Conjunctival melanoma (CM) is an exceptionally rare tumor, with a propensity for local and distant recurrence, with the lungs, skin, liver, and brain being the most common sites of metastasis. Recent progress in systemic treatments, with checkpoint inhibitors and targeted therapies blocking BRAF and MEK, has redefined the standard of care of advanced unresectable and metastatic melanoma. Although most trials did not include patients with conjunctival melanoma, its close molecular and genetic relationship to cutaneous melanoma might suggest a similar response to these novel agents. The authors describe two uncommon cases of metastatic conjunctival melanomas with distinct genetic profiles and, as such, submitted to different systemic treatments. Conjunctival melanoma (CM) is a rare tumor, with an incidence of 0.02 to 0.08 per 100,000 in white populations. It accounts for 1% to 3% of all ocular malignancies in adults and represents only 1.6% of all noncutaneous melanomas in published series, with increasing incidence in the elderly [CM usually develops within the bulbar conjunctiva, rather than the forniceal or palpebral conjunctiva, and can arise in areas of primary acquired melanosis, from a preexisting nevus or de novo .CM is an aggressive tumor, with a propensity for local and distant recurrence .Pathological features recognized as predictive of poor survival and locoregional recurrence are tumor thickness, nonepibulbar locations, and multifocal CM.In addition to spreading by lymphatics and the bloodstream, CM can undergo direct extension to the globe and orbit .The most common sites of metastasis are the lungs, skin, liver, and brain.Although a historically treatment-refractory disease, recent progress in systemic treatments, with important benefits in disease control and survival, has redefined the standard of care of advanced unresectable and metastatic melanoma .A 56-year-old Caucasian woman presented with a pigmented lesion developing on a preexisting pterygium of the right eye, with gradual growth since October 2007. Complete excisional biopsy was performed in March 2010, showing a malignant melanoma of the bulbar conjunctiva, with 1.9 mm in thickness.The patient was lost to follow-up until July 2013, when she turned to the hospital after developing nasal obstruction and fatigue. Laryngoscopy and head MRI revealed a pedunculated mass on the right oropharyngeal wall, with 31 × 13 mm in diameter.The mass was surgically removed, with a thorough pathologic assessment revealing an ulcerated metastasis of a conjunctival melanoma, with 6.5 mm in thickness and no subepithelial component.A staging PET/CT scan showed multiple pharyngeal lymphadenopathies.BRAF gene (V600) was detected by Cobas 4800.The patient was referred to our institution for further management, with the abovementioned findings being confirmed. A mutation in exon 15 of the Two months after surgery, there was evidence of local recurrence with symptoms reappearing, and a body PET/CT scan showed a de novo endophytic mass, with 31 × 22 mm, on the previously affected location .The lesion was considered unresectable, and palliative external radiotherapy, with a total dose of 20 Gy/5 fr, was performed.In April 2014, systemic therapy was initiated with vemurafenib (960 mg) twice a day.After one month of therapy, there was full symptomatic resolution.Restaging evaluation showed sustained favorable response, resulting in complete remission .There was a need for dose reduction to 480 mg twice a day because of grade 2 arthralgia, grade 2 diarrhea, and grade 1 skin rash.In February 2017, she was diagnosed with early stage, grade 2, invasive ductal carcinoma of the left breast, pT1cN0snM0, with estrogen receptors +100%, progesterone receptors +1%, HER2 1+, and Ki67 +15%.She was submitted to a left simple mastectomy with sentinel lymph node biopsy and began hormonal treatment with tamoxifen (20 mg) once a day.The patient remains free of new lesions as of the last follow-up, on August 8, 2017.A 51-year-old Caucasian man had a remote history of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections, medicated with antiretroviral therapy, with a CD4 count within the normal range and an undetectable viral load. In December 2009, he presented with a hyperpigmented lesion on the temporal conjunctiva of the right eye. Excisional biopsy showed a compound melanocytic nevus.In December 2011, the lesion recurred on the temporal, inferior, and nasal conjunctiva.Surgical excision revealed a completely excised malignant melanoma of the bulbar conjunctiva, with 2.4 mm in thickness.One year later, he was reintervened for a second recurrence on the inferior and temporal conjunctiva.He was maintained under active surveillance until July 2016, when right cervical adenopathies were noted. Excisional biopsy confirmed metastases of malignant melanoma positive for S100 and MELAN A and negative for CAM 5.2.BRAF gene was detected.The patient was then referred to our institution for further management, with pathologic reevaluation confirming the diagnosis. No mutation in the A body PET/CT scan documented cervical, submandibular, and pharyngeal adenopathies.He was submitted to a right cervical lymphadenectomy, with metastases in five of the thirty lymph nodes removed.Subsequent radiotherapy was initially planned; however, within three weeks, there was rapid disease progression, with multiple de novo subcutaneous nodes of the face and neck.In December 2016, systemic therapy was initiated with pembrolizumab (2 mg/kg) every 3 weeks.After the third cycle, there was near to complete resolution of the subcutaneous lesions.At the last follow-up, on August 23, 2017, the patient was on complete remission, with good tolerance to the ongoing treatment.We describe two seldom cases of metastatic conjunctival melanomas with distinct genetic profiles and, as such, submitted to different systemic treatments. Both patients have sustained favorable responses up until the writing of this article.BRAF V600 mutations, while programmed death-1 (PD-1) inhibitors are effective regardless of BRAF mutational status [Advances in systemic treatments, with an important enhancement in disease control and survival, have recently changed the standard of care of advanced unresectable and metastatic melanoma . Immunotl status .BRAF mutations in 23 of 78 (29%) conjunctival melanomas, a majority of which (91%) were V600E mutations.Although most trials did not include patients with conjunctival melanoma, its close molecular and genetic relationship to cutaneous melanoma might suggest a similar response to these novel agents . GriewanIn case 1, when the patient began treatment, single-agent therapy with vemurafenib or dabrafenib was the standard of care. Although the combination with BRAF and MEK inhibitors proved to have less toxicity and to be more effective regarding response rates, progression-free survival, and overall survival, it only became available in our country posteriorly , 11.Despite the known risk of the emergence of resistance , 13, givHer recent diagnosis of breast cancer raises the question whether the disease can be related to prolonged treatment with vemurafenib.BRAF wild-type cells [Secondary tumors, like cutaneous squamous cell carcinomas or keratoacanthoma, can occur in approximately 25% of patients treated with vemurafenib, as a result of paradoxical activation of the mitogen-activated protein kinase pathway in nonmelanoma pe cells .BRAF-mutated cutaneous melanoma.Novik et al.  reportedAt the present time, however, there are insufficient data to make this assumption.In case 2, there were concerns about the use of a checkpoint inhibitor in a patient with HIV, given there could be immune-related side effects specific to this population.There is scarce literature regarding this issue. As this is a generalized concern, these patients are usually excluded from trials with immunotherapy.Davar et al.  reportedThe same was observed in our patient, with treatment proving to be effective and excellently tolerated.BRAF gene mutation) and PD-1 inhibitors, seem to be valid options in the treatment of metastatic conjunctival melanoma.In conclusion, both treatments, with BRAF inhibitors (in the presence of the Prospective studies are needed to further confirm the efficacy and tolerability of these treatments in this patient population.


