Androgen receptor (AR) signaling is a major driver of prostate cancer (CaP). Although most therapies targeting AR are initially effective in CaP patients, drug resistance is inevitable, mainly because of the inappropriate re-activation of AR pathway. However, the underlying mechanisms remain largely unknown. Here, we found that phospholipase C epsilon (PLCɛ) was highly expressed in CaP samples, and was closely associated with AR signaling activities. PLCɛ depletion triggered enhanced autophagic activities via AMPK/ULK1 pathway, causing autophagy-mediated AR degradation and inhibition of AR nuclear translocation. This subsequently reduced AR signals in CaP and inhibited AR-driven cell migration/invasion. Furthermore, a positive correlation between PLCɛ and AR signaling activity was also observed in bicalutamide-resistant CaP samples and in AR-antagonist-resistant CaP cell models. PLCɛ depletion resulted in the failure to establish AR-antagonist-resistant CaP cell lines, and hindered the metastatic prowess of already established ones. These findings suggest that PLCɛ-mediated autophagic activity alteration is indispensible for the functionality of AR signaling and for CaP development. Androgen receptor (AR) pathway is critically necessary in the development of CaP. Overactivation of AR signaling promotes cell proliferation, migration, and epithelial to mesenchymal transition (EMT), favoring tumorigenesis, and tumor progression2. Persistent activity of AR signaling has been observed in CaP as well as in castration-resistant prostate cancer (CRPC)4. Therefore, therapeutics targeting the AR signaling have been extensively explored and widely used in clinical practice, but drug resistance keeps emerging. It is urgent to understand how CaP cells “manage” to maintain a high level of AR signals.Prostate cancer (CaP) is one of the most common types of malignancies in males, ranking first in estimated new cancer cases and third in cancer-related deaths among males in the US5. Notably, PLCɛ is unique in relation to other members of PLC family: it receives signaling inputs from both heterotrimeric G proteins and Ras/Rho small GTPases. Moreover, it is able to mediate sustained signaling via the generation of DAG in response to ligands that activate receptors coupled to Rho/Gα12/136. Owing to its interaction with Ras family proteins, PLCɛ is generally deemed a tumor promoter. Indeed, a large number of studies have revealed the promoting role of PLCɛ in tumor development in a variety of cancer types10. Our previous study found that PLCɛ silencing reduced AR protein expression in CaP cells11. This urges us to explore whether PLCɛ is essential in preserving AR proteins in CaP.Phospholipase C epsilon (PLCɛ) is a member of the PLC family of enzymes. All PLC enzymes possess the enzymatic activity of catalyzing hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to generate two second messengers: inositol 1,4,5-trisphosphate (IP3), a regulator of intracellular Ca2+ level, and diacylglycerol (DAG), an activator of protein kinase C (PKC) isoforms12. Recent studies have demonstrated that autophagy is also activated at basal levels and constantly programmed by numerous cellular signals, resulting in selective clearance of specific cargos13. Selective autophagy has now been reported to regulate the protein degradation of various molecules in cancer14.Autophagy is a highly conserved process of protein degradation. Initially, autophagy was considered a process of nonspecific proteolysis to provide cells with energy or molecular materials when encountering stress conditionsIn this study, we probed the relationship between PLCɛ and AR signaling in CaP. We demonstrated that PLCɛ could regulate AR protein degradation and AR nuclear translocation, both of which were achieved through AMPK/ULK1-mediated selective autophagy. Moreover, PLCɛ/AR signal axis was associated with the metastatic prowess of CaP and played a critical role in AR-antagonists resistance. Our data suggest that targeting this pathway is a potential treatment strategy for CaP and CRPC.Clinical samples were collected from patients in the Department of Urology of the First Affiliated Hospital of CQMU between July 2014 and January 2018. All samples were reevaluated by pathologists in CQMU  were administered 48 h after transfection . Reverse transcription was performed using the Prime Script RT reagent kit  according to the manufacturer’s protocols. Real-time PCR was performed using the SYBR Premix Ex Taq™ II kit . The levels of mRNA were calculated using the comparative 211. The intensity level of each protein band was quantified using Image-Pro plus 6.0. Antibody information see STable Western blot assay was performed as previously describedCells were cultured on sterile glass coverslips and subjected to different treatments. After indicated time, cells were fixed in 4% paraformaldehyde, treated with 0.1% Triton X-100, and blocked with 5% normal goat serum. Then, cells were ringed with PBS and incubated overnight at 4 °C in dilution buffer containing primary antibodies. After three times of washes with PBS, cells were incubated with an appropriate fluorochrome-conjugated secondary antibody for 1 h and subsequently stained with DAPI/4’,6-diamidino-2-phenylindole  for 15 min at 37 °C in the dark, followed by inspection using fluorescence microscopy . Antibody information and experimental steps in detail see STable Cells were harvested and washed twice with ice-cold PBS buffer. Immunoprecipitation was performed using the Pierce™ Co-Immunoprecipitation Kit  according to the manufacturer’s protocols. The inputs and the proteins bound to the resin were analyzed by western blot. Antibody information see STable 15, pFKBP51(-3)-LUC (FKBP51-1)16, pFKBP51(12)-LUC (FKBP51-2)17 , and pCMVβ encoding β-galactosidase are purified from E. coli JM109 strain cultures using QIAGEN Plasmid Maxi Kit . Transfection of LNCaP and VCaP were carried out using jetPEITM transfection reagent according to manufacturer’s protocols . Reporter gene assay was performed using TransDetect® luciferase reporter assay kit  and Luciferase assay system . The β-galactosidase-normalized LUC activities of each groups were documented. Experiments were performed at least three times.Reporter plasmids pTATA-LUC, pPSA5.8-LUCPLCɛ-silenced cells were seeded in 6 well plates and grown to 80% confluence. The “wound” was then created by scratching with a sterile 200 μL pipette and DHT (1 nM) or enzalutamide (20 μM) was added into the media. Images of wounds were captured at 0 and 24 h after scratching. Experiments were performed at least three times.4 cells/well) were added to the upper well of the chamber, which contains a membrane filter (8 μm) and is inserted in 24-well plates , and cultured with DHT (1 nM) or enzalutamide (20 μM) for 24 hours. For the invasion assay, diluted Matrigel  was used (2.5 × 104 cells/well). Cells that pass through the membrane were stained with crystal violet and counted with a light microscope. Experiments were performed at least three times.PLCɛ-silenced cells (1.5 × 103 cells/well) for 12 h (day 0) and then subjected to various treatment. At the indicated time points, each well was added with 10 μL CCK-8 reagent solution , and incubated for 2 h. Optical density was determined using a microplate reader at the absorbance of 450 nm. Each group has five replicate wells and experiments were performed at least four times.Cells were seeded in 96-well plates  for 24 h. Cells were collected and fixed with 75% ethanol. The cell cycle distribution was analyzed using flow cytometry. Experiments were performed at least three times.PLCɛ-silenced cells were seeded in 6 well plates (650 cells/well) and treated with Enzalutamide (2 μM). After 12 days of culture, adherent cells were washed twice with PBS, fixed with 4% paraformaldehyde (20 min) and stained with crystal violet solution (15 min). The colony number was counted using a light microscope. Colony-forming efficiency was calculated. (colony number/650) × %. Experiments were performed at least three times.t-test, Chi-square test, one-way ANOVA, Pearson’s correlation coefficient and Cohen’s kappa coefficient were used to evaluate the significant associations among categorical variables. The SPSS (Version 13.0) software and GraphPad (Prism 5) were used for statistical analyses. Data returning a value of P < 0.05 were considered statistically significant.All data were derived from at least three independent experiments . Student’s RT-PCR and western blot were performed on 32 CaP samples and 12 BPH samples to investigate PLCɛ expressions. The results showed that PLCɛ transcripts Fig.  and protP < 0.001)   shift in marker expression after PLCɛ depletion. As shown in Fig. 20, whereas calpain-mediated AR degradation under control of intracellular Ca2+ level is also reported to be less common in prostate cancer21. However, blocking neither ubiquitin-proteasome degradation pathway (MG132)  Fig.  or calpa32) Fig.  could no32) Fig. , suggest32) Fig. , and res32) Fig. . These r12. We observed a supra-additive effect of LC3-II level in cells simultaneously treated with PLCɛ silencing and bafilomycin A1 (autophagosome-lysosome inhibition), which suggests an enhanced formation of autophagosomes after PLCɛ silencing  in LNCaP, and it reached the apex at 36 h. Starvation groups served as positive control Fig. . Westerning Fig. . Moreoveing Fig. . A dramaing Fig. , which iity Fig. . This de22. Further study showed that PLCɛ knockdown could activate AMPK, evidenced by phosphorylation of AMPKα at threonine 172 and dephosphorylation at serine 487 , and accordingly we established two groups of LNCaP derivatives representing two types of resistances. In castration-resistant groups , although AR were gradually increased, PLCɛ remained unchanged  of bicalutamide barely affected wild-type cells, but substantially inhibited viability of PLCɛ-silenced cells Fig. . In addip = 0.003) and even higher in Bica-R-CaP . PLCɛ increase was also associated with hK2 increase in both HN-CaP  and Bica-R-CaP  (Supplementary Table We next examined PLCɛ/AR axis and CaP metastatic property in samples from patients receiving no ADT (HN-CaP) and patients who were bicalutamide-resistant (Bica-R-CaP), respectively. Data from immunohistochemical analysis upon 18 Bica-R-CaP and 26 HN-CaP samples showed that PLCɛ, AR, hK2, Vimentin and N-cadherin were increased in Bica-R-CaP compared to HN-CaP Fig. . No diff26, and this concept perfectly portrays the relationship between AR signaling and CaP. Recent studies have revealed that AR signaling plays a critical role throughout all the stages of CaP, including carcinogenesis, primary cancer, CRPC and even CRPC resistant to AR antagonists4. This addiction indicates that certain ‘fail-safe’ mechanisms must have been established in CaP in order to sustain a functional AR signaling. Because of the importance of AR pathway in normal cellular physiology, an intrinsic capability of cells to maintain AR signaling is already existent, particularly in males who only have one single copy of AR gene and in prostate which is highly dependent on the AR axis to function properly. These biological mechanisms could be readily manipulated and pathologically strengthened by CaP cells to over-activate AR pathway. In this study, we disclosed a new mechanism underlying persistent AR signaling in CaP: Increased PLCɛ level inhibits AR protein degradation and facilitates AR nuclear translocation, resulting in enhanced AR signaling activity and cellular transformation toward a pro-metastatic phenotype.The term “oncogene addiction” describes a phenomenon that a single gene functions as the persistent driver of a certain malignancy3. Similarly, high PLCɛ level is reported to facilitate tumor development and metastasis in a number of cancer types as well9. In the present work, we found that increased PLCɛ expression was also a key contributor to the metastatic prowess of CaP. Moreover, PLCɛ expression and AR signaling were closely associated in CaP samples. Certain EMT phenotypes such as impaired E-cadherin expression or aberrant induction of Vimentin and N-cadherin are related with enhanced tumor metastasis27. Our data from clinical samples revealed good correlations among PLCɛ protein, AR signaling, Vimentin and N-cadherin in both hormone-naive CaP tissues and CRPC tissues. These results were further verified in cell study. We discovered that PLCɛ depletion was sufficient to suppress AR pathway and AR-driven cell migration/invasion in different CaP cell lines. PLCɛ could induce the alterations of E-cadherin, Vimentin and N-cadherin toward a phenotype favoring tumor metastasis. These lines of evidence strongly support the indispensable role of PLCɛ in promoting AR pathway activity and AR-mediated CaP metastasis. Our data from AR-antagonist