Flexible endoscopy involves the insertion of a long narrow flexible tube into the body for diagnostic and therapeutic procedures. In the gastrointestinal (GI) tract, flexible endoscopy plays a major role in cancer screening, surveillance, and treatment programs. As a result of gas insufflation during the procedure, both upper and lower GI endoscopy procedures have been classified as aerosol generating by the guidelines issued by the respective societies during the COVID-19 pandemic—although no quantifiable data on aerosol generation currently exists. Due to the risk of COVID-19 transmission to healthcare workers, most societies halted non-emergency and diagnostic procedures during the lockdown. The long-term implications of stoppage in cancer diagnoses and treatment is predicted to lead to a large increase in preventable deaths. Robotics may play a major role in this field by allowing healthcare operators to control the flexible endoscope from a safe distance and pave a path for protecting healthcare workers through minimizing the risk of virus transmission without reducing diagnostic and therapeutic capacities. This review focuses on the needs and challenges associated with the design of robotic flexible endoscopes for use during a pandemic. The authors propose that a few minor changes to existing platforms or considerations for platforms in development could lead to significant benefits for use during infection control scenarios. There have been over 94 million cases reported and over two million fatalities worldwide  to patients and the potential for aerosol generation. Recent work has shown that endoscopists without proper face protection such as a face visor could be at an increased risk to bacterial pathogens . Many stEndoscopy is a procedure where organs and tissues inside the body can be imaged and monitored using an endoscope. An endoscope is a thin tube with a light source and camera, often with additional tools, for example ultrasound or a working channel for introduction of biopsy forceps or therapeutic equipment. Endoscopy can be used for diagnostic  and therapeutic purposes such as removing cancer tissue. An endoscope can be either rigid or flexible; with flexible endoscopes offering a multitude of advantages for navigation to target sites. Endoscopes can be inserted through natural orifices like the mouth, anus or urethra or via incisions made in the body. Flexible endoscopy is often used as a diagnostic tool for many types of cancer and diseases and thus plays a vital role in the management of multiple malignancies. There are many different types of endoscopy from bronchoscopy (monitoring the lungs) to hysteroscopy (monitoring the uterus) and cystoscopy (monitoring the bladder); however, for the purposes of this review we will limit our focus to flexible gastrointestinal (GI) endoscopy. There are a range of flexible GI endoscopies including esophagogastroduodenoscopy , colonoscopy (for assessing the large bowel), sigmoidoscopy (for the rectum and sigmoid colon), endoscopic retrograde cholangiopancreatography (ERCP–for assessing the biliary tree and pancreatic ducts), and enteroscopy .Colorectal cancer is the third most common cancer in the world in terms of mortality and fourth most common in terms of incidence reaching nearly two million cases and one million fatalities in 2018 according to some projections . ColonosConventional endoscopy uses a semi-rigid tube and manoeuvring the endoscope is performed manually by rotation of a set of wheels on the handle and by pushing, pulling, and torqueing the insertion tube of the endoscope. Many procedures are uncomfortable or painful, requiring analgesia and sedation. A typical GI endoscopy process requires multiple HCW inside the room–an endoscopist, an assistant/technician, and a nurse to monitor the patient. For general anaesthetic and fluoroscopic procedures, this could potentially include anaesthesiologists and radiographers. Typical pre-pandemic personal protective equipment (PPE) for these processes consisted of gloves, gown/apron, and eye protection.Some endoscopic procedures are considered aerosol generating procedures (AGPs). Aerosols are small particles/droplets below 5 µm that can remain airborne for an extended period of time. One of the postulated sources of aerosol generation during endoscopy procedures is related to gas insufflation. Positive insufflation is used to visualize the lumen and create space to move the instrument forward. The potential generation of aerosols during endoscopy could pose a risk to HCW.Evidence of aerosols generated during different endoscopic procedures varies and there is no homogeneity of evidence. Endoscopic procedures such as bronchoscopy have been shown to be aerosol generating along with several other patient care and operating room procedures . HoweverUpper GI procedures are considered a greater risk during the current pandemic because the virus has been shown to be transmissible through airway secretions. The risk for lower GI procedures (e.g. colonoscopy and sigmoidoscopy) is less clear, although SARS studies have shown the presence of coronavirus in stool samples and in intestinal biopsy samples  and therDespite significant advances in the imaging capabilities of endoscopes, the controls remain largely unchanged in the last 60 years. The rear steering approach of conventional colonoscopy for example stretches the bowel and surround mesentery which makes it uncomfortable and often requires sedation or analgesia. While many new devices are being developed to attempt to address the shortfalls of conventional endoscopy , COVID-1Another avenue of improvement in endoscopy is through robotic advancements, which have the potential to reduce pain and widen the availability of procedures as they often place a lower cognitive and physical (improved ergonomics) burden on the operator and thus require less training. Robotic flexible endoscopy (RFE) can offer many advantages in general and specifically when dealing with IC related to aerosol generation. During the time of COVID-19, these include introducing physical distancing between HCW and patients (enabled as a result of teleoperation) as well as reduce the number of HCW in the endoscopy suite. In this review, we explore the impact of COVID-19 on GI endoscopy processes and how robotic flexible endoscopic platforms can be designed to minimize these impacts and improve IC mechanisms.th, 2020. When compared to 2019 levels in the United Kingdom, 51% more people were waiting for colonoscopies, 46% more for flexible sigmoidoscopies and 44% more patients were waiting for gastroscopies as of July 2020 , comprehensive room and equipment cleaning and of course air circulation  which ca2 extractors or suction mechanism, they create negative pressure zones with the potential of eliminating (or significantly reducing) aerosols escaping the procedure site and travelling around the suite. None of the mentioned risk minimizing techniques have been quantified and the evidence around aerosol dispersion while using these methods has not been published. There is a clinical need to quantify aerosol generation during standard flexible endoscopic procedures as well as robot assisted approaches. Apart from that, there is a need for evaluation of mechanical barriers and extractors currently employed in the hospitals to provide best and uniform guidelines for clinicians working during a pandemic. Air filtration is another key aspect in reducing HCW exposure to any potential aerosols  than current commercial platforms and improved control. In addition, AI systems can be used for improved navigation and localization of endoscopes using data from both imaging and on-board sensing. This increased control and better visibility of the intestine would improve detection rates while allowing procedures such as tissue sampling and cancer removal to be performed. By reducing the force and torque exerted on the luminal wall, patient discomfort can be reduced in addition to sedation requirements thus lowering risk and recovery times. Finally, it is important to consider the environmental impacts of single-use endoscopes and using recyclable materials would be beneficial for future platforms.The motivation for developing robotic platforms in endoscopy is similar to the motivation in other biomedical areas such as robotically assisted surgery. These robotic platforms allow HCW to overcome the current limitations of standard endoscopic devices for diagnostic and therapeutic procedures. These platforms can be used to improve the precision and safety of the tools thus making them more reliable and effective . The chaRFE has the potential to meet the needs of enhanced IC measures during COVID-19 and future pandemics. Remotely controlled devices increase the distance between patient and operator, hence less aerosol and droplet contact and reduced infection risk for HCW . Easy toWith the potential advantage of lowering discomfort for patients, RFE can be used in cases where the patient is unable to tolerate conventional endoscopy or in cases where frailty and co-morbidity rule out the use of sedation during conventional endoscopy procedures. This will be achieved by having precise control over the force and torque applied to the luminal walls through force feedback. It will be hugely beneficial in cases where patients require repeated and regular procedures  where using a less painful procedure will enhance surveillance uptake. It can also help to improve detection rates as more control and more comfort means better visibility. Another gap that RFE could fill is in cases where conventional endoscopes are unable to complete an examination.While RFE is a potential solution for diagnostic endoscopy and therapeutic procedures in the GI tract, challenges remain due to the limited DOF of such platforms. Therefore, current RFE research focuses on increasing the manoeuvrability and control of the tools to enable more complex and varied interventions. Flexible endoscope manipulations common to most systems are shaft insertion and tip steering. These complex movements add difficulty in developing robot-assisted flexible endoscopes.Over the years many researchers have developed RFE platforms focused on various actuation and control approaches. There are several recent reviews  that covWireless endoscopes (capsules) are a great tool for diagnostic purposes and providing painless inspection of the GI tract. Introduction through the mouth and lack of wiring makes them ideal for diagnostic applications . The lesRFE can introduce physical distancing into the endoscopy units. However, one of the main challenges is related to feeding and manoeuvring the flexible endoscope. Endoscopists can be severely limited due to a lack of manoeuvrability when manually operating the endoscopes during both diagnostic and therapeutic procedures. It has been documented that during the removal of abnormal tissue (polyps) during colonoscopies, even well-experienced endoscopists can miss up to 20% of the tissue . RoboticAnother challenge is related to operating an endoscopic instrument through the working channel, making sure that no aerosols come back from that channel. The working channel of the endoscopes can be responsible for releasing aerosols or droplets into the suite during standard operating procedure . HoweverIntroducing robotic platforms to endoscopic systems simplifies the procedure. Teleoperation allows clinicians to control the endoscope from a safe distance or/and behind mechanical barriers, with reduced need for direct contact with the patient. Reduced discomfort means less monitoring is required and no need for additional anaesthesiologists or nursing staff, thus reducing the risk to HCW as can be seen in Following evidence of transmission of infection despite decontamination, via infected biofilm in endoscopes , single RFE procedures have the potential to be completed with increased speed and without sedation and fewer complications, leading to a shorter recovery time, freeing space in endoscopy suites at a crucial time where endoscopic capacity has still not recovered to pre-pandemic levels. GI endoscopic procedures have been deemed high risk of contact and droplet formation with the potential to be aerosol generating. Despite little evidence as of now as to the true aerosol generating potential of GI endoscopic procedures, enhanced IC measures are likely to continue.The ideal RFE during COVID-19 would combine teleoperation, single-use endoscopes and mechanical barriers/seals. Teleoperation allows for physical distancing between patients and HCW, single-use endoscopes would reduce the risk of contaminated scopes. A mobile device would also be extremely useful at a time when the endoscopy suite capacity is limited to make space for COVID-19 patients. RFE has significant potential in diagnostic and therapeutic endoscopy, but some challenges remain to developing the ideal pandemic-secure RFE. There is a significant need for a study to define the aerosol generation during GI endoscopy in order to tailor future guidance for both patients and HCW and maintaining capacity levels in order to avoid devastating long term implications. Even with the hope of a successful vaccine rollout, we have learned that healthcare technologies should be resilient to pandemics in general, so we believe this review will still be relevant in the future.


