Serious games for health have been gaining in popularity among scholars and practitioners. However, there remain a few questions to be addressed.This study tests the effects of a serious game and fear appeals on smoking-related outcomes. More specifically, this research aims to understand how serious games function as a more effective vehicle for a health campaign than a traditional medium, such as a print-based pamphlet. Further, while serious games utilize a variety of persuasive strategies in the game’s content, it is not clear whether fear appeals, which are widely used persuasive-message strategies for health, can be an effective strategy in serious games. Thus, we are testing the effect of fear appeals in a serious game.We created a computer game and a print brochure to educate participants about the risks of smoking. More specifically, a flash-based single-player game was developed in which players were asked to avoid cigarettes in the gameplay context. We also developed an online brochure based on existing smoking-related brochures at a university health center; antismoking messages on the computer game and in the brochure were comparable. Then, an experiment using a 2 (media type: game vs. print) x 2 (fearful image: fear vs. no-fear) between-subjects design was conducted. The study recruitment was announced to undergraduate students enrolled in a large, public Midwestern university in the United States. After a screening test, a total of 72 smokers, who reported smoking in the past 30 days, participated in the experiment.F=7.28; P=.009; η2p=0.10]. However, in the no-fear condition, there was no significant difference between the conditions . Similarly, the intention to quit smoking  and susceptibility  were also significantly different between the conditions, but only when fear appeals were used.Overall, gameplay, when compared to print-based pamphlets, had greater impacts on attitudes toward smoking and the intention to quit smoking. Further, the game’s persuasive effects were especially pronounced when messages contained fear appeals. When fearful images were presented, participants in the game condition reported significantly more negative attitudes toward social smoking than those in the print condition [This study extends fear appeal research by investigating the effects of different media types. It offers empirical evidence that a serious game can be an effective vehicle for fear appeals. Smoking is one of the most prevalent risky health behaviors. Currently, there are more than 35 million Americans that identify as smokers . AlthougSmoking is particularly serious among young adults. Statistics indicate that nearly 9 out of 10 smokers try their first cigarette by the age of 18 years . More thIn an attempt to resolve prevalent smoking behaviors, much effort has focused on antismoking campaigns and interventions. For example, researchers have investigated the smoker identity to better understand how a smoker’s self-identification is related to their attitude toward smoking . Also, tSerious games for health have been gaining popularity among scholars and practitioners. However, there remain a few questions to be addressed. First, there is a strong need to understand how serious games can be more persuasive compared to other media types, such as print pamphlets. Second, while serious games utilize a variety of persuasive strategies in the game content, it is not clear whether fear appeals, which are widely used persuasive-message strategies for health, can be an effective strategy in serious games. That is, collectively, little research has examined the effects of media types when fear appeals have been utilized in the game content. To fill in the gap, this study investigates the effectiveness of a serious game compared to a printed brochure in the context of fear appeals.Fear appeals are described as “a persuasive message that attempts to arouse the emotion of fear by depicting a personally relevant and significant threat” . That isFear appeals have specifically been used to combat health issues and produce positive outcomes; fear-based messages have been incorporated in diverse health campaign materials to persuade people to change their behaviors and attitudes toward making healthy choices . Song etAlthough fear has been studied for decades, questions remain. In particular, it is not clear whether a fear-based strategy would also work effectively in gaming contexts. The effects of fear appeals were examined in various media platforms ; however, the role of fear in interactive games  compared to other traditional media platforms  has not been fully addressed. In this regard, there is a need to understand how a fear-based strategy would work in games, compared to other traditional media platforms.Serious games are computer or video games designed for the primary purpose of educating users beyond entertainment purposes  -22. StudSeveral health education strategies are incorporated into serious games, including exposure therapy, behavioral rehearsal, and role-playing. The exposure therapy strategy provides repeated cues that trigger anxiety, often employed to diminish the anxiety or urge as a treatment for anxiety disorders such as acrophobia and arachnophobia -40. The Games, compared to traditional media, have several unique built-in features. In particular, games can provide powerful learning experiences to change real-life behaviors with structural features and mechanisms that enable individuals to have their own experiences of the daunting future. In fact, research has identified various benefits of games for learning -47. SpecSocial Cognitive Theory  explainsParticularly, smoking-related fear appeals have been primarily associated with vicarious learning  because Following the above-mentioned argument, it is important to note that serious games can offer the benefits of both enactive and vicarious learning. First, the center of action resides in the game player rather than the observations of another individual’s actions. During gameplay, a player uses an individualized avatar , and the player’s decisions and actions in the virtual environment have consequences for their avatar. In other words, players engage in enactive learning experiences through the body of the avatar. This process, conceptualized as “mediated enactive experience” , is a unSecond, serious games also offer a breadth of vicarious learning. Unlike experiences in the physical environment, game experiences are not limited in terms of space, time, and the number of potential counterparts with whom players can interact. In gameplay, players can vicariously engage in actions and behaviors that may not be easily manifested in the physical world. Accordingly, a serious game is a unique vehicle; it provides a combination of both enactive and vicarious learning, can increase effective learning outcomes from the gameplay, and has the potential to be an effective persuasive medium.Fear appeals can be effective persuasive tools; however, they can also fail to accomplish what they were intended to do. One of the main reasons fear appeals can fail is that the experience of fear sometimes leads an individual to avoid or ignore a message that generates a negative emotion rather than changing risky behaviors . ResearcE-E is a strategy that utilizes popular entertainment media  to educate audiences ,55. The For the same reason, people watch horror movies despite the negative emotions  they may experience, as the fun factor in the narratives holds an audience’s attention through the duration of the message ,58. For Messages can be more persuasive when they are tailored to the specific individual or group of people exposed to them. Tailoring is defined as “any combination of information or change strategies intended to reach one specific person based on characteristics unique to that person, related to the outcome of interest, and derived from an individual assessment” . Thus, aCompared to nontailored messages, tailored messages are more likely to be read, understood, recalled, and perceived as trustworthy; thereby, they have a greater potential for persuasion and behavioral change -69. PartAs tailoring/customization is afforded in many digital and video games , seriousIn fact, empirical research has found that people exposed to a more interactive message were more likely to change their attitudes . For insThis study compares the potential of a serious game to a traditional, print-based format regarding the use of fear appeals as a persuasive vehicle. More specifically, the current study hypothesizes that antismoking messages featured in a serious game will be more likely to positively influence smoking-related outcomes than a print-based brochure. Additionally, the study tests the role of fear appeals in serious games to understand whether media types would interplay with fear appeal strategies. To better understand the key aspects of smoking-related outcomes, the study specifically focuses on the following: attitudes toward smoking, the intention to quit smoking, and perceived susceptibility.Based on the aforementioned literature, the following 3-part hypothesis is proposed: Individuals playing an antismoking game report stronger levels of (1) negative attitudes toward social smoking, (2) intention to quit smoking, and (3) perceived susceptibility to smoking-induced risks than individuals reading an antismoking print-based pamphlet. The following research question is also proposed: What role does a fear appeal strategy play in a serious game compared to a print brochure for an antismoking health campaign?To identify eligible participants, a screening test was conducted online among undergraduate students enrolled in a large, public Midwestern university in the United States. Only individuals who reported smoking in the past 30 days were contacted. Thus, a total of 72 smokers were included in this study. Of the 72 participants, the sample comprised of 41 men (57%) and 31 women (43%), with a mean age of 21.40 (SD 5.14) years. The median smoking frequency was 2 to 3 times a week.We conducted an experiment to test the hypothesis and research question. In particular, a 2 (media type: game vs. print) x 2 (fearful image: present vs. not present) between-subjects design was employed. Participants were assigned into 1 of the 4 experimental conditions: the game-fear condition (n=16), the game–no fear condition (n=16), the print-fear condition (n=19), and the print-no-fear condition (n=21). To ensure that there were no initial differences among the experimental groups, several analyses were conducted regarding sex, age, and smoking frequency. Results found that there were no statistically significant differences between groups. Thus, group equivalence was ensured. Demographic information for each experimental group is presented in A series of steps were taken to develop materials for the experiment. First, a print brochure was created . To deveFear conditions were manipulated based on the smoker’s face. In the no-fear condition, only a young and healthy-looking face of a smoker was presented. However, in the fear condition, both a young, healthy-looking face of a smoker and an old, wrinkled face of the same smoker (which was caused by smoking) were presented.In the fear condition for the print brochure, one page of the pamphlet showed a young and healthy-looking face with the subtitle, “Go to the next page to see how smoking can change her.” On the following page, a discolored and wrinkled face was featured with a message stating, “Smoking can make you look 20 years older.” Further explanation was provided: “Besides having the lungs of a senior, by the time a smoker turns 40, they will have approximately as many wrinkles as nonsmokers in their 60s.” In the no-fear condition, only a young and healthy-looking face of a smoker was presented, and there was no mention of what smoking can change in a person’s face. Otherwise, all the textual and graphical messages remained the same as the fear condition.For the game condition , a compuIn the fear condition for the game, participants played with an avatar that had a young and healthy-looking face in Level 1. Then, they were told their avatar’s face would look aged in Level 2 due to smoking experienced in Level 1. In the no-fear condition, a young and healthy-looking face was used in both Levels 1 and 2. In order to avoid any potential confounding effects, the gender of the smoker’s face was matched with that of participants in both conditions. Also, the smoking-related content portrayed in the game was consistent with the print pamphlet.Upon the university’s Institutional Review Board’s approval, a recruitment message was distributed via the university’s email system. Interested participants contacted the researcher via an online survey system. Prior to the experiment, researchers conducted a pretest to assess baseline smoking-related attitudes and perceptions. Approximately 2 weeks after the completion of the pretest, half of the participants, who were randomly selected to a game condition, were invited to a physical lab where they played the game. The other half of the participants were invited to an online lab where they viewed the pamphlet. Upon completing the main task of the study, participants were instructed to complete a posttest.