Exercise intolerance is very common in patients with coronary heart disease (CHD). Although some researches confirming the validation of traditional Chinese medicine (TCM) on CHD treatment, the effect of TCM on improving the exercise tolerance of patients with CHD remains unclear so far. Our trial is to investigate whether the Yangxinshi (YXS) tablet can improve exercise tolerance as well as the quality of life among CHD patients.It is a randomized, double-blind, placebo-controlled, multi-center trial. A total of 90 patients with CHD from 3 hospitals in China will be enrolled and randomly assigned to one of 2 groups: YXS group, N = 45; placebo group, N = 45. The 2 groups will simultaneously receive standardized western medicine and exercise-based cardiac rehabilitation program for 12 weeks. The primary outcome measure is the exercise capacity, which will be evaluated by the cardiopulmonary exercise test and 6-minute walking test. The 2nd outcomes include symptom improvement, psychologic issues, laboratory tests, side effects, and adverse events.To our knowledge, it is the 1st randomized controlled trial to evaluate the effect of TCM YXS tablet on exercise tolerance in patients with CHD. The results will provide more evidence for future studies in this area.This study protocol was registered in Research Registry (researchregistry5752). It is responsible for one-third of all deaths worldwide, and its incidence is forecasted to increase steadily, which will impose a huge health and financial burden on the whole country.–6 Exercise capacity (EC), also called exercise tolerance, refers to the maximum aerobic capacity that can be sustained without the occurrence of pathologic symptoms and/or medical signs, which serves as a strong predictor of cardiovascular and all-cause mortality.,8 As reports, the increase of average 1.0 metabolic equivalent  will be accompanied by the 12% improvement of survival. Peak oxygen consumption (VO2 peak) or MET can be estimated or directly measured by cardiopulmonary exercise test (CPET) or 6-minute walking test (6MWT). Exercise intolerance, characterized by dyspnea or fatigue, is an independent predictive factor and associated with poor prognosis in patients with CHD. As a multidisciplinary and comprehensive program, exercise-based cardiac rehabilitation (CR) can effectively improve EC, the quality of life (QoL) in patients with CHD. It also has other potential benefits including decrease ischemia, improves endothelial function, subsequently reduces readmissions, mortality, and morbidity.–11 American Heart Association (AHA)/American College of Cardiology (ACC) recommend CR as a class Ia for patients with acute myocardial infarction or coronary revascularization. From the perspective of traditional Chinese medicine (TCM), the pathogenesis of CHD is Qi and Yin deficiency, also named as “thoracic obstruction” or “cardialgia.” TCM, as a unique medical formulate, has been used in China for more than 2000 years and plays an important role in the treatment of cardiovascular diseases.Cardiovascular disease, especially coronary heart disease (CHD), has been a major threat to human life, A previous study has already reported the effects of promoting rehabilitation after the percutaneous coronary intervention with no obvious side effect, whether it could help improve EC for patients with CHD is still unclear. Our study aims to investigate the efficacy and safety of YXS tablet on the exercise tolerance in CHD and its role in the CR program.Yangxinshi (YXS) tablet, manufactured by Qingdao Growful Pharmaceutical Co, Ltd , is composed of thirteen kinds of Chinese herbal medicines, its active ingredient ginseng is considered as a supplement for Qi. It has been widely used for the treatment of CHD, relieving symptoms of angina pectoris, and protecting myocardial ischemia.22.1The objective of this study is to assess the efficacy and safety of YXS tablet on improving the exercise tolerance of patients with CHD, to provide evidence for its application in the CR program.2.2This study is a randomized, double-blind, placebo-controlled trial, which has been registered in Research Registry (researchregistry5752). The specific flowchart is shown in Figure 2.3This study is following with the Declaration of Helsinki and been approved by the institutional ethics committee of the first affiliated hospital of Changchun Chinese Medicine University, with the ethics number CCZYFYLL2017-038. It is also performed at Tongji hospital affiliated to Shanghai Tongji University, and Jinqiu Hospital in Liaoning Province. Any revision of protocol will be submitted to these ethics committees.2.4Patients will be recruited from three hospitals. As the leader unit of this trial, we began to recruit the patients from the outpatient or inpatient in March 2018. Specialists will explain the purposes, methods, benefits, and potential side effects of this study, then all participants need to sign an informed consent form.2.52.5.11.Aged 40 to 75, with no sex limitation.2.History of CHD, with or without revascularization treatment.3.,19Coronary angiography or coronary computed tomography angiography shows at least 1 major vessel's luminal diameter stenosis >50% according to the guidelines of western medicine diagnostic criteria of CHD.4.The patient is in a stable state, without emergency issues in recent 3 months.5.Completely understand the significance of this study and volunteer to participate.6.The patient agrees to follow all the procedures of this study and sign informed consent.2.5.21.Have a history of the acute coronary syndrome over the past 3 months.2.Uncontrolled hypertension (systolic blood pressure ≥180 mm Hg and/or diastolic blood pressure ≥100 mm Hg) or hypotension (systolic blood pressure <90 mm Hg and/or diastolic blood pressure <60 mm Hg).3.Having a history of stroke  or lower extremity peripheral arterial disease over the past 6 months.4.Pregnancy, breastfeeding, preparing for pregnancy, and perinatal female.5.With episode bronchial asthma, chronic obstructive pulmonary disease, bradycardia (resting heart rate <60 bpm), cardiac pacemaker, or atrioventricular block.6.Severe allergic constitution, known or likely to be allergic to the test drug or its components.7.Known bleeding tendency or hemorrhagic disease.8.Patients with serious liver and kidney dysfunction , other life-threatening serious primary or psychiatric diseases, and malignant tumors.9.Patients participated in other clinical trials during the past 3 months.10.Any other situations which may affect the outcomes of the trial.2.62.6.1The investigator's decision to withdraw refers to the cases in which it is not appropriate to continue the trial, including the following conditions:1.Patients who have used the medicine for more than 4 weeks should be included in the efficacy statistics; discontinuation or conversion of other similar medicines after 4 weeks (including 4 weeks) should be treated as ineffective and need to be dropout;2.During the trial, patients should be withdrawn and treated as invalid if they developed myocardial infarction or angina pectoris, which could not be relieved by nitroglycerin.3.During the trial, any changes of condition which will affect the compliance and the safety of the patients can be decided to discontinue by the investigator.4.The patients seriously violate the study scheme, have poor compliance with the trial, or consume the prohibited drugs.2.6.2All the patients are allowed to withdraw from the trial at any time without any reason. In some cases, patients no longer adapt the medical treatment and refuse to be followed up, even though they do not explicitly withdraw, can also be classified as “withdrawn.” For the cases who withdraw during the trial, the investigators carefully record the reasons, the medication process, and the last medication time. For those who withdraw or fail to come to the hospital on time, the investigators should try to inquire about the reasons via phone or letter, record the medication process, as well as the last medication time in detail. The relationship between withdrawal and the trial shall be carefully analyzed and recorded in the case report form (CRF). All original materials should be preserved.2.7Reasons for discontinuation include but not limited to the following circumstances:1.the patient needs to hospitalize for revascularization therapy;2.occurrence of serious adverse events (AEs) related to experimental drugs;3.occurrence of serious safety issues;4.financial or management reasons;5.administrative authorities.Once the discontinuation occurs, the safety and rights of the subject shall be protected as far as possible. The researchers or other staff shall cooperate in the following area: submit all generated research data; resolve all data problems; calculate, check and arrange all unused research products; and review the completeness of the research records.2.8After signing informed consent, eligible participants will be randomized into the YXS group and placebo group. An investigator carries out the baseline evaluation, named visit 1, which contain the basic information, medical history, physical examination, CPET, 6MWT, electrocardiograph (ECG), Seattle Angina Questionnaire (SAQ), Hamilton anxiety rating scale (HAM-A), Hamilton depression rating scale (HAM-D), laboratory tests, etc. Visits including symptoms, vital signs, physical examination, ECG, concomitant medications and AE will be performed 3 times per week for the following 12 weeks. At the end of the trial, the same evaluations will be performed as the visit one. The schedule of enrollment, visits, and assessments is shown in Figure 2.8.12, ventilation (VE), carbon dioxide production (VCO2), ventilatory slopes for carbon dioxide production (VE/VCO2), respiratory exchange ratio, oxygen pulse, anaerobic threshold, using V-slop method, maximum ventilation (VEmax), and peak workload. This test is performed and supervised by the trained staff.We adapt the symptom-limited CPET on a bicycle ergometer with the Bruce protocol. The initial workload is 5 W with loadless pedaling and increased 10 W every minute. The pedaling rate of 60 r/min is used to maintain consistency. The test discontinues until patients reach their limit of tolerance, such as complaining of fatigue, chest pain, shortness of breath, dyspnea, or abnormal ECG records are observed. At the end of the test, patients continue cycling with free-wheeling for 3 to 5 minutes, measurements will be continued during this period. Following parameters obtained from this test: peak VO2.8.2 Participants will be instructed to walk at their peace as far as possible along a flat, straight corridor, at least 50 m with a hard surface. The distance in 6 minutes will be recorded by the staff.This test will be performed under the American Thoracic Society guidelines.2.8.3The SAQ is designed to assess the disease-specific health status related to CAD. It contains 19 items, with the score ranges from 0 to 100, in which higher scores indicate better health status.2.8.4The HAM-A is designed to assess the severity of anxiety, which contains 14 items related to a 5-point scale, with the score ranges from 0 to 4.2.8.5The HAM-D is widely adopted in clinical trials assessing the effectiveness of antidepressants in the past decades. It consists of 17 items, which designed to measure the participants’ depressive symptomology.To avoid the risk of subjective influence, all these questionnaires performed and recorded by the same investigator at the baseline and 12 weeks.2.92.9.1All the participants continue the conventional western medical treatment for CHD, such as antiplatelet, β-receptor