resistant CaP also support PLCɛ sustaining AR signaling. We found that Bica-t/Enza-t had almost unaltered PLCɛ and AR protein level, even the synthesis of AR protein seemed to be up-regulated (evidenced by increased mRNA level). Based on our data that PLCɛ protects AR protein from degradation, it is rational to hypothesized that, without concomitant high PLCɛ level, the net protein of AR is unlikely to be elevated accordingly. Whereas in fully bicalutamide/enzalutamide-resistant cells, PLCɛ and AR were simultaneously increased. Constant up-regulation of AR protein is the impetus of CRPC. Therefore, PLCɛ elevation could be playing both a triggering role and a sustaining role in AR-antagonist resistance.AR pathway is frequently implicated in CaP tumorigenesis and metastasis13. AR protein has been reported to be the cargos of autophagic degradation31. In our study, AR protein was also found to be degraded via an autophagy-dependent pathway when PLCɛ was silenced. Moreover, our data showing that PLCɛ depletion initiated p62-mediated AR degradation support the involvement of selective autophagy, because it is generally recognized that selective autophagy is regulated by autophagy adaptors/receptors, such as p62, NBR1, OPTN, NDP52, etc. Recent studies illustrate that p62 can sometimes be degraded through non-selective autophagy as well24. Therefore, we further investigated another autophagy receptor-- NBR1. The results revealed that p62 and NBR1 could function cooperatively in the regulation of AR degradation, providing one more evidence for selective autophagy. Notably, we observed that PLCɛ depletion induced the formation and accumulation of p62-AR and NBR1-AR complex in the cytoplasm. This is in accordance with previous studies reporting that p62 and ubiquitinated proteins often exert an anchoring effect on each other when the intracellular ubiquitin level is increased, which in turn causes the decreased cytoplasmic/nuclear shuttling of p62 and targeted proteins32. Therefore, owing to the cytosolic accumulation of AR complexes, PLCɛ depletion could dampen AR signaling via inhibition of nuclear translocation, even when the lysosomal degradation is blocked or lagged. This suggests that PLCɛ-mediated suppression of autophagic activity, in addition to its role in maintaining a high level of the AR protein, could facilitate AR translocation by preventing the anchoring effect, consequently preserving the functionality of AR signaling in CaP cells.Autophagy is an important way of protein degradation. Studies of recent years have been highlighting the function of selective autophagy, which induces selective clearance of specific cargos. In that case, autophagy becomes a part of the intricate networks of cellular signals instead of simply a process in reaction to stress, and its effect is largely dependent on the specific proteins that are eliminated3, IP3 receptor and PKC34. These reports are consistent with our data that PLCɛ could suppress autophagy by activating PKC pathway. Furthermore, we demonstrated that AMPK activation is essential for the enhancement of autophagic activity after PLCɛ depletion. Although PKC pathway and AMPK pathway are commonly considered calcium-regulated, we found that PLCɛ depletion could regulate AMPK pathway in a PKC-dependent but calcium-independent manner. Previous studies have shown that PKC is able to reduce AMPK activity by phosphorylating serine 487, a site that blocks threonine 172 phosphorylation and functions as a negative-feedback regulation of AMPK activation35. Therefore, it is logical to infer that PLCɛ depletion could impair the phosphorylation of serine 487 in AMPK via PKC inhibition, resulting in threonine 172 phosphorylation and subsequent AMPK activation due to decreased negative feedback suppression. This could happen while not needing a dramatic change of intracellular Ca2+ level. This mechanism is also in line with the hypothesis that PLCɛ is associated with basal autophagy. Because PLCɛ is the only member of PLC isoenzymes that is capable of triggering sustained and steady cellular signal change, while other members always mediate sudden and intensive calcium flow, which tend to induce endoplasmic reticulum stress, a process that is highly associated with non-selective autophagy.In literature, ‘PLC-related’ molecules have been implicated as autophagy inhibitors, such as IPCollectively, our study demonstrates that increased PLCɛ in CaP inhibits basal autophagic activity to a certain level of equilibrium, which is sufficient to maintain a high level of AR function, hence the increased metastatic prowess and drug resistance in CaP. Disruption of this equilibrium can result in weakened AR signaling. PLCɛ up-regulation could be both a triggering factor and a supporting element during the entire process of CaP development. Further evaluations of PLCɛ/AR axis are warranted.Supplemental Figure 1Supplemental Figure 2Supplemental Figure 3Supplemental Figure 4Supplementary figure legendsSupplemental tables


The effects of administration on the tested parameters were checked after 30 and 90 days of administration and additionally, 90 days after the end of 90 day administration. Results: Zn alone leads to an increase in serum testosterone concentrations, while the protein expression of AR in both parts of the prostate increases. Combined administration of Zn and Se eliminates the effect of Zn, which may suggest that these two elements act antagonistically. Se supplementation alone results in the same level of AR protein expression in administration and 90 days after administration periods. Conclusion: This paper presents the first report of the influence of Zn and/or Se supplementation on the protein expression of AR in the prostate. Our findings seem to indicate that simultaneous supplementation of both elements may be ineffective.Background: Zinc (Zn) and selenium (Se) play a well-documented role in cancer prevention , and their combined supplementation is often given as a recommended prophylactic agent. The aim of the study was to determine the influence of Zn and/or Se supplementation on the androgen receptor (AR) in the prostate lobes and the serum selected hormone concentrations; a hitherto unresearched topic. Methods: Male rats ( Recent years have seen a worldwide increase in various forms of cancer, including prostate cancer (PCa). Both zinc (Zn) and selenium (Se) are well documented to counter oxidative stress, thus protecting DNA from the activity of reactive oxygen species . It is nProstate gland is an organ regulated by androgens, mainly testosterone (T) and dihydrotestosterone (DHT). The role of androgens is essential to understand when considering the reasons and treatment of PCa ,6, the sStudies on both animals and prostate cell lines have shown that Zn is an inhibitor of two key enzymes involved in T metabolism: 5α-reductase, which converts T to DHT, and aromatase, which is responsible for the formation of estradiol (E2) ,13. TherUnfortunately, there is little data about the simultaneous influence of both elements on the functioning of the endocrinal system, especially in men. In addition, few studies in the literature have examined the interaction of Zn and Se ,20. HoweHence, the present in vivo animal study was performed probably for the first time to more precisely determine the effect of long-term supplementation with Zn and Se, either separately or combined, on the protein expression of AR in the prostate gland: AR is known to play a key role in maintaining the proper functioning of the prostate . The stuZinc gluconate (puriss grade) was purchased from Alfa Aesar GmbH & Co KG . Selenomethionine (puriss grade) was obtained from Sigma-Aldrich CHEMIE GmbH .-n = 21);zinc gluconate in conversion to Zn: 5 mg Zn/kg body weight (b.w.)/day (-n = 21);selenomethionine in conversion to Se: 2.8 µg Se/kg b.w./day (-n = 21).zinc gluconate and selenomethionine combined at the above doses  from the breeding colony of the Medical University of Lodz, subjected to a two-week acclimatization period. The rats were housed under controlled temperature (22 ± 1 °C), relative humidity (45–55%), and a constant 12 h light/dark cycle. The animals were fed “Murigran”—a standard pelletized diet . Food and water were supplied ad libitum throughout the study. The animals were divided randomly into three research groups receiving the following:50 ≥ 5000 mg/kg b.w., rat, oral route [50 (1/100 LD50 in conversion to Zn).The applied doses were the same as in our previous study concerning Zn and Se bioavailability in the rat prostate . In the al route ), the don = 21) consisting of animals receiving food and water ad libitum. Throughout both administration and postadministration periods, the behavior and look of all animals were observed.Zn gluconate and selenomethionine were dissolved in water and administered directly via intragastric gavage (per os) in the maximal volume of 0.5 mL per rat. Each experimental group comprised a control group  from the start of the administration of the compounds, seven animals from each group were sacrificed, respectively, by intracardiac puncture under light carbon dioxide narcosis. Samples of whole blood  and prostate gland, divided into ventral (V) and dorso-lateral (DL) parts, were collected. The blood was centrifuged, and the serum was frozen for further analysis. All tissues samples were fixed in 4% buffered formalin, and paraffin blocks were prepared.The experiments were performed with the permission of the Local Ethical Committee for Experimentation on Animals (Resolutions No 43/LB 479/2009).Immunohistochemical staining was carried out on formalin-fixed paraffin-embedded rat sections according to a standard method. Briefly, 4 µm tissue sections were deparaffinized in xylene and rehydrated through a graded alcohol series. The samples were heated in a microwave oven in target retrieval solution pH 9.0  for 30 min to retrieve antigens. Endogenous peroxidase was quenched with 3% hydrogen peroxide for 10 min. The sections were washed with TBS and incubated for 18 h with rabbit polyclonal primary antibody against androgen receptor . After washing, EnVision-HRP detection system  was used. 3,3′-diaminobenzidine was used as the chromogen. After counterstaining with Mayer’s hematoxylin, the slides were washed, dehydrated, cleared in xylene, and coverslipped. The primary antibody was replaced with antibody diluent for negative controls.All sections were independently examined and scored by two pathomorphologists, who were blinded to the clinical information. The protein expression of AR  was scored by summing the mean signal intensity and the percentage of positively stained cells using Allred scale . The AR The determination of LH, FSH, and E2 in serum were performed with an Immulite 2000 automatic chemiluminescent immunoenzymatic analyzer (Siemens) using LH-PIL2KLH-19, FSH-PIL2KFS-13, and ESTRADIOL-PIL2KE2-24, respectively. All IMMULITE kits  are based on a solid-phase competitive immunoassay labeled with a chemiluminescent enzyme.Serum T quantification was performed with the Elecsys Testosterone II test using the ECLIA electrochemiluminescence method on a Roche Elecsys 2010 Analyzer, while the quantity of DHT in serum was determined using an ELISA kit  on a Synergy ABS/HTR multisectional ELISA reader . Measurement ranges and sensitivity for the determination of the above hormones in rat serum are shown in Zn was determined by flame atomic absorption spectrometry  after mineralization. A calibration curve was obtained using a range of concentrations of a standard solution of metals . The graph obtained was linear in the concentration range of 0.05–1.5 μg/mL, and the equation of the curve was as follows: The limit of detection, calculated as concentrations corresponding with an absorption value equal to a three-fold standard deviation of the signal for the lowest concentration, was 0.031 μg/mL. Se determinations was performed on a Hitachi F-4500 spectrofluorometer according to the method described by Danch and Drozdz (1996) . The limThe intra-laboratory quality control was based on certified standard lyophilized blood—Seronorm Whole blood L−1 . The reference material contained Zn and Se at concentrations of 4.6 μg/mL and 0.069 μg/mL , respectively. The relative standard deviations obtained in the reference material determinations were 2.3% for Zn and 5.5% for Se.p-value of less than 0.05 were considered statistically significant.Statistica 10.0  was used for all statistical analyses. Bartlett’s test was used to confirm homogeneity of variance, following which Tukey’s test was used to determine the significance of the differences for the selected parameters. The statistical analysis of the immunoreactivity of AR in prostate lobes was limited to the TS and was performed based on two-factor analysis of variance. If the