Gambling Disorder (GD) has recently been reclassified from an impulse-control disorder to a behavioural addiction and, as in other addictive disorders, the dopaminergic reward system is involved. According to neuroimaging studies, alterations within the striatal dopaminergic signalling can occur in GD. However, the findings to date are controversial and there has been no agreement yet on how the reward system is affected on a molecular basis. Within the last 20 years, there has been growing evidence for a higher risk to develop GD in response to certain dopaminergic medication. Especially the dopamine agonists pramipexole and ropinirole, and the dopamine modulator aripiprazole seem to increase the likelihood for GD. The goal of this study was to examine the association between a prescription for either of the three pharmaceuticals and a GD diagnosis in a large cross-sectional study of the Swedish population. Compared to patients with any other dopaminergic drug prescription (38.7% with GD), the diagnosis was more common in patients with a dopamine agonist prescription (69.8% with GD), resulting in an odds ratio of 3.2. A similar association was found between aripiprazole prescriptions and GD diagnoses, which were analysed within the subgroup of all patients with schizophrenia or a schizotypal, delusional, or another non-mood psychotic disorder. An aripiprazole prescription increased the likelihood of GD (88.8%) in comparison to patients without an aripiprazole prescription (71.2%) with an odds ratio of 3.4. This study contributes to the increasingly reliable evidence for an association between several dopaminergic drugs and a higher risk for developing GD. Therefore, one future research goal should be a better understanding of the neurobiology in GD to be able to design more selective dopaminergic medication with less severe side effects. Additionally, this knowledge could enable the development of pharmacotherapy in GD and other addictive disorders. Gambling disorder (GD) is the permanent and severe manifestation of the gambling activity that many in the general population pursue sporadically as a hobby. Common forms are casino games, slot-machines, and lotteries, but in the last years internet gambling has grown in popularity . The preFormerly seen as an impulse-control disorder, GD has been reclassified as a behavioural addiction and been renamed from “Pathological Gambling” in the latest revisions of the International Classification of Diseases ICD 11) and the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) , 10. Thi and the One central element in the neurobiology of GD is the dopamine-based reward and reinforcement system . Its mai2 and D3 receptors has been found to positively correlate with symptom severity in GD patients but not in healthy controls, for instance higher impulsivity, increased excitement and alertness, or impaired decision making in a reward context [2 antagonist haloperidol, which acts indirectly activating on dopamine signalling through inhibition blockage, can enhance the rewarding effects and priming caused by gambling within GD patients despite no observed effect in the control group [Many contradictory studies have been published on the alterations within striatal dopamine signalling in GD, often based on positron emission tomography (PET) measurements . On the  context –23. Furt context . Ampheta context , 26. Mod context . The D2 ol group .2 and D3 receptor availability has been found to be the same in GD patients and controls [2 antagonist olanzapine has shown no effect in comparison to a placebo [On the other hand, several measurements have shown no difference between GD patients and healthy controls regarding striatal dopamine signalling. The striatal Dcontrols , 29. Add placebo , 31. HenWithin the last 20 years, the evidence for an association between developing GD and the intake of dopaminergic medication has grown. One of the first case reports was published in 2000, where 10 patients with Parkinson’s disease (PD) under levodopa treatment had developed GD and the authors speculated on the altered dopaminergic tone being the neurobiological explanation . This th3 receptor compared with the D2 receptor, which has been proposed as one possible explanation for their increased property to cause addictive and impulse-control disorders [3-selectivity over D2 in dopamine agonists.According to previous research, certain dopaminergic medication entails a higher risk for impaired impulse control and GD development than other substances. In comparison to other dopamine agonists, especially pramipexole and ropinirole seem to correlate with increased GD rates. Both pharmaceuticals were developed as agonists for the D2-like receptor family, thus activating the inhibitory pathways of dopamine signalling, and are prescribed as part of the dopamine replacement therapy in PD but also against restless legs syndrome –48. Pramisorders . Seeman 2 and D3 receptors, which could explain its impact on the development of GD.Another substance, that has been associated with an increased likelihood for developing GD, is the atypical antipsychotic aripiprazole, mainly prescribed against schizophrenia and bipolar disorder . The parEven if the connection between dopamine agonists, respectively aripiprazole, and an impaired impulse control could not be revealed on a molecular basis yet, the association has been shown and recognised increasingly within in the last years –55. NevePatient data was provided by the Swedish National Board of Health and Welfare and obtained from the Swedish National Patient Registry, which is based on in-patient and specialised out-patient health care records, and the Swedish Prescribed Drug Register. The study was approved by the Swedish Ethical Review Authority under the file number 2019–01559. Since the data was analysed anonymously, consent from the participants was not obtained. For the study population, all patients in Sweden with the diagnosis pathological gambling (F63.0 according to ICD-10) between 2005 and 2019 were selected (n = 3689). Each case was paired with two age- and gender-matched controls from the total population register by Statistics Sweden with the goal to analyse the association between the exposure to the medication and the development of gambling disorder independently of the variables age and gender. This resulted in a total study population of 11 067 individuals.The register material from the Swedish National Patient Registry included data on diagnoses according to ICD-10, age at treatment, gender, and the date of treatment. These data were combined with information from the Swedish Prescribed Drug Register containing the prescription date and the full anatomical therapeutic chemical (ATC) codes for the drug classes N02, N03, N04, N05, N06, N07B, R06AD01, R06AD02 and R06AD52.The aim of this study was to explore a possible association between the prescription for a certain medication and a GD diagnosis using the software IBM SPSS Statistics 26 to process and analyse the data. In preparation for the analysis, the different files received from the Swedish National Board of Health and Welfare were merged into one main file, containing all relevant information for each patient. Based on the ATC codes, binary variables were created for each medication group or pharmaceutical in question, for instance “pramipexole prescription/no pramipexole prescription” or “psychotropic drug prescription/no psychotropic drug prescription”. The same method was used to create binary variables for certain diagnoses, such as “GD/no GD” or “PD/no PD”, from the full list of diagnoses made in the in-patient and specialised out-patient health care.In a next step, adequate subgroups within the total study population were identified. The goal was to select patients with a similar severity of disease to make individuals with a prescription for the pharmaceuticals in question as comparable as possible to the whole reference group. Since the counts of PD and restless legs syndrome patients were very low , the subgroup for analysing dopamine agonists (pramipexole and/or ropinirole) was chosen to be all patients with any dopaminergic drug prescription , which includes dopamine agonists but also for instance levodopa. Within this group, patients resembled each other in age, gender, and a possible PD diagnosis, not influenced by whether they had a prescription for a dopamine agonist (n = 149) or only another dopaminergic drug (n = 31) see . As a reTo determine the association between a dopamine agonist or an aripiprazole prescription with a GD diagnosis, chi-square tests were performed within the chosen subgroups. The tests were based on the binary variables “dopamine agonist prescription/no dopamine agonist prescription”, respectively “aripiprazole prescription/no aripiprazole prescription”, and “GD diagnosis/no GD diagnosis”. In both cases, all expected counts were higher than 5 per cell, making a chi-square test applicable.Additionally, a binary logistic regression with the dependent variable “GD diagnosis/no GD diagnosis” was performed for each group to examine the effect size. Covariates were gender, age, dopamine agonist prescription, and psychotropic drug prescription, respectively gender, age, and aripiprazole prescription. The covariate psychotropic drug prescription was applied in the group with dopaminergic drug prescriptions to distinguish a compromised mental health status in general from GD as a specific psychiatric diagnosis. The variable included all prescriptions within psycholeptics, psychoanaleptics, drugs used in addictive disorders and antihistamines used as tranquillisers . Since the subgroup for analysing aripiprazole prescriptions was chosen according to the psychiatric diagnosis of the patients, the use of the covariate psychotropic drug prescription was assumed to be redundant in this case. The number of patients with the outcome GD diagnosis was large enough to apply four, respectively three covariates, with 116 GD patients in the group with dopaminergic prescriptions and 307 GD patients in the subgroup chosen to analyse aripiprazole prescriptions. For the chi-square tests and the logistic regressions, a significance level of 0.05 was applied and all hypothesis testing was performed two-tailed on independent samples.Considering a possible causal relation, the time that passed in between the first dopamine agonist or aripiprazole prescription and the GD diagnosis was calculated based on the earliest date of prescription and the earliest date of the GD diagnosis for each patient. This analysis was performed on all 104 patients with a dopamine agonist prescription and a GD diagnosis, and all 151 patients with an aripiprazole prescription and a GD diagnosis.The chi-square test showed a significant association between a dopamine agonist prescription and a GD diagnosis with a P-value (P) of 0.001. Of all patients with any dopaminergic drug prescription (n = 180), including dopamine agonists but also for instance levodopa, 116 (64.4%) had been diagnosed with GD. While 104 of 149 patients (69.8%) with a dopamine agonist prescription had a GD diagnosis, only 12 out of 31 patients (38.7%) without a dopamine agonist prescription had a GD diagnosis see .In the logistic regression, the covariates gender and age had no significant influence on the outcome GD diagnosis. The odds ratio (OR) for having a GD diagnosis was 3.2  = 1.4–7.6; P = 0.008) for patients who had received a dopamine agonist prescription compared to patients without a dopamine agonist prescription. In the same regression model, the OR for having a GD diagnosis when having a psychotropic drug prescription was 5.8  in comparison to patients without any psychotropic drug prescription. The complete characterisation of the applied regression model can be found in the When the date of the first dopamine agonist prescription was compared with the date of the GD diagnosis, a majority of the patients had received the prescription first (81 of 104). The time that passed between