibba L., salt treatment significantly inhibited photosystems I (PSI) and PSII activities, and decreased the overall activity of electron transport chain while stimulating ROS production. Further investigation revealed that ROS production was highly correlated to the maximal quantum yield of PSII [Duckweeds belong to the Lemnaceae family and include five genera of  species . In rece species ,3. Howev species . The red tissues ,6. React tissues ,6. In Le of PSII . Notably of PSII . OverallLandoltia punctata (G. Meyer) Les & Crawford; ABA treatment [S. polyrhiza; NH4+ toxicity [L. minor; and nitrogen starvation [L. aequinoctialis Welw. However, to the best of our knowledge, currently no RNA-seq analyses concerning salt stress have been performed in duckweeds. Therefore, it is very necessary to complement this gap to improve our understanding of the mechanisms of salt stress in this species. RNA-seq is a common approach to quantify gene expression during plant development and upon various stress stimuli, and has been widely applied to identify specific trait-associated genes and regulatory networks in plants ,11. It ireatment  in S. potoxicity  and ionitoxicity  in L. miarvation  in L. aeS. polyrhiza (strain No. 7498), which is easy to culture and handle and has high growth rate in laboratory condition, was selected to investigate the physiological changes in response to salt stress with three different concentrations. Later, RNA-seq was used to survey the dynamic changes of gene expression at different time points of salt treatment. The results will provide novel insights into the underlying mechanisms of salt stress response, and can be served as a useful foundation for improving salt tolerance of duckweeds. In this study, S. polyrhiza in response to salt stress, four traits, including fresh weight, Rubisco enzyme activity, AGPase activity, and starch content were determined under four salt concentrations  across ten time-points  in S. polyrhiza  protein levels are constant in some plants , but itsThe reaction catalyzed by AGPase is considered as the first and key step in starch biosynthesis . AGPase S. polyrhiza in response to salt stress, sodium, potassium, and calcium contents were measured under 100 mM NaCl treatment at time points of 0, 6, 12, 24, and 72 h after treatment. Results indicated that the sodium content before salt stress was quite low (0.05 %DW), however, sodium content increased to 0.47 %DW after 6 h treatment, and reached the highest level  at 24 h, and then declined to 0.49 %DW at 72 h  under 50 and 100 mM NaCl treatments. Thus, the samples of S. polyrhiza were subsequently collected at 0, 6, 12, and 24 h, respectively, under 100 mM NaCl treatment, and subjected to RNA-seq sequencing to further explore the changes of gene expression at the transcriptional level.Taken together, Rubisco enzyme activity, AGPase activity, and potassium and calcium intake in S.polyrhiza reference genome (phytozome version 2). The expressed genes were arbitrarily identified by a threshold cutoff, FPKM (Fragments Per Kilobase of exon per Million fragments mapped) > 10, to minimize the false positive rate. In total, 11,012 expressed genes, which is equivalent to about three-fifths of the annotated genes in the S. polyrhiza genome, were identified.In total, 1002 million raw reads with 150-bp in length were obtained by pair-end Illumina sequencing. After removing adapters and low-quality reads, about 868 million (86.6 %) clean reads were retained and subsequently mapped to the 2FC ≥ 1) of samples collected at different time-points, respectively. There were 1159, 1066, and 437 DE genes identified between 0 h and 6, 12, and 24 h, respectively  genes were identified by pair-wise comparison , PS II , and their related polypeptide subunits . Consistently, DE genes related to electron carrier and state transition of light reaction were also declined , hydroxymethylbilane synthase (HEMC), protochlorophyllide oxidoreductase A (PORA), and PORC, were greatly downregulated by salt stress  which catalyzes the first and rate-limiting step of starch biosynthesis, and beta-amylase 3 (BMY3) and starch transporter (TPT) located on the starch-degradation pathway was significantly decreased (SPS) located on the sucrose-synthesis pathway, and cell wall invertase 4 (cwINV4), cytosolic invertase 1 (CINV1), and fructokinase-like 1 (FLN1) located on the sucrose-degradation pathway was also significantly decreased. However, we noted that the expression levels of several genes, including AGPase (APL2) and starch branching enzyme (SBE2) of starch-synthesis pathway, cytosolic/plastidic alpha-glucan phosphorylase, alpha-amylase, beta-amylase, glucan water dikinase (SEX1), and disproportionating enzyme 2 (DPE2) of starch-degradation pathway, and sucrose synthases (SUS3 and SUS4) of sucrose-degradation pathway, were greatly induced by salt stress  the expression of genes related to starch and sucrose metabolisms, indicating a possible requirement to maintain the balance between synthesis and degradation of carbohydrate metabolism  in response to salt stress.CSLD3) and CSLD5, respectively, mutants of the former initiated root hairs that burst at their tip soon after initiation in Arabidopsis [CESA1, CESA3, and CESA6, respectively, much evidence suggested that CESA3, along with CESA1 and CESA6 were presented in the same plasma membrane complex for cellulose biosynthesis in Arabidopsis, and mutants of these three genes, respectively, exhibited cellulose defect in the primary cell wall [FLA1), of which the mutants showed defects in shoot regeneration [In total, 62 DE genes were involved in cell wall pathways, including six subgroups of cell wall protein, cellulose synthesis, cell wall precursor synthesis, cell wall modification, cell wall pectin esterase, and cell wall degradation . Amazingbidopsis , while mbidopsis ; spipo27ell wall ,29; spipneration .ATPMEPCRB in Arabidopsis, spipo23g0018000 is a homolog of salt overly sensitive 5 (SOS5), of which the Arabidopsis mutants showed thinner cell walls, abnormal siliques, and inhibited root growth under salt stress [Besides, several cell wall-related genes were identified and participated in salt response. For examples, spipo0g0068000 