assumption about the homogeneity of variance was not fulfilled (no transformations stabilizing variance were applied), the resistance of F-Snedecor’s statistics was used, because the experimental system was completely balanced (in every time point and in every group there are seven observations). During the analysis, simple effects, that is, differences in rows and columns of the presented tables were evaluated. Differences with a During the whole experiment (administration and observation periods), no mortality and changes in animals behavior, look, and feed and water intake (data not shown) were noted. As shown in AR immunoreactivity did not differ significantly from control values at any measurement point, that is, after 30 or 90 days of Zn and Se supplementation, nor after the 90 day postadministration period . MoreoveAs shown in The calculated DHT/T ratio  indirectOne of the main factors regulating the androgen action in the prostate is AR. Its strongest ligand is DHT, and after binding, the two form an active DHT–AR complex. This complex penetrates into the cell nucleus, where it plays a crucial role both in the normal growth/development of the prostate gland, and in the progression of PCa ,28. TherProstate cell proliferation is regulated by inter alia T and DHT on the one hand, and by Zrt/Irt-related protein 1 (ZIP1) transporter/Zn/citrate regulation on the other . It has Our present study based on rats subjected to Zn and Se supplementation, both separately and in combination, found a significant increase in AR-positive immunoreactivity animals, but only in the groups receiving Zn alone. However, there is very little data on how Zn influences the expression/concentration of AR. Zn-deficient rats display decreased concentrations of AR in prostatic and hepatic cells, and decreased levels of DHT, T and LH in serum ,33. Alth2, decreased AR protein expression was revealed. Interestingly, administration of this dose to mice without tumors did not result in such an effect. However, decreased AR protein expression was observed in mice without tumors after doubled dose of Zn, that is, 20 mg/kg, which turned out to be lethal to several mice with tumors [50) of nontoxic compound . Conversely, To et al. used more toxic compound  in doses toxic to animals—half and almost LD50 [However, the actions of Zn on AR protein expression seem to be not so obvious. Recently, an in vitro study found Zn to suppress proliferation in AR-retaining PCa cells, but not in AR-deficient PCa cells. Zn not only suppressed AR expression in androgen-deprived conditions, but also down-regulated androgen-stimulated AR expression. On the other hand, in vivo studies provided more ambiguous results. In mice with implanted tumors (with TRAMP-C2 cells) after administration of 10 mg/kg ZnClh tumors . The aboost LD50 . As therost LD50  proved tost LD50 .In the case of Se, it was shown that this element is able to significantly reduce the transcription of AR and expression of prostate-specific agent (PSA). This down-regulation of AR signaling by Se serves as an important basis for the chemoprevention of PCa; however, its molecular mechanism remains unclear. The activation of AR requires binding to its ligand, translocation to the nucleus, and interaction with coregulators, including coactivators and corepressors, in the AR target genes . Chun etSe has been found to reduce AR expression in rats supplemented with selenocysteine and isoflavones . In the Our findings confirm those of our previous work, indicating that simultaneous administration of Zn with Se may be not beneficial. Se affects the bioavailability of Zn to the prostate  and, as


Physiotherapy-led home rehabilitation after breast cancer surgery can protect against the development of upper limb dysfunction and other disabling consequences of surgery. A variety of barriers can limit physical rehabilitation outcomes, and patients may benefit from more support during this time. Mobile health  systems can assist patients during rehabilitation by providing exercise support, biofeedback, and information. Before designing mHealth systems for a specific population, developers must first engage with users to understand their experiences and needs.The aims of this study were to explore patients’ rehabilitation experiences and unmet needs during home rehabilitation after breast cancer surgery and to understand their experiences of mHealth technology and the requirements they desire from an mHealth system.This was the first stage of a user-centered design process for an mHealth system. We interviewed 10 breast cancer survivors under the two main topics of “Rehabilitation” and “Technology” and performed a thematic analysis on the interview data.Discussions regarding rehabilitation focused on the acute and long-term consequences of surgery; unmet needs and lack of support; self-driven rehabilitation; and visions for high-quality rehabilitation. Regarding technology, participants reported a lack of mHealth options for this clinical context and using non-cancer–specific applications and wearables. Participants requested an mHealth tool from a reliable source that provides exercise support.There are unmet needs surrounding access to physiotherapy, information, and support during home rehabilitation after breast cancer surgery that could be addressed with an mHealth system. Breast cancer survivors are open to using an mHealth system and require that it comes from a reliable source and focuses on supporting exercise performance. Breast cancer accounts for 1 in 4 cancer diagnoses in women in Europe . The advUpper limb dysfunction is a prevalent, persistent, and disabling consequence of breast cancer treatment, which is reported by up to 62% of women 6 years after treatment ,7,8. SymThe Global Observatory for electronic health  defines mobile health  as “medical and public health practice supported by mobile devices…and other wireless devices” . MHealthTo design an effective mHealth system, developers must first understand the experiences and unmet needs of the target population ,46. UnmeThe aim of this study was therefore to explore the experiences and unmet needs of women during home rehabilitation following surgery for breast cancer and to gather survivors’ perspectives on and requirements from mHealth technology for postoperative breast cancer rehabilitation.A qualitative research study design using semistructured interviews with breast cancer survivors was undertaken to explore their experiences and needs regarding rehabilitation and technology. This methodology was chosen to allow a detailed description of experiences and opinions, which can produce rich, detailed, and complex data, even when collected from a small number of respondents ,57. The Participants for this study were women with a history of breast cancer living in the wider Dublin area, who were recruited from a sports club whose members are all survivors of breast cancer. This sample was chosen to ensure the data collected represented the general perspectives of breast cancer survivors and was not influenced by where they received cancer treatment. It was important to capture how consequences of surgery and postoperative rehabilitation may have impacted participants in the medium- and long-term . TherefoA literature review of unmet needs and technology in breast cancer rehabilitation, as discussed in the introduction to this study, informed the development of a flexible, semistructured interview guide, which is presented in Topic 1: Rehabilitation experiences1.1 Please talk me through your process of rehabilitation, from the time of the surgery until now.1.2 Can you describe your awareness of how the operation would impact you, both short and long term?1.3 Tell me about a time throughout the rehabilitation process that you found difficult.1.4 What were your expectations of the physiotherapy care you would receive? How did they match the reality?1.5 How well-equipped did you feel to do the postoperative rehabilitation at home? How confident did you feel doing the exercises?1.6 Were you given exercise instructions and information? How?1.7 Is there anything that might have helped you during rehabilitation after the surgery?Topic 2: Digital technologies in breast cancer rehabilitation2.1 Did you use any modern technologies to help you during your rehabilitation?2.2 Do you use any technologies for your general health and well-being?2.3 When discussing your experiences of physiotherapy, you mentioned [insert problem]. If there was a piece of technology to help you with this issue, what would it do?The interviews were transcribed verbatim and anonymized transcripts were imported into the qualitative data analysis management software NVivo . The lead researcher applied a thematic analysis with a semantic, mixed inductive and deductive approach to analyze the data, following the process outlined by Braun and Clarke . After aIn total, 10 women participated in the semistructured interviews. Participant characteristics are summarized in Under the topic of “rehabilitation experiences,” four main themes were identified: acute and long-term consequences of surgery; unmet needs and lack of support; self-driven rehabilitation; and visions for high quality rehabilitation.Participants experienced short-term and long-term consequences of the surgery, which impacted their function and quality of life. Long-term upper limb dysfunction was described by Participant (P) 6: “This arm is definitely still a bit weaker. If I go to pick things up, picking up groceries or whatever, I don't have the same strength in this arm.” As well as physical impairments of stiffness and weakness, participants experienced feelings of overwhelm, fear, and anxiety. The prospect of developing side effects of surgery could be worrying; P8 stated, “I was terrified of lymphedema.” Most participants were surprised by how they felt emotionally after the surgery, and report not being prepared for what to expect:I had no idea that there would be such life changing things going on in my body. You kind of think, you’ll have your surgery…and then life will go back to normal, but it doesn't.P6Secondary musculoskeletal complications developed for several participants, such as frozen shoulder, bursitis, and postural problems. P2 reported how she could not move her upper limb into the required position to receive radiotherapy:The first time around, I developed a frozen shoulder. This was very evident when I was going for my radiotherapy, I couldn’t do it.Participants were not always aware of the importance of physiotherapy and rehabilitation and several felt that there was a lack of emphasis on rehabilitation throughout their hospital stay:I mean I was told, but it wasn't heavily enforced and it wasn't really stressed. I don't feel that it was high priority.P5As demonstrated in Your physiotherapist comes to you in the ward, goes through the exercises with you, but other than that, there isn’t any real physio support, you do the exercises yourself.This was felt to be insufficient, especially if concerns arose after this assessment, as P6 expressed: “I didn't really feel like I had a physiotherapist that I could ring up and talk to.” There were also challenges with how the HEP was provided. The sheet of paper containing descriptions of the exercises was carefully minded by some participants, but others, including P4, didn’t take the same approach: “you don't know where you have left it half the time.” Several participants felt that the physiotherapy assessment took place too soon after the surgery. P10 explained: “I was probably still on morphine, so I probably wasn’t paying enough attention.”As a result of the above unmet needs, P1 reported feeling uncertain about how to perform the exercises:I didn't even know whether I was doing it right or not, you know, sitting on the bed supposed to be doing this or this, or whatever, and I didn't bother then.P1Additionally, there were difficulties with the postoperative self-management advice that they had received from the hospital. The information was “very unstructured and informal” [P5] and two participants felt it was too focused on what not to do: “don’t do this, don’t do this; it wasn’t reassuring to me in any sense” [P4]. Upon discharge from hospital, treatment side effects restricted participants’ abilities to do the HEP; they reported feeling “quite weakened” from chemotherapy [P8], that radiotherapy “affected my skin and it made the stretching very sore” [P10] and that fatigue was “the biggest impediment to doing (the HEP)” [P4]. Financial barriers prevented two participants from arranging private physiotherapy appointments. Time and scheduling were additional barriers to home rehabilitation, as was a fear of movement:I think a woman is naturally terrified of it and afraid that those stretch things are going