α=.77) were measured by 6 items . Intention to quit smoking (α=.89) was assessed using 8 items . All of the responses were obtained on a 10-point Likert-type scale .Negative attitudes toward social smoking (α=.77) and intention to quit smoking (α=.89) were measured in the posttest again, and the same items were used from the pretest. Susceptibility (α=.89) was measured by 6 items focusing on the negative consequences of smoking described in the antismoking messages . Due to the specificity of the measure related to the messages in both the game and print conditions, considerable test-sensitization issues were expected. Therefore, susceptibility was asked only once in the posttest. All of the measures were developed for the study, and the responses were obtained on a 10-point Likert-type scale .Negative attitudes toward social smoking  were conducted using SPSS software  to test the proposed hypothesis and the research question. Because attitudes towards social smoking and intention to quit smoking were measured at both pretests and posttests, they were tested based on the score difference between pretests and posttests (score=posttest – pretest). Therefore, positive scores indicate that posttest scores are higher than the pretest scores. Since susceptibility was measured in the posttest only, the final index was based on the posttest score.F=5.25; P=.03; η2p=0.07]. As for the second part of the hypothesis (hypothesis 2), there was no significant difference in the intention to quit smoking between the game condition  and the print condition  . With regard to the third part of the hypothesis (hypothesis 3), individuals in the game condition  reported a greater level of susceptibility  than those in the print condition . In sum, hypothesis 1 (attitudes) and hypothesis 3 (susceptibility) were supported, but hypothesis 2 (intention) was not. Regarding the first part of the hypothesis (hypothesis 1), individuals in the game condition , compared to those in the print condition , reported significantly more negative attitudes toward social smoking . However, the differences between the pretests and posttests appeared to be greater in the game condition compared to the print condition  to reduce any potential error rate. First, when fearful images were presented, participants in the game condition reported significantly more negative attitudes toward social smoking than those in the print condition . However, in the no-fear condition, there was no significant difference between the game and the print condition . Further, there was no significant difference between the fear and no-fear condition in the game condition  and in the print condition . In all, the result indicated that the effect of fear on negative attitudes toward social smoking was stronger in the game condition than the print condition.Further analyses were conducted to examine the simple main effects of fear and media types. As 2 different comparisons were conducted in each simple test, the decision of statistical significance was made based on the adjusted P value of .035  compared to those in the print condition. However, in the no-fear condition, there was no significant difference . Further, there were no significant differences between the fear and no-fear condition in the game condition  and in the print condition . The result found that the effect of fear on the intention to quit smoking was stronger in the game condition than the print condition, although the P value was at .035.Next, simple main effects on the intention to quit smoking were tested. In the fear condition, participants in the game condition reported greater intention at a F=6.92; P=.011; η2p= 0.09]. However, when fear was not presented, there was no significant difference between the 2 media types . Further, in the game condition, there was no significant difference between the fear and no-fear condition . Finally, in the print condition, people in the no-fear condition reported greater susceptibility than those in the fear condition . The results indicated that the effect of fear on susceptibility was stronger in the game condition than the print condition. Further, in the print condition, not using fear was more effective than using fear in increasing susceptibility  was comparable, it was not possible to control the time that each participant spent in their experimental condition. In particular, due to the nature of the medium, participants in the game condition may have spent a bit more time completing their tasks than those in the print condition. Even in the game condition, some participants may have finished the gameplay faster than others. In this regard, future researchers are encouraged to further investigate this issue.Next, participants in this study were all college students. While previous research has already identified social smoking as being a prevalent behavior among college students -9, many Moreover, the study focused on short-term effects only. Participants were asked their attitudes toward smoking only after interacting with either the game or the pamphlet. Thus, it is not clear whether these interactions would have a long-term effect on the participant’s behavior. Future research should conduct a longitudinal study to assess how effective games and pamphlets are at inducing behavioral changes, such as intentions to quit smoking, over time.Lastly, although this study did not investigate the role of narratives specifically, the study acknowledges that narratives in games play an important role, especially in persuasion . With thGames have great potential to expand fear appeal research with their unique features. As with media convergence, games are gradually merged into other media such as social media and social television. Thus, it is important to further investigate ways to utilize games merged into other media to expand the fear appeal literature. In this regard, this study sheds light on the use of serious games in the context of utilizing fear appeals.


Atoh1, a developmentally expressed basic helix-loop-helix (bHLH) transcription factor, reliably expresses CRE-dependent reporter genes in ∼60% of the IH and IF MNs. We determine that CRE-dependent expression in IH and IF MNs is ectopic because an Atoh1 mouse line driving FLPo recombinase does not label these MNs although other Atoh1-lineage neurons in the intermediate spinal cord are reliably identified. Furthermore, the CRE-dependent reporter expression is enriched in the IH and IF MN pools with much sparser labeling of other limb-innervating MN pools such as the tibialis anterior (TA), gastrocnemius (GS), quadricep (Q), and adductor (Ad). Lastly, we find that ectopic reporter expression begins postnatally and labels a mixture of α and γ-MNs. Altogether, the Atoh1 CRE knock-in mouse strain might be a useful tool to explore the function and connectivity of MNs involved in fine motor control when combined with other genetic or viral strategies that can restrict labeling specifically to the IH and IF MNs. Accordingly, we provide an example of sparse labeling of IH and IF MNs using an intersectional genetic approach.Motor neurons (MNs) innervating the digit muscles of the intrinsic hand (IH) and intrinsic foot (IF) control fine motor movements. The ability to reproducibly label specifically IH and IF MNs in mice would be a beneficial tool for studies focused on fine motor control. To this end, we find that a CRE knock-in mouse line of  Atoh1, serving as a useful genetic tool for future studies of fine motor control.Motor neurons (MNs) of the intrinsic hand (IH) and intrinsic foot (IF) are reproducibly labeled in an ectopic manner postnatally using a CRE knock-in mouse line of the basic helix-loop-helix (bHLH) transcription factor Motor neurons (MNs) innervating the muscles in the digits of the hand allow for exquisite control of fine motor movements required for dexterous skills, such as writing or sewing. Primates are known for their ability to precisely control individual digits of the hand, but skillful movements are not limited to the hand, as people born without arms are able to dexterously manipulate tools with their toes . PreciseAtoh1, a transiently expressed gene in the dorsal-most part of the developing neural tube that is also known to specify excitatory (+Vglut2) spinal cord neurons that project rostrally to the hindbrain  along with a ssDNA megamer for homologous recombination . Assembling CRISPR reagents and microinjections were performed as previously described  were fixed in 4% paraformaldehyde (PFA)/PBS for 2–3 h at 4°C. Early postnatal animals  or younger) were cooled on ice, decapitated, their spinal cords dissected out, and fixed in 4% PFA/PBS for 2 h at 4°C. Mice older than P14 were anesthetized with avertin  and transcardially perfused, first with 0.012% w/v Heparin/PBS and then 4% PFA/PBS. A dorsal or ventral laminectomy exposed the spinal cord to the fixative. The spinal cords were fixed for 2 h and the brains overnight at 4°C. Tissue was washed in PBS for at least 1 d and cryoprotected in 30% sucrose dissolved in deionized water. Tissue was marked with 1% Alcian Blue in 3% acetic acid on one side to keep orientation and were then embedded in OCT . Tissue was sectioned using a Leica CM1950 Cryostat.Embryos were timed as embryonic day (E)0.5 on the day the vaginal plug was detected and P0 on the day of birth. Pregnant females were euthanized with COCryosections (30 μm) were blocked with PBS/1–3% normal goat or donkey serum/0.3% Triton X-100 (Sigma) for up to 1 h at room temperature (RT) and incubated overnight with primary antibody at 4°C. After washing three times with PBS, the appropriate secondary antibody  was incubated for 1 h at RT. Sections were rinsed three times in PBS, mounted with Aquapolymount (Polysciences Inc.), and coverslipped (Fisher). The following primary antibodies and dilutions were used: 1:500 rabbit anti-dsRed (Clontech), 1:100 goat anti-choline acetyltransferase , 1:1000 rabbit anti-matrix metalloproteinase-9 , 1:8000 rabbit anti-HB9 , 1:3000 guinea pig anti-copine-4 (CPNE4) and 1:8000 guinea pig anti-fidgetin , 1:500 mouse anti-NEUN (Millipore Sigma), 1:100 mouse anti-ERR3 (R&D Systems), 1:3000 α-bungarotoxin (BTX) 488 (Invitrogen), 1:1000 rabbit anti-Syntaxin1 , and 1:1000 guinea pig anti-vesicular glutamate transporter 1 . Sections were referenced to the Christopher Reeves Spinal Cord Atlas .Fluorescent images were taken on a Zeiss LSM710 or LSM880 confocal microscope with an appropriate optical slice (0.5–10 μm) depending on the image. Images were pseudocolored using a magenta/green/blue or magenta/yellow/cyan color scheme using Adobe Photoshop (Adobe) or Fiji .Mice aged P14 were anesthetized using isoflurane and prepared for injections into muscle. An approximate total of 500–750 nl of cholera toxin subunit B (CTB) Alexa Fluor 488 or 647 conjugate  was injected into two to three different locations in the left forepaw (IH MN pool) or hindpaw (IF MN pool) in 50.6 nl increments. For injections into the tibialis anterior (TA), gastrocnemius (GS), quadricep (Q), or adductor (Ad), the area of the skin above the muscle was shaved and 70% ethanol and betadine  applied. An incision was made above the muscle and 500–750 nl of the CTB-conjugated Alexa Fluor was injected into three to four different locations directly into the muscle. The incision was closed with surgical glue . Carprofen (5 mg/kg) was administered daily 3 d after surgery. Spinal cords were harvested 5 d after injection. For injections at P0, mice were anesthetized with isoflurane and injected with <250 nl CTB-488 or 647 in one or two different locations of the forepaw and hindpaw and harvested 3 d later.n = 2, the SEM is equal to half of the range between the two data