Ghrelin has been shown to alleviate neuropathic pain by inhibiting the release of proinflammatory cytokines. The purpose of this study was to investigate the role of GSK-3β/β-catenin signaling in mediating the effect of ghrelin on neuropathic pain and to understand the associated mechanisms. Chronic constriction injury (CCI) of the sciatic nerve was used to establish a rat model of neuropathic pain. Hyperalgesia and allodynia were evaluated by observing the mechanical withdrawal threshold and the thermal withdrawal latency. Wnt3a and β-catenin protein expression and GSK-3β phosphorylation were detected by western blotting analysis. The levels of tumor necrosis factor-α and IL-1β were determined using an enzyme-linked immunosorbent assay. In addition, we used immunohistochemical analysis to determine the levels of GSK-3β phosphorylation in the dorsal horn of the spinal cord. Intrathecal delivery of ghrelin effectively ameliorated CCI-induced mechanical allodynia and thermal hyperalgesia at 7 and 14 days and reduced the levels of tumor necrosis factor-α. Ghrelin inhibited CCI-induced GSK-3β activation and β-catenin overexpression in the spinal dorsal horn. Moreover, intrathecal injection of ghrelin suppressed the activation of GSK-3β in the spinal dorsal horn of CCI rats, as assessed by immunohistochemical analysis. Our data indicated that ghrelin could markedly alleviate neuropathic pain by inhibiting the expression of β-catenin, via the suppression of GSK-3β activation, in the spinal cord of CCI rats. NP morbidity is currently up to 7–8% and it imparts a heavy economic and psychological burden to patients3. However, currently available medication fails to adequately control the pain and is accompanied by various side effects3. A lack of knowledge of the exact mechanisms of NP is the leading cause of the inefficacy of these medications and therefore, it is important to clarify these mechanisms. Various cell signaling pathways, such as Wnt signaling pathway, have been shown to play critical roles in regulating NP.Neuropathic pain (NP), characterized by spontaneous pain, hyperalgesia, and allodynia, is a type of chronic pain defined as “pain caused by a lesion or disease of the somatosensory system”4. Wnt signaling consists of the canonical pathway (β-catenin–dependent) and non-canonical pathways (β-catenin–independent), such as the Wnt/JNK pathway7. Song et al. first demonstrated that the Wnt/β-catenin signaling pathway, both in the periphery and in the central nervous system, plays a pivotal role in the induction and maintenance of NP caused by nerve injury5. Subsequently, many studies have confirmed this effect, which may occur by regulating inflammation, synaptic plasticity, and neuronal excitability10.Wnt proteins are a family of secreted lipid-modified proteins. Wnt3a has been shown to be a stimulant of the Wnt/β-catenin signaling pathway12. Emerging evidence from recent studies suggests that GHSR is also expressed in some regions involved in controlling pain transmission, such as the hypothalamic arcuate nucleus, ventricular hypothalamic nucleus, hypophysis, hippocampus, and spinal cord13. These findings suggest that ghrelin may play an important role in pain regulation. Extensive research has shown that ghrelin is associated with anti-nociceptive activity and anti-inflammatory properties in inflammatory pain and NP by regulating the opium system, inflammatory factors, and oxidative stress19. However, the exact mechanism is not entirely clear.Ghrelin, which was first isolated from the rat stomach by Kojima in 1999, has a plethora of biological effects by interacting with growth hormone secretagogue receptor (GHSR). These effects include promoting the release of growth hormone and gastric acid and increasing insulin sensitivity23. Nevertheless, the relationship between NP, ghrelin, and the GSK-3β/β-catenin signaling pathway has not been reported. Hence, we hypothesized that ghrelin may mitigate NP by regulating the activation of GSK-3β and consequently, the Wnt3a/β-catenin signaling pathway.Glycogen synthase kinase-3β (GSK-3β) is one of the limiting enzymes of glycogen synthesis. It participates in various cellular signaling pathways and is also a contributing factor in regulating these pathways, including the Wnt3a, NF-κB, and MAPK pathways. There is a growing body of literature showing that ghrelin has a neuroprotective effect in various animal models of neurodegenerative disease by regulating GSK-3βTo test this hypothesis, we examined the effect of ghrelin in a rat model of CCI, focusing on behavioral assays. By employing western blotting and immunohistochemistry, we aimed to detect changes in GSK-3β and the Wnt3a/β-catenin signaling pathway.p > 0.05, n = 5). In the CCI group, the ipsilateral TWL significantly decreased at day 3 and the MWT significantly decreased at day 1 , but the contralateral side showed no change compared with the sham group and the normal control rats. This increased sensitivity to mechanical and thermal stimuli was sustained until at least day 14 after CCI.As shown in Fig. p = 0.05, n = 5) and these effects lasted until 14 days after CCI surgery. Neither CCI nor treatment with ghrelin affected the MWT or the TWL on the contralateral side  daily from day 1 to day 7 after CCI. Continuous intrathecal injection of ghrelin for 7 days significantly increased the TWL and MWT on the ipsilateral side compared with CCI group . However, ghrelin treatment significantly counteracted this change . The values of these semiquantitative measurements are expressed as ratios of pGSK-3β to GSK-3β.As shown in Fig. To investigate the levels of pGSK-3β in the spinal dorsal horns of CCI rats, we also immunostained spinal cord sections with anti-pGSK-3β antibodies. As shown in Fig. p < 0.05, n = 5 per group). After continuing the intrathecal injection of ghrelin for 7 days, the expression levels of β-catenin decreased significantly . However, the protein expression levels of Wnt3a were stable after ghrelin injection. These semiquantitative measurements were expressed as the ratios of β-catenin and Wnt3a to GAPDH.To confirm the change in the Wnt/β-catenin signaling pathway after CCI and intrathecal injection of ghrelin, we investigated the protein expression levels of Wnt3a and β-catenin in rat spinal dorsal horn tissues by western blotting analysis. As shown in Figs 17. However, little is known about the effect of ghrelin on NP. Although there are some studies reporting that ghrelin relieves NP due to its potent anti-inflammatory effects and regulates the endogenous opioid system25, the underlying mechanism remains unknown. To confirm that ghrelin is involved in NP, we assessed the MWT and TWL in CCI rats after intrathecal injection of ghrelin. Our results showed that after CCI, both the MWT and TWL of ipsilateral hind paws significantly decreased and intrathecal injection of ghrelin for 7 days clearly ameliorated the mechanical allodynia and thermal hyperalgesia.NP is a serious and refractory disease, but the mechanisms involved remain obscure. Previous research has found that ghrelin relieves inflammatory pain by regulating the release of inflammatory factors produced during central and peripheral inflammatory processes7. Activation of the Wnt3a/β-catenin signaling pathway results in the upregulation of many inflammatory factors, such as TNF-α and IL-1β5, whereas blocking Wnt3a/β-catenin signaling reverses this effect. In addition, Wnt3a/β-catenin signaling pathway is closely linked with the activation of astrocytes and microglial cells, which is important in NP7. Therefore, we hypothesized that ghrelin could alleviate NP by suppressing the Wnt3a/β-catenin signaling pathway. To test this hypothesis, we investigated changes in the levels of Wnt3a and β-catenin. The results of the present study showed that intrathecal injection of ghrelin suppressed the expression of β-catenin and this effect lasted for 14 days. However, we found that ghrelin had no impact on Wnt3a.The Wnt3a/β-catenin signaling pathway has been shown to be necessary for the development and maintenance of NP26. Phosphorylation at Ser9 prevents the binding of GSK-3β to its substrate, thereby inhibiting the activation of GSK-3β. Previous studies have suggested that a destructive compound is formed by the assembly of pGSK-3β, APC, axin, and β-catenin, and that this compound degrades β-catenin so that it cannot translocate to the cell nucleus to activate its target genes27. Therefore, GSK-3β is closely related to the activation of the Wnt3a/β-catenin signaling pathway. It is well known that the Wnt3a/β-catenin signaling pathway is closely related to NP, but the relationship between GSK-3β and NP has not previously been reported. However, previous studies have found that ghrelin plays an important role in neuroprotection by inactivating GSK-3β21. In brief, ghrelin phosphorylates GSK3β on Ser9 to render it inactive, which in turn promotes β-catenin stabilization and the induction of target genes. In this study, we found that ghrelin decreased GSK-3β phosphorylation at Ser9 and accelerated the degradation of β-catenin. Suppressing GSK-3β may enable the accumulated β-catenin to translocate to the nucleus. Thus, we speculate that ghrelin can suppress GSK-3β and therefore, control the Wnt3a/β-catenin signaling pathway, to relieve NP. In the present study, we found the GSK-3β activation was inhibited after CCI. However, this effect was counteracted after intrathecal delivery of ghrelin. These results demonstrated that the inhibition of the Wnt3a/β-catenin signaling pathway by ghrelin may be related to the suppression of β-catenin via the inhibition of GSK-3β activation in the spinal cord. Our results showed that ghrelin could significantly alleviate NP by inhibiting the expression of β-catenin via the suppression of GSK-3β activation in the spinal cord of rats challenged with CCI.To further explore the underlying mechanisms of the inhibition of the Wnt3a/β-catenin signaling pathway by ghrelin, we assessed the effect of ghrelin on GSK-3β. It is now well established from numerous studies that the equilibrium of activation between Tyr216 and Ser9 sites in GSK-3β determines its activity and phosphorylating the N-terminal Ser9 residue leads to the auto-inhibition of GSK-3β28. Central and peripheral administration of ghrelin increases food intake dramatically in rats and mice, which may be due to activation of various brain regions, including the feeding-related areas such as the paraventricular nucleus (PVN) in rats30. So, we cannot ignore the fact the effects of ghrelin on feeding behaviour and this may alter pain sensitivity throughout the day, which will be worth a very good direction for further study in the future.However, there are some limitations of our study. Although we showed that ghrelin inhibited the GSK-3β/β-catenin signaling pathway in the spinal cord, we are not certain whether the effect of ghrelin was due to its action on neurons, astrocytes, or microglia. In addition, ghrelin levels in the blood fluctuate throughout the day in humans, as reflected in rising before a meal and decreasing upon food consumption. Increasing evidence in clinical studies has revealed that patients with eating disorders, including food consumption, are almost entirely neglected in patients with chronic pain, which may induce a worse prognosis or a more severe pain perceptionIn summary, our data indicated that ghrelin could