is a homolog of a salt and chilling-sensitive gene t stress .Taken together, these results indicate that salt stress greatly inhibits the expression of genes involved in the biosynthesis and degradation pathways of cell wall.In total, 32 DE genes referred to abiotic stress were identified responding to salt treatment. The GO categories of these DE genes are heat stress (19 genes), drought/salt stress (nine genes), cold stress (three genes), and touch/wounding  were expressed highest at 0 h, and their expression levels were dramatically downregulated from 6 h to 24 h. A homolog (Spipo23G0037000) of ESK1, which functioned as a negative regulator of cold stress [RD22, which was mediated by abscisic acid (ABA) and responded to desiccation and salt stresses [These genes were grouped into three main clusters according to the hierarchical clustering A1–A3, . Overallbidopsis ,33, was d stress , and a hstresses , were inIn total, 61 hormone-related genes, referred to ABA, brassinosteroid (BR), auxin, cytokinin (CK), gibberellic acid (GA), ethylene, and jasmonic acid (JA), were differentially expressed under salt treatment. The hormone with the most abundant DE genes was auxin (20 genes), followed by BR (12), JA (9), ABA (9), and ethylene (7) . SubsequNCED3-like gene (Spipo5G0039400) was significantly increased from 6 h to 12 h and then declined at 24 h. Similar expression trends were observed in two ABF1-like genes (Spipo4G0008600 and Spipo7G0034500), which acts as an ABA-responsive element-binding factor to mediate the transcriptional regulation of ABA responses (Nine-cis-epoxycarotenoid dioxygenase (NCED) is a crucial enzyme in the ABA biosynthetic pathway. The expression of a esponses .ILL6-like gene (Spipo4G0007100), which encodes an IAA-amino acid conjugate hydrolase, was slightly upregulated from 6 h to 12 h but downregulated at 24 h. However, PIN1, which encodes an auxin efflux carrier, together with dozens of auxin-responsive genes, were significantly downregulated from 6 h to 24 h after salt treatment , meristem receptor-like kinase 2 (Spipo16G0046100), and EXORDIUM like 5 (Spipo17G0033100). These results indicated that the signal transduction pathway of BR was downregulated upon salt stress.Several critical genes involved in the BR biosynthesis pathway were found. Of which, the expression levels of nditions , includiGA3OX1-like gene (Spipo21G0010100) was gradually increased from 0 h to 24 h in response to salt treatment. CYTOKININ OXIDASE/DEHYDROGENASE (CKX) encode cytokinin-degrading enzymes [ACO1-like gene (Spipo23G0011700), which catalyzes the final step of ethylene biosynthesis, was greatly downregulated from 0 h to 24 h under salt treatment  3-oxidases catalyze the conversion of GA precursors to bioactive forms . The exp enzymes . Two CKXreatment . These rLOX2, LOX3, LOX5), an allene oxide synthase (AOS), an allene oxide cyclase (AOC3), and a 12-oxophytodienoate reductase (OPR1), which constitute a complete pathway of JA biosynthesis, were all significantly downregulated by salt stress. Coincidently, the expression of JAZ1, which is involved in JA signaling and transduction, was also greatly downregulated , C2H2 (11), HB (11), and MYB (11), followed by AP2/EREBP (10), and bZIP 7, . MYB96 for drought and ABA signaling and RAV1 for cold stress, were included. Many TFs related to leaf initiation and differentiation , cell differentiation (SCAP1), trichome growth (HDG2), and auxin response (ARF19) were also included in Group G6. Group G2 contained also several abiotic stress-related TFs, including CBF2 for cold and ABA, ERF53 for drought, and STZ for salt stress, in accordance with the functional category enrichment of this group (SHY2 and AUX2-11), cell differentiation (PDF2), and trichome differentiation (ZFP8 and NOK) were included in group G5. According to the expression patterns A, tens ois group . HoweverATH1 for GA biosynthesis regulation and ETT for auxin stimulus, were included in group G3. In comparison, several salt-responsive TF members, including MYB113, WRKY33, and STZ, were included in group G1 and G4, respectively.The groups with less abundant TF members were G4 (17 genes), G3 (9), and G1 (6). Several hormone-related TF members, including Taken together, these results suggest that the TF members involved in abiotic stress, cell differentiation, and hormone biosynthesis and signaling responded to salt treatment in diverse expression patterns.+ taken by the cell must be extruded from the cytoplasm to reduce toxicity [+ to the vacuole by Na+/H+ antiporters at the tonoplast (NHX) and extrusion of Na+ outside of the plasma membrane by SOS1 transporters [SOS1 gene and five NHX genes were identified in the genome of S. polyrhiza, they were mostly more abundant upon salt stress except Spipo12G0030600, which was weakly expressed in stress-free medium but downregulated to an undetectable level under salt stress  including the most predominantly expressed gene Spipo5G0038600 were downregulated at one or more time points, while the other OSCA genes were slightly upregulated. Five Ca2+/H+ antiporter genes (CAX) and four Na+/Ca2+ exchanger genes (NCL) were also identified, Spipo26G0021300 (SpNCL1) was dominant in transcripts among these genes, and it was only slightly upregulated under salt stress  play key roles in salt stress signaling . A totalt stress . This restresses . The exp+ transporter, Spipo10G0003400), and a CHX K+ antiporter gene (Spipo03G0081700), were downregulated at one to three time-points after salt stress . These results may explain partly the decrease of potassium content in the plants treated with 100 mM NaCl. Most members of the Shaker family inward rectifying potassium channel, including AKT (Spipo8G0031700 and Spipo23G0039700), SKOR (Spipo20G0014300), and HKT  between these two independent measurements.To verify the transcriptome results, in total nine genes, including  qRT-PCR . The res4+ toxicity, metal pollution, and salt stress [4+ toxicity also significantly inhibited the growth of L. minor, however, the MDA content and the activities of ROS enzymes such as SOD and POD were greatly increased [2+ and Ni2+ concentration , the growth rate, net photosynthesis rate, chlorophyll content, and