Gramicidin is a monomeric protein that is thought to non-selectively conduct cationic currents and water. Linear gramicidin is considered an antibiotic. This function is considered to be mediated by the formation of pores within the lipid membrane, thereby killing bacterial cells. The main non-psychoactive active constituent of the cannabis plant, cannabidiol (CBD), has recently gained interest, and is proposed to possess various potential therapeutic properties, including being an antibiotic. We previously determined that CBD’s activity on ion channels could be, in part, mediated by altering membrane biophysical properties, including elasticity. In this study, our goal was to determine the empirical effects of CBD on gramicidin currents in human embryonic kidney (HEK) cells, seeking to infer potential direct compound-protein interactions. Our results indicate that gramicidin, when applied to the extracellular HEK cell membrane, followed by CBD perfusion, increases the gramicidin current. Linear gramicidins are a family of antibiotics whose function is determined by increasing the cationic permeability of the membrane , 2. IncrThe rate of gramicidin channel dimerization is directly related to membrane stiffness or elasticity , 8. ThisAmphiphiles are among the compounds characterized using the gramicidin-based assays . AmphiphCannabis sativa  extracellular solution using a ramp protocol. We clamped the cell membranes at -80 mV, close to the K+ equilibrium potential (EK+). Then, we hyperpolarized the cells to -120 mV and ramped the voltage to +50 mV, which is close to ENa+. We added the gramicidin and compound combinations after about 22 minutes, when the currents were stable, then gramicidin/compound combos were added for a 5-minute interval, and the measurements were taken at the end of the interval .We show average gramicidin current density from the ratio of current amplitude to the cell membrane capacitance (pA/pF) at -120, -80, 0, and +50 mV . Our results indicate that, at negative potentials, gramicidin conducts inward currents and, as the membrane potential becomes more positive, the current becomes outward with the reversal potential (Erev) being close to 0 mV, as would be predicted for a non-specific monovalent cationic channel. We also measured the effects of 1 μM and 10 μM CBD  experiment resulted in a Erev close to 0 mV. This raises the possibility of a potential nonselective leak current component induced by gramicidin, but not carried by gramicidin, as a confounding variable. To ensure that we were recording gramicidin pore currents, we performed the same experiment with lower extracellular sodium [Na+ = 1 mM]. This experiment resulted in the same overall trends of altered gramicidin currents densities as the high [Na+] experiment, for both CBD and TX100 . As expected, reducing [Na+] lowered the gramicidin Erev to ~-80 mV (close to EK+). These results confirm our results from the high Na+ experiment and further suggest that, when both Na+ and K+ are present at high concentrations, gramicidin permeability is not highly selective for K+ over Na+ bringing the gramicidin Erev to ~0 mV. Overall, these results show that CBD increases gramicidin currents during short exposures and suggests, therefore, that CBD could be altering membrane elasticity or gramicidin channel conductance directly.The presence of a gramicidin dependent current indicates ion flux across the cell membrane. Gramicidin pores are analogous to puncturing cation-selective holes through the cell membrane. Gramicidin induced currents in the previous high Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: Partly**********  2. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: YesReviewer #2: Yes**********  3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: YesReviewer #2: Yes**********  4. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********  5. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: This is a nice observational study of cannabidiol impacts on gramicidin in human embryonic kidney cells as a test of whether CBD has effects on bulk bilayer properties in mammalian cells. This is an interesting and important topic, as CBD is poorly soluble with very high partition coefficient and affects a wide array of membrane proteins. Whether CBD's promiscuity is generally due to bilayer effects or protein binding is unclear. The use of gramicidin in patch clamped cells to test for bilayer effects is smart. The study appears to be straightforward and observational as promised. This study will have value to readers interested int he topic of CBD-membrane interactions.My major concern is that the data do not strongly support the conclusions. I suggest softening the conclusions. The CBD effect is weak, and I was not convinced that it is not a fluke of serial errors. The statement " CBD ... slightly increased gramicidin currents at ... 10 μM (p>0.05)." is logically flawed because the test indicates that the increase was not significant. The statistic of p<0.05 for 1 μM is not compelling. Additional statistical analyses and discussion of why the statistical test is appropriate could be helpful. Any information about how opportunities for serial errors were minimized between compound and cell prep could be important. A positive control for gA activation is lacking. I think this manuscript would better err on the side of caution and describe the weakness of the CBD effect, and discuss the possibility the effect could not even be real. I suggest changing the conclusion to "CBD does not appear to inhibit gA currents at concentrations where is active against other membrane proteins, and the statistical analyses suggest there may even be an activating effect."Reporting transparency:What specific gramicidin & CBD were used? Report source.Report on care taken handling of gramicidin and CBD (tubes tubing etc). Both stick to surfaces and easily depleted from solution.Report p-values rather than just p<0.05.Report if blinding and randomization were implemented and how.Report the sequence and timing of compound additions to HEK cells.Please provide a metric for assessing what the maximum tolerated voltage error was. Series resistance compensation was applied at 100% yet the accuracy of the compensation is dependent on the accuracy of the series resistance estimate itself, and these estimates can be noisy. As current densities of up to 10 nA/pF are reported the potential for error seems real.Please report whether series resistance was reassessed after every compound addition.Reviewer #2: This manuscript by Ghovanloo Goodchild and Ruben describes the effects of CBD on gramicidin channels incorporated in HEK293 cells, using patch clamp.Major commentsThe description of the methods appears insufficient. In particular, application of gramicidin on only one side of the membrane would be difficult to compare to gramicidin added in the tiny amount used when added to both side of lipid bilayers. Did the authors thought of using a low concentration of gramicidin in the pipette to let the two sides bring a small amount of gramicidin as in the bilayer experiments of Andersen’s lab? How much was the stock in DMSO? How much DMSO was added to get to 26 μM?It would be useful to see the full temporal course of the experiment. This is because the actions of gramicidin on biological membranes tend to take long to equilibrate when gramicidin is added on only one side . This will demonstrate that the drugs were always added only after the current is completely stable can the drugs be added. If this is not possible, at least give the time course of increase in conductance after application of gramicidin and how long after gramicidin application were the drugs added, and how long was the waiting time before measurements in the presence of drug.I believe the large concentrations of gramicidin required to form pores in the whole cell are the reason why opposite than expected results are observed with triton. It would be good to discuss the differences between what is observed in HEK cells and what has been described in lipid bilayers and the possibility that at these massive concentrations part of the gramicidin channels are not as well behaved as in the bilayers. I think that it is good that the authors have the results in both systems. Perhaps what happens is that large part of the channels are not in the conformation normally seen at the low concentrations used in artificial lipid bilayers.The gramicidin monomer/dimer association dissociation is not a conformational change. The channel associates and dissociates and the energetic cost for this only comes from the membrane deformation, please correct that statement.About the discussionI don’t think this results suggest that there is a different mechanism, I think this suggests that the channels formed by gramicidin are not the well behaved channels seen in artificial bilayers. This will require reviewing literature about different structures of gramicidin channels can adopt , but clearly gramicidin channel in artificial membranes form at concentrations of 10 nM not the 26 μM used in this work or to increase permeability in biological membranes. The possibility of the channel adopting strange conformations should be considered here, even if its cation selectivity remains.As presented more than indicating that CBD acts through a different mechanism than bilayer modification, the data indicates that triton may be interacting directly with the gramicidin channel. I say this, because the observation with CBD is an increase in current, as expected from the GFA. The effect of triton is not.Minor comments.3rd line of the discussion, please correct the statement on bilateral modification toThese findings indicate that CBD is a modifier of the bilayer physical properties at the tested concentrations of 1-30 μM ...The room temperature appears to be very high (27C), I am just confirming this is not a typo occurring twice.**********  6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Reviewer #1: NoReviewer #2: Nohttps://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, AttachmentThis manuscript by Ghovanloo Goodchild and Ruben describes the effects of CBD on gramicidin channels incorporated in HEK293 cells.docxSubmitted filename: Click here for additional data file. 29 Jun 2022Reviewer #1: This is a nice observational study of cannabidiol impacts on gramicidin in human embryonic kidney cells as a test of whether CBD has effects on bulk bilayer properties in mammalian cells. This is an interesting and important topic, as CBD is poorly soluble with very high partition coefficient and affects a wide array of membrane proteins. Whether CBD's promiscuity is generally due to bilayer effects or protein binding is unclear. The use of gramicidin in patch clamped cells to test for bilayer effects is smart. The study appears to be straightforward and observational as promised. This study will have value to readers interested int he topic of CBD-membrane interactions.We thank the reviewer for a positive and constructive assessment of our manuscript. My major concern is that the data do not strongly support the conclusions. I suggest softening the conclusions. The CBD effect is weak, and I was not convinced that it is not a fluke of serial errors. The statement " CBD ... slightly increased gramicidin currents at ... 