Epoxy asphalt concrete (EAC) is a widely used steel bridge deck pavement (SBDP) material. Due to the curing reaction, the EAC-based material needs a long curing period before opening to traffic, which in an inconvenience in the construction of SBDP. This study developed a cold mix high-early-strength (CHES) epoxy asphalt through the design of a compatilizer and curing agent system. The optimum formula of CHES epoxy asphalt was determined through a series of laboratory tests. By comparison of the performances of CHES EAC and some conventional EACs for SBDP, it was found that the developed CHES epoxy asphalt can significantly reduce the curing period, and the pavement performance of CHES EAC is, overall, excellent for application in SBDP. In addition, the sufficient allowable construction duration shows that the CHES EAC has a good construction workability. Epoxy asphalt concrete (EAC) is a thermosetting material with desirable mechanical properties and outstanding durability and has been widely used in the steel bridge deck pavement (SBDP) ,2,3,4. DEpoxy asphalt is usually mixed at a high temperature that is in the range from 110 °C to 130 °C for warm mix EAC, and in the range from 170 °C to 185 °C for hot mix EAC. The high-temperature paving of EAC is harmful to the environment due to the high energy consumption and emission of toxic fumes and can cause temperature effects on the steel bridge deck as well ,10,11. IThe curing reaction can transform the epoxy resin contained in the epoxy asphalt into plastic or rubber by a cross-linking process and subsequently link the molecular chains into a rigid, 3D structure. Meanwhile, the curing reaction is influenced by the temperature and curing time. Therefore, the strength development law of EAC is complicated. Qian et al. developed a curing reaction model to reveal the strength evolution law of EAC . As the The main objective of this study is to develop a cold mix high-early-strength (CHES) epoxy asphalt for SBDP. Through blending the compatilizer and optimizing the curing agent system, a CHES epoxy asphalt was designed and the optimum formula was determined. Meanwhile, the pavement performances of CHES EAC were compared with the conventional EACs in SBDP through a series of laboratory tests, and the feasibility and construction workability of the developed CHES EAC for SBDP was evaluated.Epoxy asphalt is generally comprised of an epoxy resin, curing agent, and base asphalt. E51 is a common bisphenol-A liquid epoxy resin with the epoxy value of 0.53 mol/100 g and was used to synthesize the CHES epoxy asphalt in this research. The selection and proportion of curing agent have a significant influence on the curing temperature, curing rate, and mechanical properties of EAB. Based on a literature review of recent studies on epoxy asphalt in the chemical industry, a modified aliphatic amine curing agent was selected due to the low curing temperature, fast curing rate and good flexibility for the EAB ,19,20. OThe CHES epoxy asphalt was composed of component A and component B, where the former is E51 epoxy resin and component B was prepared through mixing the curing agent, base asphalt and compatilizer. The base asphalt was heated to 120 °C for more than 30 min until it was fully fluidized. Subsequently, the base asphalt and the compatilizer were mixed in a three-necked flask, and the mixture was stirred and refluxed at 120 °C for about 1 h. The mixture was cooled to 40 °C, and the curing agent was then added and stirred for 1 h at ambient temperature to obtain component B. Finally, component A and component B were blended in suitable proportions and the blend was stirred for 5 min at ambient temperature to prepare the CHES epoxy asphalt.The compatibility between asphalt and epoxy resin with different proportion of compatilizer was evaluated by a segregation test. The following test procedure was adopted: (1) heat asphalt at 120 °C for 30 min, (2) mix the compatilizer with the fluidized asphalt, (3) cool the mixture of asphalt and compatilizer to 40 °C, (4) add the epoxy resin to the mixture and stir evenly, (5) pour the mixture into a glass tube with the lubricant coated onto the inner wall, (6) let the glass tube stand for 48 h, (7) freeze in the refrigerator for 4 h and subsequently take out the mixture specimen and (8) measure the softening points of the upper and lower parts of the asphalt-resin specimen. The softening point difference can reflect the compatibility between asphalt and epoxy resin, and the optimum proportion of compatilizer was determined to have a softening point difference of less than 1 °C.The tensile strength and elongation at break are important mechanical indexes for an epoxy asphalt, as they can reflect the strength and deformation properties. The tensile strength and elongation at break of CHES epoxy asphalt was measured through a tensile test, following a standard test procedure in ASTM D638 , as showDue to its unique thermosetting features, the viscosity of epoxy asphalt increases with the curing reaction, and the increasing rate of viscosity has a great influence on the workability and curing period of epoxy asphalt. The viscosity of CHES epoxy asphalt was measured by a Brookfield viscometer , following a standard test procedure described in AASHTO T316 . The CHEAfter the laboratory evaluation of the CHES epoxy asphalt, its effectiveness also needs to be evaluated in the context of asphalt mixture performance. The pavement-related properties of CHES epoxy asphalt mixture were evaluated by the Marshall stability test, following a standard test procedure in AASHTO T245 . Basalt (1)The CHES epoxy asphalt was mixed with aggregates at ambient temperature to form a mixture.(2)The mixture was compacted in a Marshall compactor with 75 blows on each side, and the Marshall specimens were extracted from the mold and kept at the room temperature (25 °C) for different curing periods. In addition, the volume parameters of the molded mixture were measured.(3)The specimens were immersed in the 60 °C constant-temperature water for 0.5 h, and the Marshall stability and flow values were measured subsequently.Since the specimens of CHES epoxy asphalt mixture would gain the strength and stiffness over time due to binder curing, the Marshall stability and flow values of CHES epoxy asphalt mixture after different curing periods were measured, including curing periods of 8, 10, 12, 16, 20 and 24 h. The steps to prepare and test the CHES epoxy asphalt mixture specimens are as follows:The CHES EAC should have adequate high-temperature rutting resistance, low-temperature cracking resistance, and moisture susceptibility for its application to the steel bridge deck. The wheel tracking test and bending beam test were respectively used to evaluate the high-temperature rutting resistance and low-temperature cracking resistance, and the detailed test procedures have been described in the authors’ published literature . The moiThe proportions of epoxy resin, curing agent, base asphalt, and compatilizer have a great influence on the strength, curing period, allowable construction duration, and viscosity properties of epoxy asphalt. The formula of CHES epoxy asphalt was determined based on three principles. Firstly, the compatibility between asphalt and epoxy resin should be evaluated to determine the optimum proportion of compatilizer. Secondly, the CHES epoxy asphalt should have an adequate allowable construction duration, which means the initial curing rate should not be fast. In general, the time for viscosity of epoxy asphalt increasing to 3 Pa·s is used to evaluate the allowable construction duration of epoxy asphalt . ThirdlyThe segregation test results are listed in In the curing agent system of the CHES epoxy asphalt, a modified aliphatic amine curing agent was used as the base curing agent to ensure the mechanical properties of epoxy asphalt, and a synergistic curing agent produced by Jiangsu Zhongyitong New Road Materials Co., Ltd. , was used to shorten the time for curing reaction of the epoxy asphalt. In this study, five types of modified aliphatic amine curing agents (named curing agents A1~A5) were used to prepare epoxy asphalts, and the base curing agent for CHES epoxy asphalt was selected through comparing the properties of different samples. When preparing the epoxy asphalt, the mass proportion of modified aliphatic amine curing agent, E51 epoxy resin, compatilizer, and 70# asphalt was 1:1:0.2:2.2. For each modified aliphatic amine curing agent, 10 groups of epoxy asphalt samples were prepared for testing. One group of samples was used to measure the viscosity variation with time at 25 °C, and the allowable construction duration  was determined. The other samples were used to conduct tensile tests in which the tensile strength and elongation at break of the epoxy asphalts were measured. To determine the fully-cured time of the different epoxy asphalts , the nine groups of samples were cured at 25℃ with different curing times , and the tensile strength varied with the curing time were measured. The test results of rotational viscosity test and tensile test are given in As shown in The synergistic curing agent (named SY curing agent) was used to further shorten the fully cured time of epoxy asphalt, and the test results of epoxy asphalt with different mass proportions of the base curing agent and synergistic curing agent are listed in In this section, the best proportion of epoxy resin, curing agent, and 70# asphalt was determined. Based on the previous studies and for cost reasons ,34, 16 gWith the increase of curing agent, the degree of curing reaction between the epoxy resin and curing agent improves, resulting in an increase of tensile strength but a decrease of elongation at break . However, when the mass ratio of curing agent to epoxy resin exceeds 0.8, the redundant curing agent could reduce the cross-linking degree of the cured product, resulting in a reduction of tensile strength. As the proportion of 70# asphalt increases, the tensile strength of epoxy asphalt decreases, and the toughness increases. However, when the proportion of 70# asphalt exceeds 80%, the curing reaction degree of epoxy asphalt is very low, and the tensile strength is too small. Given that the tensile strength of epoxy asphalt for SBDP should be larger than 2.0 MPa, and the elongation at break should be larger than 100%, some formula schemes in To determine the optimum formula of CHES epoxy asphalt, the epoxy asphalts with Formula III, Formula IV, and Formula VI were used to prepare the EAC and conduct the Marshall stability test. The test results are given in 3. The aggregate gradation of EAC is shown in As of today, the EACs applied to SBDP contain hot mix EAC, warm mix EAC, and cold mix EAC. The purpose of the design of CHES epoxy asphalt in this study