Obesity has become a major risk factor for developing metabolic diseases, including insulin resistance, type 2 diabetes, and hypertension. Growing pieces of evidence indicate that the Wnt/β-catenin signaling pathway plays an important role in adipogenesis and obesity. Activation of the Wnt/β-catenin signaling pathway inhibits adipogenesis by suppressing the differentiation of committed preadipocytes into mature adipocytes. CXXC5 is highly induced with suppression of Wnt/β-catenin signaling in early adipogenic differentiation. In addition, silencing CXXC5 in vitro increased β-catenin and decremented the major adipogenic differentiation markers. KY19334, a small molecule that activates the Wnt/β-catenin pathway via inhibition of CXXC5- Dishevelled (Dvl) protein–protein interaction (PPI), suppressed adipogenic differentiation. Administration of KY19334 ameliorated obesity by 26 ± 1.3% and insulin resistance by 23.45 ± 7.09% and reduced adipocyte hypertrophy by 80.87 ± 5.30% in high-fat diet (HFD)-fed mice. In addition, KY19334 accelerated the browning of adipose tissue and promoted hepatic glucose homeostasis in HFD-fed mice. In conclusion, activation of the Wnt/β-catenin signaling by inhibiting the interaction of CXXC5 and Dvl by small molecule-mediated interference is a potential therapeutic approach for treating obesity and insulin resistance. In the past, dietary and lifestyle modifications can be effective to in improving obesity and preventing diabetes. However, these approaches are challenging to maintain in the long term3. Current pharmacological approaches for treating obesity focus on reducing appetite, calorie intake, or absorption of dietary fat6. Unfortunately, anti-obesity therapies have been unsatisfactory due to the adverse effects, low safety, and poor long-term maintenance of weight loss8. Therefore, understanding the regulatory mechanisms of adipogenesis is necessary to develop better therapies for treating obesity and adipose tissue inflammation.Obesity has become a global epidemic and is associated with metabolic diseases, including type 2 diabetes, dyslipidemia, and hypertension, and is also known as a high-risk factor severe COVID-1910. Activation of the Wnt/β-catenin pathway inhibits adipogenesis by suppressing the expression of the peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα)10 which are subjected to the repression by WNT1-inducible signaling pathway protein-1 (WISP1), a direct transcriptional target of the Wnt/β-catenin signaling. Therefore, identifying a factor regulating the Wnt/β-catenin pathway, especially which drives obesity, is important for developing anti-obesity drugs.The canonical Wnt/β-catenin pathway plays important roles in the development, tissue homeostasis, cell proliferation, and cell differentiation12. CXXC5 plays various pathophysiological roles involving adult tissue regeneration at the specific pathophysiological status16. Growing evidence indicates that CXXC5 is a key driving factor for metabolic diseases17 and inhibition of CXXC5 function accelerates liver tissue regeneration with metabolic improvements18. Thus we reasoned that CXXC5 might be highly expressed with suppression of Wnt/β-catenin signaling in the early adipogenic differentiation during the development of obesity. Here, we investigated the role of the CXXC5 in inhibiting Wnt/β-catenin signaling and adipocyte differentiation by using KY19334, a small molecule that activates the Wnt/β-catenin signaling via interference in the CXXC5-Dvl interaction19. By oral administration, KY19334 improved the pathological features of obesity, including adipocyte hypertrophy and hepatic steatosis induced by a high-fat diet (HFD). The systemic effectiveness of KY19334 on the anti-obesity was acquired by blockade of the function of the aberrantly overexpressed CXXC5 and subsequent inhibition of the Wnt/β-catenin signaling suppression in the early developmental stage of obesity by HFD. Indirect activation of Wnt/β-catenin signaling, actually its restorative activation by the interference of the CXXC5 function, could be an effective and safe approach for anti-obesity.CXXC5-type zinc finger protein 5 (CXXC5) is a negative feedback regulator of the Wnt/β-catenin pathway that functions via binding to Dishevelled (Dvl)Taken together, our findings revealed that the small molecular approach for activating Wnt/β-catenin signaling via interference of the CXXC5-Dvl protein–protein interaction (PPI) provides a potential anti-obesity approach against over-nutrition involving HFD.Cxxc5−/− mice  24. To further characterize the adiposity suppression by KY19334, the KY19334 effect on browning scWAT was investigated in HFD-fed mice. The fat thickness between the skin and muscle layers was significantly decreased by KY19334 treatment compared with those revealed by vehicle- and orlistat-treated HFD mice  than epiWATThe liver tissues of KY19334-treated mice did not form lipid droplets with a reduction in Pparγ and Cebpα-positive cells compared with those of vehicle- and orlistat-treated mice on HFD Fig. A,B. The 25. Growing pieces of evidence indicate that substantial early weight loss by targeting the metabolic tissues such as adipose tissue, liver, and skeletal muscle have importance on efficacy and sustainability of anti-obesity26.The current clinically applicable pharmacological agents for anti-obesity, including orlistat, lorcaserin, liraglutide, and phentermine/topiramate, have been prescribed for weight loss with the mechanisms of reduction of calorie absorption or appetite. However, low efficacy and safety issues have resulted in their limitations in developing pharmaceutical agentsIn the present study, we provided an approach for suppressing early adipogenic differentiation during the development of obesity. By illustrating the role of Cxxc5 in inhibiting the Wnt/β-catenin pathway related to adipogenic differentiation and a small molecule-mediated interference of the Cxxc5 function up-regulating Wnt/β-catenin signaling is suggested as a new potential anti-obesity target.Cxxc5−/− MEFs as well as the knockdown of Cxxc5 in adipogenic differentiation in vitro.The pathological significance of the CXXC5 function was indicated by suppression of Wnt/β-catenin signaling with overexpression of CXXC5 in the adipocyte tissues of patients with obesity as well as the early adipogenic differentiation of preadipocytes. The role of Cxxc5, which suppress Wnt/β-catenin signaling in the adipogenesis was confirmed by the phenotypes of the 27. Fundamentally different with orlistat, KY19334 suppress inflammation with the improvement of obesity by the Wisp1-mediated suppression of expression of the Pparγ and Cebpα9. Moreover, many other factors which are directly induced by Wnt/β-catenin signaling such as TCF7L2, GLP-1, PPARγ, FGF19, and FGF21 are also attributed to the anti-obesity accompanying suppression of inflammation30, hepatic steatosis31, and activation of browning of WAT32, resulting in improvement of the multiple metabolic parameters. Furthermore, ectopic expression of UCP1 in scWAT from KY19334-treated mice was linked to protecting diet-induced obesity by increased fatty acid oxidation of scWAT.The anti-obesity effects of KY19334 could be acquired by suppression of early adipogenic differentiation, the initial event of obesity, accomplished by controlling the function of CXXC5 by the interference of the Cxxc5-Dvl PPI. Administration of KY19334, which restores the suppressed Wnt/β-catenin pathway in the HFD-induced obese mouse, showed anti-obesity effects with the suppression of insulin resistance, inflammation, and hepatic steatosis as well as the induction of adipose tissue browning. The KY19334-mediated suppression of hypertrophy of adipose tissues accompanying improvement of various metabolic factors during adipogenesis in the HFD-fed mice did not or marginally occur by administration of the orlistat, a lipase inhinitorThe role of CXXC5 in obesity was correlated with high induction of the inflammation markers and cytosolic accumulation of CXXC5 was inversely correlated with β-catenin in the metabolic tissues of the HFD-induced obese mice.Overall, CXXC5 overexpression plays a role as a major driver in the adipogenic differentiation the obesity. An approach for restoring the suppressed Wnt/β-catenin signaling via blockade of CXXC5-Dvl PPI offer a novel approach for anti-obesity with the improvement of metabolic parameters on HFD-mediated over nutrition. In addition, this approach activating the Wnt/β-catenin signaling, not by direct activation, but by release of the suppressed Wnt/β-catenin signaling by blockade of a negative feedback mechanism by CXXC5 could provide a safe anti-obesity approach effect which is a major huddle in the development of anti-obesity drugs and targeting the Wnt/β-catenin pathway. The small molecule approach interfering CXXC5 function provides a potential treatment of overall metabolic abnormalities as well as obesity involving over nutrition related to the HFD.n = 6 each group): NCD or HFD consisting of 60% calories from fat . HFD-fed mice were orally administered with KY19334 (25 mg/kg) or orlistat (25 mg/kg) for 8 weeks. Body weight and food intake were measured once a week during the study. Assessment of fasting glucose levels, glucose tolerance test (GTT) and insulin tolerance test (ITT) were described in a previous study17. All mice were maintained under temperature and light-controlled (standard 12 h light/dark cycle) conditions and provided with food and water ad libitum. All animal experiments were conducted in compliance with the guidelines which were reviewed and approved by the Institutional Review Board of Severance Hospital, Yonsei University College of Medicine (09-013). The study was carried out in compliance with the ARRIVE guidelines.Twenty-four male C57BL/6 mice (5-week-old) were purchased from KOATECH . The mice were housed in a pathogen-free facility at Yonsei University . After a 1-week acclimation period with a commercial diet and tap water, mice were weight matched and divided into four different dietary groups  imaging sequence was used for high-contrast fat imaging: TE 5.9, TR 200, RARE factor 8, flip angle 60, matrix 256 × 128 × 128, 7 × 3 × 3 cm to 9 × 3 × 5 cm . Using this protocol, a 3D-reconstructed image was produced using a maximum intensity projection algorithm with an intensity threshold that shows fat as the brightest signal and disregards signals from other tissues. The total body fat and separated specific fat deposits were calculated using thresholding and voxel count plug-ins from NIH ImageJ software and a VolumeJ plug-in to create three-dimensional fat images. An animal management system in conjunction with the MRI was used to record core, skin, ambient and water-blanket temperature measurements for monitoring during imaging. The core temperature of the animal was maintained during imaging and anesthesia induction using a water blanket maintained at 37 °C.g to obtain supernatant to measure metabolic parameters17. ELISA assay kits were used to assess serum leptin , serum resistin , serum FFA . Serum chemistry variables included total cholesterol, high-density lipoprotein (HDL)-cholesterol, glucose, TG, alanine aminotransferase (ALT), and aspartate transaminase (AST) concentrations. The calibration of serum parameters was performed using the quality control card supplied with the FUJI DRI-CHEM slides whenever slides from a new lot were used.Total blood of mice was collected by cardiac puncture after fasting. The blood was allowed to clot for 30 min and was then centrifuged for 10 min at 1000×17. Dissected tissues were fixed in 4% neutral paraformaldehyde and embedded in paraffin. The paraffin sections were cut at a thickness of 4 µm and subjected to H&E staining. The adipocyte cell size was measured in 20 randomly chosen microscopic areas from 3 independent animals using a Nikon bright-field optical microscope (Nikon TE-2000U). The average adipocyte size and dermal thickness were determined using Image J software.H&E staining was performed as previously described17. Paraffin sections of 4 µm in size were deparaffinized and rehydrated. For antigen retrieval, the slides were autoclaved in 10 mM sodium citrate buffer (pH 6.0). Sections were blocked in phosphate buffered saline (PBS) containing 10% BSA at 20 °C for 30 min. The sections were incubated overnight at 4 °C with the following dilution of primary antibodies: anti-β-catenin , anti-CXXC5 (Lab made), and anti-F4/80 . The slides were washed with PBS, incubated with Alexa Fluor 488-  or Alexa Fluor 555-conjugated IgG secondary antibody  at 20 °C for 1 h, and counterstained with DAPI . The images were captured using a LSM700 META confocal microscope (Carl Zeiss) after excitation with 405-, 488-, or 543-nm laser lines.IHC staining was performed as previously described2O2  for 10 min, then blocked in PBS containing 10% BSA at 20 °C for 30 min Sections were incubated with primary antibody overnight at 4 °C with the following dilution of primary antibodies: anti-uncoupling protein 1 , anti-β-catenin , anti-CXXC5 (Lab made), anti-PPARγ , and anti-C/EBPα . Then, sections were incubated with biotinylated anti-rabbit  or anti-mouse  secondary antibody for 1 h at 20 °C. The samples were stained with 3, 3′-diaminobenzidine  for 3–7 min and counter stained with Mayer’s hematoxylin (Muto). All incubations were conducted in humid chambers. Signals were analyzed using a bright field microscope (Nikon TE-2000U).To block endogenous peroxidase activity before peroxidase IHC analysis, tissues were incubated with 1% HLiver tissues were incubated on ice in 100 µl saline solution . Cells and tissues suspensions were assayed for TG content using a TG assay kit .17. Briefly, total RNA was extracted from ground tissue powder using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Reverse transcription was performed with M-MLV reverse transcriptase (Invitrogen) using 2 µg of total RNA. Synthesized cDNA was diluted as a concentration of 100 ng/µl. Quantitative PCR analyses were performed in the Rotor-gene Q real-time PCR cycler (Qiagen) using SYBR green reagent (Qiagen) with conditions of 95 °C for 10 min followed by 40 cycles at 95 °C for 5 s and 60 °C for 15 s. Relative quantification of mRNA levels was estimated using the comparative Ct method (∆∆Ct). All mRNA values were normalized with respect to GAPDH. The primer sequences are listed in Table Real-time PCR was performed as previously describedCxxc5/++ and Cxxc5−/− MEFs were prepared from E13.5-E14.5 embryos. The embryos were cut into small pieces and enzymatically digested using 0.5% Trypsin/EDTA (Gibco). Post-digestion, the tissue was resuspended in DMEM and pipetted through a 40 µM filter (BD) to obtain single cells. MEFs were expended and frozen.Primary Cxxc5/++ and Cxxc5−/− MEFs were maintained in DMEM containing 10% (vol/vol) heat-inactivated fetal bovine serum , 100 mg/ml penicillin/streptomycin, and 2 mM L-glutamin (Gibco). For transient transfection, the cells were grown for 1 day, transfected with the required plasmid or siRNA using the Attractene transfection reagent (Qiagen) in Opti-MEM. After 16 h, the medium was changed to DMEM supplemented with 10% CS, and cells were induced to differentiate in DMEM containing 10% FBS supplemented with MDI  with or without KY19334. After 2 days, the medium was replaced with DMEM containing 10% FBS and 167 nM insulin with or without KY19334. The medium was changed with fresh identical medium every 2 days up to day 14 post-induction. Undifferentiated control groups were maintained in DMEM supplemented with 10% CS, 100 mg/ml penicillin, and 100 mg/ml streptomycin. The cells were incubated in 5% (vol/vol) CO2 at 37 °C.3T3-L1 preadipocytes were maintained in DMEM (Gibco) containing 10% (vol/vol) heat-inactivated calf serum , 100 mg/ml penicillin (Gibco), and 100 mg/ml streptomycin (Gibco). 17. Cells were lysed using radio-immunoprecipitation assay (RIPA) buffer . Samples were separated on 12% SDS polyacrylamide gels and transferred onto PROTRAN nitrocellulose membranes (Shleicher and Schuell Co.). After blocking with PBS containing 5% nonfat dry skim milk and 0.07% (vol/vol) Tween 20, the membranes were incubated with antibody specific for β-catenin , CXXC5 , PPARγ , or C/EBPα  at 4 °C overnight. Membranes were then incubated with horseradish peroxidase-conjugated anti-rabbit  or anti-mouse  IgG secondary antibody. Protein bands were visualized with enhanced chemiluminescence  using a luminescent image analyzer, LAS-3000.Western blot analysis was performed as previously described17. Liver tissues and cells were washed with PBS, 70% isopropanol , and stained with Oil Red O solution (Sigma-Aldrich) at 20 °C overnight. Samples were washed thoroughly with distilled water. Tissues were counterstained with Mayer’s hematoxylin. Images of the Oil Red O staining were visualized with a bright field microscope (Nikon TE-2000U). For the quantification of lipid contents, the Oil Red O was eluted by the addition of 500 µl isopropanol containing 4% nonidet P-40 to each well and the absorbance was measured spectrophotometrically at 590 nm.Oil red O staining was performed as previously describedhttps://www.ncbi.nlm.nih.gov/geo/) and either of the two human omental  and subcutaneous adipose tissue  transcriptome datasets are accessible through GEO accession number GSE1657.The gene expression profile results (Gene ID: 51523) were deposited in NCBI’s GEO database .Data are presented as means ± standard deviation (SD). Statistical analyses were performed using unpaired two-tailed Student’s Supplementary Information.


Human primary liver cancers have a poor prognosis. At present, there are limited treatment alternatives, and to understand the molecular aspects of liver tumorigenesis it is essential to develop more efficient pharmaceutic modalities. Liver tumors have mutations in genes encoding key components of Wnt/β-catenin signaling, and two different molecular pathways have been identified, termed non-canonical and canonical networks. In this review, we discuss the dysregulation of the Wnt/β-catenin signaling network, especially in hepatocellular carcinoma, in cholangiocarcinoma and in patients with hepatoblastoma. Based on the current literature we propose an update of molecular mechanisms focusing on Wnt/β-catenin pathways in physiological conditions and in human primary liver carcinomas, together with an overview of preclinical and clinical studies. The clinical implication of Wnt inhibitors in association with conventional surgical and lo-co-regional therapies is reviewed.Primary liver cancers (PLCs) are steadily increasing in incidence and mortality in the world. They have a poor prognosis due to their silent nature, late discovery and resistance to common chemotherapy. At present, there are limited treatment alternatives, and the understanding of PLC molecular aspects is essential to develop more efficient drugs and therapeutic surgical and loco-regional strategies. A clear causal link with liver damage, inflammation, and regeneration has been found in the occurrence of PLC over the last few decades. Physiologically, Wingless/It (Wnt)-β-catenin signaling plays a key role in liver development, metabolic zonation and regeneration. Loss of functional homeostasis of this pathway appears to be a major driver of carcinogenesis in the liver parenchyma. In the hepatic microenvironment, molecular deregulations that exceed the Wnt signaling biological capacity can induce tumor initiation and progression. Indeed, somatic mutations are identified in key components of canonical and non-canonical Wnt signaling and in PLCs and precancerous lesions. In this review, the altered functions of Wnt/β-catenin signaling are considered in human PLCs, with emphasis on hepatocellular carcinomas (HCC), cholangiocarcinomas (CCA) and hepatoblastomas (HB). Based on recent literature, we also focused on liver cancerogenesis through Wnt deregulation. An overview of preclinical and clinical studies on approved and experimental drugs, targeting the Wnt/β-catenin cascade in PLCs, is proposed. In addition, the clinical implication of molecule inhibitors that have been shown to possess activity against the Wnt pathway in association with conventional surgical and loco-regional therapies are reviewed. Primary liver cancer (PLC) is often diagnosed at an advanced stage. Worldwide, it is the fifth most common cancer, represents the third leading cause of cancer-related death and develops at a rate of 3% to 4% in patients with liver disease ,2. DespiRisk factors for HCC and CCA are heterogeneous and linked to environmental, infectious, and dietary lifestyle origins ,10. ExpoSeveral risk factors, such as liver parasites, chronic biliary tract and liver diseases, including primary sclerosing cholangitis, choledochal cysts, Caroli’s disease and HCV cirrhosis, as well as the same aspects of lifestyle that cause chronic inflammation and cholestasis in the liver, have been linked to the development of CCA ,4,13,14.HB is a rare malignant liver cancer occurring in infants and children at the age of 2–3 years for about 0.5–2% of all pediatric tumors; its etiology remains largely unknown ,17,18. HSurgical resection is the treatment of choice for patients with well-preserved liver function. Liver transplantation is the option for patients who have underlying cirrhosis or in HB. Prevention is important for parasite-endemic and HBV-endemic regions, as well as health education, vaccination, antiviral therapies, and avoidance of toxin exposure . StandarA clear causal link with liver damage, inflammation, and regeneration has been found over the last few decades in the occurrence of PLC . ReceiviCTNNB1 gene encoding β-catenin or in components of the degradation complex, such as AXIN and APC  have been detected in PLC [The Wingless/It (Wnt)/β-catenin signaling plays a critical role in liver development, metabolic zonation and regeneration ,25,26. Md in PLC ,27,28,29d in PLC . Moreoved in PLC ,31. Oncod in PLC .Antitumor therapeutic perspectives aim to guide clinical practice in the direction of personalized treatments. The inhibition of Wnt-ligand production should be considered as a therapy in biliary diseases and primary liver cancers . To thisIn this review, the functions of Wnt/β-catenin signaling are considered in light of its physiological and pathological roles in human liver. The molecular aspects of Wnt/β-catenin cascade in liver development and new insights on Wnt/β-catenin deregulation in PLCs are explored. Although more recent studies have underlined the specific genetic alterations and the oncogenic roles of aberrant Wnt/β-catenin signaling in liver cancer, especially in HCC patients as reported by current literature, we discuss the clinical implication of approved and experimental drugs that have been shown to possess activity against Wnt pathway in association with conventional surgical and loco-regional therapies.The Wnt/β-catenin signaling controls a wide range of biological processes both during embryogenesis and in adult life. Historically, the Wnt pathway has been identified simultaneously as a guide for physiological processes of development and intercellular communication and in pathological processes such as in cancer . This side novo synthesized β-catenin to enter the nucleus. In this way, β-catenin may also drive cancer cell survival by enhancing growth factor receptor signaling, such as Epidermal Growth Factor Receptor (EGFR) [APC gene) lead to the formation of constitutive nuclear TCF/β-catenin complexes and uncontrolled transcription of target genes.Canonical Wnt/β-catenin-dependent signaling is triggered by one of the secreted Wnts by engagement of a receptor complex comprising a G-protein-coupled Frizzled family receptors (Fz) and low-density lipoprotein receptor-related protein 6 or 5 (LRP 5/6) that sends the biological signal to the Dishevelled protein within the cell ,40,41 F. The Wntr (EGFR) . Mutatio2+ pathway. Both are activated by the binding of a Wnt ligand to Fz proteins and their co-receptor tyrosine kinase-like orphan receptor 2 (ROR2) to activate Dishevelled (Dvl)  and Calmodulin, that allows the transcription factor Nuclear factor of activated T-cells (NFAT) to translocate into the nucleus. NFAT, together with other cofactors  binds DNA. This induces the transcription of several genes including those for T lymphocyte activation  [Non-canonical signaling (β-catenin-independent) can be further divided into the Wnt/Planar Cell Polarity (PCP) pathway and the Wnt/Caed (Dvl) . Non-cane., IL2) . Dysfunce., IL2) ,48,49,50The canonical Wnt/β-catenin pathway is integrated into evolutionarily conserved signaling processes during human embryogenesis. Gene expression control by Wnt/β-catenin plays a pivotal role in organogenesis driving both polarity and specific cell fate. The tight regulation of embryogenesis by the Wnt/β-catenin signal is also manifested in hepatobiliary development, hepatic maturation and zoning . Liver aIn vitro hepatic differentiation protocols demonstrate progressive loss of expression of the pluripotent marker Octamer-binding transcription factor 4 (OCT4) and increased expression of early-stage hepatic differentiation genes such as SRY-box transcription factor 17 (SOX17), GATA binding protein 4 (GATA4) and Forkhead box protein A2 (FOXA2) .In the adult liver, β-catenin is localized in the perivenous area, while its negative regulator APC is in the periportal area. This dislocation allows them to exert control over hepatic metabolic activity ,55. DuriThe function of pericentral lobule hepatocytes appears to be regulated by modulation of the gene expression pattern in components of Wnt/β-catenin signaling. It is currently believed that the molecular basis of metabolic zoning in the liver directs both hepatocyte function and the limitation of initial hepatotoxic damage in specific areas. This allows for effective regeneration and restitution responses from unaffected cells. It has also been revealed that many pathological conditions in the hepatic lobule result from spatially deregulated mechanisms of hepatocyte adaptation and repair processes . A pathoThe loss of functional homeostasis in the hepatic microenvironment also involves the network of signals relying on the Wnt/β-Catenin pathway. Indeed, the dysregulation of this pathway seems to be one of the main drivers of liver carcinogenesis. Thus, the overcoming of the biological capacity, due to the damage, may induce an excessive activation of this pathway that promotes tumor initiation and progression of PLC.β-Catenin plays an important function in promoting HCC, based on the frequency of its mutations associated with aberrant Wnt signaling , whereasIn human HCC, nuclear β-catenin positivity is observed in 37.7% of patients, with a unicentric nuclear distribution in 53% of cases. The majority of these shows a well-differentiated histology . PatientThe presence of CTNNB1 mutations is associated with an increased tumor size, microvascular invasion in HCC . Based oIn HCC cells, canonical Wnt signaling can be antagonized by non-canonical Wnt5a or even remain simultaneously activated, as occurs via PFTK1 that can interact with cyclin Y (CCNY) ,77. In HWhile HCC is human malignancy that originates from hepatocytes, CCA is a neoplasia that develops from biliary tree cells. CCA is the second most common primary liver cancer after HCC that presents activation of Wnt/β-catenin signaling. CCA development is associated with a state of chronic inflammation sustained by a prolonged exposure of the cholangiocytes to different risk factors, such as infectious agents. In bile ducts this condition leads to activation of different pathways, including Wnt signaling, and promotes cell proliferation, genetic and epigenetic mutations predisposing to CCA . More frIntrahepatic CCA is associated with cirrhosis caused by HCV, while peripheral CCA is mainly attributed to primary sclerosing cholangitis . AlthougHB might occur from hepatic stem cells. It shows various histological features  within the same tumor . Based oClassical HBs show specific hypomethylated enhancers carrying binding sites for Achaete-Scute Family BHLH Transcription Factor 2 (ASCL2), an essential factor for the gene transcription regulation in the Wnt pathway into definitive endoderm cells . ProtracInterestingly, β-catenin mutations have no prognostic value due to the similar rates of mutations found in all HB histotypes and at different tumor stages . The sevSurgical resection is the treatment of choice for PLC patients with well-preserved liver function. The overall 5-year survival rate of patients with advanced PLC is around 5-8.9% ,64. SpecPLCs are very difficult to treat. Systemic chemotherapy, with or without the association of immunotherapy, has significant side effects in PLC patients. Several additional combined therapies are currently being tested in clinical trials but there is an urgent need to identify novel potential molecules with more individual therapeutic timing. For this, all efforts must made in developing direct-acting treatment modalities in selected area of the liver. Future target therapies have to guarantee a selective action in the PLC nodule, with the aim of saving normal liver parenchyma.Activation of Wnt/β-catenin signaling may induce two different phenotypes of HCC: the “Hyde phenotype” defined by cancer stem cell feature, invasion and metastasis, and the “Jekyll phenotype” defined as the HCC subset with good prognosis . TreatmeSorafenib is an anti-angiogenic and MAP kinase inhibitor active against HCC that improves survival in patients with advanced disease ,111. In CCA treatments include radical surgery, chemotherapy, radiotherapy and their combination . SurgeryCurrent primary treatments for HBs include curative surgical resection and cisplatin-based chemotherapy, while liver transplantation represents the recommended therapeutic option in advanced stage tumors with vascular infiltration . In partTherapeutic approaches targeting Wnt/β-catenin signaling consist of antibodies against Wnt ligands and Fz receptors, or molecules that inhibit their secretion and interaction. Other options include inhibition of palmitoylation and secretion of Wnt ligands. Regarding β-catenin, some molecules have been developed to promote its degradation at the cytoplasmic level, or the inhibition of its nuclear transduction . CurrentSmall molecules inhibiting PORCN have been developed to prevent palmitoylation of Wnt proteins and their secretion, and they can shut down both canonical and non-canonical Wnt signaling ,117. PORFz receptors have a significant role in cancer progression and may contribute to the therapeutic target of PLC. Monoclonal antibodies could increase apoptosis and prevent cell proliferation by inhibiting Wnt ligands and Fz receptors. Because Fz receptors are up-regulated in HCC, they are a good molecular target. Based on these considerations, it has been reported that soluble Fz7 (sFz7) can also compete with Fz receptors for the Wnt3 ligand. Vantictumab (OMP-18R5) is a novel monoclonal antibody that interacts with these Fz receptors to block canonical Wnt signaling . It has In HB treatment, Fz receptors may serve as useful therapeutic target by gene expression inhibition .Dickkopf-1 (DKK1) is a secreted modulator of Wnt signaling, but it shows immunosuppressive effects in many cancers when overexpressed and is often associated with worse clinical outcomes. DKN-01 is a humanized IgG4 antibody used to block the activity of DKK1, enhancing innate immune responses by the immunosuppressive effects of DKK1 in the tumor microenvironment . Most clTankyrase inhibitors (TNKSi) cause stabilization of Axin 1/2 proteins, β-catenin degradasome accumulation and blockade of WNT/β-catenin signaling . TNKsi iβ-catenin signaling increases the metastatic capacity of HCC cells through upregulation of pyruvate dehydrogenase kinase isoenzyme-1 (PDK1) to stimulate the Warburg effect and energy delivery to the tumor. In contrast, PDK1 activation can be suppressed by PPARγ-coactivator-1α (PGC-1α), although PPARγ appears to be upregulated in HCC cells and human HCC tissues resistant to Sorafenib . FH535, Concerning HB treatment, there are only preclinical studies that prospect a target therapy for Wnt/β-catenin signaling, the use of small molecules in combination with selective COX2 inhibitors, or natural compounds and repurposing of drugs showing anticancer effects, such as epigallocatechin-3-gallate ,135,136.The Wnt/β-catenin signaling pathway physiologically regulates cell proliferation and differentiation in the liver parenchyma through a complex network of interactions modulated by the functional requirements of the microenvironment. These interactions are lost during cancerogenesis and tumor progression in which the Wnt/β-Catenin signaling is dysregulated.Currently, applied therapeutic protocol tend to fight human liver cancer, although it should be possible to act more selectively and save the normal liver parenchyma. To this end, it is necessary to better understand the functions of the Wnt/β-catenin pathway in physiological function and liver regeneration.Combinations of drugs, using conventional and experimental molecules, should be tested and administered using selective intrahepatic transarterial procedures rather than systemic chemotherapeutic approaches. The selection of liver parenchyma might be of great interest due to the importance of reducing the toxicity of Wnt inhibitors in normal liver tissue and increasing the inhibitory effects specifically on hepatic cancer cells. Novel therapeutic drug delivery nanotechnologies associated with directional regulation of Wnt signaling could delineate new perspectives and personalized treatment opportunities for PLC care that are urgently needed in clinical practice.


There was a reduced workload in the second half during the hot condition (p = .016), which coincided with a heightened threat state (p = .007) and more unpleasant feelings (p = .008) experienced in the hot, compared to temperate, condition. Furthermore, plasma normetanephrine (NMET) was higher at half-time (p = .012) and post-CISP (p ≤ .001). Also, plasma metanephrine (MET) was higher post-CISP (p = .009) in the hot compared to temperate condition, reflecting a heightened stress response. Our findings highlight the need for practitioners to consider the detrimental effects heat exposure can have on both physical and decision-making performance when looking to facilitate performance in hot conditions.Global warming and the globalisation of sport has increased the prevalence of sports competitions being held in hot environments. However, there is currently limited research investigating the impact of the heat on soccer-specific decision-making skills during exercise reflective of the physical demands of match-play. Therefore, the effects of heat exposure on physical and soccer-specific decision-making performance, biological markers , appraisal  and affective states, during prolonged high-intensity intermittent exercise were investigated. Nine well-trained male soccer players completed a 92-min cycling intermittent sprint protocol (CISP), whilst simultaneously responding to a series of soccer-specific decision-making trials at various time points, in two temperature conditions: hot  and temperate . Results showed that decision-making score ( Successful performance in soccer is dependent upon the simultaneous execution of a range of physical and cognitive skills. Sport-specific perceptual abilities can distinguish between elite and novice players e.g., ,2, with During the physically demanding nature of competitive soccer, physical fatigue occurs due to a depletion in energy stores , increased thermal strain, dehydration, and the alteration of central mechanisms  . Soccer Research in team sport has shown that high-intensity or prolonged exercise can improve decision-making speed ,13, but C), cardiovascular strain , dehydration, and neurochemical alterations , which could also be linked with physical and cognitive performance. The Theory of Challenge and Threat States in Athletes TCTSA; 28] offers insights into how athletic performance may be impacted by competitive stress and the appraisal of an activity. It is suggested that a challenge state is usually  associated with positively valenced emotions as well as linked with effective decision-making, higher task engagement, and positive performance. In contrast, a threat state is suggested to be associated with  negatively valenced emotions as well as emotions that are perceived as unhelpful for performance, impaired cognitive functioning, reduced task engagement and performance decrements , it is known that fatigue can impair higher level thought processing in soccer players. However, to date, no study has yet to explore how the additional stress imposed by heat exposure may impact such processes. Additionally, research has yet to investigate how heat exposure during prolonged intermittent exercise reflective of physical demands of match-play can affect a combination of biological  and psychological  responses that could have a role to play on performance  in soccer players.1). Second, it was anticipated that during exercise in 32˚C, players’ ability to engage in higher level thought processes would be reduced when retrospectively evaluating their decision, compared to during exercise at 18˚C (H2). Third, it was expected that threat appraisal about the activity demands, and unpleasant affect, would be higher in 32˚C compared to 18˚C (H3). Finally, it was believed that metanephrine and normetanephrine levels would increase in response to exercise, but more significantly in the hot, compared to the temperate conditions, reflecting a heightened physiological stress response (H4).Therefore, the aim of this research was to examine how heat exposure (32˚C) during prolonged intermittent sprint exercise affected physical work output, soccer-specific decision-making, demand and resource appraisals , affective valence , and plasma metanephrines , in comparison to a temperate condition (18˚C). As it was expected that soccer players would be under greater physiological and psychological strain in the heat, the following hypotheses were tested. First, it was hypothesised that power output  and the quality of decision-making would deteriorate during the later stages of the protocol as a result of exercise-induced fatigue, and that this deterioration would be more pronounced in 32˚C compared to 18˚C (Hn = 2), regional (n = 3) and club (n = 4) levels. An estimated sample size of nine participants was determined by a power calculation using G*Power 3.1.9.4 . All decision-making clips were projected onto a television screen positioned outside the viewing window of the environmental chamber, directly in front of the participant. An omnidirectional boundary microphone  was attached to the cycle ergometer to collect verbal responses and was connected via a small port to a laptop located outside of the chamber.Panopto software  was used to sync the video footage which was played from the laptop outside of the chamber, to the verbalised reports. Each recording was then downloaded as an MP4 file and transcribed ready for analysis in NVivo . Inductive content analysis was used to analyse the recordings obtained from the participants ,43, allord, 9th, 15th and 21st sprint in each half. At rest, participants were asked “How demanding do you expect the protocol to be?” and “How able are you to cope with the demands of the protocol?” to measure demand evaluations and resource evaluations, respectively. The tense of these questions was then adapted for during the CISP . These two items were ranked using a 6-point Likert scale anchored from 1  to 6 (extremely). A ratio was calculated by dividing demands by resources, where a value greater than 1 indicated a threat state, and less than 1 indicated a challenge state [To assess challenge and threat states, demand and resource evaluations were measured to determine a cognitive appraisal ratio  pre-CISPge state .rd, 9th, 15th and 21st sprint of each half of the CISP using the Feeling Scale [very bad) through 0  to 5 (very good). Unick et al. [Affective valence and activation were measured pre-CISP and every 3ng Scale  anchoredk et al.  also fouethylenediamine tetraacetic acid (EDTA) tubes from an indwelling venous catheter located in the arm at pre-CISP, half-time, and immediately post-CISP. These were centrifuged immediately  and plasma separated for analysis of plasma metadrenaline (MET) and normetanephrine (NMET), measured as the more stable metabolites of plasma epinephrine and norepinephrine, respectively [For measurement of plasma metanephrines, 3ml blood samples were collected in ectively . These sC was measured using a temperature sensor telemetry system in the form of a small ingestible pill. Participants ingested the thermometer pill ~6 hours prior to exercise [C was first recorded pre-trial following 15-min of seated rest to allow resting values to stabilise. TC was then recorded 1-min into each 2-min block, allowing a period for TC to stabilise following the 5-s sprint [C recordings were averaged across 5-time points for each half of the CISP  for analysis.Texercise . TC was s sprint , and wasAfter sitting for 15-min to allow resting values to stabilise, participants HR (beats per min) was recorded using a polar HR monitor . HR was then recorded immediately after every sprint throughout the CISP, then averaged across 5-time points for each half of the CISP  for analysis.After being towelled off, participants were asked to enter a secure room, undress, and step onto the Tanita scales to measure nude body mass immediately pre- and post-each CISP, to determine fluid loss from the trial . Urine ord, 9th, 15th and 21st sprint in each half of the CISP using Toner et al.’s [unbearably cold) to 8 (unbearably hot) Likert scale. This thermal sensation scale has been shown to be a valid measure of perceived heat stress, where correlations with large effect sizes (r = .72) have been shown between thermal sensation ratings and rectal temperature [Thermal sensation (TSS) was recorded every 3et al.’s  0  were taken every 3rg scale . Previourg scale .p ≤ 0.05. All data were reported as means and standard deviation (SD). For visual clarity, Figure error bars represent standard error mean (SEM).Statistical analysis was completed using IBM SPSS statistics 24.0 (IBM Corporation). After data screening and checking for normality of the data, a series of two-way  repeated measures ANOVAs were performed. Firstly, to analyse differences in indicators of physiological  and perceived psychological  strain. This was to check whether participants were under greater strain in the hot compared to the temperate condition. Then, a series of two-way repeated measures ANOVAs were performed to address our hypotheses. Specifically, to examine differences in physical  and cognitive  performance, perceived challenge and threat appraisals, affective valence, and plasma metanephrines. Homogeneity of variance was assessed using Mauchly’s test of Sphericity. Where homogeneity of variance was unable to be assumed, Greenhouse-Geisser corrections were applied. Where significant results were identified, Bonferroni pairwise comparisons post-hoc tests were performed. Two-tailed statistical significance was accepted at p2) and generalised eta-squared (η2G) are reported as the effect size for ANOVAs. Due to Cohen’s [d was reported as the effect size for pairwise comparisons. In accordance with Cohen [d values of 0.2, 0.5 and 0.8 indicated small, moderate, and large effects for pairwise comparisons, respectively.Partial eta-squared  and an interaction effect , but no effect for condition . Core temperature was higher in hot compared to the temperate condition in the final 6-min of the first half  , but was not different between conditions at any other time-point  (see A condition (2) × time  ANOVA for T.84) see .F1,8 = 13.581, p = .006, ηp2 = .629, η2G = .209), time , and condition (2) × time interaction  effects found for HR . Heart rate was significantly higher in the hot compared to temperate condition at all time-points , apart from in the first 10-min of the first half of the CISP  (See There were condition (.58) See .F1,8 = 47.901, p ≤ .001, ηp2 = .857, η2G ≤ .001) effect, whereby fluid loss was significantly higher in the hot (Mdiff = -1.60 ± 0.30 kg) compared to the temperate (Mdiff = -0.80 ± 0.40 kg) condition. For urine osmolality, no condition , time  nor condition × time interaction  effect was found. Additionally, there was a main condition effect for fluid intake whereby participants consumed more fluid (water) in the hot (1067 ± 440 ml) compared to the temperate (728 ± 168 ml) condition .There was a significant condition  ANOVA identified effects for condition  and time , but no interaction effect . RPE was significantly higher in the hot (14.54 ± 1.65) compared to the temperate  condition. In terms of time, Bonferroni pairwise comparisons identified that RPE was significantly higher toward the end of the first half  , midway through the second half   and at the end of the second half of the CISP  , compared to the start of the first half of the CISP  (10.44 ± 1.55). RPE was also significantly lower at the start of the second half  (11.83 ± 2.45) compared to the end of the second half   (see A 2 (condition) by 8  see .th-min then every 12-min intervals throughout the CISP) ANOVA found significant main effects for condition  and time , but no condition × time interaction effect  for TSS. Specifically, the effect of condition revealed that TSS was significantly higher in the hot (6.10 ± 0.42) compared to temperate  condition. For the time effect, Bonferroni pairwise comparisons identified higher TSS in the 18th  and 90th-min  compared to the 6th-min (4.72 ± 0.47) (see A 2 (condition) by 8  see .Overall, the findings from the preliminary analyses revealed that in the hot condition, participants showed greater cardiovascular strain (increased heart rate), higher fluid loss, felt hotter , perceived the exercise more demanding or exerting (higher RPE). Moreover, although no main condition effect was found for core temperature, core temperature was still higher at the end of each half of the CISP in the hot condition compared to the temperate condition. Therefore, these results are suggestive that participants were under greater physiological and perceived psychological strain during exercise in the hot condition.F1,8 = 4.976, p = .056, ηp2 = .383, η2G = .010) or condition . However, a significant condition × time interaction  was found , but was not significantly different in the second half (739.12 ± 92.73 W) compared to the first half  of the CISP in 18˚C  or second half of the CISP .A 2 (condition) by 2  ANOVA revealed no effect for time  and time , but no condition × time interaction effect    and second  halves of the CISP, compared to pre-CISP (95.17 ± 9.20%).A 2 (condition) by 3  ANOVA revealed significant main effects for condition (001) See . In term.93) see . In termFrom the inductive content analysis, 4 themes and 14 sub-themes were extracted. The themes reflected a) ‘game intelligence’, used for verbalisations which recognised critical information as it occurred such as player movement, goal-scoring opportunities, tactical awareness, and/or showed deeper planning as to what could or should occur following the initial decision; b) ‘risk management’, used for verbalisations which identified potential threat or discussed risk in relation to the available options; c) ‘difficulty’, used to categorise verbalisations which referenced how difficult the decision made would be to execute, or when participants referred to the difficulty, or showed hesitation toward the decision-making process; and finally d) ‘lack of evaluation’, used to reflect responses where the decision made lacked rationale and/or where participants simply re-described the action they would take see . Across F1,7 = 0.476, p = .513, np2 = .064, η2G = .009) or time , nor a condition × time interaction effect  for the number of themes extracted. Additionally, no condition , time  or condition × time interaction  effects were identified for total number of verbalisations , time , nor condition × time interaction  effects were observed. Within the sub-themes underpinning ‘difficulty’, there were no condition , time , nor condition × time interaction  effects for ‘difficult execution’, ‘easy execution’ and ‘uncertainty’.For ‘difficulty’, no main condition , time  or condition × time interaction  effects were noted. For the sub-themes underpinning ‘risk management’, no condition , time  or condition × time interaction  effects were found for ‘perceived threat’, ‘restricted options’, ‘risk assessment’ ‘safe options’ or ‘available time’.For ‘risk management’, no condition , time  or condition × time interaction  effects were observed. For sub-themes underpinning ‘game intelligence’, a main effect for condition  was found for ‘future planning’, but there was no time  nor condition × time interaction  effects. Specifically, a lower number of “future planning” verbalisations were made in the hot (0.99 ± 0.42) compared to the temperate condition . For ‘chance of scoring’ and ‘space and positioning’, no condition , time , or condition × time interaction  effects were found.For ‘game intelligence’, no condition  or condition × time interaction  effect was found. A main effect for time  was identified whereby significantly less ‘tactical awareness’ statements were made in the second half (0.63 ± 0.62) compared to the first half . For ‘player movement’, no main condition  or time  effects were identified. However, a condition × time interaction effect  was observed whereby ‘player movement’ verbalisations were lower in the first half in the hot compared to the temperate condition , though they were not different in the second half .For ‘tactical awareness’, no condition , where more responses lacked evaluation in the second half (0.75 ± 0.48) compared to the first half  of the CISP. There was no effect for condition , nor condition × time interaction .In terms of ‘lack of evaluation’, a main effect for time was found  ANOVA revealed significant main effects for condition  and time , but no interaction effect was identified . Post-hoc analysis revealed a higher threat state in the hot condition (1.20 ± 0.50) compared to a higher challenge state in temperate condition . For the effect of time, no significant differences were observed between time points , apart from between the 6th (.55 + .21) and 66th minute  (see A 2 (condition) by 9  see .th-min, and then 12-min intervals throughout the CISP) ANOVA found significant effects for condition  and time , but no condition × time interaction effect . Post-hoc analysis revealed that affective valence was significantly lower in 32˚C (.41 ± 1.59) in comparison to 18˚C . Bonferroni pairwise comparisons also identified lower affective valence at the start of the second half  , and in the 66th min , and 78th min , compared to rest (3.50 ± 1.50). It was also significantly lower toward the end of the second half in the 66th , 78th  and 90th min  compared to early in the first half in the 4th min (2.39 ± 1.35)  by 9  see .F1,7 = 43.637, p ≤ .001, ηp2 = .862, η2G = .191), time , and condition × time interaction  effects were observed for normetanephrine (NMET) concentration. Specifically, NMET concentration was significantly higher in the hot compared to temperate condition at half-time  and post-CISP , but was not different between the hot and temperate condition pre-CISP  (see Significant condition (.06) see .F1,7 = 5.954, p = .045, ηp2 = .460, η2G = .067), time , and condition × time interaction  effects. MET concentration was significantly higher in the hot compared to temperate condition post-CISP , but not pre-CISP  or at half-time  (see In terms of metanephrine (MET) concentration, there were significant condition (.46) see .1), whereby soccer-specific decision-making scores  was impaired in 32˚C compared to 18˚C. However, no condition × time effects were observed for decision-making scores which was not aligned to our hypotheses. As expected, intermittent sprint performance was impaired in the second half compared to the first half of the CISP in 32˚C, but did not deteriorate in 18˚C. In support of the second hypothesis (H2), ‘future planning’ verbalisations were significantly lower in 32˚C compared to 18˚C, and more responses categorised as ‘lack of evaluation’ were observed in the second half compared to the first half across conditions. This partly reflected lower-level thought processing in the hot condition and following a half of intermittent sprint activity, which partially affirmed our second hypothesis (H2). However, we did not find condition × time interactions as originally anticipated. We also found support for our third hypotheses (H3), whereby players had a heightened threat state and felt more unpleasant in 32˚C compared to 18˚C. Moreover, MET and NMET concentration were significantly higher following exercise, and these increases were greater in the hot compared to the temperate condition, which reflected a heightened physiological stress response when exercising in hot condition thereby supporting our fourth hypothesis (H4). It is worth noting that these findings were similar when analysing data with 8 participants when conditions were fully counterbalanced, and a summary of these findings can be found in the supporting information.This study aimed to investigate how soccer-specific decision-making, power output, appraisal, affect, and plasma metanephrines were affected during prolonged intermittent exercise in a hot, compared to a temperate, environment. There was partial support for our first hypothesis  [C threshold of ~38.5˚C is where cognitive function becomes impaired [C did not exceed this threshold in the majority of participants.It was anticipated that a more pronounced decline in decision-making score would be observed during the CISP in the hot condition compared to the temperate condition due to additional physiological stress experienced  ,29,31. Dhe CISP) . From anhe CISP)  found thimpaired . HoweverA ‘think-aloud’ protocol was also implemented, recording participants retrospective evaluation of each situation to understand what factors were being considered when making the decision, and if this was affected by exercise-induced fatigue or heat stress. Interestingly, thematic analysis revealed that participants verbalised significantly less ‘future planning’ evaluations when in the hot compared to the temperate condition. These findings align with previous research, which found that within 15-min of passive heat exposure  complex planning performance is significantly impaired . This deVerbalisations referencing tactical awareness and execution difficulty were also significantly lower in the second half compared to the first half of the CISP. Additionally, statements which lacked evaluation were higher in the second half compared to the first half, and reference to specific player movement were also significantly reduced in the second half in the hot condition. Support can be drawn from Casanova et al. , who fouAnother possible explanation is that the heat stress in the present research could have also potentially induced some anxiety  that may have contributed in reducing soccer players’ focus during the decision-making task. For example, based on the premises of the Integrated Model of Anxiety and Perceptual-Motor Performance  anxiety C >39°C) [C reached values lower than those typically expected when performing team sport in the heat (>~39˚C) [Heat also impaired athletes’ physical performance in this study. Power output was significantly lower during the second half compared to the first half in the hot condition (-~7%) but was not different between halves in the temperate condition. Previous research has shown that increased perceived exertion and more negative affective states induced by heat exposure can impair self-paced work rate . MoreoveC >39°C) . However(>~39˚C) . This wa(>~39˚C) .C reaching average heights of ~38.4˚C. It is suggested that when players feel their TC increasing more rapidly in hot conditions, they may deliberately or reflexively reduce their high-intensity work to reduce the rate of metabolic heat production and ensure thermal strain does not exceed tolerable limits [The present research also observed higher NMET and MET levels (measured as the more stable metabolites of norepinephrine and epinephrine) at half-time and post- exercise compared to pre-exercise, but such increases were to a much greater extent in the hot compared to temperate condition. This aligns with previous research that has observed that the release of epinephrine and norepinephrine is accelerated in response to acute heat stress . The ince limits . In thisC, HR sweat rate and dehydration) [The present study also identified that participants’ demand appraisals were higher and resource appraisals were lower in hot condition compared to the temperate condition, reflective of an overall higher threat state in the hot compared to the temperate condition. Naturally, when performing in hot environments, individuals are likely to perceive that higher effort may be required than in temperate conditions due to the widely understood exacerbated physiological responses  . Furtherdration) . Based odration) , threat Whilst this study built upon previous methods by integrating a sport-specific visual decision-making task, research should incorporate tasks which require simultaneous visual and auditory attention  to better replicate the demands of match-play. Additionally, a cycling intermittent sprint protocol was used to allow players to modulate their own high-intensity physical work output as per match-play, whilst simultaneously investigating cognitive function and central mechanisms. Despite this being widely employed within team sport literature ,40, it iC was ≤38.5˚C, suggesting that complex, situation-specific decision-making as well as physical work output can be impaired below TC thresholds proposed previously when exposed to heat during exercise [In this research, soccer-specific decision-making was impaired in the hot compared to temperate condition. Physical work output was also significantly reduced in the second half in the heat but was not affected in the temperate condition. These decrements were observed when Texercise . AthleteSoccer players and sports practitioners should consider that performing in more strenuous environments may enhance the likelihood of players making infrequent, but potentially costly, decision-making errors. Sports practitioners could look to reduce potential anticipatory effects of performing in more demanding environmental conditions by improving athletes’ perceptions of such environments, reducing their level of uncertainty, and improving their resource appraisal such as through enhancing self-efficacy and developing perceived control . The impS1 Table(DOCX)Click here for additional data file.


Strix aluco of different colour morphs in a population near the southern coast of Finland. Winter weather conditions before breeding seemed to have effects on the food availability of Tawny Owls: the depth of the snow cover showed a positive relationship, and the frequency of temperature fluctuations around the freezing point had a negative relationship. In females, intrinsic factors such as colour morph and age, as well as the body condition of the mate and the stage of the season, governed body condition. In males, only age and the stage of the season suggested associations with body condition. Probably due to the efficient use of alternative prey, the effects of fluctuations of vole populations on the body condition of Tawny Owls are only moderate.The present study examines how the variations in food supply and winter weather are reflected in the body condition of female and male Tawny Owls Strix aluco of temperate origin near the northern limit of the species’ range in southern Finland to reveal if they show similar patterns to the boreal species. Winter weather conditions before breeding seemed to have pronounced effects on the food availability of Tawny Owls. In females, intrinsic factors such as colour morph and age, as well as the body condition of the mate and the stage of the season (Julian date), governed body condition. In males, only age and Julian date showed pronounced relationships with body condition. The results suggest that deep snow cover protects vole populations through winter until spring better than a minor amount of snow and that frequent temperature fluctuations around the freezing point in early spring make voles more available for owls that are preparing for breeding. This was also reflected positively in the body condition of female owls. Probably due to the efficient use of alternative prey, the effects of fluctuating vole populations on the body condition of Tawny Owls are, in general, only moderate.The body condition of boreal species of vole-eaters seems to vary largely according to fluctuations in vole populations and weather conditions of the preceding winter. I studied females and males of the Tawny Owl  Good body condition, a favourable physiological state in terms of protein and fat ,2, is neStrix aluco is a polymorphic, stationary, nocturnal, generalist predator, occupying a relatively continuous range across most of Europe from the Mediterranean to the Nordic countries )Microtus agrestis [Arvicola amphibius is of decisive importance [As all capital breeders, Tawny Owls should exhibit good body condition before breeding. In my study area, the most important prey species responsible for this at the beginning of the breeding season is the Field Vole agrestis . Followiportance ,30.From the individual point of view, the seasonal decline in the food supply means that the body condition of birds decreases, and it is increasingly challenging for the male to fatten the female to the breeding condition. From the population point of view, this means that breeding shifts later, and the later clutches are smaller than the earlier ones . The appWinter weather conditions before breeding affect the food availability of Tawny Owls in various ways. For instance, a deep snow cover protects voles, their staple prey, through winter until spring better than a minor amount of snow, while frequent temperature fluctuations around the freezing point in early spring make voles readily available for owls. Local environmental conditions, particularly local prey supply, and the individual characteristics of males are strongly involved in determining the body condition of Tawny Owls. However, the body condition is relatively resistant to variable foraging conditions due to alternative prey, as could be expected for a generalist predator of southern origin. Thus, due to the efficient use of alternative prey, the effects of fluctuating vole populations on the body condition of the Tawny Owl are, in general, moderate.


Herein, we fabricated a PDGFB targeting, biodegradable FePt alloy assembly for magnetic resonance imaging (MRI)-guided chemotherapy and starving-enhanced chemodynamic therapy for cancer using PDGFB targeting, pH-sensitive liposome-coated FePt alloys, and GOx (pLFePt-GOx). We found that the Fenton-catalytic activity of FePt alloys was far stronger than that of traditional ultrasmall iron oxide nanoparticle (UION). Upon entry into cancer cells, pLFePt-GOx nanoliposomes degraded into many tiny FePt alloys and released GOx owing to the weakly acidic nature of the tumor microenvironment (TME). The released GOx-mediated glucose consumption not only caused a starvation status but also increased the level of cellular H2O2 and acidity, promoting Fenton reaction by FePt alloys and resulting in an increase in reactive oxygen species (ROS) accumulation in cells, which ultimately realized starving-enhanced chemodynamic process for killing tumor cells. The anticancer mechanism of pLFePt-GOx involved ROS-mediated apoptosis and ferroptosis, and glucose depletion-mediated starvation death. In the in vivo assay, the systemic delivery of pLFePt-GOx showed excellent antitumor activity with low biological toxicity and significantly enhanced T2-weighted magnetic resonance imaging (MRI) signal of the tumor, indicating that pLFePt-GOx can serve as a highly efficient theranostic tool for cancer. This work thus describes an effective, novel multi-modal cancer theranostic system.The application of chemodynamic therapy (CDT) for cancer is a serious challenge owing to the low efficiency of the Fenton catalyst and insufficient HThe online version contains supplementary material available at 10.1186/s12951-022-01482-x. Cancer is a threatening challenge to human health worldwide –3. The r2O2) to highly toxic hydroxyl radicals (·OH), whose accumulation then triggers reactive oxygen species (ROS)-mediated oxidative stress and non-programmed cell apoptosis; this process is termed chemodynamic therapy (CDT) [2 from H2O2 [2 magnetic resonance imaging (MRI) contrast [2O2 into highly toxic hydroxyl radicals and thus damage the tumor cell [2O2 compared to normal cells, the H2O2 level in cancer cells is still too low to effectively activate CDT for inducing cancer cell death [2O2 and enhance the catalytic activity of CDT agents will help improve clinical therapy and diagnosis of cancer.Recently, heavy metal ions including iron , copper py (CDT) –17. Recerom H2O2 , which pcontrast , 20. Howcontrast . In weakcontrast , 23. (2)mor cell . Althougll death . Therefo2O2 in the presence of oxygen [2O2 level. This process not only realizes starvation therapy of tumor but also promotes the Fenton catalytic efficiency, thereby strengthening tumor CDT owing to the sufficient supplementation of H2O2. However, non-targeting GOx-mediated starvation therapy may result in the distribution of GOx into normal tissue, which may further lead to oxidization of glucose and cause potential systematic toxicity to the body [Considering the rapid proliferative phenotype and increased glucose consumption of cancer cells, cutting off the nutrient supply to induce starvation status is a promising strategy for tumor suppression –28. Glucf oxygen , 30. Whethe body , 32; thithe body , 34. ThuPDGFB/PDGFR-β axis is well documented as a vital oncogenic signal and potential therapeutic target for different types of carcinomas, such as prostate cancer , pancrea2O2 into highly toxic ·OH but also released abundant Pt(II) triggered by ROS, contributing to chemotherapy and CDT. In addition, the released GOx consumed cellular glucose and produced gluconic acid and H2O2, contributing to starvation therapy and accelerating CDT. Besides, the pLFePt-GOx nanoliposome possessed excellent superparamagnetism, and its targeted delivery significantly enhanced the T2-weighted MRI signal of the tumor, which is beneficial for achieving high-quality MR images and realizing accurate diagnosis of tumor. Therefore, this work describes an effective, novel multi-modal theranostic system for cancer.In this study, we found that FePt alloys possessed stronger Fenton-catalytic activity than UION, and could be an ideal Fenton catalyst. Based on this, we fabricated a PDGFB targeting and biodegradable FePt alloy assembly for MRI-guided chemotherapy and starving-enhanced chemodynamic therapy of cancer using PDGFB targeting, pH-sensitive liposome-coated FePt alloys, and GOx (pLFePt-GOx) Scheme . Upon enThe ultra-small FePt alloys were synthesized using ferric acetylacetonate and platinum bis(acetylacetonate) as precursors via a modified high-temperature decomposition method. The prepared FePt alloys were hydrophobic and only dispersed in n-hexane solution. For their in vivo application, hydrophobic FePt alloys were converted to hydrophilic FePt alloys through ligand exchange. Additional file 2O2 solution showed significant 1:2:2:1 characteristic peaks assigned to ·OH  can be also degraded by ·OH, resulting in the formation of a colorless solution. Based on this phenomenon, the MB degradation experiment was further used to assess the catalytic activity of FePt alloys. As shown in Fig. 2O2 treated with magnetic iron oxide (MIO) and ultrasmall iron oxide (USIO) only showed a slight decrease in absorbance; In comparison to this mixed solution, the MB solution containing FePt alloys exhibited a very low absorbance, implying that FePt alloys could effectively catalyze H2O2 to ·OH, thus promoting MB degradation. In addition, the degraded ratio of MB was quantitatively analyzed using a linear fit standard curve  spectra. As shown in Fig. 2O2 level and acidity. To overcome these limitations, glucose oxidase (GOx), which catalyzes glucose into gluconic acid and H2O2 in the presence of oxygen, was integrated with FePt alloys via PDGFB targeting liposomes to form the pLFePt-GOx assembly. As shown in Fig. −1, which were assigned to C-N bending vibration and C-N stretching vibration of GOx and C-O stretching vibration and C = O stretching vibration of PDGFB-PEG, respectively. This result confirmed that GOx and FePt alloys had been successfully assembled into pLFePt-GOx through PDGFB targeting liposomes. X-ray photoelectron spectroscopy (XPS) spectra analysis was used to further analyze the composition of pLFePt-GOx  released from pLFePt-GOx significantly accelerated, indicating that Pt(II) release was closely associated with ROS production. These results demonstrate that pLFePt-GOx possesses a promising potential for inducing cancer cell death via chemotherapy.To observe the ability of pLFePt-GOx to catalyze glucose into gluconic acid and Hity Fig. : Fig. S8To evaluate the tumor-targeting effect of pLFePt and reveal how the nanotheranostic agent enters the cells, the internalization process of FITC-labeled pLFePt was observed by CLSM. As shown in Fig. 2O2, thus enhancing the H2O2 level and acidity in cells. Considering the above-mentioned phenomenon, H2O2 levels in MDA-MB-231 cells treated with pLFePt-GOx were investigated using an H2O2 detection kit and fluorescence probe. As shown in Fig. 2O2 levels in treated MDA-MB-231 cells did not significantly vary among the treatment groups, indicating that MDA-MB-231 cells had a certain ability to maintain the balance of redox-reduction substance. Notably, the H2O2 level in MDA-MB-231 cells treated with pLFePt-GOx sharply increased compared to other groups, suggesting the misbalance of redox in these cells. This result demonstrated that pLFePt-GOx indeed significantly elevates cellular H2O2 level, and this elevated H2O2 can further trigger ROS production via FePt alloy-mediated Fenton reaction. Subsequently, the acidity of cancer cells treated with pLFePt-GOx was also detected using a specific pH-sensitive BCECF-AM probe whose green fluorescence weaken with the decrease of pH. As shown in Additional file 1: Fig. S9a, the green fluorescence of 4T1 cells treated with pLFePt had no significant variation, indicating that pLFePt cannot decrease cellular pH. However, pLFePt-GOx treated cells showed a significant lower green fluorescence signal compared to control and pLFePt groups. The fluorescence of 4T1 cells treated with pLFePt-GOx was also quantitatively analyzed using a fluorescence spectrophotometer, showing a similar result with CLSM observation  were incubated with FePt, LFePt, and pLFePt, and then their viability was observed through a CCK-8 assay. The viability of THLE-3 cells treated for 24 h with LFePt and pLFePt had no significant decrease at the Pt concentration range of 0.1–5 μg/mL, but at the same concentration, FePt caused the death of cells , showing stronger T2 MRI contrast capability. It might be that the assembly of pLFePt possessed stronger magnetization, resulting in a higher T2 relaxation rate. The corresponding T2 signal images of pLFePt were also darker and showed stronger attenuation than that of FePt alloys, further confirming that pLFePt had better T2 MRI contrast performance than FePt alloys  ions from FePt alloys, contributing to cancer chemotherapy. Besides, pLFePt-GOx showed excellent T2 relaxivity and its systemic delivery significantly enhanced the MRI contrast signal of tumor, acquiring high quality of tumor MR images and promoting tumor accurate diagnosis. Therefore, this study provides information on a novel Fe-based theranostic platform, which can be applied for MRI-guided starvation-enhancing CDT of cancer.In summary, a specific tumor targeting, TME responsive nanotheranostic agent (pLFePt-GOx) has been successfully fabricated for starvation-enhancing CDT of cancer. The pLFePt-GOx showed an excellent tumor targeting ability and ultrahigh Fenton-catalytic activity. These characteristics make it a promising agent for the accurate CDT treatment of carcinomas. Upon entry into cancer cells, the pLFePt-GOx was degraded owing to the weakly acidic nature of TME, thus releasing large amounts of FePt alloys and GOx. The released GOx-mediated glucose consumption not only caused a starvation status but also increased the cellular HAdditional file 1: Fig. S1. The photo of FePt alloys solution. Fig. S2. The high-magnification TEM images of LFePt-GOx. Fig. S3. ESR spectra. Fig. S4. The degradation rate of MB solution. Fig. S5. Hydrodynamic size change of pLFePt-GOx. Fig. S6. XPS spectra of pLFePt. Fig. S7. The pH variation of pLFePt-GOx solution. Fig. S8. Pt release profile of pLFePt. Fig. S9. Cellular pH analysis. Fig. S10. Cytotoxicity of GOx and pLFePt-GOx. Fig. S11. The original images of western blot analysis. Fig. S12. Pt–DNA adduct content of 4T1 cells after different samples treatment. Fig. S13. In vivo ROS-staining images of the tumors.


Apis mellifera L. and showing several therapeutic properties . The administration of PRP extracts by conventional routes has some disadvantages, such as running off over the skin in liquid or emulsion form. When taken orally, the extracts have a strong and unpleasant taste. The aim of this work was to fabricate and characterize microneedles containing polyvinyl alcohol, polyvinylpyrrolidone, poloxamer P407, and an ethanolic or glycolic extract of PRP. Also, the obtained structures were microscopically and mechanically characterized. The results of the mechanical analysis showed that formulations containing 3% of P407 presented the highest compression values in a hard surface, which was also confirmed by the height and base values of the morphological analysis and by the microscopy images. It was possible to design MNs and select the best formulations for future tests. MNs containing an ethanolic extract of PRP showed to be better structured than MNs containing a glycolic extract of PRP. The MNs obtained in these studies proved to be a promising platform for the topical application of PRP.Microneedles (MNs) are a means to break the protective skin barrier in a minimally invasive way. By creating temporary micropores, they make biologically active agents available in the skin layers. Propolis (PRP) is a gum resin with a complex chemical composition, produced by bees  In recent decades, microneedle devices have been widely used for non-invasive dermal delivery of various drugs –3. MicroNanocarriers can be used together with polymeric MNs in a synergistic therapy. The nanocarriers can immediately come into contact with the stratum corneum with the help of polymeric MNs, enhancing the transdermal drug delivery of the drugs. Furthermore, these polymeric MNs can encapsulate several types of nanocarriers, making it a unique system with different activities . Solid MDissolvable MNs are composed of a biodegradable matrix containing the bioactive agent. When they come in contact with aqueous fluids in the skin, these needles dissolve, releasing the drug and degrading. As they are dissolvable, they do not need to be removed and do not generate residues, eliminating the risk of infections and local irritations when the microneedles are broken or incompletely removed. Hollow MNs have a reservoir inside, which contains the bioactive agent. They are used to penetrate the skin, inject their charge through the epidermis, and are later removed ,14.The application of MNs creates a transport pathway for the delivery of molecules, crossing external barriers that limit the introduction of molecules into the target tissue. Furthermore, MNs are very versatile and are considered less painful, less harmful, and more safe than conventional needles . In geneApis mellifera L.) [Propolis (PRP) has already been studied in wound healing when incorporated in many vehicles, such as ointments, emulsions, hydrogels, films, or as hydroalcoholic or glycolic extracts. PRP exhibits important pharmacological activity, already proven in several studies, in addition to being a biologically safe compound –21. Thisfera L.) ,22–23.PRP extracts have been reported to enhance some antibiotic effects, attributing the antibacterial activity of propolis mainly to flavonoids or synergisms among some components . The eth2 full-factorial design was utilized to determine the influence of P407 and PRP extract on the morphology and mechanical characteristics of MNs. They were characterized macroscopically, microscopically, and regarding size and texture, yielding an improved MN formulation for each type of PRP extract.In our previous studies, polymeric systems composed of polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), and poloxamer 407 (P407) were obtained and characterized. P407 could improve structuring and rapid dispersion of polymeric matrices, which showed promising physicochemical characteristics for potential application as nanostructured platforms for controlled drug delivery –30. To oThe PRP sample was obtained from an apiary in the northwest region of Paraná. It had a characteristic aromatic odor, resin gum appearance, and a greenish-yellow color, very characteristic of the north and northeast regions of the states of Paraná, São Paulo, and Minas Gerais –33. The When using polydimethylsiloxane (PDMS), the combination of base and catalyst resulted in a translucent mixture, which was easy to handle. However, there was intense formation of air bubbles. In order to reduce the bubbles in the final mold, the flask containing the mixture was placed in an ultrasonic bath and, subsequently, in a vacuum desiccator, until all bubbles were removed. With the mixture being completely clear, it was possible to insert the master structure for mold development  showed more integrity and were easier to be removed from the mold. MNs containing glycolic extract (GE) were more difficult to remove from the mold, as there was an increase in the concentration of extract. The formulations containing 8% (w/w) GE did not result in MNs with good integrity, due to the high amount of propylene glycol, which yielded more malleable structures. Previous studies have shown that films containing PVA and propylene glycol are more malleable due to the breaking of hydrogen bonds by the effect of propylene glycol .The factorial design chosen allowed us to evaluate the characteristics of the formulations containing different concentrations of EE and EG in addition to the concentration of P407. This made it possible to choose the best concentrations for each type of extract.In previous studies carried out by Bruschi and colleagues, the concentrations of PRP extract that would have biological activity were studied. The ranges and concentrations used in this study were chosen so that the formulation obtained has a sufficient amount of PRP extract for biological activity ,35–36.The formulations could successfully replicate the master mold shape  (p > 0.05), which was confirmed by the difficult formation of MNs containing GE. It can also be confirmed by the response surface graphs; however, greater height values were observed for smaller amounts of P407.Considering the height, the MNs containing EE and P407 exhibited a significant positive interference (p < 0.05) , from the statistical analysis of the results obtained for the measurements of microneedles are given in The equations obtained for the height and base measurement responses, as well as the values for the correlation coefficient  while the MNs E9 have the highest compression value and an aspect ratio of 1:6 (base width/height). Römgens and colleagues also observed that with an increase in the diameter of the MN tips, the force for insertion increases . When diThe 18 formulations were analyzed for compression force and compression area on a hard glass surface (Petri dish). The results are displayed in Regarding the compression force, for MNs containing EE, only the extract showed positive significant interference, where higher values of compression force were observed for MNs containing a greater amount of extract, with the highest value obtained for the E9 formulation, which contains 3% P407.Y = 0.7928 + 0.5232X1 − 0.3523X112 and Y = 0.3230 − 0.1297X1 + 0.0871X2 + 0.2187X112 for MNs containing EE or GE, respectively. The response surface plots for analysis of force and compression area are displayed in Considering MNs containing GE, both independent variables had significant interference, being negative for GE and positive for P407. The formulations that presented greater compression force were the formulations G5 and G6, containing 4% of GE and 2% and 3% of P407 respectively. The respective equations obtained were Y = 11.1227 + 7.3499X1 − 9.4281X112 + 4.0988X222 and Y = 3.5036 − 2.6528X1 + 2.3812X2 − 2.8720X1·X2 + 1.8557X112. The results of strength and compression area were essential for choosing the best formulations, in addition to showing the best structures for the amounts of extract and P407 used.For the compression area of MNs containing EE, the highest values were observed for the formulations with 12% of EE, whereas for the formulations with GE the highest values were observed for the formulations containing 4% of GE. The respective equations obtained were In previous studies of Donnelly and colleagues , MNs comAfterwards, the best formulations were chosen to proceed with further characterizations. The selected formulations were E3, E6, E9, and G6. They are composed of 3% P407 and 4%, 8%, and 12% EE, or 4% GE, respectively. These formulations were chosen because they exhibited macroscopic integrity and the formulations containing EE with 3% of P407 presented the highest values in the mechanical analysis. Although the MNs containing GE were malleable, the formulation GE6 was chosen because it showed good integrity, with values similar to those of E3. It was utilized for comparison with the formulations containing EE.The best formulations  were mechanically evaluated in compression tests on PVC film, Parafilm M, gelatin, and porcine skin. The results for the compression force of the best formulations are displayed in For all analyses, as the amount of extract in the formulations increases, the compression force also increases regardless of the surface utilized in the test. The formulation G6 displayed the lowest results, probably due to the presence of propylene glycol in the formulation. Considering that the selected formulations contained 3% P407, the amount of PRP extract was the only factor that influenced the increase in the compression force. Thus, it can be inferred that the hardness of MNs increases with the increase in the concentration of PRP extract in the formulation. MNs with GE were malleable due to the plasticizing effect of propylene glycol, which resulted in lower mechanical strength . In contIn other studies using MNs composed of PVA, Carbopol 971P, and alginic acid, it was observed that with a force of 0.03 N or greater, most MNs could penetrate the stratum corneum . Thus, tt-test evaluation of possible significant differences among the compression forces for each substrate/surface utilized. The results for the p values are displayed in All selected MN formulations were statistically compared with MNs without PRP extract and stainless-steel MNs (standard) using Students’ The present study demonstrated, for the first time, the design of MNs composed of PVA, PVP, and P407 as a polymeric platform for the delivery of alcoholic and glycolic green propolis extracts. MN formulations were characterized regarding their morphology, dimensions, and mechanical properties. Selected MN formulations  exhibited suitable mechanical strength to penetrate different substrates that mimic the human skin, making them potentially useful systems for topical propolis administration. As MNs are minimally invasive platforms, the selected systems can be easy to apply and monitor. Further studies should be conducted regarding in vitro and in vivo evaluation of the biological activity and cytotoxicity of the MNs, the vitro PRP release profile, the cutaneous permeation using porcine and human skin, and photoacoustic spectroscopy.TM 184 silicone elastomers kit  was purchased from Dow . Purified water was obtained from a water purification system . Absolute ethanol was purchased from Anidrol .Poloxamer 407 (P407), gelatin, and propylene glycol were purchased from Sigma . Polyvinyl alcohol (PVA) was obtained from Neon , polyvinylpyrrolidone (PVP), and ethanol 96° GL was purchased from Labsynth . SylgardApis mellifera L. bees, located inside a eucalyptus reserve, surrounded by native forest with predominance of Baccharis dracunculifolia (Asteraceae), in the northwest of Parana state. This research was registered in Brazil with SISGEN N° AC7A2F5. The different PRP extracts were prepared by turbo extraction. The ethanolic extract (EE) was obtained using ethanol 96° GL and the comminuted drug with a drug/solvent (w/w) ratio of 3:7. The glycolic extract (GE) was prepared using the comminuted drug and an aqueous solution of propylene glycol 50% (w/w) and the same drug/solvent (w/w) ratio of 3:7. The final dispersions were filtered through grade-3 filter paper [Brazilian green propolis (PRP) was obtained from an apiary of er paper –45.The pH determination was performed using a pHmeter calibrated with buffer solutions. The relative density of the extract was determined using a pycnometer calibrated at 20 °C in a controlled temperature environment (20 °C). To determine the dry residue, exactly 1 g of extract was weighed, the excess solvents were evaporated on a hot plate for approximately 10 min. Subsequently, the containers containing the extracts were taken to an oven (110 °C) for 40 min until constant weight. An aliquot of 12.5 mL of the extractive solution was placed in a round-bottomed flask, added to 50.0 mL of purified water and subjected to simple distillation. The distillate was collected in a 50.0 mL volumetric flask and made up to volume with purified water. The distillate density was determined, and from the density the alcohol content was determined.The determination of the total polyphenol content was performed in 25.0 mL volumetric flasks, 10 mL of purified water were added and 10.0 μL of extract were added. Then, 1.0 mL of phosphomolybdotungstic reagent R  was added and the volume was made up with 14.06% (m/V) aqueous sodium carbonate solution. The extracts were analyzed using a high-performance liquid chromatograph coupled to a UV–vis spectrophotometric detector. For the detection of polyphenols, chrysin and p-coumaric acid  at a wavelength (λ) of 310 nm were used as markers. At least three determinations were performed for each assay ,46–48.The master structure utilized to fabricate the molds was a stainless-steel microneedle system (cartridge) from DermaPen  containing 36 microneedles of 2 mm in length. The molds developed were inverse replicas of this master structure.For the preparation of the PDMS mold, suitable amounts of the catalyst agent and the silicone base were mixed, in a proportion of 1:6, using a beaker and taken to ultrasound for 5 min. Afterwards, the mixture was poured into 12-well plates and taken to ultrasound for another 5 min. The microneedle cartridge was inserted into the mixture and placed under ultrasound for 5 min to remove all air bubbles. The plate containing the silicone and the microneedle cartridge was placed in a vacuum desiccator for silicone curing. After 24 h, the cartridge was carefully removed and the mold obtained was cleaned and evaluated for integrity for later use.2, totaling nine formulations for each type of extract, rendering a total of 18 formulations  and PRP extract at 4%, 8%, and 12% (w/w) for the ethanolic extract and 2%, 4%, and 8% (w/w) for the glycolic extract, resulting in a factorial design of 3ulations .Firstly, each polymer was dispersed separately. To PVA, 8 mL of purified water were added, and the mixture was heated and stirred until completely dispersed. The suitable amount of PVP was dissolved in 3 mL of absolute ethyl alcohol, and P407 was dissolved in 2 mL of the same solvent. After obtaining the individual dispersions, the P407 was poured onto the PVP, magnetically stirred for 10 min, and this mixture was poured onto the PVA under magnetic stirring for another 10 min, after which the PRP extract was placed and the mixture was stirred for 20 min.Afterwards, the final formulations were centrifuged at 3200 rpm for 10 min to remove air bubbles. Each formulation was carefully poured onto the molds to form a complete microneedle patch. Then, they were placed in a desiccator for 48 h for complete casting. Subsequently, the MNs were carefully recovered from the molds with the help of tweezers and stored in desiccators until future use.2 full factorial design (two blocks) was utilized to determine the influence of P407 (X1) and PRP extract (X2) concentrations on height and base measurements, strength, and printing area in a hard surface (glass Petri dish) [The 3ri dish) . The indY is the response (the dependent variable) as function of X1 and X2, b0 is a constant term (the arithmetic mean response of nine batches), b1 and b2 are the estimated coefficients of linear terms, and b12 is the coefficient of the interaction effect. The polynomial terms X112 and X222 consider the non-linearity, and the interaction term X1X2 shows the response changes when two factors are simultaneously changed.where r2 and r2adj), whereas the adequacy of the model was determined by a lack-of-fit test.The experiments were randomized to minimize the influence of unexplained variability in the responses. Residual analysis was also conducted to validate the assumptions utilized in the analysis of variance and to identify outliers. The proportion of variance explained by models obtained was given by the multiple and adjusted coefficients , at 40× magnification. For analysis, a row of MNs was cut from the structure obtained for better visualization of the isolated microneedles.The elucidation of more specific morphological details of the fabricated MNs was performed by scanning electron microscopy. Each sample was placed over a circular metallic holder (with the help of double-sided tape) and fixed with colloidal gold in a hermetically closed chamber in argon atmosphere. The samples were observed using a Quanta 250 electron microscope .The effects of the amount of P407 and of amount and type of PRP extract on the MN hardness and mechanical resistance were quantitatively evaluated by a microneedle textural study. A compressive load was applied  to the polymeric MNs using a TA-XTplus texturometer . The MN patches were connected to the mobile cylindrical probe  with double-sided tape. The probe was pressed against the defined surface (hard glass surface of Petri dish) at a rate of 0.05 mm/s. The pre-test and post-test speeds were 1 mm/s, and the applied force was 0.049 N. The length at which the MNs descended in the test was 1.5 mm. From the relationship between force and time, the compression force (the maximum force during compression) and compression area (the work required to deform the sample during compression) were calculated using the software Texture Exponent 6.1.12.0 .The effect of formulation composition on the puncture force and distance was evaluated by MN insertion study. The best formulations selected from previous analysis were mechanically evaluated using different surfaces. The same texturometer and probe previously described were utilized. In compression mode, the MNs patch attached to the probe was pressed against the defined surface with a force of 0.049 N at a 0.05 mm/s using 5% gelatin or porcine skin or at rate of 0.1 mm/s using PVC film or Parafilm M [p < 0.05 was considered and the Statistica 12.0 software  was used for all analyses.The effects of the amount of P407 and of amount and type of PRP extract on the height and base measurements, strength, and printing area in the petri dish were statistically evaluated using three-way analysis of variance (ANOVA). Tukey’s post-hoc test was used to analyze significant differences. A significance level of Supporting Information features pictures of molds and macroscopic appearance of the MNs, statistical analysis of the compression force of MNs applied on different substrates , images of substrates after puncture test, statistical analysis of MN height and base measurements, and images of the MNs before and after the compression test.File 1Additional figures and tables.