Objective. To test whether keratinocyte growth factor-2 (KGF-2) can reduce oleic acid-induced inflammation in ALI of rats and explore its possible mechanism. Methods. 45 Sprague-Dawley rats were randomly divided into control group, ALI group, and ALI + KGF-2 group. The animal model of acute lung injury was established by injecting 0.1 mL/kg oleic acid into the tail vein of rats. Rats in the control group were injected with equal volume of normal saline (NS). Each group needs pretreatment 72 hours before the preparation of the acute lung injury model. The control group and ALI group were instilled with 5 ml/kg NS through the airway, and the same amount of KGF-2 was instilled in the ALI + KGF-2 group. It takes 8 hours to successfully prepare the ALI model. Observe the pathological changes of lung tissue through light microscopy, ultrastructural changes through electron microscopy, and the lung wettability/dry weight (w/d) ratio and lung permeability index (LPI). By detecting changes in inflammatory factors in lung tissue and changes in the number of BALF cells, the changes in inflammation in each group were observed. The expressions of Wnt5a, β-catenin, and APC in lung tissue were detected by immunohistochemistry and Western blot. The changes of key proteins in Wnt/β-catenin signaling pathway in the lung tissue of each group were observed. Result. Compared with the ALI group, after KGF-2 pretreatment, the degree of lung injury was reduced, the expression of inflammatory factors was reduced, and the number of red blood cells and white blood cells in BALF was reduced. It can also be observed that the expression of Wnt5a, β-catenin, and APC, a key protein in the Wnt/β-catenin signaling pathway, is reduced. The analysis showed that the number of inflammatory factors, red blood cells, and white blood cells in BALF was positively correlated with the expression of Wnt5a, β-catenin, and APC. Conclusion. KGF-2 may reduce the inflammatory response in ALI induced by oleic acid by regulating key proteins in the Wnt/β-catenin signaling pathway.Reducing inflammation can effectively relieve acute lung injury (ALI).  The Wnt5a/β-catenin classic signaling pathway is one of the Wnt/β-catenin signaling pathways. Keratinocyte growth factor-2 (KGF-2) is secreted by fibroblasts and other mesenchymal cells. It can be combined with fibroblast growth factor receptor 2IIIb (FGFR2IIIb) expressed in epithelial cells to promote epithelial cell proliferation [β-catenin signaling pathway. Initially, the mechanism of action of KGF-2 on ALI is explored.The loss of control and the outbreak of inflammatory response are the basis of the onset of ALI , 2. A sediseases , 6. Actidiseases . Wnt5a, feration –11. HoweForty-five Sprague-Dawley rats of about 220 ± 20 g were kept in a clean, temperature-controlled, and independent ventilation environment. All SD rats were free to obtain food and water. All experimental protocols were approved by the ethics committee.Forty-five SD rats were randomly divided into three groups: control group, ALI group, and ALI + KGF-2 group. The preparation of the rat ALI model was completed by injecting 0.1 mL/kg oleic acid  into the tail vein of the rat. The control group was injected with 0.1 mL/kg saline (NS) in the tail vein as a control. Three groups were instilled by tracheal infusion 72 h before the tail vein injection. The ALI + KGF-2 group was infused with 5 mg/kg of KGF-2, and the control group and ALI group were infused with 5 mg/kg of NS.μm thick slices, hematoxylin staining to take the nucleus, eosin staining to take the cytoplasm, and taking pictures under an optical microscope. Five fields of vision were taken from each slice for ALI pathology scoring [After the rat ALI model was successfully prepared, the lung tissue was quickly put into 4% paraformaldehyde to fully fix it, and then the paraformaldehyde on the surface of the lung tissue was washed away with clean water. Different concentrations of alcohol were placed in the lung tissue in turn, and then the lung tissue was placed in xylene. Finally, it was embedded in paraffin to form a wax block. Continuously it was cut into 4  scoring . One poi scoring .NS cleared the blood stains on the surface of lung tissue and took the rat lung tissue into 1% osmium tetroxide for fixation. Different concentrations of alcohol were placed in the lung tissue in turn and embedded in propylene oxide to form a wax block. After cutting into thin slices, they were dyed with uranyl acetate and lead citrate, respectively. The ultrastructure of rat lung tissue was observed under transmission electron microscope.Open the rat chest cavity and ligate the right lung. Remove the upper right lung tissue, remove water and blood stains with gauze, and weigh the lung tissue as the wet weight. Place the lung tissue in a drying cabinet at 80°C for 72 h, and weigh it as the dry weight. The wet weight/dry weight is wet-to-dry weight ratio (W/D).Connect the catheter to the trachea. Inject 2 ml NS slowly into the catheter to get the NS into the lungs for 5 times. Then slowly withdraw NS. Collect approximately 7 ml of bronchoalveolar lavage fluid (BALF). Blood was collected through the abdominal aorta of rats. Centrifuge at 3000 r/min for 15 minutes to collect blood and BALF supernatant. BCA protein assay was used to detect the protein concentration in plasma and BALF. The protein concentration in BALF/plasma protein concentration is LPI.Take an ELISA kit , set up standard wells, and establish a standard curve. Set up sample wells, and add sample and horseradish peroxidase (HRP) labeled secondary antibody to each well in turn. After incubating in a 37°C incubator for 30 minutes, each well was washed 3 times. Add substrate and stop solution to each well in turn. Finally, the value of each well is tested, and the concentration of each sample is determined according to the previously established standard curve.Drop BALF onto the slide, drip Wright's Giemsa A onto the smear, and cover the specimen with staining solution for 1 min. Then, drop Wright's Giemsa B onto Wright's Giemsa A to mix the two liquids. Finally, the running water rinses off the dyeing solution. Take pictures under an optical microscope. Count the number and proportion of white blood cells and red blood cells, respectively.μm thick slices. Repair antigen under high pressure in sodium citrate (pH = 6.0). Wnt5a antibody , β-catenin antibody , and APC rabbit antibodies  were incubated overnight at 4°C and negative controls were incubated with PBS. On the next day, horseradish peroxidase (HRP) labeled goat anti-rabbit IgG  was added dropwise. The incubation temperature was 37°C for 30 minutes and rinsed with PBS three times. Add 3,3′-diaminobenzidine (DAB)  for color development. Hematoxylin was counterstained and mounted. Observed under light microscope, the tissues were dark brown for protein positive expression. No coloration or light coloration is negative or weak positive expression. Finally, the average optical density (AOD) expressed by IPP software was analyzed. The integrated optical density (IOD)/positive expression area is AOD.Lung tissue was cut into 4 β-catenin antibody , APC antibody , and GAPDH antibody  were incubated overnight at 4°C. Wash the PVDF membrane 3 times with Tris-HCl buffered saline + Tween (TBST). A secondary antibody labeled with horseradish peroxidase (goat anti-rabbit IgG) was added for reaction. TBST washed the PVDF membrane 3 times again. Finally, with the help of ECL chemiluminescent reagents, the PVDF film shows the image. Analysis was done using Image J software.The tissue is quickly removed from the liquid nitrogen, and the lung tissue is ground into a powder. Collect lung tissue powder into a centrifuge tube, and add tissue lysate and pancreatin, respectively. The supernatant after centrifugation is a protein extract. BCA protein assay was used to determine protein concentration. Protein separation is done by polyacrylamide gel electrophoresis. The electrophoretic transfer method of wet transfer is used to transfer the protein to the PVDF membrane and block it in skim milk. Wnt5a antibody , P < 0.05 was considered statistically significant.All measured data are expressed as mean ± standard deviation. One-way analysis of variance was used to detect differences in normal distribution data between groups, and the LSD test was used for comparison between groups. One-way analysis of variance is used after variable transformation of data with uneven variance. After nonnormal distribution data is subjected to rank transformation, nonparametric test is used. For bivariate normal distribution data, straight-line correlation analysis is used, and for nonlinear trend data, curve fitting analysis is used. All statistical analysis was completed using statistical software SPSS23.0; P < 0.01). Compared with the ALI group, the LIS in the ALI + KGF-2 group was reduced (P < 0.01) but was higher than the control group (P < 0.01)  .Transmission electron microscopy observed ultrastructural changes . The conP < 0.01). After KGF-2 pretreatment, the lung LPI value and w/d ratio were significantly lower than those of the ALI model group (both P < 0.01). Compared with the control group, the lung LPI value and w/d ratio of the ALI + KGF-2 group increased (P < 0.01).LPI value and w/d ratio were used to observe the changes in lung permeability of rats in each group . Compareα and IL-10 in lung inflammation (α in the lung tissue of the ALI group increased significantly (P < 0.01), while the expression level of IL-10 decreased (P < 0.01). Compared with the ALI group, the TNF-α expression level in the lung tissue of the ALI + KGF-2 group decreased (P < 0.01), and the IL-10 expression increased (P < 0.01).ELISA was used to detect the expression levels of TNF-ammation . CompareWright's Giemsa staining was used to observe the inflammation expression in BALF . Compareβ-catenin, APC expression changes (β-catenin expression was negative (light yellow), and APC expression was weakly positive (light yellow). In