As a high-prevalence health condition, hypertension is clinically costly, difficult to manage, and often leads to severe and life-threatening diseases such as cardiovascular disease (CVD) and stroke.The aim of this study was to develop and validate prospectively a risk prediction model of incident essential hypertension within the following year.Data from individual patient electronic health records (EHRs) were extracted from the Maine Health Information Exchange network. Retrospective  and prospective  cohorts were formed. A machine learning algorithm, XGBoost, was adopted in the process of feature selection and model building. It generated an ensemble of classification trees and assigned a final predictive risk score to each individual.very high risk population mainly comprised elderly (age>50 years) individuals with multiple chronic conditions, especially those receiving medications for mental disorders. Disparities were also found in social determinants, including some community-level factors associated with higher risk and others that were protective against hypertension.The 1-year incident hypertension risk model attained areas under the curve (AUCs) of 0.917 and 0.870 in the retrospective and prospective cohorts, respectively. Risk scores were calculated and stratified into five risk categories, with 4526 out of 381,544 patients (1.19%) in the lowest risk category (score 0-0.05) and 21,050 out of 41,329 patients (50.93%) in the highest risk category (score 0.4-1) receiving a diagnosis of incident hypertension in the following 1 year. Type 2 diabetes, lipid disorders, CVDs, mental illness, clinical utilization indicators, and socioeconomic determinants were recognized as driving or associated features of incident essential hypertension. The With statewide EHR datasets, our study prospectively validated an accurate 1-year risk prediction model for incident essential hypertension. Our real-time predictive analytic model has been deployed in the state of Maine, providing implications in interventions for hypertension and related diseases and hopefully enhancing hypertension care. Hypertension affects more than 85 million American adults and over 1.1 billion people worldwide, and elevated blood pressure is one of the leading modifiable metabolic risk factors for noncommunicable diseases and global mortality . For exaCommonly identified risk factors for progression to hypertension include age, gender, body mass index, obesity, stress, lipoproteins, cholesterol, physical inactivity, smoking, and family history . EvidencIn the last two decades, more than 15 high-quality hypertension risk prediction models were constructed and compared in terms of study design, model performance, calibration ability, as well as impact on decision making and outcomes of care . These mOn the other hand, the use of electronic health records (EHRs) is becoming increasingly common in hospitals and clinics . By tracGiven that the EHR data have many dimensions and are usually sparse and subject to random errors, machine learning approaches are recognized as good choices for constructing EHR-based risk models based on their ability to select impactful predictors automatically from hundreds of features and their robustness to feature correlation and random errors -29. TherThe analyzed dataset was derived from EHRs of all patients that visited any of 35 hospitals, 34 federally qualified health centers, and more than 400 ambulatory practices in the state of Maine from January 1, 2013 to December 31, 2015. This dataset covered almost 95% of the population of Maine and is a subset of the health information exchange (HIE) network provided by the HealthInfoNet organization. This study was approved by the institutional review board of Stanford University, and all personal privacy information was well protected and removed during the process of analysis and publication. Samples were excluded if they had any diagnosis of secondary or gestational hypertension, leaving a total of 1.5 million individuals in the dataset for the analysis. The detailed inclusion and exclusion criteria were demonstrated in the study design workflow .Hypertension in this study was defined by the International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM) diagnosis codes from category 401, which refers specifically to essential (or primary) hypertension. For the prediction modeling cohort, cases in the retrospective population referred to patients who received a new diagnosis of essential hypertension during the calendar year of 2014 , whereas cases in the prospective cohort were patients receiving an incident diagnosis of essential hypertension during the 2015 calendar year . Along with the implementation of ICD-10-CM since October 1, 2015, an ICD-10-CM category of I10 was used to define the diagnosis of primary hypertension, which is equivalent to the ICD-9-CM category 401 according to the ICD-10-CM General Equivalence Mapping (GEM), a crosswalk between the two code standards maintained by the Center for Medicare Services and the Centers for Disease Control and Prevention .Given that our prediction model was constructed to predict an individual’s hypertension risk during the following 1 year based on his or her medical records from the current year, feature profiles for the retrospective cohort were extracted from the clinical and health historical record of 2013, giving a total of 823,627 patients, 92,512 of whom developed hypertension in the year 2014.Samples were excluded from the retrospective cohort if there was any record of a hypertension diagnosis before January 1, 2014. Similarly, the prospective cohort was extracted from the clinical and health historical record of 2014, offering a total of 680,810 samples and 60,065 cases with incident hypertension in the year 2015. Samples were excluded in prospective cohort if there was any record of a hypertension diagnosis before January 1, 2015.American Fact Finder category, and others were derived from the USDA website using the Web-based mapping tool, Food Environment Atlas  websites ,32. Overtlas see . Using zBefore feature selection, the k-nearest neighbors (KNN) approach was used to impute missing data. For a given patient with missing values, the KNN method identified the k-nearest patients based on Euclidean distance. Using these patients, missing values were then replaced using a majority vote for discrete variables and weighted means for continuous features. One advantage of using this method is that missing values in all features are imputed simultaneously without the need to treat features individually .R=∑Ki=1AiDi/Ti/ ∑Ki=1BiCi/Ti, where Ai, Bi, Ci, and Di are the counts of individuals in 2x2 contingency table of the i- th age group. In addition, the Cochran-Armitage trend test is used to test ordinal variables, and univariate logistic regression is used for continuous variables. After applying the criteria of P ≤.05, 798 out of 15,280 features survived from this screening procedure and were recruited in the following stages of XGBoost algorithm.Before the machine-learning algorithm, a univariate correlation filtering step was introduced to remove all features that are not directly related to the outcome variable . In geneThe retrospective cohort was utilized for construction of the prediction model. This process was accomplished in two phases: (1) a data training algorithm to generate predictive estimates and (2) a calibration stage to produce risk scores for each patient.ŷi for the i- th instance , as demonstrated in equation 1, where each ƒk corresponds to an independent classification tree:For the first stage of data training, a supervised machine learning and data mining tool, XGBoost , was appŷi= ø(xi) = ∑Kk=1ƒk(xi) (1)K equaled 500. The model at the t- th iteration was trained to minimize the following objective, where l is a differentiable convex loss function that not only measures the difference between the target yi and the prediction ŷi(t-1) of the i-th instance at the t-1- th iteration but also takes the ƒt that improves the model most into account:For this study, the depth of each tree was set to be 5 and L(t)= ∑ni=1l+ ƒt(xi)) + Ω (ƒt) (2)The term Ω penalizes the complexity of the regression tree functions to avoid issues of overfitting. The approximate greedy algorithm was adopted to split trees and sort and pick features on each node according to percentiles of distribution. Splitting points were selected to optimize purity at the next splitting level.ŷ estimates were mapped to positive predictive values (PPVs) in the retrospective cohort. A PPV of a corresponding predictive estimate ŷ was defined as the proportion of incident hypertension events in the cohort having predictive estimates the same as or larger than this ŷ. Thus, PPVs could also be interpreted as risk scores that measured probability of receiving a new diagnosis of essential hypertension within the next 1 year among individuals having predictive estimates the same as or larger than this ŷ.For the second phase of calibration, very low, low, medium, high, or very high. Performance of the proposed 1-year hypertension risk prediction model was investigated carefully within each risk category in terms of sensitivity, specificity, and PPV. The receiver operating characteristic curve and the validated AUC value were derived to evaluate the performance of the derived prediction model.In the process of model evaluation, hypertension risk score expressed as PPVs were calculated and assigned to each individual in the prospective cohort. Following that, we further ranked individuals by their risk scores from low to high and allocated them into five distinct risk categories, manifesting individuals’ chance of developing essential hypertension within the next 1 year to be either For these highly weighted features, age-sex adjusted odds ratios (ORs) between cases and controls were calculated along with 95% CI using logistic regression. Distribution patterns of impactful risk predictors were explored and compared among different risk categories, uncovering predictors’ characteristics under our constructed prediction model. Moreover, survival analyses such as multivariable Cox regression were employed for the purpose of subpopulation comparison. Spearman rank correlations were performed to assess the correlation between socioeconomic features and the next 1-year risk of hypertension.Baseline demographic features and hypertension-related disease conditions in retrospective and prospective cohort, summarized in very low risk , and <1.19%  were affected by hypertension in the next 1 year. In contrast, for 41,329 individuals identified in the very high risk category , more than 50.93%  received a confirmatory diagnosis of essential hypertension during the next 1 year. Among 60,065 patients with confirmed hypertension in the next 1 year, more than one-third  were correctly classified into the very high risk category, and only 7.54%  of them were falsely assigned to the very low risk population , giving a hazard ratio (HR) for the very high risk category as high as 60.8 (95% CI 58.8-62.8) relative to the very low risk group  were overwhelmingly dominant in the low risk and medium risk categories, with proportions of 51.04%  and 54.11% , respectively. On the contrary, individuals in the age range of 50 to 64 years occupied 34.57%  of the high risk population, and people aged ≥65 years constituted the largest subset  of the very high risk population  but decreasing to proportions of 46.89%  and 48.41%  in the high risk and very high risk groups (very high risk population mainly comprised aged (>50 years) males, who occupied almost one-third of this category.To maintain the consistency of this feature, other ages were aggregated into categories as well. In addition, this age categorization could help us capture natural differentials in age-related socioeconomic backgrounds such as work or retirement status and eligibility for pensions and medicare and investigate their impact on hypertension risk and incidence . Therefoch other . Low-ristion see . Followition see . On the k groups . In summvery high risk population had a history of multiple chronic medical conditions. Most commonly CVDs, type 2 diabetes, and disorders of lipid metabolism affected 25.08% , 20.17% , and 8.76%  of the population, respectively  and compared them with curves generated from the very low risk cohort  patients with CVD would receive diagnoses of hypertension in the next 1 year, and this probability stayed around 50% for patients with conditions of either type 2 diabetes, lipid disorders, mental illness, or CKD. Thus, a half of individuals with a chronic disease in the very high risk category would develop hypertension within the next 1 year, with CVD patients at the highest risk of hypertension during the next 1 year, whereas prehypertension patients had the lowest risk, implying different causal mechanism of hypertension under different disease scenarios