Rubisco activity were significantly inhibited, while the starch content was increased sharply attributed to the increase in AGPase and soluble starch synthase activities and the decrease in α-amylase activity [Duckweed has great potential in modern agriculture and industry, mainly due to its great abilities for municipal and industrial wastewater treatment  and its good characteristics  for bio-ethanol production. Therefore, in the past decades, many studies have focused on the physiological performances of duckweed in response to diverse treatments such as nutrient starvation, NHt stress ,15,20,43t stress . NH4+ toS. polyrhiza strain DR  under 100, 150, and 200 mM NaCl stress, and found that the growth of duckweed was significantly reduced in a dose-dependent manner. The relative fresh weights were reduced by 24% and 37% after cultured for 4 days under 100 mM and 200 mM salt stress, respectively [S. polyrhiza strain DR under long-term (6 and 12 days) 200 mM NaCl stress, and found that plants showed chlorotic effect after 4–6 days, and chlorophyls were reduced significantly after 6 days of treatment. Liu et al. (2017) studied the effect of salt stress on the removal of N and P by common duckweed, L. minor, and found that salt stress exerted injury to duckweed and reduced N and P removal, and severe salt stress (100 mM NaCl) even induced duckweed to release N and P to water [L. minor is used to remove N and P from wastewater, salinities below 75 mM NaCl is required [L. minor, and NaCl salinity below 100 mM is suggested when L. minor is used in boron removal from wastewater [S. polyrhiza strain 7160 showed the highest tolerance among the tested strains, and the NaCl concentration that reduced its growth by 50% was caculated to be 377 mM, while Wolffia globosa strain 9667 was the least tolerant strain and its NaCl concentration for 50% growth reduction was only 10.1 mM [S. polyrhiza strains mostly are more tolerant to salt stress than other duckweed species, although there existed intraspecific variations [S. polyrhiza 7498 grew well under 50 mM and 100 mM NaCl condition for about 4 days, and the fresh weights were only reduced by 7.4%, 8.2%, and 22.2% in the first day for N50, N100, and N150, respectively, and were respectively reduced by 26.3%, 58.8%, and 69.7% by the end of the experiment  performed physiological analysis of ectively  further stewater . Sree etstewater . S. polyt Day 6, A. Discolt Day 6, , which iBesides growth reduction, high salt concentration  also reduced photosynthetic pigment content, but significantly enhanced MDA content and increased the activities of several ROS enzymes including POD, SOD, APX, and CAT ,6. HowevS. polyrhiza was strongly disturbed by salt stress, and plants accumulated a significant amount of sodium ion, which was 16.4 times the normal content. At the same time, potassium and calcium contents were greatly decreased  Schrad., its SOS1 gene expression was upregulated by 1.5 and 2.5 times, while its NHX gene was upregulated by one and two times when treated with 150 and 300 mM NaCl, respectively [SOS1 genes in salinity tolerant Chrysanthemum species was more strongly induced by salt stress than those carried by the non-tolerant ones [+ and more reduction of K+ in the stress-sensitive species C. morifolium [+ detoxification in S. polyrhiza is not efficient.In a previous report , removalecreased , similart stress . These r cascade . AtOSCA1bidopsis . Eight Oegulated . Howeverectively . Similarant ones , and strrifolium . ThroughS. polyrhiza during salt stress  was even slightly downregulated  and a CHX gene (Spipo3G0081700) were significantly downregulated, while most of the rest potassium transporter genes including CHX, SKOR, and HKT genes were slightly downregulated  Moq., rubisco activity was decreased at low NaCl concentrations (100 and 250 mM) but incrased at high concentrations (400 and 500 mM) [S. polyrhiza  . This doolyrhiza  was not olyrhiza , which mMany studies have observed that salt stress significantly reduced vegetable growth, inhibited photosystems I and II activities, and influenced starch accumulation in duckweed, and the effects were concentration-dependent under salt condition ,8,9. HowIn other plants, it has been well demonstrated that the expression of cell wall-related genes was significantly downregulated under salt condition ,63. AccoIn our study, we found that the expression levels of genes involved in PSI, PSII, and their related polypeptide subunits were dramatically downregulated by salt stress. Besides, several genes related to electron carrier and state transition of light reaction were also downregulated, in agreement with a recent report that salt stress caused a significant inhibition of both PSII and PSI electron transport activities and a decrease in the number of active PSII reaction centers . We alsoAPL1) which catalyzes the first and rate-limiting step of starch biosynthesis was significantly decreased; while many genes located on the starch-degradation pathway, including cytosolic/plastidic alpha-glucan phosphorylase, alpha- and beta-amylase, glucan water dikinase (SEX1), and disproportionating enzyme 2 (DPE2), were greatly induced by salt stress. These results explained the decrease of AGPase activity and starch content of S. polyrhiza within 24 h under salt stress in our study, however, since gene expression profiles during late stage of salt stress were not investigated, the increase of AGPase activity and starch content at late stage  of salt stress , which functions as both a redox transducer and a scavenger in ABA and abiotic stress response [SOD1), which is involved in salt stress response and superoxide radicals removal [S. polyrhiza under salt stress. Salt stress usually induces rapid accumulation of ROS, which causes oxidative stress-induced toxic effects in plants ,6. The eolyrhiza ,6. In ouresponse , and sup removal , were gr2+) is also recognized as a secondary messenger and plays a key role in signal transduction under salt condition [Arabidopsis, plants over-expressing AtCPK3 and AtCPK6 showed enhanced tolerance to salt, while atcpk6 mutant exhibited no obvious phenotypes [AtCML9 was also found to be involved in salt stress tolerance through