points.All details for number of sections counted, biological replicates, and male and female tissue analyzed are given in Results. No statistical tests were required as quantitation of the percentage of particular markers in any given MN pool were not directly compared with each other. Mean ± SEM are reported throughout. For samples with Cre/+Atoh1 knock-in mice crossed to tdTomato (TOM) reporter mice ) (Cre/+Atoh1 line labeled MNs of the IH and IF in thoracic 1 (T1) and lumbar 6 (L6) areas of the spinal cord )  that subnal cord . We injee system .Cre/+Atoh1 TOM+ MNs was specific to the IH and IF MN pools, we injected CTB-488 into the TA, GS, Q, and Ad muscles and found that those MN pools had much fewer TOM+ MNs : female (F), four to eight half sections/n; IF: 64 ± 3%, n = 5, 1:4 M:F, two to eight half sections/n; TA: 7 ± 2%, n = 4, 2:2 M:F, two half sections/n; GS: 14 ± 2%, n = 4, 2:2 M:F, two to four half sections/n; Q: 13 ± 4%, n = 4, 2:2 M:F, one to three half sections/n; Ad: 13 ± 3%, n = 4, 2:2 M:F, one to six half sections/n. MN areas located by CTB-488 injection into appropriate muscle group; + MNs in the IH and IF MN pools at P19 on one side is 410 ± 72 neurons for the IH MN pool  and 337 ± 7 SEM neurons for the IF MN pool . Counts of sections represented a tenth of the MN pool, so the estimated total number of ChAT+ neurons were the final counts multiplied by ten. Because the TOM+ MNs comprised ∼60% of the IH and IF MN pools, we hypothesized that the Cre/+Atoh1 mice might be labeling a particular functional class of MNs such as fast or slow twitch MNs. However, we found that only a subset of the IH and IF TOM+ MNs were labeled by the marker for fast MNs, MMP9, indicating that the IH and IF TOM+ MNs are a mixture of fast and slow MNs  in the ventral neural tube . Therefore, to corroborate the labeling of IH and IF TOM+ MNs in the Cre/+Atoh1 mouse line, we crossed these mice to an P2A-FLPo/+Atoh1 mouse and FLPo-dependent GFP reporter mouse (FSF-EGFP/+R26) such that neurons from the Cre/+Atoh1 mouse line were labeled with tdTomato and those from the P2A-FLPo/+Atoh1 mouse were labeled with EGFP. Strikingly, while the IH and IF MN pools were TOM+, they were clearly GFP–  oligodendrocyte transcription factor 2  of IH and IF MNs, we asked whether they were demarcating a specific type of MN (α or γ). α-MNs innervate the striated extrafusal muscle, are marked by the neuronal marker, NEUN, and receive VGLUT1+ proprioceptive inputs  and syntaxin (STX1+) identifies the muscle spindle . We found that labeling of the IH and IF MNs was sparse in the IRES-FLPo/+Chat mouse line alone with only ∼30% of the MN pool being labeled  and the lateral dorsal tegmentum (LDTg)  or identify the inputs to these MNs . One approach to isolate IH and IF MNs would be to use an intersectional genetic strategy crossing the MN pools  and in ACre/+Atoh1 mouse line to isolate the IH and IF MNs. For example, FLPo driven by Hb9, Fign, or Cpne4, which we have shown colocalize with the IH and IF MNs labeled in the Cre/+Atoh1 line, but not in other Atoh1-lineage neurons in the intermediate spinal cord  and an AAV-FLPo injected into either the spinal cord or in the forepaw or hindpaw to be taken up retrogradely to the IH and IF MNs. However, a caveat of this approach is the potential damage to the spinal cord or muscles of the forepaw and hindpaw because of the needle injection. Thus, appropriate injection controls would be needed with this approach.An alternate strategy would be to use viruses to restrict labeling to just the IH or IF MN pools. For example, the Cre/+Atoh1 mouse consistently labels MNs of the IH and IF and that the Cre/+Atoh1 mouse could be used to probe the function and connectivity of MNs in fine motor control. Our findings also serve as a cautionary tale of relying on CRE-recombinase mouse lines to faithfully report endogenous gene expression and speak to the need for careful follow-up experiments to appropriately interpret reporter expression.Altogether, we present here that the


It is misdiagnosed in up to 75% of cases by nondermatologists. Although the histopathological findings are well recognized and characteristic, diagnosis by fine-needle aspiration biopsy may be quite challenging. Several reports have emphasized the challenges in cytodiagnosis of pilomatrixoma, leading to a false-positive diagnosis. The lesions may show avidity for fludeoxyglucose on positron emission tomography/computed tomography scan, raising concern of a possible malignant neoplasm. CTNNB1 mutations have been reported in a high percentage of pilomatrixomas. Expression of β-catenin, the protein encoded by CTNNB1, is also frequently observed. To determine if routine cytological specimens can be successfully used to perform additional investigation and support or confirm the diagnosis in three cases of pilomatrixoma, we performed molecular analysis and immunohistochemistry to search for CTNNB1 mutation and β-catenin, respectively. β-Catenin positivity by immunohistochemistry was observed in basaloid cells in all three cases. Exon 3 mutations in CTNNB1 were detected in all cases. In addition, we detected a fibroblast growth factor receptor 2 (FGFR2) mutation in one of the cases. We reviewed the literature and present the clinical and morphological characteristics that must be considered along with other findings to accurately achieve the correct diagnosis, in correlation with the results of the ancillary technique. In conclusion, routine cytological specimens can be successfully used to perform additional investigations and support cytodiagnosis in difficult cases.Pilomatrixoma ( The exact prevalence is unknown, but pilomatrixoma accounts for approximately less than 1% of all benign skin tumors. It presents most frequently as a firm to hard intradermal/subcutaneous small tumor, in the head and neck, during the first two decades of life, with a slight predominance in females, and is misdiagnosed in up to 75% cases by nondermatologists [Pilomatrixoma, also known as ologists .The lesion may show avidity for fludeoxyglucose (FDG) on positron emission tomography/computed tomography (PET/CT) scan, raising concerns of a possible malignant neoplasm –6.The histopathological findings are well recognized and characteristic, but diagnosis by fine-needle aspiration biopsy (FNAB) may be quite challenging. In a retrospective analysis, Woyke et al. first described the cytological features of pilomatrixoma in six cases, four of which were misdiagnosed as malignant tumors .Since then, several reports, mostly short series, and isolated cases have emphasized the difficulty in cytodiagnosis of pilomatrixoma, which may lead to a false-positive diagnosis –48.β-catenin, is also frequently detected by immunostaining, but conflicting results have been reported [CTNNB1 mutation seems to be present in almost all cases of pilomatrixomas . The proreported –55.β-catenin protein expression was also detected in cell block preparations by immunohistochemistry. Besides reporting cytomorphological findings and reviewing the literature, our aim was to confirm that routine cytological specimens can be successfully used to perform additional investigations and support or confirm the diagnosis in difficult cases.We present three cases of pilomatrixoma diagnosed by FNAB in which we were able to confirm CTNNB1 mutations using routine cytological specimens. The All three cases involved women, aged 6, 13, and 38 years, presenting discrete painless nodules in the preauricular region, measuring 1.0, 1.4, and 1.4 cm, respectively; through clinical evaluation, these were initially interpreted to be of parotid gland origin.FNAB was performed using 23–25 gauge needles and conventional techniques. Ultrasound-guided punctures were performed through three passes per nodule in all cases. We systematically used different cytological preparations from the aspirated samples, including conventional smears and liquid-based and cell block specimens .Before the final diagnosis, correlation with ultrasound findings was helpful, as nodules in all cases seemed to be superficially located, outside the parotid gland, possibly in the skin, with a hyperechoic component, which is suggestive of calcification. In fact, the presence of calcification could even be inferred by lesion consistency during the procedure.The specimens showed adequate cellularity in all cases, and many cytomorphological aspects of the lesion could be appreciated in one or more of the preparations used. Figures The most important and constant finding was the presence of small basaloid epithelial cells, predominantly clustered, without peculiar arrangement, with slight size variation, but without significant atypia. Focal transition to elongated and keratinized cells was observed. Numerous cell elements without nuclei and amorphous material, consistent with “shadow cells,” were present in different amounts, with calcium deposits. Occasional, isolated, multinucleated giant cells and mild inflammatory components were also noted. No necrosis or significant mitotic activity was observed.Diagnosis was signed out as benign, basaloid epithelial neoplasia, with a note recommending close clinical correlation, suggesting the possibility of cutaneous/dermal lesions, originating from appendages, mostly pilomatrixoma.Two cases were surgically resected, and the diagnosis of pilomatrixoma was confirmed. The surgeon and the family decided to follow conservatively but observe closely the nodule from the youngest patient.β-catenin by immunohistochemistry. Formalin-fixed and paraffin-embedded cell blocks were sectioned, and 4 μm sections were processed on the Ventana Benchmark ULTRA automated stainer using a prediluted monoclonal mouse anti-human β-catenin antibody (clone 14), ultraView Universal DAB detection kit, and hematoxylin II counterstain, as recommended . Positive and negative controls were included. Immunohistochemistry was positive in basaloid cells from all cases, with different expression patterns; one case showed exclusively membranous staining, and in the other two cases, cytoplasmic staining, with equivocal, weak nuclear positivity, was observed. The transitional cells did not show positivity in any case.Cell block specimens were used to test the expression of Data from the described cases are summarized in Targeting amplicon panel contains 26 genes related to solid tumors. Cytological smears have already been established at the Molecular Pathology Laboratory of Hospital Sírio-Libanês (publication in progress). All genes present in the panel are listed in Supplementary One conventional smear containing 3,000–15,000 cells and a range of tumor cells between 60% and 80% was selected for DNA isolation from each case.The coverslip was removed with xylene, and then the slide was washed with xylene and 100% ethanol to remove any residual mounting medium. The entire smear was scraped off the slide with a razor blade and placed on a microtube to be digested with protease for 3 h at 56°C. DNA isolation was performed with QIAamp DNA Micro Kit (Qiagen), and DNA concentration was determined using a Qubit 2.0 fluorimeter (Life Technologies).DNA quality was assessed by amplifying an endogenous gene by the real-time PCR assay, which is included in the library construction kit . ΔCt <6 was considered acceptable to proceed with testing.Library construction was performed according to the manufacturer's instructions, and the libraries were sequenced at MiSeq (Illumina) using the v2 300 cycles cartridge. Data were analyzed using the MiSeq reporter for alignment to the human genome hg19, and the variants were identified on the Amplicon DS workflow provided by Illumina. The average read depth was higher than 20,000x for all samples.CTNNB1mutations were detected in all three cases. The two cases with cytoplasmic and equivocal nuclear staining presented the same CTNNB1 p.S33Fmutation (cases 1 and 2). In the case with a membranous staining pattern, CTNNB1p.S45F mutation was detected (Case 3). In Case 1, we also detected an FGFR 2p.S252L mutation.Exon 3 CTNNB1 mutations in all three cases of pilomatrixoma, using routine cytological specimens obtained by FNAB.We were able to detect exon 3 CTNNB1 encodes the β-catenin protein, which seems to possess a multifaceted nuclear function that may significantly impact the initiation and progression of numerous human tumors.CTNNB1, which affect the N-terminal segment of β-catenin, are found in at least 75% of human pilomatrixomas, leading to a constitutively overactive encoded