significantly alleviate NP by inhibiting the expression of β-catenin via the suppression of GSK-3β activation in the spinal cord of rats challenged with CCI. This suggests the potential therapeutic application of ghrelin administration for the treatment of NP.In this study, we used healthy adult male Sprague-Dawley rats weighing 200–220 g, which were provided by the Zhejiang Academy of Medical Sciences. The temperature of the animal house was maintained at 21–23 °C using central air-conditioning and the food and water were provided in the animal facilities with filtered air. All rats were housed with a diurnal rhythm. This study was approved by the Ethics Committee of the First Affiliated Hospital of Zhejiang University School of Medicine. All experiments were performed in accordance with relevant guidelines and regulations.31. After catheter placement, 2% lidocaine was injected to verify the correct tube placement and only those animals that showed paralysis of both the bilateral posterior limb and the tail were employed in our study.Surgery was performed after the administration of 4% phenobarbital sodium . A polyethylene intrathecal catheter  was implanted into the subarachnoid space at the level of the L5 and L6 vertebrae. The opposite end of the catheter was then carefully crossed through the subcutaneous tunnel and fixed in the posterior neck area to avoid being bitten offet al.32. Two days after subarachnoid catheterization, animals in the CCI group were anesthetized with 4% phenobarbital sodium . The left sciatic nerve was then exposed at the mid-thigh level and was separated near the trifurcation, at approximately 7 mm. Four ligatures  were then tied loosely around the sciatic nerve at intervals of approximately 1 mm. Rats in the sham group received the same surgery as those in the CCI group, but without nerve ligation.We used the CCI model described by Bennett et al.18. Both the CCI group and the sham group were given a subarachnoid injection of 3 µg of ghrelin in a volume of 10 µL or the same volume of 0.9% normal saline, once daily for 7 days.Ghrelin  was diluted in 0.9% normal saline before use. The doses of ghrelin were selected based on the study of Cheng-Hua Zhou Before behavioral testing, animals were habituated to the test environment for at least 2 hours per day for 3 days. Withdrawal threshold and thermal hyperalgesia were then examined. Testing was performed 1 day before and 1, 3, 5, 7, 10, and 14 days after surgery. All behavioral testing was performed between 8:00 AM and 11:00 AM by the same person who was blinded to the group identity.33. Before testing, rats were positioned in a Plexiglas chamber on an elevated glass platform and were allowed to acclimate to the test chamber for at least 30 minutes. The hind paw plantar surface was then exposed to a radiant heat source from underneath the platform. The time taken for the rat to lift its paw was recorded as the thermal withdrawal latency (TWL). To prevent tissue damage, a cutoff time of 20 seconds was used.We evaluated thermal hyperalgesia using a Hargreaves apparatus 26. As for TWL measurements, rats were firstly placed on a wire mesh platform in a plastic enclosure for at least 30 minutes to acclimate to the test environment. A 0.8 mm diameter spring with wires connected to a force sensor was applied to simulate the plantar surface of each hind paw. The pressure was gradually increased until the rat withdrew its paw, at which point the force was recorded as the mechanical withdrawal threshold (MWT).Mechanical allodynia was assessed using an electronic Von Frey Anesthesiometer Seven and fourteen days after CCI surgery, rats were anesthetized and the operative L4–L6 spinal dorsal horn was quickly removed and stored at −80°C until assayed. For sample pretreatment, frozen samples were weighed and RIPA lysis buffer, phenylmethanesulfonyl fluoride (PMSF), and phosphatase inhibitors were added. Clear glass pestles and an ultrasonic cell disruptor were then used to grind up the spinal cord samples in order to extract protein. To collect the supernatant, the sample mixture was centrifuged at 10,600 × g for 20 minutes in a microcentrifuge at 4 °C. The BCA method was used to equalize the total protein content of the supernatants for subsequent analyses, after which 5 × SDS-PAGE loading buffer was added at a ratio of 1:4. SDS-PAGE was used to separate the proteins and they were then transferred to a PVDF membrane. The membrane was then blocked with 5% non-fat dry milk for 1 hour and incubated overnight at 4 °C with primary antibodies against GSK-3β , pGSK-3β at Ser9 , Wnt3a , β-catenin , GAPDH . The membrane was then incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG secondary antibodies for 2 hours at room temperature and visualized using an enhanced chemiluminescence system. Protein levels were expressed as the percentage of the internal control (GAPDH) immunoreactivity. The density of each specific band was calculated using Image Lab software .On day 7, rats were sacrificed and spinal dorsal horns were collected. TNF-α and IL-1β protein levels were quantified using specific rat ELISA kits, according to the manufacturer’s instructions .The L4–L6 spinal cord segment was removed and placed in 4% paraformaldehyde for 24 hours and postfixed for 6 hours before being transferred to phosphate-buffered sucrose (30%) for overnight incubation. Endogenous peroxidase was quenched with 3% (vol/vol) hydrogen peroxide for 10 minutes. Nonspecific adsorption was minimized by incubating the sections with normal goat serum in PBS for 60 minutes at room temperature. Subsequently, 30-µm free-floating transverse sections were cut using a freezing microtome. Sections were collected and incubated for 48 hours at 4°C in rabbit anti-pGSK-3β at Ser9 antibody . After washing in PBS, the sections were incubated with the appropriate biotinylated secondary antibody. The sections were examined by 2 experienced pathologists blinded to the groups. We try to image the lamina 1–3 of the superficial dorsal horn. The number of nuclear-positive cells was counted under a fluorescence microscope  and 5 separate sections per rat were used to get the average value to represent the number of immunopositive cells of an individual rat.p-value < 0.05 was used to indicate statistical significance. All analyses were performed using SPSS software .The pain threshold and protein abundance were statistically analysed by performing a one- or two-way ANOVA followed by the least signifcant diference test for multiple comparison test. The unpaired Student’s t-test was used for comparisons between two groups. Non-parametric data were analysed with the Mann-Whitney U test. All data are expressed as mean ± SD and a


This study examines the potential for point-of-decision prompts (PDPs) to promote healthier food choices among shoppers in a rural, low-income, minority community. We hypothesized that a narrowly defined PDP (focused on fresh produce) would be easier for shoppers to remember than a broadly defined PDP , resulting in a higher proportion of healthy items purchased. PDPs were placed at the entrance to a supermarket in Mission, South Dakota, United States of America, on the Rosebud Sioux Reservation for alternating time periods, July 9–10, 2017. Sales records from 653 transactions were retrieved from the supermarket, comprising periods in which PDPs were in place and control periods. We examined the proportion of selected items and proportion of total expenditures that were a) any healthy foods and b) fresh fruits and vegetables. Data were analyzed in 2018. The narrowly defined prompt consistently resulted in a higher proportion of items and expenditures on healthy foods than either the broad prompt or the control condition. Shoppers in the narrow prompt condition purchased and spent significantly more on any healthy foods and fresh produce than shoppers in the control condition. While shoppers in the narrow prompt condition purchased more healthy foods and fresh produce than shoppers in the broad prompt condition, the differences were not statistically significant. Shoppers exposed to the narrow PDP consistently purchased more healthy foods than shoppers in a control group, while shoppers in the broad PDP did not, highlighting the importance of considering cognitive processes when designing health promotion messages. Obesity, which has increased steadily in the US in recent decades , is linkObesity is not distributed evenly throughout the US population. Certain populations, including members of some ethnic groups as well as rural and poorer individuals, experience higher rates of obesity on average ,6. AfricSustained improvement of dietary quality is a priority for reducing obesity and diet-related health problems. However, access to healthy foods is not necessarily sufficient to improve dietary quality. Even when individuals gain access to healthy foods, food-purchasing patterns may not change . A burgeEfforts to promote healthier food choices at the point of decision have relied primarily on providing information, though there is a small literature examining financial incentives ,15. NutrResearch on the effect of information-based PDPs on food choice shows mixed results. The effect of nutrition facts panels on the quality of food choice in retail situations appears to be fairly minor, though improvements in certain dietary nutrients were observed . SimilarCognitive load, a state in which an individual’s cognitive system is challenged by complex decisions, attempting to retain information, or other stressors , can infA few studies suggest that PDPs that explicitly prompt individuals to consider health when making food decisions—in contrast to PDPs that simply present objective information—lead to healthier choices ,24,25. RIn this study, we report the results of a grocery store-based field experiment that examines the efficacy of PDPs that explicitly encourage shoppers to consider health while making food choices in a predominantly low-income, minority population in the rural Midwest. Because low-income individuals are more likely to experience cognitive load when shopping than higher-income individuals , we testSince the items highlighted in the narrow prompt are a subset of the items in the broad prompt, it will be easier for shoppers to find preferred healthy food items within the broader set. However, if individuals experience cognitive load, it may be easier to keep the narrow prompt in mind while shopping. We hypothesize that exposure to any PDP (narrow and broad PDPs pooled together) will increase purchases of healthy foods relative to a control condition. Additionally, we hypothesize that shoppers in the N-PDP condition will increase their purchases of healthy foods more than shoppers in the B-PDP condition, and that purchases of healthy foods in each intervention condition will be higher than items purchased by shoppers in a control condition.The University of Nebraska-Lincoln’s Institutional Review Board approved this research (#20170216932EX) and Rosebud tribal authorities approved the research. Neither written nor oral consent was obtained because researchers did not interact with research subjects or collect personally identifiable information. The University of Nebraska-Lincoln's IRB waived the informed consent requirement.The research was conducted in a supermarket on the Rosebud Indian Reservation in Mission, Todd County, South Dakota. The UNL