As evidenced by current literature, there is a crucial link between emotion regulation, attachment, personality patterns, and substance abuse. However, knowledge regarding the exact interactions of these specific parameters in terms of substance abuse development is still sparse. Therefore, this study is aimed to shed light on how two specific emotion regulation strategies  might be influenced by the relationship between attachment, structural deficits in personality organization, and addictive behaviors.A total sample of 299 non-clinical young adults  filled in the Emotion Regulation Questionnaire (ERQ) together with the Adult Attachment Scale (AAS), the Personality Organization Inventory (IPO-16), and the Alcohol, Smoking and Substance Involvement Screening (ASSIST) by means of an online survey.p < 0.01) and “Depend” ence  were negatively associated with increased use of maladaptive strategies of expressional suppression of emotion, and “Depend”  was positively associated with increased use of adaptive strategies of cognitive reappraisal.As suggested by hierarchical regression analysis, attachment specifically predicted differences in Emotion Regulation (ER), whereby the AAS subscales “Close” ness (β = −0.38, In line with our assumptions, we observed a more secure attachment system to be predictive for an increased use of adequate emotion regulation strategies. The findings support the suggestion that a focus on underlying attachment-related processes in a psychotherapeutic setting might be a promising way to promote adaptive self-regulation of emotions. Emotions are an indispensable component of living, as “they can direct attention to key features of the environment, optimize sensory intake, tune decision making, ready behavioral responses, facilitate social interactions, and enhance episodic memory” .To live a functioning and fulfilling emotional life, one has to regulate their emotions. The process of Emotion Regulation (ER) is understood as a bundle of strategies and processes that change appearance, intensity, durability, and expression of emotion . Some ofCognitive reappraisal  describes antecedent-focused strategies of cognitively changing the appraisal of situations and therefore changing their emotional impact; (2) Expressive suppression (“Suppression”) means the response modulation of inhibiting the  expression of one's emotion . The participants ranged in age from 18 to 35 years . A total of 51 (17.1%) participants reported a university degree as their highest educational level, 244 (81.6%) participants reported a high school degree, 3 (1%) participants declared a completed apprenticeship as their highest educational level, and one (0.3%) participant absolved compulsory education. Concerning the current relationship status, 156 (52.2%) reported to be single, 134 (44.8%) were in a relationship, 3 (1%) were married, 1 (0.3%) person was divorced, and five (1.7%) participants preferred not to share information about their relationship status. In terms of the question, which kind of substance was ever consummated, 227 (75.9%) participants reported to have consummated tobacco, 292 (97.7%) alcohol, 198 (66.2%) cannabis, 45 (15.1%) cocaine, 57 (19.1%) amphetamines, 23 (7.7%) inhalants, 47 (15.7%) sedatives, 53 (17.7%) hallucinogens, 19 (6.4%) opioids, and 28 (9.4%) “other drugs.” Since ASSIST is a standardized tool for estimating the severity of drug abuse and the resulting need for therapeutic intervention, the following groups were formed based on the results: (1) No intervention is needed; (2) Short-term intervention is advised; (3) Intensive treatment is advised. Participants were predominantly in the “no intervention is needed” group .r = 0.16; p < 0.01), anxiety about being rejected or unloved , and “Gender” , and significantly negatively related to comfort with closeness  and comfort with depending on others . Furthermore, the ER strategy “Reappraisal” showed a significant positive correlation with “Depend”  and “Age” , and a significant negative correlation with structural deficits in “Personality Organization”  and “Anxiety” . In addition, structural deficits in “Personality Organization” showed an association to all three scales measuring adult attachment , as well as to “Substance Use”  and “Age” . “Substance Use” was moreover related to the attachment dimension “Depend” . Within the attachment scales, “Anxiety” showed a relation to “Gender”  and “Age” . “Depend” was associated with “Gender”  and “Age” . All attachment scales were correlated with each other . Additionally, Bonferroni correction was conducted in order to control for α inflation. Hereby, the originally significant correlations between “Personality Organization” and “Reappraisal,” “Suppression” and “Anxiety,” “Depend” and “Substance Use,” “Gender” and “Depend,” as well as all statistically relevant correlations with “Age,” disappeared.As normal distribution was not given for any of the examined variables, Pearson and Spearman correlations were both calculated. The changes in the correlations were negligible, and changes in correlation appearance/disappearance only were observed for correlations not concerning the dependent variables “Suppression” and “Reappraisal.” As shown in The ASSIST was inserted into the stepwise regression, even though no correlations with ER could be found, because the ASSIST correlated with the IPO and some scales of the AAS.Although some correlations had to be classified as non-significant after performing a Bonferroni correction, the originally selected predictor variables were adhered to. This decision is based on the fact that the Bonferroni procedure can be considered very conservative. In addition, most corrections were observed regarding the “Age” variable, which was still considered of interest mainly due to the explorative nature of the study.F1(2.296) = 6.58; F2(3.295) = 4.88; F3(7.291) = 16.05; all p < 0.01]. The model summary, however, showed that model 3 explained 26.1% of the variance in “Suppression” and therefore accounted for the most variance. Furthermore, inserting the ASSIST did not show any significant change in F.However, ANOVA of the models, created through stepwise regression including, firstly, age and gender, secondly, the total score of the ASSIST, and thirdly, the global score of the IPO and the three scales of the AAS, showed that all three models predicted reliably the dependent variable “Suppression” . The model summary showed that model three explained 6.8% of the variance in “Reappraisal” and therefore accounted for the most variance. Furthermore, checking the coefficients of models two and three, only age had an impact in model two , but lost its significant effect  as the IPO and AAS scales were inserted. Regarding the coefficients of model three, the variable “Depend” (β = 0.26) showed a significant result (p < 0.01). “Close” (β = −0.14) was found to be significant by tendency but will not be further interpreted. ANOVA of the models, created through stepwise regression including, firstly, age and gender, secondly the total score of the ASSIST, and thirdly, the global score of the IPO and the three scales of the AAS, showed that models two and three significantly predicted the dependent variable “Reappraisal”  or psycDespite its limitations, this study contributes to a better understanding of how attachment, personality organization, and substance use might be connected to emotion regulation. The results suggest that attachment behavior particularly helps to explain the different selection of strategies in emotion regulation.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by University of Graz. The patients/participants provided their written informed consent to participate in this study.PB, XV, and H-FU conceptualized the study. PB and XV collected the data. PB analyzed and interpreted the data. PB and H-FU drafted the manuscript. JF and H-FU critically reviewed it. All authors gave their final approval of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


Climate change is certainly a global problem that negatively affects all nations, and thus all humans, on the globe. Nevertheless, little is known about people’s perceptions of climate change and its effects on people’s attitudinal and behavioral responses to climate change. The present study successfully addressed how hotel guests’ environment-friendly behavior intention is formed through their self-perception as a member of the global community and their psychological distance of climate change. An online survey was used to collect quantitative data from hotel guests to verify the hypotheses. Our test results supported all the hypotheses in our conceptual model. Consequently, the findings of this study satisfactorily explained how hotel guests form their intention to engage in environment-friendly behaviors while they are staying at hotels. To make tourism on the globe sustainable in the future, it is critical for every participant to be faithful to one’s role. In the lodging industry, the environment-friendly management of hotels, together with the environment-friendly behaviors of guests, is commonly regarded as the core element of green management . Making However, psychological distance has been often identified as a major barrier to act on climate change . The psyAccordingly, this study adopted the psychological distance of climate change as a key antecedent of the environment-friendly behavior intention of hotel guests. In doing so, this study used the norm activation model  as a conBased on social identity theory (SIT), global identity can be defined as identifying oneself with all humans , that isConstrual level theory explains that individuals variously perceive events and objects based on their self-relevance and likelihood . AccordiAgain, global identity refers to recognizing oneself as a part of not only all humans but also the world. The literature shows that those who have a strong global identity tend to actively engage in actions that are favorable to the earth and its people . For humHypothesis 1 (H1).Global identity negatively affects psychological distance of climate change.The norm activation model (NAM)  describeHypothesis 2 (H2).Psychological distance of climate change negatively affects ascribed responsibility for not acting environment-friendly.Personal norm, as the central element in the NAM, indicates a moral obligation for performing prosocial behaviors . AccordiHypothesis 3 (H3).Psychological distance of climate change negatively affects personal norm for environment-friendly behavior.sense of responsibility, and then this sense induces people to develop an obligation toward a certain behavior depending on their judgement about whether it is the right thing to do (or not) from their value-based conceptions, which define how people should or should not behave in certain situations [obligation leads people to engage in certain behaviors [Through the norm activation theory, Schwartz  explainstuations . Then, tehaviors . Engaginehaviors . Han et ehaviors  confirmeehaviors . TherefoHypothesis 4 (H4).Ascribed responsibility for not acting environment-friendly positively affects personal norm for environment-friendly behavior.Hypothesis 5 (H5).Personal norm for environment-friendly behavior positively affects environment-friendly behavior intention .