Our RNA sequencing results showed that several genes related to the Wnt pathway have changed in DRG and spinal cord dorsal horn (SCDH) after CCD surgery. Therefore, we detected the activation of the Wnt/β-catenin pathway in DRG and SCDH and found active β-catenin significantly upregulated in DRG and SCDH 1 day after CCD surgery and peaked on days 7-14. Immunofluorescence results also confirmed nuclear translocalization of active β-catenin in DRG and SCDH. Additionally, rats had obvious mechanical induced pain after CCD surgery and the pain was significantly alleviated after the application of the Wnt/β-catenin pathway inhibitor XAV939. Furthermore, we found that the levels of proinflammatory factors tumor necrosis factor-α (TNF-α) and interleukin-18 (IL-18) were significantly elevated in CCD rat serum, while the levels of them were correspondingly decreased after the Wnt/β-catenin pathway being inhibited. The results of Spearman correlation coefficient analysis showed that the levels of TNF-α and IL-18 were negatively correlated with the mechanical withdrawal thresholds (MWT) after CCD surgery. Collectively, our findings suggest that the Wnt/β-catenin pathway plays a critical role in the pathogenesis of NP and may be an effective target for the treatment of NP.Neuropathic pain is one of the important challenges in the clinic. Although a lot of research has been done on neuropathic pain (NP), the molecular mechanism is still elusive. We aimed to investigate whether the Wnt/ Low back pain (LBP) is the most common of all chronic pain disorders, and 80% of people experience LBP in their lifetime. LBP is often caused by lumbar foraminal stenosis (LFS) and lumbar disc herniation (LDH), causing symptoms such as motor dysfunction and NP . In mostβ-catenin pathway is one of three Wnt pathways, and active β-catenin is the key protein on the Wnt/β-catenin pathway. In the absence of Wnts, the destruction complex which is located in the cytoplasm will bind active β-catenin and phosphorylate it to inactive β-catenin and after that proteasome will degrade β-catenin. While Wnts bind Frizzled (Fzd) and LRP5/6 on the cell membrane, the destruction complex will subsequently disintegrate, and then, active β-catenin is not phosphorylated and accumulates in the cytoplasm, subsequently translocating into the nucleus, in which active β-catenin interacts with T cell/lymphoid enhancer factor (TCF/LEF) transcription factors, resulting in the target gene transcription such as TNF-α and IL-18 [β-catenin through Nestin-Cre-mediated recombination was sufficient to suppress the expression of brain-derived neurotrophic factor (BDNF), a major neurotrophic factor in mammals [β-catenin pathway may make an important impact on the pathogenesis of NP induced by CCD.The Wnt/nd IL-18 –15. Rece mammals , 17. Bes mammals , and chr mammals . Based oβ-catenin pathway in the generation and persistence of NP using the CCD model which is a well-characterized rat model. The CCD model is divided into a single nerve root compression model and two nerve roots' compression model. In this study, we chose the L4 and L5 nerve roots' compression model, because clinically, most of the LFS and LBP produce compression on multiple nerve roots instead of a single nerve root [β-catenin pathway over time in DRG and SCDH after CCD-induced NP. After that, we inhibited the activation of the Wnt/β-catenin pathway in DRG and SCDH through using XAV939, a small molecule tankyrase inhibitor which targets the Wnt/β-catenin pathway and inhibits the abnormal activation of the Wnt/β-catenin pathway without affecting normal function of cells [β-catenin signaling in the generation and persistence of NP.In our research, we first found that several genes related to the Wnt pathway have undergone significant changes through RNA sequencing analysis, and then, we investigated the role of the Wnt/rve root , so L4 aof cells , to obseof cells . FinallyFifty-nine SD (Sprague Dawley) male rats (220 g-250 g body weight) were purchased from Shanghai Jiesijie Experimental Animal Co., Ltd  2018-0004). Animals were housed in padded cages at a constant temperature (12 : 12 h light-dark cycle) and got food irradiated by the standard laboratory  and tap water with freedom. The animal experiments and handlings were approved by the Animal Ethics Committee of Tongji Hospital, Shanghai.To produce intervertebral foramen stenosis and NP, the CCD model was used. The procedure for CCD has been described , and theβ-catenin pathway , in 10% DMSO/90% 0.9% NaCl, was injected in doses of 1.25 mg/kg intraperitoneal. Besides, the sham+vehicle group and the CCD+vehicle group also received an injection (200 μl of 10% DMSO/90% 0.9% NaCl). The injections above were given at about 10 am every day for seven consecutive days.XAV939, an inhibitor of the Wnt/The rats were placed in a transparent box on the test grid at 8 am on test day, maintained at 25°C at room temperature, and adapted to the environment for 15-30 min. Von Frey hairs  were applied, according to former reports , in brieμl RNA was taken for quantification and 500 ng RNA was taken for library construction according to the quantification results. After that, the library was subjected to quality inspection using Qubit instruments . Then, Illumina HiSeq was used for sequencing, and the instrument was NovaSeq 6000 . Deseq2 software (v1.16.1) was used to screen for DEGs, and the screening criteria are P value ≤ 0.05 and ∣log2 fold change | ≥1.Rats were anesthetized with 2% pentobarbital sodium (80 mg/kg) by intraperitoneal injection 28 days after CCD and decapitated after reaching full anesthesia. The left L4 and L5 nerves were quickly exposed and were carefully extracted. Then, the enlarged DRG was cut with a blade on ice. Later, according to the positioning of the lumbar enlargement, the dorsal horn of the left spinal cord of L4-5 segment was removed. All the tissues above were, respectively, placed in centrifuge tubes containing RNA later  and were subsequently transferred to a 4°C refrigerator. RNA sequencing was completed by Shanghai Genergy Biotechnology Company, and the general protocol was as follows: 1 μm PVDF membrane . The membranes were treated with primary antibodies overnight at 4°C, after being blocked with 5% skimmed milk diluted in TBST, followed by being incubated in appropriate secondary antibodies. The following primary antibodies were used: rabbit anti-active β-catenin , mouse anti-β-tubulin , and horseradish peroxidase-linked secondary anti-rabbit or anti-mouse antibodies . Finally, the blots were visualized by DRAFT-FluorChem Q  and optical density of all bands was conducted by the ImageJ software .Rats were anesthetized with 2% pentobarbital sodium (80 mg/kg) by intraperitoneal injection and decapitated after reaching full anesthesia. The left L4-L5 DRG and SCDH segments were homogenized, respectively, by using the RIPA lysis buffer and then protein samples were quantified by applying BCA Protein Assay . The protein samples were separated by 10% SDS-PAGE  after being heated for 5 min at 99°C and subsequently transferred to 0.45 μm and 15 μm, respectively. Next, immunofluorescence staining was performed, and the steps are as follows: after being blocked with a 0.01 M PBS blocking solution containing 10% goat serum and 0.3% Triton X-100 for 1 h at 37°C, the sections were incubated with the primary antibody overnight at 4°C , followed by being incubated in goat anti-rabbit Cy3-conjugated secondary antibody  for 2 hours at 37°C in the dark. Subsequently, sections were stained with nuclear dye DAPI  for 7 minutes at 37°C in the dark. The sections were imaged with the confocal microscope  and fluorescence microscope .Rats were anesthetized with 1% pentobarbital sodium (40 mg/kg) by intraperitoneal injection. 0.9% saline and 4% paraformaldehyde were sequentially administered to these anesthetized rats. The L4 and L5 DRG were removed intact and subsequently fixed in 4% formaldehyde overnight. These tissues were then dehydrated with gradient sucrose  and frozen with OCT as they settled to the bottom in the 30% sucrose solution. After the embedding was completed, the tissues were sliced, and the thicknesses of the DRG and spinal cord sections were 10 α and IL-18 concentrations were calculated according to the OD value of the sample.The rats in the deep anesthetized with sodium pentobarbital (80 mg/kg) were subjected to blood collection from the orbital venous plexus, and then, blood collected was centrifugated to get serum at 4000 rpm for 10 min at 4°C. Detailed steps were performed according to the kit  instructions. The optical density (OD) value was measured by DENLEY DRAGON Wellscan MK 3  at 450 nm, and all OD values were calculated after subtracting the blank value. Ascent software for Multiskan  was applied to draw a standard curve, and the corresponding TNF-N) of rats and the statistical significance were stated in the figures and figure legends. The sample size was determined based on previous experience. Two-way ANOVA was used to analyze the result of behavior, and one-way ANOVA was applied to analyze the western blot and ELISA. Spearman correlation coefficient analysis was used to analyze the correlation between MWT and inflammatory factor levels, including TNF-α and IL-18. Data are represented as mean ± SD (standard deviation). P < 0.05 was considered statistically significant.GraphPad Prism 7.0 software  and SPSS 20.0 software  were applied to analyze all data. The number (P < 0.0001). By the 28th day after surgery, the MWT remained at a low level (P < 0.0001). The MWT at each time point in the CCD group was lower than that in the sham group (P < 0.0001), while no obvious allodynia was observed in the sham group (P > 0.05).To confirm that the CCD model successfully and reproducibly induced tactile allodynia , we perfFor selection of a subset of genes related to the Wnt pathway, the NCBI gene database and the Panther Classification System database system were used. “Wnt pathway” was used as a keyword to conduct a restrictive search for “rattus norvegicus.” 