prescription and diagnosis where 3.5 years as a median with an interquartile range (IQR) between 0.6 and 6.8 years, including all 104 patients regardless if prescription or diagnosis occurred first see .Even for an aripiprazole prescription the association with a GD diagnosis could be confirmed by a chi-square test with a significance of P < 0.001. Within the 389 patients with a F20-29 diagnosis, 307 (78.9%) had a GD diagnosis. Out of 170 patients with an aripiprazole prescription a total of 151 (88.8%) had a GD diagnosis, while only 156 out of 219 patients (71.2%) without the prescription had been diagnosed with GD see .The covariates gender and age had no significant effect on the outcome GD diagnosis in the logistic regression performed. However, the OR for having a GD diagnosis when having an aripiprazole prescription was 3.4 (95% CI = 1.9–6.1) with a significance of P < 0.001 in comparison to patients without an aripiprazole prescription. A full description of the regression model is included in the Most of the patients had received their first aripiprazole prescription before the GD diagnosis (101 of 151). The median time difference including all 151 patients was 1.5 years between prescription and diagnosis with an IQR between -1.3 years and 4.9 years see .The results of the statistical analysis were convincing and coherent. Dopamine agonists as well as aripiprazole had an association with an increased risk for GD. Given the study design, these findings can be expected to have a high reliability and validity. The study population was large with more than 11 000 individuals, of which 3689 were diagnosed with GD. Since the data was obtained from a nationwide public institution, it can be assumed to represent the situation in Sweden accurately. Furthermore, the reference groups for the statistical analysis were selected to reach the highest comparability possible. The controls were age- and gender-matched and the overall health state was expected to be homogenous within the reference groups. Thus, a GD diagnosis is presumably linked to the intake of medication rather than a poor health condition in general. The OR for dopamine agonists was calculated while using a psychotropic drug prescription as another covariate. Both covariates showed significant ORs, and it can therefore be assumed that GD is not only associated with a compromised mental health state but also with dopamine agonist intake independently. For analysing the effects of aripiprazole, a subgroup with similar psychiatric diagnoses was chosen. Thus, GD presumably is connected to aripiprazole intake and is not only a comorbidity to other psychiatric diagnoses.Since a GD diagnosis was treated as a binary variable, the course of disease could not be taken into consideration in this study. Future study designs should focus on the progression of the disorder under certain medication in more detail, for instance by using patient questionnaires, which could also take subclinical problematic gambling into account. Due to the cross-sectional study design, a causal relationship between the medication and GD can only be a speculation, given that most patients received their prescriptions before their diagnosis. For exploring causality, a longitudinal study would be more adequate, in which follow-up screenings for GD and impaired impulse control could be performed on patients who received the medication in question. Another limitation of this study is the missing information about the actual medication intake since the data was obtained from the prescription register. To what extent the patients made use of the prescribed medication is uncertain. Even this issue could be avoided in the future by following up on certain patients instead of using register data.3 receptor could be the possible mode of action for dopamine agonists, which would explain a dose-dependent relationship and pramipexole and ropinirole as agonists that increase the risk for GD the most. The pharmacodynamics of aripiprazole on the other hand, are more complex and still subject to debate [In contrast to the well-recognised DOMINION study on impulse-control disorders as a reaction to dopamine agonists , this sto debate . It actso debate . TherefoIn summary, our study puts additional emphasis on the possible effects of dopamine agonists and modulators on GD development. Even if institutions like the U.S. Food and Drug Administration (FDA) have recognised the problem, the knowledge needs to be established broadly in the primary care centres. One beneficial measure could be to assess a patient’s background and risk factors for GD and impulse-control disorders, before prescribing dopaminergic medication. If, for instance, the general mental health state indicates a higher likelihood to develop GD, it could be advisable to choose another pharmaceutical if possible, or to closely monitor the patient for side effects related to impaired impulse-control.The association between the intake of certain dopaminergic pharmaceuticals and the development of GD has started to become an international consensus over the last years. This study provides further evidence for GD being a possible response to dopamine agonists or aripiprazole. A causal relationship is likely, and the dopaminergic reward system seems to be the linkage between the medication and the addictive disorder. Since the molecular background of GD is only partly understood to date, it remains unclear how dopamine agonists or aripiprazole can influence its pathophysiology. The possible association with GD should be taken into consideration when prescribing dopamine agonists and aripiprazole, and for patients at risk for a compromised impulse control other medication seems more suitable. Furthermore, an extended knowledge on the matter is necessary to develop dopaminergic medication that can act more specifically on its intended target without causing impulse-control disorders and addictions. Revealing the molecular mechanisms behind GD would in addition contribute to a better understanding of addictive disorders in general and would open new possibilities for pharmacotherapy in this field.S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.S3 Appendix(DOCX)Click here for additional data file.S4 Appendix(DOCX)Click here for additional data file.S5 Appendix(DOCX)Click here for additional data file.


Craniopharyngioma is an intracranial congenital epithelial tumor growing along the pathway of the embryonic craniopharyngeal tube. The main clinical symptoms of patients with craniopharyngioma include high intracranial pressure, visual field defect, endocrine dysfunction, and hypothalamic dysfunction. At present, the preferred treatment remains the surgical treatment, but the recovery of endocrine and hypothalamic function following surgery is limited. In addition, endocrine disorders often emerge following surgery, which seriously reduces the quality of life of patients after operation. So far, research on craniopharyngioma focuses on ways to ameliorate endocrine dysfunction. This article reviews the latest research progress on pathogenesis, manifestation, significance, and treatment of endocrine disorders in patients with craniopharyngioma. Craniopharyngioma is a rare solid or mixed cystic epithelial tumor in the sellar and suprasellar region, accounting for 2–4% of intracranial tumors, which can be found in each age group . PatientIn the nineteenth century, an increasing number of young patients exhibited unexplained physical and mental symptoms, including loss or delay of sexual maturity, progressive obesity, abnormal somnolence, and dementia-like behavioral changes. These patients were reported to have large solid cystic tumors, characterized by dilatation in the funnel and third ventricle, exceeding the anatomically intact pituitary gland. Cushing chose the term “craniopharyngioma” to refer to these lesions . Recent Hypothalamic dysfunction caused by the tumor space-occupying effect is a risk factor for developing endocrine disorders , and theThe surgical treatment strategy of craniopharyngioma remains controversial . BecauseCraniopharyngiomas often involve the anterior part of the third ventricle, and its surrounding structures, such as the hypothalamus, are important. When total resection of craniopharyngiomas is difficult, adjuvant radiotherapy after subtotal resection is an effective method to control tumor growth . At presPituitary fibrosis is also a factor in developing endocrine disorders in patients with craniopharyngioma, especially in GHD. The fact that ameloblastic craniopharyngioma secretes a range of pro-inflammatory cytokines distinguishes it from other sellar tumors . Pro-infPituitary hormones include GH, ACTH, Gn, TSH, oxytocin (OT), antidiuretic hormone (ADH), and so on. These hormone deficiencies are critical in the maintenance of normal physiological function. Unfortunately, such hormones are often deficient in patients with craniopharyngioma, impairing their quality of life.Commonly used methods for determining hormones are radioimmunoassay and chemiluminescent immunoassays. Diagnostic criteria for hypopituitarism: (1) gonadal axis dysfunction: testosterone (TEST) decreased with abnormal follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL) levels in adult male patients, estradiol (E2) decreased with normal or decreased FSH and LH or abnormal PRL in female patients, and decreased FSH and LH in children . (2) TheIn the related literature reports, the incidence of various hormone defects in patients with craniopharyngioma is slightly different. We searched PubMed for keywords such as craniopharyngioma, endocrine, and hormone, and the subjects were identified as children or adults, and 11 related literatures , 49–53 wp = 0.024 by the log-rank test), but the significance was only borderline in the sensitivity analysis (p = 0.06). A meta-analysis showed that overall craniopharyngioma recurrence rate was lower among children who were treated by GHRT  compared with those who were not , the p-value comparing the two groups was <0.01  . In a noFollowing craniopharyngioma growth, the secondary mass effect can oppress the normal pituitary and stalk and lead to a deficiency of important pituitary hormones such as Gn , resultiThe TSH deficiency can cause central hypothyroidism. Typical hypothyroidism symptoms include cold intolerance, constipation, dry skin, sparse or fragile hair, weight gain, loss of energy, and bradycardia . TSH defp > 0.05). Cook et al. : an initial polyuric phase, a subsequent antidiuretic phase (the patients can temporarily concentrate urine and syndrome of inappropriate ADH secretion (SIADH) and hyponatremia develops), and a final polyuric phase that is usually chronic . To screHypernatremia and hyponatremia often occur postoperatively due to DI and SIADH. Treatment strategies for hyponatremia : (1) milThe use of glucocorticoids is the most important in the perioperative period of patients with craniopharyngioma. Patients after surgery undergo assessments serum cortisol daily morning . At mostFollow-up can find tumor recurrence in time, correct and treat water electrolyte, and endocrine state in time. Endocrine, electrolyte, liver and kidney function, and saddle MRI should be performed 14, 30 days, 3, 6 months, and 1 year postoperatively (follow-up frequency should be increased if necessary).The glucocorticoid most widely used for the cortisol replacement therapy worldwide is oral hydrocortisone in daily divided doses. At present, the clinical evaluation of the glucocorticoid replacement therapy is mainly based on the clinical evaluation. Too low glucocorticoid doses increase the risk of adrenal crisis and reduce well-being, whereas too high doses increase the risk of complications such as osteoporosis, obesity, and impaired glucose tolerance . Given tCompared with primary hypothyroidism, patients with TSH deficiency have more difficulty obtaining the best thyroid hormone replacement because they cannot be guided by serum TSH levels . CertainThere is no sign of recurrence 1–2 years after surgery, and GRHT may be considered. As mentioned earlier, GRHT does not promote tumor recurrence. In patients with permanent or confirmed GHD, a starting low rhGH dose (0.01–0.03 mg/day) to be adjusted according to IGF-1 concentrations is also widely accepted . MolitchFemale children are recommended to start the estrogen replacement therapy between the ages of 11 and 12 to mimic average physiology . Low dosAdult male patients with central hypogonadism should be accepted the TEST replacement therapy to reduce fat mass and improve bone mineral density, libido, sexual function, energy levels, sense of well-being, and muscle mass and strength . The conHormonal replacement consists of an estrogen component and a progestogen component, in females possessing a uterus . ReplaceAdequate fluid replacement, treatment of the underlying condition, and desmopressin administration are the mainstays of management . We thinCTNNB1 gene abnormality results in imbalanced Wnt pathway and nuclear β-catenin accumulation, contributing to tumor invasiveness .