Meteorus pulchricornis is a solitary endoparasitoid wasp of many larvae of lepidopteran pests. The lipid content during developmental stages of M. pulchricornis was measured; it was higher in the larval and pupal stages but declined from six-day-old pupae. Lipid accumulation constantly decreased in the adult stage, even after feeding on honey solutions. To investigate the roles of FASs in lipid synthesis in M. pulchricornis, four FAS genes (MpulFAS1~4) were identified from the transcriptome database of M. pulchricornis. All FAS genes included full-length open reading frames and shared 72–79% similarity with the sequences of Microplitis demolitor. qRT-PCR validation showed that all four FASs had the highest expression after the adult wasps were fed on honey diets. MpulFAS1 and MpulFAS2 reached their expression peaks at the adult stage but MpulFAS3 and MpulFAS4 peaked at the larval stage. To further study the function of FASs, dsRNA injection knocked down the expression of four MpulFASs and resulted in a significant decline of lipid content at the adult stage in M. pulchricornis. Results from this study suggest that M. pulchricornis adults cannot accumulate lipid content effectively and FASs may still contribute to lipid synthesis in the adult stage. This broadens the knowledge on the ability of lipid synthesis in parasitoid wasps and provides insight into the roles of FASs in insects with parasitic life-history traits.In insect parasitoids, fatty acid synthases (FASs) have received less attention and their roles associated with lipogenesis loss are far from clear.  Lipid reserves can support a large and long-term energy source for organisms; they play key roles in survival and reproduction. In insects, lipid accumulation can be stimulated and achieved by digesting a diet rich in sugars and other carbohydrates ,2. LipidPrevious studies have revealed that feeding on sugar-rich foods, such as honey or nectar, does not lead to an increase in adult lipid content in parasitic wasps ,10. ThisFASs) are highly conserved and crucial genes in the fatty acid biosynthetic pathway, especially for the synthesis of palmitic acid, which acts as a precursor for a variety of other lipid types [FASs have only been identified in a narrow range of insect species, such as the model insect Drosophila melanogaster [FASs have been much less studied. The transcription level of FASs in Nasonia vitripennis did not increase significantly even after short- and long-term sugar feeding; thus, degradation of FASs has been predicted to be associated with the loss of lipogenesis in parasitic lifecycle-based insect species [FASs may be a promising way to uncover this unusual evolutionary trait.Fatty acid synthase performs the condensation of acetyl coenzyme A (acetyl-CoA) and malonyl-CoA, using NADPH as a reducing equivalent to produce the 16-carbon saturated fatty acid palmitate . Fatty aid types . To datenogaster . In para species . AlthougSpodoptera litura (Lepidoptera: Noctuidae) is an important omnivorous insect that causes widespread economic damage to vegetables and other crops [Meteorus pulchricornis (Hymenoptera: Braconidae) is a solitary endoparasitoid wasp of S. litura and many other free-living lepidopteran larvae [M. pulchricornis include host selection strategy [M. pulchricornis was likely to lose the capacity for de novo lipid synthesis, or that at least that they cannot accumulate lipids during the adult stage. However, the detailed dynamics of lipid contents, especially at the scale of whole life stages, are still unknown. Furthermore, the molecular mechanism of lipid decline has yet to be uncovered.The common cutworm, er crops . It is cn larvae . Extensistrategy , the effstrategy ,16,17,18strategy ,20. Recestrategy  demonstrM. pulchricornis and identified candidate FAS genes from a previously constructed M. pulchricornis transcriptome dataset. We also performed a series of tests consisting of real-time quantitative RT-PCR (qRT-PCR) validations and RNA interference to investigate the functions of these FAS genes. We aimed to answer the following questions: (1) What is the lipid content dynamic throughout the life history stages of M. pulchricornis; (2) what are the functions of FASs in lipid synthesis or accumulation? The information gained from this study can improve the understanding of the biosynthetic mechanism of lipids in parasitoids.The present study measured the lipid content throughout the developmental stages of M. pulchricornis had less lipid content than emergency but higher than other ages of adult wasps. When fed on water or deprived of food, the lipid content also decreased compared to that at emergence and reached the lowest level on the third day and sixth day, respectively . All four MpulFASs contained full-length ORFs, ranging in size from 1042 to 2441 amino acid residues . MpulFAS1 and MpulFAS4 were clustered with FAS2 (MdemFAS2) and FAS1 (MdemFAS1) into separate sub-branches, respectively (A neighbor-joining (NJ) tree of the FASs was constructed using the protein sequences from ricornis . Four Mpectively .MpulFASs in three-day-old adult wasps after different diets-feeding were measured. Honey-feeding significantly promoted the expression of four MpulFASs and their expression levels were highest after feeding on honey. MpulFAS1 and MpulFAS4 followed a similar expression pattern, in which expression was highest in the honey-fed treatment, followed by starvation and water-fed treatments. The expression levels of MpulFAS2 and MpulFAS3 increased in the order of starvation, water, and honey-fed treatments  (In order to investigate the roles of ricornis . The expjection) .Neochrysocharis formosa and Diadegma insulare continuously declined [M. pulchricornis females were much lower than those at emergence even after they digested various kinds of sugary foods [The association of parasitoid wasp and its host is an excellent model system to study co-evolution between species . During declined ,23. Simiry foods .M. pulchricornis fed on a honey solution, the constant decline of lipid content at each time point demonstrated that: (1) lipid reserves were consumed throughout the adult stage and (2) no further new lipids were recruited despite continuous food digestion, or at least, the synthesis rate of lipids was much lower than the rate at which food was consumed. Similar results for lipid dynamics in wasps fed with water and deprived of food were also detected. Lipid synthesis is usually deemed to be an involuntary dose-dependent physiological process under excessive carbohydrate diets [However, studies on the lipid content of juvenile parasitoids still receive less attention. Therefore, we prolonged the measurement to immature stages and to a detailed time interval in the adult stage to track the global change in lipid content. The adult stage in te diets . In contte diets . It is pte diets . A lack te diets . This dete diets ,27,28. MM. pulchricornis, the lipid content was stable and at a higher level compared to the adult stages. Until now, less attention has been paid to the lipid dynamics at the larval and pupae stages in parasitoids [At the larval and pupal stages in asitoids . As pupaasitoids . More deM. pulchricornis, we identified four FAS genes from the M. pulchricornis transcriptome dataset [FASs was relatively low among species, including insects. For example, D. melanogaster and Lysiphlebia japonica have six and three FASs, respectively [FASs in different orders of insects are comparable. The phylogenetic analysis showed that FASs of M. pulchricornis were adjacent to M. demolitor, which also belong to the family Braconidae; therefore, FASs were more conserved within the same family.It has been demonstrated that FASs play a vital role in lipid synthesis in insect species, yet limited FASs have been identified in parasitoid wasps ,30. To f dataset . Since lectively , which iMpulFASs had higher expression in honey-fed groups than in starved or water-fed groups. This result suggested that FASs can be activated after feeding on sugary foods, but this was in contrast to the lipid dynamic in three feeding treatments and previous studies regarding FASs degradation contributing to the loss of lipogenesis. In N. vitripennis, compared to starved groups, the expression of FASs in short-term and long-term fed wasps was not significantly changed [FASs in N. vitripennis  were unregulated in the sugar-fed treatment. Conversely, D. melanogaster accelerated its fatty acid synthesis after sugar-feeding, as indicated by the upregulation of the key gene fatty acid synthase 1, suggesting that the expression pattern of FASs is variable between Hymenoptera and other insect orders.Until now, research on the expression patterns of FASs in insects, especially in parasitic wasps, has been scarce. Parasitic wasps can manipulate their hosts to produce sufficient amounts of lipids to meet the needs of developing offspring of parasitoids ,33,34; h changed . Lammers changed  also demFASs under variable feeding conditions, we validated the global expression levels of FASs from larval to adult stages with dispersed time points. The results showed that the expression patterns among developmental stages in M. pulchricornis varied greatly. The highest expression levels of MpulFAS1 and MpulFAS2 were observed in the adult stage, while MpulFAS3 and MpulFAS4 had the highest expression at the larval stage. Notably, two FASs in M. pulchricornis had higher expression in the larval stage than in other stages, which was consistent with lipid dynamics among each developmental stage. Alternatively, another two FASs were more highly expressed but with different expression peaks in the adult stage. Specifically, both MpulFAS1 and MpulFAS2 had the highest expression after 10 days of honey feeding, where the wasps had a higher lipid content at the adult stage. This divergence in the FASs expression pattern revealed functional differentiation; therefore, the explanation for FAS function in fatty acid synthesis is more complicated. Although FASs can be causally linked to lipid synthesis, they also have other important roles in insect physiology. For instance, FASs are various precursors of hormones or enzymes, suggesting that higher transcriptional levels of FASs can lead to other physiological functions in the metabolic networks of M. pulchricornis.To explain the unusual expression patterns of M. pulchricornis, we conducted the RNAi experiments to validate the expression levels of MpulFASs and the change in lipid contents. Interestingly, after knocking down, all the four MpulFASs were down-regulated and eventually resulted in decreased lipid contents in the adult stage. These results strongly suggested that FASs may still make consequences on the lipid synthesis in M. pulchricornis. Similarly, the accumulation of lipids decreased significantly in Colaphellus bowringi by silencing FAS, indicating that FASs play essential roles in lipid synthesis in C. bowringi [To further understand the roles of FASs in lipid synthesis in bowringi .FASs. Furthermore, lipid synthesis-related genes in the lipid metabolic pathway are usually antagonistic to each other. For example, high levels of malonyl-CoA can inhibit the activity of carnitine acyltransferase I (CAT1), preventing fatty acid transport to the mitochondria, thereby limiting β-oxidation. Therefore, the upregulation of FASs may inhibit other key lipid synthesis-related genes, which contributes to the lipid decrease. Based on this, further studies should be conducted to summarize a global transcriptional change of lipid metabolic genes to screen for candidate genes that are key for lipid lipogenesis loss, in addition to detailed biochemical and molecular validation.Metabolic pathway-related lipid synthesis is complicated, and several other genes can play a direct or indirect role in the fatty acid synthesis pathway, such as Acetyl-CoA Carboxylase (ACC) ,37. ACC M. pulchricornis, lipid content remains at high levels during the larval and pupal stages but declines from the end of the pupal stage through the adult stage. After honey feeding, the lipid content declined, which confirmed that M. pulchricornis adult wasps did not accumulate lipid reserves effectively. FASs in M. pulchricornis were variably expressed at all life-history stages, and the expression levels of a portion of FASs were still high even after saccharide