β = 0.802)”; “I identify myself as a global citizen (β = 0.852)”; and “I believe people should be made more aware of how connected we are to the rest of the world (β = 0.795)”) [β = 0.858)”; “serious consequences of climate change primarily impact other people (β = 0.892)”; and “climate change is a significant problem mainly for others (β = 0.900)” were used to measure social distance. “Climate change mostly affects other parts of the world (β = 0.857)”; “climate change is a significant problem mainly in distant locations (β = 0.866)”; and “impacts of climate change are primarily experienced in developing countries (β = 0.761)” were used to evaluate spatial distance. “Climate change effects will mostly occur in the future (β = 0.976)”; “serious consequences of climate change will be felt primarily in the future (β = 0.818)”; and “climate change will be more of a significant problem in the future compared with now (β = 0.554)” were employed to measure temporal distance. “I am uncertain whether the climate is changing (β = 0.803)”; “I am uncertain over the causes of climate change (β = 0.941)”; and “I am uncertain what the effects of climate change are (β = 0.968)” were used to assess hypothetical distance. Ascribed responsibility was measured with three items  while staying at a hotel” (β = 0.838)”; “I feel jointly responsibility for the negative consequences of not practicing eco-friendly activities  while staying at a hotel (β = 0.920)”; and “I feel joint responsibility for the environmental pollution and ecological damage problems caused by not practicing eco-friendly activities  while staying at a hotel (β = 0.941)”) [β = 0.839)”; “regardless of what other people do, because of my own values/principles I feel that I should behave in an environmentally friendly way while staying at a hotel (β = 0.892)”; and “I feel that it is important to engage in eco-friendly activities while staying at a hotel, reducing the harm to the environment (β = 0.924)”) [β = 0.693)”and “the next time I stay at a hotel, I will expend effort on reducing waste  (β = 0.863)” were used to measure waste reduction intention. “The next time I stay at a hotel, I am willing to engage in towel/linen reuse activities (β = 0.478)” and “the next time I stay at a hotel, I will expend effort on saving water  (β = 0.820)” were used to evaluate water saving intention. All the items were measured with a seven-point Likert scale from “strongly disagree” (1) to “strongly agree” (7). Including the measurement items mentioned above, the questionnaire was composed of a brief research introduction and queries for respondents’ demographic characteristics. A pre-test for the questionnaire was conducted with five graduate students from a hospitality program and three hotel industry professionals. Based on their feedback, a slight modification was made. Lastly, the questionnaire was finalized through the reviews of academic experts.Validated measures were adopted from previous studies to assess the research variables. Multiple measurement items were utilized to evaluate the constructs in the conceptual model. Specifically, three items were adopted to measure global identity ”) . To eval0.795)”) . “Climat0.941)”) . Three i95”) . ToTo collect the data, an online panel survey was performed through an internet-based survey firm’s system. General hotel guests in South Korea were randomly selected from the firm’s database. An e-mail invitation for the survey with a brief research introduction was sent to them. Only those who have stayed at hotels within the last nine months were invited to answer the questionnaire by clicking the survey link. The survey was collected from 13 April 2021 to 17 April 2021. A total of 1036 people initially participated in the survey. Out of them, 393 respondents completed the survey. After eliminating inconsistent responses, a total of 387 samples remained for analysis. The respondents stayed at hotels in a variety of locations and categories in South Korea.Among the 387 samples, 48.6% were male and 51.4% were female guests. On average, the respondents were 43.84 years old, ranging from 20 to 69 years old. A total of 21.7% were in their 50s, 20.7% were in their 40s, 19.9% were in their 30s, 19.4% were in their 20s, and 18.3% were in their 60s. In terms of education level, 63.3% held a bachelor’s degree, 15.0% held graduate school degrees or higher, 12.9% held a high school diploma, and 8.8% held an associate degree. Regarding monthly income, 36.7% reported an income lower than USD 2500, followed by between USD 2501 and 4500 (32.6%), USD 4501 and 6500 (16.5%), USD 6501 and 8500 (8.5%), and over USD 8500 (5.9%). When asked for their frequency of hotel stay, the respondents reported 2.2 times per year on average.2 = 592.730 ; RMSEA = 0.059, CFI = 0.956, IFI = 0.956, and TLI = 0.948). All the measurement items were significantly loaded on their associated constructs at p < 0.001. The composite reliability of the measures ranged from 0.856 to 0.982, showing higher values than the recommended cutoff of 0.700 [To generate the measurement model and evaluate the quality of the data, a confirmatory factor analysis (CFA) was performed. As shown in of 0.700 . The valof 0.700 . Thus, tof 0.700 .χ2 = 778.020 ; RMSEA = 0.072, CFI = 0.932, IFI = 0.932, and TLI = 0.922). The model showed satisfactory variance explanation power for environment-friendly behavior intention  (R2 = 76.7%). The detailed results are shown in To assess the hypothesized research model, structural equation modeling (SEM) was conducted. As shown in β = −0.253, p < 0.001), supporting H1. In turn, the psychological distance of climate change showed a significant, negative effect on ascribed responsibility , supporting H2. On the other hand, the psychological distance of climate change did not significantly affect personal norm (H3) . However, a post hoc mediation test showed that the insignificant impact was due to the full mediation effect by ascribed responsibility. Specifically, when the path from ascribed responsibility to personal norm was constrained to zero, the impact of the psychological distance of climate change on personal norm became significant ). The χ2 difference between the original model and the constrained model (Δχ2(1) = 205.74) was significant at p < 0.001, indicating that the mediation effect was highly significant. Going back to the original model, ascribed responsibility showed a significant, positive effect on personal norm  and, in turn, personal norm showed a significant, positive effect on environment-friendly behavior intention , supporting H4 and H5.Regarding the hypothesized relationships, as shown in β = 0.038, p < 0.05). The psychological distance of climate change exhibited a significant, negative indirect effect on personal norm . In turn, ascribed responsibility showed a significant positive effect on environment-friendly behavior intention . Lastly, in terms of the total effect on environment-friendly behavior intention, our findings indicated that personal norm exerted the greatest impact , followed by ascribed responsibility .As a next step, the indirect effects of the constructs were assessed. As in As mentioned in the literature review, customers’ positive global identity has been suggested to reduce the psychological distance of climate change perception ,12,17,19The one of objectives of this research was to identify the relationships between the psychological distance of climate change, ascribed responsibility, and personal norm. Han et al.  noted thThe norm activation model (NAM) theory is widely used in tourism research ,21. PrioTested in the hotel industry context, this study revealed that guests’ ascribed responsibility for not acting environment-friendly positively influenced their personal norm for environment-friendly behavior. In turn, guests’ personal norm for environment-friendly behavior positively affected their environment-friendly behavior intention . These results are consistent with the findings of previous research using the NAM on other environment-friendly behaviors, such as sustainable transport behavior , energyThe samples in this study are restricted to Korean travelers who traveled during the COVID-19 pandemic period. Therefore, responses to the same questions can be different when asked to non-Koreans during post-COVID-19 period. Depending on cultural backgrounds, some people may have more holistic views of the world than others, leading them to having stronger global identity and thus feeling other people’s problems closer to themselves. The COVID-19 pandemic may have led people to feel that all humans are closely connected. In fact, we literally have affected one another regardless of any boundaries during the pandemic period. Thus, perhaps, during the post-COVID-19 period, people may develop stronger global identity than ever before and view the earth as one system where we have to rely on one another to survive. Therefore, the generalizability of the findings of this study may be further tested in other cultures during the post-COVID-19 period.As mentioned earlier, future researchers may adopt the psychological distance of climate change instead of awareness of consequences when using or extending/modifying the NAM in explaining other types of pro-environmental behaviors to generate more specific findings. It appears that some people do not realize the apparent harmful outcomes of climate change although they are occurring in front of their eyes . Therefo


In obesity, autonomic dysfunction occurs, which may result in a loss of PVAT function and subsequent vascular disease. Accordingly, we have investigated abnormalities in obese PVAT, and the potential for exercise in restoring function.Perivascular adipose tissue (PVAT) exerts an anti-contractile effect which is vital in regulating vascular tone. This effect is mediated via sympathetic nervous stimulation of PVAT by a mechanism which involves noradrenaline uptake through organic cation transporter 3 (OCT3) and β3-adrenoceptors, OCT3 and tumour necrosis factor-α (TNFα) in PVAT.Vascular contractility to electrical field stimulation (EFS) was assessed ex vivo in the presence of pharmacological tools in ±PVAT vessels from obese and exercised obese mice. Immunohistochemistry was used to detect changes in expression of β3-adrenoceptor activation. Moreover, adiponectin no longer exerts vasodilation. Additionally, exercise reversed PVAT dysfunction in obesity by reducing inflammation of PVAT and increasing β3-adrenoceptor and OCT3 expression, which were downregulated in obesity. Furthermore, the vasodilator effects of adiponectin were restored.High fat feeding induced hypertension, hyperglycaemia, and hyperinsulinaemia, which was reversed using exercise, independent of weight loss. Obesity induced a loss of the PVAT anti-contractile effect, which could not be restored via βLoss of neutrally mediated PVAT anti-contractile function in obesity will contribute to the development of hypertension and type II diabetes. Exercise training will restore function and treat the vascular complications of obesity.The online version contains supplementary material available at 10.1007/s10557-020-07136-0. Second, excess NA is sequestered by adipocytes via organic cation transporter 3 (OCT3). In obesity, PVAT function is lost, which may contribute to development of metabolic syndrome [Perivascular adipose tissue (PVAT) is a highly metabolically active tissue which surrounds the majority of blood vessels and contributes to the modulation of vascular tone –3. Previsyndrome –7. Howev3-adrenoceptors, leading to reduced adiponectin release. Bioavailability of adiponectin has already been demonstrated to be reduced in human obesity [It is well known that in obesity, pathological over-activity of the sympathetic nervous system occurs , 9. Card obesity , hyperte obesity , and typ obesity .In obesity, PVAT morphology changes, and adipocyte size and number increase. However, there is no increase in blood supply, leading to hypoxia and chronic inflammation , 15. By The role of adiponectin in the inflammatory response may contribute to insulin insensitivity. Adiponectin is an inhibitor of the cytokine tumour necrosis factor-α TNFα), which inhibits insulin signalling –22. TherSympathetic nerves play a role in recruiting inflammatory mediators, and a number of studies have demonstrated that exercise, which is considered to be healthy sympathetic stimulation, can reduce inflammation in obesity –26. In aThe beneficial