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Long noncoding RNAs (lncRNAs) act crucial roles in the progression of vascular diseases, including atherosclerosis. This study aims to investigate the expression levels of the atherosclerosis-associated lncRNA DANCR in patients diagnosed with atherosclerosis and whether its abnormal expression affects the progress of atherosclerosis. The expression of DANCR in the serum samples of all study participants was quantified using RT-qPCR. Then, the predictive capacities of DANCR for the detection of atherosclerosis patients were evaluated via receiver operating characteristic (ROC) curve analysis. The effects of DANCR on vascular smooth muscle cells (VSMCs) proliferation and migration were then explored using cell counting kit-8 (CCK-8) and Transwell migration assays. The DANCR exhibited increased expression trends in patients with atherosclerosis than healthy controls. Moreover, there were differences in the levels of low-density lipoprotein cholesterol (LDL-C), homocysteine (Hcy), and C-reactive protein (CRP) between the healthy controls and atherosclerosis patients. The DANCR expression was positively correlated with serum LDL-C, Hcy, and CRP levels. DANCR expression could distinguish patients with atherosclerosis from healthy individuals with a high area under the ROC curve (AUC), sensitivity, and specificity. Additionally, knockdown of DANCR weakened the proliferative abilities and migration capacities of VSMCs. It was also shown that DANCR could compete with miR-335-5p binding. Herein, it appears that the LncRNA DANCR was closely associated with the progression of atherosclerosis by targeting miR-335-5p, which might be a potential detective predictor and target for the treatment of atherosclerosis.  Atherosclerosis underlies the vast majority of common cardiovascular disorders, including coronary artery disease and stroke, which is one of the leading causes of mortality burden worldwide . CarotidNon-coding RNAs include various types such as microRNA (miRNA), small nuclear RNA, and long noncoding RNA (lncRNAs) . Among tThis study aims to investigate the clinical role and functional role of DANCR in the pathogenesis of atherosclerosis and explore the potential molecular mechanisms, wherein the expression pattern of DANCR was measured in the serum of patients with atherosclerosis and healthy controls. Then, the loss-of-function experiments were conducted to test the proliferative abilities and migration capacities of VSMC cells. After that, the potential sponge of DANCR was uncovered in atherosclerosis.All experimental procedures were approved by the Ethics Committee of Dongying People’s Hospital . The study population consisted of 68 healthy individuals and 137 patients with atherosclerosis, and all the patients and healthy individuals signed written informed consent.From January 2018 to September 2020, the participants were consecutively recruited from Dongying People’s Hospital. The inclusion criteria include: 1) atherosclerosis patients diagnosed with plaque formation using carotid artery ultrasonography. 2) Patients have complete clinical characteristics. The exclusion criteria include: 1) patients with previous myocardial infarction and percutaneous coronary intervention. 2) uncontrolled malignant arrhythmia. 3) patients with acute and chronic infectious diseases and malignant tumors. 3) patients with severe liver and kidney diseases. The healthy subjects who received annual routine physical examination in our hospital were enrolled in healthy control group. The healthy control and patient groups were matched for age, sex, and body mass index (BMI). The clinical characteristics and analysis results were then collected and recorded.Blood samples were obtained from the study population and were centrifuged at 1000 × g for 15 min to collect the serum. The serum samples were then transferred to RNase/Dnase-free 2-mL EP tubes and stored at −80°C until RNA extraction.−ΔΔCt (cycle threshold) method and were normalized to the expression of GAPDH and U6, respectively.Total RNA from serum samples and cells were isolated using RNAiso Plus  and then reverse-transcribed into cDNA using a PrimeScript real-time (RT) reagent kit . The real-time quantitative PCR (RT-qPCR) was carried out using either a SYBR Green qPCR SuperMix  or a TaqMan MicroRNA assay kits  on an ABI PRISM7500 Sequence Detection System . The expThe sequences for PCR were as follows: DANCR, 5’- ACCCAGGCTGGATGGAGTAT-3’ (forward) and 5’-CAGCTGGCTACAACAGAGCT-3’ (reverse); miR-335-5p, 5’-GGGTCAAGAGCAATAACGA-3’ (forward) and 5’-CTCAACTGGTGTCGTGGA-3’ (reverse); GAPDH, 5’-TCATCTCTGCCCCCTCTGCT-3’ (forward) and 5’-CGACGCCTGCTTCACCACCT-3’ (reverse); and U6, 5’-GCTTCGGCAGCACATATACTAAAAT-3’ (forward) and 5’-CGCTTCACGAATTTGCGTGTCAT-3’ (reverse).2.A human vascular smooth muscle cell line  was purchased from the American Type Culture Collection (ATCC) and cultured as per the manufacture’s instructions. The cells were cultured in a DMEM medium  added with 10% fetal bovine serum  and were incubated at 37°C in an incubator with 5% COTo adjust the DANCR expression and explore its effects on VSMC cells, DANCR siRNA  or siRNA negative control  was transfected into VSMC cells using Lipofectamine 2000 . At indicated time point (24 h) post the transfection, cells were harvested for further cellular experiments analysis.The cell counting kit-8  was used to determine cell viability . Briefly4 cells) were re-suspended in a serum-free medium and seeded into the top chamber, and complete medium with 10%FBS was added to the the lower chambers. After 24 h, the migrated cells on the membrane were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. The numbers of cells per field was then counted under a light microscope in five random fields.The migratory abilities of VSMC cells treated with si-DANCR were measured using a 24-well Transwell migration assay . Brieflyhttp://carolina.imis.athena-innovation.gr/diana_tools/web/index.php?r=lncbasev2%2findex-experimental) was used to predict the binding sites between DANCR and miR-335-5p. Dual-luciferase reporter assay was used to validate the correlation between DANCR and miR-335-5p [The LncBase Experimental v.2 online database  normality test. All statistical analyses were carried out using SPSS 20.0  and GraphPad Prism 7.0 software . Differences between groups were compared through the unpaired student’s t-test or one-way ANOVA. Correlations of the datasets were assessed using Pearson’s correlation coefficient r analysis. The difference was considered as statistically significant when the In the current study, the expression of DANCR was detected in the serum samples of all participants using RT-qPCR. The diagnostic power of DANCR in atherosclerosis patients was analyzed using the receiver operator characteristic curve (ROC) curve. The effects of DANCR on cell viability and migration in VSMC cells were explored after transfection of si-DANCR or si-NC using CCK-8 assay and Transwell migration assay. The potential target miRNAs of lncRNAs were evaluated and confirmed using LncBase Experimental v.2 online bioinformatics database and dual-luciferase reporter assay.2 and 22.16 ± 2.83 kg/m2 of atherosclerosis patients and the healthy control group, respectively. Thus, the demographic information has no statistical difference between atherosclerosis patients and healthy control. The levels of low-density lipoprotein cholesterol (LDL-C), homocysteine (Hcy), and C-reactive protein (CRP) exhibited significant differences between the healthy control group and patients with atherosclerosis (P < 0.05). The serum total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), fasting blood glucose (FBG), triglyceride (TG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), uric acid (UA), and creatinine (Cre) levels were not significantly different between groups (P > 0.05).The demographic characteristics and clinical characteristics of the subjects are provided in The DANCR expression levels were detected in the serum samples of all participants through RT-qPCR. As displayed in To further assess the diagnostic power of DANCR in patients with atherosclerosis, the ROC curve was conducted to estimate the sensitivity and specificity of DANCR expression for distinguishing patients with atherosclerosis from healthy participants. As exhibited by P = 0.0004), Hcy , or CRP levels . As exhibited in P < 0.01). These results demonstrated that elevated LDL-C, Hcy, and CRP levels might increase the risk of atherosclerosis and were positively correlated with DANCR expression levels.Based on the median serum DANCR expression level, patients with atherosclerosis were grouped into the low- and high- DANCR expression groups. Furthermore, on account of the statistical difference of LDL-C, Hcy, and CRP between the healthy control group and atherosclerosis patient group, the corerlation between DANCR expression and LDL-C, Hcy, or CRP levels was analyzed using Pearson correlation analysis, the LDL-C, Hcy, and CRP levels were evaluated in the low- and high-expression DANCR groups. Firstly, the correlation between were analyzed using Pearson correlation analsis. The results in P < 0.001). The CCK-8 assay then revealed that DANCR knockdown could attenuate the proliferation ability of VSMCs , contributing to the occurrence of cardiovascular diseases . In the Some studies have demonstrated that non-coding RNAs have a crucial role in the process of atherosclerosis ,22. For Previous studies highlights the potential of lncRNAs could serve as clinical diagnostic or prognostic markers in multiple diseases ,32. ConsThe proliferation and migration of VSMC cells have essential roles in the progression of atherosclerosis. For instance, the viability and migration of VSMC cells could be inhibited by LOC285194, which might be a therapeutic target to treat atherosclerosis . DownregHowever, there are some limitations in the current study. Firstly, the disparity in sample size between atherosclerosis patients and healthy control was a limitation that strains the statistical analysis in the preliminary study. On the other hand, the effects of DANCR on cellular activities were explored using VSMC cells. Future studies will use more cells that contribute to atherosclerotic lesion formation  to evaluate the role of DANCR in atherosclerosis. Finally, the detailed mechanism of DANCR in atherosclerosis needs to be further explored in future studies.LncRNA DANCR was highly expressed in the serum samples of patients with atherosclerosis and was positively associated with LDL-C, Hcy, and CRP levels. DANCR could be a potential diagnostic marker to distinguish atherosclerosis patients from healthy individuals. Knockdown of DANCR could reduce proliferation capacities and migration abilities of VSMC cells through the regulation of miR-335-5p. This study uncovered that DANCR might be a diagnosis predictor and a potential target for treating atherosclerosis.


