Fibroblast growth factor 5 (FGF5) is a famous dominant inhibitor of anagen phase of hair cycle. Mutations of FGF5 gene result in a longer wool in mice, donkeys, dogs, cats, and even in human eyelashes. Sheep is an important source of wool production. How to improve the production of wool quickly and effectively is an urgent problem to be solved. In this study, we generated five FGF5-knockout Dorper sheep by the CRISPR/Cas9 system. The expression level of FGF5 mRNA in knockout (KO) sheep decreased significantly, and all FGF5 proteins were dysfunctional. The KO sheep displayed a significant increase in fine-wool and active hair-follicle density. The crosstalk between androgen and Wnt/β-catenin signaling downstream of FGF5 gene plays a key role. We established downstream signaling cascades for the first time, including FGF5, FGFR1, androgen, AR, Wnt/β-catenin, Shh/Gli2, c-MYC, and KRTs. These findings further improved the function of FGF5 gene, and provided therapeutic ideas for androgen alopecia. Many of the signaling cascades involved in embryonic hair development are redeployed during adult hair development, and hyperactivation or inappropriate maintenance of these signaling pathways can result in skin disorders and cancers.Hair is a primary characteristic of mammals. Hair-follicle development takes place during fetal skin development, and relies on tightly regulated ectodermal–mesodermal interactions. After birth, mature and actively growing hair follicles eventually become anchored in the subcutis, and periodically regenerate by spontaneously undergoing repetitive cycles. The hair-growth cycle in mammals is composed of three phases: anagen, catagen, and telogenFGF5 gene is a member of the FGF family, which exhibits a diverse array of biological activities5. Hebert et al.6 reported that deletion of the FGF5 gene abnormally prolongs anagen in mice, suggesting that its expression leads to termination of anagen and induction of catagen. Subsequent to identification of FGF5 as causative for the angora mouse phenotype, genetic variants in FGF5 have been shown to underlie hair-length regulation in several other species, including cats (Felis catus)8, dogs (Canis lupus familiaris)10, donkeys (Equus africanus asinus)11, Syrian hamster (Mesocricetus auratus)12, domestic guinea pigs (Cavia porcellus)13, and even human (Homo sapiens)14.The 21. In the previous study, we had successfully generated FGF5-knockout heterozygous sheep using the CRISPR/Cas9 system by direct one-step cytoplasmic injection of Cas9 mRNA and sgRNA into zygotes, and the relationship between FGF5 gene and wool length in sheep was proved22. In this study, we continue the previous approach, and found that FGF5 is also related to the wool and active hair-follicle density in Dorper sheep. However, the specific mechanism of the FGF5 gene in the development of hair follicles is unclear, and whether it has other effects besides promoting changes in the follicular cycle is currently elusive.Sheep are one of the most important wool animals. However, traditional breeding limits the genetic improvement of sheep breeding in the short term. Current results indicate that the CRISPR–Cas9 system can be successfully employed in a broad range of organisms24. Androgen levels are under the control of enzymes. Testosterone, as one of the androgens, can be reduced to dihydrotestosterone (DHT) by 5α-reductase (SRD5A) enzyme, which has three isotypes. SRD5A1 is predominantly expressed in skin and annexes26. Hydroxysteroid 17-beta-dehydrogenase 2 (HSD17β2) is also a key player in the inactivation of testosterone27. The importance of the Wnt/β-catenin pathway in AGA is emphasized by the demonstration of molecular crosstalk between androgens and Wnt signaling in dermal papilla cell (DPC)28. Androgen/AR complex binding to antioxidative response elements (AREs) containing promoters of target genes disrupts Wnt agonist/antagonist balance involved in DPC-inductive ability, such as dickkopf-1 (DKK1), which is a specific inhibitor of Wnt coreceptors of the LRP family29.Androgenetic alopecia (AGA) is the most common form of hair loss in humans, which is mediated mainly by androgens. Androgens regulate hair growth, sebum production, and secretion, among other physiological effects in the skinSonic hedgehog (Shh) also plays an important role in both embryonic and adult hair development. Lim et al.30 showed that hair-follicle activation in Wnt-active dermal cells promotes their fate conversion into dermal papilla (DP), the regenerative dermal niche for hair-follicle formation. Also, Veltri et al.31 believed that Shh signaling serves as a downstream pathway of Wnt/β-catenin signaling to regulate hair-follicle (HF) induction.FGF5 KO sheep and AGA? If so, does crosstalk between AR and Wnt/β-catenin also participate in the process of wool and active hair-follicle density in FGF5 KO sheep? Are there any other signaling pathways or other factors that also play a role? In this study, we revealed that the crosstalk between androgen and Wnt/β-catenin signaling plays a major role in the increase in wool and active hair-follicle density due to the activation of c-MYC and KRTs associated with inner root sheath (IRS) in FGF5 KO sheep, and the Shh pathway is also involved in this process as a downstream pathway of Wnt/β-catenin signaling.Is there an association between the increase of wool and active hair-follicle density in Since we have investigated the efficiency of detecting mutations by PCR sequencing  in the KO group compared with the control group  as well . Sacpic staining results also showed that the proportion of primary and secondary active hair follicles has no significant change.To investigate the changes in wool phenotype caused by the loss-of-function mutation in oup Fig. . Both thFGF5 KO sheep, we tested the expression levels of testosterone and DHT that cause androgenetic alopecia in the skin tissue of the KO and control groups by ELISA. As a result, the levels of testosterone in the KO and control group were comparable, while the level of DHT was significantly reduced in the KO group , while 17β-HSD2 mRNA level was significantly higher in the KO group  signaling plays a critical role in hair-follicle development and skin cancer. Since we have previously confirmed the involvement of Wnt/β-catenin signaling, as a factor downstream of the Wnt/β-catenin signaling pathway, we are also curious about the role of the Shh pathway in the increase of wool and active hair-follicle density. The same method as before, we examined the transcription of Shh, Gli1, Gli2, and Gli3. As a result, Shh mRNA level was significantly lower in the KO than in the control group (P < 0.05), Gli2 mRNA level was significantly higher in the KO group, and the expression levels of Gli1 and Gli3 had no significant change . Despite some limitations, such as efficient delivery and safety, CRISPR–Cas9 is still the most convenient tool for gene-editing purposesWnt/β-catenin signaling pathway is important for the initiation and maintenance of hair morphogenesis38, and is critical for the maintenance of DPC-inductive properties required for hair-follicle regeneration and growth of the hair shaft40. Previous research had shown that in DP cells from AGA patients, the Wnt/β-catenin signaling pathway is negatively influenced by ligand-activated AR, inhibiting HFSC differentiation28. DHT-inducible DKK1 produced by balding DP cells promotes apoptosis of neighboring follicular keratinocytes in vitro, together with the higher expression of DKK1 in balding compared with the haired scalp, suggesting that DKK1 may be one of the key factors involved in the pathogenesis of AGA41. In the present study, we associate the phenotype of increased wool density with the phenotype of AGA, and tests of testosterone and DHT speculated that androgen-induced signaling pathways were highly likely to be involved. Thus, we focused on whether the Wnt/β-catenin signaling pathway was involved. The expression of β-catenin protein in the skin tissues of the KO group was significantly higher than in the control group. DKK1 were significantly lower in the KO group in both the mRNA and protein levels. For further verification of the crosstalk between androgen and Wnt/β-catenin pathway, we added DHT, finasteride, and DMSO in the culture medium of DPCs to detect the expression of related genes. We explored the optimal concentration of DHT and finasteride in DPCs based on relevant reports42 . All surgical operations were performed under anesthesia.The control group were siblings born at the same time as the experimental group. The investigator was blinded to the group allocation during the experiment.FGF5 gene-editing sheep is shown in Fig. FGF5 single-guide RNA transcription were constructed by our laboratory before. The target site in the ovis genome for FGF5 sgRNA is shown in Fig. The process of production of FGF5 sgRNA for native gene disruption activity at its target locus was determined by DNA sequencing in microinjected individuals. The primers are as follows: forward, TGCAAGTTCAGGGAGCGATT and reverse, ATCCCTGTATGCACCAAGCA. Mutations were identified by alignment of sequenced alleles to the wild-type allele. DNAMAN software was used to perform multiple alignments.The capacity of Cas9 mRNA and http://www.rgenome.net/cas-offinder). The selected potential off-target sites were initially PCR-amplified and then subjected to a DNA sequencing assay. The information of the off-target loci and primer pairs used is listed in Supplementary Table STo determine the site-specific cleavage of the CRISPR–Cas9 system in vivo, the potential off-target loci were searched using Cas-OFFinder (https://www.ncbi.nlm.nih.gov) was used. Changes in secondary structures were predicted by PSIPRED v3.3 (http://bioinf.cs.ucl.ac.uk/psipred). The Phyre2 server (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id = index) was used for homology modeling and fold recognition.The sequence of ovis FGF5 (NM_001246263.2) in NCBI . PrimeScrip RT reagent Kit with gDNA Eraser   was used to obtain the cDNA. Real-time PCR was performed on ABI Stratagene Mx3000P instrument  using a TB Green Premix Ex Taq II . The primer sequences used in this experiment are shown in Supplementary Table SFor ELISA, skin tissue samples of the KO and control groups were obtained and homogenized by hand grinders, removing the supernatant after centrifugation. The ELISA procedure was performed as described in the product . The concentrations of androstenedione and testosterone in the skin tissue were measured using an androstenedione ELISA kit  and a testosterone ELISA kit  in accordance with the manufacturer’s protocol.For western blotting, equal amounts of protein of sheep skin tissue or DPCs were resolved on 12% SDS-polyacrylamide gel (SDS-PAGE) and transferred to the polyvinylidene difluoride membrane (PVDF). After incubation with antibodies, protein bands were detected using ECL chemiluminescence . The primary antibodies were rabbit anti-FGF5 antibody , goat anti-SRD5A1 antibody , rabbit anti-androgen receptor antibody , goat anti-DKK1 antibody , beta-catenin polyclonal antibody , c-MYC polyclonal antibody , and beta tubulin monoclonal antibody . The secondary antibodies were HRP-conjugated affinipure goat anti-rabbit IgG(H + L) , HRP-conjugated affinipure rabbit anti-goat IgG(H + L) , and HRP-conjugated affinipure goat anti-mouse IgG(H + L) .The diameters of 200 randomly chosen fiber samples were measured using an Optic Fiber Diameter Analyzer . Photographs were documented for 48 h after shaving to provide a record of wool density. Images were captured with a handheld digital microscope—HotBeauty Skin tester Ht-B20S. Photographs were analyzed by using the ruler to correct the actual field of view, and the densities of coarse and fine wool were calculated separately.Sacpic is a histological method for determining the proportion of growing hair follicles in skin samples. Skin preparations of the KO and control groups were used to illustrate structural characteristics of follicles viewed in cross section at various stages of the hair cycle. Data from skin biopsies were also used to demonstrate quantitative estimation of follicle activity.The skin tissues of the KO and control groups were digested with neutral protease  at 37 °C for 2 h. The epidermis was carefully peeled off; the hair follicles were separately clustered from other tissues with a microscopic flaw. Excess tissues between hair follicles were removed by digestion with collagenase II  at room temperature for about 30 min. Finally, the hair follicles were transferred to the culture dish; DP cells will slowly overflow in DMEM/F-12 (Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12), 20% (v/v) fetal bovine serum (FBS) supplemented with 100 units/ml penicillin and 0.1 mg/ml streptomycin . DPCs were further purified and extracted using a cloning loop. For all experiments, cells were used between passage three and six.5 cells/ml in 100-mm dishes) were seeded into 100-mm culture dishes. After a night of adherent growth, DPCs were treated with 10−7 M DHT and 10−5 M finasteride  for the next 48 h, respectively. The media were changed every 24 h. After 2 days of conditioned culture, DPCs were collected for the extraction of total RNA and protein.The effects of DHT and finasteride on DPCs in the KO and control groups were also determined. DPCs  and Hoechst 33342 (1:1000) for 1 h. Ninety-six-well plates with the fixed cells were examined using ImageXpress (Molecular Devices).DPCs were seeded in 96-well plates  in each well. Cells were fixed with 4% paraformaldehyde for 20 min at room temperature, and then permeabilized with 0.3% Triton X-100 for 10 min at room temperature. They were washed three times with PBS for 5 min. After a 20-min incubation with immunol staining blocking buffer , cells were continuously incubated with mouse anti-SMA  and rabbit anti-CD133 Statistical comparison of wool fineness and density was performed by the one-way ANOVA. Statistical analyses were performed using SAS release 9.2 .Fig S1Fig S2Fig S3Fig S4Fig S5Fig S6Supplementary Figure legendsSupplementary Table S1Supplementary Table S2Supplementary Table S3