effects of exercise on brown adipose tissue function surrounding the aorta has been well documented –31; howe3-adrenoceptors and OCT3 are reduced. These changes in the PVAT environment will be reversed by exercise. In addition, we hypothesise that the resultant symptoms of metabolic syndrome in obesity will be alleviated by exercise.The present study was designed to examine the mechanisms of PVAT dysfunction in obesity and the potential for a significant lifestyle intervention in restoring function. We tested the hypothesis that a loss of PVAT function in obesity can be reversed via exercise. We anticipate that in obesity, PVAT becomes inflamed, and expression of β2 asphyxiation. Prior to sacrifice, blood pressure was recorded in conscious, restrained animals using the CODA tail cuff blood pressure monitoring system . Immediately after sacrifice mixed blood samples were collected by severing the thoracic aorta. Blood glucose concentration was measured immediately using an automatic blood glucose system . Following euthanisation, the mesentery was exposed and removed. Epididymal fat pads, hearts, and kidneys were collected and weighed.All experiments were performed in accordance with the appropriate Home Office project licence and complied with the UK Animal (Scientific Procedures) Act 1986. Male C57BL/6j mice were randomly assigned to three groups; control, obese, and exercised obese (see below) and aged until 18–20 weeks. Animals were housed under a 12-h light/12-h dark cycle and provided with food and water ad libitum. The body weights of all groups were recorded every 2 weeks from 8 weeks of age. Mice were fasted overnight and sacrificed using COn = 20).Control mice were fed a standard chow diet ad libitum  until they were euthanised at 18–20 weeks old (n = 44). A 10–25% increase in body weight as compared with age-matched controls is considered as moderate obesity in rodents [Male C57BL/6j mice  were purchased at 6 weeks old and allowed a 2-week acclimatisation period before being placed on a 60% kcal from fat diet  ad libitum for 10–12 weeks and euthanised at 18–20 weeks .For exercised obese mice, male C57BL/6j mice followed the diet-induced obesity protocol as described above. At week five, forced swim training commenced. Mice were initially exercised for 15 min once a day, and the duration of exercise increased in daily increments of 15 min until reaching a final duration of 60 min. From week six, mice were exercised five times per week. Mice were swum in tanks with a surface area of 2,500 cmhanising  (n = 20)n = 10; Supplementary Figure n = 8; Supplementary Figure For the exercised obese model, there were two additional control groups. The first group was a diet-induced obesity group, which were high fat fed as described above, and were euthanised after 5 weeks (the time point at which exercised groups commenced training) to examine mouse phenotype and PVAT function at this time point  and were mounted on 40-μm diameter wires in a wire myograph . Arteries were allowed to equilibrate for 30 min at 37 °C in PSS bubbled with 95% air/5% CO2 to maintain a pH of 7.4. Following equilibration, vessel starting tension was standardised using a standard normalisation procedure [+ PSS . Vessels were stimulated using an electrical field stimulation protocol over a frequency range of 0.1–30 Hz  [Second-order mesenteric arteries were prepared as previously described . Arteriarocedure . Vessels3-adrenoceptor agonists and antagonists were used to delineate their role in mediating the vascular response to EFS in the presence or absence of PVAT; following the first EFS stimulation, β3-adrenoceptor agonist CL-316,243 (10 μM) or β3-adrenoceptor antagonist SR59230A (1 μM) was added to ±PVAT vessels and incubated for 30 min, before re-stimulating the vessels. The effects of globular adiponectin (5 μg/ml) were tested on only −PVAT vessels. Vessels were stimulated once and allowed to recover for 15 min before adding adiponectin. The adiponectin was incubated for 5 min before repeating the EFS protocol.Previously published pharmacological assessment which has been conducted in control vessels were repeated in obese and exercised obese vessels in this study . β3-adreTo examine the role of nitric oxide and NA transport, a third arterial preparation was used whereby a section of exogenous PVAT was suspended above a clean artery .2 before stimulating using EFS at 10 Hz for 4 s . The supernatant was collected and stored at − 80 °C. An adiponectin ELISA kit  and dissolved in PSS. Recombinant mouse globular adiponectin was obtained from Generon . Corticosterone  was dissolved in 100% ethanol. Vehicle controls confirm no effect of 100% ethanol (data not shown).3-adrenoceptor, OCT3, and tumour necrosis factor alpha (TNFα) expression was assessed in control, obese, and exercised obese PVAT by immunohistochemistry [3-adrenoceptors , OCT3 , or TNFα  overnight at 4 °C. Slides were then incubated with a biotinylated goat anti-rabbit secondary antibody  for 1 hour at room temperature before addition of vectastain avidin-biotin complex  and 3,3′-diaminobenzidine solution  for detection of antibody binding. Images were captured using a colour camera  mounted on a microscope .βhemistry . In brieMouse kidney was used as a positive control, and negative controls were conducted using samples incubated with phosphate buffered saline in place of the primary antibody. Controls are provided in Supplementary Figure P values < 0.05 were considered statistically significant. Normal distribution was confirmed using the Shapiro-Wilk normality test, supported by Skewness and Kurtosis coefficients (using an acceptable range of ± 2).Data are expressed as the mean ± SEM. All statistical analysis was performed using GraphPad Prism v7 . Differences in mouse phenotype measures including blood pressure, blood glucose, organ weights, and plasma ELISA data were analysed using a one-way analysis of variance (ANOVA) followed by a Tukey post hoc test to compare individual groups. For body weight progression, differences were tested using a two-way ANOVA. A minimum sample size of 20 was used for each group. For blood pressure, the average of the 10 measurement cycles was used from each mouse .Contractile responses were normalised to the maximum contraction elicited with KPSS, as consistent with other studies , 37, 40 3-adrenoceptors and OCT3, which were localised to adipocyte membranes, were quantified within/around five arbitrarily selected adipocytes, i.e., staining intensity per adipocyte, as opposed to using a set sized area for staining. This was to account for changes in adipocyte size. TNFα staining was quantified within the whole field of view. Correlation of adipocyte area and intensity of adipocyte membrane staining were assessed to confirm that changes in intensity were not due to changes in adipocyte size . Images were taken from five fields of view per mouse. The intensities of staining for βn = 20, obese n = 44, exercised n = 20. Control vs obese P < 0.0001, control vs exercised obese P < 0.0001). The difference in body weight became significant after 4 weeks of high fat feeding (P < 0.01). At week 6, the exercised obese group commenced swimming. This group exhibited no further significant increases in weight gain; however, these mice did not lose any weight. To confirm that changes in weight were due to increases in fat depots and not muscle mass, epididymal fat pads were removed and weighed . High fat feeding resulted in a significant increase in fat depots in both obese and exercised obese groups .Obesity was established in C57BL/6j mice by high fat feeding for 10–12 weeks. High fat feeding resulted in a significant increase in body weight in both obese and exercised obese mouse groups . There were no changes in heart or kidneys weights .High fat feeding resulted in significant elevations in both systolic and diastolic blood pressures in the obese group; however, in the exercised obese group, blood pressure was comparable with healthy control mice . Plasma insulin was significantly increased in obesity, which combined with hyperglycaemia indicating possible type II diabetes. Insulin was reduced in the exercised obese group , and plasma NA was significantly reduced in obesity, indicating autonomic dysfunction. In exercise, NA was restored to control levels .ELISA kits were used to measure plasma insulin Fig.  and NA  . In obese mice, staining intensity of TNFα in PVAT was significantly increased compared with controls (P < 0.001). In obese exercised mice, expression of TNFα was significantly reduced as compared with both obese (P < 0.0001) and control mice (P < 0.05) . Therefore, electrical activation of sympathetic nerves in obese PVAT no longer elicits an anti-contractile effect. However, electrical activation of sympathetic nerves in exercised obese PVAT elicited a significant anti-contractile effect , indicating that exercise has restored PVAT function.To examine the effects of obesity on sympathetic nerve function in PVAT, EFS profiles were generated in mesenteric resistance arteries from obese mice . In obese mice, there was no significant difference between −PVAT and +PVAT vessels , indicating that these receptors may be downregulated in obesity.In a previous study, we have indicated that PVAT function is mediated in part by βoceptors ; therefolus Fig. . CL-316,3-adrenoceptors was examined using immunohistochemistry in sections of mesenteric PVAT from control, obese, and exercised obese mice . The intensity of staining in the adipocyte membrane was quantified, and in obese mice, expression of the adrenoceptor was significantly reduced as compared with that in obese mice (P < 0.0001). In exercised obese mice, expression of β3-adrenoceptors is significantly increased compared with obese mice (P < 0.01); however, expression is still significantly lower in exercised obese mice than in control mice (P < 0.05) . Similar to previously reported data in control mice [P < 0.0001, −PVAT vs −PVAT and SR59230A P > 0.05, +PVAT vs +PVAT and SR59230A P < 0.0001, −PVAT vs +PVAT and SR59230A P < 0.0001). These results indicate that exercise restored β3-adrenoceptor-mediated PVAT function in obesity.To determine if the increase in expression of β30A Fig. ; n = 8. rol mice , SR592303-adrenoceptor-mediated anti-contractile factor release, and NA uptake into adipocytes by OCT3 [n = 5 all groups; Fig. P < 0.05) and control mice . However, in the exercised obese PVAT, OCT3 expression was significantly increased compared with both control and obese groups  . Next, the exogenous PVAT was removed again and incubated with both corticosterone and SR59230A, and the restored anti-contractile effect in exercised obese mice was completely abolished , suggesting that the normal functioning of both β3-adrenoceptors and OCT3 had been restored by exercise.PVAT denuded vessels with sections of exogenous PVAT suspended above were subjected to control EFS stimulations, before removing the exogenous PVAT and incubating with the OCT3 inhibitor corticosterone Fig. . Followi3-adrenoceptor activation [n = 12 both groups, P < 0.0001).Previously, we have reported that adiponectin is the anti-contractile factor released upon βtivation ; therefon = 22, exercised obese n = 20). Plasma adiponectin was significantly increased in obesity and was reduced back to control levels with exercise .An ELISA kit was used to measure plasma adiponectin Fig.  . However, in exercised obese mice, adiponectin did cause a vasodilation , indicating that the impaired vasodilator function of adiponectin in obesity has been restored by exercise.To determine if adiponectin presents a viable target for treating the vascular complications of obesity, the effects of globular adiponectin were tested in obese and exercised obese mice. Following control stimulations with EFS, −PVAT vessels were given 15 min to recover before incubating with exogenous adiponectin for a further 5 min. In obese vessels, exogenous application of adiponectin no longer elicits a vasodilator effect  adiponectin secretion from mesenteric PVAT is reduced in obesity; and (6) the vasodilator function of adiponectin is lost in obesity but can be rescued using exercise.This study has for the first time investigated the effects of autonomic dysfunction on the PVAT effect in obesity and the potential for exercise in restoring PVAT function in resistance arteries. The main findings of this study were (1) in obesity, PVAT becomes inflamed, and this inflammation is reversed with exercise; (2) the sympathetic nerve-mediated PVAT anti-contractile effect is lost in obesity, but can be rescued using exercise; (3) reversal of PVAT dysfunction in obesity using exercise is accompanied by normalised blood pressure, blood glucose, and plasma insulin, independent of weight loss; (4) expression of βWe found that 10–12 weeks of high fat feeding induced increased adiposity, hypertension, and hyperglycaemia. Plasma insulin was also increased threefold, which, in combination with hyperglycaemia, would suggest that these mice may have type II diabetes, although glucose tolerance testing is required to confirm. In humans, metabolic syndrome is defined by a combination of at least three out of five conditions: abdominal obesity, hypertension, type-II diabetes, hypertriglyceridemia, and hypercholesterolemia . TherefoWe reported a reduced level of circulating NA in our obese mouse model, and a similar reduction in plasma NA in obesity has been reported in human patients . However3-adrenoceptors using an agonist did not rescue loss of function, indicating either dysfunction or downregulation of the receptor. There are no agonists available for OCT3; therefore, we were unable to determine if pharmacological manipulation of this transporter could restore function. In this study, we have indicated a downregulation of both β3-adrenoceptors and OCT3 using immunohistochemistry, which is not a quantitative measure and is therefore a limitation of this study. In heart failure, over-activity of sympathetic nerves results in desensitisation and internalisation of β3-adrenoceptors [3-adrenoceptors and OCT3. It has been suggested that β3-adrenoceptors are a potential target for the treatment of hypertension in obesity, as agonists have been shown to be hypotensive in healthy canines and rodents [3-adrenoceptors in obesity make the application of an agonist redundant. Reduced expression of β3-adrenoceptors in obesity has been shown in a number of obese mouse models [Activation of βoceptors . In futu rodents , 53; howe models ; however3-adrenoceptors in obesity results in a reduction in adiponectin secretion as demonstrated in this study using an ELISA. Reduced secretion of the vasodilator adiponectin will contribute to increases in arterial tone and blood pressure. Paradoxically, we found plasma adiponectin levels to be elevated in our obese mice. Studies have shown that adiponectin clearance in obesity is impaired [The reduced expression of βimpaired ; therefoimpaired , 55, 56.To determine if administration of adiponectin would be useful in obesity, we applied exogenous adiponectin to obese arteries. However, the vasodilator effects of adiponectin were absent. This may suggest that increased plasma adiponectin may have resulted in desensitisation of receptors. In addition, adiponectin causes vasodilation via mechanisms dependent upon adenosine monophosphate-activated protein kinase (AMPK) , 59. In This is the first study to demonstrate a restoration of the PVAT anti-contractile effect in resistance arteries in obesity using exercise. Moreover, these findings were independent of weight loss. One of the beneficial effects in PVAT of exercise is reduced inflammation. There is a clear reduction in the expression of TNFα, an important inflammatory marker  in exerc3-adrenoceptors and OCT3 are increased by exercise. Expression levels of OCT3 have been returned to control levels, whereas expression of β3-adrenoceptors is still below normal. However, the efficacy of the β3-adrenoceptor antagonist in reducing the anti-contractile effect alone, and abolishing the anti-contractile effect in combination with the OCT3 inhibitor would suggest that the expression of β3-adrenoceptors has increased enough to restore the healthy PVAT anti-contractile mechanism. Therefore, healthy sympathetic activity, i.e., exercise, has restored PVAT’s ability to release an anti-contractile factor upon β3-adrenoceptor activation, and the ability to transport NA into adipocytes via OCT3, preventing the NA from reaching the blood vessel and eliciting contraction.We are the first to demonstrate that expression of β3-adrenoceptors [Many studies have indicated that exercise can induce “beiging” of white adipose tissue –65. Thisoceptors –72 and Ooceptors  have bee3-adrenoceptors and OCT3, resulting in a loss of PVAT function. Moreover, obese arteries are no longer responsive to adiponectin. Healthy sympathetic activity, i.e., exercise, restored the normal PVAT anti-contractile mechanism in obesity, independent of weight loss. Exercise reduced inflammation of PVAT and increased expression of β3-adrenoceptors and OCT3 to restore function. Moreover, the vasodilator function of adiponectin was restored. As a result of repaired PVAT function, the vascular complications of obesity, hypertension, and type II diabetes were reversed, indicating exercise as an effective treatment for obesity-related illnesses, highlighting the importance of exercise as a first line of treatment in the vascular complications of obesity.Dysfunction of the sympathetic nervous system in obese PVAT results in a loss of the PVAT anti-contractile effect, which may be contributing to the development of hypertension and type II diabetes. The over-activity of sympathetic nerves in obesity causes downregulation of βESM 1(PDF 352 kb)


Edwardsiella ictaluri is a Gram-negative facultative intracellular pathogen causing enteric septicemia of catfish (ESC), a devastating disease resulting in significant economic losses in the U.S. catfish industry. Bacterial secretion systems are involved in many bacteria's virulence, and Type VI Secretion System (T6SS) is a critical apparatus utilized by several pathogenic Gram-negative bacteria. E. ictaluri strain 93–146 genome has a complete T6SS operon with 16 genes, but the roles of these genes are still not explored. In this research, we aimed to understand the roles of two hemolysin co-regulated family proteins, Hcp1 (EvpC) and Hcp2. To achieve this goal, single and double E. ictaluri mutants  were generated and characterized. Catfish peritoneal macrophages were able to kill EiΔhcp2 better than EiΔevpC, EiΔevpCΔhcp2, and E. ictaluri wild-type (EiWT). The attachment of EiΔhcp2 and EiΔevpCΔhcp2 to ovary cells significantly decreased compared to EiWT whereas the cell invasion rates of these mutants were the same as that of EiWT. Mutants exposed to normal catfish serum in vitro showed serum resistance. The fish challenges demonstrated that EiΔevpC and EiΔevpCΔhcp2 were attenuated completely and provided excellent protection against EiWT infection in catfish fingerlings. Interestingly, EiΔhcp2 caused higher mortality than that of EiWT in catfish fingerlings, and severe clinical signs were observed. Although fry were more susceptible to vaccination with EiΔevpC and EiΔevpCΔhcp2, their attenuation and protection were significantly higher compared to EiWT and sham groups, respectively. Taken together, our data indicated that evpC (hcp1) is involved in E. ictaluri virulence in catfish while hcp2 is involved in adhesion to epithelial cells and survival inside catfish macrophages. Edwardsiella ictaluri  is the causative agent of enteric septicemia of catfish (ESC)  . Althoug species –4. At the system , 6. Howetrophils . To reple stress –10.E. ictaluri highly depends on its resistance to host stress factors and modulating the host environment. Edwardsiella ictaluri encodes urease that is activated in acidic phagosomes of macrophage to cope with low pH , Eip18 (evpC), Eip55 (evpB), Eip20 (evpA), have been first identified during the catfish host-pathogen interaction  are involved in bacteria-host interaction. Particularly, they are involved in adhesion and invasion, intracellular survival of bacteria, bacterial cytotoxicity, and virulence . In E. ieraction . The secictaluri . Fish paE. tarda . A recentivation .E. ictaluri genome has a complete T6SS operon with evpC (hcp1) while hcp2 is located outside of the T6SS operon. In this study, we evaluated the role of hcp genes in E. ictaluri-catfish interaction. Our study revealed roles of evpC and hcp2 in adhesion and invasion of catfish epithelial cells, survival and replication inside catfish peritoneal macrophages, adaptation to the stress factors, and virulence and efficacy in catfish.Edwardsiella ictaluri strain 93–146 (EiWT) and isogenic hcp mutants were grown at 30°C in Brain Heart Infusion (BHI) broth or agar. Escherichia coli (E. coli) CC118λpir, BW19851 (ΔuidA3::pir), and DH5α strains were cultured on Luria–Bertani (LB) agar or broth and incubated at 37°C. Antibiotics were added to the culture medium at the following concentrations: gentamicin (10 μg/ml), ampicillin (100 μg/ml), and colistin (12.5 μg/ml).Bacterial strains and plasmids used in this work were listed in evpC (NT01EI_RS11900) and hcp2 (NT01EI_RS14960) were obtained from the E. ictaluri 93–146 genome (GenBank accession: 95 CP001600)  . The oveach gene . Two ampach gene . The oveEiΔevpC, EiΔhcp2, EiΔevpCΔhcp2, and EiWT were streaked on sheep blood agar plates (Fisher Scientific), which were incubated at 30°C for 48 h. Hemolytic activity of the mutants was visualized using a Stuart Colony Counter with sub-stage illumination (Cole-Parmer).gfplux1 was used to construct bioluminescent EiΔevpC, EiΔhcp2, and EiΔevpCΔhcp2 strains, as described previously (E. coli SM10λpir carrying pAKgfplux1 and mutants were grown overnight and mixed at the ratio of 1:2 (donor: recipient). Mixture pellet was spotted on 0.45 μM filter paper placed on BHI agar and grown at 30°C for 24 h. Filter paper containing a mixture of bacteria was washed with BHI broth containing ampicillin and colistin, and serial dilutions were spread on selective BHI agar containing ampicillin and colistin. Ampicillin resistant mutant colonies carrying pAKgfplux1 appeared on the selective plates after 30°C for 24–48 h.pAKeviously . BrieflyEiΔevpC, EiΔhcp2, and EiΔevpCΔhcp2 strains were exposed to catfish normal serum. Bioluminescent EiWT (positive control) and E. coli DH5α (negative control) were also included in each experiment. Catfish serum was collected as previously described (600) = 1.0] was mixed with the serum and inoculated for 4 h at 30°C. The experiment included four replicates, and bioluminescence was measured by using SpectraMax M5 Multi-Mode Microplate Reader (Molecular Devices). Bioluminescent images were taken using an IVIS Lumina XRMS In Vivo Imaging System Series III (PerkinElmer).Bioluminescent escribed . Then, 1EiΔevpC, EiΔhcp2, and EiΔevpCΔhcp2 (treatments), and one tank for EiWT (positive control). After 1 week of acclimatization, the water level was reduced to 10 L, and 100 ml bacterial culture was added to each tank . Following 1 h incubation, water flow was restored in each tank. Fish were anesthetized with 100 mg/L MS222, and bioluminescence emitted from the fish body was collected for one min by using IVIS Lumina XRMS In Vivo Imaging System Series III (PerkinElmer). Following bioluminescent imaging, fish were transferred to buckets with aerated water for recovery. Bioluminescent imaging was conducted at 0, 6, 12, and 24 h post-infection, and subsequent daily intervals until 14 days.Sixteen specific-pathogen-free (SPF) catfish fingerlings  were obtained from the CVM hatchery and stocked into four tanks (4 fish/tank). Three tanks were assigned to 5 