Alzheimer’s disease (AD) is a fatal neurodegenerative disorder associated with severe dementia, progressive cognitive decline, and irreversible memory loss. Although its etiopathogenesis is still unclear, the aggregation of amyloid-β (Aβ) peptides into supramolecular structures and their accumulation in the central nervous system play a critical role in the onset and progression of the disease. On such a premise, the inhibition of the early stages of Aβ aggregation is a potential prevention strategy for the treatment of AD. Since several natural occurring compounds, as well as metal-based molecules, showed promising inhibitory activities toward Aβ aggregation, we herein characterized the interaction of an organoruthenium derivative of curcumin with Aβ(1–40) and Aβ(1–42) peptides, and we evaluated its ability to inhibit the oligomerization/fibrillogenesis processes by combining in silico and in vitro methods. In general, besides being less toxic to neuronal cells, the derivative preserved the amyloid binding ability of the parent compound in terms of equilibrium dissociation constants but (most notably) was more effective both in retarding the formation and limiting the size of amyloid aggregates by virtue of a higher hindering effect on the amyloid–amyloid elongation surface. Additionally, the complex protected neuronal cells from amyloid toxicity. More than 50 million individuals worldwide suffer from Alzheimer’s disease (AD), the most common form of fatal neurodegenerative dementia ,2,3,4 thHistologically, the accumulation of misfolded proteins as plaques in the aging brain is among the main hallmarks of AD: this triggering event causes oxidative and inflammatory damage, which, in turn, leads to energy failure and synaptic dysfunction . These pBased on these evidences, in this study, we explored the amyloid binding and anti-aggregating abilities of a bioactive half-sandwich organo-ruthenium(II) complex of curcumin  by integThe binding modes of curcumin and RuCurcumin with Aβ(1–40) or Aβ(1–42) monomers were rationalized by molecular docking studies. In terms of binding affinity, p-cym-Ru modification induced a low-to-negligible effect: in fact, irrespective of the different energy contributions to the stabilization of each complex , comparable values of binding affinities in the low micromolar range were computed . In addiStructurally, the beta-turn region of the U-shaped amyloid 1–40 monomer was calculated to be the most likely to accommodate the molecules of interest. Specifically, parent curcumin and its Ru-derivative targeted two largely overlapping regions of the amyloid 1–40 monomer, with amino acids Ala13, Glu14, Asp15, Val16, and Ser18 being common to both binding interfaces , in line with the computed lower electrostatic energy contribution.Both molecules interacted with Aβ(1–40) and Aβ(1–42) monomer peptides, specifically and reversibly , with moBased on these results and on the computational study, according to which both curcumin and RuCurcumin could interact with amyloid peptides and  antagonize the amyloid–amyloid dimerization, we performed a competitive binding assay that exploited the same amyloid-β functionalized surfaces.Interestingly, the pre-saturation of independent surface-blocked amyloid peptides with 20 μM curcumin or RuCurcumin, significantly interfered with the formation of amyloid–amyloid complexes, as evident from the general increase in the values of dissociation kinetic and equilibrium parameters  and mos, which iGlobally, RuCurcumin showed the highest destabilizing effect on amyloid 1-42 dimers, with a nearly 50-fold decrease in binding affinity.To test the effective inhibitory ability of curcumin and RuCurcumin against amyloid-β aggregation, we monitored kinetics of fibril formation using a thioflavin T (ThT) fluorescence assay. Our data proved that the aggregation into fibrils of Aβ(1–40) and Aβ(1–42) peptides was impaired in the presence of 20 µM curcumin/RuCurcumin, with respect to ligand-free control .c parameter, d and e parameters, respectively, The two molecules showed a similar anti-aggregation activity against Aβ(1–40), with the Ru-derivative being more effective in limiting fibril elongation. Conversely, more significant differences occurred with Aβ(1–42). Specifically, RuCurcumin had a minor effect on the Aβ(1–42) nucleation, as evident from the negligible shift in the point of transition with respect to ligand-free aggregation  aggregates, grown in the absence of any co-treatment, showed three bands approximately at 55 kDa, 40 kDa (corresponding to medium-size oligomers) and at 15 kDa (corresponding to tetramer). The band at 55 kDa was not detectable upon treatment with curcumin, whereas the effects of the treatment with RuCurcumin were even more evident, with only the 15 kDa band being still observed , Panel ATo obtain information on the morphology of Aβ fibril aggregates, scanning electron microscopy analyses were performed on stub-deposited amyloid aggregates grown in the presence and in the absence of 20 µM curcumin/RuCurcumin, as described in the Materials and Methods. Morphologically, large β-amyloid plaque deposits (with a mean diameter in the range 50–80 μm) were generally observed. These deposits consisted of Aβ fibril network structures with peculiar arrangements. Amyloid 1-40 and 1-42 plaques showed major differences in terms of structural organization . Specifiin) and longer permanence in the membrane, which is most likely due to the higher structural complexity. Conversely, curcumin was not significantly retained in the membrane. Irrespective of these initial differences, the internalization process globally occurred in a fully comparable timeframe for both molecules (400 min), given the faster release rate from the membrane of RuCurcumin  fluorescent probe, as previously reported . Upon cuTo evaluate the protective effects of RuCurcumin on a neuronal cell model, SH-SY5Y cells , were treated with subtoxic concentrations of RuCurcumin for 24 h. Comparative analyses revealed that the higher amyloid production   was assoThe compound [Ru(cym)(curc)Cl] was prepared by modifying the previously described procedure  Scheme .2]2  was added. The mixture was stirred for 24 h at room temperature and an orange precipitate formed, which was removed by filtration and washed with n-hexane . The residue was concentrated to 2 mL and stored at 4 °C. Red crystals formed over several days. The compound is soluble in acetone, acetonitrile, chlorinated solvents, DMF, and DMSO. Λm : 22 S cm2 mol−1. Λm ((CH3)2SO, 298 K, 10−3 mol/L): 2 S cm2 mol−1. IR : 3220 m br ν(OH), 1619 m, 1591s, 1500vs ν; 1H NMR : δ, 1.39 2 of p-cym), 2.34 , 2.98 2 of p-cym), 3.93 , 5.45 H of curc), 5.55br, 5.84br 2 of p-cym), 6.42 H of curc, 3Jtrans = 15 Hz), 6.91 H of curc), 7.00 H and CH of curc), 7.52 H of curc, 3Jtrans = 16 Hz); 13C  [(p-cym)Ru(curc)]+. IR spectra were recorded from 4000 to 30 cm−1 on a PerkinElmer Frontier FT-IR instrument. Additionally, 1H and 13C NMR spectra were recorded with a 500 Bruker AscendTM  and a 400 Mercury Plus Varian  instrument operating at room temperature, relative to TMS. {1H,13C}-HSQC and {1H,13C}-HMBC NMR spectra were recorded with a 500 Bruker AscendTM  operating at room temperature, relative to TMS. Positive and negative ion electrospray ionization mass spectra (ESI-MS) were obtained on a Series 1100 MSI detector HP spectrometer using methanol as the mobile phase. Solutions (3 mg/mL) were prepared using reagent-grade methanol. Masses and intensities were compared to those calculated using IsoPro Isotopic Abundance Simulator, version 2.1.28. Melting points are uncorrected and were recorded on a STMP3 Stuart scientific instrument and on a capillary apparatus. Samples for microanalysis were vacuum-dried to a constant weight  and analysed on a Fisons Instruments 1108 CHNS-O elemental analyser.The molecular structure of the complex was also previously reported and discussed . CurcumiHz); 13C  NMR (CDCThe molecular models of the complexes between Aβ(1–40) and Aβ(1–42), and the molecules of interest were obtained according to flexible ligand-receptor docking using Autodock 4.2 . Rutheni3COONa, pH 4.5 , then covalently coupled to independent carboxylic surfaces via the N-terminus of Lys residues. Different stock solutions of Aβ(1–4x), with concentrations in the range 50–400 μg/mL, were tested to achieve optimal surface density: 100 μg/mL was finally selected, as it minimized steric hindrance and, at the same time, prevented the dimerization between blocked peptides, with both events being likely to reduce the number of available binding sites on the sensing surface. Free carboxylic sites on the sensor surface were inactivated with 1 M ethanolamine, pH 8.5, before surface re-equilibration with PBS. The resulting shifts in the baseline (ΔR = 360 arcsec) generally indicated the assembly of a partial monolayer for a 4 kDa protein (approximately 80% surface occupancy), corresponding to a final surface density of 0.60–0.70 ng/mm2, equivalent to 4.5–5 mg/mL. Negative baseline drift signals in Aβ surfaces were not observed with time or upon multiple washes, confirming that the amyloid oligopeptides were irreversibly anchored to the sensor surface. The temperature of the sensing chamber was set at 37 °C throughout. Next, immobilized Aβ peptides were added with different concentrations of the ligands of interest , each time monitoring association kinetics up to the plateau, and assessing baseline recovery among consecutive additions.Commercial Aβ(1–40) and Aβ(1–42) were dissolved to 1 mM in HFIP and immediately aliquoted in sterile tubes; the solvent was removed, under vacuum, at −20 °C, and the resulting peptide films were stored at −80 °C until use . Bindingexc = 440 nm, λem = 485 nm). The plate was shaken for 10 s before each reading. Background ThT fluorescence was subtracted from all readings. Aggregation kinetic parameters were obtained by fitting fluorescence raw data points to the sigmoidal curve in the following five-parameter equation [t is time, a and d control the position of the first and the second horizontal asymptotes, respectively, b controls the slope of the transition between asymptotes and together with e control the rate of approach to the second asymptote, and c controls the position of the transition region.ThT fluorescence assay was used to quantitatively monitor the role of curcumin and RuCurcumin on the aggregation kinetics of amyloid fibrils. Aβ(1–40) and Aβ(1–42) films were re-dissolved to a final concentration of 100 μM in 10 mM HCl and incubated, at 37 °C, for 24 h to promote the formation fibrils . Aggregaequation  using MA® Novex 12% Bis–Tris Gel, and electroblotted onto PVDF membranes. Membranes with transferred proteins were blocked overnight at 4 °C in TTBS , containing 5% bovine serum albumin, and then incubated with an anti-oligomer A11 polyclonal antibody. The immunoblot detection was performed with an ECL Western blotting analysis system . Each gel was loaded with molecular mass markers in the range of 20–245 kDa .Aβ(1–40) and Aβ(1–42) films were re-dissolved to a final concentration of 100 μM in F12 cell media and incubated at 4 °C, for 24 h, to promote the formation of oligomers . OligomeAβ(1–40) and Aβ(1–42) peptide films were re-suspended in 10 mM HCl, to a final concentration of 10 μM, and incubated at 37 °C, for 24 h, in the presence and in the absence of 20 μM curcumin or RuCurcumin. Each resulting solution (100 μL) was loaded onto individual SEM stubs and dried by stepwise ethanol treatment to preserve the nanostructures of β-amyloid fibrils . Resultiexc = 340 nm; λem = 460 nm) was used to monitor compound cell internalization by following the changes in membrane fluidity [5 SH-SY5Y cells per mL were incubated with 1 μM TMA-DPH, then individually added with the compounds. Fluorescence anisotropy (r) was measured for 400 min, at 10-min intervals, and was calculated as previously reported [ink and outk) were derived according to a general mono-exponential model:inr and outr are the fluorescence anisotropy intervals corresponding to drug entry and exit phases from the membrane, respectively.TMA-DPH probe  and AβPP  mutations (APPmut) was performed as described elsewhere [−1. Cell viability was evaluated with the 3--2,5-diphenyltetrazolium bromide assay (MTT) [−1) was added to the culture medium without FBS and incubated, for 2 h, at 37 °C. The medium was then removed and replaced with 100 μL of DMSO. The optical density was measured at 550 nm in a microtiter plate reader. At least six cultures were utilized for each time point.SH-SY5Y cells were cultured in 1:1 Dulbecco’s modified Eagle’s medium and Nutrient Mixture F12 containing 10% foetal bovine serum (FBS), 2 mM glutamine, 100 units mLlsewhere . These cay (MTT) . Upon 24p-values < 0.05 and < 0.01 were considered significant.Results presented in this study are expressed as mean values, with their standard deviations obtained from tree separate experiments. Statistical analysis was performed with one-way ANOVA, followed by the Bonferroni test using MATLAB R2021b. AD is a multifactorial neuropathology related to several biological and physiological alterations. Currently available drugs can ameliorate cognitive, behavioural, and functional deficits, but they cannot stop the progression of AD.Herein, we tested an arene Ru(II) curcumin complex for its anti-aggregating properties toward amyloid 1-40 and 1-42 peptides. The modified curcumin was shown to reversibly bind both peptides with moderate affinity and interfere with the stacking mechanisms of the aggregation. Most interestingly, experimental evidence and molecular modelling data demonstrated that the chemical modification of curcumin improved its efficacy, most likely due to a higher hindering effect on the amyloid aggregation mechanism.These results supported the hypothesis that the biological properties of naturally occurring compounds can be properly tuned upon chemical modification and, case in point, that curcumin derivative can prevent amyloid aggregation and, consequently, reduce the cellular toxicity of Aβ aggregates. Globally, our data may pave the way for the exploration of more potent amyloid inhibitors , which are also able to disrupt existing aggregates, based on the rational modification of naturally occurring molecules.


The placenta is responsible for materno-fetal resource allocation. Placental insufficiency may disrupt fetal growth trajectory, with negative consequences for maternal and fetal health. By manipulating a growth-controlling protein named PI3K-p110α, this work, performed on mice, shows the relevance of the maternal environment and fetal genotype in determining the conceptus growth and mitochondria bioenergetic reserve in the placental transport zone. Interestingly, the disruption of maternal and/or fetal PI3K-p110α exerted these effects in a sex-specific manner, with male fetuses more affected than females. These data highlight the importance of the sex of the fetus and placental mitochondrial function in the regulation of fetal growth.Fetal growth is reliant on placental formation and function, which, in turn, requires the energy produced by the mitochondria. Prior work has shown that both mother and fetus operate via the phosphoinositol 3-kinase (PI3K)-p110α signalling pathway to modify placental development, function, and fetal growth outcomes. This study in mice used genetic inactivation of PI3K-p110α (α/+) in mothers and fetuses and high resolution respirometry to investigate the influence of maternal and fetal PI3K-p110α deficiency on fetal and placental growth, in relation to placental mitochondrial bioenergetics, for each fetal sex. The effect of PI3K-p110α deficiency on maternal body composition was also determined to understand more about the maternal-driven changes in feto-placental development. These data show that male fetuses were more sensitive than females to fetal PI3K-p110α deficiency, as they had greater reductions in fetal and placental weight, when compared to their WT littermates. Placental weight was also altered in males only of α/+ dams. In addition, α/+ male, but not female, fetuses showed an increase in mitochondrial reserve capacity, when compared to their WT littermates in α/+ dams. Finally, α/+ dams exhibited reduced adipose depot masses, compared to wild-type dams. These findings, thus, demonstrate that maternal nutrient reserves and ability to apportion nutrients to the fetus are reduced in α/+ dams. Moreover, maternal and fetal PI3K-p110α deficiency impacts conceptus growth and placental mitochondrial bioenergetic function, in a manner dependent on fetal sex. In mammals, adequate placental function is required to support fetal growth and development. Evidence from human and experimental animal models have shown that aberrant placental transport function is associated with fetal growth restriction (FGR), a condition that currently affects approximately 5–10% of pregnancies ,3. MoreoPik3ca) results in embryonic death, whilst heterozygous deficiency leads to placental dysmorphogenesis, adaptive up-regulation of nutrient transport, and FGR in mice [Several signalling pathways have been described to be involved in the control of placental growth and function ,19,20,21 in mice ,18,20,26Whole body metabolism and energy balance has been reported to be affected in genetic models with disrupted PI3K-p110α signalling . In metaThis study was performed in accordance with the UK Home Office Animals (Scientific Procedures) Act 1986 and University of Cambridge ethics committee. Mice were housed in the University of Cambridge Animal Facility under a 12:12 h light-dark cycle with free access to chow food  and water. Non-pregnant WT and heterozygous PI3K-p110α deficient (α/+) females were reciprocally mated with WT and α/+ male mice to generate pregnancies with both WT and α/+ conceptuses. All mice were on a C57BL/6 background and WT, and α/+ females were nulliparous and 4 months old at the time of mating. The day a copulatory plug was detected was designated gestational day 1 (GD1). The generation of α/+ mice has been previously described .Sry gene using the Taq Ready PCR system (Sigma) and specific primer sequences  and α/+ mice identified by genotyping using primers 5′-TTCAAGCACTGTTTCAGCT-3′ and 5′-TTATGTTCTTGCTCAAGTCCTA-3′, as previously described [On gestational day 18 (GD18), between 08:00 h and 09:00 h, dams were killed by cervical dislocation. The gravid uterus was removed, and each fetus and its corresponding placenta were weighed. Maternal organs were also dissected and weighed. Fetal tails were taken for sex and genotype determination by PCR. Fetal sex was determined by detection of the escribed ,22. Afte2+, 1 mM free Mg2+, 20 mM imidazole, 20 mM taurine, 50 mM K-MES, 0.5 mM DTT, 6.56 mM MgCl2, and 5.77 mM ATP and 15 mM phosphocreatine) containing saponin  for 20 min, followed by 3 washes of 5 min in respiratory medium . Samples were blotted on filter paper to determine wet tissue mass, and then 15–20 mg of the Lz sample was placed into a pre-calibrated chamber of an Oxygraph-2k respirometer with respiratory medium MiR05. Samples were analyzed at 37 °C, and oxygen concentration was kept between 250 and 300 μM. DatLab software  was used for real-time acquisition and analysis of oxygen consumption. A sequential substrate, inhibitor, and uncoupler titration protocol was then performed, as described previously [High resolution respirometry  was used to assess the placental capacity for mitochondrial OXPHOS, namely substrate use and electron transfer system (ETS) function, as previously described . Brieflyeviously . Additiot-test or Mann–Whitney test, based on a prior analysis of the normality and homogeneity of all variables assessed using the Shapiro-Wilk test. The effect and interaction of maternal and fetal α/+ genotype was evaluated by two-way ANOVA, followed by Tukey post hoc tests, for each fetal sex separately. Litter composition (sex and genotype) was analyzed by student t-test. Outliers were detected