protein. Transgenic mice expressing activated β-catenin are predisposed to the development of skin tumors resembling pilomatrixomas [β-catenin is a major cause of pilomatrixomas in humans.Somatic mutations in trixomas . This peCTNNB1 mutations reported in human cancers are localized in exon 3, and most of them occur at serine-threonine sites or on adjacent residues.Nearly all CTNNB1 mutations, p.S33F and p.S45F, are within the ubiquitination recognition motif of the CTNNB1 protein, at the Gsk3b phosphorylation site of β-catenin. S33F is predicted to confer a gain-of-function to the protein, as demonstrated by its nuclear accumulation [β-catenin and increases CTNNB1-dependent transcription [p.S33F mutation was detected in two cases, and p.S45F mutation was detected in another case. Both mulation –59. S45Fcription .CTNNB1 p.S45F mutation has not been reported previously in pilomatrixoma but was described in neoplasms of other sites, such as the colorectum, endometrium, stomach, liver, and central nervous system [As far as we are concerned, s system .FGFR2 p.S252L mutation in one case, which is a novel gene alteration that has not been reported previously in pilomatrixoma. FGFR comprises a family of related, but individually distinct, tyrosine kinase receptors. FGFR2 mutations have been reported in tumor specimens originating from the prostate, breast, lung, uterine, and ovarian tumors [Because our cases were submitted to a panel of genetic markers, we also detected an n tumors .UniProt.org) and has not been functionally or clinically validated but is considered to be likely oncogenic although its effect on FGFR2 protein function is unknown.FGFR2 p.S252L lies within the extracellular domain of FGFR2  family and the entry of these binding proteins into the nucleus. In the nucleus, β-catenin functions both as an activator and repressor of transcription in a context-dependent manner. It promotes cell proliferation and supports tumor growth by enhancing angiogenesis. β-Catenin-mediated signaling regulates cancer cell metabolism and has been associated with tumor-initiating cells in multiple malignancies. In addition, it functions both as a pro- and antiapoptotic factor, while also inhibiting the recruitment of inflammatory antitumor T cells [ T cells .β-catenin exists in three different pools: membranous, cytoplasmic, and nuclear. This has been demonstrated by immunohistochemistry of component cells in pilomatrixoma, presenting with membranous, cytoplasmic, and nuclear patterns, as expected.Consequently, cellular β-catenin immunoexpression in all three cases. The pattern of β-catenin expression in this type of lesion  and the cellular type  varies among different studies [β-catenin localization detected by immunostain and CTNNB1 mutation is controversial.Besides the CTNNB1 mutation, we observed  studies –55. Furtβ-catenin expression and CTNNB1 mutation since no mutations were detected in cases without β-catenin nuclear staining, suggesting that its nuclear localization could be considered a predictor of gene mutation [Moreno-Bueno et al.  and Hassmutation . Kim et mutation .β-catenin antibodies.These contrasting results may arise from technical differences, such as the stain method and/or anti-β-catenin in basaloid cells. Interestingly, in the case of p.S45F mutation, a membranous staining pattern of β-catenin was observed in the same cells.Two of our cases with p.S33F CTNNB1 mutation showed cytoplasmic and equivocal nuclear immunostaining of β-catenin [Nakamura and Fujiwara studied immunohistochemical localization of phosphorylated (pBC) and unphosphorylated (npBC) -catenin . They obβ-catenin degradation depends upon its phosphorylation in the WNT pathway.It is not possible to assess the status of the protein in our cases, whether phosphorylated or not, since we used a commercial antibody in our immunostaining, normally applied to a routine workflow for tumor diagnosis. This information could be interesting for further evaluation because β-catenin, as detected by immunohistochemistry but lead to the same gain-of-function of the protein.In the light of our observations, we speculate that another possible explanation for discrepancies of protein immunostain is that CTNNB1 mutations occurring in diverse and specific domains may confer different expression patterns of Our three cases of pilomatrixoma were diagnosed successfully by a combination of factors: awareness of the entity, usual clinical presentation, FNAB procedure, examination performed by the same cytopathologist, and maximum use of the obtained samples, through multiple preparation types, to facilitate the search for morphological findings, while still enabling additional studies.Ieni et al. discussed the limitations of FNA in a series of 25 cases, pointing out that the most dangerous mistake is diagnosing a primary or metastatic malignant cutaneous tumor , stressiA review of the literature focusing on FNA diagnosis of pilomatrixoma revealed almost 200 reported cases. The extracted data confirmed previous observations. There is a wide range of ages, from 2 months to 76 years, with a predominance of young age, mainly the first 2 decades. Females were more commonly affected, approximately twice, and the head and neck/upper extremity locations were far more common than other sites (6 times). The size of the lesions varied broadly, from 0.5 to 9.0 cm in diameter.Approximately one-third of the cases were misinterpreted, frequently as malignant 60%). Since PET/CT scan is now widely used in the workup of malignancy or suspicious lesions, and pilomatrixoma lesions may show intense abnormal FDG uptake, clinical correlation is essential because it is well established that false-positive results frequently occur secondary to nonmalignant processes that increase glucose uptakes, such as infection, inflammation, lymphoid follicular hyperplasia, and granulomatous diseases –6.0%. SinceFive publications presented a more significant series, totaling 87 cases , 39, 43.In the absence of all morphological findings or when one feature predominates, diagnosis can be very difficult , 27, 39.The use of different cytological preparations, including conventional smears and cell blocks, can be very helpful . The impFGFR2 gene mutation.Only two patients underwent surgery, and the cytological diagnosis was confirmed. Although the remaining case presented typical cytomorphological and clinical findings, and clinical follow-up with observation was initially considered, we contacted the responsible physician to communicate our results and possible long-term risks, after successfully demonstrating β-catenin mutations may result in the development of pilomatrixomas in younger patients, which over time, with additional mutations, could progress into malignancy and formation of pilomatrical carcinoma, which is reported in rare cases [Initiating re cases , 65. Pilβ-catenin is expressed in pilomatrixoma, but not in basal cell carcinoma, squamous carcinoma, and small cell carcinoma and other entities with similar morphology [β-catenin expression [Immunohistochemistry can be particularly helpful in limited biopsy specimens, including cell blocks obtained by FNAB, especially in difficult cases; for example, rphology . Similarpression .β-catenin, respectively, which could be valuable for differentiation from other entities and proper management of the patient.In conclusion, pathology is continuously incorporating knowledge through molecular diagnostics. By combining traditional and new technologies, pathologists integrate the morphological, clinical, and molecular characteristics of the disease. In recent years, the morphomolecular approach has a central role in diagnosis and therapeutics. Minimally invasive procedures such as FNAB are increasingly used to obtain tissue samples to study. We were able to establish a preoperative diagnosis of pilomatrixoma by FNA, which represents an attractive alternative for punch or excisional biopsy by being simple, noninvasive, fast, and accurate in experienced hands. In addition to routine cytopathological evaluation, FNA specimens can be successfully used to detect the molecular and protein expression of CTNNB1 and


However, higher values of the strain intensity were obtained during the deformation of the material in flat anvils. The purpose of the research was to propose assumptions for forging technology of producing a blank from AZ91 alloy with the use of flat and shaped anvils. Numerical examination for AZ91 magnesium alloy was carried out using the Forge®NxT commercial software. The rheological properties of the investigated alloy were determined on the basis of uniaxial compression tests carried out in the Gleeble 3800 metallurgical simulation system. The numerical analysis of the process of forging AZ91 alloy ingots on a press was conducted in the temperature range of 200–400 °C and at several forging passes.The paper presents the results of numerical modelling of the forging process of magnesium alloy ingots on a hydraulic press with the use of flat and shaped anvils. The use of shaped ( The use of numerical research with the finite element method in the design of engineering structures is very important from the point of view of the economics process as well as the design and implementation of the technology of manufacturing semifinished products [The unabated search for lightweight structural materials characterized by favourable strength properties in recent years results in increased interest in magnesium alloys ,7,8,9,10products ,15. Undeproducts ,17,18,19The objective of the study was to determine temperature distributions and strain fields in AZ91 magnesium alloy bars elongated with the open die forging method, in flat and shaped dies, based on the model tests of these technological processes.®NxT commercial software (version 2.0) based on the FEM, which enables tracking temperature and strain intensity changes in metal during plastic forming, at any forging stage. The analysis of numerical test results will be used as a base to develop guidelines for the production of magnesium alloys bars using the open die forging method, in flat and shaped dies. The test results should contribute to improving the structural and mechanical properties of AZ91 magnesium alloy bars through the appropriate selection of the process parameters for open die forging in flat and shaped dies.Forging was modelled using the ForgePlastometric tests were also conducted and used as a base to develop stress-true strain relationship graphs for the AZ91 alloy and to match flow stress function coefficients, which were used in the numerical study.The strain, strain rate and temperature ranges were adopted during the theoretical studies based on the characteristics of the machines used within real forging process and source literature data ,20.The material chosen for the tests was magnesium alloy AZ91 with chemical compositions as given in pσ and limit strain The basic feature characterizing the plastic formability of material in plastic working processes is the flow stress pσ, which is the stress necessary to initiate and continue the metal plastic flow in uniaxial stress conditions is a function of strain (ε), strain rate (T) and strain course history.The flow stress Determining the process plasticity characteristics is particularly difficult in hot plastic working conditions, since the material structure simultaneously experiences processes arising from the plastic strain mechanism, as well as strengthening processes, heat activated processes and processes depending on the phenomenon duration, leading to material weakening.pσ values are determined based on the adopted flow stress function. Most often, flow stress is expressed by a relationship in the form of pσ values, depending on the strain ε, temperature T and strain rate pσ value in software for the numerical modelling of plastic working processes [pσ is the flow stress, ε is the true strain, T is the temperature, m1–m9 are coefficients characterizing magnesium alloys.Within the available software intended for solving problems in the field of metal plastic flow or for calculating the strain forces and powers using the finite element method, the flow stress tion (1) . This rerocesses :(1)σp=AeIn the paper, the rheological properties of the AZ91 magnesium alloy were determined based on compression tests conducted with a Gleeble 3800 simulator  for metallurgical processes, which is owned by the Department of Plastic Working and Safety Engineering of the Częstochowa University of