526 genes related to the Wnt pathway were obtained in the NCBI gene database. The overlapping genes of these data sets and the DEGs  screened in the DRG and SCDH were analyzed using an online tool of the Venn diagram, and the results showed that 8 DEGs and 4 DEGs in DRG and SCDH were related to the Wnt pathway, respectively. Then, we performed gene ontology analysis by the Panther Classification System database system to enrich the pathways involved in proteins encoded by DEGs in DRG and SCDH and found that 4 DEGs in DRG and 1 DEG in SCDH related to the Wnt pathway were screened out. After removing the duplicated genes, we screened out 12 and 4 genes in DRG and SCDH, respectively .β-catenin pathway in DRG and SCDH in CCD rats has changed. Active β-catenin is a critical protein in this pathway and whether it undergoes nuclear translocation reflects whether Wnt/β-catenin is activated. Therefore, we detected the level of active β-catenin by western blot and nuclear translocation of active β-catenin by immunofluorescence. As is shown in western blot results, the compression of DRG resulted in a rapid rise in the expression of active β-catenin in DRG (P < 0.05). Both reached a peak of expression in 7-14 days and then gradually declined. Additionally, immunofluorescence experiments showed that after 7 days of modeling, a large number of active β-catenin positive stains (red) were seen on the left DRG (β-catenin (red) and DAPI (blue) showed that the expression of active β-catenin in the nucleus rose in CCD rats. To further explore the potential mechanism of the Wnt/β-catenin pathway affecting NP, we detected the expression levels of TNF-α and IL-18, which are the proinflammatory cytokines in serum by ELISA. The results showed that the level of TNF-α and IL-18 in rat serum increased significantly on the first day (P < 0.0001) and reached the peak at 7 days, and then, TNF-α gradually decreased, while IL-18 remained at a stable level until postoperative 28 days (P < 0.0001)  and IL-18  were negatively correlated with MWT after CCD surgery, which meant that the higher the levels of TNF-α and IL-18, the more obvious the pain in rats, and vice versa, the pain in rats was relieved.DRG and SCDH changes caused by nerve injury are critical for the generation and persistence of NP. We addressed whether the Wnt/n in DRG  and SCDHn in DRG   . The results above demonstrated that the activation of the Wnt/β-catenin pathway affected the persistence of NP and the expression of TNF-α and IL-18 was regulated by the activation of the Wnt/β-catenin pathway after CCD.Next, to investigate whether the Wnt/ 0.0001) . Further Figures . Corresp Figures  when we β-catenin pathway in the induction and continuation of NP following chronic compression of nerve to light. The main findings can be attributed to the following three aspects: (1) chronic nerve compression led to changes in the expression of multiple Wnt pathway-related genes and rapid and sustained activation of the Wnt/β-catenin pathway in DRG and SCDH; (2) inhibition of activation of the Wnt/β-catenin pathway alleviated allodynia induced by chronic nerve compression; (3) CCD led to elevating the level of TNF-α and IL-18. TNF-α and IL-18 levels were significantly correlated with MWT, while blocking the Wnt/β-catenin pathway decreased the levels of TNF-α and IL-18.LBP is one of the most common pains in the clinic. Mechanical compression and chemical stimulation of DRG may lead to ischemia, edema, and degeneration of DRG and eventually leading to the development of allodynia , 30. Howβ-catenin pathway is activated in SCDH in several NP models, such as CCI and PSL [α [α and IL-18 among many elevated inflammatory factors was that these two inflammatory factors were not only closely related to pain, but also target genes closely downstream of the Wnt/β-catenin pathway [β-catenin pathway makes an important impact on the development and progression of NP, and the activation of this pathway may participate in the pathogenesis of NP by regulating the transcription of TNF-α and IL-18, which are closely related to pain.Wnt is very important for various development processes. The Wnt pathway was first revealed by scientists in the development of Drosophila  and is a and PSL , 18. Besd PSL [α , 35 and d PSL [α . The rea pathway . These sβ-catenin was immediately and continuously upregulated in DRG and SCDH in the CCD model, and immunofluorescence results confirmed the translocation of active β-catenin into the nucleus, which can more intuitively observe the localization of active β-catenin protein in cells and more powerful than the extraction of active β-catenin protein in the nucleus to prove the nuclear translocation of active β-catenin protein and the activation of the Wnt/β-catenin pathway. This suggested the activation of the Wnt/β-catenin pathway lasted for a long time, which was consistent with previous studies in other models [α and IL-18, which are closely related to NP, were considerably raised in the serum of CCD rats; however, after inhibiting this pathway, the levels of TNF-α and IL-18 correspondingly decreased. Moreover, the levels of TNF-α and IL-18 were significantly correlated with the allodynia time of CCD rats. These results implicated blocking the Wnt/β-catenin pathway can inhibit NP by decreasing the level of IL-18 and TNF-α. These results revealed that the Wnt/β-catenin pathway takes part in the development of NP after compression of DRG and is likely to affect NP by regulating the transcription of downstream inflammatory factors. Mechanisms underlying contributions of the Wnt pathway to neuropathic pain are summarized in Our study found that active r models , 18. Addβ-catenin pathway may also induce peripheral sensitization and central sensitization by increasing neuronal excitability in DRG and increasing synaptic strength in SCDH, thereby affecting the progression of NP.What is more, many scholars have conducted extensive research on the mechanism of NP, and it is believed that NP is caused by peripheral sensitization and central sensitization , 38. Perβ-catenin pathway in the production and persistence of NP after chronic compression of DRG to light. Additionally, the Wnt/β-catenin pathway may induce NP through modulating the levels of TNF-α and IL-18. Moreover, it provides new targets and new ideas for the treatment of nerve compression diseases including LFS and LDH and may also be one of the ways for some drugs and biological treatments such as stem cells to exert analgesic effect.In summary, our research brings a pivotal role for the Wnt/


A β-catenin-driven switch in TCF/LEF transcription factor binding to DNA target sites promotes commitment of mammalian nephron progenitor cells. Published 15, February 2021We overlooked an important published study by Ramalingam et al (2018) which draws the conclusion that different levels of ß-catenin, resulting from different levels of Wnt9b ligand, determine nephron progenitor cell (NPC) outcomes. Though our study focuses on defined NPC cultures and genomic interactions amongst ß-catenin and Tcf/Lef transcriptional mediators that were not explored in Ramalingham et al., our approaches agree on the importance of ß-catenin levels in modifying the state of NPCs. Accordingly, we have corrected our manuscript below to acknowledge Ramalingam et al (2018) were appropriate and we apologize to the authors for the oversight.#1Original text:Genomic analysis of β-catenin engagement at TCF/LEF recognition motifs within enhancers linked to genes driving NPC differentiation , and subsequent transgenic studies demonstrating TCF/LEF-dependent activity of cis regulatory elements, provides strong evidence for a canonical Wnt/β-catenin/Tcf regulatory axis .Corrected text:Genetic and chemical modification of Wnt pathway components suggest different levels of β-catenin distinguish maintenance and commitment of NPCs . Genomic analysis of β-catenin engagement at TCF/LEF recognition motifs within enhancers linked to genes driving NPC differentiation , and subsequent transgenic studies demonstrating TCF/LEF-dependent activity of cis regulatory elements, provides strong evidence for a canonical Wnt/β-catenin/Tcf regulatory axis .#2Original text:Within 3 days of elevating CHIR levels (3 µM), aggregate NPC cultures show a robust signature of nephron differentiation Corrected text:Within 3 days of elevating CHIR levels (3 µM), aggregate NPC cultures show a robust signature of nephron differentiation #3Original text:Wnt4 Genetic analysis of mouse models points to a requirement for β-catenin in both the maintenance  and differentiation  of NPCs. Previous studies directly examining β-catenin association in differentiating NPCs showed direct engagement of β-catenin at enhancers regulating expression of differentiation promoting genes such as Corrected text:Wnt4 .Genetic analysis of mouse models points to a requirement for β-catenin in both the maintenance  and differentiation  of NPCs. Genetically modulating Wnt inputs and chemically modifying Wnt-pathway activity suggest Wnt-modulation of β-catenin levels is critical to these alternative outcomes . Previous studies directly examining β-catenin association in differentiating NPCs showed direct engagement of β-catenin at enhancers regulating expression of differentiation promoting genes such as #4Insert reference below into reference list:Ramalingam H, Fessler AR, Das A, Valerius MT, Basta J, Robbins L, Brown AC, Oxburgh L, McMahon AP, Rauchman M and Carroll TJ. (2018). Disparate levels of beta-catenin activity determine nephron progenitor cell fate. Developmental Biology440(1):13-21.#5 (an independent typographic error)Original text:Sub section title - Elevating β-catenin leads to activation of Tcftt/Lef-bound enhancersCorrected text:Sub-section title - Elevating β-catenin leads to activation of Tcf/Lef-bound enhancers