the ALI group, Wnt5a expression was strongly positive (dark brown), β-catenin expression was strongly positive (dark brown), and APC expression was strongly positive (dark brown). The lung tissue of ALI + KGF-2 group showed positive Wnt5a expression (brown yellow), β-catenin expression was weakly positive (light brown), and APC expression was weakly positive (light brown). Compared with the control group, the positive expressions of Wnt5a, β-catenin, and APC in the ALI group increased significantly . After KGF-2 pretreatment, the positive expression of β-catenin and APC in the lung tissue of rats was significantly lower than that of the ALI group (both P < 0.01), and there was no significant change in the positive expression of Wnt5a (P > 0.05).Immunohistochemical analysis of Wnt5a,  changes . In the β-catenin, and APC protein expression in the lung tissue of each group . After KGF-2 pretreatment, the expression of Wnt5a protein in lung tissue was not significantly lower than that of ALI model group (P > 0.05). The expression of β-catenin and APC protein was lower than that of the ALI model group (P < 0.01), but it was still higher than that of the control group.Western blot analyzed the changes of Wnt5a, ch group . Compareβ-catenin, and APC protein expression levels were positively correlated with TNF-α expression level and negatively correlated with IL-10 expression level. Wnt5a, β-catenin, and APC protein expression levels were positively correlated with the number of red blood cells and white blood cells in BALF. In ALI + KGF-2 group, β-catenin and APC protein expression levels were positively correlated with TNF-α expression level and negatively correlated with IL-10 expression level. β-Catenin and APC protein expression levels were positively correlated with the number of red blood cells and white blood cells in BALF. In ALI + KGF-2 group, Wnt5a protein expression level had no correlation with TNF-α expression level; it had a negative correlation with IL-10 expression level. There was no correlation between β-catenin and APC protein expression levels and the number of red blood cells and white blood cells in BALF.In the ALI group, Wnt5a, In this study, 0.1 ml/kg oleic acid was injected into the tail vein to induce ALI. After being injected with oleic acid, the rats gradually developed mental depression, increased breathing amplitude, and increased breathing frequency. After 8 hours, the thoracic cavity of the rat was exposed, a large amount of lung tissue was congested, and the lung tissue was significantly swollen. Under the light microscope, the alveolar interval was thickened, the alveolar space was reduced, and a large number of inflammatory cells accumulated in the alveolar cavity , 14. To ARDS is an acute respiratory failure caused by ALI, and its pathological feature is diffuse alveolar injury , 16. Thiα and IL-10 revealed that TNF-α expression increased and IL-10 expression decreased in the ALI group. As a proinflammatory mediator, TNF-α causes severe damage to pulmonary microvascular endothelial cells and alveolar epithelial cells, causing cell apoptosis, vascular damage, and thrombosis, increasing lung tissue permeability and hypoxia [α can promote the release of a variety of inflammatory mediators and further expand the inflammatory response, thereby aggravating lung tissue damage [α can also activate the blood coagulation system and complement system, so that the inflammation “cascades expansion” and spreads in the lung tissue [In ALI, the pathogenic factor can directly act on the alveolar membrane to cause ALI, and at the same time, the pathogenic factor can directly activate white blood cells and indirectly damage the lung tissue –21. Afte hypoxia , 26. At e damage , 28. TNFg tissue , 28, 29.g tissue , 31.β-catenin signaling pathway can not only regulate growth, value added, and differentiation, but also regulate inflammation, fibrosis, and other pathological processes [β-catenin signaling pathway includes Wnt family secreted protein (Wnt), β-catenin, Axin, glycogen synthase kinase 3 (GSK3), and APC protein [β-catenin, and APC proteins are the key proteins of Wnt5a/β-catenin signaling pathway. Wnt5a is an upstream protein in the classical pathway of Wnt5a/β-catenin signaling pathway. The Wnt5a/β-catenin signaling pathway is an important part of the entire Wnt/β-catenin signaling pathway family. Activation of Wnt protein can start the regulation of the entire Wnt/β-catenin signaling pathway. β-Catenin is the core regulator of Wnt/β-catenin signaling pathway, and its expression level determines the opening of the pathway [β-catenin signaling pathway. On the one hand, APC protein is involved in the recognition, phosphorylation, and targeted degradation of β-catenin. On the other hand, APC protein may act as a nuclear shuttle protein and play certain roles in the nuclear transport of β-catenin, thereby reducing its nuclear level and transcriptional activity [β-catenin, and APC in the lung tissues of each group of rats. The results showed that the expression of Wnt5a, β-catenin, and APC in the ALI group increased significantly, indicating that when ALI occurs in the body, the expression of Wnt5a, β-catenin, and APC can be upregulated. The occurrence and development of ALI may be related to the key point proteins of Wnt/β-catenin signaling pathway.Wnt/rocesses –34. Wnt/ protein –37. Amon pathway . APC proactivity . In thisβ-catenin and APC decreased significantly. The results show that KGF-2 can downregulate β-catenin and APC protein expression, but its effect is not achieved by Wnt5a activation. The Wnt5a/β-catenin signaling pathway is just one of the Wnt/β-catenin signaling pathways. The expression of Wnt/β-catenin signaling pathway can also be achieved by Wnt/β-catenin signaling pathway initiated by Wnt3a and other nonclassical Wnt/β-catenin signaling pathways.KGF-2 can combine with its receptors FGFR2-IIIb (KGFR) and FGFR1III-b to promote epithelial cell growth, differentiation, and migration . In thisβ-catenin signaling pathway, inflammatory factors, and the number of cells in BALF. The results further indicated that oleic acid-induced inflammation in rat ALI is related to the above proteins. The key proteins of the Wnt/β-catenin signaling pathway were preliminarily discussed, which laid a preliminary foundation for the next step to specifically study the mechanism of the Wnt/β-catenin signaling pathway in oleic acid-induced ALI.In this study, we analyzed the correlation between key proteins of Wnt/β-catenin signaling pathway in this article is only a preliminary discussion on the key points in this pathway. It can only be initially proved that KGF-2 reduces the inflammatory reaction of ALI induced by oleic acid in rats, which may be related to the activation of key proteins in Wnt/β-catenin signaling pathway. The in-depth study on the Wnt/β-catenin signaling pathway including the use of Wnt/β-catenin signaling pathway activators and inhibitors will be further studied on this basis. In this study, the research on white blood cells was not classified. The presented results can only represent the overall leukocyte level and cannot reflect the specific leukocyte level. At the same time, the research on lung protection in this article only stays at the blood gas barrier stage, and the impact on alveolar epithelial cells and changes in alveolar surfactants cannot be reflected.KGF-2 is a protective drug, and there is no difference between the previous airway administration research centers and the control group. Therefore, this study only set up one intervention group, namely, ALI + KGF-2 group. The drug concentration of this group of pretreatment is 5 mg/kg, and it is administered 72 h in advance. This intervention method comes from the basis of previous research . The reβ-catenin signaling pathway.In summary, this study confirmed that KGF-2 pretreatment reduced the inflammatory response in ALI rats. The mechanism may be related to the activation of key proteins in the Wnt/


Gukang capsule (GKC) is a traditional Chinese medicine formulation which has been used extensively in the clinical treatment of bone fractures. However, the mechanisms underlying its effects on fracture healing remain unclear. β-catenin and BMP/SMAD signaling pathways to study the mechanisms by which GKC promotes fracture healing.  In this study we used a rabbit radius fracture model, and we measured the serum content of bone alkaline phosphatase (ALP), calcium, and phosphorus and examined pathology of the fracture site as indicators of the fracture healing effects of GKC. SaOS-2 human osteosarcoma cells were used to measure (i) ALP activity, (ii) ornithine transcarbamylase (OTC), calcium, and mineralization levels, (iii) the expression of osteogenic-related genes, that is, runt-related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2), collagen I (COL-I), osteopontin (OPN), OTC, and osterix (Osx), and (iv) the expression of key proteins in the Wnt/β (GSK3β) phosphorylation and Smad1/5 and β-catenin protein levels, thereby activating Wnt/β-catenin and BMP/Smad signaling pathways. Inhibitors of the Wnt/β-catenin and BMP/Smad signaling pathways  suppress the osteogenic effects of GKC.  