its effects on the ABA mediated pathways [S. polyrhiza, addition of CaCl2 to the salt stressed plants increased the contents of proline, and lowered the degree of injury caused by NaCl [2+ signaling in response to salinity treatment. In this study, a total of 19 DE genes involved in Ca2+ signaling were found , which is a crucial enzyme in proline biosynthesis, was dramatically increased under salt stress, while another gene encoding GS1 (spipo15G0035800) was greatly decreased in response to salt treatment, in accordance with the result of a previous report [In addition to ROS, the calcium ion , hormone and TF genes are also recognized as important players involved in salt stress [S. polyrhiza and other plants . In addition, one SnRK2 and two PP2C genes, which respectively act as positive and negative regulators of ABA signaling in crop plants [NCED3, indicating that the two PP2C genes in S. polyrhiza might function differently compared with their homologs in other plants. ABFs are the bZIP family members that bind to ABA-responsive elements (ABREs) and regulate gene expression [SnRK2 kinases are responsible for ABA-dependent phosphorylation of ABFs [ABF1 and SnRK2 in S. polyrhiza showed similar expression patterns upon salt stress, and they were both upregulated at 6 h, but return to the normal level at 12 h , increased JA levels and enhanced salt tolerance in Arabidopsis, suggesting that JAs positively regulate salt tolerance [ACO1 in S. polyrhiza was downregulated by salt stress , one each of allene oxide synthase (AOS), allene oxide cyclase (AOC3), and 12-oxophytodienoate reductase (OPR1) that located on the JA biosynthesis pathway [EIN3 and a few bHLH TFs under drought and salinity conditions [JAZ1, EIN3, and bHLH38 was greatly downregulated upon salt stress. These results suggested that JAs act as negative regulators of salt tolerance in S. polyrhiza. Under salinity stress condition, the expression levels of lots of ethylene-responsive genes were significantly altered [Arabidopsis [TaACO1, which catalyzes the final step of ethylene biosynthesis, exhibited enhanced ethylene accumulation and increased salt sensitivity, and reduced the expression of two key TFs, DREB1B/CBF1 and DREB1A/CBF3 [ACO1, together with EIN3, which acts as a positive regulator of the ethylene signaling and a negative regulator of the CBF pathway [In addition, ethylene and JA were demonstrated to play essential roles in regulating plant response to salt stress . Transgeolerance ,86. Howet stress . It is a pathway . As a kenditions ,88,89. C altered . It seembidopsis . In addiB1A/CBF3 ,92. As e pathway , was strS. polyrhiza (strain No. 7498) was selected and cultured at the Biological Collection Centre of the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Hainan, China. To reveal the physiological changes of duckweed responding to salt stress, 0.5 g three-frond colonies were inoculated with three replicates in a 250 mL flask containing 100 mL MH medium supplemented with 0, 50, 100, and 150 mM NaCl, respectively, and cultivated under 16 h daily photoperiod at 40 μmol m−2 s−1 photosynthetic active radiation (PAR) at 25 ± 1 °C as previously described [escribed . S. polyrhiza samples were respectively collected at a total of ten time-points including 0, 3, 6, 12, 24, 48, 72, 96, 120, and 144 h after salt treatment, to survey the physiological traits including fresh weight, Rubisco activity, AGPase activity, and starch content, and each was repeated three times.Rubisco activity, AGPase activity, and starch content were measured spectrophotometrically, respectively. Briefly, 0.25 g samples were ground and homogenized in 2 mL 0.01 M PBS extraction buffer (pH7.8), then the homogenate was centrifuged for 15 min at 4000 rpm at 4 °C. The resulting supernatant was collected to determine the Rubisco activity, AGPase activity, and starch content, respectively, by using plant enzyme-linked immunosorbent assay (ELISA) kits .S. polyrhiza 7498 was cultured as described above, and 100 mM NaCl was supplemented in the MH medium. Samples were collected with six replicates at 0, 6, 12, 24, and 72 h after treatment, respectively. These samples were washed in deionized water briefly, and ground into powder in liquid nitrogen, and freeze-dried for 24 h. The contents of sodium and potassium ions were measured with a flame photometer , and the calcium ion was measured with an atomic absorption spectrometer . To investigate the ionic status during salt stress, S. polyrhiza 7498 was cultured as described above. Samples were collected with three replicates at 0, 6, 12, and 24 h after 100 mM NaCl treatment, respectively. These samples were immediately frozen in liquid nitrogen and stored at −80 °C for RNA-seq sequencing.To investigate the transcriptome changes of duckweed in response to salt stress, TM RNA sample prep Kit  with Ribo-Zero Magnetic kit for rRNA removal following the manufacturer’s recommendations, and sequenced using Illumina Hiseq 4000 in PE150 model.The total RNA extraction, transcriptome libraries preparation, and RNA sequencing were conducted by the Annoroad Gene Technology Corporation . Briefly, total RNA was extracted for each sample using RNA plant reagent kits  . The inthttp://hannonlab.cshl.edu/fastx_toolkit/). The quality of sequence was checked using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and low-quality reads were removed using customized Perl scripts. Clean reads were aligned to S. polyrhiza genome (version 2) derived from phytozome database  using Tophat v2.0.10 (http://tophat.cbcb.umd.edu/) [2FC| > 1. Gene expression levels were normalized as Fragments Per Kilobase of exon per Million fragments mapped (FPKM). Significance of expression level differences of certain genes was evaluated by ANOVA at a significance level of 0.05.As previously described ,11, adapmd.edu/) . Raw coumd.edu/) , and submd.edu/) . Differemd.edu/)  based onDuckweed genes were functionally annotated and assigned into different hierarchical categories using MapMan software . Signifi2+ signaling, ROS-scavenging, hormone metabolism, and transcription factor, were picked and analyzed by qRT-PCR (−ΔΔCt method. Pearson correlation was used to calculate the correlation coefficient between qRT-PCR and RNA-seq results. To validate the results of RNA-seq, a total of nine DE genes, related to Ca qRT-PCR . The 18S