In this research, an epoxy-based dual-cure system is developed and characterized for SLA additive manufacturing. Dual-cure systems consist of UV-curable acrylates and thermal active components. The second curing step offers an additional degree of freedom to design specific material properties. In this study, a blend of varying concentrations of an epoxy/curing agent mix, respectively, DGEBA, DICY and photocurable methacrylate, was used to create a material that is printable in the SLA process into a UV-cured or green part and subsequently thermally cured to achieve superior thermal and mechanical properties. Calorimetric measurements were performed to determine the reactivity of the thermal reaction at different concentrations of epoxy. The fully cured specimens were tested in mechanical and dynamic mechanical measurements, and the results showed a significant improvement in tensile stress and glass transition temperature with rising epoxy concentrations. Fractured surfaces from tensile testing were investigated to further characterize the failure of tested samples, and thermal degradation was determined in TGA measurements, which showed no significant changes with an increasing epoxy concentration. Stereolithography (SLA) is the oldest additive manufacturing (AM) technique and is based on the generation of three-dimensional parts by the subsequent addition of single layers onto each other, using a photochemical reaction of monomers . This laDifferent stage-curing epoxies are already a common way to produce epoxy-based prepregs for subsequent manufacturing processes. For this application, the already gelificated epoxy is undercooled to stop a further curing process . Dual-cuIn the field of dual-cure systems for use in additive manufacturing, different promising approaches have already been pursued. Next to the common pure acrylate systems, many resin systems for SLA are based on the usage of the epoxy acrylates  or linkiIn this study, a latent curing methacrylate/epoxy blend is prepared and printed. The aim is to generate a latent curing material system to achieve superior mechanical properties in combination with high thermal stability compared to a pure acrylate system. Due to their good mechanical properties, chemical and electrical resistance and thermal properties, epoxies find usage in many applications over coatings, adhesives, construction, composites and electronics materials ,29,30, mThe material contains two different resin systems, which were mixed in varying ratios. For the UV-resin, the commercial “Clear V4” (further referred to as “Clear” or “methacrylate”) resin of Formlabs  is used, consisting of a mixture of 75–90% methacrylated oligomer, 25–50% methacrylated monomers and <1% diphenyl  phosphine oxide (TMDPO) as photoinitiator. For the epoxy EPILOX A 18-00 by Leuna Harze , a diglycidyl ether of bisphenol A (DGEBA) with an epoxy equivalent weight of 175–185 g is used a. RegardIn the first step, the epoxy resin was prepared. A total of 100 phr of EPILOX A 18-00 were preheated to 60 °C to lower the viscosity and ensure a homogenous dispersion of the DICY. Next, 7 phr of Amicure were added and mixed using a mechanical stirrer for 10 min at a rotation speed of 200 rpm. This epoxy-curing-agent mixture will further be referred to as epoxy. After the mixture was cooled to 40 °C, a specified amount  of ClearThe mixture was degassed in a vacuum chamber with a pressure of 0.03 bar for 5 min to prevent oxygen-induced inhibition during UV-curing and reduction in mechanical properties due to voids.A Form 2 SLA-printer  was used for the printing process with the designated presettings for the Clear V4 resin. The printed specimens are tensile bars  and DMA testing samples . For both types of testing specimens, support structures were used with a complete raft, a density of 1.0, and a diameter of the connection point of 0.6 mm. A layer height of 0.1 mm was chosen. After printing the samples, they were first spun to remove the remaining resin and afterward, washed in IPA (concentration of 99.5%) in two stages, with each stage lasting 30 s. After the IPA cleaning, the green parts were carefully dried with pressurized air to avoid the solvent from damaging the still sensitive parts. During the printing process, the photoinitiator (PI) of the Clear resin, TMDPO, respectively, forms radicals subsequently to UV-irradiation a 32]..32].These free radicals (R) initiate a chain polymerization with the methacrylated monomers and oligomers (M) to form new radicals (RM●) repeatedly until, after a certain chain length a Polymer (P) of the length x is formed. This reaction occurs until a termination reaction occurs b 33,34,,34,35. TFor the thermal post-curing step, a forced convection oven type DKN612C  was used with a two-stage heating program. In the first stage, the parts were heated up 165 °C and pre-cured for 1 h and in the second stage, cured at 180 °C for 2 h. The temperature and time for the second stage was gathered from the Amicure CG 1200 datasheet. The first stage was added since pretests showed that heating up to 180 °C in only one step leads to a reaction that is too high, which induced cracking of parts. Exemplary DSC measurements were performed after thermal curing in the oven to secure complete curing. They showed no further curing reactions, which states the oven cycle was sufficient. The curing of DGEBA/DICY happens to be a complex process where multiple different reactions can occur. The essential reactions are, dependent on the temperature and the composition of the resin, a ring opening addition reaction of the epoxy group and the primary and secondary amin of the DICY, the addition of hydroxyl to the oxirane functionality or also called etherification of epoxy group with the hydroxyl group, homopolymerization of epoxy groups and an addition reaction of the nitrile group ,31. SincDifferential Scanning Calorimetry measurements were performed to ensure and identify the intensity of the exothermic reaction occurring between the Amicure curing agent and the DGEBA. For the DSC measurement, a type Jupiter STA 449 F3 DSC device  was used. The samples of the Clear/epoxy blends for DSC measurement were cut from UV-cured solid specimen. As a reference for this study, a liquid 100% epoxy concentration sample (100/0) and a 0% epoxy concentration sample (0/100) were added to the measurement, The heat flow of the sample was continuously measured during the heating from 50 to 200 °C, using a heating rate of 5 K/min. For the measurement, an open 85-microliter aluminum crucible was used with a weight of 19 ± 0.5 mg and a sample weight of 8 ± 1 mg. The measurements were performed under nitrogen atmosphere.Thermogravimetric analysis was performed to determine whether there is an influence of the epoxy on the beginning and speed of material degradation. The TGA measurements were conducted on a type Jupiter STA 449 F3 TGA device . The samples were prepared in a similar way to the DSC samples. For the measurement, small samples were heated to 500 °C with a heating rate of 5 K/min, while the loss of mass was determined by constant weighing of the sample. For the measurement an open 85-microliter aluminum crucible was used with a weight of 19 ± 0.5 mg and a sample weight of 8 ± 1 mg. The measurements were performed under nitrogen atmosphere.D, the tensile strength σmax and the elongation at break εr. The testing speed was set to 2.5 mm/min with a pre-force of 0.5 N. The tensile modulus was determined with an extensometer.The samples were stored before the tensile testing at standard climate  for a minimum of 48 h. Subsequently, the samples were tested with a type 1465 universal testing machine  according to DIN EN ISO 527: 2012, to measure the tensile modulus Eg of the prepared samples in dependency of the used epoxy proportion. Measurements were performed on a type RSA G-2 DMA measurement device . The load was introduced by torsion with a frequency of 1 Hz and a strain of 0.02%. While keeping mechanical stress constant, a heat ramp of 2 K/min was used, beginning at −60 to 200 °C. The determination of Tg was conducted by evaluating the peak of tan(δ).Dynamic mechanical testing (DMA) was used to measure the storage modulus E’, loss modulus E’’ and tan(δ) to determine the glass transition temperature TThe fractured surfaces of the samples obtained from the tensile testing were inspected with a scanning electron microscope type Gemini Ultra-Plus  with a voltage of 10 kV, varying working distance (WD) and a magnification of 22, 50 and 100.DSC measurement is a reliable tool for measuring and visualizing the exothermic reaction of epoxy thermosets. The results of the measurement are shown in The missing peak should also affect the results of the mechanical and thermal properties. The curves of the 30/70, 50/50 and 70/30 samples show the same reaction peak with an increasing intensity for a higher epoxy concentration and a shift to lower temperatures of the peak itself, while the onset of the peak remains at 150 °C. The reason for the increase in peak intensity is that with a higher absolute number of reactive groups, a higher share of reactions can also possibly happen. The shift to lower temperatures combined with the increase in of the gradient of the graphs for higher epoxy concentrations indicates a faster and more intense reaction. This shift can be explained by the auto-accelerating effect of the exothermic DGEBA/DICY-reaction . The purThe tensile properties of the Clear/epoxy blend, depending on the epoxy concentration, are summarized The variation of the tensile modulus with different concentrations of epoxy is shown in The examination of the fracture surfaces shown in For all the different epoxy concentrations, minor defects in the form of voids are identifiable. The reasons for this phenomenon could be insufficient gas evaporation during the degassing process due to the relatively high viscosity of the resin mixture or the consequences of shrinkage and, therefore, residual stress during thermal curing, which is typical for epoxy . Since tThe structure of the fracture surface shows three areas, which is typical for an epoxy fracture. In particular, the zones are the flat mirror zone, the transition zone, characterized by increasing surface roughness, and the final propagation zone . The 10/As shown in g, which is traced back to the two different materials in the mixture. For the 50/50 sample, the graph of tan(δ) results in a broad peak while being distinct for the 70/30 sample. The graphs for the high epoxy concentrations, on the other hand, again show the double peak structure since the broad peak of 50/50 is probably a superimposition of two single peaks. This double peak supports the initial assumption of the existence of two TThe results of the TGA measurements, shown in Only the reference sample of 100% epoxy is mostly stable until approx. 