Broiler nuggets supplemented with 2% BPP (T1) received the greatest sensory scores in terms of flavour, tenderness, and juiciness. Results of current study revealed the potential of BPP to be utilized as an effective natural source of fibre supplementation in food products along with enhanced antioxidant and anti-microbial properties.Banana peel powder is considered one of the most nutritive and effective waste product to be utilized as a functional additive in the food industry. This study aimed to determine the impact of banana peel powder at concentrations of 2%, 4%, and 6% on the nutritional composition, physicochemical parameters, antioxidant potential, cooking properties, microbial count, and organoleptic properties of functional nuggets during storage at refrigeration temperature for 21 days. Results showed a significant increase in nutritional content including ash and crude fiber ranging from 2.52 ± 0.017% to 6.45 ± 0.01% and 0.51 ± 0.01% to 2.13 ± 0.01%, respectively, whereas a significant decrease was observed in crude protein and crude fat ranging from 13.71 ± 0.02% to 8.92 ± 0.02% and 9.25 ± 0.02% to 4.51 ± 0.01%, respectively. The incorporation of banana peel powder significantly improved the Water Holding Capacity from 5.17% to 8.37%, cooking yield from 83.20 ± 0.20% to 87.73 ± 0.16% and cooking loss from 20.19 ± 0.290% to 13.98 ± 0.15%. Antioxidant potential was significantly improved as TPC of functional nuggets increased ranging from 3.73 ± 0.02 mg GAE/g to 8.53 ± 0.02 mg GAE/g while a decrease in TBARS  was observed. Furthermore, functional broiler nuggets depicted a significantly reduced total plate count (3.06–4.20 × 10 Musa balbisiana) is a tropical and subtropical crop that is the most important fruit crop after rice crop, wheat crop, and maize globally. Over the past 20 years, banana production has steadily increased, going from over 70 million tonnes in 1999 to nearly 117 million in 2019 , minerals , amino acids , and polyunsaturated fatty acids, particularly linoleic acid . Banana Dietary fiber is a non-starch polysaccharide having resistance to absorption and digestion by the enzymes present in the human gastrointestinal tract. Since the human body cannot digest and absorb dietary fiber, moisture absorption in the digestive system is affected . Fiber sNutritionally, meat is a good source of high-value protein and essential fatty acids, vitamins, and minerals . Among ai.e., bread, garlic powder, ginger powder, white pepper, soya sauce, iodized salt, and all chemicals and solvents used in this experiment were of analytical grade purchased from Descon OxyChem Ltd.  and ICI Pakistan Ltd. .The bananas were procured from the local market of Lahore, Pakistan. Boneless fresh broilers meat was purchased from the local groceries store in Lahore, Pakistan. The meat was packed in small bags of LDPE and held in a refrigerator at (4 ± 1 °C), which later was used for functional nuggets formulation. The other ingredients used in nuggets preparation, The purchased bananas were washed with water to eliminate dust and debris. The pulp and peel of the banana fruit were separated. Banana peels were cut into small pieces and treated with a 0.5% (w/v) solution of citric acid for 10 min to prevent enzymatic browning. The solution of citric acid was drained and cut pieces of peels of bananas were dried using a hot air oven  at a temperature of 40 °C for 48 h. After that, the banana peels were dried, ground, and then screened using 60 mesh (250 μm) screens, resulting in banana peel powder . The peeProximate parameters of banana peel powder, such as moisture, crude protein, crude fat, total ash, and crude fiber were determined as per standard protocols of The antioxidant potential of banana peel powder was measured by DPPH  assay following the method of , using mMinced broiler meat and bread were mixed along with spices garlic powder, ginger powder, salt, white pepper, and soya sauce according to the formulation mentioned in The proximate composition  was analyzed using standard protocols . The carThe cooking yield was calculated by subtracting the weight of cooked nuggets from the weight of raw nuggets according to the method provided by 5/g) of the sample.Microbial load  of all treatments were evaluated by using method of After air frying, five trained panellists assessed each treatment of broiler nuggets enriched with banana peel powder for organoleptic qualities. The test was conducted using a 9-point hedonic scale, where nine stood for strongly like and one for extremely dislike . Sensorypost hoc analysis were performed. p-values of (p < 0.05) were regarded as statistically significant for all tests.Physicochemical and sensory properties of broiler nuggets were examined in relation to the impacts of adding banana peel powder using descriptive statistics ), t-test, Analysis of Variance (ANOVA). Version 25.0 of Statistical Package for the Social Sciences (SPSS) software was used to analyse the data. For multiple comparisons, two-way ANOVA and least significant difference (LSDs) Nutritional composition of hot air oven-dried banana peel powder is presented in The results of the physicochemical analysis of all treatments are depicted in p < 0.05) and was observed to be in the range of 6.71 ± 0.017% to 9.92 ± 0.017%. Analysis of pH indicated that pH values of nuggets supplemented with banana peel powder were decreased to a statistically significant level (p < 0.05). An increase in total phenolic content was observed in functional nuggets (3.73 ± 0.017 mg GAE/g to 8.53 ± 0.017 mg GAE/g) as compared to banana peel powder. The highest antioxidant activity was observed in the sample with 6% banana peel powder. However, a significant decrease in the total phenolic content was observed with the storage time of 21 days and the least TPC was observed at day 21. Analysis for TBARS values indicated that it decreased significantly (p < 0.05) with the addition of banana peel powder. The least values of TBARS were observed for the T3 sample supplemented with 6% banana peel powder.Physicochemical parameters of functional nuggets containing different concentrations of banana peel powder given as 2%, 4%, and 6% are depicted in p < 0.05) on the cooking attributes of broiler meat nuggets. Results showed that cooking yield of nuggets varied from 85.08–89.16% during storage. T3 (6% BPP) showed highest cooking yield (90.80%) on day 21 and the control sample (T0) 0% BPP presented the lowest cooking yield (83.20%) at 0 day. A significant decrease (p < 0.05) was observed in cooking loss and it ranged from 20.19 ± 0.29% to 13.98 ± 0.15% for nuggets supplemented with BPP in comparison to the control. Physiochemical analysis revealed that treatment containing 6% BPP (T3) showed best results at day 21 as compared to control and all other treatments.The data regarding cooking yield and cooking loss is depicted in 5/g) was detected in nuggets having banana peel powder as compared to the control (2.81 ± CFU × 105/g). The maximum microbial load (4.20 CFU × 105/g) was observed in the control treatment without banana peel powder while the lowest total plate count (3.06 CFU × 105/g) was observed in T3. During the storage time of 21 days, the bacterial load of nugget samples depicted a significant increase (p < 0.05).The bacterial load of broiler nugget samples supplemented with banana peel powder is displayed in The sensory scoring for different organoleptic attributes of control and treated samples is illustrated in The moisture content observed in banana peel powder was in line with Similar findings were made for moisture analysis by Color scores of nuggets supplemented with banana peel powder were reduced because of the dark brown colour of banana peel powder. The taste was perceived as less in the control sample due to the presence of starch which provides a blunt flavor and hard texture. The scoring of sensory evaluation agrees with the findings of Banana peel powder is a good source of fiber and bioactive compounds having possible health-stimulating benefits and enhancing the quality of broiler nuggets. Its supplementation at concentrations of 2%, 4%, and 6% enhances the shelf life of broiler nuggets because of its antimicrobial and antioxidant potential. Proximate parameters of broiler nuggets such as moisture, crude protein, crude fat, and carbohydrates were significantly affected by the addition of banana peel powder, but ash and crude fiber showed non-significant effects during storage time of 21 days at 4 °C. Furthermore, the incorporation of banana peel powder significantly improved the quality parameters such as WHC, TPC, and cooking characteristics of the product along with considerably reduced lipid oxidation and microbial proliferation during the whole storage time. Sensory evaluation indicated that broiler nuggets made with 2% banana peel powder were most liked by the sensory panel whereas broiler nuggets made with 6% banana peel powder were liked least but in the acceptable score.10.7717/peerj.14364/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.14364/supp-2Supplemental Information 2Click here for additional data file.