We found that GKC effectively promotes radius fracture healing in rabbits and enhances ALP activity, increases OTC and calcium levels, and stimulates the formation of mineralized nodules in SaOS-2 cells. Moreover, COL-I, OTC, Osx, BMP2, and OPN expression levels were higher in SaOS-2 cells treated with GKC than control cells. GKC upregulates glycogen synthase kinase 3β-catenin and BMP/Smad signaling pathways and increasing osteoprotegerin (OPG) secretion by osteoblasts (OBs), which prevents receptor activator of nuclear factor kappa B ligand (RANKL) binding to RANK. GKC promotes fracture healing by activating the Wnt/ Seleosarcoma , 6.Fructus Psoraleae, Dipsacus asper Wall., Panax notoginseng Wall., Musa basjoo Siebold, and Oxalis corniculata Linn. It strengthens tendons and bones, nourishes the liver and kidney, and stimulates meridians to achieve pain relief according to TCM theory. GKC is commonly used as a supplementary treatment for fracture in clinical practice in China and shows good therapeutic effects [GKC is a traditional TCM formulation consisting of  effects . HoweverFracture healing is a dynamic biological process that involves restoration of the original tissue structure and bone function via complex physiological processes and the interaction of various types of bone cells, cytokines, and genes . During β. The phosphorylation of GSK3β in Ser 9 results in an increase in the amount of β-catenin in the cytoplasm; β-catenin ultimately enters the nucleus, where it interacts with T-cell factor/lymphoid enhancer factor (TCF/LEF), activates the downregulated RUNX2 gene, and promotes the Osx gene to initiate transcription of a series of target gene, thereby promoting the proliferation and differentiation of OBs [A previous study has shown that the canonical Wnt signaling pathway is closely related to the proliferation and differentiation of OBs . When thn of OBs , 13. In n of OBs . BMPs man of OBs . BMPs bin of OBs , 17.in vivo, and we used a SaOS-2 cell line to study the mechanisms by which the Wnt/β-catenin and BMP/Smad signaling pathways affect osteogenesis upon GKC treatment. In addition, given that many TCMs have multiple targets, we also examined the effects of GKC on the osteoprotegerin (OPG)/receptor activator of nuclear factor kappa B ligand (RANKL) system.Our previous study showed that GKC promoted the osteogenic differentiation and mineralization of SaOS-2 human osteosarcoma cells. However, the mechanisms underlying its effects on bone differentiation and mineralization have not been clarified. In this study, we used a rabbit radius fracture model to confirm that GKC promoted fracture healing β-catenin (ab6302), Smad4 (ab40759), GAPDH (ab8245), and GSK3β (ab93926) were from Abcam . DKK1 (PHC9214), Noggin (PHC1506), antibodies against Smad1/5 (PA5-80036), and p-Smad1/5 (MA5-15124) were obtained from Thermo fisher (American). p-GSK3β  antibody was got from Cell Signaling Technology (American). All other reagents used in the present study were of analytical grade.SaOS-2 cell was kept in the laboratory. GKC (20180521) was obtained from Guizhou Wei Kang Zi Fan Pharmaceutical Co. Ltd. . Jiegu-Qili tablet was obtained from Hunan Jin Sha Pharmaceutical Co. Ltd. . Pentobarbital Sodium (6900183) was purchased from Beijing Solarbio Science & Technology Co. Ltd. Penicillin G Sodium (170907) was from Lukang Pharmaceutical Co. Ltd. . ALP (A059-2), Pi (C006-3), and Ca (C004-2) were supplied by Nanjing Jiancheng Biotechnology Co. Ltd. . Primers of ALP, COL-I, OTC, Osterix, RUNX2, BMP2, OPN, OPG, RANKL, and GAPDH were obtained from Shanghai Generay Biotech Co. Ltd. . Antibodies against RUNX-2 (ab23981), OPG (ab73400), BMP2 (ab14933), RANKL (ab9957), Male Healthy rabbits (2.0–2.5 kg) were purchased from Chongqing Teng Xin Biotechnology Co. Ltd. . The license number for animal manufacturers SCXK(Yu)2012-0003. Rats were maintained at a controlled temperature (22 ± 2°C) and humidity (50 ± 5%) under a 12 h light/dark cycle and given free access to water and food. All experiments were carried out under the approval of the Animal Ethical Committee of Guizhou Medical University  and conformed with the guidelines of the National Institutes of Health for the Care and Use of Animals.Rabbits were anesthetized by intravenous injection of 3% sodium pentobarbital (30 mg/kg) through the ear vein. Each rabbit was then fixed on the operation bench, and the hair of the left forelimb was shaven off. The skin was disinfected with 75% ethanol. A sterile scalpel was used to cut open the skin and separate the surrounding muscle, soft tissues, and blood vessels to fully expose the radius. A small electric saw was used to induce a fracture in the bone, resulting in a complete defect of 3 mm in the middle of the left upper limb . The ulnn = 7 per group), that is, the control group, model group, Jiegu-Qili tablet group (0.14 g/kg), low-dose GKC group (0.22 g/kg), moderate-dose GKC group (0.45 g/kg), and high-dose GKC group (0.67 g/kg). Treatments were administered by oral gavage once per day, with rabbits of the control and the model group being administered equal amounts of distilled water.42 male rabbits were randomly divided into six groups  was completely dissected and sawn 1 cm away from both ends of the fracture site, the surrounding skeletal muscles and soft tissues were removed, and the bone was fixed in 10% formaldehyde solution. The fixed bone tissues were placed in acid mixed decalcifying solution (800 ml distilled water + 100 ml hydrochloric acid + 100 ml formic acid) and were left to decalcify for a week. Decalcification was considered complete if a pin could easily penetrate into the bone tissue. The decalcified bone tissues were subjected to conventional paraffin embedding, sectioning, hematoxylin and eosin (HE) staining, and light microscopy.2 at 37°C until 80% confluency was reached. The cells were then trypsinized and subcultured. Well-grown cells of passage 5–10 in the logarithmic growth phase were used for subsequent experiments.SaOS-2 human osteosarcoma cells were cultured in McCoy's 5a medium containing 10% fetal bovine serum and 1% penicillin/streptomycin in an incubator with 5% CO5 cells/ml was inoculated in a 24-well plate (500 μL/well); after 24 h, the medium was refreshed. To monitor the effect of GKC to the differentiation of SaOS-2 cells, the culturing medium was changed to osteogenic osteoinduction medium which contained 10 mM β-glycerophosphate, 10 mM dexamethasone, 50 μg/mL ascorbic acid, and GKC of different concentrations. SaOS-2 cells of the low-dose, moderate-dose, and high-dose GKC groups were treated with medium containing 10, 100, and 200 mg/l GKC, respectively. SaOS-2 cells of the GKC plus DKK1 group were treated with medium containing DKK1 at a final concentration of 0.5 μg/ml for 5 days. Then the medium was replaced with medium containing 200 mg/l GKC. SaOS-2 cells of the GKC plus Noggin group were first treated with medium containing 1.5 μg/ml Noggin for 5 days. Then the medium was replaced with medium containing 200 mg/l GKC. The control group were incubated with McCoy's 5a medium. The vitamin D3 group were incubated with medium containing 30 mg/l vitamin D3.The cytotoxicity of different concentrations of GKC on SaOS-2 cells were measured by MTT assay. Concentrations of 10, 100, 200, and 1,000 mg/l for 1, 3, and 5 days were not cytotoxic. SaOS-2 cells in the logarithmic growth phase were selected, and cell suspension of 2 × 10μl 1% Triton X-100 was added, and the cells were kept on ice for cell lysis for 10 min. The ALP activity on SaOS-2 cells was measured by disodium phenyl phosphate colorimetry. ALP activity and protein quantity were measured by ALP kit and bicinchoninic acid (BCA) assay, respectively.After 48 h of treatment of SaOS-2 cells, the culture medium was discarded, the cells were washed twice with phosphate-buffered saline (PBS), 200 Seven days after treatment of SaOS-2 cells, the culture medium was discarded, the cells were washed twice with PBS, and the cells were fixed with 95% ethanol for 15 min. The cells were washed three times with PBS, an appropriate amount of 5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt (BCIP)/nitro-blue tetrazolium chloride (NBT) staining solution was added, and samples were incubated at room temperature for 30 min in the dark. After removing the staining solution and washing twice with distilled water, the stained cells were observed by light microscopy.Fourteen days after treatment of SaOS-2 cells, the medium was discarded, the cells were washed twice with PBS, and the cells were fixed with 95% ethanol for 10 min. The cells were washed three times with distilled water, an appropriate amount of 0.1% Alizarin Red (pH 8.3)-Tris-HCl staining solution was added, and the cells were incubated for 30 min. Excess dye was removed by washing with distilled water, and the cells were air-dried and observed and photographed under a light microscope.−ΔΔCt method was used to calculate the fold change compared to the control group. −ΔΔCt method.Twenty-four hours after treatment of SaOS-2 cells, total RNA was extracted using TRIzol reagent, followed by reverse transcription using PrimeScript™ RT reagent Kit (TaKaRa). The target gene expression was quantified by SYBR Green II-based qRT-PCR, and the 2μg of each sample of SaOS-2 cells was loaded per lane for separation by SDS-PAGE. Protein was transferred to PVDF membranes, and membranes were blocked in blocking buffer  at 4°C overnight. The primary antibodies were diluted in blocking buffer according to the corresponding dilution factors. Membranes were incubated with primary antibody on a shaker at 4°C for 2 h, washed with Tris-buffered saline with 0.1% Tween 20 (TBST) five times (5 min each), and incubated with HRP-conjugated secondary antibody (diluted in TBST buffer) on a shaker at room temperature for 2 h. After washing with TBST five times (5 min each), the membranes were photographed using a gel imager. The scanned images were analyzed, and the gray value ratio of target protein to GAPDH in each lane was analyzed separately to calculate the relative protein expression levels.24 hours after treatment of SaOS-2 cells, the cells were lysed with RIPA buffer, followed by protein quantification by BCA assay. 