to, you know, hurt, so, I think there is just a barrier.P8At a time when professional advice and support was much needed, participants felt alone and unsupported:Certainly, post-surgery you feel quite isolated, whereas, in chemo you are meeting oncology nurses all the time, but post-surgery…you are on your own.P8Having little contact with health care professionals was especially difficult when participants experienced unexpected side effects, as P8 reported: “I found it quite shocking and terrifying…essentially I had nobody to turn to.”A desire and motivation to recover was expressed by all participants. Despite the unmet rehabilitation needs and barriers to home rehabilitation, 6 participants expressed they had had a strong internal motivation which led them to persevere with the HEP. P4 asserted: “if you say to me that this is what you need to do to get better, I'll do it.” Another motivating factor was personal experience of upper limb dysfunction:There is no question about it. I felt if I didn't stretch my arm…it would be very stiff and, and very, very sore.P6When participants did have follow-up physiotherapy appointments, this sense of accountability was an external motivating factor. Participants were proactive in fulfilling their own rehabilitation needs; for example, they sourced information, support and advice as required:It's hard to do nothing, therefore whatever is at hand, you will look up, research and investigate or speak to…people like to feel like they are being pro-active about their own recovery.P5There was a strong role for peer support, and speaking to other individuals with breast cancer was an effective way to gain knowledge, advice and assistance:It was very rarely involved interacting with professionals, it was almost exclusively peer to peer, asking other women, what to do about lymphoedema, what to do about, when to wear a sleeve?P5The holistic benefits of exercise were mentioned by all participants. Some participants tried new forms of exercise to address surgery-related upper body dysfunction, including yoga, Pilates, and going to the gym:My posture was horrendous, I was just completely rounded, you know, to hide… So now, through the Pilates…my posture is greatly improved.P2Overall, 3 participants reported that they did not engage in exercise prior to surgery, but continued because they saw the benefits of the particular activity. P1 stated that, “There is a whole new me, from being a couch potato all my life, to doing this.” Additionally, 8 participants discussed the social and psychological benefits of exercise, which ranged from having improved confidence and feeling calmer, to having the opportunity to get fresh air and make new friends. Regarding the sports club, participants spoke of finding “a great camaraderie” [P3] and of “meeting people who have been through something similar, you can all relate” [P4]. P10 expressed gratitude for the club and her experience as a member of that community:I also think that was the silver lining to the whole experience ‘cos now I have a whole life around that, and a whole lot of new friends, so that has been brilliant.Discussions of good experiences with health care professionals and recommendations for service improvements provided insights into patient preferences for breast cancer rehabilitation. Participants appreciated proactive care and patient education. P6 described a valuable interaction with her physiotherapist: “she wasn't forceful but she made it clear that if you don't do your exercises you will be left with less movement.” All participants recommended more access to physiotherapy services, especially follow-up assessments after discharge from hospital:I would have liked to, maybe, have gone through the exercises that I was trying to do, check that I was doing them properly.P3Several participants recommended providing patients with more information about what to expect after the surgery, regarding diet, exercise, and breast prostheses. One participant suggested this could be done through a physiotherapist-led preoperative information session, while another requested a postoperative support group:A support group for people, for the after-effects of surgery and everything else, which would include the physiotherapist plus maybe other things as well.P6The experiences of the participants with mHealth technology and the features that participants would like to see in an mHealth tool for breast cancer rehabilitation were the two distinct themes from this portion of the interviews.The digital technologies used by participants for health and rehabilitation are detailed in Technology played two main roles: first, it acted alongside health care professionals, for example to help patients understand information provided by the hospital: “[The terminology] was all new language to me, so when I came home I did google all that” [P1]. Second, it compensated for unmet needs in health care provision, for example to provide exercise advice: “I would do Pilates, I would watch an app to do that” [P6]. Overall, 6 participants used activity monitors, and they perceived them as good motivators to be physically active, providing a sense of achievement when participants reached their goals. Some disadvantages of mHealth were expressed, such as feeling overwhelmed by the volume and content of information: “you can really live in cancer-land” [P6]. Concerns about data privacy were raised: “What happens if [your personal information] somehow gets into the wrong hands?” [P7]. Additionally, it was acknowledged that technology cannot replace the role of the human in health care:As much as you want apps or websites, you also need the human bit, you need them all.P10All participants stated that they would have liked to have tried out an mHealth system during home rehabilitation if such a system had been available. Throughout the discussion, participants suggested features they would like to see in such a system . These fI don't think you would need anybody bald, you wouldn't want, necessarily, that reminder. Maybe not somebody with gorgeous locks, short hair would be fine.P5A desire for both audio and visual exercise biofeedback was expressed by P9: “If there is going to be an audio, having something visual as well, it’s there if I wanted to check, ‘Yeah, I am doing this correctly.’” It was important to many that the system should be provided from the hospital: “if it's through the hospital, then, you know, it's got that legitimacy” [P5]. A record of exercise performance was a popular feature, not only because it would provide feedback to patients, but because it would remotely provide feedback to the physiotherapist as well:I would like to know that…somebody had taken an interest and a follow-up in what I am doing…and then they would be able to intervene if they saw I wasn’t doing it properly.P1Regarding the style of the system, there was a strong preference for a “less is more” approach, with concise information that is specific to the patient’s own situation:Your concentration is shot after cancer when you are having treatment…You need to ask a question and get an answer.P2There was a preference toward content that is positive and life-affirming, delivered with an encouraging and reassuring tone:I think maybe some encouragement because it's such a difficult time, not just 'do this,' but 'how are you finding it?' You know, 'how's your day?'P5This study presented detailed insights into the physical rehabilitation experiences and unmet needs of 10 women after surgery for breast cancer. It has highlighted the barriers to home rehabilitation and presents participants’ visions for high-quality rehabilitation. Through this, the role of an mHealth system with biofeedback features to reduce barriers and improve quality of care can be seen. Additionally, this study explored patient preferences and requirements for such a system. Under the topic of “rehabilitation,” themes covering the experiences of acute and long-term consequences of surgery; participants’ unmet needs and feelings of lack of support; the motivation and need to take the lead in one’s own rehabilitation; and participants’ visions for high-quality rehabilitation were identified. Under the “technology” topic, the sample’s experience with digital technology and mHealth were outlined, and key requirements for an mHealth home rehabilitation support system were identified.The findings regarding the acute and long-term consequences of breast cancer surgery were wholly in line with previous research. Throughout the “rehabilitation” section of the interview, participants reported a desire for increased access to physiotherapy services, both before and after surgery, and at later stages of treatment. Alongside clinical practice guidelines, other research with patient populations also supports these recommendations for routine preoperative and follow-up physiotherapy services ,65,66. MThe unmet needs described by participants in this study include a need to be educated in advance about the impact of the surgery and the importance of rehabilitation; a need for more physiotherapy services; a need for timely and clearly presented information regarding postoperative care; a need for more support on discharge and the ability to contact the appropriate health care professional when concerns arise. These unmet needs highlight areas in which improvements to care could be made, either through traditional service development methods or through the use of technology. An mHealth system, such as an app, could assist in these improvements by being a portable source of postoperative support by providing information, prescribed exercises, and a method of remote contact with a physiotherapist. Improved clinical outcomes have been demonstrated by combining a standard postoperative HEP with an mHealth app and biofeedback technology ,72. AddiAn important finding of this study was the participants’ high levels of motivation to recover after surgery. Although adherence to HEPs has been found to be low in many populations ,74,75, iNo participants used mHealth technology for their postoperative rehabilitation. This reflects the lack of digital technology specifically designed for this period of breast cancer care and a lack of awareness of available apps, rather than a lack of desire to use mHealth, as all participants stated they would have used a suitable mHealth tool if it was available. Older age was a limiting factor for 1 participant for use of digital technology in daily life, but it was not a barrier to her desire to use mHealth. This is consistent with research by Abelson et al  which foOverall, participants saw value in an mHealth tool to support patients during home rehabilitation after breast cancer surgery. They requested a system which focuses on providing exercise information, feedback on progress, and guidance in an audiovisual manner. This is echoed in a 2019 exploration of breast cancer survivors’ preferences for mHealth physical activity interventions by Phillips et al , who fouFinally, it was important to participants that an mHealth system would be provided by the hospital or a health care provider, and that it would include a remote connection with the physiotherapist. Chandra et al  found thBreast cancer is a disease which affects 1 in 8 women across their lifetime, and therefore the experiences of women with breast cancer are incredibly varied and impossible to capture in a single study. The themes presented strongly across the participants and therefore appear to be highly representative of the population . SpecifiAn mHealth intervention has the potential to support patients during rehabilitation after breast cancer surgery. Understanding unmet needs of users and their perspectives on mHealth at an early phase of development enables the co-design of a user-friendly and relevant system. A thematic analysis of interviews with breast cancer survivors reflecting on their rehabilitation experiences demonstrates that, while participants were motivated to exercise, a lack of support on discharge and a lack of emphasis on rehabilitation led to uncertainty during rehabilitation and feelings of being alone and unsupported. Participants stated they would have welcomed an mHealth support tool that was reliable, provided by the hospital, and focused on rehabilitation support. The results of this analysis support a drive for improvements in breast cancer rehabilitation, which can be led by technological and nontechnological means. The impact of rehabilitation promotion and facilitation of access to postoperative physiotherapy services on rehabilitation outcomes should be investigated. These results provide motivation and rationale to develop an mHealth system with biofeedback that strives to improve outcomes for these often-undertreated consequences of breast cancer treatment.


