Metastatic breast cancer is responsible for the death of the majority of breast cancer patients. In fact, metastatic BC is the 2nd leading cause of cancer-related deaths in women in the USA and worldwide. Triple negative breast cancer (TNBC), which lacks expression of hormone receptors (ER-α and PR) and ErbB2/HER2, is especially lethal due to its highly metastatic behavior, propensity to recur rapidly, and for its resistance to standard of care therapies, through mechanisms that remain incompletely understood. WAVE3 has been established as a promoter of TNBC development and metastatic progression. In this study, we investigated the molecular mechanisms whereby WAVE3 promotes therapy-resistance and cancer stemness in TNBC, through the regulation of β-catenin stabilization.The Cancer Genome Atlas dataset was used to assess the expression of WAVE3 and β-catenin in breast cancer tumors. Kaplan–Meier Plotter analysis was used to correlate expression of WAVE3 and β-catenin with breast cancer patients’ survival probability. MTT assay was used to quantify cell survival. CRISPR/Cas9-mediated gene editing, 2D and 3D tumorsphere growth and invasion assays, Immunofluorescence, Western blotting, Semi-quantitative and real-time quantitative PCR analyses were applied to study the WAVE3/β-catenin oncogenic signaling in TNBC. Tumor xenograft assays were used to study the role of WAVE3 in mediating chemotherapy resistance of TNBC tumors.Genetic inactivation of WAVE3 in combination of chemotherapy resulted in inhibition of 2D growth and 3D tumorsphere formation and invasion of TNBC cells in vitro, as well as tumor growth and metastasis in vivo. In addition, while re-expression of phospho-active WAVE3 in the WAVE3-deficient TNBC cells restored the oncogenic activity of WAVE3, re-expression of phospho-mutant WAVE3 did not. Further studies revealed that dual blocking of WAVE3 expression or phosphorylation in combination with chemotherapy treatment inhibited the activity and expression and stabilization of β-catenin. Most importantly, the combination of WAVE3-deficiency or WAVE3-phospho-deficiency and chemotherapy suppressed the oncogenic behavior of chemoresistant TNBC cells, both in vitro and in vivo.We identified a novel WAVE3/β-catenin oncogenic signaling axis that modulates chemoresistance of TNBC. This study suggests that a targeted therapeutic strategy against WAVE3 could be effective for the treatment of chemoresistant TNBC tumors.The online version contains supplementary material available at 10.1186/s13058-023-01634-3. Identification of a novel WAVE3/β-Catenin oncogenic signaling axis that regulates chemoresistance may provide new therapeutic strategies for the treatment of TNBC by targeting WAVE3 and/or its downstream effectors.According to the latest statistics from the American Cancer Society , breast WAVE3, like all members of the WASP verprolin-homologous family of proteins, was originally identified as a regulator of actin cytoskeleton and its associated cell migration phenotype, through the regulation of membrane ruffles at the leading edge of migrating cells downstream of Rho and Rac small GTPases (reviewed in ). The laOur data established a novel WAVE3/β-catenin signaling axis that drives chemo-resistance of TNBC, and provide the impetus for developing novel potential therapeutic options that target WAVE3 and/or β-catenin for the treatment of therapy-resistant TNBC tumors.WAVE3 and the mouse Wave3 genes by lentiviral transduction as described previously [TNBC Cell lines were acquired from the American Type Culture Collection , and maintained according to the manufacturer’s established protocols. Cell lines were also routinely authenticated by short tandem repeat DNA fingerprinting analysis. WAVE3-deficient (W3-KO) cells were generated using two different and verified sgRNAs targeting the human eviously . A scrameviously . Generateviously . GFP-tageviously . The GFPeviously . For proRabbit anti-WAVE3 (1:1000) and mouse anti- β-catenin (1:1000) were from Cell Signaling Technologies . Goat horseradish peroxidase-conjugated anti-mouse IgG (1:5000) and goat horseradish peroxidase-conjugated anti-rabbit IgG (1:5000) from Calbiochem ; and Alexa 488-conjugated anti-rabbit IgG and Alexa 568-conjugated anti-mouse IgG are from Invitrogen . Vecta-shield with 4′,6-diamidino-2-phenylindole was from Vector Laboratories .3 cells were seeded into 96-well culture plates for overnight followed by treatment with either DMSO (0.1%) or the chemotherapeutic agents as indicated for 2 days.3--2,5-diphenyltetrazolium bromide (MTT) assay was performed following standard protocol. For each BC cell line, 5 × 103D multi-spheroid formation and 3D spheroid invasion assays were performed as described previously .http://rsbweb.nih.gov/ij/docs/guige/146.html).Western blotting assays were performed according to standard protocols. β-Actin, Lamin B or α-Tubulin were used as loading standards. ChemiDoc MP Imaging system (Bio-Rad) was used for image acquisition of developed gels. Signal intensity of WB bands was quantitatively analyzed by ImageJ software, as described previously  and acco6 cells per mouse, n = 5) or 4T1 cells  were implanted into the mammary fat pads of 6–8-week-old female NSG or Balb/C mice, respectively. Both the left and the right mammary fat pads were injected, resulting in 10 tumors per group. For the chemotherapy treatments, once the tumors became palpable (~ 100 mm3), mice were randomized into control and experimental groups (n = 5 mice per group) and treated with either the diluent , cisplatin (1 mg/kg i.p) or doxorubicin (1 mg/kg i.p.), twice a week for 4 weeks. Tumor growth was monitored by twice weekly measuring tumor volume with digital Vernier calipers. At the endpoint, tumors were collected and fixed with 4% paraformaldehyde. Tumors were also weighed prior to fixation. All animal studies were performed under protocols approved by the Institutional Animal Care and Use Committee.Parental, WAVE3-deficient (W3-KO), WAVE3-deficient overexpressing wild-type WAVE3 (W3-WT), or phospho-mutant WAVE3 (W3-Y4) MDA-MB-231 cells  medium , and 8-μm sections were prepared. Tumor sections were stained with the following antibodies: rabbit anti-WAVE3 and mouse anti-β-Catenin, followed by Alexa 488 anti-mouse or Alexa 568-conjugated goat anti-rabbit IgG. Lung sections were stained with hematoxylin (Vector Laboratories) and eosin  following the manufacturer’s protocol. Confocal or bright-field imaging microscopy  was used to capture the images and ImagePro Plus Capture and Analysis software  was used to analyze the stained sections from 10 to 15 independent fields/sections.Total RNA was extracted from cancer cell lines using TRIzol reagent  following the manufacturer’s instructions. Qt-RT-PCR was performed, as described previously . qPCR Prt-test. In calculating two-tailed significance levels for equality of means, equal variances were assumed for the two populations. Results were considered significant at p < 0.05.Experiments were performed in triplicate and analyzed using the Student’s p < 0.001) decrease in cell viability after treatment with cisplatin  reduction in the number of tumorspheres after treatment with cisplatin, paclitaxel, or doxorubicin. Re-expression of wild-type WAVE3 (W3-WT) restored tumorsphere growth to levels compared to the control (GFP) cells, even after treatment with the chemotherapeutic drugs. By contrast, re-expression of phosphomutant (W3-Y4) failed to activate tumorsphere growth in both the presence and absence of chemotherapy treatments. Tumorsphere invasion, as measured by the number of micro-spheres that were able to invade the Matrigel, was also inhibited in MDA-MB-231-derived tumorspheres  in MDA-MB-231 , 41 days post-injection, when the first tumors reached ~ 100 mm3, mice started receiving doxorubicin (Dox) or DMSO (Unt) injections twice a week for five more weeks, after which mice in all 8 groups were sacrificed and tumors harvested. In the untreated groups (Unt), mice injected with WAVE3-deficient MDA-MB-231 cells (W3-KO-Unt) developed tumors that were significantly (p < 0.01) smaller  inhibited in all treated four groups  compared to their untreated counterparts  smaller  MDA-MB-231 cells, their WAVE3-deficient (W3-KO), W3-KO re-expressing wildtype (W3-WT) or W3-KO re-expressing phosphomutant WAVE3 (W3-Y4) derivatives were injected in the mammary fat pads  of NSG mice ( MDA-MB-2p < 0.05) increase in expression levels of WAVE3 mRNA levels in the Cisplatin-resistant cells  induced in cisplatin-resistant  faster, and tumors were also significantly larger in mice injected with the cisplatin-resistant MDA-MB-231 cells than their parental control counterparts. Treatment with cisplatin seemed to have slowed tumor growth in both groups during the first week of the treatment with cisplatin. However, this inhibitory effect did not last for more than a week and tumors started growing faster afterwards with a faster rate for the CIS-R tumors than the parental control  smaller than both those derived from the parental or the cisplatin-resistant MDA-MB-231 cells. Tumor metastasis to the lungs was also significantly (p < 0.001) blunted in mice injected with the WAVE3-deficient-cisplatin-resistant cells , which suggested to us that the chemotherapy-mediated degradation of β-catenin may be at the posttranslational level and may involve a proteasome-mediated degradation. To this end, WAVE3-deficient (W3KO) or W3KO expressing phospho-deficient (W3Y4) MDA-231 or 4T1 cells were treated with the proteasome inhibitor GM6001 (GM) in the presence or absence of cisplatin treatment (Cis), and β-catenin expression levels were assessed by Western Blot  showed a very significant positive correlation between WAVE3  and β-catenin , and patient survival probability. Elevated expression levels of WAVE3 expression reduced BC patient survival probability by more than 50 months  showed a significant (p < 2.2e−16) positive correlation between WAVE3 and β-catenin mRNA expression levels  loss of WAVE3 expression or its phosphorylation sensitizes TNBC cell lines to chemotherapeutics both in vitro in and in vivo; (ii) WAVE3 expression is increased in TNBC cells after chronic exposure to chemotherapy; the cisplatin-resistant (CIS-R) and doxorubicin-resistant (DOX-R) MDA-MB-231 cells express significantly higher levels of WAVE3 than their control counterparts, which results in increased tumor growth of the CIR-R cells; (iii) increased WAVE3 expression results in activation of β-catenin expression and its accumulation in the nucleus, while loss of WAVE3 expression and/or phosphorylation leads to decreased β-catenin protein levels and its depletion from the nucleus, even after exposure to chemotherapy; (iv) WAVE3 is required for the stabilization of β-catenin, since loss of WAVE3 expression and/or phosphorylation results in β-catenin degradation, and treatment of WAVE3-deficient or WAVE3-phospho-deficient MDA-231 cell with proteasome degradation inhibitors rescues β-catenin expression. (v) Given the association between the accumulation of β-catenin in the nucleus and its oncogenic activity, loss of WAVE3 expression or its phosphorylation inhibited β-catenin nuclear accumulation, while chronic exposure to chemotherapy in CIS-R and DOX-R cells increased nuclear β-catenin, which is concomitant to increased WAVE3 accumulation in the nucleus; and (vi) both WAVE3 and β-catenin expression levels are increased in the aggressive TNBC cells, compared to their less aggressive ER + counterparts, and increased levels of both WAVE3 and β-catenin correlate with poor disease outcomes and decreased survival probability of BC patients.Early studies reported that WAVE3 is one of the regulators of YB1 , and YB1Additional file 1. Fig. S1: A Graphic representation of the WAVE3 functional domains and GFP-fused truncation mutants. The location of the four tyrosine residues is also shown. BR basic region, PRD proline-rich domain, VCA verprolin, cofilin, and acidic. B and C Representative Western blots of protein lysates from parental MDA-MB-231 cells (GFP), their WAVE3-deficient derivatives (W3-KO), or the W3-KO expressing either wildtype (W3-WT) or phospho-mutant (W3-Y4) WAVE3 that were subjected to immunoblotting with antibodies against WAVE3. β-Actin was used as loading control. Fig. S2: A Representative images of H&E staining of lung tissues sections of mice injected in the mammary fat pads with parental (CTRL), cisplatin resistance (CIS-R) or WAVE3-deficient CIS-R (CIS-R-W3KO) MDA-MB-231 with the treatment of cisplatin (1 mg/Kg). B Quantification of lung metastases from the mice described in (A). Data are the mean ± SD . Fig. S3: Quantitative RT-PCR of mRNA of the indicated genes from total RNA isolated from parental (CT), WAVE3-deficient (W3KO), W3KO-overexpressing wildtype WAVE3 ((W3WT) or phospho-mutant WAVE3 (W3Y4) MDA-MB-231 cells. Data are the mean ± SD .Additional file 2: Contains uncropped Western Blots.


