Acute exacerbation of chronic obstructive pulmonary disease (AECOPD) is the main factor that leads to the deterioration of the disease. Currently, the diagnosis of AECOPD mainly relies on clinical manifestations, good predictors or biomarkers are lacking. We aim to reveal specific biomarkers and potential pathogenesis of AECOPD and provide a research basis for the diagnosis and treatment.Four patients with AECOPD, four patients with stable COPD, and five control subjects were enrolled for RNA sequencing and KEGG analysis. The mRNA level of target genes was verified by quantitative real-time PCR (qPCR) with an expanded sample size . ELISA and immunofluorescence were used to identify the target proteins. Furthermore, the expression and function of WNT/β-catenin signaling pathway were assessed in animal models of COPD.P < 0.05), and β-catenin protein was significantly decreased in plasma of AECOPD and stable COPD (P < 0.01), while phosphorylated β-catenin was significantly up-regulated in peripheral blood mononuclear cells of AECOPD (P < 0.05). Similarly, WNT ligands, WNT receptors, and downstream signaling molecules were down-regulated, with an increased phosphorylated β-catenin protein in animal models of COPD. Activation of WNT/β-catenin signaling pathway by lithium chloride reduced the expression of phosphorylated β-catenin and ameliorated the COPD-like airway inflammation in mice.RNA sequencing showed that 54 genes were up-regulated and 111 genes were down-regulated in the AECOPD. Differentially expressed genes were mainly enriched in WNT signaling pathway, et al. QPCR revealed that multi-genes of the WNT/β-catenin signaling were significantly down-regulated in AECOPD (WNT/β-catenin signaling pathway is down-regulated in AECOPD patients and in animal models of COPD. Increased expression of phosphorylated β-catenin in the blood might be a potential biomarker of AECOPD. Activation of WNT/β-catenin pathway may also represent a therapeutic target for AECOPD.The online version contains supplementary material available at 10.1186/s12931-022-01950-w. Chronic obstructive pulmonary disease (COPD), characterized by persistent respiratory symptoms and restricted airflow, is a chronic airway disease that seriously endangers human health . With coAcute exacerbation of chronic obstructive pulmonary disease (AECOPD) is defined as patients who have acute exacerbation of respiratory symptoms and require additional treatment . FrequenTranscriptomics can simultaneously detect thousands of RNA and has gradually become an important tool for biomarker research. RNA sequencing (RNA-seq) is a transcriptomic analysis method using deep sequencing technology, which can detect the overall transcription activity of cells or tissues more accurately, efficiently, and widely . CurrentIn this study, we performed RNA-seq of mRNA in peripheral blood mononuclear cells (PBMC) of patients with AECOPD, stable COPD, and control subjects, screened differentially expressed genes (DEGs) and multiple signaling pathways that potentially related to the pathogenesis of AECOPD by KEGG analysis. We use quantitative real-time PCR (qPCR) to verify the transcription level of WNT/β-catenin signaling in an expanded-sample size, use ELISA and immunofluorescence to verify the differential expression of target genes at the protein level. Besides, we further elucidated the expression of WNT/β-catenin signaling pathway in a traditional COPD model with cigarette smoke (CS) exposure, and in a novel COPD model combining cigarette smoke sensitization and elastin challenge (CE).Patients with stable COPD and AECOPD and control subjects were recruited from the Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China. All participants have given written informed consent to participate in the study, which was approved by the Institutional Review Board for Human Studies of Second Affiliated Hospital of Zhejiang University School of Medicine . The definition of patients with stable COPD and AECOPD was according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD 2018) . PatientSix to 8 weeks male C57BL/6 mice were purchased from the Animal Centre of Slaccas . All mice were maintained in the animal facility of the laboratory animal center of Zhejiang University. Mice at the end of modeling were anesthetized by intraperitoneal injection of 0.2 mL 2% pentobarbital sodium and were killed by cervical dislocation, in the scientific research building of Zhejiang University. All experimental protocols were approved by the Ethical Committee for Animal Studies at Zhejiang University.CE model: Mice (n = 5) were exposed to CS in a stainless-steel chamber using a whole-body smoke exposure system  for approximately 2.5 h per day (100 cigarettes), 5 days per week, and last 2 weeks. Control subjects (n = 4) were exposed to filtered room air. Mouse elastin E6402-SPEC) was purchased from Sigma-Aldrich. 2 mg/ml elastin was suspended in sterile saline and sonicated. 100 μg elastin in 50 μl saline was administered intratracheally for 3 times at day 29, 30, and 31, and mice were sacrificed 48 h after the last elastin challenge. The detailed experimental protocol refers to the previous study [402-SPEC CS model: Mice (n = 6) were exposed to CS in a stainless-steel chamber using a whole-body smoke exposure system  for approximately 10 times per day (5 cigarettes for each time), 5 days per week, and last 3 months. Control subjects (n = 5) were exposed to filtered room air.The WNT/β-catenin signaling in the lungs from C57BL/6 mice was activated via intraperitoneal injection of lithium chloride (LiCl) (200 mg/Kg/BW/day). LiCl is a substituted compound used as activator to evaluate the participation of WNT/β-catenin signaling pathway. Like canonical WNT ligands, LiCl can suppresses the β-catenin destruction complex (Axin/ APC/ CK1/ GSK3β) by inhibiting GSK-3β [® Ultra™ RNA Library Prep Kit for Illumina®  following the manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). cDNA was synthesized by reverse transcription with a reverse transcriptase kit . The library fragments were purified with QiaQuick PCR kits and elution with EB buffer, then end repair, A-tailing, and adapter ligation were implemented. The aimed products were retrieved by agarose gel electrophoresis and PCR was performed, then the library was completed. Transcriptome sequencing was performed on the Illumina Hiseq xTen platform.Total RNA from PBMC of four patients with AECOPD, four patients with stable COPD, and five control subjects was extracted using RNAiso Reagent  according to the manufacturer’s instructions. 2 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext2Ratio|≥ 1 are identified as DEGs. The KEGG  enrichment of DEGs was implemented by the hypergeometric test, in which p-value was adjusted by multiple comparisons as q-value, which less than 0.05 was considered as significant enrichment.Raw Data was processed with Perl scripts to ensure the quality of data used in further analysis. DESeq (v1.16) was used for differential gene expression analysis between two samples with biological replicates using a model based on the negative binomial distribution. The p-value could be assigned to each gene and adjusted by Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with q ≤ 0.05 and|logLGR6, FZD4, and CTNNB1, etc.) and 11 mouse genes  were measured using specific primers  according to the manufacturer’s instructions. Reverse transcription was performed with a reverse transcriptase kit . Real-time PCR analysis of 9 human genes (CTNNB1)  in human plasma was quantified using ELISA Kit according to the manufacturer’s protocol.The concentration of β-catenin (Immunofluorescent staining was used to quantify phosphorylated β-catenin (p-β-catenin) in the PBMC of human and lung tissue of mice. PBMC was isolated according to previous study , and fixThe lung tissue was prepared with RIPA buffer  containing protease inhibitors . The supernatants of lung tissue were run on gels and incubated with relevant antibodies, p-β-catenin . Actin was used as a loading control. Quantification was performed by densitometry and analyzed using Image Studio Lite .Morphological analysis of lung tissue sections was done by H&E staining , following manufacturer recommendations. Histological changes were analyzed under microscopy at 20×.P < 0.05 was considered statistically significant.Data were analyzed using GraphPad Prism 8.0 . The Student’s t-test was used to determine the relationship between two groups. One-way analysis of variance (ANOVA) with Newman-keuls test was used to analyze the statistical differences among three or more groups. Data are expressed as mean ± SEM and 1, % Predicted) in stable COPD and AECOPD were 61.58 ± 11.19 (%) and 45.23 ± 20.97 (%), respectively, with no significant differences. Forced expiratory volume in 1 s/ forced vital capacity  in stable COPD and AECOPD were 57.46 ± 13.47 (%) and 49.10 ± 15.35 (%), respectively, with no significant differences. The control subjects had no respiratory symptoms and no previous chronic airway disease.We used 13 subjects to analyze the DEGs by RNA-seq. Basic demographic characteristics for control subjects, patients with stable COPD or AECOPD are summarized in Table 1 /Predicted in stable COPD and AECOPD were 53.33 ± 14.64 (%) and 54.71 ± 26.09 (%), respectively, with no significant differences. FEV1/FVC in stable COPD and AECOPD was 51.70 ± 11.18 (%) and 49.05 ± 14.58 (%), respectively, with no significant differences. The control subjects had no respiratory symptoms and no previous chronic airway disease.Then, 92 subjects were used for qPCR verification, including 35 control subjects, 27 patients with stable COPD and 30 patients with AECOPD , and partial WNT/β-catenin signaling components including WNT10b, FZD4, and FOSL1 were significantly down-regulated in stable COPD (P < 0.05). Compared with the stable COPD, LEF1 was significantly down-regulated in AECOPD (P < 0.001). Actually, most of the other genes were further down-regulated in AECOPD, although with no significant difference. Besides, the mRNA level of FRAT2 among the three groups has no significant difference.Previous studies have shown that down-regulation of WNT/β-catenin signaling pathway is associated with impaired lung tissue repair –10. The P < 0.01), but