is to reduce the curing period for EAC while reaching the ultimate strength. In this section, the strength increase trend of CHES EAC was compared with the conventional hot mix EAC, warm mix EAC, and cold mix EAC. In this study, the hot mix epoxy asphalt tested is the KD-BEP epoxy asphalt imported from Japan, the warm mix epoxy asphalt was a 2910-type epoxy asphalt produced by Jurong Ningwu New Material Co., Ltd.  and the cold mix epoxy asphalt was produced by Jiangsu Zhongyitong New Road Materials Co., Ltd. The basic properties of these epoxy asphalts are listed in Through Marshall stability tests, the strength variations of different EACs with time were measured, and the results are given in The high-temperature rutting resistance, low-temperature cracking resistance and moisture susceptibility of CHES EAC were evaluated and compared with the conventional EACs. The results are given in In the production of CHES EAC, the epoxy asphalt is mixed with the aggregate at ambient temperature. In a normal humid environment, the aggregate is subject to moisture. The moisture will be retained in the CHES EAC during its production, which may affect the performance of CHES EAC. Therefore, the influence of moisture content of the aggregate on the performance of CHES EAC should be studied. In addition, the allowable construction duration is a key construction factor for EAC. The allowable construction duration for CHES EAC at different ambient temperatures should be clear to evaluate the construction workability of CHES EAC.In this study, the aggregates were treated in a humidity chamber to simulate the aggregate in different humid environments. After the treatment process, the moisture content of aggregate was measured, and the aggregates were used to fabricate the CHES EAC. Through the Marshall stability test, the relationship between the aggregates’ moisture content with the performance of CHES EAC was obtained, as shown in In view of the above results and discussions, it is suggested that the moisture content of aggregate for CHES EAC should be less than 1%. If the moisture content of aggregate is high, a drying treatment should be adopted for the aggregate before producing the CHES EAC.The curing reaction begins once the two components of epoxy asphalt are mixed, therefore, the mixture’s strength is increasing during the mixture transportation, and mixture paving process. The allowable construction duration was defined as the time from the completion of mixture production to the initiation of mixture compaction. The strength of CHES EAC before compaction should not be too high to ensure its compaction quality. Therefore, the allowable construction duration of CHES EAC at different ambient temperatures should be evaluated.In this study, the ambient temperatures were selected as 10 °C, 20 °C, 30 °C, and 40 °C. The allowable construction duration was determined through the rotational viscosity test and Marshall stability test. The viscosity variations of CHES epoxy asphalt with time at different temperatures are given in (1)A new CHES epoxy asphalt was developed through blending a compatilizer and optimizing the curing agent system, and the optimum proportion of epoxy resin, curing agent, compatilizer, and 70# asphalt in the CHES epoxy asphalt is 1:0.8:0.2:3.(2)The developed CHES epoxy asphalt can significantly reduce the curing period for EAC, and the curing time for CHES EAC to reach its ultimate strength at 25 °C is about 20 h.(3)The CHES EAC has good high-temperature rutting resistance, low-temperature cracking resistance, and moisture susceptibility, and its pavement performance is, overall, excellent for application in SBDP.(4)The moisture in the aggregate has an adverse effect on the Marshall stability of CHES EAC. It is suggested that the moisture content of aggregate for CHES EAC should be less than 1%. If the moisture content of aggregate is high, a drying treatment should be applied to the aggregate before producing the CHES EAC.(5)The allowable construction duration of CHES EAC at the ambient temperatures of 10 °C and 20 °C exceeds 150 min, and the allowable construction duration at the ambient temperature of 30 °C and 40 °C is about 120 min.This study developed a CHES epoxy asphalt for SBDP. The optimum formula of CHES epoxy asphalt was determined and the performances of different CHES EAC samples were evaluated through a series of laboratory tests. The results of this study can be summarized as follows:


Here, we have analyzed the underlying mechanism. We found that VE-cadherin–α-catenin constitutively recruits the actin adaptor vinculin. However, removal of the vinculin-binding region of α-catenin did not impair the ability of VE-cadherin–α-catenin to enhance junction integrity. Searching for an alternative explanation for the junction-stabilizing mechanism, we found that an antibody-defined epitope, normally buried in a short α1-helix of the actin-binding domain (ABD) of α-catenin, is openly displayed in junctional VE-cadherin–α-catenin chimera. We found that this epitope became exposed in normal α-catenin upon triggering thrombin-induced tension across the VE-cadherin complex. These results suggest that the VE-cadherin–α-catenin chimera stabilizes endothelial junctions due to conformational changes in the ABD of α-catenin that support constitutive strong binding to actin.Cadherin-mediated cell adhesion requires anchoring via the β-catenin–α-catenin complex to the actin cytoskeleton, yet, α-catenin only binds F-actin weakly. A covalent fusion of VE-cadherin to α-catenin enhances actin anchorage in endothelial cells and strongly stabilizes endothelial junctions  Summary: There are novel antibody epitopes at the actin-binding domain of α-catenin that correlate with high affinity binding and are exposed in junction-stabilizing VE-cadherin–α-catenin fusion proteins. CTNNB1), which in turn binds to α-catenin , which links the complex to F-actin  and its mechanical coupling with the actin cytoskeleton. As for other cadherins, actin coupling is achieved by the binding of VE-cadherin to β-catenin  was recruited more efficiently to F-actin, given that detergent extractability as well as membrane mobility of the chimera were severely reduced in comparison to normal VE-cadherin . HoweverThe essential role of α-catenin for the anchoring of cadherins to the actin cytoskeleton is complex and relies on direct interactions as well as on adaptor proteins that can link α-catenin to F-actin. α-Catenin contains three major domains, the N-terminal β-catenin-binding domain, the modulation (M) domain (consisting of domains M1 to M3) and the actin-binding domain (ABD). Direct binding of α-catenin to F-actin is based on catch bond behavior , according to which the VEC-αC chimera would lack the first α-helix of the M1 domain, which affects the formation of a four-helix bundle necessary to occlude the vinculin-binding domain (VBD) in an autoinhibitory conformation.We then tested the interactions with vinculin more directly by immunoprecipitating vinculin from lysates of eEND and MDMVECs of both genotypes and probing in immunoblots for the amount of either co-immunoprecipitated VE-cadherin or of VEC-αC. We found that VEC-αC was much more efficiently co-precipitated than normal VE-cadherin, independently of the type of endothelial cells (eEND or MDMVECs) that was analyzed D–F. ThisCollectively, these results suggest that the VBD is largely masked in WT α-catenin, but constitutively accessible in the VEC-αC chimera.in vivo, we generated two equivalent knock-in mouse lines in which the VBD of VEC-αC was either deleted or modified, thereby preventing the association of VEC-αC with vinculin (Table S1), similar to what was found for the original VEC-αC mice  by injecting Evans Blue intravenously and stimulating intradermally with histamine. After 30 min, permeability was measured by extraction of the leaked dye from the excised skin area with formamide. In line with our previous study , insoluble residual material was re-extracted with an SDS-containing buffer (insoluble fraction) and the two fractions were analyzed by immunoblotting. As shown in Fig. S1).Next, we wanted to determine whether conformational changes in the ABD of VEC-αC could be found, which might be relevant for its enhanced interaction with the actin cytoskeleton in endothelial cells. Based on SMD simulations of αN-catenin, it was recently suggested that a short N-terminal α1-helix of the ABD (in αE-catenin residues 669–675) unfolds upon simulating pulling forces . FurtherFig. S2A). With these antibodies, we again found strongly enhanced staining at junctions of cells expressing VEC-αC compared to that in those expressing VEC-WT . This indicates, that the α1-helix is unfolded in VEC-αC, which is in line with the concept that the α-catenin ABD in VEC-αC is conformationally modified and resembles the high-affinity conformation of ABD, which is formed when α-catenin is under tension.Using these antibodies, we stained cultured primary MDMVECs isolated from VEC-WT or VEC-αC mice. The same cells were co-stained with antibodies against VE-cadherin and antibodies that recognized total α-catenin independent of its conformation. As shown in Next, we asked whether the VD7 antibody does indeed recognize α-catenin in a force-dependent manner. To this end, HUVECs were stimulated with thrombin in the presence or absence of the myosin II inhibitor Blebbistatin, and then fixed, permeabilized and stained with VD7 and control antibodies against total α-catenin. As shown in CTNND1) and the β-catenin-binding site of VE-cadherin. The FRET module contains YPet (donor) and mCherry (acceptor) separated by the elastic FL linker  and made use of a recently generated VE-cadherin tension sensor . In thisL linker C. TensioCollectively, these results demonstrate that the VD7 antibody recognizes epitopes that are buried in the α1-helix of α-catenin and become accessible upon exposure to force.in vitro binding of a recombinant form of the cytoplasmic part of VEC-αC (cyto-VEC-αC) to F-actin would be enhanced in comparison to α-catenin. Monomeric α-catenin is known to dimerize, which strongly enhances binding to F-actin . Dimers and monomers of α-catenin were separated at different positions of the elution profile , in line with previous reports .Since VD7 epitopes were strongly exposed in VEC-αC and these epitopes define catch bond-related conformational responses of α-catenin under mechanical load, we tested whether direct  F-actin . Therefo reports . Purity Using an F-actin co-sedimentation assay, we compared the binding of cyto-VEC-αC to F-actin with the binding efficiency of monomeric and dimeric α-catenin. For these assays, each of the three protein preparations were incubated with F-actin for 30 min, followed by an ultra-speed sedimentation. The resulting supernatants (S) and pellets (P) were separated by SDS-PAGE and stained. For controls, ultra-speed sedimentations were performed in the absence of F-actin, to determine the amount of material that would unspecifically precipitate.As expected, we found that most of α-catenin dimers bound to F-actin, whereas only a very small fraction of α-catenin monomers was co-sedimenting with F-actin A,B. UnexThis unexpected result raised the question of whether the tension-dependent VD7 epitopes of α-catenin, which we found constitutively exposed in VEC-αC molecules at endothelial junctions, would perhaps not be exposed in soluble VEC-αC molecules. To this end, α-catenin antibodies against tension-independent epitopes and VD7 antibodies were used to immunoprecipitate α-catenin and VEC-αC from cell lysates of WT-VEC- or VEC-αC-expressing MDMVECs. Immunoprecipitates were analyzed by immunoblotting with anti-α-catenin antibodies. We found that VD7 antibodies recognized solubilized VEC-αC with similar efficiency to solubilized α-catenin C. QuantiFig. S4). This shows that the VD7 epitope is clearly exposed when the ABD of α-catenin is in a high-affinity conformation. To test whether exposure of the VD7 epitope in VEC-αC at junctions requires force, we grew MDMVECs from VEC-WT or VEC-αC mice on substrate with either high stiffness (20 kPa) or low stiffness (<0.2 kPa), and stained cells with VD7 or anti-total α-catenin antibodies. As shown in Collectively, these results suggest that the α1-helix is not constitutively unfolded in soluble VEC-αC. In contrast, we found that the soluble α-catenin dimer was much more efficiently immunoprecipitated with the VD7 antibody than monomeric α-catenin  for the dynamics and behavior of epithelial adherens junctions show some similarities with the effects caused by VEC-αC. Enhanced actin binding of the α-catenin ABD containing the H1 mutation  is in agckground . In geneckground .in vitro binding results, which revealed that soluble αEcat-H1 bound efficiently to F-actin in in vitro sedimentation assays, whereas no such binding was observed for recombinant cyto-VEC-αC. A third explanation for the more dramatic embryonic defects caused by the αEcat-H1 mutant could be based on the higher propensity of this mutant to bundle F-actin, which could further reduce plasticity at cellular junctions  is missing in the VEC-αC construct. Since this helix is involved in masking of the VBD in the MI domain, and force is needed for unmasking, one could speculate that the force that is normally consumed to unmask the VBD could now immediately affect the conformation of the ABD, which would then affect the α1 helix, making the VD7 epitopes accessible. Thus, baseline forces at junctions are more exclusively directed towards unfolding the ABD of the chimera lacking the buffering effects of other structural elements.We had originally expected that the constitutive binding of vinculin to VEC-αC would contribute to the stabilizing effect of this chimera on endothelial junctions. This was based on the fact that vinculin is well documented to provide stability to cell substrate interactions at focal adhesions when tension rises . SimilarAt present, we cannot exclude that conformational changes of α-catenin in the VEC-αC chimera may also have consequences for interactions with other molecular components that could be relevant for junction stability. α-Catenin binds other actin-binding proteins, such as α-actinin , formin-Cadherin–α-catenin chimeras have been analyzed before in various experimental settings, leading to the stabilization of cellular junctions . In one In conclusion, we present here novel antibodies that allow monitoring of conformational changes of the ABD of α-catenin, which are induced by elevated mechanical load and are thought to be relevant for the tuning of catch bonding to actin, at endothelial junctions. These conformational changes were constitutively observed in junctional VEC-αC under conditions of baseline tension and are likely relevant for the enhanced anchoring of VEC-αC to junctional actin and for the stabilization of endothelial junctions against inflammation-induced opening. The second conformational change of α-catenin in VEC-αC, which caused constitutive binding of the actin-adaptor vinculin, was irrelevant for endothelial junction stability.The VEC-αC-ΔVBD fusion construct was generated by removing the vinculin-binding domain (aa 301–401 of αE-catenin) from the original VEC-αC chimera . Using t#20144; To generate VEC-αC-swapVBD cDNA, the VEC-αC-ΔVBD cDNA was digested with XhoI and the XhoI flanked mouse vinculin cDNA (aa 514–606)  of VEC-WT, VEC-αC, VEC-αC-ΔVBD or VEC-αC-swapVBD mice were isolated and cultured as previously described . EndotheThe following antibodies were used .VD7 directed against the entire α1-helix sequence of α-catenin and its flanking regions (AGQSARAIMAQLPQ) was obtained by immunization of rabbits and affinity purification using a described method    against mouse VE-cadherin , mouse monoclonal antibodies (mAb) V284  , mouse mAb hVIN-1   and rabbit mAb EPR8185   against vinculin, mouse mAb G11   and mAb alpha-CAT-7A4   against α-catenin, isotype control Ab , (IP: 5 µg/reaction), mAb against α-tubulin  . Alexa Fluor 405-, Alexa-Fluor 488-, Alexa Fluor 568-, Alexa Fluor 594- and Alexa Fluor 647-coupled secondary antibodies were purchased from Invitrogen CA, USA) . IRDye 680RD- and IRDye 800CW-coupled secondary antibodies were purchased from LI-COR Biosciences  . All other secondary antibodies were purchased from Dianova  .2. To inhibit myosin II, HUVECs were treated with 50 μM Blebbistatin  for 30 min. DMSO was used as vehicle control.HUVECs were stimulated with 1 U/ml thrombin  for 10 min at 37°C and 5% CO2, 10% glycerol, and Complete EDTA-free proteinase inhibitor cocktail ]. Lysates were cleared by centrifugation at 20,817 g for 2 min before aliquots for direct blot analysis were set aside and aliquots for immunoprecipitation were incubated for 1 h at 4°C with 4.5 μm Dynabeads  loaded with the respective antibodies. Immunocomplexes were washed five times with lysis buffer and dissolved in Laemmli sample buffer for standard western blot analysis.MDMVECs or eEND cells were lysed at 4°C for 10 min in IP-lysis buffer  and scraping all residual material off the dish. Lysis was performed for 30 min at 4°C in a head-over-end shaker and lysates were centrifuged for 30 min at 20,817 g and 4°C. Supernatants with proteins of high and low detergent solubility were subjected to SDS-PAGE and western blot analysis.Cells were rinsed twice with ice-cold PBS before extraction buffer I  and grown to confluence. Cells were washed with PBS, fixed with 4% PFA in PBS for 7 min at room temperature, and permeabilized using 0.5% Triton X-100 in PBS for 10 min at room temperature, followed by blocking with 3% BSA, 2% donkey-serum for 2 h and incubation with primary antibodies. Primary antibodies were detected with Alexa Fluor-coupled secondary antibodies. Fluorescence signals were detected using a confocal laser-scanning microscope (LSM 780/LSM 880) with online fingerprinting mode [ZEN 2.3 ]. ImageJ/Fiji or Imaris  software were used for image processing. To quantify VD7 intensity at cell–cell junctions, a colocalization channel between VD7 and total α-catenin was created, and fluorescence intensity of VD7 was measured over the colocalization channel and normalized to fluorescence intensity of total α-catenin.®  according to manufacturer's guidelines.For interference with human α-catenin expression, the siRNA 5′-ACAUGGCAGAUGUCUACAAtt-3′ produced by Life Technologies  was used. For interference with human VE-cadherin expression, CDH5 siRNA by Life Technologies was used (5′-GGGUUUUUGCAUAAUAAGCTT-3′). AllStars negative control siRNA , which does not target any known mammalian gene, was used as control. Routinely, HUVECs were transfected with 40–80 nM siRNAs for 48–72 h using INTERFERin4 cells per chamber, followed by re-expression of the VE-cadherin tension sensor (VEC-FL)   for 24 hFor the analysis of FLIM-FRET data, pixel-wise data fitting  was used with subsequent bi-exponential tail-fitting. For fluorescence intensity images, ImageJ/Fiji was used to create a mask based on total α-catenin intensity signal. This mask was further segmented with the Watershed segmentation algorithm (ImageJ/Fiji) to create different regions of interest (ROIs). Next, the plugin ‘Ratio Plus’ was used to create a pixel-by-pixel ratio image of VD7 to total α-catenin intensity. Average ratio and VEC-FL lifetime of the automatically generated ROIs were used for correlation analysis in R software (Version 1.3.1093).A modified Miles assay for the induction of vascular permeability in the skin was performed as described previously . 8–12-we6 tag. Recombinant fusion proteins were expressed in BL21 (DE3) Codon Plus E. coli cells, purified on HisTrap HP columns , and eluted with imidazole. Fusion proteins were further purified at 4°C using Superdex 200 gel filtration chromatography  in 20 mM Tris-HCl pH 8.0, 150 mM NaCl and 2.5 mM DTT. Recombinant α-catenin containing a C-terminal His6 tag was expressed and purified similarly to cyto VEC-αC, and the monomer was separated from the dimer by two consecutive rounds of gel filtration chromatography on Superdex 200.cDNA encoding the mouse cytoplasmic tail of VEC-αC (cyto VEC-αC) [aa 623–784 of VE-cadherin fused to the C-terminal two-thirds of mouse α-catenin (aa 301–906)] was generated by PCR using the original VEC-αC cDNA as a template. The cDNA encoding cyto VEC-αC or cDNA encoding full-length mouse α-catenin were inserted into the pET21a vector (Sigma-Aldrich) to generate an in-frame fusion between mouse α-catenin and a C-terminal Hisg for 30 min at 4°C in a TLA 55 rotor . Pellets were resuspended in 1× Laemmli sample buffer, separated by SDS-PAGE, and stained with Coomassie Brilliant Blue. Gels were imaged on an Epson scanner  and band intensities were quantified using the Image Studio software .The assay was performed using the Actin Binding Protein Spin-Down Biochem Kit  according to the manufacturer's instructions with minor modifications. Rabbit muscle G-actin was incubated in 1× actin polymerization buffer  for 1 h at room temperature to polymerize filaments. Gel filtered cytoplasmic tail of VEC-αC, as well as dimers and monomers of α-catenin were diluted to a final concentration of 1 µM in 1× reaction buffer , with and without F-actin and incubated for 30 min at room temperature. Actin cushion buffer  was pipetted into centrifuge tubes, then the reaction mixture was carefully loaded on top of the cushion buffer and centrifuged at 186,000 t-test, one-way ANOVA, or two-way ANOVA for independent samples. Tukey's multiple comparison test was applied to correct for multiple comparisons. GraphPad Prism8  or R (version 3.6.0) software was used for this analysis. P-values are indicated by asterisks: *P<0.05, **P<0.01, ***P<0.001, and ****P≤0.0001. Results are shown as means±s.e.m. Immunoblot signals were quantified using the software Image Studio™ (LI-COR Biosciences). For FLIM-FRET, data was analyzed and plotted using R (Version 1.3.1093) with the ggplot2 package using Spearman rank correlation test.Datasets were tested for normality (Shapiro–Wilk) and equal variance. Statistical significance was analyzed using two-tailed unpaired Student's