Gut-derived satiety hormones provide negative feedback to suppress food intake and maintain metabolic function in peripheral tissues. Despite the wealth of knowledge of the systemic effects of these hormones, very little is known concerning the mechanisms by which nutrients, such as dietary fats, can promote the expression of genes involved in L-cell hormone production. We have tested the role of various dietary fats and found that after hydrolysis into free fatty acids (FFA’s), there is a differential response in the extent to which they induce PYY gene and protein production. The effect of FFA’s also seems to relate to triglyceride (TG) re-esterification rate, with MUFA re-esterifying faster with lower PYY production. We have also found that there are differences in potency of FFA’s based on their desaturation patterns in vitro. The potency effect of FFA’s is influenced by the rate of TG re-esterification, such that the longer FFA’s are in contact with L-cells, the more PYY they produce. We found that chronic consumption of high-fat diets enables the small intestine to re-esterify FFA’s into TG faster and earlier which resulted in a blunted postprandial PYY response. Lastly, we found that FFA’s induce X-box-binding protein-1 activation (Xbp1s) in L-cells and that adenoviral delivery of Xbp1s was sufficient to induce PYY gene expression. Taken together, the present work indicates that dietary fat can induce satiety, in part, prior to re-esterification. Chronic high-fat diet consumption increases the rate of re-esterification which diminishes satiety and may lead to increased food intake. Targeting intestinal TG synthesis may prove beneficial in restoring obesity-associated reductions in postprandial satiety. Peptide tyrosine tyrosine (PYY) is a peptide hormone produced and secreted by enteroendocrine L-cells of the ileum and colon in response to nutrient supply. When released from L-cells, PYY reduces food intake through inhibitory signaling via neuronal receptors in the arcuate nucleus of the hypothalamus. In the pancreas, PYY improves insulin release and reduces exocrine pancreatic secretions. In the gut, PYY slows gastric motility, slows absorption of macro- and micronutrients, and produces systemic feelings of fullness ,2. A majWhat is known to-date regarding the mechanism of PYY production  is essentially limited to FFA-mediated cell signaling  or extraNutrient–gene interaction is a critical aspect of maintaining physiological homeostasis. It enables the organism to adapt to acute and chronic changes in metabolic flux by altering transcriptional pathways responsible for assimilating nutrients. Dietary fat has been shown to induce endoplasmic reticulum (ER) stress, with varying degrees of activation based on free fatty acid (FFA) composition ,16,17. EThe purpose of the current study was to begin to define the nutrigenetic mechanisms by which FFA’s induce PYY gene and protein expression to better understand the physiology and biochemistry of diet-gene interactions in L-cells. We found that that the longer FFA’s are in contact with intestinal L-cells, the more PYY is produced. Additionally, we hypothesized that PYY gene expression is controlled by FFA-induced Xbp1 splicing in L-cells. Direct delivery of Xbp1s into L-cells increased PYY gene and protein expression and blocking the cell stress response prevented FFA-induced PYY gene expression.p = 0.01).We knew from our previous work that fatty acid composition differentially affected PYY responses ,4,18,19.Accumulation of FFA’s in cells has been shown to activate the unfolded protein response (UPR) with specific induction of X-box-binding protein-1 (Xbp1) activation via splicing (Xbp1s). In STC-1 cells treated with SFA or MUFA, there was a significant increase in total Xbp1 mRNA (Xbp1 + Xbp1s) A. Upon fp = 0.05) with no significant effect on the cholesytokinin (CCK) expression demonstrating specificity of Xbp1s for PYY (p = 0.007) (p = 0.004) with no significant effect from 4µ8c alone. However, when Ire1α-mediated Xbp1s was blocked via 4µ8c, SFA failed to induce PYY expression .SFA treatment of STC cells induced both PYY expression and Xbp1s. To demonstrate a direct effect of Xbp1s on PYY gene expression and protein secretion, we treated cells with adenovirus expressing active Xbp1s. Gene expression of PYY was increased 13.7 ± 7.0 fold ( for PYY A. Media = 0.007) B. Next, = 0.007) . Since SIn addition to the known effects of SFA vs. MUFA on Xbp1 splicing, it is also clear that FFA→TG esterification is impacted by fatty acid composition. We and others have shown that SFA is more slowly incorporated into TG than MUFA ,21, and We and others have shown that SFA is poorly incorporated into TG and there is a preferential desaturation activity to convert SFA into MUFA via stearoyl-CoA desaturase-1 (SCD1) in tissues ,21. WhenTo test the concept that MUFA’s are absorbed faster than SFA, we fed mice ad libitum diets that were matched in macronutrient content but varying in lipid composition for 4 weeks . Food inn = 6 per group); the rationale was that chronic consumption of SFA may induce GI remodeling to reduce any potential deleterious effect of SFA. After 12 weeks, the animals were overweight . We tested the effect of oral gavage of a high SFA meal  require desaturation prior to esterification . The rolThe impact of lower satiety hormone production from MUFA may be due to increase food intake (feelings of hunger) which leads to increased body weight and obesity. Work from our lab and others has demonstrated that since MUFA’s are rapidly esterified into TG, they do not induce a cell stress-like response upon lipid loading ,26. SatuThe current studies demonstrate how satiety signaling is transmitted from dietary fat through the L-cell in normal and HF diet conditions. To date, very little is known regarding the mechanisms of nutrient–gene interactions in L-cells and these studies are some of the first of their kind. We assessed the factors involved in adaptive mechanisms of down-regulation of nutrient signaling in the gut and L-cells of HF-fed mice which can provide targets for therapeutic intervention as well as improved diet-design for individuals seeking to restrict calories to lose weight. We also determined the impact of ER stress on transducing nutrient signals into satiety hormone production in L-cells to better understand their adaptation to HF diets. The information provided from these studies helps to understand how dietary fatty acids are able to increase satiety hormone gene expression in the gut, and how obesity-associated loss of satiety occurs. Finally, it is important to consider dietary recommendations in which MUFA-rich diets are preferred over SFA-rich diets. However, this may result in weakened satiety signals and subsequently higher food intake.All experiments using mice were approved by the Texas Tech University Institutional Animal Care and Use Committee . Male and female C57BL/6 mice were purchased from Jackson Laboratories  and kept on a 12 h light/ dark cycle. Mouse research diets were from Harlan Laboratories ; SFA (TD.130051) and MUFA (TD.130379) enriched diets were provided ad libitum for 4-weeks beginning at 8 weeks of age. For experiments involving diet-induced obesity, mice were either kept on standard rodent chow (TD.8604) or fed a high fat (HF) custom research diet (TD.06414) at 8 weeks of age for 12-weeks. Nutrient composition of the test diets are provided in Sodium salts of FFA’s were purchased from Sigma . SCD1 (A939572) and Ire1α (4μ8c) inhibitors were from Calbiochem . The processed murine SREBP1c coding sequence was amplified from liver cDNA, sub-cloned into pAdTrack-CMV, sequence verified, and incorporated into pAdEasy-1 vector via homologous recombination in BJ5183 cells . Total cellular lipids were extracted according to the Folch method  and reso2 and 1% antibiotic/1% antimycotic). The cells were grown to confluence then treated with unconjugated palmitate (16:0) or oleate (18:1) as FFA. In initial experiments, we found that BSA conjugated FFA’s do not induce PYY expression, yet the unconjugated Na-salts of FFA were effective. Cells were treated for indicated times in serum-free DMEM + 1% P/S to exclude effects from confounding lipids provided by horse or fetal bovine serum.STC-1 cells were obtained from Dr. Douglas Hanahan via ATCC  and grown in the recommended culture conditions   and cDNA synthesis was performed using high capacity cDNA synthesis kits . Real-time PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems). Gene-specific primers were designed using Integrated DNA Technologies software (DNA.com)  and all t-tests were performed using SAS software . All values represent means ± SE of three independent experiments; * denotes a significant difference from the respective control (p < 0.05).ANOVA or 2-tailed


Our research focused on the identification of easily available and sensitive markers for early prediction of acute kidney allograft rejection (AR). We aimed to investigate the association between neutrophil-to-lymphocyte ratio (NLR) and AR in kidney transplant patients.  The medical records of 51 kidney transplant patients  were evaluated retrospectively. We considered a cut-off value of >2.5 as high NLR.  A total of 22 biopsy-proven AR patients and 29 controls were evaluated. The AR group had a higher NLR compared to the controls (P < 0.001). NLR levels over 2.5  were significantly associated with AR in univariate analysis. The NLR levels were the only significant factor associated with AR in multivariate models, in model 1 (adjusted by age and sex) , and in model 2  .  Our data showed that higher NLR values (>2.5) are associated with AR in kidney transplant patients, leading to the conclusion that NLR might be an easily available and useful marker option for detection of AR in this patient population.  