cells) were transferred into a 96-well plate (Evergreen Scientific), and bioluminescent E. ictaluri strains were added at a 1:1 ratio and mixed gently by pipetting up and down. The final volume of the cell-bacteria mixture was 200 μl in each well, and the plate included four replicate wells for each treatment and negative control (cell only). The plate was centrifuged at 1,500 rpm for 5 min at 24°C to compact the cells and bacteria at the bottom. The plate was then incubated for 1 h at 30°C to allow the invasion of catfish peritoneal macrophages by bioluminescent mutants and EiWT. Following the first incubation, the cell-bacteria mixture was centrifuged at 2,000 rpm for 7 min, and the media was removed. After this, CCMM containing 100 μg/ml gentamicin were added, and cells were incubated an additional 1 h at 30°C to kill non-phagocyted E. ictaluri. At the end of incubation, each well was washed three times with PBS, and peritoneal macrophages were suspended in CCMM with 10 μg/ml gentamicin. Finally, cells were transferred to black 96-well-plates (Fisher Scientific), and the plate was placed in Cytation 5 (BioTek) where the cells were incubated for 48 h under 5% CO2 at 30°C. Bioluminescence was captured every hour, and data were analyzed to determine the number of survived bioluminescent bacteria in catfish peritoneal macrophages.The bacterial killing assay was performed as previously described , 31, 32.7 cells ml−1. Bioluminescent mutants and EiWT were mixed with CCO cells at a 1:1 ratio and placed in a 24-well-plate. The cell-bacteria mixture's final volume was 1 ml in each well, and the plate included four replicate wells for each treatment and negative control (no bacteria). The plate was incubated 1 h at 28°C for the attachment of mutants and EiWT to CCO. After that, the cell suspensions were incubated in DMEM containing 100 μg/ml gentamicin for 1 h to kill the external bacteria. The plate was washed with PBS three times, and the invasion of E. ictaluri strains was determined by imaging IVIS Lumina XRMS In Vivo Imaging System Series III (PerkinElmer).Attachment and invasion assays were performed by using channel catfish ovary (CCO) cell line, as described previously . Briefly2O2) (Sigma) and nitrosative stress in sodium nitroprusside (SNP) (Sigma) were tested in BHI (rich medium) and low phosphate minimal medium at pH 5.5 (MM19-P) (600 adjusted to 0.5 for each culture. Five microliter of bacteria from each strain were inoculated into 195 μl of BHI and MM19-P broth containing 0.75 mM H2O2 (diluted from 30% stock solution) and 5 mM SNP. Each 96-well-black plate included three replicates for each mutant and EiWT as a positive control. The mean photon counts for each stress treatment were measured after 4, 8, 12, and 24 h incubation at 30°C by using IVIS Lumina XRMS In Vivo Imaging System Series III (PerkinElmer).The mutants' survival in oxidative stress in hydrogen peroxide (H(MM19-P) . BacteriEiΔevpC, EiΔhcp2, EiΔevpCΔhcp2 (vaccination), EiWT (positive control), and BHI (sham) groups. Each treatment had three replicates. Immersion vaccination was applied by lowering the water level in each tank to 10-L, and by adding 100 ml of bacterial culture . After 1 h, water flow (1 liter/min) was restored to each tank. Mortalities were recorded daily for 21 days, and the percent mortalities were calculated for each group. To assess the protective capabilities of mutants, all fish that survived the EiΔevpC, EiΔhcp2, and EiΔevpCΔhcp2 vaccination were re-challenged with EiWT (2.8 × 107 CFU/ml) 21 days post-vaccination as described above. Fish mortalities were recorded daily, and the experiment was terminated when no fish mortalities were observed for three consecutive days.Vaccination and efficacy were performed as previously described . Brieflyp < 0.05.The significance of the differences between treatment means was established by one-way ANOVA and two-way ANOVA procedures with Tukey's test in SAS for Windows 9.4 . The level of significance for all tests was set at EiΔevpC, EiΔhcp2, and EiΔevpCΔhcp2 was similar to EiWT . In addition to the attachment, invasion of all mutants was reduced, but there were no significant differences compared to EiWT  and EiΔevpCΔhcp2  were completely attenuated in catfish fingerlings in comparison to EiWT  (p < 0.05). Interestingly, EiΔhcp2 caused a severe and rapid death  in catfish fingerlings  and EiΔevpCΔhcp2  were significantly better than EiWT  and EiΔevpCΔhcp2  decreased significantly in comparison to EiWT  (p < 0.05). These mutants showed protection in fry compared to the sham-vaccinated group  was better than that of EiΔevpCΔhcp2 .The immersion challenge was used to evaluate mutants' virulence and efficacy in catfish fingerlings and fry . EiΔevpCgerlings . The prohan EiWT . In catfed group . The prohcp1 (evpC) and hcp2 of T6SS in E. ictaluri virulence in catfish. To achieve this, EiΔevpC, EiΔhcp2, and EiΔevpCΔhcp2 mutants were constructed and persistence in catfish, survival and replication inside catfish peritoneal macrophages, attachment and invasion capabilities in catfish epithelial cells, adaptation and survival to stress factors, and virulence and efficacy in catfish were investigated  are involved in adhesion and invasion, intracellular survival of bacteria, bacterial cytotoxicity, and virulence  and EiΔevpCΔhcp2 (3.6 × 104 photons−1 cm−2 steradian−1) was reached the peak at 48 h post-infection, after which bacterial clearance from catfish was observed. However, bioluminescence from EiΔhcp2 (1.4 × 105 photons−1 cm−2 steradian−1) and EiWT (1.6 × 105 photons−1 cm−2 steradian−1) was gradually increased, even after 48 h post-infection, until the fish dies. Our result showed that EiΔevpC and EiΔevpCΔhcp2 had no mortalities for 14 days, although the bioluminescence quantity of EiΔevpC and EiΔevpCΔhcp2 started to increase earlier than EiΔhcp2 and EiWT. Our bioluminescent imaging data were corroborated with our virulence and efficacy study showing that persistence and replication of EiΔevpC and EiΔevpCΔhcp2 in catfish up to 48 h post-infection and decrease afterward may stimulate catfish an immune response, hence the survival of catfish.The real-time bioluminescent imaging is a quantification method that allows detection  catfish , 41. Thehcp reduced survival of Burkholderia pseudomallei in macrophages  away from evpC . Thus, these two proteins classified in the Hcp protein family may have a different role in E. ictaluri.Although rom evpC . It is pE. ictaluri genome seems to have diverse roles in E. ictaluri pathogenesis. hcp2 is important in adherence to epithelial cells and replication within macrophages. However, evpC plays a crucial role in E. ictaluri virulence in catfish. Therefore, secretion of potential evpC and hcp2 dependent effector proteins via T6SS need more investigation.In conclusion, the two Hcp family proteins found in the The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The animal study was reviewed and approved by Institutional Animal Care and Use Committee at Mississippi State University.SK, AOK, LP, and AK conceived and designed the experiments. SK, HA, and AOK performed the experiments. SK wrote the manuscript. All authors reviewed and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The effectsof bending and hydrogen passivation on the potential terms are discussed.The presented SAIP provides a noticeable improvement in the state-of-the-artdescription of Au–C interfaces. Furthermore, its functionalform is suitable to describe the interfacial interaction between other2D and bulk materials.Wepresent a semi-anisotropicinterfacial potential (SAIP) designedto classically describe the interaction between gold and two-dimensional(2D) carbon allotropes such as graphene, fullerenes, or hydrocarbonmolecules. The potential is able to accurately reproduce dispersion-correcteddensity functional theory (DFT+D3) calculations performed over selectedconfigurations: a flat graphene sheet, a benzene molecule, and a C Gold and graphene are twovery stable materials that can be produced with a high level of crystallinityand cleanness over large surface areas. For these reasons, the gold–grapheneinterface has been the prototypical choice for a large number of casestudies, including diffusing and sliding gold nanoclusters,16 a simple two-body Lennard-Jones (LJ) potential, often expressedin the form VLJ(r) =4ϵ[(σ/r)12 – (σ/r)6], has been so far employed and parametrizedin order to fit observations from specific experimental setups.7 However, the oversimplified nature of the LJ potential, with justϵ and σ setting the two-body dissociation energy and equilibriumlength, respectively, makes it extremely challenging to fit more thana few among the several physical quantities impacting the static anddynamical properties of a gold–graphene assembly. As a consequence,previous studies have employed very different parametrizations tomatch some particular quantities of interest, with ϵ varyingfrom 2.5 to 90.0 meV and σ varying from 2.5 to 3.2 Å,17 making a straightforward comparison amongthese studies often impractical.Despite the vast interest expressed bythe scientific communityin this composite system, to date, no reliable classical force fieldis available for computational simulations involving interfaces ofgold with graphitic systems. Since the interaction between grapheneand metals is mainly governed by van der Waals forces,19 This exemplifies the need for an anisotropic potential to describethe gold–graphene interfacial energy landscape.Beside quantitative errors,even a qualitative description in LJterms is questionable. For a single gold atom residing on a graphenesurface, a typical pairwise isotropic potential , such as the LJ potential,would find the minimum energy position, where the gold atom is locatedover a graphene hexagon center (hollow position). Nevertheless, experimentsand first-principle calculations have demonstrated that the atop position,where the Au atom resides over a C atom, is energetically favorable.20 Extended contacts,on the other hand, were predicted to prefer intermediate misfit angles0 < θ < 30°.21 This, inturn, is expected to be manifested in the interfacial tribologicalproperties, where the combination of the weak Au–C interactionsand the strong internal cohesive forces of gold and graphene may leadto exceptionally low friction coefficients (<10–3)—a condition often referred to as structural superlubricity.23 In superlubric systems, the corrugationenergy (CE)—the energy barrier resisting sliding—candecrease to values much below meV per interface atom. Therefore, studiesaiming at describing superlubricity, often considering a combinationof metals and graphitic materials,26 are criticallysensitive to the chosen experimental or theoretical setup.Beyondthe single-atom contact, when an extended gold–grapheneinterface is considered, the sliding energy landscape becomes muchmore complex due to the intrinsic incommensurability of the contact.In fact, nanoscale graphene flakes and ribbons residing over the (111)surface of gold can be found in both the epitaxially aligned R0 andthe R30 tilted orientations.Ea = 7687.1 mJ/m2 in the case of graphene-covered gold nanoparticles,27 while a pull-off force of Fa = 45.7 ± 5.1 nN was measured by Li et al. for gold-coated atomicforce microscopy probes forming a ∼200 nm2 contactwith a graphite substrate.23 Other experimentsprovide only indirect information based on empirically fitted modelsor simulations.28 The comparison with experiments is furthercomplicated by the presence of surface reconstruction at the goldsurface, usually neglected in first-principles calculations due tothe large supercell size required to encompass its long (∼6.3nm) wavelength. Leaving out such reconstruction effects potentiallyoverestimates the computed interaction energies, but in most cases,this approximation yields only minor structural modifications in themodel systems.29Itis, therefore, evident