Technology . The GleThe advantage of an uniaxial compression test at an elevated temperature is the fact that the data on the true stress relative to the true strain can be obtained for a significantly wider strain range compared to the strains studied, e.g., by the tensile test.The phenomenon of friction occurs in almost each case of the uniaxial compression tests, and the upsetting tools are subject to slight cooling. Using Gleeble 3800 simulator enables minimizing friction through the application of lubricants and a simultaneous reheating of the tools.sample temperature: 200 °C, 300 °C, 400 °C,−1, 1 s−1, 10 s−1,strain rate: 0.1 strue strain: max. 0.8.The plastometric tests were conducted with the following parameters:The samples were heated at a constant rate of 5 °C/s, up to a preset temperature, held at this temperature for 20 s and then compressed. The diagram for conducted tests is shown in Graphs showing the dependence of flow stress from true strain for the AZ91 magnesium alloy were developed based on the conducted plastometric tests a–c. AfteThe data showed in The first stage of engineering a technological forging process is the proper selection of shape and dimension parameters of the dies. The geometric shape and dimensions of the die forging tools were selected based on a literature study for the purposes of the research paper subject and previous studies conducted at the Department of Plastic Working and Safety Engineering of the Czestochowa University of Technology ,31,32,33Shapes and dimensions of dies used for the theoretical tests of the forging process are shown in Main process parameters of the forging operation were selected based on our previous research and source literature ,31,32,33®NxT software, which allows for the thermo-mechanical simulation of plastic working processes, was selected for the numerical modelling of the AZ91 magnesium alloy forging process. A detailed description of the temperature, power, and stress and strain functionalities, as well as the thermo-mechanical and friction laws used for the calculations can be found in the research paper [Forgech paper . The Galch paper , whereasThe simulations for the purposes of the research paper utilized a thermo-viscoplastic model of a deformed body, based on the theory of high plastic strains.l = 80 mm. The number of nodes adopted for the simulation equalled 9116, while the number of tetrahedral finite elements was 41,568.The simulation adopted a cylindrical reference batch with a square section of 80 mm × 80 mm and a length The friction coefficient value adopted pursuant to Coulomb’s law was μ = 0.3.1 = 10,000 W/m2K, while the coefficient of heat transfer between the metal and the environment was λ2 = 10 W/m2K. The study adopted an ambient temperature of 25 °C and a die temperature of 25 °C. The initial batch temperature before the elongation, throughout its entire volume was adopted as uniform and equal to 400 °C [The assumed coefficient of heat transfer between the dies and material amounted to λo 400 °C .The paper analyses the metal strain intensity and temperature distributions within the hot forging process involving AZ91 magnesium alloy bars. The results of temperature distribution from numerical tests are shown in The objective of the conducted analysis was to determine the temperature changes in a forged alloy, especially in areas adjacent to the areas interfacing with dies. Too high material cooling in the contact zone may cause the appearance of cracks and delaminations of the deformed material. Furthermore, the analysis also included the temperature distribution throughout the volume of the elongated bar, in order to determine the material temperature changes in the course of the forging process. Excessive decrease of the material temperature hinders the plastic flow of the metal within the open die forging processes and contributes to the formation of cracks.The data included in l = 80 mm.Analysis of the data in The data in When analysing the temperature field distribution throughout the surface of a forged forging c, it canThe data in In the initial forging operations, prior to rotating the forging by 90°, as a result of using trapezoid-rhombic dies, it was impossible to introduce higher forging reductions for bars with a diameter φ = 80 mm, because the hammer block would already be in contact with the lower die. This is associated with their geometry and dimensions, compared to flat dies, where there are only two parallel working surfaces. In the case of trapezoid-rhombic dies, the material-tools contact surface expands, the forging free surface is significantly smaller and the dies also impact the lateral surfaces of the strained bar a. Only aWhen analysing the data in The forging in trapezoid-rhombic dies, due to the restricted free flow of the material caused by die shape, can be compared to the die forging process.The analysis of the data in The data in Based on the analysis of temperature distributions presented in The data in Based on the data included in The data in Based on the analysis of the data in The data in Based on the data in The data in Based on the data in The data in The analysis of the data in The data included in The analysis of the data shown in In economic and process terms, it is rational to conduct AZ91 magnesium alloy bar elongation operations in trapezoid-rhombic dies (designed by the authors), since it leads to reducing the number of forging passes from six in the case of flat dies, to four in the case of shaped dies. A reduced number of forging passes does not influence the end dimension of a finished product.The application of shaped dies for elongation operations, compared to conducting the process in flat dies, leads to obtaining a more uniform temperature distribution between the central forging part and the alloy areas by the material-tool contact surfaces, which impact a better plastic flow of the material.When conducting elongation operations in both flat, as well as shaped dies, there was a significant drop in the bar temperature in areas adjacent to the material-tool contact surface. The cause behind this decrease was transferring heat to unheated dies. The forging areas near the action zones of dies, after cooling and further heat treatment are treated as machining losses.A very uniform distribution of the strain intensity value can be found throughout the entire bar during elongation in trapezoid-rhombic dies. However, strain intensity values are lower compared to the values obtained when elongation in flat dies. Higher strain intensity value proves good material processing, which results in obtaining better mechanical properties for the finished product.The following final conclusions were drawn based on analysing the results of conducted tests:trapezoid-rhombic dies should be used for the initial forging forming stages, since they can provide a uniform distribution of strain intensity,it seems justified to use flat dies for the ultimate forging forming stages, since they provide higher strain intensity values within the volume of the strained bar,a forging reduction of 40% shall be applied for the initial forging forming stages, due to the shape of used dies,a forging reduction of 40% can be applied in the final stages,a relative feed of 0.8 shall be applied for the elongation operations,the initial bar temperature assumed for the numerical simulations proved correct, since there was no significant material cooling observed in the course of the elongation operation, which could result in decreased flow stress of the strained alloy,when elongation using both flat, as well as trapezoid-rhombic dies, apply forging turning of 90°.The following assumptions for the forging technology were suggested when analysing the AZ91 alloy bar elongation in flat and trapezoid-rhombic dies:


Sylvia atricapilla) during their autumn migration, and quantified their crown reflectance. We detected robust correlations between migratory and molt condition indices, and the correlation structure was similar in the two sexes. Furthermore, the across‐season measure of body condition was positively related to the darkness of the black crown in males, while being unrelated to reflectance traits of the reddish crown in females. Our results reinforce the possibility that some melanin‐based ornaments may be year‐round indicators of individual quality via their dependence on nutritional condition.Sexually dimorphic ornamental traits are widely regarded as indicators of nutritional condition. However, variation of nutritional condition outside the reproductive and the ornament production seasons has rarely been considered, although it affects the generality of information content, especially for ornaments that may be used across the year. We measured several indicators of migratory and molt condition in male and female blackcaps ( We found a positive correlation between the expression of a melanin‐based plumage ornament and an across‐season measure of body condition. These findings support a mixed interpretation of the information content of melanin ornamentation, with the simultaneous presence of condition‐dependence and pleiotropy. Costs of mate choice  necessitate benefits that outweigh these costs. A major group of benefits arises from condition‐dependence, which means the adjustment of signals to the physiological state of the individual, as a consequence of the costs of expressing elaborate signal values  during their autumn migration  in relation to aspects of body condition during the summer molt and actual body condition at migration. Blackcap cap reflectance has rarely been examined as a signal trait and little is therefore known about its phenotypic variability and possible information content .2.1We sampled migrating blackcaps at Ócsa Bird Ringing Station, Hungary . The second measure taken from these feathers was feather mass, a measure of overall material investment into a single rectrix by the bird . Feather mass as a second feather growth axis may provide important additional information over feather growth rate, for example, due to the repeatedly demonstrated trade‐off between growth rate and feather quality  in the middle section of the feather under direct illumination, using a calliper (nearest 0.1 mm). Feather growth bar width is an index of feather growth rate and thereby body condition during molt . We first constructed the correlation matrix of all six condition measures across seasons for males and females, and compared these matrices between sexes using the software CPC . These variables were calculated directly from raw spectral data instead of relying on the results of the spectral band PCA because, while well representing the main directions of reflectance differences, spectral band PCs seem to provide an inaccurate representation of the relative contributions of particular wavelengths to spectral shape variation . Before using the first condition PC as an independent variable across sexes, it was standardized  for year in females and for the whole group in males. The second condition PC was standardized for year. We finally assessed the relationship between condition PCs and the reflectance measures using Pearson correlations  and a general linear model . The latter model consisted of brightness as a dependent variable, sex as a factor, the two standardized condition PCs as continuous predictors, and the two‐way interactions between the sex and the PCs. Statistical analyses were done in Statistica 5.5 .We included body mass, fat score, and muscle score as measures of actual, migratory condition . PC1 (standardized as described in the Methods) showed a significant interaction with sex in determining crown brightness , but not significant in females . PC2 (year‐standardized), on the other hand, showed little relationship with crown brightness in either sex  was unrelated to our main condition axis. Interestingly, our finding is consistent with the stimulatory effects of experimental antioxidant intake on male but not female crown color in the same species  and pheomelanin in females ; Data curation ; Formal analysis ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Supervision ; Writing‐original draft ; Writing‐review & editing . Miklós Laczi: Conceptualization ; Data curation ; Formal analysis ; Investigation ; Writing‐review & editing . Dóra Kötél: Data curation ; Investigation ; Writing‐review & editing . Tibor Csörgő: Data curation ; Investigation ; Writing‐review & editing .Appendix S1Click here for additional data file.