Stress (hyperdopaminergic) is delivered when dopamine replacement therapy leads to a relative overdosing of the already-sensitized ventral striatum. These alterations are consistent with consecutive changes in reinforcement mechanisms, which stimulate learning from reward and impede learning from punishment, thereby fostering the development of impulse-control disorders. This vulnerability-stress model might also provide important insights into the development of addictions in the non-Parkinsonian population.Impulse-control disorders are commonly observed during dopamine-replacement therapy in Parkinson’s disease, but the majority of patients seems “immune” to this side effect. Epidemiological evidence suggests that a major risk factor may be a specific difference in the layout of the dopaminergic-reinforcement system, of which the ventral striatum is a central player. A series of imaging studies of the dopaminergic system point toward a presynaptic reduction of dopamine-reuptake transporter density and dopamine synthesis capacity. Here, we review current evidence for a  A key feature of addiction is the reduced ability to control behavior , despite of their obviously harmful effects2. There are substance-related and non-substance-related addictions, such as pathological gambling, which are also called behavioral addictions or impulse-control disorders (ICDs). It is conceivable that for the development of ICDs, both predisposing traits and triggering mechanisms play a role. These traits and triggers may have identifiable biological substrates. The main pathophysiological mechanism in these disorders has been linked to disrupted dopamine homeostasis2. While longitudinal data covering the entire development of ICDs are almost impossible to come by in the general population, a unique opportunity is provided in Parkinson’s disease (PD). In a large multicenter study, Weintraub et al. demonstrated that ICDs occur as a side effect of dopamine replacement therapy (DRT) in 14% of PD patients5. Newer research points out that the incidence of ICDs in PD could be much higher (up to 46%)7. Therefore, the development of ICDs in PD could serve as a model for all addictions8 because seemingly mentally healthy PD patients develop ICDs in a very short period of time.Addiction is a global social, economic, and health problem. So far, there is no effective treatment and pathophysiology is insufficiently understoodoverdose theory: among the basal ganglia loops, the motor loop is mainly affected by neurodegeneration in PD. Hence, when DRT is administered, the dopaminergic tone in the motor loop is balanced, but the relatively intact limbic loop is overdosed, which leads to a hyperdopaminergic state in the ventral striatum10. The dopamine signal of the limbic basal ganglia loop modulates conditional learning and has motivational impact on a person’s behavior: via dopaminergic signaling, we learn implicitly to approach stimuli with positive outcomes (Go-Learning) and to avoid the opposite (NoGo-Learning)11. Accordingly, patients with ICDs show altered dopamine-modulated behavior in the form of impulsivity, risk proneness, and overengagement in rewarding behavior as well as deficits in inhibitory control14. While this concept is attractive, it does not explain why only a fraction of patients develop the ICD phenotype. Furthermore, findings of several imaging and rodent studies leave doubts about the hyperdopaminergic concept for ICDs in PD and hint at hypodopaminergic changes in these patients, which may represent a premorbid biological vulnerability.The most popular pathophysiological concept for the development of ICDs in PD is the so-called hypodopaminergic findings with the hyperdopaminergic overdose theory in the form of a vulnerability-stress model for the development of ICDs in PD. In general, this model states that persons have an intrinsic vulnerability , leading in combination with an extrinsic stressor  to the development of mental illness16.The aim of this review is to consolidate Additionally, we want to shed light on the relationship between apathy and ICDs since both conditions might underlie comparable changes within the dopaminergic reinforcement system.hypodopaminergic changes in the ventral striatum of PD patients with ICDs: a reduced dopamine transporter (DAT) density in 123I-FP-CIT-SPECT20, a reduced dopamine synthesis capacity in 18F-DOPA-PET at rest21, a reduced BOLD activation at rest22, and a reduced D2/D3 receptor availability at rest25 . When considering general PD populations, Fazio et al. found that 36% of early PD patients had a reduced DAT density in the ventral striatum26 and early PD patients showed reduced dopamine synthesis capacity as compared with healthy controls in the ventral striatum27. Further evidence for the possibility of a premorbid vulnerability, that may be unrelated to dopaminergic neurodegeneration, comes from various studies reporting ICD development in non-PD populations  with DRT29. These hypodopaminergic changes were also found in non-PD populations with behavioral or substance addictions: pathological gamblers30, alcoholics31, tobacco, and cannabis addicts32 showed a reduced DAT signal in the ventral striatum. Young people with internet addiction had a lower DAT binding33 and a reduced D2 receptor availability34 in the ventral striatum. Interestingly, a reduced DAT density was also found in healthy individuals with higher trait impulsivity35. Furthermore, a reduced dopamine synthesis capacity was also found in cocaine addicts36, cannabis users37, and binge eaters38.Human imaging studies found several 20.An important study in drug-naive PD patients has shed further light on the question of whether these changes pre-existed before medication is started. In total, 31 de novo, drug-naive PD patients underwent DAT single-photon emission computed tomography (SPECT) and were screened for ICDs. After an average follow-up of 32 months, 11 had developed ICD symptoms without having any at baseline. These patients showed significantly lower baseline DAT binding ratios in the right ventral striatum, right anterior dorsal striatum, and right posterior putamen. Additionally, the severity of ICD symptoms at follow-up correlated negatively with baseline DAT availability18F-DOPA-PET to detect changes in dopamine synthesis capacity21. We found a negative correlation between the dopamine synthesis capacity and ICD severity in the ventral striatum at rest. Consequently, a predominant reduction of dopaminergic projections per se seems a more likely scenario. Interestingly, a postmortem study found no differences in tyrosine hydroxylase staining and α-synuclein load in the ventral striatum between PD patients with and without ICDs, indicating that the results from imaging studies could rather present functional changes than pure cell loss due to neurodegeneration39.A reduced signal in DAT-SPECT in the ventral striatum could be due to reduced DAT density per dopaminergic terminal or due to a reduction of dopaminergic projections from the midbrain or a combination thereof. For further clarification, a recently published paper by our group used vulnerability to develop ICDs.All in all, the results from the above-mentioned studies in PD and non-PD populations point to a weaker dopaminergic input to the ventral striatum as a premorbid 40 and trait impulsivity42, which in turn are associated with ICDs. Indeed, a PET study in de novo PD patients with apathy and depression did demonstrate a relative serotonergic denervation40. While these patients may be seen as “at-risk” to develop ICDs when medicated, there currently is no molecular serotoninergic imaging study in ICDs in PD. However, in non-PD binge eaters Majuri et al. found a reduced serotonin -transporter density in the ventral striatum43. Serotonin depletion in humans44 and rodents45 can lead to impulsive behavior, and polymorphisms in the serotonin-transporter protein are associated with addiction46. Moreover, perfusate serotonin increases dopamine release in the nucleus accumbens47. In sum, evidence is still lacking in PD, but a reduced serotoninergic input to the ventral striatum would be a plausible hypothesis. In a PET-study, a reduced μ-opioid receptor density38 was found in binge eaters in the ventral striatum. Furthermore, μ-opioid receptor stimulation in the nucleus accumbens amplifies hedonic wanting48. Interestingly, polymorphisms in the κ-opioid receptor were negatively associated with ICDs49. An animal study with microdialysis revealed that stimulation of these opioid receptors has an effect on striatal dopamine release50. Furthermore, an increase in glutaminergic projections from the prefrontal cortex to the ventral striatum leads to drug seeking51. Engeli et al. found a reduction of glutamate in the nucleus accumbens in cocaine addicts at rest and an increase in glutamate levels during cue-induced craving compared with healthy controls in a magnetic resonance spectroscopy paradigm52. All in all, changes in other neurotransmitter systems seem to influence the dopamine metabolism in the ventral striatum and might be associated with a hypodopaminergic state in the ventral striatum as described above.Although in this review we focus on dopamine, other neurotransmitter systems may play an important role for the development of ICDs. Serotonergic neurons project from the raphe nucleus to the ventral striatum and to the prefrontal cortex. Low serotonergic levels are associated with depression53 and seems to be the key player for the development of addictions. An important feature of addiction and compulsion is that an action becomes habitual. The key player for habit formation is commonly seen in the dorsolateral, not the ventral striatum. Belin and Everitt could show that the ventral striatum is important for the initiation of drug seeking, while the dorsal striatum is more involved in sustaining it55. Interestingly, two studies found that a reduction in DAT density in the putamen and the anterior dorsal striatum20 as well as a reduced D2/D3 density in this area are associated with ICDs in PD24. In a recently published paper by our group, we found a negative correlation between dopamine synthesis capacity in the caudate and the severity of ICDs21. Other studies could show a reduced connectivity between the anterior cingulate cortex and the left putamen57. All in all, ICDs seem to be associated with alterations in dorsal striatum. A region that is more affected from neurodegeneration in PD than the ventral striatum. To sum up, we reason that the dopaminergic loss in the ventral striatum may be critical for the initiation of ICDs and that the dopaminergic loss in the dorsal striatum might play an important role for the long-term persistence of this behavior.Most of the imaging studies concerning ICDs in PD reported alterations in the ventral striatum. This mesolimbic reward circuit, including the ventral tegmental area and the nucleus accumbens, is crucial for mediating reward and the calculation of a reward prediction errorGo-Learning). On the other hand, punishment or the omission of an expected reward leads to a dopaminergic dip and NoGo-Learning via D2/D3 receptors is fostered11. D1 receptors are activated after phasic dopamine release, whereas the activation of D2/D3 receptors (having a higher affinity to dopamine than D1 receptors) is dominated by tonic dopaminergic levels60. D3 receptors have the highest affinity to dopamine and are mainly located in limbic areas such as the ventral striatum61. Therefore, there have been speculations that D3 receptors are primarily involved in the development of ICDs. Interestingly, the influence of phasic dopamine release seems strongly affected by DAT activity, whereas the tonic dopamine release is mainly affected by the overall activity of a dopaminergic neuron population per se63. See Fig. Dopamine release in the striatum can conceptually be divided into two relatively independent forms, tonic and phasic dopamine release, which relate to tonic and phasic activities of dopaminergic neurons, and have distinguishable roles in learning from outcomes. On the one hand, an unexpected reward leads to a phasic dopamine release from the ventral tegmental area to the ventral striatum which is then followed by D1 receptor activation , or a combination of both. In light of the reduced dopamine synthesis capacity at rest21, we may interpret a reduced D2/D3 receptor availability as primarily reflecting a reduction in receptor density. This fits well with a post mortem study, showing lower levels of D3 receptors in the ventral striatum of PD patients with ICDs. This study could not find changes regarding D2 receptors39. A reduced density of D2/D3 receptors in combination with an additional administration of dopamine agonists would hamper learning from negative feedback and lead as a consequence to ICDs .A possible downstream effect of reduced dopamine synthesis capacity could be a reduced tonic stimulation  of postsynaptic D2/D3 receptors. However, imaging studies found a reduced postsynaptic D2/D3 receptor availability in the ventral striatum at rest in PD patients with ICDsCDs Fig. 11. A con65. Guo et al. found that individuals with the 10-repeat allele of the DAT1 had significantly more sexual partners66; and individuals with the 10-repeat allele had lower binding in DAT-SPECT as compared with patients with the 9-repeat allele67. Moreover, Volkow et al. found a reincrease in DAT density in abstinent cocaine addicts68. In animal studies DAT blocker enhances reactions to reward predicting cues69 and DAT knock-out mice show a greater locomotor sensitization to drugs, i.e., a greater progressive and persistent enhancement of the motor-stimulant effects of cocaine and ethanol70. Having in mind that the phasic dopamine release is mainly affected by the reuptake capacity of DAT in combination with the reduction of DAT density, as described above, we would measure more dopamine in the synaptic cleft as a consequence of a phasic dopamine release under reward conditions. In line with this hypothesis, imaging studies found a reduced D2 availability under reward conditions in PD patients with ICDs as compared with normal PD patients71. This would involve an increased dopamine release as well as a reduction in D2 receptors.Genetic studies point out that a reduction of DAT expression (polymorphism in DAT1 gene) is associated with addiction, PD, and ADHD73. According to a recently published review74, presynaptic D2 autoreceptors have three different possibilities to modulate dopamine metabolism: (1) reduction of the exocytotic dopamine release after a prior release, (2) regulating the dopamine uptake via an increase of DAT expression, and (3) downregulation of tyrosine hydroxylase (reduced filling of dopamine vesicles). Ray et al. found a reduced activation of these autoreceptors in PD patients with pathological gambling75, which could also explain increased phasic dopamine release. See Fig. 76.Furthermore, a chronic underexpression of DAT leads to a reduced function of midbrain D2 autoreceptors which may evoke higher extracellular dopamine levels77 and behavioral adaptions after negative feedback78. The lateral orbitofrontal cortex is responsible for punishment-based decision-making79 and is important for suppression of previously rewarded behavior80. Voon et al. found a reduced BOLD activation of the anterior cingulate cortex in ICD in PD during risk-taking14. Another study reported a reduced activation of the anterior cingulate cortex and lateral orbitofrontal cortex in PD gamblers as compared with PD controls in H2O-PET81. Several other studies found a reduced connectivity between the anterior cingulate cortex and the ventral striatum78. Another PET study found a higher availability of D2 and D3 receptors in this area, which could indicate low levels of synaptic dopamine in PD patients with75. All in all, imaging studies point out that there is a diminished top-down control of inhibitory cortical areas in ICDs5.Besides from changes in striatal regions, the prefrontal cortex plays an important role for development of ICDs. The anterior cingulate cortex is crucial for error monitoring4.In case of L-DOPA, 7.2% of PD patients develop ICDs, 14% in case of dopamine agonists, and 17.7% in case of bothhypodopaminergic changes and the associated mechanisms, the system becomes vulnerable to relatively small alterations in dopaminergic levels. The system is adjusted to low levels of tonic dopamine and reduced D2 receptors. Then, as a consequence of DRT administration, the system becomes easily overdosed. Thereby, phasic effects are boosted and dips in dopamine release are drowned by the tonic D2/D3 overstimulation82.According to our proposed theory, due to 75, which downregulate phasic striatal dopamine release. Chronic treatment with dopamine agonists may lead to a desensitization of D2 autoreceptors in the midbrain with consecutive dysregulation of phasic dopamine release83. Furthermore, dopamine agonists reduce the activity of inhibitory control areas in PD patients with ICDs, whereas they increase the activity in these areas in PD controls81. Likewise, dopamine agonszts diminish reward processing in the lateral orbitofrontal cortex during negative errors of reward prediction82.Interestingly, patients taking dopamine agonists have twice the risk for ICD than patients taking L-DOPA alone. A reason could be the altered function of D2 autoreceptors in the midbrainIn the same vein, a combination therapy with agonszts and L-DOPA will lead to the highest prevalence of ICDs because increased D1 effects (higher dopamine release because of L-DOPA and low D2 autoreceptor function) and D2/D3 overstimulation (dopamine agonists) are combined.84. A reason could be the association between the cumulative dopamine agonist dose and the development of ICDs6. Likewise, Perez et al. could find a correlation between agonist dose and ICDs85 as opposed to Weintraub et al. using a different pharmacological model4. So, in every prone PD patient, there might be an individual (cumulative) dose threshold. After discontinuation of dopamine agonists, ICDs resolve in about 50% of the patients6. Two longitudinal studies could show an improvement of ICDs after reduction of agonists or a switch to L-DOPA87, whereas personality traits associated with ICDs persisted. So far, variable rates of relapse or remission are not fully understood and further research is needed.Astonishingly, time onset of ICD diagnosis after the initiation of DRT is highly variable (from 3 months up to 10 years)88. While there potentially are multiple mechanisms leading to apathy, it is interesting that some forms of apathy are clearly temporally correlated with a reduction of dopaminergic stimulation. Apathy occurs following deep-brain stimulation, especially when DRT is reduced to a large degree89. Apathy can also be found as a part of dopamine-agonist withdrawal syndrome (DAWS). Intriguingly, dopamine agonist withdrawal in PD patients with DAWS almost always was preceded by ICDs, and in patients without ICDs, dopamine agonist withdrawal did not lead to apathy90. Additionally, when comparing PD patients with these two PD subgroups, overlaps in behavior were found92. Scott et al. showed in a recently published cohort study that more than a third of PD patients with apathy also suffer from ICDs. Interestingly, these were the patients with the longest disease duration93.Arguably, one could arrange the motivational spectrum of behavior in such a way that ICDs would be at the positive end and apathy at the negative end of the spectrum. Apathy generally is even more prevalent in PD patients than ICDs, including early stages of the disease95 as well as human imaging studies96 hint at a hypodopaminergic state in the striatum predisposing for apathy. Therefore, apathy and ICD might share the same pathophysiological principle, i.e., hypodopaminergic changes in the striatum, which then leads to either ICDs or apathy, depending on DRT. Sierra et al. use the term “Ying and Yang” of dopamine-dependent behavior97. Figure 98.In addition, rodent studies99. In other designs, dopaminergic lesions were set in the posterior VTA. After the submission of DRT, the animals showed impulsive behavior in a place-preference task101. Holtz et al. also used 6-OHDA to produce a dopaminergic lesion in the striatum. In a delay-discounting task, rats showed risk-taking behavior when pramipexole was administered. Interestingly, mirtazapine leads to a reduction of risk-taking102. In another design, 6-OHDA was administered at the substantia nigra. After surgery, animals took less from a rewarding sucrose solution. As a more general claim, rats became apathetic, which was fully reversed after the intake of pramipexole94, which supports our theory-comparable changes within the dopaminergic reinforcement system in apathy and ICD described above.Rodent studies of the dopaminergic reward system show interesting insights into the development of ICDs. Lesions with 6-hydroxydopamine (6-OHDA) can imitate our theory of a premorbid vulnerability: Cardinal et al. produced a dopaminergic lesion in the nucleus accumbens with 6-OHDA, leading to impulsiveness in a delay-discounting taskstress in our model, can be derived from chemogenetics. “Designer receptor exclusively activated by designer drugs” (DREADDs) can be used to activate or inactivate certain types of dopaminergic receptors. Boender et al. injected a D1-activating DREADD in the nucleus accumbens of rats, leading to an increased intake of sucrose pellets, which was annulated by the administration of a D1 antagonist103. Zhu and colleges injected D2 receptor activating and inhibiting DREADDs in the nucleus accumbens of rats. D2 activation reduced locomotion and running, whereas D2 inhibition had the opposite effect104.Interesting insights into the effect of dopaminergic medication, vulnerability-stress model of ICD development.In all, rodent studies—despite their limitations in comparability—corroborate and validate biological concepts of the hypodopaminergic changes in the ventral striatum that would act as a biological vulnerability toward addictive behavior. These alterations predispose the dopaminergic system (vulnerability), which, in combination with DRT (stress), leads to ICDs. As the most likely scenario, a reduction of dopaminergic projections in combination with a reduced DAT density and autoreceptor function results in adjustment processes at the postsynaptic membrane. Furthermore, it comes to a diminished top-down control of inhibitory cortical areas. As a consequence, DRT overwhelms the prone system. So, a combination of a premorbid vulnerability and overdosing could lead to ICDs in PD and can be seen as a vulnerability-stress model. It is tempting to speculate that similar biological processes may underlie other drug or non-substance addictions in the non-PD population.We discuss a hypothetical model of hypodopaminergic state in striatal regions and therefore with an increased sensibility to DRT. There is an overlap in patients suffering from both ICDs and apathy.Apathy is associated with a reduced DAT density in the dorsal striatum, whereas patients with ICDs have also a reduction of DAT in the ventral striatum. We assume that apathy and ICDs go along with a hypodopaminergic changes in the ventral striatum, leading to a vulnerability for DRT and thereby to ICDs, is only hypothetic and, of course, a simplification of the complex development of ICDs. So, there are some limitations to consider. In this review, we mainly shed light on the so-called dopaminergic reinforcement system, but also other neurotransmitters, as mentioned above, play an important role.The model of Additionally, we do not have de novo data concerning the postsynaptic membrane. We do not know whether the reduced D2 availability might predate the presynaptic changes. Hence, theoretically, the model described above could be vice versa. Changes in D2 density could lead to a hypodopaminergic state in the striatum.105. In addition, Boileau et al. could not find differences in D2/D3 availability between pathological gamblers and healthy controls106. Similarly, the results of genetic studies in PD with ICDs are not consistent107. Altogether, despite many converging results around a premorbid biological vulnerability, more rigorous studies with larger samples are needed to consolidate the genetic and molecular features of this vulnerability.Furthermore, imaging data do not always point in the same direction: One study found an increase in dopamine synthesis capacity in impulsive PD patients18F-DOPA-PET studies measure baseline dopamine synthesis capacity. This would help to classify the reduced D2 receptor availability under reward conditions. Furthermore, a validation of our vulnerability-stress model in a rodent model combined with PET imaging would be of great interest for the understanding of ICDs in PD in particular but also for the development of addiction in general.It would be interesting to measure D2 receptor availability and DAT density in de novo PD patients and after the development of ICDs under DRT. With this approach, it would be possible to check if postsynaptic changes also predate the development of ICDs. In addition, it would be interesting to measure dopamine synthesis capacity under reward conditions since all existing Further information on research design is available in the Reporting Summary