no significant difference between the stable COPD and AECOPD   Fig. B, C.Fig.WNT10b, WNT2, LRP6, LGR6, FZD4, CTNNB1, and LEF1, were significantly down-regulated in CE mice (P < 0.05), except FOSL1  . The mRNA of WNT10b was significantly up-regulated in CS mice (P < 0.05). No significant difference was observed for WNT2, LEF1, and FOSL1 between CS mice and air control mice . Importantly, after treatment with LiCl, the level of p-β-catenin was significantly down-regulated in CE + LiCl mice compared with CE + NS mice (P < 0.05)  and was again markedly down-regulated in CE + LiCl mice compared with CE + NS mice (P < 0.05) , as a WNT/β-catenin pathway activator, can inhibit the p-β-catenin through GSK3β. The dephosphorylated β-catenin increased in cytoplasm transfers to the nucleus, which mediates downstream signal transduction and gene expression . Immunof05) Fig. A–D. MoreIn this study, RNA-seq was used to obtain DEGs in the blood of different stages of COPD. Combined with bioinformatics analysis, multiple signaling pathways were found to participate in the pathogenesis of AECOPD, which is consistent with previous studies , 12–14. In the present study, RNA-seq was used to identify DEGs in the blood of patients with stable COPD and AECOPD, as well as control subjects. The number of DEGs obtained by RNA-seq was correlated with the number of biological replicates, the sequencing depth and the fold change. Studies have found that when the number of biological replicates was 3, 5 and 10, the statistical power to detect DEGs of twofold change was 87%, 98% and 100%, respectively . For a c1 in COPD patients [One of the aims of the present study was to identify and annotate the DEGs. Thousands of DEGs were revealed from the PBMC between different groups. And 54 up-regulated genes and 111 down-regulated genes in AECOPD were considered as disease-related genes, according to the characteristics of disease progression from stable to acute exacerbation of COPD. To elucidate the biological processes involved in these genes, we used KEGG database for enrichment analysis and identified 16 signaling pathways that may be related to the pathogenesis of AECOPD. Some previous studies have demonstrated the correlation between the signaling pathways and the pathological process of COPD. PI3K signaling pathway is significantly activated and is associated with increased susceptibility to pulmonary infection in patients with COPD. Cigarette smoke extract can induce Akt degradation, activate the death-signaling pathway, and lead to cell death in normal human lung fibroblasts , 17. ECMpatients , 19, 20.Except for the above signaling pathways, we found that 3 DEGs enriched in WNT signaling pathway. The WNT family includes 19 glycoproteins, which interact with transmembrane receptors to mediate the signal transduction from cytoplasm to nucleus, thus regulating gene expression, causing cytoskeleton rearrangement, and changing cell polarity . The canWNT10b, WNT2), WNT receptors  and downstream signaling molecules  were down regulated in the PBMC of AECOPD patients. Importantly, to our knowledge, it is for the first time to identify that the expression of p-β-catenin protein was up-regulated in the PBMC of AECOPD patients, which suggests its potential value as a novel biomarker of AECOPD.Since RNA-Seq was conducted on a relatively small set of patients to reveal the transcriptome profiles, which provided the general understanding of the AECOPD patients compared with the controls and came out the key targets for the following validations and functional investigations. To validate the critical targets identified from our RNA-Seq analysis, validation examined by a more significant number of patients was performed. In addition, it will aid in concluding relatively reliable conclusions on our findings of these main targets. By qPCR, we found that the mRNA expression of WNT ligands (To further evaluate the impact of WNT/β-catenin signaling pathway during acute airway inflammation and stable COPD, we applied two different animal models, CE and CS exposure, respectively. The CE model has been demonstrated with much higher levels of neutrophilic airway inflammation, mucus hyperproduction, and predominant Th17 response than traditional CS model, somehow mimicking the bronchitis phenotype or AECOPD , 24, 25.Some limitations of this study are also worth discussing. Firstly, although the sample size of RNA-seq in this study was able to make the detection rate of DEGs close to 98%, further studies with a larger sample size were needed to confirm our results. Secondly, it is worth further investigating the expression of WNT/β-catenin pathway-related proteins in sputum of AECOPD patients. Thirdly, whether the value of p-β-catenin as a potential biomarker of AECOPD is better than clinical indicators still needs to be explored, and it should be noted, immunostaining of p-β-catenin is not as convenient as other common methods like ELISA for its clinical application. Finally, except the WNT/β-catenin pathway, the role of other signaling pathways obtained by RNA-seq in the pathogenesis of AECOPD is worth further exploring.In conclusion, we find 16 potential signaling pathways related to the pathogenesis of AECOPD, including PI3K/Akt signaling pathway, MAPK signaling pathway, and ECM- receptor interaction, et al. Remarkable, we demonstrated, for the first time, that the WNT/β-catenin signaling pathway is decreased in patients with AECOPD and CE mouse model. Phosphorylated β-catenin protein is up-regulated in the blood of patients with AECOPD. Therapeutic activation of WNT/β-catenin signaling pathway can alleviate lung inflammation in CE mouse model. As a new potential biomarker of AECOPD, p-β-catenin is expected to provide a new research target for the early diagnosis and treatment of AECOPD.Additional file 1: Table S1. The primers sequence for qPCR of human blood. Table S2. The primers sequence for qPCR of mice lung tissue.


Teeth play essential roles in life. Their development relies on reciprocal interactions between the ectoderm-derived dental epithelium and the underlying neural crest-originated mesenchyme. This odontogenic process serves as a prototype model for the development of ectodermal appendages. In the mouse, developing teeth go through distinct morphological phases that are tightly controlled by epithelial signaling centers. Crucial molecular regulators of odontogenesis include the evolutionarily conserved Wnt, BMP, FGF and sonic hedgehog (Shh) pathways. These signaling modules do not act on their own, but are closely intertwined during tooth development, thereby outlining the path to be taken by specific cell populations including the resident dental stem cells. Recently, pivotal Wnt-Shh interaction and feedback loops have been uncovered during odontogenesis, showing conservation in other developing ectodermal appendages. This review provides an integrated overview of the interplay between canonical Wnt and Shh throughout mouse tooth formation stages, extending from the initiation of dental placode to the fully formed adult tooth. Cdkn1a) and secretion of a variety of signaling molecules, in particular members of the sonic hedgehog (Shh), wingless-type MMTV integration site (Wnt), fibroblast growth factor (FGF), and bone morphogenetic protein (BMP) families, that affect the surrounding epithelium and mesenchyme (Mouse tooth development initiates around embryonic day 11 (E11), when localized epithelial thickenings in the oral ectoderm form and establish the molar and incisor dental placodes at E11.5 . Subsequsenchyme .Cdkn1a-expressing cells, thus comparable to the pEK. Moreover, identical signaling molecules were found in pEK and IK (Eda (Tabby mice), which encodes a transmembrane protein of the tumor necrosis factor (TNF) family and an important regulator of tooth size and shape , termed the initiation knot (IK). First described in incisors  and onlyK and IK . Whereasnd shape , show a nd shape . Interes or longitudinally (incisors), thus extending into and around the underlying mesenchyme and forming the cervical loops (CLs) on both sides of the condensed mesenchyme, now referred to as the dental papilla . WhereasAt the end of the cap stage, the pEK of both incisors and molars undergoes apoptosis . In monoDuring the transition from cap to bell stage, the epithelium folds, and is further compartmentalized into morphologically and functionally distinct inner and outer enamel epithelia . The IEE lies adjacent to the dental papilla, while the OEE is located in the periphery of the future enamel organ . The zonFollowing further invagination and elongation of the CLs into the dental mesenchyme, the crown and root are established, and the dental epithelial and mesenchymal cells further differentiate. These differentiation processes are dependent on intimate epithelial-mesenchymal interplay. Under influence of the IEE, the mesenchyme differentiates into odontoblasts that produce dentin, while the mesenchyme directs the IEE into differentiation toward ameloblasts that produce enamel . The OEEIn molars, the CLs elongate apically into the underlying mesenchyme to form Hertwig’s epithelial root sheath (HERS), a transient bilayer structure involved in epithelial-mesenchymal interactions necessary for tooth root formation and sandwiched in between the dental pulp and follicle  Li et a, 2017. ITooth development is tightly controlled by multiple evolutionarily conserved signaling pathways . In partvia binding of the Lrp co-receptors  . The mameceptors . On the eceptors .Shh is one of the three secreted peptides [in addition to Indian and Desert hedgehog (Hh)] that mediate Hh signaling. In the absence of ligand, the Hh receptor Patched (Ptch) accumulates in the cell membrane and represses the other receptor Smoothened (Smo). Upon binding of Hh ligands to Ptch, the complex is internalized and degraded, thereby terminating Smo inhibition and allowing it to accumulate at the ciliary membrane. This activated Smo leads to the processing and subsequent activation of glioma-associated oncogene (Gli1-3) transcription factors which regulate the downstream expression of Shh target genes. Shh signaling is self-regulating with pathway activity modulating the expression of Ptch and Gli transcription factors