feeding. More importantly, FASs may still contribute to lipid synthesis at the adult stage based on RNAi experiments. The present study suggests that the loss of lipid accumulation may be related to other functional genes or more complicated mechanisms.We demonstrated that, during the life history of M. pulchricornis is thelytokous, the populations are all l females [S. litura larvae collected in soybean fields at the campus of Jiangsu University of Science and Technology in Zhenjiang City, Jiangsu Province, China. Wasps were maintained using L3–L4 S. litura larvae as the hosts. Adult wasps were reared in a glass tube  and provided with a honey solution (10% w/v) once a day. The host species, S. litura, were reared on an artificial diet [M. pulchricornis  were included. Because infant and median instar parasitoid larvae were difficult to collect for biochemical and molecular assays, we chose the final instar (L3) parasitoid larvae to conduct assays.Since  females . Parasitial diet . Adult mial diet . All theM. pulchricornis.We compared the lipid dynamics of adult wasps under three feeding conditions: starvation, water-feeding, and honey-feeding treatments. Preliminary tests showed that the adults can live no more than three or seven days without any food supply or only feeding on water, respectively. Therefore, the lipid contents on the first and third day of starved adult wasps and on the third and sixth day of water-fed adult wasps were determined. Then, to illustrate the detailed lipid dynamic, we measured a time series of lipid content at the adult stage, including at emergence, 10, 20, 30, 40, and 50 days old. Meanwhile, the lipid content of larvae (L3) and pupae (from the first day to the sixth day) were determined to compare the global change of lipid dynamics in v/v = 1:2) in a 1.5 mL centrifuge tube. After centrifugation at 16,000× g for two min, the supernatant was transferred into a new 1.5-mL centrifuge tube. Then, the supernatant was heated at 90 °C for complete evaporation of the solution and 40 μL of 95–98% sulfuric acid was added and heated at 90 °C for 2 min. After that, 960 μL of the vanillin-phosphoric acid reagent was added and left to react at room temperature for 25 min. The solution absorbance at 525 nm was read using a spectrophotometer  and compared to lipid standards for triglyceride [The lipid content of individual wasps was determined using the vanillin assay . Each inlyceride . For eacM. pulchricornis transcriptome dataset (GenBank accession number: SRR8981255) [E-value < 1 × 10−5) was used to identify candidate unigenes encoding putative FASs with the available sequences of these proteins from Microplitis demolitor, Nasonia vitripennis, Drosophila melanogaster, and Tribolium castaneum. All candidate genes were manually checked using BlastX against the GenBank non-redundant (nr) protein database at NCBI. The open reading frames (ORFs) of the putative FAS genes were predicted using an ORF finder (https://www.ncbi.nlm.nih.gov/orffinder/). The theoretical isoelectric point (pI) and molecular weight (Mw) were predicted using ExPASy (https://web.expasy.org/compute_pi/). A phylogenetic tree was constructed by MEGA7.0 software using the neighbor-joining method with 1000 bootstrap replications [cDNA sequences encoding FASs were retrieved from a previously constructed 8981255) . The tBLications .Total RNA samples were extracted using the Trizol reagent  following the manufacture’s protocol, and the residual DNA was removed by RNase-Free DNase I . Approximately 5 survivors were pooled for preparing one sample. RNA quality and concentration were determined by Nanodrop 2000 spectrometer . All RNA samples were reverse transcribed using the PrimeScript RT reagent Kit with gDNA Eraser .2O. The reaction process was as follows: 5 min at 95 °C, followed by 45 cycles for 15 s at 95 °C, 60 °C for 31 s, and dissociation. A fluorescence melting curve from 55 to 95 °C was used to ensure a single gene-specific peak and the absence of primer-dimer peaks [qRT-PCR was performed using the QuantStudio™ Real-Time PCR system . The reaction volume was 20 μL, containing 10 μL SYBR Premix Ex Tap II , 0.4 μL each of the forward and reverse primer, 0.4 μL ROX Reference Dye (50×), 1.5 μL cDNA template, and 7.3 μL ddHer peaks . Each sabeta-actin gene was used as the reference gene to normalize the expression of target genes and correct sample-to-sample variation. The primers were designed using the primer designing tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and synthesized by Sangon Biotech . Each sample was analyzed in triplicate from three biological replicates, and the relative expression levels of MpulFAS genes among the different samples were measured using the 2−ΔΔCt method [The primers used in the qRT-PCR analysis are listed in t method .M. pulchricornis. Gene-specific primers for dsRNA synthesis were designed with BLOCK-iT™ RNAi Designer (https://rnaidesigner.thermofisher.com/). According to the manufacturer’s steps, four single-stranded Oligos of each MpulFAS were synthesized and are listed in RNA interference (RNAi) was conducted to further analyze the functions of FASs in MpulFASs were injected to 10-day-old adults using a Nanoject II microinjector , respectively. Fifty wasps were injected for each treatment. Total RNA was extracted after 24 h and 48 h to check the efficiency of RNAi by qRT-PCR. According to the qRT-PCR results, the lipid content per individual wasp was measured based on the time point with the highest interference efficiency and the method was the same as lipid content analysis in Fifty ng dsRNA of four p < 0.05) was used to check for significant differences in the expression levels of each target gene among the treatments. The data analysis was carried out in R 3.4.0 [Since the assumption of normality was not satisfied, the non-parametric Kruskal–Wallis test was used to detect the significant differences of lipid content among developmental stages, followed by pairwise comparisons using the pgirmess package in R software (version 3.4.0). A one-way analysis of variance (ANOVA)  with Tukey’s post-hoc test ( R 3.4.0 .


LgDel mice that carry a heterozygous deletion parallel to that associated with DiGeorge/22q11.2 deletion syndrome (22q11.2DS). Disrupted coordination of tongue movement in LgDel mouse pups may contribute to suckling, feeding, and swallowing (S/F/S) disruptions that parallel pediatric dysphagia in infants and toddlers with 22q11.2DS. Using an in vitro rhythmically active medullary slice preparation, we found spontaneous firing as well as IPSC frequency differed significantly in neonatal LgDel versus wild-type (WT) protruder and retractor CNXII MNs that were identified by retrograde tracing from their target muscles. In response to respiration-related activity, initiation and decay of transiently increased firing in WT protruder MNs is delayed in LgDel, accompanied by altered excitatory/inhibitory (E/I) balance. In addition, LgDel retractor MNs have a transient increase in firing with diminished IPSC frequency that is not seen in WT. There were no significant differences in cell body volume of either XII class in WT and LgDel. Sholl analysis showed the total numbers of dendritic intersections (at 50- and 90-μm radii from the cell soma) were significantly greater for LgDel versus WT retractor MNs. Thus, the physiological, synaptic and cellular properties of distinct classes of CNXII MNs that coordinate tongue movement in neonatal WT mice are altered in LgDel. Such changes could contribute to sub-optimal coordination of S/F/S that underlies pediatric dysphagia in 22q11.2DS.We asked whether the physiological and morphologic properties of hypoglossal motor neurons (CNXII MNs) that innervate protruder or retractor tongue muscles are disrupted in neonatal  LgDel mouse model of 22q11DS, the only established genetic model for pediatric dysphagia. We found significant differences in firing activity, synaptic inputs, and morphology when comparing protruder and retractor CNXII MNs from LgDel and wild-type (WT) animals. Our observations provide a foundation for understanding infant hypoglossal nerve as well as tongue function or dysfunction, and their contribution to perinatal feeding and swallowing abnormalities in 22q11.2DS.Pediatric dysphagia is a frequent and serious clinical complication in up to 85% clinically defined neurodevelopmental disorders, including DiGeorge/22q11.2 deletion syndrome (22q11.2DS). Heterozygous deletion of the full set of mouse orthologues of human 22q11 genes disrupts key physiological properties of distinct classes of hypoglossal motor neurons (CNXII MNs) that coordinate oropharyngeal function in the LgDel mouse pups, a genomically accurate model  mice. These observations provide a foundation for understanding the role of diminished 22q11.2 gene dosage in potential disruption of tongue movements, their coordination with respiratory cycles and altered CNXII function. Such changes in cranial nerve MN circuits could contribute to perinatal S/F/S difficulties in 22q11DS.To better understand the functional consequences of disrupted cranial nerve and circuit development critical for optimal S/F/S in LgDel males (C57BL6N background) were bred to C57BL6N WT females. A total of 81 pups  from 26 litters were used. Fluorescent tracers were injected into the tongue of postnatal day (P)1 pups  to retrogradely label protruder and retractor CNXII MNs . Heterozygous adult NXII MNs . Electron = 3). Retractor hypoglossal MNs were identified simultaneously by fluorescent tracer  and specificity confirmed by absence of labeled brainstem neurons when CTB-Alexa Fluor 488 was injected into one side of the tongue after the lateral branch of CNXII has been sectioned (n = 3).To identify protruder and retractor CNXII MNs for recording and injection, a two-stage procedure was used. In an initial surgery, P1 pups were anesthetized with the short acting inhalation anesthetic isoflurane, tongues pulled gently from the mouth, and cholera toxin subunit B (CTB) conjugated to Alexa Fluor 555 or 488  injected into the base or side of the tongue, respectively A. Protru2-saturated buffer: 140 mm NaCl, 5 mm KCl, 2 mMCaCl2, 5 mm glucose, and 10 mm HEPES. The brains were oriented for vibratome sectioning with caudal ends up and rostral sides attached to an agar block perpendicular to the blade. A single 700-μm slice of the medulla was prepared containing the fundamental respiratory control circuit: hypoglossal motor nucleus and rootlets, intermediate reticular formation interneurons, and pre-Bötzinger complex , perfused with room temperature artificial CSF (aCSF):125 mm NaCl, 3 mm KCl, 2 mm CaCl2, 26 mm NaHCO3, 5 mm glucose, and 5 mm HEPES equilibrated with 95% O2 and 5% CO2, pH 7.4 . In a subset of slices, intracellular injections of a highly photostable far-red (excitation laser 640 nm) biocytin fluorophore conjugated dye, CF640R were made  to visualize cellular and dendritic morphology.At P3–P6, pups injected with fluorescent tracers on P0 were anesthetized by isoflurane and killed by rapid cervical dislocation. Brains were submerged in 4°C, continuously 100% O complex . Slices m KCl, 4 mm MgCl2, 10 mm EGTA, 2 mm Na-ATP, and 10 mm HEPES or 150 mm K-gluconic acid, 10 mm HEPES, 10 mm EGTA, 1 mm MgCl2, and 1 mm CaCl2 at pH 7.3. A biocytin-conjugated fluorophore, CF640R (2%), was included in the patch solution to label neurons for confocal imaging. Spontaneous and inspiratory related AP activity was analyzed in current clamp; spontaneous and inspiratory related synaptic events were recorded in voltage clamp at −80-mV holding potential. An PV830 Pneumatic PicoPump pressure delivery system (WPI) was used for focal ejection of drugs from a patch pipette positioned within 30 μm from the patched hypoglossal MNs. Prior work has shown this approach effectively delivers drugs 100–120 μm from the pipette tip forward and considerably less behind the drug pipette (m) to block GABAergic IPSCs and strychnine (1 μm) to block glycinergic IPSCs. GABAergic IPSCs were isolated by focally applying strychnine (1 μm) to block glycinergic IPSCs and D(-)-2-amino-5-phosphopentanoic acid  and 6-cyano-7-nitroquinoxaline-2,3-dione  to block glutamatergic excitatory neurotransmission. At the end of each experiment gabazine, or APV and CNQX, were applied to block and confirm the isolation of GABAergic IPSCs and