IRB waived the informed consent requirement. The Rosebud Indian Reservation is the home of the Rosebud Sioux Tribe—over 90% of Reservation residents are fully or partially Native American . ResidenThe supermarket experiment was conducted on consecutive days, a weekend day and a weekday, July 9–10, 2017. The research was limited to two days for two reasons. Most importantly, store management only agreed to provide data on purchases for two days due to the logistically and time intensive nature of generating purchase data. The data were only accessible by printing out itemized receipts and obtaining data for two days resulted in approximately 1,000 printed pages. Second, researchers only had access to one supermarket. To get a clean measure of the effect of the intervention, we needed to avoid exposing shoppers to multiple conditions on repeat visits, which could lead to a loss of control over consumer behavior in the experiment. Each condition was implemented for two hours each day and rotated so that one day the B-PDP condition occurred first, followed by a control condition, and finally the N-PDP condition, while the other day the conditions were reversed.For a healthy diet, try to buy at least five fruits and vegetables. Food is Good Medicine.” The B-PDP message read “For a healthy diet, try to buy at least five healthy food items. The Food Is Good Medicine label can help you identify healthy food choices”  if the shopper did not purchase any food items, or 2) if the shopper purchased only single-serving, ready-to-eat foods and/or beverages from the supermarket deli. Non-food items were subtracted from the total item count for receipts that included food and non-food items. Researchers coded each purchased food item as healthy or unhealthy based on the FIGM system and recorded the total number of all food items, all healthy items, and all F&V as well as expenditures on all food items, on all healthy items, and on F&V per transaction.Researchers combined the data on the number and amount of money spent on all healthy foods and on F&V with data on total food items purchased and total amount spent on food to create variables that capture all healthy items or all F&V as a proportion of all items purchased and of total food expenditures. Proportions control for variation in the number of items purchased and amount spent from one transaction to another. We used these variables as our key outcomes to evaluate the effect of the pooled PDPs and the separate N-PDP and B-PDP conditions. Because the data were generated by the supermarket’s sales management software, we do not have access to typical demographic control variables, such as gender, education, age, or income. However, we also avoid concerns about researcher demand effects since researchers did not interact with shoppers during the research . As we cFirst, we compare the pooled PDP purchases to purchases in the control condition . The fouThe proportion of healthy items purchased by shoppers in the control condition (no PDP displayed) was 0.114. For shoppers in a PDP condition, the proportion was 0.156. This represents a 37% increase in the proportion of healthy food items purchased and is statistically significant (p<0.01). For fresh fruits and vegetables, the proportion purchased increased from 0.068 in the control condition to 0.095 in the PDP condition (p<0.05). The proportion of food expenditures demonstrates very similar patterns. Expenditures on all healthy items increased from 0.099 in the control condition to 0.135 in the PDP condition (p<0.05), while expenditures on fruits and vegetables increases from 0.063 in the control condition to 0.087 (p<0.05). In each case, the proportion of healthy purchases or expenditures is between 36 and 40 percent higher in the PDP condition.We break the pooled PDP data into the narrow (N-PDP) and broad (B-PDP) message conditions to evaluate whether one condition drove the increase in the proportion of healthy purchases observed in the first analysis. Again, we compare purchases in the two PDP conditions to the control condition . The proNext, we examine the proportion of F&V purchased by condition. In the control condition, 0.068 of shoppers’ items were F&V. In the B-PDP condition, the proportion was 0.075, while in the N-PDP condition, the proportion was 0.109. The proportion of F&V purchased in the N-PDP condition was significantly higher than in the control condition (p < 0.03).Expenditures on healthy items follow a pattern similar to the number of items purchased. In the control condition, the proportion of expenditures on healthy food items was 0.099, while the proportion of expenditures on healthy foods was 0.112 in the B-PDP condition. Shoppers in the N-PDP condition spent a significantly higher proportion, 0.151, of total expenditures on healthy items than shoppers in than the control condition (p = 0.01).The proportion of expenditures on F&V is slightly lower than the proportion of F&V purchased but shows the same relationship across conditions. The lowest proportion of total expenditures on F&V occurred in the control condition (0.063), followed by the B-PDP condition (0.067). In the N-PDP condition, the proportion spent on F&V was highest: 0.102. Expenditures on F&V in the N-PDP condition were significantly higher than the proportion of F&V expenditures in the control condition (p < 0.05).We use one additional measure to assess whether shoppers’ responses to the PDPs are consistent with the PDP’s message. If shoppers respond to the prompt, shoppers in the N-PDP condition should increase purchases of F&V relatively more than shoppers in B-PDP. For this measure, we calculate the change in each of the four variables we examined above between the control and both PDP conditions. We calculated this measure for all four variables and for both PDP conditions. We then look at the percentage of the difference in healthy food purchases/expenditures attributable to fruits and vegetables. For instance, we take the proportion of F&V purchased in N-PDP minus the proportion of F&V purchased in the control condition, and the proportion of all healthy foods purchased in N-PDP minus the proportion of all healthy foods purchased in the control condition. We then divide the difference in F&V by the difference in all healthy foods. The results suggest that shoppers did respond differently to the two prompts . In the Concerns about the relationship between diet, obesity and health outcomes have arisen in recent decades, especially for low-income populations. Most efforts to change behavior at the point of decision have provided information about the nutritional content of packaged and prepared foods. The majority of research on information-based PDPs find no effect ,17. The In our grocery store-based field study, we examined the effect of two PDP prompts encouraging shoppers to set a healthy food purchase goal. The shoppers were from a low-income community with high rates of obesity and diet-related health problems, and the prompts were designed to test whether interaction between naturally occurring cognitive load and the prompt message influences PDP effectiveness. A narrow prompt encouraged shoppers to set a goal of purchasing five F&V (N-PDP), while the broad prompt condition encouraged shoppers to set a goal of purchasing any five healthy items (B-PDP). We consistently find that shoppers purchase the greatest amount and spend the most on healthy food items in the N-PDP condition. While the number of items purchased and amount spent in the B-PDP condition for healthy foods is higher than the control condition, the differences are not statistically significant. Shoppers in the N-PDP condition purchased a higher proportion of healthy foods overall (49% higher) and F&V (60.3% higher) than the control condition. The N-PDP condition yielded more purchases than the B-PDP condition of all healthy foods (25% higher) and F&V (45% higher). Expenditures show a similar pattern. Shoppers in the N-PDP condition spent more than shoppers in the control condition on all healthy items (52.5% higher) and on F&V (61.9% higher). Shoppers also spent a higher percentage in the N-PDP condition than in the B-PDP condition .The observed patterns support our hypotheses about the interaction between prompt design and cognitive load. Shoppers in the N-PDP consistently purchased more F&V than shoppers in the B-PDP or control condition, leading to higher overall purchases of healthy items. Shoppers in the B-PDP did not increase purchases of other healthy items enough to keep pace with the increases in healthy purchases of shoppers in N-PDP.Our findings yield multiple important points. First, a simple, inexpensive, poster-based prompt can lead to an immediate, significant increase healthy food purchases in a low-income community at high risk of obesity and diet-related diseases. This is important because research findings frequently suggest that it is difficult to change the shopping habits of low-income consumers ,15, thouThird, our results suggest that the design of prompts and other point-of-decision materials needs to consider the possibility of a target audience experiencing cognitive load. We find that shoppers purchase a significantly higher percentage of healthy foods and fresh fruits and vegetables when exposed to a PDP message focused on a limited set of products (N-PDP) compared to a control group, while shoppers exposed to a PDP message encouraging the purchase of a broader range of products (B-PDP) did not significantly increase their healthy food purchases. This is despite the fact that shoppers have access to more products available for purchase in the B-PDP condition than in the N-PDP condition—since items identified in N-PDP are a subset of items in B-PDP. This finding may result from the narrow prompt being easier to keep in mind than the broad prompt if shoppers experience cognitive load.There are limitations to this study, which require further research. The most significant limitation is the duration of the study. Our results demonstrate short-term impacts of the PDPs in a field setting, but future research should investigate long-term effects of these PDPs, which would require implementation across multiple stores. Research suggests that information-based PDPs may be overlooked once individuals become habituated to the materials , though Despite study limitations, this research identifies a simple, low-cost PDP that significantly increased the relative purchase of and amount spent on healthy foods and on fresh fruits and vegetables in a low-income community at high risk of obesity and diet-related diseases. Though there is little preliminary research on PDPs that encourage individuals to consider health when making shopping decisions, these results identify the approach as a potentially effective, inexpensive means by which to increase the healthfulness of retail food purchases.Our study provides evidence of the feasibility of using a low-cost point-of-decision prompt (PDP) to increase healthy food purchases in a low-income, minority community. Our research also emphasizes the importance of considering the impact of cognitive stressors—such as those introduced while individuals are actively thinking about what they need to buy and how much they can spend—on the effectiveness of the PDP. These results suggest that PDPs may need to be carefully designed to help shoppers retain and respond to the message.