by the Prisms Grubbs’ test. Data are shown as mean ± SEM and individual points when possible. Data were considered statistically significant at values of p < 0.05.Statistical analyses were performed using GraphPad Prism version 9 . Maternal body composition and litter size composition (sex and genotype) were analysed using Student Female α/+ mice were lighter at the start of the pregnancy and showed a tendency for lighter hysterectomised weight at the end of the pregnancy . PregnanThere was no difference in litter size, genotype, or sex frequency at GD18 between the WTxα/+ and α/+xWT parental crosses . There wp < 0.001; p < 0.001), and this was reflected by an interaction between fetal and maternal α/+ genotype. In female fetuses only, there was an overall effect of maternal α/+ to reduce Lz weight by 11% (p < 0.001). Together, these data indicate that the effect of fetal and maternal p110α deficiency on fetal-placental growth is modified by fetal sex, with more dramatic effects observed in male fetuses.In females, there was not an effect of fetal or maternal α/+ on placental weight B. Howevet by 11% C. There t by 11% D. HoweveOXPHOS) was significantly greater for male α/+ fetuses, compared to their respective WT littermates in α/+ dams  and CI + CIIP OXPHOS in males (PInt = 0.0628)  . These rUsing genetic manipulations, this study demonstrates that a deficiency in PI3K-p110α signalling in the mother and/or fetus modified, in a sex-specific manner, the conceptus mass and mitochondrial bioenergetic capacity in the mouse placental Lz. For instance, male fetuses were more sensitive than females to fetal PI3K-p110α deficiency, as they presented less fetal, placental, and Lz weight, when compared to their WT littermates. Placental weight was also altered in males only of α/+ dams. In addition, α/+ male fetuses showed an increase in Lz mitochondrial reserve capacity, when compared to their WT male littermates in α/+ dams. Sexual dimorphism, in response to genetic and/or gestational perturbations in placental function, has been extensively reported in the recent years ,31,32,33OXPHOS-related oxygen flux. The mitochondrial reserve capacity is a parameter used to describe the amount of extra ATP that can be produced by OXPHOS, in case of a sudden increase in energy demand [Previous data reported that PI3K-p110α deficiency in the fetus negatively affects fetal growth and placental development ,18. Thisy demand . This iny demand . Interesy demand . There ay demand ,37. Togey demand ,20 or try demand  isolatedy demand  been repPrevious data has also identified that placental development is influenced by whether the mother herself carries the p110α mutation (α/+) . PlacentIn summary, this work, using genetic manipulations of the growth and metabolic regulator PI3K-p110α, has established that changes in the fetal genetic ability to grow, as well as the genetically-determined maternal environment, affect feto-placental development and placental respiratory function in a sex-specific manner. Moreover, the effects detected in mitochondria respiratory function seem to depend on an interaction between the mother and/or fetus carrying the mutation for each sex. These findings may have importance for understanding the fetal outcomes in pregnancies reporting alterations in the PI3K-AKT signalling pathway in the placenta ,41, and/


In vitro and in vivo release of PTX and CMPT were investigated to find the release mechanism from nanofibers compared to direct administration of pristine PTX and CMPT. The mean fiber diameter for composite and core–shell nanofibers with shell feeding rates of 0.3, 0.5, and 0.7 mL h−1 was about 225, 330, 520, and 640 nm, respectively. In vivo release studies indicated that the blood concentration of CMPT and PTX for rats fed with core–shell nanofibers reached the highest values of 26.8 ± 0.04 μg mL−1, and 26.5 ± 0.05 μg mL−1 in 36 h, and 24 h and reduced slowly within 84 h, and 48 h, respectively. The maximum cytotoxicity was 75% in the presence PVA/κ-carrageenan/CMPT/Au/pegylated-PU/PTX core–shell nanofibers. In vivo antitumor activity results confirmed the synergic effect of Au, CMPT and PTX anticancer drugs on the reduction of tumor volume without change in mouse weight by the PVA/κ-carrageenan/CMPT/Au/pegylated PU/PTX core–shell nanofibers. The obtained results indicated that the simultaneous loading of CMPT and PTX anticancer drugs and Au nanoparticles is more beneficial for lung cancer treatment.Gold nanoparticles, paclitaxel (PTX), and camptothecin (CMPT) were loaded into the PVA/κ-carrageenan/pegylated-PU composite and core–shell nanofibers prepared by two-nozzle and coaxial electrospinning methods. The capability of composite and core–shell nanofibers was investigated for the targeted delivery of anticancer drugs in lung cancer treatment.  in vivo release from core–shell nanofibers against lung cancer.Core–shell nanofibers and  In other study, the combined effect of PTX and CMPT was investigated against melanoma,3 breast, pancreatic,4 and medullary thyroid carcinomas.5 Chen et al.6 attached PTX and CMPT anticancer drugs on the 2-nitroimidazole against hypoxic tumors. In another study, CMPT and PTX were conjugated with two cyclic cell-penetrating peptides to inhibit MCF-7 breast cancer cells.7 Hariri et al.8 investigated the targeted delivery of PTX and CMPT from a cross-linked sponge against lung cancer. The use of targeted drug delivery systems such as liposomes,9,10 microspheres,11,12 nanoparticles,13,14 and nanofibers15–18 could provide the sustained delivery of anticancer drugs.The combination of paclitaxel (PTX) and camptothecin (CMPT) could provide several advantages over monotherapy. In previous studies, better treatment of various cancers, including lung, breast, and melanoma, is demonstrated by the co-delivery of PTX and CMPT compared to the monotherapy of PTX or CMPT1-4. For instance, the concentrations of PTX and CMPT could be controlled by their loading into the liposome.19–23 Carrageenan could also interact with inorganic materials such as graphene oxide (GO), metal–organic frameworks (MOFs) and proteins. κ-carrageenan, and κ-carrageenan-based composites have been used in various drug delivery systems. For instance, Sun et al.20 interacted κ-carrageenan with zein nanoparticles. Javanbakht et al.21 synthesized κ-carrageenan/MOFs composite hydrogel for oral delivery. Vinothini et al.22 used κ-carrageenan-grafted graphene oxide for drug delivery of DOX. Tort & Acartürk23 investigated the capability of electrospun κ-carrageenan nanofibers for oral mucositis treatment. The use of nanofibers prepared by electrospinning technique is an effective strategy for loading the high content of drugs due to their high porosity, high surface area, small size, good flexibility, and interconnecting channels.24 However, there are still limitations for electrospun nanofibers, due to their rapid degradation over time. The various nanofibers, including composite, multi-layered,25 and core–shell nanofibers,17,18 could provide the prolonged-release profiles. In recent studies, the hydrophobic polymers such as poly(ε-caprolactone),26 polyurethane (PU),27 poly(lactic acid) (PLA),28 and poly(lactic-co-glycolic acid) (PLGA)29 were used to increase the stability of polysaccharide nanofibers in aquatic systems.κ-Carrageenan, an anionic sulfonated polysaccharide, is extracted from red algae and used for biomedical applications such as food, tissue engineering and drug delivery systems.30 The degradation rate of PUs is mainly due to the cleavage of hydrolytic bonds present in their soft segments. The composition of the soft segments could control the degradation rate of the PUs. The degradation rate of PUs with hydrophilic soft segments such as poly(ethylene glycol) is faster than PUs with hydrophobic soft segments such as polycaprolactone diol.31 Loading of anticancer drugs into the PUs, and its derivates could increase the availability of the anticancer agents at the tumor sites, suppress the cancer cells that did not kill by the initial dose of drug31,32 and decrease the adverse side effects. For instance, Gajbhiye et al.33 reviewed the performance of biodegradable PU nano-constructs for the targeted delivery of anticancer drugs. Yin et al.34 indicated that PTX molecules were released slowly from liposome-encapsulated PU scaffolds compared to PTX release from PU. Liu et al.35 investigated the potential PU functionalized with amine, and carboxyl group micelles for incorporating of doxorubicin hydrochloride (DOX) and PTX. In previous studies, the capability of drug-loaded PU nanofibers for various cancers treatment was studied by our group.17,18,36,37 Recently, inorganic materials such as metal oxides, zeolites, MOFs and noble metals were loaded into the nanofibrous matrix to increase the delivery of anticancer drugs on the surface of the cancerous tissues. Gold-based nanomaterials with various shapes, including nanoparticles,38–40 nanocages,41,42 and nanorods43–45 are incorporated into the different forms of drug delivery systems for targeted delivery and imaging in cancer therapy. Gold nanoparticles (Au NPs), due to their high stability, and easier synthesis were used to improve the attachment of cancerous cells and increase the therapeutic efficacy.46,47Most PUs are non-degradable and have little use in biomedical applications. On the other hand, the natural/synthetic biodegradable polymers are stiff with low flexibility or soft with poor mechanical properties. To overcome these challenges, the biodegradable PUs, composed of a hard segment, soft segment, and chain extender, have been introduced as one of the most popular polymers due to their low cost, high biocompatibility, and excellent efficiency for drug delivery systems.In the present study, PTX and CMPT were loaded into the κ-carrageenan/Au/pegylated-PU composite nanofibers prepared by two-nozzle and coaxial electrospinning methods for targeted delivery of anticancer drugs against lung cancer treatment. It was hypothesized