blockers, angiotensin-converting enzyme inhibitors, angiotensin receptor inhibitors, statins, and nitroglycerin. Other kinds of medicines that would not affect the results, such as antibiotics, anti-inflammatory drugs, vitamins, laxatives, hypnotics, etc, can also be allowed to be taken during the trial. Moreover, information about these medicines or concomitant treatments must be recorded in the CRF for future analysis.2.9.22-receptor agonist, tricyclic antidepressants, benzyprodil,pixicillin, sildenafil, vardenafil, and similar medicines are prohibited during the trial. If the patients’ condition gets worsen and has to take these above medicines, the case should be regarded as withdrawal or drop-out, and need to be recorded in the CRF.The following medicine including trimetazidine, any other TCM except for YXS tablet, antiarrhythmic, amiodarone, digitalis, β2.9.3 The control group receives placebo treatment, which mainly consists of starch without any effective ingredient. The placebo is provided by the same manufacturer as YXS. It is a dextrin that matches as much as possible in appearance, shape, color, size, package, and the taste with YXS table with no side effects on human health. All these medicines are consistent with the Chinese Medicine Standards of the State Food and Drug Administration.About 1.8 g of YXS tablet (0.3 g/tablet) will be taken in the experimental group, 3 times a day, orally, while 1.8 g of placebo will be taken in the control group with the same usage both for 12 successive weeks. YXS tablet is manufacture by Qingdao Growful Pharmaceutical Co Ltd. The main components are list in Table 2.9.4 The training intensity is determined based on the heart rate recorded at the anaerobic threshold assessed by CPET to customize a personalized exercise prescription. The detailed exercise prescription is shown in Table All the patients participate in the aerobic exercise for 12 weeks (3 times weekly) performed in the outpatients with no time limitation. Each session last 30 minutes, which includes 5 minutes warm-up, 20 minutes aerobic exercise on a cycle ergometer or treadmill, and a 5 minutes cool-down according to the exercise-based guideline in CR.2.10Compliance and adherence have a significant influence on the outcomes of CR. To ensure the compliance of exercise, the investigator checks the CRF weekly with medical and exercise performed, and makes telephone calls follow-up if necessary.At each visit time point, patients are also asked to return the remaining drugs; thus, the investigator calculates the drug compliance with the following formula: 2.11The randomization sequences of the population and drug are generated by the computer-based software program. The drugs are distributed according to the serial number, and the patients are randomly allocated (as 1:1 ratio) to the experimental or control group. Drug distribution is supervised by the main research coordinator, who had no role in the evaluation and management, is also responsible for supplying enough materials and inventories timely for each center. Randomization sequence and intervention allocation will be kept in the Qingdao Growful Pharmaceutical Co Ltd so as it can be unblinded when necessary.It is a double-blinded clinical trial, patients, investigators, and statistical staffs involved in are unaware of the treatment allocations until the study is completed in all the research centers.2.122 peak in the experimental group is estimated to be 20 mL/kg/min, while that in the control group is 18.5 mL/kg/min. The sample size is calculated using the PASS 15.0 software. A sample size of 39 achieving 90.487% power is sufficient to reject the null hypothesis, indicating that when the mean difference in population is 1.5 mL/kg/min, a standard deviation for both groups would be 2.0 mL/kg/min, with a significance level (a) of 0.05. Allowing for the shedding rate of 10% to 20%, a total of 90 cases need to be enrolled as a type I error rate of a = 0.05, a power of 80%, with each center responsible for 30 cases.The sample size calculation was based on the primary endpoint (EC). According to the previous report, the mean value of VOThis calculator uses the following formulas to calculate sample size and power, respectively: 2.13t test. Categorical variables will be presented as percentages and analyzed by the Chi-squared test. Variance analysis will be used to compare the changes in the SAQ, HAM-A, and HAM-D scores between the YXS group and the placebo group from baseline to 12 weeks. All tests are 2-sided tests and P < .05 is considered statistically significant. Intent-to-treat analysis of effectiveness and safety will also be carried out.Statistical analysis will be performed by the Statistical Analysis System (SAS) software . Continuous variables will be presented as the mean ± standard deviation, along with the analysis of the 2.14Data will be collected and recorded by the investigator on the CRF at each visit time points . Each center securely stores the data in its computer system. The data is anonymous, only the main investigator has access to the data. Once the trial is completed, all the data will be frozen respectively. All the co-investigators are responsible for data monitoring.2.152.15.12 peak and 6MWT from baseline to 12 weeks between 2 groups will be compared.The primary outcome of this trial is EC. Changes of the VO2.15.2The secondary outcome includes left ventricular ejection fraction, lipid level , SAQ, HAM-A, and HAM-D scores.2.16Symptoms, vital sign , physical examination, blood routine , urine routine, liver and renal function , and ECG will be performed for consideration of safety.2.17 The supervisor will carefully record all the details, such as time of occurrence, degree of AE, and probable causes on CRF. Once AE occurs, researchers should unblind and provide optimal treatment immediately. Any AE need to be submitted to the ethics committee within 24 hours.Treatment-related AE will be assessed by CTCAE4.0 at each visit.3On the base of exercise-based CR, whether the YXS tablet combined with basic treatment can improve exercise tolerance as well as the QoL in patients with CHD will be investigated in this trial. To our best knowledge, this is the first randomized controlled trial evaluating the effects of TCM in the CR program; hence, the outcomes will provide the evidence for future, large-scale, clinical trials. For CHD patients, Qi deficiency and blood stasis are the main pathogenesis, which further limits EC.,31 The unbalance between oxygen delivery and demand response for exercise intolerance. Although traditional Chinese methods, such as Taiji and Ba Duanjin, have been adopted in the exercise prescription, Chinese herbs still lack enough evidence. As a certified Chinese patent medicine developed and manufactured by Shanghai Pharmaceuticals Holding Co Ltd, YXS tablet contains various complicated components that play an important role in supplementing Qi, such as Astragalus membranaceus, rhizoma anemarrhenae, rhizoma corydalis, etc. It has been illustrated in previous researches that YXS could benefit rehabilitation after percutaneous coronary intervention and myocardial ischemia.,33 We speculate that the active compounds of YXS for supplying Qi could improve myocardial energy metabolism, further improve the EC for CHD patients, this still requires confirmation by evidence-based clinical trials.According to traditional Chinese theory, Qi, as the most basic substance constituting and sustaining the human body, could activate blood circulation, remove blood stasis, and relieve painfulness.–36 Previous studies reported the effects of YXS on improving depression-like behaviors, its role in CR still needs further investigation. The safety of the YXS tablet will be evaluated as patients’ symptoms and laboratory tests, such as blood routine, urine routine, liver and renal function, and echocardiography.The EC serves as the primary endpoint in the study, and SAQ, HAM-A, HAM-D are also observed. CR as a multidisciplinary and promising program can also effectively relieve depression and anxiety, thus improve QoL in patients with CHD.To improve the validity and representativeness of the samples and then reduce the risk of selection bias, 3 comprehensive 3rd-grade 1st-class hospitals in mainland China participate. This study is a double-blind, randomized, placebo-controlled clinical trial, which will provide higher quality evidence regarding YXS application in the CR program.The authors thank Tongji hospital affiliated to Shanghai Tongji University, and Jinqiu Hospital in Liaoning Province for participating.Conception and design: Sisi Zhang, Xiaoping MengData curation: Zhijia Zhao.Manuscript writing: Sisi ZhangMethodology: Jiaojiao Li.Recruiting the participants: Sisi Zhang, and Yang YangReview and editing: Xiaoping MengSupervision and coordination: Congying Liang, Zhijia Zhao, Jiaojiao LiSupervision: Congying Liang.Validation: Yang Yang.Writing – review & editing: Xiaoping Meng.


The lymph nodal invasion diagnosis is critical for therapeutic-decision and follows up in gastric cancer. However, the number of nodes to be examined for nodal invasion diagnosis is still under controversy, and the model for quantifying risk of missing positive node is currently not reported yet. We analyzed the nodal invasion status of 13,857 gastric cancer samples with records of primary tumor stage, the number of examined and positive lymph nodes in the surveillance, epidemiology, and end results (SEER) database, fitting a beta-binomial model. The nodes need to be examined with different primary tumor stage were determined based on the model. Overall, examining 11 lymph nodes reduces the probability of missing positive nodes to <10%, and the currently median nodes dissected is adequate (12 nodes). While the number of nodes demands to be dissected for T1, T2, T3, and T4 subgroups are 6, 19, 40, and 66, respectively. The currently implemented median value for these samples was 12, 12, 13, and 16, separately. It implies that the number of nodes to be examined is sufficient for early gastric cancer (T1), but it is inadequate for middle and advanced gastric cancer (T2–T3). The clinical significance of nodal staging score was validated with survival information. In summary, we first quantified the lymph nodes to be examined during surgery using a beta-binomial model, and validated with survival information. Patients with lymph nodal invasion significantly benefit–4 from radiation, while not for lymph node-negative patients. On the other hand, drawbacks of radiation have been widely reported, such as secondary malignancy.,6Gastric cancer is among most prevalent cancers worldwide, with 679,100 new cases and subsequently 498,000 deaths due to gastric cancer in China, 2015. Report suggests that at least 18 lymph nodal should be warranted for advanced node-negative gastric cancer, while another two studies suggest 15.,10 However, the exact number of nodal to be examined for patients with different primary tumor stages has not been reported hitherto.As lymph nodal invasion status is one of the important clinical indicators for post-operational radiation therapy, adequate lymph nodes dissection and examination are necessary for accurate diagnosis. According to previous studies, pre-operative staging is an important indicator for guiding lymph nodal dissection.In our work, the relationships between missing positive possibility and tumor stage was investigated using the number of examined lymph nodes and invaded nodes from the surveillance, epidemiology, and end results (SEER) database , fitting a beta-binomial model. Our results indicate that the minimum number of nodes