Background: The results have been inconsistent with regards to the impact of uric acid (UA) on clinical outcomes both in the general population and in patients with chronic kidney disease. The aim of this study was to study the influence of serum UA levels on mortality in patients undergoing continuous ambulatory peritoneal dialysis. Methods: Data on 492 patients from a single peritoneal dialysis unit were retrospectively analyzed. The mean age of the patients was 53.5 ± 15.3 years, with 52% being female (n = 255). The concomitant comorbidities at the start of continuous ambulatory peritoneal dialysis (CAPD) encompassed diabetes mellitus , hypertension , and cardiovascular disease . The study cohort was divided into sex-specific tertiles according to baseline UA level. A Cox proportional hazard model was used to calculate hazard ratios (HRs) of all-cause, cardiovascular, and infection-associated mortality with adjustments for demographic and laboratory data, medications, and comorbidities. Results: Multivariate Cox regression analysis showed that, using UA tertile 1 as the reference, the adjusted HR of all-cause, cardiovascular, and infection-associated mortality for tertile 3 was 0.4  0.24–0.68, p = 0.001), 0.4 , and 0.47 . In the fully adjusted model, the adjusted HRs of all-cause, cardiovascular, and infection-associated mortality for each 1-mg/dL increase in UA level were 0.84 , 0.79 , and 0.79  for men and 0.57 , 0.6 , and 0.41  for women, respectively. Conclusions: Higher UA levels are associated with lower risks of all-cause, cardiovascular and infection-associated mortality in women treated with continuous ambulatory peritoneal dialysis. Uric acid (UA) is the final product of a purine metabolism generated by xanthine oxidase or xanthine dehydrogenase in humans . Rather The burden on healthcare systems caused by chronic kidney disease (CKD) is increasing worldwide. Patients with CKD, including both those requiring hemodialysis and peritoneal dialysis, are at high-risk of cardiovascular (CV) events, and approximately 40% of patient deaths are due to cardiovascular disease (CVD) annually ,4. From To date, only a handful of studies have investigated the association between UA and mortality in patients with end-stage renal disease (ESRD) treated with dialysis. Therefore, the purpose of the present study was to investigate the possible link between UA and mortality in patients undergoing continuous ambulatory peritoneal dialysis (CAPD) after adjusting for a variety of crucial variables. In addition, we also evaluated possible differences in sex with regards to the association between UA and mortality.This study was carried out retrospectively at a single peritoneal dialysis (PD) center of Changhua Christian Hospital in Taiwan. From 1 January 2003 to 31 December 2014, patients who started CAPD as renal replacement therapy for ESRD were screened for this study. The enrollment criteria included incident patients of at least 18 years of age who had survived on CAPD for at least 3 months. Eligible patients were followed up from the date of commencing CAPD and the end of the study was on 31 December 2015 when the clinical outcomes were censored. CVD and infection were the two leading causes of death in our patients. Therefore, the primary outcome of interest was all-cause mortality, and the second outcomes of interest were CV and infection-associated mortality. CV mortality was defined if a death was attributed to any of the following cardiovascular events: coronary events, cardiomyopathy, cardiac dysrhythmia, sudden cardiac death, congestive heart failure, ischemic brain injury, cerebrovascular accident and peripheral vascular disease. Infection was the cause of death if a patient directly died from sepsis. The cause of death was determined by reviewing the discharge diagnosis and hospitalization summary in electronic medical records. The study protocol was approved by the institutional review board of our hospital, and the study was conducted in compliance with the declaration of Helsinki.2, was derived from averaging the values of creatinine and urea clearance and standardized for body surface area.Baseline characteristics of the whole cohort were obtained from the hospital’s electronic database. Demographic and clinical data were recorded at the initiation of PD, and included age, sex, status before PD , body mass index (BMI), medications, comorbidities, and smoking status. The comorbidities included diabetes mellitus (DM), hypertension, CVD and Charlson Comorbidity Index, which is one of the most commonly used scoring systems to predict the risk of mortality. Blood tests, consisting of white blood cell (WBC) counts, hemoglobin, total cholesterol, triglyceride, calcium, phosphate, alkaline phosphate (ALP), uric acid, glutamic-pyruvic transaminase (GPT), intact parathyroid hormone (iPTH), ferritin, transferrin saturation, serum albumin, blood urea nitrogen (BUN), and creatinine were checked at the laboratory of Changhua Christian Hospital. The use of medications was recorded based on prescriptions, and included angiotensin-converting enzyme (ACE) inhibitor, angiotensin II receptor blocker (ARB), hypouricemic agent , erythropoiesis stimulating agents (ESA), loop diuretic (furosemide), calcium channel blocker (CCB), vitamin D, and statins. Information on PD-related parameters included daily ultrafiltration, total Kt/V urea, residual renal function (RRF), daily urine volume, normalized protein nitrogen appearance (nPNA) and dialysate-to-plasma creatinine ratio (D/P creatinine) at 4 h. RRF, in milliliters per minute per 1.73 mn) and percentage (%) for categorical variables, unless otherwise indicated. Differences in categorical variables among the three groups were tested using the chi-square test or Fisher’s exact test. Continuous data with normal distribution were compared by analysis of variance (ANOVA), and skewed continuous data by the Kruskal-Wallis test.The patients were classified into sex-specific tertiles according to the serum UA level: for men, tertile 1 ≤ 6.8 mg/dL; tertile 2 = 6.9–8 mg/dL; tertile 3 ≥ 8.1 mg/dL; and for women, tertile 1 ≤ 6.5 mg/dL; tertile 2 = 6.6–7.6 mg/dL; tertile 3 ≥ 7.7 mg/dL. Summary statistics are presented as mean ± standard deviation (SD) for continuous variables of normal distribution, median with interquartile range (IQR) for continuous variables of skewed distribution, or as number  and 95% confidence intervals (CIs).p value < 0.05 for the analysis. All statistical analyses were conducted using IBM SPSS Statistics for Windows, Version 22.0 .To strengthen our findings, we conducted three sensitivity tests. First, Cox regression analysis was repeated with UA as a continuous variable with HRs presented per 1-mg/dL increase in serum UA level by sex. Second, all of the patients were dichotomized into either hyperuricemia or normouricemia groups by sex to examine the relationship between hyperuricemia and mortality. Hyperuricemia was defined as a UA level >7 mg/dL for men and >6 mg/dL for women. Third, the relationship between UA and mortality was calculated after excluding the patients taking allopurinol at entry to the study to mitigate the confounding effect of hypouricemic agents on serum UA level. In addition, we also performed stratified analyses by nutritional markers, including BMI, cholesterol, albumin, nPNA, phosphorus, and creatinine. Statistical significance was set at a n = 255). The concomitant comorbidities at the start of CAPD encompassed diabetes mellitus , hypertension , and cardiovascular disease . The median UA level was 7.2  mg/dL for all patients, and the men had a significantly higher UA level than the women (7.4 (6.6–8.3) mg/dL vs. 7 (6.1–8) mg/dL, respectively; p = 0.004). A total of 126 participants (25.6%) were taking hypouricemic agent  at baseline. One hundred and twenty-seven patients died during the study period, including 74 from cardiovascular disease and 41 from infection.Four hundred and ninety-two CAPD patients were enrolled in this study, with a median follow-up period of 36.4 months. Most (81.9%) of our study participants commenced CAPD as the first renal replacement therapy, i.e., pre-dialysis status. The baseline patient characteristics according to the sex-specific tertiles of UA are shown in p < 0.001). The associations between UA tertile and all-cause mortality by Cox regression analysis are shown in p = 0.001).As shown in p = 0.002). The associations between UA tertiles and CV mortality are shown in p = 0.01) with respect to CV mortality.As shown in p = 0.007). The associations between UA tertiles and infection-associated mortality are shown in As shown in We also evaluated the relationship between mortality and UA by non-sex-specific tertiles. As shown in p = 0.07), 0.79 , and 0.79  for men and 0.57 , 0.6 , and 0.41  for women, respectively. After excluding 126 patients who received allopurinol treatment, the significant association remained in the women but not in the men  was associated with a lower risk of all-cause, CV, and infection-associated mortality only in the female ESRD patients treated with CAPD, independent of several confounding factors. This observation was further verified by similar results noted when UA was treated as a dichotomous or continuous variable. Furthermore, the association remained significant after excluding the patients taking hypouricemic agents. These findings are in contrast to previously published studies on PD patients, and imply that hyperuricemia per se may protect against the risk of mortality in PD patients.To date, only few investigations have evaluated the impact of UA on mortality in PD patients. One study, conducted in Sweden, comprised 294 subjects starting renal replacement therapy, 161 of whom were started on peritoneal dialysis and the rest were started on hemodialysis . All theThe reasons for the differences in the impact of UA on patient survival between male and female patients have yet to be clarified, and may be due to patient ethnicity, differences in the follow-up period, differences in the adjusted confounders, and differences in serum UA levels between men and women. A study reported a significant relationship between UA and CV mortality both in women and men after a longer follow-up period, while the previous study of the same cohort only reported the association merely in women . Sex horThe contradictory results relating UA to outcomes were partly attributed to the dual effects of UA on CV outcomes. Several mechanisms were proposed to explain the detrimental effects of UA. First, hyperuricemia is closely related to the established conventional cardiovascular risk factors, such as DM, hypertension, older age, and male gender. Increasing evidence shows that UA may cause kidney disease, hypertension, high oxidative stress, and metabolic syndrome ,24,25. SOn the other hand, four possible explanations may explain why lower UA levels are associated with higher mortality rates. First, serum UA level may reflect the nutritional status because the major source of UA is derived from dietary intake of purines and nucleotides. Therefore, a low serum UA may indicate malnourished status, which in turn accounts for susceptibility to sepsis and impaired immune function . In thisThe strengths of our study include a medium-size population of CAPD patients. Since detailed information was collected regarding patient demographic features, comorbidities, medications, and laboratory data, we were able to conduct a comprehensive analysis to minimize the potential bias in observational studies. Some potential limitations of the present study should nonetheless be mentioned. First, all the patients were recruited from a single PD center and thus it does not allow the generalization of the results to the entire population of CAPD patients due to the diverse differences in cultural and demographic factors. Second, residual confounding factors cannot be completely excluded. For example, insulin resistance, which has been shown to be a predictor of cardiovascular mortality in ESRD patients, was not evaluated in our study. Third, due to the retrospective nature of this study, we were unable to determine whether the low UA level contributes to the reduced antioxidant capacity. Whether UA per se is a causative factor or merely a marker of nutrition, inflammation or oxidative stress cannot be answered from this study. Fourth, a single measurement of UA at baseline, which did not cover acute change in UA during the study period, might underestimate or over-estimate the true relationship with study outcomes. Despite these limitations, we believe this study contributes to the rather limited knowledge of the predictive value of UA in CAPD patients.In conclusion, we found an inverse relationship between UA level and all-cause, CV, and infection-associated mortality in female patients on CAPD. Based on our findings, the beneficial impact associated with UA appears to outweigh its harmful effects, and may thus have a protective role in CAPD patients. This significant association should alert clinicians to pay more attention to women on CAPD with low UA levels. This UA paradox may be explained by the association between a low level of UA and poor nutrition and possibly increased oxidative stress. However, whether a low level of UA is the cause or consequence of these conditions remains to be elucidated. Further prospective studies of larger sample size are needed to confirm this relationship and to clarify the underlying mechanisms such as the role of UA in antioxidant capacity.