Kidney transplantation is the best therapeutic option for end-stage renal disease (ESRD) patients. Health-related quality of life and patient survival are assumed to improve with kidney transplantation compared to conventional hemodialysis . Acute kidney allograft rejection (AR) is the most important risk factor for graft loss after transplantation, and some kidneys do not recover function even with intense antirejection therapy. Many other variables contribute significantly to the chances of rejection of kidney transplants, such as donor source, age, sex, creatinine level, blood group, Rh type, waiting time, duration of hospitalization, and vascular complications . The use of a large array of immunosuppressant drugs reduced the incidence of AR episodes, but long-term graft survival is still insufficient in this patient population. The neutrophil-to-lymphocyte ratio (NLR) was first described in 1967 (5) and was reported as an easy metric to assess inflammatory state. There are several studies that give evidence for its efficacy as a predictor of prognosis and/or mortality in various inflammatory states, such as cancer , acute coronary syndrome (8–10), and infections and postoperative complications (11–13). Numerous previous studies have demonstrated that an increase in NLR is associated with delayed and poor kidney graft function (14–16). In this retrospective study, we aimed to evaluate the association between NLR and acute kidney allograft rejection, which is an important inflammatory state in kidney transplant patients. We retrospectively investigated kidney transplant patients who were under follow-up treatment from September 1996 to January 2018 at the Yüksek İhtisas University Faculty of Medicine and Gazi University Faculty of Medicine. This study was conducted according to the Helsinki Declaration and the institutional review board of each university approved the study protocol. Inclusion criteria were receiving an ABO-compatible first-time kidney transplantation and medical chart achievability. Exclusion criteria were being under the age of 18, receiving a second transplantation, any proven history of cardiovascular disease or cancer, any diagnosis of BK nephropathy or CMV positivity, obesity, and active systemic, urinary, or local infection proven by a culture during the rejection. We also excluded patients who were not using steroids to eliminate the effect of steroids on neutrophil and lymphocyte distribution and to provide homogeneity for the population. The immunosuppression protocol used at our institutions consists of an antithymocyte globulin and methylprednisolone induction regimen, followed by maintenance therapy with mycophenolate mofetil/sodium or azathioprine, prednisolone, and a calcineurin inhibitor (tacrolimus or cyclosporine). Medical records were reviewed to find the data for the patients’ medical history, age, sex, and kidney biopsy pathology reports during AR. Acute rejection was defined by an increase of creatinine of 30% above baseline that was not attributable to any other causes, and AR was detected via Banff criteria by allograft biopsy in all cases (17). NLR was calculated with the formula of neutrophil count divided by lymphocyte count. We used blood drawn on the day of the application to the clinic for the acute rejection group and on the last outpatient clinic visit day for the control group. Different values of NLR are used in the literature in different disease states. Currently there is no universally accepted reference value, but a ratio above 3 is accepted as a high value in some disease states. In our study, serial receiver operating characteristic (ROC) curve analysis of NLRs revealed the 75th quartile (>2.5) to be the most sensitive and specific determinant of AR, leading us to consider values of 2.5 or greater as elevated. We used age-matched kidney transplant patients who had no rejection episode as a control group. We also retrospectively obtained serum biochemistry results, uric acid levels, and erythrocyte sedimentation rates. All immunosuppressive regimens were noted. All collected data were analyzed with SPSS 24.0 for Mac . P < 0.05 was considered statistically significant. All comparison analyses were performed by nonparametric tests due to restricted sample size. Characteristics were compared using the Wilcoxon rank sum test for baseline continuous variables and the chic-square test for categorical variables. Spearman’s test was used for the detection of correlation between variables. The ROC curve was calculated and the area under the curve (AUC) was analyzed for determination of the cut-off value of NLR. Results were expressed as median and interquartile range (IQR). Binary logistic regression analysis was used to investigate factors affecting AR. Because of the limitation of our sample size and to avoid overfitting, 3 independent variables were included in regression analyses in multivariate models. We included the results of 51 kidney transplant patients who matched our inclusion/exclusion criteria in our analysis. Of those patients, 9 received deceased and 42 received living donor kidney transplantation. All patients in the study groups received a standardized immunosuppressive regimen consisting of calcineurin inhibitors and mycophenolate mofetil/sodium or azathioprine and steroids. Acute allograft rejection occurred in 22 of those subjects (41%). 21 experienced acute cellular rejection, and 1 experienced antibody-mediated rejection. The median time in the AR group between renal transplantation and acute rejection was 7.5 (0.25–193) months. The median time of required follow-up was 8 (3–25) months for the AR group and 25 (8–60) months for the control group. Characteristics of the study population are provided in Table 1.Comparisons of the demographics and clinical features between the AR and control groups are summarized in Table 2. The patients were similar in terms of age, sex, and laboratory assessments of baseline creatinine, white blood cell count, and erythrocyte sedimentation rates. The median serum uric acid level was statistically significantly higher in the AR group (P = 0.043). There was 1 patient with diabetes mellitus in the control group but none in the AR group, and the control group had more hypertensive patients than the AR group.The median NLR level was statistically significantly higher in the AR group, with a median of 4.06 (3.11–7.03) in the AR group versus a median of 1.24 (1.08–1.57) in the control group (P < 0.001) . Univariate binary logistic regression analysis was performed to analyze each factor affecting AR. Serum uric acid levels  and NLR >2.5  were statistically significantly associated with AR. Also, having living donor transplantation seemed to reduce the chances of rejection with marginal statistical significance  (Table 3). For multivariate analysis, we created 2 models, each with a maximum of 3 independent variables due to the restricted sample size. Only NLR was statistically significantly associated with AR  in the first multivariate model (adjusted by age and sex). The statistically significant association between serum uric acid level and AR disappeared in this model (Table 3). The second model was adjusted by steroid dosage, serum uric acid level, and NLR. In this model, steroid dosage was included to exclude a possible effect on NLR. We also checked the association between steroid dosage and NLR to determine the confounder effect (interaction) for model 2 and found no statistically significant association between steroid dosage and NLR . The second multivariate model revealed that only NLR was statistically significantly associated with AR  (Table 3). In this study, we retrospectively examined kidney transplant patients who had AR and analyzed the association with their NLR levels obtained on the day of application to the clinic. We also included age-matched control patients who had not experienced AR in our analysis and found that the NLR in the AR group was significantly higher compared to the control group. Both univariate and multivariate analyses showed that NLR of >2.5 was statistically significantly associated with AR. The NLR is an inflammatory marker that is inexpensive and easily available in routine clinical practice. There are several reports in the literature providing evidence that NLR is strongly associated with morbidity or mortality in several inflammatory conditions such as cancer and cardiovascular disease, as well as being a predictor for infectious and postoperative conditions . NLR levels were positively correlated with inflammatory cytokines in ESRD patients (18). Another study showed that kidney transplant patients had a higher NLR than healthy subjects (19) and concluded that the higher values were due to the ongoing inflammation in these patients. A prospective study by Cankaya et al. also reported that mean NLR levels were improved at the first year after transplantation but did not reach the levels found in healthy controls, which is further evidence for chronic inflammation as permanent in kidney transplantation (15). AR is the major cause of graft dysfunction in these patients. Some of the patients do not recover even with high-dose antirejection treatment. Most AR episodes are overlooked by both clinicians and patients, since they are mostly asymptomatic, but even if this is the case AR episodes have a negative impact on long-term graft survival. It is important to use an easily applicable marker for both the prediction and early detection of AR for this population. A rise in serum creatinine is an important and widely used laboratory test for predicting AR, but creatinine increases indicate significant histological damage in the kidney, which is a reflection of the late course of the rejection episode. Our results present favorable findings regarding using NLR as a simple, easily available, inexpensive marker for the assessment and prediction of AR. An NLR level above 2.5 appears to be a sensitive and specific predictior of AR, according to ROC curve analysis. Both univariate and multivariate logistic regression analysis found it independently associated with AR. We believe the increase in NLR in AR patients was unrelated to corticosteroid dosage, because all the patients were using the same maintenance dosage and our interaction analysis did not show any significant association as a confounder effect on the prediction model. Halazun et al. also found that elevated preoperative NLR levels were associated with a higher risk of developing delayed graft function, another state that has a shared casual mechanism with AR episodes, after kidney transplantation (16). They concluded that the preoperative active inflammatory response that was detected by higher NLR levels might affect the immediate graft function. They found that NLR over 3.5 acted as a predictor for delayed graft function. However, in our study, patients with NLR over 2.5 were more likely to develop AR. There is no universal cut-off level for NLR values; most studies have reported the levels according to the median and higher quartile. A study from a nongeriatric and healthy population reported that a normal value for NLR is between 0.78 and 3.58 (20). In other disease states such as cancer, and in cardiovascular and surgical patients, NLR values over 3.5 were accepted as a prediction value for each disease . NLR over 2.5 in heart transplantation and over 3.5 in kidney transplantation were found as predictors for RRT and graft survival in several studies . We found that the NLR cut-off value of 2.5 has 90% specificity and 60% sensitivity for detection of AR. There are several limitations of our study. First, the small sample size impedes generalizability of results. The retrospective design limits our interpretation of causality. A single measurement of NLR might not accurately reflect the relationship over time. Future studies that obtain serial changes of NLR would be useful to clarify to role of NLR in the follow-up of AR. We still believe that NLR is a practical marker in both inpatient and outpatient settings, and the accumulation of studies reporting the usefulness of practical and inexpensive markers for either diagnosis or follow-up of chronic disorders is important. The reduction of expenditure is of great of importance in health care all over the world. In conclusion, even with these limitations, our present study was able to detect NLR as being higher in AR patients compared to those without rejection, which might indicate an increased risk of AR associated with higher NLR levels. The findings from this retrospective study must be confirmed. Larger, prospective, controlled, and long-term follow-up studies are needed to further determine the sensitivity, specificity, and predictability of this inexpensive, convenient test in the early diagnosis of acute graft rejection. Further validation and feasibility studies are required before it can be considered for routine clinical use.Part of this study was presented as a poster presentation at the 30th National Congress of Nephrology, Hypertension, Dialysis, and Transplantation in Antalya, Turkey (2012).