examined for primary tumor (T1–T4) were 6, 19, 40, and 66, respectively, to reach <10% probability of missing nodal.22.1The data is all retrieved from SEER database, thus no ethics committee or institutional review board approval is needed for this study. After pre-processing of records in the SEER database, 13,857 samples were involved in this study. The detailed information of primary tumor locations and histological type was shown in Table 2.2Using our model, the parameters of beta-binomial model, α and β, were estimated to be 1.0058 (95% CI 0.9724–1.0402) and 1.1518 (95% CI 1.1100–1.1950). The overall probability of missing nodal invasion (1-sensitivity), as a function of the counts of examined nodes was calculated, as shown in Figure 2.3The primary tumor stage is the most important indicator for nodes examination. The probability of missing nodal was also estimated in different primary tumor stage T1–T4). To minimize the probability of missing nodal below 20%, the least number of examined nodes are 2, 8, 17, and 29 for T1, T2, T3, and T4, respectively , 19 (95% CI: 17.1–20.0), 39 (95% CI: 34.5–43.2) and 67 (95% CI: 65.3–73.5), which consistent with our results.2.4The follow-up information was not used in our model, and it is independent from our nodal staging score (NSS). The NSS in diagnosed N0 stage (no invaded lymph nodes detected) in primary tumor stages was divided by quantiles, and survival difference was compared Fig. . The NSS2.5In addition to primary tumor stage, differentiation grade was also used to evaluate the NSS. To reach <10% nodal missing possibility, 7, 19, 39, and 36 nodes need to be examined . The result indicates that the differentiation is also an important indicator for nodal dissection.3,12 Hence, lymph node invasion is crucial for therapeutic decision-making of gastric cancer patients, including radiation therapy selection and drug usage.,13 However, the number of examined lymph nodes is controversial, as inadequate nodes examination may lead to the miss of invaded nodes, while excessive number of nodes examination would reduce the immune activity. In the past years, efforts have been devoted to optimize the examined nodes.–17 However, the exact nodes number required for dissection in gastric cancer is not suggest. Using beta-binominal model, our work has shown that currently implemented number of nodes dissected is sufficient for early gastric cancer (T1) but not for middle and advanced gastric cancer (T4).Lymph node invasion is strongly associated with recurrence, metastasis, and poor prognosis in gastric cancer.–21 Another assumption in this work is that all examined nodal status is correct, which means, no false positive or false negative exist. This is considered to be reasonable as it is claimed by SEER database that all diagnoses were made by expertise pathologists. Clinical information including pathological information impact on cancer development and migration predict nodal invasion. Results of NSS based on differentiation grade were consistent with this.In this study, we chose primary tumor stage as the indicator to determine the number of lymph nodes to be dissected and examined. In present model, a few hypotheses were employed. First, the beta-binomial distribution of nodal invasion is assumed. However, the lymph nodes invasion rate of disease on different anatomic locations varies, which may have brought bias for the model. Experimentally, we did not see the lack of the model, based on previous studies in other cancers.Limits of this study also exist. It is also a retrospective study, though multicenter. Important clinical variables, such as drug usage, time to metastasis/recurrence, and surgery type are not available for this study, which makes the lack of enrollment control and may bring some bias. On the other aspect, the impact the NSS on treatment, including adjuvant therapy/neoadjuvant therapy cannot be found in SEERstat database, which makes the implication evaluation of NSS on therapy unavailable.44.1The SEER database includes 17 registries covering 26% population in the United States. Only samples with gastric cancer as the primary cancer were retained (resident tumor). Patients without records related to number of regional nodes examination, or positive nodes number were discarded. Furthermore, records with missing values in all these entities were also excluded. The primary tumor stages were divided into four categories . Totally, 13,857 samples enrolled in this study.Limitation of this study is that the data retrieved from SEER database was retrospective and detailed clinical variables, including time to metastasis and local recurrence, were not included in the records. Thus, recurrence-free survival and metastasis-free survival information are unavailable with this dataset.4.2 using the beta-binomial distribution to estimate the possibility of missing the invasion-positive as a function of total lymph nodal dissected and examined. The true positive indicates that the true status of lymph node was invaded, and the results showed at least one lymph node was invaded. The true negative means that the true status was uninvaded, and the examination results also showed no lymph invasion. The false negative defines that the true status is invaded, while the results showed no invasion.A previously described mathematical model was adoptedThree hypotheses employed in this model:1.All nodes examined are correct, which means, no false-negative or false-positive result exist.2.The distribution of lymph nodes is exchangeable. That means, the possibility of the examined lymph nodes has all the same chance to be invaded, which enables us to calculate the invasion possibility across patients. Biologically, this assumption is not reasonable, especially in small studies with different clinicopathological characteristics. However, if a large cohort employed, the assumption is reasonable, as most previous studies stated.3.The sensitivity of true positive and false negative is same, which enables us to generalize the results to pathologically node-negative samples. Sensitivities only can be calculated in node-positive samples.The procedure of model development and validation:1.–24The proportion of the number of positive lymph nodes (non-N0 stage) and the number of total nodes dissected/examined was used to estimate the coefficients of beta-binomial (α and β) distribution. In this step, samples were limited to these with at least two lymph nodes examined, because by definition, the node-positive rate would be 100% if only one lymph node examined. In gastric cancer, primary tumor stages are used as indicators for surgery and adjuvant therapy.2.False-negative rates were estimated according to the model and coefficient estimated, in the overall datasets and sub-datasets (different primary tumor stage), and the observed and corrected prevalence was calculated using the following function: kadj, indicates adjusted false negatives; FN is observed false negative; TPkadj, is the number of true positive; T indicates the primary tumor stage categories. FN, TP, TN means false negative, true positive, true negative, respectively.where FN3.Considering overall survival information is independent from lymph nodes dissection and NSS, we used it for validation. Tumors with no lymph nodes invasion was selected. Samples of each primary tumor stage category (T1–T4) were divided based on the quartile of NSS, and survival difference was compared using Log Rank test.4.3All analyses involved in this study were implemented on R software (v3.1.0) and R packages. The VGAM (v1.0-3) and bbmle (v1.0.18) package were used to evaluate α and β parameters of beta-binomial model. The survival analysis was completed with R package “survival” using Kaplan-Meier method.Liping Sun, Qiaohong Liu and He Ren paticipate in experimet design, Liping Sun and Lining Sun is responsable for funding acquisition, Liping Sun, Qiaohong Liu, He Ren and Ping Li analyzed and visualized the data, all authors intepreted the results, wrote the draft and revised the manuscript.


Multiple randomized controlled trials have shown that acupuncture (ACU) work well in the treating mammary gland hyperplasia, which has been widely used in hospitals of China. Although the choices of ACU treatments varies in practice, most are based on experience or preference. Therefore, we outline a plan to assess and rank the efficacy of the various ACU methods to formulate a prioritized regimen for mammary gland hyperplasia in ACU therapy.We will make a comprehensive retrieval in 7 databases as following: PubMed, Embase, Cochrane Library, China BioMedical Literature, China National Knowledge Infrastructure, Chinese Scientific Journals Database, and Wanfang database. The time is limited from the construction of the library to June 2020. We will evaluate the quality and the evidence of the included randomized controlled trials by the risk of bias tool and grading of recommendations assessment, development and evaluation, respectively. Bayesian network meta-analysis will be conducted using Stata16.0 and WinBUGS V.1.4.3.The results of this study will be published in a peer-reviewed journal.Our study is expected to provide high-quality, evidence-based recommendations on further treatment of MGH for clinicians.PROSPERO (registration number CRD42020158743). MGH is a common disease among young and middle-aged women, accounting for more than 70% of all cases of breast disease, which seriously affects the normal life of patients.Mammary gland hyperplasia (MGH) is a noninflammatory proliferative disease mostly occurring in women aged 25 to 45. In the United States, the rate of breast cancer in women whose biopsies showed proliferative disease with atypia is nearly 3-fold that of the women who had nonproliferative disease. Thus, the treatment of MGH is believed to be an effective approach for breast cancer prevention. Perhaps due to the fast pace of modern life, alongside increasing work-related stress and competitive pressure, the rate of occurrence of MGH has increased. The incidence of MGH in our country reached 25.5%, among which the number of people suffering from MGH accounted for the first place of mammary gland disease, reaching 78.6% of the number of people suffering from mammary gland disease, which was the high incidence among healthy people.,9In recent years, more and more attention has been paid to the carcinogenesis tendency of MGH. The incidence of cancerization is 2% to 4%. Modern medicine generally considers MGH to be related to sex hormone disorders secondary to dysfunction of the hypothalamus-pituitary-ovarian axis.Nowadays, it has become a generally accepted view that the occurrence of this disease is related to the endocrine disorder and mental factor. Periodic hormonal imbalance and(or) increased sensitivity of breast tissue to hormones are the leading causes of this disease. Hormone therapy or endocrinal therapy is often used to relieve symptoms over a short period. However, the side effects and complications of those treatments are severe, and the curative effect in long term application is also doubtful, such as delayed menstruation or menopause, increased leucorrhea, hot flashes, nausea, and so on. Vitamins are safe, easy to take and cheap, but the pain recurrence rate of it is high. Bromocriptine(a kind of prolactin inhibitors) is highly effective, but a long-term high dose of it can cause pulmonary, pleural, and retroperitoneal fibrosis. MGH itself has no indication of surgical treatment, and the operation often involves unnecessary injury or sacrifice of the breast, which is hardly accepted by the majority with a high recurrence