10 μM (p>0.05)." is logically flawed because the test indicates that the increase was not significant. The statistic of p<0.05 for 1 μM is not compelling. Additional statistical analyses and discussion of why the statistical test is appropriate could be helpful. Any information about how opportunities for serial errors were minimized between compound and cell prep could be important. A positive control for gA activation is lacking. I think this manuscript would better err on the side of caution and describe the weakness of the CBD effect, and discuss the possibility the effect could not even be real. I suggest changing the conclusion to "CBD does not appear to inhibit gA currents at concentrations where is active against other membrane proteins, and the statistical analyses suggest there may even be an activating effect.We thank the reviewer for raising these points and suggestions. The statistical tests that were used in this study are outlined in the Methods section, in which each drug condition was compared with the gA (no drug) condition. The automated patch clamp system that we used takes measurements, head-to-head over the time of a given experiment, and therefore, the effect of a given drug and/or concentration on a given cell was taken at the exact same time. We agree with the reviewer that the effect of CBD is not a strong one. We have added/reflected this onto the revised manuscript. This new addition is under the Discussion section.Reporting transparency:What specific gramicidin & CBD were used? Report source. This information has been added to the Methods section. CBD powder was purchased from Cayman Chemicals. Gramicidin was obtained Sigma.Report on care taken handling of gramicidin and CBD (tubes tubing etc). Both stick to surfaces and easily depleted from solution. In this study, we used nonbinding plates and tubes for preparing and the experiments with CBD and gramicidin. This information has been added to the Methods section. Report p-values rather than just p<0.05. Done. Report if blinding and randomization were implemented and how. This info has been added to methods: The Sophion Qube is an automated electrophysiology instrument that is blinded to cell selections and experimentation, and selection was performed in a randomized manner. All subsequent data filtering and analysis was performed in a non-biased manner, in which automated filters were applied to the entire dataset from a given Qube run.Report the sequence and timing of compound additions to HEK cells. Gramicidin was first perfused followed by 5 mins of waiting followed by CBD/TX100 addition. Please provide a metric for assessing what the maximum tolerated voltage error was. Series resistance compensation was applied at 100% yet the accuracy of the compensation is dependent on the accuracy of the series resistance estimate itself, and these estimates can be noisy. As current densities of up to 10 nA/pF are reported the potential for error seems real. https://sophion.com/products/rs-compensation-qube-384/The Qube uses a proprietary algorithm to perform 100% compensation, after accurately measuring series resistance, as outlined below. Information can be found here: Please report whether series resistance was reassessed after every compound addition. The Sophion Qube actively measures series resistance at every experimental block. The compensation is applied at each measurement. Reviewer #2: This manuscript by Ghovanloo Goodchild and Ruben describes the effects of CBD on gramicidin channels incorporated in HEK293 cells, using patch clamp.Major commentsThe description of the methods appears insufficient. In particular, application of gramicidin on only one side of the membrane would be difficult to compare to gramicidin added in the tiny amount used when added to both side of lipid bilayers. Did the authors thought of using a low concentration of gramicidin in the pipette to let the two sides bring a small amount of gramicidin as in the bilayer experiments of Andersen’s lab? How much was the stock in DMSO? How much DMSO was added to get to 26 μM?We thank the reviewer for a positive and constructive assessment of our manuscript. “We made fresh gramicidin stock from powder (5 mg/100 µL) in DMSO (26.56 mM). Gramicidin was dissolved in 100% DMSO, and the final concentration of 26 µM. CBD was purchased from Cayman Chemicals (No. 90080) and gramicidin was obtained from Sigma-Aldrich (CAS 11029-61-1).”It would be useful to see the full temporal course of the experiment. This is because the actions of gramicidin on biological membranes tend to take long to equilibrate when gramicidin is added on only one side . This will demonstrate that the drugs were always added only after the current is completely stable can the drugs be added. If this is not possible, at least give the time course of increase in conductance after application of gramicidin and how long after gramicidin application were the drugs added, and how long was the waiting time before measurements in the presence of drug. We added a supplementary figure and new section on this, as follows:“We added the gramicidin and compound combinations after about 22 minutes, when the currents were stable, then gramicidin/compound combos were added for a 5-minute interval, and the measurements were taken at the end of the interval .”I believe the large concentrations of gramicidin required to form pores in the whole cell are the reason why opposite than expected results are observed with triton. It would be good to discuss the differences between what is observed in HEK cells and what has been described in lipid bilayers and the possibility that at these massive concentrations part of the gramicidin channels are not as well behaved as in the bilayers. I think that it is good that the authors have the results in both systems. Perhaps what happens is that large part of the channels are not in the conformation normally seen at the low concentrations used in artificial lipid bilayers. Done. We thank the reviewer for this point. We have added this to the discussion section. The gramicidin monomer/dimer association dissociation is not a conformational change. The channel associates and dissociates and the energetic cost for this only comes from the membrane deformation, please correct that statement. Done.About the discussionI don’t think this results suggest that there is a different mechanism, I think this suggests that the channels formed by gramicidin are not the well behaved channels seen in artificial bilayers. This will require reviewing literature about different structures of gramicidin channels can adopt , but clearly gramicidin channel in artificial membranes form at concentrations of 10 nM not the 26 μM used in this work or to increase permeability in biological membranes. The possibility of the channel adopting strange conformations should be considered here, even if its cation selectivity remains. Done. We have added a section on the possibility of other conformations of gramicidin. As presented more than indicating that CBD acts through a different mechanism than bilayer modification, the data indicates that triton may be interacting directly with the gramicidin channel. I say this, because the observation with CBD is an increase in current, as expected from the GFA. The effect of triton is not. Done. We added this important point to the manuscript. Minor comments.3rd line of the discussion, please correct the statement on bilateral modification toThese findings indicate that CBD is a modifier of the bilayer physical properties at the tested concentrations of 1-30 μM ... Done.The room temperature appears to be very high (27C), I am just confirming this is not a typo occurring twice. Yes, the temperature on our patch-clamp setup was 27 degrees. 8 Jul 2022Cannabidiol increases gramicidin current in human embryonic kidney cells: An observational studyPONE-D-22-02483R1Dear Dr. Ruben,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Mark S. ShapiroAcademic EditorPLOS ONEAdditional Editor Comments :I believe the authors have satisfactorily addressed the comments and concerns of the reviewers.Reviewers' comments: 14 Jul 2022PONE-D-22-02483R1 Cannabidiol increases gramicidin current in human embryonic kidney cells: An observational study Dear Dr. Ruben:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Mark S. Shapiro Academic EditorPLOS ONE


Leukaemia and lymphoma are common malignancies. The Wnt pathway is a complex network of proteins regulating cell proliferation and differentiation, as well as cancer development, and is divided into the Wnt/β-catenin signalling pathway  and the noncanonical Wnt signalling pathway. The Wnt/β-catenin signalling pathway is highly conserved evolutionarily, and activation or inhibition of either of the pathways may lead to cancer development and progression. The aim of this review is to analyse the mechanisms of action of related molecules in the Wnt/β-catenin pathway in haematologic malignancies and their feasibility as therapeutic targets. Common haematologic malignancies include various types of leukaemia, malignant lymphoma, and multiple myeloma. Physico-chemical, biological and genetic factors play an important role in the development of malignant diseases of the haematological system. For example, people who are exposed to formaldehyde and radiation are 20 to 30 times more likely to develop leukaemia than the general population. Human T-lymphotropic virus type I causes T-cell leukaemia/lymphoma in adults, and more than 80% of patients with Burkitt’s lymphoma have significantly higher titres of EBV antibodies in their sera , 2.With the continuous development of society, the occurrence of malignant neoplasms in the haematological system has also increased each year. In addition to genetic factors , the WntIn 1982, Nusse and Varmus isolated the first Wnt gene in the mouse genome, and subsequent studies found that ectopic expression of Wnt induced mammary carcinogenesis in mice and defects in insect embryonic development . In the Although exciting results about the Wnt classical signalling pathway have been obtained from studies of the aforementioned solid cancers, its roles are poorly understood in haematologic malignancies, likely because most haematologic neoplasms are not solid cancers, which makes research difficult. This review summarizes and outlines recent advances in the understanding of the Wnt/β-catenin signalling pathway in haematologic malignancies and explores potential therapeutic targets identified in recent years.The Wnt signalling pathway is critical for human development. The Wnt family consists of at least 19 secreted glycoproteins with 22–24 conserved cysteine residues, and these proteins are associated with human development and disease occurrence . The Wnt+ pathway, which activates PLC and PKC [The noncanonical Wnt signalling pathway includes (1) the planar cell polarity pathway, which is involved in the activation of JNK and cytoskeletal rearrangement; (2) the Wnt/ Ca and PKC ; and (3)Here, we focus on the canonical Wnt signalling pathway . β-CatenHaematopoietic stem cells are the stem cells of the adult blood system and have the functions of self-renewal and differentiation into various mature blood cells. During their differentiation, adverse intrinsic or extrinsic stimuli may lead to the development of malignant diseases of the blood system , and theIn addition, CBP and P300 are essential for the self-renewal and normal differentiation of haematopoietic stem cells, and mutations in P300 and CBP are found in many malignancies. In acute myeloid leukaemia, chromosomal translocations affecting CBP and, less commonly, P300 are found. Mouse models have also confirmed that P300 and CBP are inhibitors of haematologic neoplasm formation–44. The Mll-af9, Hoxa9 and meisla can induce the transformation of haematopoietic stem cells into leukaemic stem cells. In-depth studies have revealed that fusion genes can induce differentiated progenitor cells to become LSCs by activating self-renewal signalling pathways, such as the β-catenin signalling pathway [Cancer stem cells, by functional