as well as of negative regulators such as Hh-interacting protein (Hhip1) and growth-arrest specific 1 (Gas1). Importantly, Sostdc1 is a Shh target gene and at the same time a Wnt inhibitor, supporting a negative feedback loop between Shh and Wnt signaling .Wnt10b is essentially restricted to the dental epithelial thickenings in the oral ectoderm, while Wnt4, Wnt6 and Fzd6 are expressed throughout the oral ectoderm  in the developing dental placodes and the underlying dental mesenchyme, as well as in the dental lamina   .Shh, initially expressed throughout the oral epithelium, becomes restricted to the early dental placodes  spatial transcriptomics; . Together, they form the earliest known signals of tooth initiation  transcription via a TCF/LEF1 binding site in the Cdh1 gene promoter  (Sufu), a major negative regulator of the Shh pathway, from the dental mesenchyme results in delayed bud-to-cap transition of tooth germs due to a defective pEK  . Targetetive pEK . Sufu muansition , 2019.Lrp4 mutant mice phenocopy Sostdc1 mutants, i.e., generating fused molars and supernumerary incisors and molars   exhibiting differing dosages of Shh and/or Wnt/β-catenin signaling, it was established that a properly balanced Shh and Wnt/β-catenin activity is paramount  . Secondlgnaling) . Moreove Sostdc1 . Furthern levels . All finreceptor   display sustained development of R2, a rudimentary tooth bud located distally from the first molar in the diastema  during prior developmental stages  or deletion of Sostdc1 narrows the intercuspal distance and results in supernumerary cusp structures , enamel-producing ameloblasts start to differentiate from the IEE and dentin-generating odontoblasts from the adjacent dental papillary cells, both separated by a basement membrane, through an active interplay. The ameloblast lifecycle consists of four stages, defined by morphological and functional hallmarks.In the first (presecretory) stage, IEE cells (pre-ameloblasts) initiate the cytodifferentiation process by inducing progression of adjacent dental papilla into odontoblasts which deposit a fine layer of pre-dentin at the future dentino-enamel junction DEJ;  Bei, 20. Pre-denDuring the second episode, secretory-stage ameloblasts start to secrete enamel matrix proteins such as amelogenin, enamelin, and ameloblastin to form a protein-rich and soft enamel matrix. Ameloblasts are also equipped with a variety of mineral and bicarbonate transporters to drive mineral growth within the matrix . In addiIn the third (transition) stage, ameloblasts retract their Tomes’ processes, contract again and deposit a new basal lamina. Ultimately, in the fourth and final (maturation) stage, ameloblasts cycle between two morphologies at the enamel surface: ruffle-ended and smooth-ended. The ameloblasts degrade and reabsorb the enamel protein matrix through secretion of proteolytic enzymes , while the enamel crystallites continue to grow and expand. Eventually, the fully formed enamel is highly mineralized with very little amount of protein remaining, thereby forming the hardest substance in the body.Wls), a chaperone protein required for proper cellular secretion of Wnt ligands , as well as for tooth root development (see below)  and decorin  contain TCF/LEF binding sites and have been identified as Wnt/β-catenin target genes during odontoblast differentiation  . In anal-catenin  resulted-catenin . Moreoventiation .Notum severely disrupted formation of both crown and root dentin in molars and of crown dentin in incisors, but did not affect amelogenesis (Smad4), a key regulator of transforming growth factor beta (TGFβ)/BMP signaling, in the dental mesenchyme, resulting in downregulation of the Wnt inhibitors Dkk1 and Sfrp1 and thus Wnt pathway activation, caused impaired odontoblast differentiation and dentinogenesis, but had no effect on ameloblast differentiation  development.Interestingly, Wnt activation by ubiquitous genetic deletion of the secreted Wnt inhibitor ogenesis . In inciogenesis . Similarntiation . MoreoveWls in the Shh-expressing dental epithelial lineage leads to defective ameloblast development, suggested to be largely due to decreased expression of Shh pathway components such as Shh, Gli1, and Ptch1  binding regions in the er cells . Constiter cells . Thus, aer cells ], and MmDifferent from the tooth crown, which is shielded by enamel, tooth root is covered by the much softer cementum. Tooth root development starts once the crown is formed, i.e., when the enamel reaches the future junction with the cementum layer [cemento-enamel junction (CEJ)], the prospective boundary between crown and root  Figure . The CL-via direct contribution to the PDL lineage after undergoing EMT and secretion of signaling molecules  and directly contribute to the cementoblast population . Importaolecules . Some deolecules . These colecules . The fibolecules .Axin2 expression, which is strongly associated with HERS, surrounding apical dental papilla, root odontoblasts and cementocytes , can severely impact crown/root development , a key regulator of extracellular diphosphate levels in mineralizing tissues], β-catenin and TCF/LEF activity is highly increased, thus reinforcing Wnt/β-catenin’s key role in this process , similar to the phenotype observed in humans with WNT10a mutations   .Shh, Ptch1, and Gli1 is gradually restricted to the apical IEE and eventually to HERS during crown-to-root transition, while Ptch1 and Gli1 are also detected in the surrounding mesenchyme , a key transcription factor during root development , Sostdc1 (as well as Dkk1) expression is elevated and Wnt/β-catenin activity reduced resulting in decreased cementum formation and mineralization odontoblasts along HERS, where it may be involved in odontoblast differentiation, based on its capacity to activate expression of dentin sialophosphoprotein (Dspp), a key non-collagenous component of dentin, as observed in vitro in a mesodermal cell line   but strongly persists in the tip of the laCL, where SOX2+ stem cells remain present throughout the animal’s life  can be traced back to the earliest stages of odontogenesis. During establishment of the dental placodes, the PITX22+ cells . The SOXd molars . Around nd laCL) . In the l’s life . In the  and ERM . Throughan teeth .In vivo lineage tracing has identified DESCs as slow-cycling label [genetic or bromodeoxyuridine (BrdU)]-retaining cells. These studies have uncovered numerous stem cell markers in addition to Sox2 such as the Shh components Gli1 (Ptch1 (Lgr5 (Bmi1)  (Lrig1) (Igfbp5)  stem cell populations  , integris Cd49f) , leucine (Lrig1)  and insu(Igfbp5) . Interesulations .Lrig1-expressing cells have been advanced as a subset of Gli1-expressing DESCs, and both Ptch1- and Lgr5-expressing cells have been found to be subsets of Sox2-expressing DESCs, with Lgr5+/Sox2+ cells representing the smaller subpopulation  detected after tamoxifen induction , whereas other subtypes required longer time (∼months) to give rise to descendants of all the epithelial lineages, thus representing more quiescent DESC subtypes   .Sox2, Lrig1, and Lgr5), including rare Sox2+/Lgr5+, Sox2+/Lrig1+, and Sox2+/Lgr5+/Lrig1+ subtypes. In addition, the study identified a novel Acta2+  stem cell population located predominantly in the outer SR and OEE  and in mature ameloblasts (after 1–2 months), thus contributing to all dental epithelial lineages , however, still requiring functional validation, as well as a long-lived Egr+ (early growth response 1)/Fos+ /Sox2– progenitor cell population specifically contributing to OEE cells  and drastically contributed to the proliferating progenitor cell pool as well as the ameloblast layer . Hh signaling constitutes an essential driver for the continued generation of enamel-forming ameloblasts in the mouse incisor, but not for DESC self-renewal   , whereas1 (Ldb1) . The Wnt2+ DESCs . Lineage2+ DESCs . Sc tran2+ DESCs . Given todontoma .+ DESCs remain localized in the dental lamina [also referred to as the successional dental lamina (SDL)] throughout odontogenesis and are competent for successional tooth formation  the second and third molars  are mutually exclusive in the SDL, being conserved throughout the animal kingdom, with activated Wnt/β-catenin in the tip of the SDL where Sox2 expression is excluded  and in mouse SOX2+ RSDL (through expression of constitutively activated β-catenin) results in a supernumerary tooth phenotype  feedback signals from the niche , indicate the presence of a complex interactive system regulating stemness phenotype and function in the incisor epithelium. Of note, some studies have reported conflicting results, indicating activation (and not inhibition) of both canonical and non-canonical Wnt/β-catenin pathway in SOX2+ DESCs from mouse molars and incisors  that SOX2 may exert an inhibitory effect on Wnt/β-catenin signaling, further underwrites the notion that Wnt/β-catenin inhibition is essential to keep the continuously renewing incisor DESCs in check . Intrigu context . The finincisors .Together with the knowledge that, at previous developmental stages, both Wnt (hyper)activation and inhibition severely impair odontogenesis, and the fact that we currently lack data on the effect of Wnt inhibition on DESCs, it remains premature to draw firm conclusions on the role of Wnt/β-catenin signaling in DESCs.It was long thought that the complete dental mesenchyme is solely derived from the cranial neural crest-derived ectomesenchyme . HoweverGli1+ DMSCs surround the incisor arteries and neurovascular bundle, and sensory neuron-derived Shh was shown to be a key regulator of DMSC homeostasis   . Moreovechanisms .Gli1+ DMSCs which activates Wnt/β-catenin signaling  in TA cells via the IGF1 receptor (IGF1R), resulting in TA cell proliferation  -like orphan receptor 2] from Gli1+ DMSCs both resulted in a reduction of Gli1+ DMSCs and label-retaining cells, as well as disturbed dentinogenesis  is secreted by feration . Deletior Wnt5a) . Deletioogenesis . Of noteed roots . Togethehe tooth .It is clear that Wnt/β-catenin and Shh signaling must be tightly balanced throughout the different stages of mouse tooth development. Both inactivation and hyperactivation of each one of these pathways lead to dental aberrations resulting in dysmorphic, missing or