rate. Thus, many patients need complementary and alternative therapy, such as acupuncture (ACU), which is generally considered to be safe and efficient by the populace.Western medicine commonly used in the treatment of MGH include hormone preparations, hormone receptor inhibitors, Vitamins, and prolactin inhibitors. Modern research and clinical experiences of Traditional Chinese Medicine (TCM) doctors have also shown that the diseased kidney and conception and thoroughfare vessels are the root cause, that liver Qi stagnation, blood stasis, and phlegm are the symptomatic manifestations, and that they have a mutual cause-and-effect relationship.MGH, also known as breast dysphasia, is classified under the “Rupi” category in Chinese medicine. Based on the traditional Chinese medicine theory, the pathogenesis is the disharmony of Chong and Ren, the stagnation of the liver, and the stasis of phlegm and blood. The treatment principles are to balance Chong and Ren, improve liver and kidney, and dredge phlegm and blood stasis. Also, it can effectively speed up the blood flow in the lesion area, reduce the resistance index of blood flow, and improve the blood circulation of breast tissue.Nowadays, there is increasing scientific evidence suggests that ACU is beneficial for MGH. It has been found that ACU can regulate estrogen secretion level, make a benign effect on estrogen signal transduction pathway, inhibit estrogen promoting breast hyperplasia, thereby inhibiting the replication of breast proliferative cells, slowing down cell proliferation, and promoting cell apoptosis. It compared the effectiveness of ACU and other therapies for MGH, not between various ACU treatments. Besides, it had deficiencies such as few outcome indicators and single search only in Chinese database. Hence, the aim of this study is to renew, improve, and strictly compare various ACU therapies to optimize ACU treatment of MGH and provide a foundational reference for clinical guide.Presently, there was a network meta-analysis (NMA) of ACU for MGH, which is an English article.22.1 It is registered on the International Prospective Register of Systematic Reviews  statement.2.22.2.1All the randomized controlled trials which is stated the “randomization” phrase will be included, regardless of allocation concealment, or used of blinding. The language will be restricted in Chinese or English.2.2.2All patients, who are over 18, have MGH (as diagnosed using any authoritative diagnostic criteria) regardless of age, sex, race, duration of disease, weight, or education.2.2.3ACU treatments include moxibustion, catgut embedding, electro-ACU, transcutaneous electrical acupoint stimulation, auricular ACU, scalp ACU, warm needling, manual ACU, acupoint injection, regardless of needling techniques and stimulation method.2.2.4The control group is treated with sham-ACU, placebo, pharmacotherapy, which is recommended in international or domestic authorized clinical guidelines, or no treatment. When studies combine ACU treatments with other active therapy, both the experimental and the control groups are required to use the same active therapy.2.2.52.2.5.1The effective rate.2.2.5.2Secondary outcomes will contain:(1)Numerical rating scale: in the most painful day of breast pain before treatment and after treatment.(2)Breast lumps size score: compare the size of the target mass before treatment and after treatment.(3)Symptom disappearance rate .(4)Quality of life (QoL) measures of general health status, for example, the MOS 36-Item Short Form Health Survey (SF-36).(5)Adverse effects .2.3The exclusion certain contain the following items:(1)Patients with acute medical conditions or pregnancy.(2)Intervention combined with any complementary therapy will be excluded, for example, Chinese herb decoction, ACU, and other complementary therapy.(3)Non-randomized controlled trials reviews, animal experiments, case report, expert experience, and conference article.(4)Incomplete data or information.(5)Repeatedly checked or published literature.2.4We will search the following electronic bibliographic databases: PubMed, Cochrane Library, EMBASE, Chinese National Knowledge Infrastructure, Chinese Biomedical Literature Database, Wanfang Database, the Chongqing VIP Chinese Science and Technology Periodical Database. All of them will be searched from inception to June 2020. The retrieval mode used will be a combination of free words and medical subject headings terms, including “fibrocystic breast disease,” “fibrocystic disease of breast,” “breast hyperplastic disease,” “hyperplasia of mammary glands,” “mammary glands hyperplasia,” “cyclomastopathy,” “acupuncture,” “acupuncture therapy,” “electroacupuncture,” “auriculotherapy,” “acupoint,” “acupoint injection,” “acupoint catgut embedding,” “moxibustion,” “needle,” “warm needle,” “temperature needle” “randomized controlled trial,” “randomized controlled,” “randomized, controlled,” “clinical trial.” The search strategy takes PubMed as an example, as shown in Table 2.52.5.1The selection, which includes literature screening, data extraction, and examination, will be conducted by 2 reviewers (QSH and JX). If there is any disagreement, a third reviewer (YFJ) will participate in the consultation to reach a consensus. The titles and abstracts of retrieved studies should be read first to rule out unrelated or repeated studies. Then, the remaining articles will be reviewed in full text by the reviewer according to the inclusion criteria, and the researches that meet the criteria will be selected at last. We will use the PRISMA flowchart to represent the complete process, which is shown in a PRISMA flow chart Fig. .2.5.2Two independent reviewers (QSH and HFH) shall design a standardized data extraction form, which including identification information , characteristic of trial , participants , interventions of both the observation group and control group , outcomes , adverse effects, duration of follow-up, type and source of financial support. In case of any discrepancies between 2 reviewers, we will discuss and reach a consensus, and if necessary, seek arbitration from a third reviewer (JX).2.5.3If there are ambiguous or unreported data, we will contact the corresponding authors for the missing data to get specific information by telephone or email. If the missing information still cannot be obtained that missing information, we will exclude it from the analysis.2.6 The main contents comprise 7 items: random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, selective reporting, and other sources of bias. The studies will be evaluated as being of “low risk of bias,” “high risk of bias,” or “unclear risk of bias.” Inconsistency will be consulted with the third review author (HLL).The assessment will be conducted by 2 reviewers (XHZ and KL) with the risk-of-bias assessment method from Cochrane Reviewer's Handbook 5.0.24.2.72.7.1I2 statistic will be used to assess levels of the heterogeneity of each pairwise comparison. If I2 < 50%, a fixed-effect model will be used, or else a random-effect model will be used. When the results are substantial heterogeneity or considerable heterogeneity, sensitivity analysis or meta-regression and subgroup analysis will be made to explore possible sources. When there is no explanation for statistical heterogeneity, a random-effects model will be used with a test level of a = 0.05.Continuous outcomes will be pooled with standardized mean differences with 95% confidence interval (95% CI), and dichotomous outcomes will be analyzed by calculating odds ratios with 95% CI. The 2.7.2 The convergence of the stimulation is tested by the potential scale reduction parameter, when the closer potential scale reduction parameter will be to 1, the better the model convergence is and more reliable the conclusion is. The selection of the final model will depend on the deviance information criterion value. Generally, a model with a smaller deviance information criterion value is better. Numerical variables will be presented as standardized mean differences with 95% CI. We will draw a surface under the cumulative ranking (SUCRA) for each outcome indicator to predict the order of curative effect of treatment measures, the larger the area under the curve, the better the treatment measures.STATA16.0 software will be used to through the GeMTC package will be used to perform NMA to synthesize direct and indirect evidence. The NMA will be undertaken primarily in WinBUGS1.4.3 software, using the Bayesian Markov Chain Monte Carlo random effect model.,26We will conduct the consistency model analysis of the primary outcome indexes and the probability ranking of the best treatment measures. If there is a closed ring, the node splitting method will be used to evaluate the inconsistency of each loop.2.7.3We will explore sources of heterogeneity by performing a network meta-regression using a random-effects network meta-regression model. If the necessary data are available, subgroup analysis will be carried out according to different factors as follows: the duration or dosage of ACU, duration of disease, period of treatment, and the type of intervention in the control group. A sensitivity analysis will be performed when there is significant heterogeneity according to the following aspects: sample size, heterogeneity qualities, methodological elements, and characteristic of research. If heterogeneity is reduced after low-quality or small sample studies are excluded, and we must be more cautious in concluding.2.7.4Publication bias will be evaluated using an Egger regression test which will help avoid observation bias and produce a funnel plot indicating a digitally based modeling result.2.7.5 All studies will be rated as 4 levels as following: high, moderate, low or very low, which according to the 5 aspects .The grading of evidence quality will be conducted by 2 independent reviewers using the grading of recommendations assessment, development, and evaluation instrument.3 which can make clear about the inferences of efficacy and help to compare efficacy of available therapies. Therefore, we would like to see an NMA, which is made in strict accordance with the NMA procedures, so as to provide more evidence to support the beneficial use of ACU and encourage more extensive use of ACU as an alternative therapy for MGH.ACU as an effective technique of TCM has been accepted for MGH in China. Still, due to the lack of direct comparison between different ACU methods, clinicians cannot choose the best one. As a result, clinicians tend to combine several ACU-based methods from their experience to determine which are most suitable for patients, which increases the burden of time and capital input, and results in inefficient utilization of medical resources. NMA can be used to integrate direct and indirect comparisons,The study also has some defects as follows: low quality of original researches, possible occurrence of false positive or false negative results, various duration of disease, different dosage and frequency of intervention, language restriction, and so on. All of these will lead to some bias and influence the results of evaluation, which may ultimately affect the reliability of this study.Conceptualization: Fanghui Hua, Jun Xiong.Data curation: Fanghui Hua, Jie Xiang, Yunfeng Jiang, Shouqiang Huang, Kai Liao.Formal analysis: Shouqiang Huang, Jie Xiang.Investigation: Jun Xiong, Honglian Li.Methodology: XiaoHong Zhou, Shouqiang Huang, Jie Xiang, Meihua Liu.Software: Kai Liao, Honglian Li, Meihua Liu.Supervision: Jun Xiong, Honglian Li, Yunfeng Jiang.Writing – original draft: Jun Xiong, Honglian Li.Writing – review & editing: Jun Xiong, Honglian Li.