Epoxy-based biocomposites are a good alternative for metals in lightweight applications. This research has been focused on the effect of accelerated weathering on the mechanical, thermal, contact angle, and water absorption behavior of neat epoxy, individual kenaf and sisal, and kenaf/sisal hybrid epoxy composites. The composite was fabricated by hand layup method. Among the various composites studied, sisal/kenaf/sisal hybrid epoxy composites showed the best properties and retained the thermo-mechanical properties with the lowest water absorption properties even after the weathering test. Thus, composites with hybridized kenaf and sisal with sisal outer layer are encouraging semistructural materials in outdoor applications. Epoxy resins are one of the important thermosetting polymers used for making numerous materials for many applications. Recently, several studies on epoxy thermoset are focused on developing advanced composites, shape memory polymers, flexible polymer, self-healing polymers, etc. ,4,5,6,7.Recently, several approaches have been adopted for the replacement of synthetic fibers, in one such approach, Giridharan  hybridizThus, from the literature reports, natural fiber-reinforced epoxy composites have attracted increasing interest over the last few years. This is because of the many advantages of using natural fiber in epoxy composites. First, the amount of non-biodegradable epoxy resin used for the manufacturing of the products can be reduced with the incorporation of natural fiber fabric. Second, for long-term applications, the natural fiber fabric-based composites can provide better mechanical properties. Third, the reuse, recycling, or degradability of synthetic fiber after the service life is difficult since the epoxy resin is warped over it. On the other hand, natural fibers are cheap, easy to process, eco-friendly, and biodegradable. Fourth, this method facilitates the development of cheap and more eco-friendly composites. However, comparing with synthetic fibers-based composites, natural fiber-based composites are prone to degradation when used for outdoor applications due to moisture absorption, microbial attack, temperature change, and ultraviolet radiation. The water absorption of the natural fiber-reinforced composites is reported to be higher than synthetic fiber-reinforced composites; moreover, the fiber degradation in the matrix may initiate microcracks, which will eventually deteriorate the performance of the composites. To address these issues few of the recent studies are focused on the accelerated weathering analysis of natural fiber-reinforced epoxy composites ,24,25,26The epoxy resin (Epotec YD-535 LV) and hardener (Epotec TH-7257) were kindly supplied by Aditya Birla Chemicals Ltd., Rayong, Thailand. The epoxy resin has a density of 1.1–1.2 gm/cc and a viscosity of 1000–1500 mPa s at 25 °C. The Epotec TH-7257 is a polyamine-based hardener . The che2 and a thickness of 3 mm. The samples were cured at room temperature for 24 h, followed by post-curing at 80 °C for 24 h. For the preparation of epoxy composites, first, the fiber fabrics were dried at 60 °C for 24 h in an air oven and then cut it to fit in the mold size of 300 × 300 mm2. Second, the epoxy/hardener mixture was prepared. Third, the three layers of the fabric were placed in the mold and were applied with the epoxy hardener mixture by hand layup method. This was followed by curing at room temperature for 24 h, followed by post-curing at 80 °C for 24 h. Irrespective of the composites, the fiber volume fraction used was ~18%. The average standard deviation in thickness of the prepared composites varies between 0.02 to 0.15 mm. The cured samples were later cut into small size for testing using a diamond-tipped saw cutter based on ASTM standards. Before testing, sandpaper was used to remove the defects and to make the samples more uniform. The schematic of the prepared samples and the photographs of the cut samples (before the application of sandpaper) is given in The neat epoxy and epoxy composites were prepared as follows . For theEpoxy composites have been used in many outdoor applications such as automobile, aerospace, and construction and building. These composites are exposed to harsh environmental conditions like temperature, humidity, and UV radiation during their service. The accelerated weathering instrument can replicate various real environmental conditions using a special weathering chamber. The instrument accelerates the weathering process and provides information on the durability of the composites. The epoxy composites are indeed coated with UV resistant paints before outdoor applications. However, in this study, the accelerated weathering test was used to predict the usefulness of epoxy fiber–fabric composites for long-term applications. As temperature, humidity, and light conditions may influence the structural properties of the composites, the accelerated weathering experiment was carried out in a Q-Sun Xe-3 weathering tester machine, Q-Lab Corporation, Westlake, Ohio, USA, according to ASTM standard G155-13 cycle 1, as in our previous study . The xenTg) of the composites was evaluated using a DSC 3-Mettler Toledo, Schwerzenbach, Switzerland. Approximately 5 mg of the sample was tested in the temperature range −50 to 200 °C at a heating rate of 20 °C/min. The viscoelastic properties of the composites were measured using a DMA/SDTA861e, Mettler Toledo, the test was carried out from −30 to 140 °C at a heating rate of 2 K/min and frequency 1 Hz. A OCA 15LJ data physics-contact angle instrument was used to measure the contact angle of the composites. To measure the contact angle, the water droplets were placed at different positions of the same sample and an average of 6–10 measurements were taken as the contact angle value. The water absorption behavior of the composites was studied based on ASTM D570. The samples were carefully cut into a small disc of 50 mm diameter and thickness 3 mm before the water absorption studies. The edges are not isolated in this experiment. The composite samples were immersed in distilled water for 120 days and the weight change is measured for every 10 days.A M1 type Comtech tensile testing machine was used for testing the mechanical properties of the samples based on ASTM D3039. The test was carried out at a loading rate of 2.5 mm/min and the load cell used was 2.5 KN. Five samples were tested for each composition and the average was taken as the final value. A Zwick/Roell pendulum impact tester was used to study the impact properties of the composite based on ASTM D256-06. Five samples were tested for each composition and the average was taken as the final value. A Hitachi TM4000Plus scanning electronic microscope was used to examine the fracture surface of the composites. The thermal stability of the composites was measured using a TGA 2-Mettler Toledo TGA/DSC 3+ HT/1600, Schwerzenbach, Switzerland. The test was carried out from 30 to 700 °C, at a heating rate of 10 °C/min. The glass transition temperature , final decomposition temperature (Tf), and maximum decomposition temperature (Tmax) obtained from the thermogram are given in Ton, Tmax, and Tf values of composites before weathering are stable with only marginal changes with the incorporation of fiber fabrics. Similarly, irrespective of the introduction of the fibers, fiber type, hybridization, and stacking sequence, the neat epoxy and epoxy composites show no major change in the values of Ton, Tmax, and Tf after the weathering test. These results prove that the composites are thermally stable before and after the weathering test. It is important to point out that the neat epoxy system and composites show two peaks: a shoulder and the main peak in the DTG curve  repels water and reduces the water absorption. Similarly, if the contact angle is less than 10°, the composite will be superhydrophilic with higher wettability and good self-cleaning ability ,39,40. FThe water absorption behavior of the neat epoxy, individual composites, and hybrid composites before and after weathering composite samples was studied up to 120 days. The water absorption behavior of the composites before and after weathering is shown in Tg. The Tg is followed by the rubbery region [Tg is maximum for neat epoxy resin, but the drop is not very pronounced for the composites before and after aging. This is due to the presence of stiffer fiber fabric in the rubbery region. Among the various composites studied, the SKS hybrid composites show reasonably good thermal stability, mechanical properties, and water absorption properties. Therefore, the viscoelastic properties of neat epoxy and SKS hybrid composites before and after weathering samples are studied. The viscoelastic properties of neat epoxy and SKS composite before and after weathering are shown in y region . As in oy region , the droE′G = storage modulus at the glassy state (at 35 °C)E′R = storage modulus at the rubbery state (at 120 °C).The reinforcement effect of fiber fabric with the epoxy matrix can be measured by calculating the reinforcement effectiveness concept (C) the using the Equation (1)  and the If the value of C is less than 1, it represents the interaction between fabric and epoxy matrix. The C value of SKS composite is less than 1, which shows the existence of the interaction between fabric and epoxy matrix. As there is no increase in the modulus of the epoxy glassy state with the incorporation of the fabrics, rather than the reinforcement effect, the stiffer fiber fabric in the rubbery region could be the cause for the lower C value.Tg with the incorporation of fiber fabric. The neat epoxy system after the weathering test shows lower Tg and higher loss modulus peak height. The high loss modulus peak values showed that the intermolecular bonding is destroyed during the accelerated weathering test [Tg was not affected with the accelerated weathering test; moreover, the loss modulus peak height is lower when compared with neat epoxy system showing that the fiber fabrics prevent the degradation of the epoxy matrix at the macro-level.The loss modulus profile of the neat epoxy system and SKS composite is shown in ing test . It is wThis study aimed at investigating the influence of accelerated weathering on the performance of natural fiber fabric-reinforced epoxy composites. The mechanical, thermal, morphology, contact angle, and water absorption properties were evaluated. Among the composites prepared, hybrid SKS composites showed reasonably good thermo-mechanical, contact angle, and water absorption properties. From the obtained results, it can be suggested that the hybridization of sisal and kenaf fiber fabrics may be used for the fabrication of semi-structural epoxy composites.