Inactivating mutations of the tumor suppressor Adenomatosis Polyposis Coli (APC), which are found in familial adenomatosis polyposis and in 80% of sporadic colorectal cancers (CRC), result in constitutive activation of the Wnt/β-catenin pathway and tumor development in the intestine. These mutations disconnect the Wnt/β-catenin pathway from its Wnt extracellular signal by inactivating the APC/GSK3-β/axin destruction complex of β-catenin. This results in sustained nuclear accumulation of β-catenin, followed by β-catenin-dependent co-transcriptional activation of Wnt/β-catenin target genes. Thus, mechanisms acting downstream of APC, such as those controlling β-catenin stability and/or co-transcriptional activity, are attractive targets for CRC treatment. Protein Kinase C-α (PKCα) phosphorylates the orphan receptor RORα that then inhibits β-catenin co-transcriptional activity. PKCα also phosphorylates β-catenin, leading to its degradation by the proteasome. Here, using both in vitro (DLD-1 cells) and in vivo (C57BL/6J mice) PKCα knock-in models, we investigated whether enhancing PKCα function could be beneficial in CRC treatment. We found that PKCα is infrequently mutated in CRC samples, and that inducing PKCα function is not deleterious for the normal intestinal epithelium. Conversely, di-terpene ester-induced PKCα activity triggers CRC cell death. Together, these data indicate that PKCα is a relevant drug target for CRC treatment.  Variation in the activity of the Wnt/β-catenin signaling pathway along the intestinal epithelium crypt/surface axis is a key regulatory element of intestinal homeostasis . Indeed,APC (Adenomatous Polyposis Coli) result in constitutive activation of Wnt/β-catenin signaling. These alterations include loss of heterozygosity (in about 30–40% of CRC) and point mutations that lead to the expression of truncated versions of the APC protein without the C-terminal region, a critical region for β-catenin destruction complex scaffolding [MYC that is considered the master gene responsible for the oncogenic activity of this pathway [In inherited familial adenomatosis polyposis—the major hereditary disease leading to colorectal cancer (CRC)—and in up to 80% of sporadic CRC, genetic alterations of ffolding  , PKC role as oncogenes or tumor suppressors has been the subject of discussion for a long time. This could be one of the main reasons why PKC is not considered to be a prime target for cancer therapy. Significant evidence indicates that PKCα acts as a tumor suppressor in intestinal epithelial cells: (i) PKCα inactivating mutations have been detected in CRC [PRKCA (the gene encoding mouse PKCα) knock-out in mice is associated with the development of spontaneous intestinal polyps, and with higher tumor aggressiveness and reduced survival in the APCMin/+ background [No therapy targeting the activity of the Wnt/β-catenin pathway is currently used to treat CRC, partly due to the lack of targets downstream of APC. However, several observations indicate that Protein Kinase C-α (PKCα) might fulfill this criterion. PKCα is a member of the Protein Kinase C (PKC) family that includes ten serine/threonine kinases encoded by nine distinct genes. The first members were isolated 41 years ago from mammalian brain extracts  and rapid in CRC ; (ii) PKd in CRC ,11;  PRckground . Therefockground ,15,16,17ckground  reportedckground  demonstrhttps://clinicaltrials.gov/). However, despite the findings suggesting that PKCα is a potential drug target candidate for inhibiting the constitutively activated Wnt/β-catenin pathway in CRC, it is not known whether increasing PKCα activity is beneficial for fighting CRC. Using both in vitro and in vivo PKCα knock-in models, we provide evidence that PKCα is a relevant drug target to be stimulated in CRC. An additional attractiveness of PKC as a therapeutic target is the availability of many pharmacological compounds that can modulate its activity, many of which have been used in clinical trials to evaluate their potential anti-tumor effects (http://cancer.sanger.ac.uk/cosmic) (http://dcc.icgc.org). In agreement, sequencing analysis of the eight human CRC cell lines used in this study showed that HCT116, RKO and HT29 cells carried silent PKCα mutations, whereas only DLD1 cells exhibited a non-silent mutation within the PKCα coding sequence, resulting in a non-previously described methionine to isoleucine substitution at position 299 of the protein  . Most of protein . Given t protein , it can PRKCA knock-out has been associated with higher tendency to develop spontaneous intestinal tumors in wild type mice, and with more aggressive tumors and reduced survival rate in APCmin/+ mice [PRKCA cDNA ROSA26 knock-in transgenic mice  that weKCαTg/Tg  , we con570 nm) relative to untreated cells (control)  in the presence or absence of 1 μg/mL doxycycline and/or 1 nM PMA. After 10 days, we stained cultures with crystal violet A, and qucontrol) C. The recontrol) D. IncubaThe previous results demonstrated that doxycycline-induced PKCα overexpression inhibits CRC cell growth and promotes their death upon PMA stimulation. Although PKCα is infrequently mutated in CRC cells, it has been reported that PKCα expression is decreased in CRC , raisingDespite significant prevention and screening measures implemented in recent years, CRC remains the third leading cause of cancer-related deaths in industrialized countries .The constitutively activated Wnt/β-catenin signaling pathway is the prime target in CRC; however, no therapy to antagonize this pathway is currently available in the clinic, despite extensive research in this field . Zhang ePRKCA mutations can be found in many human tumors, sometimes with high frequencies  . However, these observations must be tempered by the results displayed in the TCGA database  that identify PRKCA mutations in only 4.48% of skin cancers. This discrepancy can be explained by the use of distinct sequencing strategies i.e., Whole Genome Sequencing (WGS) for ICGC and Whole Exome Sequencing (WXS) for TGCA. Besides, in a recent study, Rosenberg et al. identified a recurrent point mutation in PRKCA as a hallmark of chordoid gliomas [PRKCA mutations are infrequent and heterozygous in CRC. This indicates that PKCα integrity is preserved and that PKCα activity could be stimulated in CRC cells.  gliomas . ConversPRKCA knock-out is sufficient to induce tumor formation in the intestine of wild-type mice and to promote tumor progression in the intestine of the APCmin/+ mice, indicating that PKCα plays a significant role in maintaining the intestinal epithelium homeostasis [PRKCA cDNA was knocked-in only in the epidermis of transgenic mice where PKCα overexpression did not have any significant effect, except upon PMA induction of its activity that resulted in a striking inflammatory response, without tumor promotion [PRKCA cDNA knock-in in intestinal epithelial cells resulted in a significant increase of PKCα expression at the plasma membrane of differentiated and functionally mature cells in both the small intestine villi and the colon epithelium surface. This indicates that differently from what was reported for the epidermis, PKCα overexpression in the intestinal epithelium correlates with an increase of its activity. The increase of PKCα function in the intestine did not lead to deleterious phenotypic changes, detectable alterations of the intestinal function, and/or effects on food intake. eostasis . Conversromotion . Here, wPKCα overexpression is not sufficient to increase PKCα activity in CRC cells, as indicated by its expression mainly in the cytoplasm and not at the plasma membrane  were cultured in DMEM medium  supplemented with 10% fetal bovine serum (FBS)  and 1× penicillin/streptomycin (Gibco).DLD-1 cells in which PKCα overexpression was induced with doxycycline were established by lentiviral infection with a vector carrying the pTRIPZ inducible system  and the cDNA of interest instead of the turbo red fluorescent protein. Puromycin-resistant clones were selected and maintained in DMEM medium containing 10% FBS and 10 µg/mL puromycin . All experiments were conducted in DMEM medium containing 10% FBS and 10 µg/mL puromycin without (control) or with 1 μg/mL doxycycline . PMA (Sigma-Aldrich) or PEP005  were added at the concentrations and for the times indicated in the legends to figures.570nm measurements  of cells seeded in 96-well plates (50 cells/well), stained with crystal violet  and lysed in 1% SDS. Cell number, volume and diameter were measured using the ScepterTM Automated Cell Counter . Cell viability was quantified by counting living and trypan blue-stained dead cells using a Countess II automated cell counter , according to the manufacturer’s instructions.Cell growth was evaluated after 11 days of cell culture by image analysis (ImageJ Software) of scanned clones grown from 1000 cells/10 cm dish and stained by crystal violet or by OD6 DLD-1-PKCα cells grown with/without (doxycycline + PMA) for 24 h, were washed with 1× PBS, trypsinized and centrifuged at 2000 rpm at 4 °C for 5 min. The resulting pellets were resuspended in 500 µL 1× PBS and fixed on ice in 1.5 mL of cold absolute ethanol for 10 min. Fixed cells were centrifuged