In all the STPs, the removal efficiencies for the stimulant caffeine, NSAIDs , and the antibiotic cefradine were high; they were removed mainly by biological processes. Certain compounds, such as the NSAID ketoprofen, contrast agent iopromide, lipid regulator gemfibrozil, and antibiotic sulfamethoxazole, showed varying removal efficiencies depending on the contribution of biodegradation and sludge sorption. In addition, a quantitative meta-analysis was performed to compare the pharmaceutical removal efficiencies of the biological treatment processes in the four STPs, which were a membrane bioreactor (MBR) process, sequencing batch reactor (SBR) process, anaerobic–anoxic–oxic (A2O) process, and moving-bed biofilm reactor (MBBR) process. Among the biological processes, the removal efficiency was in the order of MBR > SBR > A2O > MBBR. Among the tertiary treatment processes investigated, powdered activated carbon showed the highest removal efficiency of 18%–63% for gemfibrozil, ibuprofen, ketoprofen, atenolol, cimetidine, and trimethoprim.In this study, we analyzed 27 pharmaceuticals in liquid and solid phase samples collected from the unit processes of four different sewage treatment plants (STPs) to evaluate their distribution and behavior of the pharmaceuticals. The examination of the relative distributions of various categories of pharmaceuticals in the influent showed that non-steroidal anti-inflammatory drugs (NSAIDs) were the most dominant. While the relative distribution of antibiotics in the influent was not high , it increased to 14%–30% in the effluent. In the four STPs, the mass load of the target pharmaceuticals was reduced by 88%–95% mainly in the biological treatment process, whereas the ratio of pharmaceuticals in waste sludge to those in the influent ( With rising living standards, the introduction of new chemical substances, and the development of medical technology, new trace contaminants are being detected in water environments, which has drawn significant social attention ,2. Among3/d or more in South Korea. Based on the type of biological treatment process, these plants can be classified into plants using sequencing batch reactors (SBR) (35%), A2O (26%), MBBR (23%), and MBR (9%) [Nevertheless, previous studies calculated the removal efficiency of pharmaceuticals by STPs simply based on the concentrations of the chemicals in influent sewage and effluent. However, recent studies quantitatively analyzed not only the influent of STPs, but also the substances adsorbed to the solid phase, such as suspended solids (SS) composed of organic solids, clays, and silts in process water and waste sludge and activated sludge in the bioreactors ,14. AnalMBR (9%) . TherefoAccordingly, this study investigated the contents of the target substances, namely 27 types of pharmaceuticals including antibiotics, NSAIDs, and antiarrhythmic agents, in the liquid and solid phase samples obtained from each unit operation of STPs. We then calculated the mass load of each substance, classified them according to their pharmacological use, and analyzed their fate for each unit operation. The target STPs were selected from plants in which the four types of treatment processes mentioned above comprised 93% of all the sewage treatment processes. We investigated the main removal mechanisms of the target substances through the analysis of their mass balance, and accordingly compared the removal efficiencies of the sewage treatment processes through meta-analysis. In addition, we evaluated the removal efficiencies of the tertiary treatment processes, including full-scale coagulation, ultraviolet (UV) treatment, and PAC treatment, in removing pharmaceuticals, and evaluated their degree of contribution to the overall treatment efficiency.Based on the marketing data of pharmaceuticals, 57 types of pharmaceuticals were initially selected as target compounds in our preliminary study. Out of the 57 compounds, we selected 27 compounds as the targets in this study based on their influent concentrations and detection frequencies. In total 27 types of pharmaceuticals, including 11 antibiotics, 6 anti-inflammatory/NSAIDs, 2 antiarrhythmic drugs, and 1 stimulant were selected as the target substances. We selected four STPs in Gyeonggi-province, South Korea, using MBR, SBR, A2O, or MBBR as a main sewage treatment process. Samples were collected three times, i.e., in June–July 2018, September–October 2018, and December 2018–January 2019, during normal operation of each STP. The primary sludge, waste sludge, and reject water were collected using the grab sampling method, whereas the 24 h composite sampling method was used for all other samples. In the STPs under study, the wastewater generating from the sludge-thickening and -dewatering processes is re-entered into their main-stream processes. The returned wastewater is referred to as reject water and contains a larger amount of pollutants such as nitrogen and phosphorus. Pharmaceutical compounds are also present at higher concentrations in the reject water, possibly having a negative impact on the treatment stability of a STP. Therefore, the pharmaceutical concentrations in the thickening water and dewatering water in STP A, washing water and dewatering water in STP B, and dewatering water in STP C were investigated.Equal volume discrete sample aliquots (250 mL) were collected manually from predetermined points of each STP , using a2EDTA; 10 μL; Sigma-Aldrich, Saint Louis, MO, USA) at a concentration of 40 mg/mL, and isotopically labeled standards (10 μL) at a concentration of 10 μg/L were added. The quick, easy, cheap, effective, rugged, and safe (QuEChERS) method was modified to extract target compounds from the solid phase samples, the details of which are shown in 2EDTA (0.2 mL) at a concentration of 40 mg/mL, and isotopically labeled standards (100 μL) at a concentration of 100 μg/L were added and mixed using a vortex for 1 min. Then, the mixture was subjected to solid-liquid separation at 4000 rpm for 5 min. One milliliter of the acetonitrile layer was collected in a glass tube, concentrated using a nitrogen concentrator, and dissolved in a solvent mixture of methanol and 0.1% formic acid at a ratio of 1:9. Finally, the sample was filtered using a syringe filter with a 0.45 μm pore size PVDF membrane  to create the final sample.As pharmaceuticals may exist in a liquid phase or solid phase in STPs, different pretreatment processes were applied to the two types of samples. A schematic diagram of analytical methods for determination of pharmaceuticals is shown in The sludge in the solid phase was subjected to solid-liquid separation at 3000 rpm for 5 min using a centrifuge. The supernatant obtained after centrifugation of the sludge was filtered using a syringe filter with a 0.45 μm pore size PVDF membrane and processed using the same procedure as that for the liquid phase sample. One gram of wet sludge remaining in the glass centrifuge tube was collected, and the solid samples were extracted by the same process as that for the SS. The analysis was performed in triplicate; The limit of detection and limit of quantification (LOQ) were calculated to have a S/N ratio of 3 or more and 10 or more, respectively, on the chromatogram . Table Sinfluent (g/d) = flow rate (m3/d) × influent concentration of each pharmaceutical (ng/L) × 10−6; Meffluent (g/d) = flow rate (m3/d) × effluent concentration of each pharmaceutical (ng/L) × 10−6; Msorption (g/d) = sludge production rate (m3/d) × sludge concentration of each pharmaceutical (ng/L) × 10−6; and Mbiodegradation (g/d) was calculated from the mass loads of the influent, effluent, and sorption.We calculated the daily mass load in the unit operation of each STP and used Equation (1) to analyze the mass balance .Minfluex is the removal efficiency of each substance, μ is the mean removal efficiency of the substance in all sewage treatment processes, σ is the standard deviation of the substance in all sewage treatment processes, n is the number of data points in each sewage treatment process, and N is the total number of data points in all sewage treatment processes.In the biological treatment process of the STP, it may be difficult to accurately evaluate the removal of pharmaceuticals owing to various factors such as the characteristics of the substance and the operating conditions of the treatment process. Meta-analysis has proven very useful for integrating and interpreting large amounts of data, and has been extensively applied in various fields such as medicine, ecology, and toxicology . This stp < 0.05.All statistical analyses were performed using GraphPad Prism 5.0 . For determination of significant variance between treatment processes and removal efficiencies of pharmaceuticals, a one-way analysis of variance (ANOVA) with Tukey’s multiple-comparison post hoc test was performed. Significant variance was achieved with Among the 27 types of substances in the influent, 16 showed 100% detection frequency and 25 showed at least 50% detection frequency. Acetaminophen (NSAID) had the highest concentration at 30,000 ng/L, followed by caffeine 26,000 ng/L), acetylsalicylic acid (6500 ng/L), ibuprofen (5300 ng/L), and cimetidine 2800 ng/L) a. Previo,000 ng/L00 ng/L aThe detection frequency of eight types of substances  was 100% in the effluent, and the detection frequency of 25 types of substances, excluding oxolinic acid and sulfamethazine, was 50% or greater. Sulfamethazine was found to be below the LOQ, which was similar to that in the influent. Cimetidine showed the highest concentration of 2100 ng/L; the substances in decreasing order of their concentrations were ibuprofen (1300 ng/L), clarithromycin (740 ng/L), iopromide (730 ng/L), roxithromycin (290 ng/L), and diclofenac (250 ng/L) b. A meanWith the exception of sulfamethazine, 26 types of substances were detected at a frequency of at least 50%, and 12 were detected with 100% frequency. Ibuprofen, cimetidine, acetaminophen, and acetylsalicylic acid were detected at concentrations of 1000 ng/L or more, and 13 types of substances including caffeine, ofloxacin, and iopromide showed a concentration range of 100–1000 ng/L c.We evaluated the ratio of the reject water concentration to the influent concentration (R/I ratio) . If thisTwenty of the 27 types of substances were detected at least once in the SS in the influent. Caffeine and naproxen showed 100% detection frequency, and 13 types of substances including acetylsalicylic acid, clarithromycin, and roxithromycin showed at least 50% detection frequency. In contrast, the concentrations of carbamazepine, oxolinic acid, propranolol, sildenafil, sulfamethazine, sulfamethoxazole, and trimethoprim were below the LOQ. Acetylsalicylic acid showed the highest concentration at 641 ng/g. The other substances in decreasing order of their concentrations were ibuprofen (410 ng/g), caffeine (280 ng/g), acetaminophen (220 ng/g), atenolol (210 ng/g), and clarithromycin (150 ng/g) d. Ashfaqp > 0.05).In the activated sludge collected from the aerobic tank, 25 types of substances were detected at least once and 16 types of substances showed at least 50% detection frequency. Only ofloxacin showed a 100% detection rate among the target substances. The concentration of ibuprofen was the highest at 1300 ng/g, followed by cefradine (1100 ng/g), acetylsalicylic acid (890 ng/g), ofloxacin (790 ng/g), clarithromycin (440 ng/g), and cimetidine (430 ng/g) e. Other The pharmaceuticals detected in the target STPs were classified into four types, namely stimulants, NSAIDs, antibiotics, and others, in order to assess their relative distributions and mass loads by use . The masFor caffeine (classified as a stimulant), the influent mass load was 990 g/d for STP A, 1197 g/d for STP B, 833 g/d for STP C, and 2253 g/d for STP D with the relative distribution ranging from 18–45%, which was the second highest after NSAIDs. The effluent mass load for stimulants decreased by at least 99% in comparison with the influent mass load in the four STPs, and their relative distribution in the effluent was less than 1%. Gao et al. 2016) also reported that 3848 g/d of caffeine flowed into the WWTP, more than 99% of which was present in the liquid phase . In the 16 also row), which indicates low hydrophobicity. However, they reportedly have zwitterionic properties and high adsorption owing to electrostatic interactions [ow values, thereby indicating that they are more hydrophobic than the other substances. Tran et al. (2016) reported that adsorption occurs between the positively charged dimethylamino group and the negatively charged sludge surface [The mass loads of the activated sludge and return sludge were 2226 g/d and 4811 g/d in STP A, 1720 g/d and 1203 g/d in STP B, 959 g/d and 6006 g/d in STP C, and 675 g/d and 1279 g/d in STP D, respectively. Ibuprofen , cimetidine , acetylsalicylic acid , and ofloxacin  showed high mass loads. With the exception of STP B, the mass loads of the return sludge were higher than those