Improving quality of life (QoL) is the goal of behavior analytic services, but there can be barriers to assessing the QoL of autistic children due to characteristics inherent in the condition. Given that happiness is a fundamental element of QoL, previous research has relied on behavioral indicators of mood  to evaluate the overall QoL of disabled individuals. However, the use of these traditional indices may not accurately reflect the emotional well-being of autistic individuals, who are known to engage in idiosyncratic mood indicators. The current study replicated selected procedures from Parsons et al.  to identThe online version contains supplementary material available at 10.1007/s40617-022-00710-y. There is some research to suggest that individuals on the autism spectrum experience a much lower quality of life (QoL) than nonautistic individuals across the lifespan  have a diagnosis of autism spectrum disorder (ASD) from an independent clinical psychologist; (2) have functional communication skills ; and (3) be able to select an item between two or more items or pictures.M = 4.7 years). According to school records, six of the participants presented with a secondary diagnosis at the time of the study. Five of the boys communicated with PECS, whereas Daniel and Jack communicated with both PECS and one-word vocal requests. Louis could make requests using short phrases and sentences, whereas Trevor was the only participant that communicated using an AAC device.Nine young boys met the inclusion criteria , and sad. The emojis were colored green, yellow, and red, respectively, similar to the emotion thermometer  completed the Indices of Happiness and Unhappiness Questionnaire Appendix ppendix AThe responses to the first two questions were compared. Any indices of happiness or indices of unhappiness that were agreed upon by at least two adults were selected for observation. The researcher then conducted 1 or 2 days of informal observation with each participant to confirm the presence of these happy and unhappy indicators. During these observations, the researcher observed the children during their regularly scheduled school activities and noted the topography of their idiosyncratic mood indicators as they naturally occurred. Based on these observations, the indices of happiness and unhappiness were operationally defined for each participant.For Louis, only two indices of happiness were agreed upon by the adults familiar with him . However, during his informal observation, the researcher noted that Louis rarely engaged in positive talk, but quite frequently engaged in smiling during preferred activities. As a result, a follow-up questionnaire was given to his respondents to complete. The adults were asked if Louis would smile in situations he was deemed to be happy, and they were expected to respond with “yes,” “no,” or “sometimes.” As all three adults responded with “yes” or “sometimes,” smiling was included as a third indicator of happiness for this participant. The individualized indices of happiness and unhappiness for each participant are operationally defined in Table A second trained observer  independently watched the video recordings and collected data for at least 26% of sessions within each condition for each participant, in line with the recommendations made by Wolery et al. . InterobThere were two conditions for each participant, which closely mimicked their idiosyncratic happy and unhappy situations identified by the questionnaire. To compare the effects of each condition on the children’s indices of happiness and unhappiness, an alternating treatments design without a baseline was employed. The order of the conditions was randomly alternated across sessions, then counterbalanced to ensure that neither condition was conducted for more than two consecutive sessions. The only exception to this was the first session, in which the happy condition was conducted with all participants. As the researcher was unfamiliar to the children, conducting an initial happy session with the participants was deemed appropriate because the presence of the researcher could have otherwise been associated with nonpreferred or aversive activities. This may have inadvertently led to avoidance behaviors in subsequent sessions, regardless of the condition. Two sessions were conducted with each participant daily and they were separated by at least 1 h to control for carryover effects. For Jesse only, the study comprised of 18 sessions due to his availability. For the remaining eight participants, the study comprised of 15 sessions.To validate the individualized indices of happiness and unhappiness identified for each participant, the children were exposed to two different conditions that represented their idiosyncratic happy and unhappy situations. These conditions were identified based on the responses to the latter two questions of the Indices of Happiness and Unhappiness Questionnaire. Any setting or situation that was agreed upon by two or more respondents was selected by the researcher. If there was more than one situation identified for either the happy or unhappy condition, the researcher selected the one that required less staff resources. For instance, if a situation required two or more staff members , it was not selected because it would have limited the availability of the teaching assistants for other students in the classroom. Furthermore, the first unhappy situation identified for both Daniel and Ryan was eliminated due to ethical concerns. Daniel and Ryan were identical twins, and the respondents all agreed that the most common unhappy situation for each child was when the other brother was crying. The researcher could not ethically evoke crying in one brother in order to conduct the unhappy condition with the other. Therefore, this situation was not selected for either of the children and the second agreed upon situation was selected instead.Sessions were conducted by the teaching assistant who was most familiar with each child. During both conditions, the children were exposed to their idiosyncratic environmental events for a total of 5 min. If a participant was informally observed to be engaging in any of their mood indicators prior to the start of a session, the session was delayed by at least 5 min until the child was displaying neutral behaviors. Both conditions involved naturally occurring situations that the children would regularly encounter in a typical school day. To minimize any distress during the unhappy condition, the sessions were kept brief, and they were to be terminated based on individualized criteria for each participant. For example, the session termination criterion for Jacob was two or more instances of head banging. Although these criteria were put into place, none of the unhappy sessions had to be terminated for any of the participants. The happy and unhappy conditions selected for each participant are listed in Table According to Green and Reid , the mosAs the effects of choice were to be examined in a subsequent study with the same participants  was calculated to help quantify the differences between the two conditions. PND was determined by comparing the two conditions on a point-by-point basis. For example, the first data point of the happy condition was compared to the first data point of the unhappy condition, the second with second, third with third, and so forth  relative to the unhappy condition . Although his indices of happiness were highly variable during the happy condition, they were only present at a low level during one session of the unhappy condition. The happy condition was superior to the unhappy condition in six out of seven comparisons, yielding a PND score of 85.7%.The indices of happiness for each participant are displayed in Fig. M = 41.4%, range: 16.7%–83.3%) relative to the happy condition . However, there was a highly variable, increasing trend present in both conditions. Although his indices of happiness occurred at a similar level during both conditions, there was a notable increase in these behaviors during the last two sessions of the unhappy condition. The unhappy condition was superior to the happy condition in only four of the seven comparisons, yielding a PND score of 57.1%.Daniel showed more indices of happiness during the unhappy condition  relative to the unhappy condition . Despite the high variability present during the happy condition, his indices of happiness occurred at a moderate-to-high level. Meanwhile, there was a decreasing trend evident during the unhappy condition. There was only one point of overlap between the two conditions, but a PND score of 100% showed clear superiority of the happy condition.Ryan engaged in more indices of happiness during the happy condition  relative to the unhappy condition . His indices of happiness were variable in both conditions, but they occurred at a moderate-to-high level during the happy condition. Jacob’s indices of happiness were not present, or they only occurred at a low level, during the unhappy condition. The only exception to this finding was session 5. The happy condition was superior to the unhappy condition in six out of seven comparisons, yielding a PND score of 85.7%.Jacob demonstrated more indices of happiness during the happy condition  relative to the unhappy condition . Although there was a decreasing trend during the happy condition, there was only one point of overlap between the two conditions. Joel’s indices of happiness were present at a mostly moderate level during the happy condition, whereas they occurred at a low level during the unhappy condition. There was a PND score of 100%, which showed clear superiority of the happy condition over the unhappy condition.Joel had more indices of happiness during the happy condition  relative to the unhappy condition . His indices of happiness occurred at a low level during both conditions, but they were more frequent during the happy condition in comparison to the unhappy condition. There was no overlap between the two conditions, resulting in a PND score of 100%. This demonstrated clear superiority of the happy condition over the unhappy condition.Louis engaged in more indices of happiness during the happy condition  relative to the unhappy condition . With the exception of session 10, his indices of happiness were stable at a high level during the happy condition. During the unhappy condition, his indices of happiness only occurred at a low level. Although there was one point of overlap between the two conditions, the happy condition was clearly superior to the unhappy condition with a PND score of 100%.Seth demonstrated more indices of happiness during the happy condition  relative to the unhappy condition . Although his indices of happiness were highly variable during both conditions, they occurred at a slightly higher level during the happy condition in comparison to the unhappy condition. The happy condition was superior to the unhappy condition, with only one point of overlap between the two. This resulted in a PND score of 85.7%.Jack had