at 2000 rpm at 4 °C for 10 min, washed in 1× PBS, and centrifuged at 2000 rpm at 4 °C for 10 min. They were then stained in 500 µL 1× PBS containing 40 µg/µL PI and 100 µg/µL RNase A at room temperature for 20 min. Cells were analyzed with a Gallios flow cytometer .Cell cycle was analyzed using a Propidium Iodide (PI) (Sigma-Aldrich) flow cytometry assay. Briefly, 2 × 10The rabbit anti-PKCα and mouse anti-β-actin (AC-15) antibodies and phalloidin were from Sigma-Aldrich. The mouse anti-E-cadherin and mouse anti-β-catenin antibodies were from BD Transduction Laboratories . The mouse anti-active β-catenin was from Millipore. The mouse anti-α-tubulin was from Santa-Cruz . The Alexa Fluor 488-conjugated F(ab’)2 fragment of goat anti-rabbit IgG and Alexa Fluor 543-conjugated F(ab’)2 fragment of goat anti-mouse IgG and Hoechst were from ThermoFisher Scientific. HRP-conjugated anti-mouse and anti-rabbit antibodies were from Millipore.PRKCA cDNA between a floxed Neo/Stop cassette and a pIRES-EGFP sequence in the pRosa26-DEST vector [XhoI digestion before electroporation in 129/Sv ES cells. G418-resistant ES clones containing the recombinant allele (PRKCA-KI) within the Rosa26 locus were identified by PCR (Tg/wt) were then inter-crossed to produce F2 homozygous R26-PKCαTg/Tg mice. The established R26-PKCαTg/Tg line was finally bred with villin-Cre mice [PKCA knocked-in gene in the intestinal epithelium upon excision of the Neo/Stop cassette. The resulting heterozygous villin-Cre; R26-PKCαTg/wt mice were backcrossed with R26-PKCαTg/Tg mice to obtain homozygous villin-Cre; R26-PKCαTg/Tg mice.The knock-in construct was made by using the GateWay-compatible vector technology  to insert the human T vector . After id by PCR  using thd by PCR . F1 germCre mice  to speciTg/Tg and villin-Cre;R26-PKCαTg/Tg littermates were monitored and weighted weekly from weaning to 34 weeks post-partum. Whole intestines of 14- and up to 76-week-old mice were collected, fixed as rolls, and embedded in paraffin. Then, 3 μm thick tissue sections were stained with hematoxilin and eosin (H&E) and processed for immunofluorescence analysis of PKCα expression. H&E-stained tissue sections were scanned (×20 magnification) using the Nanozoomer technology . The number of villi and crypts in 2 μm was counted in each intestinal section (n = 4 areas per section) and their size (n = 10 per section) was calculated using the NDP.view2 software . Fresh intestinal epithelium was also collected by scraping the mice inner intestinal layer and frozen for western blot analysis. Four males and four females R26-PKCαTM AF1 Plus DAPI mounting medium  and observed with a Leica DM6000 microscope (Objective ×40).H&E staining and immunohistochemistry were performed on tissue sections deparaffinized in xylene and gradually rehydrated in 100%, 96%, 70%, 30% alcohol. For H&E staining, tissue sections were incubated in 0.1% Mayer hematoxylin, decolorized in 70% and 95% alcohol, counterstained in 0.5% eosin, dehydrated in 100% alcohol, and mounted in Mountex (Sigma-Aldrich). For immunohistochemistry, tissue sections were unmasked in 10 mM citrate buffer pH6 at 99 °C for 30 min, and permeabilized in 0.2% Triton X-100. For immunocytochemistry, 50,000 cells were seeded on 12 mm round coverslips, cultured for 48 h, fixed in 4% paraformaldehyde and permeabilized in 0.2% Triton X-100. Unspecific binding was blocked by incubation in 1× PBS/0.1% Triton X-100/10% BSA. Cells were incubated with the primary antibodies at 4 °C overnight, and with Alexa Fluor-conjugated secondary antibodies at room temperature for 1 h. Cells were mounted in CityFluorEqual amounts of total protein extracts from mouse intestinal epithelium samples (50 μg) were separated on 10% acrylamide/Bis-acrylamide gels and then transferred onto PVDF membranes that were then blocked in 10% milk/1× PBS. Primary antibodies were added at 4 °C overnight, followed by HRP-conjugated secondary antibodies at room temperature for 1 h. Immunoblots were revealed using ECL western blotting detection reagents . t-test). Statistical analyses were performed using the PRISM version 5.0, GraphPad software . Data are the mean ± SEM . Protocols were approved by the institute ethics committee.The constitutively activated Wnt/β-catenin signaling pathway is the prime target in CRC but no therapy to antagonize this pathway is currently available in the clinic. Considering that PKCα has been shown to be both a physiological enhancer of β-catenin degradation  and a re


Extrinsic noise, on the other hand, is restricted by β-catenin–mediated regulation of major cellular stress pathways.Molecular noise refers to fluctuations of biological signals that facilitate phenotypic heterogeneity in a population. While endogenous mechanisms exist to limit genetic noise in biological systems, such restrictions are sometimes removed to propel phenotypic variability as an adaptive strategy. Herein, we review evidence for the potential role of β-catenin in restricting gene expression noise by transcriptional and post-transcriptional mechanisms. We discuss mechanisms that restrict intrinsic noise subsequent to nuclear mobilization of β-catenin. Nuclear β-catenin promotes initiation of transcription but buffers against the resultant noise by restraining transcription elongation. Acceleration of cell cycle, mediated  In biological systems, noise can be defined as variability in transmission of a signal between two sources. The variability often manifests in the unpredictability of emergence of a certain outcome. Such unpredictability is a direct consequence of variations in biological signals that maintain the genotype/phenotype continuum. Within physiological limits, phenotypic heterogeneity that is driven by biological noise enriches the population and provides a basis for natural selection. It must be emphasized, however, that phenotypic heterogeneity mediated by noise contrasts sharply, and acts synergistically, with encoded (genotypic) heterogeneity. While genetic noise could propel phenotypic diversity in the absence of genotypic differences, the existence of such genomic variability amplifies the impact of noise.Bacillus subtilis is a prime example of such noise-mediated adaptation to stressors  activity are a major source of intrinsic noise . The provia binding to the chromatin remodeling factor, CHD8  in a population of cells would approach a minimum (Smin) if the copy numbers in individual cells converge toward a central value:By these mechanisms, β-catenin reduces cell–cell variability and hence the standard deviation of molecular signals in a population. Reduction of the standard deviation can be interpreted as establishing the upper and lower limits of transcriptional noise. In other words, standard deviation (S) of a protein copy number (xxi) is established by stabilizing the transcribed mRNAs. The upper limit (max xi) is maintained by β-catenin–mediated buffering of transcriptional bursts as described above.The lower limit  is one of the key genes that is ignaling . Myc, inignaling . It is, ignaling . Myc binignaling . Myc alsignaling . In addiignaling . While eignaling , couplinignaling  restrictignaling . β-catentrans-activated by nuclear β-catenin. As such, enhanced β-catenin signaling accelerates interphase by shortening G1 phase of cycle , and Skp, Cullin, F-box–containing complex (SCF complex) . The APCoff cells, β-catenin acts as an adaptor that directs post-translational degradation of Hippo cascade mediators and hence reduces noise in this signaling pathway. In addition to interaction with intrinsic sources of noise, the buffering capacity of β-catenin is bolstered by its particular mode of communication with extrinsic inducers of gene expression noise.While β-catenin is a major target of glycogen synthase kinase 3β (Gsk-3β) and the associated destruction complex, other substrates are also directed to proteosomal degradation by activity of this complex . These iOn route to degradation, β-catenin is enzymatically altered by proteins including Gsk-3β that also sense cellular stress. Therefore, activation of stress adaptation pathways in response to genetic noise  could poCascade-level associations with other signaling pathways remarkably amplify the capacity of β-catenin in buffering genetic noise . Once inon) blocks the destruction of β-catenin and enhances noise buffering capacity of the protein under special circumstances  . In yeasvia β-Catenin could enhance robustness of developmental self-organization.It may be concluded that evolution of β-catenin, in addition to its documented role in organization of cytoskeleton, provided metazoan life forms with a capacity to modulate gene expression noise. Buffering of noise is required to stabilize the evolutionary adaptations that characterize various species. This feature, referred to as Waddington’s canalization , improveRF: conception of the work. SR-L and RF: extensive literature search and manuscript drafting. NH and RF: critical revision of the work. RF and NH: final version approval.This study was supported by NIDCR Grant R01 DE015272 and Australian National Health and Medical Research Council Grant 512524.3.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.