Use of rapid molecular diagnostic panels in the evaluation of diarrhea provides increased sensitivity for organism identification and decreased time to results. However, their inappropriate use can lead to unnecessary expenditures and antimicrobial exposures. We aimed to characterize the appropriateness of testing using the BioFire® FilmArray® Gastrointestinal Panel (SFA) in different clinical settings and to describe the impact of SFA results on patient care. Retrospective study of adult patients presenting to hospitals part of an integrated health system in Des Moines, Iowa, between July 30 and September 30, 2019, and who had a SFA ordered and collected in the Emergency Department (ED) or an inpatient service. The appropriateness of SFA testing was determined according to adherence to a local algorithm available through the hospital’s intranet . Reason for testing, appropriateness of SFA test, molecular targets identified, and antibiotic exposures were collected. p< 0.0001). Documented indications for SFA are presented in Table 1. Among ED patients testing was most frequently considered inappropriate due to absence of diarrhea on the day of test collection (41.3%), and among inpatients due to the use of SFA for assessment of hospital-onset diarrhea (47.0%) (Table 2). Overall, there were 94 (36.6%) positive SFA . Among ED patients, the percentage of positive SFA samples was 30.4% and 50.8% for inappropriate and appropriate testing respectively (p=0.03), while for inpatients it was 33.0% for inappropriate orders and 30.4% for appropriate orders (p=0.76). Antibiotics were prescribed to 28.2% and 28.1% of patients tested in the ED and inpatient service respectively.We identified 257 patients, 111 (43.2%) who had SFA done in the ED and 146 (56.8%) as inpatients. Testing was deemed inappropriate in 46 (41.4%) of ED patients compared to 100 (68.5%) of inpatients (High proportions of inappropriate SFA testing were identified both in the ED and inpatient services, with distinct issues in each site. Characterization of the reasons underlying inappropriate use of SFA can aid in the design of diagnostic stewardship interventions tailored to each clinical setting. All Authors: No reported disclosures


Background: The coracoclavicular joint (CCJ) is an anomalous articulation between the surfaces of the inferior clavicle and superior coracoid and its etiology is controversial. Reportedly, symptomatic patients demonstrate significant functional limitations including shoulder abduction loss and potential for brachial plexus compression and impingement.Purpose: To determine the prevalence of CCJ across age, gender and ethnicity, and to identify clinically useful morphological characteristics.Methods: 2,724 subjects with intact clavicles and scapulae from the Hamann-Todd Osteological Collection were evaluated for the presence of CCJ. Logistic regression was used to determine the effect of age, height, gender, and race on prevalence of CCJ. 354 clavicles with CCJ were measured for size and location of the CCJ facet.Results: CCJ was observed in 9% of subjects. CCJ was more prevalent in African-Americans (12%) than Caucasian-Americans (6%) (p < 0.001) and more prevalent in females (11%) than males (8%) (p = 0.055). Facet location along clavicle length was consistent . But, facet location along clavicle width varied , with males having a more posterior location. For every 10-year increase in age, facet elevation (p = 0.001) and surface area (p < 0.001) increased.Conclusions: CCJ prevalence was 9% in our large osseous population, found more commonly in African-Americans and females. Facet location is predictable with respect to clavicle length, but less so along clavicle width. The clavicular facet may develop at some point in life and continue to grow in size after its appearance.Clinical Relevance: Presence of a CCJ represents a potential overlooked source of anterior shoulder pain and supracoracoid impingement. Epidemiologic and morphological characteristics presented in our study can aid in the identification, clinical understanding, and surgical excision of a symptomatic CCJ. Level of Evidence: Level IV. The coracoclavicular joint (CCJ) is an anomalous articulation between the horizontal part of the coracoid process of the scapula and the inferior surface of the clavicle. This articulation was first observed in the 19th century through cadaveric dissection and osteological studies have labeled the presence of a round and bulging osseous clavicular facet on the conoid tubercle distinct from the cone-shaped ossification of the conoid ligament usually with an associated osseous facet on the superior surface of the horizontal part of the coracoid process as a CCJ . SubsequA CCJ as a source of anterior shoulder pain may be encountered clinically more often than previously appreciated and some authors have proposed that symptomatic cases are grossly underreported . This coPrevious osteological studies have compared the morphometric variables of the clavicle and scapula in individuals with and without a CCJ. One study found that individuals with CCJ have larger scapulae, longer clavicles, and longer first ribs . A laterDue to the limited and contradictory data currently available in the literature, our study's purpose is to (1) estimate the prevalence and population trends of CCJ based on a large sample of osseous specimens, to (2) describe morphological characteristics of the CCJ, and to (3) determine if there is a relationship between CCJ prevalence and increasing age.Clavicles and scapulae were obtained from the Hamann-Todd Osteological Collection at the Cleveland Museum of Natural History . This collection consists of complete, cleaned human skeletons of unclaimed status from the Cleveland city morgue between 1912 and 1938. Use of these specimens does not require institutional review board approval as it is a publicly available collection. All specimens with a known age and race were included. 245 specimens had to be excluded due to significant specimen damage, a missing clavicle or scapula, evidence of fracture, or extensive arthritic changes to the clavicle precluding measurements. A total of 2,724 subjects  were included in this study. One specimen with bilateral CCJ facets had a damaged clavicle that did not allow for measurement. This clavicle was included in the prevalence analysis, but was excluded from the morphological analysis. The Hamann-Todd Collection provided clinical data at time of death for the samples including age, gender, height, and race . A repreTwo authors (LS and CT) performed the assessments and measurements. The clavicles of all specimens were examined for the presence of a CCJ facet. This was defined as a round and bulging structure on the conoid tubercle of the clavicle different from the smaller cone-shaped ossified structure representing conoid ligament using methodology similar to that of Gumina et al. . For spefL) and breadth (fB) were measured on the longest and shortest axes to approximate facet surface area by treating the facet as an ellipse where area is equal to (fL/2)(fB/2)π. A third axis was measured to describe the elevation or height of the facet from the normal surface of the clavicle (The distance was measured from the acromioclavicular (AC) joint facet to the center of the observed clavicular facet (CC facet) along the long axis of the clavicle. The clavicular width at the center location of the CC facet was measured. Along the same anterior-posterior axis of the clavicular width, the distance was measured from the anterior margin of the clavicle to the center of the facet. Facet length  was modeled using univariate and multivariable logistic regression to determine the effect of age, height, gender, and race. The best multivariable model was chosen from among all possible subsets including all two-way interactions based on the model that had the lowest Schwarz's Bayesian Information Criterion (BIC). Age was treated as a dichotomous variable with a cutoff of 30 years; an age at which a person's joints would be theoretically fully developed unless influenced by environmental effects. The same analysis strategy was used for modeling the prevalence of bilateral CCJ.Size and location of CC facets were measured at the clavicle level on those clavicles for which there was a facet present. Size was measured by elevation and surface area, which were normalized using clavicle length. Location was measured by the midpoint of the facet along clavicle length and the midpoint of the facet along clavicle width, which were normalized using clavicle length and width, respectively. Results obtained were then multiplied by 100 to increase readability and interpretability after the normalization process. The effects of age, gender, and race on morphologic variables were estimated with regression models utilizing generalized estimating equations (GEE) with an exchangeable correlation structure to account for the expected correlation within subjects who had more than one facet. In this analysis, age was treated as a continuous variable because the size of the facet would be expected to increase over time (if age is an important predictor of size). All statistical analyses were performed using R .p < 0.001]. A bilateral CCJ was present in 4.5% of individuals examined. Males had a statistically significantly lower prevalence of bilateral CCJ (4.1%) than females (6.4%) . African-Americans (6.6%) had a significantly higher prevalence of bilateral CCJ than Caucasians (3.0%) . There was no significant difference in prevalence of CCJ according to gender in the multivariable model.A CCJ was observed in 233 (8.6%) of individuals examined. Of the individuals having a CCJ, 122 (52.4%) expressed bilaterally, 60 (25.8%) were right unilateral, and 51 (21.9%) were left unilateral . The prep = 0.017), but not with respect to age or race. CC facet position along the width of the clavicle varied in all groups (range 10–90%), but was located consistently more posteriorly in males. With regard to CC facet midpoint along clavicle length  using the AC joint as the reference point, there were no significant differences according to age, race or gender. However, its location was more consistent, approximately 25% along the length on average from the AC joint (range 15–35%) .p < 0.001]  was significantly different from the prevalence in all other groups (6.5%)  . The resp = 0.001). Normalized surface area of the CC facet is on average approximately 50% of clavicle length, and increased about 4% for a 10-year increase in age (p < 0.001). No significant gender or race effects were found for size.Interestingly, normalized elevation of the CC facet is on average