more indices of happiness during the happy condition  relative to the unhappy condition . There was an increasing trend present in the happy condition, whereas his indices of happiness remained at a low level during the unhappy condition. The happy condition was superior to the unhappy condition in six out of seven comparisons, yielding a PND score of 85.7%.Trevor demonstrated more indices of happiness during the happy condition  relative to the happy condition . His indices of unhappiness were highly variable during the unhappy condition, but they occurred at a moderate-to-high level. Meanwhile, Jesse’s indices of unhappiness only occurred at a low level during two sessions of the happy condition. There was no overlap between the two conditions, resulting in a PND score of 100%. This demonstrated clear superiority of the unhappy condition.The indices of unhappiness for each participant are displayed in Fig. M = 27.1%, range: 10%–43.3%). During this condition, his indices of unhappiness were variable at a mostly low level. There was clear superiority of the unhappy condition over the unhappy condition, with a PND score of 100%.Daniel’s indices of unhappiness were only present during the unhappy condition . During this session, his indices of unhappiness occurred at a moderate level. The unhappy condition was superior to the happy condition in only one of the six comparisons, resulting in a PND score of 16.7%.Ryan’s indices of unhappiness were rare, as they only occurred during one session of the unhappy condition . During this condition, his indices of unhappiness were variable at a low level. The superiority of the unhappy condition was clear, as there was no overlap between the two conditions, which resulted in a PND score of 100%.Jacob’s indices of unhappiness only occurred during the unhappy condition  relative to the happy condition . His indices of unhappiness were variable at a low level during the unhappy condition, whereas they were only observed during one session of the happy condition. The unhappy condition was superior to the happy condition in only three of the seven comparisons, yielding a PND score of 42.9%.Joel had more indices of unhappiness during the unhappy condition . During this session, his indices of unhappiness occurred at a low level. The unhappy condition was superior to the happy condition in only one of the seven comparisons, resulting in a PND score of 14.3%.Louis’s indices of unhappiness were also rare, as they only occurred during one session of the unhappy condition  relative to the happy condition . His indices of unhappiness were rare in both conditions. Nevertheless, Jack’s indices of unhappiness were slightly more frequent during the unhappy condition. During these two sessions, his indices of unhappiness occurred at a low-to-moderate level. The unhappy condition was superior to the happy condition in only two of the seven comparisons, yielding a PND score of 28.6%.Jack demonstrated more indices of unhappiness during the unhappy condition . During this condition, his indices of unhappiness were highly variable at a low level. The unhappy condition was superior to the happy condition in four out of seven comparisons, yielding a PND score of 57.1%.Trevor’s indices of unhappiness only occurred during the unhappy condition . A similar outcome was seen in Jack. This participant responded during five of the opportunities, but only two of these responses corresponded with the condition . However, his indices of happiness during sessions 6 and 9 were observed at a low level. Therefore, it could be argued that these responses did not corroborate the data from these two sessions. Trevor responded to the mood scale following four of the sessions, but these responses did not coincide with the presented condition. During all four opportunities, he responded with happy following the unhappy condition.For example, Joel only responded to the mood scale following six of his 15 sessions. Only two of these responses seemed to align with the utilized condition , the superiority of the unhappy condition was evident. For many of the participants, their indices of unhappiness rarely occurred during the unhappy condition, and if they did, they were present at a low level. However, these indices were completely absent during the happy condition for six of the participants. Although the absence of the unhappy indicators during both conditions led to more overlap between the data paths, this finding could be considered more socially valid. The procedures were effective in identifying and validating the unhappy indicators for most participants without needing to evoke high frequencies of these behaviors. Most importantly, none of the unhappy conditions involved contrived aversive situations that the participants would not typically encounter on a daily basis.It should be noted that Seth’s indices of unhappiness could not be validated, as he did not demonstrate any unhappy indicators during the sessions. One explanation for this finding was that the selected situation for Seth’s unhappy condition  was no longer aversive to the participant. It was mentioned by his teacher that sharing with peers was a social skill that the teaching team had previously targeted for improvement, as Seth often engaged in tantrums while playing with peers. To teach this skill, his teaching assistants would praise Seth each time he shared with a peer or cooperated during a game in which he had to take turns. Although this was a socially significant skill to teach, this history of reinforcement likely reduced the aversiveness of the play context. It is possible that the delivery of praise acted as an abolishing operation for Seth’s avoidance behaviors, and in turn, his indices of unhappiness.The researcher was also unable to complete the secondary validation measure with Seth. In particular, the presentation of the mood scale had inadvertently become an aversive stimulus for this participant, so it was discontinued after session 10. When the visual was presented after the happy sessions, Seth engaged in aggressive and avoidance behaviors that included hitting and running away from the researcher. It was theorized that the visual had become a reflexive conditioned motivating operation . Although the researcher tried to control for satiation by suggesting that different cars were used across the happy sessions, this may have been ineffective. Frequent access to his preferred stimuli may have inadvertently reduced his indices of happiness, because there is some research to suggest that emotions can be the product of motivating operations  before they began to cry. It is also worth noting that the indices of happiness identified for some participants , were found to be unhappy indicators for other children . This underscores the importance of individualizing the operational definitions of mood for different children who may not be able to express their emotions in conventional ways.Most importantly, this study proved that the indices of happiness and unhappiness identified for each child could be systematically manipulated through environmental arrangements. In view of this, these indices could be measured as direct outcomes during intervention. By directly measuring these indices, practitioners have a more objective approach to evaluating the happiness of individuals with disabilities during interventions intended to improve their overall QoL. This is crucial when examining the QoL of individuals who may struggle to communicate their emotions, such as children on the autism spectrum


Mycoplasma hyorhinis and Salmonella Typhi, the protozoan parasite Toxoplasma gondii, the fungus Aspergillus flavus, and liver flukes such as Clonorchissinensis or Opisthorchis viverrini, may induce malignant transformation in hepatocytes or in target cells of the biliary tract through aberrant Wnt signaling activation. This review focuses on new insights into infectious agents implicated in the deregulation of Wnt signaling and PLC development. Since the Wnt/β-catenin pathway is a driver of cancer following viral and bacterial infections, molecules inhibiting the complex axis of Wnt signaling could represent novel therapeutic approaches in PLC treatment. Interaction between infectious agents and liver tissue, as well as repeated and extreme biological events beyond adaptive capacities, may result in pathological conditions predisposing people to development of primary liver cancers (PLCs). In adults, PLCs mainly comprise hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Various infectious agents in the hepatic microenvironment can destabilize normal liver cell functions by modulating the Wnt/β-catenin pathway components. Among them, hepatotropic viruses B, C, and D are involved in Wnt/β-catenin signaling dysregulation. Other microbial agents, including oncogenic viruses such as Epstein–Barr virus (EBV) and human papilloma virus (HPV), bacteria, e.g.,  According to global cancer 2020 estimates, primary liver cancer (PLC) is the sixth most common cancer worldwide and represents the third cause of tumor death . In adulDifferent risk factors are involved in HCC and CCA pathogenesis and are related to diet, environmental exposure, lifestyle, and genetic predisposition ,7,8. IndPatients affected with NAFLD show a higher risk of developing bacterial, fungal, and viral infections, but the exact mechanism by which this occurs is still unknown . HepatitMultiple risk factors have been linked to the development of CCA. These include hepatic parasites, chronic biliary tract, and liver diseases, including primary sclerosing cholangitis, choledochal cysts, Caroli’s disease and HCV cirrhosis, as well as lifestyle factors associated with chronic hepatic inflammation and cholestasis ,14,15,16In recent decades, a clear causal link has been found between liver damage, inflammation and regeneration and the occurrence of PLCs. Chronic inflammation determined by long-term infections is observed in more than 80% of the HCCs and represents an important factor able to modify the hepatic microenvironment . The livThe Wingless/It (Wnt)/β-catenin signaling pathway is a pathway preserved throughout animal evolution. It is involved in the regulation of cells’ differentiative fates both during embryonic development and in the homeostasis of tissue regeneration in the adult organism ,29. GiveWnt signaling plays key physiological functions in the liver, from development to metabolic zonation and regeneration after damage ,37. SpecAXIN and APC genes, and the CTNNB1 gene encoding β-catenin, have been detected in PLCs [Upregulation of Wnt ligands and their receptors/coreceptors, as well as downregulation of Wnt/β-catenin signaling inhibitors, have been reported in liver cancer and its adjacent precancerous lesions . Hepatic in PLCs ,44,45,46Infectious agents implicated in PLC development can modulate Wnt pathway molecular components to destabilize normal cell functions . In factA better understanding of the events that lead to dysregulation of the Wnt signal following infection by