glutamatergic EPSCs, respectively.Individual-labeled protruder (red) or retractor (green) CNXII MNs were identified by epifluorescence microscopy, then imaged for recording using differential interference contrast optics . Infrared illumination and high-resolution infrared-sensitive video imaging was used to position the patch pipette on the surface of identified protruder or retractor MNs. To isolate inhibitory or excitatory currents, patch electrodes  were filled with 150 m pipette . Glutamaz-step size of 2.4 μm to produce volumes allowing full reconstruction of the biocytin injected MN. In all cases, inclusive reconstruction of the MNs’ dendritic tree required several adjacent volumes to be stitched together. Biocytin injected cells were captured in photon counting regime, an approach allowing for high-dynamic range images to be collected. This was necessary since the injected cell body and the fine distal dendrites represented dramatically different fluorescence signal, which cannot be captured without saturation or missing signal, using more conventional analog photomultipliers. Images were saved in the “Lif” format, then processed and analyzed using Imaris software (Bitplane Inc.).Morphologic properties of protruder and retractor CNXII MNs filled intracellularly with biocytin (CF640R) were analyzed in confocal images stacks obtained at low (10×) and high (20×) magnification. Biocytin fluorophore was confined to the soma and dendrites of the recorded and filled cell, with negligible spillover in the extracellular space. MNs were excluded from analysis if the whole cell patch-clamp recording was stable for <20 min, the cell soma was not clearly intact, or the dendrites were shorter than 20 μm. At the end of each electrophysiology experiment, the slices were fixed in 4% paraformaldehyde  overnight, dehydrated through ascending concentrations of methanol and cleared in 1:2 benzyl alcohol /benzyl benzoate . The slices were mounted in depression glass slides, coverslipped and imaged with a Leica TCS SP8 multiphoton confocal microscope equipped with supercontinuum white laser source and single molecule detection hybrid detectors . A DFC365FX camera at 2048 × 2048-pixel resolution was used for navigation through the sample. Final images for analysis were taken at 10× from the entire slice, to position the injected neurons and verify its hypoglossal MN by a distinct axon coursing in the intramedullar portion of the XII nerve. Then z-stacks were taken with 20×/0.75 oil-immersion objective, at LgDel CNXII protruder and retractor MNs entire dendritic arborization from three-dimensional confocal stacks . Somatic volume of biocytin filled cell bodies as well as retrograde-labeled CTB-Alexa Fluor 555 and 488 fluorescent cell bodies were directly measured from the confocal-generated volumes. Sholl analysis of branching was performed using an Imaris algorithm for branching analysis. The soma of each labeled cell was identified as the center for this analysis. All dendrites from each cell were traced manually through three dimensions, assisted by the AutoPath protocol. The number of dendritic intersections with a series of concentric spheres at intervals of 10 μm from the soma center point was determine using the Imaris’ Sholl analysis algorithm.Several morphologic properties: cell body sizes, regional variation in cell body sizes, dendritic branching, total dendritic length and dendrtic complexity evaluated by Sholl analysis were analyzed after tracing both WT and LgDel comparisons, we used unpaired Student’s t tests. For within group comparisons before, during, and postburst, we used one-way ANOVA with repeated measures and Dunnett’s post hoc analysis. Two-way RM ANOVA with Bonferroni post hoc tests were applied for between group comparisons. For all comparisons, the significance level was set at p < 0.05.All electrophysiological data were collected and digitized using Clampex  and analyzed with Clampfit . CNXII rootlet inspiratory bursting activity, MN AP firing and synaptic events were detected using MiniAnalysis (version 6.0.7 Synaptosoft) with the minimal acceptable spike amplitude at 3 V, 60 mV, and 10 pA, respectively. AP firing and synaptic events occurring during inspiratory bursting epoch were defined as bursting related or phasic activity; AP firing and synaptic events not coincident with the bursting period were considered basal activity. Burst-related AP and postsynaptic currents were grouped into 13 consecutive 1-s epochs. The first 3–5 s before burst onset were control (before burst), followed by three 1-s burst epochs and a 3- to 5-s recovery (postburst). All data were within a normal distribution with equal variances using frequency distribution and Bartlett’s corrected statistic test, respectively. Statistical analysis was performed using GraphPad Prism 5.0 (GraphPad Software), Microcal Origin 6.1 (OriginLabs), and Microsoft Excel (Microsoft). Data are presented as mean ± SEM. For WT versus LgDel, we identified protruder and retractor CVXII MNs by applying retrograde tracers with different fluorescence characteristics into protruder and retractor muscles at the posterior and lateral aspect of the tongue, respectively . Each recorded XII MN was identified based on its retrograde label as protruder or retractor before electrophysiological recording.Hypoglossal MNs (XII MNs) that innervate protruder and retractor muscles of the tongue are located in the hypoglossal nucleus (CNXII) in the dorsomedial medulla A-C. To dectively A,B. Protectively C, red, wectively C, green.ectively . SeverinMovie 1.This video shows a detailed 3-D rendering of retrogradely labeled hypoglossal protruder motorneurons (in red) and retractor motorneurons (in green). Protruders were mainly in the rostral (anterior) and ventral parts of the hypoglossal nucleus, whereas retractors were localized more to the posterior  and dorsal areas within the XII nucleus.10.1523/ENEURO.0520-19.2020.video.1LgDel pups in current clamp mode. In both genotypes, the XII MNs had similar baseline resting membrane potentials, typically between −50 and −65 mV, with AP spike amplitudes of 70–80 mV. The mean spontaneous basal AP frequencies did not differ between WT protruder and retractor XII MNs  firing frequencies for protruder versus retractor CNXII MNs in P3–P6 WT and  XII MNs A-C. In cctor MNs A-C. Signotruders A-C. ThusLgDel CNXII retractor MN AP firing frequency, we studied excitatory and inhibitory synaptic inputs to CNXII MNs. There were no significant differences in either frequency nor amplitude of EPSCs that could possibly contribute to increased firing rates observed in LgDel CNXII retractor MN (data not shown). In contrast, frequency, but not amplitude, of GABAergic IPSCs was significantly diminished in LgDel MN compared with WT  with no inhibitory counterbalance during each of the 3-s burst epochs  and LgDel (n = 6) protruder XII MNs (LgDel). The total dendritic length did not differ for WT  versus LgDel  protruder CNXII MNs.We first assessed the cell body volume distribution by first applying 3-D segmentation of the retrogradely labeled XII protruder and retractor WT and nd LgDel A,B. We nxtension A,B. Shol XII MNs C did notLgDel. The total numbers of dendritic intersections at 50- and 90-μm radii from the cell soma were significantly greater for LgDel versus WT retractor MNs .In contrast, the dendritic architecture of retractor CNXII MNs differed between the WT and LgDel pups, in which several aspects of S/F/S, including nasopharyngeal aspiration are parallel to pediatric dysphagia seen with high frequency in infants and toddlers with 22q11.2DS. The basal spontaneous and inspiration-related AP firing rates as well as synaptic properties of protruder versus retractor XII MNs in LgDel mouse pups differ from those in WT. The physiological differences in LgDel retractor MNs are more substantial than those between other XII MN classes. In addition, LgDel retractor MN dendritic arbors are more branched. These distinctions in physiological, network, and cellular properties of LgDel CNXII retractor and protruder MNs may underlie anomalous coordination of tongue movement critical for S/F/S early in life, as well as formation and oropharyngeal transit of food boluses subsequently. Thus, pediatric dysphagic symptoms that result from heterozygous 22q11.2 gene deletion may reflect altered development and function XII MNs and circuits in addition to anomalies in differentiation of oropharyngeal structures critical for optimal S/F/S at birth and throughout life.The coordination of XII protruder and retractor MN activity is altered in LgDel mouse pups is abnormally elevated compared either to LgDel protruder MNs or WT retractor MNs. This is consistent with elevated firing frequency reported in LgDel CNXII MNs whose projection targets were not identified , and absence of an opposing increase in inhibitory GABAergic inputs. The resulting protracted period of higher frequency firing because of this change in excitatory/inhibitory (E/I) balance may increase retraction of the tongue during the suck/breathe/swallow cycle, reduce milk flow into the esophagus, diminish total milk intake during infancy and disrupt food bolus formation at later ages. These changes could also prevent appropriate tongue position during S/F/S, blocking the epiglottis from closing to protect the nasopharynx from aspiration (LgDel mouse pups (WT retractor XII MNs excitatory synaptic inputs during putative inspiratory bursts are counterbalanced by enhanced inhibitory inputs, which presumably act to maintain consistent firing frequencies during inspiratory bursts. In contrast, piration  and may use pups .LgDel versus WT protruder and retractor MNs suggests that some changes may reflect altered interactions between XII MNs and dysmorphic targets. Indeed, substantial changes in LgDel retractor MN synaptic and circuit properties are accompanied by substantial changes in dendritic size and branching. Further assessment of 22q11.2 gene dosage-dependent modulation of CNXII MN and interneuron differentiation, tongue muscle differentiation, and resulting afferent/target interactions that might influence circuit differentiation will further define pathogenic vulnerability of brainstem circuits underlying perinatal dysphagia in 22q11.2DS.This selective disruption of E/I balance may be a direct, local effect of 22q11.2 deletion on the differentiation of XII MNs and associated interneurons  or an inLgDel protruder XII MNs also are significantly different their WT counterparts. Spontaneous AP firing frequency is significantly less in LgDel protruder XII MNs than that in WT. During putative inspiratory related bursting in WT, protruder AP firing only increases during the first second from the start of the burst, whereas in LgDel, protruder firing activity is delayed. LgDel protruder AP firing increases significantly in the second 1-s epoch after the start of the burst, likely because of delayed excitatory inputs to LgDel protruders. The combined consequences of significantly enhanced, temporally anomalous CNXII retractor MN firing and temporally dysregulated CNXII protruder MN activity may degrade the coherence of coordinated neural control of tongue movement critical for optimal S/F/S.LgDel mouse pups in vivo; however, additional observations (LgDel mice. Disrupted coordination of the suck/swallow/breath sequence in infants and toddlers with 22q11.2DS slows or interrupts feedings because of gagging and/or regurgitation (LgDel protruder versus retractor hypoglossal MN outputs is a prime candidate for disrupted S/F/S as well as additional oromotor dysfunction. Although altered protruder MN activity likely complicates S/F/S in 22q11.2DS, retractor MN output changes are more pronounced. These results might predict that E/I imbalance and elevated retractor hypoglossal MN firing alters tongue compression and backward movement (We did not examine the function of protruder and retractor muscles in WT or rvations  indicategitation . Older cgitation . Dysarthgitation . Obstrucgitation . Differemovement , thus th