Therefore, it could be likely that S1P-S1PR1 signaling might take part in the maintenance of continuous bone turnover under physiological conditions, while lead to the pathogenesis of bone deformities during inflammation. In this review, we summarized the immunological regulation of S1P-S1PR1 signal axis during bone remodeling with an emphasis on how osteo-immune regulators are affected by inflammation, an issue with relevance to chronical bone disorders such as rheumatoid arthritis, spondyloarthritis and periodontitis.The fundamental interaction between the immune and skeletal systems, termed as osteoimmunology, has been demonstrated to play indispensable roles in the maintenance of balance between bone resorption and formation. The pleiotropic sphingolipid metabolite, sphingosine 1-phosphate (S1P), together with its cognate receptor, sphingosine-1-phosphate receptor-1 (S1PR1), are known as key players in osteoimmunology due to the regulation on both immune system and bone remodeling. The role of S1P-S1PR1 signaling in bone remodeling can be directly targeting both osteoclastogenesis and osteogenesis. Meanwhile, inflammatory cell function and polarization in both adaptive immune (T cell subsets) and innate immune cells (macrophages) are also regulated by this signaling axis, suggesting that S1P-S1PR1 signaling could aslo indirectly regulate bone remodeling  Osteoimmunology, a term first coined at the beginning of this century   forms 3-keto-dihydrosphingosine  , 17. Theingosine , 17, whiingosine . The desingosine , the ceningosine , which cingosine , 22. Sphingosine , 23. S1Pingosine , 24–27.  in vivo . The exc in vivo , and ery in vivo . This ab in vivo –33. Cell in vivo .via targeting TNF receptor-associated factor 2 (TRAF2), therefore participating the inflammatory, anti-apoptotic and immune processes (The phosphorylation of sphingosine is performed by sphingosine kinases 1 and 2 (SPHK1 and SPHK2) . SPHK1 irocesses . Once S1rocesses , 43. Thirocesses . Transporocesses , 45–49. rocesses , 51. Esprocesses –57. Underocesses , 58–60, rocesses . Furtherrocesses .The secreted S1P regulates pleiotropic biological functions by binding with its receptors . Upon acS1pr1 gene deletion, which results in defective vascular maturation and then embryonic lethality , limited blood flow, and vascular leakage -derived osteoclasts are considered as the major type of cells responsible for bone resorption . OsteoclOsteoblasts are the major producer of RANKL and M-CSF , indicatBone remodeling is a strictly regulated process that must maintain bone formation at a rate equal to that of bone resorption . SkeletaS1P has been found to induce both osteoclastogenesis and osteogenesis, a dual role that makes S1P-S1PR1 signaling more intriguing.in vitro and in vivo. S1P-S1PR1 signaling is thought to regulate osteoclast precursor trafficking to and from bone surface, where the precursor cells fuse and differentiate into osteoclasts, a process which dynamically regulates bone mineral homeostasis and osteoclastogenesis  1/2-ERK1/2 pathway activation . Anotherignaling . Conditiignaling . These pt manner . In a moogenesis .Interestingly, intracellular S1P, which is produced during osteoclastogenesis, also inhibits this process, by suppressing p38-MAPK signaling, a key signaling pathway downstream of RANK . This isin vivo gene modification, results in a condition characterized by an increased number of osteoblasts and bone formation, as well as an increased number of dysfunctional osteoclasts and impaired bone resorption  is an enzyme that is involved in bone degradation which, when specifically deleted in osteoclast lineage by targeted sorption . The in nd S1PR3 .via SPL inhibition  in vivo, and this resulted in increased bone mass and enhanced bone strength, accompanied with induced OPG expression and reduced osteoclastogenesis in mice  cells, such as IFNγ and granulocyte-macrophage colony-stimulating factor (GM-CSF), suppress osteoclastogenesis by interrupting the RANK signaling  102–105105. Howein vivo . Other cin vivo –110. IL-in vivo –113. IL-in vivo , 115, anin vivo , 117. Thin vivo , inhibitin vivo . Anotherin vivo , 121, a in vivo . Howeverin vivo .Cells from the innate immune system also contribute to the regulation of osteoclastogenesis. Macrophages, the major components of innate immunity, constitute three sub-populations of cells: (1) non-activated M0 macrophages; (2) pro-inflammatory M1 macrophages, which are classically activated by LPS or Th1 cell cytokines such as IFNγ; and (3) M2 macrophages, which is alternatively activated by Th2 cell cytokines, such as IL-4 or IL-13, and are classified as anti-inflammatory macrophages –125. Macin vivo via STAT3 signaling, a further indication of the ability of IL-6 to affect osteogenesis  deficient mice show reduced bone healing, evidence for its critical role in osteogenesis  and suppress their maturation and function. MSCs also induce macrophage polarization to the M2 phenotype and interfere with T cell proliferation, cytokine production and polarization, in particular the promotion of Treg cell differentiation  are activated by LPS, IFN-α/γ, or TNF-α, MSCs can respond by producing pro-inflammatory cytokines  such as When S1P binds with S1PR1, it forms a complex that governs a diverse range of immune cell activities, such as cell migration, proliferation, and differentiation . This imin situ retention , neutrophils, mast cells, T and B lymphocytes, natural killer T (NKT) cells , 186–193etention , which tetention .