approximately 1% of the clavicle length and, for every 10-year increase in age, elevation increased by about 0.1% on average  and grade 2  . InteresThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.EH data analysis and manuscript writing. LS and CT data collection and manuscript writing. BD and JI critical editing of the manuscript. SM supervisor, oversaw data analysis, drafted, and critically revised manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Dead space after rectal resection in colorectal surgery is an area with a high risk of complications. In this study, our goal was to develop a novel 3D implant based on composite hydrogels enriched with fractionalized nanofibers. We employed, as a novel approach in abdominal surgery, the application of agarose gels functionalized with fractionalized nanofibers on pieces dozens of microns large with a well-preserved nano-substructure. This retained excellent cell accommodation and proliferation, while nanofiber structures in separated islets allowed cells a free migration throughout the gel. We found these low-concentrated fractionalized nanofibers to be a good tool for structural and biomechanical optimization of the 3D hydrogel implants. In addition, this nano-structuralized system can serve as a convenient drug delivery system for a controlled release of encapsulated bioactive substances from the nanofiber core. Thus, we present novel 3D nanofiber-based gels for controlled release, with a possibility to modify both their biomechanical properties and drug release intended for 3D lesions healing after a rectal extirpation, hysterectomy, or pelvic exenteration. Pelvic surgeries, especially those that are extensive such as extirpation of the rectum, hysterectomy, or pelvic exenteration, result in the development of dead space. This is often a reason for postoperative complications, such as bowel obstruction due to a relocation of small bowels into the dead space and their adhesion, hematoma, abscess, or perineal hernia. The fact that bony walls of the dead space are not compressible hinders the solution to most biomechanical problems of the filling materials. Optimal fillers should also stimulate cell proliferation, serve as antibacterial protection, and act against bowel (tissue) adhesion. Up to the present, autologous materials, predominantly omental and myofascial flaps, have mostly been used. The disadvantages of these matrices are that the omentum or muscle flaps’ length, number, and mobility are limited and often insufficient ,2,3 and Both synthetic and natural meshes have been tested to reconstruct the pelvic floor. Synthetic meshes like polytetrafluoroethylene or fully resorbable copolymer meshes are available in spray form only, and a risen thin layer is far from a suitable material capable of filling up the relatively large 3D dead spaces involved ,9. MoreoThe main aim of this study was to develop applicable fillers for the dead space meeting the following minimal requirements: 3D material with optimal biomechanical properties, minimal long-term adhesion formation, active against bacterial colonization, and promoting cell proliferation. To achieve these goals, agarose gels were chosen as an example of a biocompatible 3D scaffold, and fractionalized nanofibers were investigated as an active part of the composite. Agarose fillers are known in the field of aesthetic medicine , and sevv/v) fetal bovine serum  and antibiotic–antimycotic solution . MilliQ water was used for the preparation of all aqueous solutions . All reagents and chemicals used in experiments were of analytical grade and were used as received.Poly (PVA), Poly(ε-caprolactone) (PCL), Agar-Agar , Standard agarose , Fluorescein Isothiocyanate-Dextran  (FSC), H3PO4, and Glyoxal  were used. In vitro experiments were performed on immortalized BALB/3T3 mouse fibroblast cell lines obtained from the American Type Culture Collection . Mouse embryo fibroblast BALB/3T3 clone A31 cells (ATCC CCL-163) were cultured as adherent monolayers in plastic Petri dishes Nunc™  in Dulbecco’s modified eagle medium  supplemented with 10%  and a wire collector covered with polypropylene nonwoven textile with an electrical field intensity of 25 kV/cm. The PVA nanofibers were crosslinked using glyoxal for 72 h at a temperature of 60 °C to prevent the dissolution of nanofibers in the wet environment.Nanofibers were prepared using the electrospinning method. PVA  was dissolved in distilled water at 70 °C. A 10% w/w solution was used for spinning. The fibers adhered to the nonwoven fabric throughout the spinning process.PCL nanofibers were also prepared by electrospinning using the Nanospider Elmarco NS 500 spinner. PCL  was dissolved in chloroform at 23 °C. A 24% Two types of nanofibers have been tested, namely PCL and PVA. PCL is based on a monomer caprolactone, a cyclic ester that is miscible, but hardly soluble in water, and possesses a seven-membered ring. This property determines its biological and medical application as a material with a long degradation time in aqueous environments, including the human body. PVA is a water-soluble synthetic polymer. It has the idealized formula [CH2CH(OH)]n. PVA is used in various medical applications because of its biocompatibility, low tendency for protein adhesion, and low toxicity. PVA is a hydrophilic water-soluble substance broadly used in medicine and cosmetics. It has a short degradation half-time in water, which is possible to regulate via degree of polymerization. PVA and PCL fibers were cut into small pieces with scissors. They were then divided into two parts. Each working cycle included 10 grinding (1 min) and recooling periods (1 min). The applied impact frequency was 10 Hz. Nanofibers were ground in liquid nitrogen using a cryogenic grinder  with small vials.Nanofiber morphology of PCL and PVA nanofibers as well as fractionalized nanofibers were examined by electron microscopy. First, samples were sputtered with a thin gold layer using a rotary pumped coater . Magnification of the electron microscope  varied from 500× to 2000×, and a secondary electron (SE) detector at an accelerating voltage of 10 kV was used. Subsequently, the average fiber diameter was measured for 50 randomly selected fibers using Tescan software.For preparing different sample mixtures of Agar-Agar, standard agarose was put into a heating nest (Heating Nest LTHS for 1000 mL), which was heated to 150 °C, and the gels were taken out whilst boiling. The gels were then poured into plastic petri dishes with diameters of about 3.5 cm, dept 0.5 cm, slowly cooled down to the laboratory temperature in a humid atmosphere, and stored at 5 °C.Agar and agarose concentrations were selected based on the standard concentration for agar and agarose medium preparation. Other agarose concentrations (1% and 3%) were also prepared before the desired concentration (1.5%).Agarose (0.15 g + PCL 0.15 g)/10 mL distilled water, (0.15 g + PCL 0.015 g)/10 mL distilled water and Agarose (0.15 g + PVA 0.15 g)/10 mL distilled water, (0.15 g + PVA 0.015 g)/10 mL distilled water–agarose gels were prepared using the above methods. However, 0.15 g of fibers per 10 mL of gel were added to the gel after cooling to approximately 60 °C. The reason for the reduced temperature was that nanofibers are not damaged at this temperature rate and their functionality is preserved.The samples of gels were divided into 6 pieces with a sterile surgical scalpel measuring approximately 7 × 7 × 4 mm. Half of the pieces were used for the constant force load test-creep, and the other half for the destructive simple pressure test. The exact thickness of the individual samples was measured throughout the loading process. The unloaded cross section of each sample was measured using a calibrated photograph in the Autocad 2014 program. The instantaneous average cross section of the samples was calculated on the basis of the incompressibility condition, i.e., a constant sample volume, and Poisson’s constant was estimated as 0.5.E [Pa], modulus of elasticity from Kelvin modelη [-], viscosity from the Kelvin modelτ [s], relaxation time from Kelvin modelY [Pa], Young’s modulus of elasticity of a simple pressure testEσ [Pa], internal tension S [Pa], limit of simple pressure testσε [-], relative elongation of the simple pressure testS [-], maximum relative elongation of the simple pressure testεT [mm], thickness of unloaded sample2], cross section of unloaded sampleS [mm3], volume of the unloaded sampleV  and the relative elongation ε [-] on time were determined. The stress was calculated from the applied force at each time point according to the instantaneous cross section of the sample. The cross section was calculated from the instantaneously measured sample thickness and assuming incompressibility, i.e., constant volume. From these calculated data, the functional dependences of the internal stress on the relative elongation σ (ε) were created. The mechanical characteristics of the individual gel samples were calculated from the graph of this dependence: Young’s modulus of elasticity Egression .The tests were performed on a 1-axis loading device. The device was powered by a Kistler NCFH 10 actuator capable of developing a force of up to 10 kN with a working stroke of 200 mm. The drive was controlled by the Kistler MaXYmos system with a minimum step of 0.01 mm and precise control of the feed rate. Measurements of displacement, length, and elongation of the samples were provided by two independent incremental sensors with a step of 0.001 mm, which were in perfect agreement throughout the tests. We estimate the maximum absolute error of the sample elongation measurement to be ±0.01 mm. The tensile force on the sample was measured by two complementary connected 3-axis piezoelectric calibrated dynamometers Kistler 9317c. Their signal was processed by Dewetron DAQP ChargeB amplifiers  and AD converter NI PCI 6281 . Position and force were recorded and analyzed in Dewesoft 7.1 software ; further postprocessing, calculations, analysis, and statistical processing were performed in MS Excel 2010 and OriginPro 2020. The accuracy of force gauges is given <1%, measuring range was ±8 N, and division size was <0.0001 N. Our estimation of force measurement error was ±2% of the magnitude of the quantity. The frequency of scanning all quantities was 2000 Hz; for analysis the data were reduced to a frequency of 200 Hz by selecting every 10 values. No hardware or software signal filters were used in the measurement or signal processing.The samples were loaded with a constant force. The results were converted to a constant tension. To determine the rheological properties, the Kelvin viscoelastic model, a parallel connection of the damper and the spring, was chosen .σ=E.