Whispering gallery mode resonators such as silica microtoroids can be used as sensitive biochemical sensors. One sensing modality is mode-splitting, where the binding of individual targets to the resonator breaks the degeneracy between clockwise and counter-clockwise resonant modes. Compared to other sensing modalities, mode-splitting is attractive because the signal shift is theoretically insensitive to the polar coordinate where the target binds. However, this theory relies on several assumptions, and previous experimental and numerical results have shown some discrepancies with analytical theory. More accurate numerical modeling techniques could help to elucidate the underlying physics, but efficient 3D electromagnetic finite-element method simulations of large microtoroid (diameter ~90 µm) and their resonance features have previously been intractable. In addition, applications of mode-splitting often involve bacteria or viruses, which are too large to be accurately described by the existing analytical dipole approximation theory. A numerical simulation approach could accurately explain mode splitting induced by these larger particles. Here, we simulate mode-splitting in a large microtoroid using a beam envelope method with periodic boundary conditions in a wedge-shaped domain. We show that particle sizing is accurate to within 11% for radii  Whispering-gallery mode (WGM) optical microresonators possess ultrahigh quality (Q) factor and subwavelength mode volume, enabling them to probe nanoparticles and their properties ,2,3. LocMode-splitting in microtoroids has been experimentally verified to size nanoparticles as small as 30 nm in radius . In theoλ/10 , and (2)λ/10 ,13. Theoλ/10 , an addiλ/10 , and somλ/10 , dependiFinite element method models are generally quite accessible and straightforward to implement in microresonator systems. They have been used to simulate mode splitting in small microresonators ,19. HoweExperimentally, large microresonators show good spectral resolution and sensitivity because they have ultrahigh Q-factors and compact mode volumes. Furthermore, they have large capture areas that make analyte detection events more likely. These attributes make their accurate simulation a significant goal. In 2013, Kaplan et al. simulated a 3D microtoroid coupled to a gold nanorod and predicted the near-field enhancement for biosensing. The model is reduced to a small wedge using perfect electrical conductor (PEC) boundary conditions [Here, we use a 3D periodic wedge-shaped beam envelope method (BEM) model with periodic boundary conditions to simulate mode-splitting and particle sizing using a 90-µm diameter microtoroid. Advantages of finite element BEM modeling over more common finite difference time domain (FDTD) models include lower memory requirements due to larger mesh sizes enabled by the BEM approach, faster computation times due to modeling in the frequency domain rather than time domain, and more accurate results due to more conformal polygonal mesh cells rather than a fixed rectangular grid. We have previously used a related, but different, Floquet boundary condition method to model coupling of free-space light with traveling waves within the microtoroid . Unlike When the microtoroid is only weakly perturbed, the splitting of the two degenerate modes is less than their linewidth, and the spectral response is dominated by the shift in resonance. For larger perturbations, the degenerate travelling wave modes split into two standing wave modes. According to the position of the nanoparticle, these modes redistribute themselves as a symmetric (SM) mode and an antisymmetric (ASM) mode . Dipole a0≪λ) ,24. In tplitting ,16. As iplitting ,16, for ts e.g., e regardiquencies . ComputiThe advantages of mode-splitting can be seen from this equation, where the particle size only depends on the particle-WGM interaction quantified by A 2D axisymmetric eigenfrequency simulation is run to probe the resonance features of the bare microtoroid see a. In theTo run efficiently, the 3D BEM requires an approximate phase specification ; in this2π/m see b. The we2π/m see b with ce−1 nm−3 in water and −1 nm−3 in air. The condition for mode splitting to be resolvable is The following are the 3D simulation results for mode splitting and nanosphere sizing. Mode splitting occurs when the microtoroid is locally perturbed by a polystyrene nanosphere with refractive index of To compare the contributions to Q-factor from particle-induced losses with the intrinsic microtoroid Q-factor, we calculate the Q-factors of the coupled WGMs using two methods, both resulting from 3D eigenfrequency simulations: particle . In the In theory, the mode splitting In summary, we used a computationally efficient 3D simulation method to simulate mode splitting induced by a nanoparticle positioned in the evanescent field of a large microtoroid. Q-factors can be recovered from a single simulation run. We find excellent agreement between analytical theory and simulation for nanoparticles of radius up to 70 nm in water and up to 90 nm in air, which both correspond to


The obtained results show that the particle size and loading of feldspar particles (from 25 wt% to 70 wt%) do not influence the glass transition temperature of the model epoxy-amine coating significantly. In general, the smaller the particle size the lower the glass transition temperature of the coating but this depression in gT seems negligible when seen relative to the change in extender particle size. Similar observations are reported for two model coatings having the same lamda (Λ) value but with silica of very different particle sizes .Inorganic extenders are important constituents of 2K thermosetting epoxy-amine coatings and their physical properties play an important role in the final properties of the organic coatings. The effects of extender particle size and loading  on the glass transition temperature ( Among thermosetting polymers, 70% of the market share is captured by epoxy resins (excluding polyurethanes) and 60% of the globally produced epoxy resins is employed in the coatings industry due to their low shrinkage during cure, good adhesion with various substrates, excellent corrosion and chemical resistance, high strength and durability, and good compatibility with numerous commercially important materials. Commercial epoxy coatings can be 1K or 2K . 1K epoxy coatings contain anionic or cationic initiators (in one package), which under heat or photoirradiation, can initiate the polymerization of the epoxy monomer. In 2K epoxy coatings, generally, the component containing the epoxy resin , pigment(s), defoaming agent etc.) is termed as the Component A while the second component, termed as Component B, contains the hardener or curing agent. Component A and B are mixed before application. Thermosetting 2K epoxy formulations can be cured by compounds that are reactive towards the epoxy functional group such as, amines, thiols, organic acids, anhydrides, alcohols, and phenols. For liquid epoxy coatings intended to cure at ambient temperatures and below, amine functional curing agents are most widely used. The type of the epoxy resin and curing agent used along with the curing conditions can have a significant effect on the final properties of the coating [Commercial epoxy coatings contain additives and fillers which may help in easier processing during production and application, cost reduction, and improving the final properties of the coating . Among Most of the research work investigating the role of extender properties in impacting mechanical and thermal properties of cured epoxies is related to nanocomposites. The particle size of the extenders  becomes important, especially, in the nanometer (nm) range. It is believed that in coatings and nanocomposites an immobilized polymer layer of about 1 nm is formed around the surface of the extender particles. In this layer, known as the interaction region, the conformational entropy and chain kinetics are significantly different from the bulk of the material. For nano-sized extender particles the volume fraction of this interaction region is higher than that for micron-sized particles and, therefore, the properties of coatings and composites containing nanoparticles are different from those materials having their micron-sized analogs as extenders ,7.gT) is an important thermal property of organic coatings which contributes in determining their application area and service life properties. Various researchers have investigated the influence of extender particle size on the gT of thermosetting epoxies. Sun et al. [gT of cured epoxy containing nanoparticles was lower than the gT of the epoxy containing a similar type of microparticles which was attributed to the increase in free volume due to the enhanced resin-filler interface using nanoparticles. Liu et al. [gT first decreased with up to 30% nanosilica amount  followed by an increase in gT with up to 50% nanosilica amount. At 70% nanosilica content, no gT was observed. The authors suggested that at low extender content, gT decreased due to the plasticization effect of nanosilica whereas at high extender content the motion of the polymer chains was highly hindered by the particles leading to an increase in gT. According to Chen et al. [gT of the cured model nanocomposite (in comparison to the system without nanosilica). Dittanet et al. [gT of epoxy cured with piperidine by changing the volume concentration of nanoparticles from 0% to 30%. Note that the silica nanoparticles used by these authors were surface treated with organosilane. No other information was provided about the type of organosilane used as it would be important to understand whether the organosilane played a role in the observed gT independence of silica particle size or not. Similar results were obtained with respect to the gT of the nanocomposite when glass spheres (average diameter of 42 µm) were combined with silica nanoparticles to simulate a bimodal particle size distribution [gT of low molecular weight DGEBA based epoxy, cured with a polyamide curing agent. The amount of silica with the same particle size was varied in the range of 3.3 to 9.2 vol%. The authors showed that the gT of the cured resin filled with silica is lower in comparison to neat cured epoxy up to a silica content of 6.4 vol% followed by an increase at a silica content of 9.2 vol%. It was proposed by the authors that the initial gT depression (up to 6.4 vol% of filler content) was probably due to the ability of the silica nanoparticles to prevent a complete cure of the epoxy which lead to a reduction in crosslinking degree. In the case of incomplete reaction during the curing period, DSC heating scans show an exothermic event which can be attributed to the heat released during the reaction of epoxy with the curing agent. No such exothermic events were presented by the authors in DSC heating scans as indication of incomplete cure of the used epoxy resin. At the highest filler content, the gT was increased due to the increased physical immobilization of polymer chains near the filler surface. Recently, El-Fattah et al. [gT of room temperature cured epoxy coating. By using FTIR, the authors showed that silanol groups on the silica surface chemically react with the epoxy groups due to the local heat produced during ultrasonication and that the extent of reaction in smaller particles (average particle size of 7 nm) was higher than that in larger particles (average particle size of 30 nm). However, the effect of silica particle size on the gT of coatings was not significant as was expected due to this reaction. At similar amounts of silica nanoparticles, the gT of the coating containing 7 nm diameter particles was found to be 7.5% lower than the gT of the neat epoxy, whereas, this difference was 4.5% for the coating containing 30 nm diameter particles. Reduction in gT was noticed up to 2 wt% of both silica types after which the gT of the coating was found to be similar to the neat epoxy coating. The authors attributed this behavior to poor dispersion at higher nanosilica amounts.The glass transition temperature  which may lead to different dispersibility characteristics of these silica types [gT of model epoxy-amine coatings by using commercially important extenders. Furthermore, the effect of extender particle size on the gT of model systems with the same lamda value (Λ) was also studied. Λ is the ratio of pigment volume concentration (PVC) to critical pigment volume concentration (CPVC). PVC indicates the volumetric concentration of extenders (or pigments) in the coating. CPVC is representative of the PVC corresponding to the random