+T cells  is a group of several inner-related disorders: psoriatic arthritis, arthritis related to inflammatory bowel disease, reactive arthritis, a subgroup of juvenile idiopathic arthritis, as well as ankylosing spondylitis (the prototypic subtype) . SpondylS1P is also strongly associated with the pathogenesis of infection-related inflammatory bone loss, as seen in periodontitis and periapical lesions: an inflammatory condition caused by teeth-related bacterial infections that erodes alveolar bone. In a mouse periodontitis model, the ablation of SPHK1 can significantly attenuate alveolar bone loss and is accompanied by a reduction in the numbers of leukocytes and osteoclasts in the periodontal tissues . S1P-S1PAlthough S1P has been studied for years, many questions still remain un-resolved regarding its role in bone remodeling. For instance, the actual outcome of S1P-S1PR1 signaling-derived modulation on bone remodeling is unknown, since it is found to induce both osteoclastogenesis and osteogenesis. The role of S1P-S1PR1 signaling in osteoimmunology is even more complicated, as its downstream signaling pathway, mTOR, has a dual role in immune system, that in Th cells it directs the polarization toward inflammatory phenotype, while in macrophages it directs the anti-inflammatory M2 polarization , 227–229In summary, S1P, a key coupling factor for osteoclasts and osteoblasts, plays a complex role in bone remodeling by targeting both osteoclastogenesis and osteogenesis. The immunomodulatory feature of S1P-S1PR1 signaling further indicates that favors the inflammatory cell phenotypes in the adaptive immune system (T cell subsets), while induces macrophage polarization toward the anti-inflammatory phenotype. This dual role in immune system indicates that S1P-S1PR1 signaling might take part in the maintenance of continuous bone turnover under physiological conditions, while lead to the pathogenesis of bone deformities during inflammation. Further investigation of the S1P-S1PR1 signaling pathway should help to get a better understanding about osteoimmunology and therefore benefit the clinical approach for inflammatory bone disorders.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. LX involved in the concept and design of the article, wrote the manuscript. YZ involved in the conception and design of the article, reviewed the manuscript. TF assisted with manuscript preparation. KB reviewed the manuscript. YX involved in the conception and design of the article, and reviewed the 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.


Occipital horn syndrome (OHS) is a rare connective tissue disorder caused by pathogenic variants in ATP7A, encoding a copper transporter. The main clinical features, including cutis laxa, bony exostoses, and bladder diverticula are attributed to a decreased activity of lysyl oxidase (LOX), a cupro-enzyme involved in collagen crosslinking. The absence of large case series and natural history studies precludes efficient diagnosis and management of OHS patients. This study describes the clinical and molecular characteristics of two new patients and 32 patients previously reported in the literature. We report on the need for long-term specialized care and follow-up, in which MR angiography, echocardiography and spirometry should be incorporated into standard follow-up guidelines for OHS patients, next to neurodevelopmental, orthopedic and urological follow-up. Furthermore, we report on ultrastructural abnormalities including increased collagen diameter, mild elastic fiber abnormalities and multiple autophagolysosomes reflecting the role of lysyl oxidase and defective ATP7A trafficking as pathomechanisms of OHS. ATP7A [Occipital horn syndrome , previously known as Ehlers–Danlos syndrome type IX or X-linked cutis laxa, is a rare disorder characterized by prominent connective tissue abnormalities including cutis laxa, hernias, joint laxity and bladder diverticula, and pathognomonic exostoses with “occipital horns” or downward pointing exostoses situated in the tendinous insertions of the sternocleidomastoid and trapezius muscles. In addition, patients may show mild to moderate intellectual disability ,3,4,5,6.ATP7A ,9,10.ATP7A gene maps to Xq13.3 and spans a genomic region of ~140 kb. Its predominant transcription product contains 23 exons [+/K+-, H+/K+- and Ca2+-pumps), as well as lipid and cationic homeostasis [ATP7A mediates (1) copper transport from the gastrointestinal tract into the bloodstream, (2) intracellular delivery of copper to cupro-enzymes in the secretory pathway and (3) efflux of excess copper from the cell ,12. Intr23 exons ,18,19,20eostasis , includieostasis . P-type eostasis ,25.ATP7A, mostly “leaky” splice-site variants resulting in exon-skipping, which permit the production of a small amount of normal ATP7A mRNA [MD is mostly caused by truncating variants, including nonsense variants, frameshift variants and splice-site variants that lead to out of frame transcripts and large deletions, which result in very low to absent ATP7A levels ,26. On tP7A mRNA ,28,29,30P7A mRNA . ConnectP7A mRNA . PatientP7A mRNA ,34.Due to the rarity of OHS and the limited number of cases scattered in literature, the natural history and clinical phenotype remain incompletely studied. This study reports the clinical, molecular and ultrastructural data for two new cases of OHS. In addition, we performed a clinical review of 32 previously described patients ,49,50,51ATP7A pathogenic variants in the Pubmed and Embase databases, only including studies with ample clinical data. Selection of the reports was undertaken independently by two different authors (A.B and K.V.M). Clinical data from the literature were evaluated using the same methodology  was born at term after an uncomplicated pregnancy. At birth, his weight was 2400 g, length 45.5 cm and head circumference 32.4 cm. Developmental milestones were achieved normally. At three years of age, he presented with micturition problems evolving to urinary retention, which led to the discovery of giant bladder diverticula. Over subsequent years, several diverticulectomies were performed, followed by continent diversion and intermittent catheterization.Clinical examination at 11 years of age showed mild facial dysmorphism  with a fAdditional examinations included a total skeletal radiography and MRI/MRA . X-rays Subject 2 (F2:II-1) was evaluated at the age of 30. He was born at term after an uncomplicated pregnancy. He developed an inguinal hernia at six weeks of age. Since then, he had multiple inguinal and femoral hernias requiring repeated mesh repairs. Starting from two years of age, he had severe bladder problems, including recurrent urinary tract infections and urinary retention, due to multiple bladder diverticula. After