ε+ηThe measured relative elongation time curve was interpolated by the function:v/v ratio was mixed with 0.042% Fluorescein Isothiocyanate-Dextran (FSC), . Noncrosslinked nanofibers were prepared by electrospinning from a stock solution without any further treatment. Crosslinked nanofibers were prepared from the stock solution mixed with 0.6% H3PO4 and 0.4% Glyoxal  and crosslinking was induced by 82 h incubation at 60 °C and 15% relative humidity. Nanofiber degradation was monitored by dissolving fluorescent labeled nanofiber scaffold in a 0.9% NaCl aqueous solution. Six identical fluorescent labeled samples with a weight of 0.280 g ± 0.003 g were put into glass bottles with 177 mL of 0.9% NaCl aqueous solution. The bottles with samples were placed in a shaking incubator with a shaking frequency of 60 Hz and a temperature of 37 °C. Aqueous solution samples of 100 µL from each glass bottle were taken at predetermined time intervals and analyzed using SYNERGY H1  at an emission wavelength of 520 nm and excitation wavelength of 490 nm. Degradation of the functionalized PVA loaded with FSC was statistically determined using a calibration curve prepared by dissolving 0.007 g, 0.014 g, 0.028 g, 0.056 g, 0.084 g, 0.112 g, 0.14 g, 0.0168 g, 0.196 g, 0.224 g, 0.252 g, and 0.28 g of noncrosslinked fluorescent labeled samples loaded in 177 mL of 0.9% NaCl aqueous solution.PVA nanofibers were produced from a 10% PVA stock polyvinyl alcohol solution. For fluorescent labeling, a solution of PVA and 5-88 (Merck) at 1:1 2. Each cell was supplemented with 20 µL of MTS substrate  and 100 µL of growth media. The cells were cultivated for 1 h under normal cell culture conditions . Then, absorbance was spectrophotometrically measured (100 µL of product media at wavelength 490 nm) with a reference wavelength of 690 nm. Cells cultivated in a culture medium without incubation on gels were used as a negative control. Proliferation was measured on the 1st, 3rd, and 7th day of the experiment. Cell concentrations were determined by microscopy using a Burker chamber. On the 1st, 3rd, and 7th day of the experiment, samples were visualized using phase contrast microscopy . Cells were seeded in a Petri dish with gel at a density of 3000 cells/cm2. The cells were cultivated under normal cell culture conditions . After 72 h of cultivation, cells on gels were stained by the LIVE/DEAD® Viability/Cytotoxicity Kit  following protocol .The MTS test was used to evaluate the influence of gels on cells proliferation. Samples comprising 2 mL of growth media (DMEM with FBS and ATB) were incubated for 48 h on the tested gels. 3T3 cells were seeded in 96-well culture plates at a density of 3000 cells/cmAgar and agarose gels were selected in this study as a basis for the preparation of 3D implants. All gels were prepared as described in the methods section. Importantly, maturation was found to be an essential step towards implant biomechanical property formation. We compared two freshly prepared samples of agar and agarose with maturated samples kept for one week at 5 °C under 100% humidity. The relative deformation ε at temperatures ranging between 31 °C and 45 °C was measured. A phase transition between 30 °C and 37 °C was identified in the agar sample . This inNanofibers from PVA and PCL were prepared by electrospinning as described in the methods section, and their structure was visualized by scanning electron microscopy a,b. SEM Clearly, the diameter distribution showed significantly different structural characteristics for each nanofiber mesh. Different structural characteristics of nanofibers were observed for different polymers and for different physical parameters employed during the electrospinning process. Consequently, determination of structural characteristics belongs among the key parameters for reproducible production of the nanofiber-based drug delivery system. The average diameters of the PCL and PVA were determined immediately after electrospinning  and remaNanofiber meshes prepared as described above are 2D-like materials due to their minimal thickness. This is hardly acceptable for filling large 3D lesions. To solve this problem, we prepared nanofibrous materials suitable for dispersion in liquids or gels. First, nanofiber mesh was fractionalized on micro-sized particles at a temperature below the gel phase transition. It was then mixed with a suitable gel. Such a 3D system maintained all the advantages of nanofibers, as fractionalization at liquid nitrogen temperatures yielded a fine powder with preserved nanofiber internal substructure c,d. The average diameter of the PCL and PVA nanofibers was determined from scanning electron microscopy images by TESCAN software. The average diameter of the PVA nanofibers was smaller than the average diameter of the PCL nanofibers because, with used fabrication technology, the higher viscosity of the used PCL solution limits the stretching of the jet during formation and therefore affects the diameter.Gel functionalization was performed with fractionalized nanofibers to develop a novel generation of smart scaffolds with modifiable biomechanical and chemical properties. First, we tested the effect of fractionalized nanofibers on gel structural properties. The optimal description of biomechanical properties is a stress–strain diagram reflecting properties over a wide range of applied stress. We determined the stress–strain diagram for the agarose gel enriched with either PCL or PVA nanofibers . We provNevertheless, owing to the relatively narrow intervals of temperature and stress values to which the ideal implant should be exposed, the determination of the Young’s modulus of elasticity  was chosen to be a sufficient experimental approximation. Gel deformation as a stress reaction was measured at 23 °C and 43 °C, and the Young’s modulus of elasticity was calculated. Implant structure should resist at least the pressure applied during laparoscopic surgery. A set of gels withstanding the recommended pressure was prepared, and their biomimetic parameters were evaluated by surgeons. Based on the surgeons’ experience, the gel with Young’s modulus of elasticity characteristic for the 1.5% agarose was also identified as the most biomimetic gel resembling the native tissue macroscopically, as indicated by a blue arrow in Biomechanical gel properties belong indisputably among the key parameters of the optimal implant. The effect of fractionalized nanofibers on gel rigidity was considered as potentially significant and, thus, was carefully studied. The thick line indicates the average of the measurements, the field standard deviation. The samples were loaded with a constant force; the internal stress was calculated according to the instantaneous average cross section of the samples. The tests were performed in order to obtain the rheological properties of the gels, the modulus of elasticity, and the viscosity. These quantities were calculated using the Kelvin rheological model. From the graph and the calculated values, it can be deduced that the admixture of nanofibers causes a reduction in the modulus of elasticity and viscosity, more significantly in the case of PCL. The added gels will therefore be softer, more fluid, and at the same time withstand higher loads and deformations.Clearly, the presence of the fractionalized nanofibers significantly influenced the Young’s modulus of elasticity even at very low concentrations. The PCL nanofibers affected the relative elongation of gels more than the PVA . Both PVFirst, fractionalized nanofibers were found as essential for gel rigidification. However, gel functionalization by degradable nanofibers could also open the door for the development of gels with the controlled release of encapsulated bioactive substances. Thus, the effect of nanofibers with a controlled degradation was examined and PVA nanofibers were used as a model system. Clearly, PVA as a hydrophilic substance is easily degradable in an aqueous environment. However, controlled crosslinking can prolong their degradation half-life in water. PVA nanofibers have been produced from polymers of different degrees of crosslinking, and their degradation was measured in distilled deionized water at a temperature of 36 °C on a vibrating heated platform with a vibrational frequency of 51 Hz. Fluorescein Isothiocyanate-Dextran was used as a marker for determination of the degradation half-time and nanofiber labeling was performed and analyzed as described in the methods section. Measured data clearly show that crosslinking can significantly prolong the degradation half-time . While tCell proliferation was used for testing of the fractionalized nanofibers’ biocompatibility. Both PCL and PVA fractionalized nanofibers were tested, and cells cultivated in a culture medium without incubation on gels were used as a negative control.® Viability/Cytotoxicity Kit.The cell cytotoxicity test did not show significant differences between the standard culture medium used for cell culture experiments, PCL-conditioned medium, and PVA-conditioned medium . TherefoDead space after rectal extirpation in colorectal surgery is an area with a high risk of complications. Unfortunately, optimal methods for avoiding these complications have been missing. In the present study, we aimed to develop a novel 3D implant based on composite hydrogels with fractionalized nanofibers. Agarose and agar were chosen as the hydrogel standard for testing nanofiber effects. Despite the fact that the agarose and agar gels have been studied for a long time and have been shown as biocompatible, very little information about their exploitation in abdominal surgery has been reported ,23. ReceMedical application of nanofibers has several clear advantages. Among the more striking advantages is their biomimetic character, which resembles the structure of an extracellular matrix. In combination with a suitable gel, such a composite system has specifically required features, such as adherence to a target location, the possibility of microshaping, and the high surface area to volume ratio, which favors cell adhesion. Generally, prepared nanofiber meshes are usually very thin, 2D-like materials. This is hardly acceptable for filling large 3D lesions. To eliminate this problem, we prepared nanofibrous materials suitable for dispersion in liquids or gels. We used PVA and PCL, respectively, as polymers for nanofiber fabrication. Though PVA is approved by medical authorities, it is a slight irritant ,26. PCL Nanofiber mesh  was fractionalized to form micro-sized particles at a temperature below the gel phase transition. The microparticles were prepared with their average size comparable with the nanofiber mesh thickness. This step led to the production of an interesting material that could be mixed with the macro 3D systems . Such a composite material has several advantages, their application to endoscopic or laparoscopic surgery undoubtedly being among the most important. The addition of nanofibers significantly affects the yield strength and relative elongation. In the case of PVA, the yield strength increased by about 80% and thus allowed the use of up to 10 mmHg of operating pressure ,29,30; uNanofibers allow an increase in the yield strength and a greater relative compression of the material of about 0.39 in the case of PVA and about 0.35 in the case of PCL, i.e., an increase of 25 resp. 17 percent.The results of determining rheological properties using creep are very interesting. With the admixture of nanofibers, the modulus of elasticity decreases significantly, and at the same time, the viscosity of the materials decreases significantly. Together with the simple pressure test results, these data prove that adding nanocomposites to the gels makes them become softer and more fluid, and at the same time withstand higher loads and deformations. In the case of the addition of PVA, the limits of possible stress increased more significantly, while the elasticity and viscosity increased only slightly. By adding PCL fibers, we did not achieve great durability, but the material softened more significantly.The unique properties of nanofibers, such as the high surface-to-volume ratio, porous structure, and nanoscale microarchitecture, make them ideal candidates for tissue engineering applications . SurfaceIn our study, fractionalized nanofibers were found to be an ideal additive for innovative 3D hydrogel-based implants. Besides their effect on biomechanical properties, low-concentrated nanofibers have been shown to be effective as a drug delivery system for a controlled release of encapsulated bioactive substances from their core. The scoring system was developed for the implant characterization and its further optimization, namely for a specific application .In this study, we employed a combination of fractionalized nanofibers on pieces several microns or dozens of microns large. These pieces preserve their nano-substructure, which is important for cell accommodation, and their size simultaneously allows them obstacle-free migration throughout the gel. Thus, we could create three-dimensional nanofiber-based gels and exploit all the advantages of nanofibers for healing 3D lesions such as those following rectal extirpation. In summary, hydrogels enriched with fractionalized nanofibers functionalized bioactive substances  in their core.