The diagnostic procedure for upper aerodigestive tract (UADT) tumours is by white light endoscopy (WLE) combined with biopsy. However, WLE has difficulty identifying minute epithelial changes which hinders early diagnosis. Storz Professional Image Enhancement System (SPIES) is designed to enhance the visualization of microvasculature on the mucosal surface and detect any epithelial changes. In this study, we aimed to evaluate the use of Ni endoscopic classification with SPIES endoscopy in the detection of UADT tumours. Fifty-nine patients with suspected UADT tumours underwent WLE followed by SPIES endoscopy. All the tumours were biopsied and sent for histopathological examination (HPE). The kappa index (κ) was used to evaluate the agreement between the methods. The level of agreement between SPIES using Ni classification and HPE showed almost perfect agreement as compared to moderate agreement between WLE and HPE. The sensitivity and specificity for WLE and HPE were 77.5% and 84.2% respectively with positive predictive value (PPV) of 91.2% and negative predictive value (NPV) of 64%. The sensitivity and specificity for SPIES endoscopy using Ni classification and HPE were 97.5% and 94.7% respectively with PPV of 97.5% and NPV of 94.7%. SPIES endoscopy using Ni classification is a valid tool for earlier tumour detection. Subepithelial and epithelial microvascular irregularities will appear due to alterations of the vessel architecture and it depends on the degree of dysplasia. The superficial changes of neoangiogenesis created by the UADT lesions can be enhanced and recognized by using narrow band imaging (NBI) and Storz Professional Image Enhancement System (SPIES)2.The gold standard in diagnosing upper aerodigestive tract (UADT) tumours is by identification of suspicious lesions by white light endoscopy (WLE) combined with the histopathological examination (HPE) findings. However, WLE can only provide poor quality images causing difficulty to identify minute epithelial changes and making it difficult to differentiate benign from malignant tumours. Neoangiogenesis is a prerequisite for the progression of precancerous and cancerous lesions of the UADT1. While Clara and Chroma modes are used mainly for endoscopic procedures to gain better clarity of anatomical structures, Spectra A and Spectra B modes are used to visualize the vascular arrangements in the mucosa of UADT. SPIES endoscopy aids in visualizing early changes of the vascular arrangements and improves the diagnostic capability in early cancer detection. Early detection of malignant change improves the prognosis as immediate definitive treatment can be instituted. In comparison to NBI , SPIES uses the different light wavelengths obtained with the standard white light sources to enhance the image quality2. The technological difference between NBI and SPIES purely relies on the different design of the light filtering process. Although both use filter wavelengths, pre-processing of image occurs in NBI, while in SPIES it happens in the post-processing phase. Additionally, NBI uses one kind of filter, while SPIES uses a software system to create different light wavelengths and contrast in the production of images. SPIES endoscopy is a useful technique to obtain clearer images and reduces the chances of missed diagnosis. It also enables complete resection of tumour by delineating the margin of healthy and tumour tissue.SPIES  uses a high definition camera system with image enhanced endoscopy for better endoscopic visualization. In the assessment of the microvasculature’s pattern on the mucosal surface, the endoscopic images can be visualized by using 5 spectral modes of “Clara, Chroma, combined Clara and Chroma, Spectra A and Spectra B”3. There are two main classifications based on the epithelial microvascular changes being used for NBI endoscopy to differentiate benign and malignant lesions of UADT. By identifying the capillary loop arrangements on the mucosal vessels of the oral cavity, Takano et al.4 classified their findings as 4 types; type 1 when there is presence of “normal mucosa and regular brown dots”, type 2 when there is “capillary dilation and crossing”, type 3 when there is “capillary elongation and meandering” and type 4 when there is “capillary destruction and angiogenesis”. Their classification is actually a modification of Inoue classification5, which is a classification based on microvascular changes seen on the pharyngoesophageal epithelium. However, the most used classification system of vascular changes seen in laryngeal and pharyngeal lesions is the Ni classification7 based on the microvasculature pattern of the abnormal capillary loop arrangements. Ni classification6 categorized the microvascular changes into five different types; types I to III demonstrating longitudinal oblique or branching vessels but no intraepithelial papillary capillary loop, while types IV and V representing visible perpendicular intraepithelial papillary capillary loops with increasing irregularity. Type I-IV are considered as benign lesion while type V is considered as malignant lesion7. Bertino et al.8 compared both classifications for NBI endoscopy and found that Ni6 has better sensitivity and specificity as compared to modified Inoue classification4 in detecting benign and malignant lesions of UADT.The presence of dilated and abnormal intraepithelial capillary loops in a brownish area with dark spots is the typical finding of NBI in patients with precancerous lesions9. Thus, any classifications used and valid for NBI should also be applicable for SPIES as well. However, there was no study done on the concordance and validation of the Ni classification system for SPIES endoscopy application. There was also no previous study done using SPIES endoscopy with Ni classification system to identify UADT tumours as most studies were specifically for laryngeal and hypopharyngeal lesions. Therefore, we aimed to use and validate the Ni endoscopic classification with SPIES endoscopy system in the detection of UADT tumours.It has been established that both NBI and SPIES endoscopy systems are comparable in recognition and analysis of vascular patterns that is typical for benign and malignant lesions4 and the second according to the classification of the microvascular endoscopic patterns of Ni classification6 . Biopsy of the mass was taken, fixed in 4% formalin, and submitted for HPE. The HPE results were compared with WLE and SPIES endoscopic findings.The study protocol was approved by Human Research Ethics Committee USM [USM/JEPeM/16110492], performed in adherence with the Declaration of Helsinki and approved guidelines. Patients who attended the otorhinolaryngology clinic were recruited from March 2017 until March 2018. Inclusion criteria were patients aged 18 years old and above with suspicion of UADT tumours including sinonasal, nasopharygeal, oral, oropharyngeal or laryngeal tumours. Patients who had undergone surgery involving the UADT, prior treatment with chemoradiotherapy and pregnant women were excluded. Informed consent was obtained from all patients before their participation in this study. The patients’ demographic data and clinical history were recorded in the patient’s proforma. All patients with suspected UADT tumours underwent both WLE and SPIES endoscopic examination. WLE was performed initially, followed by SPIES endoscopy, which was easily switched between each modality by using a button on the camera head. SPIES endoscopy in a Spectra A mode was performed using 0 degree or 70 degrees, 4 mm rigid endoscope  and connected to Image 1 high-definition camera system . The UADT was locally anaesthetized before introducing the rigid endoscope. 10% cocaine hydrochloride solution was used as a topical anaesthetic, with vasoconstrictors for nasal endoscopic procedures while 10% lignocaine spray was used for oral or laryngeal endoscopic examination. The procedure was done under local anaesthesia except for patients who could not tolerate the procedure or patients with laryngeal mass who required biopsy under general anaesthesia. The videos of all endoscopic examinations were recorded and saved on a personal computer. On WLE, lesions were considered suspicious for cancer when there were changes such as leucoplakia, erythroplakia, exophytic, or ulcerated lesions seen. Then, the SPIES endoscopy findings were evaluated. The first evaluation was made according to modified Inohue classificationData were stored in a computer based filling system, and analysed using the PASW Statistics 22 software . The kappa index (κ) was used to evaluate the agreement of WLE or SPIES results with HPE findings and agreement between the methods. The value of kappa less than 0.20 is considered as no agreement, 0.21 to 0.39 as minimal, 0.40 to 0.59 as weak, 0.60 to 0.79 as moderate, 0.80 to 0.90 as strong and above 0.90 as almost perfect agreement. The sensitivity, specificity, and positive and negative predictive values for WLE and SPIES endoscopy were calculated. A p value of < 0.05 was considered statistically significant.A total of 59 patients,41 males and 18 females, were enrolled with the mean age of 52.2 years. The youngest was 18 years old and the oldest was 94 years old. There were 19 lesions from the larynx, 17 lesions from the nasal cavity, 11 lesions from the nasopharynx, 9 lesions from the oral cavity and 3 lesions from the oropharynx. The demography is shown in Table Based on the HPE findings, there were 40 benign lesions and 19 malignant lesions from a total of 59 lesions Table . Out of κ = 0.592 (95% CI 0.368–0.755)(p < 0.001) which showed moderate agreement. The level of agreement between SPIES endoscopy and histological assessment was 86.7%, kappa index κ = 0.753 (95% CI 0.482–0.916) (p < 0.001) for modified Inohue classification and the agreement was 95.0%, kappa index κ = 0.927 (95% CI 0.762–1.000) (p < 0.001) for Ni classification. The agreement was confirmed as strong for modified Inohue classification while almost perfect agreement for Ni classification.Results of WLE and SPIES endoscopy (modified Inohue and Ni classifications) with the HPE were compared. The level of agreement between WLE and histological assessment was 78.3%, kappa index The sensitivity and specificity in the differentiation between WLE and HPE were 77.5% and 84.2% respectively with positive predictive value of 91.2% and negative predictive value of 64%. The sensitivity in the differentiation between SPIES endoscopy using modified Inohue classification and HPE was 87.5% while specificity was 89.5% with positive predictive value of 94.6% and negative predictive value of 77.3%  has proposed an approach to distinguish between benign  and malignant (perpendicular vessels) lesions instead of a classification of vascular pattern correlated with their histopathologic findings done by Ni and colleagues11. Nevertheless, the Ni classification and the ELS classification are almost similar, as the Ni types I, II, and III represent changes in the longitudinal vessels, whereas the Ni types IV and V represent the perpendicular vessels for ELS classification7. Among the classifications, ELS is the easiest and more practical to be taught, remembered, and systematically applied.The features and organisation of blood vessels are dynamic and may undergo change during the transition from a precancerous to a malignant state, as the microvascular architecture becomes more dilated, elongated, distorted, or replaced by neoplastic vasculature due to the epithelial cancerogenetic stimulus6 demonstrate a high sensitivity of 97.5% and specificity of 94.7%, with positive and negative predictive values of 97.5%, and 94.7% respectively in the detection of UADT tumours. In comparison, the conventional WLE has a sensitivity of 77.5% and a specificity of 84.2% whereas SPIES endoscopy using modified Inohue classification has a sensitivity of 87.5% and a specificity of 89.5%. Our findings on Ni classification6 used with SPIES endoscopy are consistent with another study that assessed its use with NBI endoscopy8. As there was an almost perfect agreement of SPIES endoscopy using Ni classification6 with the HPE results , SPIES endoscopy is comparable to the results obtained by NBI endoscopy in the early assessment of UADT tumours. Hence, our results showed that both NBI and SPIES have equal property in picking up and diagnosing UADT tumours.Our data showed SPIES endoscopy using Ni classificationet al. reported that in the assessment of intraepithelial papillary capillary loop features using NBI endoscopy, interference is possible in the presence of necrotic tissue or a thick white patch overlying the lesions, resulting in false negative findings6. Therefore, surrounding regions and areas with a thinner white patch need to be evaluated, in order to delineate any dots or irregular, tortuous, line-like shapes which may aid classification6. In addition, bleeding during the rigid endoscope insertion may affect the evaluation of intraepithelial papillary capillary loop features. The presence of blood on mucosa will be shown as generalized black lesion as haemoglobin absorbs blue light. Thus, touching and injuring the mucosal surface should be avoided during the endoscopic procedure. The result of our study showed, that SPIES endoscopy in combination with Ni classification6 could detect accurately all squamous cell carcinomas but not diffuse B cell lymphoma as there was no alteration of the microvascular pattern within the epithelial and mucosal lining of the lesion.In our study, SPIES endoscopy revealed only one false positive result in the case of granulation tissue and one false negative result in the case of diffuse B cell lymphoma. In the case of granulation tissue, it becomes falsely interpreted as malignancy due to the presence of necrotic tissue that obscured the lesion. In the case of diffuse B cell lymphoma, there was no change of the microvascular pattern within the epithelial and mucosal lining and this was rightly diagnosed as normal epithelium. Ni 12 is to emphasize the role of NBI endoscopy as their strategy for an earlier tumour detection. As both NBI endoscopy and SPIES endoscopy methods are comparable in recognition and analysis of benign and malignant lesions9, SPIES endoscopy should also be recommended as one of the strategy for an earlier diagnosis of UADT tumours. Both NBI and SPIES endoscopy systems form an optimal strategy for preventing the progression of the cancer and improving the prognosis by earlier detection. Furthermore, early detection may help in minimally invasive treatment such as endoscopic resection with curative intent. Watanabe et al.13 conducted a study on patients with the suspicion of having laryngeal cancer and they found that NBI was superior in the early detection of abnormal microvascular changes when compared to WLE. Their study reported a sensitivity of 91% and specificity of 92% in the differentiation between low-grade and high-grade dysplasia13. Another study by Piazza et al.14 on patients undergoing treatment or follow-up for laryngeal cancer, revealed overall sensitivity, specificity, positive predictive value and negative predictive value of 98%, 90%, 86%, and 88% respectively. The outcome of their studies using NBI endoscopy are comparable to our results with SPIES endoscopy.One of the recommendations by National Institute for Health and Care Excellence (NICE)Ni classification is valid to be used for SPIES endoscopy in the distinction between benign and malignant tumours. It offers a high sensitivity and specificity rate in the detection of tumours and a reliable diagnostic tool for earlier UADT tumours recognition.