definition, are a small fraction of cancer cells with stem cell-like functions in neoplastic tissue that maintain neoplastic cell proliferation, invasion, and metastasis. Cancer stem cells make up a very small percentage of neoplastic cells and have their own unique and specific molecular markers. Compared with neoplastic cells, which are negative for specific molecular markers, cancer stem cells have a significantly enhanced ability to invade and metastasize. For example, CD133 + hepatocellular carcinoma cells have higher clonogenicity in vivo and tumorigenicity in vitro than their CD133- counterparts . ALDH is pathway , 55. The pathway . Bmi1, a pathway . There iLeukaemias are a group of malignant clonal diseases of haematopoietic stem and progenitor cells. Leukaemic cells have strong proliferative capacity that can inhibit normal haematopoietic function, but they stop developing at different stages because their proliferation is uncontrolled, they have impaired differentiation and impaired apoptosis, and they lack normal functions. Leukaemias are divided into two categories, acute and chronic, according to the degree of differentiation and maturation of the cells. Acute leukaemia is divided into acute lymphoblastic leukaemia and acute myeloid leukaemia. Chronic leukaemia is divided into chronic myeloid leukaemia, chronic lymphocytic leukaemia, and there are other rare types of leukaemia. Several mechanisms can lead to overactivation of the Wnt/β-catenin signalling pathway that causes leukaemia, such as abnormal expression of Wnt protein and dysfunction of the destruction complex Table .The Wnt/β-catenin pathway, like the PI3K-Akt pathway, is very commonly mutated in malignant diseases. LSCs are the main component responsible for resistance to leukaemia treatment. In ALL, Akt can activate β-catenin by inhibiting GSK3β. Reducing the immune resistance of LSCs by inhibiting Akt-activated β-catenin is a good strategy for cancer treatment and preventing immune evasion . LY29400In T-ALL, the PI3K/Akt/mTOR pathway increases HIF-1α activity in hypoxic cancer cells . MoreoveMYCN belongs to a small gene family that also includes the c-myc and l-myc genes . MYCN waIn AML, FLT3 mutations are often found. FLT3 is a type 3 receptor tyrosine kinase. It is the most commonly mutated gene in acute myeloid leukaemia . AberranCEBPA is a key regulator of myeloid differentiation and regulates many protein-coding genes. CEBPA upregulates the expression of the lncRNA PLIN2, and PLIN2 promotes the occurrence and development of CML by upregulating the expression of β-catenin, providing a theoretical basis for targeting the CEBPA/PLIN2 axis for the treatment of CML .Experiments have confirmed that transferrin conjugated to doxorubicin has significantly increases cytotoxicity compared with the free drug. Transferrin has successfully been used as a carrier molecule to deliver doxorubicin to resistant leukaemia cells . SevofluNoncoding RNAs, including miRNAs and lncRNAs, have been popular research topics for many years and have been found to play an important role in leukaemia in recent studies. For example, the miR-29b/Sp1/FUT4 regulatory axis may promote the progression of acute myeloid leukaemia through fucosylation and CD44-mediated Wnt/β-catenin signalling. Glycosylation is a very common form of posttranslational protein modification, and fucosylation is one of the most important types of glycosylation in cancer. Aberrant fucosylation of proteins is a well-known hallmark of cancer , 80. In The long noncoding RNA LOC101928834 is linked to the Wnt/β-catenin signalling pathway via FRAT2. FRAT2, a weak Wnt signalling activator, binds to GSK3β and promotes the activation of Wnt signalling, leading to the expression of a series of downstream genes . FRAT2 pIn conclusion, by targeting various factors related to the Wnt/β-catenin pathway, these newly discovered molecules affect the proliferation and/or apoptosis of leukaemic cells in different ways. Strategies targeting these molecules to treat leukaemia are worthy of further study, and some of them have shown promising therapeutic effects in clinical trials.Lymphomas are malignant neoplasms of the blood system that originate in lymph nodes and extranodal lymphoid tissue. They are histopathologically classified into Hodgkin’s lymphoma and non-Hodgkin’s lymphoma. Lymphoma is a large group, and its pathogenesis varies. Physicochemical factors, immune factors, and genetic factors all play an important role in lymphoma morbidity. Despite the wide variety of lymphomas, studies of the Wnt/β-catenin signalling pathway in relation to lymphoma have focused on diffuse large B-cell lymphomas Table .Research has found that the long noncoding RNA Smad5-AS1 can act as a competitive RNA for miR-135B-5p to upregulate APC expression and inhibit the proliferation of diffuse large B cell lymphoma cells , 87. CirBy regulating the nuclear translocation of β-catenin, long noncoding RNAs play a substantial role in the development of lymphoma. MYC expression is upregulated in DLBCL cells and positively regulates the lncRNA functional intergenic repeat RNA elements (FIRRE). When FIRRE is activated, it promotes diffuse large B-cell lymphoma by inducing nuclear translocation of β-catenin. When the FIRRE gene is knocked down, cell proliferation is reduced, and apoptosis is increased . FOXM1, Other molecular pathways also regulate nuclear translocation. T-cell immunoglobulin and mucin-containing structural domain 4 is an important factor in various malignant diseases. TIMD4 exerts its function by promoting the nuclear translocation of β-catenin . The resIn DLBCL, the transcription factor Foxp1 promotes acetylation of β-catenin by CBP, which increases the transcription of β-catenin/TCF7L2 target genes. Thus, Foxp1 is an active enhancer of the Wnt/β-catenin signalling pathway, but the exact mechanism by which Foxp1 recruits CBP is unclear . By diffMoreover, some new discoveries have built on previous findings. GPNMB is a transmembrane glycoprotein that is highly expressed in a melanoma cell line and has low metastatic properties . It has Multiple myeloma is a malignant proliferative disease of plasma cells. Abnormal proliferation of clonal plasma cells in the bone marrow and secretion of monoclonal immunoglobulins or their fragment M proteins results in damage to organs or tissues. The natural course of MM is highly heterogeneous, with a median survival of approximately 3–4 years, and few patients survive for more than 10 years. Numerous factors affect the prognosis of MM patients, and the Wnt/β-catenin pathway plays a substantial role in the development of multiple myeloma. The utility of targeting this pathway to treat MM has been validated in various trials   phosphorylation and β-catenin accumulation in the nucleus, enhanced BZM-induced apoptosis, and promoted BZM-induced cell cycle arrest . FurtherBC2059 is a novel Wnt/beta-catenin pathway inhibitor. It can disrupt the binding of β-catenin to TBL1 and its related protein TBLR1, facilitating its destruction. BC2059 was confirmed to decrease β-catenin protein levels and the expression of downstream target genes. Furthermore, BC2059 was shown to synergize with low doses of the proteasomal inhibitor BZM in killing MM cells and was effective in a murine xenograft model of human MM, thus providing a rationale for further evaluation of the drug in the treatment of MM .Panobinostat was approved by the FDA in 2015 for patients with relapsed MM. A recent study indicated that panobinostat and the β-catenin inhibitor Tegavivint had a favourable toxicity profile both in vitro and in vivo. Given the significant anti-MM effect of this novel combination, the strategy warrants further evaluation as a treatment for MM patients with relapsed and refractory MM .In conclusion, the role of the canonical Wnt signalling pathway in malignant haematologic diseases is unquestionable. There are various ways to modulate this pathway. Abnormal expression of Wnt ligands; loss of function due to mutations in factors that make up the destruction complex, such as APC and AXIN; and abnormalities in the activation and nuclear translocation of β-catenin can all contribute to cancer occurrence. Therapeutic approaches targeting Wnt/β-catenin are effective, and many treatment options have been mentioned in this article. However, these treatments have a variety of adverse effects. For example, CWP232291 treatment has been found to result in gastrointestinal symptoms , infusion-related reactions, and myalgia . BC2059


Wnt/β-catenin signaling pathway is closely related to the pathogenesis Osteonecrosis of the femoral head (ONFH). β-catenin, as a major component of Wnt signaling pathway, plays a vital role in the proliferation of osteoblasts. But the effect of altering β-catenin level on the early diagnosis and staging of ONFH has not been studied. Our purpose is to investigate the role of β-catenin level in the progress of ONFH.One hundred and one patients with three stages of ONFH and fifty healthy controls were recruited between May 2016 and November 2016. We divided the patients into 32 cases of stage II, 41 cases of stage III and 28 cases of stage IV according to the Association Research Circulation Osseous (ARCO) classification. We evaluated the clinical bone histomorphology, expression position and level of β-catenin as well as the plasma β-catenin level. We investigated the level of β-catenin from the serum and tissue samples using ELISA and Western Blot assay. We also evaluated the expression of β-catenin in bone tissue by immunohistochemistry. Data were analyzed by independent t-test and ANOVA.P < 0.001). Moreover, the β-catenin levels were 49.30 ± 4.649 ng/ml, 72.54 ± 4.864 ng/ml and 79.10 ± 4.773 ng/ml in the ONFH patients with ARCO stage II, stage III and stage IV respectively, showing significant difference among them (P < 0.001). We also found that the area under the curve (AUC) calculated by ROC curve analysis to determine the values for β-catenin levels in ONFH compared with those in the control group was 0.9358 (P < 0.001), where the sensitivity was 77.23% and specificity was 98.00%.We found that the mean (± SD) serum level of β-catenin was 66.99 ± 3.032 ng/ml in the ONFH patients, which was higher than 20.14 ± 1.715 ng/ml observed in the control group (Our results indicate that the increased β-catenin may play a vital role in the progress of ONFH and the level of β-catenin is correlated with ARCO stages. The cut-off concentration may be used as one of the sensitive marks to assess the disease process of ONFH.The online version contains supplementary material available at 10.1186/s12891-022-05399-2. Osteonecrosis of the femoral head (ONFH) is a common orthopedic disease with high disability rate ,  and thWnt pathways are critical in regulating cell proliferation, apoptosis and differentiation, and they contribute to the pathologies of many diseases . The cann = 18 totals, 3 cases of stage II, 4 cases of stage III and 11 cases of stage IV) were obtained after THA, while bone samples in the control group with femoral neck fracture  were collected. The weight-bearing zone in cartilage tissue was chosen for analysis. Bone samples were collected from healthy tissue or subchondral necrotic zone at 1-4 mm below the cartilage [Patients from the First Affiliated Hospital of Guangzhou University of Chinese Medicine who were diagnosed ONFH via medical history, physical examinations, X-ray and MRI between May 2016 and November 2016 were initially included. And then some of the patients with such concurrent conditions as congenital diseases, smoking, renal dysfunction, HIV infection, diabetes mellitus, cancer and cardiovascular disease were excluded. According to the ARCO