After N0, attention decreased significantly at DR in comparison with BR (p < 0.05) and AR (p < 0.001). BD and TD improved after N35 compared to N0 at BR , DR , and AR . A significant increase in attention was observed after N35 in comparison with N0 at DR (p < 0.01) and AR (p < 0.01). However, no changes were found for the perception of mood states, stress, sleep, muscle soreness, and the FI during the 5mSRT. Also, N35 was better than N0 for RPE at DR (p < 0.05), feelings at AR (p < 0.05), and fatigue estimation at AR (p < 0.01). A 35-min nap opportunity may have beneficial effects on physical and cognitive performances before, during, and after Ramadan.Ramadan observance is characterized by several changes in behaviors, such as food and sleep, which could affect physical and cognitive performance. The aim of the present study was to investigate the effects of a 35-min nap (N35) opportunity on physical performance during the 5-m shuttle run test (5mSRT); attention; feelings; mood states; and perceptual measures of stress, fatigue, and muscle soreness during Ramadan observance. Fourteen physically active men  were tested after a no-nap condition (N0), N35 15 days before Ramadan (BR), the last 10 days of Ramadan (DR), and 20 days after Ramadan (AR). Measures included the digit cancellation test (attention estimation), the profile of mood state (POMS), and the Hooper questionnaires. After a 5-min standard warm-up, participants performed the 5mSRT , total distance (TD), and fatigue index (FI) were recorded), along with the rating of perceived exertion (RPE) after each test repetition. After the 5mSRT test, participants responded to the feeling scale (FS). The results showed that TD and FI during the 5mSRT were not affected by Ramadan observance. However, BD was significantly lower than DR compared to AR after N0 (∆ = −4.3 ± 1.3%;  Intermittent fasting is an obligation for healthy pubescent Muslims during the month of Ramadan (observance lasting between 29 and 30 days) . During Taraweeh)) have considerable effects on sleep [Changes in eating schedules as well as other activities  [Boukhris et al.  reporteday fast) ; it may ay fast)  suggesteay fast) ,20 and pay fast) ,22 perfoay fast) , recent ay fast) . When reay fast)  reporteday fast) ,27 and 4ay fast) ). For exay fast)  and Abdeay fast)  suggesteay fast)  reportedTherefore, the aim of the present study was to investigate the effects of a 35-min nap before, during, and after Ramadan on physical and cognitive performance in physically active men. In a recent study, Hsouna et al.  showed tA minimum of eleven participants was required for inclusion in the present study. This sample size was calculated using the software G*power  and based on the effect size (= 0.46) shown by Herrera et al. . Due to All participants were familiarized to the protocol and tests of the present study. They then participated randomly in two testing sessions: a no-nap opportunity (N0) and a 35-min nap opportunity (N35). Each testing session occurred during three separate occasions: 15 days before Ramadan (BR), the last 10 days of Ramadan (DR), and 20 days after Ramadan (AR). At least 72 h was required between successive testing sessions. During each condition, participants came to the laboratory for a period of time between 13h45 and 18h30. For the nap, participants were allotted 15 min to be accustomed to the new place of sleep in the sleep laboratory. Following the 15 min acclimation period, participants completed the 35-min nap in dark and quiet sleep rooms. After N35 and during N0, participants did not engage in physical activity but were allowed to perform leisure activities such as watching TV, playing video games, etc. in a comfortable armchair. At 17h00, participants completed the digit cancellation test (assessment of attention), 5-min of standardized warm-up, and the 5mSRT (6 × 30 s with 35 s in between). The digit cancellation test consists of deleting target numbers  on a sheet of randomly arranged possibilities; the sum of correct responses was recorded for analysis.During BR, DR, and AR (after the nap conditions), after the end of the 5mSRT, the Hooper questionnaire was used to estimate sleep, stress, fatigue, and muscle soreness using subjective scales ranging from “1” (very low or good) to “7” (very high or bad) . During Rating of perceived exertion (RPE) was estimated after each repetition of the 5mSRT on a scale ranging from “0” (very light) to “10” (very hard) . The meaDuring BR, DR, and AR, participants were instructed to record estimated quantities of all food and beverages consumed for ten days during each period of physical testing. The amount and type of food and fluid consumed were calculated using the software program Bilnut (Nutrisoft Bilnut: Food Survey Program version 2.01) and the food-composition tables of the Tunisian National Institute of Statistics (1978) by an expert nutritionist after interviewing all participants.Furthermore, participants were allowed to bring their own food and drink and consume ad libitum.Sleep duration, sleep quality, sleep latency, sleep efficiency, sleep disturbances, the use of sleeping medications, and daytime dysfunction were estimated using the Pittsburgh Sleep Quality Index (PSQI), containing 19 questions . The totThe collection of the digit cancelation test, POMS, and PSQI data took place in the laboratory, while the 5mSRT, RPE, and FS data collection took place on a sport field.During the study, participants fasted for ~15–16 h per day. During BR, DR, and AR, the temperature and relative humidity were respectively 28 °C and 50%, 32 °C and 49%, and 31 °C and 47%.As described by Boukhris et al. , particiTotal distance (TD) = Sum of distances in meter covered during the 6 × 30-s shuttlesBest distance (BD) = The higher distance in meter realized during the 6 × 30-s shuttlesFatigue index (FI):During the test, the following parameters were calculated:p2). However, when the normality was not confirmed, a Friedman test was performed [p ≤ 0.05. Due to the high number of dependent variables, only significant differences are reported.Data were presented as mean ± standard deviation (SD) and analyzed using STATISTICA 12.0 Software . Normality of the distribution was confirmed using Shapiro Wilk’s test. TD, PSQI, carbohydrates, and lipids data were normally distributed. A two-factor ANOVA (period × nap) was performed for TD, and a one-factor ANOVA (period) was performed for PSQI, carbohydrates, and lipids data. Post hoc comparisons were completed using the Bonferroni test; effect sizes were calculated as partial eta-squared  (p < 0.01), DR , and AR .The repeated measures ANOVA revealed a significant main effect of nap  . The Bonp < 0.001, Kendall’s W = 0.32) (p < 0.05), DR , and AR . However, BD was lower at DR in comparison with AR in N0  and N35 .The Friedman test revealed a significant main effect  . Pairwisp > 0.05, Kendall’s W = 0.13)  .Attention scores are presented in p < 0.001, Kendall’s W = 0.5). The Wilcoxon post hoc test showed that attention scores were significantly lower after N0 at DR in comparison with BR (p < 0.05) and AR (p < 0.01). After N35, attention scores were higher at AR in comparison with BR (p < 0.01). However, pairwise comparisons revealed a significant increase in attention after N35 in comparison with N0 at DR (p < 0.01) and AR (p < 0.01)  .p < 0.001, Kendall’s W = 0.32). Pairwise comparisons revealed a significant decrease of RPE scores recorded after N0 at AR when compared with BR (p < 0.01) and DR (p < 0.01). Also, RPE scores were significantly lower after N35 in comparison with N0 at DR (p < 0.05) (RPE scores are presented in  < 0.05) .p < 0.01, Kendall’s W = 0.28). The Wilcoxon post hoc test showed that feelings scores significantly decreased at DR in comparison with BR after N0 (p < 0.05) and after N35 (p < 0.05). After N35, feelings scores were significantly higher at AR than DR (p < 0.05). Feelings scores were significantly higher at AR after N35 in comparison with N0 (p < 0.05) (Feelings scores are presented in  < 0.05) .p > 0.05; Kendall’s W = 0.05), proteins  and lipids  was reported. However, statistical analyses revealed a significant effect for carbohydrates   , whereinPSQI parameters are presented in p > 0.05, Kendall’s W = 0.07), sleep efficiency , or sleep disturbances   .p < 0.0005, Kendall’s W = 0.53). The sleep quality scores increased significantly at DR (p < 0.01) and AR (p < 0.05) in comparison with BR.A significant effect was reported  with decrease in sleep duration DR in comparison with BR (p < 0.05).There was a significant effect  with higher sleep dysfunction scores DR in comparison with BR (p < 0.05).There was a significant effect . Total PSQI scores were significantly higher in DR (p < 0.001) and AR (p < 0.05) in comparison with BR. The statistical analysis revealed a significant effect , stress , or muscle soreness   .p < 0.05, Kendall’s W = 0.16). Pairwise comparisons revealed significant increases in fatigue scores for AR compared with BR (p < 0.05) after N0. At AR, the fatigue scores were significantly lower after N35 compared to N0 (p < 0.01).For the fatigue scores, the Friedman test revealed a significant main effect , tension , anger , confusion , depression , fatigue , and vigor   .i) TD and BD significantly improved after N35 in comparison with N0 at BR, DR, and AR and (ii) attention was significantly higher after N35 in comparison with N0 at DR and AR.The purpose of this study was to investigate the effects of a 35-min nap on physical performance during the 5mSRT; attention; feelings; mood states; and the perception of stress, muscle soreness, and fatigue before, during and after Ramadan observance in young physically active men. The main findings of the present study were as follows:  only in some periods of study: attention at DR and AR, RPE at DR, feelings at AR, and fatigue estimation at AR. In contradiction, Hsouna et al.  did not Although the present study presented important practical recommendations, some limitations exist. First, the present study lacks an objective measurement of the night and the nap sleep . Future studies should utilize registration of the previous night’s sleep and the nap during the experimental period. In addition, a specific sleep scale relating to quality of sleep during the nap should be used for future studies. Moreover, due to a technical issue, the current study lacks control of the luminosity level during the sleeping period; this drawback should be avoided in future studies. In addition, to confirm the results of the present study, further studies should compare different nap durations outside and during Ramadan.The results revealed that TD and FI during the 5mSRT were not affected by Ramadan observance. However, BD was significantly lower in DR compared to AR after N0 and N35. During N0 condition, attention decreased significantly in DR in comparison with BR and AR. BD and TD were better after N35 compared to N0 at BR, DR, and AR. A significant increase of attention after N35 in comparison with N0 at DR and AR was observed. However, no changes have been reported for the perception of mood states, stress, sleep, muscle soreness, and FI during the 5mSRT. Additionally, N35 was better than N0 for attention at DR and AR, RPE at DR, feelings at AR, and fatigue estimation at AR. We suggested that further increases of the nap duration during Ramadan would allow for significant effects of the nap in all parameters before, during, and after the fasting month.Therefore, coaches and athletes should plan some periods of 35-min daytime naps during Ramadan observance to overcome any sleep loss and/or to improve physical and cognitive performances.