30 P < 0.05 was considered to indicate a statistically significant difference.Statistical analyses were carried out using SPSS13.0 . Data were compared by one-way ANOVA. Results are presented as mean ± standard deviation, and The pathological sections of the fracture gaps  showed tP < 0.01). Comparison of serum bone ALP levels between the GKC groups and the model group has been made. It showed no significant difference between the low-dose (0.22 g/kg) GKC group and the model group; however, the serum ALP levels of the moderate-dose (0.45 g/kg) and high-dose Gukang groups were significantly higher than the model group (P < 0.01). In addition, the calcium content of the GKC groups was higher than that of the model group, with a significant difference between the high-dose GKC group and the model group (P < 0.05).As shown in To further explore the effects of GKC in promoting osteogenesis and osteogenic differentiation in SaOS-2 cells, we performed staining and quantitative analysis of ALP and mineralized nodules. Our results showed that as the concentrations of GKC increased, the ALP staining intensity of the GKC groups also increased. Analogous phenomenon was observed in MC3T3-E1 cells as well . CompareALP, COL-I, OPN, OTC, RUNX2, BMP2, and OPG were generally increased after 24 h of GKC treatment (P < 0.05), and ALP mRNA changed most (P < 0.05). GKC also boosted the Osx mRNA expression, but no significant difference was found compared to the control group (P > 0.05). GKC had no significant effect on RANKL mRNA expression in vitro.BMP2 and RUNX2 mRNA expression in GKC-treated SaOS-2 cells. GKC  significantly increased the OPG/RANKL protein ratio, and 200 mg/l had the greatest effect on the OPG/RANKL ratio.Western blot analysis  showed tβ-catenin signaling pathway showed that after 24 h of GKC treatment, β-catenin mRNA, and protein expression levels were increased in a dose-dependent manner, and the phosphorylation of GSK3β protein was effectively upregulated . However, GKC had no significant impact on total Smad1/5 and Smad4 protein expression , thereby inhibiting the degradation of β-catenin. Hence, β-catenin gradually accumulates in the cytoplasm. Once β-catenin enters the nucleus, it interacts with the nuclear transcription factor TCF/LEF, activating the expression of the downstream genes RUNX2 and Osx, thereby initiating transcription of a series of related target genes, and promoting OB proliferation and differentiation [β binding to the free β-catenin in the cytoplasm, resulting in the phosphorylation and degradation of β-catenin. Therefore, GSK3β is an important negative regulator in the Wnt/β-catenin signaling pathway [β-catenin signaling pathway. It inhibits Wnt signaling by competing with the ligand Wnt for receptor LRP5/6 and affects the expression of downstream genes that play a role in osteogenic differentiation.Wnt/ntiation . Blockad pathway . DKK1 isβ-catenin signaling, several signaling pathways are involved in the expression or activation of key transcription factors, such as RUNX2, which regulate OB differentiation and osteogenesis. Among them, the most important key transcription factor is the BMP/Smad signaling pathway [In addition to Wnt/ pathway , 30. BMP pathway .β-catenin, a key factor in the Wnt/β-catenin signaling pathway, dose-dependently increased in SaOS-2 cells treated with GKC. GKC also upregulated the phosphorylation level of GSK3β protein in SaOS-2 cells. In addition, GKC upregulated the phosphorylation level of Smad1/5 in the BMP/Smad signaling pathway. The Wnt/β-catenin and BMP/Smad signaling pathway inhibitors DKK1 and Noggin reversed the inhibitory effect of GKC on ALP activity and the formation of mineralized nodules, indicating that GKC promoted OB differentiation through BMP/Smad and Wnt/β-catenin signaling. Previous studies had shown that BMP/Smad and Wnt/β-catenin signaling cooperated and promoted each other's function during OB differentiation and maturation [β-catenin plays a role in the regulation of BMP2 protein expression, and activation of the Wnt/β-catenin signaling pathway effectively promoted BMP2 gene expression. Activation of the BMP/Smad signaling pathway increases the expression of membrane receptors and ligand proteins in the Wnt/β-catenin signaling pathway, inhibiting the degradation of cytoplasmic β-catenin. As a result, the Wnt/β-catenin signaling pathway exhibits a stronger ability to regulate osteogenic differentiation [β-catenin, BMP2, and RUNX2 were significantly upregulated in the SaOS-2 cells treated with GKC. Therefore, the osteogenic effects of GKC in SaOS-2 cells are most likely jointly mediated by the Wnt/β-catenin and BMP/Smad signaling pathways.In this study, we showed that the expression of turation , 33; β-cntiation . In thisThe OPG/RANK/RANKL system plays an important role in maintaining the dynamic balance of bone tissue in the body. OPG is a member of the tumor necrosis factor receptor superfamily. It is the only factor that is known to directly downregulate the function of osteoclasts, and it inhibits their activation and differentiation. Therefore, OPG is also known as osteoclastogenesis inhibitory factor. OPG content is a direct measure of bone resorption activity. RANKL is mainly secreted by OBs and bone matrix. It is a member of the tumor necrosis factor superfamily. RANKL binds to RANK on the surface of osteoclasts to promote the differentiation and maturation of osteoclasts. In addition, RANKL inhibits the apoptosis of osteoclasts, thereby promoting osteoclastic bone resorption . OPG comin vivo. Besides, a SaOS-2 cell line was used to study the mechanisms, and we found that GKC promoted fracture healing through enhancing osteoblast differentiation and triggering the Wnt/β-catenin and BMP/Smad signaling pathways. In addition, given that many TCMs have multiple targets, we also examined the effects of GKC on the OPG/RANKL system, and the results showed that GKC increased OPG secretion by OBs, which prevents RANKL binding to RANK.In summary, we used a rabbit radius fracture model to confirm that GKC promoted fracture healing


Developing polyols derived from natural sources and recycling materials attracts great interest for use in replacing petroleum-based polyols in polyurethane production. In this study, rigid polyurethane (PUR) foams with various isocyanate indices were obtained from polyols based on rapeseed oil and polyethylene terephthalate (RO/PET). The various properties of the prepared PUR foams were investigated, and the effect of the isocyanate index was evaluated. The closed-cell content and water absorption were not impacted by the change of the isocyanate index. The most significant effect of increasing the isocyanate index was on the dimensional stability of the resulting foams. This is due to the increased crosslink density, as evidenced by the increased formation of isocyanurate and increase of the glass transition temperature. Additionally, the influence on compression strength, modulus, and long-term thermal conductivity were evaluated and compared with reference PUR foams from commercially available polyols. Rigid PUR foams from RO/PET polyol were found to be competitive with reference materials and could be used as thermal insulation material. Polyurethanes (PURs) have been widely used for different applications, such as coatings, adhesives, sealants, elastomers, resins, and foams. Most of these PUR materials are obtained from petrochemicals which are non-renewable, have low sustainability, and cause environmental concerns for society . Due to Over the past few decades, oils and fats of vegetable origin have served as a viable alternative to petroleum resources . ResearcTypical methods used to produce polyols from vegetable oils are applied for RO too: epoxidation and opening of oxirane rings ,17, tranPET is one of the most versatile commodity thermoplastics, widely used in a broad range of applications, like fibres and disposable soft-drink bottles. The high popularity of PET as packaging material and its non-biodegradability creates huge amounts of waste. Therefore, effective recycling of PET is crucial ,24. PET One of the chemical recycling products of PET, that is suitable for obtaining PUR, is an aromatic polyester polyol. There are various chemical recycling pathways for aromatic polyester polyol synthesis: hydrolysis, aminolysis, methanolysis, and glycolysis . GlycolyRigid PUR and polyisocyanurate (PIR) foams are one of the most energy-efficient thermal insulation materials, which are mainly used in civil engineering as well as in the refrigeration industry. PUR and PIR foams are also applied as impact absorption materials for structural and functional engineering apllications . PURs arg), dimensional stability, thermal stability, as well as thermal insulation with increasing isocyanate index value is reported [The amount of PUR bonds and isocyanurate rings is closely related to a parameter called isocyanate index, which is the ratio of the equivalent amount of isocyanate used relative to the theoretical equivalent amount times 100. A theoretical equivalent amount is equal to one equivalent isocyanate per equivalent OH group. Higher isocyanate index incorporates more isocyanurate rings and increases the non-renewable material concentration in the end-product. The amount of isocyanurate rings in the polymer matrix affects the performance of PUR foams. The increase of glass transition temperature  were used for development of PUR foam formulations. The various properties  of the prepared PUR foams were investigated. For comparison, three series of reference rigid PUR foams were obtained from commercially available polyols. The isocyanate index effect on the foams’ performance was evaluated, as well as optimal isocyanate index as a compromise of sustainable material content, and foam performance was determined.Aromatic polyester polyol based on RO and PET  with RO/PET molar ratio 1:4 was synthesized, as described in our previous work . Three bRigid PUR foams were prepared by reacting synthesized RO/PET or reference polyols with different amounts of polymeric diphenylmethane diisocyanate (PMDI) (NCO group content = 31.5%) purchased from BASF. Other reagents used were: high functional polyether polyol Lupranol 3422 (OH = 490 mg KOH/g) from BASF, catalysts CAT NP-10 and 30 wt% solution of potassium acetate (KAc) in DEG from Performance Chemicals Handels, surfactant NIAX Silicone L6915 from Momentive Performance Materials, flame retardant Levagard PP (tris (2-chloroisopropyl)-phosphate)) from Lanxess, and physical blowing agent Solkane 365/227 (pentafluorobutane:heptafluoropropane = 87:13) from Solvay. These materials were used without any additional prior treatment. Rigid PUR foam series RO/PET was prepared from synthesized RO/PET polyol. In addition, three series of reference foams were prepared. As mentioned before, three base polyols for reference foams were chosen: Lupranol 3300, NEO 380, and RO/TEA. The obtained foam series were named LUPR, NEO, and RO/TEA, respectively. Chosen isocyanate indices were 110, 120, 130, 150, and 180. Foam samples were named SERIES NAME II = n, where n is the relevant isocyanate index. For example, RO/PET II = 120 is an RO/PET series rigid PUR foam with an isocyanate index of 120. Polyols’ systems were prepared by mixing all of its components according to the formulations presented in II is the isocyanate index; PMDIm, nm, and H2Om refer to the mass of PMDI, each polyol, and summary water, respectively; NCOw refers to the content of NCO groups of PMDI (31.5%); and nOH is the hydroxyl value of each polyol (mg KOH/g).The necessary amount of PMDI was calculated according to the equation:The calculated amount of PMDI was added to a polyol system, and the resulting mixture was stirred vigorously at 2000 rpm for 10 s. Afterward, it was instantaneously poured into an open mold with the dimensions 30 × 30 × 10 cm to obtain free-rise foams. After preparing all foam samples, they were allowed to cure at ambient conditions for 24 h before any further tests.