of the activated sludge; the return rate of STP B was 25% (0.25Q), which was at least 2 times lower than that of the other treatment plants. In the activated sludge, substances classified as “others,” such as cimetidine, carbamazepine, and iopromide, were distributed at a higher rate in the liquid phase than in the solid phase except for in STP A, while the other types of substances were contained at a much higher rate in the solid phase. At least 98% of the stimulants and 93% of the antibiotics were contained in the solid phase of the return sludge in the four STPs. All types of substances were distributed at a much higher rate in the solid phase than in the liquid phase. Ofloxacin had a mass load of 620 g/d and ciprofloxacin had a mass load of 47 g/d in samples taken between the anoxic and aerobic tanks, and at least 99% of both substances was found in the solid phase [ractions . Macroli surface ,41.ow value of 4.8, more gemfibrozil tended to be adsorbed and removed in the sludge than other substances, even though it had a low removal efficiency overall. Unlike the abovementioned substances, iopromide had the highest removal efficiency in the SBR process (81%). As a hospital and medical complex were located near STP B, which used the SBR process, the influent mass load of iopromide (contrast agent) was at least 10 times higher than that in the other treatment plants, thereby resulting in higher removal efficiency. The removal of iopromide by sludge sorption was insignificant. It was mainly removed by biodegradation, the results of which were consistent with those reported by Joss et al. (2005) [We calculated the mass loads of substances flowing into and discharged from the bioreactor, as well as their mass loads in the waste sludge. Among the 27 types of substances flowing into the bioreactor, we evaluated the removal mechanism by biodegradation and sorption for 20 types . Seven s. (2005) . An evalThe removal efficiencies of clarithromycin and roxithromycin ranged from −47% to 60% and −60% to 59%, respectively, according to the treatment process. The two compounds were relatively hydrophobic and showed a higher tendency to be removed by sludge sorption. In the A2O and MBBR processes, the mass loads discharged from the bioreactor were higher than the mass loads flowing into the bioreactor, thereby resulting in negative removal efficiencies. Similar to the results in this study, the removal efficiency of clarithromycin was −340% to 15% in three WWTPs using activated sludge and BNR processes, thereby indicating that the substances were discharged without treatment in some WWTPs . Lin et p < 0.05; ANOVA)  a. The op; ANOVA) . The SRTThe removal of pharmaceuticals during the biological treatment process depends on not only the operating conditions but also the diversity and function of microbial communities. The changes in operating conditions are considerably related to microbial population dynamics in biological treatment processes. The shifts of microbial communities strongly impact on the removal of pharmaceuticals, as reported by recent studies ,57. This3/d, 62% are equipped to remove total phosphorus, among which disk filters and filtration systems account for 53% and sedimentation/flotation account for 39%. Therefore, we compared the pharmaceutical removal efficiencies in total phosphorus treatments based on coagulation, such as coagulation/disk filter (CD), coagulation/sedimentation (CS), and rapid coagulation-sedimentation (RCS) processes, in addition to comparing the pharmaceutical removal efficiencies with the removal efficiencies of five types of tertiary treatment processes, including UV and PAC treatments, for disinfection and reuse . In the tertiary treatment processes, the SRE decreased in the order of PAC > CD > CS > RCS > UV. The SRE values were similar for the CD and CS processes in the three total phosphorus treatment plants, whereas it was lower for the RCS process. In coagulation plants used for the removal of organic substances and total phosphorus, the removal of pharmaceuticals has not been observed to have a significant effect [ow value of 4 or higher have been reported to improve the removal efficiency [ow value was higher than 4, did not improve the removal efficiencies of the three total phosphorus treatment plants despite being hydrophobic. A similar removal efficiency was observed for CS even after passing through two disk filters (fiber disk type) after coagulation treatment in a CD, thereby indicating that filtration through disk filters did not have a significant influence on the removal of pharmaceuticals. This was because the molecular size of the pharmaceuticals was much smaller than the pore size of the disc filters, thereby making disc filters ineffective at removing these compounds [We evaluated the performance of the tertiary treatment process for 16 types of substances whose influent mass loads were greater than 1 g/d and whose removal efficiencies were not significantly negative in the tertiary treatment. In South Korea, the water quality standard for total phosphorus in the effluents from public STPs was strengthened from 2.0 mg/L by 0.2 mg/L to 0.5 mg/L in 2012 to suppress the occurrence of algae in rivers and lakes. Of the 604 STPs with a treatment capacity of more than 500 mnd reuse b. A metaficiency . Gemfibrompounds . Similarow value of 3.1–4.8), such as gemfibrozil, ibuprofen, and ketoprofen, increased by 18%–63% through the PAC treatment. On the other hand, the removal efficiencies of atenolol, cimetidine, and trimethoprim, which have low hydrophobicity , increased by 42%, 35%, and 20%, respectively. Similarly, Guillossou et al. (2019) compared the effluent concentrations of a WWTP and a pilot-scale activated carbon treatment [The removal efficiency of PAC was the highest among the five types of tertiary treatment processes evaluated. The removal efficiencies for compounds with relatively high hydrophobicity  is smaller than those of conventional activated sludge processes (1.2–1.6 m2∙m−3∙d) [Since it is presumed that pharmaceuticals should, if discharged, negatively affect the water environment, they need to be degraded in a STP. As presented above, however, it has been turned out that pharmaceuticals could not be completely removed in none of the STPs where sampling was performed. Moreover, it is difficult to conclude which compounds are efficiently removed by which STP. Nonetheless, our results showed that two processes  could more effectively treat pharmaceuticals than other treatment processes. Several studies have demonstrated that MBR and PAC can be cost-competitive when the effluent quality level, footprint, and operational costs are collectively considered. For example, the average footprint of an MBR (0.9 m2∙m−3∙d) . Activat2∙m−3∙d) . In part2∙m−3∙d) . Thus, wIn this study, the behavior and distribution of 27 pharmaceuticals for liquid and solid phase samples in four STPs were investigated. A comparative evaluation on removal efficiencies of representative treatment processes used in South Korea was also performed based on the mass balance and meta-analysis.Most substances were present in the liquid phase in the four STPs, and the proportion distributed in the solid phase in the influent was less than 2%. After biological treatment, only 5%–12% of the influent mass loads remained, and the mass loads discharged owing to waste sludge in all processes were less than 2%.Caffeine, acetaminophen, naproxen, acetylsalicylic acid, and cefradine were removed to a great extent by biodegradation in all the STPs. The removal efficiencies of ketoprofen, iopromide, gemfibrozil, and sulfamethoxazole varied depending on the contributions of biodegradation and sludge sorption.A comparison of the SREs of the pharmaceuticals in the four types of biological treatments showed that the SRE decreased in the order of MBR > SBR > A2O > MBBR. The difference between the removal efficiencies of the MBR and MBBR processes was statistically significant.Among five types of tertiary treatment processes, PAC showed the most efficient performance, with an increase of 18%–63% in the removal efficiency for compounds including gemfibrozil, ibuprofen, ketoprofen, atenolol, cimetidine, and trimethoprim.Overall, our findings provide an overview of the elimination and behavior of pharmaceuticals in different unit processes of STPs in South Korea. We believe this paper will provide useful information to those who design or operate STPs regarding occurrence and fate of pharmaceuticals in sewage.


This study aimed to identify certain occupational risk factors for stress among healthcare workers (HCWs) during the COVID-19 pandemic. Using a multistage random sampling approach, an online questionnaire was applied to collect data on role conflict and ambiguity, self-esteem and social support from 1378 HCWs working in primary health centers  across Saudi Arabia. The results showed that stress correlated positively with role conflict and ambiguity and negatively with social support. HCWs in fever clinics exhibited significantly more stress and role conflict and ambiguity than those who were working in regular primary healthcare centers. In conclusion, role conflict and ambiguity and social support were determinants for stress among HCWs, especially those working in fever clinics. Coronavirus Disease-2019 (COVID-19) is a disease caused by Severe Acute Respiratory Syndrome that was firstly detected in Wuhan, China in December 2019 . The incWith regards to Saudi Arabia, the first case was officially reported on 2 March 2020 ,6,7. AccPrevious national and international studies, however, have shown high levels of stress among HCWs, whether in regular conditions or during the COVID-19 pandemic ,13,14,15In addition to sociodemographic characteristics, several work-related factors were suggested to affect the stress levels of HCWs, including role conflict and ambiguity, self-esteem and social support ,22,23,24However, no study, to date, has investigated the environmental and personal impacts on the levels of stress among HCWs in Saudi Arabia during the COVID-19 pandemic. We also have no information on whether HCWs in fever clinics specialized to manage patients with COVID-19 symptoms experience the same stress levels and personal and environmental stressors as their counterparts in regular primary healthcare centers in Saudi Arabia. Thus, this research was conducted to identify the association between role conflict and ambiguity, self-esteem and social support with stress levels among HCWs in primary healthcare centers in Saudi Arabia. We also objected to assess the differences in stress, role conflict and ambiguity, self-esteem and social support between HCWs in regular healthcare centers and fever clinics.n = 20). Each directorate supervises regular primary healthcare centers (n = 2070) and fever clinics (n = 119). In this study, we randomly selected six regular health centers and two fever clinics from all directorates and sent emails to all workers in the selected centers and clinics, including electronic questionnaires, before sending reminders one week later. Data collection was started on 27 September 2020 and continued for three weeks. The anticipated time to complete the survey was approximately 10 min. Based on power analysis with G*Power software edition 3.1.9.7 for linear multiple regression analysis , the required sample size is between 235 and 245.This cross-sectional study was conducted on HCWs in the primary healthcare centers in Saudi Arabia using a multistage random sampling technique. Saudi Arabia is divided geographically into five regions; Central, Eastern, Western, Northern and Southern regions. The Saudi Minister of Health is represented in each region by the Directorates of Health Affairs  and other workers  who worked in the primary healthcare centers were the target population. All employees working  in each randomly selected center were invited to participate in the study by using the official emails from the Ministry of Health. The total number of invited persons was 2007, of whom 1378 replied, representing a response rate of 69%. The head of the primary healthcare center in each Health Affairs Directorate coordinated the data collection process in each health directorate. The principal author was available to contact when having any questions or facing any difficulties. The head of the primary healthcare center in each directorate of health affairs emailed the self-administrated survey link to the targeted participants. During the time frame of data collection, a weekly reminder was mailed to the targeted participants.This study had one dependent variable (perceived stress) and several independent variables . Other covariates included age, gender, smoking, marital status, nationality, educational level, the type and the site of the healthcare center (regions) and job.The Perceived Stress Scale (PSS) was used to measure stress in this study. The PSS has been vastly utilized as a measure of stress