P = 0.026). Among the corneal areas measured using specular microscopy, the superior area, where the Ex-PRESS tube was inserted, had significantly more severe corneal ECD reduction than the inferior area after 2 years . More cataract progression occurred in the trabeculectomy group than in the Ex-PRESS group (P = 0.04). Twelve eyes (37.5%) in the trabeculectomy group and 4 eyes (12.5%) in the Ex-PRESS group underwent cataract surgery (P = 0.019). The total number of other postoperative complications between 3 months and 2 years was significantly higher in the trabeculectomy group than in the Ex-PRESS group (P = 0.02). Although Ex-PRESS implantation might be associated with an increased rate of corneal endothelial cell loss compared with trabeculectomy, it is beneficial for preventing cataract progression after filtering surgery.We compared complications between Ex-PRESS implantation and trabeculectomy for 2 years after surgery. Sixty-four open-angle glaucoma eyes were randomly assigned to treatment with trabeculectomy (n = 32) or Ex-PRESS implantation (n = 32). The primary outcomes were postoperative complications, including reduction of the endothelial cell density (ECD) of the cornea, cataract progression and the frequency of other late postoperative complications. The Ex-PRESS group had significantly greater reduction of postoperative corneal ECD than the trabeculectomy group did at 2 years after surgery ( Especially, early postoperative complications due to over-filtration include a flat/shallow anterior chamber, choroidal detachment, suprachoroidal haemorrhage and hypotensive maculopathy, which are often associated with visual disturbance after surgery5. Filtering surgery using the Ex-PRESS glaucoma filtration device  is a surgical option to reduce the frequency of complications resulting from over-filtration because the device is believed to offer more stable filtration compared with limbal tissue excision during trabeculectomy7. Previously, our clinical trial evaluating complications following Ex-PRESS implantation versus trabeculectomy in medically uncontrolled open-angle glaucoma (OAG) eyes, called ‘Complications Postoperatively of Ex-PRESS versus Trabeculectomy Study (CPETS)’, showed that Ex-PRESS implantation reduced the frequency of some early postoperative complications, including hyphema and flare elevation in the anterior chamber, compared with trabeculectomy8. Another prospective study comparing Ex-PRESS implantation with trabeculectomy reported fewer early postoperative complications with Ex-PRESS implantation9.Trabeculectomy is a common filtering surgery for glaucoma patients with medically uncontrollable intraocular pressure (IOP). However, complications often develop after the surgery12. Recently, a case report described the occurrence of corneal decompensation after Ex-PRESS implantation in an eye with exfoliation glaucoma13. Loss of corneal endothelial cells frequently occurs after tube implantation in the anterior chamber when using the Baerveldt glaucoma implant and the Ahmed glaucoma valve15. Additionally, cataract progression is a common late complication in trabeculectomy. The complication seems to be related to peripheral iridectomy during the surgery because laser and incisional peripheral iridectomies are also associated with cataract progression17. Cataract progression was also compared in CPETS because phakic OAG eyes were included in the study8. The present study reports a comparison of the postoperative complications over 2 years, including quantification of the reduction of corneal endothelial cell density (ECD) and cataract progression following the two procedures.Despite the advantage of fewer early postoperative complications following Ex-PRESS implantation, late postoperative complications after the two procedures have not been well investigated. Corneal decompensation is a serious late complication after filtering surgery8. CPETS was approved by the Institutional Review Board of Fukui University Hospital, Fukui, Japan, and written informed consents were obtained from all the patients. The protocol conformed to the tenets of Declaration of Helsinki. Patients were recruited between August 30, 2012 and February 13, 2015 at Fukui University Hospital. Japanese patients over 20 years with primary OAG (POAG) or exfoliation glaucoma with IOP ≥ 18 mmHg were enrolled in the study. They had phakic eyes without prior intraocular surgery. Eligible eyes were randomly assigned to undergo trabeculectomy (the trabeculectomy group) or Ex-PRESS implantation (the Ex-PRESS group). Mitomycin-C (0.4 mg/mL) was applied for 4 minutes during the surgery in all the eyes. Follow-up visits for the clinical trial were at 3, 6, 12 and 24 months after the surgery. Each pre- and postoperative examination included measurements of the slit lamp biomicroscopy, IOP measurement with Goldmann applanation tonometry, best corrected visual acuity and corneal specular microscopy.The present study protocol has been described in detail elsewhere18. The light scattering intensity of the lens was evaluated using an anterior eye segment analysis system . A camera unit was used to obtain a two-dimensional linear image of the cross-section of the anterior segment along 0° with a 200 W light source. The light scattering intensity  of the images was analysed using ImageJ.The primary outcomes were postoperative complications, which included postoperative percent reduction of the corneal ECD, the coefficient of variance (CV) and hexagonal cell appearance rate (6A) of corneal endothelial cells compared with preoperative corneal ECD, CV and 6A, nuclear cataract progression and the frequency of postoperative complications between 3 and 24 months. The change in corneal ECD, CV and 6A of corneal endothelial cells between preoperative and postoperative visits was quantified in five areas  photographed with a non-contact specular microscope  by a single experienced examiner (S.A.). The patients who underwent cataract surgery or reoperation due to insufficient IOP reduction were excluded from the analysis of corneal ECD. Nuclear cataract progression was quantified using the Lens Opacification Classification System III (LOCS-III)The secondary outcomes were the dependency of the corneal ECD reduction on the five photographed areas after surgery within each group, the comparisons of IOP and visual acuity between the two groups.2 test. P values were considered statistically significant if less than 0.05. The sample size was found to provide 80% power to prove (at a one-sided level of 0.05) the superiority of a significant outcome between the trabeculectomy and Ex-PRESS groups for an effect size of 0.7.JMP version 10.0  was used for statistical analysis. Univariate analysis was performed with Wilcoxon’s non-parametrical test, the paired t-test with Bonferroni correction and the χMedical Information Network Clinical Trials Registry of Japan .8, 32 eyes in the trabeculectomy group and 32 eyes in the Ex-PRESS group were postoperatively analysed. Table P = 1.0), whereas the 2-year follow-up evaluation was completed for 25 eyes (78.1%) in the trabeculectomy group and 28 eyes (87.5%) in the Ex-PRESS group (P = 0.3).As shown in our previous report about early postoperative complications in the CPET study2 in the trabeculectomy group and 2,564 ± 329 cell/mm2 in the Ex-PRESS group before surgery. The Ex-PRESS group had a significantly greater percent reduction in the postoperative corneal ECD than the trabeculectomy group did at 6, 12, and 24 months after surgery . The CV of corneal endothelial cell calculated by the average of the five areas was 0.369 ± 0.061 and 0.370 ± 0.072 in the trabeculectomy and Ex-PRESS groups, respectively, before surgery (P = 0.70). The postoperative percent reduction of CV during the follow-up period also exhibited no significant differences between the two groups. . The 6A of corneal endothelial cells calculated by average of the five areas before surgery was 54.6% ± 9.02% and 56.3% ± 12.3% in the trabeculectomy and Ex-PRESS groups, respectively (P = 0.61). The postoperative percent reduction of 6A during the follow-up period also showed no significant differences between the two groups .The corneal ECD (average of the five areas) was 2,487 ± 275 cell/mmP = 0.89; Fig. P = 0.02 at 12 months; 136.8 ± 42.5 cct vs 101.2 ± 41.9 cct, P = 0.04 at 24 months in the trabeculectomy vs the Ex-PRESS groups, respectively). Despite no significant preoperative difference in the nuclear colour grades between the two groups according to LOCS-III, the trabeculectomy group had a significantly more nuclear colour grade than the Ex-PRESS group did at 24 months , and 12 (37.5%) of the 32 eyes in the trabeculectomy group and 4 (12.5%) of the 32 eyes in the Ex-PRESS group underwent cataract surgery within 2 years (P = 0.019).There was no significant difference in the preoperative light scattering intensities of the lens nuclei between the two groups .As for the corneal ECD of the patients who had undergone cataract surgery during the follow-up period, the corneal ECD decreased significantly more in the Ex-PRESS group than in the trabeculectomy group followed by cataract surgery .Other postoperative complications after trabeculectomy and Ex-PRESS implantation are reported in Table P = 0.64).There were no significant differences in the IOPs between the trabeculectomy and Ex-PRESS groups at any postoperative visits Table . The numP = 0.41 at 6 months, 0.60 ± 0.65 and 0.58 ± 0.52; P = 0.69 at 12 months, 0.49 ± 0.60 and 0.51 ± 0.50; P = 0.38 at 24 months in the trabeculectomy and Ex-PRESS groups, respectively). The postoperative visual acuity of the patients who had not undergone cataract surgery during the follow-up period also showed no significant differences between the two groups .The postoperative visual acuities, which were calculated as LogMAR values, showed no significant differences between the two groups  . Subgroup analysis in the Ex-PRESS group showed a greater reduction of corneal ECD in the superior area than the inferior area . The postoperative cataract progression was more intense in the trabeculectomy group than in the Ex-PRESS group . More eyes in the trabeculectomy group were postoperatively followed by cataract surgery than eyes in the Ex-PRESS group . The number of other postoperative complications in 2 years was significantly higher in the trabeculectomy group than in the Ex-PRESS group (P = 0.02).The main aim of our present study was to compare postoperative complications following trabeculectomy and Ex-PRESS implantation over 2 years. There was significantly more reduction of corneal ECD after Ex-PRESS implantation than after trabeculectomy  or Ex-PRESS (n = 32)22. One patient in the Ex-PRESS group and five in the trabeculectomy group underwent cataract surgery within 3 years. Our study is unique because the comparison is focused on the complications after more than 1 year including quantification of corneal ECD reduction and cataract progression.Despite the many reports25. Regarding Ex-PRESS implantation, two previous studies have compared corneal ECD between Ex-PRESS implantation and trabeculectomy. The corneal ECD reduction was compared among trabeculectomy (n = 22), Ex-PRESS implantation (n = 24), and Ahmed glaucoma valve implantation (n = 18) for 3 months after surgery26. Ex-PRESS implantation did not significantly reduce corneal ECD within 3 months while trabeculectomy and Ahmed glaucoma valve implantation resulted in a significant loss of corneal ECD. Compared with trabeculectomy, the Ex-PRESS filtration device is designed to reduce over-filtration during surgery and in the early postoperative period. The lower loss of corneal ECD with Ex-PRESS implantation in the previous study might reflect endothelial protection from the lack of over-filtration in the early postoperative period. Another randomised study compared corneal ECD between phaco-Ex-PRESS and phacotrabeculectomy for 1 year after surgery. The study showed significantly greater loss of corneal ECD in the phaco-Ex-PRESS group21. Although combined phacoemulsification might have