350 °C before it also drops rapidly. It stands out that the 0/100 and the 10/90 sample both show two clear steps in the material degradation, probably due to the two different types of methacrylate used in the Clear resin. This two-step degradation is still present for the 30/70 sample but absent for higher epoxy concentrations. With an increasing epoxy concentration, the double peak structure morphs into a local linear decrease in mass, probably due to the superimposition of the two degradation processes of Clear resin and epoxy composition. The 100/0 sample shows only one clear degradation step. Besides the change to only one degradation step with the increasing epoxy content, the speed of the degradation process is also decelerated until the crossover with the graph of the 100/0 sample. After this crossover, the speed of degradation is accelerated with an increasing epoxy contend. It stands out that the samples show different residuals that are proportional to their related content of epoxy. While the 0/100 sample shows no residuals, they are increasing until the 100/0 pure epoxy sample.UV-resin blended with different amounts of epoxy, DGEBA/DICY, respectively, for resin-based additive manufacturing was prepared, successfully printed and investigated. With the used printing-system and the standard parameter set, a concentration of a maximum of 70 wt.-% of epoxy was achievable. Higher concentrations led to an insufficient layer adhesion followed by misprints. However, it was found that the first layers, the so-called base layers, were able to be printed at higher concentrations. The printer automatically uses higher exposure settings for these early layers to achieve a good layer adhesion to the building platform. Consequently, adjusting the exposure settings for the following layers should also allow higher concentrations to be printed. DSC measurements showed that a reaction is prevented for low concentrations of epoxy, probably by a separation of reactive groups due to the poor mobility of the system and the low absolute number of reactive groups. This effect was also present in the analysis of mechanical and thermomechanical properties, where no significant changes were found for low concentrations up to 30 wt.-%. After a critical concentration of 50% epoxy, a reaction of DGEBA and DICY occurred, which led to changes in the material properties of the manufactured samples. While not significantly changing the elongation at break, the addition of 70% epoxy led to an increase in the tensile stress from 48 to 83 MPa and a shift of glass transition temperature from 90 to 140 °C compared to the benchmark pure UV-resin parts considered under perfect manufacturing conditions. The structure of the fracture surface also resembled a shift to a more ductile behavior. For thermal degradation, no significant changes could be measured. Since the aim of this work was to achieve an increase in mechanical and thermal properties, the results can be stated as successful. However, the samples showed a critical concentration after which an exponential increase in the tensile stress, elongation at break and glass transition temperature occurred. An epoxy concentration above 70% has a high chance of leading to another significant increase in the mechanical properties, which will be studied in the following investigations.


Drug addiction is considered maladaptive learning, and drug-related memories aroused by the presence of drug related stimuli (drug context or drug-associated cues) promote recurring craving and reinstatement of drug seeking. The mammalian target of rapamycin signaling pathway is involved in reconsolidation of drug memories in conditioned place preference and alcohol self-administration (SA) paradigms. Here, we explored the effect of mTOR inhibition on reconsolidation of addiction memory using cocaine self-administration paradigm. Rats received intravenous cocaine self-administration training for 10 consecutive days, during which a light/tone conditioned stimulus was paired with each cocaine infusion. After acquisition of the stable cocaine self-administration behaviors, rats were subjected to nosepoke extinction (11 days) to extinguish their behaviors, and then received a 15 min retrieval trial with or without the cocaine-paired tone/light cue delivery or without. Immediately or 6 h after the retrieval trial, rapamycin (10 mg/kg) was administered intraperitoneally. Finally, cue-induced reinstatement, cocaine-priming-induced reinstatement and spontaneous recovery of cocaine-seeking behaviors were assessed in rapamycin previously treated animals, respectively. We found that rapamycin treatment immediately after a retrieval trial decreased subsequent reinstatement of cocaine seeking induced by cues or cocaine itself, and these effects lasted at least for 28 days. In contrast, delayed intraperitoneal injection of rapamycin 6 h after retrieval or rapamycin injection without retrieval had no effects on cocaine-seeking behaviors. These findings indicated that mTOR inhibition within the reconsolidation time-window impairs the reconsolidation of cocaine associated memory, reduces cocaine-seeking behavior and prevents relapse, and these effects are retrieval-dependent and temporal-specific. Drug addiction is defined as a chronic psychiatric disease with a high rate of relapse even after a long period of withdrawal. Addicted individuals experience intense drug craving that can persist long after withdrawal, leading to high relapse rates. Additionally, they exert compulsive seeking and drug-taking behaviors that are difficult to control despite the serious adverse consequences of drug abuse. The critical problem of drug addiction is to prevent and reduce relapse after withdrawal . To dateThe formation of conditioned associations between repeated drug abuse and associative context is essential for the generation and maintenance of addiction-related behaviors. Exploring the neural mechanisms underlying the associated learning process in the formation of drug memories will help us understand addictive behaviors from the perspective of maladaptive learning and memory and develop new treatments to prevent relapse . Chronicde novo protein synthesis to be restabilized  signaling pathway is well known to exert complex physiological functions proteins . Reconsoproteins . Therefoproteins . The mTOproteins . HoweverIt is generally accepted that rapamycin is the specific inhibitor of mammalian target of rapamycin . In the Male Sprague Dawley rats  were obtained from the Tianqin Laboratory Animal Technology Co. Ltd., China. Rats were housed in groups of five under controlled temperature (23 ± 2°C) and humidity (50 ± 5%), and maintained on a 12 h light/dark cycle  with access to food and water ad libitum. All of the rats were handled 3 min per day for 5 days before the surgeries. All animal procedures were performed in accordance with the Guide of Hunan province for the Care and Use of Laboratory Animals, and the experiments were approved by the Local Committee on Animal Care and Use and Protection of the Hunan Normal University. All the experiments were performed during the dark phase.Rats (weighing 300–320 g at the time of surgery) were anesthetized with sodium pentobarbital . Catheters were inserted into the right jugular vein with the tip terminating at the opening of the right atrium as described previously . The canThe operant chambers used  were equipped with two nosepoke operandi  located 5 cm above the chamber floor. Nosepokes in the left side (active) operandum led to cocaine infusions that were accompanied by a 5 s tone-light cue. Nosepokes in the right side (inactive) operandum were also recorded but had no programmed consequences. Rats were trained to self-administer cocaine hydrochloride (0.75 mg/kg/infusion) during three 1 h daily sessions separated by 5 min over 10 days. The sessions began at the onset of the dark cycle (8:00–20:00). A fixed-ratio one reinforcement schedule was used, with a 40 s timeout period after each infusion. Each session began with the illumination of a houselight that remained on for the entire session. The number of drug infusions was limited to 20 per hour to prevent death by overdose . We exclDuring extinction, the conditions were the same as during cocaine self-administration training, except that cocaine was no longer available and without the tone-light cue .A 15 min retrieval to reactivate cocaine-associated memories began 24 h after the last nosepoke extinction session. The retrieval conditions were the same as during cocaine self-administration training except that active nosepokes with no reward cocaine.During extinction, the conditions were the same as during cocaine self-administration training, with the exception that no cocaine injections were given with the delivery of cue (tone/light).Rats were returned to the same self-administration context and recorded the number of nosepokes to both operandi (active and inactive) for 1 h. The conditions were the same as during cocaine self-administration training except that cocaine injections were not given upon active nosepoking.For priming-induced reinstatement test, rats were injected with cocaine  5 min before the start of the session. Rats then were placed in the same self-administration context and recorded the number of nosepokes to both operandi (active and inactive) was recorded for 1 h. The conditions were the same as during cocaine self-administration training except that active nosepokes were not reinforced with cocaine.For a spontaneous recovery test, active and inactive operandi nosepokes were recorded for 1 h after 28 days withdrawal. The testing conditions were similar to those during the cue-induced reinstatement test.Rats received three 1-h daily sessions of intravenous cocaine self-administration training for 10 days, and 24 h after training, all rats then underwent 11 consecutive days of daily nosepoke extinction training. 