Anterior cervical discectomy and fusion (ACDF) is an often performed procedure in spine neurosurgery. These are often performed using an operating microscope (OM) for better illumination and visualization. But its use is limited to the surgeon and the assistant. There is difficulty in maneuvering long surgical instruments due to the limited space available. Exoscope (EX) has been used as an alternative to microscopes and endoscopes. We used an EX in patients undergoing ACDF for cervical spondylotic myelopathy.A prospective comparative trial was conducted to test the safety and usability of a low-cost EX compared to a conventional surgical binocular OM in ACDF. Twenty-six patients with degenerative cervical myelopathy symptoms were operated by ACDF assisted by the EX and OM between December 2021 and June 2022. The authors collected and compared data on operative time, intraoperative hemorrhage, hospital admission, and complications in the two groups.There were no statistically significant differences between the two groups in mean operative time, hospital stay, or postoperative complications. The average intraoperative blood loss was significantly more in the OM group. There were no surgical complications related to the use of the EX or OM. The comfort level, preoperative setup and intraoperative adjustment of position and angle of the EX were rated higher than the OM group. The image quality, depth perception, and illumination were rated as inferior to that of the OM. The low-cost EX was rated to be superior to that of the OM with regard to education and training purposes.Our study showed that the low-cost EX appears to be a safe and effective alternative for OM-assisted ACDF with great comfort and ergonomics and serves as an essential tool for education and training purposes. However, some limitations of our EX included slightly inferior image quality and illumination when compared with the OM. Anterior cervical discectomy and fusion (ACDF) is considered the standard treatment of disc herniation in the context of degenerative cervical myelopathy (DCM) causing anterior neural compression , 2. It iSince the introduction of exoscope (EX), it has been used as an alternative to microscopes and endoscopes , 3, 5, 7High purchase costs prevent the widespread use of OM and EX in resource-constrained areas. We have previously described a more cost-friendly and simple EX constructed of industrial digital microscope parts with enough capacity for magnification and luminescence during both cranial and spinal surgeries , howeverWe used an EX in patients with an ACDF approach for disc herniation in the context of DCM.A prospective comparative trial was conducted to test the safety and usability of a self made low-cost EX compared to a conventional OM in ACDF. The study was performed in the Department of Spine Surgery at the Hospital of the Russian Academy of Sciences.From 32 patients that underwent single-level myelopathy between December 2021 and June 2022, a total of 26 patients were enrolled, 13 of whom underwent EX assisted ACDF and the rest with OM. Patients were chosen in a randomized fashion. All had DCM presentations that were identified and confirmed by magnetic resonance imaging (MRI). All patients underwent single-level ACDF with the assistance of either the OM or EX. Patients with persistent myelopathic symptoms despite 6 weeks of conservative treatment were also included, in contrast, those with radiculopathy, tumors, infections, trauma, deformity, and a past history of cervical spine surgery were excluded.TCTR20221103003.All the surgeries were performed by a senior neurosurgeon who was assisted by a resident. Operations in both groups were performed by the same surgeons to avoid bias. All the surgeons received 2 weeks of mandatory training in EX before using it in a real patient. Patients with degenerative disease planned for single-level ACDF using an EX were enrolled in a prospective cohort study. Similar age, sex and procedure-matched patients planned for OM-assisted ACDF served as the control group. We were granted ethical committee approval and Thai clinical trials registry number The patient demographic characteristics, radiological features, operative records, and complications were recorded. Postoperatively, the patients were assessed for new or worsened sensory or motor deficits and bladder bowel dysfunction.Immediately at the end of each procedure, a survey was conducted where the participants had to answer a standardized questionnaire that included questions regarding the image quality of the EX, the illumination of the operative field, adjustment of magnification, focal length, depth perception, position and working angle of the EX, intraoperative handling of the instrument, convenience for the operating surgeon, assistant and scrub nurse, and the need to convert to OM during the surgery. Finally, we inquired if in the future, they would continue using the EX and recommend it to their colleagues. Postoperatively, the video recordings were analyzed to measure the operative time and evaluate the image quality when the EX or OM was used.t-test if normally distributed. The p value of <0.05 was considered statistically significant.Statistical analysis was implemented using SPSS 26.0 statistical software . Categorical variables were compared using Chi-square test. Continuous variables were expressed as mean and standard deviation and compared using We used a previously described device , this EXA total of 26 subjects with DCM were included in this study, consisting of the levels C3–C4 (three cases), C4–C5 (11 cases), C5–C6 (11 cases) and C6–C7 (one case). In the totality of patients, the compression was successfully removed. The mean age was 51.6 ± 10.9 years (range of 34–71 years); The mean operation time was 113.8 ± 14.7 min (range of 88–138 min); The mean surgical blood loss was 30.4 ± 8.2 ml (range of 20–50 ml); The mean hospital stay was 3.2 ± 0.5 days (range of 3–5 days). The patient demographics, diagnosis, and operative data are shown in p = 0.021). The mean hospital stays were longer in the EX group compared to the OM group (3.38 ± 0.650 vs. 3.08 ± 0.277). No intraoperative complications occurred in either group. The comparison between the two groups is shown in Twenty-six operations were performed. Each surgery was performed by either two consultants or a consultant and a resident. There was no interruption during the procedure due to any technical problem. There was no conversion from the EX to the OM in any procedure. The operative time was less in the EX group when compared to the OM group although not statistically significant. The average intraoperative blood loss was significantly more in the OM group compared to the EX group . Verification of the low-cost EX was done prior to starting the procedure by confirming the support arm movement, zoom of the camera and light intensity. As previously described  illuminaVariable positioning of the camera can sometimes be a challenging consequence of the low versatility in the arm support movement, something that could be fixed by adapting another type of arm, however, spine surgery does not require significant movement as the camera remained in the same position most of the surgery.The 32GB disc space allowed only 5 h of recording time, making it imperative to transfer files between surgeries. We found no intraoperative complications. The operating room team experience, including surgeon, assistant, nurses and anesthesiologist responded positively to the use of the low-cost EX. By everybody looking at the screen, superiority in teaching and feeling of inclusiveness in comparison with OM were the main consensus by the team.We interviewed 10 neurosurgeons. Of them, 60% were residents and the remaining consultants. Most (70%) of them found the brightness inferior to the conventional OM and the remaining 30% found the brightness similar to the OM. All agreed that the image quality of the surgical field was inferior to OM, nevertheless, zooming received a neutral response, we recommend taking into account the type of surgery before using the EX , 2.About 70% of them felt that the ease of using the EX was similar to the OM. The same percentage of residents and scrub nurses strongly agreed that it was better for them as compared to a conventional OM, this was similar to the response to teaching, where 80% strongly agreed it was easier when using the EX. This follows previous reports of the main strength when compared with OM is that allows participation of every member in the operating room .Almost all (90%) agreed that it was easier to set up the EX compared to OM preoperatively. The adjustment during surgery got a positive response, with almost 60% agreeing it was easier to adjust the position and angle of the EX than conventional OM.None of the operating surgeons felt the need to switch to conventional OM during the procedure. All of them unanimously approved the low-cost EX and agreed to recommend or use it again.OM has become an indispensable part of spine surgery since its introduction in 1977 , 6. It iIn the present study, there was no conversion from the EX to the OM in any procedure. The mean operative time and intraoperative blood loss were less in the EX group compared to the OM group. The mean hospital stay was more in the EX group compared to the OM group. There were no surgical complications related to the use of the EX or OM. These results were similar to previously published reports on the use of EX systems in neurosurgery –7.We were able to achieve superior operative results with our low-cost EX compared to the conventional binocular OM. This device has gained widespread popularity due to ergonomics and improvement in the surgeon's comfort , 8, 11. Our study revealed some significant limitations of this system, including slightly inferior image quality and illumination compared with the conventional binocular OM. About 70% of the surgeons found the brightness inferior to the conventional OM and almost all of them agreed that the image quality of the surgical field was inferior to binocular OM. Our findings are consistent with previous literature showing that depth perception, quality of the image, and illumination of the EX were rated as inferior to the OM in ACDF procedures , 6. HoweAccording to our senior neurosurgeons users, the lack of stereopsis was a major drawback, substantiating previous findings in literature , 8, 11. The study aim was to evaluate our operative experience with an initial patient cohort. The size of the sample size makes statistical analysis not possible. We elected this specific surgical procedure because of the relative simplicity and familiarity for neurosurgeons worldwide. However, using the low-cost EX for surgeries with higher complexity needs further investigation. In addition, some bias could be present as the survey was filled by some authors that are included in the article. We believe that it is imperative to continue the investigation into the potential uses of the low-cost EX to determine its reliability and efficacy when compared to the OM. Larger cohort of patients could prove indispensable to establish this.Our study demonstrated that the low-cost EX it's a safe and effective alternative for OM-assisted ACDF with the benefit of great ergonomy, comfort and simplicity and furthermore serves as an essential tool for education and training purposes. Notwithstanding, our study revealed some crucial limitations of this technology, including slightly lower image quality and illumination compared with the conventional binocular OM. The low cost exoscope can be used as a surrogate for OM in spinal surgery to accomplish great surgical outcomes.


The WNT–β-catenin signaling pathway has a major role in regulating cell proliferation and differentiation. Aberrant activation of the pathway contributes to various human cancer types. Because casein kinase CK1α-initiated phosphorylation of β-catenin is a key first step to restrain WNT signaling, effective restoration of CK1α activity represents an innovative strategy to combat WNT-driven cancer. A recent study in JBC reveals the anthelmintic pyrvinium directly binds to CK1α as an activator and also stabilizes CK1α protein, doubling against WNT-driven cancer activity. Wnt gene discovery about 40 years ago, evolutionarily conserved Wnt signaling has attracted tremendous attention due to its essential roles in regulating embryonic development and governing stem cell proliferation and differentiation  and the β-catenin–dependent pathway . While less understood, the noncanonical pathway is involved in cytoskeleton rearrangement, cell polarity, and migration  or in promoting proliferation and antagonizing the canonical pathway through the WNT/Ca2+ branch. On the other hand, strongly associated with human diseases and cancer, the canonical pathway has been the focus of most WNT-related studies. In this pathway, WNT binds to one of 10 Frizzled (FZD) receptors and the coreceptors LDL-receptor–related proteins 5 and 6 (LRP5 and LRP6), transduces the signal through the FZD-associated scaffolding protein Dishevelled (DVL), and recruits binding protein AXIN to the cytoplasm membrane, leading to the release of β-catenin from the dissembled destruction complex containing AXIN, adenomatous polyposis coli (APC), glycogen synthase kinase 3β, casein kinase 1α (CK1α), the E3-ubiquitin ligase β-TrCP, and protein phosphatase 2A (c-Myc and cyclin D1 by displacing TCF/LEF-bound corepressors and/or recruiting coactivators (Since the first ntiation . The sigtivators  2).Figu.FiguWnt The essence of canonical WNT signaling activation is the stabilization of β-catenin protein. Effective elimination of free cytoplasmic β-catenin is the key to keeping the canonical pathway in check under physiological conditions. The “priming” step of β-catenin degradation involves the CK1α-initiated phosphorylation of β-catenin at its Ser45 residue, which enables glycogen synthase kinase 3β to phosphorylate the Ser33, Ser37, and Thr41 residues, creating the binding site for the adapter protein β-TrCP that complexes with other proteins to promote β-catenin ubiquitination and subsequent proteasomal destruction. Interestingly, as a critical WNT negative regulator, CK1α stability is negatively regulated by WNT through E3-ubiquitin ligase component Cereblon (CRBN)-initiated degradation (APC or AXIN or gain-of-function/oncogenic mutations of β-catenin (Perhaps, upward of half of all human solid tumors and leukemias harbor aberrant constitutive activation of canonical WNT signaling through genetic and/or epigenetic alterations of the key players in the pathway, such as loss-of-function mutations of -catenin , 5. ThusXenopus laevis eggs (Repurposing current FDA-approved drugs is a time-saving and cost-effective alternative to cancer drug discovery. One such FDA-approved drug, pyrvinium, was identified as a potent inhibitor of WNT signaling through a biological function-based screen for compounds that could stabilize AXIN and promote β-catenin turnover in vis eggs . Mechanivis eggs . Pyrvinivis eggs . Pyrvinivis eggs . Since ivis eggs .et al. (via interaction with the E3-ubiquitin ligase component Cereblon. Specifically, through a series of biochemical and cell biology assays, pyrvinium was shown to disrupt the interaction between Cereblon and CK1α at the immunomodulatory drug– binding pocket within the Cereblon–CK1α complex (More recently, Shen et al.  revealed complex , which w complex . These i complex .While the evidence surrounding pyrvinium’s activation of CK1α and subsequent inhibition of WNT signaling is convincing, it is conceivable that, like many other small molecule drugs, pyrvinium may exert its anticancer activity by targeting other cellular processes in addition to WNT. Pyrvinium indeed has been shown to target mitochondria under nutrient-depleted conditions, impair JAK/STAT signaling, downregulate AKT/PKB signaling, or target autophagy addiction as a part of its anticancer mechanisms, which indicates pyrvinium might be a home run. However, a major hurdle for pyrvinium’s clinical anticancer use is its low bioavailability, which may demand innovative delivery strategies. Nonetheless, pyrvinium presents an exciting prospect of being one of the first potent WNT-targeting agents for cancer treatment.The author declares that they have no conflicts of interest with the contents of this article.