microbial agents in PLCs could lead to a targeted therapeutic approach in liver cancer treatment. Therefore, it is important to identify the infectious agents that alter the Wnt pathway in liver cells, with the aim to understand the mechanisms used by bacteria or viruses or parasites to modify the Wnt signal and evaluate their involvement in the pathogenesis of HCC and CCA. This review considers what is currently known about deregulation of Wnt signaling due to the presence of infectious agents in the hepatic microenvironment as a driver of liver carcinogenesis. Comprehension of the cellular and molecular events occurring in the microenvironment in the presence of infectious agents could improve targeted therapeutic approaches in the treatment of PLCs. APC, AXIN1 and AXIN2 genes or methylation in APC aberrantly modify the Wnt signaling response [AXIN1 mutations are less frequently associated with CTNNB1 ones than those found in HCV-related HCC [AXIN1, AXIN2, CTNNB1, and WNT2 genes seem to confer a different HBV-dependent HCC susceptibility and progression [WNT2 and WNT1 gene expression can be considered as diagnostic or prognostic biomarkers, respectively, while Wnt-3a protein expression could be a novel HCC target in early diagnosis [CTNNB1-mutant and TP53-mutant HCCs were found to be differentially expressed and associated with the loss of epithelial phenotype. This condition most likely resulted from altered phosphorylation of proteins implicated in the actin filament organization or decreased expression of the epithelial markers E-cadherin (CDH1) and Keratin-19 (KRT19). CTNNB1-mutant HCCs are often low grade and linked with the immune-desert phenotype, while TP53-mutant HCCs are related to HBV infection [In approximately 80% of HCCs, the development of disease is associated with HBV and/or HCV chronic infections . Withoutresponse ,64. In Hated HCC ,65. Polygression . In addiiagnosis ,68. Actiiagnosis . HBV caniagnosis . In HBV-iagnosis . Recentlnfection . In addinfection ,47.Chronic HCV infection affects an estimated 115 million people worldwide; this disease is more common in East/Central Asia and North Africa than in Europe . CDH1 gene promoter hypermethylation, with consequent dissociation of the β-catenin/E-cadherin complex, and stimulates tumor cell growth by GSK-3β inactivation and Wnt3a release [c-MYC and Cyclin D1 (CCND1), and different nuclear levels of β-catenin in cells infected, according to various HCV subtypes [HCV core protein, non-structural protein 4B (NS4B) and 5A (NS5A) are able to activate Wnt/β-catenin signaling in HCC pathogenesis . The cor release ,77. A stsubtypes .NS4B activates the Wnt3a-induced Wnt/β-catenin signaling in HCC Huh7 cells and normal human liver LO2 cell lines ,79. NS5AThe progression from chronic HCV hepatitis to HCC is favored by binding of Wnt1 and Wnt5a to Fzd, which transactivates the EGFR pathway . In a moDownregulation of APC or Axin2 induced by HCV increases de-phospho-β-catenin, which translocates to the nucleus to activate the target genes and induce malignant changes in hepatocytes . ChronicHDV is a defective RNA virus that needs co-infection with HBV. At a global level, approximately 5% (15–20 million) of chronic HBV carriers are co-infected with HDV. HDV infection may occur with HBV co-infection or superinfection in persistent HBV hepatitis . HDV proEBV has a high global prevalence and is present in more than 90% of adults . In HCV-Prevalence and distribution of over 207 HPV genotypes vary according to different populations and geographical areas . Some reToxoplasma gondii, a protozoan parasite, is involved in liver disease and fibrosis, with a global distribution. Indeed, in China, the highest seroprevalence of T. gondii was found among patients with liver disease, who ranged in age between 41–50 years [T. gondii secretes GRA18, a protein exported to the cytoplasm of infected cells, in which it is a potential inhibitor of the β-catenin destruction complex [Echinococcus granulosus seems to significantly extend the survival and play a protective effect in HCC patients [Mycoplasma hyorhinis, which predominantly infects swine, has been also detected in human cancer tissues [M. hyorhinis infection promotes HCC progression and cell migration. The interaction between mycoplasma p37 membrane protein and the epithelial cell adhesion molecule (EpCAM) involves the Akt/mTOR pathway [M. hyorhinis infection has been reported to induce nuclear β-catenin accumulation and increase its target gene expression [M. hyorhinis in hepatocarcinogenesis, by Wnt signaling dysregulation. Regarding the role of non-viral agents in liver carcinogenesis, it has been reported that 50 years . T. gond complex . In the  complex . Echinocpatients . The pre tissues . M. hyor pathway , which ipression , furtherAspergillus flavus has a high prevalence and is frequently isolated in the respiratory tract [Aspergillus fungi (flavus), is a genotoxic compound responsible for HCC. Persistent AFB1 exposure activates the Wnt pathway [A. flavus, A. fumigatus, and Candida albicans strongly activate Wnt signaling [A. flavus. Finally, AFB1 may increase the HCC risk in the presence of HBV or HCV infections [In some countries, such as India, Mexico, Pakistan, Sudan, and Saudi Arabia, ry tract . Aflatox pathway . Other s pathway ,114. Morignaling . These cfections ,117. The onset of CCA is related to a chronic inflammatory condition due to long exposure of the cholangiocytes to predisposing risk factors, such as transmissible agents . In bileCTNNB1 (1.5%), AXIN1 (4%) and APC (2%) genes are the most commonly identified [In this tumor, mutations in the entified . The hosentified ,119. Morentified . Tumor-aentified . In factentified . Other sentified . TERT) mRNA transcription in CCA cell lines [HBV genes are present in the genome of CCA. HBx protein may be implicated in cholangiocarcinogenesis, since HBx gene insertion induces cis-activation of Telomerase reverse transcriptase  is more frequent than human polyomavirus 6 (HPyV6) and oncogenic Merkel cell polyomavirus (MCPyV) [In CCA lesions and adjacent peritumoral cells of bile duct epithelium, the occurrence of  (MCPyV) . EBV can synergistically cooperate with HBV in the onset of a rare subtype of intrahepatic CCA (EBVaICC), a tumoral form distinguished from others by exclusive clinicopathological and genetic alterations  and probClonorchissinensis, Opisthorchis viverrini and Opisthorchis felineus are trematodes of the family Opisthorchiidae. In particular, the genera Clonorchis and Opisthorchis are responsible for CCA [Mechanisms leading to CCA development are dependent on genetic alterations induced by different trematodes. Liver flukes such as  for CCA ,131.C. sinensis determines clonorchiasis, which is correlated with hyperplasia of biliary epithelium and metaplasia of mucin-secreting cells in bile ducts, probably through upregulation of Wnt7b, Fzd6 and cell cycle controllers, which leads to CCA development [C. sinensis, the growth factor-like protein termed granulin, seems to be involved in cell migration and invasion in CCA as well as in HCC, with activation of EMT by upregulation of β-catenin and decline of E-cadherin [C. sinensis infection, macrophages showed variations in polarization and phenotype during different phases of disease [O. viverrini contain growth factors and other molecules, including liver fluke granulin. In Opisthorchiasis, chronic inflammation is induced by mechanical damage to biliary epithelia caused by O. viverrini, which continuously exposes cholangiocytes to granulin and other molecules [elopment . An excrcadherin . To supp disease . On the olecules . The chrolecules . O. viverrini-infected hamsters, increased expression of Wnt3, Wnt3a, Wnt5a, Wnt7b as well as β-catenin nuclear translocation were found [In particular, the development of opisthorchiasis-dependent CCA is associated with upregulation of the Wnt/β-catenin signaling pathway. In cholangiocarcinogenesis of re found . Similarre found . Cryptosporidium parvum has been isolated in human immunodeficiency virus (HIV) cholangiopathy. C. parvum induces a Toll-like receptor 4-dependent response that is inhibited by HIV-1 trans-activator of transcription (Tat) protein [C. parvum from the small intestine to biliary ducts favors a pro-inflammatory cholangiopathy that involves molecules such as micro-RNA let-7i, NF-kB p50-CCAAT/enhancer-binding protein beta (C/EBPbeta) and NRAS [C. parvum through activation of the Fas/Fas ligand pathway [C. parvum infection predisposes humans to sclerosing cholangitis, which may evolve to CCA [ protein . Possibland NRAS . Moreove pathway . In indie to CCA . Wnt/β-ce to CCA . Helicobacter pylori was found to be associated with opisthorchiasis and hepatobiliary disease [ disease . It indu disease .Salmonella typhi. Chronic carriers for S. typhi show a 200-fold risk of developing CCA [Salmonella enterica serovar Typhimurium increases mRNA expression of Wnt2, Wnt3, Wnt6, Wnt9a, Wnt11 as well as Fzd2, Fzd4, Fzd6, Fzd7, Fzd8, and Fzd9 [S. enterica serovar Typhimurium could dysregulate the Wnt/β-catenin signaling by the translocation of antigens through the gut–vascular barrier, with a mechanism based on its pathogenicity island (Spi) 2–encoded type III secretion system that permits its dissemination in the liver and bloodstream [Salmonella in the activation of the Wnt pathway in CCA.ping CCA . In inteand Fzd9 ,146,147.odstream . Inductiodstream . These dKinases involved in the Wnt signaling pathway could represent attractive molecular targets for their inhibitors and antiviral agents in the treatment of infectious agents responsible for liver cancer. A novel therapeutic target in HCV-infected patients could be GSK-3, which constitutively phosphorylates cytosolic β-catenin. GSK-3 is expressed as GSK-3α and GSK-3β isozymes, which are structurally similar in catalytic domains . Under pOther interesting therapeutic perspective is represented by targeting of the β-catenin destruction complex  with the aim to induce the inactivation of Wnt signaling in HCC. The protein destruction complex induces degradation through CK1- and GSK3-mediated phosphorylation of β-catenin, followed by enrollment of the beta-transducin repeats-containing protein (β-TrCP) ligase . TargetiThe identification of microbial DUBs, capable of interfering with the molecular pathways of the host cell, could be important for the development of inhibitory drugs. Therefore, it would be interesting to understand whether infectious agents, related to the occurrence of primary liver tumors, produce DUBs that inhibit the proteasomal degradation of β-catenin. Pathogens alter Wnt-associated processes to increase both infection and survival in the human host. In conserved eukaryotes, Wnt signaling is involved in the interplay between the human host and extracellular and intracellular bacterial pathogens . These aA better insight into the molecular mechanisms leading to dysregulation of Wnt signaling during infections could open up new perspectives in clinical practice with integrated therapies in PLC patients.