estimate with strong psychometric properties . It inclr = 0.142, p < 0.01).Self-esteem is defined by Rosenberg  as a posr = −0.220, p < 0.01).Social support is explained as “understood or real instrumental and/or meaningful provisions supplied by the group of society, social networks and confiding” . The Mulr = 0.472, p < 0.01).Role conflict is defined as the concurrent event of at least two job pressures so that the consistency with one makes it very hard to respond to the next . Bowlingr = 0.436, p < 0.01).This variable is defined as “reflecting certainty about duties, authority, allocation of time and relationships with others; the clarity or existence of guides, directives, policies; and the ability to predict sanctions as outcomes of behavior” . The AmbFirst, age was measured in this study as a continuous variable. Second, gender was coded in this study to be male (1) and female (0). Third, the nationality of the health care workers was coded to be a dichotomous variable as Saudi (1) and non-Saudi (0). Fourth, marital status was a dichotomous variable as married (1) and not married (0). Fifth, education level was coded to be high school or less (0) and a university degree (1). Sixth, smoking in this study was coded as yes (1) and no (0). Seventh, type of job was recoded as medical and health job (1) and non-medical and health job (0). Eight, type of the health center was coded as regular health care center (1) and fever clinic (0). Ninth, regions were dummy coded into four dummy variables as following: Eastern (dummy 1), Western (dummy 2), Northern (dummy 3) and Southern (dummy 4). The Central region was used as a reference variable.This study was approved by the Central Institutional Review Board of the Ministry of Health in Saudi Arabia. The informed consent was on the first page of the electronic survey and included a description of the study, including the purpose and the names of the researchers. Anonymity was ensured since the names and I.P addresses were not collected. Also, to protect the confidentiality of the participants’ data, the data were saved on the primary researcher’s password-protected personal computer and no one had access to the data except the research team. Participants had to select “agree to participate” and “submit response” for their participation to be considered.Data were imported into the statistical software Statistical Package for the Social Sciences  to analyze the study data. This study used a variety of statistical analysis procedures to analyze the data and the level of statistical significance was set to 0.05 for all used statistical tests. First, sociodemographic and occupational data were described. Second, Pearson correlation was used to examine the relationships between stress and all sociodemographic and occupational characteristics in addition to the workplace challenges. Third, linear regression analysis was used to assess the relationship between stress and the statistically significant factors in the correlation using two steps; step 1: control variables were entered and step 2: role conflict, role ambiguity, social support and self-esteem were entered. Fourth, independent sample t-tests were used to examine the differences between HCWs in regular and fever clinics in the levels of stress, role conflict and ambiguity, self-esteem and social support.The mean age (standard deviation) of participants was 38.9 (8.12) years; 908 (65.9%) were aged 31 to 45 years old, 771 (56.0%) were males, 1158 (84.0%) were married and 1171 (85.0%) were citizens. Regarding their job, 306 (22.2%) were physicians, 432 (31.3%) were nurses, 131 (9.5%) were other HCWs, 509 (39.9%) were administrative workers, 1101 (79.9%) were working in a regular primary healthcare center and 277 (20.1%) were working in fever clinics .r = 0.472, 0.436, 0.142 and −0.220; respectively, p-values <0.01 in all correlations). Stress also correlated with other control variables; positively with a job and Eastern and Western regions  and negatively with age, gender and center type   . Severalβ = −0.234, p < 0.01), followed by gender , nationality  and center type . These factors explained 13% of stress. In step 2, age  and gender  remained associated with stress. In addition, role conflict , role ambiguity , social support  and self-esteem  were also associated with stress. Eventually, all of these factors together explained 35.4% of perceived stress, therefore, it could be concluded that workplace challenges in the form of role conflict, role ambiguity, social support and self-esteem contributed to 22.4% of stress  versus 22.85 (6.86), p-value= 0.038 and role ambiguity: 17.56 (8.74) versus 16.19 (8.32), p-value= 0.016, respectively  for fever clinics versus regular healthcare centers were as follows: stress: 18.70 (7.62) versus 17.33 (7.54), ectively .This study indicated that role conflict and ambiguity, self-esteem and social support were significant predictors for stress among HCWs in Saudi Arabia during the COVID-19 pandemic. Besides, HCWs who were working in fever clinics specialized to manage people with COVID-19 in Saudi Arabia had higher stress levels and role conflict and ambiguity than their counterparts who were working at regular primary healthcare centers.The increased stress levels among HCWs in fever clinics who were on the frontlines during the COVID-19 pandemic came in line with previous national and international studies assessing stress among similar study populations. In a study conducted on 426 HCWs from Saudi Arabia and Egypt  on the frontlines fighting COVID-19, more than half 55.9%) of them had stress .9% of th. One stuIn comparison with the general population or the HCWs managing diseases other than COVID-19, HCWs who inspect and treat patients with COVID-19 are more vulnerable to psychological deficits, including stress, for many reasons ,59,60. FFurthermore, we could find that HCWs in fever clinics had higher levels of role conflict and role ambiguity than other HCWs in regular primary healthcare centers in Saudi Arabia. To the best of our knowledge, this is the first study to assess role conflict and ambiguity among HCWs from Saudi Arabia during the COVID-19 pandemic. The fever clinics were recently initiated in Saudi Arabia as a response measure to tackle the spread of COVID-19 across the country; therefore, it is not unexpected that HCWs in these new clinics do not have enough details on their job descriptions. They also might have incomplete knowledge of infection control precautions. Above all, the changing guidelines regarding COVID-19 precautionary and management protocols can be perplexing for HCWs managing COVID-19 patients. On the other hand, HCWs in other regular primary healthcare centers should have been occupying their places for years, so they are expected to have clear job descriptions, enough knowledge of the diseases they manage and strict protocols regulating their work. This discrepancy in job descriptions and knowledge of diseases could be the reason behind the differences in role conflict and role ambiguity between HCWs in fever clinics and regular primary healthcare centers in Saudi Arabia.Moreover, our results demonstrated that role conflict and role ambiguity reported by HCWs in fever clinics were associated with their stress levels. This finding agrees with the results of previous studies conducted on workers from various disciplines. A self-report questionnaire administered to 66 health professionals in Italy showed that role ambiguity constituted a psychosocial deficit that influenced workers’ wellbeing and led to emotional exhaustion . In a crAlso, we could detect a negative correlation between social support and stress among the studied HCWs. In a previous study, Saudi HCWs who lacked emotional support from family and society during the COVID-19 pandemic showed higher levels of stress compared with their counterparts who received family and social support . This fiUnexpectedly, we could notice a positive association between stress and self-esteem, which contradicted previous findings among HCWs ,65. WhilThis study poses many strengths that should be addressed, such as examining for the first time the status of role conflict and ambiguity among HCWs in Saudi Arabia during the COVID-19 and determining their association with stress, investigating a large sample, using a random sampling procedure to guarantee representativeness of the study population and allow extrapolation of results and assessing the exposures and outcomes using validated questionnaires. However, some limitations should be considered. First, the cross-sectional design of this study does not imply causality; therefore, prospective cohort studies are advised to confirm our findings. Second, some confounding variables that might have affected the results were not assessed, such as the availability of personal protective equipment and medical specialty. Third, due to the strict measures of social distancing, we had to access participants via e-mails; therefore, the possibility of non-response bias cannot be excluded since the sociodemographic and occupational characteristics of responders might have differed from non-responders. For example, HCWs with a busy schedule who probably have higher stress levels might have less time to check their e-mails and respond to the questionnaire .This study revealed higher levels of stress and role conflict and ambiguity among HCWs in fever clinics during the COVID-19 pandemic than HCWs in regular primary healthcare centers in Saudi Arabia. Besides, role conflict and ambiguity, social support and self-esteem were found to be major determinants of stress among HCWs during the COVID-19 pandemics. Future programs and interventions should be considered to minimize role conflict and role ambiguity in fever clinics in Saudi Arabia to deliver the most efficient health services to patients with COVID-19.


Octopus vulgaris, it has been demonstrated that animals housed in an enriched environment show adult neurogenesis in specific brain areas. Firstly, we evaluated the optimal acclimatization period needed for an O. vulgaris before starting a cognitive stimulation experiment. Subsequently, we analyzed differential gene expression in specific brain areas in adult animals kept in tested (enriched environment), wild , and control conditions (unenriched environment). We selected and sequenced three protocadherin genes (PCDHs) involved in the development and maintenance of the nervous system; three Pax genes that control cell specification and tissue differentiation; the Elav gene, an earliest marker for neural cells; and the Zic1 gene, involved in early neural formation in the brain. In this paper, we evaluated gene expression levels in O. vulgaris under different cognitive stimulations. Our data shows that Oct-PCDHs genes are upregulated in the learning and lower motor centers in the brain of both tested and wild animals (higher in the latter). Combining these results with our previous studies on O. vulgaris neurogenesis, we proposed that PCDH genes may be involved in adult neurogenesis processes, and related with their cognitive abilities.Octopuses are unique invertebrates, with sophisticated and flexible behaviors controlled by a high degree of brain plasticity, learning, and memory. Moreover, in  Octopuses have considerable skills that show them as “intelligent” animals. They express high flexibility in solving demanding problems, and they have been observed using objects as tools. Octopuses also learn very fast when faced with artificial tasks ,6,7,8,9.Octopus vulgaris cognition and learning abilities are also linked to adult neurogenesis [Octopus vulgaris.Recently, it has been observed that ogenesis : Animalsogenesis ,11. Octoogenesis . Moreoveogenesis . What reNautilus, which lacks the elaborated Coleoid nervous system [O. vulgaris and O. bimaculoides, suggesting that the PCDHs’ expansion occurred before the speciation [O. vulgaris [In cephalopods, assembled transcriptomes reveal a substantial expansion of protocadherin genes (PCDHs) except for s system ,14. The eciation . StrikinThe octopus genome encodes 168 multi-exonic PCDH genes, nearly three-quarters of which are found in tandem clusters on the genome, which is 10 times more than many vertebrates, and more than twice as many as humans and other mammals. PCDHs are expressed in adult mammalian brains, especially in the hippocampus, cerebellum, and cortex ,18,19,20pax3 and pax7 contribute to the development of the nervous system [Drosophila [pax3/7, pax2/5/8, and pax6 in the nervous system development, in the sensory structure formation, and in the eye morphogenesis, respectively. In particular, among Cephalopods, in Sepia officinalis, Sof-pax6 expression was largely distributed in the central nervous system (CNS) and in the brachial nervous chord [Sof-pax6 expression at the distal tip of growing arms, which is described as a growing/proliferation region in Octopus [Euprymna [pax6 in the promoting proliferation mechanisms underlying the growth of the arms. Furthermore Sof-pax3/7 neural expression occurs later, suggesting that these genes are not involved in early neurogenesis, but it is restricted to the ventral brain in “motor” areas controlling the arms [Sof-pax2/5/8 might be involved in early steps of locomotor structures’ development derived from the ancestor mollusk foot [Additionally, Pax genes encode for a family of metazoan transcription factors, which are essential for cell specification and tissue differentiation, including the nervous system ,24,25,26s system ,28,29; tosophila ,31 are eosophila  and are osophila . In the osophila ,35, Anneosophila , Molluscosophila ,39,40,41osophila , and Nemosophila , the expus chord . Likewis Octopus  and EuprEuprymna , suggestthe arms . Moreoveusk foot . They cousk foot .Drosophila, ELAV is the earliest marker for neural cells as they just exit the cell cycle and start to differentiate into neurons [Drosophila, ELAV is necessary for neuronal differentiation [Sepia, Sof-elav appears to be involved in neurogenesis during embryogenesis [In annelid and  neurons ,48. The  neurons . In addi neurons . In Drosntiation , and it ogenesis .zic homologs have been identified in chordates, arthropods, and nematodes [zic family genes are expressed in a subset of the developing brain and mesoderm derivatives in annelids [ZIC family members, instead, play key roles in early neural patterning and the development of the neural crest, visual system, and cerebellum in mammals ,54. zic ematodes . Moreoveannelids .Octopus vulgaris, many genes have been hypothesized to have a role in its cognitive abilities, but up to date, their roles have been suggested just based on genomic and transcriptomic analysis [In analysis . NerveleWe than sequenced two isoforms of Oct-PCDH18 and one of Oct-PCDH15. The two isoforms of PCDH18 found in zebrafish play a crucial role in the brain: PCDH18a has a role in cell adhesion and migration , whereasOct-pax2/5/8, Oct-pax3/7, Oct-pax6, Oct-elav, and Oct-zic1. We then analyzed for all these selected genes their differential expression patterns under different behavioral cognitive stimulations.As potential co-actors of PCDHs in octopus cognitive processes, we sequenced Octopus vulgaris specimens , collected in Bay of Naples, were transferred to the Department of Biology [ Biology .Carcinus mediterraneus) or mussels  once a day.Animals were housed in individual tanks to prevent aggressive social interactions and cannibalism. They were housed in PVC tanks (50 × 50 × 50 cm), covered with a Plexiglas lid to avoid animals’ escape, equipped with a den, natural sand, and shells. Water and room temperature were maintained at 18 °C, and the light/dark cycle was set to the natural photoperiod. Water was treated with biological filters and protein skimmers. First days of captivity were considered as the acclimatization period, during which several physiological and behavioral parameters were monitored to verify the welfare and healthiness of the octopuses ,60. DuriOctopuses were anaesthetized by isoflurane insufflation  and braiOur research conformed to European Directive 2010/63 EU L276, the Italian DL. 4/03/2014, no. 26, and the ethical principles of Reduction, Refinement, and Replacement .In order to experimentally quantify the acclimatization time required for octopuses, we performed an initial experiment to determine the time needed by octopuses to feel comfortable and ready to face the challenge of opening two jars containing food.Octopuses  were tested at different times after they arrived in the lab for 3 consecutive days, once a day, with two plastic jars closed with a screw lid containing a live prey. During experimental days, octopuses had no feeding opportunities except to open a jar. The first group was tested since day 1, the second starting on day 2, the third on day 6, the fourth on day 14, and the fifth on day 19, measuring the average of opened jars during the training period. Jar position was constant through the experiments, and behavioral quantification was restricted to measuring the average number of opened jars.Following the results of the acclimatization experiment, we set up a period 14 days for the next experiments.Novel animals (N = 18) were used in three experimental groups: Control, tested, and wild. The control animal group (N = 6) was not tested, and they were fed regularly without any task for 17 days . The tested animal group (N = 6) was tested for the consecutive 3 days after the acclimatization period (14 days).We altered the standard housing conditions providing a cognitive challenge. During 3 experimental days, once a day, octopuses were tested with two jars containing a live prey and closed with a screw lid. During experimental days, octopuses had no feeding opportunities except to open the jars to reach the prey . The wilAll experiments (acclimatization and cognitive stimulation) were conducted once per day and recorded for at least 1 h with a digital camera  positioned on the front of the aquarium (20 cm), to analyze the octopus’s choice and behavioral responses, such as exploring, opening the jar, and eating.® Reverse Transcription Kit (Qiagen). Three members of the PCDH family , pax2/5/8, pax3/7, pax6, elav, and zic1 were characterized in an EST library and initial amplification primers for polymerase chain reaction (PCR) were synthesized using the software Geneious 9.1 (http://www.geneious.com). PCR products were analyzed with GenBank BLASTn and BLASTx . The analysis of the sequencing confirmed the identity of the fragments. All sequence data generated in this study were deposited in GenBank (accession numbers MN138036-43).Total RNA was extracted using the RNeasy minikit . The quality and amount of purified RNA were analyzed spectrophotometrically with Qubit 3.0 . RNA of 1000 ng was reverse transcribed with the QuantiTectious 9.1 . PCRs weubiquitin gene was used for normalization of the relative expression. At the end of each test, a melting curve analysis was done (plate read every 0.5 °C from 55 to 95 °C) to determine the formation of the specific products. Each sample was tested and run in duplicate.Additionally, we performed a real-time PCR using the QuantiTect SYBR Green PCR Kit (Qiagen). PCR was performed in a final volume of 25 µL, with 50 ng of cDNA, 1 mM of each primer, and 12.5 µL of QuantiFast SYBR Green PCR Master Mix (2×). The PCR cycling profile consisted of a cycle at 95 °C for 5 min, 40 three-step cycles at 95 °C for 15 s, at 60 °C for 20 s, and at 72 °C for 20 s. Quantitative RT-PCR analysis was conducted by using the 2-(ΔΔCt) method . RT-PCR p-values < 0.05 were considered statistically significant.The control and the treatment groups in various assays were compared and analyzed using a Wilcoxon two group test and data with We measured the efficiency of the acclimatization process as the average of jars opened after different numbers of days . The firOf the control group animals, we evaluated the transcription activities of the selected genes  in three brain regions, the central part of the supraesophageal mass, the subesophageal mass, and the optic lobes, including the olfactory and peduncle lobes (OOP) on the optic tracts .The subesophageal mass, compared to the supraesophageal mass, showed a higher expression for Oct-PCDH18a and b, Oct-pax2/5/8, Oct-pax3/7, and Oct-elav genes, and lower gene expression for Oct-pax6 and Oct-zic1 genes; there were no significant differences for the Oct-PCDH15 gene . The OOPThe tested animal group (N = 6) was acclimatized for 14 days and animals were trained for 3 consecutive days using two plastic jars. All octopuses were considered to have the same behavioral performance, responding to the test stimulation, opening both jars from the first day of the experiment, progressively decreasing the time spent opening the jar during the 3 days of training.Comparing the results among wild, tested, and control animals, it is possible to observe that in the supraesophageal mass , tested In the OOP, tested and wild animals showed an upregulation for Oct-PCDH18a, Oct-PCDH18b, Oct-pax6, and Oct-zic1 genes . Oct-PCDIn the subesophageal mass, tested animals showed a synergistic upregulation of all genes considered, except for Oct-pax3/7 . This paO. vulgaris possesses high cognitive capabilities, and just recently, it has never been shown that they have adult neurogenesis [Adult neurogenesis can be considered as an intrinsic factor to the maintenance of a healthy brain and to memory assessment . O. vulgogenesis ,12. We nOur study is the first to describe how the enhancement of the environmental conditions, in wild and tested octopuses, affects the RNA expression level in genes involved in cell proliferation and synaptogenesis, compared to control animals, that lack any environmental stimulation.O. vulgaris [Interestingly, we observed that, among the analyzed genes possibly involved in adult neurogenesis, wild animals show higher PCDH expression either than tested or control animals. In all examined brain areas, their expression level showed a positive trend from the control, tested, and wild animal groups . Since evulgaris ,12, thesOctopus sp. [The supraesophageal mass contains the vertical lobe and the superior frontal lobe (VL-SFL); this system plays an important role in learning and memory. Furthermore, the supraesophageal mass is also the site of visual learning and tactile memory, respectively localized in the vertical/subvertical/superior frontal lobes and inferior frontal lobe . These bopus sp. ,14,15. Iopus sp. .For the other genes considered, we did not observe a similar trend . Oct-paxSepia officinalis, this gene and PAX6 gene do not seem involved in neurogenesis of the supraesophageal mass, but they are restricted to the “motor” areas controlling the arms in the subesophageal mass [Furthermore, the Oct-pax3/7 gene is downregulated in both tested and wild animals. In eal mass . The OctThe lower motor centers of the octopus’s brain, responsible for accurate motor control and coordination, are the anterior part of the subesophageal mass that is involved in the actions of arms and suckers, while the posterior part is involved in the actions of the mantle and viscera ,69,70. IInterestingly, wild animals showed a significantly higher expression level for the Oct-pax3/7 gene, probably because they face more stimuli coming from the natural environment than the control and tested animals, and it did not appear to be affected by the specific proposed task. On the contrary, Oct-pax2/5/8 and Oct-pax6 genes were upregulated only in the tested octopus, suggesting a specific involvement in the implementation of the nervous network necessary for tuning fine movements, such as those needed to open screw-lid jars .In this brain area, the Oct-elav gene expression also upregulated in tested animals while significantly downexpressed in wild ones. Different from what was observed in supraesophageal mass, in tested octopus, the higher expression of Oct-elav allows us to hypothesize that this gene is specifically involved in the interneuron proliferation. This contributes to the fine motor coordination, supporting our previous finding on adult neurogenesis in the subesophageal mass . Wild ocThe optic and olfactory lobes integrate sensory stimuli detected by sensory organs from the environment while the spine of the peduncle lobe is considered an analog of the vertebrate cerebellum. These lobes are involved in the coding of the visual and chemical inputs as well as the coordination of voluntary movements, such as posture resulting in smooth and balanced muscular activity supporting the neurophysiological bases of octopus’ sophisticated behavior ,72,73,74In wild and tested animals, we found upregulation for Oct-PCDHs, Oct-pax6, Oct-elav, and Oct-zic1 genes, suggesting that these genes are related to sensory integration that is strictly connected to the increase of the neuronal structure at adult neurogenesis as previously found in this area ,12. On tO. vulgaris’ brain [After the discovery that cognitive stimulation positively affects adult neurogenesis in s’ brain , we propWith a detailed analysis of expression levels for the selected genes, we found a clearer pair of adult neurogenesis occurring after cognitive stimulation with Oct-PCDHs, which appeared to be upregulated in both tested and wild octopuses. Our study showed that the gene expression level of PCDHs could represent a valuable tool to detect and measure adult neurogenetic processes, including synaptic plasticity. The selected three non-clustered PCDHs are known to be involved during early stages, such as axon outgrowth and path finding ,21,22.Furthermore, the next steps could be directed to expand the gene marker panel in order to deeply understand the adult neurogenesis mechanism. We then proposed a novel approach that combining cognitive stimulation and evaluation of the pattern of differential gene expression could give information about remote and proximate causes of animal behavior.