supernumerary teeth. Unfortunately, studies supporting these in-depth mouse-based findings in humans are sparse and are typically limited to gene and protein expression analysis, lacking mechanistic insight. However, also in humans, Wnt/β-catenin-activating and -inhibiting mutations, as well as mutations in components of the Shh pathway, have been linked to various tooth phenotypes , therebyOur integrative review may also fertilize the field of dental tissue engineering and regeneration in which the discussed Wnt/β-catenin-Shh principles can be applied to achieve biomimetic structures . However, as shown by the numerous studies discussed in the review, both pathways are very precisely regulated in time and space which will make clinical translation very challenging. Finally, given the highly similar development of tooth and other ectoderm-derived tissues , we expect that aspects and principles highlighted and discussed here will also be valuable for these other fields.FH collected all the information, wrote the manuscript, and co-designed the figures. LH designed the figures and critically revised the manuscript. IL critically revised the manuscript. AB co-wrote and critically revised the manuscript. HV actively co-wrote, critically revised, and finalized the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We also consider the role that systemic activation of the CX3CL1-CX3CR1 axis in renal disease contributes to CKD-associated cardiovascular disease. Based on this evidence, we highlight the potential for therapies targeting CX3CL1 or CX3CR1 to benefit people living with CKD.Innate immune cells are key contributors to kidney inflammation and fibrosis. Infiltration of the renal parenchyma by innate immune cells is governed by multiple signalling pathways. Since the discovery of the chemokine fractalkine (CX3CL1) and its receptor, CX3CR1 over twenty years ago, a wealth of evidence has emerged linking CX3CL1-CX3CR1 signalling to renal pathologies in both acute and chronic kidney diseases (CKD). However, despite the extent of data indicating a pathogenic role for this pathway in kidney disease and its complications, no human trials of targeted therapeutic agents have been reported. Although acute autoimmune kidney disease is often successfully treated with immunomodulatory medications, there is a notable lack of treatment options for patients with progressive fibrotic CKD. In this article we revisit the CX3CL1-CX3CR1 axis and its functional roles. Furthermore we review the accumulating evidence that CX3CL1-CX3CR1 interactions mediate important events in the intra-renal pathophysiology of CKD progression, particularly  We also consider the role that systemic activation of the CX3CL1-CX3CR1 axis in renal disease contributes to CKD-associated cardiovascular disease. Based on this evidence, we highlight the potential for therapies targeting CX3CL1 or CX3CR1 to benefit people living with CKD.Innate immune cells are key contributors to kidney inflammation and fibrosis. Infiltration of the renal parenchyma by innate immune cells is governed by multiple signalling pathways. Since the discovery of the chemokine fractalkine (CX3CL1) and its receptor, CX3CR1 over twenty years ago, a wealth of evidence has emerged linking CX3CL1-CX3CR1 signalling to renal pathologies in both acute and chronic kidney diseases (CKD). However, despite the extent of data indicating a pathogenic role for this pathway in kidney disease and its complications, no human trials of targeted therapeutic agents have been reported. Although acute autoimmune kidney disease is often successfully treated with immunomodulatory medications, there is a notable lack of treatment options for patients with progressive fibrotic CKD. In this article we revisit the CX3CL1-CX3CR1 axis and its functional roles. Furthermore we review the accumulating evidence that CX3CL1-CX3CR1 interactions mediate important events in the intra-renal pathophysiology of CKD progression, particularly Drosophilia melanogaster and this term has therefore not remained in extensive use . Both groups identified a cDNA clone which corresponded to a large protein containing a sequence of hydrophobic residues suggestive of a transmembrane domain. Pan et al. reported that messenger RNA for CX3CL1 was identified in most human tissues with the notable exception of peripheral blood cells but corresponding analyses of protein expression were not reported in this paper. Human endothelial and epithelial cell lines transfected with the coding region of CX3CL1 were found to express CX3CL1 on the cell surface, consistent the hypothesis that the newly discovered protein was membrane-bound. Bazan et al. showed that CX3CL1 could also be released into cell culture supernatants. The authors described disparate results for chemotactic assays with CX3CL1 which was reported to induce in vitro CX3CL1-induced chemotaxis  or the pro-inflammatory cytokines tumour necrosis factor-alpha (TNF-α) and interleukin-1 (IL-1)  . It was 1 (IL-1) .In relation to the kidney, the signalling pathways involved in TNF-α−mediated CX3CL1 expression within mesangial cells were first dissected by Chen et al. . Mesangigfp/+ mice to demonstrate that Ly6Clo monocytes crawl on the luminal surface of blood vessels and extensively patrol the endothelial surface , a chemokine analogue produced by human herpes virus-8, in experimental anti-GBM glomerulonephritis in Wistar-Kyoto rats . This stin vitro work demonstrated that proximal tubular epithelial cells (PTEC) upregulate CX3CL1 expression after TNF-α stimulation and that this upregulation is capable of supporting adhesion of either THP-1 cells or ex vivo natural killer (NK) cells  or with acute transplant rejection, using either normal tissue obtained at the time of tumour nephrectomy or biopsies showing minimal change disease as negative controls. At gene expression level, mRNA for CX3CL1 was increased in the glomeruli of patients with acute AAV and increased to a lesser extent in patients with acute transplant rejection. In the tubulointerstitium, mRNA for CX3CL1 was increased in both groups compared to control tissue but to a greater degree in transplant rejection. At protein level, CX3CL1 expression, determined by immunohistochemistry, was higher in biopsies with renal inflammation. Serial section analysis demonstrated that CX3CL1 positivity was localised to areas of T-cell or macrophage infiltrates . RelatedK) cells .+ monocyte/macrophages. CX3CR1 was highly expressed and, similarly, corresponded to the location of monocyte/macrophage or T-cell infiltration. Of note, these and other early studies in animal models and human kidney samples focused primarily on expression patterns and associations of the CX3CL1-CX3CR1 axis in acute settings. Although clearly of potential mechanistic importance in the intense inflammation associated with acute kidney diseases, insights into the expression and functional significance of CX3CL1/CX3CR1 signalling in CKD was lacking. For the remainder of this article, we focus on subsequent evidence that CX3CL1 interaction with CX3CR1 is of distinct pathophysiological importance to the progression of fibrosis and impaired renal function in CKD.Segerer et al. further studied the expression of CX3CR1 by infiltrating cells in human kidney disease using similar control samples . Serial in-vitro exposure to high concentrations of glucose, TNF-α or advanced glycation end-products (AGEs). These data demonstrate that CX3CL1 expression is induced in DN and may be modulated by therapeutic strategies already in use -RNA-mediated reduction of CX3CL1 expression attenuated these changes. These data highlight that CX3CL1 signalling via CX3CR1 contributes to macrophage infiltration and renal fibrosis in mouse models of DN and that mesangial cells both produce and respond to CX3CL1. Such findings related to mesangial cells in DN are consistent with reports in an acute glomerulonephritis model, indicating that CX3CL1, produced by mesangial cells plays a role in monocyte recruitment to the kidney . Kikuchi et al.  showed, y in use . The stry in use . Renal fe kidney  and in i fashion .-/- mice. However, collagen deposition was reduced in the CX3CR1-/- animals, as was intra-renal macrophage infiltration while lymphocyte and neutrophil numbers were unaffected. Overall, this work was consistent with a role for CX3CR1-dependent macrophage-mediated fibrosis following IRI. To account for the possibility of developmental differences in macrophage lineages in CX3CR1-/- mice, Furuichi et al. also used pharmacological blockade by administering a CX3CR1-neutralising antibody following IRI. Animals treated with the blocking antibody had significantly reduced post-IRI renal fibrosis compared to controls  model. In this study, IRI resulted in increased expression of CX3CL1 protein in the kidney but did not demonstrate a corresponding change in mRNA levels. Furthermore, the location of CX3CL1 expression within the kidneys changed, being limited to endothelial cells in sham-operated animals while also detected on tubular epithelial cells and in association with infiltrating cells following IRI. Renal expression of CX3CR1 was increased at both protein and mRNA levels following IRI with protein expression mostly observed in association with infiltrating cells. The lack of correlation between mRNA and protein expression may simply reflect a technical limitation given the correlation between mRNA and protein level expression described by other authors. The severity of acute tubular necrosis following IRI did not differ between wild type (WT) and CX3CR1controls . OverallOh et al. also demonstrated increased CX3CL1 expression after IRI, using immunoblotting and immunohistochemistry. In this study, blockade of CX3CR1 was associated with protection against acute renal impairment, as assessed by histology and blood urea and creatinine levels, and with reduced macrophage infiltration . Another+ macrophage infiltration of the kidneys with interstitial fibrosis and these changes, along with intra-renal TGFβ1 level, were ameliorated in CX3CR1-deficient mice  and Ly6Clo/CX3CR1hi  monocytes are recruited to the kidney after obstruction, albeit within different time-frames. The authors reported that, while Ly6Chi monocytes are the major producers of the proinflammatory cytokines TNF-α and IL-1β, Ly6Clo monocytes produce more of the pro-fibrotic cytokines TGF-β and platelet-derived growth factor (PDGF). Similar to other reported models, renal fibrosis was attenuated in CX3CR1-deficient mice after UUO. Further analyses demonstrated a specific lack of nonclassical monocyte accumulation within the obstructed kidneys of CX3CR1-deficient mice. Interestingly, this deficit was due to increased apoptosis rather than impaired recruitment, in keeping with the role of CX3CR1 signalling in cell survival outlined  model, Peng et al. investigated the role of CX3CL1 in recruitment of different monocyte subsets into the obstructed kidney . This mooutlined . Severale damage . More sp+ (T-cell) infiltrates and S100A4, a protein expressed by fibroblasts and associated with epithelial-to-mesenchymal transition  and dendritic cells (CD11c+) also express CX3CR1. In keeping with this, human renal fibroblast cell lines also express CX3CR1 and migrate towards a CX3CL1 gradient suggesting a functional role for the CX3CL1- CX3CR1 in promoting renal fibrogenesis through fibroblast recruitment  and for their pro-fibrotic effects in human kidneys. For example, Kassianos et al. used flow cytometric analysis of leukocytes within human fibrotic and non-fibrotic kidneys to demonstrate that CD1car cells . In vitrOf potential relevance to these tissue-based observations, a number of groups have investigated the association between single nucleotide polymorphisms (SNPs) within the CX3CR1 gene and the presence of CKD or end stage kidney failure (ESKF). One such polymorphism is V249I. The frequency of the I allele and the II genotype were found to be increased in ESKF by two groups , 69. In It is well established that atherosclerosis is accelerated and cardiovascular morbidity and mortality increased among people with CKD , 74. Onehi intermediate and nonclassical monocytes. Increased CX3CR1 expression has been found in the arterial walls of people with CKD undergoing renal transplantation  cell polarization within atherosclerotic plaques and that this was necessary for the development of CKD-driven atherosclerosis. Adoptive transfer of CX3CR1+ T-cells restored atherosclerosis progression in mice with renal impairment, suggesting a specific contribution of CX3CR1+ T-cells to atherosclerosis in setting. This is in keeping with the study of Yadav et al. which an association between intima-medial thickening of the carotid artery and CX3CR1+/CD4+ T-cells in patients with CKD  cells also showed that treatment with angiotensin increased CX3CR1 expression and that this was reduced by concomitant treatment with losartan. The induced CX3CR1 was functional, leading to increased migration of angiotensin-treated MOVAS cells compared to untreated cells and to cells treated with angiotensin + losartan  has been reported and may be a mechanism by which they contribute to renal protection in CKD. This was highlighted in the animal studies above regarding RAS blockade . Mesench effects . Studies effects , 92. The effects . Another effects + cell lines to CX3CL1 in vitro. Furthermore, the authors demonstrated that in vivo CX3CR1 antagonism with F1 led to reduced leukocyte recruitment in a mouse model of thioglycolate induced peritonitis. Neutralising antibodies are commonly used in vitro or in vivo to establish the pathogenic significance of a biological pathway and several experimental studies described in previous sections have utilized anti-CX3CR1 antibodies  Programme, supported by the Wellcome Trust and the Health Research Board (Grant Number 203930/B/16/Z), the Health Service Executive National Doctors Training and Planning and the Health and Social Care, Research and Development Division, Northern Ireland. MG was supported by grants from the European Commission [Horizon 2020 Collaborative Health Project NEPHSTROM (grant number 634086) and FP7 Collaborative Health Project VISICORT (grant number 602470)] and from Science Foundation Ireland [CÚRAM Research Centre (grant number 13/RC/2073)] and by the European Regional Development Fund.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Organic wastes represent an increasing pollution problem due to the exponential growth of their presence in the waste stream. Among these, waste flour cannot be easily reused by transforming it into high-value-added products. Another major problem is represented by epoxy-based thermosets, which have wide use but also poor recyclability. The object of the present paper is, therefore, to analyze both of these problems and come up with innovative solutions. Indeed, we propose a completely new approach, aimed at reusing the organic waste flour, by converting it into high-value epoxy-based thermosets that could be fully recycled into a reusable plastic matrix when added to the waste epoxy-based thermosets. Throughout the research activity, the organic waste was transformed into an epoxidized prepolymer, which was then mixed with a bio-based monomer cured with a cleavable ammine. The latter reactant was based on Recyclamine™ by Connora Technologies, and in this paper, we demonstrate that this original approach could work with the synthetized epoxy prepolymers derived from the waste flour. The cured epoxies were fully characterized in terms of their thermal, rheological, and flexural properties. The results obtained showed optimal recyclability of the new resin developed. The use of epoxy-based composites is widely accepted in different fields. In the aerospace sector, epoxy resins are used because of its low cost and suitability for producing large structures. Recent studies reported novel technologies for producing enhanced composites for the aeronautical field. Zotti et al.  developeRybicka et al.  describe2O2/acetone mixed solution heated between 60 and 150 °C. The degraded products were analyzed, showing mainly bisphenol A and its derivatives, such as phenol derivatives, which are generated during the decomposition of the epoxy network. Wang et al. [3COOH solution selectively cleaved the C−N bond, allowing for obtaining oligomers from the epoxy resins. These papers demonstrated the possibility to cleave epoxy networks, but the reuse strategies for the recovered oligomers were not assessed.Chemical recycling is emerging as a viable approach to recover clean and undamaged reinforcing fibers while allowing for the recovery of monomers or oligomers that can be reused. Xu et al.  presenteg et al.  developeBack in 2012, the company Connora Technologies presented a novel class of ammine reagents named Recyclamine™, designed to be selectively cleaved in an aqueous solution with acetic acid, using mild conditions , thus yielding clean reinforcing fibers and a reusable thermoplastic matrix from the epoxy network. The Recyclamine™ reactants were characterized in terms of their aging resistance , and mix12 t, and it is expected to rise up to 2.2 × 1012 t per year by 2025 , 2017).The use of petroleum-based raw materials for the synthesis of epoxy monomers is another limit of the epoxy-based composites used nowadays. To overcome this limit, several researchers developed bio-based epoxy monomers synthetized from vegetable oils , naturalThe huge amount of organic waste is becoming an increasing issue for the waste management of modern cities, while the technology developed by Esposito Corcione et al. ,19 can tIn the present paper, epoxidized monomers synthetized from waste flour were mixed with bio-based epoxy precursors; then the epoxy blends obtained were cured using a cleavable ammine to develop a fully recyclable bio-based epoxy thermoset. The resins were fully characterized in terms of thermal and mechanical properties to optimize their final performances. The optimized formulation was recycled using only an acidic aqueous solution under mild conditions to demonstrate the possibility to recover a reusable plastic from the cured epoxy resin.Polar Bear , a bio-based epoxy system designed specifically for the composite processing.Recyclamine R-101 , is a recyclable epoxy cure agent for composite manufacturing. Polar Bear and Recyclamine R-101 are both liquid at room temperature.2, was used for waste flour treatment.Waste flour (WF) was obtained from the processing waste of the pasta factories. Epoxidized waste flour (EWF) was obtained by contemporary exposure to UV radiations and ozone for 5 h following the method reported in a previous work  and in a−1, 128 scans, and 4 cm−1 of resolution by using a germanium round crystal window. The spectra acquisition was carried out before and after the UV/ozone and after curing.FTIR analysis, performed with an FTIR , was used to assess the presence of epoxy groups after UV/ozone exposure. Infrared spectra were recorded in the wavelength range between 400 and 4000 cmAs reported in our previous article , the epoDifferent samples were produced by mixing the Polar Bear resin with specific amounts of epoxidized waste flour and Recyclamine R-101. First, different blends were produced by varying the ratio between Polar Bear and waste flour, keeping a constant amine content . The iniAfter choosing the optimal ratio between Polar Bear and waste flour, the amine content was varied by adding different phr’s of Recyclamine R-101  in orderAll the mixtures were degassed by applying vacuum at room temperature, then pouring in silicon molds and curing for 24 h at room temperature. The cure was followed by a postcure process for 2 h in a static oven. The postcure temperature was varied between 120, 150, and 160 °C, as shown in TM is an epoxy hardener developed by Connora Technologies that allows for obtaining a recyclable thermoset that can be converted into a meltable thermoplastic. Its recyclability key factor is based on the presence of amino-acid-cleavable groups that allow the cleavage of the crosslink points of the epoxy-cured network [Recyclamine network .The resin system selected for the recycling trials was EWF50_A15, which showed the best properties among all the resin systems tested, as it will be shown in the paper. The chemical recycling procedure is schematically drawn in 3COOH) at 80 °C for 1 h. The obtained mixture was rotoevaporated at 60 °C at a pressure ranging between 110 and 60 mbar and at a rotation speed of 3500 rpm. The distilled acetic acid was stocked, being reusable for a new chemical recycling treatment, while the concentrated solution obtained (about 75 mL) was neutralized in 300 mL of 50 %vol ammonium hydroxide solution. During this phase, a whitish compound started to precipitate, which was the recycled thermoplastic of interest. Then, the solution containing the precipitate was centrifuged for 5 min at 3000 rpm and, at the end, the supernatant removed. The solid phase at the bottom of the test tube was recovered and washed in ionized water to remove any residual traces of acetic acid and ammonium hydroxide solutions. Eventually, it was dried in a vacuum stove for 24 h at 50 °C. The thermoplastic obtained is a brown compact solid shown in the panel of A sample of 5 g of the epoxy system EWF50_A15 was solubilized in 300 mL of 25 %vol acid acetic solution , and frequency (1 Hz). The tests consisted of a temperature ramp from room temperature to 130 °C.DSC analysis was performed on a Mettler Toledo 622 differential scanning calorimeter (DSC). Samples were heated from 25 to 250 °C at 20 °C/min in air.The thermal stability of the films was assessed by TGA, with a TA Instruments SDT Q600 . The samples were heated in an alumina holder from 20 to 600 °C at a heating rate of 10 °C/min under air atmosphere; three measurements were performed on each sample.The flexural properties of each cured sample were measured using a dynamometer, Lloyd LR5K, according to ASTM D790  (three points bending with the specimen dimension: 80 mm × 10 mm × 4 mm). Five replicates were performed on each sample.−1; the samples were cooled down naturally and reheated up to 180 °C at 5 °C min−1. Similar techniques were also reported by Carlier et al. [Dynamic mechanical analysis was carried out on a dynamic mechanical thermal analyzer  by single cantilever geometry. The recycled polymers, after 1 day drying at 40 °C, were tested in their powder form using the pocket DMA approach, a technique used for testing powders in the pharmaceutical field  and for r et al.  for orgap-value. The p-value is the probability of the test statistic to be at least as extreme as the one observed, given that the null hypothesis is true. A small p-value is an indication that the null hypothesis is false. It is good practice to decide in advance of the test how small a p-value is required to reject the test, that is, to choose a significance level, α, for the test. For example, it can be decided to reject the null hypothesis if the test statistic exceeds the critical value (for α = 0.05) or, analogously, to reject the null hypothesis if the p-value is smaller than 0.05.Analysis of variance (ANOVA) was used to highlight the statistical significance of different parameters, as the different amounts of EWF and Polar resin, on the mechanic properties. For this purpose, the F value, which is defined as the ratio of the variation between sample means to the variation within the samples, was calculated from the measured data. Then, being “a” the number of levels of the variance factor and “n” the number of tests for each level, the critical F value, FCV, α), can be estimated. FCV represents the value of F distribution with degrees of freedom (a-1) and a(n-1), which, at a confidence level, α, corresponds to the null hypothesis . Therefore, F < FCV indicates that the population means are equivalent, whereas F > FCV indicates that the population means are significantly different. Another quantitative measure for reporting the result of a test of hypothesis is the −1 assigned to –CH2 bending and –COO stretch, 1075, 1048 cm−1 and 1022 cm−1 assigned to the crystalline and amorphous regions of starch, respectively, and 1164 cm−1 assigned to vibrations of the glucosidic C–O–C bond and the whole glucose ring that can present different modes of vibrations and bending conformations.The FTIR spectra on waste flour are reported in −1. This indicates that the treatment allows for obtaining epoxidized waste flour (EWF).A contemporary exposure of waste flour to UV/ozone radiation involves the appearance of the typical signals of the epoxy ring at 1260, 890, and 827 cm−1 can be again attributed to the bending vibration of residual glucose.After curing of the waste flour in the presence of the amine, the peaks due to the epoxy rings disappear, confirming epoxy curing reaction. The strong peak at 1075 cmThe viscosity curves of the commercial system and its blend with 50% of EWF are reported in However, as shown in In order to choose the optimal postcure temperature, DSC scans were carried out on systems postcured at three different temperatures  after the cure at room temperature for 24 h.The DSC curves in the temperature range between 30 and 170 °C, reported in With the aim of developing an epoxy system that could find use in different industrial applications, a glass transition at around 140–150 °C is high enough to guarantee good performances and stability of properties. On the other hand, the lower Tg represents a limit of the developed system.Therefore, our further efforts were aimed at increasing the lower Tg signal. In the following analysis, we will only focus on the lower Tg, neglecting potential changes of the higher Tg.In Thermogravimetric analyses, shown in In p-value, which was then compared with the confidence level, α = 0.05. According to ANOVA, p > α corresponds to the null hypothesis , whereas p < α indicates that the population means were significantly different. For flexural strength and modulus, p = 0.0013 and p = 0.01, respectively, indicate the statistically relevant effect of the addition of EWF on the corresponding property of the blend. In contrast, for strain at break, p = 0.39 indicates that the effect of EWF was not statistically significant. This was confirmed by one-way analysis of variance (ANOVA). Considering the amount of EWF as the source of variation, with four levels, and three degrees of freedom, its significance on flexural modulus, strength, and strain at break was tested by calculating the F value as the ratio of the variance between the means to the variance of the experimental error. The F value was then used in order to calculate the corresponding p = 0.85 and p = 0.50, respectively, indicates that the corresponding mechanical properties were not influenced by the amount of EWF in the tested range of compositions. This is consistent with the fact that, in On the other hand, limiting the ANOVA to the blends, and therefore neglecting the sample of commercial epoxy, with three levels, and two degrees of freedom, for flexural strength and modulus, p = 2.1 × 10−11, p = 8.3 × 10−6, and p = 8.2 × 10−4, respectively, indicate that the amount of amine had a statistically significant effect on each of the mechanical properties. Referring to the data in In p = 0.43, p = 0.8, and p = 0.61 highlight that the two samples were not statistically different. This is consistent with the observation from DSC analysis, which showed that, for the sample at 15 phr amine, a relatively high Tg was found, which was only 15 °C lower than that of the commercial system. All the ANOVA findings are summarized in In addition, ANOVA was used to compare only the commercial system with the blend at 15 phr of ammine. With two levels, and one degree of freedom, for flexural strength, modulus, and strain at break, The effect of the lower Tg of the blends on the flexural strength and modulus is also highlighted in The recycled polymer obtained from the chemical recycling of the epoxy network was characterized by DMA and DSC. The tan δ vs. temperature was measured at three different frequencies: 1, 10, and 30 Hz. The results  clearly An exemplary bio-based epoxy resin formulation showing full recyclability was presented in this paper. Two different epoxy resin precursors were mixed with a cleavable ammine: a commercial bio-based epoxy and a novel epoxidized waste flour. The latter chemical reactant was obtained using a green approach based on the use of a UV/ozone treatment. Mixing the two epoxy monomers allowed for obtaining formulations easy to mix at room temperature showing low viscosity in the unreacted state that could be cured at temperatures varying between 120, 150, and 160 °C. The glass transition temperature of the epoxy formulation was optimized by varying the ratio between the two epoxy monomers and the amount of the cleavable amine. When the ammine was added at 22 phr, the glass transition temperature was fixed at about 54 °C with no significant change varying the bio-based epoxy content. However, reducing the ammine content down to 15 phr improved the glass transition temperature up to 76.5 °C, while increasing the ammine content to 30 phr reduced the Tg down to 45.4 °C. Similar results were obtained when considering the mechanical properties with the formulation cured with 15 phr ammine content. This hardener amount was chosen as optimal since a further reduction of the ammine content resulted in high viscosity systems.The resin developed showed optimal recyclability when treated with an acidic solution under mild conditions , obtaining a plastic material with a Tg of 69 °C by DMA and about 50 °C by DSC. The plastic material obtained from the recycling is unique compared with those obtained before as it was derived from a valuable thermoset formulated using 50 wt% of an epoxy monomer synthetized from waste flours.The use of materials derived from organic waste as valuable thermoset prepolymer, which, after curing, can also be recycled at the end of their life, can truly revolutionize the recycling strategies for organic wastes, avoiding negative impacts on the environment. At the same time, the possibility to recover a high percentage  of this product into a reusable plastic matrix can guarantee the respect of the environment. However, more efforts will be required to further optimize the thermal and mechanical properties of the cured thermosets. These efforts should be focused on the synthesis of reactants, leading to a balanced stoichiometry and with a stiffer structure to improve the glass transition temperature and the mechanical properties.Esposito Corcione, C., Greco, A., Visconti, P., Striani, R., Ferrari, F., 2019. Process for the production of bio-resins and bio-resins thus obtained. 102019000016151. IT.