We aimed to analyze the prevalence, causes, and clinical settings of 4-year critical radiologic reports (CRRs) notified from the musculoskeletal section of the radiology department. Then, we investigated the communication outcomes.This study was approved by our institutional review board. We retrospectively included 175 musculoskeletal CRRs from our database between January 2017 and December 2020. The CRRs were analyzed by two musculoskeletal radiologists, who categorized the CRRs by clinical setting (emergency department(ED) patient, outpatient, and inpatient), body part, type of image modality, reason for CRR, incidental lesion, and clinical outcome. The clinical outcome was retrieved from the electronic medical records.n = 3217) available in the study period. Most CRRs  corresponded to the musculoskeletal system, while the remaining ones  corresponded to the non-musculoskeletal system. In addition, the spine, extremities, and thoracic cage accounted for 52.6%, 40.6%, and 1.7% of the musculoskeletal CRRs, respectively. Moreover, most patients presented to the ED , followed by inpatients , and outpatients . The CRR reasons included missed fracture (54.3%), suspected malignancy (16%), clinical emergency (10.3%), unexpected infection/inflammation (11.4%), and others (8%). Furthermore, 11 (6.3%) incidental lesions were not related to the primary imaging purpose. Referring clinicians actively acknowledged 80% of the CRRs. The loss to follow-up action was the highest in the ED patients , being significantly higher than that in outpatients  and inpatients .The 175 musculoskeletal CRRs accounted for 5.4% of the CRRs (Missed fractures were the most common cause of musculoskeletal CRRs. ED showed prevalence in musculoskeletal CRRs and reflected the highest loss to follow-up action. ED physicians should pay more attention to CRRs to enhance patient care. Medical imaging comprises an examination, a verified report, and the report communication . CurrentRadiologic reports with clinical significance or requiring immediate action should be promptly informed to the referring physician to ensure the continuity of patient treatment. The Joint Commission has mandated compliance with the communication of critical test results among caregivers on a timely basis as an important part of the National Patient Safety Goals .Non-routine communication varies depending on the institution, and many medical centers have implemented electronic systems for non-routine communication –6. Thus,In this study, we analyzed the prevalence, reasons, clinical settings, and follow-up rate related to CRRs in the musculoskeletal section and identified the impact of electronic notification systems and communication outcomes.This retrospective study was approved by the Gil Medical Center institutional review board and the requirement for informed consent was waived given the retrospective nature of this study.Fig 1).Our institution is a tertiary-referral academic medical institution with 1700 beds and a national regional trauma center. An electronic alert notification system was embedded in PACS for CRRs in 2015. In total, 3217 consecutive CRRs were retrieved from the electronic medical record (EMR) system  from January 2017 to December 2020. Metadata including patient identification, patient age, referring department, and study name were automatically extracted during database search, obtaining 180 CRRs from the musculoskeletal section of the radiology department. As 5 cases showed no clinical significance , we used 175 musculoskeletal CRRs for analysis  was documented in the corresponding EMRs. During the study period of 4 years, five radiologists worked in the musculoskeletal section, and they reported CRRs for emergency findings that needed urgent management or for clinically important findings  that were considered unrecognized by the referring physician.Two musculoskeletal radiologists with 12 and 4 years of experience reviewed the EMRs and magnetic resonance imaging (MRI) reports with image findings of each musculoskeletal CRR case. We categorized the data by clinical setting (emergency department(ED) patient, inpatient, outpatient), body part , imaging modality , ultrasound, X-ray), reason for CRR , and incidental lesion or not.The CRR was considered as actively acknowledged when clinical notes were available about the radiology result notification described by physicians in the EMR or when additional management  was performed after the CRR notification. The clinical outcomes of each CRR were obtained from the corresponding EMRs and classified as surgical treatment/intervention, medical treatment, conservative management, and telephonic notification to patient. On the other hand, loss to follow-up action was considered for unacknowledged CRRs.p < 0.05.Descriptive statistics were compared among the causes, clinical settings, and follow-up cases using chi-squared tests implemented in SPSS . Statistical significance was set at Table 1. The age range of the subjects was 7–84 years with mean of 54.8 years. In the clinical setting, most patients were presented to the ED , followed by inpatients  and outpatients . Radiography  was the most frequent imaging modality followed by MRI , CT , and ultrasound .The characteristics of the 175 study subjects  are listed in n = 175) of the available CRRs (n = 3217). Most musculoskeletal CRRs  corresponded to the musculoskeletal system, and the remaining CRRs  corresponded to the non-musculoskeletal system . In the musculoskeletal system, the spine cases  outnumbered cases in the extremities .The CRRs from the musculoskeletal imaging accounted for 5.4%  were the most common reasons followed by suspected malignancy , unexpected infection/inflammation , clinical emergency , and others . However, there were significant differences in the reasons according to the clinical setting. Missed fractures were significantly more numerous in the ED than in other clinical settings (p < 0.001). The most common reason per clinical setting was missed fractures (78/88) in ED patients, clinical emergency (14/54) in inpatients, and suspected malignancy (12/33) in outpatients.Table 3 lists the missed fractures according to the body part. Most fractures were missed in the spine  followed by lower extremity , upper extremity , and thoracic cage . The C-spine  was the most common site of a missed spinal fracture.Table 4 and Fig 6).From the cases, 11 (6.3%) incidental lesions in the brain, lung, retroperitoneum, and musculoskeletal system were detected from spine MRI, spine CT, and shoulder CT , with loss to follow-up action occurring for the remaining 20% (35/175) of cases (p < 0.001). All the 13 telephonic notifications were identified only in the ED patients. The details about the cases of loss to follow-up action are shown in of cases . The losWe analyzed the usage of an alert notification system for CRRs in the musculoskeletal section. The analysis included prevalence, causes, clinical settings, and whether the appropriate follow-up action was taken after the CRR. The results revealed that musculoskeletal CRRs accounted for 5.4% of the total CRRs. Missed fractures were the most common cause of musculoskeletal CRRs, with ED patients showing most of these cases. The active acknowledgement rate of musculoskeletal CRRs was 80%, whereas follow-up action was lost in 20% of CRRs. However, the results differed depending on the clinical setting . The rate of loss to follow-up action was the highest in the ED.Few studies have reported that musculoskeletal imaging shows a relatively low prevalence of CRRs  of incidental lesions in the brain, lung, retroperitoneum, and bone and joint were detected on spine MRI, CT, and shoulder CT. In approximately half of these cases, CRRs were generated by suspected malignancy. To prevent unexpected radiologic findings deriving in mortality or morbidity, CRRs due to incidentally suspected malignancy are important for patient safety. With the development of imaging techniques, the diagnostic performance has improved, and the frequency of incidental findings that are unrelated to the primary purpose of examination has increased . In wholp < 0.001) than in inpatients or outpatients. Therefore, clinicians being acquainted with missed lesions during emergencies can help to reduce the loss to follow-up action rate. Telephonic follow-up is being increasingly focused on patient management, particularly in the ED [We found a 20% loss to follow-up action, possibly due to ineffective communication between the radiologists and referring physicians or to the patient’s disagreement to subsequent work-up or treatment. Roy et al.  reportedn the ED , 30. In This study has some limitations to be considered. First, since this study targeted the CRRs generated in musculoskeletal imaging studies read by musculoskeletal attending radiologists, we did not include critical musculoskeletal lesions detected on non-musculoskeletal imaging . Therefore, our study likely substantially underestimates the proportion of all CRRs that are missed fractures. Second, telephonic and direct communications between radiologists and referring physicians were not included for the CRRs owing to problem solving and lack of electronic records. Third, as no detailed manual with specific examples of radiologic CRR is available in our institution, the CRR frequency may have been different depending on the radiologist. Fourth, as radiologists are usually not informed about the clinical outcome of their CRRs, we estimated it from the EMRs. Finally, this study was performed in a single institution with regional trauma center, the incidence of traumatic injury is relatively high. Hence, the results of this study may not reflect the reality of institutions without a trauma center. Nevertheless, the findings allowed us to learn about traumatic lesions that clinicians often miss.In conclusion, missed fractures were the most common cause of musculoskeletal CRRs. In addition, the ED had the highest prevalence and rate of loss to follow-up action in patients with musculoskeletal CRRs. Physicians in the ED must pay more attention to CRRs, and radiologists should effectively communicate with the referring physicians and provide accurate and timely radiologic reports. A reliable standardized CRR manual is needed, and deployment is necessary across clinical practice.