enhanced corneal ECD loss after filtering surgery, the data is consistent with our present study. A single arm case series for eyes treated with Ex-PRESS implantation also showed a significant reduction in the corneal ECD27. The mechanism underlying the damage to the corneal endothelial cells following Ex-PRESS implantation remains unknown. Other glaucoma implants inserted into the anterior chamber also reduce corneal ECD during a long-term follow-up period30. A secondary outcome in our study was that the superior area had higher ECD reduction than the inferior area in the Ex-PRESS group whereas no area dependency was observed in the trabeculectomy group. The data suggest that the mechanism underlying the damage to the corneal epithelium following Ex-PRESS implantation might be different from that of corneal ECD loss after trabeculectomy. It is possible that the damage might be caused by foreign body reaction or immune reactions due to the stainless material used for the tube, the changed flow of the aqueous humour around the tip of the tube, or mechanical ablation between the corneal endothelium and the tube. Interestingly, among eyes that underwent cataract surgery32, the corneal ECD decreased more significantly in the eyes with Ex-PRESS implantation than in the eyes with trabeculectomy. Similar to the data, corneal decompensation frequently occurs after cataract surgery in eyes treated with tube shunt surgery. Taken together, cataract surgery might be associated with damage to corneal endothelial cells, which are compromised by the Ex-PRESS implantation.Corneal ECD reduction is frequently reported as a postoperative complication after filtering surgery37. The reason for cataract progression after glaucoma surgery is not clearly understood. Eyes with trabeculectomy have a higher risk of cataract progression compared to those with non-penetrating deep sclerectomy38. A comparison of trabeculectomy and viscocanalostomy39 revealed a tendency for cataract progression in eyes treated with trabeculectomy (P = 0.066). The data suggest that hypotony in eyes with trabeculectomy is related to cataract progression. However, in our clinical trial, both postoperative IOPs and the frequency of hypotonic complications such as a shallow anterior chamber, choroidal detachment, and hypotonic maculopathy in the trabeculectomy group were comparable to those in the Ex-PRESS group in the early8 as well as the 2-year-postoperative periods. A recent cohort study for prophylactic laser peripheral iridotomy for eyes suspected of primary angle closure showed that eyes treated with prophylactic laser peripheral iridotomy were more frequently associated with cataract progression than eyes suspected of primary angle closure that had never undergone prophylactic laser peripheral iridotomy40. The results suggest that another opening site for aqueous humour in the iris facilitates cataract progression. Because iridectomy is not required for Ex-PRESS implantation, unlike trabeculectomy, Ex-PRESS implantation might result in a lower incidence of cataract progression compared to trabeculectomy.Many previous studies have reported a higher progression of cataracts after trabeculectomyP = 0.02) in the group. A retrospective assessment of bleb morphologic features after Ex-PRESS implantation versus trabeculectomy revealed a more diffuse bleb area in the Ex-PRESS group6. Diffuse bleb formation might be related to a lower frequency of leaking bleb in the Ex-PRESS group.The tendency for more frequent bleb leaks in the trabeculectomy group (P = 0.08) might contribute to the significantly higher frequency of postoperative complications (41. CPETS has been monitored for 5 years after surgery. Further follow-up periods might provide more information about postoperative complications for Ex-PRESS implantation.The present study had some limitations. Firstly, we could not obtain significantly better postoperative visual acuity in the Ex-PRESS group despite the greater cataract progression in the trabeculectomy group. The discrepancy may be associated with the unrestricted inclusion criteria for preoperative visual acuity. Variation of preoperative data in each group results in a lower possibility of significant differences in the outcome. Eyes treated with cataract surgery postoperatively were excluded from the comparison of postoperative visual acuity between the two groups. This bias might have resulted in insufficient power to prove the superiority of the significant outcome. Second, the present data still do not offer long-term results for postoperative complications after 5 years or longer. Tube erosion or corneal decompensation is reported to be associated with Ex-PRESS implantationIn conclusion, 2 years of follow-up for the comparison of postoperative complications between trabeculectomy and Ex-PRESS implantation revealed that Ex-PRESS implantation is associated with a significantly greater reduction of corneal ECD, especially in the corneal area around the inserted tube, but less cataract progression than trabeculectomy. Ex-PRESS implantation is beneficial for preventing postoperative cataract progression. However, caution should be employed to prevent adverse effects against corneal endothelial cells. Therefore, Ex-PRESS implantation should not be considered for eyes with compromised corneal function.CONSORT 2010 ChecklistProtocol


Calcification of human VIC cultures was induced by cultivation in an osteogenic medium and the effect of co-incubation with 1μM XAV-939 was monitored. Calcification was quantified when mineral deposits were visible in culture and was histologically verified by von Kossa or Alizarin red staining and by IR-spectroscopy. Protein expression of alkaline phosphatase, Axin, β-catenin and Sox9 were quantified by western blotting. In 58% of the VIC preparations, calcification was induced in an osteogenic culture medium and was accompanied by upregulation of alkaline phosphatase. The calcification induction was prevented by the XAV-939 co-treatment and the alkaline phosphatase upregulation was suppressed. As expected, Axin was upregulated, but the levels of active non-phospho-β-catenin were also enhanced. Sox9 was induced during XAV-939 treatment but apparently not as a result of downregulation of β-catenin signalling. XAV-939 was therefore able to prevent calcification of human VIC cultures, and XAV-939 treatment was accompanied by upregulation of active non-phospho-β-catenin. Although XAV-939 does not downregulate active β-catenin, treatment with XAV-939 results in Sox9 upregulation that may prevent the calcification process.The development of a substance or inhibitor-based treatment strategy for the prevention of aortic valve stenosis is a challenge and a main focus of medical research in this area. One strategy may be to use the tankyrase inhibitor XAV-939, which leads to Axin stabilisation and subsequent destruction of the β-catenin complex and dephosphorylation of β-catenin. The dephosphorylated active form of β-catenin (non-phospho-β-catenin) then promotes nuclear transcription that leads to osteogenesis. The aims of the present study were to develop an experimental system for inducing  In developed countries, calcific aortic valve (AV) stenosis occurs in 1.7% of the population older than 65 years . Developin vitro. Media formulations that are used for differentiation of mesenchymal stem cells, such as those containing ascorbic acid phosphate, dexamethasone, and β-glycerophosphate, have been tested previously [The cell-driven or cell-affecting processes related to CAVD include lipid infiltration, inflammation, the endothelial-to-mesenchymal transition, fibrosis and osteogenesis , 8. Of peviously . Ascorbieviously .in vitro and in vivo and LRP5/Wnt signal and β-catenin are related with cardiovascular calcification [The signalling pathways involved in AV osteogenic processes are Notch, bone morphogenetic protein and Wnt/β-catenin signalling . The Wntfication , 17–19. fication . Stimulafication . Similarfication . Porcinefication .Fang et al. linked the Wnt/β-catenin pathway to Sox9, a master regulator of chondrogenic lineage, and revealed that β-catenin limits the expression and nuclear localisation of Sox9 in cultured chicken aortic valves and in adult mice aortic valve nodules , 16, 23.XAV-939 inhibits the Wnt/β-catenin signalling pathway by stabilising Axin , a scaffIn addition to Wnt/β-catenin signalling, the Notch pathway has been implicated in regulation of Sox9, although Sox9 does not seem to be a direct Notch target . Notch ain vitro in human VIC cultures by incubation with a combination of ascorbic acid phosphate, dexamethasone and β-glycerophosphate and the mineral deposits were characterised.The aim of the present project was to analyse the potential anti-calcific effect of XAV-939 and the expression of Axin, β-catenin and Sox9 in human VIC cultures in terms of potential regulatory mechanisms for osteogenic differentiation via Wnt/β-catenin signalling versus Sox9 driven chondrogenesis. Calcification was induced Ethikkomission an der TU Dresden, registration number EK429102015). The valves were replaced with prostheses in the daily routine of cardiac surgery. Thirteen patients were male and six were female (average age 65.2 ± 11.8 years). Three of the 19 valves prepared for the present study were bicuspid and 16 were tricuspid.Nineteen aortic valves were used to isolate VICs for cell culture after the written informed consent form was signed by patients (no minor patients). The study was approved by the ethics committee of the Dresden University . Calcification was induced by seeding 2000 human VICs/cm2 (passages 1–6) in 6-well plates (not collagen coated). After 72 hours, the culture medium was replaced with ADG medium . ADGM and ADGM including XAV-939 were freshly prepared weekly and replaced every 2–3 days until mineral precipitations were visible (the maximum incubation time was 35 days without passaging). Control cells were co-cultured in DMEM. As soon as mineralisation was visible in the ADGM-culture, the experiment was stopped and the end points were analysed. XAV-939 was solubilised in DMSO and freshly diluted to a final concentration of 1 μM in culture medium. Due to the high dilution factor (1:6000), DMSO was not added to the DMEM control wells. Two plates were prepared in parallel for calcium quantification and to isolate protein extracts using RIPA buffer. Impact of XAV-939 inhibitor treatment on cell viability of human VICs was investigated by MTT-assay  after 72h treatment at concentrations of 10, 1 and 0.1 μM revealing (n = 4). To perform MTT-assay cells were seeded at a density of 2000 cells per well of a 96-well-plate. After 72h XAV-939 containing media were added and replaced after additional 24h. MTT-assay was performed after 72h of inhibitor incubation according to the manufacturer’s instructions in a 96-well format.Human VICs were isolated as described previously . After eThe calcified cell layer in histological setups was analysed by washing the 6-well plates containing the ADGM induced cell monolayers and the DMEM controls twice with DPBS , adding 400 μl DPBS and scraping and separating the cell layers from the wells. The separated cell material was transferred to a reaction tube and centrifuged at 200 g for 10 min to precipitate the material. The pellet was fixed in 4% buffered formalin solution. Samples were paraffin embedded and sliced. Movat Pentachrom staining was performed according to the manufacturer’s (Morphisto) instructions. Calcification was visualised by staining the slices with von Kossa and Alizarin red stain according to standard methods. As a control, sliced specimens were decalcified after rehydration by overnight incubation in Osteosoft solution. Images were acquired using a Slide Scanner (Axio ScanZ.1 by ZEISS) or Zeiss Observer Z.1.2 object slide. Sections of cell culture pellet sample were analysed and compared to a formaldehyde-fixed, calcified human AV sample.One induced cell culture preparation was used for initial investigation of the precipitates via Fourier Transformed Infrared (FT-IR) spectroscopy. The cell culture well was washed twice with DPBS and the cells were carefully scraped from culture plastics and centrifuged at 300 g. The resulting pellet was directly transferred to embedding media and frozen at -80°C. Cryosections with a thickness of 20 μm were transferred to a CaF2. The compositions of individual infrared images were captured, subject to the size of the area investigated. Background spectroscopy images were recorded from the pure CaF2 object slide before the tissue sections were investigated. For all measurements, 100 interferograms (scans) were co-added. The interferograms were Fourier transformed applying Happ-Genzel apodisation and a zero filling factor of 1. Spectra at a resolution of 6 cm-1 of the sample image were rationed against the spectra of the background image and transformed into absorbance values.IR spectroscopy images were collected in transmission mode using a Vertex 70 FT-IR spectrometer coupled with a Hyperion 3000 infrared microscope . The imaging detector was a Santa Barbara focal plane MCT 64 x 64 array detector. The 15× Cassegrainian objective with a numerical aperture of 0.4 imaged a sample area of approx. 