staging system , the patartilage –14. The Necrotic bone samples collected from necrotic area and healthy control femoral head were mounted in 4% formaldehyde for 24 h at room temperature. After subsequent decalcification with 10% EDTA over 2 weeks, the samples were embedded in paraffin wax. Specimens cut longitudinally into 5-μm sections were stained with hematoxylin–eosin(H&E). The stained sections were observed through the microscope  –14.2O2 for 1 h to quench the endogenous peroxidase activity, the sections were incubated with primary rabbit anti-human β-catenin antibody  in a solution consisting of 1% bovine serum albumin and 0.05% sodium azide in 0.1 M PBS for 24 h at 4 °C. After three washes with PBS, the specimens were exposed to biotinylated goat anti-rabbit IgG diluted 1:200 in PBS for 4 h at room temperature. Next, the peroxidase reaction was developed for 10 min in 0.05 M Tris buffer (pH 7.6). Slides were covered with DEPEX mounting medium, and observed under a microscope  [Immunohistochemistry was used to further analyze the expression of protein β-catenin. The femoral head tissue sections obtained from previous sections/2.2 were processed with avidin–biotin-peroxidase complex (ABC) to detect the presence of activated β-catenin. After being deparaffinized in xylene and rehydrated by a graded series of alcohols to water, and then incubated in 0.3% HOlympus) –14.The bone samples were washed with 0.9% NaCl and PBS and lysed by NET-Triton lysis buffer. Aliquots of lysates were electrophoresed on SDS-PAGE with Tris–glycine running buffer and then the proteins were transferred to poly membranes (BioRad). Nonspecific binding of the antibodies to the membrane was blocked by a one hour incubation with TBS/Tween20  containing 5% w/v non-fat dried milk for 1 h and were then incubated in TBS/Tween20 with 5% w/v non-fat dried milk supplemented with specific rabbit anti-human β-catenin antibody  overnight at 4 °C. Horseradish peroxidase-conjugated anti-β-actin  was used as loading control. Signals were detected using a diluted (1:5000) secondary polyclonal antibody (goat anti-rabbit conjugated with peroxidase) and the membranes were immersed in ECL detection solution . The protein bands were quantified using an Epson GT-8000 laser scanner –14.Levels of β-catenin in plasma were analyzed by a commercial sandwich enzyme-linked immunosorbent assay (ELISA) . The concentration of β-catenin in the samples was determined by comparing the O.D. of the samples to the standard curve. ROC curves were used to draw data from the results obtained in this study, and the cut-off value was set to provide optimal diagnostic accuracy and likelihood ratios for the level of β-catenin –14.P < 0.05 were used for statistical analysis.Statistical analysis was performed using SPSS 23.0 and GraphPad Prism v7.0. Unpaired t-tests with the significance level of P < 0.001). Although no differences were found between stage III and stage IV (P > 0.05), the ratio of empty lacunae in both stages III and IV were higher than that in stage II (P < 0.001).Figure Both necrotic femoral head region and healthy region of the femoral head positive for β-catenin were evaluated by immunohistochemical staining Fig. . Intact P < 0.001). Among the three ARCO stages, the level of β-catenin in the ONFH patients increased with the severity of X-ray findings increased.The expression of β-catenin was determined by Western blotting Fig. . The levP < 0.001) . According to multiple comparisons, difference of plasma β-catenin level was found between stage II and stage III , and between stage II and stage IV   , where the sensitivity was 77.23% and specificity was 98.00%   Fig. A. MoreovIn the present study, we investigated the relationship between β-catenin level and disease process in the patients with ONFH. We found that at both protein level and plasma level, β-catenin was significantly higher in the ONFH group, especially in the post-collapse patients than those in the control group, and it was positively associated with ARCO stages, but not with etiology. To the best of our knowledge, this is the first study effectively explaining the correlation between β-catenin level and the disease severity of ONFH. Our finding suggests that β-catenin could possibly be used as a biomarker to assess the progress of ONFH.Wnt/β-catenin signaling pathway which regulates the differentiation of bone marrow mesenchymal stem cells is closely related to the regulation of bone balance . β-catenIn addition, the results from the X-ray film, observations of the femoral head section as well as the HE staining showed that the pathological features of ONFH are apoptosis of osteocytes and rising number of lacunae. The ratio of lacunae was increased as the disease proceeded, confirming that the samples collected are trustworthy and capable of proving the pathological alterations in different stages. Although the exact mechanisms of ONFH are now still in dispute, but the femoral head cartilage damage caused by osteocyte apoptosis is seen highly associated with osteonecrosis .There are certain limitations in this study. First, we have not evaluated the levels of β-catenin in stage I, due to the difficulty to enroll patients at an early stage of ONFH. Second, a relatively small sample size has limited the accuracy of the research. Even with these limitations, our study is the first one demonstrating that β-catenin could possibly be used as a marker for disease progression of ONFH.Our findings indicate that the increased β-catenin may play a vital role in the progress of ONFH and the level of β-catenin is correlated with ARCO stages. The cut-off concentration may be used as one of the sensitive markers to assess the disease process of ONFH.Additional file 1.


In this study, we generated skin-specific overexpression of ovine β-catenin in transgenic mice by pronuclear microinjection. Results of polymerase chain reaction (PCR) testing and Southern blot showed that the ovine β-catenin gene was successfully transferred into mice, and the exogenous β-catenin gene was passed down from the first to sixth generations. Furthermore, real-time fluorescent quantitative PCR (qRT-PCR) and western blot analysis showed that β-catenin mRNA was specifically expressed in the skin of transgenic mice. The analysis of F6 phenotypes showed that overexpression of β-catenin could increase hair follicle density by prematurely promoting the catagen-to-anagen transition. The results showed that ovine β-catenin could also promote hair follicle development in mice. We, therefore, demonstrate domestication traits in animals. TGF-β2)  . In addielopment .β-Catenin has two main functions. It activates the expression of target genes downstream of the Wnt pathway by activating the pathway and also mediates cell-cell adhesion . The “bulge-activation hypothesis” considers that when a signal is transferred from the dermal papilla and activates the stem cells in the bulge of the hair follicle, proliferation of the hair follicle stem cells is stimulated, and the follicles enter a new growth period. β-catenin plays a crucial role in this process .The β-catenin-IRES-EGFP, then the restricted DNA products were resuspended in sterile ddH2O at a concentration of 20 ng/μL. Then, the products were injected into the male pronuclei of zygote of C57BL/6 by Shanghai Research Center for Model Organisms. The F1 generation mouse were produced by mating positive founder mouse (carry K14-β-catenin-IRES-EGFP fragments) with wild type.In order to generate the linear DNA fragments, the AseⅠ was used to digest the plasmid K14-β-catenin DNA sequence. The PCR procedure: 5 min at 95°C, 35 cycles of 30 s for 95°C, 30 s for 58°C and 72°C for 1 min, 72°C for 10 min and hold at 4°C forever. The further southern blot assay, digestion: the BstEⅡ was selected for digested the DNA , which is obtained from mouse tail. The probe (739 bp) amplified by primer . PCR assay was performed by using the primers shown in β-catenin expression was evaluated by quantitative real-time PCR using real-time PCR detection system . The β-actin was used as internal references. The results of β-catenin expression related β-actin were test in triplicate. The final results were generated using the 2−△△CT method  were isolated by using an RNAprep Pure Tissue Kit . According to the guidelines in the kit, 1–2 μg total RNA was used to synthesized the first-stand cDNA using ImPromⅡ Reverse Transcription System . qRT-PCR was performed . For western blot analysis, according to the kit guidelines of RIPA buffer , protein was extracted from different tissues of transgenic and control mouse. The concentration of extracted protein was measured by BCA Protein Assay Kit  and the bovine serum albumin was used as a standard. Each lane contained an equal amount of protein (20 μg per lane) and was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then transferred onto the polyvinyl-difluoride membranes at 120 V for 90min. The membranes were incubated with 1:1000 primary anti-β-catenin , and β-actin . After 1 hour blocking, wash five times with Tris-buffered saline, each time last 5 min. Horseradish peroxidase-conjugated secondary antibody  was used to incubated the membranes . Then, the membranes were visualized by using enhanced chemiluminescence system after wased six times with TBST (each time last 5 min).The 8 micron-thick of skin sections were obtained by freezing microtome . The hematoxylin and eosin staining was performed using a Hematoxylin and Eosin Staining Kit . For analysis of the hair follicle density, the Zeiss dissecting microscope and Zesiss light microscope  were used, the hair follicle numbers of per one square millimeter of mice back skin section was counted and the average of three areas of each samples were identified as the hair follicle density.p values were identified significant while lower than 0.05.Differences between transgenic and non-transgenic mice groups were tested using independence Student’s t-test. In this study, two-sided β-catenin gene was further confirmed by Southern blot    . We inve < 0.01) . The resβ-catenin protein could be detected in the heart, liver, lung, kidney, skin, muscle, and adipose tissues (β-catenin protein was observed in the skin compared with that in other tissues (p < 0.01). These results indicated that the β-catenin protein expression level in the skin of transgenic mice of line 59 was significantly higher than that in the other lines (p < 0.01). In addition, the β-catenin protein was expressed at significantly higher levels in the skin of transgenic mice than in that of non-transgenic mice  was expressed in the skin tissue in mice from line 59 but not in the other lines . The wes tissues . The higer lines  (p < 0.0nic mice .β-catenin) between transgenic and non-transgenic mice, the more significant the differences in β-catenin expression  of the dorsal skin  from Aohan fine wool sheep. Homology analysis showed that the β-catenin gene is highly conserved among species, and the amino acid sequence of the β-catenin protein is 99% conserved among species  mediated the exogenous gene and GFP expression together, which could improve the detection accuracy of transgenic-positive animals  . As a keollicles . Transiee growth . Frozen research . Immunohollicles . Previouvolution . The skiic sheep , which aβ-catenin. The recombinant plasmid exhibited good genetic stability, and the exogenous gene could be transmitted from F1 to F6. The transgenic mice were healthy and had no defects. We demonstrated that transgenic mice overexpressing ovine β-catenin showed a high density of hair follicles and an early-entered anagen (In conclusion, we successfully generated transgenic mice with skin-specific overexpression of ovine d anagen .