This work investigates the molecular interactions within the main triacylglycerols constitutive of palm oil, all having a key role in the multi-step dry fractionation process. Identification of these interactions is possible thanks to the establishment of binary and ternary phase diagrams, using differential scanning calorimetry (DSC) and powder X-ray diffraction (XRD) at variable temperature. The following systems were selected: PPP-POP, PPP-OPP, PPP-POO, POP-OPP, POP-POO, OPP-POO, PPP-POP-POO and PPP-OPP-POO , and analyzed in direct mode , and after tempering for three months at 20 °C (tempered mode). DSC makes it possible to bring out crystallization and melting phenomena associated to polymorphic transitions, which are further characterized  by XRD. The results show that unsaturated are poorly soluble in fully saturated triacylglycerols, that the intersolubility decreases in proportion to the number of unsaturated fatty acids, that positional isomerism (POP/OPP) has a major impact, that OPP may induce formation of molecular compounds and that co-crystallization properties are highly modified by tempering depending on the polymorphic properties of the systems. This provides a better understanding and allows for effective control of the palm oil dry fractionation process. In palm oil, tripalmitin (PPP), oleo-dipalmitin (P2O), palmito-diolein (PO2) and trilolein (OOO) are the most abundant TAGs of each class. Mono- and di-unsaturated TAGs are found to be symmetrical (StUSt and UStU) or asymmetrical (UStSt and StUU); their relative proportion in crude palm oil is generally constant [2U, StU2 or UUU; their TAG composition can be quantified by reverse phase high performance liquid chromatography (RP-HPLC). The iodine value (IV) refers to the unsaturation degree of a fat and can be determined by titration or calculated from gas chromatography (GC); typical IV for palm oil ranges from 51 to 53 while the IV of the highest melting point fraction (super-stearin) is usually below 15 and the IV of the most liquid fraction (top-olein) can exceed 72 [Palm oil is the most widely consumed edible oil, with Indonesia and Malaysia by far the largest producing countries. Most of the oil is used for food applications such as frying, cooking, salad oils, margarines, spreads, confectionary fats, etc. Palm oil is a semi-solid fat with a melting point above 35 °C. Both palmitic P: C16:0) and oleic O: C18:1) fatty acids contribute for about 85% of the total fatty acid composition. Triacylglycerols (TAGs) can be classified according to their saturation: tri-saturated (StStSt), mono-unsaturated  was a Supelco  product. Purity, verified by gas chromatography (GC), was above 99%. Furthermore, 2-oleo-dipalmitin (POP), 1-oleo-dipalmitin (OPP) and 1-palmito-diolein (POO) were synthesized and purified at Gembloux Agro-Bio Tech  based on a procedure described by Elisabettini et al. ; purity,v/v) with a flow rate of 1.2 mL/min; the injection volume was 20 µL. The samples were dissolved in methanol/chloroform (1/1 v/v) and a differential refractometer was used for the detection. This method allows quantification of individual triacylglycerols which are eluted according to their partition number (PN). PN = CN − 2(DB), where CN is the total carbon number and DB is the total double bonds for each fatty acid. For example, in the group of PN = 48, OOO, PO2, P2O and PPP elute separately and in sequence. The assignment was confirmed by comparing retention times with those of some standards . Peaks were integrated with Empower Pro “Apex Track” algorithm; peak areas below 4000 area counts  were not taken into account.The triacylglycerol composition was analyzed by reverse phase HLPC based on the AOCS Ce 5b-89 recommended method which does not distinguish between positional isomers . Analyse2 at a pressure of 9.08 PSI and a flow of 0.7 mL/min. The injection volume was 1 µL. Before injection, the triacylglycerols were converted into fatty acid methyl esters based on the AOCS Ce 2-66 recommended method and the iodine value was calculated according to the AOCS Cd 1c-85 recommended method [The iodine value was determined using gas chromatography (GC) based on the AOCS Ce-1h-05 recommended method . Analysed method ,36.DSC measurements were performed with a Setaram DSC 111 , using liquid nitrogen as cooling device. Samples of about 10 mg were used, with an empty pan as reference. Calibration was made with indium and nitrogen was used as purge gas. Melting profiles were recorded at 5 °C/min according to two different procedures (direct mode and tempered mode).Endothermic and exothermic temperatures reported in the phase diagrams correspond to maximal temperature of each peak (Tp).Samples were first heated up to 80 °C for 10 min to ensure complete melting and thermal memory erasure. Afterwards, they were quickly frozen (quenched) at −40 °C (−25 °C/min) and kept for 10 min at this temperature for complete solidification. The reported melting profiles were recorded from −40 to 80 °C at a heating rate of 5 °C/min.The DSC profiles of the same samples were recorded after three months of tempering at 20 °C. The objective of this tempering was not to stabilize all blends in their most stable polymorphic form but to compare crystallization properties evolution in identical operating conditions. For that purpose, samples were first heated up to 80 °C for 10 min, quickly frozen (quenched) at −40 °C (−25 °C/min), kept for 10 min at this temperature and heated to 20 °C at 5 °C/min. DSC pans were then removed and tempered in an oven stabilized at 20 ± 1 °C. After three months, the pans were further placed in the DSC, quickly frozen (quenching) at −40 °C (−25 °C/min), kept for 10 min at this temperature and melted; the reported melting profiles were recorded from −40 to 80 °C at a heating rate of 5 °C/min.α Cu: 1.54178 Å) equipped with a temperature control system  was used. Temperature control was performed with a TTK Anton Paar system  coupled with cold ethanol circulation using a Huber HS 60 cryostat . Aluminum sample pans contained about 100 mg of material. A gaseous nitrogen flow prevented condensation of water during measurements at low temperatures. Polymorphism was identified at selected temperatures based on the corresponding DSC profiles (direct and tempered modes). Short  and long  spacings were analyzed in separate runs. Short spacings make it possible to differentiate polymorphic forms  while long spacings give information on the type of stacking (double-chain length L-2 or triple-chain length L-3) [Polymorphism was investigated by powder X-ray diffraction. A Philips  PW 1050 diffractometer (Kgth L-3) ,38. Thisgth L-3) . The L-2gth L-3) ,41. A scgth L-3) .The investigated binary and ternary mixtures were prepared by mixing melted pure triacylglycerols in the appropriate weight, directly in the DSC pans; pans were immediately sealed. The compositions reported are expressed as weight fractions. The phase diagrams were constructed thanks to DSC heating thermograms (melting and transition temperatures) and X-ray powder diffraction patterns (short and long spacings) The black circles in the binary diagrams correspond to melting temperatures (endotherms) and the open squares on circles to transition temperatures (exotherms). The ternary diagrams are presented as melting iso-lines; they connect high melting and low melting points of binary and ternary systems.2O and PO2, the main representatives of mono-saturated, mono- unsaturated and di-unsaturated TAGs, are reported in 2O; the solid fractions (stearin and super-stearin) are particularly enriched in PPP and the palm mid fractions concentrate P2O.The industrial multi-step dry fractionation process of palm oil involves three steps and can be conducted along three routes : the liq2O play an essential role in what concerns the physicochemical characteristics of the palm liquid fractions: improving the cold stability is possible thanks to PPP removal in the olein and to P2O reduction in the super- and top-olein [2O and its reduction in the super-olein; this is characterized by quite important heat release that cannot be easily balanced by the cooling medium of the fractionation equipment. A similar critical zone is observed during hard PMF production, sometimes limiting the performance of this route [In practice, PPP and Pop-olein . On the op-olein ; this isis route . The POPMelting and transition temperatures and polymorphic forms observed in direct mode, last melting temperatures and polymorphic forms observed in tempered mode for pure PPP, POP, OPP and POO are presented in ′-2 to β-2 transitions; the α-2 to β′-2 transition is melt-mediated, in accordance with literature data [′-2; the α-2 to β′-2 transition does not appear to be melt-mediated. The X-ray powder diffraction pattern shows the existence of two types of β′ forms, both in double-chain length: sub-β′-2 and β′-2. These β′ forms were already reported by Lutton et al. [′-3, in accordance with data reported by Minato et al. [′-3 transition is melt-mediated. POO crystallizes in sub-α-3, α-3 and β′-3; only one β′-3 is observed under the investigated conditions, contrary to Bayes-Garcia et al. who reported 2 distinct β′ forms [Upon heating, PPP shows a succession of endo- and exotherms corresponding to α-2 to βure data . Only onure data . In the n et al. , and latn et al. . The asyo et al.  and Mizoo et al. . The α-3β′ forms ,49. The In direct mode, β polymorphism (double-chain length L-2) is only observed for the fully saturated PPP; the symmetrical POP crystallizes in double-chain length while the asymmetrical OPP and POO solidify in triple-chain length L-3. Various molecular arrangements derived from powder XRD are proposed in ′-2 to β-2; the last melting point in β-2 is almost not changed. The final melting temperatures evolve from 27.5 °C for POP in β′-2 and 31.2 °C for OPP in β′-3, to 34.2 °C for POP in β-3 (Δ = 6.7 °C) and to 32.6 °C for OPP in a more compact β′3 form (Δ = 1.4 °C)  .The β-3 In order to elucidate the co-crystallization properties of PPP, POP, OPP and POO, the next binary systems: PPP/POP, PPP/OPP, PPP/POO, POP/OPP, POP/POO and OPP/POO were studied both in direct and in tempered mode. The fatty acids involved are differentiated by the chain length (C:16 vs. C:18) and the presence of a cis double bond (O vs. P), the positional isomerism and the number of mono-unsaturated fatty acids on the glycerol.The PPP-POP, PPP-OPP, PPP-POO, POP-OPP, POP-POO and OPP-POO phase diagrams obtained in direct mode are presented in SS (D), which is transformed into β′-2 (C) upon heating. A monotectic line is observed at ~27 °C which corresponds to the melting of significant amounts of POP β′-2 and the occurrence of mixed crystals (MC): (PPP-POP) MC in β′-2 (B) with liquid. These mixed crystals are then transformed into (PPP-POP) MC β-2 (A) (transition point) before final melting. Quantities of POP co-crystallizing with PPP in (PPP-POP) MC β′-2 and in (PPP-POP) MC β-2 are ~15% (blue dotted circles).As pure components, these two triacylglycerols crystallize in double-chain length and the difference in melting point is higher than 35 °C. It is therefore not surprising that all the blends also solidify in L-2, showing monotectic behavior for the final