The kinetics of the corrosion reactions were evaluated using potentiodynamic polarization (PDP) tests. In addition, electrochemical impedance spectroscopy (EIS) tests were carried out at open circuit potentials (OCPs). It was demonstrated that the glass fiber-reinforced polymer coating system offered the best protection against corrosion, with a high stability against deterioration when compared with epoxy and Kevlar fiber-reinforced polymer coating systems. Scanning electron microscopy images revealed cracks and deteriorated embedded fibers due to acid attack, sustained/assisted by the diffusion of the corrosion species.Corrosion remains one of the major and most costly challenges faced by the steel industry. Various fiber-reinforced polymer coating systems have been proposed to protect metallic piping distribution networks against corrosion. Despite increasing interest among scientific and industrial communities, there is only limited predictive capability for selecting the optimum composite system for a given corrosive condition. In this study, we present a comprehensive evaluation of the electrochemical behavior of two different fiber-reinforced polymer composite systems against the corrosion of carbon steel pipes under a wide range of acidic and corrosive solutions. The composites were made of glass and Kevlar fibers with an epoxy resin matrix and were subjected to corrosive solutions of 0.5 M NaCl, 0.5 M HCl, and 0.5 M H Corrosion is considered to be the leading cause of failure in pipelines transporting gases and liquids . The priFurthermore, the degradation influences the composite components—namely, the matrix, the fibers, and the interface ,34. For To highlight the importance of this study, the electrochemical behavior of different coatings and FRP systems as anti-corrosive coatings for carbon steel (CS) in different solutions and corrosive mediums reported in recent literature are summarized in Cleaned EN10130 carbon steel sheet specimens with a thickness of 1.5 mm, length of 8 cm, and width of 4 cm were used as the substrate for epoxy coatings and FRP layers. The chemical composition of the EN10130 carbon steel sheet is given in Four types of specimens were prepared using the hand layup process, as shown in Potentiodynamic polarization testing was used to evaluate the kinetics of the corrosion reactions and the corrosion rates and potentials concerning the anodic reactions, the cathodic reactions, and passivity . The pot2SO4. The open-circuit potentials were monitored  the nature of the interfacial interactions on the protective epoxy-based systems, across them, and beneath them at the steel/layer interface; (ii) the mechanisms of corrosion and mass transport; (iii) the deterioration of the epoxy or FRP; (iv) the distribution of the anodic and cathodic reactions with time, and concerning the environmental conditions and physical properties of the layer system, and in comparison to the response of a steel substrate without protection. In addition, the results were used to corroborate the kinetic results of the OCP and potentiodynamic polarization. Regardless of the solution (medium-pH (0.5 M NaCl) or low-pH (0.5 M HCl and 0.5 M H2SO4)), the anodic reaction involves the dissolution of the steel substrate, forming ferrous ions. Those ions migrate and accumulate first within possible pores reacting with hydroxide and O2 to form iron hydroxide (Fe(OH)2), iron carbonate (FeCO3), and iron oxides (Fe2O3 and Fe3O4) [2SO4 solutions than in the NaCl solutions. The anodic dissolution with time becomes high as a result.The electrochemical impedance spectroscopy response was monitored across periods of 5, 10, 15, 20, and 25 h of free immersion in 0.5 M NaCl, 0.5 M HCl, and 0.5 M Hd Fe3O4) . Howeverω is the angular frequency and n is the CPE exponent.Nyquist and Bode plots were used to elucidate the interactions and their physical effects on the substrate and protective layer with time. It was determined that in all of the solutions, regardless of the physical conditions at the interface (bare surface or coated), the interactions proceeded with the exact mechanism and in a time-independent fashion. The equivalent circuit of the configuration, {R(Q(R(QR)))}, was correctly fitted to the experimental data across the entire frequency range, from the high frequency (charge transfer and surface interface) to the low frequency (epoxy layer and bulk solution). The suitability of the equivalent circuit to the bare steel surface related interestingly to the developing corrosion products, which with time achieved a significance similar to that of the original protective layer. To account for the heterogeneities, the capacitance of the double layer, corrosion products, and coating system was calculated as a constant phase element (CPE), with admittance expressed as .(1)Y = The equivalent electrical circuits were fitted using the Gamry Echem Analyst software. 2 to 16 MΩ·cm2, indefinitely with time. The admittance did not change, at nearly 190 × 10−9 S.sn/cm2, with exponent n values of nearly 0.7 at the double layer. This indicated the significance of the passive film in enhancing the protectiveness, in a pseudocapacitive manner, with pores continuously filled with protective film. The Kevlar/epoxy system, in agreement with the OCP and polarization data, showed the lowest corrosion resistance, which was decreasing with time, from nearly 6.5 MΩ·cm2 to 30 kΩ·cm2, with a higher admittance (that did not change with time) of nearly 150 × 10−12 S.sn/cm2. This underlines the deterioration of the Kevlar-based epoxy layer, thinning for greater permeation of the chloride and hydroxyl ions to accelerate the deterioration of the layer and dissolution of the substrate, leading to the formation of porous passive films. The epoxy-only layer exhibited a continuously decreasing corrosion resistance before it increased at the end of testing, indicating its reliability against corrosion in the long run without interference from glass or Kevlar. It had the lowest permeability value as it was across the double layer, and the epoxy layer was more capacitive than the glass/epoxy and Kevlar/epoxy systems. From the Bode diagrams, the phase peak values did not change, nor did they shift in frequency with time, indicating the single-phase constant response and similarity in the governing mechanisms of interactions, similar to the findings in [The EIS response represented by the Nyquist and Bode profiles in the 0.5 M NaCl solution is shown in dings in ,47. The 2SO4 solutions shown in 2, although showing a steady decrease with time to as low as 0.2 MΩ·cm2. The Kevlar/epoxy system, on the contrary, had less resistance. However, it increased with time from 45 kΩ·cm2 to 0.5 MΩ·cm2 and to 4 MΩ·cm2, underlining the significance of Kevlar in promoting the formation of passive films that, with time, decelerate dissolution and prove the significance of glass in stabilizing the epoxy system that initially protects the system. Interestingly, the epoxy-only system did not show a net change in resistance; it increased only after 10 h to decrease steadily back to nearly the initial resistance values of the charge transfer and protective layer at nearly 1.2 MΩ·cm2. Thus, the epoxy-only layer exhibited comparable reliability to that of the glass/epoxy layer against corrosion during the total time of the experiment. Furthermore, the phase peaks in The EIS response underwent the exact same governing mechanism of interfacial interactions in the acidic solutions as the one that governed the coated systems in the NaCl solutions (of mild pH). The dominance and rate of the cathodic reactions for the direct reduction of hydrogen protons onto and across the diffusion channels of the coated systems were higher at the beginning of the immersion. However, with time, this incentivized the anodic dissolution, passivation, and partial weakening or partial deterioration of the coating layer. The Nyquist profiles of the 0.5 M HCl and 0.5 M H2SO4 solutions, the hydrogen generation was more vigorous. However, for a glass/epoxy system, it incentivized the anodic reactions to form protective films at rates high enough to surpass the disruption from the hydrogen generation, precipitate and decelerate dissolution, and promote the resistance of the layer. The resistance was higher than that of the HCl solutions, and it increased with time from nearly 3 MΩ·cm2 to 10 MΩ·cm2. In the Kevlar/epoxy system, however, the resistance was expectedly lower, and it decreased with time. The epoxy deteriorated with Kevlar fibers, not facilitating precipitation of the influential passive films on the heavily attacked surface with a system resistance of nearly 0.1 MΩ·cm2. Its Bode phase peaks, as shown in 2. The physical properties of the reinforcing elements of glass and Kevlar, if associated with the adherence of the passive films in acidic conditions in future studies, could be used to better predict the reliability of coating systems in the long run.In H2SO4 solutions, slight localized changes in color were observed on the fibers. Kevlar samples showed deterioration in epoxy, and the appearance of pores and the color change was mostly in the H2SO4 solution. The epoxy-only system showed evidence of minor pores acting as diffusion channels, but it showed stability regardless of the environmental conditions. Comparably, the steel samples were heavily attacked, most severely in the acidic solutions.The SEM images presented in 2SO4. Generally, the GFRP coating system offered the best protection, with a high stability against deterioration, promoted by the glass fiber and epoxy compared with the epoxy and KFRP coating systems. The following conclusions can be drawn:2SO4 solution comparable to an exposed substrate after nearly 50 ks.As demonstrated by the open circuit potential results, the Kevlar/epoxy system interestingly showed deterioration in the HThe corrosion rates for the coated substrates were 1 million to 10 million times less than that of the exposed substrates, as reported by the PDP tests.The equivalent circuit of the configuration, {R(Q(R(QR)))}, was correctly fitted to the experimental data across the entire frequency range.The peaks of the glass/epoxy and epoxy coating systems in the Bode diagrams indicated reliability against activity at both the double and coating layers, appearing at frequencies two orders of magnitude higher than those of the Kevlar/epoxy system.The resistance of the glass/epoxy system in the HCl solution was the highest, although it showed a steady decrease with time. The Kevlar/epoxy system, on the contrary, had less resistance, but it noticeably increased with time. This result underlines the significance of Kevlar in promoting the formation of passive films that, with time, decelerate dissolution and confirm the significance of glass in stabilizing the epoxy system that initially protects the system.The SEM images revealed cracks and the exposure of the embedded fibers from under the epoxy subjected to deterioration from the acid attack, sustained/assisted by the diffusion of the corrosion species.The results of this comprehensive study indicate the high reliability of the coating systems involving glass, Kevlar, and epoxy against the corrosion of the carbon steel substrate in the various corrosive solutions studied: 0.5 M NaCl, 0.5 M HCl, and 0.5 M H


Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults with poor prognosis. Especially for AML-M5 type, due to the strong cell migration ability, the possibility of extramedullary invasion is large and widespread, which leads to poor therapeutic effect. Previous studies have found that protein arginine methyltransferase 5 (PRMT5) could promote the proliferation and differentiation of leukemic cells in AML, but its regulation on the invasive ability of AML cells remains unclear. This study was designed to explore the role of PRMT5 in regulating the invasion of AML cells and to investigate the mechanisms. Patient samples were collected for detection of PRMT5 expression level. AML cells were used for exploring the function of PRMT5. The results of clinical samples showed that the expression of PRMT5 was significantly increased in newly diagnosed and recurrent AML patients, and the expression of leukocyte immunoglobulin-like receptor B4 (LILRB4) was positively correlated with the level of PRMT5. In the cell experiment in vitro, we found that when PRMT5 was knocked down, the invasion, migration, and adhesion capacities of MV-4-11 cells and THP-1 cells were decreased, and the mRNA and protein levels of LILRB4 were also decreased. Moreover, we screened related signaling pathways and found that PRMT5 affected the expression of downstream LILRB4 by activating mTOR pathway, which in turn enhanced the invasive ability of AML cells. Taken together, PRMT5 plays an important role in the invasion of AML, which acts via regulating the expression of LILRB4. PRMT5 could act as a potential therapeutic candidate for AML. Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. Despite the various therapeutic discoveries on AML with the deepening research –4, the “Protein arginine methyltransferases (PRMTs) are a type of enzymes that transfer methyl from cofactor S-Adenosylmethionine (SAM) to substrate protein arginine omega-nitrogen. According to its specificity, PRMTs can be divided into three subgroups: I, II, and III, which are asymmetric dicarboxylic esters, symmetric dicarboxylic esters, and monomethylated substrates, respectively . ProteinLeukocyte Ig-like receptor subfamily B (LILRB) is a group of type I transmembrane glycoproteins with ligand-bound extracellular Ig-like domain and intracellular immune receptor tyrosine inhibitory motif (ITIMs) and can recruit tyrosine phosphatase SHP-1, SHP-2, or inositol phosphatase SHIP, with immunosuppressive function –26. ThusIn this study, we investigated the changes of PRMT5 expression in AML-M5 patients, MV-4-11, and THP-1 and further explored the effects of PRMT5 on the invasion and migration in AML. Moreover, we also examined the correlation between PRMT5 and LILRB4 in relation with AML infiltration and additionally assessed the pathway underlying the effects of PRMT5 methylation on the expression of LILRB4, aiming to provide experimental evidence for the treatment of AML.Bone marrow samples were collected from 30 (AML-M5 or AML-M4) patients from the Affiliated Hospital of Guiyang Medical University  from May 2018 to April 2020 . All AML2.Human THP-1 (AML-M5), kasumi-1 (AML-M2), HL-60 (human promyelocytic leukemia cell), and MV-4-11 (AML-M4) cells were cultured in RPMI1640 medium and IMDM medium containing 10% fetal bovine serum (FBS), respectively. The primordial cells of patients with AML were taken from the blood samples of patients from the Affiliated Hospital of Guizhou Medical University and stored in the leukemia cell bank of the Affiliated Hospital of Guizhou Medical University. All cells were incubated at 37°C and 5% COκB (K002162P), anti-phosph-NF-κB (K006209P), anti-mTOR (K003351P), and anti-phosph-mTOR (K006205P) antibodies were purchased from Solei Bao Technology (Beijing). Anti-ERK (4695T) and anti-phosph-ERK(4370T) were purchased from Cell Signaling Technology .EPZ015666 and mTOR inhibitor-3 were purchased from MCE (China). Antibody against PRMT5 (YN3030) was purchased from Immunoway . Anti-LILRB4 (K004989P), anti-PI3K (K106692P), anti-phosph-PI3K (K006379P), anti-AKT (K101311P), anti-phosph-AKT (K006214P), anti-NF-μg) was separated by 10% SDS-PAGE gel and transferred to the PVDF membrane . Membranes were subsequently blocked with 5% skim milk diluted in TBST for 2 h and incubated with primary antibody (1 : 1000) overnight, followed by incubation with fluorescent coupled secondary antibody (1: 2000). β-Actin was used as the loading control and for normalization. The signal strength of the film was measured by Tanon .After washing with saline twice, cells were lysed in RIPA buffer containing PMSF and proteasome inhibitor to extract the total protein , followed by quantification of protein concentration by BCA protein concentration assay kit . The same amount of protein  and a PRISM 7500 real-time PCR detection system . The expression level of target gene was normalized to that of siRNA against PRMT5 (si-PRMT5) was used to suppress the expression of PRMT5 in cells. Sasastic siRNA (si-NC) was used as the negative control. The siRNA sequence was designed and synthesized by Quan yang Biotechnology Co., Ltd. .The sequence is as follows: si-PRMT5: 5′-GGGACUGGAAUACGCUAAUTT—3′.μl/hole) were mixed and further added to the ThP-1 and MV-4-11 cells, followed by incubation for 48 h. Western blot was used to detect the downregulation efficiency of siRNA.The final concentration per siRNA was 2.5 nmol. According to the commodity specification, Lipo8000TM transdye reagent and siRNA raw solution (250 4 cells/ml) were added to the polycarbonate membrane  for migration assay and polycarbonate membrane with fibronectin coating  for invasion assay, respectively, and 600 μl RPMI1640 medium was added to the lower chamber of Transwell. After incubation at 37°C for 24 h, cells were observed under microscope (100x) by randomly selecting nine views from each well, aiming to count the number of cells penetrating into the lower cavity and to observe cell morphology.siRNA was used to downregulate the expression of PRMT5 in MV-4-11 and THP-1 cells, followed by assessment of the migration and invasion ability of the corresponding cells. In brief, leukemia cells  was purchased to detect the cell adhesion ability of THP-1 and MV-4-11 cells after downregulating PRMT5 and the corresponding control cells at the same time. Briefly, 100 t-test. P < 0.05 was considered as statistically significant.All results were analyzed using the GraphPad Prism 7.0 software. Data were presented as mean ± SD and analyzed by either one-way ANOVA or Student's Blood samples from 30 patients with AML-M5 or AML-M4 and 20 normal controls were collected from the Hematopoietic Stem Cell Laboratory of the Affiliated Hospital of Guizhou Medical University from May 2018 to April 2020. The clinical characteristics of the patients are summarized in 10X was used to calculate the mRNA level of PRMT5 and LILRB4. According to the R value, there was a positive correlation between PRMT5 and LILRB4  and normal human samples by Western blot. As a result, the expression of PRMT5 in cell lines was higher than in normal human samples . MoreoveAlthough the increased expression of PRMT5 has been confirmed to be associated with the pathogenesis and prognosis of AML and to affect the proliferation and differentiation of AML cells, however, the effects of PRMT5 on the invasion and migration of AML cells were not well-studied. To confirm the roles of PRMT5 expression in the adhesion, invasion, and migration of AML cells, we assessed the adhesion, invasion, and migration abilities of MV-4-11  and THP-To further validate the results, PRMT5 inhibitors (EPZ015666) were purchased to inhibit PRMT5 expression in MV-4-11 and THP-1 cells. And Western blot was used to detect PRMT5 expression in MV-4-11 and THP-1 cells, showing that the expression of PRMT5 in the EP015666 group was significantly decreased Figures . We furtIn order to verify whether PRMT5 affected the invasiveness of AML cells by downregulating the expression of LILRB4, protein samples were extracted from AML cells after downregulating PRMT5. As a result, the expression of LILRB4 was significantly lower in the si-PRMT5 group than in the CON group of MV-4-11 cells , and theκB, and mTOR pathways [κB pathway was only affected in THP-1  and the expression of LILRB4 was inhibited (P < 0.05). Meanwhile, we observed the same changes in THP-1 cell lines  to inhibit PRMT5 expression in both MV-4-11 and THP-1 cells. Western blot was used to detect the changes of mTOR pathway and LILRB4 at protein level in MV-4-11 , which r Figures .In order to explore the relationship between the above changes and LILRB4, we detected the proteins of PRMT5 and LILRB4 in AML cells after blocking mTOR pathway. Therefore, after blocking mTOR pathway, the protein abundance of LILRB4 in the mTOR inhibitor-3 group was significantly lower than that in the CON group of both MV-4-11  and THP-Some studies have shown that PRMT5 is necessary for the survival of AML cells and is highly expressed in many types of leukemia, especially in M5 leukemia , 37. HowPRMT5 exists in the nucleus and cytoplasm. A great deal of evidence shows that PRMT5 activates or suppresses the transcription via the methylation of arginine on histone and promotes cancer by inhibiting the expression of tumor suppressor gene , 17, 20.2Recent studies have shown that LILRB4 is vitally involved in the pathogenesis and development of AML, and the overexpression of LILRB4 in AML-M5 is closely associated with the invasive ability of AML-M5 . We furtκB signaling pathway [In addition, the mechanism underlying the effects of PRMT5 on downstream gene expression and phenomena was also discussed. To be specific, in lung cancer, inhibition of PRMT5 expression can alleviate the trigger of enolase-1, thus reducing the invasion of lung adenocarcinoma . Previou pathway , 20, 34. pathway . In this pathway . Wei et PRMT5 is highly expressed in AML, which is a marker of poor prognosis for AML. PRMT5 plays an important role in the invasive capability of AML cells. Inhibition of PRMT5 can alleviate the invasion and migration of AML cells and improve the prognosis of AML, which is associated with the change of LILRB4 induced by PRMT5. The effects of PRMT5 on LILRB4 may be associated with its regulation on mTOR. The overexpression of PRMT5 is critically involved in the invasion and migration of AML, which could act as a new therapeutic target for AML.