Epigenetic regulation is essential in regulating gene expression across a variety of biological processes. Many high-throughput sequencing technologies have been widely used to generate epigenetic data, such as histone modification, transcription factor binding sites, DNA modifications, chromatin accessibility, and etc. A large scale of epigenetic data is stored in NCBI Gene Expression Omnibus (GEO). However, it is a great challenge to reanalyze these large scale and complex data, especially for researchers who do not specialize in bioinformatics skills or do not have access to expensive computational infrastructure.GsmPlot can simply accept GSM IDs to automatically download NCBI data or can accept user’s private bigwig files as input to plot the concerned data on promoters, exons or any other user-defined genome locations and generate UCSC visualization tracks. By linking public data repository and private data, GsmPlot can spark data-driven ideas and hence promote the epigenetic research.https://gsmplot.deqiangsun.org/.GsmPlot web server allows convenient visualization and efficient exploration of any NCBI epigenetic data in any genomic region without need of any bioinformatics skills or special computing resources. GsmPlot is freely available at  The public epigenetic data stored in NCBI is essential for biomedical research but an easy-to-use tool with the quick visualization function is missing.We present GsmPlot, a user-friendly web server to allow scientists without any bioinformatics expertise, or any high-performance computational resources to easily visualize public epigenetic data in NCBI.GsmPlot can be used to study the crosstalk between histones, DNA modifications, co-binding of TFs, and other epigenetic factors at any functional genomic regions or user defined regions.GsmPlot supports user-server interactions which allow users to choose their concerned regions to further explore different epigenetic factor interactions among multiple samplesEpigenetic mechanisms alter phenotypes by regulating gene expression patterns without altering the DNA sequences in response to physiological or pathological signals . Due to Although DaVIE , OctopusTo alleviate these limitations, we developed GsmPlot, a user-friendly web server to easily generate customized visualizations for the public data in GEO and additionally provide interactive explorations. GsmPlot is convenient to use as it need only GSM IDs or the bigwig files provided by user. GsmPlot can conveniently generate profile plots on functional genome elements  or visualization on one specifically concerned region through UCSC genome browser integration. Moreover, GsmPlot allows interactive selection of regions with specific epigenetic patterns in the heatmap for further explorative study.GsmPlot provides two flexible methods for the user to query the data: GSM IDs or bigwig files on user computer. GsmPlot automatically downloads the bigwig/wig/bedgraph file from GEO or from the user computer to the web server. Users can profile the data along user-defined genome intervals by providing BED files or along user-defined gene sets by providing gene names  and hydroxymethylation (5hmC) data for human and mouse ES cells –19 . TheFigure GsmPlot also can be used to investigate the relationship between TFs and DNA methylation or hydroxymethylation. Figure Epigenetic data from different sources are usually generated and normalized differently, preventing such data to be compared directly. To circumvent this problem, we can use z-score to replace raw wig signal to allow direct comparison. For each sample, we calculate the average bigwig signal in bins of user-defined size along concerned regions. Then, we calculate z-scores of the corresponding wig values for each bin in each region . These results indicate that 5hmC may play different roles in terms of how heart related TFs bind to CGIs with single or dual transcriptional directions.As an example, to illustrate that GsmPlot has the potential to shape novel biological hypothesis or discoveries, we explored the potential roles of DNA hydroxymethylation (5hmC) around CGI regions in heart development. We used mouse heart DNA hydroxymethylation data (CMS-IP) from wildtype GSM3466904) and Tet2/3 knockout (GSM3466906) mice . We alsoelopment , 26. Our466904 anBiomedical data stored in NCBI is valuable for biomedical researchers. However, most researchers and physicians do not have computation skills or infrastructure, and hence this “treasure” could not be used immediately. Even for bioinformaticians, complicated procedures including download, computation, aggregation, hosting of data are required to visualize NCBI data. We developed a web server, GsmPlot, which can download, compute, visualize and compare data. The most important feature of GsmPlot is the ability of performing multiple omics integration studies, such as RNA-seq, Bis-seq, ChIP-seq, ATAC-seq with simply GSM IDs from NCBI. Private data sequenced by users in proper visualization format can be fed into GsmPlot to compare with public data. Compared with other good epigenome analysis platforms, such as EpiMINE, GsmPlot have many advantages. GsmPlot does not need users to download public data manually. GsmPlot do not depend on users’ computer capacity especially for computation intensive bis-seq data, which cannot be handled on a desktop computer. In addition, installation problems, such as software compatibility, software version, could be a big headache for many researchers, but can be completely avoided using GsmPlot. Moreover, interesting regions with certain epigenetic features can be extracted using interactive heatmap, which can be fed into GsmPlot again to explore if there are new epigenetic factors in these interesting regions. Importantly, we have successfully proved GsmPlot’s reliability and its potential ability of making novel biological ideas from three case studies. Above all, GsmPlot is a user-friendly and reliable tool to investigate public epigenetic data, especially for those biomedical researchers who do not have any computation skills.Although GsmPlot has an email alert for those large data tasks, GsmPlot will add more CPUs to further improve the speed of calculation in the future depending on the demand. The figure’s format, label sizes and other features will be added as user options which will allow users to generate publication quality figures using GsmPlot.We have presented GsmPlot, a user-friendly web server for quick visualization and exploration of public NCBI data. To our best knowledge, this is the first webserver that can automatically download data from GEO, transform data, generate images, and support user interaction. Users can easily and quickly visualize and explore any public epigenetic data without requiring of any special training or computing resources, and hence can study the epigenetic mechanism efficiently. The three applications presented above confirmed that GsmPlot can be a huge driver to accelerate the research process by providing convenient visualization of both public and private data, and hence promoting data driven ideas. GsmPlot will dramatically improve the efficiency of utilization of public epigenetic data and further promote the research in epigenetic community.deepTools [rows as regions and columns as bins are generated, and the column mean values are plotted as aggregated profile. By transforming the wig signal to z-score, we also plot all the z-scores in one bin as a boxplot and so for all bins, as illustrated by the Additional file https://matplotlib.org/) and R (https://www.r-project.org/). (3). GsmPlot web interface is implemented using HTML, CSS , and JavaScript. The backend of GsmPlot is based on Django web framework (https://www.djangoproject.com/). The interactive functions between users and GsmPlot web server are implemented using jQuery (https://jquery.com). For large data which takes long time to finish the calculation, we include an email alert function by using django.cor.mail function. Due to the limited computing resources, we currently only allow one task for each user at a time. GsmPlot has been tested in Firefox, Chrome, Safari, and Edge.GsmPlot server is composed of three parts: web crawler, data process and web interface. (1). Web crawler was coded in Python 3.5 and specifically designed for NCBI to automatically detect the URLs and download files with bigwig, wig and BedGraph format. We also include genome reference version check in web crawler. Data process include two parts: calculation and visualizations. (2). For data calculation, we wrapped eepTools  to calcuThe flowchart of GsmPlot is in Additional file Project name: GsmPlot.Project home page:https://gsmplot.deqiangsun.org/Operating System: Platform independent.Programming language: Python.License: GNU GPL.Other requirements: Internet Explorer 10 or later.https://groups.google.com/d/forum/moabs_msuiteDiscussion Group: Support email: moabs_msuite@googlegroups.com.Any restrictions to use by non-academics: None.Additional file 1: Figure S1. Scheme for the structure of GsmPlot web server.Additional file 2: Figure S2. Illustration of the data matrix for the profile curve and the z-score boxplots (left), and illustration of the data matrix for the heatmap (right).Additional file 3: Figure S3. UCSC genome browser visualization for RNA-Seq, H3K27Ac, H3K4me3 on an example region for human H1 ESC.Additional file 4: Figure S4. A, the average ChIP-Seq signal along the CTCF binding sites with red curve for the CTCF signal and blue curve for the 5hmc signal. B, The UCSC genome browser visualization for CTCF peak, DNA methylation and DNA hydroxymethylation on an example region. The yellow highlight areas showed the depletion of 5mC at the center of the CTCF peak.Additional file 5: Figure S5. GsmPlot default figures for the average signal curve along the gene body regions. Blue: H3K4me3; Green: H3K27ac; Red: H3K27me3.Additional file 6: Table S1. Statistics for datasets with bigwig/wig/bedgraph format available in GEO. Table S2. Processing time for variable files sizes. Table S3. Processing time of GsmPlot and EpiMINE.