A receptors, the azobenzene-nitrazepam-based photochromic compound (Azo-NZ1), has been described. In the present study, using patch-clamp technique in heterologous system and in mice brain slices, site-directed mutagenesis and molecular modeling we provide evidence of the interaction of Azo-NZ1 with glycine receptors (GlyRs) and determine the molecular basis of this interaction. Glycinergic synaptic neurotransmission determines an important inhibitory drive in the vertebrate nervous system and plays a crucial role in the control of neuronal circuits in the spinal cord and brain stem. GlyRs are involved in locomotion, pain sensation, breathing, and auditory function, as well as in the development of such disorders as hyperekplexia, epilepsy, and autism. Here, we demonstrate that Azo-NZ1 blocks in a UV-dependent manner the activity of α2 GlyRs (GlyR2), while being barely active on α1 GlyRs (GlyR1). The site of Azo-NZ1 action is in the chloride-selective pore of GlyR at the 2’ position of transmembrane helix 2 and amino acids forming this site determine the difference in Azo-NZ1 blocking activity between GlyR2 and GlyR1. This subunit-specific modulation is also shown on motoneurons of brainstem slices from neonatal mice that switch during development from expressing “fetal” GlyR2 to “adult” GlyR1 receptors.Photopharmacology is a unique approach that through a combination of photochemistry methods and advanced life science techniques allows the study and control of specific biological processes, ranging from intracellular pathways to brain circuits. Recently, a first photochromic channel blocker of anion-selective GABA Photochromic molecules are becoming widely used for studying and modulating various biological processes. Successful application of these compounds, whose activity can be controlled with light, potentially provides a promising tool for future therapeutic approaches. The main advantage of such compounds is their precise spatial and temporal selectivity, a property that favors specific drug action and diminishes their side effects. In the present study, we describe in detail the interaction of the novel azobenzene-nitrazepam-based photochromic compound (Azo-NZ1) with glycine receptors (GlyRs) and determine its subunit-specific blocking activity in the Cl-selective pore of GlyRs. This compound offers a new strategy for specific control of glycinergic circuits and stepping stone for design of new GlyR-active drugs.A receptors were developed as well , the azobenzene derivative of nitrazepam, blocks in a UV-dependent manner the ion channel pore of GABAcurrents . These eGlyRs are ligand-gated anion-permeable ion channels that belong to the Cys-loop receptor superfamily and provide the main inhibitory drive in the brain stem and spinal cord of vertebrates, as well as regulate network excitability in the retina, hippocampus and amygdala . GlyRs pGinkgo biloba leaves  and ginkgolide groups have more complicate patterns of interaction with GlyRs of different subunit composition, but they have in common a strong dependence on the β subunit presence . Such a trans-conformation, while nearly inactive in cis-form. Using mutagenesis analysis and molecular modeling, we have shown that the amino acid at 2’ position of the ion channel pore is crucial for Azo-NZ1 blocking action.Here, we present a detailed characterization of different GlyRs subtypes interacting with Azo-NZ1, the first photo-switchable ion channel blocker of gamma-aminobutyric acid receptors (GABARs) and GlyRs. We have demonstrated that Azo-NZ1 inhibits GlyRs in a subunit specific manner, being a potent blocker of α2 GlyRs in the Synthesis and photochemical characterization of Azo-NZ1 were performed as previously reported .m), to reduce the detrimental alterations of membrane potential because of activation of overexpressed GlyRs by glycine present in culture medium. Electrophysiological recordings were conducted on fluorescent cells 24–72 h after transfection.GlyRs of different subunit composition were heterologously expressed in cultured Chinese hamster ovary (CHO) cells obtained from the American type Tissue Culture Collection (ATCC) that were maintained as previously described . Transfem NaCl, 2 mm CaCl2, 2.8 mm KCl, 4 mm MgCl2, 20 mm HEPES, and 10 mm glucose . Intracellular solution used for filling recording patch pipettes contained the following: 140 mm KCl, 2 mm MgCl2, 2 mm MgATP, and 2 mm BAPTA  . Recording pipettes were pulled from borosilicate glass capillaries (Harvard Apparatus Ltd) and had resistances of 5–10 MΩ. Rapid replacement of solutions was provided by fast application system , placed 40–50 μm above the recorded cell. Cells with low input resistance (<150 MΩ) and a rapid run-down (>30% with repetitive application) were excluded from analysis.Whole-cell patch-clamp recordings were held at room temperature (20–25°C) using an EPC-9 amplifier (HEKA Elektronik). Cells were continuously superfused with external solution containing the following: 140 mSingle-channel recordings were performed in the outside-out configuration, at room temperature using an EPC-9 amplifier (HEKA Elektronik). External and internal solutions were the same as those used for whole-cell recordings.Guide for the Care and Use of Laboratory Animals  and European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes . All animal protocols and experimental procedures were approved by the Local Ethics Committee of Kazan State Medical University . Mice had free access to food and water and were kept under natural day length fluctuations.Experiments were performed on white laboratory ICR outbred mice of both sexes at postnatal day (P)4–P8. Use of animals was conducted in accordance with the + solution, containing the following: 120 mm K-gluconate, 10 mm HEPES‐acid, 15 mm Na-gluconate, 0.2 mm EGTA, and 4 mm NaCl . After cutting, slices were placed for 10 min at room temperature in a choline‐based solution, containing the following: 110 mm choline chloride, 2.5 mm KCl, 1.25 mm NaH2PO4, 10 mm MgCl2, 0.5 mm CaCl2, 25 mm NaHCO3, 10 mm glucose, and 5 mm sodium pyruvate . Before experiments slices were incubated for 1 h in a chamber filled with an oxygenated artificial CSF (aCSF) containing the following: 126 mm NaCl, 3.5 mm KCl, 2 mm CaCl2, 1.3 mm MgCl2, 1.2 mm NaH2PO4, 10 mm glucose, and 25 mm NaHCO3 .For electrophysiological experiments, coronal slices containing hypoglossal nucleus were obtained. Mice were decapitated; the brainstems were removed and sliced into 350-μm-thick sections using a tissue slicer . Sections were prepared in an ice-cold high Km), APV (40 μm), and bicuculline (20 μm) were routinely added to aCSF to block the glutamatergic and GABAergic synaptic transmission. Recording was conducted at room temperature in the whole-cell configuration with the holding potential of 0 mV using the EPC‐10 patch-clamp amplifier (HEKA Elektronik). Patch electrodes were filled by the intracellular solution containing the following: 130 mm Cs-gluconate, 4 mm MgATP, 10 mm phosphocreatine, 0.3 mm GTP, 10 mm HEPES, 5 mm EGTA, and 4 mm MgCl2 . Effects of Azo‐NZ1 on the amplitudes of glycinergic eIPSCs were examined at visible light and under illumination of aCSF by diode emitting 365 nm UV (Thorlabs).Glycinergic evoked IPSCs (eIPSCs) were obtained from the motor neurons of the hypoglossal nucleus, as previously described . The DS3I is the normalized current amplitude induced by the agonist at concentration [A], maxI is a maximal current induced at given cell, Hn is the Hill coefficient and EC50 is the concentrations at which a half-maximum response was induced.To analyze the results of patch-clamp recordings, PatchMaster (HEKA Electronic), Origin 7.5 (OriginLabs), Excel 2016 (Microsoft), and Igor Pro 6.02 (WaveMetrics) software were employed to accomplish a statistical analysis of the data and to plot the graphs. Concentration-response curves using different concentrations of glycine were fitted using a nonlinear fitting routine of the Origin 7.5 software with the Hill equation:1I is the amplitude of the current before application of Azo-NZ1 (control) and 2I is the amplitude of the current during the application of Azo-NZ1.To quantify the inhibitory effect of Azo-NZ1, the following equation was used:1I is the amplitude of the current before application of Azo-NZ1 (control), 3I is the amplitude of the current during the application of Azo-NZ1 on UV illumination.To quantify the effect of UV illumination, we used the following equation:Taking into the account that, in the case of α2Z, α2M/βM, and α1H G254A receptors, desensitization was present, we suggested to estimate the UV effect with respect to the washout current. However, the hump current that appeared during the washing and the potentiation effect recorded on UV illumination  made the correct estimation of UV effect impossible. Because of these complications and the visibly prominent UV effect, we have arbitrarily fixed it at the level of the control current.Concentration dependences of responses to different doses of Azo-NZ1 were fitted using a nonlinear fitting routine of the Origin 7.5 software (OriginLabs) with the Hill equation:m was taken arbitrarily.The effect of Azo-NZ1 at each concentration was estimated through normalization of the current amplitude with respect to the amplitude of the control current. The point corresponding to the 1000 μt tests, paired sample Wilcoxon signed-rank test. Differences were considered significant at p < 0.05.Data are represented as mean ± SEM. Significance of differences was assessed using paired and unpaired Data were sampled at 10 kHz using PatchMaster (HEKA Electronic) software. Further analysis was performed with the Nest-o-Patch program.o) was calculated as the total open time of the channel during a recording divided by total time of the recording (The amplitude histograms of simple openings were fitted with the sum of Gaussian functions (for α2M GlyRs) or built based on the amplitudes of only completely resolved events (α1 and α1H G254A mutant GlyRs). Kinetic parameters have to be considered as “apparent” because of the effects of undetected shutting and opening . The opeecording .m) was prepared using dimethylsulfoxide (DMSO) and then diluted to the final concentration by extracellular solution. All other drugs were obtained from Hello Bio and Tocris or Sigma-Aldrich. Stock solutions were prepared using DMSO or MilliQ water and kept at −20°C.Stock solution of Azo-NZ1 (10 min silico using the molefacture tool (version 1.3) in VMD . Structures of these GlyRs were modelled in the open channel state. For homomeric α1Z GlyR, we used the cryo-EM structure with glycine bound (PDB code 3JAE) from reference . For homHomology models were generated for homomeric α2H and heteromeric α2M/βM GlyRs using SWISS-MODEL . For thecis- and trans- isomers) were created employing Avogadro  were considered for docking. Optimized ligand structures were taken from  or exit the pore (reaching the bath solution). The outcome will depend on the mean opening time of the channel and the dissociation rate of cis-Azo-NZ1. With the data at hand, we cannot distinguish between the two aforementioned possibilities. Therefore, we decided to perform docking calculations not only with trans-Azo-NZ1, but also with the cis isomer, to model the