that there is an urgent need to developa new force field able to reliably describe the interaction betweengraphene and gold. Unfortunately, there is only a handful of experimentalstudies providing data that can be directly employed to parametrizesuch a force field. For example, Torres et al. measured an adhesionenergy (AE) Due to the lackof direct experimental measurements and accuratecomputational reference data for the adhesion and corrugation energiesof graphene–gold interfaces, we performed dispersion correcteddensity functional theory (DFT+D3) calculations on the R30 tiltedgraphene–gold heterojunction. This reference dataset allowedus to parametrize a newly developed semi-anisotropic interfacial potential(SAIP) that is able to simultaneously reproduce the DFT+D3 AE curvesand sliding potential energy surfaces (PES). The developed SAIP andits suggested parameterization present a significant advancement withrespect to the present stand of classical description of the interfacialinteractions in gold and graphene junctions. Furthermore, the SAIPformulation provides a general tool for describing interfaces formedbetween two-dimensional (2D) materials and bulk solids.234 The potential consists of two terms: an isotropic term that describesthe long-range attractive dispersive interactions and an anisotropicterm that describes the Pauli-type repulsion between the grapheneπ electrons and the gold surface electron density. The dispersiveterm treats long-range van der Waals interactions via a C6/r6 LJ type potential, dampenedin the short range with a Fermi–Dirac-type function similarto that introduced in dispersion-corrected DFT calculations to avoiddouble counting of correlation effects35rij is the distance between carbon orhydrogenatom i and gold atom j, d and sR are unit–lessparameters determining the steepness and onset of the short-rangeFermi-type dampening function, and rijeff and Cij6, are the sumof effective atomic radii and the pair-wise dispersion coefficients,respectively. The Tap(rij) function provides a continuous (up to 3rd derivative) long-rangecutoff at rij = Rcut to the potential aiming to reduce computationalburden36The presented SAIP is based on theconcept of anisotropic interlayerpotentials for 2D materials.30 scheme, the anisotropic term of the potential is constructed froma Morse-like exponential isotropic term, multiplied by an anisotropiccorrection, in which the orientation of graphene is described by normalvectors associated with each carbon or hydrogen atomrij) is thecutoff smoothing function of ij andβij set the slope and range of thepotential, and γij sets the widthof the Gaussian decay factors in the anisotropic correction term andthus determines the sensitivity to the transverse distance, ρij, between carbon or hydrogen atom i and gold atom j  serve to calculatethe transverse distance ρij betweenpairs of carbon or hydrogen atom (i) and gold atom(j)Following the Kolmogorov–Crespiom j see 1. C and ni defines the local normal direction to the graphenesheet (or to the benzene molecule) at the position of atom i /(∥rkl∥ ∥rkm∥) [see xy plane, all definitionsof the normal vector should give ni = ẑ, making the specific choice irrelevant.In the case of curved graphene, such as for nanotubes, all normalvector definitions should produce very similar results except forextremely small radii of curvature.37Each normal vector om i see 1. Note tkm∥) see 1a. In ourij. Notably, this assumption is suitable for manybulk material surfaces, for example, for systems possessing s-typevalence orbitals or metallic surfaces, whose valence electrons aremostly delocalized, such that their Pauli repulsions with the electronsof adjacent surfaces are isotropic. Caution should be used in thecase of very small gold contacts, for example, nanoclusters, whereedge effects may become relevant.To accountfor the isotropic nature of the isolated gold atom electroncloud, their corresponding normal vectors are assumed to lie alongthe interatomic vector nj∥rij, one getsρji = 0. The latter simplificationreduces the anisotropic termto the following final formFollowing the above assumption, 33.120 The secondis composed of a benzene molecule residing over three Au(111) layers.DFT spin-polarized calculations were performed by the Perdew–Burke–Ernzerhof(PBE) exchange–correlation functional within the generalizedgradient approximation augmented by Grimme’s D3 long-rangedispersion correction and the Rappe–Rabe–Kaxiras–Joannopoulosultrasoft core-corrected pseudopotentials, as implemented in the QuantumESPRESSO software.41 Previous benchmark DFT calculations of metal–organic frameworksindicate that the PBE exchange correlation density functional approximationwith the D3 dispersion correction can accurately describe the energeticsof complex systems involving organic and inorganic components.43 However, whether this is thebest DFT approach  for the system under considerationcannot currently be concluded mainly due to the lack of experimentalresults or high-accuracy computational reference data.As model systems forthe parameterization process, we choose the two interfaces depictedin k-points for the Au(111)–grapheneinterface and 3 × 3 × 1 k-points for theAu(111)/benzene system (see below). Convergence of total energy overthe above cutoffs and k-points grid was established.Periodic boundary conditions were applied in all directions. In allthe calculations, graphene or benzene were kept flat15 and the gold atoms were maintained in the fcc bulk configuration.Kinetic-energy cutoffs of 600 eV for the wavefunctions and 4952eV for the density were employed, with a reciprocal-space mesh of7 × 7 × 1 a = b = 4.965638Å, c = 30 Å, α = β = 90°,and γ = 60° /benzeneand Au(111)/C60 cases, we have employed a supercell with a = b = 14.896914 Å, c = 30 Å, α = β = 90°, and γ = 60°–graphene case, the lateral stress was minimizedby iteratively rescaling the system size. The final supercell vectorlength and angles were  60° see 2a,b. The 60°see 2c,d.Equ3.2M = Mb + Ms DFT datasetsincluding Mb AE curves and Ms sliding PESs. The AE curves were calculated for fivehigh-symmetry stacking modes  distance. The slidingPESs were obtained at a fixed vertical (z) distanceof 3.5 Å, by rigidly shifting the adsorbate along the lateral(x–y) directions with respectto the gold substrate. The single-point total energy at each of the441 points of a uniform mesh grid was recorded. The origin  configurationsof the PESs correspond to those presented in The parameters ofthe SAIP were determined against reference 2 isthe Euclidean norm (2-norm) that measures the difference between theSAIP predictions and the DFT reference data, ξ represents theset of potential parameters, and Emband Ems represent the Mb AE curves and Ms slidingPES data sets, respectively. wmb and wms are the corresponding weighting coefficients.The reference DFT interfacial energies, Emb,DFT and Ems,DFT, are obtainedas follows: for any given configuration m of theheterostructure, the total energy is first obtained from DFT+D3 calculation: EmDFT,total. Then, the energies of the isolatedgraphene, EmDFT,graphene, and of isolatedgold, EmDFT,gold, are computedseparately using the same cell as that of the composite system. TheDFT interfacial energy appearing in Optimal SAIP parameters were obtained by minimizing the followingobjective function that quantifies the difference between the DFTreference data and the potential predictionswmb = 1 ,and those of the sliding PESs were chosen as 45 , the energy weights for the AE curves were setto  in refs  and 37)wmb = 1  has to be applied alonga distance of ≃33 nm, way beyond the interlayer interactionrange. This indicates a discrepancy between the two experimentallymeasured values. We note that our calculated AE of about 0.5 eV/cell(375 mJ/m2) obtained for the trilayer gold–grapheneinterface model 15 and 394 mJ/m2 recently calculated for the Cu(111)–graphene interface50 that is also dominated by vdW interactions.As mentioned earlier, comparison with experimentis challenging due to the limited availability of measured AE valuesfor the graphene–gold interface. Specifically, Torres et al.found an AE of ce model 3a is low22 We wouldlike to note that we obtain CE and AE values using energy differencesof nearly identical systems, thus we expect a very high numericalaccuracy. Furthermore, in the case of rigid contacts considered herein,such small CE ensues from the approximation employed to describe thelattice mismatch between gold and graphene. A truly incommensuratelattice mismatch, only obtainable in the thermodynamic limit of aninfinite supercell size, would in fact yield a vanishing CE regardlessof the potential parameters.The sliding PESs calculated using the SAIP parametrization showgood agreement with the reference DFT+D3 data see 4, as wel6 Notably, an LJ fittingof the AE curves results in substantially different values, ϵ= 8.50 meV and σ = 3.37 Å, confirming that the isotropicLJ description is incapable of simultaneously describing the bindingand sliding physics of vdW interfaces.An LJ fittingof the DFT+D3tri-layer gold–graphene PES resulted in ϵ = 14.86 meVand σ = 3.8 Å, in agreement with previous semi-empiricalLJ parameterizations used to reproduce experimental results of goldcluster diffusion on graphene.4.251Going beyond theperiodic interface, we next consider the case of a benzene moleculeresiding atop a gold surface. C6 coefficient and a larger d value than those of the corresponding Au–C parameters, suggestinga weaker and shorter-range dispersion term for the former. Furthermore,the Au–H anisotropic repulsive term  of the adjacent surfaces,they should be captured by the SAIP with the parameterization presentedin 60 fullerene physisorbed over an Au(111)surface in two different orientations, marked as α and β,as depicted in 60 AE islarger than that of benzene by about 80%, thus supporting the needto provide a separate parameterization for curved systems. This isin agreement with previous calculations indicating an increased reactionenergy for nanotubes of a smaller curvature radius.52 The increased surface reactivity is manifested also inthe equilibrium distance from the gold surface, which reduces from3.48 Å for flat graphene to 2.6 and 2.8 Å for C60 at the α and β configurations, respectively , indicating that the Au–C interaction is weakly dependenton shear motion in this highly curved system and that the slidingenergy surface corrugation is expected to be very small. Similar considerationscould be made in the case of very small-diameter carbon nanotubes(CNTs) on gold, for which 54 ispresently under investigation.Finally, wechallenge our developed SAIP against extremely bent graphitic systems.In this case, one might expect significant deviations of the interfacialenergy profiles from the ideally flat graphene case discussed above.In particular, it is known that mechanical bending can alter the reactivityof graphene sheetsvely see 6e. The s560–goldinteractions, as obtained from DFT+D3 calculations. While the functionalform of the SAIP is suitable to treat many interfaces between graphiticsystems and gold surfaces, system specific parametrizations of theSAIP are recommended in order to obtain optimal accuracy. However,the presented potential is expected to describe the structural anddynamical response to external forces of a large number of prototypicalsystems, such as gold nanoclusters on graphite,4 graphenenanoribbons on Au(111),9 and C60 on Au(111),56 among others, with much improved accuracy with respect to previousclassical models. Furthermore, our formulation can be generalizedto describe a wide variety of interfaces between hexagonal 2D materialsand bulk solids, such as MoS2/Au,25 h-BN/Au,26 or graphene/Ag.15 This, in turn, will considerably increase the scope ofmaterial interfaces that can be treated using reliable dedicated classicalforce-fields.The results presented above indicate that the proposed semi-anisotropicinterface potential (SAIP) is able to accurately reproduce the energeticsof graphene–gold, benzene–gold, and C