−1 resolution for each spectrum. Dynamic mechanical analysis (DMA) was carried out with Mettler Toledo DMA/SDTA861e: temperature range from 25 to 200 °C, ramp rate of 3 °C/min, frequency of 1 Hz, amplitude 40 μm, and maximal force of 2 N. Compression oscillation mode was used. Three cylinder specimens with a diameter of ≈16 mm and a height of ≈8 mm were tested for each composition. Specimens were cut with a drill press using a crown drill bit. The maximum of the peak in the viscoelastic parameter tan δ was used to define Tg.The process of rigid PUR foam formation was monitored by measuring foaming parameters: the duration of cream time, gel time, and tack-free time. The closed-cell content and apparent density were measured according to the standards ISO 4590 and ISO 845, respectively. The compression strength of rigid PUR foams were tested on testing machine Zwick/Roell Z100 . Cylinder specimens with diameter and height of ≈20 mm were cut with a drill press using a crown drill bit. Water absorption was tested immersing PUR specimens into the water for seven days, according to ISO 2896. The dimensional stability measurements were acquired according to ISO 2796. Two modes were chosen: 28 days at 70 °C, ambient relative humidity (R.H.); and 28 days at 70 °C, R.H. = 97%. The initial thermal conductivity of the rigid PUR foams was measured, as well as thermal conductivity after 24 weeks. These tests were carried out with Linseis Heat Flow Meter 200 according to the ISO 8301 standard. The temperature range was +10...+30 °C, and dimensions of specimens were 200 × 200 × 30 mm. The FTIR data was collected using an attenuated total reflectance technique with a ZnSe and Diamond crystals on a Thermo Fisher Nicolet iS50 spectrometer. A total of 32 scans were averaged at 4 cmRigid PUR foams with different isocyanate indices were obtained by the free-rise method. Renewable and recycled material content was calculated and is shown in The amount of renewable and recycled materials in RO/PET foams is between 11% to 14%. In comparison, reference LUPR series foams contain up to 8%; NEO series, 15%; and RO/TEA series, 20%. The difference in sustainable material content between series of PUR foams depends mainly on the sustainability of the base polyol. The largest quantity of non-renewables in PUR formulations is made by PMDI. Hence, the sustainable material content decreases by rising isocyanate index. 3), NEO (ρ = 40–45 kg/m3), and RO/TEA (ρ = 41–48 kg/m3) series, showed slightly lower closed-cell content: 93 vol.%, 95 vol.%, and 93 vol.% on average, respectively. Closed-cell content 92–98 vol.% is suggested as a typical rigid PUR foam characteristic [The obtained rigid PUR foams were characterized by apparent density and closed-cell content . Closed-teristic . All obtReactivity of the polyol system is characterized by foaming parameters, such as cream time (the visible increase of foam volume), gel time (the transition from liquid to solid), and the tack-free time (when the outer surface of the foam is not sticky anymore). Gel time is the starting point of a stable network formation by intensive allophanate crosslinking as well as urethane and urea linkages . Table 3As mentioned earlier, the amount of foaming catalyst for reference foams was adjusted according to the reactivity of their polyol mixes. The series RO/TEA and RO/PET polyol systems were required to add less catalyst, as they showed higher reactivity. The higher reactivity was due to a tertiary amine group in the main polyol structure .c and an increase in elasticity. Additionally, the increase in Tg is expected for higher crosslinking [It is expected that at a higher isocyanate index, the crosslinking density will increase. Two effects contribute to this. First, the functionality of PMDI is 2.7 and its molecular mass is relatively small (aprox. 381 g/mol), so it will work as a crosslinking reagent by itself. Second, when an excess of isocyanate is added, allophanates, biurets, and isocyanurates can be formed via side reactions. That will add additional crosslink points in the PUR polymer matrix, which gives a decrease in Mslinking .-1, and it is shown in FTIR analysis was carried out for RO/PET series to judge the amount of isocyanurate formation. An isocyanurate band appears at 1410 cmg was determined from damping factor curves, as shown in g of PUR foams for RO/PET II = 120 and II = 130 are similar: 121 °C and 129 °C, respectively. A higher increase of Tg is observed by increasing the isocyanate index, and it reached 172 °C for RO/PET II = 180. The peak intensity for foams with II = 110–130 is very similar. It is clearly seen that for higher isocyanate indices, more isocyanurates are formed. As no trimerization agent is added for formulations with II = 110–130, for those foams, crosslinking due to the formation of isocyanurate is closely similar. It agrees with the results of DMA. TFTIR and DMA results prove that crosslink density increases because more crosslinks are formed due to the formation of isocyanurates. Additionally, an increase in elasticity can be expected .3 using the equations of Hawkins et al [Compression strength is strongly affected by apparent density. Therefore, compression strength and modulus values for all samples were normalized for an apparent density of 45 kg/mns et al . Relatinns et al . Additions et al . In our case, the compression strength of RO/PET PUR foams slightly increases with an increase in isocyanate index a. It raiDue to the enhancement of foam elasticity, anisotropic ratio  for RO-PET 1/4 II = 180 is smaller (AR = 1.79) than for samples with lower isocyanate index (AR = 1.85–2.16). RO/PET shows higher compression strength than reference foams, LUPR and RO/TEA series, but slightly lower than the NEO series. NEO performance could be explained by the higher number of hard segments from recycled PET in its PUR foam matrix. Reference foams show an unexpected decrease in compression strength and modulus with an increase in isocyanate index from 110 to 130. It could be related to the lack of isocyanate trimerization catalyst in their formulations, hence no addition crosslinks are formed and unreacted isocyanate remains in foams. Nevertheless, all samples are within commercially acceptable limits. According to Javni et al., typical industrial PUR rigid insulating foams have compression strength at 10% strain between 0.15 and 0.25 MPa .The results of water absorption are presented in The increase of the isocyanate index improves dimensional stability due to the incorporation of the isocyanurate ring into the PUR foam matrix . The resAlthough RO/PET polyols show better compatibility with blowing agents , which c3 was measured, as well as thermal conductivity, after 24 weeks. The initial values are given in Initial thermal conductivity of rigid PUR foams with apparent density 42 ± 3 kg/mTo evaluate the retention of thermal conductivity in the long term, No impact of isocyanate index on the retention of thermal conductivity for rigid PUR foams series NEO, RO/PET, and LUPR is observed. Only for PUR foams based on RO/TEA polyol, does the increase of isocyanate index improve retention. RO/TEA polyol chemical structure introduces long dangling chains into the PUR polymer matrix, which causes a low packing degree of PUR macromolecules . The excRigid PUR foams with isocyanate index 110–180 were obtained from polyols based on rapeseed oil and polyethylene terephthalate. Additionally, reference PUR foams were obtained from commercially available polyols: Lupranol 3300, Neopolyol 380, and rapeseed oil/triethanolamine polyol. Closed-cell content of rapeseed oil/polyethylene terephthalate PUR foams was ≈97 vol.% and was not impacted by the isocyanate index. Additionally, no effect of the isocyanate index on water absorption was observed.The values of foaming parameters increased with an increase in isocyanate index. The increasing tendency of gel time was due to the lower molecular weight polymers formed, which are typically exposed to the free surfaces, causing adhesiveness and prolonging tack-free time. The increase of compression strength, compression modulus, and dimensional stability of RO/PET PUR foams with the increase of isocyanate index were caused by the higher ratio of crosslinking due to the formation of isocyanurate (evidenced by FTIR and DMA).Overall, the optimum complex of the physical and mechanical properties of RO/PET PUR foams was achieved for formulations with isocyanate indices of 130–180. Therefore, it is possible to obtain commercially competitive rigid PUR foams with sustainable material content up to 13%.