system immediately after UV irradiation. These calculations are aimed at investigating the change in the receptor-ligand binding mode right after trans-cis isomerization .We would like to note here that, on UV light irradiation, In order to explore the possibility of Azo-NZ1 binding to other regions of the GlyR channel, we also performed “blind” docking calculations, in which the search space includes the whole receptor. The same docking protocol as for the calculations focused on the M2 helices of the pore was used. This strategy was recently used to uncover the putative binding site of another photochromic ligand, Glyght, to GlyRs .trans/P and cis/P results are discussed in the main text. The results of the corresponding M conformers are almost identical (data not shown) and, in the case of the cis isomer, the contribution of the M conformer is expected to be minor, as it is 3.5 kcal/mol less stable than cis/P. We used two different approaches to pinpoint the exact binding site of Azo-NZ1 in the M2 ion channel pore. First, we analyzed the number density of the sulfonate group of the ligand poses. Previous studies have successfully used this type of analysis to identify ligand binding sites in other ion channels (https://github.com/luisico/clustering) and then the representative structure of the most populated cluster(s) was analyzed using the Binana algorithm  were pooled together to carry out this analysis, to have more sampling. All the images of the modeling section were generated with either the UCSF Chimera package  GlyRs expressed in CHO cells. The ECμm n = 6;B, which μm n = 6;.m) with subsaturating concentration of glycine (20 μm) resulted in a decrease of current amplitude by 34 ± 2% of control .Analysis of concentration dependencies revealed that ECμm n = 6;B. As thim) in trans-conformation, to 9 ± 5% , while currents mediated by homomeric α2M GlyRs were inhibited to 23 ± 3%  in trans-state provoked a slight decrease of the open probability (NPo) of the channels (from 0.013 ± 0.002 to 0.011 ± 0.002), which was strengthened by UV illumination  in trans-state induced a prominent decrease of the open probability of channels, which was accompanied by an increase in the flickering of channels openings. UV illumination caused restoration of NPo and even its transient increase, particularly, in the case of application of Azo-NZ1 at 50 μm concentration. We have estimated NPo of α2 GlyR channels in all conditions: in control, it was 0.64 ± 0.09; on co-application of glycine with 20 μm Azo-NZ1, it decreased to 0.19 ± 0.02; UV light restored NPo almost to the control level, to 0.47 ± 0.07. Application of Azo-NZ1 in higher concentration (50 μm) decreased NPo to 0.18 ± 0.06, while UV light increased it to 1.14 ± 0.3  completely abolished eIPSCs, confirming the glycinergic nature of the currents (The effect of Azo-NZ1 on the amplitude of evoked glycinergic IPSCs (eIPSC) was analyzed in the presence of glutamate receptor and GABA% n = 8; A,B. Strycurrents C.trans-Azo-NZ1 concentrations on glycinergic eIPSCs showed that the amplitude of currents decreased with elevation of the doses. However, the inhibition did not exceed 50% even at high concentrations of the compound (mtrans-Azo-NZ1 (n = 6).Analysis of the effect of different compound C,D. Thustrans-Azo-NZ1 . This effect was abolished by UV illumination (It is well established that the amino acid sequences of ion pore-forming transmembrane domains (TMDs) of α1 and α2 subunits differ by one amino acid, which results in subunit-specific action of such inhibitory compounds as NFA and cyanotriphenylborate CTB; . To clarmination B–D, simimination .m) decreased NPo of α1H G254A channels from 0.14 ± 0.019 to 0.022 ± 0.002, while UV light induced elevation of channels activity, and NPo increased 10-fold to 0.26 ± 0.025  and heteromeric α2M/βM . All receptors were modelled in the open channel state. In view of the importance of the 2’ pore-lining amino acids indicated by the experimental data , the doc10.1523/ENEURO.0294-20.2020.f7-1Extended Data Figure 7-1trans-Azo-NZ1 bound in the transmembrane part of the pore of α1, α1G254A, and α2/β GlyRs. Regions of continuous density correspond to higher sulfonate occupancy and indicate tighter sulfonate binding. Each contour line corresponds to one particle/nm3, where “particle” refers to the sulfonate group in a given binding pose and is used to follow Azo-NZ1 binding. Longitudinal view of the pore: the front subunit is not displayed to reveal the interior of the pore. The S/T16’ and G/A/P2’ regions are marked with dashed lines. A higher percentage of binding poses is observed in these two regions; the percentage of poses (between parentheses) where the sulfonate is placed in the 16’ region (up configuration) is 40%, 47%, and 34%, whereas 21%, 21%, and 26% of binding poses placed the sulfonate in the 2’ region (down configuration) for α1, α1G254A, and α2/β GlyRs, respectively. Download Figure 7-1, TIF file.Density map of the sulfonate group of 10.1523/ENEURO.0294-20.2020.f7-2Extended Data Figure 7-2trans-Azo-NZ1 in the “up” binding pose for (A) α1, (B) α1G254A, and (C) α2/β GlyRs. A longitudinal view of the pore is shown, with the pore lining residues represented as ball-and-sticks and their carbon atoms colored in gray. Trans-Azo-NZ1 is represented as sticks with carbon atoms in orange. Nitrogen and oxygen atoms are colored in blue and red, respectively. Hydrogen bonds are marked with red dashed lines and hydrophobic interactions are represented as yellow surfaces. Download Figure 7-2, TIF file.Interactions of 10.1523/ENEURO.0294-20.2020.f7-3Extended Data Figure 7-3trans-Azo-NZ1 in the “down” binding pose for (A) α1, (B) α1G254A, and (C) α2/β GlyRs. Representations and color code are the same as in Extended Data Figure 7-3, TIF file.Interactions of 10.1523/ENEURO.0294-20.2020.f7-4Extended Data Figure 7-4cis-Azo-NZ1 bound in the transmembrane part of the pore for (A) α1, (B) α1G254A, and (C) α2/β GlyR’s. A longitudinal view of the pore is shown; the front subunit is not displayed to reveal the interior of the pore. The T/S13’ and G/A/P2’ regions are marked with dashed lines. A higher percentage of binding poses is observed in the 13’ region for the first two receptors, while for the heteromeric α2/β GlyR binding poses are more dispersed all over the 16’–2’ region. The percentage of binding poses (between parentheses) where the sulfonate is placed in the 13’ region is 60%, 60%, and 65% for α1, α1G254A, and α2/β GlyR’s, respectively. Only the the heteromeric α2/β GlyR shows a significant percentage of poses with the sulfonate bound at 2’ (down configuration), i.e., 34%. Download Figure 7-4, TIF file.Density map of the sulfonate group of 10.1523/ENEURO.0294-20.2020.f7-5Extended Data Figure 7-5cis-Azo-NZ1 in the “up” binding pose for (A) α1, (B) α1G254A, and (C) α2/β GlyRs. A longitudinal view of the pore is shown, with the pore lining residues represented as ball-and-sticks and their carbon atoms are colored in grey. Cis-Azo-NZ1 is represented as sticks with carbon atoms in purple. Nitrogen and oxygen atoms are colored in blue and red, respectively. Hydrogen bonds are marked with red dashed lines and hydrophobic interactions are represented as yellow surfaces. Download Figure 7-5, TIF file.Interactions of 10.1523/ENEURO.0294-20.2020.f7-6Extended Data Figure 7-6cis-Azo-NZ1 in the “down” binding pose for (A) α1, (B) α1G254A, and (C) α2/β GlyRs. Representations and color code are the same as in Extended Data Figure 7-4A), and thus, we expect that the probability of having cis-Azo-NZ1 bound in the down configuration for this receptor is very low. Download Figure 7-6, TIF file.Interactions of 10.1523/ENEURO.0294-20.2020.f7-7Extended Data Figure 7-7A, B) α1 and  α2 GlyRs. Regions of continuous density correspond to higher sulfonate occupancy and indicate tighter sulfonate binding. Each contour line corresponds to 0.0006 particle/nm3, where “particle” refers to the sulfonate group in a given binding pose and is used to follow the putative interaction regions of Azo-NZ1. Longitudinal view of the receptor: the front subunit is not displayed for the sake of clarity. The pore region (between positions S/T16’ and G/A2’), as well as the interface region between the ECD-TMD, are marked with dashed lines. The residue at position 2’ is displayed as black spheres. Trans-Azo-NZ1 binds preferentially inside the 16’–2’ region for both GlyRs . Instead, cis-Azo-NZ1 shows two possible interaction sites, either inside the pore or at the ECD-TMD interface. The relative percentage of poses in the two regions varies significantly between the two GlyR types. For α1 GlyR, cis-Azo-NZ1 has a higher probability to interact with the ECD-TMD interface (35%) than with the pore (28%). In contrast, for α2 GlyR, the sulfonate density in the interface region is more dispersed, and the associated probability is lower (28%) than the one corresponding to binding in the pore (46%). Download Figure 7-7, TIF file.Density map of the sulfonate group of Azo-NZ1 resulting from the blind docking to . Moreover, the number of interactions with the receptor is decreased for cis-Azo-NZ1 compared with the trans isomer , indicating weaker binding. Hence, we will focus on the trans-Azo-NZ1 docking poses  appear to have a stronger contribution to the binding of trans-Azo-NZ1 in GlyRs than in GABARs.Out of the two aforementioned orientations of ng poses B where tp of T6’ B. The leeceptors  is importrans-Azo-NZ1 for α1 GlyR. The Ala2’ residue in α2 GlyR is placing five methyl groups, one from each of the five M2 helixes, in the narrowest part of the pore, compared with five hydrogen atoms for the Gly2’ ring in α1 GlyR. This decreases the diameter of the pore at the 2’ position by at least 1 Å in α2 GlyR, and thus makes it easier for the sulfonate group to clog the pore. Interestingly, the negatively charged sulfonate group of trans-Azo-NZ1 (down pose) occupies the same position as the chloride ion trapped in x-ray structures of GABAAR  by Pro (β) reduces the diameter of the pore at this position by ∼2 Å, it still allows sulfonate binding.The results for the heteromeric α2/β GlyR are similar to the G254A α1 mutant GlyR, for both trans-Azo-NZ1 on α2 GlyRs compared with α1 GlyR and the relief of the pore blocking-mediated inhibition of α2 GlyRs on cis isomerization. However, α1 GlyRs show an unexpected behavior, in that they are weakly inhibited by trans-Azo-NZ1 but inhibition increases slightly on UV irradiation  and TMD (Extended Data cis-Azo-NZ1 has a slightly higher probability to interact with ECD-TMD region (35%) than the pore (28%) in α1 GlyR, whereas in α2 GlyR it mostly binds in the pore (46%). Altogether, we surmise that cis-Azo-NZ1 might be able to inhibit α1 GlyR by binding to the ECD-TMD interface.The docking results obtained rationalize the stronger blocking effect of adiation . Considetrans-configuration, Azo-NZ1 caused strong inhibition of glycine-induced currents on cells expressing homomeric α2 GlyRs, while it was not able to modulate α1 GlyRs responses at saturating concentrations of glycine; (2) in cis-configuration, induced by UV illumination, Azo-NZ1 completely lost the ability to inhibit α2 GlyRs.This study presents the experimental and modeling analysis of the action of Azo-NZ1 on α1 and α2 subunits of