at the very high risk stage. In contrast, in the very low risk category, more than 85% of patients were free from development of hypertension in the next 1 year, regardless of chronic conditions, where the survival curve dropped fastest for patients with prehypertension but went down slowly for the cardiovascular subgroup, opposite to the trends at the very high risk stage.To further explore real-time hypertension events in the k cohort . In the very high risk group for hypertension, with proportions of 22.21% , 13.40% , and 2.71% , respectively, but much less prevalent in the very low risk group , a diagnosis of depression would double the 1-year hazard of hypertension even if there were no other chronic conditions, regardless of gender.By using multivariable Cox proportional hazards models, the contribution of depression to the hazard for hypertension was assessed after adjustment for age and gender . In the very low risk group but gradually increased from low to high risk groups, with the highest values in the very high risk population  in the very high risk population  cohort, these five identified risk categories showed distinct HRs for incident hypertension within the next 1 year  cohorts, respectively. On the basis of the risk model, patients were ultimately stratified into five distinct risk categories, ranking the hypertension risk as t 1 year , indicatUnlike traditional prediction methods, our study adopted a machine learning algorithm called XGBoost for feature selection and model construction. This supervised machine learning technique is designed to discover statistical patterns in high-dimensional and multivariate datasets and is able to handle nonlinear correlations and random errors both in input features and the output variable .Variable correlation is another common issue when using a large number of variables spontaneously to construct a model. For instance, in our study, potential spatial correlation may exist among demographic or socioeconomic features. Most regression algorithms such as generalized linear model assume feature independence and could become less accurate if variable correlation or multicollinearity exist. On the contrary, decision trees (including XGBoost) are nonparametric algorithms  that do very high risk category. In the prospective cohort, 50.93%  of people in the very high risk group were diagnosed with hypertension during the next 1 year. Such a high incidence of hypertension in the very high risk subgroup made us believe that a short-term (1 year) prediction of hypertension was necessary to identify a high-risk cohort, as well as promote their follow-up intervention or prevention.As a result, XGBoost provided a more accurate prediction model (AUC of 0.87 in the prospective cohort) than prior models with no extra cost by capturing previously ignored but potentially powerful predictors from patients’ current health conditions, chronic disease and medication history, clinical utilization measures, and social determinants. Specifically, individuals with the highest risk in our study were typically older (>50 years) and suffered from multiple chronic conditions . In the socioeconomic domain, low-income and low-educated people who had public insurance and lived near food stores but far away from parks were enriched in the very high risk group suffered from other medical conditions, and only 1.56%  had no other chronic medical conditions, whereas the majority  of the very low risk population had no other chronic disease diagnosis. Moreover, as the number of people with diagnostic chronic diseases increased, those with multiple chronic conditions (MCCs), also known as concurrent chronic conditions [very high risk category  of elderly (≥65 years) patients having no chronic conditions but undergoing treatment for depression would develop hypertension within the next 1 year. EHR data in the state of Maine coding mental health diagnoses were masked for privacy protection; only consumed mental disorder drugs could be accessed as proxies. Therefore, as a limitation of the study, we cannot directly ascertain the effect of mental health disorders or the treatment thereof on the progression of hypertension. However, we can examine the relationship between mental illness diagnoses or therapeutics and hypertension development in several different ways according to the large amount of previous studies exploring this relationship -52,55-59Social determinants of health are defined as “the structural determinants and conditions in which people are born, grow, live, work, and age” ,61 and ivery high risk category, 21.26%  of the population would receive a diagnosis of hypertension within the first 3 months of next year, and this number would gradually increase to 50.93%  after the 1-year period. Therefore, monitoring those high-risk patients as soon as possible and developing a personalized longitudinal intervention plan is important to prevent or delay the development of incident hypertension, as well as to reduce corresponding health care expenditures. Given the fact that 98.44%  people in the very high risk cohort had other chronic diseases, one possible early intervention plan could be more active monitoring and treatment of relevant diseases, blocking possible pathways from chronic diseases to hypertension development. For instance, strong evidence has illustrated that patients with depressive disorders can be effectively treated in primary care by using either low-cost remote cognitive behavioral therapy or face-to-face psychological interventions, which may ultimately benefit the prevention of hypertension [Beyond the prediction of hypertension risk itself, subsequent actions to prevent and intervene are necessary in high-risk patients. In our rtension . MoreoveOur study has several limitations. First, missing data is inevitable in EHR records, and thus, data organization and extraction are critical. The KNN approach was used in our data preprocessing for missing data imputation. One limitation of this approach is that, when most variables are missing for a certain patient, the KNN cannot be identified appropriately, and therefore, the imputation can be less accurate and cause bias . Second,very low, low, medium, high, to very high. Our real-time predictive analytic model has already been deployed in the state of Maine. Integration of such predictive analysis into clinical prescriptive solutions may help health care providers target high-risk populations, tailor the prescription and intensity of treatment solutions to such high-risk cohorts, improve decision making and patient adherence to prescribed intervention, and eventually benefit individuals’ health and quality of life while reducing health care costs.In summary, we have constructed and prospectively validated a risk prediction model of future 1-year incident essential hypertension using EHR data derived from more than 1.5 million people in the state of Maine. The EHR-based model achieved 0.917 and 0.870 predictive accuracy in retrospective and prospective  cohorts, respectively, and successfully stratified patients into five distinct risk categories from


After 12 weeks, the verum group achieved significantly greater improvements in the total PSQ30 score compared with the placebo group. In the verum group, serum taurine concentration, folic acid concentration, urinary magnesium excretion, and the ratio of l-tryptophan to the sum of competing amino acids rose significantly. In the placebo group, serum concentrations of serotonin, protein, and magnesium decreased significantly, whereas the cardiometabolic risk parameters body weight, body mass index, waist circumference, and waist-to-height ratio increased significantly. Compared with placebo, the verum supplementation resulted in a higher improvement in perceived stress. Beneficial effects on the serotonergic system and preventive effects on magnesium homeostasis and some cardiometabolic risk factors were supposed. Additional effects might be caused by the optimized food intake.Chronic work-life stress leads to dysfunction of the hypothalamus–pituitary–adrenal axis, the autonomic nervous system, and the serotonergic system, with resultant impairment of overall well-being. Aim of the study was to improve perceived stress by a specific amino acid composition with micronutrients in the verum versus placebo group. A total of 59 participants (18–65 years) with self-reported perceived chronic stress and exhaustion conditions participated in this randomized, double-blind, placebo-controlled study. The Perceived Stress Questionnaire (PSQ Chronic psychosocial stress has adverse effects on the physical and mental health of individuals and organizational effectiveness. It is recognized as a major challenge worldwide ,2,3. In The brain is the key organ for the stress response. Exposure to stress causes the activation of the sympathetic–adrenal–medullary (SAM) axis, resulting in the release of epinephrine and norepinephrine, which affect the heart rate and other autonomic changes, as well as the hypothalamus–pituitary–adrenal (HPA) axis, leading to the release of glucocorticoids such as cortisol ,15. Duril-tryptophan (l-Trp) with the rate limiting step being catalyzed by the enzyme tryptophan hydroxylase. The precursor l-Trp is transported across the blood–brain barrier. Several amino acids, the branched-chain amino acids , and the aromatic l-tyrosine and l-phenylalanine, collectively known as the competing amino acids (CAA), are transported via the same carrier system [l-Trp to the sum of CAA results in an uptake of l-Trp by the brain, suggesting that this condition may increase the synthesis of serotonin [l-ornithine supplementation has shown potential to relieve stress, reduce HPA axis activity, and improve fatigue-related sleep quality [6, and B12 are directly involved in neurotransmitter synthesis and homocysteine metabolism [The mesolimbic dopaminergic system and other brain regions involved in stress/motivation circuits may play important roles in chronic stress-induced food intake, food preference and reward sensitivity . Howeverr system . Studieserotonin ,31. l-or quality . Moreove quality ,34. An atabolism ,36. Vitatabolism ,38. Zinctabolism . Individtabolism . A previtabolism .l-ornithine, l-phenylalanine, and l-tyrosine) with micronutrients  on perceived stress (primary outcome), frequency of neurovegetative symptoms, and endocrine–cardiometabolic risk factors in women and men with chronic psychological stress and exhaustion conditions. The hypothesis of the study was that the verum supplementation results in a lower total Perceived Stress Questionnaire (PSQ30) score in participants with chronic stress and exhaustion conditions as compared with the placebo supplementation after 12 weeks.The aim of this randomized, placebo-controlled, double-blind clinical trial was to evaluate the impact of a daily supplementation with a specific amino acid composition ” and “the most intense sensation imaginable (5 points).” In addition, five cartoon faces from happy face to sad face depicted 1–5 points. The participants were asked to mark the score on points 1–5 or the associated face on the line how they currently felt. The points of each line were summed up to the total VAS points. The psychometric properties of the VAS and Likert-type scale are specified by Hasson and Arnetz .30 [30 score above 0.50 were included into the study. Exclusion criteria for participation were supplementation or therapy with dietary supplements or drugs that contained l-Trp, vitamins, or minerals within four weeks prior to and for the duration of the study, therapy with antidepressants (monoamine oxidase inhibitors), hypertension , organic fatigue, phenylketonuria, and chronic diarrhea. The trial profile is shown in Participants with subjectively perceived chronic stress and exhaustion conditions, such as symptoms of burnout, were recruited via local media and online advertising, internal mail systems, flyers and printed media distributed in the Cologne/Bonn region, Germany. Perceived chronic psychological stress was defined by an elevated level of the standardized PSQ30 . At the 30 score of 0.30 ± 0.15 in the general population. In this study, with the inclusion criterion of a total PSQ30 score above 0.50, a mean total PSQ30 score of 0.60 ± 0.20 or more was expected at baseline. After 12 weeks of dietary intervention with verum, a broad normalization of mean total PSQ30 score, meaning a reduction of at least 0.25 in the verum group, was suspected. With a reduction in total PSQ30 score of at least 0.10 in the placebo group, a Cohen’s effect size of 0.75 was assumed. Primary outcome measures were the intergroup comparisons of the pre-post changes of the PSQ30 after eight and 12 weeks.This randomized, double-blind, placebo-controlled intervention trial was performed at the University Hospital Bonn between October 2014 and January 2016. A seven-day pre-randomization run-in period (adaptation phase) with