Persistent hair loss is a major cause of psychological distress and compromised quality of life in millions of people worldwide. Remarkable progress has been made in understanding the molecular basis of hair loss and identifying valid intracellular targets for designing effective therapies for hair loss treatment. Whereas a variety of growth factors and signaling pathways have been implicated in hair cycling process, the activation of Wnt/β-catenin signaling plays a central role in hair follicle regeneration. Several plant-derived chemicals have been reported to promote hair growth by activating Wnt/β-catenin signaling in various in vitro and in vivo studies. This mini-review sheds light on the role of Wnt/β-catenin in promoting hair growth and the current progress in designing hair loss therapies by targeting this signaling pathway. Hair loss is characterized by either the loss of hair density or hair thinning, or both, and it may result from both hormonal and non-hormonal causes. The loss of hair caused by the excess responsiveness of testosterone and its active metabolite dihydrotestosterone is known as androgenic alopecia, which has been extensively investigated for the understanding of its pathophysiology and designing therapies for hair growth promotion. Non-hormonal causes of hair loss include aging, scalp inflammatory disorders, auto-immune disorder, and mechanical stress, which are known as senescent alopecia, cicatricial alopecia, alopecia areata, and traction alopecia, respectively. Focal hair loss, resulting secondarily from an underlying medical disorder, may either be non-scarring patchy hair loss, such as tinea captitis, alopecia areata, or may be a rare scarring type cicatricial alopecia, for example hair loss resulting from lupus erythematosus . On the Anagen effluvium is a diffuse non-scaring hair loss mostly associated with the use of chemotherapeutic agents, while telogen effluvium is linked with stress and involves loss of clumps of hair during hair brushing. Tinea capitis is also a patchy type hair loss that is associated with a dermatophyte infection and commonly occurs in children . IrrespeSeveral lines of therapeutic modalities are currently available to promote hair re-growth or to prevent hair loss. These include the use of 2% or 5% topical minoxidil for androgenic alopecia and anagen effluvium; triamcinolone acetonide for alopecia areata; and antifungal agents  for tinea capitis. Finasteride, which blocks the conversion of testosterone to dihydrotestosterone, is also used to treat androgenic alopecia in patients unresponsive to minoxidil. However, these drugs have limited therapeutic efficacy and possess serious side effects ,7,8. ForThe hair follicle (HF) is an integral part of the skin that protects the internal organs and plays a role in body temperature regulation. HFs are specialized organelles that are constantly recycling. In general, a human scalp has about 120,000 HFs, which regulates the hair cycling and the constant maintenance of scalp hair. Structurally, HF extends from the epidermis into the deeper layer of the dermis where it takes a tubular shape making a bulb-like structure at the base. This follicular bulb encircles the dermal papillae (DP), which is composed of dermal papillary cells (DPC), connective tissues, and a capillary network. The DPCs constitute a special mesenchymal component that regulates periodic regeneration of HF . A layerThe hair shaft is originated from the follicular epithelial matrix. The three distinct stages of hair development are induction, organogenesis, and cytodifferentiation. The induction phase involves the thickening of epithelial cells to form a placode, followed by the organogenesis stage, when the epithelial cells first send signals to dermal cells to proliferate and form dermal condensate. This dermal condensate then directs epithelial cells to proliferate and migrate towards dermis. During cytodifferentiation stage, the dermal condensate forms the dermal papilla upon being encapsulated by the follicular epithelial cells. Several intra- and intercellular signaling molecules including various morphogens and growth factors play a key role in generating the HF .The human hair cycle consists of three phases: the anagen, catagen, and telogen phases. The anagen phase that lasts between two and eight years is the active hair growth phase, whereas the catagen is the regression phase that ranges 2–3 weeks. The telogen is an about three-month resting phase that ends through the original hair falling out followed by the appearance of new hair ,19. A syWI), causes the secretion of Wnt, which is expressed in developing hair placodes, embryonic epidermis and HF [The hair development process requires a controlled cross talk between the mesenchymal and epithelial cells in the HF, which receives signals from epidermal and dermal compartments . Althougs and HF . Figure s and HF ,31. Kishs and HF .The concept of the involvement of Wnt signals in follicular development stemmed from the early findings of the presence of nuclear β-catenin in chick feather prior to placode formation  as well Wl, alternatively known as Gpr177. Genetic ablation of epithelial Wls expression inhibited hair placode formation at E14.5, and diminished the expression of placode markers, such as Eda, BMP2, BMP4, and SHH, and that of nuclear β-catenin. Restoration of the stabilized β-catenin expression led to the epithelial placode formation but failed to initiate dermal condensate and dermal papilla formation, suggesting that epithelial Wnt signaling is capable of epithelial hair formation but not inducing a dermal response. These authors further demonstrated that dermis-specific deletion of Wl inhibited β-catenin reporter activity in vitro and reduced dermal cell proliferation, although β-catenin expression in vivo remained unchanged and epithelial placode formation was present [The Wnt protein expression and secretion are regulated by the gene  present . These f present  demonstr present . A previ present . In cont present .There are multiple lines of evidence suggesting that various Wnts promote hair cycling and regeneration via the activation of β-catenin signaling. For instance, intradermal injection of Wnt1a-enriched conditioned media from bone marrow mesenchymal stem cells in to depilated mouse skin accelerated HF progression from telogen to anagen, increased the amount of hair, and elevated the expression of hair induction-related genes, such as Lef1, versican, and Gli1 . Wnt10b Factors that regulate Wnt-β-catenin signaling in HF include androgen , BMP 5252, solubA recently conducted gene ontology analysis of microarray gene expression data collected from bald frontal and haired occipital scalps of five men with androgenic alopecia revealed that all downregulated genes belong to the Wnt and TGF-β signaling pathways, while upregulated genes fall under the oxidative stress pathway . Exposur2) on depilated dorsal skin of healthy or diabetic mice markedly induced epidermal hair follicle development and early anagen induction, as evidenced by change of skin color from pink to black. Mechanistically, TRF inhibited the expression of epidermal E-cadherin, resulting in the increased nuclear translocation of β-catenin and its interaction with Tcf3. Co-treatment of mouse skin with a pharmacological inhibitor of β-catenin arrested TRF-induced anagen hair cycling [Prunus mira Koehne, a Tibetan medicinal plant, has long been used traditionally to care hairs and eyebrows. High performance liquid chromatography-based chemical analysis of P. mira nut oil revealed the presence of a-tocopherol, β-sitosterol, vitamin E, linoleic acid, and oleic acid. Topical application of P. mira nut oil (0.47–60.26 mg) on depilated telogenic (pink color) dorsal skin of C57BL/6 mice significantly increased the dermal thickness, number of HFs, hair weight, and hair length as compared to that of minoxidil. P. mira nut oil also increased the mRNA and protein expression of β-catenin. Skin irritation assay showed that application of P. mira nut oil on New Zealand rabbit skin caused mild erythema, which disappeared within 1 h of treatment [The well-defined role of Wnt/β-catenin activation in hair morphogenesis has led researchers to find activators of this signaling pathway as potential therapy for hair loss treatment. Until now, many natural as well as synthetic compounds have been reported to promote hair regrowth via activation of Wnt/β-catenin signaling . The fol cycling . The seereatment . Truong reatment .Several terpenoid compounds have been reported to exhibit hair growth promotion by activating Wnt/β-catenin signaling pathway. Loliolide, a monoterpenoid hydroxyl lactone obtained from brown or red algae, increased the viability of human hair follicle dermal papilla (HDP) cells without causing any cytotoxicity until a dose of 100 μg/mL. Loliolide (20 μg/mL) significantly increased the size of HDP spheroids in 3D culture and enhanced the expression of growth factors, such as vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), and keratinocyte growth factor (KGF) in HDP spheroids. These effects of loliolide was mediated through increased nuclear localization of β-catenin and enhanced activation of TCF/Lef transcriptional activity via activation of Akt and phosphorylation-dependent inactivation of GSK-3β. Pharmacological inhibition of Akt abrogated loliolide-induced mRNA expression of Wnt5a and Lef1, suggesting that the β-catenin activation by loliolide was mediated through Akt activation. Moreover, loliolide upregulated the expression of DP signature genes, such as alkaline phosphatase (ALP), versican, and BMP2, in HDP spheroids. These studies further demonstrated that loliolide-treated HDP spheroids secretes VEGF, IGF, and KGF, and the culturing of HaCaT keratinocytes using conditioned media from loliolide-treated HDP spheroids increased the proliferation and migration of keratinocytes 9 . LikewisSilybum marianum, is an antioxidant and anti-inflammatory compound known to cure various skin disorders. Silibinin induced human hair follicle dermal papilla (HDP) cells spheroid formation in 3D culture. Treatment with silibinin elevated the phosphorylation of Akt, increased the expression of Wnt5a and Lef1, and promoted the TCF/Lef reporter activity. Co-treatment with an Akt inhibitor abrogated silibinin-induced Akt phosphorylation and TCF/Lef activity. In addition, silibinin increased the mRNA levels of DPC markers ALP, FGF7, and BMP2, suggesting the potential of silibinin in promoting hair growth [According to current literature, several naturally occurring flavonoids and chalcones also stimulate hair growth by targeting Wnt/β-catenin pathway. Baicalin is a glycosyloxyflavone abundantly present in many medicinal plant species. Xing et al.  examinedr growth .Ginkgo biloba extract, which is widely used for the treatment of various cardiac and neuronal diseases [Ginko boliba, ginkgolide B and bilobalide have been shown to increase the viability of mink dermal papilla cells, which was associated with the phosphorylation of Akt, ERK (for only ginkolide), and GSK-3β. These ginkolides, at a concentration of 50 µM, also elevated the expression of β-catenin and downregulated the expression of DKK1 in DP cells. However, the hair growth promoting potential of these compounds in vivo is yet to be reported [Cornus officinalis, possesses diverse biological activities. Treatment of outer root sheath cells isolated from human scalp with morroniside at 1 or 10 