Endoscopy is practical both in the diagnosis and treatment of various pathologies. Technological advances, especially in endoscopy, gradually progress and discover many possibilities which allow rapid advancement. Endoscopy development aims to assess human orifice that has not been inspected, probed, and examined over the centuries. Endoscopy over these decades is improving, which led to new problem solving using advanced technological approaches. Thus, a surgeon can solve any issues from examination, diagnosis, and treatment using progressive endoscopy evolution. This review delivers a brief history of advances in surgical endoscopy and describes current endoscopy development.Surgeons have a role in observing, detect abnormalities, disease, and other deficiencies in function which could be treated. Diagnosing and treating back days were challenging for many reasons. However, technology's innovation enhances surgeons' ability to treat their patients. The term  •The advancement of endoscopies are the breakthrough to the upcoming possibilities in treating patients.•Each step ahead in various approaches of endoscopy gain further improvement of patient outcomes.•Diagnostic endoscopy is shifting as a new therapeutic modality to resolve patient's issues. It was used to observe the rectal fistula by using a rectal speculum  [[1], 2. The iss75 BC. Itlichtleiter (light conductor), a tin tube illuminated by a candle and reflected using an angled mirror  deserved the most credit as the pioneer of modern endoscopy ,3. The B–1809 desd mirror  6]. Thi. Thilichcystique . Other dcystique ,6.Fig. 2Significant development of endoscopy is originated by Maximilian Nitze (1848–1906), a German urologist who collaborated with Wilhelm Deicke and Louis Beneche to produce a miniature telescope that magnified the image of the bladder using water-cooled platinum wire. Later in 1880, Thomas A. Edison's invention of light was being used by Maximilian to place a small lamp at the end of a cystoscope .The semi-flexible tube endoscope, firstly developed by Georg Kelling in 1898, a surgeon, performed peritoneoscopy by placing a camera at the endoscope's tip. Later, it was improved by Rudolf Schindler in collaborated with Georg Wolf and considered as the “father of gastroscopy.” In 1957, Basil Hirschowitz produced a glass fiber gastroscope and upgraded it into a bundle of fiber called fiberoptic endoscopy  & Fig. 422.1The first endoscopic myotomy for achalasia was performed in 1980 by Ortega et al. For decades, this technique wasn't well improved, and there was no further report of the procedure. For the first time in Japan in 2008, Inoue et al. performed Per-Oral Endoscopic Myotomy (POEM) in a human being . This tep < 0.0003 and the resting lower esophageal sphincter (LES) pressure decreased from mean 52.4 to 19.9 mmHg; p < 0.0001. The short-term outcome (5 months) of POEM was excellent, but further long-term studies are required , .   15] BrieThis technique was originally developed for children, while nowadays, it is widely used for all ages of patients. Common disease states are responsible for these disorders, such as esophageal cancer, oropharyngeal cancer, esophageal dysmotility, and neurologic conditions  that impairs or weakens swallowing . RoughlyA review of 150 cases by Ponsky et al. described complications in 15 patients (10%). Superficial wound infections around the catheter were found in 7 patients. Nonetheless, administrating a single preoperative dose of cephalosporin successfully prevented wound infections in 125 cases. Altogether of 150 PEGs, there were 0% mortality and 10% morbidity rates . Another2.3Endoscopic Retrograde Cholangiopancreatography (ERCP) was introduced in 1968 by Dr. William S. McCune, an obstetrician who performed ERCP using a fiber duodenoscope for a diagnostic tool. Later in 1972, Dr. Peter Cotton introduced cannulation in ERCP. In the following years, Dr. Meinhard Classen in Germany and Keiichi Kawai in Japan discovered a therapeutical potential of ERCP with endoscopic sphincterotomy . BrieflyThe advances of high-resolution imaging modalities such as magnetic resonance imaging with magnetic resonance cholangiopancreatography (MRCP), pancreatic protocol computed tomography scans, endoscopic ultrasound (EUS) have substituted ERCP as a diagnostic tool. Thus, the therapeutic/interventional approach becomes the main focus of ERCP .The indications of ERCP may vary in clinical situations. Most commonly is used to treat biliary problems  than pancreatic issues such as pancreatitis, pancreatic fistule, pancreatic fluid collections, etc. ,28 EndosVarious complications of ERCP might occur, such as post-ERCP pancreatitis (PEP), hemorrhage, perforation, cholangitis, cholecystitis, cardio-pulmonary depression, asymptomatic hyperamylasemia, aspiration, bleeding, hypoxia, sepsis, and death ,27,28. I2.4Endoscopic ultrasound (EUS) has advanced from a diagnostic imaging modality discovered by Dimagno et al. in the 1980s to an interventional procedure . The advDiagnostic EUS uses an echo-endoscope with several variants, such as radial or linear, and developed in 1991, referred to as EUS-fine-needle aspiration (EUS-FNA) . On the Current EUS with radial and linear echo-endoscope has a non-flexible transducer which produces a more rigid tip, 3–5 cm length, of echo-endoscope. Furthermore, an oblique endoscopic view, the echo-endoscope insertion, and advancements of the instruments create a semi-manual maneuver; thus, adverse events, though rare, include perforation, bleeding, and infection. A study involving 43,852 subjects reported only 16 (0.03%) cervical esophageal perforation with only one death within the EUS procedures . A prosp2.5en bloc resection of lesions [The history of endoscopic resection began in 1955 when Rosenberg introduced endoscopic mucosal resection (EMR) by creating a plane for fulguration of sigmoid and rectal polyps. Then in 1973, Dehyle utilized submucosal injection to the sessile or flat lesions for complete removal. At first, EMR was unpopular and regarded as a risky procedure. In the meantime, it became accepted in 1980, but sometimes EMR isn't dependable to ensure the complete resection of the tumors. Hence, endoscopic submucosal dissection (ESD) was developed in the 1990s for  lesions .en bloc removal of large tumor above 20 mm and flat GI lesion  tract . The maxI lesion . Normal en bloc resection, the complete resection, and the recurrence rates based on Lee et al. comparing EMR and ESR with a result of 42.9% vs 92.7%, 32.9% vs 87.6%, and 25.9% vs 0.8%, respectively [Both EMR and ESD are required for definitive therapy of early-stage (T1mN0) and malignant lesions of the GI tract. Another function of EMR and ESR is to obtain larger histological specimens and provide an accurate histologic T staging ,49. The ectively . Therefoectively .2.6Acute colonic, such as acute colonic pseudo-obstruction (ACPO), colonic volvulus, and malignant obstruction, is a medical emergency with high morbidity and mortality. Colonic decompression is one of the established treatment strategies . The firThe advancing methods of colonic decompression expand the choices included radiologic placement of decompression tubes under fluoroscopy guidance and colonoscopic decompression with or without placement of a decompression tube. These techniques are recommended by the European Society of Gastrointestinal Endoscopy (ESGE) if the cecal diameter is more significant than 12 cm ,60. For A retrospective cohort study of 53 patients reported a clinical success rate of up to 92.5% and an additional decompression tube at an 86% success rate. The complication rate was 3.8% with one perforation . Bode et2.7trans-gastric NOTES appendectomy was demonstrated by Rao and Reddy [trans-esophageal, trans-gastric, trans-vaginal, trans-vesical, and trans-colonic. The main aims of the NOTES technique are to minimalize cosmetic alteration, avoid abdominal incisions, and reduce invasiveness compared to traditional laparotomy or laparoscopic surgery [Natural orifice transluminal endoscopic surgery (NOTES), widely known as a “scarless surgery,” has withdrawn attention in these recent years as a novel surgical method to develop minimally invasive surgery ,66. Kallnd Reddy . Origina surgery .The procedure begins with a standard single-channel gastroscopy and placement of an overtube. Clearance of the gastric contents by suction and gastric lavage, then flush the stomach with an antibiotic. Cefazolin is the recommended option for maximum results. The cleansing step has not been studied; however, this procedure is required to maintain the sterility of the peritoneal cavity. An incision is made in the anterior gastric wall as the most common site. Various instruments, including the needle-knife, insulation tipped (IT) knife, controlled radial expansion (CRE) balloon, or pull-type sphincterotome, can be used for gastrostomy. Subsequently, a gastroscope is inserted into the peritoneal cavity and visualizes abdominal organs , Fig. 14As a novel method, NOTES provides many advantages compared to traditional laparotomies, such as zero incision and scar, less pain, injury, a reduced dosage of analgesic and anesthetic, rapid recovery, reduce hospitalization, and decrease inpatient complications  ,73. Cons2.8Obesity, type 2 diabetes mellitus, and cardiovascular disease dominate the epidemic proportions . Bariatr2 had lost 14.4% of total BMI at the first six months, 17.6% at 12 months, and 20.9% at 24 months, alongside with significant reduction in hemoglobinA1c (p = 0.01), systolic blood pressure (p = 0.02), waist circumference (p < 0.001) and serum triglycerides (p = 0.02) [2 at one year with no significant adverse events [2 after one month, three months, and six months mean weight loss of 11.5 kg, 19.4 kg, and 33 kg no adverse events noted [Endoscopic sleeve gastroplasty is an incision-less and minimally invasive technique that intentionally reduces the functional volume of the stomach by 80% using an interrupted triangular suture pattern created along the greater curvature of the stomach , Fig. 16 = 0.02) . Lopez-Ne events . Other sts noted . Thus, E3Inventions and developments of surgical techniques in endoscopy have evolved within decades. Surgeons collaborate to generate something according to patient conditions to minimize adverse events, enhance life quality, and improve safety. Alongside technology's advancement and surgeons' capability, recent methods are improving and promising a genuine endoscopy; thus, each step ahead alters the function of endoscopy into branches.Author does not require ethical approval in this article.Author declares no funding of this article.Author contributes in study concept, data collection, and writing the paper of this article.Author does not need consent in this article.Author does not need registration of research studies.Reno Rudiman acts as the author and the guarantor of this article. Author is fully responsible for the work, the conduct of the study, data accessibility and decision to publish.The author reports no conflicts of interest in this work. This paper abstract was presented at the 11th Global Gastroenterologists Meeting named Recent Advances in Surgical Endoscopy as a poster presentation. The poster abstract was published in “Poster Abstracts” in the Journal of Gastrointestinal & Digestive System. DOI: 10.4172/2161-069X–C1-049.Author declares no conflict of interest.


Depression is characterized by impairments in adult neurogenesis. Reduced hippocampal function, which is suggestive of neurogenesis impairments, is associated with depression-related phenotypes. As adult neurogenesis operates in an activity-dependent manner, disruption of hippocampal neurogenesis in depression may be a consequence of neural circuitry impairments. In particular, the entorhinal cortex is known to have a regulatory effect on the neural circuitry related to hippocampal function and adult neurogenesis. However, a comprehensive understanding of how disruption of the neural circuitry can lead to neurogenesis impairments in depression remains unclear with respect to the regulatory role of the entorhinal cortex. This review highlights recent findings suggesting neural circuitry-regulated neurogenesis, with a focus on the potential role of the entorhinal cortex in hippocampal neurogenesis in depression-related cognitive and emotional phenotypes. Taken together, these findings may provide a better understanding of the entorhinal cortex-regulated hippocampal neurogenesis model of depression. Globally, depression is one of the leading causes of disability . The worDefective hippocampal neurogenesis is a hallmark of depression. Ample evidence emphasizes a varying spectrum of neuropathology, from defects in hippocampal activity and volumes to alterations in activity-dependent gene expression, all of which suggest defective neurogenesis in the hippocampal dentate gyrus ,16,17. GOur hypothetical concept of the entorhinal cortex-regulated hippocampal neurogenesis model of depression is based on both the neurobiological knowledge of adult neurogenesis and results of recent studies that not only demonstrate adult neurogenesis but also recapitulate the influence of neural circuitry and neurogenesis on depression-related phenotypes. We explored the neurobiological phenomenon of the co-operative engagement of the entorhinal cortex and hippocampus in adult neurogenesis ,27,28,29Episodic memory deterioration is a key cognitive phenotype in depression ,44,45,46The entorhinal cortex-hippocampal neural circuitry is regarded as the memory center of the mammalian brain and is mainly involved in the regulation of episodic memory, including object, spatial, and temporal information ,59,60,61In addition, studies in mice have shown that deep brain stimulation of the entorhinal cortex enhances spatial memory and learning, which are accompanied by improved neurogenesis in the dentate gyrus a. In parIn addition to deep brain stimulation, a preclinical study employed optogenetics to access the detailed mechanisms of memory regulated by the entorhinal–hippocampal circuitry. Robinson et al. addressed the question of whether the entorhinal–hippocampal circuitry modulates temporal memory, which is coded in the principal cells of the hippocampal CA1, also known as time cells . They exPattern separation defects are another hallmark of cognitive dysfunction in depression. The ability to distinguish between similar contexts is reliant on hippocampal neurogenesis ,74,75,76Functional brain imaging supports the idea that the entorhinal cortex is involved in the upstream neural circuitry of neurogenesis to drive pattern separation. Earlier studies using older human subjects examined the relationship between entorhinal circuitry defects and cognitive deficits, given that volumetric reductions in the medial temporal lobe, including the entorhinal cortex, have been reported to provoke cognitive deficits in both depression  and aginAmong the earliest studies exploring the impact of neurogenesis defects on pattern separation, X-ray irradiation of the hippocampus or synaptic plasticity disruption in dentate granule cells was mainly utilized to recapitulate neural stem cell niche defects to inspect impairment in the discrimination of a similar, safe context from a foot-shock context ,88,89,90Trip8b, whose knockdown excites hippocampal neurons, thereby enabling neurogenesis in the dentate gyrus. In the knockdown mice, entorhinal glutamatergic stimulation resulted in hippocampal neurogenesis, such as dendritic maturation of adult-born neurons by augmenting the inherent excitability of stellate cells in the entorhinal cortex. A contextual fear conditioning task was then implemented to inspect the pattern separation ability of the knockdown mice to distinguish a foot shock-related context from a similar context. The knockdown mice (Trip8b-shRNA) showed 50% more freezing behavior in the foot shock-related context than the control mice (SCR-shRNA), thereby demonstrating the improvement in pattern separation following entorhinal glutamatergic stimulation. In addition, X-ray irradiation of the dentate gyrus, leading to ablated neurogenesis, significantly diminished the effect elicited by entorhinal-targeted Trip8b knockdown. Taken together, the innovative work using a chemogenetic approach suggests that entorhinal cortex-regulated hippocampal neurogenesis regulates pattern separation. Nevertheless, the entorhinal–hippocampal circuitry-regulated neurogenesis mechanism underlying pattern separation still remains unclear; therefore, further preclinical research using brain stimulation and optogenetic or chemogenetic approaches to explain the entorhinal–hippocampal circuitry mechanism regulating pattern separation is required.Recently, Yun et al. used a chemogenetic approach to generate transgenic mice with entorhinal cortex-targeted knockdown of a stress-provoked protein using an adeno-associated virus-mediated transfer gene b 25]. A. A25]. AHippocampal neurogenesis is associated with the inherent depression pathophysiology as well as the response to antidepressants ,92. HippCurrently, there are no available studies that have used superficial or deep brain stimulation aimed at the entorhinal cortex to link hippocampal neurogenesis and anti-depressive effects. Most studies have focused on other brain regions, including the prefrontal cortex ,113,114,Trip8b, a specific stress-provoked protein that alters hippocampal neuron activity. Indeed, Trip8b-knockout mice demonstrated higher hippocampal neuronal firing frequency and increased neurogenesis with neuronal maturation than those in controls, mainly in the temporal dentate gyrus, which is known as a hippocampal region responsible for emotional processing and stress valance . T. T25]. T valance ,141. Theosterone ,143. Und mCherry  in the el defeat . These rPioneering preclinical studies examining entorhinal cortex-regulated hippocampal neurogenesis and cognitive and emotional symptoms of depression have led to initial advances in the neural circuitry–neurogenesis model of depression. Gomes-Leal et al.  suggesteSecond, future studies are also required to scrutinize complex upstream hippocampal circuitries that include diverse brain subregions related to the pathogenesis of depression, while also considering hippocampal neurogenesis. Recent evidence suggests that various brain subregions, in addition to the entorhinal cortex, are also associated with dentate gyrus neurogenesis, indicating that complex neural circuitries may be implicated in hippocampus-dependent tasks as well ,154. ForPreclinical and clinical studies suggest that the upstream hippocampal circuitry may link the entorhinal cortex with adult neurogenesis, which in turn suggests that the neural circuitry neurogenesis mechanism can be a tenable concept to use when considering approaches to address defects in memory, pattern separation, and emotion, all of which are hampered in depression. In the treatment of depression, the neural stem cell niche in the hippocampus is considered an essential target of antidepressants, in addition to brain stimulation approaches to facilitate maturation of dentate gyrus neurons. Considering the link between hippocampal neurogenesis and antidepressant effects, the entorhinal–hippocampal circuitry is not only a valid neurophysiological concept, but also a novel area to explore in the pathogenesis of depression, with a particular emphasis on the potential role of the entorhinal cortex in regulating hippocampal neurogenesis, inducing improvements in memory, pattern separation, and anti-depressive behaviors. Therefore, entorhinal cortex-regulated hippocampal neurogenesis is a tenable example of the neural circuitry neurogenesis model that further expands our knowledge of depression pathogenesis related to cognitive and emotional symptoms in patients with depression, and ultimately aids in the development of novel therapeutic approaches.


Accelerated dental pulp mineralization is a common complication in avulsed/luxated teeth, although the mechanisms underlying this remain unclear. We hypothesized that hypoxia due to vascular severance may induce osteo/odontoblast differentiation of dental pulp stem cells (DPSCs). This study examined the role of B-cell CLL/lymphoma 9 (BCL9), which is downstream of hypoxia-inducible factor 1α (HIF1α) and a Wnt/β-catenin transcriptional cofactor, in the osteo/odontoblastic differentiation of human DPSCs (hDPSCs) under hypoxic conditions. hDPSCs were isolated from extracted healthy wisdom teeth. Hypoxic conditions and HIF1α overexpression induced significant upregulation of mRNAs for osteo/odontoblast markers , BCL9, and Wnt/β-catenin signaling target genes  in hDPSCs. Overexpression and suppression of BCL9 in hDPSCs up- and downregulated, respectively, the mRNAs for AXIN2, TCF1, and the osteo/odontoblast markers. Hypoxic-cultured mouse pulp tissue explants showed the promotion of HIF1α, BCL9, and β-catenin expression and BCL9-β-catenin co-localization. In addition, BCL9 formed a complex with β-catenin in hDPSCs in vitro. This study demonstrated that hypoxia/HIF1α-induced osteo/odontoblast differentiation of hDPSCs was partially dependent on Wnt/β-catenin signaling, where BCL9 acted as a key mediator between HIF1α and Wnt/β-catenin signaling. These findings may reveal part of the mechanisms of dental pulp mineralization after traumatic dental injury. Experimental replantation or transplantation of rat or mouse molars also induces mineralization in this pulp tissue5. Hypoxia resulting from ischemia due to severance of the vasculature is a major pathological event that takes place in the pulp tissue following tooth replantation/transplantation7. Hypoxia induces higher mineralization activity in human dental pulp cells than normoxia does8. When the oxygen level in rat molar pulp was reduced by arresting the blood supply through implantation of a mini-screw into the inferior dental nerve canal, mRNA expression of osteocalcin (OCN) and dentin sialophosphoprotein (DSPP) increased and OCN- and DSPP-positive cells were localized in the odontoblastic layer9. These findings suggest that the impact of hypoxia on the accelerated mineralization of traumatized dental pulp is of considerable importance, although the mechanisms behind the hypoxia-induced mineralization require detailed investigation.The dental pulp is a neural crest-derived non-mineralized connective tissue surrounded by mineralized tissues such as enamel and dentin. Accelerated formation of bone-like and/or dentin-like mineralized tissues in dental pulp is a common complication in enamel/dentin damage, caries lesions, and avulsed/luxated teeth11. Under normoxic conditions, HIF1α is effectively degraded after hydroxylation of one of its two proline residues by prolyl hydroxylase domain proteins, followed by ubiquitination and degradation by the proteasome12. However, hypoxic conditions reduce the activity