placebo-capsules was used to exclude subjects at risk of poor compliance by capsule counting. All participants were instructed to consume one capsule with 200 mL of water two times a day after breakfast and dinner for seven days. The following 12-week intervention phase was planned with a tolerance range of 84 ± 2 days. Inclusion and exclusion criteria were examined before the run-in period and at the beginning of the dietary intervention phase (baseline). Participants were assigned to one of two intervention groups via computer-generated randomization with a block size of four  During the dietary intervention phase, participants were required to take the content of a sachet of supplement (9.4 g of powder dissolved in 200 mL of water) daily in the morning. The composition of the verum and placebo products is shown in 30 [30 score above 0.50 was assessed at the beginning of the run-in-phase and at baseline. Participants answered the PNF at baseline and after 12 weeks of dietary intervention. The VAS was completed at baseline and after eight and 12 weeks.The stress experienced during the past 4 weeks was measured by the German version of the PSQ30  at the e2) was calculated by dividing weight (kg) by height (m) squared. Waist circumference (WC) was measured via flexible tape at the midpoint between the lower margin of the least palpable rib and the top of the iliac crest [Anthropometric and clinical measurements were performed in the morning after an overnight fasting period of at least 12 h and under standardized conditions at baseline and after 12 weeks. Body weight and height were determined on a personal floor scale with a height measuring stick . Body mass index . Salivary cortisol concentrations were determined using Cortisol ELISA  and the microplate reader SUNRISE . Venous blood samples were obtained after an overnight fasting period of at least 12 h at baseline and after 12 weeks. Serum glucose (hexokinase method), glycated hemoglobin , insulin (ECLIA), gamma-glutamyltransferase , total cholesterol , low-density lipoprotein-cholesterol (LDL-C) and high-density lipoprotein-cholesterol , triglycerides  and sensitive C-reactive protein  were measured using a Roche analyzer. Diagnosis of the metabolic syndrome was performed [Laboratory Schottdorf MVZ GmbH  conducted almost all laboratory analyses. Saliva samples were collected in the evening (between 10 and 11 p.m.) and morning (30 min after awakening) prior to dietary intervention and after 12 weeks. The collections were performed according to the laboratory instructions. Subjects were requested in advance not to eat, drink, or smoke at least 30 min before saliva collection in the evening and in the morning to collect after an overnight fasting period. All saliva samples were provided prior to tooth brushing by using Cortisol-Salivetteerformed . InsulinThe 24-h urinary sodium and chloride excretion were analyzed by an ion sensitive electrode . Photometry and atomic absorption spectrometry (AAS) were used to detect the 24-h urinary calcium  and magnesium concentration , respectively. Urinary pH value was analyzed at the laboratory of the University Stone Centre, Department of Urology, University of Bonn, Germany.The self-reported dietary intake (three-day food records) data were reviewed with each participant at baseline and after 12 weeks. These food records were kept in the week prior to baseline and in week 12. PRODI 6.4 basis software  with database BLS 3.02 was used to calculate the nutrient content of foods. The average of three days was assessed.® for Windows . Data are presented as mean ± standard deviation (SD) unless otherwise stated. Medians and interquartile ranges of the outcome measures are reported  population and by using SPSSreported . Statistn = 31; 23 women and eight men) and the placebo group , of whom 59 participants completed all visits and were included into the ITT population , thyroid drugs , antihypertensive drugs , cholesterol lowering medications (placebo group: n = 1) and anti-diabetic medications (placebo group: n = 1). There was no significant group difference in the use of medication.A total of 62 women and men were randomized into the verum group . Four participants with a low morning cortisol concentration (<4 ng/mL) of whom two participants additionally had a low evening cortisol concentration (<2 ng/mL) were detected only in the verum group. For these participants, adrenal impaired function with burnout occurrence could be assumed. At baseline, no significant intergroup differences were found for the PSQ30 score, morning and evening salivary cortisol concentrations, serum serotonin, and anthropometric, clinical, and cardiometabolic risk parameters.Among all participants, 48% were overweight or obese, 46% had abdominal obesity, and 19% had an elevated BP. At baseline, the level of impulsion  and the difference in salivary cortisol concentrations (morning–evening) were significantly lower in the verum group than in the placebo group.n = 4) and the placebo group (n = 7) led to exclusions from the per protocol (PP) population without significant group differences , and under-consumption was observed for one of the participants in the placebo group. For seven participants , the duration of study differed slightly from protocol by an average of 1.9 days due to short-term postponements of the final study visit by these chronically stressed participants. As a direct consequence, underconsumption was additionally presented by one of the participants in the placebo group.Protocol violations in the verum group (ferences : An over30 score remained constant in both groups (data not shown). After the 12-week dietary intervention phase, subjectively perceived stress (PSQ30) and neurovegetative symptoms (PNF) improved significantly in the verum and placebo groups  and 12 weeks , respectively. However, participants in the verum group experienced a significantly lower total PSQ30 score than participants of the placebo group after 12 weeks (see the assumed Cohen’s effect size). The influence of baseline and repeated measurements after eight and 12 weeks, respectively, showed no change in the statistical evaluation of the group effects . At the end of the trial, the mean total PSQ30 score was below the inclusion criterion of > 0.50 only in the verum group. After eight weeks, no significant group difference was observed for change in total PSQ30 score, but a significant group difference was found in the overload scale . The improvements in perceived overload after eight and 12 weeks and of perceived irritability, lack of joy, fatigue and tension after 12 weeks were significantly greater in the verum group than in the placebo group. Moreover, participants in the verum group achieved the initially suspected reduction in total PSQ30 score of at least 0.25 at significantly higher rates than participants of the placebo group after eight  and 12 weeks .Notably, in the run-in phase, the total PSQo groups . ReducedAfter the 12-week dietary intervention phase, the total PNF points improved significantly in both the verum and placebo groups without group differences . The cat30, PNF, and VAS correlated significantly in both groups , placebo group: −0.03 ± 0.05 mmol/L (p = 0.004); U = 283.0, p = 0.021). Moreover, after the verum treatment, participants showed a significantly higher taurine concentration (p = 0.005) and the serum taurine concentration  during the 12-week intervention phase (age-adjusted).Salivary cortisol concentration in the morning and evening were not significantly changed in the verum and placebo groups . After 1ntration , serum fntration . SignifiU = 11.5, p = 0.011). These results demonstrate positive effects on HPA axis activity in men. In the placebo group, body weight, BMI, WC, and WHtR increased significantly during 12 weeks, whereas participants in the verum group were unaffected , the prevalence rate was estimated to be 14.5% for an elevated stress experience  and 3.1% for a high stress experience  [30 score of 0.669 ± 0.119 in the present ITT population at baseline, this study included a specially selected group of participants with high subjective perceived stress as expected. The longitudinal data from Lindgren et al. [30 score above 0.34 (the moderate and high stress group) had significantly smaller hippocampal volumes, which may be a vulnerability factor for stress-related disorders, compared with those in the low perceived stress group. Kato et al. [30 subscales of overload, irritability, lack of joy, fatigue, and tension than the placebo group. After 12 weeks, 25% more participants from the verum group compared with the placebo group achieved total PSQ30 scores below the corresponding inclusion criterion . Thus, the analysis revealed that significantly more participants in the verum group reported a reduction of at least 0.25 of the total PSQ30 score compared with participants in the placebo group after 8 and 12 weeks. The PSQ is also associated with neurovegetative complaints [30 scores were correlated significantly and positively with the changes in the total points of the PNF and VAS after 12 weeks. However, group differences were not yet observed at these parameters during the 12-week dietary intervention, but the subscales impulsion (PNF) and productivity (VAS) were more favorably affected in the verum group compared with the placebo group. Thus, the present results suggest that the intake of the specific amino acid composition with micronutrients decreased the perception of chronic stress in a complex way.The PSQ in original and reduced size is one of the most frequently used instruments for assessing subjectively perceived stress in men and women ,52. Prev ≥ 0.60) ,55. Whenn et al.  revealedo et al.  suggesteo et al. , burnouto et al. . HoweverThe HPA axis is a vital part of the human stress response system. Therefore, cortisol regulation is a key topic in psychobiological stress research . In the l-Trp to the sum of CAA. This quotient can be increased by l-Trp treatment [l-Trp concentration because binding to albumin is inhibited [l-Trp/BCAA-ratio and l-Trp/CAA-ratio significantly increased after dietary intervention in the verum group, although the present verum supplement contained the two CAA l-tyrosine and l-phenylalanine. Thus, a stimulation of central serotonin synthesis could be assumed. However, the reason for this increase is uncertain. The verum supplement did not contain l-Trp and, as expected, no change was detected in the serum l-Trp concentration. However, a 40–70% increase in the plasma l-Trp/CAA-ratio of chronically stressed subjects may be required to stimulate central serotonin synthesis [Serotonin is one of the most powerful neurotransmitters, with widespread effects in behavioural and neuropsychological processes including mood, cognition, perception, stress responses, appetite, memory, and sleep/circadian rhythms. In the periphery, serotonin acts as a gastrointestinal regulating agent and a modulator of vasoconstriction/dilation . The maireatment ,70; consreatment ,72,73. Inhibited ,75,76. Tynthesis ,78. The p = 0.025). Thus, in comparison with the results in the verum group, the verum supplement seemed to have preventive effects on plasma magnesium homeostasis and serotonin synthesis in participants with chronic stress. However, the specific combination of amino acids, vitamins and minerals, including magnesium and several other important cofactors of the serotonin synthesis [The relationship between peripheral and central serotonergic systems has been discussed in mice and humans . Some stynthesis , is suppp = 0.024). Plasma taurine concentration increased significantly in the verum group and did not change in the placebo group. Thus, an effect of taurine on central serotonin synthesis and enhanced stress tolerance were supposed in the verum group. Potential beneficial actions of taurine in hypertension, atherosclerosis, and diabetic cardiomyopathy have been discussed [Serotonin metabolism is also controlled by taurine, glycine, GABA, and glutamate in vitro . A signiiscussed ,83. No ciscussed . Thus, tBrain serotonin dysfunction is not only associated with psycho-neurovegetative symptoms, mood disorders, and impaired stress coping but also with decreased control of overall energy intake . High leDuring the 12-week dietary intervention phase, the significant change in plasma ferritin concentration in the verum group could not be explained by gender segregation or age groups (data not shown). The verum supplement did not contain iron but zinc. In previous studies, an inhibitory effect of oral zinc supplementation on iron absorption was supposed, possibly due to the antagonistic action of zinc during iron absorption from the gastrointestinal tract ,98,99. I30 for participant selection and assessment of the primary outcome was necessary, because of the small sample size of the present pilot study and the fact that a large number of study participants without an improvement potential