more than 20 surgical bladder repairs, he started self-catheterization at 8 years of age. Aged 20 years, he underwent left nephrectomy for a non-functioning kidney. He reported a mild motor delay and walked at two years of age. Joint hypermobility resulted in repeated shoulder and elbow subluxations. He was of normal intelligence and pursued higher education. He suffered from migraine, worsening musculoskeletal pain, fatigue, and depression for which he was treated with gabapentin and amitriptyline. He had a history of mitral valve prolapse, orthostatic hypotension, palpitations and a prolonged QT interval on electrocardiogram. Finally, he had a longstanding history of upper and lower gastrointestinal symptoms including nausea, post-prandial vomiting and chronic diarrhea, possibly in the context of dysautonomia. He was diagnosed with asthma.His height was 169 cm, his weight was 50.6 kg and his head circumference was 58.5 cm. Craniofacial features consisted of dolichocephaly, a high forehead, biparietal narrowing, downslanting eyes, a convex nasal ridge, midfacial hypoplasia, micrognathia and low-set ears. He had lax and hyperextensible skin . He had FBLN5 , LTBP4  and ATP7A  gene sequencing was performed without the identification of a pathogenic mutation. Total RNA was extracted from cultured fibroblasts using the RNeasy Kit  and cDNA was synthesized with the iScript cDNA Synthesis Kit . ATP7A cDNA analysis was performed followed by confirmation with long-range RT-PCR and next-generation sequencing . For individual 2, array comparative genome hybridization was normal after which ATP7A gene sequencing (GenBank RefSeq accession number NM_000052.7) was performed.For individual 1, genomic DNA was extracted from blood leukocytes using standard procedures. Array comparative genomic hybridization  showed a 300 kb deletion of chromosome band 1q31.1 (1q31.1.1q31.1(188407205-188707983)x1)(hg19/GRCh37), which does not contain any known genes. 4 with K3Fe(CN)6 in 0.1 M NaCacodylate buffer, pH 7.2. After washing in double-distilled H2O, samples were subsequently dehydrated through a graded ethanol series, including bulk staining with 2% uranyl acetate at the 50% ethanol step, followed by embedding in Spurr’s resin. To select the area of interest on the block and in order to have an overview of the phenotype, semi-thin sections were first cut at 0.5 µm and stained with toluidine blue. Ultrathin sections of a gold interference color were cut using an ultramicrotome , followed by post-staining in a Leica EM AC20 for 40 min in uranyl acetate at 20 °C and for 10 min in lead stain at 20 °C. Sections were collected on formvar-coated copper slot grids. Grids were viewed with a JEM 1400plus transmission electron microscope  operating at 80 kV.For transmission electron microscopy, 3 mm skin fragments from both individuals and an age- and sex-matched control were immersed in a fixative solution of 2.5% glutaraldehyde and 4% formaldehyde in Na-Cacodylate buffer 0.1 M, placed in a vacuum oven for 30 min and left rotating for 3 hours at room temperature. This solution was later replaced with fresh fixative and samples were left rotating over night at 4 °C. After washing, samples were post-fixed in 1% OsOFibroblast cultures were started from skin biopsies from individuals 1 and 2, and a matched control. Biochemical (pro)collagen sodium dodecyl sulfate polyacrylamide gel electrophoresis  was performed on the medium and cellular fractions of the cultured skin fibroblasts. When confluent, cells were labeled with 14C-proline . After SWe describe the clinical characteristics of two new individuals with OHS and 32 patients described in the literature . All patInitial presentations leading to the diagnosis are presented in Patients displayed variable craniofacial features, including a long face , large ears , sagging cheeks  and hair abnormalities . Trichoscopy confirmed pili torti in 7 out of 10 examined individuals. Connective tissue abnormalities were reported in all subjects. Cutis laxa was present in the majority of patients , as well as inguinal hernias . An umbilical hernia and hiatal hernia were observed in only one patient each . Wound healing was reported to be normal after trauma or surgery. Occipital horns were reported in nearly all cases . Other recurrent skeletal manifestations included hammer-shaped claviculae , scoliosis , pectus deformities , radial/tibial exostoses , coxa valga  and genua valga . Dislocations, mainly of the radial heads and joint hypermobility were present in about two-thirds of individuals, 12/20 (60%) and 16/26 (62%), respectively. Less frequently reported skeletal manifestations include bowing of the long bones, mid-diaphyseal broadening, metaphyseal spurring and rounding of the iliac wings. Osteopenia or osteoporosis was reported in four patients and fractures were observed in two subjects.Mild, mainly motor, developmental delay, was observed in 17 subjects . Intellectual disability was present in an equal number of patients  of whom seven had mild, four had moderate and three had severe intellectual disability. In three patients, the severity of intellectual disability was not specified. Several subjects displayed muscle hypotonia, especially at a young age (before the age of two). Seizures were documented in five patients . One individual was diagnosed with a spontaneous intracerebral hemorrhage at the age of 15, resulting in aphasia and spastic quadriparesis. MRI brain studies were performed in 11 patients and documented to be abnormal in 7 subjects, showing mild atrophy and/or delayed myelination. Intracranial tortuosity was present in two-thirds of patients . Tortuosity of extracranial arteries was found in three cases and was located in the cervical, splenic and hepatic arteries. Aneurysm formation was found in five patients, mainly affecting the same arteries. One individual was diagnosed with a type A aortic dissection at the age of 39, which was successfully surgically manage. Two patients showed dilatation of the large veins. Orthostatic hypotension, temperature instability, chronic diarrhea, and other symptoms of dysautonomia were seen in a high number of patients . Severe urological complications are an important