GlyR heterologously expressed in cultured cells and also on glycinergic synaptic currents in whole-cell recordings from hypoglossal motoneurons of brainstem slices. Our observations demonstrate that Azo-NZ1 is an efficient and subunit-specific photoswitchable inhibitor of GlyRs with the following main differences between its action on α1 and α2 GlyR subunits: (1) in 50 = 9 μm) is much higher than the one of GlyRα1 and GABAARs  situated in 2’ position of the pore-forming TM2 domain of α1 receptors was sufficient to switch the mode of Azo-NZ1 action, which became similar to those observed for α2 GlyRs B,C. An ay effect .trans-Azo-NZ1 is because of two essential features of the photochromic molecule. First, the negative charge of the sulfonate group, which mimics that of the chloride ion. Second, its longitude (14.1 Å) which allows Azo-NZ1 to form hydrogen bonds between the amide group of the nitrazepam core and T13’ and place the sulfonate in the 2’ region, thus spanning the whole pore  concentration of Azo-NZ1 produced a strong transient increase of the open probability of α2 channels. Apparently Azo-NZ1 while being switched from trans- to cis-states is able to induce elevation of the open probability of α2 channels. This effect was completely absent at application of Azo-NZ1 to α1 GlyRs, confirming the mismatch of their interaction sites. The molecular mechanism of this transient potentiation of α2 GlyRs needs to be clarified.Analysis of single-channels recordings of α2 GlyRs activity on application of Azo-NZ1 disclosed another very interesting aspect of their interaction. UV illumination at high (50 μtrans-Azo-NZ1 inhibited the amplitude of glycinergic eIPSCs only by ∼50% (m (trans-Azo-NZ1 (Our electrophysiological analysis in the mouse hypoglossal motoneuron, proved Azo-NZ1 to be a photoswitchable modulator of the glycinergic eIPSCs and further support the suggestion that the photochrome is a selective modulator of synaptic transmission mediated by α2 GlyRs. We show that at the high concentrations causing complete block of α2-containing GlyRs D, trans- by ∼50% C,D. This by ∼50% . At the  by ∼50% . It is a ∼50% (m . At thes-Azo-NZ1 B and theIn conclusion, we present here the detailed analysis of the novel photochromic compound Azo-NZ1 action on different GlyRs expressed heterologously and on synaptic glycinergic currents in brain slices. Experimental observations, supported by modeling analysis, demonstrate that Azo-NZ1 is a potent TM2 pore blocker of α2 GlyRs. This subunit-specific light-controlled modulator of GlyRs may be a useful tool for controlling neuronal activity in spinal cord, brainstem, cerebellum and other parts of nervous system with high expression of glycinergic synapses.


Existing research has indicated that dangerous objects may conflict with an individual’s prepared motor actions and thus slow responses. This phenomenon is called the motor interference effect from dangerous objects. However, its origin remains arguable. The current study aimed to preclude an alternative origin and to investigate whether the efficiency of processing a prepared response toward a dangerous object could benefit from increasing the perceptual salience of the object by painting the object red. The design used a shape categorization task to emphasize the dangerous elements of target objects and manipulated target color (gray versus red), target dangerousness (safe versus dangerous) and prime-target congruency (congruent versus incongruent). The null effect of N2 amplitudes between the dangerous and safe conditions precluded the alternative origin and suggested that the motor interference effect did not originate from response inhibition. Furthermore, the results indicated a modulation effect of the motor interference effect in different colors. The classic motor interference effect was observed in the gray target condition, but it diminished in the red target condition. The underlying cognitive processes were reflected in ERPs. More positive P2 and frontal P3 amplitudes were identified in the red target condition than in the gray target condition, which indicated that deeper feature detection was assigned to and more attentional resources were automatically recruited for the red targets than for the gray targets. Analysis of the parietal P3 amplitudes identified a similar result pattern as the mean RTs. A more positive P3 amplitude was identified in the dangerous condition than in the safe condition when the targets were painted gray. In contrast, the P3 amplitudes were identical between the dangerous condition and the safe condition when the targets were painted red. The results indicated that the increased attentional resources facilitated the evaluation of red target dangerousness and thus accelerated reactions to the red dangerous targets; the reaction speeds to those targets were close to those for the reaction speeds to the red safe targets. Detailed processes that underline these components are discussed. In human life, we constantly interact with objects. We can touch or grasp objects voluntarily, but objects also imply potential action information that might evoke motor responses  technique adopted by We expected that if the shape categorization task forced participants to attend to the periphery of the targets in early processing, a significant difference between the dangerous and safe conditions should emerge in the frontal P2 component. Specifically, a more positive P2 amplitude should emerge in the dangerous than the safe condition at the frontal area. For the N2 component, if the dangerous objects directly inhibited the prepared responses elicited by the prime, a more negative N2 amplitude should emerge in the dangerous condition than in the safe condition, or if processing of the dangerous objects only recruited more attentional resources for evaluation without inhibiting the prepared response, as suggested by A priori sample size estimation with a large effect size (f = 0.4) and 0.95 statistical power was conducted by G∗Power to determine the sample size required for the experiment  and a significant difference between the gray and red conditions . The results indicated that the rectangular saw blade and the circular saw blade were assessed as more dangerous than the smooth rectangle and circle. Moreover, the red targets were assessed as more dangerous than the gray targets.The experimental stimuli included primes and targets. The primes  were identical to those in The experiment was run with E-Prime software  on a standard PC linked to a 17-inch CRT monitor (60-Hz refresh rate). Electroencephalogram (EEG) data were recorded by a NeuroScan system . A Neuroscan Synamp 2 amplifier with a 64 Ag/AgCl electrode cap mounted according to the extended international 10–20 system was used to continuously record EEG data (sampling rate at 500 Hz).The participants were seated in a dimly lit room with a computer screen placed 60 cm in front of their eyes, and they were instructed to maintain their central eye fixation throughout the experiment. The trial procedure of the experiment is presented in The design manipulated the target color (red versus gray), the target dangerousness (safe versus dangerous) and the prime-target congruency (congruent versus incongruent). The experiment consisted of 480 trials, which included 2 types of target color (red versus gray) × 2 types of target dangerousness (safe versus dangerous) × 2 types of prime-target congruency (congruent versus incongruent) × 60 repetitions. Furthermore, the handedness of the prime (left versus right) and the target shape (rectangular versus round) were assigned in equal proportions in each experimental condition. Prior to the formal experiment, a 16-trial practice was conducted, and the formal experiment did not begin until the participant achieved 85% accuracy in the practice phase. The participants were given a break of at least 2 min after every 80 trials and were encouraged to take longer breaks when necessary.In recording EEG, the signals were bandpass-filtered at 0.05–100 Hz and referenced to the tip of the nose. The electrode impedance was maintained at less than 5 kΩ throughout the experiment. The EEG data were preprocessed using the EEGLAB toolbox  accordinBefore grand-averaging, the artifact-free data were resegmented and initiated from the 200 ms before and the 800 ms after the target onset and were referenced to the baseline . Then, the extracted average waveforms for each participant and condition were used to calculate the grand-average waveforms.ps > 0.32), but the error rates deviated from normality (ps < 0.003). Accordingly, the mean RTs and logarithmic correct rates  were analyzed by three-way repeated-measures ANOVAs . F-ratio were corrected using the Greenhouse–Geisser method, and multiple comparisons were adjusted by the Bonferroni method in the analyses. The effect sizes are presented as partial eta-squared values  and N2 (the most negative peak amplitude between 200 and 300 ms) components and the mean amplitudes for the frontal P3 (the mean amplitude between 300 and 400 ms) and parietal P3 (the mean amplitude between 260 and 390 ms) components. Kolmogorov–Smirnov tests of normality were also performed on the amplitudes at each condition. The results indicated that the distributions of the amplitudes did not deviate from normality  = 10.52, p = 0.004, post hoc analysis indicated that the mean RTs for the incongruent condition (408 ± 63 ms) were longer than those for the congruent condition (400 ± 61 ms). Moreover, a significant two-way interaction between the target color and the target dangerousness  in the gray color condition. In contrast, the mean RTs for the dangerous condition (398 ± 61 ms) did not significantly differ from those for the safe condition  in the red color condition. However, analysis of the log-transformed correct rates identified non-significant main effects and interactions . The results of the RTs were more convincing because the mean errors for each condition were less than 2.27%, which may have caused a ceiling effect.For the RTs  = 5.77, p = 0.03, post hoc analysis indicated that the P2 amplitude for the red target condition (1.43 ± 0.64 μV) was more positive than that for the gray target condition (0.99 ± 0.56 μV). Moreover, a significant main effect of target dangerousness was identified  in the gray color condition. In contrast, the frontal P3 amplitude for the dangerous condition (0.36 ± 4.76 μV) did not significantly differ from that for the safe condition  in the red color condition. Additionally, the results identified a significant two-way interaction between target dangerousness and prime-target congruency  and a non-significant difference between the dangerous and safe conditions in the incongruent condition .Analysis of the frontal P3 amplitudes revealed a significant main effect of color  in the gray target condition. In contrast, the parietal P3 amplitude for the dangerous condition (2.84 ± 4.88 μV) did not significantly differ from that for the safe condition  in the red target condition.Analysis of the parietal P3 amplitudes revealed a significant main effect of the channel distribution . However, the mean RT for the dangerous targets was slightly shorter (nearly significant) in the red color condition than in the