melting. After quenching, PPP/POP blends solidify in α-2 as a solid solution (SS): (PPP-POP) ′-2 to β-2 transition of mixed crystals (transition point); however, contrary to POP, OPP exhibits an L-3 polymorphism. Despite this, after quenching, PPP/OPP blends first solidify in sub-α-2 as (PPP-OPP) SS (F) which transforms into α-2 (E) and then β′2 (D), upon heating. In this case, exothermic DSC peaks are observed just below the monotectic line and attributed to a transformation of (PPP-OPP) SS β′2 into a mix of (PPP-OPP) MC β′-2 (B) and (OPP- PPP) MC β′-3 (C), already in the solid state. This melts upon heating and (PPP-OPP) MC β′-2 (B) further transforms into β–2 (A) before final melting. Quantities of OPP co-crystallizing with PPP in (PPP-OPP) MC β′-2 and β-2 reach ~25–45% (blue dotted circles) that is far above the maximal content of POP in PPP.The PPP/OPP phase diagram is somewhat similar to PPP/POP in that they are both of monotectic type (similar difference in melting point) with a β′-2 into β-2 transition is no longer observed in the monotectic zone. The monotectic line (at ~18 °C) goes along with the occurrence of (PPP-POO) MC (A) directly crystallizing in β-2. Indeed, after melting and quenching, PPP/POO blends solidify as (PPP-POO) SS sub-α-2 (D) which transform into α-2 (C) and into β′-2 (B) upon heating; this solid solution quickly transforms into (PPP-POO) MC β-2. Quantity of POO co-crystallizing with PPP in (PPP-POO) MC β-2 is very low: ~5% (blue dotted circles).POO solidifies in L-3 like OPP but the difference in melting point with PPP is higher (superior to 45 °C); this makes the interactions also of monotectic type but the βMoC crystallizing in β′ double-chain length (melting point ~22 °C) (MC in β′-2 (A) and liquid. On the right side of the molecular compound, the solid solution α-2 (D) transforms into (OPP/POP) MC β′-2 (C) becoming β–2 later (B) in the presence of liquid.The difference in melting point between POP and OPP is lower than 4 °C. POP solidifies in L-2 while OPP does in L-3. Specificity of this binary phase diagram is a minimum observed in melting temperature at ~50/50 concentration ratio, which can be attributed to the formation of a molecular compound (OPP-POP)  ~22 °C) . On the ′-2 .Difference in melting point between POP and POO is ~15 °C; POP exhibit L-2 and POO L-3 polymorphism. Surprisingly, after quenching, the blends crystallize in L-2 as solid solution  all overMoC β′-2 (MoC β′-2 (B) and to the occurrence of (OPP/POO) MC β′-2 (A) and liquid. Contrariwise, the right side presents a kind of eutectic interaction, another stable structure, at higher amounts of POO. A line can be drawn at ~12 °C corresponding to the melting of (OPP-POO) SS β′-2 (C) and its transformation into (OPP/POO) MoC β′-2 (B) and liquid. It should be noted that, although both triacylglycerols solidify in L-3 as pure, the triple chain length structure is only observed after quenching at low temperature, in sub–α and in α: (OPP-POO) SS sub-α-3 (F) and (OPP-POO) SS α-3 (E); this solid solution transforms into α-2 (D) and β′-2 (C) upon heating.This binary phase diagram is significantly more complex compared to POP/POO. The melting point difference is ~12 °C and both pure OPP and POO solidify in L-3. The OPP/POO binary system shows some peculiar crystallization features, and in this case, at two specific compositions: at 50/50 and 25/75 concentration ratios. The first peculiar behavior appears as a break in the melting curve (50/50) and the second (25/75) as a minimum. Melting behaviors on the left and right sides of the 50/50 concentration which correspond to a molecular compound (OPP/POO) MoC β′-2  are compThe PPP-POP, PPP-OPP, PPP-POO, POP-OPP, POP-POO and OPP-POO phase diagrams obtained in tempered mode are presented in ′-2 into β–3 while PPP stays in α-2 at the tempering temperature (20 °C). Global effect is that α-2 mixed crystals, β–2 mixed crystals and blends of α-2/β-2 mixed crystals and β′-2/β-2 mixed crystals are observed within well-defined areas . At high concentration (right side of the phase diagram), POP is excluded from (PPP-POP) MC β′-2 and crystallizes separately as (POP-PPP) MC β-3. Solubility of POP within (PPP-POP) MC is comparable to direct mode: ~45–55% in the β′ and β forms (blue dotted circles).With the tempered conditions used, the PPP/POP binary system remains of the monotectic type with a transition line at ~35 °C (and a transition point). POP transforms from β′-3 form. As for PPP/POP binary system, α-2 and β-2 mixed crystals, blends of α-2/β-2 and of β′-2/β-2 mixed crystals  are observed within well-defined areas. At higher concentration, OPP is excluded from (PPP-OPP) MC β′-2 and crystallizes separately as (OPP-PPP) MC β-3. Solubility of OPP within (PPP-OPP) MC in β′ and β is increased to 45 and 60%, respectively (blue dotted circles).The PPP/OPP binary system is also of monotectic type in the tempered mode, with a transition line at ~35 °C (and a transition point). The tempering used does not highly affect OPP polymorphism which remains in β′-3 forms  (consequence of quenching of the liquid POO). It is mixed with (PPP-POO) MC β-2 (A). A small portion at the left of the phase diagram is showing (PPP-POO) MC β′-2 (B). The solubility of POO in PPP (~10%) is somewhat increased after tempering (blue dotted circles).Considering that PPP remains in α-2 form and that POO is liquid at the tempering temperature (20 °C), the tempered PPP/POO binary system looks very similar to the non-tempered one, excepted that POO crystallizes separately under sub-α-3, α-3 and β′-2 into β–3 and OPP stabilizes in β′-3. As for the non-tempered system, there is still a difference between the left and the right side of the phase diagram, the cut-off also being at ~50/50 ratio, composition of the molecular compound (OPP-POP) MoC observed in dynamic mode. Melting point (~29 °C) of this (OPP-POP) MoC is higher compared to the non-tempered (OPP-POP) MoC (~16 °C), due to β-3 stabilization. One single endotherm is observed at the left side of the molecular compound and two endotherms at the right side. L-3 packings are present within the whole compositional range, mostly corresponding to (POP-OPP) MC β-3 (A) and (OPP-POP) MC β′-3 (B).After the tempering used, both POP and OPP crystallize in L-3: POP transforms from β′-3(C) mainly at the right part of the system, due to solidification upon quenching. For other compositions, POP/POO ss β′-2 clearly transforms into β′-3: (POP-POO) MC β′-3 (B) is formed. The melting line (~24 °C) (and transition point) corresponds to the transformation into (POP-POO) MC β-3 (A). Solubility of POO into POP as β′-3 MC is ~10% (blue dotted lines).POP stabilizes in β-3, but POO and part of the POO enriched blends were liquid at the tempering temperature (20 °C). This explains the occurrence of POO in sub-α-3 (E), α-3 (D) and β′-3 at the tempering temperature and part of the blends is partially liquid. The result is the occurrence of (OPP-POO) MC β′-3 (A), without transition line as is observed for POP/POO. Upon cooling, the blends crystallize in sub-α-2, α-2 and β′-2 as solid solutions , as it is observed for POP/POO. Contrary to POP/POO system, the last melting is (OPP/POO) MC β′-3; no β polymorphic form is detected. Solubility of POO into OPP as β′-3 MC is ~10% (blue dotted lines).The OPP/POO system is very similar to the POP/POO one and highly simplified compared to direct mode. OPP stabilizes in βTernary blends were also investigated: ternary diagrams were drawn to supplement the above information. High and low melting iso-lines are reported for the PPP/POP/POO and PPP/OPP/POO systems.′ melting) between 16 and 27 °C depending on POP and POO relative contents. The high melting iso-lines  between 40 and 65 °C depending on PPP content and a second series (βso-lines a correspso-lines b corresp′-2 melting iso-lines (30–31 °C) are observed at OPP/POO enriched compositions. Contrary to PPP/POP/POO, there are clearly two series of low melting iso-lines (′-2 polymorphism) and the second series (up to 39 °C) corresponds to the melting of OPP enriched blends (β′-3 polymorphism).PPP/OPP/POO ternary blends also exhibit two series of iso-lines. The high melting iso-lines c correspso-lines d. The fi′2 to the higher melting point β-3 form. Overall, all these modifications create a steep melting area in the enriched POP part of the ternary diagram, which shows β′-3 and β-3 polymorphism.The high melting iso-lines a are not′-2 polymorphism). On the contrary, and as for the PPP/POP/POO system, the low melting iso-lines  although OPP and POO exhibit a L-3 polymorphism. The binary blends PPP-POP crystallize as L-2 for all the compositions: SS β′-2 and β-2 forms, the solubility of OPP in PPP is higher than that of POP and POO. OPP and POP form a β′-2 molecular compound at 50/50 concentration ratio, POP and POO exhibit a complete miscibility although OPP and POO also show one molecular compound at 50/50 and one eutectic interaction at 75/25 concentration ratios. OPP tends to induce the formation of molecular compounds when it is blended with POP or with POO.Overall, for the studied systems analyzed in direct mode, PPP can be considered as an inducer of L-2 structures, when it is mixed with POP, OPP or POO; for the last melting form, all binary systems co-crystallize with PPP in L-2 configuration whatever the position or the number of oleic acid on the glycerol. This observation is important since it helps to understand the role of palm oil (as PPP source) to stabilize the crystallization of palm olein in the production of super-olein (step 2) by dry fractionation. In βThe tendency of these triacylglycerol blends to form molecular compounds which are stable entities releasing significant heat when crystallizing can explain the typical crystallization behaviors of the olein for the production of super-olein (step 2) and of the soft PMF for the production of hard PMF (step 3) by dry fractionation.