would obliterate the therapeutic effect. However, this procedure can introduce bias. While other measures, such as salivary cortisol, provide a complimentary and adequate approach for examining stress, the manner in which cortisol was measured and the reliability of subject-collected saliva limited its utility in this study. Therefore, saliva collection should be performed under more controlled conditions. Besides plasma folic acid and magnesium concentrations, no other biochemical blood measures of vitamins and minerals were measured. For example, the concentrations of B vitamins and zinc, as well as their relation to changes in perceived stress, plasma serotonin, cardiometabolic risk factors, and plasma ferritin, would be of great interest. These would clarify the interpretation of the extent to which the verum supplement was related to primary and secondary outcomes. The change in plasma ferritin should be examined in other studies with comparable supplements. During further studies, changes in physical activity and personal situation should be assessed in addition to the nutrition status. More studies with a larger cohort are necessary to determine further associations and effects.The small number of cases is a limiting factor. The use of the PSQ30 subscales of overload, irritability, lack of joy, fatigue, and tension were reduced to a greater extent than those in the placebo group. Improvements in neurovegetative symptoms were similar in both groups. However, the HPA axis was not significantly affected. In the verum group, favorable effects on the serotonergic system were assumed by the increase in the l-Trp/CAA ratio, the l-Trp/BCAA ratio, and several verum ingredients that act synergistically as cofactors in serotonin synthesis. In addition, the verum treatment had preventive effects on serum magnesium homeostasis, which was disturbed in the placebo group due to decreased serum concentration. Cardiometabolic risk factors, such as body weight, BMI, WC, and WHtR, increased significantly in the placebo group and resulted in a doubling of subjects with metabolic syndrome after 12 weeks compared with baseline. The cardiometabolic risk of participants in the verum group did not significantly deteriorate. Additional positive effects of the optimized food intake of fruits, vegetables, and animal-based foods causally related to the verum treatment or lifestyle changes were supposed. Further research about the complex interaction between specific nutrients and other lifestyle factors, such as food choice, would be of interest.Daily supplementation with a specific amino acid composition with micronutrients in participants with chronic perceived stress and exhaustion conditions resulted in more beneficial effects compared with the placebo group after 12 weeks. In the verum group, the reported subjectively perceived stress and the PSQ


Colaptes melanochloros (2n = 84), Colaptes campestris (2n = 84) and Melanerpes candidus (2n = 64)–by means of fluorescence in situ hybridization. Clusters of 18S rDNA were found in one microchromosome pair in each of the three species, coinciding to a region of (CGG)10 sequence accumulation. Interstitial telomeric sequences were found in some macrochromosomes pairs, indicating possible regions of fusions, which can be related to variation of diploid number in the family. Only one, from the 11 different microsatellite sequences used, did not produce any signals. Both species of genus Colaptes showed a similar distribution of microsatellite sequences, with some difference when compared to M. candidus. Microsatellites were found preferentially in the centromeric and telomeric regions of micro and macrochromosomes. However, some sequences produced patterns of interstitial bands in the Z chromosome, which corresponds to the largest element of the karyotype in all three species. This was not observed in the W chromosome of Colaptes melanochloros, which is heterochromatic in most of its length, but was not hybridized by any of the sequences used. These results highlight the importance of microsatellite sequences in differentiation of sex chromosomes, and the accumulation of these sequences is probably responsible for the enlargement of the Z chromosome.Birds are characterized by a low proportion of repetitive DNA in their genome when compared to other vertebrates. Among birds, species belonging to Piciformes order, such as woodpeckers, show a relatively higher amount of these sequences. The aim of this study was to analyze the distribution of different classes of repetitive DNA—including microsatellites, telomere sequences and 18S rDNA—in the karyotype of three Picidae species — It is known that the avian genome is extremely compact, corresponding to approximately one third of the typical mammalian DNA content . This dePicoides pubescens, where the transposable element CR1 is one of the most important components [So far, the Picidae family (woodpeckers) shows the highest proportion of repetitive sequences in genomes among birds, reaching up to 22% of total DNA amount in the species mponents . In addimponents –15.Gallus gallus motifs (GA)15 and (GAG)10 were detected mainly in the W chromosome and, at low frequency, in the Z and autosomes. However, the organization of repetitive sequences in the genome of birds, especially their chromosomal location, is practically unknown.In other groups of organisms, analyses of repetitive sequences have related their accumulation to the process of sex chromosomes differentiation, as in plants, reptiles and, in a greater proportion, fish –18. In tThe Picidae family showed a large karyotype variation in their 2n and a high amount of repetitive sequences in their genomes in comparison with other birds. Therefore, in this study we aim to characterize the chromosomal distribution of 13 classes of repetitive DNA sequences in three Picidae species, focusing on the association of these sequences with karyotype evolution and the ZW sex system differentiation.Colaptes campestris, five female Colaptes melanochloros and one male Melanerpes candidus  were used in this study: three male and one female candidus . AnimalsChromosome preparations were obtained using short term cultivation of bone marrow  or fibroChromosome biometry was performed using the software Micromeasure 3.3 . C-bandi5′-GTA GTC ATA TGC TTG TCT C-3′, NS8 5′-TCC GCA GGT TCA CCT ACG GA-3′ and nuclear DNA of Ocyurus chrysurus  [n were obtained by PCR in the absence of a DNA template, using primers (TTAGGG)5 and (CCCTAA)5 [18S rDNA fragments were amplified by PCR using primers NS1 janidae) . AfterwaCCCTAA)5 , labeledCCCTAA)5 .15, (CAA)10, (CAC)10, (CAG)10, (CAT)10, (CG)15, (CGG)10, (GA)15, (GAA)10, (GAG)10 and (TA)15 were directly labeled with Cy3 during synthesis , as described by Kubat et al. [Oligonucleotide probes containing microsatellite sequences (CA)t et al. .At least 30 metaphase spreads per individual were analyzed to confirm the 2n, karyotype structure and FISH results. Metaphases were analyzed in an epifluorescent microscope , and images were captured with the software Axiovision 4.8 . Final editing of images used Corel Photo Paint X5.Colaptes, while Melanerpes candidus showed 2n = 64  observed in the centromeric region of pairs 1–3 in both  species  and paircandidus .15 did not produce signals in any of the species analyzed. The same hybridization patterns were observed in both Colaptes species, while M. candidus showed a distinct pattern  pattern . AccumulColaptes (10 and (CAG)10 in Colaptes, while M. candidus showed bands with (CAC)10, (GC)15 and (GAA)10 in chromosome 2. For microchromosomes, two different patterns of microsatellite distribution were observed: some accumulated along the total length of the microchromosomes (such as (CA)15 and (CGG)10), and the rest were observed in the terminal region of the chromosome arms (such as (GAA)10, (CG)15 and (CAT)10).Macrochromosomes showed a preferential accumulation of microsatellites in centromeric and telomeric regions in pairs 1, 2, 3 and 5 in all species, while pair 4 showed signals only in Colaptes . At inte10, (GAG)10 and (GA)15 sequences were found at the three species analyzed, showing three different bands along this chromosome: (GAG)10 and (GA)15 were found in three bands in Colaptes, while in M. candidus they formed four distinct bands in the Z chromosome. And (CGG)10, (CAT)10 and (CA)15 were found only in M. candidus, in the terminal region of the Z chromosome romosome .Birds belonging to the Picidae family showed an interesting chromosomal variation, with 2n ranging from 64 to 108 . This, aC. campestris and C. melanochloros have similar karyotypes in both, morphology and number of chromosomes, with 2n = 84 each. This same 2n has also been found in another species of this family, Picoides mahrattensis, although the only other species from the same genus, Colaptes auratus, showed 2n = 90 [C. auratus is more basal than both, C. campestris and C. melanochloros [Our data suggest that  2n = 90 . Consideochloros , thus, tochloros .Colaptes, and considering that C. auratus (2n = 90) is placed in a more basal position in relation to C. campestris and C. melanochloros, both with 2n = 84, it can be argued that ITS found in the centromeric region of submetacentric pairs 1, 2 and 3 in species with 2n = 84 confirm the occurrence of three centric fusions, which would decrease the hypothetical basal 2n from 90 to 84. However, we also need the occurrence of a pericentric inversion to explain the difference in the number of arms (92 in C. auratus to 90 in C. campestris and C. melanochoros), but all of these rearrangements can be confirmed only by comparative chromosome painting.Although in birds, as in other groups, the occurrence of ITS in the centromeric region may represent the accumulation of repetitive sequences, coincidently similar to telomeres , it may Rhea americana, Crypturellus tataupa, Tinamus solitarius and Pterocnemia pennata [10 was found in chromosomes possessing the secondary constriction, bearers of 18S rDNA clusters. A similar result was found in the fish Triportheus trifurcatus , where this sequence exist in the W chromosome, which also bears 18S rDNA [Despite their phylogenetic position, the species analyzed in this study retained the plesiomorphic character of showing 18S rDNA clusters in only one microchromosome pair, as it is in the majority of the bird species analyzed so far, including some basal groups such as Paleognathas  pennata , 31, 32.18S rDNA .Microsatellite sequences were found in both, macrochromosomes, including the Z and in microchromosomes, with some differences between species and in each of the sequences. Despite the existence of interstitial blocks of repetitive DNA, accumulations of microsatellites were also found in centromeric and telomeric regions of the chromosomes.10 is accumulated in the W chromosome of Gallus gallus [Colaptes. Instead, some of these sequences were found accumulated in both Z chromosome arms which could explain the fact that it is the largest chromosome in these species. It represents an unusual example of the Z accumulating more repetitive DNAs than the W chromosome. Similar results have been found in the fish Hoplias malabaricus, in which the X chromosome was the preferred site for repetitive DNA accumulation in comparison with the Y [Repetitive sequences play an important role in the differentiation of sex chromosomes in different groups, including birds, despite the small amount of these classes of DNA in their genome. For instance, it was found that the sequence (GAG)s gallus . Howeverth the Y . Thus, tOverall, it may be said that our FISH analysis showed that several microsatellite sequences are found amplified on the Z chromosome of three species belonging to the Picidae family. This may explain the fact that the Z is the largest element of the karyotype, and that their genome contains the highest number of repetitive sequences compared to other groups of birds. Interestingly, none of the sequences were found accumulated on the W chromosome, although they play an important role in the differentiation of sex chromosomes, and are usually found amplified on Y/W chromosomes. These results suggest that, despite the common origin proposed for the ZW sex system in birds, these chromosomes followed different evolutionary trajectories in each species, indicating a high plasticity for sex chromosome differentiation in this group. Amplifications of microsatellite motifs were also found in macrochromosomes and microchromosomes. However, considering the lack of information concerning the genomic distribution of these sequences, it is not yet possible to make a comparison with other birds.This work is the first step towards clarifying the role of satellites and microsatellite sequences in the differentiation of sex chromosomes. Future studies involving other groups of birds are needed to increase our knowledge in processes of evolution and differentiation of these chromosomes.


Our findings provide a better understanding of the pathogenesis of PS and RA, suggesting potential strategies for treating and preventing both diseases. This study may also provide a new paradigm for illuminating the common pathogenesis of different diseases.Psoriasis (PS) and rheumatoid arthritis (RA) are immune-mediated inflammatory diseases. Previous studies showed that these two diseases had a common pathogenesis, but the precise molecular mechanism remains unclear. In this study, RNA sequencing of peripheral blood mononuclear cells was employed to explore both the differentially expressed genes (DEGs) of 10 PS and 10 RA patients compared with those of 10 healthy volunteers and the shared DEGs between these two diseases. Bioinformatics network analysis was used to reveal the connections among the shared DEGs and the corresponding molecular mechanism. In total, 120 and 212 DEGs were identified in PS and RA, respectively, and 31 shared DEGs were identified. Bioinformatics analysis indicated that the cytokine imbalance relevant to key molecules , p38 mitogen-activated protein kinase (MAPK), tumor necrosis factor (TNF), colony-stimulating factor 3 (CSF3), interleukin- (IL-) 6, and interferon gene (IFNG)) and canonical signaling pathways (such as the complement system, antigen presentation, macropinocytosis signaling, nuclear factor-kappa B (NF- PS is characterized by epidermal hyperplasia, and the predominant pathological feature of RA is the destruction of synovial joints. Studies have increasingly suggested that patients suffering from PS or RA have similarly increased risks of certain disorders, such as major adverse cardiovascular events, malignancy, and liver fatty changes, compared to the general population –6. Additpatients , 9. For patients . An incrDue to the progress in high-throughput techniques for biological research, next-generation sequencing (NGS) platforms are often used to explore the gene profile; these platforms have the advantages of greater sensitivity and more precise quantification, thus providing a more complete picture of the transcriptome in studies of gene expression than that obtained by microarrays . MeasureThe present study applied RNA sequencing technology to the peripheral blood mononuclear cell (PBMC) RNA of PS and RA patients and healthy volunteers, and differentially expressed genes (DEGs) were explored among the groups. Furthermore, bioinformatics analysis was performed to identify the key molecules and signaling pathways relevant to RA and PS as well as the upstream regulators related to the identified genes. This study aimed to obtain a comprehensive understanding of the cytokine imbalance in RA and PS based on DEGs, which may provide new insights into the pathogenesis of and suitable prevention strategies for these two diseases.PS patients, RA patients, and healthy volunteers were recruited from the China-Japan Friendship Hospital in Beijing City of China at the dermatology clinic, the rheumatology clinic, and the health screening center, respectively. The diagnosis of PS was consistent with the guidelines of the care for the management of psoriasis from the American Academy of Dermatology and the guidelines for the treatment of psoriasis from the Psoriasis Study Group of Chinese Medical Association , 15. AddThe following subjects were excluded: individuals who were ≥65 years old and ≤18 years old; individuals with complications, such as cardiovascular and cerebrovascular diseases, respiratory, digestive, urinary, and hematological diseases, metabolic syndrome, and mental disturbances; individuals who were pregnant, lactating, or who planned to become pregnant within a year; individuals who received topical treatments (such as corticosteroids or retinoic acid) within 2 weeks, systemic therapy within 4 weeks, or biological therapy within 12 weeks; PS patients with a concurrent RA diagnosis; and RA patients diagnosed with any type of PS.In summary, 10 female PS patients, 10 female RA patients, and 10 female healthy controls were enrolled into this study. All protocols involving human subjects were approved by the ethics committee of the China-Japan Friendship Hospital (ethics ID: 2014-58), and informed consent was signed by all participants before the study began.7 PBMCs)  and stored at −80°C for the subsequent testing. Total RNA in PBMC sample was isolated using the Trizol extraction method, and it was quantified with a NanoDrop ND-1000 spectrophotometer . The RNA Integrity Number was greater than 7.0, and acceptable quality values accorded with A260/A280 ratios ranging from 1.8 to 2.2 for each total RNA sample.In all, 3 mL peripheral fasting blood samples were collected from all subjects in the morning. PBMCs were isolated using density gradient centrifugation. Specifically, based on Ficoll-Hypaque gradient solution , 3 mL of heparinized whole blood was diluted to 6 mL with phosphate-buffered saline , layered on top of 3 mL of Histopaque and centrifuged for 30 min at 400 ×g. PBMCs were aspirated, washed twice, suspended in PBS, and counted with a hemocytometer. PBMCs were lysed in Trizol reagent . Only the tags with perfect matches or one mismatch were further considered and annotated based on the reference genes. The expression level of each gene was estimated by the frequency of clean tags and then normalized to TPM (number of transcripts per million clean tags), which is a standard method extensively used in DEG analysis . The numhttp://www.ingenuity.com). The “Core Analysis” module in IPA was utilized to analyze and visualize interactions of the shared DEGs. These interactions were characterized by specific canonical pathways and molecular networks. Analytical score was the negative base 10 logarithm of Fisher's exact test p value in canonical pathway analysis. Significance for biological functions of each network was symbolized by a p value for the enrichment of the genes in the network by comparison with the entire Ingenuity Pathway Knowledge Base.The information of shared DEGs identified in PS and RA was uploaded into the Ingenuity Pathways Analysis system . As shown in One hundred and twenty genes in PS and 212 genes in RA were identified as DEGs when compared with the controls  more than 2.00E + 00 represented the most significantly relevant pathways related to the merged network and included the complement system, antigen presentation, macropinocytosis signaling, acute phase response signaling, nuclear factor-kappa B (NF-κB) signaling, IL-6 signaling, IL-17 signaling, and p38 MAPK signaling , IL-6, FOS, p38 MAPK, and TNF, and the connection between every regulator and the corresponding target molecule is presented in Thirty-six upstream regulators were identified by biomolecular network analysis, with the majority being cytokine molecules, that is, 52.78% of them  were associated with an imbalance of cytokines; this imbalance may provide new clues for a better understanding of PS and RA.PS and RA are immune-mediated inflammatory diseases. An increasing number of studies have reported a correlation between PS and RA, but the exact common molecular mechanisms have not been elucidated. With the development of high-throughput detection and analysis techniques, including genomics and bioinformatics, the exploration of these comprehensive mechanisms has become feasible. PBMCs can be extracted from whole blood and consist of lymphocytes  and monocytes. Identifying gene expression in PBMCs is an important strategy to determine disease-specific genes in holism . Methodoα inhibitors exert their effectiveness in these two diseases [γ and IL-17A, regulate macropinocytosis in macrophages [γ) mediate the acute phase response [The complement system is an essential component of innate immunity, and it plays an important role in modulating adaptive immunity. Its activation contributes to the pathogenesis of autoimmune and inflammatory diseases, such as PS and RA . Reductidiseases . In thisdiseases . Increasdiseases . This stdiseases . In addidiseases . This stdiseases . The resdiseases . Antigendiseases . Cytokinrophages . Cytokinresponse . In shorα, is the best-known member of TNF superfamily. As a cytokine, TNF stimulates cell proliferation and cell differentiation and plays a key role in the pathogenesis of PS and RA [γ protein. IFNγ is a cytokine secreted by Th cells  and is an important activator of macrophages. IFNγ has been implicated in the initiation/maintenance of inflammation. A study showed that the pathogenesis of RA was correlated with reduced frequencies of IFNγ producers [γ, as well as the corresponding signaling pathways are involved in the pathogenesis of PS and RA, which not only verified previous discoveries but also reflected the importance of these cytokines. In brief, an imbalance in these cytokines plays a crucial role in the pathogenesis of PS and RA, and targeting these cytokines is a key strategy for prevention and treatment.IL-6 is a proinflammatory cytokine that induces activation of Th cells and controls the balance between Treg cells and Th17 cells. In lesional psoriatic skin, IL-6 is markedly elevated, and T lymphocytes encounter high IL-6 levels, thus allowing cutaneous T cells to avoid Treg suppression and increasing the Th17 inflammatory activity . TargetiS and RA . A lack S and RA . CSF3 acS and RA . IFNG enroducers . This stκB is essential for the expression of proinflammatory cytokines and controls a number of essential cellular functions, including the immune response, cell proliferation, and apoptosis. Loss of normal NF-κB signaling regulation is a major contributor to a variety of inflammatory and autoimmune diseases, such as PS and RA [κB, and p38 MAPK signaling activation is an important characteristic of the inflammatory response in activated macrophages in RA, and p38 MAPK signaling is involved in the process of RA angiogenesis [α and IL-6 through inhibiting the activation of NF-κB and ERK1/2 is an important strategy for treating RA [κB, Fos, p38 MAPK, and ERK1/2, are highly linked molecules or upstream regulators that are closely associated with the shared DEGs in PS and RA and therefore regulated cytokines; these four biomolecules represent novel targets to prevent and treat these two diseases.NF-S and RA , 43. ActS and RA . The immS and RA . ERK1/2 S and RA , 47. IL-ogenesis . In addiating RA . This stκB signaling, IL-6 signaling, IL-17 signaling, and p38 MAPK signaling, reflected this type of imbalance. The new findings in this study provided a new molecular understanding of PS and RA and could pave the road for the discovery of new strategies for treating PS and RA.The common pathogenesis of PS and RA was characterized by a cytokine imbalance. The deregulation of certain key molecules, such as ERK1/2, CSF3, FOS, IFNG, and TNF, as well as the perturbation of signaling pathways, including the complement system, antigen presentation, macropinocytosis signaling, NF-In Supplementary Material, identified DEGs in PS vs. Control and RA vs. Control, functions and upstream regulators of the shared DEGs between PS and RA, as well as signaling pathways relevant to the merged bionetwork were listed in table in detail.


The most obvious increase or decrease in physical parameters was found under the combined stress condition. Moreover, to identify proteins differentially regulated by the three stress conditions at the eight-leaf stage, total proteins from the maize leaves were identified and quantified using multiplex iTRAQ-based quantitative proteomic and LC-MS/MS methods. In summary, the expression levels of 135, 65, and 201 proteins were significantly changed under the heat, drought and combined stress conditions, respectively. Of the 135, 65, and 201 differentially expressed proteins, 61, 28, and 16 responded exclusively to drought stress, heat stress, and combined stress, respectively. Bioinformatics analysis implied that chaperone proteins and proteases play important roles in the adaptive response of maize to heat stress and combined stress, and that the leaf senescence promoted by ethylene-responsive protein and ripening-related protein may play active roles in maize tolerance to combined drought and heat stress. The signaling pathways related to differentially expressed proteins were obviously different under all three stress conditions. Thus, the functional characterization of these differentially expressed proteins will be helpful for discovering new targets to enhance maize tolerance to stress.At the eight-leaf stage, maize is highly sensitive to stresses such as drought, heat, and their combination, which greatly affect its yield. At present, few studies have analyzed maize response to combined drought and heat stress at the eight-leaf stage. In this study, we measured certain physical parameters of maize at the eight-leaf stage when it was exposed to drought, heat, and their combination. The results showed an increase in the content of H Under field conditions, crops are often subjected to a combination of several stresses, which have an adverse effect or may even prove lethal. Recently, researchers have begun to pay more attention to the potential molecular mechanisms involved in crop endurance to combined stress  methods and quantitative proteomic and LC-MS/MS (liquid chromatography/tandem mass) assays  not only constitutes a major cereal crop, and food for both humans and animals, but has also become a critical resource for industrial use and for bio-energy production throughout the world. Maize is highly productive under suitable growth conditions. However, in many regions of the world, maize is mainly grown in semi-arid environments characterized by water scarcity, high temperature, and a combination of these conditions in the field. Maize originated from the tropics but is still sensitive to drought and heat, particularly after reaching the eight-leaf stage  was measured using an OS-30p Chlorophyll Fluorometer  on the eighth fully expanded leaf.Malondialdehyde (MDA) content was measured according to the method described by Hodges et al. : 50 mg fg for 20 min at 4°C and the supernatant was immediately used for antioxidant enzyme assays.According to the method we described previously  was visualized at the subcellular level using cerium(III) chloride (CeCl3) for localization  were excised from the treated and untreated leaves and incubated in freshly prepared 5 mM CeCl3 in 50 mM 3-(N-morpholino) propanesulfonic acid (MOPS) at pH 7.2 for 1 h. The leaf sections were then fixed in 1.25% (v/v) glutaraldehyde and 1.25% (v/v) paraformaldehyde in 50 mM sodium cacodylate buffer, pH 7.2, for 1 h. After fixation, tissues were washed twice for 10 min in the same buffer and post-fixed for 45 min in 1% (v/v) osmium tetroxide, and then dehydrated in a graded ethanol series  and embedded in Eponaraldite . After 12 h in pure resin, followed by a change of fresh resin for 4 h, the samples were polymerized at 60°C for 48 h. Blocks were sectioned (70~90 nm) on a Reichert-Ultracut E microtome, and mounted on uncoated copper grids (300 mesh). Sections were examined using a transmission electron microscope at an accelerating voltage of 75 kV.Hydrogen peroxide  and 4 ml phenol , then 1 mM phenylmethanesulfonyl fluoride (PMSF) and PhosSTOP phosphatase inhibitor cocktail  was added to inhibit protease and phosphatase activity. The mixture was thoroughly vortexed for 30 s and the phenol phase was separated by centrifugation at 14,000 × g and 4°C for 15 min. The upper phenol phase was pipetted into new 10 ml tubes, and four-fold volumes of cold methanol plus 100 mM ammonium acetate were added. After centrifugation at 14,000 × g and 4°C for 15 min, the supernatant was carefully discarded and the precipitated proteins were washed twice with cold acetone. Finally, the protein mixtures were harvested by centrifugation. Protein concentrations were measured using a 2-D Quant Kit , with bovine serum albumin  as the standard. To enhance the quantitative accuracy, extracted proteins from every biological replicate were adjusted to the same concentration for the subsequent analysis.As reported in our earlier study  procedure described by Wiśniewski et al.  and Lv eFor labeling, each iTRAQ reagent was dissolved in 70 μl of ethanol and added to the respective peptide mixture. The samples were referred to as control (under no stress), drought, heat, and combined drought and heat stress and were labeled with reagent and vacuum dried.2PO4 in 25% of Acetonitrile, pH 2.7) and loaded onto a PolySULFOETHYL 4.6 × 100 mm column . The peptides were eluted at a flow rate of 1 ml/min with a gradient of 0–10% buffer B  for 2 min, 10–20% buffer B for 25 min, 20–45% buffer B for 5 min, and 50–100% buffer B for 5 min. The elution was monitored by absorbance at 214 nm, and fractions were collected every 1 min. The collected fractions (about 30 fractions) were finally combined into 10 pools and desalted on C18 cartridges . Each pool was concentrated by vacuum centrifugation and reconstituted in 40 μl of 0.1% (v/v) trifluoroacetic acid. All samples were stored at −80°C until LC-MS/MS analysis.iTRAQ-labeled peptides were fractionated by strong cation exchange (SCX) chromatography using the AKTA Purifier system . The dried peptide mixture was reconstituted and acidified with 2 ml buffer A . Ten microliters of each fraction was injected for nanoLC-MS/MS analysis. The peptide mixture (5 μg) was loaded onto a C18 reversed-phase column  in buffer A (0.1% formic acid) and separated with a linear gradient of buffer B (80% acetonitrile and 0.1% formic acid) at a flow rate of 250 nl/min controlled by IntelliFlow technology over 140 min. MS data was acquired using a data-dependent “top10” method, which dynamically chooses the most abundant precursor ions from the survey scan (300–1800 m/z) for HCD (higher collision dissociation) fragmentation. Determination of the target value is based on predictive automatic gain control (pAGC). Dynamic exclusion duration was 60 s. Survey scans were acquired at a resolution of 70,000 at m/z 200, and resolution for HCD spectra was set to 17,500 at m/z 200. Normalized collision energy was 30 eV and the underfill ratio, which specifies the minimum percentage of the target value likely to be reached at maximum fill time, was defined as 0.1%. The instrument was run with peptide recognition mode enabled.MS/MS spectra were searched using Mascot 2.2 (Matrix Science) embedded in Proteome Discoverer 1.4 against the uniprot_Zea_mays_87227_20150504.fasta  and the decoy database. For protein identification, the following options were used: peptide mass tolerance, 20 ppm; MS/MS tolerance, 0.1 Da; enzyme, trypsin; missed cleavage, 2; fixed modification Carbamidomethyl (C), iTRAQ 4-plex (K), iTRAQ 4-plex (N-term); variable modification Oxidation (M), FDR ≤ 0.01.The protein and peptide probabilities were set at 50 and 60%, respectively. Only proteins with at least two unique peptides with a Mascot score of at least 25 and detected in at least two replicates were further analyzed.z-scores to normalize the data.For each replicate of proteomics, iTRAQ ratios between drought/heat/combined stress and controls for each run were converted to http://wolfpsort.org). Protein–protein interaction networks were predicted using the publicly available program STRING (http://string-db.org/). STRING is a database of known and predicted protein–protein interactions. The interactions include direct  and indirect  associations, and they are derived from four sources: the genomic context, high-throughput experiments, co-expression, and previous knowledge. STRING quantitatively integrates the interaction data from these sources for a large number of organisms, and where applicable, transfers information between these organisms.The molecular functions of the identified proteins were classified according to their gene ontology annotations and their biological functions. The subcellular localization of the proteins identified in this study were predicted using the publicly available program WolfPsort (http://www.blast2go.com), specific groups of proteins were selected and analyzed on the basis of, for example, stimulus responses, chloroplasts proteins and enzymes.According to the known or predicted cellular localization and molecular function of the proteins, as determined by Blast2Go  and a threshold of ≥ 1.5-fold or ≤ 0.66-fold change ratio of stress-induced protein expression levels compared with control: under the heat stress, the expression level of 135 proteins showed significant changes, of which 67 were up-regulated and 68 were down-regulated; under the drought stress, the expression level of 68 proteins showed significant changes, of which 46 were up-regulated and 22 were down-regulated; and under the combined stress, the expression level of 201 proteins showed significant changes, of which 113 were up-regulated and 88 were down-regulated  of 1%. In detail, based on a significant linear regression . Under all three stress conditions, ribonucleoprotein A and fatty acid desaturase were down-regulated in common (Table Table 3), 36 proteins were up-regulated in common, of which 20 were shock proteins (HSPs), including 14 small HSPs (sHSPs). All of these HSPs were obviously up-regulated by the heat stress and combined stress, but were only slightly affected by the drought stress (Table Table 3). Of particular note was the finding that the expression levels of abscisic acid (ABA) stress ripening protein 2, ethylene-responsive protein, and ABA-, stress- and ripening-inducible-like protein were significantly up-regulated under the combined stress (Table Table 3).In this study, the expression level of 19, 39, and 59 proteins related to stimulus response showed significant changes under the drought, heat, and combined stress conditions, respectively  responded to the stresses. Under the drought stress, 12 enzymes, including one kinase and one phosphatase, were identified as differentially expressed. Under the heat stress, 27 enzymes, including two kinases and one phosphatase, were identified. Under the combined stress, 38 enzymes, including four kinases and one phosphatase, were identified Table . Three i5). Under the drought stress and the combined stress, asparagine synthetase (4332506) was found to interact with decarboxylase (4329593). In fact, for all three stress conditions, extensive interactions were found among all of the chloroplast proteins . Under the heat stress  was reduced by the heat stress and combined stress Table . The exp8, respectively. Most of the annotated molecular functions were found to relate to binding and catalytic activity, while most of the annotated biological processes were found to relate to cellular and metabolic processes.All identified proteins were classified by gene ontology (GO) annotation software and then classified as three functional groups: molecular function, biological process, and cellular component. The results of the GO analyses for the drought, heat and combined stress conditions are shown in Figures On the basis of biological process analysis using the BLAST2GO program, among differentially expressed proteins: for the drought stress, 19 proteins were classified as “response to stimulus,” two were involved in transport, and 11 were classified as binding proteins involved in DNA binding, protein binding and nucleotide binding Figures ; Table 2In the light of KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis: under the drought stress, the differentially expressed proteins were found to be mainly involved in the galactose metabolism, photosynthesis, and carbon metabolism pathways Figure ; under tThe final physiological response is dictated by the growth stage and plant tissue type, along with the severity and duration of the stress exerted on the plants. After developing to the eight-leaf stage, maize is sensitive to heat stress, especially to combined drought, and heat stress. In this study, we measured the changes in physical parameters and comprehensively analyzed the differentially expressed proteins in maize leaves in response to drought, heat, and their combination using iTRAQ-based quantitative proteomic and LC-MS/MS methods. The combined stress caused very significant changes in the level of protein expression in the maize leaf, and some changes exclusively resulted from the combined drought and heat stress.2O2 and MDA, and the activities of SOD, APX, and GR, were enhanced by these three stress treatments, especially by the heat stress and combined stress. Our results indicated that all three stress conditions induced and aggravated membrane injury; in addition, they showed that the maize plants triggered an anti-oxidative defense mechanism to alleviate the ROS, which enhanced their tolerance to stress.The generation of reactive oxygen species (ROS) often leads to the destruction of cellular structures, which ultimately causes cell death. MDA is widely used as a marker of oxidative lipid injury. In this study, the accumulation of HAbiotic stresses bring about serious damage to plant photosynthetic systems. In photosynthetic systems, photosystem II (ΦPSII) is one of the most sensitive components under drought and heat stresses. In soybean, 25 differentially expressed proteins of photosynthesis were involved in RuBisCO regulation, electron transport, the Calvin cycle, and carbon fixation under drought and heat stress conditions  related to chlorophyll syntheses were down-regulated, while PSI reaction center subunit V/N, HSP101, and FtsH protease were up-regulated by the heat stress and combined stress. FtsH protease is an ATP-dependent metalloprotease. In soybean, FtsH protease was up-regulated under the heat stress  is an important organelle responsible for proteostasis. The accumulation of misfolded proteins in the ER disturbs ER homeostasis and thus brings about ER stress. Misfolded proteins may bind to chaperone BiP and be degraded through the proteasome  under the heat stress and combined stress. The results suggest that the depression of PDI expression may cause the accumulation of misfolded proteins in ER. Thus, the expression of HSPs was significantly elevated in order to eliminate misfolded proteins. It is important to uncover the role of HSPs in protein turnover under heat stress and combined stress.bak1 (BRI1-associated receptor kinase 1) has been found to lose more water than wild-type and to be insensitive to ABA in stomatal closure, suggesting that BAK1 is involved in stomatal closure induced by ABA  belongs to a multigene family that regulates various different physiological responses  are primary channels that mediate the transfer of water and other small molecules across vacuolar and plasma membranes, and are associated with plant tolerance to stress. In this study, drought stress and combined stress up-regulated the expression of aquaporin PIP2-6. Other results have shown that the over-expression of MzPIP2:1 in + is involved in many cellular processes, including enzyme activation, protein synthesis, and osmotic regulation (Anschütz et al., Arabidopsis responses to drought, it is essential to regulate the homeostasis of intracellular K+. KZM2 has a voltage-gated K+ channel activity (Shabala and Pottosin, KArabidopsis leaves, the carrier protein SWEET17 is a major factor controlling fructose metabolism. The decrease of SWEET17 expression by stress causes fructose to accumulate in leaves (Chardon et al., Arabidopsis tolerance to stress (Klemens et al., Under abiotic stress conditions, carbohydrates accumulate in plant cells. In Owing in part to climate change, food resources are being challenged by drought, heat, and the combination of these factors. Plants apply different mechanisms to adapt to combined stresses than to adapt to a single stress. Among the drought, heat, and combined drought and heat stress conditions, we found more similar proteins between the heat stress and combined stress conditions. HSPs, especially sHSPs, showed abundant expression under the heat stress and combined stress, and were found to play a role in extensive signaling pathways, suggesting that HSPs play a crucial role in maize tolerance to heat stress and combined stress. Even though similar signaling pathways were found in response to the heat stress and combined stress, relative to the drought stress and heat stress, the combined stress led to the greater expression of chloroplast proteins, enzymes and stimuli response proteins, which led to the development of more extensive signaling pathways and protein interaction networks. Our results also implied that ethylene-responsive protein and ripening-related proteins, which promote leaf senescence, may also have a potential role in maize endurance to combined drought and heat stress. Therefore, our results could be used to further our understanding of the mechanisms of crop response to combined stresses.XH conceived and designed the research. FZ, YZ, and FT performed the experiments. DZ, HY, and XH analyzed the data. WW and CL contributed reagents/materials/analysis tools. XH and DZ wrote the paper. All authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


For the expanding population of bladder cancer survivors in Korea, the development of subsequent cancers is a significant concern. Here, we provide the second primary cancer incidence rates and types in Korean patients with bladder cancer.Using population-based data from the Korea Central Cancer Registry from 1993 to 2013, we studied the standardized incidence ratios among 48,875 individuals with an initial diagnosis of bladder cancer. Standardized incidence ratios for second primary cancers were evaluated according to age at diagnosis, latency, diagnostic year, and treatment.p < 0.05). For specific cancers, the standardized incidence ratios for stomach, colon, liver, and non-Hodgkin lymphoma were significantly lower in patients with bladder cancer. However, the risk of prostate and kidney cancer in patients with bladder cancer were significantly increased. The risk of lung squamous cell carcinoma and lung adenocarcinoma as second primary cancers was significantly elevated in patients with bladder cancer.Over the same period, the overall risk of a second primary cancer was reduced by 6% in patients with bladder cancer compared with the development of a new malignancy in the general population  contains supplementary material, which is available to authorized users. Bladder cancer (BC) is the 9th most frequent cancer worldwide  and the There is a long-term survival concern in patients with BC, especially those with second primary cancer (SPC). For Western patients, compared with the general population, BC survivors are more likely to develop SPCs, which frequently occur in the lungs or neck , 5. HoweThe purpose of this population-based cohort study was to calculate the incidence of SPC in Korean patients with BC and to estimate the effect of SPC on survival using a nationwide population-based cancer registry. The primary goal was to produce useful data for managing patients with BC.A total of 48,875 patients diagnosed with BC were evaluated between 1993 and 2013 as documented in the KCCR. The KCCR gathers information on ~ 80–90% of cancer cases across 180 hospitals across South Korea. In 1999, the scope of the KCCR was expanded to cover the entire South Korean population using the Population-Based Cancer Registry Program [To ensure that SPC remains distinct from primary BC recurrences and metastases, the KCCR uses coding rules based on the histological or topographical classifications of the International Classification of Diseases for Oncology 3rd edition  and the The first primary BC included patients with a single primary BC and the first BC in patients with multiple primary cancers. We excluded the following first primary BC cases: 1) age at diagnosis, unknown; and 2) BC reported at death. In addition, because SPCs diagnosed within two months of the first primary cancer diagnosis are considered synchronous, these cases were excluded to reduce the misclassification of undetected synchronous cancers and metastases.Ethical approval for the research protocol was provided by the institutional review board of the National Cancer Center (NCC2017–0182).Standardized incidence ratios (SIR) were used to compare the relative risk of the SPC incidence rates with those of the general population at baseline. We estimated cancer incidence for each cancer type according to age at diagnosis, latency, and diagnostic year, which was multiplied by the cumulative number of years at risk to calculate the number of cancer outbreaks expected for each stratum. SIR was estimated by dividing the observed number of SPCs in patients with BC by the number of patients at risk of developing a new malignancy in the general population. The 95% confidence intervals for the SIRs were estimated using Byar’s exact approximation to the accurate Poisson distribution of the observed number. The person-years at risk were calculated from two months after the initial BC diagnosis until death, the date of last known survival, or the study completion date .Results were classified based on age at the time of initial diagnosis with BC , year of first BC diagnosis (1993–2000 or 2001–2013), latency time among first BC diagnosis and subsequent primary cancer , and treatment type .P-value < 0.05, and were two-sided. The SIR and 95% CI calculations were performed using SEER*Stat . Survival analyses and log-rank tests were performed using STATA .Survival curves using the Kaplan-Meier method were calculated for BC patients with or without a subsequent cancer. The log-rank test was employed to verify the difference between groups of survival curves. All of the statistical tests were determined statistically significant at We obtained data from 48,875 patients, including 39,351 men (80.5%) and 9524 women (19.5%), with a median age at diagnosis of 67 years. The cohort characteristics are shown in Table Significantly lower SIRs were observed for cancers of the tongue , tonsil , stomach , colon , and liver , and for non-Hodgkin lymphoma . However, the risks of prostate cancer and kidney cancer in patients with BC increased significantly  . The 5- and 15-year OS rates for the SPC group were 72.3 and 28.3%, respectively, whereas those for the non-SPC group were 64.8 and 43.8%, respectively.At 21 years follow-up, 22,036 of the 48,875 BC patients had died. The 10-year overall survival (OS) rates were 46.2 and 52.6% in the SPC and non-SPC groups, respectively . Before 2.5 years, the presence of SPC accounted for 5.38% of distant stage cases in the non-SPC group, and 3.45% of cases in the SPC group (p = 0.183). Afterwards, the proportions of distant staging were 0.48% in SPC and 0.82% in non-SPC group (p = 0.719)  and non-Hodgkin lymphoma were less likely to occur as SPCs in patients with BC. However, these findings were incongruent with those reported previously , 5, 11. A previous study that evaluated Korean patients with prostate cancer and kidney cancer using similar methods revealed SPC SIRs of 0.75 and 1.13, respectively , 7. In tIn a study examining associations between urinary tract cancers, Kinoshita et al. demonstrated that BC and prostate cancer share similar traits such as DNA repair and N-acetyl transferase polymorphism . Kellen In this study, we interestingly found that the risk for tongue and tonsil cancer significantly decreased in patients with BC, and the result for tongue cancer is significantly lower in those over 60 compared to those aged 40–59. Chemical factors like tobacco and alcohol, biological factors like human papillomavirus (HPV), syphilis, oro-dental factors, dietary deficiencies, chronic candidiasis and viruses have been known to be significantly associated with oral cancer . The mecSmoking is a well-known risk factor for BC, kidney, lung, mouth, and pharynx cancers  and has The risk of cancer caused by radiation follows the individual exposed to radiation and continues to increase throughout the individual’s lifetime . StudiesTo our knowledge, this is the first study to evaluate the histological subtypes of lung cancers as an SPC. Cigarette smoking is an established risk factor for lung cancer, but the severity of its association with other histologic types is unclear. Khuder , 26 repoCumulative survival curves of patients with or without SPC were estimated to investigate whether SPC affects the survival rate of patients who have BC. In particular, for the first 8 years, the SPC group had superior survival rates compared with the non-SPC group. Overall, this study demonstrated that patients in the non-SPC group had significantly more advanced BC at the time of diagnosis. Therefore, the survival rate of the non-SPC group was lower than that of the SPC group for the first 2.5 years after diagnosis of BC. Conversely, after 2.5 years the reverse was noted with survival in the SPC group being inferior to that in the non-SPC group. These findings suggested that the SPC group would require more attentive and systemic surveillance after 2.5 years of follow-up.The present study has several limitations. First, information concerning several potential confounding variables including smoking, alcohol consumption, obesity, and familial cancer history were not available. Second, there was limited data available concerning genetic factors and specific cancer stages among the patients, making it impossible to evaluate the correlation between disease severity and SPC incidence. Third, the higher incidence of SPC might be associated with close surveillance or misclassifications because BC, prostate cancer, and kidney cancer often develop synchronously , 16. FouThe risk is lower among Korean survivors of BC compared to the expected risk of developing SPC in the general population. However, patients with BC remain at increased risk of some cancers, particularly prostate and kidney, lung squamous cell carcinoma, and lung adenocarcinoma. Therefore, longer and closer surveillance could be recommended for the early detection of SPC.Additional file 1:Figure S1. Kaplan-Meier curve: survival after bladder cancer (BC) according to the incidence of a second primary cancer (SPC) in all patients (2006–2013). (TIF 1011 kb)Additional file 2:Figure S2. Stage distributions by bladder cancer occurrence and follow-up years. (JPG 184 kb)