gray color condition . Although the result of the paired t-test was marginally significant, the results supported the first possibility and suggested that the RTs were slightly accelerated in the dangerous target condition when the targets were red compared to when they were gray. Additionally, the behavioral results identified a classic congruency effect, which was evidenced by longer RTs for the incongruent condition than for the congruent condition. The results suggested that processing the left- or right-hand primes influenced the target responses. This finding of a significant congruency effect contradicts an argument that target processing is not under motor readiness status because the left- or right-hand prime, which aims to activate response readiness, is not related to the target response; thus, participants might ignore the prime. If this argument holds true, the target responses could not be accelerated in the congruent condition compared to the incongruent condition. Obviously, the significant congruency effect contradicts this argument and suggests that target responses are accelerated in the congruent condition compared to the incongruent condition, although the response rules are not related to the primes.The behavioral results identified a classic motor interference effect, which was evidenced by a longer RT for the dangerous target condition than for the safe target condition when the targets were painted gray. In contrast, the motor interference effect diminished in the red target condition, which was evidenced by a non-significant RT difference between the safe and dangerous conditions. The behavioral results support the hypothesis and suggest that relative to painting the target gray, painting the target red diminishes the RT difference in responding to safe and dangerous targets. However, two reasons might explain the diminished RT difference: (1) the RTs were accelerated in the dangerous target condition when the targets were red compared to when they were gray, or (2) the RTs were delayed in the safe target condition when the targets were red compared to when they were gray. To distinguish between these two possibilities, differences in the mean RTs between the red and gray conditions were separately tested in the safe and dangerous conditions via paired The ERP results further clarified the underlying cognitive processes. Analysis of the P2 component identified a more positive P2 amplitude for the dangerous condition than for the safe condition at the frontal area. Moreover, a more positive frontal P2 amplitude was identified for the red target condition than for the gray target condition. As stated in the introduction, the P2 component reflects the processing of object identification , with thThe frontal P3  is a stimulus-driven component that originates from frontal attention mechanisms . This coThe parietal P3  component maximally emerged in central-parietal areas , and itsIn summary, the current study first confirmed that the origin of the motor interference effect does not originate from direct response inhibition from dangerous objects, as indicated by a null effect between the dangerous and safe conditions in the N2 amplitudes. Furthermore, this study investigated whether processing a prepared response toward a dangerous object could benefit from increasing the perceptual salience of the object by painting the dangerous object red. Both the behavioral and ERP results indicated that the participants were sensitive to the color of the targets and that target dangerousness was differentially processed in the gray and red target conditions. In the gray target condition, a classic motor interference effect, as indicated by faster responses and larger P3 amplitudes in the dangerous condition than in the safe condition, was identified. In the red target condition, deeper feature detection was assigned to the red targets than to the gray targets, as reflected by the P2 amplitudes in early processing. Thus, more attentional resources were automatically recruited for the red targets than for the gray targets, which was evidenced by the frontal P3 amplitudes. The pre-recruited attentional resources facilitated the evaluation of dangerousness and thus accelerated reactions to the red dangerous targets, reaching reaction speeds similar to those of the red safe targets. Accordingly, RTs for the red dangerous condition were identical to those for the red safe condition. Moreover, sufficient attentional resources were recruited to evaluate the dangerousness of the safe and dangerous targets when they were painted red. Therefore, parietal P3 amplitudes for the red dangerous target condition were identical to those for the red safe target condition. The practical value of this study is that it might provide a reference for safety management by showing that increasing the perceptual salience of a dangerous object is useful for increasing efficiency in processing a prepared response toward that dangerous object. For example, painting the body of a machine in gray and painting dangerous elements of the machine in red could help operators quickly attend to dangerous elements and react to them faster.The datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by School of Public Management ethics committee. The patients/participants provided their written informed consent to participate in this study.PL designed the experiment, and created the stimulus materials and analyzed the data. GC and PL collected the data. All authors contributed to the writing process and agreed on the final text.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Gynecologic malignancies, including ovarian cancer, endometrial cancer, and cervical cancer, affect hundreds of thousands of women worldwide every year. Wnt signaling, specifically Wnt/β-catenin signaling, has been found to play an essential role in many oncogenic processes in gynecologic malignancies, including tumorigenesis, metastasis, recurrence, and chemotherapy resistance. As such, the Wnt/β-catenin signaling pathway has the potential to be a target for effective treatment, improving patient outcomes. In this review, we discuss the evidence supporting the importance of the Wnt signaling pathways in the development, progression, and treatment of gynecologic malignancies.  Wnt signaling is one of the most studied signaling pathways and plays a critical role in multiple biological processes, including cell differentiation, proliferation, survival, and migration. There is significant evidence that Wnt signaling also plays a pivotal role in tumorigenesis and other oncogenic processes ,2,3. TheMore recently, there is growing research evaluating the role of Wnt signaling in gynecologic malignancies. Specifically, Wnt signaling promotes metastasis and therapy resistance in ovarian cancer, plays a crucial role in tumorigenesis and recurrence in endometrial cancer, and participates in human papillomavirus (HPV) -related tumorigenesis and metastasis in cervical cancer ,23,24,25There are three well-defined Wnt signaling pathways separated into two categories: canonical (β-catenin-dependent) and non-canonical (β-catenin-independent). The canonical pathway is a fundamental growth control pathway known to have important roles in many fields, including cancer. The non-canonical pathways mediate cell polarity and regulate levels of intracellular calcium .There are 19 mammalian protein-encoding Wnt genes, many with unique functions. Wnt proteins undergo several post-translational modifications, including palmitoleoylation, which is accomplished by the palmitoyl transferase, Porcupine (PORCN). These modifications are critical for Wnt extracellular signaling, rendering the proteins hydrophobic so that they may be secreted from the cell. The transmembrane protein Wntless then traffics the Wnt proteins to the plasma membrane where they are secreted to signal targeted cells nearby. Wnt signaling then continues at the target cell membrane of the targeted cell where Wnt proteins bind to the Frizzled (FZD) transmembrane receptor and recruit the Dishevelled protein (DVL). There are 10 FZD receptors and at least 5 co-receptors . The specific FZD and co-receptors engaged by the Wnt ligand tailor the initiation of intracellular signaling and determine which Wnt signaling pathway, canonical or non-canonical, proceeds ,34,35. The non-canonical Wnt signaling pathways are introduced here. However, as the majority of Wnt signaling aberrations in gynecologic malignancies are in the canonical pathway, the majority of this review will focus on the canonical Wnt signaling pathway. There are six main components of the Planar Cell Polarity (PCP) pathway known as the core module. These components consist of three transmembrane proteins  and three cytoplasmic proteins , and Diversin). These components ultimately activate the small Rho GTPase effector molecules, c-Jun N-terminal kinase (JNK) and Nemo-like kinase (NLK). The PCP Wnt pathway components are arranged asymmetrically within the cell in order to influence cell polarity . DetermiThe calcium-dependent Wnt pathway controls gene expression by modulating intracellular levels of calcium . This inAt the initiation of the intracellular canonical Wnt signaling pathway , Wnt proteins bind to FZD as part of a larger receptor complex at the cell membrane. LGR4, LGR5, and LGR6 are members of the 7-transmembrane receptor family and are able to bind R-spondins (RSPO) with high affinity in order to enhance the Wnt signal when there is a low dose of Wnt ligand . The recThe Wnt/β-catenin signaling pathway has multiple identified regulatory mechanisms in addition to phosphorylation and ubiquitination of β-catenin by the degradation complex, as previously described . NegativCTNNB1 gene (encodes for the β-catenin protein) alter the N-terminus of β-catenin and prevent its phosphorylation and degradation by the destruction complex. Concerning gynecologic malignancies, CTNNB1 mutations are detected in uterine endometrial, ovarian endometrioid, and ovarian clear cell carcinomas [Numerous mechanisms of Wnt/β-catenin hyperactivation have been described. Overexpression of Wnt ligands results in increased Wnt signaling . Intracercinomas ,47,48. Hrcinomas ,49,50.Epithelial ovarian cancer (EOC) is the 5th leading cause of cancer-related death in women in the United States . High grTP53 and MYC, [Wnt/β-catenin signaling plays a role in HGSOC tumorigenesis, metastasis, and therapy resistance. According to The Cancer Genome Atlas (TCGA), while mutations in the pathway are rare, gene amplification or deletions of Wnt signaling components  are detand MYC, . For insBoth canonical and non-canonical Wnt signaling can promote ovarian cancer cell survival in specific contexts. Wnt/β-catenin signaling promotes survival in HGSOC cells and its inhibition leads to impaired proliferation and migration . For exaThe regulation of canonical and non-canonical Wnt signaling is complex and dysregulation resulting in hyperactivation or loss of different components of the signaling pathways can ultimately lead to tumorigenesis or disease progression. For example, activation of non-canonical Wnt signaling by overexpression of WNT5A  in ascites fluid promotes metastatic stem-cell like behavior in HGSOC cells and results in worse