that the co-delivery of PTX and CMPT from nanofibers and the use of gold nanoparticles in the nanofibers structure could enhance the targeted delivery of anticancer drugs on cancerous tissues.2.2.1Mn = 2000 Da, PEG) supplied from Sigma-Aldrich (USA); hexamethylene diisocyanate (HDI) and 1,4-butanediol (BDO) purchased from Merck Company (Germany) were used to synthesize the pegylated polyurethane. Polyvinyl alcohol  and κ-carrageenan were used to prepare PVA/κ-carrageenan nanofibers. Paclitaxel and camptothecin supplied from Sigma-Aldrich (USA) were utilized as anticancer drugs. HAuCl4, and sodium citrate provided from Sigma-Aldrich (USA) were applied to synthesize the gold nanoparticles.Poly(ethylene glycol) . To load PTX molecules into the pegylated PU, 1 and 2 mg mL−1 PTX were added into the pegylated-PU solution under sonication for 30 min.The shell solution was the PTX-loaded pegylated polyurethane (PU). The pegylated polyurethane was synthesized by reacting PEG, HDI, and BDO as described previously by Yao 4 was used to synthesize Au nanoparticles.37 2 wt% κ-carrageenan (dissolved in distilled water) and 10 wt% PVA (dissolved in distilled water) were mixed under stirring for 6 h (3 : 7 v/v). To load CMPT and Au nanoparticles into the PVA/κ-carrageenan solution, 2 wt% Au suspension (with respect to the PVA/κ-carrageenan solution v/v) and 1, 2 mg mL−1 CMPT were added into the PVA/κ-carrageenan solution under sonication for 30 min.The core solution was the CMPT/Au-loaded PVA/κ-carrageenan solution. The citrate reduction of HAuCl2.3−1 for both solutions to produce the PVA/κ-carrageenan/CMPT/Au/pegylated PU/PTX composite nanofibers under an electrospinning time of 4 h.To prepare PVA/κ-carrageenan/CMPT/Au/pegylated PU/PTX composite nanofibers, the prepared pegylated PU/PTX and PVA/κ-carrageenan/CMPT/Au solutions were separately transferred into the two syringes (needle tip: 19 gauge) and then placed at both counter sides of the cylindrical collector which was placed an aluminum foil on the collector. The applied high voltage was varied between 15 and 25 kV. Tip–collector distance was constant at 12 cm. The feeding rate was between 0.3–1 mL h−1. The shell solution feeding rate varied between 0.3 and 0.7 mL h−1.To prepare the PVA/κ-carrageenan/CMPT/Au/pegylated PU/PTX core–shell nanofibers, the prepared core and shell solutions were transferred into the two syringes (tip needle: 27 and 14 gauges). The voltage, tip–collector distance, and core solution feeding rate were constant at 20 kV, 12 cm, and 0.3 mL h2.4−1 with a resolution of 2 cm−1 on the Fourier-transform infrared spectroscopy . X-ray diffraction (XRD) analysis ranged from 5° to 80° using an X-ray diffractometer . Morphology of nanoparticles and nanofibers was carried out using a scanning electron microscope  and a transmission electron microscope .Fourier transform infrared (FTIR) analysis was implemented ranging from 500–4000 cm2.5To evaluate the drug loading measurements in the nanofibers, drug entrapment efficiency (DEE%) and drug loading efficiency (DLE%) were calculated as follows:2.6In vitro release studies were implemented by immersion of 100 mg drug-loaded nanofibers in 500 mL of phosphate-buffered saline (PBS) solutions with a rotation rate of 50 rpm at the temperature of 37 °C, and pH values of 5.5 and 7.4. At preplanned times, 2 mL of aliquot was withdrawn, and 2 mL of fresh PBS was added. The final concentrations of PTX and CMPT were determined using High-performance liquid chromatography  and UV/Vis spectrophotometer  at 230 nm, and 360 nm, respectively. HPLC conditions are: a reversed-phase HPLC method, column: C-18 column, mobile phase: acetonitrile, methanol and water , temperature: 25 °C and flow rate of 1.0 mL min−1. Experiments were repeated three times, and the results were reported as mean ± S.D. The experimental data was statistically analyzed using an analysis of variance (ANOVA) as well as a t-test (P < 0.05).Q(t) = K0t), Higuchi (Q(t) = KHt0.5), and Korsmeyer–Peppas (Q(t) = Atn) equations.The release data were fitted by pharmacokinetic models, including zero order  that were maintained under a maximum isolation environment for 6 weeks based on Institutional Animals Ethics Committee (94/PO/ReBi/S/1999/CPCSEA). All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Tehran University and approved by the Animal Ethics Committee of National Institute for Medical Research Development (NIMAD), Tehran, Iran. Then, nanofibers-implemented 15 old mice were divided into five groups of three . The mice dose of PTX and CMPT was 5 mg kg−1. The nanofibrous scaffolds were implanted into the lower right flank of each mouse. At predetermined intervals , 0.5 mL of blood samples were withdrawn and were stored in a centrifuge tube. The samples were centrifuged at 5000 rpm for 5 min to separate the plasma that was frozen at −20 °C until HPLC and UV assays to determine the concentrations of PTX and CMPT, respectively.For investigating the 2.82 and 37 °C for 24, 48 and 72 h. 5 × 104 cells per well were transferred in a 48-well plate and treated with 1 and 2 mg mL−1 concentrations of pure drugs and their equivalent doses of drugs-loaded nanofibers. The MTT assay was implemented by using a microplate reader  at the wavelength of 570 nm.27 Briefly, the MTT assay was carried out as follows: 20 μL of MTT solution with a concentration of 5 mg mL−1 in PBS (pH 7.4) was added to each well, and then, were incubated for a further 4 h. Then, the solution was aspirated cautiously from each well. After treating the cells with Sorenson buffer, the optical density of each well was readied at 570 nm.To investigate the biocompatibility, and cytotoxicity of nanofibers, the pure drugs and drugs-loaded nanofibers were cut into flakes and placed in each well of L929 murine fibroblast and A549 lung cancer cell lines. Cells were purchased from the Institute Pasteur of Iran, and cultured in RPMI-1640 media under 5% CO2.96 A549 lung cancer cells were subcutaneously injected into the left hind flank of each anesthetized mice after isolation for 6 weeks. When the tumor volume reached 100 mm3, the mice were treated with nanofibrous samples, and experiments were continued for 20 days. The relative tumor volume with respect to the initial volume of tumor (V0 = 100 mm3) before treatment was calculated as V/V0. Briefly, animals were anesthetized by intra-peritoneal injection of pentobarbital at 20 mg kg−1, and a small incision was made on the skin to expose the tumor. A small incision was made into the tumor, and synthesized fibers were inserted into the tumor and then the wound was closed using subcutaneous suturing. The tumor volume is calculated as length × width × (length + width)/2.The tumor-bearing mice were divided into 5 groups of three. 2 × 103.3.1−1, 2920 cm−1, 1690 cm−1, 1425 cm−1, 1080 cm−1 and 887 cm−1 were assigned to the –OH stretching, CH2 asymmetric stretching, C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O stretching, CH2 bending, C–O stretching, and C–C stretching, respectively. For pure κ-carrageenan, the observed peaks at around 3320 cm−1, 1420 cm−1, 1220 cm−1, 1150 cm−1, 930 cm−1, and 840 cm−1 were due to the –OH stretching, C–O–H bending, OSO stretching, C–O–C absorption, and O–SO3 stretching bands, respectively. The new peaks between 500–700 cm−1 after loading Au nanoparticles into the PVA/κ-carrageenan confirmed the presence of Au nanoparticles in the nanofibrous matrix. The shift of carbonyl stretching vibrations correspond to the ketone groups from 1610 cm−1 to 1660 cm−1, and the lactone groups from 1720 cm−1 to 1745 cm−1 demonstrated the interaction of CMPT molecules with nanofibrous matrix. For pegylated PU, the appeared peaks at 3350 cm−1, 2930 cm−1, 2840 cm−1, 2225 cm−1, 1730 cm−1, 1644 cm−1, 1550 cm−1, 1460 cm−1, and 1105 cm−1 were related to the N–H stretching, asymmetric CH2 stretching, symmetric CH2 stretching, –NC–O absorption, CO stretching, urea absorption, N–H bending, CH2 bending, and C–O–C absorption, respectively. The detected peaks, in the FTIR spectrum of pure paclitaxel, at 3630 cm−1, 3095 cm−1, 1721 cm−1, 1650 cm−1, 1245 cm−1 and 1078 cm−1 were due to the presence of O–H, CH2, CO, C–C, C–N and C–O groups. The prominent peaks of pegylated PU and paclitaxel have overlapped.FTIR spectra of PVA, κ-carrageenan, Au, CMPT, PVA/κ-carrageenan/CMPT/Au, pegylated-PU, PTX, and pegylated-PU/PTX are illustrated in −1) are presented in −1 led to increase the fiber diameter from 225 nm to 470 nm. Therefore, an applied voltage of 20 kV, tip–collector distance of 12 cm and feeding rate of 0.3 mL h−1 were selected to fabricate composite nanofibers for further experiments. SEM images from PVA/κ-carrageenan/CMPT/Au/pegylated PU/PTX core–shell nanofibers with different shell feeding rates , core feeding rate of 0.3 mL h−1, the voltage of 20 kV, and the tip–collector distance of 12 cm are presented in −1 to 0.5 mL h−1, and 0.7 mL h−1 led to increased fiber diameter from 330 nm to 520 and 640 nm, respectively. The comparison of the PVA/κ-carrageenan/CMPT/Au/pegylated PU/PTX composite and core–shell nanofibers indicated that the composite nanofibers had a sharp distribution of fibers compared with broader distribution of core–shell nanofibers at studied condition. However, the non-alignment of fibers was detected for both composite and core–shell nanofibers, and thus have a randomly distributed nanofibrous structure for synthesized nanofibers. TEM image from core–shell nanofibers demonstrated the core-sheath structure of nanofibers prepared by coaxial electrospinning.SEM images of PVA/κ-carrageenan/CMPT/Au/pegylated PU/PTX composite nanofibers under different applied voltages  and feeding rates   were smaller than 0.45 from core–shell nanofibers which indicated that CMPT molecules were fled from the core–shell nanofibers following the Fickian mechanism. The linear release achieved for CMPT release from core–shell nanofibers demonstrated an excellent fitting of release data with the zero-order pharmacokinetic model. The “n” values for PTX release from both composite and core–shell nanofibers and “n” values higher than 0.45 for CMPT release from composite nanofibers confirmed the non-Fickian diffusion mechanism.DEE (%) and DLE (%) are evaluated for PTX and CMPT-loaded nanofibers which results are presented in pH: 7.4) . The obtequation . The valin vivo PTX and CMPT (10 mg of each drug) release profiles from nanofibers are presented in −1 PTX and CMPT solutions were used as control. Using pure PTX and CMPT solutions, plasma concentrations of PTX and CMPT came to their highest within 1 h and lower to average level at about 6 h for both anticancer drugs. When rats were fed with composite nanofibers, the blood concentration of CMPT and PTX reached the highest values of 29.3 ± 0.05 μg mL−1 and 26.8 ± 0.03 μg mL−1 in 6 h and 12 h and reduced slowly within 24 h and 48 h, respectively. Additionally, owing to the drug concentration gradient distribution from the inner layer to shell layer and then granted by core–shell nanofibers, which stores most drug in the inner layer, the curve of CMPT and PTX release from core–shell nanofibers exhibited a sustained release for about 84 h and 48 h, respectively due to the continuous release of drugs from core–shell nanofibers.The 3.3−1). The sustained release of CMPT and PTX from nanofibers resulted in more cytotoxicity of core–shell nanofibers than composite nanofibers. In vitro cytotoxicity results showed that its inhibition effect on tumor cells by the PVA/κ-carrageenan/CMPT/Au/pegylated PU/PTX core–shell nanofibers was remarkable, but the toxicity for normal cells was slight. Similar trends are reported by other researchers.49,50 The simultaneous incorporation of Au nanoparticles and co-delivery of PTX and CMPT into the core–shell nanofibers led to effective treatment of A549 lung cancer cells.The cell viability of synthesized composite and core–shell nanofibers toward L929 normal cells is implemented to investigate the biocompatibility of synthesized nanofibers. The results are illustrated in 3.4in vivo antitumor efficacy of the PVA/κ-carrageenan/CMPT/Au/pegylated PU/PTX composite and core–shell nanofibers was investigated on the A549 tumor-bearing mice with an initial tumor volume of 100 mm3 which the results of tumor volume change and the weight of mice after treating with nanofibers are presented in 's body weight after therapy revealed that no significant change occurred for all mice after injecting tumors for 20 days -loaded poly(l-lactic acid) nanofibers (PLLA/DOX@MSNs) against human spca-1 lung cancer cells. The maximum cell viability was 73.10%. The release trend of DOX from PLLA/DOX@MSNs composite nanofiber was similar to the release profiles of CMPT and PTX from PVA/κ-carrageenan/CMPT/Au/pegylated PU/PTX composite nanofibers . The inhibition rate of titanocene dichloride-loaded PLLA nanofibers was 68.2% (240 mg L−1 drug concentration).52 The maximum cell viability of A549 lung cancer cells by the chitosan/PLLA/TiO2/DOX/GO nanofiber and a magnetic field was about 82% after 86 h.53 Therefore, the co-incorporation of Au nanoparticles and anticancer drugs into the nanofibers demonstrated an optimal therapy effect on the tumor inhibition without changing the body weight.The  20 days . For com4.−1 were optimum values for the fabrication of PVA/κ-carrageenan/CMPT/Au/pegylated PU/PTX composite nanofibers with an average diameter of 225 nm. The mean fiber diameter for core–shell nanofibers produced under shell feeding rates of 0.3, 0.5, and 0.7 mL h−1 were 330, 520 and 640 nm, respectively. The higher DEE than 95% for PTX and CMPT confirmed an effective loading of anticancer drugs into the nanofibers. Time durations for PTX vb. CMPT release from core–shell nanofibers with 0.3, 0.5 and 0.7 mL h−1 shell feeding rates were 60, 48, 40 h vs. 96, 72, 60 h, respectively. The linear release is achieved for CMPT release from core–shell nanofibers. The PTX release data of composite, core–shell nanofibers and CMP release data of composite nanofibers were best described using the Peppas' equation. In vivo release studies indicated that rats fed with core–shell nanofibers, the blood concentration of CMPT and PTX reached the highest values of 26.8 ± 0.04 μg mL−1 and 26.5 ± 0.05 μg mL−1 in 36 h and 24 h and kept in the constant values between 36–84 h and 24–48 and finally reduced after 84 h and 48 h, respectively. The L929 cell viability higher than 90% for both composite and core–shell nanofibers confirmed the high biocompatibility of the synthesized composite and core–shell nanofibrous samples. The maximum cytotoxicity was 75% in the presence PVA/κ-carrageenan/CMPT/Au/pegylated PU/PTX core–shell nanofibers. In vivo antitumor efficacy results of A549 tumor-bearing mice treated with composite and core–shell nanofibers demonstrated the best effect on the reduction of tumor volume and enhancement of tumor inhibition without changing the mice's weight in the presence PVA/κ-carrageenan/CMPT/Au/pegylated PU/PTX core–shell nanofibers.In this work, Au nanoparticles, CMPT and PTX anticancer drugs were successfully incorporated into the PVA/κ-carrageenan/pegylated PU composite and core–shell nanofibers. The applied voltage of 20 kV, tip–collector distance of 12 cm and feeding rate of 0.3 mL hANOVAAnalysis of varianceBDO1,4-ButanediolCMPTCamptothecinDOXDoxorubicin hydrochlorideDEEDrug entrapment efficiencyDLEDrug loading efficiencyFTIRFourier transform infraredAu NPsGold nanoparticlesGOGraphene oxideHDIHexamethylene diisocyanateHPLCHigh-performance liquid chromatographyMOFsMetal–organic frameworksDMFN,N-DimethylacetamidePTXPaclitaxelPBSPhosphate buffered salinePEGPoly(ethylene glycol)PLAPoly(lactic acid)PLGAco-glycolic acid)Poly(lactic-PUPolyurethanePVAPolyvinyl alcoholSEMScanning electron microscopeTEMTransmission electron microscopeXRDX-ray diffractionThere are no conflicts to declare.


Rotaviruses (RVs) are a significant cause of severe diarrheal illness in infants and young animals, including pigs. Group C rotavirus (RVC) is an emerging pathogen increasingly reported in pigs and humans worldwide, and is currently recognized as the major cause of gastroenteritis in neonatal piglets that results in substantial economic losses to the pork industry. However, little is known about RVC pathogenesis due to the lack of a robust cell culture system, with the exception of the RVC Cowden strain. Here, we evaluated the permissiveness of porcine crypt-derived 3D and 2D intestinal enteroid (PIE) culture systems for RVC infection. Differentiated 3D and 2D PIEs were infected with porcine RVC (PRVC) Cowden G1P[1], PRVC104 G3P[18], and PRVC143 G6P[5] virulent strains, and the virus replication was measured by qRT-PCR. Our results demonstrated that all RVC strains replicated in 2D-PIEs poorly, while 3D-PIEs supported a higher level of replication, suggesting that RVC selectively infects terminally differentiated enterocytes, which were less abundant in the 2D vs. 3D PIE cultures. While cellular receptors for RVC are unknown, target cell surface carbohydrates, including histo-blood-group antigens (HBGAs) and sialic acids (SAs), are believed to play a role in cell attachment/entry. The evaluation of the selective binding of RVCs to different HBGAs revealed that PRVC Cowden G1P[1] replicated to the highest titers in the HBGA-A PIEs, while PRVC104 or PRVC143 achieved the highest titers in the HBGA-H PIEs. Further, contrasting outcomes were observed following sialidase treatment , which significantly enhanced Cowden and RVC143 replication, but inhibited the growth of PRVC104. These observations suggest that different RVC strains may recognize terminal (PRVC104) as well as internal (Cowden and RVC143) SAs on gangliosides. Finally, several cell culture additives, such as diethylaminoethyl (DEAE)-dextran, cholesterol, and bile extract, were tested to establish if they could enhance RVC replication. We observed that only DEAE-dextran significantly enhanced RVC attachment, but it had no effect on RVC replication. Additionally, the depletion of cellular cholesterol by MβCD inhibited Cowden replication, while the restoration of the cellular cholesterol partially reversed the MβCD effects. These results suggest that cellular cholesterol plays an important role in the replication of the PRVC strain tested. Overall, our study has established a novel robust and physiologically relevant system to investigate RVC pathogenesis. We also generated novel, experimentally derived evidence regarding the role of host glycans, DEAE, and cholesterol in RVC replication, which is critical for the development of control strategies. Rotaviruses (RVs) are a significant cause of severe diarrheal illness in infants and young animals, including pigs . In chilhttps://talk.ictvonline.org/taxonomy/, accessed on 16 August 2022), designated as RVA, RVB, RVC, RVD, RVF, RVG, RVH, RVI, and RVJ  and RV143 G6P[5] and Cowden G1P[1] PRVC strains were used in this study. The contents were diluted 1:10 in sterile Minimal Essential Medium , clarified by centrifugation at 2000× escribed ,42. ImmeCrypt cells were extracted from the small intestine (ileum) as described previously , with sl® solution. An aliquot of Corning® Matrigel® was thawed and kept on ice until use. An appropriate amount of cold (2–8 °C) Dulbecco’s phosphate-buffered saline (DPBS) was dispensed at 100 µL/well in 96-well plates. Then, thawed Matrigel® was added to the cold DPBS (1 μL of Matrigel® to 49 μL DPBS), mixed well, and kept on ice. The diluted Matrigel® solution was used immediately to coat the tissue culture-treated culture wares. Then, the culture plates were incubated at 37 °C for 1 h before use. The excess Matrigel® solution was removed using a serological pipette ensuring that the coated surface was not scratched. We used previously established protocols for growing PIEs as monolayers , with soTwo-dimensional monolayer growth medium (2D-MM) was prepared as follows (example is for preparing 120 mL of monolayer growth medium): 50 mL of IntestiCult™ Component A + 50 mL of IntestiCult™ Component B + 100 µL Y-27632 + 25 µL CHIR99021 + 20 mL fetal bovine serum . The desired amount of antibiotics was added immediately before use. 4/well. The cell numbers of 3D PIE generally reached ~1–2 × 105 cells/dome. Sufficient numbers of domes were harvested to achieve the desired seeding density. One mL of gentle cell-dissociation reagent  was added to each well containing a dome to be harvested; then, the domes were incubated at room temperature (20–25 °C) for 1 min, followed by vigorous pipetting using a 1000 μL pipettor to disrupt the Matrigel® dome. The fragmented PIEs were then incubated at room temperature for 10 min with gentle rocking. Then, the harvested PIE fragments were pooled and centrifuged at 400× g for 5 min at 4 °C. The supernatant was discarded and 3 mL of ice-cold CMGF- was added, followed by vortexing and centrifuging at 400× g for 5 min at 4 °C. This step was repeated twice. Then, the supernatant was gently removed, followed by resuspending the PIE fragments in 1 mL of warm (37 °C) trypsin-ethylenediaminetetraacetic acid , vigorous vortexing, and incubation at 37 °C for 10 min. Following that, 3 mL of CMGF- supplemented with 10% FBS was added and mixed thoroughly by pipetting, and centrifuged at 900× g for 5 min at 4 °C. Then, the supernatant was removed, and the PIE fragments were resuspended in 1 mL of the 2D-MM, as indicated above. The PIE fragments were then passed through a 25 G needle multiple times to disrupt their structure to achieve single-cell suspensions. After cell counting and adjustment to the desired concentration, the cell suspension was slowly and gently added to each well and incubated at 37 °C under 5% CO2, and the medium was replaced every 2–3 days until monolayers were formed. If differentiated PIEs were desired, the medium was changed from 2D-MM to DM two days after seeding, and the monolayers were incubated in it for two days.For plating PIEs as a monolayer, the seeding density of PIEs in 96-well plates was 1.0–2.5 × 102O (three times for 1 h each). Then, the coverslips were air-dried and sterilized under UV light for at least 4 h. One cover glass was added to each well of 6-well cell culture plates, coated with 1 mL of diluted Matrigel, and 2D PIEs were grown on glass coverslips as described above. On the day of staining, the 2D culture medium was removed from the plate and washed twice with phosphate-buffered saline (PBS). Then, 1 mL of 4% paraformaldehyde in PBS (pH 7.4) was added, incubated for 10 min at room temperature, and washed three times with PBS, followed by antigen retrieval steps. Antigen retrieval, permeabilization, blocking, and antibody staining were conducted as described previously [The IF staining of 3D PIEs was conducted as described previously . The IF n PBS pH .4 was adWe used the protocol described previously with slight modifications . Briefly4 FFU/mL to achieve an MOI of 0.1. After that, we added 50 µL of loading solution and 50 µL of virus inoculum into the wells of 96-well plates and incubated them at 37 degrees for 2 h. The virus inoculum was then removed, and the plates were washed twice using CMGF- medium. Fresh CMGF- was added to the plates, the plates were placed in an incubator at 37 °C, and the virus was harvested at 2 and 48 h post-infection.For 2D infection, the conditional medium from differentiated PIEs was removed, CMGF- was added, and plates were incubated for 3 h at 37 °C. The RVC inocula were pre-activated with 10 µg/mL trypsin at 37 °C for 30 min. The loading solution was prepared by adding pancreatin (Invitrogen) to CMGF- at a final concentration of 0.25 mg/mL. The virus stock was then diluted (using loading solution) to 1 × 102, and 0.02% NaN3: TNC buffer) of sialidase/neuraminidase (NA) from Arthrobacter ureafaciens  for 1 h at 37 °C before inoculation. TNC buffer was used as a negative control for sialidase treatment. Following sialidase treatment, the PIEs were infected with RVCs and harvested as described above.Briefly, 2F-peracetyl-fucose (2F) is a cell-permeable fluorinated fucose derivative that acts as an inhibitor of fucosyltransferases following its uptake and metabolic transformation into a GDP-fucose mimetic ,45,46. WDEAE-dextran , porcine bile extract , and water-soluble cholesterol  dissolved in DPBS or Milli-Q water (cholesterol only) were added to PIEs to achieve final concentrations of 50 µg/mL, 20 µg/mL, and 10 µg/mL, respectively. CMGF- containing the above additives or CMGF- alone (used as a negative control) were removed following 1 h of incubation at 37 °C, and the PIEs were washed twice with CMGF- medium and then infected with RVCs as described above. Methyl-β-cyclodextrin , which has a high affinity for inclusion in the cell wall, competing with cholesterol and causing its cellular depletion, was used in the cholesterol-depletion assay. The cholesterol-depletion protocol has been described previously, and we used it with slight modifications . Briefly2 (Sigma), 0.45% IGEPAL (Sigma), 0.45% Tween-20 , and DEPC-treated water (Promega) was used for the total RNA extraction. Briefly, 200 µL of lysis buffer was added to each well of 96-well plates that contained PIEs and was incubated for 30 min at 37 °C. Then, 100 µL of the PIE–lysis buffer mixture was transferred to a 96-well PCR plate, followed by incubation in a PCR cycler with the following parameters: 30 min at 65 °C and 2 min at 98 °C, and the samples were then cooled down to 4 °C. The RNA concentration was measured by NanoDrop2000c and adjusted to the same level. A Qiagen one-step RT-PCR kit was used for RVC detection using the primers, probe, and protocol described previously [2 = 0.98).As described previously , lysis beviously . The Ct t-test unless specified. Differences were considered statistically significant when p  ≤  0.05.GraphPad Prism v 5.0  was used for data analysis. Comparison of the viral titers between different treatments was done using Student’s In this study, we first evaluated whether 3D-PIEs support RVC replication and if PIEs expressing different HBGA types support RVC replication to different levels. To evaluate the replication efficiency of RVCs in PIEs expressing different HBGA types  , differeTo further confirm the role of HBGAs in RVC infection, a fucosyltransferase inhibitor, 2F ,45,46, wBecause we observed no inhibitory effects of the 2F treatment on RVC replication, we wanted to further explore the role of another receptor—Sas—in RVC attachment/entry. The replication of Cowden, PRVC104, and PRVC143 was evaluated in PIEs that were pre-treated with 10 mU of NA or TNC buffer . Our results demonstrated that the replication of PRVC104 was significantly decreased following NA treatment, suggesting the sialidase-sensitive nature of the RV104 strain B. In conCell culture additives, such as DEAE-dextran, bile acids, and cholesterol, were previously reported to increase the propagation of RVs and other viruses ,38,39,40Many studies have demonstrated that cellular cholesterol is required for multiple viruses, such as porcine reproductive and respiratory syndrome virus, transmissible gastroenteritis virus, bovine herpesvirus type 1, bovine RV, murine leukemia virus, and simian virus 40 ,50,51,52Recently, 3D-back-to-2D enteroid monolayer cultures were established and found to recapitulate many of the features of 3D cultures . AlthougTo explore why 2D-PIEs failed to support RVC infection, we compared the expression of SOX9 (stem cell marker) and villin (enterocyte marker) in 3D cultures vs. 2D monolayers by IF ; Table 1Robust in vitro models are prerequisites for studies of molecular mechanisms associated with virus cell entry, replication, and the pathogenesis of many endemic and emerging viruses. As it threatens the pork industry and public health, RVC represents an important virus for further studies. However, our knowledge of RVC entry and host/tissue restriction factors is limited due to the lack of a suitable cell culture system. Several studies have reported successful serial passaging for some of the RVC strains in several cell lines, including MA104 ,55 and sOne study suggested that transition from 3D PIEs to 2D monolayers may alter the relative expression levels of different epithelial cell markers, indicating a decrease in goblet and Paneth cells in the monolayers . Our datHBGAs ,60,61,62Cell culture supplements and additives compensate for characteristics that the basal medium lacks and provide optimal cell-growth conditions. Our current results suggest that DEAE-dextran may affect RVC attachment, but not replication. Earlier studies indicated that the presence of polycations enhanced absorption, but also interfered with the release of reovirus . This maAlthough the addition of external cholesterol did not seem to affect RVC replication, the physiological levels of cellular cholesterol seemed to be essential for the efficient infection of RVC Cowden. More importantly, replenishing the cellular cholesterol levels partially rescued the titer decrease caused by MβCD. These findings suggest that cellular cholesterol plays an important role in RVC infection. As an essential part of the lipid raft membrane, cellular cholesterol is associated with various physiological processes, such as lipid sorting, protein trafficking, cell polarization, and signal transduction ,72,73. COur study provides the first evidence that several virulent RVC strains can be efficiently propagated in the 3D-PIEs culture. The 2F treatment and NA treatment assays revealed that HBGAs may serve as co-factors, while SAs could play an important role in RVC infection. Finally, our experiments confirmed that, similar to RVAs, cellular cholesterol is one of the essential factors for RVC replication. Overall, our study has generated a robust tool to investigate RVC pathogenesis and has broadened our knowledge regarding the roles of SAs and cholesterol in RVC replication, which may be important for the development of preventative and interventional strategies currently unavailable for RVCs.


O-caffeoylquinicacid was the most predominant, accounting for 14–39% of totalquantified phenolic contents (TPA) determined in this study. The variationswere mainly cultivar dependent. The genetic background effect on thechemical compositions is complex, and breeding selections from thesame parental cultivars varied dramatically in chemical compositions.Putative perry pears contained more 4-O-caffeoylquinicacid, 5-O-caffeoylquinic acid, caffeoyl N-trytophan, caffeoylshikimic acid, coumaroylquinic acid isomer, syringicacid hexoside, procyanidin dimer B2, (+)-catechin, and malic acid,whereas putative dessert pears had higher esters, alcohols, and aldehydes.The results will be helpful in providing industry with phytochemicalcompositional information, assisting pear selections in commercialutilization.The phenolic profilesand other major metabolites in juices madefrom fruits of 17 cultivars and selections of European pears wereinvestigated using UHPLC-DAD-ESI-QTOF-MS and GC-FID, respectively.A total of 39 phenolic compounds were detected, including hydroxybenzoicacids, hydroxycinnamic acids, flavan-3-ols, procyanidins, flavonols,and arbutin. Among these compounds, 5- The annual production of pears is approximately23.1 million tons globally in 2020, of which 2.8 million tons wasproduced in Europe and mainly consisted of European  pear(P. communis L.).1 Contrary to the crispy Asian pears , the European pears typically have asoft and smooth flesh texture.2 Unavoidably,a large amount of pear fruits are wasted annually, as they do notreach the fresh markets due to the low fruit quality or logisticalissues. Approximately 45% of the global fruit and vegetable productionis lost yearly.1 The losses of pear fruitsmay be ascribed to the high temporal and local variation at the farm(5–25%) and storage (8–29%) levels in the fruit supplychain.3 The juiced fraction, e.g., fruitswith external defects, low internal quality, or a wrong maturationtime for a target market, still contains high nutritional value withnotable amounts of sugars, minerals, amino acids, and phenolic compounds.4 They also have higher levels of dietary fibersbut lower calorie contents than some of the most common fruits andvegetables, as previously reported.6 Thus, fromthe points of view of commerce and sustainability, it is essentialto transfer the wasted fruits into value-added products, such as freshjuices, canned jellies, canned jams, alcoholic beverages, and dryfruits.9Pear  is thereforeimportant for reducing fruit loss and waste.The Nordic countries produce 7 million kg ofpear in total, lessthan 1% of the European pear production.12 For example, “Fausset”,“De Cloche”, and “Plant de Blanc” areused as perry pears, whereas “Conference” and “Williams”are widely used as dessert pears in European countries.6 Phenolic compounds were reported to influencethe sensory properties of fruits positively or negatively, especiallythe color, flavor, and astringency.13 Inpear juices, the predominant polyphenolic constituents are mainlyhydroxycinnamic acids, including caffeic acid, p-coumaricacid, ferulic acid, and their derivatives.14 Amongthese compounds, chlorogenic acid has been reported to be the dominantphenolic acid in the pear juices made from five Australian-grown pearvarieties.9 A number of flavan-3-ols, procyanidins,and flavonol glycosides were also found in pear fruits, as well assimple phenolics, such as arbutin.15 Inaddition, the sugar/acid ratio varied among pear cultivars and significantlyaffected the sour and sweet taste of fruits. No significant differencesin total quantified sugar contents were found between apples and pears,whereas the total quantified organic acid contents of pears were significantlylower than those of apples.16 Pear hasbeen reported to contain the highest amount of sorbitol among certainfruit juices .16 Aroma compounds alsoplayed an important role in affecting overall flavor of pear fruits,which determined the consumer perception and acceptability of thefinal pear products. Esters and alcohols were detected as the mainaroma compounds as reported in European  pears and Asiatic pears, such as “Niitaka”(P. pyrifolia) andKorla pear (P. bretschneideri Rehd.).19Fruit quality canbe described as the combination of organolepticand nutritional aspects tightly correlated with the bioactive compoundsas well as shelf life, fruit size, and juiciness, which are highlydependent on the cultivar. New pear cultivars can be bred by crossingtwo cultivars and selecting new cultivar candidates from their offspring.However, hidden genetic variation in the parent cultivars may resultin unexpected variation of fruit quality traits in the breeding progenies.In general, dessert pears are popular due to their pleasant taste,high nutritional properties, and good storability, whereas perry pearsare smaller, bitter, and more astringent with high concentrationsof polyphenols.20 In the current work, qualitative and quantitativeanalyses of pear phenolic compounds, including phenolic acids, flavan-3-ols,procyanidins, flavonols, and arbutin (hydroquinone), were conductedwith ultrahigh-performance liquid chromatography equipped with a diodearray and an electrospray quadrupole/time-of-flight tandem mass spectrometer(UHPLC-DAD-ESI-QTOF) and UHPLC-DAD. To the best of our knowledge,studies on the variability of phenolic composition among pear juicesproduced from fruits of different cultivars are scarce in the currentliterature. This is also the first report to characterize the rangeof variation in the phenolic profiles and composition among breedingselections from controlled crosses between dessert cultivars. Moreover,the profiles of sugars, organic acids, and main volatiles relatedto the overall quality of pear juices were also characterized by usinga gas chromatograph equipped with a flame ionization detector (GC-FID).Multivariate models, including principal component analysis (PCA)and partial least squares-discriminant analysis (PLS-DA), were alsoapplied to study the relationships between the key chemical variablesand the samples and/or sample grouping developed by breeders. Thegenetic background effect has been also considered in the study, makingthis study a significant starting point for future investigationson the heritability and genetic determinants of fruit biochemicalcomposition and its variation available in European pear. The findingsof this study can help breeders in the more targeted application ofbiochemical analyses in selection of new cultivars and meeting thebreeding targets of fruit quality suitable for multiple or additionaluse, such as juice and perry making. In addition, the current studyprovides the fruit industry with important compositional informationon phytochemicals, assisting in the selection of pear cultivars forcommercial utilization.The main aim of the present study was to characterize thechemicalprofiles of pear juices made from fruits of breeding selections andtest cultivars and to investigate their potential for juice and perryuses. Two commercial dessert pear cultivars (“Conference”and “Clara Frijs”), with pleasant flavor, juiciness,and aroma, were included as external standard cultivars. Moreover,“Conference” is the most commonly grown cultivar andone of the most important produced fruits in European countries.22.1O-caffeoylquinic acid, 5-O-caffeoylquinicacid, p-coumaric acid, gallic acid-4-O-glucoside, and caffeic acid were provided by Sigma-Aldrich Co. . Quercetin-3-O-glucose,kaempferol-3-O-glucose, and procyanidin B2 were obtainedfrom Extrasynthese .LC and LC–MS gradechemicals were purchased from VWR International Oy .Ethanol (≥99.7%) was purchased from Altia Oyj .The standards of ethyl acetate, acetaldehyde, butan-1-ol, and aceticacid were obtained from Sigma-Aldrich .The standards of myo-inositol, xylose, fructose, glucose, sorbitol,sucrose, tartaric acid, malic acid, succinic acid, citric acid, quinicacid, and ascorbic acid were purchased from Extrasynthese . (−)-Epicatechin, (+)-catechin, arbutin, 3-2.2Pyrus communisL.) (P. communis seedling rootstock (two test cultivars)or supported by their own roots (13 breeding selections). The treeswere drip irrigated and trained in a modified central leader witha tree spacing of 3.5 × 1 m. The ground at the tree rows wascovered with woven polypropylene groundcover (MyPex), and the inter-rowspaces were covered by grass. Two chemical control sprays againstscab (caused by Venturia spp.) were applied duringthe season. Fertilization was distributed on the ground surface oradded via drip irrigation, and the amount and product selection werecalculated based on nutrient requirements as supported by the soiltest. Additionally, pear fruits of the cultivar “Conference”(Con) produced in the Netherlands and “Clara Frijs”(Cla) produced elsewhere in southern Finland were purchased from thelocal supermarket , two test cultivars (“Stolishnaia”and “Krupnoplodnaja Susova”), and 13 unreleased breedingselections from the pear breeding program of Natural Resources InstituteFinland (Luke) 1. The brmunisL.) 1, and thermarket 1.The fruit samples were stored in a fruit storage chamber  and assessed for fruit qualityand maturity. At the eating ripeness of each sample, fruits for juicingwere carefully selected for fruit size, maturity, and absence of externalor internal damage. The fruits were washed, sliced into small pieces,and then crushed into juices with a juice presser. For each cultivar,the juices were pressed in triplicate and stored at −20 °Cimmediately until further chemical analyses.2.321 Briefly, phenolics were extracted from pear juices (25mL) with 20 mL of ethyl acetate, assisted by sonication (20 min) andcentrifugation  for four times.All the supernatants were combined and evaporated at 35 °C untilcompletely dry. The residue was dissolved in 1.5 mL of methanol andfiltered through a 0.2 μm PTFE filter before injection.Extractionof phenolic compounds was carried out according to the method reportedby a previous study with slight modifications.22 Theidentification was performed via a UHPLC-DAD-ESI-QTOF system  consisting of a Bruker ultrahigh-performanceliquid chromatograph (UHPLC) in combination with an Elute diode-arraydetector (DAD), an electrospray ion (ESI) source, and an Ultra-HighResolution Impact II quadrupole/time-of-flight (Q-TOF) tandem massspectrometer. The column used was an Aeris peptide XB-C18 column  from Phenomenex . Mobilephase A consisted of 0.1% formic acid in water, and mobile phase Bconsisted of 0.1% formic acid in acetonitrile. The gradient used wasas follows: 2–4% B, 0–5 min; 4–7% B, 5–10min; 7–8% B, 10–15 min; 8–10% B, 15–20min; 10–18%, 20–30 min; 18–20% B, 20–35min; 20–25% B, 35–40 min; 25–35% B, 40–45min; 35–40% B, 45–46 min; 40–70% B, 46–49min; 70–2% B, 49–51 min; 2% B, 51–53 min, sequentially.The flow rate was 1.0 mL/min at 25 °C. After splitting, 0.4 mL/minof LC eluent was directly flown into the MS system. The mass spectrometerwas operated under both negative and positive ion modes with the followingsource settings: end plate offset, 500 V; nebulizer gas pressure,2.5 bar; drying gas flow, 11 L/min; drying gas temperature, 280 °C;capillary voltage, 4.5 kV (positive mode) and 3.5 kV (negative mode);quadrupole ion energy, 5.0 eV. The mass was scanned across the rangeof m/z 20–2000, and the rangeof collision energy was set as 5.0–12.5 eV. A sodium formatesolution (10 mM) was continually introduced to the system as internalcalibration for high-accuracy mass calibration. The MS data were collectedand analyzed by Compass Data analysis software 4.4 .Liquid chromatography separation was performed according to a publishedmethod with slight modifications.O-caffeoylquinic acid, (+)-catechin, procyanidinB2, and quercetin-3-O-glucoside. Flavan-3-ols andhydroxybenzoic acids were recorded at 280 nm; hydroxycinnamic acidswere recorded at 320 nm; and flavonols were recorded at 360 nm. Thequantification was calculated using external standards. (+)-Catechinand procyanidin B2 were used to quantify monomeric flavan-3-ols andprocyanidins, respectively. 