Periodontal lesions are common and frustrating diseases that impact life quality. Efforts in this aspect aim at developing local drug delivery systems with better efficacy and less toxicity. Herein, inspired by the sting separation behavior of bees, we conduct novel reactive oxygen species (ROS)-responsive detachable microneedles (MNs) that carry antibiotic metronidazole (Met) for controllable periodontal drug delivery and periodontitis treatment. Benefiting from the needle-base separation ability, such MNs can penetrate through the healthy gingival to reach the gingival sulcus's bottom while offering minimal impact to oral function. Besides, as the drug-encapsulated cores were protected by poly (lactic-co-glycolic acid) (PLGA) shells in MNs, the surrounding normal gingival tissue is not affected by Met, resulting in excellent local biosafety. Additionally, with the ROS-responsive PLGA-thioketal-polyethylene glycol MN tips, they can be unlocked to release Met directly around the pathogen under the high ROS in the periodontitis sulcus, bringing about improved therapeutic effects. Based on these characteristics, the proposed bioinspired MNs show good therapeutic results in treating a rat model with periodontitis, implying their potential in periodontal disease. Porphyromonas gingivalis (P.g.) and Fusobacterium nucleatum (F.n.) are the primary culprits [Periodontitis is a common oral disease worldwide that brings great trouble to human health and life, being considered the leading cause of tooth loss in adults ,2. Generculprits . To defeculprits , ornidazculprits , and metculprits . Among tculprits . Despiteculprits –10. In aculprits ,12. ThusHere, inspired by the sting separation behavior of bees, we propose detachable microneedles (MNs) with Met loading and inflammation-responsive ability to eliminate anaerobic bacteria and treat periodontal disease Fig. . MNs, anIn this paper, getting inspiration from the separable sting bees, we present the Met-loaded, reactive oxygen species (ROS)-responsive and detachable MNs to deliver drugs into the gingival sulcus for combating anaerobic bacteria to treat periodontitis. Poly  (PVA) was chosen as the back of the MNs because it could promptly dissolve and detach from the tissue-penetrated MNs , thus re2 for 24 h, and the scanning electron microscopy showed that the tips were porous, indicating the ROS-responsive ability of MNs , oral mucosa cells (HaCaT), and human umbilical vein endothelial cells (HUVECs) were taken as regular cell lines to incubate with MNs for cell viability assessments. It was demonstrated by the calcein-acetoxymethyl staining that all the cells grew gradually and well whether they were cultured with MNs or not Fig. . The CelThe MN patch for periodontal treatment is rare but efficient, as the oral mucosa is easy to penetrate and adhere to. To verify the therapeutic effect of MNs, the periodontitis model was established in Sprague Dawley rats with tying silk thread. After 3 weeks of treatment, the micro-computed tomography (micro-CT) was used to detect the periodontal tissue. As in Fig. It is crucial to consider the biosafety of biomaterials, especially implanted biomaterials. To check the regional tissue after MN application, we collected the regional draining lymph node and the adjacent tissue, upper lip, and lower lip to check the status after application. The H&E staining results exhibited no noticeable pathological change after MN insertion with or without the drug Fig. , indicatIn summary, we have presented a bee-inspired Met-loaded separable MN that could ROS-responsively release drugs into the gingival sulcus to treat periodontists. To ensure proper oral function, drug-laden microcarriers like nanoplatform ,37, spunHerein, inspired by the behavior of the bee sting separation, we have fabricated separable MNs with ideal responsive drug releasing and antiperiodontal pathogen abilities to conquer the above problems. Owing to the direct penetration of MNs into the sulcus, the drug could be injected into the bottom of the sulcus to kill the infiltrating bacteria. Also, the separation between MNs and the MN base minimized the effects of MNs on normal oral function as the space taken by MNs was reduced sharply. Besides, due to the slowly degraded protective PLGA shells and ROS-responsive PLGA-TK-PEG tips, these MNs achieved inflammation-responsive local drug delivery and thus effectively avoided the systemic adverse reactions caused by antianaerobic antibiotics. In addition, the MNs showed an outstanding performance in antibacteria and bone loss inhibition in the rat periodontitis model. These properties make the drug-loaded MNs potential candidates for periodontal diseases and other oral applications.Polyvinyl acetate, fetal bovine serum, and cell culture medium (high-glucose Dulbecco’s modified Eagle medium) were obtained from Adamas-beta . Fluorescent polystyrene nanoparticles (L2153 and L9777) were from Sigma. The CCK-8 was purchased from Bimake.2O2 was used to mimic the high ROS environment. After incubation for 24 h, a scanning electron microscopy test was conducted to analyze the change of tips.The MNs were developed with a modified polydimethylsiloxane mold as previously described . The tipThe force collection is launched when the sensor contacts the tips at a certain speed (0.2 mm/s). To test the MNs' penetration ability, MNs were pressed into a rat’s gingival. The tissue was dealt with gentian violet (1%) for 5 min to make the change obvious. After that, the tissue was collected for H&E staining.To test the stability of MNs, a degradation detection was tested in simulated human saliva. The chips of MNs were weighed precisely, and after continuous vibration under 37 °C, the chips were dried and their mass was measured. The volume change was also detected by the measurement of MNs’ diameter and height.The FITC-BSA was used to mimic the drug in the MNs. After storage in the artificial saliva for a predetermined time, the supernatant was taken out to detect the spectrum at 520 nm under the excitation of 494 nm. The concentration was calculated through the curve of the standard substance.P.g. is the leading pathogen resulting in the periodontists. To test the antibacterial capacity on P.g., the P.g. was seeded on a plate. The MNs were sterilized with ultraviolet irradiation for 24 hours. After MN application, bacteria were cultured for 7 d before checking the colony formation. Also, the MNs were incubated with bacteria in liquid nutrient medium for 24 h, after which the supernatants were recoated on a plate.The 3T3, HaCaT, and HUVEC cell lines were used as model cells to test the toxicity. The cells were added in a 96-well plate, with 3,000 cells per well, respectively. After attachment, the MNs were put into the culture system after sterilization. Then, the CCK-8 test and live/dead staining were conducted on the first, second, and third day. The calcein-acetoxymethyl/propidium iodide (PI) were added to detect the live/dead status following the manual with fluorescence microscopy.All the animal experiments were under the approval of the Animal Investigation Ethics Committee of The Affiliated Drum Tower Hospital of Nanjing University Medical School (2021AE02007). For rat models of periodontitis, female Sprague Dawley rats were used. After anesthesia, silk sutures (4-0) were inserted and knotted around the subgingival area of the maxillary M2 (second molar). A high-sugar drink was applied during the model's build to accelerate the process. After confirming the model establishment with micro-CT, the silk was removed, followed by the treatment. The animal was divided into 3 groups: (a) control group (untreated); (b) empty MNs (without drug); and (c) MNs with drug. After the rat was anesthetized with 2% isoflurane, the MNs were inserted into the gingival sulcus through the gingival, horizontally, and the bases were removed when separating from the tips. The patches were checked daily to ensure that the MNs were in place. The MNs were reported when missing, and the rats' weights were measured every other day.The rats were sacrificed under anesthesia when the treatment was over. The major organs and local tissue in the oral cavity were collected for pathological examination. The tissues were fixed with 4% paraformaldehyde overnight, followed by embedment in paraffin. The maxillary bones were fixed in 4% paraformaldehyde. After micro-CT analysis, they were decalcified in a decalcifying solution (10% EDTA and 1% NaOH) for 20 d. After puncture confirmation of decalcification, tissue fixation and embedding were performed. The serial sections of rat maxillary were obtained along the mesiodistal direction, followed by H&E staining. IHC staining was conducted separately on maxillary sections with TNF-α, IL-6, iNOS, and Arg-1.t test between 2 groups and 1-way analysis of variance for more than 2 groups.All data in the figures were demonstrated as means with standard deviation (mean ± SD). The statistics were analyzed using GraphPad software by the Student


In conclusion, it seems that WNT signaling system has functional role in the preimplantation goat embryos, and inhibition of this pathway during the period of compact morula to blastocyst transition (D4-D7) can improve preimplantation embryonic development.The specific role of the canonical WNT/β-catenin signaling pathway during the preimplantation development of goat remains unclear. Our objective was to investigate the expression of β-CATENIN, one of the critical components of Wnt signaling pathway, in IVF embryos and compare it with SCNT embryos in goat. In addition, we evaluated the consequence of inhibition of β-catenin using IWR1. Initially, we observed cytoplasmic expression of β-CATENIN in 2 and 8–16 cell stage embryos and membranous expression of β-CATENIN in compact morula and blastocyst stages. Furthermore, while we observed exclusively membranous localization of β-catenin in IVF blastocysts, we observed both membranous and cytoplasmic localization in SCNT blastocysts. We observed that Inhibition of WNT signaling by IWR1 during compact morula to blastocyst transition (from day 4 till day 7 of  TheWnt signaling pathway is a highly regulated and complex signaling pathway which is regulated by various WNT ligands, and these ligands interact and bind with a variety of receptors, including frizzled (FZD), receptor tyrosine kinase-like orphan receptor (ROR), receptor tyrosine kinase-related tyrosine kinase (RYK) and protein tyrosine kinase-7 (PTK7) and also co-receptors including low-density lipoprotein receptor-related proteins 5 and 6 (LRP5 and LRP6) –26.Currently, three different WNT pathways are introduced to be activated upon WNT-receptor interaction , 27–30. 