Increased long-chain C20:0 ceramides have been found in the serum of patients with depression. Moreover, ceramides are linked with increased microglia reactivity and inflammatory cytokine production, which are associated with depression. Since ceramides can readily cross the blood brain barrier, peripheral C20:0 ceramides could enter the brain, activate microglia, and induce depressive-like behavior. In this study, we determined whether localized infusion of C20:0 ceramides into the ventral hippocampus (VH) of rats is sufficient to activate microglia and induce depressive-like behaviors. Adult male and female rats received infusions of C20:0 ceramides or vehicle solution every other day for 2 weeks. After the third infusion, C20:0-infused animals showed reduced sucrose preference suggesting anhedonia-like behavior. In contrast, infusions of C20:0 ceramides did not affect immobility in the forced swim test or sucrose grooming suggesting that the behavioral effects of ceramides are task dependent. Furthermore, C20:0-infusions did not increase Iba-1 + microglia or inflammatory markers in the VH suggesting that localized increases in C20:0 ceramides in the VH are sufficient to induce anhedonia-like behavior without microglia activation. As central molecules in the metabolism of sphingolipids, ceramides contribute to cell membrane structure and signaling to trigger the cellular effects produced by cytokines . High coLittle is known about the relationship between long-chain ceramides and microglial activation. The accumulation of ceramides can produce inflammatory or anti-inflammatory responses depending on the acyl chain length and can affect neuronal development and synaptic activity . Short-cIn this study we examined if increased long-chain C20:0 ceramides in the ventral hippocampus (VH) are sufficient to induce the expression of microglia-related genes and depressive-like behaviors. To assess this, we infused long-chain C20:0 ceramides into the VH of adult male and female rats and evaluated anhedonia-like and despair-like behavior. The male and female rats showed anhedonia-like behavior but not despair-like behavior after three C20:0 ceramide infusions. However, C20:0 ceramide infusions did not increase the expression of microglia-related genes in the VH. Our findings suggest that C20:0 ceramides selectively produce anhedonia-like behavior without activating hippocampal microglia.ad libitum.All animals used for behavioral experiments and tissue collection for molecular analysis were treated according to the legal and ethics requirements of the Institutional Animal Care and Use Committee (IACUC) from Ponce Health Sciences University (PHSU). Male and female 53 to 60-days-old Sprague Dawley rats were obtained from PHSU/PRI Animal Facilities and maintained in standard laboratory conditions , with food and water provided Our study consisted of two different experimental protocols. The aim of the first experiment was to determine if infusions of exogenous C20:0 ceramides into the VH induce short- and long-term anhedonia-like and despair-like behavior. In this experiment, adult female and male rats  were surgically implanted with bilateral cannulas in the VH , which were fixed in place using anchor screws and dental cement. After cannula implantation, rats recovered for 2 weeks, during which they received handling during the last 7 days to habituate them to cannula manipulation and reduce stress during infusions. Fourteen days after the VH cannula implantation, male and female rats received seven infusions of either 100 μM long-chain C20:0 ceramides or a vehicle solution every other day (48 h between each infusion). The C20:0  ceramide solution was prepared by diluting the drug in alcohol to produce a 1 mM C20:0 ceramide stock solution in 100% ethanol. The stock solution was diluted in saline 0.9% to a final concentration of a 100 μM C20:0 ceramide and 10% ethanol prior to infusion. The vehicle solution was composed of 10% ethanol in saline 0.9%. Infusions were given at a rate of 0.5 μL per minute per side over a period of 2 min. Solutions were made fresh the day of each infusion.The second experiment was to determine if VH C20:0 ceramide infusions affect behavioral despair, sucrose grooming, or microglia activation. The experimental protocol is the same as in the first experiment, but animals were administered four infusions of 100 μM long-chain C20:0 ceramides or a vehicle solution.Rats received a SPT 24 h after every infusion of long-chain ceramides or vehicle solution for a total of seven sessions of the SPT to assess anhedonia-like behaviors. First, rats went through 4 days of acclimation to the sucrose solution. On days 12 and 13, male and female rats were exposed to two bottles of water in their home cage. On day 14, one of the bottles of water was switched to a bottle containing a 1% sucrose solution so that the animals acclimated to the sucrose. On day 15, the bottles were changed, so animals did not develop preference for the side where the bottles are located. The day of the test, rats were water and food deprived for 5 h and then the test was performed. This test consisted of giving the rats access to a water bottle and to another bottle that contained 1% of sucrose solution for 1 h. Sucrose solution consumption was measured for 1 h and expressed as percent of sucrose intake over water intake to assess anhedonia-like behavior.The FST assessed despair-like behavior in the rats by measuring the time spent immobile, swimming, or struggling. Immobility was considered when rats were practically immobile and only doing slight movements to keep the head above water surface. Swimming was evaluated when rats moved all their paws to stay afloat and struggling was considered when rats moved the anterior and posterior paws, but the movement of the upper paws break the water surface. In this test we put the rats in a cylinder . Slides were rinsed in 10% PBS and then heated while incubating with antigen retrieval 0.01 M Citrate-EDTA buffer (pH = 6.2) at 90–95 °C for 40 min. Tissues were washed with deionized water and remained in PBS for 5 min. Non-specific binding was prevented by adding protein block  for 15 min. Tissues were incubated with an Anti-Iba-1 rabbit polyclonal primary antibody  overnight in a humidified chamber at 4°C. A negative control was included by adding PBS instead of primary antibody. On the second day, slides were washed twice with PBS and incubated with Goat Anti-Rabbit IgG Secondary Antibody conjugated with Alexa Fluor 555  for 30 min at room temperature. This was followed by two PBS washes and incubation with DAPI for 5 min to label the nuclei. Tissues were washed with PBS twice, and slides were mounted with ProLong Gold antifade . Two to three representative images per brain sample were taken using an Olympus System Microscope Model BX60 (Olympus Life Sciences Solution). ImageJ software was used to measure the mean fluorescence intensity  and to pFor this, VH was dissected, and the tissue was homogenized by diluting with 700 μL of TRIzol reagent and centrifuging 3 min using magnetic beads. RNA was isolated using miRNeasy Micro Kit from Qiagen (Cat No. 217084). An RNA concentration and purity was estimated using the Thermo Scientific NanoDrop 2000 to calculate appropriate concentration for cDNA synthesis as specified in the manufacture’s manual. Complementary DNA was synthesized using the iScript cDNA Synthesis Kit from Bio-Rad (Cat No. 1708891). Then, the cDNA samples were diluted by a 1:20 factor and RT-PCR was performed using iQ SYBR Green Supermix from BioRad (Cat No. 1708882) to evaluate mRNA expression of Iba-1  and inflammatory genes . The RT-PCR sequences of each primer are shown in the t-test with Welch’s correction was used to compare between treatments, vehicle vs. ceramide infused animals for the behavioral analysis, immunofluorescence staining quantification and mRNA expression analysis on VH. All analyses were performed using GraphPad Prism 9.2.0 for Windows.All data was reported as the mean ± standard error of the mean. Ordinary two-way ANOVA with Tukey’s multiple comparison test was used to analyze the sucrose preference test. Unpaired parametric F = 4.824, p = 0.0385]. Moreover, the animals infused with C20:0 ceramides showed a reduction in the average sucrose preference across the seven C20:0 infusions  further suggesting that elevated C20:0 ceramides in the VH are sufficient to induce anhedonia-like behavior. Four to 6 days after the last assessment of sucrose preference, rats were exposed to the SGT to assess apathy-like behavior and FST to assess despair-like behavior. The VH infusion of C20:0 ceramides did not affect the grooming activity . Like the SGT, the FST did not show differences in immobility , swimming , or struggling .To determine if the localized infusion of C20:0 ceramides into the VH of rats would cause the development of anhedonia-like behavior, adult male and female rats were implanted with cannulas in the VH and infused with 100 μM C20:0 ceramide or vehicle every other day for 2 weeks. Anhedonia-like behavior was assessed with the SPT 24 h after each VH infusion of C20:0 ceramides as shown in the experimental timeline in t(13.27) = 1.578, p = 0.1381], swimming , or struggling time  in the FST after the third infusion of C20:0 ceramides. In addition, C20:0 ceramides did not affect sucrose grooming after the fourth infusion . These data suggest that the behavioral tasks show different sensitivities to the C20:0 ceramide infusions. After the sucrose grooming task, animals were sacrificed, and ventral hippocampal tissue was processed for molecular analysis.The lack of effect on the sucrose grooming and behavioral despair in the first experiment could be due to the reversal of the effects of the C20:0 ceramide infusions or to a difference in the sensitivity of the tasks to the ceramides. To distinguish between these possibilities, we exposed a different cohort of animals to the FST and the SGT within 1 day of C20:0 ceramide infusion. Rats received four infusions of C20:0 ceramides every 48 h. Since the sucrose preference in the first experiment was observed the day after the third ceramide infusion, rats were exposed to the FST the day after the third infusion and the SGT after the fourth infusion of ceramides . The VH t(16.90) = 1.566 p = 0.1358] or the number of Iba-1 + microglia in the VH  suggesting that the C20:0 ceramides did not activate VH microglia.Since ceramides affect cultured microglia  and micrt(12.09) = 1.230, p = 0.2421]. Furthermore, C20:0 infusions did not increase the mRNA expression of the inflammatory cytokine TNF-α  or the inflammasome NLRP3 . Moreover, we measured the mRNA expression of the high mobility group box-1 (HMGB-1), which can signal inflammation through activation of the inflammasome and NF-kB signaling cascade (t(15.43) = 0.1176, p = 0.9079]. Lastly, we found that the infusion of ceramides did not affect the mRNA expression of the inflammatory modulator TLR-4  suggesting that the infusion of C20:0 ceramides into the VH did not induce neuroinflammation.In addition to quantifying the VH microglia, we also assessed whether the neuroinflammatory profile in the VH was increased by the infusion of C20:0 ceramides. Consistent with the lack of change in overall Iba-1 protein expression, we found that the ceramide infusion did not change Iba-1 mRNA expression [ cascade  and founAlthough clinical findings suggest that long-chain C20:0 ceramides may contribute to depression , it is nResearch suggests that long-chain ceramides in the hippocampus contribute to depressive-like behaviors in rodents. A previous study found strong evidence that fluoxetine and amitriptyline produce their antidepressant effects by reducing acid sphingomyelinase production of hippocampal ceramides in male mice . FurtherAlthough microglia are associated with anhedonia-like behavior  and can In conclusion, our findings suggest that localized increases in C20:0 ceramides in the VH are sufficient to induce anhedonia-like behavior through a mechanism that does not require microglial activation. Further studies are needed to explore the mechanisms by which different long-chain ceramides produce their behavioral effects.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.This animal study was reviewed and approved by the Institutional Animal Care and Use Committee from Ponce Health Sciences University.LS-E: conceptualization, methodology, investigation, validation, formal analysis, resources, supervision, writing – original draft, and writing – review and editing. AF-Q: investigation, formal analysis, and writing – original draft. LT-C: investigation, writing – original draft, and writing – review and editing. CS: investigation and writing – review and editing. DP-C and WF-F: writing – original draft. NI-M, AH-L, and MC-R: investigation. JP: conceptualization, methodology, validation, formal analysis, resources, supervision, writing – original draft, and writing – review and editing. All authors contributed to the article and approved the submitted version.


Balance deficits are heterogeneous among children with Developmental Coordination Disorder (DCD). Balance performance depends on different balance domains, each associated with specific underlying neurological systems. In DCD, any of these domains can be affected, but the control mechanisms are poorly understood. The mirror neuron system (MNS) seems to play a key-role in DCD-related deficits. To understand the role of MNS as a control mechanism underlying the balance deficits, simultaneous registration of cortical MNS activity while performing balance tasks is imperative. Therefore, a protocol for combining real-time registration of cortical MNS activity during functional balance tasks in children with DCD, CP and TD is introduced. Methods: Children with DCD, CP and TD (n=108) aged 5-10yr perform preselected tasks of the Kids-BESTest, representing specific balance domains (mixed design): leaning with eyes closed , single-leg-stance, alternate stair touching , in-place response, compensatory stepping backward  and walking over obstacles (gait stability). Simultaneously, functional Near-Infrared Spectroscopy (fNIRS) monitors cortical activity involving the MNS: premotor, inferior and superior parietal cortex and supplementary motor area. An 8-8-optode bundle, making 22 channels, targets this region of interest. Outcome measures are: (de)oxygenated hemoglobin concentration changes per task per channel. Results: In this ongoing research, the protocol was already feasible in 19 children (7.52±1.19). Conclusion: Simultaneous registration of cortical MNS activity (fNIRS) and Kids-BESTest scores will help increase the understanding of the control mechanisms underlying the heterogeneous balance problems in DCD. Consequently, first steps are made to confirm whether DCD shows deviant or delayed development.No significant relationships.


Alternaria solani, is an important foliar disease that causes major yield losses of potato. Effector proteins secreted by pathogens to host cells can inhibit host immune response to pathogens. Currently, the function of effector proteins secreted by A. solani during infection is poorly understood. In this study, we identified and characterized a novel candidate effector protein, AsCEP50. AsCEP50 is a secreted protein that is highly expressed throughout the infection stages of A. solani. Agrobacterium tumefaciens-mediated transient expression in Nicotiana benthamiana and tomato demonstrated that AsCEP50 is located on the plasma membrane of N. benthamiana and regulates senescence-related genes, resulting in the chlorosis of N. benthamiana and tomato leaves. Δ50 mutants were unaffected in vegetative growth, spore formation and mycelium morphology. However, the deletion of AsCEP50 significantly reduced virulence, melanin production and penetration of A. solani. These results strongly supported that AsCEP50 is an important pathogenic factor at the infection stage and contributes to the virulence of Alternaria solani.Early blight, caused by the necrotrophic fungus  Alternaria solani is a necrotrophic fungus that can cause early blight disease of tomato, potato, tobacco, and many other vegetables and crops, and lead to huge losses in agricultural production [A. solani digests host cell inclusions by producing germ tubes and secreting various metabolites, thereby penetrating host tissues or directly invading stomata or wounds adjacent to epidermal cells [A. solani also reduces the photosynthetic rate in infected leaves by inhibiting the photosystem II activity and reducing the chlorophyll and other photosynthetic pigment contents, resulting in a general reduction in plant growth and a decline in yield [oduction –3. The tal cells , 5. In tin yield , 7.Botrytis cinerea, Alternaria solani, Plectosphaerella cucumerina, and Sclerotinia sclerotiorum all belong to BHNs [Necrotrophic pathogens extract nutrients from dead hosts by destroying host tissues cells. Thus, cell death also occurs early in necrotrophs infection and is an indicator of successful infection –10. Necr to BHNs –18. In cA. solani effectors is of great significance to further explore the pathogenic mechanism of necrotrophic pathogens.Many necrotrophic pathogens utilize plant defense responses to enhance pathogenicity and induce host cell death by secreting toxic secondary metabolites , 20. SomAlternaria solani, we selected a novel candidate effector protein, AsCEP50, with high expression levels. The AsCEP50 was localized to the host plasma membrane and its expression level increased continuously during infection. Transient expression assays in Nicotiana benthamiana and tomato showed that AsCEP50 regulated host senescence-related genes, resulting in chlorosis. In addition, we compared the phenotypic and pathogenic differences between wild-type, Δ50 mutant and revertant strains. These results elucidate the role of AsCEP50 in the vegetative growth and host infection stages of A. solani, and provide insight into the molecular interactions between A. solani and its host.Based on the transcriptome and genome results of Nicotiana benthamiana and tomato were grown in a greenhouse at 23°C to 25°C with a cycle of 16-h light/8-h darkness. Potato ‘Favorita’ was grown in a greenhouse at 19°C to 22°C. The wild-type strain HWC-168 of A. solani was collected from leaves infected with potato early blight in Weichang County, Hebei Province, China. The HWC-168 was maintained in our laboratory and grown on potato dextrose agar (PDA) medium at 25°C. Escherichia coli strain DH5α was cultured in Luria-Bertani (LB) medium at 37°C and used for plasmid amplification. Agrobacterium tumefaciens strain EHA105 was cultured in LB medium at 28°C for Agrobacterium-mediated transient expression in N. benthamiana leaves.All the plants were grown on the campus of Hebei Agricultural University . http://www.cbs.dtu.dk/services/SignalP/), SMART (http://smart.embl-heidelberg.de/), ProParam (https://web.expasy.org/protparam/) were used to predict protein signal peptides, structural domains, molecular masses and theoretical isoelectric points.The Online tools SignalP 5.0 Server (AsCEP50 were PCR amplified from the cDNA of potato leaves infected with HWC-168 using an Easy Pure Plant RNA Kit (TransGen Biotech) and a TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech). The hygromycin B resistance gene (Hyg) was amplified from the pUC-Hyg vector. The upstream and downstream sequences of AsCEP50 gene were amplified from HWC-168 genomic DNA. The pCAMBIA1301::EGFP vector was digested with restriction enzymes SalⅠ and BamHⅠ. Recombinant Agrobacterium was constructed by ligating AsCEP50 (FL and NSP) to the linearized pCAMBIA1301::EGFP vector using a pEASY-Uni Seamless Cloning and Assembly Kit (TransGen Biotech) for Agrobacterium-mediated transient expression in N. benthamiana. The Hyg gene, the upstream and downstream fragments of AsCEP50 gene were connected with the pUC19 cloning vector with restriction enzymes BamHⅠ and EcoRⅠ for the construction of deletion strains. The AsCEP50 gene was ligated with the KN vector containing the neomycin resistance gene (Neo) with restriction enzymes BamHⅠ and EcoRⅠ for the construction of revertant strains.A full-length open reading frame (referred to as FL) and a truncated coding region without the signal peptide sequence but with an engineered ATG start codon (referred to as NSP) of A. solani, we selected a relatively stable housekeeping gene "Actin" as the internal reference gene [Actin and AsCEP50 were designed using the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/). PP2A was selected as a housekeeping gene in N. benthamiana [https://www.uniprot.org/), and their specific primers were designed using Primer 3 Plus (https://www.primer3plus.com/). The cDNAs of the hyphae and growing on potato and N. benthamiana leaves were serial diluted to detect primer specificity. The RNAs were extracted using an Easy Pure Plant RNA Kit and cDNA were synthesized using a TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix. The cDNAs were obtained using an Easy Pure Plant RNA Kit and a TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix. All the primers used in qPCR were tested for amplification efficiency by serial dilution. The qRT-PCR was performed using the Bio-Rad CFX384 Touch Real-Time System (Bio-Rad), and the total reaction volumn was 20 μL containing 2 μL gene specific primers, 6 μL of cDNA and 10 μL of 2×Magic SYBR Mixture . The qRT-PCR was performed under the following conditions: 95°C for 30 s, followed by 40 cycles at 95°C for 5 s and 59°C for 30 s. The threshold cycle (CT) values were used to determine the fold change in transcript accumulation with the 2−ΔΔCt method [According to the research on nce gene , 28. Spethamiana . The SEN-1 MgCl2, and then resuspended with an infiltration buffer containing 10 mmol L-1 MgCl2, 10 mmol L-1 2-(N-morpholino) ethanesulfonic acid (pH 5.6) and 200 μmol L-1 acetosyringone. The optical density (OD)600 value of the A. tumefaciens suspension was adjusted to 0.6–0.8, and infiltrated into the leaves of N. benthamiana [Recombinant Agrobacterium was cultured at 28°C, washed once with 10 mmol Lthamiana . Empty vN. benthamiana and tomato were cultured at 23°C for 5 days until symptoms appeared. Whole N. benthamiana and tomato leaves were immersed in 3,3’-diaminobenzidine (DAB) solution  at room temperature. After 12 hours, the leaves were soaked in 95% ethanol and boiled for 30 min and photographed. Each treatment was repeated six times.To detect HR, the strain expressing AsCEP50 protein was infiltrated into the right side of each leaf and EV was infiltrated into the left side. The AsCEP50-NSP expression vectors were separately infiltrated into 5-week-old N. benthamiana leaves. At 48-72 hours post inoculation (hpi), the leaves epidermal cells were visualized with excitation at 488 nm and emission at 507 nm using a confocal laser scanning microscope .For subcellular localization, EV and The function of