concern in OHS, with bladder diverticula in 83% (25/30) of reported patients, of which three were eventually diagnosed with a bladder rupture. Recurrent urinary tract infections based on incomplete emptying of the bladder were seen in 74% (17/23) of patients. One-third of patients had vesicourethral reflux (VUR), sometimes complicated by secondary renal problems. Asthma was reported in four cases. Low levels of serum copper and ceruloplasmin were found in two-thirds of cases, 20/28 (71%) and 20/27 (74%), respectively.ATP7A gene sequencing, cDNA analysis showed a deletion of exon 10 in subject F1:II-1. Subsequent confirmation with long-range RT-PCR revealed a deep intronic c.2407-516G>T variant, underlying the exon 10 skipping, likely due to the activation of a cryptic splice acceptor. In patient F2:II-1, ATP7A gene sequencing identified a hemizygous c.2917-4A>G splice-site variant in ATP7A.After normal ATP7A pathogenic variants were reported  findings in skin samples of subjects F1:II-1, F2:II-1 and a matched control are shown in Considering the collagen abnormalities observed in both individuals with transmission electron microscopy, SDS-PAGE analysis of the intracellular and secreted (pro)collagen proteins produced by skin fibroblasts was performed for subjects 1 and 2 and a matched control. In both cases, we observed normal SDS-PAGE migration patterns .The absence of large case series and natural history studies precludes efficient diagnosis and management of OHS patients. This study describes the clinical and molecular characteristics of two new patients and 32 patients previously reported in the literature. OHS is considered a mild but rare allelic variant of Menkes disease. OHS patients show prominent connective tissue abnormalities and have a better prognosis and survival rate than patients with MD [The clinical characteristics of OHS are listed in Concerning the skeletal features, the pathognomonic occipital horns are present in all patients but one, which is probably an age-dependent discrepancy ,54,55. OUrological complications, mostly giant bladder diverticula and vesicourethal reflux, are found in over 80% of subjects. Secondary problems such urinary tract infections, urinary retention, bladder rupture, or VUR-mediated renal failure, which are all related to the bladder diverticula, are of great concern. Surgical interventions removing the diverticula often fail, with frequent relapse of the diverticula. Self-catheterization, vesicostomy, or continent diversion are often required for proper emptying of the bladder.Previous reports describe a mild neurological phenotype limited to slight generalized muscle weakness and symptoms of dysautonomia ,27, howeBrain imaging studies are rarely performed in OHS patients and are often reported as normal. However, intracranial arterial tortuosity are noted in approximately two thirds of patients. Extracranial arterial tortuosity is less frequently observed, but has been reported in the cervical, splenic and hepatic vasculature. Aneurysm formation may complicate pre-existing tortuosity and may affect the arterial and venous circulation.FBLN5- and LTBP4-related cutis laxa (OMIM#219100 and #613177) [FBLN5- and LTBP4- related cutis laxa, nor easy bruising or severe skin fragility as in the dermatosparaxis type of EDS. ATP6V0A2-related cutis laxa (OMIM#219200) may also present with mild intellectual impairment but does not commonly show bladder diverticula or exostoses. Later on the exostoses can be mistaken for hereditary multiple exostoses due to defects in EXT1 or EXT2 (OMIM#133700 and #133701) [OHS is often diagnosed upon the identification of connective tissue anomalies, including cutis laxa, joint hypermobility with hypotonia, mild intellectual impairment and bladder diverticula. Later on, the pathognomonic occipital horns become more evident. The initial differential diagnosis includes other forms of cutis laxa, including #613177)  and the #613177) , which m#133701) .Concerning follow-up, routine evaluation of neurodevelopment and early intervention with physiotherapy is recommended, as well as regular urological evaluation, including video urodynamic studies of bladder function. Any pain, or vascular or neurological symptoms that may relate to exostoses should be promptly investigated. To date, there are no data on a possible risk for chondrosarcomas in exostoses although these have been described in hereditary multiple exostoses . It coulATP7A causing OHS mainly included splice-site variants, while missense and frameshift variants were less frequently observed. This observation may fit the hypothesis that a low amount of normal ATP7A mRNA and residual ATP7A functioning is sufficient to prevent the more severe MD [The molecular data confirmed that pathogenic variants in evere MD ,29,31,60evere MD . Variantevere MD ,21,61,62Our transmission electron microscopy data in skin samples of OHS individuals show larger diameters of collagen fibrils that were more loosely packed compared to those of the control sample. Elastic fibers were affected to a lesser extent but are fragmented and/or surrounded by extracellular matrix debris. OHS is associated with decreased activity of the copper dependent lysyl oxidase (LOX), that oxidates lysyl and hydroxylysyl residues in collagen and lysyl residues in elastin to aldehydes . These aThe observation of major fibroblast abnormalities, with multiple autophagolysosomes containing sickle-shaped electron dense deposits, is novel and might point to defective trafficking and trapping of proteins needing cuproenzyme-dependent processing in the post-Golgi network ,15,21.In conclusion, this study sheds light on the natural history of OHS by describing two new patients and 32 patients reported in the literature. Mortality in adulthood warrants long-term specialized care and follow-up, incorporating MR angiography, echocardiograpy and spirometry into standard follow-up guidelines for OHS patients, next to orthopedic and urological follow-up. Ultrastructural abnormalities including increased collagen diameter, mild elastic fiber abnormalities, and multiple autophagolysosomes reflect the role of LOX and defective ATP7A trafficking in the pathomechanism of OHS.