175 × 175 μm-1 was considered for data volume minimisation. Data pre-processing involves a removal of outliers, a baseline correction and a normalisation of each absorbance value of a spectrum to the integral absorbance. Outliers are spectra that are obviously not associated with tissue or spectra with a maximum absorbance value larger than 1.5. The baseline of each spectrum was corrected using the msbackadj function of the Statistics Toolbox of Matlab. Afterwards, the spectra were area-normalised to eradicate spectral differences due to sample thickness or variation in the density of cellulose fibres. K-means cluster analysis of with four clusters was performed with the k-means function of the Statistics Toolbox.The spectral data were evaluated using the Matlab Package . Only the so-called fingerprint region between 950 and 1800 cmWhole cell protein was isolated from the wells after removal of the medium and washing the culture twice with DPBS. A 200 μl volume of cold (4°C) RIPA buffer containing protease inhibitors and phosphatase inhibitors was added to each well and incubated for 10 min on ice. Cellular material was scraped, transferred to tubes, sonicated to disrupt the cells and pelleted by centrifugation. The protein content of the supernatant was determined in a 96-well format using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.https://www.licor.com/bio/products/software/image_studio_lite), with expression in DMEM control wells set as 100%.Proteins were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes (Roth). The membranes were blocked overnight at 4°C or for 1.5 h at room temperature (RT) in TBS or PBS, respectively, containing 0.1% Tween20 and 5% milk powder. After three washes with buffers supplemented with 0.1% Tween20, the membranes were exposed to the following antibodies in their respective dilutions: Anti-Alkaline Phosphatase , Anti-Axin1 , Anti-β-catenin , Anti-Non-Phospho (Ser33/37/Thr41) β-catenin , Anti-Sox9  and Anti-GAPDH . Primary antibodies were incubated for 1 h at RT or overnight at 4°C. Membranes were washed, incubated with secondary anti-mouse , anti-rabbit  or anti-goat  immunoglobulin/HRP  and treated with western blotting luminol reagent (Merck) for 1 min. The luminol signal was detected using a PHASE-Detection camera (PHASE) at defined time points. Semiquantitative analyses of expression levels relative to GAPDH were performed with Image Studio Lite software  was used at an equivalent concentration. After washing of the slides, a secondary donkey Anti-Rabbit IgG 488  antibody in PBS/BSA was incubated for 1 h at RT. The cells were DAPI stained and washed with PBS twice and then embedded in fluorescent mounting medium . Staining was evaluated and images were acquired using a Zeiss Observer Z.1 Apotome fluorescence microscope. Fluorescent nuclei were counted in relation to DAPI-stained nuclei and the percentages of Sox9-positive nuclei were evaluated.Human VIC cultures were seeded on glass slides at a density of 3 × 10The calcium content in the induced cell culture wells was determined by replacing the medium, washing the dish twice with calcium free DPBS and adding 1 ml 0.1 M nitric acid. The sample was incubated at RT overnight and calcium ions were quantified using a Spektroquant Calcium-Test Kit , according to the manufacturer’s instructions and after relevant dilutions.The calcium content was related to total protein by removing the nitric acid solution, washing the wells twice with DPBS, adding 500 μl of a 0.05 M NaOH/0.1% SDS solution per well and incubating overnight at RT. Protein concentration was determined as described above. For better comparability, the results were calculated in units of mol/(kg protein).The Wilcoxon matched pairs test was used to test statistical significance of calcium content of DMEM vs ADGM cell culture samples.A two tailed paired t-test was used to test alkaline phosphatase expression levels in the ADGM vs DMEM conditions, and a Wilcoxon matched pairs test was used to compare DMEM-XAV-939 vs ADGM-XAV-939. The statistical significance of SOX9 nuclear localisation was tested with a paired t-test.One-way-ANOVA and the Newman Keuls multiple comparison test were applied to Axin1, β-catenin and SOX9 western blot data and to quantify the calcium content in the experiments with the four arms: DMEM, ADGM, DMEM-XAV-939 and ADGM-XAV-939.Analyses were performed using Graph Pad Prism 5 software . Data are presented as mean ± SD. Significant differences are indicated by asterisks.Nineteen individual human VIC cultures were used to study calcification processes induced by culture in ADGM. The ADGM-induced cultures showed a light to brownish precipitate after different culture times from 14 to 33 days  in the ATwo of the inducible VIC preparations were isolated from valves from female patients and nine from male individuals. The cell preparations that did not calcify resulted from aortic valve tissue from four female and four male patients. Nine and seven of the aortic valves the inducible vs. non-inducible VIC preparations resulted from were tricuspid, respectively. There was no statistically significant relationship of these patient parameters to the calcification potential.Histological analysis of ADGM-induced VIC cultures, when compared to a homogeneous ECM . Incubat-1 and 1237 cm-1, respectively, and increased carbonyl stretching mode of phospholipid esters at 1744 cm-1 [-1 were assigned to CHx groups of lipids [-1 [A calcified VIC cell culture was prepared for IR-spectroscopy analysis by scraping the material, pelleting the cells by centrifugation and cryo-sectioning. A patient’s AV was analysed in parallel for comparison of the spectra. 744 cm-1 –37. Othef lipids . Spectrapids [-1 , 36 withHistological analysis after von Kossa staining of the cell pellet revealed a positive calcification signal. Respective cluster analysis of this sample is shown in In vitro calcification was induced from 0.2 ± 0.1 mol/kg protein in the DMEM controls to 3.3 ± 3.0 mol/kg protein (p<0.05) after culturing in ADGM. When XAV-939 was added to the ADGM culture wells, no calcification was apparent  on MTT-based viability of VICs was not detected . The impapparent  and the apparent . Therefoapparent .Upregulation of β-catenin and Sox9 expression is involved in osteoblast and/or chondrocyte differentiation. Inhibition of tankyrase by XAV-939 stabilises Axin1, which is one member of the β-catenin destruction complex. The expression levels of Axin1, β-catenin and Sox9 were analysed by western blotting of protein lysates of VIC standard cultures and compared to ADGM-induced calcified cells and cells co-incubated with XAV-939.As expected, Axin1 was stabilised in VIC cultures after co-incubation with XAV-939 . A signiHuman VICs cultured on glass slides in DMEM, ADGM, DMEM-XAV-939 and ADGM-XAV-939 were stained for Sox9 expression in an immunofluorescence setup. Evaluation of Sox9 nuclear localisation revealed a rate of 32.1 ± 19.3% in VICs cultured in DMEM and a significant reduction to 11.2 ± 11.1% in the ADGM condition . Incubatin vitro induction of calcification in human VIC culture preparations. As expected, XAV-939 treatment resulted in the stabilisation of Axin, a member of β-catenin degradation complex [The tankyrase inhibitor XAV-939 is able to prevent  complex ; therefo complex ; however complex ), but thThe Sox9 protein expression is upregulated when cultures are incubated with XAV-939 in the DMEM and ADGM conditions, but this Sox9 protein upregulation does not correlate with a reduced expression of the non-phospho-β-catenin. The induction of Sox9 expression in the DMEM-XAV-939 condition vs the DMEM control, where non-phospho-β-catenin is expressed and where Wnt signalling seems to be active, is of particular interest. The total protein level indicates a significant reduction in the rate of Sox9 nuclear localisation detected by immunofluorescence in the ADGM-induced calcified samples. The addition of XAV-939 to the ADGM samples abrogated this reduction when compared to the DMEM-XAV-939 control. As expected, the higher SOX9 activity resulted in no induction of calcification. The relationship to Wnt/β-catenin signalling in this context and the phosphorylation status of Sox9 needs further investigation, as the phosphorylated protein form is active and localised in the cell nucleus .in vitro settings in basic research studies to define mechanistically important processes.XAV-939 is one member of a large set of small-molecule inhibitors of the Wnt/β-catenin pathway , 39, 40.in vitro cultured human VICs showing induced calcification with ADGM. Only 58% of the VIC preparations showed an induction of calcification during an incubation time of up to 35 days. Cell preparations are heterogeneous, consisting of various subpopulations, so the in vitro calcification processes are expected to differ. The rate of calcification induction can depend on individual culture preparations and their responsiveness to ADGM, as well as the time in culture. In addition aspects like gender of patients the VICs originate from and bi-vs. tricuspidal valve development have to be considered in studies investigating a larger sample set.The model system analysed in the present study was in vitro [in vitro calcification processes will require further culture experiments.Calcified AV exhibit an increased prevalence of cells positive for smooth muscle cell (SMC) markers . SMCs ofin vitro –48 and ain vitro . Other vin vitro . DetermiLimitations of the present study include the use of human VICs isolated from diseased human AV tissue and the heterogeneity of the isolated cell population. However, the use of human VICs instead of cells of porcine, bovine or ovine origin isolated from very young individuals may be advantageous, as most of the mechanistic studies on VICs have been performed on cellular material from other species , 50, 51.in vitro calcification of VICs induced by ADGM and is therefore a substance of interest, on the one hand, for the investigation of relevant molecular pathways involved in osteogenesis and, on the other hand, as a small molecule inhibitor that can be implemented in in vitro and in vivo analyses for further drug development.Both active β-catenin and Sox9 are downregulated in calcifying VICs and upregulated in VICs co-treated with XAV-939. Therefore, discovery of the regulatory molecular processes of this human VIC model will require evaluation of alternative regulatory models  as a next step. In addition, a need to identify the actual cell culture subpopulations that are responsible for the calcification process is envisioned. XAV-939 is able to prevent the S1 FigMTT-assay cell viability was not statistically significant altered after XAV-939 inhibitor incubation of VICs at different concentrations and an incubation time of 72h.(TIF)Click here for additional data file.S2 FigEquivalent to (TIF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.