the predicted signal peptides was verified by the yeast secretion system. To determine the signal peptide secretory activity, the signal peptide of AsCEP50 was cloned into vector pSUC2 using specific primers. The recombinant vector was transformed into yeast strain YTK12. The positive colonies were screened on a CMD−W medium . To test for invertase secretion, successfully transformed yeast strains were grown on YPRA agar .AsCEP50 gene was replaced with Hyg using homologous recombination technology. Approximately 1-kb upstream and downstream fragments from the wild-type HWC-168 genomic DNA were amplified and linked to Hyg gene to construct the Δ50 deletion mutant strains. The fusion fragments were used for the protoplast mediated transformation of HWC-168.The -1 NaCl solution 2–3 times. The hyphae were transferred to a 50-mL centrifuge tube using tweezers, and digested with a mixture of enzymes, 0.1 mg of snailase , 0.1 mg of driselase  and 0.1 mg of lysing enzyme  in 10 mL of 0.7 mol L-1 NaCl. The protoplasts were resuspended in STC buffer  and the fusion fragments mixtures were added to the PTC buffer . The transformants were screened on Regeneration PDA medium containing ampicillin (50 μg mL–1) and hygromycin (50 μg mL–1), and PCR was performed using specific primers of Hyg, upstream and downstream fragments to confirm the transformants.The wild-type HWC-168 hyphae were inoculated into 200-mL flasks containing 100 mL Potato Dextrose Broth (PDB) medium, incubated at 25°C and shaken at 120 rpm for 36 h. The mycelia were filtered through three layers of gauze and thoroughly rinsed with 0.7 mol LΔ50 mutant strains. The recombinant KN vector was transformed into the protoplasts of mutant strains by PEG-mediated protoplast transformation. The neomycin resistance was used to screen the revertant strains.After obtaining the mutant strains, the protoplasts were obtained from the Δ50 mutant and revertant strains with a diameter of 5 mm were inoculated independently into the 350-mL flasks containing 100 mL PDB medium, incubated at 25°C and shaken at 150 rpm for 5 d. The solutions of the wild-type, Δ50 mutant and revertant strains were filtered with gauze, and the absorbance of filtrate was measured at 400 nm.The colonies of the wild-type, Sterile cellophane was spread on the PDA medium, and then, wild-type, deletion mutant and revertant strains were placed on the PDA medium at 25°C for 3 d in the dark. Then, the cellophane was uncovered and the strains were cultured for 4-5 d.Δ50 mutant and revertant strains were prepared and their concentrations were adjusted to 105 mL-1. Five-week-old potato leaves were sterilized with 75% alcohol, rinsed with sterile water, and then the petioles were inserted into water agar medium. Approximately 20 μL of each suspension was inoculated onto potato leaves in vitro. The leaves were incubated at 25°C under a 16-h photoperiod. The accumulation of H2O2 was detected by DAB staining [The HWC-168 strain was inoculated into tomato agar medium. After the hyphae were overgrown, mechanical damage was performed, and sporulation was stimulated by ultraviolet light for 15 min. Spore suspensions of HWC-168 wild-type, staining . The symAsCEP50 gene (GenBank accession numbers: OM735615) with effector characteristics. The signal peptide of AsCEP50 gene correponded to 1-19 amino acids (aa) using SignalP analysis  in N. benthamiana as a positive control [Fungal pathogens manipulate the host immune response to facilitate infection by secreting specific effector proteins. Transient expression analysis was performed to determine whether AsCEP50 directly induced host cell death, suppressed host immune responses, or promoted host cell death in  control . When AsAgrobacterium AsCEP50-NSP and AsCEP50-FL alone. At the same time, we repeated this experiment on tomato leaves and found that Agrobacterium AsCEP50-NSP and AsCEP50-FL also caused tomato leaf chlorosis. Furthermore, we investigated the ability of AsCEP50-NSP and AsCEP50-FL to stimulate hydrogen peroxide (H2O2) in N. benthamiana and tomato leaves. The results showed that AsCEP50-NSP and AsCEP50-FL accumulated more H2O2 than controls at 5 dpi  were determined in EV and AsCEP50-infiltrated leaves. After treatment with AsCEP50, the expression levels of senescence-related genes were significantly up-regulated compared with EV treatment. The expression level of DHAR1 was 16 times higher than EV treatment. These results suggested that AsCEP50 induced plant leaf chlorosis by regulating senescence-related genes.Many genes that regulate senescence during plant growth have been defined as senescence related genes. The expression levels of three senescence-related genes (AsCEP50 gene was replaced by Hyg gene using PEG-mediated protoplast transformation. Two hygromycin-resistant A. solani strains were obtained by three independent transformation processes on selective plates containing ampicillin (100 μg/mL) and hygromycin (100 μg/mL). The Hyg gene was successfully amplified from two AsCEP50 deletion strains by RT-PCR using Hyg-specific primers, but no Hyg gene was amplified in wild-type strain  in the host also increase significantly in 72 h, indicating that the substances secreted by A. solani cause serious damage to defense-related responses of the host [AsCEP50 expression may indicate that AsCEP50 plays an important pathogenic role in attacking host defense systems and digesting host cells.Although not all secreted proteins were associated with pathogenicity, studies have shown that secreted proteins expressed during the infection phase are more likely to affect pathogenicity of pathogens . Specifithe host . TherefoSclerotinia sclerotiorum not only secretes SsCP1 effector to induce the host cell death, but also secretes SsITL effector to inhibit the host immune responses [N. benthamiana leaves showed no direct induction of cell death and inhibition of cell death. The leaf chlorosis symptoms of N. benthamiana and tomato and the expression of senescence-related genes in the host indicated that AsCEP50 promoted leaf chlorosis by regulating the expression of senescence-related genes. There are many pattern recognition receptors (PRRs) in the host cell membrane, which play an unparalleled role in resistance to pathogen infection [A. solani on the SA signaling pathway in host defense and the localization of the candidate effector AsCEP50 secreted by A. solani to the cell membrane of N. benthamiana suggest that AsCEP50 is likely to play an important role in infection.Phytopathogenic effectors are often overexpressed in plants to induce phenotypes that reflect their level of virulence activity. Effectors with different functions may directly induce, inhibit or promote host cell death in the infection . For exaesponses , 44. Thenfection , 46. Furnfection . The effAsCEP50 reduced the melanin content of the mutant strains and decreased hyphal penetration. Likewise, the pathogenicity of isolated potato leaves inoculated with wild-type, deletion and revertant strains showed that deletion of the AsCEP50 gene resulted in reduced spot area and lessened virulence of Alternaria solani. The effector proteins such as CfEC92 in Colletotrichum fructicola, F12 in Fusarium graminearum and AvrPtoB in Pseudomona all play an important roles in promoting the virulence of the pathogens themselves [Melanin is widely found in fungi, and it greatly increases the fungal tolerance to external conditions, including ultraviolet radiation, enzymatic hydrolysis and extreme temperatures , 49. Altemselves –55.AsCEP50 gene of the A. solani candidate effector as having a critical role in infection. The AsCEP50 gene was highly expressed throughout the infection process of A. solani, and was localized to the host cell membrane. The gene promoted host leaf chlorosis by regulating host senescence-related genes. In addition, the comparison of wild-type, mutant and revertant strains found that AsCEP50 not only affected the melanin density and penetration of A. solani, but was also crucial for the pathogenicity of A. solani. We will further investigate the targets and interaction modes of AsCEP50 in the host to explain the molecular basis of necrotrophic pathogen-host interactions.In this study, we identified the S1 FigAsCEP50 protein 1-19 is a signal peptide sequence.(TIF)Click here for additional data file.S2 FigThe AsCEP50 protein contains domains Pro_Al_protease and Trypsin.(TIF)Click here for additional data file.S3 FigSP with yeast signal trap assay. The YTK12 yeast strain containing pSUC2 is able to grow on a CMD−W medium without tryptophan, but not on YPRAA medium. AsCEP50SP can grow on both CMD−W and YPRAA media. The SP of Avr1b was used as positive control.The validation of the function of AsCEP50(TIF)Click here for additional data file.S4 FigThe upper left and upper right corners of the leaf were injected with control (EV) and AsCEP50 (FL/NSP), respectively. The lower left and right corners were respectively injected with INF1 and AsCEP50 (FL/NSP) coupled with INF1.(TIF)Click here for additional data file.S5 FigAsCEP50 gene in the genomes of mutant and revertant strains (RT).Verification of the (TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S7 FigAlternaria solani wild-type (left), AsCEP50 mutant (middle) and revertant (right) strains cultured on PDA medium for 7 d at 25°C in the dark.The colony phenotypes of (TIF)Click here for additional data file.S8 FigΔ50 mutant strains , wild-type strains  and revertant strains .The isolated potato leaves were inoculated with the spore suspensions of (TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 TableActin, SEN4, SAG12 and DHAR1 in N. benthamiana leaves injected with empty vector and Agrobacterium containing AsCEP50.The expression of (PDF)Click here for additional data file.S3 TableN. benthamiana leaves injected with empty vector and Agrobacterium containing AsCEP50.Lesion length and width of (PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.