2+ signaling pathway. Each pathway is activated by specific ligands and receptors that can be varied among different cell types or different physiological conditions, even in a defined cell type. These differences are related to multifaceted interactions between various ligands and receptors, and regulatory molecules in intercellular and intracellular microenvironments. Thus, specific ligand-receptor interactions can have various consequences in different cell types [The two other pathways are non-canonical pathways and are independent of β-catenin. A typical example of a non-canonical pathway is the planar cell polarity pathway (PCP). The other non-canonical pathway is the Call types , 27, 29.The canonical WNT/ß-Catenin pathway is initiated by the interacting WNT ligands with FRZ/LRP5 and LRP6 co-receptors. This complex recruits the dishevelled (Dvl), and subsequently Dvl traps the destruction complex containing Axin/APC/CK1/GSK-3β. This event prevents phosphorylation and degradation of β-catenin and then leads to the accumulation of β-catenin in the cytoplasm and translocation to the nucleus, where it activates transcription factors in the nucleus –33.Canonical WNT/ß-Catenin is a maternally-derived pathway that seems to be essential for the preimplantation development of embryos . StudiesWhile, up to our knowledge, there is no report about the role of WNT signaling pathway in the preimplantation development of goat, the results in bovine, led us to interpret the role of the WNT pathway by pharmacological inhibition of Wnt/β-catenin using a Tankyrase inhibitor, IWR1 in IVF- and SCNT derived embryos and investigating its effect on preimplantation embryo development.All methods used in this study were performed under the Institutional Review Board and Institutional Ethical Committee of the Royan Institute guidelines and regulations (IR.ACECR.ROYAN.REC.1399.074). In addition, the slaughterhouse ovaries were transported to the embryology laboratory with the permission of the manager of the slaughterhouse and the agreement of the veterinary organization.All materials and media used in this study were obtained from Sigma-Aldrich Company  and Gibco , respectively, unless otherwise indicated.The experimental design of this study included the following steps:FRIZZLED and β-CATENIN in 2 cell, 8–16 cell, compact morula and blastocyst embryos derived from IVF in goat. In this experiment, embryos were collected at each stage at a specific and defined time point. The 2 cell embryos were collected at 30-hour post insemination (hpi), 8–16 cell stage at 72 hpi, compact morula stage at 96 hpi and blastocyst stage at 144–156 hpi. The mRNA expression was assessed using real-time RT-PCR in three independent replications. In addition, the protein expression was assessed using β-CATENIN monoclonal antibody with immunocytochemistry (ICC) technique in stages mentioned above of goat IVF embryos in three independent replications.In experiment 1, we first assessed the mRNA expression of in vitro culture (IVC) from day 5 (D5) to D7 (D5-D7). Then, we extended the treatment time from D4 to D7 (D4-D7). On day 7 post-fertilization the blastocyst rate, blastomere allocation (in D4-D7 group), protein expression of β-CATENIN using immunostaining (ICC) (in D4-D7 group) and mRNA expression of OCT4, NANOG, CDX2, and β-CATENIN using real-time RT-PCR (in D4-D7 group) were assessed.Experiment 2 was designed to determine the effect of IWR1, a Wnt/β-catenin signaling inhibitor, on the development of goat IVF-treated embryos. First, the IVF embryos were treated with IWR1  for three days during FRIZZLED and β-CATENIN using real-time RT-PCR and protein expression of β-CATENIN using ICC between IVF and somatic cell nuclear transfer (SCNT) derived blastocysts.Experiment 3 was carried out to compare the mRNA expression of OCT4, NANOG, CDX2, and β-CATENIN were assessed in the D4-D7 group.Finally, in experiment 4, to assess the effect of supplementation of IVC with IWR1 on blastocyst rate of SCNT embryos, the SCNT embryos were treated with IWR1  from D4 to D7 (D4-D7). Then, at day seven, blastocyst rate, blastomere allocation, protein expression of β-CATENIN and mRNA expression of Commercially available IWR1  was dissolved in DMSO (10 mg in 489 μl DMSO), which was formulated in 50 mM stock solution and stored at -20°C. The desired working solutions including, 1.25, 2.5 and 5 μM, were made by diluting the stock solution with synthetic oviduct solution (SOF) . In 5 μMIn this study, goat ovaries were collected from two local slaughterhouses (Khomeini Shahr and Fasaran) and transferred to the embryology laboratory. After the arrival of the ovaries, the surrounding tissues were separated, and the ovaries were kept in the normal saline supplemented with penicillin/streptomycin at 15° C for overnight .in vitro maturation (IVM) [In the next day, after washing the ovaries, cumulus-oocyte complexes (COCs) were retrieved from follicles with diameter of 2–6 mm with a disposable 21-gauge needle connected to a vacuum pump (60–70 mm Hg). The aspiration medium was the HEPES-buffered tissue culture medium 199  supplemented with 10% fetal bovine serum (FBS) and heparin (10 μl/ml). The aspirated medium was transferred to a 10-cm culture dish, and only grade I and II oocytes were selected for on (IVM) .The procon (IVM) , 43. The6/ml motile sperms in droplets of fertilization medium at 38.5°C, 6.5% CO2 and humidified atmosphere. Eighteen hours post insemination, presumptive zygotes were mechanically denuded of cumulus cells, and cultured in synthetic oviduct fluid (SOF) supplemented with ITS  and Myo-inositol (modified SOF: mSOF) without glucose and serum (mSOF-) for 3 days at 38.5°C, 6.5% CO2 and 5% O2 in humidified air under mineral oil. On Day 3, the cleavage rate was assessed. After that, the embryos were transferred to mSOF medium supplemented with charcoal stripped serum (mSOF+), and the blastocyst rate was evaluated on Day 7. The derived embryos at 2 cell, 8–16 cell, compact morula and blastocyst stages were used for evaluating the mRNA and protein expression of target gene(s) (experiments 1 and 3). In addition, in the periods mentioned above , the IVC medium (D5-D7 or D4-D7) was supplemented with IWR1  to assess the inhibition of WNT pathway on preimplantation development of IVF embryos.The IVF procedure was performed similar to our previous studies . To thisGoat ear fibroblast cells (GEFs) were obtained from the Sannen breed, similar to our previous papers .The spec®, Australia) were used for oocyte-cell fusion. Then, the oocyte-cell couplets were activated using ionomycin (5 μM) for 1 min and then cultured in 2 mM 6-DMAP at 38.5°C, 6.5% CO2, and maximum humidified atmosphere for 2 h under mineral oil. Finally, SCNT embryos were cultured in mSOF+ for 7 days at 38.5°C, 6.5% CO2, 5.5% O2, and humidified atmosphere under mineral oil [ß-CATENIN/ß-CATENIN (experiment 3). In addition, the IVC medium of SCNT embryos was supplemented with IWR1  during D4 to D7 (D4-D7) to assess the inhibition of WNT pathway on preimplantation development of SCNT embryos.Twenty hours after IVM, cumulus cells were removed from matured COCs with hyaluronidase (300 IU/ml). Then, the zona pellucida was dissolved by treating with pronase (2.5 mg/ml) for up to 30 secs. A finely handmade pulled Pasteur pipette was used for enucleation of the matured oocytes . Demecoleral oil . The SCNThe quality of the IVF and SCNT-derived blastocysts from various treatments was evaluated by differential staining allowing us to distinguish and quantify the blastomere allocation to trophectoderm (TE) and inner cell mass (ICM) that appeared pink or blue under UV light, respectively. Briefly, blastocysts at day 7 were washed in HTCM199 and 5 mg/mL bovine serum albumin (BSA). After that the blastocysts were treated with 0.5% Triton X-100 for 20 secs and then with 30 mg/mL propidium iodide for 30 secs. In the end, blastocysts were stained and fixed for 15 minutes in ice-chilled ethanol supplemented with 10 μg/ml Hoechst 33342. After mounting on the slide, the blastocysts were examined using a fluorescent microscope . At least 20 blastocysts  in three replicates were used for differential staining.Embryos were washed three times in phosphate-buffered saline/polyvinyl alcohol (PBS/PVA), fixed in 4% Paraformaldehyde (PFA) for 20 min. After washing extensively, embryos were permeabilized with 1% Triton-X-100 for 30 min at RT. Then, the unspecific binding sites in embryos were blocked by treating with blocking solution (PBS/PVA containing 0.1% Triton-X-100 + 1% BSA + 10% goat serum). Embryos were then incubated for overnight at 4°C with ß-CATENIN primary antibody (Anti-B-catenin antibody (E5); sc-7963, Santa Cruz). An appropriate negative control was carried out by omitting the primary antibody and using mouse IgG . After w-, collected in pools of 20, frozen and stored at -80°C until RNA extraction. All embryo pools used for RNA extractions were collected and analyzed in triplicates. RNA extractions of the embryo pools were then performed using the RNeasy Plus Micro Kit  according to the manufacturer’s protocol. Total RNA was reverse transcribed using a cDNA Synthesis kit (Biotech rabbit) according to the manufacturer’s protocol. The quality and integrity of cDNA were checked using PCR and the housekeeping primer (B-ACTIN) as a reference gene. Real time PCR (qPCR) was performed using the SYBR Green Technology. Analysis of qPCR was performed in a 10 μl reaction volume by adding 1 μl cDNA in the PCR mix containing gene specific primers  and 5 μl SYBR Green. qPCR conditions were 5 min at 95°C and 40 cycles of 20 seconds at 95°C and 20 seconds at specific annealing temperature for each specific paired primer. At the end of every reaction, a melt curve analysis was performed to ensure the specificity of the products. The mean cycle threshold (Ct) was calculated after performing three technical replicates for each sample. Relative expression was computed using mean Ct values which were normalized against B-ACTIN. Fold change in gene expression was calculated using 2-ΔΔCT. All the primers were designed by the Primer 3 program (http://primer3.ut.ee/), and their characteristics are listed in The mRNA expression of target genes see  was analP< 0.05. One-way analyses of variance (ANOVA) was applied to compare the effect of the treatments between various groups (α = 0.05), followed by the Tukey post-hoc test. The comparison of mRNA and protein expression of β-CATENIN between IVF and SCNT groups and also the mRNA expression of target genes in IVF and SCNT blastocysts derived from IWR1 (5 μM) treated groups compared to their control groups were analyzed by independent samples t-test. GraphPad Prism (v.6.0.1) and IBM SPSS program  were used for creating graphs and statistical analysis, respectively.All assessments were carried out at least three times. Data are presented as mean ± S.E.M. Statistical significance was set at FRIZZLED and β-CATENIN, two crucial components of the WNT signaling pathway, in IVF preimplantation goat embryos . The mRNA expression level of both genes was similar between 2 cell and 8–16 cell stage (P> 0.05) and also between the compact morula and blastocyst stages (P> 0.05), but it was significantly lower in compact morula and blastocyst stage as compared to 2 cell and 8–16 cell stage embryos (P< 0.05).In the first step of this experiment, we assessed the relative mRNA expression of st stage , P< 0.05P< 0.05, P< 0.05, β-CATENIN in blastocyst embryos.In the next step, we immunocytochemically assessed the protein expression of β-CATENIN, the main component of the WNT signaling pathway in preimplantation IVF goat embryos. The immunolabelling of β-CATENIN revealed cytoplasmic localization of β-CATENIN in 2-cell, 8–16 cell and compact morula stage . InteresP> 0.05) the blastocyst rate as compared to embryos cultured without IWR1.This experiment (Experiment 2) was carried out to assess if inhibition of canonical WNT signaling using IWR1 promoted the blastocyst formation of IVF goat embryos. Based on previous studies, we added various concentrations of IWR1   to the IP< 0.05). While the blastocyst rate in the 1.25 and 2.5 μM groups was higher than the control group but it did not reach a significant level (P> 0.05).In this study, treatment of goat IVF embryos was started at 5 dpi based on a study in bovine. While the compaction of goat embryos starts one day earlier (at 4 dpi) as compared to bovine embryos, we extended the time of treatment, and IWR1 was added to the IVC medium at 4 dpi) till 7 dpi (D4-D7 group) to assess the developmental competence of IWR1 treated embryos. As shown in nt level P> 0.05.P> 0.05).Then, we assessed