24 h after the last nosepoke extinction session, the rats were divided into two groups: 1) Intraperitoneal injection of rapamycin  immediately after a 15 min retrieval trial ; 2) Intraperitoneal injection of vehicle  immediately after a 15 min retrieval trial . The doses of rapamycin were chosen based on previous studies . On Day Rats received three 1 h daily sessions of intravenous cocaine self-administration training for 10 days, and 24 h after training, all rats then underwent 11 consecutive days of daily nosepoke extinction training. 24 h after the last nosepoke extinction session, the rats were divided into two groups: 1) Intraperitoneal injection of rapamycin  immediately after a 15 min retrieval trial ; 2) Intraperitoneal injection of vehicle  immediately after a 15 min retrieval trial . 24 h after the last extinction session, rats underwent the cue-induced reinstatement test. To confirm the long-term inhibition effect of rapamycin on cocaine-seeking behavior in rats, 28 days after the cue-induced reinstatement test, all rats were tested for spontaneous recovery on Day 58 .The experimental procedure for Experiment 3 was the same as in Experiment 1, except that injection of rapamycin was made 24 h after the last nosepoke extinction session without the retrieval trial session .The experimental procedure for Experiment 4 was the same as in Experiment 1, except that rapamycin was administered intraperitoneally 6 h after the retrieval trial session .post-hoc test in each experiment (see results). The values are presented as the mean ± S.E.M. Values of p < 0.05 were considered statistically significant.The data were analyzed using repeated measures ANOVAs with between-subjects factor of treatment condition and within-subjects factor of test condition followed by Tukey’s  = 36.620, p < 0.001; main effect of the treatment condition: F = 0.035, p = 0.854; interaction of training day × treatment condition: F = 0.299, p = 0.974]  = 32.410, p < 0.001; main effect of the treatment condition: F = 0.508, p = 0.488; interaction of extinction day × treatment condition: F = 0.331, p = 0.971]  = 42.61, p < 0.001; main effect of the treatment condition: F = 0.01549, p = 0.9027; interaction of different nosepokes × treatment condition: F = 0.03614, p = 0.8519]  = 45.940, p < 0.001; main effect of the treatment condition: F = 28.342, p < 0.001; interaction of test condition × treatment condition: F = 19.710, p = 0.001]; Post hoc shown that drug-seeking in the Retrieval + Rapa group was significantly reduced compared to the Retrieval + Veh group in the cue-induced reinstatement test (p < 0.05)  = 1.575, p = 0.230; main effect of the treatment condition: F = 0.172, p = 0.684; interaction of test condition × treatment condition: F = 0.080, p = 0.781]  = 88.352, p < 0.001; main effect of the treatment condition: F = 11.98, p = 0.004; interaction of test condition × treatment condition: F = 10.560, p = 0.006; ]; Post hoc shown that drug-seeking in the Retrieval + Rapa group was significantly reduced compared to the Retrieval + Veh group in the priming-induced reinstatement test (p < 0.05)  = 0.654, p = 0.432; main effect of the treatment condition: F = 0.066, p = 0.801; interaction of test condition × treatment condition: F = 0.001, p = 0.975]  = 41.518, p < 0.001; main effect of the treatment condition: F = 0.077, p = 0.785; interaction of training day × treatment condition: F = 0.757, p = 0.656]  = 30.187, p < 0.001; main effect of the treatment condition: F = 0.193, p = 0.665; interaction of extinction day × treatment condition: F = 0.265, p = 0.988]  = 29.68, p < 0.001; main effect of the treatment condition: F = 0.2486, p = 0.6238; interaction of different nosepokes × treatment condition: F(119) = 0.0484, p = 0.8282]  = 55.265, p < 0.001; main effect of the treatment condition: F = 19.091, p < 0.001; interaction of test condition × treatment condition: F = 14.283, p = 0.001]; Post hoc shown that drug-seeking in the Retrieval + Rapa group was significantly reduced as compared to the Retrieval + Veh group during the cue-induced reinstatement test (p < 0.05)  < 0.001, p = 0.989; main effect of the treatment condition: F = 0.295, p = 0.593; interaction of test condition × treatment condition: F = 0.085, p = 0.773]  = 60.320, p < 0.001; main effect of the treatment condition: F = 11.220, p = 0.003; interaction of test condition × treatment condition: F = 15.444, p = 0.001]; Post hoc shown that drug-seeking in the Retrieval + Rapa group was significantly reduced as compared to the Retrieval + Veh group in the spontaneous recovery test (p < 0.05)  = 0.987, p < 0.001; main effect of the treatment condition: F = 0.150, p = 0.703; interaction of test condition × treatment condition: F = 0.012, p = 0.915]  = 29.498, p < 0.001; main effect of the treatment condition: F = 2.014, p = 0.175; interaction of training day × treatment condition: F = 0.943, p = 0.490]  = 18.906, p < 0.001; main effect of the treatment condition: F = 0.044, p = 0.837; interaction of extinction day × treatment condition: F = 0.735, p = 0.691]  = 39.240, p < 0.001; main effect of the treatment condition: F = 0.122, p = 0.732; interaction of test condition × treatment condition: F = 0.074, p = 0.789]  = 1.493, p = 0.239; main effect of the treatment condition: F = 2.419, p = 0.139; interaction of test condition × treatment condition: F = 0.620, p = 0.443]  = 75.577, p < 0.001; main effect of the treatment condition: F = 0.152, p = 0.701; interaction of test condition × treatment condition: F = 0.053, p = 0.821]  = 10.826, p = 0.005; main effect of the treatment condition: F = 0.031, p = 0.862; interaction of test condition × treatment condition: F = 1.111, p = 0.307] (p > 0.05). Therefore, the results of Experiment 3 indicate that the effect of rapamycin in Experiment 1 depends on the retrieval trial and that rapamycin administration without a retrieval trial has no effects on subsequent cue-induced and priming-induced reinstatement of cocaine-seeking behavior.In Experiment 3, we examined whether rapamycin disrupting reconsolidation of cocaine memory was retrieval dependent. After the cocaine self-administer acquisition and extinction, rats administered rapamycin or vehicle but without a retrieval trial . There w= 0.490] . Further= 0.691] . In addi = 15.687, p < 0.001; main effect of the treatment condition: F = 0.701, p = 0.418; interaction of training day × treatment condition: F = 1.021, p = 0.427]  = 17.667, p < 0.001; main effect of the treatment condition: F = 1.088, p = 0.316; interaction of extinction day × treatment condition: F = 1.080, p = 0.383]  = 57.10, p < 0.001; main effect of the treatment condition: F = 0.06455, p = 0.8034; interaction of different nosepokes × treatment condition: F = 0.7395, p = 0.4054]  = 49.409, p < 0.001; main effect of the treatment condition: F = 0.040, p = 0.845; interaction of test condition × treatment condition: F = 0.382, p = 0.547]  = 0.693, p = 0.420; main effect of the treatment condition: F = 0.620, p = 0.445; interaction of test condition × treatment condition: F = 0.028, p = 0.870]  = 43.008, p < 0.001; main effect of the treatment condition: F = 0.349, p = 0.565; interaction of test condition × treatment condition: F = 0.058, p = 0.814]  = 11.956, p = 0.004; main effect of the treatment condition: F = 0.690, p = 0.421; interaction of test condition × treatment condition: F = 0.204, p = 0.659] (p > 0.05). These findings indicate that the effect of mTOR kinase inhibition via rapamycin on blocking reinstatement of cocaine seeking is time-limited, that is, this effect must occur within 6 h after retrieval.In Experiment 4, we examined whether the rapamycin treatment outside the sensitive time window could disrupted the reconsolidation of cocaine memory. We assessed the effect of rapamycin administration 6 h after retrieval of cocaine cue memory on cue-induced and priming-induced reinstatement of cocaine seeking behaviors . During = 0.427] . Further= 0.383] . For the 0.4054] . For theThe study first identified the critical role of mTOR in reconsolidation in the classic cocaine cue memory model. The main findings of this study are following: 1) Systemic administration of rapamycin immediately after retrieval of cocaine cue memory effectively reduced the cocaine-seeking behavior induced by drug-associated cues and the reinstatement of cocaine seeking induced by cocaine in rats; 2) The inhibitory effect of systemic rapamycin administration immediately after retrieval of cocaine cue memory on cocaine-seeking behavior persisted for least 28 days; 3) rapamycin when administered intraperitoneally with a 6 h delay or without a retrieval trial had no effects on cue-induced and cocaine-priming-induced reinstatement of cocaine-seeking behavior, indicating that the inhibitory effects of rapamycin on cocaine-seeking behavior is retrieval-dependent and temporal-specific.de novo protein-dependent process, which is completed within 6 h after retrieval. The effectiveness of interventions that destroy reconsolidation depends on the time window and the manipulation of memory retrieval  and ribosomal protein S6 kinases (S6Ks), it thus plays a critical role in the protein translation process . mTOR alIn addition, the mTOR signaling pathway can also affect the protein’s translation, synthesis, apoptosis, and the occurrence and proliferation of tumor cells by regulating ribosome synthesis and downstream gene transcription . FurtherPrevious studies have also shown that immediate intraperitoneal rapamycin injection after reactivating memory by exposure to drug-associated environment can disrupt reconsolidation of cocaine, morphine, and alcohol-induced addiction memory in the CPP conditioned place preference paradigm . In the In summary, our findings demonstrated that the mTOR activity plays a critical role in the reconsolidation of cocaine cue memory using the operational cocaine SA paradigm. Administration of rapamycin immediately after memory reactivation disrupts reconsolidation of the cocaine cue memory, leading to a reduction in reinstatement of cocaine seeking behavior in rats. The present study identifies the mTOR signaling pathway as a promising target for treating cocaine addiction and preventing relapse.


The fields of pediatric gastrointestinal endoscopy and sedation are critically important to the diagnosis and treatment of gastrointestinal (GI) disease in children. Since its inception in the 1970s, pediatric endoscopy has benefitted from tremendous technological innovation related to the design of the endoscope and its associated equipment. Not only that, but expertise among pediatric gastroenterologists has moved the field forward to include a full complement of diagnostic and therapeutic endoscopic procedures in children. In this review, we discuss the remarkable history of pediatric endoscopy and highlight current limitations and future advances in the practice and technology of pediatric endoscopy and sedation. Pediatric endoscopy came into existence nearly 170 years after Dr. Phillip Bozzini developed the first “endoscope” in 1805 known as the “Lichleiter” candle . Then, iIn 1969 the Hopkins rod-lens system permitted miniaturization of the endoscope. This revolutionary system paved the way for development of the pediatric specific fiber endoscope , 6. In tSedation for pediatric gastrointestinal endoscopy is an important component for patient comfort and procedural success. Pediatric developmental and physiologic considerations, however, require a specialized approach to avoid serious complications. Although the occurrence of serious complications from pediatric procedural sedation performed by experienced practitioners in a culture of safety is <2% , life-thCardiopulmonary and sedation-related adverse events may account for up to 60% of periprocedural complications from pediatric endoscopy . PatientGiven the anxiety and discomfort associated with pediatric endoscopy, deep sedation or general anesthesia administered by a dedicated provider or anesthesiologist are typically required . SedatioIncreased attention has focused on expanding options for non-anesthesiologist-administered sedation including with propofol. Propofol is a potent amnestic and hypnotic agent with rapid onset and short duration allowing for rapid titration to a targeted depth of sedation without gastrointestinal side effects. Its primary disadvantage its narrow therapeutic window and ease of moving quickly between levels of sedation into general anesthesia with potential airway, respiratory and hemodynamic compromise. With proper training and institutional support, usually aligned with practice recommendations from the ASA , credentPediatric sedation for gastrointestinal endoscopy results in rare but sometimes serious adverse events and requires a specialized approach. It is important to determine sedation risk based on the patient's profile and their ASA physical status score.Pediatric sedation is technically challenging and requires the expertise of pediatric anesthesiologists or an approved hospitalist led sedation team in accordance with ASA guidelines, institutional regulation, and applicable legislation. Endoscopist performed sedation is not advised as it results in decreased patient satisfaction, increased procedure time, and leads to higher risk for cardiopulmonary adverse events.As with anesthesia, selection of the proper endoscopic tools in pediatric endoscopy is necessary for procedural success and patient safety. Endoscopes are presently manufactured in a range of sizes to permit access into the gastrointestinal tract of children. Diagnostic endoscopy includes the acquisition of endoscopic images and sampling of mucosal tissue and includes both esophagogastroduodenoscopy (EGD), and colonoscopy. These procedures can be performed by an adequately trained pediatric gastroenterologist with an appropriately sized endoscope.Depending upon the pathology, therapeutic procedures are sometimes indicated. A range of endoscopic therapies are available but only a handful are typically employed by general pediatric gastroenterologists. Some of these procedures include stricture dilation, variceal ablation, polypectomy, foreign body management, hemostatic therapy, and transnasal endoscopy. However, options for some of these remain limited in ultrathin pediatric gastroscopes. These scopes, including trans-nasal endoscopes, range from 4.9 to 5.9 mm in diameter, contain a single 2.0–2.4 mm working channel and are requisite in children weighing <5 kg. Due to these size restrictions some therapies like endoscopic balloon dilation, use of large retrieval devices, and application of topical hemostatic agents are not possible , 18.Slim gastroscopes have an insertion diameter of 7.8–9.0 mm and are typically used in children weighing <10–15 kg. Standard gastroscopes range from 9.0 to 10.0 mm in diameter and are useful in children weighing more than 20 kg . The priEsophageal stricture dilation is an important procedure for pediatric endoscopists . BalloonManagement of gastrointestinal foreign bodies in children is a unique and important aspect of pediatric gastroenterology. Some solid ingestions like esophageal button battery, multiple magnets, or sharps necessitate rapid resolution . In addiSignificant gastrointestinal bleeding is rare in the pediatric population, but this represents an important indication for endoscopy. Appropriate endoscopy unit planning and stocking is required to manage these events effectively. Hemostatic therapies broadly include, mechanical, thermal, topical, and injection methods . UltrathTNE offers pediatric endoscopists the ability to perform non-sedated endoscopy in the clinic setting. However, free-standing gastroenterology offices may encounter logistical difficulties related to scope reprocessing. TNE has historically been used in adult patients but is garnering attention in pediatrics in part because of disorders like eosinophilic esophagitis (EOE) which require serial endoscopies. A major benefit of TNE is that it overcomes the need for sedation and can be performed within the clinic setting  thereby The smaller working channel found in ultrathin endoscopes can present therapeutic challenges for certain disease states.Pediatric endoscopy centers should maintain appropriate quantities of endoscopic tools for foreign body management and hemostasis in both standard and ultrathin pediatric scopes.Limit accidental unpackaging of inappropriate sized equipment by clearly designating scope size requirements.To successfully employ TNE within the gastroenterology clinic it is important to consider the logistics of daily scope reprocessing. TNE is beneficial for patients who require serial esophagoscopy and it can be helpful to utilize non-pharmacologic methods for anxiolysis such as virtual reality goggles.Advanced endoscopic procedures including endoscopic ultrasound (EUS), endoscopic retrograde cholangiopancreatography (ERCP), and per-oral endoscopic myotomy (POEM) are becoming more widely available to children, but many pediatric centers still cannot offer these therapies. These therapies provide minimally invasive solutions for patients with illnesses that previously would have been surgically managed , 29. A rERCP in pediatrics has been increasing steadily over the past 20–30 years and has shifted from a diagnostic to a therapeutic procedure. For most patients weighting >10 kg a standard adult duodenoscope can be utilized. However, for smaller patients a pediatric duodenoscope must be used . ERCP isEndoscopic ultrasound (EUS) has diagnostic and therapeutic relevance in pediatrics along with promising patient safety data –39. EUS POEM has become an important procedure for the management of pediatric achalasia and is being performed by both surgeons and gastroenterologists. Achalasia has long been managed with pneumatic balloon dilation and surgical correction but a meta-analysis from 2019 demonstrated the superiority of POEM for all three achalasia subtypes . POEM waThe future of pediatric advanced endoscopy is bright with new advances in training, pediatric specific endoscopes, and instrument development. However, to continue to advance the field of pediatric advanced endoscopy it is crucial to maintain a collegial relationship with our adult GI colleagues .Pediatric centers are frequently unable to provide advanced endoscopic procedures, however, adult advanced gastroenterologists may be able to help manage some patients. Establish and maintain a collegial relationship with local advanced endoscopists who are willing and able to care for pediatric patients.Pediatric endoscopists may acquire advanced endoscopic skills through formal or informal training programs in the United States and globally. This will help meet needs at institutions where advanced procedures are limited.EUS should be performed in cases where choledocholithiasis is uncertain as it frequently avoids unnecessary ERCP.POEM has become an important treatment option for pediatric achalasia and is being offered at numerous centers. POEM can be considered for first line treatment but may also be considered after unsuccessful surgical myotomy.Adverse events (AEs) related to pediatric endoscopy are rare but high-quality large-scale data remains scant. However, recent publications and clinical practice guidelines have helped inform practicing gastroenterologists and guide quality improvement measures within endoscopy units.Until recently, the largest studies detailing AEs in pediatric endoscopy came from retrospective multi-center datasets. The 2006 PEDS-CORI report acquired data during or immediately following pediatric gastroduodenoscopies and cited a 2.3% (1.6% anesthesia related and 0.7% endoscopy related) overall AE rate with no deaths or perforations . This stA more recent study prospectively evaluated AEs within the 72 h following endoscopy over 4 years and reported a 2.6% cumulative AE rate from all diagnostic and therapeutic endoscopies. Medically significant AEs related to infection, bleeding, and perforation were encountered in only 0.28%, and therapeutic procedures accounted for most of these cases . This stDespite the low rate of serious AEs, these remain a concern and warrant attention. A review from 2018 cited various studies which identified endoscopist experience, pre-procedural assessment, identification of appropriate equipment, and CO2 insufflation as the most frequently proposed counter measures to reduce procedural complications . The NorFinally, it is important that post-procedural complications including fever and abdominal pain are handled appropriately. In most cases these symptoms are unrelated to serious AEs and providing reassurance can help allay caregiver anxiety. Recently, data from a clinical care guideline aimed at improving post-endoscopy fever management showed a reduction in health care overutilization by nearly 40% . The guiEndoscopy centers should develop ways to systematically record adverse events during and within the 72 h following procedures. AE data may reveal unique challenges for individual centers, and these should be used to inform quality improvement efforts.CO2 should be considered for pediatric endoscopy because it reduces post-procedural discomfort. This may reduce caregiver anxiety and lead to reduced emergency department overutilization.Institutions should develop post-procedural guidelines to triage and advise patients who develop post-procedural fever or pain as this can reduce emergency department overutilization.The future of pediatric endoscopy and endoscopy in general involves technological developments that will advance the field of gastroenterology and may gradually shift the role of an endoscopist. Artificial intelligence, robotic assistance, and disposable endoscopes are being developed to improve efficiency, diagnostic accuracy, increase procedure tolerability, and reduce the transmission of infectious disease.AI is revolutionizing most industries because of its ability for complex data processing. Though it has been a “hot topic” for decades, AI in medicine is now taking shape largely because of the robust technological infrastructure currently in place . MedicalAI has also shown the potential to improve the sensitivity of pill endoscopy while saving time for gastroenterologists. Recent studies involving convolutional neural networks demonstrate how computer assisted diagnosis using pill endoscopy outperforms human readers in the detection rate of pathology 88.39–99.98% vs. 74.57% and in exam completion time: 5.9 vs. 96.6 min, respectively , 64. AI Improving efficiency for clinicians is one of the most exciting improvements, but AI is not limited to high-skill tasks such as diagnostics. Documentation consumes substantial amounts of physician time and some have proposed incorporating AI into generation of procedure reports. Investigators have trained systems to recognize anatomic location, endoscopic tools, and the goodness of cleanout –67. LeveTM Model D  which achieved equal cannulation compared with standard reusable duodenoscopes in low complexity ERCPs (To address the growing concerns of exogenous infection using reprocessed endoscopes, including Carbapenem-resistant Enterobacteriaceae , the Amety ERCPs . MultidrFinally, robotically assisted magnetic capsule endoscopy is an emerging technology that represents a potential paradigm shift in the way endoscopy may be performed in the future. This novel strategy employs a robotic arm wielding an electromagnet, which guides a tethered, pill-shaped endoscope through the intestine. An important patient advantage of this machine is the reduction in shearing forces on the bowel wall , 72. ForCommercially available AI for endoscopy is available through multiple major manufacturers but has not been studied in pediatrics. AI for pill endoscopy will soon be available. These technologies represent an important area for future pediatric research.Disposable duodenoscopes are commercially available and should be considered for use in pediatric patients undergoing ERCP at adult hospitals to limit exposure to multidrug resistant bacteria.The practice of endoscopy is a cornerstone in the field of pediatric gastroenterology and has evolved over the last 50 years to include an array of advanced diagnostic and therapeutic procedures. However, despite a host of improvements, limitations related to patient safety, procedure tolerability, and diagnostic accuracy still exist. While it is not possible to know exactly how the field of pediatric endoscopy will evolve in the ensuing decades, the ongoing surge in innovation offers hope that many of today's limitations will become tomorrow's history.CC, TL, and JK co-wrote the first draft of the manuscript and performed the literature review. GH conceived the manuscript, made significant contributions, and revised the final manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Wnt/β‐catenin signaling is an ancient pathway that regulates key aspects of embryonic development, cell differentiation, proliferation, and adult stem cell homeostasis. Work from different laboratories has shed light on the molecular mechanisms underlying the Wnt pathway, including structural details of ligand–receptor interactions. One key aspect that has emerged from multiple studies is that endocytosis of the receptor complex plays a crucial role in fine‐tuning Wnt/β‐catenin signaling. Endocytosis is a key process involved in both activation as well as attenuation of Wnt signaling, but how this is regulated is still poorly understood. Importantly, recent findings show that Wnt also regulates central metabolic pathways such as the acquisition of nutrients through actin‐driven endocytic mechanisms. In this review, we propose that the Wnt pathway displays diverse characteristics that go beyond the regulation of gene expression, through a connection with the endocytic machinery. In this review, we analyze the role that endocytosis plays in regulating Wnt signaling. We also discuss recent findings on how Wnt regulates metabolic processes, through endocytic mechanisms. Because the mutant flies did not develop wings, the gene was named wingless (wg)  was discovered while studying mouse models of mammary carcinomas induced by DNA integrations of the mouse mammary tumor virus (MMTV)  receptors are a shared component of all Wnt pathways, coreceptors such as the low‐density lipoprotein receptor‐related protein 5 and 6   inhibition. Finally, we will cover the most recent and unexpected findings showing that Wnt can modulate cell metabolism and nutrient acquisition through macropinocytosis.2Drosophila Armadillo), a component of adherens junctions that is involved in the activation of Wnt transcriptional targets , CK1γ, Axin1, and GSK3β . Binding to the receptors promotes the recruitment of several downstream effectors such as the adaptor Dishevelled , in complex with the Fzd cysteine‐rich domain (CRD) at a resolution of 3.25 Å  that establishes hydrophobic bonds with key amino‐acid residues of Fzd site 2. Some of these site 2 contact residues are substituted in other Fzd receptors, likely providing some degree of discrimination for different Wnt ligands , depends on the cytosolic adaptor Dvl, which is relocated to the plasma membrane and forms a complex with Fzd early after Wnt stimulation  prevents Lrp6 phosphorylation  or Rab5 , markedly reduced Wg target transcription in vivo  in its DIX domain . Accordingly, Y1522A mutation blocked Lrp6–AP2 interaction and decreased Wnt‐induced reporter activity   and in mouse intestinal crypts. Notably, Wnt signaling is high in the crypts and is fundamental for the proliferation and maintenance of ISCs  in the presence or absence of Wnt3a ligand  was shown to be part of the signalosome, by interacting with Lrp6 and Fzd  , of which PRMT‐1 alone accounts for 85% of total protein arginine methylation , a protein involved in lipid metabolism  that would promote protein degradation upon phosphorylation ‐like L63/PFTK, which then primes Lrp6 for subsequent CK1γ activating phosphorylations , via protein arginine methylation , concomitantly with Wnt activation. This effect was dependent on β‐catenin transcriptional activity, confirming a direct link between canonical Wnt and macropinocytosis. Stimulation of Wnt signaling with recombinant Wnt3a protein, or through knockdown of APC or Axin1 similarly increased macropinocytic activity and uptake of fluorescent dextran, a commonly used probe for macropinocytosis  or loss of either Axin1 or APC all promoted a strong increase in uptake of extracellular proteins by macropinocytosis and their digestion in the lysosome (Albrecht et al., +/H+ exchanger inhibitor EIPA, Wnt/β‐catenin reporter and β‐catenin nuclear accumulation were strongly inhibited (Table A key observation is that macropinocytosis itself is required for Wnt signaling (Tejeda‐Munoz et al., ed Table  (Tejeda‐ed Table . While ied Table . β‐catened Table , as welled Table . From thed Table . Of noteed Table . This obed Table , as β‐caXenopus embryos, by binding to Lrp6 (Kim et al., On the other hand, macropinocytosis could increase recycling and trafficking of Wnt receptors to the plasma membrane, as has already been observed for EGFR (Chiasson‐MacKenzie et al., 12There is mounting evidence showing that Wnt does not only operate through transcriptional changes, but profoundly affects membranous compartments, protein stability, cell metabolism, and nutrient acquisition, via regulation of various endocytic mechanisms. The growing list of signaling pathways able to directly regulate nutrient uptake, to which Wnt has been added as a new member, is a clear demonstration that dysregulation of growth factors not only changes gene expression dynamics in cancer cells, but also provides a growth advantage in nutrient‐poor niches. Understanding how the connection between Wnt, endocytosis, and metabolism works at a molecular level will be of importance for developing new therapeutic approaches for treating Wnt‐driven cancers.


The inactivation of β‐catenin in HERS led to interrupted root elongation due to premature disruption of HERS. This phenotype was accompanied by reduced cell‐cell adhesion and decreased expression of junctional proteins, as well as increased epithelial‐to‐mesenchymal transition of HERS cells upon β‐catenin depletion. Accordingly, stabilization of β‐catenin in HERS (Catnbi∆shh) led to the formation of unfragmented HERS and resulted in the failure of HERS dissociation, with increased expression of junctional proteins. Our results suggest that fine control of β‐catenin is important for HERS to guide root formation through regulating its structural integrity.β‐catenin, a key mediator of Wnt signaling, plays multiple roles in tooth development. However, the role of β‐catenin in Hertwig's epithelial root sheath (HERS) during root formation remains unclear. In this study, we generated inducible tissue‐specific  We demonstrated that tooth root elongation was impaired by premature disruption of Hertwig's epithelial root sheath (HERS) following the inducible inactivation of ß‐catenin in HERS, and that HERS disruption was failed following the induction of ß‐catenin stabilization in HERS. Our results suggest that fine control of ß‐catenin is important for HERS to guide root formation through regulating its structural integrity. After the formation of tooth crown, the dental epithelium leads root formation. Around postnatal Day 4 (P4) in mice, the inner and outer enamel epithelia fuse at the lower edge of the enamel organ and form a bilayer tissue called Hertwig's epithelial root sheath  gene in mice , ShhcreERT2, and R26R reporter mice /+ mice, respectively  and ShhcreERT2:Catnblox(ex3)/+ (Catnbi∆shh) mice, tamoxifen was injected intraperitoneally with a single injection at 150 mg/kg body weight. Age‐matched Ctnnb1CO/CO or Catnblox(ex3)/+ mice were used as the control. At least five independent littermates were used for each experimental group . To analyze the Cre activity induced in ShhcreERT2 mice, ShhcreERT2 mice were crossed with R26R mice, and tamoxifen was injected intraperitoneally to modulate β‐catenin in HERS at the indicated stage. Then, using a previously described method  was dissolved in corn oil (Sigma‐Aldrich) at 20 mg/ml. To induce Cre‐recombination in 2.3The mice were killed, and their mandibles were carefully dissected for histological analysis. Tissues were fixed in 4% paraformaldehyde (Sigma‐Aldrich) and decalcified in 10% EDTA for 2 to 4 weeks at 4°C. The decalcified tissues were dehydrated through a graded ethanol series, embedded in paraffin, and sectioned at 5‐μm using standard histological procedures as formerly described , cytokeratin 14 , E‐cadherin , laminin , tissue nonspecific alkaline phosphatase , osterix , and nestin . A rabbit‐specific HRP/DAB detection IHC kit (Abcam) was used according to the manufacturer's instructions. For immunofluorescence staining, tissue sections were permeabilized by incubation in 0.2% Triton X‐100 for 10 min at room temperature. After rinsing with phosphate‐buffered saline (PBS), nonspecific binding sites on the cells were blocked with 5% bovine serum albumin in PBS for 30 min at room temperature. Tissue sections were then incubated with primary antibodies against β‐catenin and CK14  for 16 h at 4°C. Normal rabbit or mouse immunoglobulin (Santa Cruz Biotechnology) was used as a negative control for the primary antibodies. Alexa Fluor®‐conjugated secondary antibodies (Invitrogen) were used for detection. DAPI was used for counterstaining. Cell staining was evaluated using a fluorescence microscope (Leica Microsystems).2.5Ctnnb1 (TG500280) and control shRNA (TR30013) were purchased from OriGene Technologies (Rockville). The establishment of stable cell lines by viral transductions were conducted as previously described . DNA constructs were transfected by using LipofectamineTM LTX and PLUS reagent (Invitrogen) according to the manufacturer's instructions.HERS01a, a mouse HERS cell line, was cultured as described previously  was induced by tamoxifen administration at P4 and analyzed at P10 and P14 to determine the dynamics and fate of HERS during root formation. The subsequent β‐catenin inactivation in HERS was confirmed using immunostaining of dental tissues at P10  were verified by comparing the protein level of β‐catenin  and messenger RNA (mRNA) levels of its transcriptional targets (Lef1 and Axin2) with those from cells transfected with the negative control shRNA . Interestingly, the Ctnnb1‐knockdown HERS01a cells (shCtnnb1) exhibited lower cell‐cell contact during cell culture than the control . As expected, HERS01a cells with shCtnnb1 exhibited significantly lower mRNA expression levels of junctional proteins, including zonula occludens‐1, occludin, cingulin, integrin αν, integrin α10, vinculin, nectin1, and nectin4, compared to the control . We also analyzed the proliferation status of HERS cells using the immunoreactivity of Ki67. Few proliferating cells were detected in the HERS of Ctnnb1i∆shh mice at P10, whereas the cells apically located in the HERS of the controls were strongly positive for Ki67 . The lower proliferation rate of HERS cells with β‐catenin inactivation was also confirmed using an in vitro proliferation assay comparing Ctnnb1‐knockdown HERS01a cells (shCtnnb1) with the control . We used quantitative real‐time PCR to examine the expression of EMT‐associated molecules and found that compared with the control, Ctnnb1‐knockdown HERS01a cells retained significantly higher expression levels of EMT markers, such as Zeb1 and Snail2, lower expression of E‐Cad and connexin 43 (Cx43), which are epithelial and junctional proteins, respectively, and higher expression of N‐cadherin (N‐Cad) and vimentin, which are well‐known molecular markers of mesenchymal cells  and control cells (shNC‐CM) were administered to MDPC‐23 cells. Interestingly, the MDPC‐23 cells treated with conditional medium from Ctnnb1‐knockdown HERS01a cells (shCtnnb1‐CM) exhibited significantly reduced mRNA expression levels of genes, such as Osx, Nfic, Msx1, and Msx2 compared with cells treated with conditioned medium from the control cells  at P10 and P14 . To avoid those unexpected results, we adjusted the timing of the tamoxifen to induce stabilization of β‐catenin in HERS to the point at which the dissociation of HERS should occur. Based on our findings from the inactivation of β‐catenin, we expected the activity of β‐catenin in HERS to become weaker during dissociation. After generating appropriate mice by crossing Catnblox(ex3)/+ and ShhcreERT2 mice (Catnbi∆shh), based on our results for the stage‐specific reporter expression of ShhcreERT2  and mRNA levels of Lef1 and β‐catenin  are recognized as important for determining the number, length, and shape of the root and also for the development of the periodontium, including the periodontal ligament and cementum  to investigate the gain‐of‐function in HERS during disintegration, in which less cell‐cell adhesion between HERS cells is expected. As anticipated, stabilized β‐catenin led to the failure of HERS dissociation, which is contextually similar to our results from β‐catenin inactivation.To understand the stage‐ and process‐specific mechanisms through which β‐catenin exerts its in vivo regulatory activity in HERS during root development, we here applied different timing of tamoxifen induction in the β‐catenin is the central player in the Wnt signaling pathway (Cadigan & Nusse, β‐catenin (Bienz, β‐catenin as a component of the cell adhesion complex leads to severe developmental failures (Junghans et al., In this study, we demonstrated the roles of β‐catenin in the cell‐cell adhesions of HERS during tooth development, which has not before been carefully observed. n Bienz, . Increasn Bienz, . Conversβ‐catenin in HERS led to premature disruption of HERS and shorter roots through impaired odontoblast differentiation for root elongation. On the other hand, stabilization of β‐catenin in HERS led to the failure of HERS dissociation. Our results suggest the functional influence of β‐catenin in regulating the structural integrity of HERS and postnatal root formation, implying that Wnt/β‐catenin signaling plays a crucial role in the cell dynamics of HERS during root development. These findings could provide new insight into the molecular and cellular events that underlie root formation and offer potential instruction for future tissue engineering applications.In summary, we modulated the activity of β‐catenin in HERS during root development using inducible gene targeting technologies. Inactivation of The authors declare that there are no conflict of interests.Siqin Yang and Hwajung Choi conceived and designed the research. Tak‐Heun Kim and Siqin Yang performed the animal experiments. The histological tissue analysis and scoring were conducted by Siqin Yang, Tak‐Heun Kim, Ju‐Kyung Jeong, Yudong Liu, and Hidemitsu Harada provides HERS01a cell line. The statistical analysis and interpretation of the results were performed by Siqin Yang, Hwajung Choi, Tak‐Heun Kim, Ju‐Kyung Jeong, Yudong Liu, and Eui‐Sic Cho. The manuscript was principally written and revised by Siqin Yang, Hwajung Choi, and Eui‐Sic Cho. All the authors critically reviewed the manuscript for important intellectual content and approved the final submitted manuscript.