tightest possible packing of the extender (or pigment) particles and the minimum amount of binder necessary to fill the interstices between extender particles. Λ value less than 1  typically indicates that the coating film is continuous and contains only polymer (or binder) and pigments. Λ values very close to or higher than 1  indicate that the coating film has air pockets around the extender particles due to lack of polymer. The importance and role of PVC and CPVC in coating properties was reviewed by Perera et al. [gT of the model coatings.The above-mentioned literature review shows that the effect of extender particle size on the ca types ,14 and, −1). Phenalkamine curing agent used to cure the epoxy was obtained from the Cardolite Corporation, USA, with active hydrogen equivalent weight (AHEW) of 162 g·eq−1. Extenders used in this study included feldspar , calcite , alumina , talc , Spherilex DP- 0115 amorphous silica , and nanosilica . Coatings grade of xylene and n-butanol were used as solvents. Defoaming agent BYK-A 530 was obtained from BYK, Germany.Epoxy resin used in this work was Epokukdo YD-128 obtained from the Kukdo Chemical Co., Ltd., Korea, which is a liquid type standard epoxy resin derived from bisphenol-A with epoxy equivalent weight (EEW) of 187 g per equivalent (g·eq50 and D98 for each extender.Particle size distribution (PSD) of selected extenders was obtained according to ISO 13320. Samples were collected from thoroughly mixed containers using a spoon. Particle size measurements were obtained on a Mastersizer 3000 instrument using Hydro G dispersion unit with water (23 °C) as dispersing agent. Samples were manually pre-dispersed in a small beaker by using a pipette before being added to the Hydro G. All extenders were pre-dispersed in water except talc which was pre- dispersed in 70% EtOH. Samples were added to Hydro G until 5–15% obscuration was reached. During measurement 20% ultrasound was applied continuously and the stirrer/pump speed was set to 2400 rpm. The samples were analyzed by manual mode and measurement duration set to 10 s. Consecutive measurements were made until stable results were achieved. The PSDs of different extenders are shown in gT of the samples. All samples were analyzed by conventional heat/cool/heat cycle with heating and cooling rates of 10 °C·min−1 under continuous nitrogen flow , then cooled down to −50 °C at a cooling rate of 10 °C·min−1, followed by a heating scan −50 °C to 150 °C at a heating rate of 10 °C·min−1 (referred to as the second DSC heat scan in this work). The gT of the samples was estimated from the heating scans as the mid- point of the glass transition region by using Universal Analysis software from TA instruments.Differential Scanning Calorimetry (DSC) analysis of the samples was performed on a Q200 DSC instrument from TA Instruments. 5 to 10 mg of sample was placed in a hermetic aluminum DSC cell. A hole was made in the lid of the DSC cell prior to running DSC analysis in order to avoid effects of evaporating species on the −1. The obtained data was analyzed by using Universal Analysis software from TA instruments.DMA analysis was performed on a Q800 DMA model from TA Instruments with film tension clam and DMA Multi-Frequency—Strain mode. For all samples, oscillation amplitude and force track were kept at 5.0 µm and 125.0%, respectively. Each sample film was first cooled down to −50 °C followed by heating up to 150 °C or 200 °C at a constant heating rate of 4 °C·minFor morphology of the extenders used, a Hitachi SU3500 Scanning electron microscope was used.gT) region of the coating with neat epoxy is different from that of the epoxy coatings filled with extenders. This is shown by using the peak endothermic temperature (pT) which is 93.45 °C for the neat epoxy sample whereas for coatings filled with extenders pT is close to 101 °C and shows some dependence on particle size of the used extenders. Overall, the first DSC heating scans suggest that addition of extenders increases the gT of the epoxy coatings in comparison to the epoxy coating without any extender and that the gT shows almost no dependence on the particle size of the used extender  of filled coatings.nder see . This in systems . It is igT of epoxy-amine coatings, it should be noted that while the morphology of the used extenders is different . Two grades of the same extender also ensured that the extender chemistry has been kept very similar. All other parameters  were kept as for the samples mentioned above.With respect to the effect of the particle size of extenders on the rent see  their PSrent see  but simirent see e were usgT for each sample shown in gT values estimated from 1st and 2nd heating DSC curves (see gT of the epoxy cured with anhydride remains unchanged (in comparison to neat cured epoxy). However, with nanometer sized silica, the gT of the cured epoxy decreased at higher loadings .rves see  do not stan δ curves of the selected samples with different extender loadings and particle sizes. Observations from DMA analysis are similar to those made from the DSC analysis i.e., (i) extenders decrease the gT of the cured epoxy-amine system, and (ii) a slight effect of particle size on the gT of the cured system can be noticed and one might conclude that at the same extender loading (or pigment volume concentration (PVC)) the gT of the coating with smaller extender particles is lower than that of the coating with larger particles. However, considering the 3-fold change in particle size and loading this change in gT of the coating is probably insignificant. Nevertheless, it is important to mention that long term service life properties of epoxy coatings with extenders of different particle sizes might be different as the physical and chemical ageing behavior of these coatings may be affected by such differences in the physical properties of the extenders.gT of epoxy-amine coatings, two more formulations were prepared where the nanosilica and micronsized silica were used as extenders but lamda (Λ) of the final coating  was kept constant at 0.51. For these coatings, all other components  and extender(s) particle size effect(s) on the gT of the coatings need to be separated.In order to further investigate the effect of particle size on nterface  but thisgT) of model epoxy-amine coatings was investigated by selecting extenders of commercial importance with different particle sizes and particle size distributions. DSC and DMA analysis of the cured epoxy- amine systems showed that the particle size of the extenders  slightly impacts the gT of the cured epoxy-amine coatings i.e., the smaller the size of the extender particles the lower the gT of the final cured coating. However, the extent of gT depression is not proportional to the extent of change in extender particle size i.e., a three-fold reduction in average particle size of the extenders used, reduced the gT of the coating (obtained from the first heating curve of the DSC) by a maximum of 3 °C (at the same PVC). Therefore, it can be concluded that changes in average particle size of the epoxy-amine coatings do not have a significant influence on the gT of the cured coating. Increasing the extender amount up to 70 wt% also showed no significant influence on the gT of the coating.The effect of the particle size of extenders on the glass transition temperature (


Adenomatous Polyposis Coli (APC). APC is an essential component of a cytoplasmic protein complex that targets β-catenin for destruction. Following Wnt ligand presentation, this complex is inhibited. However, a role for APC in this inhibition has not been shown. Here, we utilized Wnt3a-beads to locally activate Wnt co-receptors. In response, the endogenous β-catenin destruction complex reoriented toward the local Wnt cue in CRC cells with full-length APC, but not if APC was truncated or depleted. Non-transformed human colon epithelial cells displayed similar Wnt-induced destruction complex localization which appeared to be dependent on APC and less so on Axin. Our results expand the current model of Wnt/β-catenin signaling such that in response to Wnt, the β-catenin destruction complex: (1) maintains composition and binding to β-catenin, (2) moves toward the plasma membrane, and (3) requires full-length APC for this relocalization.Wnt/β-catenin signaling is essential for intestinal homeostasis and is aberrantly activated in most colorectal cancers (CRC) through mutation of the tumor suppressor  As a result, deregulation of Wnt signaling is often associated with cancer and other diseases3. Notably, over 90% of colorectal cancers (CRC) have mutations that activate the Wnt pathway with over 80% containing mutations in the Wnt antagonist Adenomatous Polyposis Coli (APC)4. Human APC mutations commonly arise in a central region of the open reading frame, referred to as the “mutation cluster region” , Axin interaction sites (SAMP), nuclear localization sequences, and a C-terminal basic region which mediates cytoskeletal interactions  and low-density lipoprotein receptor 5/6 (LRP5/6) to form the heterotrimeric “signalosome,” the β-catenin destruction complex is inhibited through an incompletely resolved mechanism14. While the downstream effects of Wnt signaling have been extensively analyzed, the specific molecular events which cause destruction complex inhibition are not well understood. Further, the exact roles of APC in normal destruction complex function have remained elusive, and it has not been determined whether APC is also involved in promoting the transduction of a Wnt signal.The canonical Wnt pathway serves to regulate the level of transcriptional coactivator β-catenin17. Traditionally, these intestinal Wnt ligands have been thought to diffuse from the crypt base toward the luminal surface to form a signaling gradient. However, Wnt was recently shown to predominantly engage with FZD receptors on the ISCs that were immediately adjacent to Wnt-secreting cells and not those that were further removed from the Wnt source. These findings are inconsistent with a Wnt diffusion model, but instead support a model whereby Wnt is initially bound to receptors following secretion and forms a gradient through receptor turnover and cell division18. Therefore, intestinal Wnt signaling may convey positional information within the crypt and direct intracellular protein localization based on the location of the Wnt source. In the current study, we will examine the effect of a local, immobilized Wnt signal on colon epithelial cells.Wnt ligands are required for intestinal stem cell (ISC) self-renewal and crypt homeostasisApcMin/+ mouse model, much focus has been directed to the small intestine rather than the colon, the site of most tumor-initiating APC mutations in humans19. Further, very little is known about Wnt signaling in colon epithelial cells that are from non-malignant origin, as most studies utilize only cultured CRC cell lines. Finally, prior research has mostly relied on overexpression of specific Wnt pathway components or cells treated with soluble Wnt in the media, limiting the ability to elucidate endogenous β-catenin destruction complex dynamics in response to a local Wnt signal (reviewed in3).Several challenges have historically limited our understanding of Wnt signaling dynamics as it relates to intestinal homeostasis in the normal and cancerous state. Due to the widespread use of the Here, we examine the response of endogenous β-catenin destruction complex components to a locally presented Wnt signal in human colon epithelial cells of both malignant and non-malignant origin. We demonstrate for the first time that a localized Wnt source can recruit the signalosome and β-catenin destruction complex in colon epithelial cells and find that this Wnt-induced recruitment requires full-length APC. Our work identifies a novel role for APC in the regulation of destruction complex movement toward the membrane following Wnt exposure.APC is mutated in >80% of colorectal cancers and is a major scaffolding protein for the β-catenin destruction complex, we first asked whether APC redistributes toward a localized Wnt3a signal in a panel of human CRC cell lines. Three colon cancer cell lines, each with a different Wnt pathway status, were used: RKO , HCT116βm 20, and DLD1 cells  that were isolated from normal tissue and propagated in culture under near physiological conditions (low