Although oncological advances have improved survival rates of female cancer patients, they often suffer a reduced fertility due to treatment side effects. In the present study, we evaluated the potential fertoprotective effects of the specific inhibitor of SIRT1, EX-527, on the gonadotoxic action exerted by cyclophosphamide (CPM) on loss of primordial follicles (PFs).The effects of the CPM metabolite phosphoramide mustard (PM) on follicle activation, growth and viability and the protective action of EX-527 against PM effects were evaluated on bovine ovarian cortical strips in vitro cultured for 1 or 6 days. To understand whether PFs exposed to PM plus EX-527 were able to activate and grow to the secondary stage after suspension of the treatment, strips cultured for 3 days in PM plus EX-527 for 3 days were transferred to plain medium until day 6. Follicle growth and health were evaluated through histology and viability assay at a confocal microscope. In order to investigate the molecular pathways underlying the ovarian response to PM in the presence of EX-527, we analysed the protein level of SIRT1, HuR, PARP1 and SOD2 after 1 day of in vitro culture.We found that (1) PM, the main CPM active metabolite, promotes PF activation; (2) the ovarian stress response induced by PM includes a SIRT1-dependent pathway; and (3) EX-527 reduces PF activation and growth induced by PM.SIRT1 can represent a candidate molecule to be targeted to protect ovarian follicles from alkylating agents and EX-527 could represent a potential fertoprotective agent for cancer patients. Although oncological advances have improved survival rates of female cancer patients, after completing therapies many patients experience a reduced quality of life due to treatment side effects such as reduced fertility , 2. In aAlkylating agents are the most toxic to the ovaries, and cyclophosphamide (CPM), which is routinely used against solid and haematological cancers, is associated with the highest rate of ovarian toxicity . CPM canAlthough CPM differently impacts on ovarian oocytes and somatic cells depending on animal age and follicle stage , 11, the+) for their activity  polymerase 1 (PARP-1) antibody (sc-74479) were purchased from Santa Cruz Biotechnology Inc. . Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (TA802519) was purchased from OriGene Technologies Inc. . HRP-conjugated anti-rabbit antibody (BA1054) was purchased from Boster Biological Technology Co., Ltd . ECL kit was purchased from Thermo Scientific .Lumox gas-permeable 50-mm culture dishes (PD) were purchased from Sarstedt . Leibovitz’s L-15, α-MEM GlutaMax, Insulin-Transferrin-Selenium (ITS) 100 × and Live/Dead Fixable Far Red Stain were purchased from Invitrogen . Bovine ovaries were collected at the time of slaughter from individuals aged 8–24 months  and transported within 2 h to the laboratory in Leibovitz’s L-15, 2 mM glutamine, 3 mg/mL BSA, 1% Pen-Strep and 1 μg/mL amphotericin B (handling medium) at 4 °C. Cortical slices (~ 0.5 mm thick) were manually dissected from bovine ovaries in handling medium at room temperature (RT), avoiding regions with visible antral follicles. Cortical slices were homogeneously minced into 1 mm × 1 mm × 0.5 mm strips using a tissue chopper , pooled and mixed by gentle agitation in a 10-cm Petri dish and washed twice in fresh handling medium, and 10 cortical strips were randomly distributed into 5 mL of medium for culture in PD. Fresh strips from each sample were processed for histology and viability assessment as controls.2 and 95% humidity in air for 1, 3 and 6 days. These time points were selected on the basis of previous studies [In each experiment, strips from the same ovary were cultured in PD in α-MEM, 3 mM glutamine, 0.1% BSA, 1% Pen-Strep, 1% ITS , 1 μg/mL amphotericin B, 50 μg/mL ascorbic acid at 37 °C, 5% CO studies  showing  studies . Half men = 3) was aimed to evaluate the effects of the active cyclophosphamide (CPM) metabolite PM  on  on n = 3n = 3) was aimed to understand whether PFs exposed for 3 days to PM plus EX-527 were able to activate and grow during culture in medium alone for 3 additional days. Ovarian strips were cultured in (1) medium alone for 3 days (3D) or (2) 6 days (6D); (3) PM 10 µM plus EX-527 10 µM for 3 days (PM + EX 3D); and (4) PM 10 µM plus EX-527 10 µM 3 days followed by culture in medium alone for 3 days (PM + EX 3D + M 3D).Experiment 2 (n = 3) was aimed to evaluate the effects of exposure to PM, EX-527, PM plus EX-527 or medium alone on expression of SIRT1, HuR, PARP1 and SOD2. To this end, ovarian strips were cultured as in experiment 1 for 1 day and then processed for protein extraction, SDS page and western blotting. Parallel samples were processed for histology and viability.Experiment 3 , the supernatants were collected and stored at − 80 °C until processed for protein analysis. Protein concentration was determined by BCA protein assay kit. Thirty micrograms of proteins was resolved by 10% sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis, transferred to a PVDF membrane and blocked with 5% BSA in Tris-buffered saline containing 0.05% Tween 20 (TBS-T) for 1 h at room temperature. Then, membranes were incubated with polyclonal rabbit anti-SIRT1 antibody (1:700), anti-SOD2 antibody (1:1000), mouse monoclonal anti-HuR (1:250), anti-PARP-1 (1:300) or anti-GAPDH (1:750) overnight at 4 °C, followed by incubation with HRP-conjugated anti-rabbit (1:3000) or anti-mouse secondary antibody (1:2000) for 1 h at room temperature. After washing, the immunoreactive bands were stained by ECL kit and immediately detected by Uvitec Cambridge system . The images obtained by Uvitec were analysed by ImageJ 1.44p software to obtain values of band intensity. Data obtained were placed in a spreadsheet. The intensity of bands of the proteins of interest was normalised for GAPDH and values were given as relative units (RU). The experiment was performed in triplicate.t test or one-way ANOVA followed by the Tukey HSD test for multiple comparison. Analyses were performed using the SigmaStat software (Jandel Scientific Corporation). p values < 0.05 were considered statistically significant.Histological and viability data are presented as cumulative percentages. Statistical analysis was performed by Fisher’s exact test for pairwise comparisons when overall significance was detected. Western blotting data are presented as mean ± SEM. Statistical analysis was assessed by n = 3) was aimed to evaluate the effects of PM and EX-527 used alone or in combination on follicle activation, growth and health during bovine ovarian tissue in vitro culture. Overall, follicle staging . Culture with EX-527 for 6 days (EX D6) reduces PF activation and growth  compared to both control day 6 (p < 0.01) and PM D6 (p < 0.01).Experiment 1 .Follicle quality Fig.  was not p < 0.05; PM + EX vs D0, p < 0.01). In sharp contrast, a marked decrease of follicle viability was observed in tissues cultured in EX-527 compared to D0, D6, PM D6 and PM + EX D6 (p < 0.01). Overall, data indicates that PM enhances follicle activation and growth, and the combined treatment with EX-527 is able to prevent them without affecting follicle quality and viability. Although treatment with EX-527 alone is also able to prevent follicle activation and growth, it exerts a detrimental action on follicle quality and viability with respect to its use in conjunction with PM.Follicle viability Fig.  in tissun = 3) has been addressed to understand whether follicles whose activation and growth were prevented by combined treatment with PM and EX-527 were able to activate and grow to the secondary stage after suspension of the treatment. To this end, bovine ovarian tissue was cultured in medium for 3 (D3) or 6 days or under combined treatment with PM and EX-527 for 3 days (PM + EX D3), followed or less by culture in medium alone for further 3 days (PM + EX D3 + M D3). Overall, follicle staging  was aimed to investigate the molecular pathways underlying the ovarian response to PM observed after a 6-day in vitro culture in the presence or absence of the SIRT1 inhibitor. To this end, we analysed the protein level of SIRT1, HuR, PARP1 and SOD2 after a 1-day in vitro culture of bovine ovarian strips. Parallel samples were processed to evaluate follicle staging and health after culture for 1 day (D1).Experiment 3 . Follicle viability (not shown) exceeded 90% in all samples.Follicle quality Fig.  was not Anticancer therapy is known to be associated with increased risk of premature ovarian failure (POF) and infertility. As an early effect, gonadotoxic treatment induces amenorrhea and the loss of follicles, and the long-term is represented by the permanent loss of the ovarian reserve as recently reviewed . Therefo+-dependent deacetylase with numerous regulatory roles in the ovary [Herein, the bovine has been adopted as a model for human folliculogenesis as in both species one dominant follicle is selected from a cohort of 5–30 follicles, all other recruited follicles become atretic, the time of development from primordial to the ovulatory follicle stage is similar, and the number of early growing follicles slowly declines with increasing age , 39, 40.he ovary , can be As previously demonstrated, the culture system used in this study efficiently supports PF activation and enhances the rate of secondary follicles developed after in vitro culture of bovine ovarian cortical strips by means of optimization of oxygen availability through in vitro culture in gas-permeable dishes . Under tThe hypothesis that SIRT1 is involved in the ovarian response to CPM arises from the finding that that CPM administration induces early upregulation of ovarian SIRT1 protein in the mouse. We have previously demonstrated that the ovary activates an adaptive response based on upregulation of SIRT1 protein that reaches its peak at 24 h . TherefoEX-527 is a specific SIRT1 inhibitor showing 100-fold selectivity for SIRT1 over SIRT2/3, along with metabolic stability and membrane penetrability . We repo+ ratio, a condition favouring SIRT1 activation. Overall, SIRT1, being a regulator of both follicle dormancy and ovarian stress response to CPM, is a good candidate as a sentinel molecule directly involved in the loss of ovarian reserve induced by alkylating agents.Based on the considerations above, our findings are consistent with recent research demonstrating the role of SIRT1 as a regulator of the PF activation process in the mouse model . Indeed,+ level, and its hyper-activation may deplete intracellular NAD+ and ATP levels [To test whether ovarian adaptive response to PM involves DDR and/or OSR signalling, we analysed the protein expression level of PARP1 and SOD2, as main components of DDR and OSR, respectively. PARP1 regulates primary repair mechanisms to resolve DNA lesions and plays a critical role in the maintenance of chromosome stability at key stages of meiosis in the female germ line –52. PARPP levels . In rat P levels , 20. SODP levels . Here weP levels .Here we proposed for the first time the hypothesis that SIRT1 can be a candidate as a molecule to be targeted to protect ovarian follicles from alkylating agents. This conclusion is based on the findings that the ovarian stress response induced by PM includes a SIRT1-dependent pathway and that the SIRT1 inhibitor EX-527 reduces PF activation and growth induced by PM. Nevertheless, further studies are needed to better characterise the beneficial effects of EX-527 against the molecular damage by CPM. Moreover, that this inhibitor does not counteract the anticancer effect exerted by this alkylating agent remains to be demonstrated. EX-527 therapeutic potential has been evaluated in animal models for several pathologies, including cancer. EX-527 safety was demonstrated in phase I clinical trials and it is currently under investigation as a therapeutic agent in women with unexplained infertility . Taken together, these data support the potential use of EX-527 as a fertoprotective agent for cancer patients.