survival . Our groTP53-mutation lesion in the fimbriated end of the FT, then causes cancer cells to accumulate there and subsequently exfoliate, resist anoikis, and colonize the peritoneal cavity. This metastatic process appears to be mostly independent of the vasculature and lymphatics [As previously mentioned, HGSOC is usually diagnosed at a late stage in which the tumor has metastasized to multiple sites. Most HGSOC arise via a non-classical metastatic process, which starts in a mphatics . The epithelial to mesenchymal transition (EMT) is the process by which epithelial cells transform into a more motile, invasive mesenchymal phenotype. It conveys resistance to anoikis in cancer cells, thus promoting cancer cell migration and peritoneal metastasis in HGSOC . Wnt/β-cWNT5A gene results in significantly reduced peritoneal metastasis [Peritoneal metastasis of HGSOC is also promoted by WNT5A, which is abundant in ascites ,62. As ptastasis . Wnt/β-catenin signaling is involved in HGSOC chemotherapy resistance ,12,13. LAnother essential therapy in the management of HGSOC is PARP inhibitors (PARPi), which are approved for upfront maintenance therapy in all advanced cases ,66,67,68Tumor immune response plays a significant role in patient outcomes and can affect possible treatment strategies. Immunologically “hot” tumors refer to tumors that have immune cell infiltration, while “cold” tumors lack this immune response . SpecifiCD274 (PD-L1) [Immune checkpoint blockade (ICB) strategies  have conveyed limited benefit in patients with HGSOC compared to those with melanoma or lung cancer, but Wnt/β-catenin signaling may serve as a targetable pathway to improve response to ICB. For instance, in triple-negative breast cancer, Wnt/β-catenin signaling directly promotes the expression of  (PD-L1) . While fBeyond ICB, recent evidence suggests that the Wnt inhibitor DKK1 may serve as an immunotherapeutic target in ovarian cancer . OverexpCTNNB1 mutations [CTNNB1 mutational analysis of 149 ovarian cancer samples detected 16% of endometrioid tumors harbored activating CTNNB1 mutations [CTNNB1 mutations, often called hotspot mutations, are focused around serine and threonine residues in exon 3, which encode known sites on β-catenin that are phosphorylated by GSK-3β. The most commonly mutated residues are Ser33 and Ser37, in which serine is changed to cystine, phenylalanine or tyrosine [MYC, APC, and CREBBP, promote cell malignant transformation in ovarian endometrioid carcinoma [While mutations in the Wnt/β-catenin signaling pathway are rare in HGSOC, they have been observed in other histotypes of ovarian cancer, namely endometrioid and mucinous carcinomas . In muciutations . A CTNNButations . The CTNtyrosine ,83,84,85arcinoma . Dapper1arcinoma , and ovearcinoma . All of Endometrial cancer (EC) is the most common gynecologic malignancy in the United States and is one of the only cancers with an increasing incidence and mortality ,88,89. CCTNNB1 exon 3 mutations, as well as microsatellite stability and overall low mutation rates. Following TCGA, multiple studies independently reproduced the molecular classification of EC into the same four distinct subgroups with the same clinical outcomes, emphasizing the prognostic implications of this molecular classification system [One of the first and most comprehensive studies to molecularly classify EC was TCGA, which evaluated 373 cases of EC using whole-genome sequencing, exome sequencing, microsatellite instability assays, copy-number analyses, and DNA methylation testing . TCGA idn system ,92,93,94CTNNB1 gene occur in approximately 20–25% of endometrioid EC [CTNNB1-mutant EC have a low overall genomic mutational rate, indicating that the CTNNB1 mutations have autonomous oncogenic relevance [CTNNB1 mutations have been associated with low-risk histopathologic features, including low-grade histology, lack of lymph node metastasis, absence of LVSI and lower rates of deep myometrial invasion [CTNNB1 mutations are also associated with younger age at diagnosis, which is also considered a low-risk factor in current risk-stratification [Exon 3 mutation in the rioid EC ,95,96,97elevance ,83. Amoninvasion ,94. CTNNfication ,15,98.CTNNB1-mutant EC has poorer clinical outcomes [CTNNB1 mutations were associated with decreased recurrence-free survival in low grade, early-stage EC [CTNNB1 mutations occurred at significantly higher rates in the recurrent EC [CTNNB1-mutant low-grade EC exhibits upregulation of the Wnt/β-catenin pathway and poorer overall survival [CTNNB1 mutations was also demonstrated in a proteogenomic analysis of 95 EC by Yongchao et al. [CTNNB1 mutations need to be further evaluated as a marker for risk-stratification among low-risk EC. Despite the association with histopathologic and clinical features considered to be low-risk in the current EC risk-stratification system, outcomes ,14,15,98rrent EC . Liu et o et al. . Based oCTNNB1 mutations result in the accumulation of the β-catenin protein, and subsequent translocation into the nucleus and increased transcriptional activity [CTNNB1 mutations and can distinguish CTNNB1-mutant EC from wildtype with a sensitivity of 91% and 85% and a specificity of 89% and 100%, respectively [CTNNB1 mutations in EC [As previously described, activity ,85. Ιmmuectively ,99. IHC ns in EC ,99. Wnt/β-catenin signaling is involved in both the regulation of the normal endometrium and the aberrant development of endometrial hyperplasia or malignancy ,95,100. Wnt/β-catenin signaling is present in the endometrium and is regulated in the same cyclical fashion by estradiol (E2) and progesterone in both patient endometrium samples and EC cell lines . SpecifiCorrect regulation of Wnt/β-catenin signaling in this cyclical fashion is required for normal endometrial function ,95,100, Other components of the Wnt/β-catenin signaling pathway have been implicated in the EC tumorigenesis. WNT7A is a Wnt/β-catenin signaling protein and is overexpressed in EC compared to normal endometrium and benign endometrial lesions. This overexpression is associated with worse clinical outcomes  . These fCervical cancer (CC) is the most common gynecologic malignancy in women worldwide and the third most common in the United States ,102,103.Wnt/β-catenin signaling is hyperactivated in CC and plays an important role in HPV-dependent tumorigenesis . WNT5A , as wellβ-catenin is differentially expressed between CC and normal cervix, with multiple studies showing β-catenin expression in the nucleus and cytoplasm of CC cells compared to at the cell membrane in normal cervical tissue ,111, sugCTNNB1 promoter and trans-activating expression of CTNNB1 [GSK3B, preventing its expression and reducing the phosphorylation and proteasomal degradation of β-catenin [CTNNB1 [In CC, there is crosstalk between Wnt/β-catenin and other oncogenic signaling pathways. Sal-like 4 (SALL4) is overexpressed in CC and promotes cell proliferation and tumor formation . SALL4 if CTNNB1 . Treatmef CTNNB1 . KIF18B f CTNNB1 . Long nof CTNNB1 . Treatmef CTNNB1 . DAX1 prf CTNNB1  and tran-catenin . Express-catenin  and rest [CTNNB1 . All of CC is a disease that typically spreads by local extension. As such, the majority of CC cases that present with extra-cervical disease have local metastases to other pelvic organs. Distant metastases at the time of diagnosis are uncommon in CC (approximately 2%) and confer a poor prognosis with a 5-year survival rate of 16.5%. When distant metastases do occur, common sites include lung, bones, liver, and brain ,120. Wnt/β-catenin signaling has been found to play a role in the migration and invasion of CC. Both WNT5A and WNT11 promote CC cell proliferation and invasion, and both are also associated with CC metastasis and recurrence ,22,121. As described, Wnt/β-catenin signaling plays a pivotal role in tumorigenesis, metastasis, recurrence, and chemoresistance of EOC, EC, and CC, suggesting that it may serve as a targetable pathway in these cancers. Several inhibitors have been developed that different target nodes of this pathway .PORCN is essential for Wnt ligand secretion. Several inhibitors that target PORCN in the endoplasmic reticulum prevent the palmitoylation of Wnt proteins, which in turn prevents their secretion ,35,122. CGX1321, another PORCN inhibitor, inhibits both canonical and non-canonical Wnt signaling pathways . A phaseWnt signaling may also be inhibited by direct binding to and inhibition of Wnt ligands and FZD receptors. Ipafricept  is a recombinant fusion protein that competes with the FZD8 receptor and binds directly to Wnt ligands ,126. IPADVL is important for transducing Wnt signals by recruiting components of the destruction complex to the cell membrane ,131. In in vivo, and these effects are enhanced when pyrvinium is combined with paclitaxel. Pyrvinium has these effects on EOC by decreasing β-catenin levels and suppressing β-catenin-mediated transcription; when β-catenin is stabilized or overexpressed, EOC cells are no longer impacted by pyrvinium [Stabilizing the β-catenin destruction complex can lead to increased ubiquitination of β-catenin, making it an attractive drug target. Pyrvinium, an FDA approved drug, binds all CK1 family members in vitro, selectively potentiating CK1α kinase activity . Colon cyrvinium . Pyrvinium pamoate (Pyr. Pam.) is also an FDA approved drug that stabilizes GSK-3β potentially through inhibition of Akt/PI3K, leading to decreased levels of β-catenin and its downstream targets . Pyr. PaTNKS proteins are PARPs that can regulate the destruction complex . TNKS poThere are a number of co-activators of β-catenin-dependent transcription, including CREB binding protein (CBP) . In PhasSeveral small molecules have been designed that inhibit CDC-like kinase (CLK) activity , therebyAs Wnt signaling is highly conserved and complex, playing an important role in many biological processes, targeting the Wnt signaling pathways carries a risk for significant side effects and toxicities ,143. TheWnt signaling impacts EOC, EC, and CC in various ways, differing between cancer types and disease phases. Although the exact mechanisms are not yet clear, it is evident that Wnt/β-catenin signaling plays a vital role in EOC’s therapy resistance, recurrence in EC, metastasis in EOC and CC, and tumorigenesis for all three cancer types. Studies that have demonstrated Wnt/β-catenin signaling activity in gynecologic malignancies have not only illuminated important behaviors of these cancers but have also shed light on possible targets in the Wnt/β-catenin signaling pathway for future treatments. Targeted therapies against Wnt/β-catenin signaling are beginning to be evaluated in various cancer types, but further research evaluating Wnt/β-catenin signaling inhibitors in gynecologic malignancies is needed. Looking toward the future of inhibiting Wnt signaling in gynecologic malignancies and reducing systemic toxicities, research is needed for improved targeting of drugs to specific tissue. For instance, utilizing antibody–drug conjugates that target specific FZD receptors could increase cancer cell specificity and inhibit tumor progression. Further, given the contribution of Wnt signaling to immune cell response, there is strong rational to evaluate combinatorial immunotherapies and Wnt inhibitors.