oxygen and sera). Shay and colleagues isolated HCECs from routine colonoscopy and immortalized them with expression of cyclin-dependent kinase 4 (Cdk4) and human telomerase (hTERT)KO cells  and then determined localization of β-catenin, Axin1 and GSK3β. APC knock-down was efficient in HCEC 1CT and resulted in increased β-catenin protein levels, as expected Fig. . In celllls Fig. .Figure 7Like APC, Axin1 is a key scaffolding protein in the β-catenin destruction complex, and thus, might control proper localization of the complex. Using HCEC 1CT cells, we depleted Axin1 with siRNA (siAxin1) and determined the localization of APC and β-catenin. Both APC and β-catenin still localized toward the Wnt-bead in Axin1-depleted HCEC 1CT cells Fig. . HoweverTogether, these data demonstrate that the β-catenin destruction complex retains the ability to orient toward a localized Wnt source in HCEC 1CT cells and reveal a requirement and novel function of full-length APC in destruction complex trafficking toward a Wnt cue.KO cells. We extended this study to a non-transformed human colon epithelial cell line which exhibits stem cell characteristics and found that Wnt-directed destruction complex localization also occurs in colon epithelial cells of non-malignant origin. When compared to the protein distribution in DLD1 cells, which were already limited in signalosome and destruction complex reorientation in response to localized Wnt, APC depletion in the non-transformed human colon epithelial cells completely abolished Wnt-induced destruction complex localization. Combined with a previous report that the truncated APC found in DLD1 remains associated with destruction complex protein Axin125, it appears that APC utilizes its function in scaffolding as well as interactions in the C-terminus to fully traffic the destruction complex toward a Wnt cue.To investigate the role of APC in Wnt-directed destruction complex localization, we first explored signalosome and destruction complex response in commonly used CRC cell lines with varying Wnt pathway status. The data from these experiments provide novel findings that the β-catenin destruction complex re-localizes toward Wnt in colon epithelial cells and that this redistribution is impaired in DLD1 cells expressing a truncated form of APC. Truncated APC is present in the majority of human CRC, and results in loss of the C-terminal half of APC, which contains domains involved in destruction complex assembly, nuclear localization, and cytoskeletal interactions. Further, we demonstrate that APC loss ablates Wnt-induced destruction complex localization. Unlike the parental RKO cell line, Axin1, GSK-3β and β-catenin are unable to localize toward a Wnt-bead in RKO-APC36. However, other studies have demonstrated that some of the destruction complex may be targeted to the plasma membrane following receptor activation38. Recently, an elegant study exposed mouse embryonic stem cells to Wnt3a-conjugated beads, resulting in APC, β-catenin, and LRP6 localization toward a Wnt-bead and also found that the local Wnt cue could trigger asymmetric cell division39. To our knowledge, this is the only study examining the orientation of signalosome and destruction complex components in response to localized Wnt and was performed in pluripotent cells of nonhuman origin and limited to three endogenous proteins.Numerous models of the events following Wnt receptor activation have been proposed, however the specific molecular proceedings that occur are still debated due to the use of overexpression of Wnt pathway components, global addition of Wnt to culture media, or the wide range of cell and tissue types used. Many models propose that the destruction complex is either inactivated or partially disassembled following Wnt/co-receptor interaction42. It is possible that these differences reflect more spatially restricted Wnt contact in our study compared to global Wnt exposure. It also seems likely that the bead attached to Wnt ligand in our study was so large as to prohibit endocytosis. Potentially related, recent evidence points to a role for the central region of APC in preventing clathrin-mediated endocytosis in the Wnt-off state31. Over time, the complexity of potential APC functions related to Wnt signaling has been gradually revealed. Previous work by our lab and others demonstrated that APC performs roles outside of its classically defined scaffolding function in the β-catenin destruction complex. For example, APC interaction with nuclear β-catenin leads to repression of Wnt target genes through several potential mechanisms: providing access to the transcriptional corepressor CtBP or the E3 ligase β-TrCP, β-catenin sequestration from transcriptional coactivator LEF-1/TCF, or facilitating β-catenin’s nuclear export47.Our study builds upon the current model of Wnt signal transduction and brings to light a new role for APC. Here, we report the requirement of APC for proper Wnt-induced localization of the β-catenin destruction complex in both malignant CRC cell lines and in non-transformed colon epithelial cells. In contrast to our findings, others have demonstrated that key members of the complex are degraded or endocytosed following prolonged Wnt exposure25. Another possibility, is that Wnt stimulation leads to an APC-dependent membrane orientation of the destruction complex which results in complex inactivation. Upon removal of Wnt signal, this already assembled complex would be unlocked and able to process β-catenin for destruction. These two models are not mutually exclusive and might even be interdependent.Together, our results suggest a novel mechanism of β-catenin destruction complex regulation by the APC protein Fig. . In addi48. Based on the compromised destruction complex localization observed in DLD1 cells and APC-depleted HCEC 1CT cells, we suggest that APC is required for Axin docking to membrane-associated Dvl following Wnt exposure. In this light, our results strongly support the model proposed by Tacchelly-Benites et al., based on studies in Drosophila, that APC plays a major role in regulating Axin’s signalosome recruitment in response to Wnt signaling by facilitating phosphorylation of Axin by GSK-3β49. The compromised localization observed in DLD1 cells suggests that C-terminal domains contribute to this trafficking, potentially through cytoskeletal interactions51. The mechanism by which APC moves the β-catenin destruction complex toward the signalosome is a necessary focus for future studies in order to provide insight into colon epithelial cell biology and further elucidate unknown aspects of Wnt signal transduction. Overall, our findings provide additional mechanistic details of destruction complex behavior following Wnt exposure, and uncover a novel role for APC in Wnt signal transduction.Future studies are necessary to clarify the precise mechanism of APC-regulated destruction complex trafficking to the membrane. Or study does not support an absolute requirement for Axin1 in Wnt-mediated destruction complex relocalization. Perhaps Axin2, a Wnt target, is able to compensate for Axin1 in this capacity. Axin has been postulated to localize to Wnt receptors following Wnt activation through a Dvl-dependent mechanismKO cells were cultured in DMEM  supplemented with 10% FBS and were maintained at 37 °C and 5% CO2. HCEC 1CT cells were generously provided by Dr. Jerry Shay, UT-Southwestern and cultured as described previously in X-media (4:1 DMEM/Medium 199) supplemented with EGF (20 ng/ml), hydrocortisone (1 μg/ml), insulin (10 μg/ml), apo-transferrin (2 μg/ml), sodium selenite (5 nM), and 2% cosmic calf serum32. Cells were maintained at 37 °C in low-oxygen incubators to reduce stress-induced senescence from normal atmospheric oxygen tension and to better recapitulate physiological conditions, as described previously52. For immunofluorescence assays, cells were seeded onto fibronectin/Poly-D-Lysine coated coverglass (Neuvitro) at 25% confluence and incubated at least 24 hrs prior to treatment with 5 μl beads/well for 12–14 hrs. HCEC 1CT cells were seeded at 10–15% confluence prior to siRNA transfection and Wnt-bead treatment.RKO, HCT116βm, DLD1, and RKO-APCFor siRNA-mediated inhibition, HCEC 1CT cells were transfected using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions with 37.5 nM of each siRNA targeting human APC , human Axin1  or nontargeting siControl siRNA (Dharmacon). Cell media was changed one day following siRNA transfection, and cells were grown 48 hrs prior to Wnt-bead or Unloaded-bead treatment.39. Briefly, 2.8 μm Dynabeads M-270 Carboxylic Acid (Invitrogen) were activated by NHS/EDC  then washed three times with cold MES buffer. Wnt immobilization was performed by diluting 0.5 μg of purified Wnt3a protein  in cold MES buffer and incubated at room temperature (RT) for 1 hr. To quench non-reactive carboxylic acid groups, beads were incubated with 50 mM Tris pH 7.4 at RT for 15 min. Beads were washed twice in PBS pH 7.4 before final resuspension in 400 μl PBS/0.5% BSA and stored at 4 °C. Unloaded-beads were prepared in parallel by incubating 1 hr in MES without Wnt. Wnt3a activity following bead immobilization was verified using a TOPflash luciferase reporter assay53.Wnt3a was immobilized onto Dynabeads as described previously2, 1 mM EGTA) for 20 min at RT, washed two times in PBS prior to permeabilization in TBS/0.2% Triton X-100 for 5 min. Cells were washed in TBS two times prior to incubation for 1 hr at RT in blocking buffer containing TBS/0.2% Triton X-100, 1% BSA, and 3% Normal Goat Serum. Primary and secondary antibodies were incubated for 1 hr at RT. Cells were washed in TBS three times following primary and secondary antibody incubations. Coverslips were mounted and counterstained with Prolong Diamond Antifade Mountant with DAPI (Invitrogen). Staining of FZD7 was performed without cell permeabilization or use of detergent to keep the cell membrane intact. Nuclei were counterstained with 5 μg/ml Hoechst 33342 (Invitrogen) in PBS for 5–10 min prior to mounting with Prolong Antifade (Invitrogen). Antibodies were diluted in blocking buffer as follows: anti-APC-M2 rabbit pAb , anti-Axin1 rabbit mAb , anti-β-catenin mouse mAb , anti-CK1α rabbit pAb , anti-GSK3β rabbit pAb , anti-β-TrCP rabbit pAb , and anti-Frizzled-7 rabbit pAb , Alexa Fluor 488 and 568 conjugated secondary antibodies . Stained cells were examined using an Axioplan microscope (Zeiss) with a X100 objective. Images of stained cells were captured using an Orca R2 digital camera (Hamamatsu).Cells were briefly rinsed in PBS prior to fixation. Cells were fixed in 4% PFA in Brinkley’s Buffer 1980 . Scraped cells were transferred to Eppendorf tubes, heated at 95 °C for 1 min, pulled through an insulin syringe three times, and heated again. Samples were separated on 7.5% SDS-PAGE  using Tris-Glycine running buffer and transferred to a nitrocellulose membrane (GE) with a 0.45-μm pore size. Antibodies were diluted in Odyssey Blocking Buffer TBS (LI-COR) as follows: anti-APC-M2 rabbit pAb , anti-β-catenin mouse mAb , anti-Axin1 goat , and anti-β-actin mouse mAb (Sigma) , IRDye 680LT and 800CW anti-rabbit or anti-mouse secondary antibodies . Immunoblots were imaged on a LI-COR Odyssey CLx imaging system.25. After addition of lysis buffer, cells were scraped into 1.5 ml tubes and rotated at 4 °C for 30 min. Beads were isolated using a magnet and the supernatant was transferred to a new tube. Beads were washed three times in 500 μl of cold lysis buffer. Following the final wash, beads were resuspended in 40 μl cold PBS and 20 μl 3X SDS sample buffer. Samples were analyzed by western blot as described above.Cells were grown in 6-well tissue culture plates and treated with 40 μl Unloaded-beads or Wnt-beads for four hours. Following bead treatment, cells were briefly washed in 1x PBS prior to lysis in 200 μl lysis buffer, described previously ImageJ/FIJI  was used for line scan analyses to quantify protein localization in relation to a Wnt- or Unloaded-bead. Line width was set to 50, corresponding to approximately the size of a Dynabead. Lines were drawn beginning at the bead and across the cell, moving through and across the center of the nucleus. For graphical presentation in Fig. , each scBlinded scoring was performed on a subset of samples using the scoring system presented in Fig. Supplementary figures and legends.