the effect of IWR1 on the quality of derived blastocyst. While the proportion of cleaved embryos that reach to blastocyst rate increased following treatment with IWR1, IWR1 did not significantly affect the ICM, TE and TCN , P> 0.05In addition, we assessed the protein expression of β-CATENIN in derived blastocysts. As depicted in OCT4, NANOG, CDX2, and β-CATENIN remained unchanged after treatment with 5 μM IWR1 in D4-D7 group as compared to control group (P> 0.05).Then, blastocyst embryos at day 7 were assessed mRNA expression of target genes using real-time RT-PCR. The results  revealedFRIZZLED and β-CATENIN in blastocysts derived from IVF and SCNT groups. As depicted in FRIZZLED and β-CATENIN was similar between IVF and SCNT embryos at blastocyst stage (P> 0.05).This experiment was designed to assess the relative mRNA expression of st stage , P> 0.05P> 0.05). Interestingly, we observed that while the localization of β-CATENIN in IVF blastocysts is exclusively membranous, its localization in SCNT blastocysts is both membranous and cytoplasmic.In addition, the protein expression of β-CATENIN was assessed by immunocytochemistry in IVF and SCNT blastocysts. Quantification of fluorescence intensity of FITC-labeled β-CATENIN revealed a similar level of fluorescence intensity between IVF and SCNT embryos at the blastocyst stage , P> 0.05P< 0.05) in the percentage of cleaved embryos that developed to blastocyst stage at day 7.This experiment (Experiment 4) was carried out to assess if inhibition of canonical WNT signaling using IWR1 promoted the blastocyst formation of SCNT goat embryos. Based on results from IVF embryos (Experiment 2), we added 1.25 and 5 μM IWR1 to the IVC medium at day 4 post activation (4 dpa) till 7 dpa (SCNT D4-D7 group) to assess the blastocyst developmental rate. As depicted in P> 0.05).Then, we assessed the effect of IWR1 on blastomere allocation at the blastocyst stage. While the proportion of cleaved embryos that reached to blastocyst stage increased following treatment with IWR1, IWR1 did not affect the ICM, TE and TCN , P> 0.05P< 0.05). Interestingly, we observed that treatment of SCNT embryos with IWR1 shifted the localization pattern of β-CATENIN from both cytoplasmic and membranous to the only membranous pattern at the blastocyst stage .Then, assessing the mRNA expression of target genes in day 7 blastocysts revealed that, similar to IVF blastocysts , mRNA exol group , P> 0.05-CATENIN is required for the process of compaction, and its subsequent reduction is required to maintain the state of pluripotency, at least in ungulates, to prevent ICM differentiation during dormancy or before implantation [β-CATENIN in the whole embryo and in TE and ICM post IWR1 treatment. Previous studies have shown that different types of cell-to-cell adhesion, including Ca2+ dependent cell adhesion, gap junction, and tight junction, developed during the formation of compact morula and blastocyst embryos and is one of the perquisites for this stage [The canonical WNT pathway is one of the main signaling pathways involved in the early embryonic development of mammals, and little is known regarding its role in preimplantation embryos . In geneantation , 55. Theis stage –58. Withis stage , 60, whiis stage .in vitro conditions, including IVF procedure, may dysregulate these pathways and hamper proper embryonic development. IWR1 is an inhibitor of the WNT pathway that acts intracellularly to inhibit Tankyrase (TNKS) and stabilize the AXIN, leading to an increase in the destruction of ß-catenin and the inactivation of this pathway [E-cadherin is associated with β-catenin, and these two proteins accumulate together in the cell membrane. This association stabilizes the β-catenin by inhibition of the binding of the destruction complex to the β-catenin . It has  pathway . We obsein vivo condition, during compact morula formation, the endometrium express DKK1, which is an inhibitor for the WNT/β-catenin pathway [In vitro studies in bovine demonstrated that activating this pathway (using AMBMP) at day 5 decreased the blastocyst rate and reduced numbers of trophectoderm and inner cell mass cells. Further, they showed that DKK1 (inhibitor of the WNT/β-catenin pathway) could block the action of AMBMP and reverse its detrimental effect. However, DKK1, in the absence of AMBMP, did not improve the development, which is inconsistent with our result that may be related to species difference or the action of inhibitor of the WNT pathway (IWR1 vs DKK1) [It has been shown that in  pathway , 62. In vs DKK1) , 63. TheFRIZZLED and β-CATENIN and protein expression of β-CATENIN in goat IVF and SCNT blastocysts. The relative mRNA expression of FRIZZLED and β-CATENIN was similar between IVF and SCNT blastocysts. There are very limited studies about the status of this pathway in SCNT embryos. In contrast to our results, a recent study demonstrated the downregulation of DKK1 in SCNT compared to IVF blastocysts in buffalo which indicates the higher activity of the WNT/β-catenin pathway in SCNT blastocysts in buffalo as compared to their IVF counterparts [Additionally, we assessed the protein expression of the β-CATENIN in the treated embryos to monitor if treatment with IWR1 during D4-D7 of IVC, affects the expression of the β-CATENIN protein. The quantification of ICC images revealed effective inhibition of the WNT pathway by downregulation of the β-CATENIN protein in derived blastocysts. Next, we asked if inhibition of the WNT/β-catenin pathway affects blastomere allocation and, or gene expression. Our results revealed no difference in the blastomere allocation, pluripotency and trophectodermal markers in the derived blastocysts. Previous studies also showed that DKK1 (antagonist of the WNT/β-catenin pathway) did not affect the number of ICM or TE cells, suggesting that ICM proliferation is regulated through a mechanism independent of β-catenin, which needs to be elucidated . In the terparts . Then, wSo, in the following experiment, similar to IVF embryos, SCNT embryos were treated with IWR1 from 4 dpa till 7 dpa. Similar to IVF embryos, we observed a higher blastocyst rate in IWR1 treated embryos. In addition, ICC images showed that IWR1 treatment changed the localization of β-CATENIN and exhibited only a membranous pattern, which is an indicator of the inactivation of the WNT/β-catenin pathway in SCNT blastocysts. Consequently, we hypothesized that maybe extended treatment of SCNT embryos with IWR1 during all days of IVC (D0-D7) may increasingly enhance the blastocyst rate. Interestingly, we observed that prolonged treatment of SCNT embryos (D0-D7) with IWR1 insignificantly decreased the blastocyst rate  that, agTaken together, our data revealed activation of the WNT/β-Catenin pathway in the early stage of preimplantation development of IVF goat embryos and inactivation of this pathway around compact morula and blastocyst embryos. In addition, we demonstrated that supplement of IVC medium with IWR1, an inhibitor of the canonical WNT signaling pathway, before EGA but not after EGA promotes blastocyst formation rate in both IVF and SCNT goat embryos. Furthermore, IWR1 changed the localization of β-catenin from cytoplasmic to the membranous pattern in SCNT goat embryos. It is hypothesized that the action of IWR1 modulates WNT/β-catenin signaling in developing embryos in goat and may accelerate further development during postimplantation.S1 FigScale bars represent 200 μm.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S1 TableP> 0.05).At least three replications were performed for each treatment. Developmental rates of treated embryos were monitored as cleavage and blastocyst rates at day 3 and 7, respectively. Within a column, developmental rates with similar superscripts are not significantly different from each other ((DOCX)Click here for additional data file.S2 TableAt least three replications were performed for each treatment. Developmental rates of treated embryos were monitored as cleavage and blastocyst rates at day 3 and 7, respectively. Within a column, developmental rates with different superscripts (a and b) are significantly different from each other (P< 0.05).(DOCX)Click here for additional data file.S3 TableAt least three replications were performed for each treatment. Developmental rates of treated embryos were monitored as cleavage and blastocyst rates at day 3 and 7, respectively. Within a column, developmental rates with different superscripts  are significantly different from each other (P< 0.05).(DOCX)Click here for additional data file.


Tumor-initiating cells (TICs) are rare, stem-like, and highly malignant. Although intravenous hepatitis B and C immunoglobulins have been used for HBV and HCV neutralization in patients, their tumor-inhibitory effects have not yet been examined. Hepatitis B immunoglobulin (HBIG) therapy is employed to reduce hepatocellular carcinoma (HCC) recurrence in patients after living donor liver transplantations (LDLT).We hypothesized that patient-derived intravenous immunoglobulin (IVIG) binding to HCC associated TICs will reduce self-renewal and cell viability driven by β-CATENIN-downstream pathways. β-CATENIN activity protected TICs from IVIG effects.The effects of HBIG and HCIG binding to TICs were evaluated for cell viability and self-renewal.EGLN3-AS1 and lnc-β-CatM. Co-expression of constitutively active β-CATENIN with NANOG promoted self-renewal ability of TICs through EGLN3 induction. β-CATENIN-induced lncRNAs stabilized HIF2 to maintain self-renewal of TICs. Targeting of EGLN3-AS1 resulted in destabilization of EZH2-dependent β-CATENIN activity and synergized cell-killing of TICs by HBIG or HCIG immunotherapy.Inhibition of β-CATENIN pathway(s) augmented TIC susceptibility to HBIG- and HCIG-immunotherapy. HBV X protein (HBx) upregulates both β-CATENIN and NANOG expression. The co-expression of constitutively active β-CATENIN with NANOG promotes self-renewal ability and tumor-initiating ability of hepatoblasts. HBIG bound to HBV+ cells led to growth inhibition in a TIC subset that expressed hepatitis B surface antigen. The HBx protein transformed cells through β-CATENIN-inducible lncRNAs Taken together, WNT and stemness pathways induced HIF2 of TICs via cooperating lncRNAs resulting in resistance to cancer immunotherapy. Therefore, therapeutic use of IVIG may suppress tumor recurrence through inhibition of TICs. Hepatocellular carcinoma (HCC) is the fourth most common cancer, accounting for 80% of liver cancer cases, and is the third deadliest form of cancer –3. Clinie antigen (+) as an adjunct following living donor liver transplantation (LDLT)  . This HBn (LDLT) . FurtherAs a contributor to more than 50% of HCC cases worldwide , hepatitin vivo how HBV-mediated beta-CATENIN signaling promotes stemness and tumor recurrence/therapy resistance. The interplay between HBx, β-CATENIN, and other signaling molecules to promote malignancy and TIC formation has not been fully elucidated. β-CATENIN/CBP signaling promotes HCC tumor occurrences through β-CATENIN/CBP-target gene expression, including MYC, CYCLIN D1 and OCT4. It is important to determine in vitro and in vivo. Intravenous immunoglobulin (IVIG) immunotherapy is an established practice. Cancer resistance to therapy is seen in the clinic and one of several contributory driver pathways is β-CATENIN signaling. The mechanism(s) as to how beta-CATENIN signaling promotes immunotherapy resistance and IVIG treatment resistance mechanisms are not fully understood. The pluripotency transcription factor NANOG promotes stemness and reprograms metabolism which promote therapy resistance and self-renewal  and maintained 10 months after DEN injection and euthanized. These mice