Mother-to-child-transmission (MTCT) of human T-cell lymphotropic virus type-1(HTLV-1) contributes disproportionately to the burden of HTLV-1 associated diseases. All preventive measures to avoid MTCT rely on the identification of infected mothers. However, the impact of pregnancy on HTLV-1 diagnosis has not been clearly assessed. Paired samples from 21 HTLV-1 infected women taken during pregnancy and while not pregnant were analysed by CMIA and PCR. The signal-to-cut-off values (S/CO) were higher during pregnancy than in the paired non-pregnant samples. HTLV-1 proviral load did not alter significantly by pregnant state. S/CO positively correlated with HTLV proviral load. Pregnancy does not impair the diagnosis of HTLV-1/2, by either immunological (CMIA) or molecular (qPCR/nPCR) tests. Human T-cell lymphotropic virus (HTLV) can be transmitted from mother to child, mainly by breastfeeding. All preventive measures to avoid HTLV mother to child transmission depend on the identification of infected pregnant women. HTLV diagnosis is based on serological screening tests, confirmed by serology and/or molecular assays. The aim of the study was to verify whether pregnancy adversely impacts the identification of HTLV infection. Using paired samples from 21 women living with HTLV-1/2 obtained during pregnancy and while not pregnant we demonstrate that both serology and molecular assays perform equally well in both settings and can be use for the diagnosis of HTLV infection during pregnancy. At least 5–10 million individuals are living with human T-cell lymphotropic virus type 1 (HTLV-1) in the world . InfectiThis study was conducted under the auspices of the communicable diseases research tissue bank and approved by the NRES Committee South Central–Oxford C (15/SC/0089). All participants signed a written informed consent.Twenty-one women attending the National Centre for Human Retrovirology, St. Mary’s Hospital, London, with either HTLV-1 or -2 infection donated blood samples. HTLV infection had in all cases been confirmed by Western Blot (Genelabs HTLV 2.4) in accordance with the manufacturer’s instructions, 19 had HTLV-1 and two HTLV-2 infection. Paired sera obtained during a pregnancy and whilst not pregnant were analysed according to the manufacturer’s instructions for anti-HTLV-1/2 antibodies detection using Abbott Architect rHTLV-I/II platform, a fully automated third generation CMIA that includes HTLV-1/2 recombinant proteins. The optical density (OD) of each sample compared to the negative/reactive cut-off value for each time point was recorded. HTLV-1 proviral load (PVL) in peripheral blood mononuclear cells (PBMCs) was determined by quantitative real time PCR (qPCR) targeting HTLV tax gene and human betaglobin gene as previously reported . In thosD'Agostino & Pearson omnibus normality test was used to verify whether the results had a Gaussian distribution. Paired t test and Wilcoxon matched pairs test were used to compare groups (pregnant and not pregnant) of parametric and non-parametric data, respectively. Spearman test was used to verify if there was a correlation between S/CO values and PVL.The clinical status, the trimester of testing and the serological and molecular results are presented in There was no significant difference in HTLV PVL in samples collected during pregnancy and while not pregnant (Median PVL (Interquartile range) HTLV-1 DNA copies per 100 PBMCs: Not pregnant 0.6 (3.85); pregnant: 0.6 (7.65); p = 0.8). The PVL was quantifiable in 17 women in whom it was lower during pregnancy in seven, the same in four and higher in six. Four women (3 HTLV-1 and 1 HTLV-2) did not have quantifiable HTLV PVL DNA in either sample of which: two women had HTLV-1 provirus DNA amplified and detected by nested PCR only in the sample from pregnancy; one did not have detectable HTLV-1 DNA in either sample, whilst in the patient with HTLV-2 proviral DNA was detected in 1/4 replicates, only in the sample obtained whilst pregnant.HTLV PVL positively correlated with the antibody titre (S/CO) in both settings .Pregnancy is a very specific physiological state in which the woman must tolerate the foetus by undergoing many immune and morphophysiological changes . ImmunogOnce HTLV-1/2 reactivity has been detected the importance of further tests both to confirm and type HTLV-1/2 infection is well documented. Whilst more specific immunological tests such as WB and Immunofluorescence have been used as confirmatory tests for HTLV-1 infection (3) molecular tests are sometimes required especially for investigating indeterminate serology. In a recent study from Southern Brazil of 643 pregnant women Medeiros et al 18) reported that 0.6% tested positive in CMIA, of which 50% were confirmed by PCR. They found that negative-PCR samples had low S/CO values (1.3 and 2.5) in the Architect assay whereas the PCR-positive samples had high S/CO values (78.3 and 137.5) consistent with previously reported UK findings [ reportedIn a Japanese cohort HTLV-1 PVL was stable during pregnancy, with a slight increase after birth . In our Strong correlation between antibody reactivity and PVL in blood was already described among pregnant women at delivery  and in nIn conclusion, this study shows that pregnancy does not impair the diagnosis of HTLV-1/2, by either immunological (CMIA) or molecular (qPCR/nPCR) tests. Therefore, currently available tests can be used for antenatal screening and for confirmation of HTLV-1/2 infection allowing women to make informed decisions particularly in regard to infant feeding and onward transmission to their offspring.


An exercise pill contains bioactive compounds that mimic the beneficial effects of exercise without exercising3. Major efforts have focused on approaches to understand the molecular mechanisms underlying fatty acid oxidation, mitochondrial biogenesis, and oxidative fiber-type transformation induced by activation of peroxisome proliferator-activated receptor gamma coactivator-1α to determine whether compounds such as AICAR, GW1516, and resveratrol mimic the health effects of aerobic exercise4. According to a systematic review by Katashimaet al.,5 ursolic acid (UA) showed the same beneficial effects of aerobic and resistance exercises on sarcopenia and obesity.“If we had a pill that contained all the benefits of exercise, it would be the most widely prescribed drug in the world,” stated Ronald M. Davis, former president of the American Medical Association. Recently, efforts to search for an exercise pill has increased4,6. In regard to muscle mass, UA has beneficial effects similar to those of resistance exercise. UA improves insulin/insulin-like growth factor-1 (IGF-1) signaling, reduces atrophy by decreasing the expression of muscle RING-finger protein-1 (MuRF1) and muscle atrophy F-box (MAFbx)7, and induces muscle hypertrophy7-10. The effects of UA treatment when used alone have not been demonstrated in an animal model of atrophy with hindlimb immobilization (IM), but UA in combination with other treatments such as low-intensity treadmill exercise is effective in increasing skeletal muscle mass11. If UA shows beneficial effects similar to those exhibited by resistance exercise, it can be proposed as an exercise pill for the prevention of osteoporosis because resistance exercise is one of the best methods to strengthen bones12. Beginning with a study reporting that UA increases bone-forming activity13, several studies report that UA inhibits osteoclast production14,15 and bone loss16,17. However, no research has been conducted on the potential effects of UA as a resistance exercise mimetic that simultaneously ameliorates muscle atrophy and the deleterious effect on bones.UA is a pentacyclic triterpenoid present in many plants such as rosemary, basil fruit, apple, and coffee. It has a range of biological activitiesIn this study, the effects of dietary UA on muscle mass and bone microstructure were investigated using a Sprague-Dawley (SD) rat model of atrophy. The aim of this study was to demonstrate the potential of UA as a resistance exercise mimetic.This study used 18 8-week-old male Sprague-Dawley (SD) rats . Following an adaptation period of 1 week in a laboratory setting with constant temperature (21℃) and humidity (40–60%), IM was performed on the left hindlimb of SD rats to induce muscle atrophy and the right hindlimb was used as an internal control (IC). After a 10 day IM period, six rats were euthanized and examined for IM-induced atrophy and the corresponding levels of bone mineral density (BMD). SD rats were divided into two groups after immobilization, SED  and UA (n=6) groups, and treated with UA for the following 8 weeks. Body weight was measured using an electronic balance  once a week between 9 and 10 am before UA injection, and the skeletal muscles and bones were extracted from the euthanized rats after 8 weeks of treatment for analysis. Rats were euthanized by cervical dislocation under anesthesia. All rats were allowed free access to food  and water without restrictions.18. Rats had plaster casts on the left hindlimb only and the right hindlimb was reserved for use as IC, thus no treatment was given. During the IM period, rats were monitored daily for clinical conditions of the hindlimb such as edema and discoloration, and SD rats with such issues were excluded from the experiment.Rats were anesthetized with pentobarbital sodium  and immobilized using plaster casts11. UA was dissolved in distilled water containing 0.1% Tween 80  and administered by intraperitoneal injection (5 mg/kg) once per day . The SED group received the same dose of vehicle (distilled water containing 0.1% Tween 80) by intraperitoneal injection.UA treatment was carried out as per the same method used in a previous studyTissue collection was performed under anesthesia using pentobarbital sodium . Skeletal muscles such as gastrocnemius (GAS), soleus (SOL), tibialis anterior (TA), and extensor digitorum longus (EDL) were extracted and weighed using an electronic balance . The weight measurements were used to calculate the recovery rate for the IC.After skeletal muscles were collected, the hip joint area was cut and fixed in 10% paraformaldehyde, followed by micro-CT  imaging taken for bone microstructure assessment. For the cancellous bone of proximal tibia, CT Analyzer software  was used to analyze the BMD, trabecular bone volume fraction (BV/TV), trabecular number (Tb.N), and trabecular separation (Tb.Sp) within the region of interest (ROI) of the proximal growth plate. Three-dimensional (3D) images were generated using CT Vol Realistic Visualization software .Data are presented as mean ± standard error of the mean (SEM) using SPSS 12.0 . An unpaired t-test was conducted to compare skeletal muscle mass during the 10-day IM, and a one-way ANOVA was carried out to evaluate the improvements in skeletal muscle mass after 8 weeks of UA treatment. In addition, two-way ANOVA was used to determine the bone-related effects of 8-week UA treatment between the treatment conditions (SED and UA) and internal control (IC) or immobilization (IM). A Bonferroni post hoc test was performed, and the significance level was p<0.05.p<0.05). BMD was reduced by 49% (p<0.05).The effects of IM-induced hindlimb muscle atrophy on BMD were examined to evaluate the beneficial effects of UA treatment on muscle mass and the deleterious effect on bones caused by IM . Ten dayp<0.05) was only observed in the GAS muscle.p<0.05) due to IM, but no statistical difference was observed between IC and IM concerning the 8-week UA treatment (reatment . These rreatment . HoweverThe aim of this study was to demonstrate the beneficial effects of UA on muscle mass and bone microstructure, rather than explore concrete UA mechanisms for alleviating the deleterious effects of skeletal muscle atrophy on the bone.19-21. Therefore, resistance exercise for building muscle can be a highly effective treatment to preserve and increase BMD and bone strength22,23. However, due to the high risk of musculoskeletal injury24, resistance exercise has limited applications, especially in the elderly with less physical strength. The discovery of a substance that mimics the effect of resistance exercise, allowing users to achieve the positive effects of high-intensity resistance exercise with low-intensity exercise, will benefit many individuals who require resistance exercise, such as the one designed to alleviate the deleterious effect of muscle atrophy on the bone.Skeletal muscle-bone interaction involves crosstalk7,8,10. Recent studies show that UA prevents retinoic acid-induced bone loss16 and ameliorates the deleterious effect on bones in streptozotocin-induced diabetic mice17. Such beneficial effects of UA involve bone formation induced by the regulated expression of osteoblast-specific genes, such as mitogen-activated protein kinases, nuclear factor NF-κB, and activator protein-113. These effects are based on enhanced osteogenesis caused by NF-κB signaling15, tryptophan hydroxylase 125, and suppressed osteoclast differentiation. These effects of UA indicate potential as a bioactive compound that mimics the beneficial effects of resistance exercise on the bone. However, these discoveries alone are insufficient for UA to be accepted as a resistance exercise mimetic. To date, there have been no studies that determine whether UA can simultaneously decrease atrophy and alleviate the deleterious effects of atrophy on the bone, as is the case with resistance exercise. This study offers key findings in support of the suggestion that UA has potential as a resistance exercise mimetic. First, the effects of 8 weeks of UA treatment on atrophic skeletal muscles induced by hindlimb IM (UA reduces atrophy by inhibiting the expression of hypertrophy- and atrophy-related genes MuRF1 and MAFbx, and increasing the activity of the insulin/IGF-1 signaling pathwaydlimb IM  were exadlimb IM . In regadlimb IM . In the dlimb IM . In summary, this study showed that UA mimicked the beneficial effects of resistance exercise, but this discovery alone is inadequate for presenting UA as a resistance exercise mimetic. However, analysis of the relevant tissue pathways, which were not covered in this study, in combination with other modified conditions such as UA treatment period and dose, will allow a more definite conclusion to be drawn. Moreover, such a conclusion will be of great assistance in demonstrating that UA has potential as a resistance exercise mimetic for those who require resistance exercise but have limited capabilities.