Dysregulation of the Wnt/β-catenin signaling pathway is critically involved in gastric cancer (GC) progression. However, current Wnt pathway inhibitors being studied in preclinical or clinical settings for other cancers such as colorectal and pancreatic cancers are either too cytotoxic or insufficiently efficacious for GC. Thus, we screened new potent targets from β-catenin destruction complex associated with GC progression from clinical samples, and found that scaffolding protein RACK1 deficiency plays a significant role in GC progression, but not APC, AXIN, and GSK3β. Then, we identified its upstream regulator UBE2T which promotes GC progression via hyperactivating the Wnt/β-catenin signaling pathway through the ubiquitination and degradation of RACK1 at the lysine K172, K225, and K257 residues independent of an E3 ligase. Indeed, UBE2T protein level is negatively associated with prognosis in GC patients, suggesting that UBE2T is a promising target for GC therapy. Furthermore, we identified a novel UBE2T inhibitor, M435-1279, and suggested that M435-1279 acts inhibit the Wnt/β-catenin signaling pathway hyperactivation through blocking UBE2T-mediated degradation of RACK1, resulting in suppression of GC progression with lower cytotoxicity in the meantime. Overall, we found that increased UBE2T levels promote GC progression via the ubiquitination of RACK1 and identified a novel potent inhibitor providing a balance between growth inhibition and cytotoxicity as well, which offer a new opportunity for the specific GC patients with aberrant Wnt/β-catenin signaling. As one of the most globally prevalent cancers, gastric cancer (GC) has an incidence in the world ranking fifth and mortality ranking third in all kinds of cancers according to “National Cancer Report 2019” published by the National Cancer Center . For unrThe Wnt/β-catenin signaling pathway is one of the key cascades that is essential to many physiological processes, such as cell proliferation and differentiation, stem cell renewal, embryogenesis, and tissue homeostasis , 8. GeneIn this study, we found that current Wnt pathway inhibitors in preclinical or clinical trials for other cancers are either of high cytotoxicity or low efficiency for GC cells. Then, we found a critical ubiquitin-conjugating enzyme E2T (UBE2T) that catalyzes the proteasomal degradation of RACK1 and then induces hyperactivation of Wnt/β-catenin signaling. Moreover, we developed an inhibitor that blocks the UBE2T-RACK1-Wnt/β-catenin axis and inhibits the GC progression. Together, our findings could offer new opportunities for Wnt/β-catenin signaling aberration GC patients.It is well-known that dysregulation of the Wnt/β-catenin signaling pathway is critically correlated with cancer initiation and progression . In ordeNext, we investigate whether previously developed Wnt pathway inhibitors in preclinical or clinical trials possess inhibitory effects on GC cell growth. We chose to test the effects of nine inhibitors in clinical or preclinical trials for other cancers on cell viability in HGC27, MKN45, and AGS cells, using GES-1  as control. Our data showed that three kinds of the inhibitors  have obvious cytotoxicity to GES-1 Fig. , whereasRACK1 with CRISPR/Cas9 technology in HGC27, AGS, and MKN45 cells , glycogen synthase kinase 3 (GSK3), and AXIN tightly controls the fate of β-catenin . TherefoIn order to determine how RACK1 protein level was downregulated in GC tissues, we tested the mRNA expression of RACK1 in 24 paired gastric cancer and para-carcinoma tissues. We found that RACK1 expression level was similar between gastric cancer tissues and paired para-carcinoma tissues Fig. , which iUBE2T−/− AGS cells had higher RACK1 protein levels than wild AGS cells  supplemented with 10% fetal bovine serum, penicillin (100 mg/ml), and streptomycin (100 mg/ml) and incubated at 37° C in a humidified chamber with 5% CONuclear Protein Extraction kit  was used to segregate the cytoplasm and nuclear protein. The Lamin B1 and GAPDH as the references of nuclear and cytoplasm protein, respectively.Tissue of patients and mice specimens were formalin-fixed and paraffin-embedded, and the process of immunohistochemistry was conducted referring to the article of Deng et al. . All cli490 after reaction 1 h with CellTiter 96® AQueous One Solution Reagent .Approximately 4000 cells/well were seeded in a 96-well plate and treated with 11.8 μM M435-1279 for 48 h. Cell viability was measured OD6 MKN45 cells were suspended in 100 μl of PBS and 100 μl of Matrigel for intratumor injection. For small-molecule inhibitor, when the tumor size reached 75–100 mm3, mice were subjected to intratumor injections of 0.9% NaCl with DMSO, 5 mg/kg/day M435-1279 dissolve in 0.9% NaCl, respectively. All procedures involving mice and experimental protocols were approved by the Ethics Committee.The BALB/C nude mice were purchased from the Beijing Charles River company. In total, 4 × 10Cells expressing HA-Ubiquitin, no-tag-UBE2T, and flag-RACK1 were lysed by lysis buffer and then incubated with Flag-gel beads at 4 °C for 2 h. For ubiquitination assay in denaturing condition, cells were lysed by SDS lysis buffer  containing 2% SDS for 5 min at 95 °C. Then, the lysates were diluted fourfold with dilution buffer  , and the2O. Second, incubate the reaction at 37 °C for 2 h, add stop buffer and SDS-buffer. Finally, boil it for 5 min and perform a western blot assay.First, add reaction buffer  (10×) to solution contain UBE1 , UBE2T , RACK1  and Ubiquitin . Add a solution of Mg-ATP, and then bring the volume to 20 μl with ddHT test, or paired t test were used to compare the difference between groups for categorical and continuous data, respectively. And the data were expressed as percentages, mean ± standard or median . P < 0.05 was considered significant.SPSS version 26.0  and GraphPad Prism 8.0  were used to perform statistical analyses and graphing, respectively. Pearson chi-square tests, independent sample Primer sequence and antibody information are showed in Supplementary Tables Fig. S1Fig. S2Fig. S3Fig. S4Fig. S5Fig. S6Fig. S7Fig. S8Fig. S9Fig. S10Fig. S11Table S1Table S2Table S3Table S4Table S5Supplementary Materials and Methods


The mechanistic target of rapamycin (mTOR) and wingless-related integration site (Wnt) signal transduction networks are evolutionarily conserved mammalian growth and cellular development networks. Most cells express many of the proteins in both pathways, and this review will briefly describe only the key proteins and their intra- and extracellular crosstalk. These complex interactions will be discussed in relation to cancer development, drug resistance, and stem cell exhaustion. This review will also highlight the tumor-suppressive tuberous sclerosis complex (TSC) mutated, mTOR-hyperactive lung disease of women, lymphangioleiomyomatosis (LAM). We will summarize recent advances in the targeting of these pathways by monotherapy or combination therapy, as well as future potential treatments. The mechanistic target of rapamycin (mTOR) protein is a phosphatidylinositol-3-kinase (PI3) kinase-related serine/threonine kinase, which is the basis for two intracellular protein complexes, namely mTORC1 and mTORC2. When activated, these protein complexes are primarily associated with lysosomes .Genetic or epigenetic changes to key mTOR pathway proteins may lead to cellular metabolic instability and human diseases, particularly cancers ,6. ProteWingless-related integration site (Wnt) signaling is a complex cell communication system that controls aspects of organ development, stem cell proliferation, injury and repair responses, and homeostatic maintenance of tissues ,11. HypeDuring their cellular co-evolution, the highly conserved mTOR and Wnt pathways become intertwined with each other, and with other growth pathways, resulting in tangled extracellular and intracellular crosstalk. In many cancer cells, the mTOR and Wnt pathways are synergistic for growth, and when one pathway is inhibited by drug treatment, the other becomes activated.A critical and common element of both mTOR and Wnt signaling is glycogen synthase kinase-3 (GSK3), an over-achieving serine/threonine kinase with a plethora of protein substrates and wide biological roles . Rarely There are two mTOR complexes made up of the mTOR kinase as the central catalytic protein, with several common proteins, including the regulatory Dep domain-containing mTOR-interacting protein (Deptor), and several distinguishing proteins, namely the regulatory protein associated with mTOR (Raptor) for mTORC1 and the rapamycin-insensitive companion of mTOR (Rictor) for mTORC2. Raptor and Rictor are essential for the kinase substrate specificity of mTORC1 and mTORC2, respectively. TSC2, when complexed with TSC1, functions as a GTPase activating protein (GAP) for the small G protein Ras homolog enriched in the brain (Rheb). Rheb, in the GDP-bound form, is then unable to increase mTORC1 kinase activity. When TSC2 is inactivated, by mutation, for example in the GAP domain, or by growth factor signaling, or amino acid stimulation of Ras-Related GTPases (RAG GTPases) occurs, mTORC1 translocates to lysosomes. On lysosomal membranes Rheb–GTP activates mTORC1 [Two of the key proteins phosphorylated by mTORC1 are S6K1 and initiation factor 4E-binding protein (4E-BP), the former promoting ribosomal synthesis and the latter releasing eukaryotic initiation factor 4E (eIF4E), the rate-limiting protein in the initiation of the translation of capped mRNAs, particularly those with a complex secondary structure. In addition, activation of mTORC1 results in increased nucleotide and lipid synthesis and inhibition of autophagy. When nutrients are scarce, another important energy-sensing regulatory kinase, AMP-activated kinase (AMPK), inhibits mTORC1. Therefore, it is not surprising that mTORC1 signaling has been linked to metabolic disease, neurodegeneration, aging, and cancer . This reTM) was originally used as an immunosuppressant, but with the characterization of the mTOR growth pathway and its role in diseases, particularly cancer, its use widened. Unfortunately, as monotherapy in cancer, rapamycin and other rapalogs are only weakly effective [The critical role of mTORC1 in cell growth was demonstrated by the discovery that the natural product rapamycin  binds to the peptidyl-prolyl cis-trans isomerase FK506-binding protein 12 (FKBP12), which, in turn, binds to an allosteric site on mTOR and inhibits mTORC1 activity. Chronic treatment with rapalogs may lead to indirect secondary inhibition of mTORC2 . RapamycIn contrast, in LAM, the use of rapamycin has been the difference between life and death for many women. As a result of a phenomenal collaboration between patients, clinicians, researchers, the LAM Foundation in the U.S., and worldwide LAM clinics, sirolimus was recognized as a therapy for LAM and was approved by the United States Food and Drug Administration (FDA) in 2015 for use in LAM in the U.S. . HoweverWnt1, a homolog of wingless in Drosophila, was first discovered in mammalian cells as an oncogene identified through investigation of a mouse mammary tumor virus integration site. A delightful retrospective treatise including the original discovery and expansion of mammalian Wnt signaling provides an excellent introduction to this field . A more Nineteen cysteine-rich glycosylated secreted Wnt family proteins can activate Wnt signaling through ten different cell surface, seven transmembrane proteins known as frizzled (FZD) receptors. These FZD receptors are associated with single transmembrane low-density lipoprotein-related proteins 5 and 6 (LRP5/6) co-receptors, which are required for Wnt/FZD signaling . Wnt ligIn the cytoplasm, newly formed β-catenin is bound to a degradation complex composed of Axin; Adenomatous Polyposis Coli (APC); casein kinase 1a (CK1a); non-phosphorylated, active GSK3β; and an E3 ligase. In the absence of Wnt proteins, β-catenin is targeted for proteolysis through phosphorylation by CK1a and GSK3β. In the presence of canonical Wnt ligands, the scaffold protein Disheveled (DVL) is activated, which induces the dissociation of the β-catenin–ubiquitin E3 ligase degradation complex and non-phosphorylated β-catenin. β-catenin accumulates in the cytoplasm and translocates to the nucleus ,12,20. NFurther complicating any analysis of canonical Wnt/β-catenin signaling is that particularly in epithelial cells, β-catenin binds to cadherin on the plasma membrane at the cell adhesion junctions . While tThe non-canonical Wnt-activated pathways that do not activate FZD receptors or directly stabilize β-catenin are less well studied . The besThe mTOR and Wnt cancer cell signaling pathways, and indeed all major mammalian cell growth pathways, have diverse extracellular activating and inhibiting ligands and multiple points of intersection. Intracellular crosstalk between these pathways is complex and has been demonstrated in many cells. The mTOR and Wnt signal transduction mechanisms were initially discovered over 30 years ago. Since then, there has been somewhat confusing literature on the crosstalk between these important growth and developmental pathways. Resistance to pathway inhibitors has mainly been related to activation/inhibition by phosphorylation and dephosphorylation of key proteins under specific in vitro/in vivo conditions. In the majority of cancer cells, stimulation of the Wnt/β-catenin pathway directly activates the transcription of genes coding for specific growth and proliferative mRNAs. Activation of the mTOR pathway, via many growth factors, importantly including the insulin growth factor family, results in increased translation, particularly of capped mRNAs, with complex 5-prime structures, including Wnt-activated genes such as c-Myc.Many growth networks are cooperative in cancer cells, and when one is inhibited or mutated, the other is activated, resulting in resistance and continued growth. An example of cooperative growth pathways for mTOR and Wnt was demonstrated in breast cancer cells in transgenic mice overexpressing the glycoprotein Nmb, having increased β-catenin transcriptional activity and phosphorylation of mTOR and 4E-BP1 . An examIn stem cells, hyperactive mTORC1 generally inhibits the Wnt/β-catenin pathway, resulting in stem cell exhaustion . mTORC1 There are multiple ways a cell can respond to stimulation of canonical Wnt and mTORC1 pathways. We highlight this in a highly simplified cartoon, the Wnt/β-catenin activation of transcriptional events and mTORC1 activation of translational events . This reIn both TSC and sporadic (non-inherited) LAM patients, mutations in TSC2 have been related to increased active β-catenin in kidney angiomyolipomas and pulmonary LAM cells ,41. An iTsc2 gene, specifically in mouse lung mesenchymal progenitor cells, resulting in progressive age-dependent enlargement of alveolar spaces, as well as pulmonary lung function decline [Wnt3a, Wnt4, Wnt5a, and Wnt10b, encoding for Wnt ligands, Fzd1, Fzd2, and Fzd8, encoding for FZD receptors, were elevated in the Tsc2-knockout lung mesenchyme, while Wnt3a and Wnt5a gene expression appeared to be downregulated by in vivo rapamycin treatment. Rapamycin, however, did not reverse but slowed down the alveolar space enlargement phenotype. We highlighted elevated crosstalk between Tsc2-deficient mesenchymal-derived cells and surrounding epithelial cells via canonical Wnt ligand/receptor signaling [cttnb1), in addition to mTORC1 activation in the mouse lung mesenchyme, reversed age-dependent alveolar enlargement. However, Wnt pathway activation by genetically stabilized β-catenin in the lung mesenchyme was not sufficient for the development of the observed phenotype in this model [Our research showed upregulation of the Wnt pathway in LAM-lung-specific mesenchymal cells . To foll decline . Selecteignaling . Geneticis model . TogetheTsc2-deficient breeding females. We found that select genes, including Wnt3a and Wnt5a, were differentially upregulated specifically in females, suggesting the involvement of estrogen-dependent regulation. Indeed, the stimulating effect of estrogen on Wnt3a and Wnt5a was reported in earlier studies [Tsc2-null mouse lung fibroblasts. Besides the activation by estrogen, ERα is activated via phosphorylation by S6K1, an enzyme that is directly activated and phosphorylated by mTORC1 [LAM occurs almost exclusively in women and is exacerbated by pregnancies. Consistent with these observations of LAM susceptibility, the most severe lung structure changes and lung function decline was observed in lung mesenchymal  studies . Additio studies . It is oy mTORC1   is the original mTOR pathway inhibitor, as discussed previously. This allosteric mTOR inhibitor binds specifically to FKBP12 protein, which, in turn, binds to a site on mTOR in context with the mTORC1 complex. Sirolimus has long-lasting pharmacokinetics and a reasonable toxicity profile at 1–2 mg daily . This prMany close analogs of sirolimus have been developed for clinical use, including everolimus (RAD001) and temsirolimus . These sTsc2-null cells, we showed that omipalisib inhibited phosphorylation of Akt and both protein synthesis arms downstream of mTORC1 activation, namely S6K and 4EBP1 [There are many PI3K/mTOR mixed inhibitors, but only a few have advanced in clinical cancer trials due to pharmacokinetic, tolerability, and safety issues. An example of a promising PI3K/mTOR inhibitor with phase 1 clinical data in idiopathic pulmonary fibrosis is GSK2126458  . In precnd 4EBP1 . It is nnd 4EBP1 . Rapamycnd 4EBP1 , mainly nd 4EBP1 .Another important kinase sensor of the extracellular environment growth conditions is AMPK, which phosphorylates and activates TSC2 suppression of mTOR, and one clinically available AMPK inhibitor metformin has been repurposed for cancer treatments . HoweverExcellent basic research on the mechanism of growth of LAM cells has resulted in repurposing several older drugs for LAM. To date, only phase I clinical tolerability studies, including with the cyclooxygenase-2 inhibitor celecoxib, the HMGCoA reductase inhibitor simvastatin, and the autophagy inhibitor hydroxychloroquine, have been completed ,58. A seNo specific Wnt pathway inhibitors have been clinically approved. However, some multi-target inhibitors, such as lithium chloride via inhibition of GSK3-β or inhibitors of cyclooxygenase-2, a gene upregulated by β-catenin, have shown benefits, which may be partially due to the Wnt/β-catenin pathway inhibition . Such inβ-Catenin requires many transcriptional co-activators to generate a transcriptionally activated complex, including cyclic AMP response element-binding protein (CBP) and histone acetyltransferase EIA-binding protein p300. It has been shown that genes transcribed from the CBP/β-catenin interaction are mainly proliferative, whereas those transcribed from the p300/β-catenin interaction are mainly differentiation genes . ICG-001An intriguing link between mTOR and Wnt pathways is the mTOR complex protein Deptor that has been shown to be a tumor promoter in CRC and a downstream target of canonical Wnt and c-Myc signaling . This isIn many oncology indications, targeting a single growth pathway by monotherapy usually results in therapeutic resistance by compensatory upregulation of the signaling of other growth pathways. Therefore, combination therapies may result in additive or synergistic inhibition of tumor growth. Several preclinical studies with combination mTOR/Wnt therapies have shown efficacy. For example, dual PORCN inhibitor and PI3K/mTOR inhibitor treatment showed added efficacy in Wnt-driven pancreatic cancer models and RSPO3-translocated cholangiocarcinoma cells . In miceTsc2-knockout mouse model, the genetic deletion of β-catenin also in mesenchymal lung cell progenitors prevented the alveolar structural changes induced by hyperactivation of mTORC1 in these cells [As outlined above, autocrine and paracrine interaction between the mTOR and Wnt/β-catenin pathways can result in complex crosstalk that is dependent on specific cell types and external conditions. Therefore, to support cross-pathway regulation, in a specific setting, there need to be multiple lines of evidence. One of these methods is in vitro and in vivo genetic ablation or overexpression, and another is the use of specific pathway modulators. As shown by our research, in our in vivo lung mesenchymal progenitor se cells .Tsc2 knockout tumors, with vascular anomalies, Gal-3 was overexpressed [To follow the clinical profile of new therapies, it is critical to identify non-invasive biomarkers reflecting both the specific pathway modulation and the relationship to disease progression. For LAM, the only recommended non-invasive biomarker is VEGF-D . Treatmexpressed , and in A difficult challenge for scientists and clinicians is to design clinical trials in an orphan disease with a small number of patients. Using the patients as their own control, or adaptive designs, as is done in many cancer trials, may give the most meaningful data and require fewer patients ,80. OverThe mTOR and Wnt pathways have evolved together, forming convoluted crosstalk both intracellularly and extracellularly. The great number of nodes controlled by phosphorylation and dephosphorylation provides a potential plethora of drug targets. However, the exquisite crosstalk between cellular growth pathways makes it unlikely to be able to inhibit any one kinase without encountering feedback mechanisms. For example, inhibition of S6K1, a key substrate of mTORC1, results in activation of the kinase Akt. In addition, while it has been possible to design selective ATP-competitive kinase inhibitors, many of them have significant pharmacokinetic, tolerability, and safety issues. This is an age-old problem for medicinal chemists and biologists .One of the ways to speed up the discovery of selective compounds with good metabolic profiles is to use artificial intelligence algorithms that use the chemical shape of small molecules both as to fit and selectivity with the target and a lack of shape characteristics that result in poor pharmacokinetics and toxicity . AnotherQuantum advances in mAb discovery and development have occurred before and during the Corona Virus Disease (COVID-19) era . These iNew therapeutic opportunities involve the modification of DNA or RNA targets. Genome editing, particularly by CRISPR/Cas9 technologies, holds promise for ex vivo and in vivo genome editing of diseased genes, but still, many hurdles need to be cleared to enhance the safety of these therapies . AnotherMany of these novel therapies will require sizable investments to develop. Therefore, for an orphan disease like LAM, the pragmatic, mechanism-driven repurposing of approved drugs may provide more immediate clinical benefit. As has been quoted many times before, the best way to discover a new drug is to start with an old one. However, it is necessary to note that many of the older drugs are less selective than more recent compounds, so understanding their mechanism of action, correct clinical doses, and possible toxicities in different patient populations will be critical for therapeutic success.CrossTORC and Wntegration of convoluted cell signaling cascades increase the challenges of finding safe and effective therapeutics and will require productive collaborations between patients, scientists, clinicians, and the pharmaceutical industry. A shining example of such a collaboration is in the orphan disease LAM, where such a cooperative community partnership resulted in the development of sirolimus as an approved therapy for LAM.Diseases, particularly cancers, which result from dysfunction in both mTOR and Wnt pathways, await breakthroughs in new compounds, antibodies, nucleic acid drugs, and bioengineering advances for the clinical development of the next generation of therapeutics. We conclude with a touch of levity that