µM concentrations elevated nuclear localization of β-catenin and increased the proliferation and migration of these cells in culture, which was diminished upon co-treatment with DKK1. Morroniside also upregulated the expression of Wnt10b, β-catenin, and lef1 in outer root sheath cells. Injection of morroniside (100 µM) in depilated mouse skin increased the skin thickness and immunohistochemical analysis revealed the elevated expression of β-catenin in epidermis, outer root sheath cells, and matrix during telogen–anagen transition. The compound markedly reduced catagen entry as compared to control [polygonum multiflorum [Aconitie ciliare tuber [Thuja orientalis [Malva verticilata [Caesalpinia sappan L. (Leguminosae), increased the expression of cyclin-dependent kinase-4 (Cdk4), FGF, and vascular VEGF, and promoted anagen phase hair growth in C57BL/6 mice via STST3 activation. This study also demonstrated that 3-DSC reduced the phosphorylation of β-catenin and increased the total β-catenin level without affecting its mRNA script [Broussonetia papyrifera showed promising hair growth potential. Treatment with the extract decreased the phosphorylation of β-catenin and increased expression of total β-catenin in human DPC cells.A well-known herbal medicinal resource is diseases . Among treported . Morroni control . Severaltiflorum , Aconitire tuber , Thuja oientalis , Malva vticilata , and ricticilata , have beA script . In our Despite enormous effort in developing a safe and effective therapy for hair loss, there are few options available for clinical practice. The limited efficacy and remarkable side effects profiles of the existing therapies have led to extensive research on understanding hair biology, especially the molecular mechanisms underlying hair loss and the hair follicle regeneration process. Of the diverse intracellular signaling pathways implicated in hair biology, Wnt/β-catenin signaling plays a key role in stimulating hair follicle stem cells and hair regeneration. Thus, efforts have been made to develop therapies for hair loss treatment by targeting this signaling pathway. While this mini review is designed to shed light on the role of Wnt/β-catenin signaling in hair growth promotion and summarizing the current status of drug development by targeting this pathway, there have been other molecular targets  for designing drugs for hair loss treatment ,82. As i


Wnt signaling is an evolutionarily conserved metazoan cell communication pathway required for proper animal development. Of the myriad of signaling events that have been ascribed to cellular activation by Wnt ligands, the canonical Wnt/β-catenin pathway has been the most studied and best understood. Misregulation of Wnt/β-catenin signaling has been implicated in developmental defects in the embryo and major diseases in the adult. Despite the latter, no drugs that inhibit the Wnt/β-catenin pathway have been approved by the FDA. In this review, we explore the least understood step in the Wnt/β-catenin pathway—nuclear regulation of Wnt target gene transcription. We initially describe our current understanding of the importation of β-catenin into the nucleus. We then focus on the mechanism of action of the major nuclear proteins implicated in driving gene transcription. Finally, we explore the concept of a nuclear Wnt enhanceosome and propose a modified model that describes the necessary components for the transcription of Wnt target genes. Wnt signaling is an evolutionarily conserved cellular communication pathway that plays critical roles in development, tissue homeostasis, and, when misregulated, human disease . Wnt ligThe current model of Wnt/β-catenin signaling includes the assembly of two distinct large protein complexes . In the Binding of Wnt ligands to the co-receptors Frizzled and LRP5/6 and the cytoplasmic protein Dishevelled (Dvl) induces a conformational change that is transmitted to the intracellular domain of Wnt receptors via a mechanism that remains poorly defined. The formation of a large oligomerized Frizzled-LRP5/6 complex or “signalosome” is thought to be critical for formation of an active receptor complex ,4. PhospThe entry of β-catenin into the nucleus is a major signaling step in the Wnt pathway, although a consensus regarding its mechanism of action remains elusive. The FG repeats of nucleoporins normally block nuclear entry for proteins larger than 40 kDa . In the β-catenin has no identifiable NLS , and numVarious “piggyback” or “chaperone” models have also been proposed for the nuclear transport of β-catenin. One candidate is APC, which binds tightly to β-catenin and is localized to both the nucleus and cytoplasm . A similDrosophila and mice [Recently, the guanine nucleotide exchange factor, RAPGEF5, and its associated Rap1a/b GTPases have been shown to play a role in β-catenin nuclear import. This new pathway has been proposed to act in a manner that parallels the Ran/Importin-β transport system . In addiand mice . It remaand mice .Several proteins that regulate the transcription of Wnt target genes have been identified. In this section, we discuss the best characterized of these proteins: β-catenin, T-cell factor/Lymphoid enhancer factor (TCF/LEF), Groucho/Transducing-Like Enhancer (Gro/TLE), Pygopus (Pygo), and B-cell CLL/lymphoma 9 (BCL9). Some of these proteins, however, may not be absolutely required for Wnt signaling and instead may only function in certain contexts. We also discuss β-catenin-mediated transcription of genes that involves non-TCF/LEF partners, but are, nevertheless, dependent on Wnt signaling.β-catenin is often referred to as the master regulator of Wnt signaling because it is the transcriptional switch that shifts TCF/LEF from repressor to activator. The general role of β-catenin is to facilitate 1) the recruitment of the Mediator complex essential for RNA Polymerase II-mediated transcription  and 2) tβ-catenin has multiple nuclear binding partners that play critical roles in the activation of Wnt target genes, including TCF/LEF transcription factors , BCL9/BCTCF7 gene), TCF3 (TCF7L1 gene), TCF4 (TCF7L2 gene), and LEF1 [The TCF/LEF transcription factors are the major mediators of Wnt-responsive transcription. TCF/LEFs are highly conserved; most invertebrates have a single ortholog, whereas in most vertebrates they are encoded by four genes . The fouand LEF1 . TCF/LEFand LEF1 . In addiDrosophila, there is a single copy of TCF, which exhibits both activator and repressor function [In function . Howeverfunction ,28.Gro/TLE family members are transcriptional co-repressors that bind TCF/LEF ,30. Gro/According to the prevailing model, the transition from constitutive transcriptional repression to Wnt/β-catenin-mediated activation is marked by the replacement of Gro/TLE by β-catenin on TCF/LEF. Early models suggest simple competition between β-catenin and TLE for binding to TCF/Lef . SubsequThe interaction between Gro/TLE and TCF/LEF may also be regulated by ubiquitination. The E3 ligase X-linked inhibitor of apoptosis (XIAP), upon phosphorylation by GSK3 in a Wnt-dependent manner, has been shown to ubiquitinate TLE, thereby decreasing its affinity for TCF/LEF ,38. AnotDrosophila [Pygo, a Plant Homology Domain (PHD) domain protein, was identified as an essential Wingless (Wg)/Wnt signaling component in osophila ,40,41,42osophila . Pygo haosophila . Anotherosophila ,46. Inteosophila .Drosophila. In humans and mice, Pygo is encoded by two genes, Pygo1 and Pygo2. Although Pygo2 knockout mice die shortly after birth, they have a reduction in overall Wnt signaling and defects in some Wnt-dependent tissues  [Pygo1 knockout mice did not exhibit an observed phenotype [Pygo1/2 double knockouts and Pygo2 knockouts have similar phenotypes. Further evidence that Pygo may not be required for all Wnt signaling comes from a study demonstrating that Pygo-containing complexes promote transcription of certain Wnt target genes, but not others  [Drosophila, the role of Pygo in mammals is context-dependent and may function to modulate Wnt signaling responses rather than being an essential component.The role of Pygo in Wnt signaling in mammals appears to be much more complicated than in d lungs) . Surprishenotype . Consist tissue) . Interes tissue) . These, Drosophila, which contain a single version of BCL9, Legless (Lgs) with 3 homology domains (HD1-3). The HD1 domain of Lgs was shown to bind to Pygo, whereas the HD2 domain was shown to bind directly to β-catenin. Although Lgs has an HD3 domain, it was found to be dispensable for Wingless signaling [Drosophila. In contrast with core Wnt pathway components, the specification of intestinal stem cells does not require BCL9. However, loss of BCL9 results in reduced expression of stem cell markers and reduced regeneration of the intestinal epithelium after injury [Similar to Pygo, early studies of Lgs/BCL9 were performed in ignaling . In mammignaling . Like thr injury . Based oDrosophila as well. For example, the HD4 and HD5 domains of vertebrate BCL9, which are missing in Lgs, are required for Wnt transcriptional activation in mammalian cells [Drosophila counterpart, mammalian BCL9 has been proposed to act as a dynamic scaffolding protein during Wnt signaling [There appear to be differences in the function of BCL9 and Lgs in Wnt signaling between vertebrates and an cells . Althougignaling . BCL9 inignaling . BCL9 haignaling .Hypoxia Inducible Factor (HIF)-1α controls cellular responses to low oxygen; in the absence of oxygen, HIF-1α is stabilized and acts as a transcription factor for hypoxia-responsive genes . β-catenA similar phenomenon has been observed with the Forkhead Box O (FOXO) proteins, which are involved in insulin signaling and in oxidative stress . FOXO prSox (Sry-related HMG box) transcription factors are another class of transcription factors that interact with β-catenin. Sox proteins play essential roles in animal development and in the maintenance of adult tissues . Early eDrosophila, nearly 20% of all Wg-responsive genes are predicted to be repressed by Wg signaling [ADAM12, ITGB1, APP, DSP, and 15-PGDH) have also been shown to be directly inhibited by β-catenin in response to Wnt signaling [Wnt/β-catenin signaling commonly results in conversion of a target gene from a repressed to an activated state. However, for certain genes, transcription is activated in the absence of Wnt signaling, and Wnt pathway activation represses gene transcription. In ignaling . Wnt-repignaling . Many ofignaling . Similarignaling . Severalignaling ,69,70.By analogy to the large molecular size Wnt receptor signalosome at the plasma membrane and the β-catenin destruction complex in the cytoplasm, Fiedler et al.  coined tDrosophila for a context-dependent enhanceosome comes from studies of Earthbound/Jerky and Erect wing (Ewg) [Unlike the signalosome and the β-catenin destruction complex, the universality of the ChiLS-enhanceosome model in Wnt signaling is unclear, given context dependent differences in enhanceosome components and the controversial role of Pygo as an essential component in vertebrate Wnt signaling ,49. Thisng (Ewg) . Earthbong (Ewg) . Similarng (Ewg) . Althougng (Ewg)  is neverDrosophila, we propose that these proteins are the universal Wnt enhanceosome components [Based on studies indicating that β-catenin and TCF/LEF are absolutely required for transcription of all Wg target genes in mponents . In ordeConstitutive activation of Wnt signaling is associated with the development of numerous cancers . A major