The microstructures, morphologies, and electrochemical performances of MnO2NFs/NrGO were studied using X-ray diffraction (XRD), scanning electron microscopy (SEM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS), respectively. The electrochemical experiments showed that the MnO2NFs/NrGO composites have the largest effective electroactive area and lowest charge transfer resistance. MnO2NFs/NrGO nanocomposites displayed superior catalytic capacity toward the electro-oxidation of DA and UA due to the synergistic effect from MnO2NFs and NrGO. The anodic peak currents of DA and UA increase linearly with their concentrations varying from 0.2 μM to 6.0 μM. However, the anodic peak currents of DA and UA are highly correlated to the Napierian logarithm of their concentrations ranging from 6.0 μM to 100 μM. The detection limits are 0.036 μM and 0.029 μM for DA and UA, respectively. Furthermore, the DA and UA levels of human serum samples were accurately detected by the proposed sensor. Combining with prominent advantages such as facile preparation, good sensitivity, and high selectivity, the proposed MnO2NFs/NrGO nanocomposites have become the most promising candidates for the simultaneous determination of DA and UA from various actual samples.This study reports facile synthesis of MnO Dopamine (DA) and uric acid (UA) often coexist in the biological fluids, such as blood serum, urine, and extracellular fluids, which play a vitally significant role on the regulation of human physiological functions and metabolic activities . As an eUp until now, various detection approaches have been reported for detecting DA and UA, such as chemiluminescent , HPLC 1,11, fluo2 has been intensively utilized in energy storage, catalysis, and sensors because of its peculiar properties including low-cost, more abundance, high-catalytic activity, and environmental friendliness. Until now, a variety of nanostructured MnO2 such as nanowires , K3[Fe(CN)6], MnSO4, KMnO4, NaOH, H3PO4, and absolute ethanol were supplied by Sinopharm Chemical Reagent Co. Ltd. . All of the chemicals were analytically pure and used as received. NrGO was supplied by Nanjing Xianfeng NANO Material Tech Co. Ltd. . Human serum samples were provided by Zhuzhou People’s Hospital . The human samples are a mixture of residual serum from various individuals after clinical examination. Deionized water with the resistivity of 18.2 MΩ was used in all of the experiments.UA, DA, NaH2NFs and MnO2NFs/NrGO were investigated by powder X-ray diffractometry (XRD) and scanning electron microscopy (SEM), respectively. SEM images were taken from a cold field-emission SEM . The XRD patterns of MnO2NFs were collected using a powder XRD system  with monochromatized Cu Kα radiation (λ = 0.1542 nm), which was operated at 40 kV and 40 mA. Crystalline structures and surface morphologies of MnO2NFs was prepared by a slow addition of MnSO4 into KMnO4 solution followed by a simple stirring procedure. Typically, 1 mmol of KMnO4 and 1.5 mmol of MnSO4 were adequately dissolved into 20 mL deionized water, separately. Then, the MnSO4 solution was added dropwise into KMnO4 solution at a rate of 1 mL min−1, and agitated continuously at room temperature for 2 h. The resultant product was collected by centrifugation at 12,000 rpm, followed by cleaning alternately with absolute alcohol and deionized water three times, and dried at 60 °C in a vacuum oven overnight. Obviously, this route is time-saving and more convenient when compared with the conventional hydrothermal method. The MnO2NFs/NrGO composites were prepared as follows. Firstly, 10 mg MnO2NFs were uniformly dispersed in 10 mL deionized water under an ultrasonication bath for 0.5 h. Then 0.2 g NrGO nanosheets were added into the above MnO2NFs dispersion (1 mg mL−1) and dispersed under ultrasonication for 1 h. The MnO2NFs/NrGO were stored at 4 °C in a refrigerator when not used. To ensure good reproducibility, the MnO2NFs/NrGO were subjected to ultrasonication for 0.5 h before each modification.MnO2NFs/NrGO-modified GCE (MnO2NFs/NrGO/GCE) was prepared via a simple drop-casting approach. Specifically, 5 μL MnO2NFs/NrGO dispersion was carefully dropped and casted on the GCE surface with a micropipette, then dried with an infrared lamp to form a sensing film. For comparison, MnO2NFs and NrGO-modified GCEs  were also prepared via similar procedures.The bare glassy carbon electrodes (GCEs) were carefully polished using 0.3 μm and 0.05 μm alumina slurry, then alternately washed by anhydrous alcohol and deionized water several times, and allowed to dry under an infrared lamp. The MnO6]3−/4− as redox probe couples. EIS plots for different electrodes were recorded at open circuit potential using 5 mV (rms) AC sinusoid signal at a frequency range from 100,000 Hz to 0.1 Hz. The voltammetric responses of 10 μM DA and UA at different electrodes were tested by CV. After a suitable accumulation, linear scanning voltammetry (LSV) was performed for the determination of DA and UA. The potentials were scanned from 0 V to 0.8 V at 100 mVs−1 for both CV and LSV. For all the electrochemical tests, a typical three-electrode assemble was immersed into a 10 mL electrochemical cell, in which a bare or modified GCE was worked as the working electrode. Saturated calomel electrode (SCE) and platinum wire were used as a reference electrode and auxiliary electrode, respectively. To evaluate the electrochemical performance of various modified electrodes, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) was measured in the 0.1 M phosphate buffered solution , using 0.5 mM 3−/4− and 0.1 M KCl . It indicates that MnO2NFs/NrGO significantly improved electrochemical performances. It is well-known that the effective electroactive area is a critical factor that directly influences the electrochemical sensing performances. The effective electroactive areas of different electrodes were also calculated, using the Randles–Sevcik equation as follows  (7.6 × 10−6 cm2 s−1  concentration (mol cm−3). The effective electroactive areas were estimated to be 0.0770, 0.3183, 0.3958, and 0.7496 cm2 for the bare GCE, MnO2NFs/GCE, NrGO/GCE, and MnO2NFs/NrGO/GCE, respectively. The effective electroactive area of MnO2NFs/NrGO/GCE is about 9-fold higher than that of the bare GCE approximately. The results suggest that the MnO2NFs/NrGO nanocomposites significantly enlarged the effective electroactive surface area, which promoted the accumulation of target analysts and thus increased the response electrochemical signals. In order to assess the electrochemical performances, CVs for various modified electrodes were measured in a mixture solution of 0.5 mM 3−/4−. The results demonstrate that the MnO2NFs/NrGO can effectively decrease the Rct. EIS has intensively been used to investigate interfacial properties of various electrochemical sensors ,40,41,42 of NrGO ,36. As eipa(DA) = 1.126 μA, ipa(UA) = 0.385 μA), demonstrating sluggish kinetics for the electrooxidation of DA and UA. After modification of the GCE by MnO2NFs, the ipa(DA) increased a little (ipa(DA) = 3.766 μA) while the ipa(UA) was enhanced significantly (ipa(UA) = 12.73 μA), showing MnO2NFs have good electrocatalytic toward the oxidation of UA because of the presence of Mn4+/Mn3+ as an electron mediator. Moreover, the high-specific surface area also contributed to the obvious enhancement on the ipa(UA). When GCE was modified with NrGO, the ipa(DA) and ipa(UA) increased to 7.029 μA and 16.43 μA respectively, suggesting superb electrocatalytic activity toward the oxidation of DA and UA. The superb electrocatalytic activity of NrGO can explain the following facts. Nitrogen atoms in NrGO sheets may interact with target biomolecules via hydrogen bond, which can activate the amine and hydroxy groups and expedite the charge transfer process. Meanwhile, the π–π interactions between NrGO and target biomolecules can also facilitate the charge transfer process [2NFs/NrGO/GCE, and the anodic peak currents enhanced remarkably (ipa(DA) = 14.8 μA, ipa(UA) = 36.3 μA). The synergistic effect between MnO2NFs and NrGO sheets was mainly responsible for the enhanced response peak currents. Specially, MnO2NFs had higher catalytic activity toward the oxidation of DA and UA when the electrical conductivity was improved by coupling with NrGO sheets. Meanwhile, the hydrogen bond and π–π interactions between NrGO sheets and target biomolecules can also facilitate the charge transfer process. It is worth noting the biggest peak potential separation (about 150 mV) at MnO2NFs/NrGO/GCE, rendering this composite more selective for the simultaneous detection of UA and DA. Besides, the largest background current was also obtained at the MnO2NFs/NrGO nanocomposites, due to the high-specific capacitance of the MnO2NFs/NrGO nanocomposites [CV responses of 10 μM DA and UA (1:1) were measured at different electrodes in 0.1 M PBS (pH 3.93) . When thmposites .ipa(DA) gradually increased with the increase of pH, then decreased slowly as the pH rose to 7.01, and suddenly decreased when the pH exceeded 7.01. Obviously, the maximal ipa(DA) was achieved at pH 3.93. As for UA, the ipa(UA) show a downward trend, with pH varying from 2.58 to 8.52. To ensure the highest possible anodic peak current for DA and UA, pH 3.93 was selected for the following experiments. Moreover, the anodic peak potentials of DA and UA linearly decreased as pH was rising (Epa versus pH can be expressed as Epa(DA) = −0.0685 pH + 0.839 (R2 = 0.974) and Epa(UA) = −0.0639 pH + 0.679 (R2 = 0.976), respectively. Their slopes (68.5 pH/mV and 63.9 pH/mV) are close to 59 mV/pH, demonstrating the equal numbers of electron (e−) and protons (H+) involved in their electrooxidation processes [−) and two protons (2H+). The electrochemical oxidation process of DA and UA at the MnO2NFs/NrGO/GCE are illustrated in As known to all, the voltammetric responses of DA and UA highly depend on the solution pH. Therefore, it’s worthwhile to optimize pH. The dependences of pH on the anodic peak currents of DA and UA are shown in s rising B. The lirocesses . As reporocesses . Hence, −1 was recommended as the optimal scanning rate. As shown in v1/2), suggesting the electrooxidation of DA is a diffusion-controlled process. There was also a good relationship between the anodic peak currents of UA and the square root of the scanning rate . In this case, only the concentrations of the target substance were varied, while the concentrations of the other substance were kept unchanged. As illustrated in ipa(DA) and DA concentrations ranging from 0.4 μM to 10 μM. The linear equation is ipa(DA) = 1.3090CDA − 0.1953 (R2 = 0.989). However, the ipa(DA) were positively proportional to the Napierian logarithm of DA concentrations (lnCDA) in the concentration range of 10 μM–100 μM (ipa(DA) = 19.1371lnCDA − 32.3044 (R2 = 0.993). The LOD was estimated as 0.054 μM. Regarding the individual determination of UA, the ipa(UA) were well-linear to the working concentrations, varying from 0.4 μM to 6.0 μM (ipa(UA) = 2.4934CDA − 0.9302 (R2 = 0.989). At the higher concentration region (6.0 μM to 100 μM), the ipa(UA) were positively correlated to the Napierian logarithm of UA concentrations (lnCUA). The corresponding linear equation is ipa(UA) = 44.3228lnCUA − 65.7789 (R2 = 