Paleoplatyura Meunier, 1899, is recorded from Burmese amber for the first time. This represents a rather rare case of the presence of an extant insect genus in the Mesozoic. Three new species of Paleoplatyura are described, indicating that this genus was relatively diverse already in the Cretaceous.Burmese amber is very rich in perfectly preserved insects. Consequently, it is an invaluable source of information for taxonomic and evolutionary studies. Moreover, it forms a unique connection between the Jurassic and Cretaceousfaunas and documents the first representatives of modern genera. In this paper, a primitive genus of Keroplatidae, Paleoplatyura Meunier, 1899, i.e., Paleoplatyura agnieszkae sp. nov., P. miae sp. nov., and P. magnifica sp. nov., are described and figured. The concept of the genus is briefly discussed, and its systematic position is clarified. A key to fossil species is provided. The genus Paleoplatyura is described from the Eocene Baltic amber. It is concluded that, in Baltic amber, this group is represented only by the type species, and the identity of the other two species is problematic. No additional specimens have been found so far in this amber. Therefore, the presence of as many as three new species in Burmese amber, certainly belonging to Paleoplatyura, is a confirmation of its occurrence already in the Mesozoic. Three new species of  Paleoplatyura Meunier, 1899 [Paleoplatyura shows the most plesiomorphic wing venation among fungus gnats (Sciaroidea) and can be considered as a “living fossil” [Platyura marginata Meigen, 1803 [Platyura pectoralis Coquillett, 1895 [Paleoplatyura melanderi Fisher, 1941 [Paleoplatyura), while Paleoplatyura johnsoni Johannsen, 1910 [The fossil record of the family Keroplatidae (Diptera: Bibionomorpha) is still relatively scarce, especially from the Mesozoic, with only several taxa formally described ,2. This er, 1899  ; althouer, 1899 ) pointed fossil” . Mantič  fossil” , for theen, 1803 , and thett, 1895 , and Paler, 1941   eocenica Cockerell, 1921 [P. aldrichii, P. melanderi, P. loewi and P. eocenica in the genus Paleoplatyura is questionable and most probably wrong. The holotype of P. aldrichii is probably lost [P. johnsoni” , indicating that P. aldrichii probably does not belong to true Paleoplatyura. Concerning P. melanderi, Mantič et al. [Paleoplatyura, because of R2+3 ending in R1 and its considerable molecular distance from P. johnsoni. Additionally, P. loewi does not belong to Paleoplatyura, because this species lacks the transverse r-m vein characteristic for this genus and possesses the more apomorphic r-m fusion, typical of most keroplatids. This can be seen exactly in the drawing of the wing given in the work of Meunier [1+2 merges at some distance with the vein Rs. The identity of P. eocenica is most obscure, because nothing important can be inferred from the original description and it is even unclear if the species belongs to Keroplatidae. Cockerell [Paleoplatyura, providing a question mark after the genus name in the original description. The genus en, 1909 ; P. johnen, 1910 , and P. er, 1941 , and threw, 1850 ), P. loebly lost , and thebly lost  is very bly lost  mentionePaleoplatyura and describe three new Cretaceous species of this remarkable keroplatid genus. In this paper, we aim to clarify the taxonomic concept of the genus Specimens were examined using a Nikon  SMZ25 stereomicroscope, equipped with a Nikon DS-Ri2 digital camera. Photomicrographs are focus stacks captured using this system and processed using NIS-Elements Imaging Software . Line drawings were produced by tracing photographs. The terminology follows Ševčík et al. , where tThis published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The LSID for this publication is: urn:lsid:zoobank.org:pub:90A627F2-9ECB-4ACE-81FC-9AC006D90656. Order Diptera Linnaeus, 1758 .Infraorder Bibionomorpha Hennig, 1948 .Superfamily Sciaroidea Billberg, 1820 .Family Keroplatidae Rondani, 1856 .Paleoplatyura Meunier, 1899 [Genus Mycetobia macrocera Loew, 1850 = Paleoplatyura macrocera  [Type species: w, 1850) .P. johnsoni, and four fossil species, i.e., P. macrocera; P. miae sp. nov., P. magnifica sp. nov., P. agnieszkae sp. nov.The genus includes one extant Holarctic species, 1+2 and Cu; R2+3 oblique and ending in C, anal vein strong and reaching wing margin; gonostylus narrow and apically bifurcated.Diagnosis: Cross-vein r-m present; basal part of Mb clearly visible; cross-vein m-cu situated in between MPaleoplatyura macrocera.Mycetobia macrocera Loew, 1850—Baltic amber (about 42 MA).1; R4+5 almost 3× longer than Rs and almost equal in length to R2+3+4+5; cross-vein r-m short; M1 almost 4× longer than M1+2; m-cu distinctly beyond fork of Mb.Amended diagnosis: Antennae reach almost 2/3 of the wing length, probably 16 segmented; wing 2.5× longer than wide; Sc vein ends distinctly before Rb forks into Rs and RP. macrocera is the type species of the genus Paleoplatyura. It was described on the basis of a single female inclusion in the Baltic amber. Unfortunately, the study is made more difficult by the missing holotype and the fact that despite the analysis of numerous specimens classified to Sciaroidea in various Baltic amber collections, no specimen belonging to this species was found. Therefore, the concept of the genus is based on the figure provided in the work of Meunier , No. MP/4288, Burmese amber; deposited in the collection of ISEA PAS.1 and Rs; R1 ends opposite half the length of R2+3, near tip of R2+3; R2+3 distinctly waved at mid-length; R4+5 more than 3× longer than Rs and 2× longer than R2+3+4; r-m short, equals ⅕ of length of Rs, located in ⅓ length of M1+2; Mb present, distinctly visible; M1 about 2.5× longer than M1+2; m-cu just beyond fork of Mb, located between M3+4 and Cu; Cu slightly arched; pseudovein (ps) clearly visible; A1 visible only in basal part .Material examined: Holotype , No. MP/4075, Burmese amber; deposited in the collection of the ISEA PAS.1 and Rs; approximately opposite ⅓ of length of Rs; R1 ends opposite half the length of R2+3, near tip of R2+3; R2+3 nearly straight; R4+5 more than 3× longer than Rs and almost 2× longer than R2+3+4; r-m short, equals ⅙ of length of Rs, located distinctively before middle of length of M1+2; Mb present, clearly visible; M1 more than 4× longer than M1+2; m-cu just beyond fork of Mb, located between M3+4 and Cu; Cu at end strongly waved; pseudovein (ps) clearly visible; A1 with apical half strongly bent to wing margin .Etymology: The specific epithet is given to emphasize the large size of the specimen , No. MP/4076—Burmese amber; deposited in the collection of the ISEA PAS.1 and Rs, opposite ⅔ of length of Rs; R1 ends opposite half the length of R2+3, near the tip of R2+3; R2+3 distinctly waved at basal part; R4+5 more than 3× longer than Rs and about 2× longer than R2+3+4; r-m short, equals ⅛ of length of Rs, located close to middle of length of M1+2; Mb present, clearly visible; M1 3× longer than M1+2; m-cu more than its own length beyond fork of Mb, situated between M3+4 and Cu; Cu at end strongly bent; pseudovein (ps) clearly visible; A1 slightly wavy at the middle of its length 1. Sc ends before Rb forks into R1 and and Rs ………………………………… 2  - Sc ends after Rb forks into RP. miae sp. nov.2. Pattern of tibial spurs 1:1:2 …………………………………………   - Pattern of tibial spurs 1:2:2 ……………………………………… ………… 34+5 and M1; tibial spurs of same length; apical processes strongly sclerotized ………… P. magnifica sp. nov.3. Costa produced to less than ¼ of the distance between tips of R4+5 and M1; tibial spurs of different length; gonostylus narrow throughout its length; apical processes not sclerotized ……………………………………………………… P. agnieszkae sp. nov.  - Costa produced to about ¼ of the distance between tips of RPaleoplatyura, P. macrocera, from Baltic amber, differs from all congeners in Burmese amber by the short vein Sc, which ends clearly before the bifurcation of Rb into R1 and Rs. Surprisingly, the Burmese amber species of Paleoplatyura are thus more similar in this respect to the extant species P. johnsoni than to the Baltic amber type species. However, the relative length of Sc also differs among various species of Macrocera Meigen, 1803 [Paleoplatyura as defined in this paper; which means including intrageneric variation of the length of Sc, considering the well-known fact that higher taxonomic categories, like genus or subfamily, are usually more or less subjective, and their concept (breadth) may differ, even within one family. The subjective concept of genera, with different breadth defined by various authors, may also be a key to the understanding why Mesozoic genera, such as Paleoplatyura, are (seemingly) present in both the Tertiary and extant fauna.The type species of the genus en, 1803 , and simApalocnemis canadambris Grimaldi and Cumming [Antocha lapra Podenas and Poinar [Helius lebanensis Kania, Krzemiński and Azar [Nemopalpus quadrispiculatus Stebner et al. [Phlebotomus vetus Stebner et al. [Elater Linnaeus [Protanyderus Handlirsch, 1909 (family Tanyderidae), in the Upper Jurassic of Mongolia [The presence of extant genera in the Mesozoic fauna is a remarkable phenomenon itself. Recent studies of Burmese amber inclusions increasingly indicate the origin of modern genera as early as in the Cretaceous. Examples of extant insect genera found in the Mesozoic are well documented, though not common. Grimaldi and Cumming  stated t Cumming  . It is  studies .Paleoplatyura are found in the genus Asynaphleba Matile, 1974 [Paleoplatyura only by the absence of the vein Mb and by shorter anal vein, not reaching the wing margin. Thus, it seems that the presence of both Mb and the cross-vein r-m are good diagnostic characters for the genus Paleoplatyura. Taking into consideration only a distinct vein Mb, as a clear plesiomorphic character state, it is not unique to Paleoplatyura within keroplatids, but it is present also in several genera of Macrocerinae, including the species-rich genus Macrocera, and also in the South African genus Schizocyttara Matile, 1974 [Paleoplatyura johnsoni by Mantič et al. [Arachnocampa Edwards, 1924 [Paleoplatyura in some other characters, such as the absence of R2+3 and different proportion of wing veins.A similar wing venation and structure of the male terminalia as in le, 1974 , containle, 1974 , which wds, 1924 , from thPaleoplatyura represents one of the most ancient and plesiomorphic genera of Keroplatidae, with the most complete wing venation representing a ground plan within the family (together with genus Arachnocampa). This group possesses also the most plesiomorphic structure of male terminalia, which appears to be simple with long and apically forked gonostyli. Molecular data indicate a close relationship of Paleoplatyura with Macrocerinae [Paleoplatyura with that of Macrocera, we can see many similarities, confirming the evolutionary trend to the reduction of radio-medial cross-vein to the so-called radio-medial fusion, typical of most keroplatids, together with prolongation of the antennae in Macrocera and elimination of some wing veins in Lygistorrhininae. The basal part of medial vein is still retained in most Macrocerinae, while it is absent in almost all species of the more apomorphic subfamilies of Keroplatinae and Lygistorrhininae. The overall structure of male terminalia is also very similar in Paleoplatyura and most Macrocerinae.Genus ocerinae ; additio


Diabetic patients with liver cirrhosis had higher Aβ42 levels , which is in accordance with previous reports. Present analysis showed that the cirrhotic liver does not eliminate Aβ42. Further studies are needed to explore the association of liver cirrhosis, Aβ42 levels, and cognitive dysfunction.Amyloid-beta (Aβ) deposition in the brain is the main pathological hallmark of Alzheimer disease. Peripheral clearance of Aβ may possibly also lower brain levels. Recent evidence suggested that hepatic clearance of Aβ42 is impaired in liver cirrhosis. To further test this hypothesis, serum Aβ42 was measured by ELISA in portal venous serum (PVS), systemic venous serum (SVS), and hepatic venous serum (HVS) of 20 patients with liver cirrhosis. Mean Aβ42 level was 24.7 ± 20.4 pg/mL in PVS, 21.2 ± 16.7 pg/mL in HVS, and 19.2 ± 11.7 pg/mL in SVS. Similar levels in the three blood compartments suggested that the cirrhotic liver does not clear Aβ42. Aβ42 was neither associated with the model of end-stage liver disease score nor the Child–Pugh score. Patients with abnormal creatinine or bilirubin levels or prolonged prothrombin time did not display higher Aβ42 levels. Patients with massive ascites and patients with large varices had serum Aβ42 levels similar to patients without these complications. Serum Aβ42 was negatively associated with connective tissue growth factor levels (r = −0.580,  Amyloid-beta (Aβ) peptides of variable length are produced by proteolysis of amyloid precursor protein (APP). The two most common isoforms of Aβ are 40 and 42 amino acid peptides . CerebraRecent evidence showed that the kidney, the skin, the gastrointestinal tract, and the liver contribute to peripheral Aβ clearance . Aβ was A separate study showed that Aβ levels in axillary lymph nodes of Alzheimer transgenic mice were as high as its brain concentrations and assumed that brain-derived Aβ40 and Aβ42 are cleared by lymphoid tissues. The mice used in this study expressed a human isoform of APP with the Swedish mutation, which causes high levels of Aβ and early onset of Alzheimer disease. Interestingly, Aβ40 and Aβ42 levels in the liver and kidney of these mice were hardly detectable .Hepatic catabolism of Aβ42 was demonstrated in mice, and about 60% of the radiolabeled Aβ peptides accumulated in the liver . Aβ42 pr® analysis detected 2.5–7.5 pg/mL Aβ42 levels, which declined about 10 fold in the cirrhotic liver [Impaired hepatic removal of blood Aβ42 can explain high plasma Aβ42 levels in patients with liver cirrhosis . This maic liver . Of noteic liver . Studiesic liver . Whetheric liver ,15.It is important to note that Aβ42 exerts protective functions in the liver. Uptake of Aβ42 peptides by murine hepatic stellate cells suppressed the expression of fibrotic proteins such as transforming growth factor beta (TGFbeta) . TGFbetaAβ further induced endothelial nitric oxide (NO) synthase in human SV40 immortalized hepatic sinusoidal endothelial cells . NO is aThere is some evidence that patients with chronic liver diseases have an increased risk for Alzheimer disease . Non-alcAnalysis of protein levels in portal venous serum (PVS), hepatic venous serum (HVS), and systemic venous serum (SVS) gives some information about hepatic clearance and synthesis of different proteins and metabolites . The livAn increase of sCD163, which is almost exclusively produced by macrophages, from the portal to the hepatic vein showed that liver macrophages released this protein ,34. PlasResistin in humans is also mainly released by macrophages, and serum levels were induced in liver cirrhosis. Resistin concentrations did, however, not differ between SVS, HVS, and PVS, suggesting that higher serum levels in liver cirrhosis are not a marker of Kupffer cell activation and may be related to the dysfunction of monocytes/macrophages in liver cirrhosis .Chemerin is abundantly expressed in the liver and levels were higher in the hepatic than the portal vein ,37,38. SVisfatin is an inflammatory and pro-fibrotic protein, and levels were higher in HVS and PVS when compared to SVS. Whether serum visfatin is changed in patients with liver cirrhosis or is related to liver disease severity has not been finally clarified .Chronic inflammation contributes to the pathogenesis of Alzheimer disease and liver cirrhosis and, thereby, may link liver diseases with cognitive impairment ,35. C-reHere, HVS, PVS, and SVS of patients with liver cirrhosis were used to measure Aβ42. Only a little serum was available, and either Aβ40 or Aβ42 could be analyzed. Regarding that systemic Aβ42 is more strongly related to cognitive status , and thaIt was hypothesized that serum Aβ42 is lower in HVS than PVS because of hepatic extraction. Clearance of Aβ42 by the liver may also contribute to lower serum levels. Moreover, it was postulated that Aβ42 levels increase in patients with more severe liver disease because of impaired removal by the injured liver.Twenty patients with clinically diagnosed liver cirrhosis were included in the study. Etiology of liver disease was alcoholic in 18 and hepatitis C infection in two patients. Patients were treated by TIPS implantation due to complications of liver cirrhosis. This procedure has been described earlier, and TIPS was inserted in the fasted state . During Standard laboratory values , aspartate aminotransferase (AST), or albumin) were measured by the Institute for Clinical Chemistry and Laboratory Medicine at our hospital. The study complied with the Declaration of Helsinki. All patients gave written, informed consent and the study was approved by the ethical committee of the University Hospital of Regensburg.G*Power3.1.6 analysis using the values of plasma Aβ42 in patients with cirrhosis (excluding patients with hepatitis B virus) and healthy controls published by Wang et al.  indicateHuman Amyloid β (aa1-42) Immunoassay was from R&D Systems . Serum was used undiluted. All of the blood samples were analyzed at one day in parallel. Therefore, inter-assay coefficient of variation (CV) could not be calculated. Intra-assay CV for samples with Aβ42 < 10 pg/mL was 12.4%, for samples with Aβ42 < 20 pg/mL was 12.3%, for samples with Aβ42 < 30 pg/mL was 8.1%, and for samples with Aβ42 < 100 pg/mL was 7.2%. The lowest value of the standard curve was 7.8 pg/mL, and almost all serum levels were greater. Median levels of Aβ42 were, indeed, about 20 pg/mL and were nearly three-fold higher than the lowest standard. Therefore, 50% of the analyzed sera had Aβ42 levels above the concentration of the second standard (15.6 pg/mL).Interleukin (IL)-6, connective tissue growth factor (CTGF), chemerin, resistin, visfatin, arginine, and asymmetric dimethylarginine (ADMA) were already measured in patients with liver cirrhosis, and results were published ,31,36,45Standards and samples were measured in duplicate, and the means were used for statistical analyses.t-test was used for calculation. Spearman correlations were analyzed by the IBM SPSS Statistics software.Data are shown as box plots . Shapiro–Wilk test showed that Aβ42 and chemerin were not normally distributed in the different blood compartments. Statistical differences were, therefore, calculated by non-parametric tests, namely the Mann–Whitney U Test, the Kruskal–Wallis Test, the Wilcoxon Test, or Friedman test (IBM SPSS Statistics 26.0). IL-6 was normally distributed, and paired Serum of 20 patients suffering from clinically diagnosed liver cirrhosis was available for this study. Patients’ characteristics are given in t-test; Aβ42 levels were similar in SVS, HVS, and PVS . Median Aβ42 level in SVS of the two patients infected with hepatitis C virus was 16.1 pg/mL and was 19.7 pg/mL in the patients with alcoholic cirrhosis, suggesting that serum Aβ42 was not changed much by hepatitis C infection.It has to be noted that systemic Aβ42 levels did not correlate with patients’ ages  of 0.702, suggesting that about 70% of HVS chemerin can be explained by its PVS levels. R2 for PVS and HVS Aβ42 was 0.347, and about 65% of the variability in HVS must be related to other factors than the Aβ42 PVS levels [In general, there is a very good correlation between the metabolite concentrations in SVS, HVS, and PVS . This waS levels .The calculation of the Child–Pugh score uses bilirubin, albumin, international normalized ratio (INR), ascites, and encephalopathy. The model for end-stage liver disease (MELD) score is calculated from bilirubin, INR, and creatinine . Levels p = 0.047) but not in PVS (p = 0.069) and SVS (p = 0.181)  did not differ between diabetic and non-diabetic patients.Type 2 diabetes increases the risk for Alzheimer disease ,48. In oSystemic inflammation contributes to Alzheimer disease ,50. HoweThere was evidence that Aβ42 induced endothelial NO synthase production in the liver . Serum ap = 0.283) or PVS  levels of these proteins were not correlated. Of note, HVS/PVS Aβ42 ratio negatively correlated with CTGF in PVS , HVS , and SVS .Recent studies showed anti-fibrotic effects of Aβ42 . CTGF wap = 0.358 for SVS; r =−0.77, p = 0.747 for HVS; r = −0.311, p = 0.182 for PVS). Esophageal varices and ascites are common complications of portal hypertension [Increased splanchnic and reduced hepatic NO production in liver cirrhosis contribute to portal hypertension . Aβ42 dirtension . SystemiThis study showed that serum Aβ42 levels were similar in portal, hepatic, and systemic serum and were not related to measures of liver function in patients with liver cirrhosis.Impaired elimination of Aβ has been proposed to contribute to elevated serum and brain levels, and thereby progresses in Alzheimer disease ,56. ReceWhatever the underlying mechanisms are, the present findings indicated that levels of Aβ42 were not significantly reduced when passing the cirrhotic liver. Impaired liver function is associated with a diminished elimination of cytokines such as IL-6, and, accordingly, IL-6 levels increased with higher Child–Pugh score ,57. Aβ42It may well be that the healthy liver contributes to Aβ42 elimination. It is, however, difficult to obtain HVS and PVS blood from liver healthy donors. Wang et al. showed increased plasma Aβ42 levels in patients with liver cirrhosis in comparison to healthy controls . This inIt may well be that hepatic Aβ42 release is enhanced in patients with liver cirrhosis, and this may also reduce levels in the liver tissues and increase systemic concentrations of this peptide ,13.In an experimental murine NAFLD model, plasma Aβ42 even declined, and analysis of hepatic pathways involved in Aβ synthesis, catabolism, and clearance revealed that all of them were reduced in NAFLD . Beta-seAβ42 levels were about three fold higher in the cirrhosis patients studied by Wang et al. in comparison to our cohort. About 30% of their patients had chronic hepatitis B (HBV) . Plasma The study by Wang et al. described associations of plasma Aβ42 with bilirubin, albumin, and AST concentrations . The corHigher age is a risk factor for liver cirrhosis and Alzheimer disease ,35. Aβ42Liver cirrhosis is often associated with chronic inflammation, and various cytokines were found induced in serum of these patients . Plasma There is evidence that Aβ42 is increased in obesity and diabetes. Obese mice had elevated plasma Aβ42 levels . In diabAβ42 was also shown to impair endothelium-dependent and -independent vasodilation and was associated with a lower NO bioavailability . In the An interesting finding was the negative association of serum CTGF with Aβ42 levels. Serum CTGF was highest in patients with ongoing fibrogenesis . Transfo® Aβ42 Advantage Kit, is a further limitation of this study. As mentioned above, Aβ42 levels detected in serum of our study cohort were comparable to the levels described in a previous analysis [The main limitation of this study is that only 20 patients were included. Thus, it was not possible to prove small differences. The use of an ELISA, instead of a highly sensitive technique such as the Single Molecule Array (Simoa)analysis . Moreoveanalysis . BecauseThe present analysis showed that there is no net effect of the cirrhotic liver on Aβ42 levels in the circulation. Further studies are needed to explore the association of Aβ42 levels, ongoing fibrogenesis, and cognitive dysfunction in patients with liver cirrhosis.