′-3 as mixed crystals. These phase diagrams are highly similar, the main differences being that the β′ → β transition of the mixed crystals is only observed for the POP/POO system; solubility of POO in POP and in OPP is similar. In the PPP containing systems, the changes are not highly pronounced from direct to tempered mode. Larger variations are observed in the systems containing POP, OPP and POO: interactions are simplified in the POP/OPP system, the POP/POO system becomes more complex while the OPP/POO system varies the least.In tempered mode, PPP/POP and PPP/OPP systems show quite similar interactions. The solubility of POP in PPP is significantly increased while that of OPP in PPP does not change much. Polymorphism is L-2, except in the part of the diagrams rich in POP or in OPP. The PPP/POO system is not really modified by the tempering, since at the tempering temperature most of the blends are already in a stable β–2 form or are liquid; solubility of POO in PPP is slightly increased compared to the direct mode. The POP/OPP system evolves into a simpler one, the entire binary system stabilizes in L-3 with a β–3 molecular compound at 50/50 concentration ratio. Last melting of POP/POO and OPP/POO systems also correspond to L-3 structure: β-3 or β′-3 for the OPP. An L-3 area is also observed in both systems for the low melting iso-lines: β-3 and β′-3 for PPP/POP/POO and β-3 for PPP/OPP/POO.Regarding the ternary phase diagrams, PPP/POP/POO systems, investigated in direct mode, behave quite similarly to the PPP/OPP/POO ones, in what concerns the highest melting iso-lines. Polymorphism is L-2 all over the phase diagrams. However, POP-POO and OPP-POO blends present different melting behaviors, creating huge differences in the low-melting iso-lines. Although PPP/POP/POO low melting iso-lines all correspond to L-2 polymorphism, low-melting iso-lines of part of the PPP/OPP/POO blends correspond to L-3 polymorphism. In tempered mode, both PPP/POP/POO and PPP/OPP/POO ternary diagrams present an area showing L-3 polymorphism for the high melting iso-lines: β-3 for the POP system and β2O and PO2. The difficulty to control the heat release observed during olein crystallization and during soft PMF crystallization has been shown to be linked to the formation of molecular compounds at certain concentration ratio in systems containing POP and OPP. Any solution  allowing to modify this ratio in palm oil or fractions are in favor of better control of the molecular interactions involved.The results show that the unsaturated triacylglycerols are poorly soluble in the fully saturated one (PPP), that intersolubility decreases in proportion to the number of unsaturated fatty acids, that positional isomerism (POP/OPP) has a major impact, that OPP may induce formation of molecular compounds and that co-crystallization properties are highly modified by tempering depending on the polymorphic properties of the systems. The results also explain some crystallization behaviors observed during the dry fractionation of palm oil. Adding palm oil as PPP source is useful to ensure a reproducible olein crystallization since PPP induces stable L-2 structures in systems containing P


As dysregulation or mutation of this pathway causes many diseases, including cancer, the β-Catenin level is carefully regulated by the destruction complex in the Wnt signalling pathway. However, the mechanisms underlying the regulation of β-Catenin ubiquitination and degradation remain unclear. Here, we find that WNK (With No Lysine [K]) kinase is a potential regulator of the Wnt signalling pathway. We show that WNK protects the interaction between β-Catenin and the Glucose-Induced degradation Deficient (GID) complex, which includes an E3 ubiquitin ligase targeting β-Catenin, and that WNK regulates the β-Catenin level. Furthermore, we show that WNK inhibitors induced β-Catenin degradation and that one of these inhibitors suppressed xenograft tumour development in mice. These results suggest that WNK is a previously unrecognized regulator of β-Catenin and a therapeutic target of cancer. Sato et al. find that WNK (With No Lysine [K]) acts as a positive regulator of the Wnt signaling pathway by attenuating the interaction between β-Catenin and the Glucose Induce degradation Deficient (GID) complex, and show that a WNK inhibitor also functions as a Wnt inhibitor, suppressing xenograft tumor development in mice. These findings suggest that WNK is a regulator of β-Catenin and a potential therapeutic target Dysregulation or mutations of this pathway are related to many diseases, such as type II diabetes4, autosomal dominant polycystic kidney5 and many cancer types6. The Wnt signalling pathway is divided into three classes: the canonical pathway, planar cell polarity pathway and Ca pathway. In the canonical Wnt pathway, β-Catenin is a core component whose protein regulation is a key process in the Wnt signalling pathway. In the absence of Wnt, the destruction complex formed by Axin1, Adenomatous Polyposis Coli (APC) and Glycogen Synthase Kinase 3β (GSK3β) phosphorylates the N-terminus of β-Catenin, which is in turn recognized and ubiquitinated by βTrCP E3 ubiquitin ligases7. Ubiquitinated β-Catenin is degraded by the proteasome, which regulates the basal β-Catenin levels. The Wnt signalling pathway is activated by the binding of Wnt ligand to a receptor complex9, which leads to Dishevelled (DVL) recruitment, where DVL polymerizes with Axin and the remaining components of the destruction complex. This polymerization inactivates the destruction complex, inducing β-Catenin accumulation in the cytoplasm. Accumulated β-Catenin translocates to the nucleus and regulates the transcription of target genes10. However, because several ubiquitination enzymes of β-Catenin have been identified and shown to be Wnt-independent17, the mechanisms underlying the regulation of β-Catenin ubiquitination remain unclear.The Wnt signalling pathway is an essential pathway in multicellular organisms that has already been identified to function in cell proliferation, differentiation and homeostasis20 and comprise four members. WNK1 and WNK4 have been identified as the causative genes of pseudohypoaldosteronism type II21, and WNK1 is also a causative gene of hereditary sensory and autonomic neuropathy type 2A22. WNK kinases are required for epidermal growth factor-mediated extracellular signal-regulated kinase 5 activation, and WNK family members are also involved in proliferation, migration and differentiation25. Previously, we found that WNK1 and WNK4 induce the expression of Lhx8 and are important for neural specification through GSK3β27. WNK was also identified as a positive regulator of the Wnt signalling pathway; however, the detailed mechanism is unknown28. Thus, WNK has several functions associated with many developmental processes.The WNK (With No Lysine [K]) kinases are atypical serine/threonine kinase family members that are conserved among many speciesHere, we demonstrated that WNK is a positive regulator of the Wnt signalling pathway. WNK attenuates the interaction between β-Catenin and the glucose-induced degradation deficient (GID) complex, which is an E3 ubiquitin ligase of β-Catenin, suggesting that WNK might regulate β-Catenin levels. Furthermore, we showed that the WNK inhibitor also functions as a Wnt inhibitor, suppressing xenograft tumour development in mice. These findings suggest that WNK is a regulator of β-Catenin and might be a therapeutic cancer target.27. GSK3β acts as a negative regulator of the Wnt signalling pathway7. Additionally, WNK was shown to be involved in the phosphorylation of Dsh (fly homologue of DVL) under Wnt stimulation in Drosophila and to work as a positive regulator of the Wnt signalling pathway in HEK293T cells28. Thus, we considered whether WNK interacts with the Wnt signalling pathway. First, we examined whether the knockdown of WNK affects the Wnt signalling pathway. We performed RT-PCR to examine the expression of the Wnt target genes AXIN2 (early response gene) and c-Jun (late response gene) under the knockdown of WNK1 and WNK4 in HEK293T cells. We found that the expression levels of AXIN2 and c-Jun were activated by Wnt stimulation  deletes the phosphorylation and ubiquitination sites, β-Catenin ΔN is no longer degraded and functions as a constitutively active form31. Because WNK is related to β-Catenin ubiquitination, we next examined the β-Catenin ΔN level under conditions with the knockdown of both WNK1 and WNK4. Surprisingly, similar to the normal β-Catenin level, the β-Catenin ΔN level was also reduced by the knockdown of both WNK1 and WNK4  in SW480 cells lls Fig. . These f16. Among the components of the GID complex, RMND5A and MAEA  encode E3 ubiquitin ligases35. We confirmed that RMND5A and MAEA ubiquitinated β-Catenin directly  reduced the β-Catenin level, but STOCK1S-50699 (hereafter referred to as 50699) did not  also reduced the β-Catenin level  and polyethyleneimine (Polysciences), respectively. The siRNA target sequences are summarized in Supplementary Table The cell lines used in this study were HEK293T, SW480, HCT116 and DLD1. Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (FBS) was used to culture HEK293 and SW480 cells, McCoy’s 5A supplemented with 10% FBS was used to culture HCT116 cells and RPMI 1640 supplemented with 10% FBS was used to culture DLD1 cells. For the co-transfection of short interfering RNA (siRNA) and plasmids, we used the The antibodies used in this study were as follows: rabbit anti-β-Catenin (Abcam), rabbit anti-Flag, rabbit anti-HA, rabbit anti-myc , rabbit anti-WNK1, rabbit anti-WNK4, mouse anti-myc  and anti-rabbit HRP-conjugated (GE) antibodies.STOCKS2S-26016 was purchased from Tocris and #13 was synthesized at the Kagechika laboratory. MG132 was purchased from Merck.GAPDH was used to normalize the cDNA samples. The sequences of the PCR primer pairs are summarized in Supplementary Table Total RNA was isolated using TRIzol reagent (Invitrogen). cDNA synthesis was carried out using Moloney murine leukaemia virus reverse transcriptase (Invitrogen). PCR Cycler (ABI) and THUNDERBIRD SYBR qPCR Mix (TOYOBO). GAPDH was used to normalize the cDNA samples.Quantitative PCR was performed using the Applied Biosystems 7300 Real-Time 26. DVL1, DVL2, DVL3, β-Catenin, RMND5A and MAEA cDNA were obtained by RT-PCR and were constructed in the pRK5 vector. To construct the constitutively active form of β-Catenin, as well as dominant-negative forms of RMND5A and MAEA, we performed site-directed mutagenesis.We made several WNK1 or WNK4 constructs with different molecular tags  from WNK1 or WNK4 constructs with Flag-tagHEK293T cells were transfected with the indicated expression vector. Lysates were prepared from transfected cells and were immunoprecipitated with the indicated antibodies and Protein A/G PLUS-agarose (Santa Cruz Biotechnology). Immunoprecipitates were subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis and western blotting, and were then detected using the image analyser LAS-4000 Mini (GE).4 cells) in 96-well plates were treated with the indicated chemicals for 24 h, and WST1 was added to each well. The absorbance (450 or 630 nm) was measured using a microplate reader, and the viability of WNK inhibitor-treated cells was determined. For cell growth, SW480 cells (1 × 105) were seeded in six-well plates and were treated with WNK inhibitor #13. The number of #13-treated cells was counted by Vi-CELL (Beckman) on the indicated days, and cell growth curves were created.Cell viability was analysed using WST1 assays (Takara). SW480 cells  were subcutaneously injected into the mice. When the tumour size reached approximately 200 mm3, all of the mice were injected intraperitoneally with the indicated chemicals twice weekly for 3 weeks at a dose of 5 or 10 mg/kg. The tumour sizes were measured before each injection. At the end of the experiments after injection for 3 weeks, the animals were sacrificed and the tumour weights were measured. Collected tumours were homogenized and applied for immunoblotting assays to analyse β-Catenin expression.Five-week-old BALB/cAJ1-All experiments were conducted at least three times independently. The data were analysed using Microsoft Excel (Microsoft) and StatPlus . Values and error bars express mean ± standard deviation (SD) and are representative of at least three independent experiments.Further information on the research design is available in the Nature Research Reporting Summary linked to this article.Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary