Microneedles (MNs) are minimally invasive tridimensional biomedical devices that bypass the skin barrier resulting in systemic and localized pharmacological effects. Historically, biomaterials such as carbohydrates, due to their physicochemical properties, have been used widely to fabricate MNs. Owing to their broad spectrum of functional groups, carbohydrates permit designing and engineering with tunable properties and functionalities. This has led the carbohydrate-based microarrays possessing the great potential to take a futuristic step in detecting, drug delivery, and retorting to biologicals. In this review, the crucial and extensive summary of carbohydrates such as hyaluronic acid, chitin, chitosan, chondroitin sulfate, cellulose, and starch has been discussed systematically, using PRISMA guidelines. It also discusses different approaches for drug delivery and the mechanical properties of biomaterial-based MNs, till date, progress has been achieved in clinical translation of carbohydrate-based MNs, and regulatory requirements for their commercialization. In conclusion, it describes a brief perspective on the future prospects of carbohydrate-based MNs referred to as the new class of topical drug delivery systems. The transdermal route can be a promising approach for drug delivery.Microneedles (MNs), a novel and important transdermal drug delivery system (TDDS), are discussed.Prisma guidelines are utilized to review carbohydrate-based MNs for medical applications.More innovative applications are predicted owing to their physiological features. Over the last few decades, we have witnessed captivating technology development in drug delivery systems. This technology improvement has enhanced the therapeutic efficiency of biotherapeutics  and hydrophilic drugs . The theEmerging techniques, like the transdermal drug delivery system (TDDS), mainly focus on delivering therapeutic agents through the skin. Universally known, the transdermal route evades the first-pass metabolism, drug–food, and drug–drug interaction, thus enhancing the bioavailability of the therapeutic agents , 6. An iA three-dimensional, needle-like biomedical device that efficiently delivers the drug through the skin barrier without disturbing and overcoming the conventional method’s limitation, microneedles present themselves as a promising development for TDDS, comprising micron-size arrays organized in a small area , 8. ThisA systematic review and meta-analyses (Prisma) are an evidence-based minimum set of records for disclosure of systematic review and meta-analysis. The preferred reporting records emphasize the propagation of studies by analyzing randomly assigned records and can be utilized as a critical element for disclosing systematic reviews of different research types, predominantly estimating the intrusion. These guidelines are designed to assist authors to enhance the presentation of systematic review and meta-analysis and used for journal peer reviewers and editors for critical evaluation of the published systematic review. It also delivers comprehensive procedures relating to the technique, including eligibility criteria, information source, search strategy to execute a systematic review, and meta-analysis .In the past, a wide range of materials were used for the fabrication of MNs. Initially, MNs were fabricated using silicon –12, titaA literature search was performed on English language papers using the PRISMA guideline . Peer-reA search of the PubMed database identified studies published until May 2020. MEDLINE, Scopus, Google Scholar, Web of Science, and gray literature were incorporated into the search strategy. The search terms included ‘microneedle drug delivery’ and ‘carbohydrate-based microneedle’. It also included terms like clinical practices, bioavailability, fabrication, microneedle array, and patient compliance.The article quality and references of relevant articles were assessed individually according to the PRISMA protocol criteria. The quality criteria used to describe the studies were biopolymers, design, evaluation, and safety of the microneedle, preclinical, and clinical presence. A detailed description of the criteria used for the qualitative assessment of the records is given in Table A literature search was conducted using PRISMA guidelines. Search strategies were set with different keywords and specific assessment criteria for inclusion and exclusion of records, as shown in Fig. Microneedles are microscale arrays designed to penetrate the skin’s stratum corneum, forming microchannels through which active molecules diffuse passively Fig.  24–26].–26.24–26Generally, MNs are categorized based on their assembly Fig. , design Microneedle arrays are optimized by choosing the array dimensions and design, which might inflict less pain, good mechanical strength, and drug delivery . Gills HThe delivery of drug-using MNs is characterized according to five different types and related forms of drug delivery Fig. , Table 2The first method is to poke the epidermis layer by microarray, which produces microchannels for drug delivery, subsequently detaching the microarray. Then, a transdermal patch is applied above it. Furthermore, to improve permeability, the electro field may be used. Solid microneedles follow this approach for the enhancement of skin permeability.The second way is to coat the microneedle arrays with the drug dispersion. The coated drug dissolves after piercing the coated microneedles into the skin. Microarrays can be covered with several compounds, including small molecules, proteins, plasmid DNA, virus particles, polymer nanoparticles, and water-soluble compounds.As a novel alternative to coated microneedles, the drug is encapsulated within microneedles made up of polymers and polysaccharides for rapid and sustained release in the skin. As a result, after piercing into the skin, the microneedles dissolve or degrade, releasing the encapsulated drug in a controlled manner.In contrast to solid microneedles, hollow microneedles offer extra abilities and fewer difficulties. Hollow microneedles have complex fabrication processes with intrinsically weaker and sophisticated geometry. They permit the liquid formulations, which allow diffusion of the drug rapidly or with controlled flow rates, as required, but the rate-limiting step in hollow micro-arrays is the maintenance of flow rate.Mechanical characterization is a crucial step in the successful fabrication of MNs. Extensive stress during the insertion and removal of MNs is applied. Hence, it is essential to perform mechanical characterization and analyze the safety of MNs . A varieIn this test, force is applied to the MNs perpendicular to the base plate Fig.  31]. Th. Th31]. Due to skin irregularities and elasticity, frequent incomplete insertion and bending of MNs arrays were observed. Thus, a transverse fracture force test is essential to investigate the performance of MN during application , 33. TheAssessment of base-plate strength is crucial as the above-mentioned mechanical strength. Fracture of the base plates during piercing to the skin is not acceptable. Hence, base plates should be flexible enough to consent to the skin structure deprived of rupturing Fig. . A threeSeveral techniques are applied to evaluate the insertion of MNs into the skin. Staining is a commonly used qualitative technique that stains epidermis cells, not microchannels created in the stratum corneum Fig.  37–39].–39.37–39Factors like different materials and geometry of MN interrupt the insertion studies, using various experimental protocols and skin models. Thus, the standard protocol is crucial to assess mechanical performance.Microneedles are based on the principle of piercing the outermost skin layer, which is not similar to the conventional transdermal patch. The suitability of microneedles has been evaluated and predicted on the degree of adverse effects, namely, itching, scaling, prickling, stinging, tightness, burning, edema, and erythema. No one has reported carbohydrate microneedle-related local infection. The disturbance may occur during the piercing or compatibility of the material used for fabrication with skin or residual solvents in the needles. The microneedle is designed to avoid pain while piercing. A formed microchannel must be reclosed in the short term to eradicate the contamination. The microneedles and formulation should be biocompatible with the skin. Modepalli et al. studied the safety and toxicity of hydrogel-forming microneedles on the human skin. They stated no toxicity in cell lines due to the microneedle . Sodium In certain cases, MNs pierce the epidermis and dermis layer, generally sterile areas of the body . Thus, MCarbohydrates play a quintessential role in the fabrication of MNs, and thus form the emphasis of this review. Carbohydrates, such as sugars, hyaluronic acid , 45, celCarbohydrates reveal an enormous structural disparity and several intrinsic characteristics, which impact the mechanical properties and invasion ability of MNs and influence the biological potency and durability of the therapeutic agent –57. CertSugars present an attractive resource for the fabrication of biomolecules delivering MNs. Also, the combination with other polymers may enhance the mechanical strength and stability of MNs. Maltose is the most widely used sugar for the fabrication of MNs and is classified as GRAS . AdditioHyaluronic acid is a crucial element of the extracellular matrix and cartilage, which has mucoadhesive properties , 54. HA Calmette–Guerin bacillus-loaded HA MNs, for tuberculosis prevention, which dissolved within 10–15 mins. As stated by the World Health Organization (WHO), the Calmette–Guerin bacillus vaccine is stable at 2–8 °C. This research reported good stability at ambient temperature and quick onset of action for the undeveloped area. For nanomedicine delivery, Wang et al. [In a separate study, Chen et al.  fabricatg et al.  formulatAnother study involved direct incorporation of cells and polymer, like the adipose-derived stromal vascular fraction cells carrying HA MNs, for diabetic wound healing. Cells loaded with HA MNs depicted improved wound healing compared with HA MNs and isolated sections, proposing an alternative approach . In a diIn another approach to customizing drug delivery, Kim et al.  designedCross-linking of HA may cause swelling and tailor the degradation or drug delivery , 71. ZhaThe incorporation of proteins and peptides was also studied. Monkare et al. developed HA MNs loaded with IgG, which recovered 82% of the protein with no change in the tertiary structure after fabrication . Liu et In different prospects, HA’s feasibility was demonstrated by e-beam sterilization, which impeded the ascorbic acid 2-glucoside degradation of HA MNs as the dissolution rate and drug release, structural features, and fracture force remained unaffected, without activity loss of capsulated active elements .Sodium chondroitin sulfate is a hydrophilic component  of the eHydrophilic polymers like carboxymethyl cellulose and (hydroxypropyl) methylcellulose are predominantly utilized because of their several features. These biopolymers are used as thickening agents, binders, stabilizers, and film-forming agents , 78. KimOn the other hand, Zaric et al.  fabricatChitin and chitosan, after cellulose derivatives, are the widely used biopolymers produced by the deacetylation process. Chitosan comprises a high-density positive charge due to its acidic nature, allowing tissue and mucoadhesion –84. DegrJin et al. formulated chitin MNs coated with antigens, a convenient diagnostic tool for tuberculosis. Chitin MNs were water-insoluble, mechanically robust, physiologically inert, and exhibited a lower swelling index. Chen et al.  preparedStarch is a biodegradable polymer extensively used due to its ease of processing and filmogenic ability for biomedical purposes . While fAureobasidium pullulans in starch and sugar cultures, plays a crucial role in biomedical applications. This biopolymer has unique characteristics, such as oxygen-barrier property, mechanical robustness, chemically inert, transparent, and filmogenic ability with no toxicity or mutagenicity. Hence, PL is an outstanding candidate for the fabrication of dissolvable MNs. Vora et al. fabricated PL MNs to deliver proteins and peptides, namely FITC-BSA and insulin. Ex vivo studies demonstrate protein/peptides detected within 15 mins when applied to porcine skin [Pullulan (PL), a hydrophilic biopolymer formed by the yeast-like fungus ine skin .Materials, length, sharpness, needle characteristics, and geometry should be correctly designedThe MN arrays should retain satisfactory microbiological standardsThe MN arrays should keep the uniformity of contentQuality manufacturing, safety, and packaging should be maintainedSafe and nonhazardous procedures should be drawn for MN arraysA self-disabling mechanism assuring a single use may require the MN arrays to avoid reuse by patients or others.If MN arrays are reusable, cleaning information should be providedFor long-term use, material safety should be considered with precisionLabeling should contain instructions for useMN arrays should be used with a proper applicator for proper insertion and pain-free delivery. Also, arrays should give reproducible results without complicationsImmunological safety assurance should be consideredMany nonclinical studies were conducted for carbohydrate-based microneedles, which revealed enhancement in efficiency and permeability, but only a few accomplished positive clinical testing results. Table Drug delivery via the transdermal route has seen incredible development, among which the microneedle arrays facilitating drug infusion via forming several microchannels in the stratum corneum have grown exponentially over the past two decades. In the past fifteen years, the aspect of material science has transformed radically, and the utilization of natural polymers for recuperative purposes would be a critical success , 92, 93.Carbohydrate usage presents an inclusive array of benefits owing to their intrinsic physiological compatibility, degradability, nontoxic, and sustainable nature. The inherent variation in carbohydrates, stemming primarily due to animal and plant origin, absence of standardization, and variation in appearance, with time or development, are few aspects that play a significant drawback role during commercialization . The appRestructuring the study protocols as per the properties of the carbohydrates is essential. For instance, in moisture-absorbing polymers, assessments forecasting storage in water-resistant packages should be conducted. Along the same lines, considering the probabilities of utilizing these microarrays in states with high RH like the WHO climatic zone IV, deficiency of availability, and setups, appropriate contingencies should be planned . The criThe actual application of MNs delayed as the regulatory guidelines are ongoing. Still, the academy is bringing novel instigating records in this zone, which highlights the difficulties during developments. The formation of policies to manufacture and analyze MNs is authoritative to ensure specifications and precision during production. Larraneta et al.  explaineSterilization of the MNs is a crucial step that impacts the stability of the active pharmaceutical agent. Concerning the storage condition, drug-loaded MNs must be stable at ambient temperature for their intact mechanical properties and pharmacological performance. Another critical aspect emphasizes MN arrays’ disposal, which may transfer blood-borne disease through blood or ISF . NanocarIn conclusion, sophisticated design and impressive fusion of carbohydrate-based MNs effectively deliver several active pharmaceutical agents with a broad spectrum of applications in various zones. Carbohydrate-based MNs have a fundamental part in wellness management, which provides stimulating revolutions in drug delivery. In these arrays, it is anticipated that such designs might be legalized in clinical practice, fetching motivation from nature into day-to-day drug delivery applications in a couple of years.


High-intensity interval training (HIIT) promotes various biological processes and metabolic effects in multiple organs, but the role of extracellular vesicles (EVs) released from a variety of cells is not fully understood during HIIT exercise (HIIT-Ex). We investigated the changes in circulating number and proteomic profile of EVs to assess the effect of HIIT-Ex.0), at 30 min (T30), and at 120 min after HIIT-Ex. The pulse rate and systolic blood pressure were measured. Circulating EVs were characterized, and EV proteins were detected via nano liquid chromatography tandem mass spectrometry.Seventeen young men  were enrolled in the study. Total duration of the HIIT-Ex was 4 min. Blood samples were collected from before HIIT-Ex (pre-HIIT-Ex), at the immediate conclusion of HIIT-Ex (T0 to pre-HIIT-Ex were significantly higher. Circulating EV number was significantly altered throughout the HIIT-Ex, and the source of circulating EVs included skeletal muscle, hepatocytes, and adipose tissue. Proteomic analysis identified a total of 558 proteins within isolated circulating EVs from pre-HIIT-Ex, T0, and T30. Twenty proteins in total were significantly changed at pre-HIIT-Ex, T0, and T30 and are involved in a variety of pathways, such as activation of coagulation cascades, cellular oxidant detoxification, and correction of acid–base imbalance. Catalase and peroxiredoxin II were increased at T0.The pulse rate and systolic blood pressure at TThe circulating EV composition can be immediately changed by particularly a short time of HIIT-Ex, indicating that EVs may intercommunicate across various organs rapidly in response to HIIT-Ex. HIIT pr2max) . HIIT is2max) , b. HIIT2max) . HIIT ha2max) , indicatExtracellular vesicles (EVs) including exosomes as endosomal origins and microvesicles as blebbing from plasma membrane, which have been shown to be released from a variety of cells, are composed of a lipid bilayer embedded with membrane proteins and an aqueous core containing water-soluble proteins and nucleic acids . EVs cirThere have been several studies concerning EV release in human exercise models  such as In this study, we investigate the association between circulating EVs and a multiorgan response, including lipid and glucose metabolism during HIIT exercise (HIIT-Ex). We also provide data on the prompt change in circulating EV numbers and EV protein composition after HIIT-Ex with short duration.Seventeen young male individuals  enrolled in the study. They were students of Mie University  majoring in health and physical education. They were physically active and were trained as players of the Mie University baseball team. The basic characteristics of the study participants are shown in 2max value for each participant was obtained by gas exchange analysis during bicycle ergometer tests. A number of protocols exist for estimating VO2max. In the present study, initial power output was set at 30 W, and the output was then gradually increased by 2 W every 4 s afterward up to all-out max effort. The pedal cycling speed was fixed at 60 revolutions per minute (rpm) throughout the analysis.Exercise was undertaken on a programmable bicycle ergometer . Prior to the HIIT-Ex, VO2max for 20 s) and rest for 10 s were performed during the HIIT session. Serum was separated from whole blood and was immediately frozen at −80°C until analysis.The protocol of HIIT-Ex is shown in 0), at 30 min (T30), and at 120 min (T120) after HIIT-Ex. Serum was separated from blood and immediately stored at −80°C for analysis.We measured anthropometric data including body mass index (BMI), lean body mass, skeletal muscle volume (SMV), fat mass, pulse rate, systolic blood pressure (BP), and diastolic BP. Lean body mass, SMV, and fat mass were measured using multiple-frequency bioimpedance technology . Measurements of pulse rate, systolic BP, and diastolic BP, as well as blood collection were performed before HIIT-Ex (pre-HIIT-Ex), at the immediate conclusion of HIIT-Ex (TSerum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transpeptidase (γ-GTP), low-density lipoprotein cholesterol (LDL-Chol), triglyceride (TG), creatinine (Cr), fasting blood glucose (FBG), free fatty acid (FFA), creatine phosphokinase (CPK), and fasting immunoreactive insulin (IRI) were measured at SRL Inc. . The homeostatic model assessment of insulin resistance (HOMA-IR) was calculated as [fasting IRI (μU/ml) × FBG (mg/dl)]/405 . The homg for 10 min to eliminate any aggregations. Circulating EVs in 2 μl of serum were stained with 4 μg/ml of calcein-AM  for 30 min in the dark at room temperature. The number of EVs was determined by flow cytometry  with flow cytometry alignment beads  by triplication in each sample, and data were analyzed using FlowJo software .Serum was centrifuged at 2,000 × g for 10 min to eliminate any aggregations. Circulating EVs were isolated from an equal amount (500 μl) of serum between samples via qEV columns  according to the manufacturer’s instruction. All fractions, fractions 1–30, were collected with 500 μl each, and protein amount was measured at OD280 according to the manufacturer’s instruction. Fractions 6–10 were concentrated with Amicon Ultracel-3K . The diameter of isolated circulating EVs was measured via nanoparticle tracking analysis, Nanosight LA10  by triplication in each sample.Serum was centrifuged at 2,000 × 0 compared to pre-HIIT-Ex. Briefly, circulating EVs were isolated from an equal amount (500 μl) of plasma between samples via qEV columns (Izon Science) according to the manufacturer’s instruction. Fractions 6–10 were collected from the columns, and EVs were precipitated using trichloroacetic acid, followed by reduction, alkylation with iodoacetamide, and trypsinization using an equal amount of protein between samples. Samples were separated using nano-LC-MS/MS, EASY-nLC 1200 (Thermo Fisher Scientific), and Q Exactive Plus (Thermo Fisher Scientific) at APRO Science Institute . Data were analyzed using Scaffold4  against the SwissProt database at APRO Science Institute. Quantitative value  on Scaffold4  was used for heatmap and protein interactions, which were generated using Heatmapper software . Blotted membranes were incubated with blocking reagent for Can Get signal  and primary antibodies followed by peroxidase-conjugated secondary antibody  in Can Get solutions. The primary antibodies used were anti-CD9 , anti-CD81 , anti-TSG101 , anti-syntenin-1 , anti-asialoglycoprotein receptor 1 (ASGPR1) , anti-perilipin , anti-α-skeletal muscle actin , anti-peroxiredoxin II (PRDX-2) , and anti-catalase (CAT) . The membrane was treated with Stripping buffer  or azide-TBST to remove horseradish peroxidase (HRP). Protein bands were visualized using enhanced chemiluminescence reagents  and digitized using a charge-coupled device camera . Expression intensity was quantified by Multi Gauge software (Fuji).Isolated EVs (10.4 μg) were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer resolved in TGX0, T30, and T120 were compared using Kruskal–Wallis test. For proteomic data, parameter values in the entire three time points  were tested via mixed model repeated-measure analysis to determine the effect of time. Values with p < 0.05 were considered statistically significant. Statistical analysis was done using Multivariable Analysis for Mac version 3.0 software  and SPSS version 22.0 J software .Data are expressed as medians with ranges. Parameter values determined at pre-HIIT-Ex, T2)  , elevate2) , and ano0 when compared to the values at pre-HIIT-Ex   when compared to the values at pre-HIIT-Ex , at T0 (p < 0.01), and at T30 (p < 0.01) . EV numbers at T0 was not significantly different from the values at pre-HIIT-Ex, although we found that circulating EV number was promptly increased in 11 out of 17 (64.7%) subjects at T0 when compared to pre-HIIT-Ex.The protocol of HIIT-Ex is shown in  < 0.05) . Diastol-HIIT-Ex . Median nificant . Serum l < 0.01) . Next, w < 0.01) . The med0  (30 compared to pre-HIIT-Ex (p < 0.05)   (0) (each p < 0.05)  . An anti < 0.05) . CA-1, aHIIT-Ex) . An antiHIIT-Ex) . We list < 0.05) .0 and T30 . It was surprising that EVs were promptly released into the systemic circulatory pathway in response to only 4 min of exercise intervention. Although we did not observe significant elevation of circulating EV number in all subjects, this result would be expected because HIIT-Ex was performed only for 4 min and circulating EV number at pre-HIIT-Ex was influenced by healthier condition, such as TG levels and inflammation. Furthermore, the efficacy of cardiopulmonary and skeletal muscle loads was different in each subject. Circulating EV number was significantly reduced at T0. HIIT-Ex is known to promote changes in hemostatic and fibrinolytic properties. Short-duration HIIT-Ex significantly increased plasma concentrations of tissue factor, tissue factor pathway inhibitor, thrombin–anti-thrombin complex, and D-dimer , acid–base homeostasis (CA-1), and antioxidant (CAT and PRDX-2). FGG, which is involved in coagulation cascades including fibrinolysis, was elevated at T D-dimer , resultitraining . Therefo2O). Our results were consistent with the previous report in which it was demonstrated that plasma CAT levels were significantly increased at 3 and 24 h after HIIT-Ex  in patients with type 2 diabetes. Conversely, another study reported that there was no change in skeletal muscle PRDX-2 expression in patients with type 2 diabetes after training   . The curCA and PRDX-2 have been identified as significant elevated EV-packed proteins in different types of Ex, such as 2-km run  and 1 h 0, and T30 was performed on three subjects. We detected catalase and PRX2 in the remaining 14 subjects by Western blotting. In the future, we need to validate the current results with proteomics analysis in a larger number of samples.The present study has some limitations. (1) The current study was a prospective single-arm study, not a randomized study with a control group. (2) The sample number of proteomics analysis was limited due to expensive cost. In the current study, proteomics analysis at pre-HIIT-Ex, TIn conclusion, the circulating EV numbers and protein composition can be immediately changed after 0 and/or 30 min by a particularly short time of HIIT-Ex. A part of circulating EVs were derived from multiple tissues and cells including the skeletal muscle, hepatocytes, and adipose tissue as a function of HIIT-Ex. Furthermore, the protein composition of EVs involved in activating coagulation cascades, removal of oxidative stress, and correction of acid–base imbalance was identified by proteomic analysis. These results indicated that EVs intercommunicate across various organs rapidly in response to HIIT-Ex .https://doi.org/10.6084/m9.figshare.13480317.v1.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The studies involving human participants were reviewed and approved by the Ethics Committee at Mie University. The patients/participants provided their written informed consent to participate in this study.AE, YK, and KT contributed to conception and design of the study. YK, AE, MI, and YTak organized the database. YTam, MT, and KI performed the experiments. SF performed the statistical analysis. YK wrote the first draft of the manuscript. AE and SF wrote sections of the manuscript. All authors contributed to manuscript revision, read, 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.


In NEC, activation of TLR4 by LPS leads to downregulation of the canonical Wnt/β-catenin signaling and upregulation of PPARγ. This review aims to shed light on the complex intracellular mechanisms involved in this pathophysiological profile by examining additional pathways such as the GSK-3β, NF-κB, TGF-β/Smads, and PI3K-Akt pathways.Necrotizing enterocolitis (NEC) represents one of the major causes of morbidity and mortality in premature infants. Several recent studies, however, have contributed to a better understanding of the pathophysiology of this dreadful disease. Numerous intracellular pathways play a key role in NEC, namely: bacterial lipopolysaccharide (LPS), LPS toll-like receptor 4 (TLR4), canonical Wnt/β-catenin signaling and PPARγ. In a large number of pathologies, canonical Wnt/β-catenin signaling and PPARγ operate in opposition to one another, so that when one of the two pathways is overexpressed the other is downregulated and  Four major pathways play a central role in the pathophysiology of NEC: bacterial lipopolysaccharide (LPS), toll-like receptor 4 (TLR4), canonical Wnt/β-catenin signaling and PPARγ. LPS activates TLR4 which in turn downregulates the Wnt/β-catenin pathway and upregulates PPARγ. Other pathways must also be discussed, i.e., NF-κB, TGF- β/Smads and PI3K-Akt pathways. Usually, canonical Wnt/β-catenin signaling and PPARγ are expressed in opposing ways so that when one pathway is upregulated, the other is downregulated and vice versa (Necrotizing enterocolitis (NEC) is the most common and major cause of morbidity and mortality in premature infants –3. Morbice versa . It appece versa –9. InherThe intestinal epithelium represents the first barrier of defense against luminal agents. It remains in permanent turnover and is progressively replaced by intestinal stem cells (ISCs). These cells are localized within the intestinal crypts and maintain the integrity and viability of the epithelium, necessary for gut regeneration. During NEC, loss of ISCs leads to severe gut damages . In premNEC is rare in full term neonates and can appear in a context of ischemia . This suLipopolysaccharides (LPS) are endotoxins found in the outer membrane of Gram-negative bacteria. They consist of three parts: O antigen, core oligosaccharide and lipid A, which is considered to be toxic on epithelial cells and granulocytes. LPS induces a strong response from the immune system. LPS binds with the TLR4 receptor and promotes the release of several pro-inflammatory cytokines. LPS plays a key role in NEC and patients with NEC present high levels of plasmatic LPS. Intraperitoneal injection of LPS in mice and rats induces intestinal injury. LPS is the ligand for TLR4 that mediates NEC , 35, 36.TLR4 is a transmembrane protein and member of the toll-like receptor family, which belongs to the pattern recognition receptor (PRR) family. Activated TLR4 leads to activation of the NF-κB pathway and production of numerous inflammatory cytokines. TLR4 is an important mediator of the innate immunity. TLR4 drives the inflammatory response by activation of pro-inflammatory agents . TLR4 plTLR4 is expressed in the intestinal epithelium. NEC is characterized by a marked defect in the migration of enterocytes, leading to serious alterations of the intestinal mucosa , 49–51. The loss of enterocytes initiates the migration of enterocytes from healthy to diseased areas. This induces the proliferation of enterocytes from stem cells located in the intestinal crypts. However, this proliferation is reduced during NEC , 55. NECin vivo restores proliferation and reduces apoptosis in NEC.During intestinal aggression, the regular and rapid turnover primarily involves stem cells or progenitors located at the base of the intestinal crypts , Bmi1 6–62, and In enterocytes, TLR4 promotes enterocyte apoptosis. In experimental NEC, a significant decrease in the number of enterocytes is observed at an early stage due to an increase in apoptosis processes , 68–73. In human infant NEC, there is an increased expression of TLR4 while inhibitors of TLR signaling, such as SIGIRR, are decreased –34, 76. The canonical Wnt/β-catenin pathway plays a key role in cell fate, metabolism, epithelial-mesenchymal transition (EMT) and embryogenesis –82. In tIn enterocytes, LPS activates GSK-3β and increases the phosphorylation of β-catenin, leading to its proteasomal degradation. Phosphorylation of GSK-3β is mediated by the phosphorylation of the PI3K-Akt pathway . LPS cauPPARγ is a transcriptional factor belonging to the nuclear hormone receptor superfamily. It heterodimerizes with the retinoid X receptor. PPARγ is expressed in numerous cell types, such as adipose tissues, muscles, brain, and immune cells. It regulates innate immune responses, insulin sensitivity, inflammation, glucose and lipid homeostasis, and cell fate , 89. PPAIn numerous diseases, the canonical Wnt pathway is generally regulated in an opposing manner to that of PPARγ . If one Some diseases are characterized by downregulation of the canonical Wnt/β-catenin signaling and upregulation of PPARγ. Conversely, numerous diseases are characterized by upregulation of the canonical Wnt/β-catenin signaling and downregulation of PPARγ , 91, 99.-β signaling by preventing the complex formation between Smad2 or Smad3 and Smad4 and the nuclear accumulation of Smad2 and Smad3 in response to TGF-β signaling . These subunits heterodimerize to form the active factor NF-κB . p50-p50The TLR4 pathway blocks PI3K-Akt phosphorylation, allowing GSK-3β to remain active  and to phosphorylate β-catenin leading to its destruction into the proteasome . InhibitThe first link between TLR4 and β-catenin was established by Ireland et al. . In a trin vivo, inhibition of TLR4 signaling reverses these effects and restores levels of enterocyte proliferation in experimental NEC. TLR4 inhibits enterocyte proliferation via the inhibition of β-catenin signaling. LPS causes a dose-dependent decrease in β-catenin expression in the nucleus. This occurs via an increase in the dephosphorylated form of GSK-3β, which is restricted to the small intestine of newborn mice  were given a single administration of formula milk via an orogastric catheter . This NEIn an adult mouse model of NEC  model of NEC), activation of PPARγ induces a protective effect on the small bowel during I/R-induced gut injury . The PPAIn premature infants, a genetic predisposition to NEC has been observed –9. GenetκB genes. This may favor the pathogenesis of NEC   . In IEC- GSK-3β) –160. In IEC mice . This shGSK-3β/β-catenin signaling plays a key role in determining the enterocyte proliferation that occurs in response to EGFR activation .Inactivation of GSK-3β by phosphorylation at serine 9, negatively affects the NF-κB activation –164, thuIn a neonatal preterm rat model, the PPARγ agonist pioglitazone (PIO) reduces the development of NEC . PIO in Administration of Wnt7b results in the maintenance of intestinal epithelial homeostasis and the avoidance of NEC intestinal injury in mice . IntestiPI3K-Akt phosphorylates GSK-3β and upregulates β-catenin activity.The TLR4 pathway blocks the PI3K-Akt phosphorylation that allows GSK-3β to remain active (unphosphorylated) and to phosphorylate β-catenin, leading to its destruction into the proteasome. Lithium chloride phosphorylates GSK-3β. Inhibition of GSK-3β with lithium chloride prevents the negative effects of LPS on β-catenin and restores the proliferation of IEC-6 cells .TGF-β2 is sequestered in preterm human milk by chondroitin sulfate proteoglycans . EnteralThis review of NEC sheds light on the complexity of the pathophysiology of the disease. In particular, the downregulation of the canonical Wnt/β-catenin pathway induced by activation of the LPS-TLR4 systems makes it possible to understand why canonical Wnt agonists attenuate the severity of the disease in experimental NEC models. Some PPARγ agonists can also minimize the deleterious effects of this disease by downregulating NF-κB signaling. This provides us with the hope that current research will lead to the development of new therapeutic avenues to treat this particularly serious disease.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


The FAIR-2 (‘Frankfurter Aufmerksamkeitsinventar’) is a pen-and-paper test of visual attention in which participants have to search for targets among distractors. For similar pen-and-paper tests of attention , the repetition of the test causes large improvements in performance that threaten both its (retest) reliability and validity. We investigated the size and possible sources of practice effects in the FAIR-2 in three experiments. In Experiments 1 and 2, participants were tested twice using the original FAIR-2. We compared how performance changed after 2 weeks (Experiment 1) or 3 months (Experiment 2), when the test was repeated (complete repetition), or when targets and distractors changed their roles . For Experiment 3, we used self-constructed versions of the FAIR that allowed for a third neutral condition  without any stimulus overlap between the two tests. The complete repetition condition produced strong performance gains (25–35%) that persisted for 3 months. For the complete-alternation condition, we observed small to moderate improvements, suggesting that stimulus-independent learning had occurred in session 1. Finally, performance did not differ between test reversal and complete alternation, therefore, suggesting that improvements in target processing had caused the large improvements in the complete-repetition condition.The online version contains supplementary material available at 10.1007/s00426-021-01481-x. Working in an attentive and concentrated manner is an important prerequisite for successful behavior in many areas of life, including school and the workplace. To assess a person’s ability to work in an attentive and concentrated manner, psychologists have developed pen-and-paper tests examining selective attention and concentration, such as the d2 test . The L measure  is the sum of hits and represents the number of correctly detected targets. The Q measure  is the percentage of correctly inspected items in relation to the number of all inspected items (including errors). Finally, the K measure  is computed from L × Q and claims to reflect the stability of performance.The FAIR-2 , in the sense of contextual cueing, is not possible in the FAIR-2 test.The empirical evidence suggests that practice may affect visual search at different stages, including changes in the perceptual processing of target stimuli  and changes in the attentional guidance of visual search  laboratory research has not yet settled the issue of what exactly causes practice effects in visual search, (b) we are lacking empirical studies on practice effects in pen-and-paper tests of visual search, and (c) there are a lot of methodological differences between search tasks in the laboratory and pen-and-paper tests. These problems prevent us from making strong predictions for our experiments from the literature.In Experiment 1, we investigated changes in performance when participants did the FAIR-2 test twice within 2 weeks. This study had three goals. First, we wanted to investigate the size of performance improvements that arise from a repetition of the test. Second, we wanted to investigate changes in performance within a single session with the FAIR-2. Therefore, we assessed and analyzed performance separately for the two pages of the test booklet in each session, rather than summarizing across the two pages. Third, we wanted to investigate how reversing the roles of targets and distractors affects performance in the second session. In contrast to the d2, there are two complementary versions of the FAIR-2 test that can be used to compare the effects of a role reversal to the effects of a test repetition. On the basis of previous studies, we expected that performance in the (second session of the) role-reversal condition would be worse than performance in a complete-repetition condition  claims to reflect the stability of performance. Whilst the usual computation of test scores in the FAIR-2 involves summarizing across both pages of the test, we computed test scores separately for each test page. Besides testing for within-test changes in performance, analyzing the scores separately for each page allows for a more sensitive analysis of practice and transfer effects across sessions because the impact of learning in session 1 may affect performance particularly strongly in the first part of session 2.We planned to analyze the effects of three independent variables, CONDITION , SESSION (1 or 2) and TEST PAGE (1 or 2) on three dependent variables in separate three-factorial Analyses of Variance (ANOVA). The independent variable CONDITION was varied between participants, whereas the independent variables SESSION and TEST PAGE were varied within participants. The three dependent variables were the L, Q and K measures. The t tests are parametric tests that require normally distributed data in each condition. We used Shapiro–Wilk tests to check for the normality of data distributions in each condition before performing omnibus analyses and pair-wise tests. Note, however, that we continued computing ANOVAs as omnibus tests even in the absence of normality  for two reasons. First, previous studies have shown that the ANOVA is robust against violations of the normality assumption  tests, and d as an effect-size measure for pair-wise comparisons . Note that there are no generally accepted criteria for evaluating an observed effect size. For example, Cohen  to detect a strong effect . Most of the participants were women (N = 47) and enrolled in psychology or educational science. Participants were randomly assigned to one of the conditions . The two groups were similar in age  and had similar ratios of men and women.With Experiment 1, we were mainly interested in the main effect of SESSION and the two-way interaction between CONDITION and SESSION . Previous studies on repetition benefits (using the d2 test) suggested very strong effects.We used the regular test booklets of the FAIR-2 . In the role-reversal condition, 25 participants were tested with two different versions of the FAIR-2 .The procedure for the FAIR-2 test was as follows: Participants first filled out their demographic information  listed on the title page. Then participants silently read the instructions for the test (on page 2) and worked through a practice line on page 3. Participants had to read the instructions silently because two different versions of the test  were distributed equally among the participants. The tester confirmed that all participants had read and understood the instructions. He or she was equipped with a stopwatch to control the time provided for each page. The tester started the test by saying “Start now!” (in German). After 3 min, he or she said “Stop. Turn the page.” (in German), and participants started working on the second test page. After 3 more minutes, the command “Stop now!” (in German) ended the test . Shapiro–Wilk tests revealed that the distribution of L was consistent with the normality assumption in all cells of the design, all ps > 0.25. Next, the individual L scores in each cell of the design were subjected to a three-way ANOVA for mixed designs with CONDITION, SESSION and TEST PAGE as independent variables. The numerical results of the ANOVA are presented in Table 2 (cf. Appendix 1). All three main effects were significant. The main effect of CONDITION reflected a higher L in the complete-repetition condition  as compared to the role-reversal condition . The main effect of SESSION reflected an increase of L from the first session  to the second . The main effect of TEST PAGE reflected an increase of L from the first page  to the second .One participant was omitted from the analysis because he/she skipped several lines in one test. Hence, 49 datasets eventually entered into the analysis. Figure L scores between the two sessions separately for the two conditions. These comparisons revealed that L increased significantly from session 1  to session 2  in the complete-repetition condition, t(23) = 8.48, p < 0.001, d = 1.730. In contrast, in the role-reversal condition, a numerical increase of L from session 1  to session 2  was not significant, t(24) = 1.28, p = 0.212, d = 0.257. The remaining F tests were also not significant (cf. Table 2).The most important result was a significant two-way interaction of CONDITION × SESSION (cf. Table 2). To identify the source of the interaction, we compared the Q for the eight cells of our experimental design. Shapiro–Wilk tests revealed that the distribution of Q deviated significantly from normality in seven of the eight cells in our design. We subjected our Q data to a three-factorial ANOVA for mixed designs with CONDITION, SESSION and TEST PAGE as independent variables. The numerical results of the ANOVA are presented in Table 3 (cf. Appendix 1). The only significant F tests were the main effect of SESSION and the CONDITION × SESSION interaction. The main effect of SESSION reflected an increase of Q from session 1  to session 2 .Table 1 (in Appendix 1) illustrates the means of Q scores between the two sessions separately for the two conditions. For the complete-repetition condition, a Wilcoxon test revealed that Q significantly increased from session 1  to session 2 , W = 18.0, p < 0.001, d = 0.759. In contrast, for the role-reversal condition, the numerical decrease of Q from session 1  to session 2  was not significant, W = 210.0, p = 0.210, d = − 0.339. The remaining F tests were also not significant (cf. Table 3).To clarify the source of the two-way interaction, we compared the K was consistent with the normality assumption for all eight cells in the design (see Table 1 for descriptive statistics). We subjected our K data to a three-factorial ANOVA for mixed designs with CONDITION, SESSION and TEST PAGE as independent variables. The numerical results of the ANOVA are presented in Table 4 (cf. Appendix 1). All three main effects were significant. First, a significant main effect of CONDITION reflected higher K values for the complete-repetition condition  than for the role-reversal condition . Second, a significant main effect of SESSION reflected an increase of K from session 1  to session 2 . Third, a significant main effect of TEST PAGE indicated an increase of K from page 1  to page 2 .Shapiro–Wilk tests revealed that the distribution of K scores between the two sessions separately for the two conditions. For the complete-repetition condition, t tests revealed that K significantly increased from session 1  to session 2 , t(23) = 8.517, p < 0.001, d = 1.739. In contrast, for the role-reversal condition, the numerical increase of K from session 1  to session 2  was not significant, t(24) = -0.873, p = 0.392, d = − 0.175. The remaining F tests were also not significant (cf. Table 4).The only significant interaction was the two-way interaction of CONDITION × SESSION. To clarify the source of this two-way interaction, we compared the L measure , and performance improved moderately in the Q measure . After 2 weeks, the repetition benefits in the FAIR-2 are, therefore, at least as large as the repetition benefits that previous studies observed for the d2 . Hence, a role reversal between sessions neither improved nor impaired performance in the second session, as compared to the first session , but not in Q. The latter observations contrast with those of Steinborn et al. . If the practice effects, which had occurred after 2 weeks, were no longer visible after 3 months, then the after-effects of a previous encounter with the FAIR-2 would not be meaningful to most practical applications of the test.L, Q, K) in separate three-factorial ANOVAs. The independent variable CONDITION was varied between participants, whilst the independent variables SESSION and TEST PAGE were varied within participants.The design of Experiment 2 was the same as for Experiment 1. Hence, we planned to analyze the effects of three independent variables, namely CONDITION , SESSION (1 or 2) and TEST PAGE (1 or 2) on three dependent variables , G*Power . Participants were randomly assigned to one of the two conditions . The two groups were similar in age  and had similar ratios of men and women. Fifty-seven participants (71%) completed both testing sessions. Due to this dropout rate, actual power decreased to β = 0.91.We used the effect size of the critical CONDITION × SESSION interaction . In the role-reversal condition, 30 participants were tested with two different versions of the FAIR-2 . The testing sessions were conducted as prescribed in the manual.L for the eight cells of our 2 × 2 × 2 design . Shapiro–Wilk tests demonstrated that the distribution of L was consistent with the normality assumption in all cells of our design, all ps > 0.18. Next, the individual L scores in each cell of the design were subjected to a three-way ANOVA for mixed designs with CONDITION, SESSION and TEST PAGE as independent variables. The numerical results of the ANOVA are presented in Table 6 (in Appendix 1). The main effects of CONDITION and SESSION were significant. The main effect of CONDITION reflected a higher L in the complete-repetition condition  compared to the role-reversal condition . The main effect of SESSION reflected an increase of L from the first session  to the second .Figure L scores between the two sessions separately for the two conditions; these comparisons revealed that L increased significantly between sessions for both the complete-repetition condition , t(26) = 9.86, p < 0.001, d = 1.900, and the role-reversal condition , t(29) = 4.23, p < 0.001, d = 0.772. Consequently, the interaction resulted from the fact that L increased more strongly in the complete-repetition condition (difference = 51) than in the role-reversal condition (difference = 24).Again, the most important result was a significant two-way interaction of CONDITION × SESSION. To identify the source of this interaction, we compared the L increased significantly in session 1 from page 1  to page 2 , t(56) = 2.068 p = 0.043, d = 0.274. In contrast, L did not change from test page 1  to page 2  in session 2, t(56) = − 0.800 p = 0.427, d = − 0.106. The remaining F tests were not significant (cf. Table 6).There was also a significant two-way interaction of SESSION × TEST PAGE. Follow-up tests revealed that Q for the eight cells of our experimental design. Shapiro–Wilk tests revealed that the distribution of Q deviated significantly from normal in seven of the eight cells in our design. Despite this finding, we subjected the Q data to a three-factorial ANOVA. The numerical results of the ANOVA are presented in Table 7 (in Appendix 1). A significant main effect of CONDITION reflected a higher Q score in the complete-repetition condition  than in the role-reversal condition . A significant main effect of TEST PAGE reflected a decrease of Q from page 1  to page 2 . The main effect of SESSION was not significant.Table 5 (in Appendix 1) shows the means of Q scores between the two sessions separately for the two conditions. For the complete-repetition condition, a Wilcoxon test revealed that the numerical increase of Q from sessions 1  to 2  was not significant, W = 124, p = 0.121, d = 0.355. Similarly, for the role-reversal condition, a Wilcoxon test showed that the numerical decrease of Q from session 1  to session 2  was not significant, W = 321, p = 0.070, d = − 0.361. The F tests for the remaining interactions were not significant.The only significant interaction was the two-way interaction of CONDITION × SESSION. To clarify the source of the two-way interaction, we compared the K was consistent with the normality assumption for all cells in the design (see Table 5 in Appendix 1 for descriptive statistics). We subjected our K data to a three-factorial ANOVA for mixed designs with CONDITION, SESSION and TEST PAGE as independent variables. The numerical results of the ANOVA are presented in Table 8 (in Appendix 1). A significant main effect of CONDITION reflected higher K values for the complete-repetition condition  than for the role-reversal condition . A significant main effect of SESSION reflected an increase of K from session 1  to session 2 . The main effect of TEST PAGE was not significant.Shapiro–Wilk tests revealed that the distribution of K scores between the two sessions separately for each condition. For the complete-repetition condition, K significantly increased from session 1  to session 2 , t(26) = 9.346, p < 0.001, d = 1.799. Moreover, K also increased significantly from session 1  to session 2  for the role-reversal condition, t(29) = 3.317, p = 0.002, d = 0.606. Hence, the significant interaction reflected the fact that the increase in K from session 1 to session 2 was larger in the former condition than in the latter.There were significant two-way interactions for CONDITION × SESSION, as well as for SESSION x TEST PAGE. To clarify the source of the significant CONDITION × SESSION interaction, we compared the K scores between the two pages separately for each session. However, neither the small increase of K from page 1 to page 2 in the first session, t(56) = 1.290, p = 0.202, d = 0.171, nor the small decrease of K from page 1 to page 2 in the second session, t(56) = − 1.634, p = 0.108, d = − 0.216, was significant. Hence, the significant interaction resulted from the fact that changes in K within sessions went in opposite directions. The remaining F tests were not significant.To clarify the source of the significant SESSION × TEST PAGE interaction, we compared the L , and a small improvement in Q . Hence, a repetition of the FAIR-2 seems to produce quite persistent improvements in performance. Furthermore, the large performance improvements in the complete-repetition condition again contrasted with the role-reversal condition: Here, we observed a moderate improvement from session 1 to session 2 in L , whereas Q remained relatively constant across sessions . The moderate improvements seen in the role-reversal condition suggest that, after 3 months, the positive effects of stimulus-independent learning in the first session had a stronger impact on performance in the second session than the negative effects of stimulus-dependent learning in the first session.The most important result of Experiment 2 was that the pattern of performance changes between two administrations of the FAIR-2, which had been observed when the two tests were held 2 weeks apart in Experiment 1, still occurred when the two tests were separated by 3 months. A repetition of the test again produced a strong performance improvement in For Experiment 3, we developed eight new versions of the FAIR-2 instead of using the regular test. In contrast to the regular versions of the test, in which each feature dimension  has two values, in our new test versions each of the two feature dimensions had four values (cf. Table 9 in Appendix 1). Increasing the number of feature values allowed for the establishment of a new “neutral” condition , in which two consecutive tests had no stimulus features in common, in addition to a complete-repetition and a role-reversal condition.With the new test materials, we pursued two goals in Experiment 3. First, Experiments 1 and 2 had only provided indirect evidence for the notion that stimulus-independent learning in session 1 can improve performance in session 2, and we wanted to test this notion more directly in Experiment 3. Accordingly, we predicted a significant improvement from session 1 to session 2 in the complete-alternation condition.Second, we made a first attempt to investigate the subject of stimulus-dependent learning in the FAIR-2. As previously described, stimulus-dependent learning in the FAIR-2 might concern (a) the selective improvement of target processing  the role-reversal condition should be worse than performance in (the second session of) the complete-alternation condition because targets and distractors switch roles in the role-reversal condition, whereas new targets and distractors with intermediate attention-attraction strengths are presented in the complete-alternation condition cf. Fig. c. AlternL, Q and K) in separate three-factorial ANOVAs. The independent variable CONDITION was varied between participants, whilst the independent variables TEST PAGE and SESSION were varied within participants. In contrast to Experiments 1 and 2, the CONDITION factor had three levels instead of two.For Experiment 3, we planned to analyze the impact of three independent variables, CONDITION , SESSION (1 or 2) and TEST PAGE (1 or 2) on three dependent variables (β = 0.95) to detect a strong effect , aged between 18 and 36 years . Participants were randomly assigned to one of the three conditions. The three groups were similar in age  and had similar ratios of men and women. Sixty-nine participants (96%) completed both testing sessions.Similar to Experiment 1, we opted a priori for a sample size of 24 participants per condition in Experiment 3. This sample size gives the experiment sufficient power . In the complete-repetition condition, 24 participants were tested twice with the same test . In the complete-alternation condition, 23 participants were tested with two tests that contained different sets of targets and distractors. Hence, in the complete-alternation condition, four pairs of tests  were administered in two possible orders. Finally, in the role-reversal condition, 22 participants were tested using two different tests in which targets and distractors were switched. Hence, in the role-reversal condition, four new pairs of tests  were administered in two possible orders. Otherwise, the testing sessions were conducted as instructed in the manual.N = 68 . Table 10 (in Appendix 1) shows the means of L, Q and K for all cells in the three-factorial design. Figure L in all combinations of two sessions and three conditions, averaged across test pages because this variable did not have any significant effect in Experiment 3. Shapiro–Wilk tests determined that the distribution of L was consistent with the normality assumption in all cells of the design, all ps > 0.06. Next, the individual L scores in each cell in the design were subjected to a three-way ANOVA for mixed designs with CONDITION, SESSION, and TEST PAGE as independent variables. The numerical results of the ANOVA are presented in Table 11 (in Appendix 1). A significant main effect of SESSION indicated an increase in L from session 1  to session 2 .Due to many line errors , the results of one participant were excluded from the data analysis. Thus, the sample was reduced to L was much larger in the complete-repetition condition  compared to the complete-alternation condition  and the role-reversal condition , with the two latter conditions not differing very much. The improvements in performance were statistically different from zero for both the complete-repetition condition, t(23) = 14.48, p < 0.001, d = 2.956, and the complete-alternation condition: t(22) = 3.453, p = 0.002, d = 0.720. In contrast, the numerical improvement in the role-reversal condition failed to achieve significance, t(20) = 1.784, p = 0.090, d = 0.389. Pair-wise comparisons further showed that the improvement in the complete-repetition condition was larger than changes in the complete-alternation condition, t(45) = 6.270, p < 0.001, d = 1.830, and the role-reversal condition, t(43) = 5.040, p < 0.001, d = 1.507. In contrast, however, changes in the complete-alternation condition and the role-reversal condition did not differ, t(42) = 0.344, p = 0.733, d = 0.104.A second significant finding concerned the two-way interaction of CONDITION × SESSION. The increase in Q (cf. Table 10 in Appendix 1) to a three-factorial ANOVA, with CONDITION, SESSION and TEST PAGE serving as independent variables, although Shapiro–Wilk tests had revealed that the distribution of Q was not normal in most cells of the design. The numerical results of the ANOVA are presented in Table 12 (in Appendix 1). The only significant effect was the two-way interaction of CONDITION × SESSION. Indeed, Q increased from the first to the second session in the complete-repetition condition , but decreased in both the complete-alternation condition  and in the role-reversal condition . In fact, only the improvement in the complete-repetition condition was significantly different from zero, t(23) = 3.01, p = 0.006, d = 0.615. The small decreases in performance in the complete-alternation condition, t(22) = − 0.206, p = 0.839, d = − 0.043 and the role-reversal condition, t(20) = − 0.344, p = 0.735, d = − 0.075, were not significantly different from zero. In addition, pair-wise comparisons showed that the improvement in the complete-repetition condition was significantly larger than the change observed in the complete-alternation condition, t(45) = 2.660, p < 0.011, d = 0.776, but only numerically larger than the change observed in the role-reversal condition, t(43) = 1.95, p = 0.058, d = 0.583.We subjected the means of K was consistent with the normality assumption for ten of the twelve design cells. The means are shown in Table 10 (Appendix 1). We subjected our K data to a three-factorial ANOVA for mixed designs with CONDITION, SESSION and TEST PAGE as independent variables. The numerical results of the ANOVA are presented in Table 13 (in Appendix 1). The only significant main effect of SESSION reflected an increase of K from session 1  to session 2 .Shapiro–Wilk tests revealed that the distribution of K scores between the two sessions separately for each condition. In numerical terms, the increase in K was much larger in the complete-repetition condition  compared to the complete-alternation condition , as well as the role-reversal condition , with the latter two conditions being broadly similar. The improvements in performance were statistically different from zero for the complete-repetition condition, t(23) = 13.671, p < 0.001, d = 2.791, and the complete-alternation condition, t(22) = 3.450, p = 0.002, d = 0.719. In contrast, the numerical improvement in the role-reversal condition missed significance, t(20) = 1.649, p = 0.115, d = 0.360. Pair-wise comparisons further showed that the improvement in the complete-repetition condition was larger than the changes in the complete-alternation condition, t(45) = 6.715, p < 0.001, d = 1.959, and the role-reversal condition, t(43) = 4.882, p < 0.001, d = 1.459. In contrast, changes in the complete-alternation condition and the role-reversal condition did not differ, t(42) = 0.226, p = 0.822, d = 0.068. The remaining F tests were not significant.The only significant interaction was the CONDITION × SESSION interaction. To clarify the source thereof, we compared the L measure , while performance in the role-reversal condition showed only a small numerical improvement . Second, in the complete-alternation condition, we observed a moderate improvement of performance from the first to the second session in the L measure . This observation provides (novel) direct evidence for the notion that stimulus-independent learning can occur in the (first session of a) FAIR test. Third, when compared directly, the role-reversal condition and the complete-alternation condition showed similar changes in performance from the first to the second session. This finding suggests that stimulus-dependent learning in the first session with the FAIR test mainly improved the processing of target stimuli.In Experiment 3, we used eight self-constructed versions of the FAIR-2 test that allowed the creation of a complete-alternation condition, which did not involve overlap between the stimuli in two tests, in addition to a complete-repetition condition and a role-reversal condition. Experiment 3 produced three notable results. First, with regard to the findings of Experiment 1, we replicated the strong performance improvement in the complete-repetition condition in the The main purpose of the present study was to investigate the effects of repeated testing with the FAIR-2 test, a pen-and-paper test of selective visual attention. In the following sections, we summarize our main findings and discuss a number of theoretical and practical implications thereof.L), and small benefits in search accuracy , when the test is repeated. In particular, concerning search speed (L), we observed large repetition benefits in Experiments 1  and 2 . Concerning search accuracy (Q), we observed a moderate repetition benefit in Experiment 1  and a small numerical benefit in Experiment 2 . Given the fact that repetition benefits have previously been reported for other pen-and-paper tests of selective attention  suggest that participants acquire and improve both stimulus-dependent and stimulus-independent search capabilities , whereas search accuracy remained the same . The results disproved the hypothesis of symmetrical transfer effects of stimulus-dependent learning in the complete-repetition and role-reversal conditions because performance (changes) in the complete-alternation condition was not in between the performance changes observed in the former conditions. Rather, performance (changes) in the complete-alternation condition were not significantly different from performance (changes) in the role-reversal condition.In Experiment 3, we established a “neutral” or complete-alternation condition between two sessions with our self-constructed versions of the FAIR test. The results showed a moderate improvement in search speed  the selective improvement of target processing , a moderate improvement in the complete-alternation condition  and only a numerical improvement in the role-reversal condition . It should be highlighted that the performance improvement in the complete-repetition condition was larger than in the remaining conditions, whereas changes in the complete-alternation and role-reversal conditions did not differ. A similar pattern of findings has previously been observed  in the complete-repetition condition  differ from the pattern of performance changes observed after 2 weeks (cf. Appendix 2). The persistence of practice effects in the FAIR-2 has implications for research and practice. An obvious implication for research on learning in visual search is the insight that practice effects, which are often investigated and observed for only very short temporal intervals in the laboratory  from the first to the second test page, whereas Q remained constant within a session. In Experiment 2 (3 months), we observed small decrements in L  during the first session only, whereas Q decreased  during both sessions. This pattern may suggest that, in Experiment 2, participants changed their speed-accuracy ratio during the first session in favor of more speed, while sacrificing accuracy. Finally, in Experiment 3, neither speed (L) nor accuracy (Q) changed significantly during a test session.Besides investigating the changes in performance between two testing sessions, we also addressed the changes in performance within a session with the FAIR-2. Therefore, we compared performance in the first half of the test  with performance in the second half . These comparisons revealed rather mixed results, however. In Experiment In summary, our findings concerning changes in performance within a session with the FAIR-2 test are different from those reported for the d2 by Steinborn et al. . These aThe fact that repeated testing with the FAIR-2 causes large and persistent improvements in test performance creates a problem for both the reliability and the validity of the test Supplementary file2 (DOCX 25 KB)Below is the link to the electronic supplementary material.


However, their poor toughness, resulting from the high crosslinking density of the epoxy network, must be improved to expand their application to the manufacturing of flexible products. In this study, ductile epoxy thermosetting was produced using thiol compounds with functionalities of 2 and 3 as curing agents. The mechanical properties of the epoxy were further enhanced by incorporating fumed silica into it. To increase the filler dispersion, epoxide-terminated polydimethylsiloxane was synthesized and used as a composite component. Thanks to the polysiloxane–silica interaction, the nanosilica was uniformly dispersed in the epoxy composites, and their mechanical properties improved with increasing fumed silica content up to 5 phr (parts per hundred parts of epoxy resin). The toughness and impact strength of the composite containing 5 phr nanosilica were 5.17 (±0.13) MJ/m Owing to their high density in crosslinking , epoxy t−1 for Si–H stretching [1H NMR spectrum of ep-PDMS, the proton resonances for Si–CH2 formed via the hydrosilylation reaction appeared at around 0.6 ppm. The proton peaks for oxirane were observed at 2.9 ppm and 3.6 ppm, indicating that oxirane was intact despite the hydrosilylation reaction. The presence of siloxanes was confirmed by the two signals observed at 7.59 ppm (for CH2Si(CH3)2O) and −21.80 ppm (for OSi(CH3)2O) in the 29Si NMR spectrum of ep-PDMS.Epoxide-terminated polydimethylsiloxane (ep-PDMS) was synthesized by reacting an allyl epoxide with a hydride-terminated PDMS through the platinum-catalyzed hydrosilylation reaction  Figure . The comretching  . When the samples were heated in a differential scanning calorimeter, one exothermic peak stemming from epoxy curing was observed, and the peak temperature (pT) of the exothermic curve increased with increasing β  were added to the epoxy resin. The thiol-based curing agent comprised EDT and TMPMP with functionalities of 2 and 3, respectively, at the EDT-to-TMPMP weight ratio of 1 to 1. Because the base-catalyzed epoxy-thiol curing reaction is complex , the degeasing β . β/pT2) and 1/pT, the apparent activation energy (aE) and pre-exponential factor (A) were calculated based on the Kissinger equation (Equation (1)) [aE and A values of the epoxy/thiol/MI curing were calculated to be 67.76 KJ/mol and 2.26 × 108 min−1 from the slope and y-intercept of the plotting line of 1/pT vs. ln(β/pT2). The apparent activation energy was also calculated based on the Ozawa equation (Equation (2)) [pT and ln(β) was obtained, and the aE value was calculated to be 70.25 KJ/mol from the slope of the line.β is the heating rate, K/min; pT is the exothermic peak temperature, K; aE is the apparent activation energy, J/mol; R is the perfect gas constant of 8.314 J/(mol·K); and F(a) is a constant function.After data obtained from the DSC analysis were converted to ln(ion (1))  )   of the epoxy/thiol/MI curing was determined as 0.93 using the averaged aE value of 69.00 KJ/mol according to the simplified Crane equation (Equation (3)) [α) of the epoxy/thiol/MI system over time at various temperatures was obtained based on Equation (4) [Next, the reaction order (ion (3)) . Finallytion (4) , and theNext, nanocomposites were fabricated by mixing neat epoxy and fumed silica. Thanks to the possible interactions between the (Si–O) backbone of ep-PDMS and the silanol groups of the nanosilica, the filler loading reached 7 phr (parts per hundred parts of epoxy) in the composites. The effect of nanosilica loading on the rheological properties of the composites was investigated by measuring the frequency dependence of complex viscosity a. The co3. It is interesting that the strength and toughness of NC-4 were lower than those of NC-3 despite higher filler loading. Given that clusters of fumed silica aggregates were formed with increased filler content, the density in the filler-rich phase was high in NC-4; thus, the applied energy during the tensile test was concentrated on the fillers and not transferred to the matrix, causing fracture to occur at the low elongation.The effect of the nanosilica content on the mechanical properties of the nanocomposites was investigated through a tensile test. Dog-bone-shaped specimens were pulled in the tensile direction until they were broken. The mechanical properties are summarized in Next, to investigate the effect of ep-PDMS on the mechanical properties of the composites, a control composite containing 5 phr fumed silica was fabricated without ep-PDMS. The mechanical properties of the control sample are summarized in 2, and this high value is rare for epoxy composites [2 [The effect of fumed silica content on the impact strength of composites was investigated through a notched Izod impact test. Rectangular specimens with a V-shaped notch were struck by a hammer, and the absorbed energy was recorded. The results are summarized in mposites ,28,29,30sites [2 . On the Next, the thermal stability of the neat epoxy and the nanocomposites was investigated by thermogravimetric analysis (TGA) in temperatures ranging from 50 °C to 450 °C . All speBisphenol A diglycidyl ether  was purchased from Kukdo Chemical Co., Ltd . 1-methylimidazole (MI) was purchased from Tokyo Chemical Industry Co., Ltd. . Hydride-terminated poly(dimethylsiloxane) , platinum(0)-1,3-divinyl-1,1,3,3-tetramethyldisiloxane complex solution in xylene (Pt cat), 2,2′-(ethylenedioxy)diethanethiol (EDT), trimethylolpropane tirs(3-mercaptopropioante) (TMPMP), and anhydrous magnesium sulfate were purchased from Sigma-Aldrich Korea Ltd. . Toluene was purchased from Samchun Chemical . Fumed silica (K-200) of silica aggregates consisting of 7 to 40 nm primary particles was purchased from OCI . All chemicals were used as received without purification.1H NMR, 13C NMR, and 29Si NMR spectra were measured on an NMR spectrometer equipped with Bruker Top Spin 3.2 software . The Fourier transform infrared (FTIR) spectra in the range from 500 to 4000 cm−1 were obtained through the attenuated total reflectance method using an FTIR spectrophotometer . A tensile test was carried out using a universal testing machine (HZ-1003A/B(1T), MMS Tech, Bucheon, Korea). A non-isothermal DSC analysis was performed using a DSC-4000 . Izod impact strength was measured using an Izod impact tester . The surface morphologies of the fractured composites were analyzed using a field emission scanning electron microscope (FESEM). The An allyl epoxide was synthesized following the previously reported method . A flask1H NMR  data: δ: 7.15–7.13 , 6.93–6.90 , 6.83–6.81 , 4.25–3.80 , 3.66–3.62 , 2.90–2.88 , 2.78–2.65 , 1.66 , 0.75–0.55 , 0.30–0.00 .13C NMR  data: δ: 156.4, 131.5, 130.1, 126.9, 121.0, 111.4, 68.6, 50.4, 44.6, 34.0, 23.7, 18.4, 0.99, 0.79.29Si NMR  data: δ: 7.59 (CH2Si(CH3)2O), −21.80 (OSi(CH3)2O).Each composition of neat epoxy and nanocomposites is summarized in The as-prepared compositions were poured into a dog-bone-shaped Teflon mold and heated at 140 °C for 1 h. Then, the specimens were placed in a tensile tester and pulled along the z-axis until they were broken.Izod impact strength was measured following an ASTM D256 standard test method using a pendulum-C type hammer. The as-prepared compositions of neat epoxy and nanocomposites were poured into a Teflon mold with dimensions of 63.5 mm × 12.7 mm × 6.35 mm (width × length × thickness) and kept at 140 °C in a convection oven. After 1 hour, all specimens were cooled to room temperature, and a V-notch with a depth of 2.54 mm was made at the center of them using a cutter.3 and 69.8 (±1.3) KJ/m2, respectively. The mechanical properties of the composite without ep-PDMS containing 5 phr nanosilica were inferior to those of NC-3 despite having the same filler content because of the poor dispersion of fumed silica. In conclusion, the employment of ep-PDMS is expected to be an effective way to realize super-toughened fumed-silica epoxy composites.In this work, epoxide-terminated PDMS (ep-PDMS) was synthesized and used to produce thiol-epoxy composites containing fumed silica. Thanks to the polysiloxane–silica interaction, the mechanical properties of the epoxy composites containing ep-PDMS improved with increasing fumed silica content up to 5 phr. The toughness and impact strength of NC-3 were 5.17 (±0.13) MJ/m


The canonical WNT/β-catenin pathway is upregulated in cancers and plays a major role in proliferation, invasion, apoptosis and angiogenesis. Recent studies have shown that cancer processes are involved under normoxic conditions. These findings completely change the way of approaching the study of the cancer process. In this review, we focus on the fact that, under normoxic conditions, the overstimulation of the WNT/β-catenin pathway leads to modifications in the tumor micro-environment and the activation of the Warburg effect, i.e., aerobic glycolysis, autophagy and glutaminolysis, which in turn participate in tumor growth.The canonical WNT/β-catenin pathway is upregulated in cancers and plays a major role in proliferation, invasion, apoptosis and angiogenesis. Nuclear β-catenin accumulation is associated with cancer. Hypoxic mechanisms lead to the activation of the hypoxia-inducible factor (HIF)-1α, promoting glycolytic and energetic metabolism and angiogenesis. However, HIF-1α is degraded by the HIF prolyl hydroxylase under normoxia, conditions under which the WNT/β-catenin pathway can activate HIF-1α. This review is therefore focused on the interaction between the upregulated WNT/β-catenin pathway and the metabolic processes underlying cancer mechanisms under normoxic conditions. The WNT pathway stimulates the PI3K/Akt pathway, the STAT3 pathway and the transduction of WNT/β-catenin target genes (such as c-Myc) to activate HIF-1α activity in a hypoxia-independent manner. In cancers, stimulation of the WNT/β-catenin pathway induces many glycolytic enzymes, which in turn induce metabolic reprogramming, known as the Warburg effect or aerobic glycolysis, leading to lactate overproduction. The activation of the Wnt/β-catenin pathway induces gene transactivation via WNT target genes, c-Myc and cyclin D1, or via HIF-1α. This in turn encodes aerobic glycolysis enzymes, including glucose transporter, hexokinase 2, pyruvate kinase M2, pyruvate dehydrogenase kinase 1 and lactate dehydrogenase-A, leading to lactate production. The increase in lactate production is associated with modifications to the tumor microenvironment and tumor growth under normoxic conditions. Moreover, increased lactate production is associated with overexpression of VEGF, a key inducer of angiogenesis. Thus, under normoxic conditions, overstimulation of the WNT/β-catenin pathway leads to modifications of the tumor microenvironment and activation of the Warburg effect, autophagy and glutaminolysis, which in turn participate in tumor growth. Metabolic reprogramming is one of the main characteristics of cancer cell activity. Cancer metabolism reprogramming not only provides ATP (adenosine triphosphate) for cancer cells, but also essential macromolecules for its protein and nucleotide biosynthesis. Several studies have shown that cancer cell metabolic reprogramming is controlled by a number of different factors ,2, amongMetabolic reprogramming in cancers is characterized by two processes involving autophagy and the Warburg glycolysis. Autophagy in cancers impairs mitochondrial energy homeostasis and favors inflammation and oxidative stress . During The canonical WNT/β-catenin pathway modulates several signaling processes involved in tissue homeostasis. It is now well established that activation of the canonical WNT/β-catenin pathway is necessary for cancer cell survival and maintenance, making it a promising target for an anticancer therapy regimen . An aberIn recent years, many studies have observed the increase in WNT/β-catenin signaling under normoxic conditions, leading to the enhancement of different pathways involved in the processes of cancer progression ,20. Thus2, which is the blood concentration of O2 multiplied by blood flow [2 absorption rates depend on the availability of O2 and therefore on the effectiveness of blood flow [2 absorption levels [2 consumption levels as the temperature rises to a maximum of around 42 °C. The temperature coefficient Q10 obtained  is 2.3, thus clearly showing fully functional mitochondrion [2 in the arterial blood feeding a tumor is a function of either the partial O2 arterial pressure (pO2), the hemoglobin content (cHb) or in combination. Modifications to each of these parameters influence the availability of oxygen as well as the rate of O2 absorption by tumors [For decades, the Warburg effect  has been described as the sequelae of damaged mitochondria  and as aood flow ,23. O2 aood flow . An incrn levels . Isolatehondrion . The cony tumors .The Warburg effect still attracts considerable interest in research based on mutations in TCA (tricarboxylic acid) cycle enzymes  and the The aerobic glycolysis may not simply be due to an increase in the glycolytic mechanism, but also to a decrease in the activity of mitochondria. This phenomenon is stimulated by the transcription factor HIF-1α, which can be increased under both hypoxic and normoxic conditions. HIF is considered to be a major modulator of the Warburg effect. HIF-1α can decrease the mitochondrial mechanism by direct stimulation of pyruvate dehydrogenase kinase 1 (PDK-1), which in turn inhibits the PDH (pyruvate dehydrogenase). Pyruvate  cannot enter the TCA cycle, which results in a reduction in OXPHOS (oxidative phosphorylation), cellular oxygen consumption and ROS generation ,28. A seMetabolic reprogramming, a consequence of metabolism plasticity, has been observed as one of the major mechanisms in cancer processes . This prIn parallel with the limited supply of ATP, the Warburg effect produces antioxidant fragments (2 NADH per glucose mole) and enables the diversion of glycolytic intermediates to numerous biosynthesis pathways , thus supporting biosynthesis pathways as long as adequate glucose intake is controlled. These biosynthesis pathways are caused by a decrease in the role of the pyruvate kinase enzyme, which catalyzes the final stage of the Warburg effect, converting phosphoenolpyruvate into pyruvate. Aerobic glycolysis can decrease the production of ROSs  which, aHigh rates of lactate release in TME result from increased regulation of aerobic glycolysis, and c-Myc-induced glutaminolysis, another major feature of cancer metabolic reprogramming. For increased lactate production in the Warburg effect, activation of both HIF-1α- and c-Myc increases the expression of PDK-1 and lactate dehydrogenase A (LDH-A), which converts pyruvate into lactate, contributing to the Warburg effect phenotype . The incThe transformation of the TME occurs in order to initiate angiogenesis. To increase molecular mechanisms and cellular movements, the cellular environment is modified. High lactate release rates lead to numerous cancerous mechanisms, including microenvironment transformation , cellulaThe activation of glycolysis alters cellular pH and osmolarity by stimulating lactate production ,51. The Lactate acid stimulates the production of metalloproteases and plasmin, disrupting the ECM, collagen and basal membrane ,37,55. SLactate activates the production of vascular growth factors by tumor cells, macrophages and fibroblasts . LactateNumerous genetic mutations (including mutated genes that encode the colony stimulating factor (CSF) receptor), the epidermal growth factor receptor (EGFR), p53 and PTEN (phosphatase and TENsin homolog), result in HIF-1α stabilization and stimulate angiogenesis through the upward control of angiogenic factors ,66,67,68The Canonical WNT/β-Catenin PathwayDrosophila melanogaster and its mouse homolog Int. The WNT/β-catenin pathway is involved in numerous signals and molecular pathways, including embryogenesis, cell proliferation, cell migration and cell polarity, apoptosis and organogenesis [The WNT name is derived from Wingless ogenesis . Neverthogenesis .The WNT pathway belongs to the family of secreted lipid-modified glycoproteins  Figure . WNT ligAt the transcriptional level, WNT signaling is primarily mediated by a family of transcription factors known as the β-catenin/T-cell factor/lymphoid enhancer factors (TCF/LEF). The accumulation in the cytoplasm of β-catenin is generated by the destruction complex AXIN, tumor suppressor adenomatous polyposis coli (APC) and glycogen synthase kinase-3 (GSK-3β). In the absence of WNT ligands, the destruction complex enhances phosphorylation of the cytoplasmic β-catenin and induces its degradation in the proteasome. In the presence of the WNT ligands, β-catenin binds to Frizzled (FZL) and LDL receptor-related protein 5/6 (LRP 5/6) , therebyGSK-3β is a major negative modulator of WNT/β-catenin signaling ,108,109.Glucose is the main source of energy for cells. Glucose is metabolized to generate ATP (as energy) by cytoplasmic glycolytic mechanisms and oxygen-dependent mitochondrial processes . Tumor cells are dependent on glycolysis for their primary source of energy , with acThe STAT3  belongs to the family of cytosolic transcription factors that allow the activation of nuclear pathways . The STAA STAT3 phosphorylated by IL-6 or LIF (leukemia inhibitory factor) is associated with activation of HIF-1α in normoxia . The β-cThe receptor tyrosine kinase (RTK) and the EGFR are enhanced in cell division, migration, adhesion, differentiation and apoptosis ,140. EGFThe PI3K/Akt pathway plays a key role in the control of cellular metabolism, growth, proliferation and cell survival ,148. ThePI3K activates mTORC1 through a dependent Akt pathway. mTORC1 decreases the tuberous sclerosis complex (TSC) ,154. TSCHIF-1α expression in human bodies enhances angiogenesis by transcriptionally stimulating several angiogenic genes and their receptors, including VEGF . HIF-1 iActivation of HIF-1α leads to the activation of gene transcription encoding several glycolysis enzymes  ,170,171 Glucose-transporters, Glut-1 and Glut-3, have their main roles in the insulin-sensitive homeostasis of glucose transport . Glut-1 HK2 activation leads to the transformation of glucose into glucose-6-phosphate. HK2 is stimulated by both c-Myc and HIF-1α ,124,186.The final step of glucose metabolism is the conversion of phosphoenolpyruvate and adenosine diphosphate (ADP) into both ATP and pyruvate. The enzyme pyruvate kinase (PK) is responsible for this phenomenon. Four isozymes of PK are defined: PKL, PKR, PKM1 and PKM2 . PKM2 isPDK-1 acts as a major modulator of the glycolysis metabolism by directly phosphorylating the PDH complex to downregulate the mitochondrial conversion of pyruvate to acetyl-CoA . PDK-1 cLDH is a tetrameric enzyme that belongs to the 2-hydroxy acid oxidoreductase family. LDH stimulates the levels of both the inter-conversion of pyruvate to lactate and the nicotinamide adenine dinucleotide (NADH) into NAD+ . Four LDIndependently of hypoxia, acidic extracellular pH, caused by the increased lactate production, can upregulate IL-8 and VEGF ,210,211.It is widely known that autophagy is damaged in cancer processes. Autophagy represents a major mechanism in preserving stem cell homeostasis by finely tuning stem cell maintenance and differentiation ,213. In Autophagy (from the Greek word for “self-eating”) is a phenomenon that depends on energy, and one that is highly regulated and conserved. Autophagy is responsible for the sequestration and recycling of redundant or altered cell organelles, long-lived proteins and even pathogens . A basalThree types of autophagy may be identified at the cellular level: micro-autophagy, macro-autophagy and chaperone-mediated autophagy (CMA) . Micro-autophagy follows the direct invagination of the lysosomal membrane, which forms an autophagic body and causes the degradation of the sequestered part of the cytosol . The CMAAutophagy can be investigated at various stages: induction and initiation of autophagy, nucleation of the phagophore, expansion of the phagophore, targeting of cargo, completion of autophagosomes and fusion with lysosomes, and degradation of content . AutophaAs a crucial compound of cell defense mechanisms, autophagy has an important role in the destruction mechanism of damaged organs, dysregulated proteins and metabolite recycling in order to maintain the stability of the internal environment . In cancHowever, while autophagy slows down the tumor initiation process, other studies have suggested that decreased autophagy may contribute to the tumor initiation process. Genetic ablation of essential autophagy genes in genetically modified mouse models for tumors has revealed the major role of autophagy in promoting malignant tumors ,237. AutDuring tumor growth, cancer cells use metabolic reprogramming to maintain growth, survival, proliferation and migration processes. To meet different metabolic needs, autophagy is modified. Currently, the relationship between autophagy and glycolytic metabolism is subject to extensive investigation. Glucose is the major source of nutrients for the production of cellular energy and a constant supply of glucose is required for the production of ATP. As a major biological mechanism, glycolysis is consubstantial with autophagy. Metabolic reprogramming induces cancer progression to regulate autophagy. Thus, glycolysis controls autophagy and participates fully in the survival of tumor cells. When the glycolytic process is decreased, as in the context of aerobic glycolysis, autophagy is improved and becomes a driving force for oxidative phosphorylation to support the production of ATP . PyruvatThe interaction between autophagy and distinct signaling, such as that associated with the WNT/β-catenin, SHH and TGF-β pathways, has a vital role in several cellular mechanisms  Figure . Numerous studies have shown that activated WNT/β-catenin is a negative controller of autophagy based on the evidence that high rates of autophagy are not compatible with cellular proliferation and survival enhanced by the WNT/β-catenin pathway . ActivatThus, the WNT/β-catenin pathway exercises a negative control on autophagy. The nuclear accumulation of β-catenin leads to the inhibition of the p62/SQSTM1 promoter, which enhances autophagy diminution . ConversDVL is involved in the regulatory feedback process between autophagy and the WNT/β-catenin pathway . DVL is Conversely, in vitro studies have shown that the WNT/β-catenin pathway and autophagy can be stimulated at the same moment . Thus thIn parallel to the shift toward the Warburg effect, tumor cells rely on the provision of bulk quantities of amino acids as major enhancers for cellular survival and growth, in order to complete the TCA cycle . StimulaThe WNT/β-catenin pathway plays an important role in glutamine metabolism. Research has shown that β-catenin target genes are involved in glutamine uptake and metabolism . While tAlthough it has been suggested that the NF-κB pathway is a modulator of glutaminolysis in breast tumor cells, the role of the WNT/β-catenin pathway in this control is still unclear .GSH, a direct product of glutamine metabolism, plays a key role in the chemoresistance of tumor cells  and cancYes-associated protein 1 (YAP) / transcription coactivator and PDZ-binding motif (TAZ) are transcription co-activators of the Hippo kinase complex . The HipThere is interplay between the WNT/β-catenin signaling and the YAP/TAZ signaling. The YAP/TAZ pathway is responsible for WNT-induced biological mechanisms ,280, whiAPC binds with SAV1 and LATS1, which are upstream controllers of YAP/TAZ. The modulation of the YAP/TAZ pathway by APC is a major reason for intestinal tumor progression. Furthermore, DVL is required for the nucleocytoplasmic shuttling of YAP . WNT actActivation of YAP/TAZ leads to stimulation of glutamine levels, to the enhancement of nucleotide synthesis, and to the promotion of glutamine synthetase (GS) expression . The stiThe stimulation of YAP/TAZ leads to the enhancement of glycolysis in cancer cells. YAP-5SA activates YAP , stimulaThe WNT/β-catenin pathway is over-activated in several cancers and plays a major role in proliferation, invasion, apoptosis and angiogenesis. Moreover, the nuclear β-catenin levels are associated with malignancies. Hypoxia is a well-known process that activates HIF-1α to promote the glycolysis metabolism. The WNT/β-catenin pathway stimulates the PI3K/Akt and STAT3 pathways and the transduction of several target genes, including c-Myc, which can stimulate HIF-1α in a hypoxia-independent manner. In cancers, the stimulation of the WNT/β-catenin pathway leads to many glycolytic enzymes that in turn induce metabolic reprogramming (the Warburg effect) in association with autophagy modification. This metabolic reprogramming leads to the production of lactate, as the primary alternative of ATP, at all oxygen rates, even under normoxia. An increase in lactate production is correlated with the modification of the tumor microenvironment, the Warburg effect, autophagy and glutaminolysis, leading to tumor growth under normoxic conditions.