Highly regulated intracellular calcium entry affectsnumerous cellularphysiological events. External regulation of intracellular calciumsignaling presents a great opportunity for the artificial regulationof cellular activity. Calcium entry can be mediated by STIM proteinsinteracting with Orai calcium channels; therefore, the STIM1–Orai1pair has become a tool for artificially modulating calcium entry.We report on an innovative genetically engineered protease-activatedOrai activator called PACE. CAD self-dimerization and activation wereinhibited with a coiled-coil forming peptide pair linked to CAD viaa protease cleavage site. PACE generated sustained calcium entry afterits activation with a reconstituted split protease. We also generatedPACE, whose transcriptional activation of NFAT was triggered by PPVor TEV protease. Using PACE, we successfully activated the nativeNFAT signaling pathway and the production of cytokines in a T-cellline. PACE represents a useful tool for generating sustained calciumentry to initiate calcium-dependent protein translation. PACE providesa promising template for the construction of links between variousprotease activation pathways and calcium signaling. Ca2+/calcineurin/nuclear factorof activated T-cells (NFAT) signaling modulates immune response withthe transcriptional regulation of cytokines and chemokines in immunecells; NFAT activity is critical for Th cell differentiation.2 An assembly of plasma membrane- and endoplasmicreticulum (ER)-based ion channels and transporters orchestrates theconcentration of intracellular Ca2+. The duration and amplitudeof the Ca2+ signal produced by opening these channels governa Ca2+-dependent signaling response.3Intracellular free calcium ions  and the plasma membrane Ca2+ release-activatedCa2+ (CRAC) channel Orai1. Depletion of Ca2+ in the ER leads to conformational changes in the STIM1 ER domain,resulting in the exposure of the effector domain of the molecule,the CRAC activating domain .5 This domain binds to the intercellular C-terminus of the Orai1 channeland renders the channel open.6 Studieshave shown that CAD alone or even its truncated version STIM/Oraiactivating region (amino acid residues 344–442) is sufficientto fully activate the channel.7 The monomericCAD forms a distinct letter R shape, which consists of four α-helicalregions. Structural and mutagenic analysis of the domain has alsoshown that CAD dimerization is vital to Orai activation, and identifiedimportant residues enabling dimerization.8 In the same study, it has also been shown that the mutation of residuesL347, W350, and L351 of α helix 1 (CC2) and W430, I433, andL436 of α helix 4 (CC3), which form interactions between thehelices, hinders the ability to activate Orai1.Store-operated Ca2+ to activate transcriptionalactivity has already been used in the field of optogenetics, in whichlight-sensitive protein domains serve as switches to activate theeffector domain from the ER calcium sensor protein STIM111 or the CRAC channel Orai1.12 The roleof Ca2+ as a transcriptional regulator has been exploredin the combination with NFAT as a Ca2+-regulatory domain.14 The activation of the NFAT signalingpathway depends on long Ca2+ signals that keep the dephosphorylatedNFAT in the cell nucleus.15 As CRAC currentis used to maintain such signals,16 itscomponents are promising tools for the activation of the artificialNFAT pathway. To date, the modulation of the STIM1 pathway has involvedthe use of intact STIM1 domain architecture. We seek to explore thepossibility of regulating the intermolecular contacts of the CAD domain.Proteolytic activity has already been described as a fast and modularoutput of a synthetic circuit.18 Proteolytic cleavageis an irreversible process, and the proteolytically processed effectorscould facilitate the sustained signals. Although light as in optogeneticsprovides good regulation of effectors’ activity, for sustainedactivity a prolonged exposure to light is required.10 Therefore, we reasoned that designing inactive variantsof the CAD domain that could help regain its biological activity throughproteolytic cleavage might be possible.The designedmobilization of Ca21 genetically fused to a CAD activating domain, was introduced toinhibit CAD activity. A tobacco etch virus protease cleavage site(TEVs) or a plum pox virus protease cleavage site (PPVs) introduced between CAD and a coiled-coil segment enabledthe activation of PACE by a defined protease that could be chemicallyregulated. The chemically reconstituted split protease activated PACEand triggered the long sustained Ca2+ entry and transcriptionalactivity of NFAT. Thus, PACE represents a useful tool for regulatingCa2+ entry and initiating physiologically relevant NFATactivation, which was demonstrated with the expression of cytokinesin Jurkat T-cells.In this study, we developedthe proteolytically activated CRACeffector (PACE), a genetically engineered mediator between proteolyticactivity and calcium influx. The coiled-coil peptide pair P3:P4,2+ sensing domain, transmembrane helix, cytosolicCAD domain inhibited by an inhibitory domain composed of three alpha-helices,and an unstructured lysine-rich C-terminal domain.22 A Ca2+-dependent structural rearrangement ofthe STIM1 dimer exposes the CAD domain that activates Orai. A CADdomain alone tends to bind and open Orai channels; therefore, we usedit as a template to design an engineered Orai activator. The bindingof alpha-helices α1 (CC2) and α4 (CC3) and the subsequentdimerization of the domain are recognized to be crucial for the abilityof CAD to bind to the channel.23 To preventspontaneous CAD dimerization and activation, we used intramolecularP3 and P4 coiled-coil forming peptides as steric hindrances to forceCAD into an inactive conformation.24 P3peptide was fused to the N-terminus of CC2, and P4 was added to theC-terminus of CC3 . The addition of a P3 peptide to theN terminus (P3-CAD) or of P4 to the C terminus of CAD (CAD-P4) didnot prevent the spontaneous activation of Orai and the transcriptionalactivation of mNFAT:TALE:VP16:KRΦ . No improvementin activity was determined when a rigid helix was inserted betweenCAD and P4 peptide in P3-TEVs-CAD-hP4 (S2B), although the TEV protease effectively cleaved the P3 peptide fromCAD .To extendthe distance between α1 and α4 helices and thus preventthe spontaneous formation of active CAD, the peptide EEEEKKKKEEEEKKwith a high helical propensityectively2A, 2B. Wprotease 2A; S2A. -CAD-hP4 2B; S2B, S2C; P3h-TEVs-CAD-TEVs-P4; S2D; P3-TEVs-CAD-TEVs-hP4). However, it also increased the backgroundsignal. A rigid helix was then inserted at the N-terminal site ofthe P4 peptide, providing a P3h-TEVs-CAD-TEVs-hP4 construct, which we named PACE (S2E). The TEVprotease-cleaved PACE, as shown by Western blot, with two bands presentingthe product, cleaved at the first or second TEVs , as it is characteristicof CAD.27 The observed bright spots of PACE, whichwere probably the results of the intermolecular binding of coils P3and P4, were also detected in the presence of TEV protease, althoughthe removal of mCherry:P3 and P4 was expected after processing withprotease (S4B). On the basis of the optimization of constructscombining CC segments, a single α-helical segment, and a proteasecleavage site, we managed to obtain a PACE construct characterizedby a low background signal and as high as 15.5-fold activation inthe presence of a TEV protease  or to ABI and PYL1 (theheterodimerization of which is inducible with abscisic acid [ABA])17 3A, 3B. E2+ influx regulators is their potential to trigger fastresponses.The kinetics of Ca2+ influx after PACE activation weremonitored in vivo in HEK293 cells using the calcium indicator CalRed.While ionophore produced an immediate and lasting influx of calciuminto the cytosol compared with untreated cells , RAPA or ABA induced a smaller but significantCa2+ influx 300 s after the addition of a chemical inductor.One of the important advantages of chemically regulatedCaed cells S5A, S5B,2+-responsive NFAT-based transcription factor. The inductionof reporter expression was detected only in the presence of PACE andRAPA or ABA , with as high as a 15-fold increase with theaddition of 3 μM RAPA and a 3-fold increase with the additionof ABA. The addition of gadolinium chloride blocked transcriptionalactivation , demonstratingthat the PACE effector activated calcium-dependent ion channels, mostlikely through Orai.Next, we examined whether ABA or RAPA couldbe used to induce theactivation of PACE and the transcriptional activation of the syntheticCa28 To expand the toolbox of PACE effectors, the TEVs recognition site was changed to a PPVs cleavage site(NVVVHQA). The lengths of TEVs and PPVs arethe same, but the proteases are orthogonal.29 Three additional PACE variants were prepared, and they containedPPVs at the first, second, or both cleavage sites (S7A). Surprisingly, we observedsignificant differences in the background activation of the PACE variants.No inhibition of CAD activity was observed for unactivated PACE variantswith the PPVs positioned between P3 and CAD . On the other hand, a type ofprotease cleavage site between CAD and P4 peptide had no impact onbackground activity . We also assessed the activityof the PACE with two different sites in combination with one or bothproteases present. Although a single cleavage was sufficient to promoteCa2+ signaling (10-fold for either TEV or PPV protease),the removal of both inhibitory coiled-coil peptides from CAD rendersadditional activation to CAD about 23-fold (S7E).TEV protease is commonly used in synthetic biology; it does notinterfere with cellular processes in human or bacterial cells becauseof its high specificity for the cleavage site.ge sites B, S7A. S and CAD S7B, S7C. and CAD S7B, S7C. and CAD S7B, S7C.s-CAD-PPV-hP4 (PACE-OR)was combinedwith the ABA-inducible split TEVp and RAPA-inducible splitPPVp proteases , aversatile cytokine responsible for T-cell growth, the differentiationof regulatory T-cells, and the mediation of activation-induced celldeath.31 Transcription of IL-2 is NFATand AP-1 dependent; therefore, an additional coactivator, phorbol12-myristate 13-acetate (PMA), is needed to induce upregulation ofIL-2 transcription.33 The electroporated Jurkat T-cellswere subjected to PMA and ABA or RAPA stimulation 19 did not improve the fold ofactivation (data not shown), which likely suggests multiple weak interhelicalinteractions.The inactive PACEis mainly a cytosol-based protein with minorinteractions with membrane-based Orai channels. While designed forintramolecular interactions, P3 and P4 coiled-coil forming peptidescould also make intermolecular