exhibited NANOG and β-CATENIN levels that were both reduced compared to the DEN+ Tg05 line which expressed an intact HBV genome  (+ Tg05 and DEN+ Tg08), the β-CATENIN and NANOG staining appeared to overlap. The DEN+ Tg05 sample β-CATENIN staining was positive near the portal triad, whereas in the DEN+ Tg08 sample β-CATENIN staining was positive near fatty liver and dysplastic tissue ; this confirmed that Polycomb repressive complex 2 (PRC2) complexes were significant NANOG regulators , which suppresses gene expression by facilitating chromatin condensation and recruitment of several demethylases to remove active histone marks (H3K4me3). We validated the predicted synthetic lethality targets of these PRC2 components, as previously identified by GeCKO-lentivirus library screening against a mutant arid1A background. We hypothesize that the loss of ARID1A activity leads to the elevated PRC2 activity resulting in inactivation of polycomb target genes (including OXPHOS genes) through H3K27-trimethylation. The latter chromatin modification would lead to the hyperactivation of the stem cell gene programs, including NANOG induction. H3K27 trimethylation represses PRC2 target genes  increase PRC2 complex activity (including expression of the EZH2 component) and induce an HCV/alcohol-mediated stem cell program (slow growing cells), leading to TIC-initiated HCC development  is known to activate the WNT pathway . We obseP<0.05) in liver tumors compared to normal livers (P<0.05).Previous studies have shown a significant increase in TLR4 and two NANOG-target genes , includil livers  was valiThe transcription factor TCF  gene encodes a member of the T-cell factor/lymphoid enhancer-binding factor family of high mobility group (HMG) box transcriptional activators. This gene is expressed predominantly in T-cells and plays a critical role in natural killer cell and innate lymphoid cell development. The encoded protein forms a complex with β-CATENIN and activates transcription through a WNT/β-CATENIN signaling pathway.Next, we analyzed the ChIP-seq data for EZH2 and TCF, which formed a complex with β-CATENIN, within regions where both the proteins colocalize . The BasProlyl hydroxylase domain-containing protein 2 (PHD2) catalyzes the post-translational formation of 4-hydroxyproline in HIF-1α proteins and leads to destabilization of HIF-1α whereas PHD3 mainly destabilizes the HIF-2α isoform –34. HIF Huh7 cells were infected with the lentivirus expressing sh-lnc-β-CatM. Knockdown of lnc-β-CatM reduced the expression level of the antisense RNA EGLN3-AS1, which binds EGLN-3 mRNA to inhibit protein expression . Accordilnc-β-CatM, q-PCR was done to measure the efficiency of knockdown; for comparison, cells infected with lentivirus expressing scrambled shRNA (Scr) were used as control. The knockdown efficiency of the sh-RNA was high, with 96% reduction in lnc-β-CatM  is a prolyl hydroxylase activity which downregulates HIFs by increasing protein turnover and is a suppressor of HIF-2α. In cancer cells, the expression of HIF target genes is activated under hypoxic conditions (EGLN3-AS1 show that the gene is activated in cancer. We hypothesized that β-CATENIN and EZH2 transactivate EGLN3-AS1 to downregulate EGLN3 in HepG2 cells (resulting in constitutively active β-CATENIN). ChIP-seq data for H3K4me3 and H3K27ac from the HepG2 cell line showed peaks in the upstream region of EGLN3-AS1 (EGLN3-AS1 promoter activity in Huh7 cells (wild type β-CATENIN) (EGLN3-AS1 promoter activity shown by luciferase reporter assay in HepG2 cells (constitutively active mutant of β-CATENIN) , notable among these genes analyzed were EZH2  treatment (EGLN3-AS1 gene (We performed CRISPR/Cas9-mediated knockout of endogenous PHD3 and replacement by PHD3 wild type and a mutant (PHD3.Y98N: phosphatase domain mutant) which were confirmed by immunoblots  compared to HCC  and HBx + NANOG + Scr groups had the highest tumor burden, while the other groups showed little to no tumor burden (As β-CATENIN + NANOG and YAP1 transactivate the BIRC5 promoter (one of the WNT/β-CATENIN-target genes), we tested whether combinations of these proteins have cooperative transactivation abilities. In p53r burden 8.+ patient sera (100-200 donors) with or without prior HBIG treatment could be used for this purpose.Based on the previous results, stopping self-renewal of TICs by antagonizing β-CATENIN activity should allow clearance of these weakened TICs due to loss of self-renewal. This could be accomplished with immunoglobulins that capture TICs to clear this cell population in patients or humanized mice harboring human HCCs. TICs may be recognized by intravenous, immunoglobulin obtained from HBV patients since cancer-driver mutation-targeted immune responses are known to be triggered by neoantigens or other TIC-specific cell surface markers. Use of pooled HBV3H-uridine incorporation and spheroid colony formation assays after treatment with HBIG antibodies. Indeed 3H-uridine was reduced in CD133+/CD49f+ TICs incubated with HBIG  or HBIG incubation. A significant decline of expression levels for NANOG, SOX2, OCT4, and CD133 was detected in HBIG with high affinity for CD133high cells from HBV associated tumors (patients 1 and 2) after HBIG incubation. Whereas TLR4 and KRT19 underwent significant reduction in TICs from patient #1. No significant differences for SOX2, OCT 4, TLR4, and KRT19 were detected in the low-affinity TICs (patients #3 and #4). NANOG and CD133 expression was, however, significantly reduced in the low affinity TICs from patient 3 but not in the TICs from patient 4. Expression of ALB and E-CAD were generally not remarkably different between CD133+/CD49f+ TICs and control CD133-/CD49f+ cells and did not change when incubated with either IVIG or HBIG. In high-affinity TICs derived from patients 1 and 2, negligible amounts of cells expressed stemness marker NANOG after HBIG treatment  as determined by qRT-PCR (+ population that were isolated from HCV-associated HCCs bound HCIG (HBIG binds hepatitis B antigens (such as HBs or HBc)-expressing hepatocytes, including transformed hepatocytes  and , we sequenced the HBIG-resistant TICs. Although HBIG treatment did not completely reduce the derived TICs, the major HCC driver genes were nonetheless sequenced. The drug insensitive population may be due to the fact that HBsAg was not expressed on those cells. The HBIG-resistant tumors harbored a CTNNB1 mutation in exon 3 (mutation hotspot region) that stabilized β-CATENIN protein , proliferation and viability of TICs. HBIG causes significant but partial regression of liver tumors in tumor-bearing mice. Clinical evidence revealed development of resistance  to these HBIG treatments in HCC patients who underwent liver transplantation in a clinical trial . The proHow does HBIG bind to chronically HBV infected patient samples? HBIG or HCIG may bind circulating tumor cells (CTC) more efficiently rather than tumor mass to capture and eliminate the possible metastatic tumor spread or recurrence to inactivate before CTCs extravasate and colonize in distal organs or intrahepatic metastasis. Efficient binding of HBIG or HCIG to disseminated cells from primary HCCs rather than the tumor mass itself would be an ideal characteristic to capture all CTCs before they reach distal organ or intrahepatic HCC spread.The mechanism by which Ig binding reduces expression of NANOG is unknown. The first possible explanation is that antibody binding to the cell surface inhibits cell aggregation or cell-cell interaction to block self-renewal ability, since growing TICs have an aggregated phenotype as opposed to quiescent TICs  is required before both components move into the nucleus and regulate their downstream targets ; Target: EZH2; Biosample summary. Homo sapiens HepG2; Biosample Type: cell line.Experiment summary for ENCSR000ARIencodeproject.orghttps://www.encodeproject.org, experimentsSample GSM4133289Accession number: H3K27Ac_rep1_HepG2_ChIPsephttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM4133289RELACS HepG2 H3K27acGSM3019929RELACS HepG2 H3K4me3GSM3019930Nov 16, 2010 — Homo sapiens HepG2. Biosample Type: cell line. Replication type: isogenic. Description: ChIP-seq on human HepG2 EZH2. Nucleic acid type: DNA.Hepatitis B immunoglobulin  and control 10% intravenous immunoglobulin  antibody preparations were obtained from Biotest Pharmaceuticals Corporation.Cultured TICs were treated with HBIG, HCIG or control IVIG for 48 hours at different concentrations and examined for HBIG or HCIG binding as well as NANOG expression.For the 0 h time point, TICs were harvested and incubated with HBIG or HCIG for 30 min. For HBIG and HCIG binding, we pre-labeled HCIG/HBIG and HBs Ag/E2 with APC and PE before FACS. Cells were permeabilized and processed for anti-NANOG antibody staining, followed by secondary antibody staining.To quantify the levels of β-catenin and NANOG staining in the mouse tissue samples, an immunoreactivity score was calculated, as per Koomagi et al. . The firTo determine if HBIG antibodies bind to propagated tumor stem cells, FACS analysis was used. Cultured TICs were treated with HBIG or control IVIG for 48 hours at different concentrations and examined for HBIG binding as well as NANOG expression. For the 0-hour time point, TICs were harvested and incubated with HBIG for 30 minutes. For HBIG binding, HBIG and HBsAg were pre-labeled with APC and PE for 30 minutes before FACS. Cells were permeabilized and processed for anti-NANOG antibody staining, followed by secondary antibody staining.TICs  were injected at day 0. HBIG was injected intraperitoneally. Control mice were injected with either PBS or HBIG at indicated doses, once a week before TIC injection and from day +7 to day +88 as previously described . Tumor vhttp://rsb.info.nih.gov/ij). For immunohistochemistry, the primary antibodies used were mouse anti-β-catenin  and rabbit anti-NANOG . Secondary antibodies were conjugated to HRP and developed using 3,3’-diaminobenzidine with hematoxylin (Invitrogen) or goat anti-mouse fluorescein isothiocyanate  and donkey anti-rabbit R-Phycoerythrin , depending on the application.Transgenic mouse tissue samples were prepared for protein isolation in RIPA buffer as previously described . ProteinPlasmids used were pBABE-YAP1 , pMXs-NANOG , and pBABE- β-catenin S33Y HA . pCMV-HBx HA  was also used.P-value < 0.05 and was carried out using a two-tailed unpaired Student t-test.All experiments, excluding the Western blot analysis, were carried out in triplicate. The presented data were the averages of three readings ± the standard deviation. Statistical significance was determined from a https://www.encodeproject.org ›experiments Accession Number: Sample GSM4133289 H3K27Ac_rep1_HepG2_ChIPsep https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM4133289 RELACS HepG2 H3K27ac GSM3019929 RELACS HepG2 H3K4me3 GSM3019930.The original contributions presented in the study are publicly available. This data can be found here: Experiment summary for ENCSR000ARI encodeproject.org Ethical approval was not required for the studies on humans in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used. The animal study was approved by institutional animal care and use committee (IACUC). The study was conducted in accordance with the local legislation and institutional requirements.CN, KM, and CC conceived of the study. MN, CN, KM, DK, RM obtained the data. PF, and LM performed the immunostaining data analysis, provided drugs. LM, PF, JO, LS provided HCC tissues. J-HO provided HBV transgenic mouse tissues. KM, MN, CN, SMT, and CC conducted the data analysis and drafted the report. All authors contributed to the article and approved the submitted version.