0.995). The LOD is 0.062 μM for the individual determination of UA. It is noteworthy that the addition of the target biomolecule does not have a notable interference on the electrochemical response signals of the other biomolecule. The results firmly imply that DA and UA can be sensitively and selectively detected at MnO2NFs/NrGO/GCE in the mixture of DA and UA. For individual detection of DA and UA at the MnOM–100 μM C,D. The o 6.0 μM A,B, with2NFs/NrGO enables simultaneous detection of DA and UA using the LSV method , the ipa(DA) and ipa(UA) enhanced linearly with their concentrations increasing (ipa(DA) = 3.0627CDA − 0.2848 (R2 = 0.991), and ipa(UA) = 3.0627CUA − 0.2848 (R2 = 0.990), respectively. However, the ipa(DA) and ipa(UA) are positively proportional to the Napierian logarithm of the DA and UA concentrations (lnCDA and lnCUA) at the higher concentration region from 6.0 μM to 100 μM (ipa(DA) = 16.2222lnCDA − 12.4506 and ipa(UA) = 37.7032lnCUA − 48.2926, respectively. The correlation coefficient is 0.990 for both DA and UA. The LODs are calculated to be 0.036 μM and 0.029 μM for DA and UA, respectively. All of the results indicate that the proposed MnO2NFs/NrGO/GCE featured wider linear detection ranges and a lower LOD for the electrochemical oxidation of DA and UA. The analytical performances were compared to those in previous works  were compared. The relative errors (less than 5%) were accepted even in the presence of 100-fold the above interfering species, demonstrating good selectivity (Ep = 0.260 V). To check the repeatability, eight successive determinations of 1 μM DA and UA (1:1) were also performed. The relative standard deviation (RSD) for DA and UA were 6.35% and 5.32%, respectively, indicating good repeatability. To examine electrode reproducibility, the anodic peak currents of 1 μM DA and UA were recorded at five MnO2NFs/NrGO/GCEs, which were prepared by similar procedure. The RSD of the anodic peaks were 6.02% and 4.70% for DA and UA respectively, showing that the electrode preparation have excellent reproducibility.Before real sample detection, the selectivity and repeatability, as well as reproducibility were also evaluated. To assess the anti-interfering ability of the proposed MnOectivity . It is n2NFs/NrGO/GCE. The determination results were calculated from the calibration curves (To validate practicability, the concentrations of DA and UA in human serum samples were also detected on MnOn curves . To furt2NFs/NrGO nanocomposites and their application on the simultaneous determination of DA and UA. The electrochemical measurements indicated that the MnO2NFs/NrGO composites possess a large, effective electroactive area and low-charge transfer resistance. MnO2NFs/NrGO/GCE showed superb catalytic capacity toward the electrooxidation of DA and UA, attributing to the synergistic effect from MnO2NFs and NrGO sheets. The anodic peak currents of DA and UA increased linearly with their concentrations varying from 0.2 μM to 6.0 μM. However, their anodic peak currents were highly correlated to the Napierian logarithm of their concentrations, ranging from 6.0 μM to 100 μM. The LODs were 0.036 μM and 0.029 μM for DA and UA, respectively. Furthermore, the proposed sensor successfully realized DA and UA detection in human serum samples with satisfactory recovery. Combining with prominent advantages such as facile preparation, high sensitivity, good selectivity, repeatability, and reproducibility, the proposed MnO2NFs/NrGO nanocomposites have become the most competitive candidates for the simultaneous determination of DA and UA in various real samples.In summary, this paper reported the facile synthesis of MnO


Human T cell lymphotropic virus 1 (HTLV-1) is a human retrovirus and infects approximately 10–20 million people worldwide. While the majority of infected people are asymptomatic carriers of HTLV-1, only 4% of infected people develop HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HAM/TSP is a chronic, progressive, neurological disease which usually progresses slowly without remission, and is characterized by perivascular inflammatory infiltrates in chronic inflammatory lesions of the central nervous system (CNS), primarily affecting the spinal cord. A high HTLV-1 proviral load, high levels of antibodies against HTLV-1 antigens, and elevated concentration of proteins are detected in cerebrospinal fluid (CSF) of HAM/TSP patients. These chronically activated immune responses against HTLV-1 and infiltration of inflammatory cells including HTLV-1 infected cells into the CNS contribute to clinical disability and underlie the pathogenesis of HAM/TSP. Since the disease development of HAM/TSP mainly occurs in adults, with a mean age at onset of 40–50 years, it is important for HTLV-1-infected carriers and HAM/TSP patients to be monitored throughout the disease process. Recent advances in technologies and findings provide new insights to virological and immunological aspects in both the CNS as well as in peripheral blood. In this review, we focus on understanding the inflammatory milieu in the CNS and discuss the immunopathogenic process in HTLV-1-associated neurologic diseases. Human T lymphotropic virus type 1 (HTLV-1) is a human retrovirus that is associated with a persistent infection in humans , HTLV-1 The central nervous system (CNS) was thought as an immune-privileged site with no lymphatic drainage but it is now recognized to mount robust immune responses to various viral infections of the CNS that is unique from the immune response in peripheral tissues. The pathogenesis of HAM/TSP has been demonstrated to involve strong inflammatory responses in the CNS, with perivascular inflammatory infiltrates in the brain and spinal cord . Unlike Before the discovery of HTLV-1, clinical observations with unusual prevalence of spastic paraplegia have been reported mainly from the Caribbean islands. In the mid 1980s, seroprevalence studies in the Caribbean islands and Japan demonstrated that HTLV-1 specific antibodies existed in high proportion of patients with the disorder, subsequently designated as HAM/TSP , 4. CurrAlthough Europe and North America are often considered as nonendemic areas for HTLV-1 infection, high rates of HTLV-1 infection have been reported in some regions of Europe and North America where most HAM/TSP patients originated from HTLV-1 endemic areas, such as the West Indies, Africa, Caribbean and South America . Recentl+ T cell responses to the virus, which control HTLV-1 provirus load (PVL) in the host and influence the susceptibility to HTLV-1-associated diseases. The HLA class I genes, HLA-A*02 and HLA-Cw*08, were associated with significant reduction in PVL and protective effect from HAM/TSP in Southern parts of Japan [HLA-A*02 was also observed in Brazil [HLA-A*02 and HLA-Cw*08, showed stronger binding of an HTLV-1 basic leucine zipper factor (HBZ) peptide, which was associated with lower HTLV-1 PVL and risk of HAM/TSP [HLA-B*07 and HLA-B*5401, and the class II allele, HLA-DRB1*0101, were associated with a higher susceptibility to HAM/TSP [Unlike human immunodeficiency virus-1 (HIV-1), the genetic variations of HTLV-1 are minimal both within and between hosts and there is no HTLV-1 strain or sequence variants directly associated with any disease outcome , 18. In of Japan , 22. A pn Brazil . In addi HAM/TSP . In cont HAM/TSP , 22, 25.IL10-592A) was associated with a two-fold reduction in the odds of developing HAM/TSP in Japan [IL6-634C) was detected at a higher frequency in HAM/TSP patients than in asymptomatic carriers while the association of IL10-592A polymorphism was not observed [TNF-863A) of the inflammatory cytokine tumor necrosis factor (TNF), has been reported to be also associated with the risk of HAM/TSP [SDF1 + 801A) and IL-15 (IL-15 + 191C) were associated with reduction in the risk of developing HAM/TSP [Analysis of single nucleotide polymorphisms (SNPs) demonstrated the associations of some host genes with the outcome of an HTLV-1 infection. A polymorphism in the promoter of the immunosuppressive cytokine Interleukin-10 , a ligand of CXCR3 [+CD4+ T cells into the CNS of HAM/TSP patients.CD4ng IFN-γ . CD4+CD2patients . Furtherseverity , 63, 64.of CXCR3  and sugg+ T cells (Treg) that constitutively express CD25 (the IL-2 receptor α chain) are engaged in the maintenance of immunologic self-tolerance by suppressing the activation and expansion of self-reactive lymphocytes that may cause autoimmune diseases [+CD25+ T cells due to decreased demethylation of the Foxp3 gene, which caused the decreased suppressive capacity of CD4+CD25+ T cells and stimulation of HTLV-1 Tax-specific CD8+ T cells in HAM/TSP patients [+ T cells [+CD25+ T cells of HAM/TSP patients [Regulatory CD4diseases . Howeverpatients , 67, 68.patients . Helios, T cells . A coinhpatients .+ T cells are routinely exposed in vivo to alterations in the microenvironment, which was associated with enhanced production of various soluble factors as well as expression of cell adhesion molecules due to activation of HTLV-1 expression. Since interaction of cell adhesion molecules induces recruitment and extravasation of lymphocytes through the BBB, increased expression of cell adhesion molecules may facilitate the migration of HTLV-1-infected lymphocytes across the BBB endothelium. Tax has been demonstrated to regulate cell adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and cell adhesion molecule 1 (CADM1/TSLC1) in HAM/TSP patients [+CD4+ T cells expressed higher coinhibitory molecule, T cell immunoglobulin and ITIM domain (TIGIT), in HAM/TSP patients compared to healthy controls [+CD25+ T cells from HAM/TSP patients [The CNS is protected from the entry of pathogens, circulating immune cells, and factors within the blood by physiological structure called the blood–brain barrier (BBB) which is maintained by the endothelial cells of cerebral microvessels with tight junctions. CD4patients –73. CADMcontrols . Activatpatients .