connections, which can likely explainthe higher-order structures of PACE, detected as bright spots. Despitethe formation of higher-ordered structures, the desired regulationwas nevertheless achieved, and the activity of PACE was not significantlyreduced, nor did such interactions increase unwanted Orai activation,which means that such assemblies still inhibit CAD activity. The useof coiled-coil peptides with 29 Interestingly, the replacement of TEVs at thefirst site between P3 and CAD with PPVs reduced the signal-to-backgroundratio. On the other hand, the substitution of the second TEVs site between CAD and P4 with PPVs maintained a high signal-to-backgroundratio. The difference between the first and second cleavage sitesprobably occurred because the second cleavage site is flanked by aGly-Pro-Gly and Gly-Ser-Gly peptide sequence, which enables substantialflexibility. The first cleavage site was retained without flankingpeptides, as the TEVs cleavage site sequence proved tobe sufficient. By combining TEVs at the first proteasecleavage site and PPVs at the second one, an effective“OR” logic gate was created with an added feature ofa 2-fold higher signal when cleaved at both sites. This can be explainedby the fact that for full activation of PACE, a cleavage at both sites,between P3 and CAD, and between CAD and P4, is necessary. During designingPACE, it became evident that activated PACE with two protease cleavagesites generates more efficient transcriptional activation probablydue to more effective activation of Orai channels. Activation by twopossible inputs enables the design of more complex synthetic biologicalcircuits. This was exploited with the inducible activation of PACE-ORin combination with ABA or RAPA-induced TEVp or PPVp protease. As proteases can be activated in a variety of ways,PACE enables the existing protease-based circuits36 to be coupledwith Ca2+ signaling networks of the cell.PACE also provided a template for the variationof protease recognitionsites. In addition to the TEV protease cleavage site, we tested theactivation of PACE with an orthogonal PPV protease.2+ influx triggered by ionophore, PACE activation is slower (8–16min). The delay in Ca2+ influx can be attributed to thetime required for RAPA or ABA entry and to the reconstitution of splitprotease reconstitution. The PACE activation time is comparable tothe activation time of cyclic luciferase reporter using ABA or RAPA-induciblesplit TEV proteases.17 Compared to activationof optogenetic constructs by light, PACE response to activation isdelayed also due to the lack of the polybasic region, which is presentat the C-terminus of native STIM and anchor active CAD in the proximityof Orai channel.11 A lower amplitude ofCa2+ was observed for PACE compared with ionophore; however,this could be beneficial, as it causes less damage to cells and activatesdifferent cell responses.37 The selectionand evaluation of PACE constructs were based on their ability to activateNFAT based signaling; therefore, the intracellular Ca2+ amplitude and the rate of Orai activation were not considered inthe selection process as they were secondary to the sustainabilityof Ca2+ signals.With externallyactivated split proteases, we have determined thekinetics of PACE activation. Compared with the rapid Ca33 TNFα expression, in whichNFAT1 transcription is sufficient, was also enhanced with PMA.38 However, protein expression using PACE was comparableto the existing optogenetic CRAC activators in both luciferase assay10 and IL-2.39 In T-cells,the transcriptional activation of NFAT with RAPA was less effectivethan that with ABA because of the immunosuppressing abilities of theRAPA upstream of the AP-1 signaling pathway.40As the construction of PACE wasbased on its ability to activateNFAT-based transcription factors, we tested its ability to activatenative NFAT pathways in T-cells. PACE activated NFAT in a T-cell line,as detected with the secretion of IL-2 and TNFα. The expressionof IL-2 requires the presence of PMA-activating AP1 in addition toNFAT.41 Each of them, however, is a standalone element and doesnot allow integration into larger and more elaborate synthetic networks.The PACE constructs presented in this study and dual protease sitevariant PACE-OR bridge this gap between existing synthetic circuitsthat regulate protease activity and the calcium current, and theyenable the construction of larger and more elaborate biological circuits.With the possible switching of protease cleavage sites, PACE providesa robust scaffold for coupling not only synthetic circuits but alsoany existing well-defined protease activity, such as caspases or cathepsins,to calcium influx.Synthetic biology research, especially optogenetics, has developeda varied toolbox of calcium current effectors.http://n2t.net/addgene:73564; RRID: Addgene_73564; Addgene plasmid # 73566; http://n2t.net/addgene:73566; RRID: Addgene_73566, respectively). All plasmids were constructedusing the Gibson assembly method, and their sequences are listed in Table S6.Orai1-GCaMP6f andmCherry-CAD were gifts from Michael Cahalan  supplemented with 10% fetal bovineserum (Gibco) was used. Jurkat cells were cultured in Roswell ParkMemorial Institute 1640 Medium  supplemented with10% fetal bovine serum.The embryonic kidney HEK293T and HEK293cell lines (ATCC) were cultured in Dulbecco’s modified Eagle’smedium  supplemented with 10% fetal bovine serum(Gibco) at 37 °C in a 5% CO4 HEK293T cells wereseeded per well in 96-well plates (Corning). For confocal microscopyexperiments, 5 × 104 HEK293T cells were seeded perwell in an eight-well chamber slide (Ibidi). For Western blot experiments,5 × 105 HEK293T cells were seeded per well in six-wellplates (Corning). At 50–70% confluence, HEK293T cells weretransfected with a mixture of DNA and polyethylenimine . Per 500 ng DNA, 6 μLof PEI stock solution  was used. Twenty-fourhours after transfection, the culture medium was replaced with a freshmedium, and the cells were stimulated with the indicated concentrationsof ionophore , RAPA (Sigma-Aldrich),or ABA (Goldbio) for 6 h. Calcium ionophore A23187 was prepared asa 10 mM stock solution in DMSO, RAPA as 1 mM stock, and ABA as 50mM stock. The phRL-TK plasmid (Promega), encoding the Renilla luciferase,was used as a transfection efficiency control in the luciferase experiments.An empty pcDNA3 plasmid (Invitrogen) was used to equalize the totalDNA amounts under different experimental conditions.Fordual luciferase assays, 2 × 107 cells/mL with three pulsesof 1600 V using 100 μL tips.Jurkatcells were electroporated using the Neon Transfection System at aconcentration of 2 × 10Cells were lysed 24 h aftertransfection or 6 h after induction of RAPA, ABA, or ionophore using25 μL of 1 × Passive lysis buffer (Promega) per well. Fireflyluciferase (fLuc) and Renilla luciferase (rLuc) activities were measuredusing the dual luciferase assay (Promega) on an Orion II microplatereader (Berthold Technologies). Relative luciferase units were calculatedby normalizing fLuc to the constitutive rLuc in each sample.5) were seeded in 12-well plates (Techno Plastic Products). The nextday, at a confluence of 50–70%, the cells were transientlytransfected with a mixture of DNA and PEI (8 μL PEI/1000 ngDNA). Forty-eight hours post-transfection, the cells were washed with1 mL PBS and lysed in 100 μL 1 × Passive lysis buffer (Promega).The cells were lysed for 20 min on ice and centrifuged for 15 minat 14 000 rpm to remove cell debris. The total protein concentrationin the supernatant was determined using the BCA assay.HEK293T cells  and transferred to a nitrocellulose membrane .Membrane blocking, antibody binding, and membrane washing were performedusing an iBind Flex Western device (ThermoFisher) according to themanufacturer’s protocol. The primary antibodies were mouse-antiMyc . The secondaryantibodies were HRP-conjugated goat antimouse IgG, diluted 1:3000(Jackson ImmunoResearch 115-035-003). The secondary antibodies weredetected with an ECL Western blotting detection reagent  according to the manufacturer’s protocol.For the analyses of fluorescentprotein-expressing cells, live cells were imaged 1 day after transfection.During microscopy, the cells were kept in a chamber at 37 °C.To maintain the physiological pH, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid (HEPES) pH 7.4 (from 1 M stock solution) was added to the media.Microscopic images were obtained using a Leica TCS SP5 inverted laserscanning microscope on a Leica DMI 6000 CS module equipped with anHCX Plane-Apochromat lambda blue 63× objective and a numericalaperture of 1.4 (Leica Microsystems). A 50 mW 405 nm diode laser wasused for GCaMP6f and excitation (emission between 420 and 460 nm),and a 10 mW 543 nm laser was used for mCherry excitation (emissionbetween 550 and 650 nm).2+ unbound state between 497 and 545 nm, emission inthe Ca2+ bound state between 617 and 696 nm). Leica LASAF software was used for acquisition, and ImageJ software  was used for image processing.For calcium imaging, we used CalRedratiometric dye (ATT Bioquest 20591), which was added to the cellsfor 1 h at a concentration of 10 g/mL before exchanging it for theusual medium with an addition of 10 mM HEPES. Microscopic timelapseimages were obtained using a Leica TCS SP5 inverted laser scanningmicroscope on a Leica DMI 6000 CS module equipped with an HCX Plane-ApochromatCS 40× objective and a numerical aperture of 1.25 (Leica Microsystems).A 10 mW 488 nm laser was used for CalRed excitation (emission in theCa6 cell/mL with the addition of 50 ng/mL PMA (Sigma-AldrichP1585), RAPA, ABA, or ionophore. After 24 h, the cells were centrifuged,and the supernatant was taken and used for ELISA for Il2  and TNFα, which were conducted following the manufacturer’sinstructions. Absorbance was measured with a Synergy Mx automatedmicroplate reader (BioTek) at 450 and 620 nm using Gen5 software.Absorbance at 620 nm was used for background correction and was subtractedfrom the absorbance at 450 nm.Jurkat cells were seeded into a 96-well plate24 h after electroporation at a concentration of 1.5 × 10t test was used, with 0 ng of constructs serving as the control forcomparison. For the analysis of protease-based constructs withoutactive protease, one-way ANOVA was used with Dunnett’s multiplecomparisons test as a post hoc analysis. The mean of each conditionwas compared with the mean of the control (0 ng of the construct added).For the analysis of the activity of the activated constructs, an unpairedStudent t test was used between the same concentrationof constructs in the presence and absence of active protease. Statisticalanalysis is depicted in Table S5.The data are presented as meanvalues ± s.d. of four independent biological repeats within thesame experiment. Graphs and statistical analyses were prepared inGraphPad Prism 8. For the analysis of the activity of variants withdifferent protease recognition sites, unpaired Student


Melanoma is the most lethal form of skin cancer, resulting from the malignant transformation of epidermal melanocytes. Recent revolutionary progress in targeted therapy and immunotherapy has prominently improved the treatment outcome, but the survival of melanoma patients remains suboptimal. Ferroptosis is greatly involved in cancer pathogenesis and can execute the outcome of immunotherapy. However, the detailed regulatory mechanisms of melanoma cell ferroptosis remain elusive. Herein, we report that Wnt/β-catenin signaling regulates ferroptosis and melanoma immunotherapy efficacy via the regulation of MITF. First of all, we found that Wnt/β-catenin signaling was prominently suppressed in melanoma cell ferroptosis. Then, we proved that targeting β-catenin exacerbated melanoma cell ferroptosis by promoting the generation of lipid peroxidation both in vitro and in vivo. Subsequent mechanistic studies revealed that MITF mediated the effect of Wnt/β-catenin signaling on melanoma cell ferroptosis, and PGC1α and SCD1 were documented as two main effectors downstream of Wnt/β-catenin-MITF pathway. Ultimately, pharmacological inhibition of β-catenin or MITF increased the efficacy of anti-PD-1 immunotherapy in preclinical xenograft tumor model by promoting ferroptosis. Taken together, Wnt/β-catenin signaling deficiency exacerbates ferroptosis in melanoma via the regulation of MITF. Targeting Wnt/β-catenin-MITF pathway could be a promising strategy to potentiate ferroptosis and increase the efficacy of anti-PD-1 immunotherapy. The most fatal type of skin cancer is melanoma, originating from the epidermal melanocyte-related malignant transformation in the skin, and its prevalence is steadily rising globally . While r+ T-cells in the tumor microenvironment improves the efficacy of anti-PD-1 immunotherapy , i, iPPARGCnt group g. Additint group h. In totPrevious reports have demonstrated that SCD1 is greatly implicated in protecting cancer cells against the induction of ferroptosis . Since w+ T-cells in TME can trigger tumor cell ferroptosis upon anti-PD-1 antibody treatment, and targeting Wnt/β-catenin signaling can exacerbate ferroptosis according to our results, we proposed that targeting Wnt/β-catenin might increase the effectiveness of anti-PD-1 antibody by the induction of tumor cell ferroptosis. To produce a xenograft tumor model, B16F10 mouse melanoma cells were injected subcutaneously into immune-competent C57BL/6 mice (Treatment of melanoma with anti-PD-1 immunotherapy is significantly effective, while poor responsiveness and resistance to the treatment considerably reduce its effectiveness . A previL/6 mice a. As shoL/6 mice b–d. ThisL/6 mice b–d. In iL/6 mice e. In parL/6 mice e.Apart from this, we also employed the B16F10 xenograft tumor model to test whether targeting MITF could enhance the effectiveness of anti-PD-1 antibodies via ferroptosis a. It wasWe first discovered in the current research that Wnt/β-catenin signaling was significantly ameliorated in response to the induction of ferroptosis. Then, it was proved that targeting Wnt/β-catenin signaling exacerbated melanoma cell ferroptosis by enhancing lipid peroxidation production. Subsequently, through the employment of RNA sequencing technology and a panel of biochemical assays, melanocytic-specific transcriptional factor MITF was identified to mediate the influence of Wnt/β-catenin signaling in melanoma cell ferroptosis. In particular, PGC1α and SCD1 were proved as two main effectors downstream of the Wnt/β-catenin-MITF pathway in melanoma cell ferroptosis. Ultimately, the results of the preclinical mouse model demonstrated that targeting Wnt/β-catenin or MITF could significantly enhance the effectiveness of anti-PD-1 antibody via ferroptosis induction. Collectively, these findings established that targeting Wnt/β-catenin signaling exacerbates ferroptosis in melanoma via the regulation of MITF . The int+ T-cells in response to anti-PD-1 immunotherapy can promote the secretion of IFN-γ that suppresses the expression of the Xc-system in the tumor, so that to trigger tumor cell ferroptosis [Iron-dependent lipid peroxidation is a hallmark of a newly found cell death mode known as ferroptosis. Earlier research has demonstrated that ferroptosis participated in the regulation of tumor cell metastasis and the effectiveness of targeted therapy ,48. Moreroptosis . Thereforoptosis ,15,16, aroptosis . Herein,roptosis . DiffereSignificant evidence has emerged to demonstrate that MITF coordinates melanocyte and melanoma biology in ways more than just increasing melanocyte cell identity and melanosomal gene regulation . High MI+ T-cells mediates the influence of melanoma immunotherapy [The blockade of PD-1/PD-L1 has significantly improved the prognosis of patients with melanoma . Of noteotherapy ,13. Our otherapy ,49. Eithotherapy . Then, Dotherapy . NeverthThe present study reveals that Wnt/β-catenin signaling was prominently impaired in response to ferroptosis induction. Either pharmacological inhibition or the genetic knockdown of β-catenin could exacerbate ferroptosis via the regulation of MITF expression, with the downstream effector PGC1α and SCD1 greatly implicating it. What is more important is that pharmacological inhibition of either β-catenin or MITF could increase the treatment effect of anti-PD-1 antibodies in a preclinical mouse model by facilitating melanoma cell ferroptosis. Together, targeting the Wnt/β-catenin-MITF pathway might be an effective strategy for potentiating ferroptosis and enhancing the effectiveness of anti-PD-1 melanoma immunotherapy.


Pseudomonas aeruginosa phage vB_PaeM-SMS29, a phage with poor antibiofilm properties, was incorporated into polyvinyl alcohol  supplemented with 0.1% (v/v) of glycerol, and cast onto two different microneedle arrays varying in geometry. The dissolving microneedles were thoroughly characterized by microscopy, force-displacement, swelling, phage release and stability. Furthermore, 48 h-old biofilms were formed using the colony biofilm procedure (absence of broth), and the antibiofilm efficacy of the phage-loaded microneedles was evaluated by viable cell counts and microscopy and compared to free phages. The phages in microneedles were fairly stable for six months when stored at 4 °C, with minor decreases in phage titers observed. The geometry of the microneedles influenced the penetration and force-displacement characteristics but not the antimicrobial efficacy against biofilms. The two PVA microneedles loaded with phages reduced P. aeruginosa PAO1 biofilms by 2.44 to 2.76 log10 CFU·cm−2 at 24 h. These values are significantly higher than the result obtained after the treatment with the free phage (1.09 log10 CFU·cm−2). Overall, this study shows that the distribution of phages caused by the mechanical disruption of biofilms using dissolving microneedles can be an effective delivery method against topical biofilm-related skin infections.Resistant bacteria prevail in most chronic skin wounds and other biofilm-related topical skin infections. Bacteriophages (phages) have proven their antimicrobial effectiveness for treating different antibiotic-resistant and multidrug-resistant bacterial infections, but not all phages are effective against biofilms. Phages possessing depolymerases can reach different biofilm layers; however, those that do not have depolymerase activity struggle to penetrate and navigate in the intricate 3D biofilm structure and mainly infect bacteria lodged in the outer biofilm layers. To address this,  Pseudomonas aeruginosa and Staphylococcus aureus being the most commonly isolated  [Phage SMS29 was amplified using the plate lysis and elution procedure . The pha/v agar] . The tai/v agar]  to identw/w] were hydrated in SM buffer , under agitation (350 rpm) at room temperature (RT) for 4 h. Afterwards, the PVAs were dissolved under agitation (350 rpm) at 85 °C or 90 °C, according to their hydrolysis degree, until complete dissolution (approximately 2–3 h). The temperature of the PVA solutions was allowed to reduce to 64 °C before the addition of phages in order not to inactivate them. PVA containing phage SMS29 were produced with 10% v/v of phages.Different polyvinyl alcohols  varying in molecular weight and hydrolysis degree were tested . The difv/v PVP:PVA], and PVA with glycerol 0.1% v/v. Briefly, 10% v/v of phage ·mL−1) was added to the solutions  and stored at 4 °C. Samples were removed for five days for phage titration as previously described [Phage SMS29 stability studies were performed in liquid PVA, PVA with polyvinylpyrrolidone (PVP) [1:7 escribed . In-house microneedle arrays were produced as described previously . In brie®184, Dow Corning, Midland, MI, USA) was mixed at a 10:1 w/w elastomer to curing agent ratio. PDMS molds were obtained after pouring PDMS in the silicone master, degassing under vacuum for 10 min, and curing at 65 °C for 1 h 30 min. The other PDMS molds were acquired from BlueAcre Technology . These BlueAcre PDMS molds consist of 11 × 11 needles with a height of 600 µm, a base of 300 µm, and 600 µm from tip to tip. Two types of polydimethylsiloxane (PDMS) molds were used in this work. INL PDMS molds were those obtained by replica molding of the in-house microfabricated silicon masters as described previously ,36. The v/v of glycerol (PVAG) to have a final concentration of 1 × 109 PFU/mL. Twenty five µL of this mixture were placed in the PDMS molds, and then vacuum was applied . The molds were centrifuged at 1000 rpm  to fill all needle cavities and eliminate entrapped air bubbles. Additional 50 µL of the mixture were placed on the PDMS molds, and the vacuum and centrifugation steps were repeated. The volume on the mold surface surrounding the MN area was removed with adhesive tape (3M), and the MNs were dried for 2 h at 40 °C in a ventilated oven . After, PVAG without phages was placed on the molds (100 µL) and dried for 2 h at 40 °C in a ventilated oven to create a thicker upper area for easier handling of the MNs. MNs in their respective PDMS molds were stored in vacuum-sealed bags at 4 °C or RT until their manual removal from the PDMS molds for further use. According to their fabrication place, the MNs were designated as INL  and BA (BlueAcre).Polyvinyl alcohol  was used to fabricate MNs, with and without phage SMS29, using a molding technique. Phage in SM buffer was mixed with PVA 4:88 with 0.1% The stability of phage SMS29 in the MNs was assessed over time after storage at 4 °C and RT. Briefly, MNs containing phages were removed from the PDMS molds and placed in 10 mL of SM buffer for 24 h at 37 °C under agitation (120 rpm) . After 24 h, the solutions were vortexed thoroughly and serially 10-fold diluted. The release of phages was studied using a procedure described previously . The dat0). The films were then placed in distilled water, and samples were taken every 2 min. The excess water was removed carefully using tissue paper, and the weight of the sample was measured (W1). The wet films were dried at 40 °C and placed in a desiccator, and the weight was measured (W2). The degree of swelling (%) and the solubility (%) were determined as follows: Swelling (%) = (W1 − W0)/W1 × 100, Solubility (%) = (W0 − W2)/W2 × 100. Two independent experiments were performed in duplicate. Swelling and solubility studies were performed with films instead of MNs. PVA solutions dissolved as described above were poured (20 mL) onto 90 mm Petri dishes and degassed. The films were dried at 40 °C , cut into 2 × 2 cm pieces, placed in a desiccator for 24 h, and after, the weight was measured (WG MNs (before and after force-displacement and perforation studies) were visualized by SEM  after gold sputtering . Also, MNs were visualized with a wide-field upright optical microscope  coupled with a one-color camera . The in-house INL master mold and PVAg, 4 °C) and the pellet was washed twice with tap water. The samples was centrifuged once more . Phages were deposited on 400 mesh copper grids and stained with 2% (w/v) uranyl acetate (pH 4.0). The grids were imaged at 200 kV using a JEM-2100-HT electron microscope .Phage particles were centrifuged . Briefly, a metallic probe was used, and the height aligned before any force was applied. MNs were glued to a plastic support with double-sided tape. The metallic probe moved down at 1.25 mm/s at a Fmax force of 3600× g until touching the array. The force-displacement plots were divided by the number of MNs. Five to six MNs of each geometry were analyzed.The MNs were subjected to axial force mechanical studies using a force-displacement system  was folded in six layers and perforated with the different MNs. The thickness of the layers was measured with a Schut electronic outside micrometer . The number of pores in the different layers was measured as previously described [Parafilmescribed  using a v/v) (Sigma-Aldrich), the excess stain was removed with a wipe, and the skin samples were photographed. Porcine skin excised from the abdomen was generously supplied by ICVS-Life and Health Sciences Research Institute . The hair was removed using scissors, and the fat was excised using a scalpel. Samples were then stored in vacuum-sealed bags at −20 °C and thawed only before use. The polymeric MNs were pressed onto the skin (thumb pressed) and removed after 5 min. The microneedle insertion sites were stained with Trypan blue 0.4% (P. aeruginosa PAO1 produced using the colony biofilm procedure [9 colony forming units (CFU)·mL−1 and diluted 10-fold. Sterile polycarbonate membrane  were cut to the size of the MN arrays and placed on TSA plates with the shiny side facing up, and inoculated with 20 µL of this culture. The polycarbonate membranes were incubated at 37 °C, under static conditions, for 24 h and transferred to fresh TSA plates and incubated for another 24 h at 37 °C. Free phages  or SM buffer (50 µL) in the case of control samples were added to the membranes and incubated at 37 °C with samples removed at different time points for CFU determination. Unloaded and phage-loaded MNs were placed on the 48 h-old biofilms formed on the polycarbonate membranes. Also, a control with only P. aeruginosa PAO1 was analyzed. The TSA plates were incubated at 37 °C, and samples were removed for CFU determination after 8 h and 24 h. The polycarbonate membrane was removed at each sampling point and placed in 10 mL of sterile saline (0.9% w/v NaCl). Also, the TSA below the polycarbonate membrane was removed using a 5 mL syringe , which was previously cut to remove the tip and sterilized by autoclaving . The TSA block was removed and added to a new 10 mL of sterile saline to determine if this contained cells. The samples were vortexed, serially diluted in saline containing 10 mM ferrous ammonium sulfate [The antibacterial activity of free phage SMS29 and phage-loaded MNS was assessed in 48 h-old biofilms of rocedure , with so sulfate , and pla sulfate . p < 0.05 .Statistical analysis of the results was performed using GraphPad Prism 6 . The independent experiments and the results are presented as mean ± standard deviation (SD). Differences between control and treated biofilms were assessed using one-way ANOVA followed by Tukey’s multiple comparison statistical tests. Differences were considered statistically significant if Pbunavirus genus of the Caudoviricetes class, having a capsid of 70.0 ± 4.2 nm and a tail with a length of 139.0 ± 1.5 nm . This method is, in our opinion, more appropriate to produce biofilms resembling those present in chronic wounds than biofilms produced in microtiter plates. The treatment with free phage SMS29 was performed for 24 h  [P. aeruginosa biofilms by 1 log10 CFU·cm−2, respectively [Pbunavirus vB_PaeM_CEB_DP1, where 26% of the survivors had become resistant [In this work, we used the colony-biofilm procedure to grow for 24 h b. SMS29 duction) , while pectively . Furtheresistant .A search for depolymerase domains in the tail-related proteins of SMS29’s genome (ORFs SMS29_031 and SMS29_045) showed that none of these proteins had domains with depolymerase function. P. aeruginosa killing. The poor antibiofilm efficacy allied to a lack of depolymerase activity makes this phage a good candidate for loading onto microneedles to assess if this delivery option allows its distribution throughout the 3D biofilms and enhances PVA dissolves at temperatures >80 °C, and, therefore, it is important to evaluate if the solution can be cooled to 65 °C and if it remains liquid so that phage SMS29 can be incorporated. Unfortunately, PVAs with high molecular weights and viscosities, when cooled to 65 °C, resulted in partial gelation  and had v/v) and PVP (1:7 v/v ratio) was tested in order to improve the stability by increasing plasticity and reducing the crystallinity of the PVA molecules.Films of the PVA solutions that remained liquid at ≤65 °C were further prepared, and based on the uniform aspect, PVA 4-88 was selected for further studies . After iv/v) of glycerol to PVA 4-88 maintained the phage concentration higher than PVA 4-88 alone, while the addition of PVP significantly decreased the concentration of phage SMS29, even when combined with 0.1% v/v of glycerol  glycerol to PVA and PVA:PVA resulted in marginally lower degrees of swelling compared to PVA and PVA:PVA without glycerol, these were not statistically significant. Other studies have added glycerol to PVA hydrogels and PVA:PVP patches and observed a reduction in swelling compared to samples without glycerol [v/v) (0.75–3% (v/v) glycerol for PVA hydrogels and 0.37–1.5% (v/v) glycerol PVA:PVP patches). The increase of glycerol concentration would render the MNs fabricated in our work impossible to use as they would become too bendable, not being able to perforate the skin. The maximum swelling percentage before the disintegration of the samples started varied slightly between 61.9% (PVA:glycerol) and 67.1% (PVA:PVP). Although the addition of 0.1%  were also observed by optical and SEM microscopy  chitosan MNs (8.12% reduction) and the most approximate to the master mold was a height of 899.89 ± 1.66 µm with poly  (PLGA) (0.01% reduction) [w/w) of sodium alginate  to the most similar when fabricated with PLGA (250.39 ± 1.12 µm). Also, Bonfante et al. described reductions in height and base diameter of approximately 10–19% with hyaluronic acid and carboxymethyl cellulose [The PVAicon MNs  and 14.1duction) . Also, t®M perforation   and PEG 10,000 (7.5% w/w) [®M puncture tests showed that MNs with a 600 µm height resulted in an insertion depth between approximately 290 µm to 480 µm. These results are similar to those obtained with the BA MNs. Cordeiro et al. tested seven different geometries [®M insertion tests, the number of holes in the different layers reduced immediately when the cylindrical MN with a conical tip was used. On the other hand, the number of holes using the conical MN decreased only after an insertion depth of 381 µm. These results are in agreement with those obtained in this work, where we show a greater perforation capability with the BA MN geometry than with the INL MNs. MN perforation studies were carried out in porcine skin and ParafilmM layers c,d. Fresrs (v/v) c. The Pa®M layer . A signiayer [®M g. Larrañ.5% w/w) . One wasometries , includiPhage SMS29 stability in INL and BA MNs was assessed after storage for different times by determining the phage titers .The concentration of SMS29 stored in SM buffer at 4 °C remained stable throughout the experiment (10 PFU·mL−1 (INL MNs) and 1.28 log10 PFU·mL−1 (BA MNs) compared to day 0. SMS29 loaded MNs stored at 4 °C hardly lost activity during the first days of storage, but after 180 days, the phage concentration had reduced by 0.95 log10 PFU·mL−1 (BA) and 4.76 log10 PFU·mL−1 (INL) by the end of the experiment . On the other hand, the treatment of biofilms with phage SMS29 in INL and BA MNs decreased the viable biofilm cell counts significantly (p < 0.05). Although the results obtained do not vary according to the MN geometry used, we expected some differences. We hypothesized that a larger MN base diameter (BA MNs) would allow phages to reach and kill a higher number of cells due to the 90 µm larger base diameter in the BA MNs. After 8 h, the phages in MNs had reduced the number of cells by approximately 1.84 log10 CFU·cm−2 (BA) and 2.14 log10 CFU·cm−2 (INL) compared to their respective controls (p < 0.05). The number of cells continued to decrease, and after 24 h of treatment, they were 2.76 log10 CFU·mL−2 (BA) and 2.44 log10 CFU·mL−2 (INL) lower than in the controls. Overall, these reductions were significantly higher than those obtained when free SMS29 was used [1.31 log10 CFU·cm−2 (8 h) and 1.09 log10 CFU·cm−2 (24 h), The control and the non-phage-loaded MNs showed similar cell counts after 8 h and 24 h   to 16.7%. P. aeruginosa. On the other hand, the samples after treatment with phage-loaded MNs showed significantly fewer cells (The 48 h-old biofilms treated with phages for 24 h treatment, and the respective controls were imaged by SEM . The coner cells d1,2–e1,2er cells d1,e1, aner cells e2, corroer cells .Pbunavirus genus, in dissolving, biodegradable MNs. These MNs were made with PVA, a polymer frequently used in pharmaceutical applications due to its biocompatible and toxicologically safe characteristics. Storage at refrigerated conditions kept SMS29 in MNs at very high concentrations, and these started releasing the phage within the first minutes due to PVA’s dissolution. We have successfully validated the use of phage-loaded MNs for antibiofilm purposes, showing that this strategy enhanced the overall antimicrobial activity of this particular Pbunavirus compared to its free application to biofilms. This phage delivery strategy might, in the future, allow the use of other phages with poor antibiofilm activity for the treatment of topical biofilm-related wounds, particularly those colonized with antibiotic resistant pathogens. To our knowledge, this is the first study that incorporates a phage into dissolving MNs and provides a thorough characterization. Further studies using ex vivo and in vivo models and phage combinations may provide valuable knowledge for future phage therapy applications for biofilm-related skin infections.In this work, we have encapsulated SMS29, a phage belonging to the


The main goal of vital pulp therapy (VPT) is to preserve the vitality of the pulp tissue, even when it is exposed due to bacterial invasion, iatrogenic mechanical preparation, or trauma. The type of new dentin formed as a result of VPT can differ in its cellular origin, its microstructure, and its barrier function. It is generally agreed that the new dentin produced by odontoblasts (reactionary dentin) has a tubular structure, while the dentin produced by pulp cells (reparative dentin) does not or has less. Thus, even VPT aims to maintain the vitality of the pulp. It does not regenerate the dentin pulp complex integrity. Therefore, many studies have sought to identify new therapeutic strategies to successfully regenerate the dentin pulp complex. Among them is a Wnt protein-based strategy based on the fact that Wnt proteins seem to be powerful stem cell factors that allow control of the self-renewal and proliferation of multiple adult stem cell populations, suitable for homeostasis maintenance, tissue healing, and regeneration promotion. Thus, this review outlines the different agents targeting the Wnt signaling that could be applied in a tooth environment, and could be a potential therapy for dentin pulp complex and bone regeneration. The main goal of vital pulp therapy (VPT) is to preserve the vitality of the pulp tissue, even when it is exposed due to bacterial invasion, iatrogenic mechanical preparation, or trauma . VPT proHowever, the poor quality of the resulting dentin bridge and its lack of sealing within the dentin walls explain why some authors prefer the use of Tricalcium silicate-based (TCS-based) cements ,5, such VPT aims to maintain and, if possible, regenerate the vitality of dental pulp. This last objective is not achieved using the conventional VPT procedures. Therefore, many studies have sought to identify new therapeutic strategies to successfully regenerate the dentin pulp complex. Two different ways are currently being tested to achieve this enormous objective. The first involves the introduction of stem or progenitor cells into a site of damage, and the second aims to activate endogenous stem cells to promote tissue regeneration. This last strategy, in which endogenous stem cells are activated, avoids the risk associated with the implementation of different cells in nature or reprogrammed cells into the human body. For this purpose, a Wnt protein-based strategy, based on the fact that Wnt proteins are powerful stem cell factors , allows The secreted family Wnt proteins participate in the regulation of cell differentiation, proliferation, and apoptosis, and through these mechanisms, play a key role in tissue generation, regeneration, and self-renewal . Wnts inAs the regenerative capacity of multiple mammalian tissues has been shown depend-ing on Wnt/β- catenin signaling and its activation. In the context of pulp dentin, complex regeneration the role of canonical pathway seems obvious. Numerous studies have shown this role in reactionary dentinogenesis . In repaHowever, the specific role of Wnt/β-catenin signaling on odontoblast-like differentiation of hDPSCs is not completely known. Indeed, some authors such as Scheller et al. demonstrated that canonical Wnt signaling inhibited odontogenic differentiation of hDPSCs . In addiIn this review, we aimed to recapitulate new biologically based strategies to enhance this natural repair response by regulating Wnt signaling via modulatory molecules. These molecules could be a potential therapy target for dentin pulp complex.The treatment of dental caries that results in pulp exposure is currently managed by replacing lost dentine with inorganic calcium-containing materials such as CH, MTA or Biodentine that remain in the crown. Since this dentine is formed directly from new odontoblast-like cells that differentiate from resident stem cells in the pulp , it is iIn the context of response to damage, the pulp dentin complex can induce a Axin2 increase repair process in odontoblast-like cells that subsequently form reparative dentin . GlycogeTideglusib is the most studied GSK3 drug that has to date been shown to be safe in patients . DeliverNP928 is a new GSK3 inhibitor small-molecule drug that has increased aqueous solubility compared to other thiadiazolidinone (TDZD) drugs and can activate Wnt/β- catenin pathway similarly to tideglusib. Therefore, NP928 is a modified version of Tideglusib that removes the naphthyl moiety and increases solubility. The reparative potency and clinical usability of NP928 was evaluated in microdose concentrations of loaded MA-HA hydrogels in a pulp damage model in wild-type mice, and showed more reparative dentine was in tested groups than in controls .® to be transferred to clinic, on the basis of it being ready for its first human clinical trials.The use of hydrogel looks superior to the sponge delivery, and the overall simpler user experience for the clinician. The use of hydrogel looks superior to the sponge delivery, and the overall simpler user experience for the clinician. These results allowed a drug called ReDentA tooth cavity is a good therapeutic site for the use of a small molecule, used at very tiny concentrations. They should have a short half-life and limited range of action. They do not activate cells in the roots, for example.Tivantinib is a small molecule that inhibits c-Met receptor tyrosine kinase. It is a non-ATP competitive inhibitor. GSK3α and GSK3β, two structurally isoforms of GSK3, have been identified as new targets for this molecule . These iLithium compounds are inhibitors of GSK3β, used among other things to treat the bipolar patients and inhibit cancer cell metastasis . Many inRecombinant proteins such as the Wnt agonist R-spondin  have beeWedelolactone is a natural plant compound that has been shown to have anti-inflammatory, anticancer, and antiosteoporosis effects. The effect of wedelolactone has also been evaluated for dental treatment. For that purpose, DPSCs were treated with wedelolactone in vitro . This exSema3A has been shown to be an osteoprotective factor by inhibiting osteoclastic bone resorption and promoting osteoblastic bone formation through canonical Wnt/β-catenin signaling . When NeIn vitro, the effects of a continuous activation of Wnt/β-catenin signaling by the addition of Wnt3a on the mineralization and differentiation of pulp cells demonstrated that Wnt3a induced marked increases in the expression of Dmp1, Dspp, and Bsp, compared to controls between days 10 and 17 , and higSclerostin, is a secreted glycoprotein which is largely produced by osteocytes under a physiologic environment. It is an antagonist of the Wnt-BMPs signaling pathway through its binding to LRP 5/6receptor which is present on the membrane osteoblast . It has Secretion of sclerostin by odontoblasts has been demonstrated during tooth development ,58. ManyThe canonical Wnt signaling pathway is considered as an important regulator of stemness ,23,63 anCertain systemic conditions compromised the capacity of proliferation and odontogenic differentiation of human DPSCs. Diabetes is one of these pathological conditions. The reduction in human pulpal mesenchymal cells stemness in diabetic patients could affect the regenerative capacity of pulp-dentin complex and the formation of the dentin bridge. Based on these data, some authors studied the potential role of Wnt signaling in a high glucose-induced senescence model (close to diabetic conditions) . InteresFamotidine is a competitive inhibitor of the histamine receptor, which is the dominant receptor involved in gastric acid secretion. This binding prevents the activation of adenylate cyclase normally induced by histamines. Many studies have investigated the Famotidine potential anti GSK3 effect. It seems that this inhibiting role could be attributed to the hypoglycemic aspect. Indeed, H2-receptor inhibitors could affect glucose metabolism and its role in the decrease in the glycemic response curve in vivo through binging with GSK3β . Its bioOlanzapine used to be a particular pharmacological psychiatric drug used for the treatment of schizophrenia. Its potential anti GSK3 effect has been sought by many authors. As Famotidine, it seems that this inhibiting role could be due to the hypoglycemic effect. Its biocompatibility and low cytotoxicity have also been demonstrated on progenitors’ murine cells . This moThe Wnt/b catenin pathway is a very complex intra cellular pathway.Several studies have shown that Wnt signals are necessary for pulp dentin complex formation and repair, and in other studies, some research groups have succeeded in demonstrating that a Wnt stimulus is sufficient to induce tissue regeneration. Small-molecule drugs that stimulate Wnt/β-catenin have shown promise as a novel biological therapy for treating exposed pulpal lesions. It is necessary to consider the need of local application to avoid systemic side effects of the highly potent small molecules acting as Wnt agonists at very tiny concentrations. Interestingly, they are very cheap to make. Strong proof-of-concept data are still needed, however, along with well-crafted safety plans, to make regenerative dental medicine a reality.After pulp exposure, molecules enhancing Wnt/β-catenin can be applied in contact with the pulp on different supports such as hydrogels or sponges . These m


Psoriasis is a common chronic inflammatory skin disease with multifactor etiology, characterized by abnormal proliferation and differentiation of keratinocytes. Huang-Lian Jie-Du decoction (HLJDD) is a traditional Chinese medicine prescription with good clinical curative effect on psoriasis. However, its therapeutic mechanisms are still unclear. β-catenin, and c-Myc in psoriasis mice. Western blot was used to examine the expressions of Frizzled-2, LRP5/6, GSK-3β, APC, Axin2, TCF4, LEF1, cyclin D1, TBX3, EPHB2, and NOTUM enzyme.  The psoriasis model of SKH-1 nude mice was established by imiquimod-induced and HLJDD gavage was given. Hematoxylin and eosin staining were used to evaluate pathological morphologies, and immunohistochemistry was used to detect the expressions of Wnt1, β-catenin, and c-Myc. Western blot results showed that HLJDD reduced the expressions of Wnt receptors Frizzled-2 and LRP5/6, and Wnt downstream target genes TCF4, LEF1, cyclin D1, TBX3, and EPHB2, while upregulated destruction complex proteins GSK-3β, APC, and Axin2.  In this study, HLJDD reduced skin erythema and lesions, decreased the thickness of epidermal and downregulated the expressions of Wnt1, β-catenin signaling pathway at multiple stages. HLJDD can effectively treat psoriasis and inhibit the Wnt/ Psoriasis is a chronic inflammatory skin disease that affects about 2–5% of the population worldwide –3. CliniDue to the complex pathophysiology of psoriasis, the clinical effect of current treatment is not ideal for most patients. Traditional treatments for psoriasis include local treatment, phototherapy, and systemic treatment . HoweverPsoriasis is an ancient disease, and traditional medicines have been used to treat it for centuries . Traditiβ-catenin signaling pathway [β-catenin signaling pathway regulates the proliferation and differentiation of epidermal keratinocytes [β-catenin signaling pathway in psoriasis mice, in order to provide a theoretical basis for clinical rational application of HLJDD.Abnormal expression of signal transduction pathway may be the molecular basis of excessive proliferation and inflammatory infiltration in psoriatic epidermis . Various pathway . Wnt pro pathway . In matuinocytes . In thisRubia yunnanensis (30 g), Zaocys dhumnades (15 g), Poria cocos Wolf (30 g), Tripterygium hypoglaucum (15 g), and Sanguisorba officinalis (15 g) mixture was refluxed with water  for 1 hours, filtered with a 60-mesh strainer. Filtrates were collected, and the residues were then refluxed in water  again for 1 hours. Filtrates were collected, and the residues were refluxed in water  for 30 minutes. The extract solutions were combined and concentrated to 700 mL, then filtered through a 200-mesh filter. Purified water was added to 1000 mL and mixed well.Huang-Lian Jie-Du Decoction mixture were made by the Preparation Center of Yunnan Provincial Hospital of Traditional Chinese Medicine  followed the modified HLJDD prescription. Lithospermum L. (30 g), Rhizoma Coptidis (10 g), Radix Scutellariae (15 g), Cortex Phellodendri (15 g), Fructus Gradeniae (15 g), Buffalo horn (30 g), n = 24), control group, which were applied with an equal amount of Vaseline cream; IMQ group, only applied with IMQ cream; IMQ group treated with HLJDD intragastric administration  began on day 7 and lasted for 7 days, and IMQ group treated with QD (0.01 g/mL) intragastric administration began on day 7 for 7 days. Mice were euthanized by cervical dislocation and skin samples were fixed in 4% paraformaldehyde and the other was frozen with liquid nitrogen and stored at −80°C.SKH-1 nude mice were purchased from Laboratory Animal Center, Hangzhou Normal University . All animals were maintained in a specific pathogen-free (SPF) environment at 25°C with a standard 12 hours light/12 hours cycle. All experiments were approved by the Laboratory Animal Ethical Committee of Yunnan Traditional Chinese Medicine University for mice experiments (license no. SCXK (Zhe) 2016–0004). Male mice at 6–8 weeks of age received applications of 5% imiquimod (IMQ) daily for 13 days . Indigo Naturalis (QD) is another TCM commonly used to treat inflammatory skin diseases, which has been shown to significantly improve psoriasis ,29. Fu-Fμm using a paraffin slicer , dewaxed with xylene and stained with hematoxylin and eosin (H&E) , and examined under a light microscope .Skin samples were embedded in paraffin and sliced into sections with a thickness of 5 β-catenin  overnight at 4°C. Then, the sections were incubated with secondary antibodies and colored with a diaminobenzidine (DAB) kit  and counterstained with hematoxylin. Images were captured on an Olympus BX53 fluorescence microscope .The tissue sections were dewaxed, hydrated, and then treated with 3% hydrogen peroxide for 30 minutes to block endogenous peroxidase activity. After blocked with a 5% goat serum solution  for 1 hour at 25°C, the sections were incubated with primary antibodies against Wnt1 , c-Myc , and β-actin antibody  was used as an internal control. The blots were measured with ECL chromogenic substrate . The following primary antibodies were used, Frizzled-2 , LRP5 , LRP6 , GSK-3β , APC , Axin2 , TCF4 , LEF1 , cyclin D1 , TBX3 , NOTUM , and EPHB2 .Skin samples were cut up with sterile surgical scissors on ice and ultrasonically homogenized for 20 seconds, then lysed with RIPA lysis buffer  for 20 minutes, and the lysates were centrifuged at 12 000 rpm for 10 minutes at 4°C. Total protein content was measured using the BCA Protein Assay Kit . SDS-PAGE electrophoresis was performed using a 10% separation gel and 5% concentrated gel, and PVDF membrane transfer was performed by the electrotransfer method. The membrane was blocked with a 5% BSA  for 1 hour at room temperature and incubated with primary antibodies at 4°C overnight followed by an HRP-labeled secondary antibody . Anti-t-test or one-way ANOVA using GraphPad Prism 9.0.0. Statistical significance was assumed if P ˂0.05.All experiments were separately repeated three times. All data presented as the mean ± SD. Each bar represents the mean ± SD of three independent experiments. Statistical significance between two or multiple groups was analyzed by Firstly, we explored the effects of HLJDD on psoriasis mice. We used IMQ to induce psoriasis lesions in nude mice to establish a mouse model of psoriasis . IMQ is β-catenin signaling pathway overexpression promotes inflammatory response [β-catenin signaling pathway. In addition, the number of positive cells expressing Wnt1 decreased dose-dependently after HLJDD treatment . The tumor suppressor protein Axin acts as a DC scaffold interacting with β-catenin, the tumor suppressor protein APC, and two constitutively active serine-threonine kinases (CK1 and GSK3). Hence, we further examined several DC-related proteins GSK-3β, APC, and Axin2 of the Wnt/β-catenin signaling pathway by WB . The expressions of TCF4, LEF1, and cyclin D1 increased significantly after 6 days of IMQ stimulation, whereas they decreased dose-dependently after HLJDD treatment. Although the expressions of TBX3 and EPHB2 were not significantly regulated by IMQ, HLJDD, and QD, they increased after IMQ application and similarly showed a decreasing trend after HLJDD treatment. Although the trends of the expressions of the five target genes affected by IMQ and HLJDD differed, the overall trends were similar . The resκB pathways and significantly improved the symptoms of atopic dermatitis in mice [HLJDD, as a traditional Chinese medicine, has been proved to be effective in the clinical treatment of inflammation, hypertension, gastrointestinal disorders, liver dysfunction, and cerebrovascular diseases . Studies in mice , and HLJ in mice . HLJDD hβ-catenin signaling pathway is associated with abnormal cell proliferation, and it has been demonstrated that β-catenin increases in keratinocytes nuclei in both skin lesions and nonskin lesions in psoriasis patients compared with healthy controls [β-catenin signaling pathway by HLJDD in the treatment of psoriasis in mice. Our results indicated that HLJDD can effectively reduce skin erythema and lesions, reduce epidermal thickness, and relieve psoriasis symptoms in a dose-dependent manner by inhibiting Wnt/β-catenin signaling pathway in psoriatic mice.The underlying mechanisms of psoriasis are complex as it involves inflammatory mediators, angiogenesis, and epidermal keratinocyte proliferation . It is gcontrols . We inveWnt protein is an intercellular signal, and during its synthesis, Wnt protein is modified by the attachment of palmitoleic acid , which iβ-TrCP can degrade β-catenin and thereby block further transmission of Wnt/β-catenin signals. Our WB results showed that the expression of GSK-3β, APC, and Axin2 all increased significantly after 7 days of HLJDD treatment, while immunohistochemical results of β-catenin expression showed a decrease. These results suggested that HLJDD promoted the expression of destruction complex, thus promoting β-catenin degradation, while inhibiting the β-catenin accumulation and transfer to the nucleus.A destruction complex of proteins consisting of Axin, APC, Ser/Thr kinases GSK-3 and CK1, protein phosphatase 2A (PP2A), and E3-ubiquitin ligase β-catenin/TCF (T-cell factor) target genes. After activation of the Wnt pathway, β-catenin accumulates in the cytoplasm and transfer to the nucleus, where it binds and activates TCF transcription factors to drive downstream genes expression. Another downstream mediator of the Wnt/β-catenin signaling pathway, lymphoid enhancer-binding factor 1 (LEF1) is a member of the TCF/LEF1 family of high-mobility group transcription factors [The final cellular response of canonical Wnt signal is determined by activating  factors , which wβ-catenin signaling pathway at multiple stages to ameliorate psoriasis in mouse. Outside and on the membrane, HLJDD inhibited the expression of Wnt1 signaling proteins as well as Wnt receptor transmembrane proteins. In the cytoplasm, HLJDD could promote the expression of DCs components, phosphorylates β-catenin, and then degrade it, resulting in a decrease in the level of dissociated β-catenin and thus inhibiting its entry into the nucleus. In the nucleus, HLJDD could inhibit the expression of Wnt target genes and the activation of nuclear transcription factors such as TCF4, LEF1, and TBX3 by β-catenin, thereby suppressing the activation of downstream transcription and inhibiting the proliferation and nuclear differentiation of keratinocytes. In summary, HLJDD could ameliorate psoriasis through multitarget inhibition of the Wnt/β-catenin signaling pathway.In conclusion, our results indicated that HLJDD, as a Chinese herbal prescription with multiple active components, could inhibit Wnt/