of prolyl hydroxylase13, which stabilizes HIF1α in the cytoplasm leading to its translocation to the nucleus. There, HIF1α forms a heterodimer with HIF1β, which is constitutively expressed. The resulting HIF1α and β complex binds to the promoter region of hypoxia-responsive genes, such as vascular endothelial growth factor (VEGF), which further activates the transcription of target genes14. Hypoxia-responsive genes possess a cis-acting element, which is called the hypoxia-response element with the core sequence 5′-RCGTG-3′ 15. HIF regulates the expression of multiple genes and is involved in the regulation of energy metabolism11, hematopoietic stem cell maintenance17, angiogenesis18, and cancer cell proliferation, apoptosis, invasion, and metastasis19. HIF1α signaling is reported to promote osteo/odontoblast differentiation via bone morphogenetic protein (BMP) signaling in stem cells from human exfoliated deciduous teeth extracted from fibrodysplasia ossificans progressiva patients20.Hypoxia-inducible factor 1α (HIF1α) is a subunit of the heterodimeric HIF1, which is a specific transcription factor activated under hypoxic conditions and plays an integral role in various intracellular responses to hypoxia21. In the absence of the ligand Wnt, β-catenin is phosphorylated and degraded by various other factors such as glycogen synthase kinase 3β (GSK3β) and AXIN, but when Wnt binds to the receptor Frizzled, β-catenin in the cytoplasm is stabilized and translocates into the nucleus. β-Catenin then binds to the transcription factors T-cell factor (TCF)/lymphoid enhancer binding factor (LEF) and promotes transcription of target genes such as AXIN2 and TCF121. Studies have disclosed that β-catenin-stabilized mice show excessive dentin and cementum formation22, and that Wnt/β-catenin signaling is essential during tooth development23. Moreover, direct capping of exposed dental pulp with GSK3β antagonists induces the formation of more reparative dentin than that when using collagen sponges or MTA cement24. These findings support the notion that Wnt/β-catenin signaling is crucial to regulate osteo/odontoblast differentiation and the formation of mineralized tissues such as bone and dentin.Wnt/β-catenin signaling plays a pivotal role in cell proliferation, cell differentiation, and tissue homeostasis25. HIF1α is also involved in carcinogenesis and tumor growth through the regulation of angiogenesis, glycolytic metabolism, and other biological mechanisms26. Recently, hypoxia and HIF1α have been reported to activate B-cell chronic lymphocytic leukemia/lymphoma 9 (BCL9), which is an essential component of canonical Wnt/β-catenin signaling28. BCL9 acts as a transcriptional cofactor in Wnt/β-catenin signaling, and BCL9 binds to β-catenin and promotes the formation of the β-catenin–TCF complex, which in turn induces the transcription of target genes29. BCL9 is also required for activation of the Wnt/β-catenin cascade in adult mammalian myogenic progenitors30. BCL9 possesses hypoxia-response elements in its promoter region, and hypoxia and HIF1α induce BCL9 expression in liver, colon, and prostate cancer cells28. Thus, hypoxia and HIF1α activate Wnt/β-catenin signaling via BCL9, resulting in cancer cell proliferation and metastasis27.Wnt/β-catenin signaling is involved in carcinogenesis not only in colorectal cancer but also in many other cancer entitiesWe hypothesized that hypoxia and HIF1α are involved in the osteo/odontoblast differentiation of human dental pulp stem cells (hDPSCs) by activating Wnt/β-catenin signaling via BCL9 induced by hypoxia and HIF1α. The aim of this study was to elucidate the role of BCL9 in HIF1α-induced osteo/odontoblast differentiation of hDPSCs.The hDPSCs used in this study showed high expression of mesenchymal stem cell markers, CD44, CD73, CD90, CD105, and CD146. In contrast, CD34-expressing cells were rarely detected  promoted the mRNA expression of RUNX2, ALP, and OC in hDPSCs, which are representative osteo/odontoblast differentiation markers31 . This led us to the notion that mechanisms other than BMP signaling are involved in the hypoxia/HIF1α-dependent osteo/odontoblast differentiation in hDPSCs. Then, we focused on Wnt/β-catenin signaling, which is one of the essential signaling pathways involved in the differentiation of mineralized tissue-forming cells including hDPSCs24. Our study revealed that the mRNA expression of TCF1, a target gene of Wnt/β-catenin signaling, was promoted in hDPSCs cultured under hypoxic conditions and HIF1α-overexpressing hDPSCs . Hypoxic conditions were maintained with a gas mixture of 1% O2, 5% CO2, and 94% N2 (referred to as 1% O2) for 48 h. All methods used in this study were in accordance with the Declaration of Helsinki.All procedures were approved by the Ethical Committee of Tokyo Medical and Dental University (#D2014-039-03) and performed in accordance with the Ethical Guidelines for Clinical Studies. Informed consent was obtained from all participants in accordance with the Ethical Guidelines for Clinical Studies. Human dental pulp stem cells (hDPSCs) were obtained from freshly extracted wisdom teeth and were cultured in alpha-modified minimum essential medium  containing 10% fetal bovine serum  and an antibiotic and antifungal solution  at 37 °C and 5% CO5 cells) were suspended in FACS buffer [phosphate-buffered saline (PBS) containing 2% FBS]. Fluorescein isothiocyanate (FITC)-labeled mouse IgG1κ isotype control antibody , FITC-labeled anti-CD34 , anti-CD44 , anti-CD73 , anti-CD90 , anti-CD105 , and anti-CD146  were incubated with hDPSCs for 20 min on ice, and cells were applied to a flow cytometer .hDPSCs  coated with fibronectin  were cultured in a neurogenic differentiation medium containing B-27 supplement  for 3 days. Neurogenic differentiation was evaluated by the expression of neuronal markers, neurofilament medium (NF-M) and glia fibrillary acidic protein (GFAP), which were detected by immunohistochemical staining using anti-NF-M mouse IgG1 isotype monoclonal antibody  and anti-GFAP rabbit polyclonal antibody .3 cells) were cultured in an adipogenic differentiation medium containing methylisobutylxanthine , dexamethasone , and recombinant human insulin  for 25 days. Oil Red O  staining was performed to identify adipogenic differentiation.hDPSCs (6 × 103 cells) seeded in spheroid-forming culture plates  were cultured in a chondrogenic differentiation medium  for 25 days. Alcian Blue  staining was performed on the frozen sections (5 μm thickness).hDPSCs (1 × 104 cells) were cultured in an osteogenic differentiation medium containing l-ascorbic acid-2-phosphate , beta-glycerophosphate , dexamethasone , and BMP-2  for 21 days. Mineralized nodules were stained with Alizarin Red S .hDPSCs (5 × 1044 were provided by Dr. Tetsu Akiyama from the Institute for Quantitative Biosciences at The University of Tokyo. We made a β-catenin expression vector (pEF-V5-β-catenin) with the Gateway cloning system (Invitrogen/Thermo Fisher Scientific). A luciferase reporter vector, pGL4.49 [luc2P/TCF-LEF RE/Hygro] vector , was used to measure the TCF/LEF activity. An enhanced green fluorescent protein (EGFP) expression vector  containing cytomegalovirus promoter was used as a control. All vectors were transfected into hDPSCs using FuGENE (Promega), in accordance with the manufacturer’s instructions.Mammalian expression vectors containing cytomegalovirus promoters, HA-HIF1α-P402A/P564A mutant-pcDNA3 , were used to induce the transient expression of HIF1α. Double point mutations, P402A and P564A, in mtHIF1α prevent degradation and facilitate the transcriptional activation of HIF1α. BCL9 overexpression vectors [pEGFP-BCL9-full and pCS2(+)-FLAG-BCL9-full]BCL9-specific siRNAs (Ambion/Thermo Fisher Scientific) were transfected into hDPSCs with Lipofectamine RNAiMAX Reagent (Invitrogen/Thermo Fisher Scientific). Scrambled siRNA (Ambion/Thermo Fisher Scientific) was used as a control.Wnt inhibitor  was dissolved in dimethyl sulfoxide (Sigma-Aldrich) and used at a concentration of 10 μM.5 cells/well in a six-well culture plate. Total RNA was isolated with QuickGene-Mini80 , and cDNA was synthesized from 150 ng of total RNA using PrimeScript™ RT Master Mix . Quantitative RT-PCR was performed with GoTaq qPCR Master Mix (Promega). β-Actin was used as an internal control. The specific primers used in this study are shown in Table hDPSCs were plated at a concentration of 2.0 × 104 cells/well in a 24-well plate and were lysed 24 h after transfection using 100 μL of lysis buffer . Luciferase activity was measured using a luciferase assay substrate  and a luminometer .hDPSCs were plated at a concentration of 5.0 × 104 cells/well) in 24-well plates were lysed in 100 μl of radioimmunoprecipitation (RIPA) buffer  containing a protease inhibitor cocktail  and a phosphatase inhibitor cocktail . Cell lysates for nuclear and cytoplasmic fractions were prepared with NE-PER nuclear and cytoplasmic extraction reagent (Thermo Fisher Scientific), in accordance with the manufacturer’s instructions. Samples were separated with 10% SDS-PAGE and transferred to Immobilon PVDF transfer membranes . After blocking with PVDF blocking reagent , the membrane was incubated with anti-HIF1α rabbit polyclonal antibody , anti-BCL9 rabbit polyclonal antibody , anti-β-catenin rabbit IgG monoclonal antibody , horseradish peroxidase (HRP)-labeled anti-β-actin antibody  and anti-Lamin B1 mouse monoclonal antibody  overnight at 4 °C. After washing with Tris-buffered saline containing Tween 20 (0.1% v/v), the membrane was incubated with HRP-conjugated anti-rabbit IgG  for 1 h at room temperature, except for the HRP-labeled anti-β-actin antibody-applied membrane. All protein bands were visualized using a chemiluminescent HRP substrate , and chemical luminescence of the membranes was captured by the digital imaging system LAS 3000 Mini .hDPSCs (5 × 105 cells/dish) in 60 mm dishes were lysed in 1 ml of RIPA buffer after the overexpression of BCL9 (FLAG Tag) and β-catenin (V5 Tag) for 24 h. V5 mouse IgG2bκ isotype antibody  was added as a primary antibody and reacted at 4 °C for 1 h, and then reacted with Protein G PLUS-Agarose (Santa Cruz Biotechnology) at 4 °C overnight under rotation. The samples were then centrifuged at 2500 rpm for 5 min, and the supernatant was aspirated and washed four times. The immunoprecipitated samples were checked for protein expression by western blotting using FLAG mouse IgG2aκ isotype antibody .hDPSCs  and in compliance with the ARRIVE guidelines. All experimental protocols were approved by Institutional Animal Care and Use Committee of the TMDU (Reference number A2021-251A). Six-week-old ICR mice  were sacrificed under CO® O.C.T. Compound . Six-micrometer-thick frozen sections were prepared. Anti-HIF1α rabbit polyclonal antibody , anti-BCL9 rabbit polyclonal antibody , and anti-β-catenin mouse IgG1κ monoclonal antibody  were used for primary antibodies, and samples were incubated with one of the primary antibodies overnight at 4 °C. Alexa Fluor 488-conjugated goat anti-rabbit IgG  or Alexa Fluor 594-conjugated goat anti-mouse IgG  was used as a secondary antibody and applied for 60 min at room temperature. Mounting Medium with DAPI  was used for nuclear staining.Pulp tissues were fixed with 4% paraformaldehyde at 4 °C overnight, after which they were embedded in Tissue-TekF test, Mann–Whitney’s U test, and Student’s t test were performed with significance set at p < 0.05.Statistical analysis was conducted using GraphPad Prism 6 . Supplementary Figures.


Androgenetic alopecia (AGA) remains an unsolved problem for the well-being of humankind, although multiple important involvements in hair growth have been discovered. Up until now, there is no ideal therapy in clinical practice in terms of efficacy and safety. Ultimately, there is a strong need for developing a feasible remedy for preventing and treating AGA. The Wnt/β-catenin signaling pathway is critical in hair restoration. Thus, AGA treatment via modulating this pathway is rational, although challenging. Dickkopf-related protein 1 (DKK1) is distinctly identified as an inhibitor of canonical Wnt/β-catenin signaling. Thus, in order to stimulate the Wnt/β-catenin signaling pathway, inhibition of DKK1 is greatly demanding. Studying DKK1-targeting microRNAs (miRNAs) involved in the Wnt/β-catenin signaling pathway may lay the groundwork for the promotion of hair growth. Bearing in mind that DKK1 inhibition in the balding scalp of AGA certainly makes sense, this review sheds light on the perspectives of miRNA-mediated hair growth for treating AGA via regulating DKK1 and, eventually, modulating Wnt/β-catenin signaling. Consequently, certain miRNAs regulating the Wnt/β-catenin signaling pathway via DKK1 inhibition might represent attractive candidates for further studies focusing on promoting hair growth and AGA therapy. Hair growth is a relatively complex process. The human hair cycle comprises three main phases: anagen, catagen, and telogen . Anagen Hair growth is controlled and influenced by various endogenous factors, including intracellular and intercellular signaling molecules. Some of the influencing factors have inhibitory and some stimulatory effects on the hair cycle. The initiation of the anagen phase is inhibited by the activation of bone morphogenetic proteins (BMP) as well as transforming growth factor β (TGF-β) signaling . On the According to the aforementioned evidence, it is prudent to postulate that inhibition of DKK1—a canonical Wnt/β-catenin signaling inhibitor—is one of the key factors to promote hair growth and develop a sustainable remedy for AGA. Indeed, there are certain microRNAs (miRNAs) that target DKK1 and are involved in hair growth-related pathways. Thereby, in this review, the possible application of miRNAs for AGA therapy via targeting upregulated DKK1 expression in the balding scalp and activating Wnt/β-catenin is postulated.The canonical Wnt/β-catenin signaling is one of the most studied pathways in biology as it is responsible for a number of vital physiological processes in the body . ControlDuring the canonical Wnt/β-catenin signaling, Wnt ligands  bind the frizzled receptor and LRP5/6 co-receptor outside of the plasma membrane , which iAlthough SFRP2 is also considered to be the inhibitor of the Wnt/β-catenin signaling pathway, some conflicting data exist. Kwack et al. have studied the influence of SFRP2 on cultured human DP cells obtained from patients undergoing hair transplantation via punch biopsy. The results showed that treatment with recombinant human SFRP2 remarkably increased Wnt/β-catenin signaling . Thus, DDKK genes encode secreted proteins that regulate the Wnt/β-catenin signaling pathway by antagonizing it . RemarkaDKK1 exhibits a dual role in the normal hair cycle. On the one hand, DKK1 induces attenuation of the hair growth process by inhibiting Wnt/β-catenin signaling via the LRP5/6 co-receptor and, on the other hand, it promotes apoptosis of keratinocytes, which are key cells involved in hair growth ,40. HencDKK1 inhibits Wnt/β-catenin signaling via binding to LRP5 and LRP6, which prevents the interaction between Wnt and the other transmembrane receptor frizzled . The inhAccording to the abovementioned information, the study of DKK1 inhibition strategies is an important tool for maintaining the Wnt/β-catenin signaling within the normal range. Indeed, some studies investigated the effect of certain natural compounds on the canonical Wnt/β-catenin signaling pathway for promoting hair growth. Zhou et al. have observed the impact of morroniside—a natural compound found in cornelian cherry—on the Wnt/β-catenin signaling pathway in cultured human ORS cells. In their results, morroniside indeed increased the proliferation of cells as well as the concentration of β-catenin. Concomitantly, the outcome of morroniside treatment was retrieved by the influence of DKK1 . AnotherC. elegans that could downregulate the level of Lin-14 protein mRNA [miRNA was first identified in 1993 by Victor Ambros and colleagues, who found a short RNA molecule Lin-4 of 22–61 nucleotides in ein mRNA . Seven yein mRNA ,63. In 2ein mRNA . Currentein mRNA ,66. Howeein mRNA , Givosirein mRNA , and Lumein mRNA , while tein mRNA  for siRNein mRNA ,72, it sImportantly, miRNAs play a crucial role in hair growth regulation . They arUp until now, there are no clinical trials regarding miRNA therapy for hair growth. To develop the miRNA-based drug for AGA treatment, the appropriate miRNA candidates should be carefully selected, which is the main purpose of this manuscript. Before getting straight to that point, other available approaches are also discussed. Notably, there was no commercially available mRNA-based vaccine before the COVID-19 outbreak. However, effective mRNA vaccines that are being widely used nowadays are hailed as a milestone in vaccinology. Similarly, miRNA-based drugs may become another breakthrough in drug development in the near future. Moreover, some miRNAs are already in clinical trials .Among the currently accessible methods of AGA treatment, transplantation is considered to be the conventional technique, e.g., platelet-rich plasma therapy, follicular unit extraction, and strip harvesting. However, the effectiveness still needs to be studied properly . In sevemiRNAs are already evidenced to play a substantial role in HF development. The functions of miRNAs are very diverse. The same miRNA has the ability to regulate multiple gene expression. Except for hair growth, miRNAs are related to having a strong influence on osteogenesis, the wound-healing process, tumorigenesis, etc. miRNAs are considered strong gene regulators as they control gene expression. Primary miRNA (pri-miRNA)—the initial form of these small non-coding RNA molecule—is transcribed from introns, then cleaved by an enzyme, Drosha . As a reSince the first identification of miRNAs in the human body, the link between miRNAs and hair growth regulation was also soon discovered . HoweverAccording to the abovementioned description, hair growth is a complex process that consists of three main phases. Normally, in the telogen phase, hair falls from the scalp and a new anagen starts to induce a new hair growth cycle. In patients with AGA, the transition from the telogen to anagen phase is hindered. The shifting process is controlled by various signaling pathways that can induce or delay transition between phases . miRNAs Although their principal function in the body is already evidenced, there are still aspects that need to be elucidated. There are a number of miRNAs that play certain roles in different stages of HF development . Indeed,Interestingly, miR-125 is abundantly expressed in balding DP cells . MoreovemiRNAs are greatly multifunctional agents and the evidence from different sources is often puzzling, e.g., chi-miR-130-5p positively affects the Zhongwei goat hair development , while tmiRNAs are versatile in terms of gene regulation. At the same time, the same miRNA targets a number of proteins. miR-29a is an example of accommodating diverse functions. miR-29s are encoded by two gene clusters and are transcribed by RNA polymerase II. The miR-29 family comprises three mature members: miR-29a, miR-29b, and miR-29c . The seeThe studies have demonstrated that miR-29a inhibits the expression of DKK1, KRM2, and SFRP2, and activates Wnt/β-catenin signaling ,167. HsuHpHr  mice. These mice exhibit hair loss and overexpression of protein hairless. The study demonstrated that the miR-31 level was significantly downregulated in the skin of HpHr/HpHr mice [miR-31 has a multifunctional capacity as it targets a number of genes and pathways . It is hrHp mice . Chen et al. have studied the miR-31-5p regulatory role in osteosarcoma cells via the Wnt/β-catenin signaling pathway. The study demonstrated that miR-31-5p targets AXIN1, which is part of the destruction complex and takes part in the reduction of β-catenin levels, and thus inactivates the canonical Wnt/β-catenin signaling pathway. Thereby, the downregulation of miR-31-5p has an inhibitory effect on the proliferation of osteosarcoma cells via overexpression of AXIN1 . Except miR-103 is homologous to miR-107 . miR-107These results support the assumption that miR-103/107 may promote hair growth via activating Wnt/β-catenin signaling by targeting DKK1 and AXIN2, and therefore, these miRNAs deserve attention for further studies on AGA. In addition, Wang et al. have demonstrated that miRs-103/107 are expressed in the epidermis and HFs of mice and are downregulated with age. The authors suggest that miR-103/107 may be one of the key factors in sustaining the HF stemness with age .Notably, miR-152 with miRNAs 199a, 126, 143, and 214 have been abundantly found in HFs ,156. MormiR-203 is among the most abundantly expressed miRNAs in epidermis . InteresmiR-218 via its role in Wnt/β-catenin signaling represents a hot spot molecule in cancer studies, as dysregulation of this miRNA is strongly associated with different cancers ,215,216.RNA therapeutics need an effective technique to be delivered to the target cells, avoiding in vivo nuclease-mediated degradation. This is one of the major challenges in miRNA-based treatment along with specificity, stability, immune activation, and toxicity in vivo and in vitro . Local dCollectively, DKK1 has a critical role in the development of human AGA. Inhibition of DKK1 levels in the balding area of the scalp in populations that are at high risk of developing AGA might prevent the further progression of this disorder. On the other hand, it might even overcome the problem of hair regrowth via stimulating Wnt/β-catenin signaling. Therefore, assessment of DKK1-targeting miRNAs that are expressed in human HFs and involved in the Wnt/β-catenin signaling pathway may lay the groundwork for developing strategies of promoting hair regeneration and treatment of AGA. Nevertheless, further studies are needed to validate the described phenomenon. Additionally, miR-103/107, miR-203, and miR-218 among other miRNAs might represent the attractive therapeutic candidates for further studies focusing on modulation of the Wnt/β-catenin signaling pathway via regulating DKK1. Prominently, the functional spectrum of miRNAs is wide. Although certain miRNAs are capable to inhibit the Wnt antagonist, they might also target other key molecules that are instrumental in the regulation of this pathway. miR-29a ideally exemplifies the described complexity ,168. Thu