+ T cells are an important immune population that serve as the major reservoir of HTLV-1 due to HTLV-1 expression and can also have dynamic functional changes including cell migration, activation and dysregulation.Thus, CD4+ T cells (CTLs) [+ T cells play a crucial role in immunity against HTLV-1 through their ability to secrete various factors that suppress viral replication and kill infected target cells in HTLV-1-infected subjects [+ T cells are more prevalent than CD8+ T cells in CSF lymphocytes of healthy individuals, in the CSF of HAM/TSP patients there was an increased predominance of CD8+ T cells over CD4+ T cells [+ T cells was higher in CSF than in peripheral blood, and was correlated with HTLV-1 PVL [+ T cells as well as CD4+ T cells expressing HTLV-1 Tax proteins were detected in the parenchyma of HAM/TSP spinal cords, suggesting that the interaction between HTLV-1-specific CTLs and HTLV-1-infected CD4+ T cells may cause bystander damage to resident cells in the CNS [+ T cells, a low frequency of HTLV-1 HBZ-specific CD8+ T cells are detected in peripheral blood of asymptomatic carriers and HAM/TSP patients, and HBZ-specific CTL clones were able to lyse naturally infected cells [+ T cells play in the pathogenesis of HAM/TSP.Tax is an immunodominant antigen recognized by HTLV-1-specific cytotoxic CD8s (CTLs) . CD8+ T subjects , 78. How T cells . ImportaLV-1 PVL , 80, 81. the CNS . In conted cells . Howevered cells , 83. It + T cells and HTLV-1 Tax-specific cytotoxic CD8+ T cells. The common γ chain family of cytokines including IL-2, IL-7, IL-9, IL-15, and IL-21 play an important role in lymphocyte proliferation, survival and function during immune responses and homeostasis. Tax has been shown to transactivate a number of the common γ chain family of cytokines and the receptors, such as IL-2/IL-2R, and IL-15/IL-15R [+ T cells, it has been suggested that IL-2/IL-2R and IL-15/IL-15R autocrine loop may contribute to the pathogenesis of HAM/TSP [+ T cells, stem cell-like memory T cells (Tscm), has been reported to be identified as a naïve phenotype but express increased level of CD95, IL-2Rβ, CXCR3 and LFA-1 and have similar functions to memory T cells including the ability to proliferate rapidly and release inflammatory cytokines in response to antigen re-exposure [+ T cells might be sustained through cytokine-driven homeostatic proliferation to achieve long-lived protection against chronic HTLV-1 infection [One of the most striking features of the cellular immune response in HAM/TSP patients is the increased numbers of memory and/or effector CD85/IL-15R . Since bexposure . Recentlnfection . Using anfection . Since t+ T cells initially acquire effector functions but gradually become less functional as the infection progresses. In HTLV-1 infection, although HTLV-1-specific CTL responses were detected in both asymptomatic carriers and HAM/TSP patients, high expression of IFN-γ in CD8+ T cells specifically in HAM/TSP patients compared to asymptomatic carriers have been reported to be induced by interaction with virus-infected CD4+ T cells and CD8+ T cells [+ T cells in patients with HAM/TSP, but not in asymptomatic carriers, were demonstrated to spontaneously degranulate and produce IFN-γ [IL-15 mRNA and IL-15 protein is up-regulated in non-T cells and CD14+ monocytes more than those in healthy controls or asymptomatic carriers [+ T cells showed higher expression of IL-15Rα, compared with CMV pp65-specific CD8+ T cells [+ T cells, but not CMV pp65-specific CD8+ T cells, to induce degranulation and IFN-γ expression [+ T cells.During chronic viral infection, antigen-specific CD8 T cells –91. Intece IFN-γ . In HAM/carriers , 92. In  T cells . It was + T cell responses during chronic viral infection are regulated by complex patterns of co-expressed stimulatory and inhibitory receptors. A number of costimulatory and coinhibitory receptors has been reported to be involved with HAM/TSP. The expression of CD244 (2B4), a signaling lymphocyte activation molecule (SLAM) family receptor, was significantly higher on CD8+ T cells in both asymptomatic carriers and HAM/TSP patients, than those on healthy controls [+ T cells as well as CMV-specific CD8+ T cells in a patient with HAM/TSP. Importantly, SLAM-associated protein (SAP) which play a role in controlling the active transduction was overexpressed in HAM/TSP patients compared to asymptomatic carriers and healthy controls whereas there was no significant differences of expression of EAT-2, a SLAM-related inhibitory protein. Blockade of CD244 and SAP inhibited degranulation and IFN-γ production in CD8+ T cells of HAM/TSP patients, suggesting that CD244/SAP pathway might be involved in the active regulation of HTLV-1-specific CD8+ T cells of HAM/TSP patients [+ T cells and HTLV-1-specific CD8+ T cells of HAM/TSP patients [+ T cells were compared among healthy controls, patients with HAM/TSP and Progressive Multifocal Leukoencephalopathy (PML) which is a rare, often fatal, demyelinating disease caused by reactivation of the ubiquitous JC virus, HAM/TSP patients as well as PML patients showed a significant higher percentage of PD-1 expression on CD4+ and CD8+ T cells in CSF than healthy controls [+ T cells in HAM/TSP patients remains to be determined, restoration or modulation of virus-specific CD8+ T cells in HAM/TSP patients may be important to prevent further tissue damage in the CNS and progression of HAM/TSP.CD8controls . High CDpatients . Furtherpatients , 96. Altpatients –98. A recontrols . AlthougIntrathecal antibody synthesis is a well-documented phenomenon in infectious and demyelinating neurological diseases. As definitive laboratory diagnosis of HAM/TSP is based on the presence of anti-HTLV-1 antibodies in the blood and CSF. Robust humoral immune responses against HTLV-1 antigens have been reported in peripheral blood and CSF of HAM/TSP patients. Intrathecal antibody synthesis against HTLV-1 has been demonstrated, as evidenced by the presence of HTLV-1-specific antibodies and oligoclonal IgG bands in CSF of patients . Intrath+ and CD8+ T cells were detected in spinal cords of HAM/TSP patients and the frequency of these T cell populations varied depending on the duration of illness. B cells were only rarely observed in HAM/TSP spinal cords by in situ histopathological studies [+CD25+ T cells significantly correlated with antibody secreting B cells and HTLV-1 PVL in the CSF of HAM/TSP patients [+ T cells (Tfh), which promote B cell growth, differentiation and class switching, were decreased in the CSF of HAM/TSP patients [Regardless of the presence of HTLV-1-specific antibodies and oligoclonal IgG bands in CSF of HAM/TSP patients, little is known about the CNS microenvironment related to this increased humoral immune response in HTLV-1-infected individuals. In vivo, T cells including both CD4 studies . A recen studies . Antibod studies . By comppatients . These fpatients . B cell As HTLV-1-specific antibody responses have been associated with the pathogenesis of HTLV-1-related diseases, strong antibody responses against HTLV-1 antigens have been reported in both serum and CSF of HAM/TSP patients. Analysis of HTLV-1 specific antibodies in CSF of HAM/TSP against synthetic peptides of HTLV-1 Gag and Env proteins demonstrated that a diverse intrathecal immune response to several HTLV-1 synthetic peptides, most frequently against Gag p19 (100–130), Env gp21 (458–488), and Env gp46 (175–199 and 288–317) , 103. Mo+ T cells and promotes the development of effector and memory T cells. Higher levels of soluble OX40 was detected in the CSF of HAM/TSP patients with rapid progression, and OX40 was overexpressed in spinal cord infiltrating mononuclear cells in a clinically progressive HAM/TSP patient with a short duration of illness [Other than HTLV-1-specific antibodies, several useful or prognostic biomarkers have been reported in HAM/TSP patients. Neopterin, a derivative of pyrimidine metabolism and a useful marker of activated monocytes and macrophages, has been reported to be elevated in CSF of HAM/TSP patients . The con illness .+ cells have been also detected in the CSF of HAM/TSP patients [HTLV-1 Tax protein directly induces upregulation of various cytokines/chemokines. Increased concentrations of IL-1β, IL-6, GM-CSF and IFN-γ has been reported in HAM/TSP CSF –111. In patients . In the patients .+ astrocytes were detected in the spinal cord lesions of patients with HAM/TSP than in control patients, suggesting that in the HAM/TSP spinal cords, astrocytes are the main producers of CXCL10 [Some chemokines, such as CXCL9, CXCL10, CCL3, CCL5 and CCL11, have been reported to be elevated in the CSF of HAM/TSP compared to those of HTLV-1 infected individuals or other noninflammatory neurologic diseases , 114–116f CXCL10 . In addif CXCL10 . Followif CXCL10 . Changes+CD25+ T cells ex vivo and sensitize target cells for lysis of HTLV-1 specific cytotoxic CD8+ T cells [Recent evidences demonstrate that extracellular vesicles, including exosomes, play critical roles in viral pathogenesis and control of host immune responses to viral infection. These microvesicles contain host and viral components, including proteins, mRNA, and microRNA . HTLV-1  T cells . Importa T cells . These fRecently, new evidences are accumulating that a proportion of HTLV-1-infected individuals also have neurological symptoms without fulfilling criteria for HAM/TSP. It has been reported that early neurological disorders were present in 24% of HTLV-1-infected individuals who were initially considered asymptomatic. These patients had sufficient signs and symptoms to classify them in a novel category of disease, called intermediate syndrome . In addi−/−γc−/− (Rag1) and Bone marrow Liver Thymic (BLT) mouse models for engraftment of human CD34+ hematopoietic stem cells demonstrated susceptibility to HTLV-1 infection with the presence of Tax in spleen and the CNS [Animal models provide a useful tool for the studies of infection, pathogenesis, treatment and prevention. Various stages of HTLV-1 infection and disease development have been studied using several animal models including naturally infected nonhuman primates and experimental animals such as rabbits, rats and mice . Mouse m the CNS . Howevertax mRNA, the frequency of HTLV-1-specific CD8+ T cells and spontaneous lymphoproliferation. Although HTLV-1 PVL remained stable, some measures of motor function were improved [+ T cells have been used and demonstrated some improvements in HAM/TSP patients. A humanized monoclonal antibody against IL-2 receptor α chain (anti-Tac) demonstrated reductions of HTLV-1 PVL in peripheral blood and spontaneous lymphoproliferation in HAM/TSP patients [+ T cells, demonstrated inhibition of aberrant CD8+ T cell functions including spontaneous lymphoproliferation and degranulation and IFN-γ expression [To date, several trials of antiretroviral drugs and immunomodulatory therapies have been reported in HAM/TSP. Corticosteroids are most commonly used for therapy of HAM/TSP patients, which showed some beneficial effects, such as reduction of inflammation at early stage and improvement of motor disability . The twoimproved . A numbepatients . A recenpatients . A humanpression .While not an exhaustive review of all trials in HAM/TSP, no therapy has been shown to dramatically clear HTLV-1 infection and significantly modify the long-term disability associated with HAM/TSP. The failure to detect any clinical improvement after therapy may be due to the long disease duration in HAM/TSP patients. After such prolonged periods of time, the neurological damages may not be reversible. In addition, the progression rate of HAM/TSP varies widely among patients. Therefore, early diagnosis and prompt treatment are necessary for successful disease prevention and long-term improvement of motor disability and quality of life for HAM/TSP patients.Regulation of the local immune response is crucial in protecting the CNS from viral infection and immunopathologically mediated tissue damage. Characterization of HTLV-1 infection and CSF immune responses that are associated with a neuroinflammatory milieu may provide evidence for a pathogenic signature of an immunopathogenic process in HAM/TSP. These findings may contribute to identifying biomarkers that could detect disease progression in the early stages in HTLV-1-infected individuals. Elimination of HTLV-1 infection and control of HTLV-1 reactivation from latency remain a goal for HAM/TSP. Symptomatic management directed at the immune response to HTLV-1 is also important for patients with HAM/TSP.


Standard Compression-After-Impact test devices show a weakening effect on thin-walled specimens due to a free panel edge that is required for compression. As a result, thin-walled undamaged samples do not break in the free measuring area but near the free edge and along the supports. They also show a strength reduction due to the free edge which can become potentially relevant for very weakly damaged panels. In order to reduce the free edge influence on the measured strength, a modified Compression-After-Impact test device has been developed. In an experimental investigation with carbon fiber reinforced plastics, the modified device is compared with a standard device. It is shown that thin-walled undamaged specimens investigated with the modified device now mainly break within the free measuring area and no longer at the free edge and along the bearings as it is the case for standard test devices. The modified device does not cause a free edge weakening effect in comparison to standard devices. The modified device is therefore more suitable for determining the compression strengths of undamaged thin-walled composite plates. Thin-walled fiber-reinforced plastics, in particular, carbon fiber reinforced plastics (CFRP), are very susceptible to reductions in strength due to low velocity impact damages. In aeronautics, this strength loss has to be considered during construction by a sufficient damage tolerance leading to weight penalties. Therefore, it is important to understand the failure mechanisms of impact damaged composite structures in order to improve the damage tolerance of CFRP structures. The influence of impact damages on the static behavior is typically investigated by Compression-After-Impact (CAI) testing as the residual compressive strength is one major property that significantly decreases due to impact damages. It is a well standardized test  or by CIf the in-plane compression strength of undamaged samples is compared to the residual CAI strength of damaged ones on the one hand, a structural strength value is compared with an in-plane material characteristic. The in-plane material characteristic ideally lacks completely an out-of-plane deformation since the free measuring length in the load direction is small with about 10 to 25 mm according to ISO 14126 . HoweverIf the CAI strength is intended to be used for undamaged specimens to quantify the damage severity on the other hand, the measured CAI strength of the undamaged panel is usually reduced due to a free sliding edge which is required for compressing the panel. For a plate damaged with sufficient impact energy, this is generally unproblematic as the damage weakens the plate more than the free edge. The plate then breaks in the area of the damage. However, in the case of an undamaged or only very slightly damaged plate, the weakening of the plate due to the damage may be less than that caused by the free edge. For this reason, the fracture of such samples almost always occurs in the area of the free edge . ConseqVarious modifications to CAI test devices are proposed in scientific literature. These differ primarily as a result of the different requirements that CAI testing equipment must meet.Many articles deal intensively with the prevention of global buckling by the use of adequately modified CAI devices ,16,17,18Global buckling is basically shifted to higher buckling loads by two main measures. One reduces the free area in the CAI equipment. With the other, additional bearings are applied without changing sample geometry. Typically, the geometry still corresponds to the dimensions according to standardized CAI tests in this case.The reduction of the free area is achieved either by applying double-sided stabilizing plates  of the top part of the specimen is loaded whereas the bottom part is completely restrained and does not move. The standard test device under consideration for the investigation is the Instron “Airbus” Compression Impact Fixture  which agrees with the standards AITM 1-0010 . This deCAI testing using the CAI-standard for undamaged thin-walled composite specimens typically leads to failure at the top clamped boundary starting usually at the free edge of the specimens. Failure does not typically occur in the free range of the specimen like it is the case for damaged specimens. This is due to the fact that the weakening effect of the free edges is greater than the one of plate imperfections. E.g., geometric plate imperfections lead to a theoretical maximum plate bending in the middle of the free measuring area. Since the free edges at the two upper and two lower corners as well as their support along the width significantly reduce the failure load of undamaged specimens, these areas have been modified in a new CAI testing device which is called CAI-modified below. It is a CAI testing device where the boundaries at all four corners are changed in order to stiffen the free edge area. The free edges in the lower part of the device are not required for compression. Therefore, they are replaced by clamped supports. a is selected, the stiffer the plate becomes near the free edge. The edge with length b primarily acts as a simple support allowing a rotation around the global y-axis. Clamping consequently leads to the constraint of the rotational degree of freedom and thus to stiffening. The dimension c of the clamped support influences the rotational degrees of freedom around the x- and y-axis in the area where the lateral knife edge support acts. The further the clamping extends into the area between the lateral simple supports  as well as a twill weave epoxy prepreg  which is processed by compression moulding through Clip Carbono  to plane plates with thickness of 2.2 mm. Both outer layers are composed of twill carbon weave (CW200-0/TW2/2 with 200 g/mrding to ). Carbonrding to ) are posrding to . The fibSpecimens are investigated in an undamaged condition because it is intended to check whether the modifications of the CAI-modified device lead to failure within the free range of the specimens and not at the supports of the loaded edges where it is expected for the CAI-standard. Five specimens are tested each with the CAI-standard respective CAI-modified device. Sample geometry for CAI-standard testing agrees with the one according to , except Measurements using the CAI-standard device are carried out with a universal material testing device . The compression is displacement controlled with a rate of 0.5 mm/min. The displacement data is obtained from the optical strain system Shimadzu Digital Video Extensometer TRViewX . The data recording rate amounts to 1 Hz. The shortening of the samples is measured between two lines having a distance of 50 mm each to the plate center respective middle of the measuring range. The CAI-modified testing is performed with a servo-hydraulic material testing device  equipped with Instron measuring instrumentation . The compression is load controlled with a rate of 0.25 kN/s leading to an averaged displacement rate of about 0.5 mm/min. Displacements are measured with the stereo pair camera digital imaging correlation (DIC) system ARAMIS . Displacements are detected on the sample surface as far as both cameras have free view on the sample. The data recording rate is 1 Hz.The fracture of the specimens tested with the CAI-standard device is illustrated in F is used as failure load. It is computed by the measured failure load divided by the specimen area. The red lines in The fracture of the specimens tested with the CAI-modified device is illustrated in This is also valid for specimen P2-S10 although the red lines are not completely connected on the surface. Fracture that is limited to the free range of the plate occurs for the specimens P2-S3, P2-S5, P2-S8 and P2-S9. Partially, fracture can be observed at the clamped support (for specimens P2-S5 and P2-S9) but these fractures are isolated and are not linked to the fracture line running through the free range of the plates. Contrary to that, specimen P2-S10 exhibits a mixed fracture, i.e., the fracture line runs at the clamped support into the free range of the plate. Its failure stress is lower than the ones for the specimens with fracture in the free range only see . But theF as well as the corresponding standard deviation s are computed based on the data according to The mean values of failure stress σThe mean stress versus the shortening of the samples is shown in In principle, all samples exhibit a linear trend between mean stress and shortening at the beginning of the measurement. According to DIC data of the CAI-modified testing, a global buckle starts to occur typically at 100 MPa which is then fully established of about 140 MPa in the plate center. From there on, the shape of the buckle remains constant. Only the absolute value of the out-of-plane displacement increases till failure . Therefore, a thin-walled specimen investigated with the CAI-modified device will probably exhibit a higher failure load so that the increase of the mean failure stress is expected to be partly caused by the reduced free measuring area. However, this is probably of minor influence as similar quasi-isotropic CFRP laminates according to 12] respeThe standard deviation of the mean failure stress is increased in CAI-modified testing compared to CAI-standard from 2.8% to 3.7% of the mean value. However, this increase is mainly caused by an undesirable failure mode in CAI-modified testing where the rupture runs partially along the loaded upper support (cp. sample P2-S10 in The fractures lines of the specimens tested with the CAI-modified device are no longer positioned at the bearings but within the free measuring area also indicating that the weakening effect of the free edge no longer is relevant. But it has to be outlined that these lines are not positioned in or near the center of the free measuring area although their occurrence might be expected there due to the theoretical maximum bending loading. They occur in the upper part of the specimens. But no rupture is observed in the lower part. This phenomenon is unclear, in particular, as fracture modes of undamaged plates concerning CAI testing are not reported in open literature. However, there is a high probability that this is at least partly influenced by the device. The methodology followed is easily extensible to other combinations of fabrics or matrices, so its study is proposed for future work . Due to the modification of the CAI test device at the loaded edge, the place of fracture of thin-walled CFRP samples no longer occurs at the clamped support only as it can be observed for standard CAI testing devices. The fracture lines usually run through the free range of the specimens. Furthermore, the failure stress is clearly increased indicating that the free edge of standard CAI devices weakens thin-walled samples in CAI testing. The intended aim of the research work is therefore achieved by the CAI-modified test device. The modified CAI testing device enables a more accurate measurement of CAI strengths of undamaged specimens. The modifications can be easily transformed to other CAI testing equipment as these usually work with similar loading edges.


Hypoxia-mimicking conditions, which include NiCl2, CoCl2, and DMOG, an inhibitor of 2-oxoglutarate-dependent enzymes, also selectively inhibited TNF-α-induced TSLP expression. These results suggested that inactivation of prolyl hydroxylase by hypoxia and hypoxia-mimicking conditions is involved in the repression of TNF-α-induced TSLP expression. Interestingly, the inhibition of TSLP production by hypoxic treatment was significantly reversed by treatment with the HIF-2α antagonist but not with the HIF-1α inhibitor. DMOG-induced inhibition of TSLP promoter activity was dependent on the -71 to +185 bp promoter region, suggesting that the binding of HIF-2 to hypoxia response element (HRE) in this region repressed the TSLP expression. These results indicated that hypoxia and hypoxia-mimicking conditions inhibited TSLP expression via HIF-2 and HRE-dependent mechanisms. Therefore, PHD and HIF-2α could be a new strategy for treatment of atopic dermatitis and psoriasis.The expression of thymic stromal lymphopoietin (TSLP), a cytokine which greatly contributes to the induction of type I allergy, is upregulated in chronic inflammation such as atopic dermatitis and psoriasis. As hypoxia in the epidermis is important for maintaining skin homeostasis, we examined the regulation of TSLP expression by hypoxic conditions in normal skin epithelial tissues. TNF-α-induced expression of TSLP in human keratinocyte HaCaT and in mouse keratinocyte PAM212 cell lines were inhibited under hypoxic condition (1% O Thymic stromal lymphopoietin (TSLP) is considered a master switch for allergic inflammation . TSLP is5′-GACATG-3′ [TM Bullet kit) were purchased from Nissui Pharmaceutical Co , Life Technologies  and Lonza , respectively. FBS was purchased from Biowest . Antibodies used were anti-HIF-1α , anti-HIF-2α , and anti-actin antibodies . TSLP promoter reporter plasmid [Human and mouse recombinant TNF-α proteins were purchased from Peprotech . Nickel chloride NiCl, cobalt  plasmid  was a gi2−95% air in 10% FBS-DMEM and 10% FBS-MEM-α, respectively. A total of 0.7 × 105 HaCaT cells/ml in KGM were seeded into 24 well plates. Two days later, fresh medium was added. In the next day, the cells were incubated for 8 h or indicated time under hypoxia (1% O2-5% CO2-94% N2) or normoxia (5% CO2-95% Air), or pretreated with NiCl2, CoCl2 or DMOG at the indicated concentrations for 2 to 24 h, and then stimulated with TNF-α (100 ng/ml). After HaCaT cells were pretreated under hypoxia, the cells were stimulated by TNF-α under hypoxia. PAM212 cells were seeded in 10% FBS-MEM-α in 24 well plates. Under 80% confluent conditions, PAM212 cells were treated the same way as with HaCaT cells.The human keratinocyte HaCaT  and the mouse keratinocyte PAM212 cell lines  were used in this study. HaCaT cells and PAM212 cells were cultured at 37°C under a humidified atmosphere of 5% CO5′-CTATCCAGCGTACTCCAAAG-3′ and R: 5′-GAAAGACCAGTCCTTCCTGA-3′, mouse β2-microglobulin F: 5′-TTCTGGTGCTTGTCTCACTGA-3′ and R: 5′-CAGTATGTTCGGCTTCCCATTC-3′, human TSLP F: 5′-GATTACATATATGAGTGGGAC-3′ and R: 5′-TTCATTGCCTGAGTAGCAT-3′, human Vascular Endothelial Growth Factor A (human VEGF-A) F: 5´-AGGCCAGCACATAGGAGAGA-3´ and R: 5´-TTTCTTGCGCTTTCGTTTTT-3´, IL-8 F: 5´-CAATCCTAGTTTGATACTCCC-3´ and R: 5´-AATTACTAATATTGACTGTGGAG-3´, Filaggrin F: 5´-CAATCAGGCACTCATCACAC-3´ and R: 5´- ACTGTTAGTGACCTGACTACC-3´, IL-6 F: 5´-AACAACCTGAACCTTCCAAAGA-3´ and R: 5´-TCAAACTCCAAAAGACCAGTGA-3´, Monocyte Chemotactic Protein-1 (MCP-1) F: 5′-GATCTCAGTGCAGAGGCTCG-3′ and R: 5′-TGCTTGTCCAGGTGGTCCAT-3´, TNF-α F: 5′-TGTAGCCCATGTTGTAGCAAC-3′ and R: 5´-TTGAAGAGGACCTGGGAGTAGA-3´, mouse TSLP F: 5´-CGAGCAAATCGAGGACTGTGAG-3´ and R: 5´-GCAGTGGTCATTGAGGGCTTC-3´, mouse Vascular Endothelial Growth Factor (mouse VEGF) F: 5´-CTGGCTTTACTGCTGTACCTC-3´ and R: 5´-CATGGTGATGTTGCTCTCTGAC-3´.Total RNA of the stimulated cells was extracted with RNAiso Plus  according to the manufacturer's instructions. Total RNA was reverse-transcribed using the PrimeScript RT reagent kit (Takara) and then PCR-amplified with Takara PCR Thermal Cycler Dice (Takara) using SYBR Premix Ex Taq II (Takara). The following oligonucleotides, F (forward) and R (reverse), were used for PCR: human β2-microglobulin F: After the stimulation with TNF-α, the medium was changed to KGM (250 μl/well), and MTT was added (25 μl/well). Four hours later, the medium was removed and DMSO was added into each well (250 μl/well) to dissolve the insoluble formazan product. Absorbance at 595 nm was determined.2 and CoCl2 for 8 h, the cells were lysed in the lysis buffer that contained protease inhibitor cocktail (20 mM HEPES buffer including 1% (v/v) Triton-X 100, 10% (v/v) glycerol, 1 mM EDTA, 10 μg/ml leupeptin, 10 μg/ml phenylmethylsulfonyl fluoride, 50 mM NaF, 1 mM Na3VO4 and 2.5 mM p-nitrophenyl phosphate). HIF-1α, HIF-2α and actin were detected by Western blot.After the treatment with Hypoxia, NiCl5′-GCCCTGTAGGAGAAAGACACTGGTATC-3′, (-3732 bp forward) 5′-TTTTCTTAATCCAAAGAGACAGATCTCCC-3′, (-3214 bp forward) 5′-CAAACTACGTATGCAGATACTGTTCAC-3′, (-1330 bp forward) 5′-AGGAACTTCCCAAGGACCAG-3′ and (reverse) 5′-TATCGATAGAGAAATGTTCTGGCACCTGC-3′, (Deletion -71 to +185 bp forward) 5′-CTCGAGATCTGCGATCTAAGTAA-3′ and (Deletion -71 to +185 bp reverse) 5′-CCTTTATAGAATTCTGAATTGATGATGTG-3′.We used the reporter plasmid which includes -4102 to +185 bp TSLP promoter region. The deletion of TSLP promoter region was performed by using KOD -Plus- Mutagenesis Kit . The used primers were as follows; (-4102 bp forward) 5 cells/ml) were seeded onto 24-well plates. Two days later, the culture medium was removed and the cells were transfected with TSLP promoter reporter plasmid (250 ng/well) or HRE reporter plasmids along with CMV (10 ng/well) or TK (50 ng/well) reporter plasmids for 24 h by X-tremeGENE HP DNA Transfection Reagent (Roche). HaCaT cells were then lysed with 100 μl of lysis buffer , and 10 μl of the lysate was used for measurement of Firefly and Renilla luciferase activities.The Dual-luciferase reporter assay system  was used for the measurement of Firefly and Renilla luciferase activities. The measurements were conducted according to the manufacturer’s protocols. HaCaT cells . Comparisons of groups were evaluated by Student’s t-test. Multiple comparisons were performed with Bonferroni, Dunnett’s and Student-Newman-Keuls tests.2) or normoxia and then stimulated with TNF-α for 2 h. As shown in The stimulation of HaCaT cells with TNF-α (100 ng/ml) induced the expression of TSLP mRNA at 2 h. To analyze the effects of hypoxia on TSLP mRNA levels, HaCaT cells were incubated for 8 h or indicated time under hypoxia  on TSLP expression were mediated mainly by HIF-2α.Treatment of HaCaT cells under hypoxia condition or, in presence of NiCln levels . Therefon levels , by NiClevels  , cystic 2). Since the oxygen concentration in adult human and rodent epithelia is reported as 0.5–5% [The expression of a barrier protein, filaggrin, in keratinocytes was upregulated under a hypoxic condition  Fig 3C)Fig 3C) as 0.5–5% , it is ls 0.5–5% . ConversIn conclusion, this study revealed that TSLP expression was downregulated in hypoxia via HIF-2 and the -62 to -57 bp TSLP promoter region-dependent ways. Therefore, PHD and HIF-2 in the epidermis might be a novel therapeutic approach for treating atopic dermatitis.S1 Fig2 at the indicated concentrations. The concentrations of TSLP in the medium were determined by Enzyme-linked immunoassay (ELISA) and cell viability was determined by the MTT assay. Data are indicated as means ± SEM from 4 samples. Statistical significance: ** p < 0.01 vs. corresponding TNF-α control.PAM212 cells were incubated for 24 h in medium containing TNF-α in the presence or absence of NiCl(PDF)Click here for additional data file.S2 Fig2 (1 mM) for 8 h and stimulated with TNF-α (100 ng/ml) for 2 h. Data are indicated as means ± SEM from 3 samples.HaCaT cells were pretreated with HIF-1α inhibitor  and NiCl(PDF)Click here for additional data file.S3 FigPutative HRE site, 5′-GACATG-3′, is indicated. HRE was searched by the TRANSFAC program (Match -1.0 Public).(PDF)Click here for additional data file.


Survival analysis is an important part of cancer studies. In addition to the existing Cox proportional hazards model, deep learning models have recently been proposed in survival prediction, which directly integrates multi-omics data of a large number of genes using the fully connected dense deep neural network layers, which are hard to interpret. On the other hand, cancer signaling pathways are important and interpretable concepts that define the signaling cascades regulating cancer development and drug resistance. Thus, it is important to investigate potential associations between patient survival and individual signaling pathways, which can help domain experts to understand deep learning models making specific predictions.In this exploratory study, we proposed to investigate the relevance and influence of a set of core cancer signaling pathways in the survival analysis of cancer patients. Specifically, we built a simplified and partially biologically meaningful deep neural network, DeepSigSurvNet, for survival prediction. In the model, the gene expression and copy number data of 1967 genes from 46 major signaling pathways were integrated in the model. We applied the model to four types of cancer and investigated the influence of the 46 signaling pathways in the cancers. Interestingly, the interpretable analysis identified the distinct patterns of these signaling pathways, which are helpful in understanding the relevance of signaling pathways in terms of their application to the prediction of cancer patients’ survival time. These highly relevant signaling pathways, when combined with other essential signaling pathways inhibitors, can be novel targets for drug and drug combination prediction to improve cancer patients’ survival time.The proposed DeepSigSurvNet model can facilitate the understanding of the implications of signaling pathways on cancer patients’ survival by integrating multi-omics data and clinical factors. Survival analysis based on clinical factors  is crucial for cancer prognosis. However, it is just as important to identify and understand essential biomarkers given large-scale genomics data . The cox proportional hazards model (Cox PH) model  is the cCompared with the Cox PH model, the deep learning models showed improved prediction accuracy by flexibly integrating a large number of genomics features without strong parametric assumptions. For example, the DeepSurv  model usCompared with existing models, we aimed to investigate the relevance or influence of individual cancer signaling pathways (pathway level) to the survival time prediction in cancer patients. In another word, instead of using multi-omics data of a large number of genes, a set of cancer signaling pathways were modeled using a simplified and partially biological meaningful deep neural network architecture, which has not been well investigated. In cancer studies, many dysfunctional signaling pathways that play important roles in tumor development and drug response are identified. For example, the analysis of ten signaling pathways using the TCGA cancer samples indicated that many genetic biomarkers were included in the ten signaling pathways . Such caFrom the UCSC Xena data server, the mean-normalized log2 scaled RSEM  values (KEGG (Kyoto Encyclopedia of Genes and Genomes)  is a datDeepSigSurvNet model. In the ‘input layer’, there were two input features, i.e., normalized gene expression across TCGA samples and integer copy number variation, for each gene. Genes that have zero expression among training dataset will be excluded from input. In the model, gene expression and copy number variation information were first linked to individual genes to compute gene state respectively for each gene. Then, the genes’ state were connected to the 46 signaling pathways only if a gene was included in a signaling pathway (not a full connection layer). The gene connection matrix and pathway connection matrix were used to design the connections. The output of the 46 signaling pathways was used as the input for the convolution and inception [Figure nception  layers  metric was used. The c-index is defined as follows. Let We compared the proposed model with the random forest model, which is available as RandomForestRegression in the scikit-learn package. We trained the random forest model using the same training and test dataset settings for the four types of cancer. The “n_estimator” and “max_depth” parameters were fine-tuned to find the best performance of the random forest models. For the DeepSigSurvNet model, we used the same architecture for all four types of cancer, but different dropout rates, regularization values and epoch numbers for each cancer type. Tables Considering the heterogeneity in TCGA dataset, multiple sampling at different ratios were also performed for evaluation of robustness. Specifically, we have tested different ratios of training data varying from 50, 60, 70, 80 and 90%, and repeated 50 times. The epoch time was set to 25, and the average c-index value of the 50 testing was used. The results are shown in Table To further test the influence of the number of pathways, the model was tested using the 10, 20, 30 and 40 signaling pathways. Specifically, a number of signaling pathways, e.g., 10 signaling pathways, were randomly selected for 50 times, using the 80% of the data as training and 20% of the data as the test data. The average c-index values were listed in Table As discussed, it is interesting to investigate and understand how the individual signaling pathways contribute to the cancer patients’ survival prediction. After training the deep learning models, we employed the ‘iNNvestigate’ package to calculate the relevance scores of the individual signaling pathways on individual cancer patients in each of the four types of cancer. Figures Specifically, the BRCA, mTOR, Hedgehog, PI3K-Akt, TGF-beta, AMPK, VEGF, Apelin, Adipocytokine and Oxytocin signaling pathways have the strongest relevance scores. P53, Wnt, Notch, NF-Kaapa B, FoxO, cGMP-PKG, cAMP, Chemokine, Sphingolipid, Relaxin, and Thyroid hormone signaling pathways have relatively high relevance scores. Surprisingly, the MAPK, ErbB, Ras, Rap1, and JAK-STAT signaling pathways as well as the cell cycle are not well associated with patients’ survival outcome, even though it is well known that these signaling pathways play important roles in cancer development. However, they can be separated in BRCA cancer samples and identified as the essential signaling pathways for patients’ survival outcome prediction. For LUDA, the patterns of density distribution are different from BRCA. More signaling pathways show high but not very strong relevance scores. For example, the MAPK, Ras, Rap1, cGMP-PKG, HIF-1, mTOR, PI3K-Akt, Wnt, Notch Hedgehog, C-type lectin receptor, GnRH, Neurotrophin, and Thyroid hormone signaling pathways have relatively high and consistent relevance scores. On the other hand, the AMPK, Hippo and NOD-like signaling pathways have zero-mean values but with great variance. Thus, it is hard to evaluate their relative importance in cancer patients’ survival prediction analysis. For GBM, the Ras, p53, mTOR, PI3K-Akt, Notch, Hippo, TNF, Estrogen, Thyroid hormone and Relaxin signaling pathways have relatively high relevance scores; the other signaling pathways are not correlated with patients’ survival. For SKCM, the patterns are similar to the LUAD cancer samples. The Ras, Calcium, cGMP-PKG, NF-Kappa B, HIF-1, FoxO, Sphingolipid, Phospholipase D, p53, mTOR, Wnt, Hedgehog, NOD-like receptor, Estrogen, Prolactin, and Thyroid hormone signaling pathways have relatively high and consistent relevance scores. Whereas, the MAPK, Rap1, PI3K-Akt, AMPK, and VEGF signaling pathways have zero-mean values but with great variance.In summary, the probability density distribution patterns of all 46 signaling pathways vary significantly among the four types of cancer. For example, the p53 and mTOR signaling pathways are strongly relevant to patients’ survival outcomes in BRCA, GBM, and SKCM cancer patients, but not in the LUDA cancer patients. The MAPK, RAS, Rap1, and ErBB signaling pathways are known as the important signaling pathways in cancer, but they are not strongly correlated with cancer patients’ survival outcome in the prediction models. This might be because all of these important signaling pathways are always activated in cancer patients. Thus, they are important targets for cancer therapy, but not informative in terms of the survival time prediction. Also, the cell cycle signaling does not play an important role in the survival time prediction. Moreover, a small set of signaling pathways  do not show important contributions to the survival of cancer patients across all four types of cancer. Also, for each type of cancer, less than half of the signaling pathways have strong effects on the survival prediction. Thus, drugs and drug combinations that can inhibit these essential signaling pathways as well as the signaling pathways with strong relevance scores for each type of cancer might be effective in improving cancer patients’ survival time and outcome.Survival prediction is important in cancer studies. Deep learning models that integrate multi-omics data have been proposed for survival prediction and have outperformed the classic Cox PH model. Signaling pathways are important in cancer research to understand the signaling cascades regulating cancer development and drug response. However, it is challenging to understand the contributions of individual genes considering the non-linear combinations of a large number of genomic features, e.g., gene expression, copy number variation. Instead of using a large number of genomics features, in this study, we proposed a relatively biologically meaningful and simplified deep learning model, DeepSigSurvNet, for survival prediction. In the model, the gene expression and copy number data of 1967 genes from 46 major signaling pathways were used. The deep learning model analysis on four types of cancer can identify the distinct patterns of these signaling pathways, which are helpful in understanding the relevance of the signaling pathways in the context of survival analysis. These pathways can also be novel targets for drug and drug combination prediction to improve cancer patients’ survival outcome.There are some improvements to the proposed model that need to be further investigated. In addition to the 46 signaling pathways, other KEGG pathways, like metabolism pathways, will be further evaluated. Moreover, Gene oncology  (GO) terIn this study, we proposed a biologically meaningful and simplified deep learning model, DeepSigSurvNet, based on a set of signaling pathways to model cancer patients’ survival. Multi-omics data and clinical factors can be integrated into the model in a relatively meaningful manner compared with existing deep learning models, and the model is robust for testing data. The interpretable analysis can help researchers understand the effects of individual signaling pathways and identify new therapeutic drugs that target the top correlated signaling pathways relevant to patient survival time and outcome.


The fibroblast growth factor (FGF) family is one of the largest growth factor families, which consists of 18 receptor-binging, intrinsic tissue regulatory polypeptides that share similarity in structures and amino acid sequences , gain-of-function mutations in the FGFR tyrosine kinases, and possibly other unidentified mechanisms.canonical and non-canonical signals. The canonical signals include FRS2α-dependent activation of ERK and PI3K, as well as FRS2α-independent activation of PLCγ  and transforming growth factor β-activated kinase 1 (TAK1), which increases the stability, as well as the activity of these enzymes . The expression of this intrinsic FGF signaling axis likely cross talked with the TNFα pathway. Overexpression of FGF21 in mice alleviated toxin-induced inflammation and damages in the liver. However, treating hepatic stellate cells with FGF1 that bound to FGFR1 independent of KLB activated the non-canonical TAK1-mediated TNFα pathway and exacerbated inflammation reaction in the cells. The results demonstrate that the intrinsic FGF21-FGFR1/KLB signaling axis and ectopic FGF1-FGFR1 elicit functional opposite signals with respect to regulating inflammation.Inflammation is a double-edged sword for the host. Overactive inflammation exacerbates injuries, including acute kidney injury and liver damage induced by concanavalin A. Koledova et al. tested two engineered FGF2 that have low heparin-binding activities with respect to stability, heparin dependence, and dynamics of activating ERK. They found that the two FGF2 derivatives, FGF2-STAB1 and FGF2-STAB2, had a higher mitogenic activity than did the wildtype FGF2. They also induced ERK phosphorylation more potently and at a faster dynamic than wildtype FGF2. Since the engineered FGF2s do not bind to heparin and have a high activity in activating the canonical ERK pathway, they are of transitional values for activating the FGFR-ERK signaling pathway in the precision medicine (Li et al., Many members of the FGF family have the heparan sulfate/heparin binding activity (Li et al., In summary, the FGF signaling is complicated, and in many cases, the signaling specificity and intensity is not only FGF and FGFR isoform-dependent, but also heparan sulfate and host cells-dependent. It can be either a foe or friend, and the only determinant of what they are is when, where, and how the signals are elicited. Yet, how resident FGF-FGFR signaling axis elicits canonical or non-canonical signals to regulate multiple cellular activities and how ectopic FGF signaling causes diseases largely remain an enigma. Solving this enigma is needed for developing FGF-based precision therapies to treat diseases caused by aberrant FGF signals.FW, XL, and CW conceived and wrote the editorial. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.