5-O-Caffeoylquinic acidand gallic-4-O-gllucoside were used to quantify hydroxycinnamicacids and hydroxybenzoic acids, respectively, and quercetin-3-O-glucoside was used to quantify flavonols. Moreover, arbutinwas also employed as an external standard to quantify the concentrationof arbutin. The list of external standard curves is shown in Table S1.Quantification of phenolic compounds with authenticstandards wasperformed in a Shimadzu UHPLC-DAD system using 5-2.423 The juice yields werecalculated by dividing the volume of juice collected (mL) by the massof pear fruit sample (kg). All the samples were centrifuged at 3000× g for 10 min to remove precipitates beforechemical analysis.The pH value of the obtainedjuices was monitored by a pH meter . Total solublesolids (°Brix) were measured by a portable °Brix meter . An Evolution UV–visible 300 spectrophotometer was used for color measurements. Sampleswere analyzed in a 1 cm path length quartz cell at absorbances of420, 520, and 620 nm. The color intensity was calculated as the sumof three absorbances , and the tonality (hue)was determined by calculating the ratio between the absorbance of420 and 520 nm.2.524 A gas chromatographinstrument together with a flame ionization detector  was used in the study. An SPB-1 column  was used as the column. Myo-inositoland tartaric acid were used as internal standards of sugars and organicacids, respectively. The studied compounds were identified by comparingthe retention times with those of the authentic standards. The totalquantified contents of sugars and organic acids were expressed asthe sum concentration of quantified individual sugar and organic acidcompounds. The total sweetness index (TSI) was calculated by multiplyingthe average amounts of each sugar and their relative sweetness respect to sucrose based on the followingequations:sucC, gluC, and fruC arethe average amounts of sucrose, glucose, and fructose, respectively.The contribution of each carbohydrate was calculated according tothe assumption that the sweetness of fructose and sucrose are 2.30and 1.35 times sweeter than glucose.25Individual sugars and organic acids wereanalyzed as trimethylsilyl(TMS) derivatives by the GC-FID method based on our previous methodwith slight modifications.2.622 The same GC-FIDsystem was used for the analysis of major volatiles, and an HP-INNOWaxcolumn  column was usedin the study. The temperature program started from 40 °C andwas held for 8 min; then, the temperature was increased to 240 °C(10 °C/min) and then held at 240 °C for 2 min. The injectortemperature was at 220 °C, and the samples were injected automatically(1 μL) with a split ratio of 1:25. The detector temperaturewas set at 280 °C. The carrier gas was helium with a flow rateof 1 mL/min. All the samples were filtered through 0.2 μm PTFEfilters before injection. The contents of major volatile compoundswere determined with standard curves of ethyl acetate, butan-1-ol,acetaldehyde, and acetic acid. The external standard curves are shownin Table S1.TheGC-FID method of volatile compound analysis was optimized basedon our previous study.2.7The resultsare presented as the mean values ± standard deviation of triplicateobservations. Statistical analysis was performed via SPSS 27 . Significant differencesamong samples were determined using one-way ANOVA and Tukey’stest. Principal component analysis models of full-cross validation(including PCA and PLS-DA) were used to investigate the correlationsbetween chemical compositions and different pear cultivars. To visualizethe bivariate correlations between the selected chemical compounds, a supervised hierarchical clusteringanalysis based on Pearson’s correlation coefficient was usedvia online software MetaboAnalyst 5.0 .33.1Table S2). The highest juice yields werefound in the two test cultivars “Sto” (84%) and “Kru”(81%), which might be preferred by the juice-pressing industry basedon economic and practical concerns. In addition, selection “Py7”from the breeding program also contained a high juice yield of 75%.The highest values were found in the cultivar “Con”(pH 4.6) and the cultivar “Cla” (pH 4.5), whereas thelowest values were found in “Sto” (pH 3.3) and “Kru”(pH 3.7). According to previous reports,21 pH was used to indicate the sourness when evaluating different fruitjuices and fruit wines. The juice made from the pear selections derivedfrom the breeding program had lower pH in general, indicating thatthese developmental pear cultivars were sourer than the studied dessertcultivars. Apart from the pH values, the °Brix value has beenreported to determine the internal quality attributes and is an importantindicator of soluble single sugars or organic acids. In general, fruitjuices with higher °Brix values are perceived sweeter and consequentlyappreciated by consumers. The pear cultivar “Py8” showedthe highest °Brix value (16.7) whereas the cultivar “Sto”showed the lowest °Brix value (8.5). Interestingly, the pearselections of the breeding program sharing same parental cultivars(Table S2) did not share similar physiologicalcharacteristics, as expected by the complex inheritance and low ormoderate heritability of these traits in pear.28The juice yield, pH values,total soluble solids (°Brix),color tonality, and color intensity were analyzed from the processedjuices obtained from 17 different pear cultivars and breeding selections(3.235 primarily as hydroxybenzoic acids (3 compounds), hydroxycinnamicacids (18 compounds), monomeric flavan-3-ols (2 compounds), procyanidins(5 compounds), flavonols (10 compounds), and arbutin. The qualitativeresults and LC chromatograms are shown in Figure S1, respectively. As shown in p-coumaric acid. For the hydroxybenzoic acids, only syringic acidand its glycosylated derivatives were detected. In the flavan-3-olgroup, (+)-catechin and (−)-epicatechin were detected by comparingtheir retention times, UV–vis, and MS spectra with those ofreference standards. The procyanidins in the studied pear juices wereidentified mostly as B-type procyanidins. A dimer of A-type procyanidinwas also detected in the study. Flavonols were identified as glycosidesof quercetin, kaempferol, and isorhamnetin.In the present work, the identification analysis ofphenolic compounds in 17 pear cultivars was conducted by UHPLC-DAD-ESI-QTOFin both positive and negative ionization modes. Altogether, 39 phenoliccompounds were identified via the comparison of the retention times,UV–vis spectra, and mass spectra with those of the referencecompounds and the results reported in the literature,3.337 The totalquantified phenolic contents  of the studied pear juices ranged from 172.9 mg/Lin cultivar “Con” to 714.6 mg/L in cultivar “Py10”.The variation in phenolic profiles might be explained by the differentgenetic backgrounds and maturity levels of fruit cultivars.38 The test cultivar “Sto” (tentativelyused as juice/perry pears) contained a high TPA of 654.3 mg/L, whereaslower TPA were found in the dessert pear groups, such as the testcultivar “Kru” (177.5 mg/L) as well as the two commercialdessert cultivars “Con” (172.9 mg/L) and “Cla”(209.7 mg/L). Pear breeding selections sharing the same parental cultivarsdid not always have similar total quantified phenolic contents. Forexample, the TPA of the pear selections derived from the cross “Pepi× Lück” ranged from 335.4 (“Py4”)to 688.9 mg/L (“Py2”), whereas two pear selections (“Py6”and “Py7”) derived from the cross “Alna ×Lück” shared similar TPA. Thus, the genetic effect onthe phenolic profiles of pear is complex, as already indicated bya moderate heritability of skin bitterness.25The phenolic profiles of pear juices madefrom 17 cultivars aresummarized in p < 0.05)from each other (Table S3). In terms ofhydroxybenzoic acids, the test perry pear “Sto” containedthe highest contents of total quantified hydroxybenzoic acids (TBA)at 127.2 mg/L, and the lowest content was found in the commercialdessert pear cultivar “Con” (18.9 mg/L). The TBA rangedfrom 48.5 (“Cla”) to 355.9 mg/L (“Sto”)in the studied pear juices. The primary hydroxycinnamic acid in thestudied pear juices, 5-O-caffeoylquinic acid (peak15), accounted for 15–40% of TPA. Tanriöven and Ekşialso found that 5-O-caffeoylquinic acid representedthe main phenolic compound in the pear juices made from seven differenttypes of Turkish pear cultivars.37 Thetwo commercial dessert pear cultivars (“Con” and “Cla”)as well as the test dessert pear cultivar “Kru” containedlower concentrations of 5-O-caffeoylquinic acid inthe current study, whereas the highest content of 5-O-caffeoylquinic acid was found in the test perry pear cultivar “Sto”(217.4 mg/L) followed by selection “Py3” derived from“Pepi × Lück” (201.4 mg/L) and selection“Py10” derived from “Karmla × Pakurlan Päärynä”(200.7 mg/L).The contentsof individual phenolic compounds in different pearcultivars also differed significantly  ranged from 15.1 (“Py5”)to 120.3 mg/L (“Py10”) in the studied pear cultivars rangedfrom 11.9 mg/L (“Kru”) to 87.6 mg/L (“Py3”)in the studied pear fruits. The predominant flavan-3-ol monomer detectedin the studied pear juices could be either (+)-catechin or (−)-epicatechin,which was mainly dependent on the pear cultivars. For example, thetest cultivar “Sto” contained a high amount of (−)-epicatechin,whereas no (+)-catechin was found in this cultivar. “Py3”,“Py5”, “Py7”, “Py11”, and“Cla” were also dominated by (−)-epicatechin,whereas the other cultivars contained slightly higher (+)-catechinor similar concentrations of (+)-catechin and (−)-epicatechin.A previous study also found that (−)-epicatechin was the predominantflavan-3-ol monomer in most of the European and Tunisian pear fruitsexcept for certain cultivars, such as “Abate” and “Comice”.ultivars3. Cultiv40 The highest amount of arbutin (29.5 mg/L) was found in“Py2”, and the lowest (3.3 mg/L) was found in cultivar“Kru”.In termsof flavonols, “Py12” contained only 9.3mg/mL of TFO, whereas extremely high amounts of flavonols were foundin “Py9” (137.02 mg/L). The differences in TFO werehighly dependent on the pear cultivars. Among the studied pears, quercetinderivatives (27–50%) and isorhamnetin derivatives (11–49%)were found in high concentrations, whereas kaempferol derivatives(0–18%) were only detected in trace amounts. Arbutin was reportedas a characteristic phenolic present in pear juices.3.416 As shown in 41 Fructose contents varied from 42.1 to 84.4 g/L, being the highestin “Py3” and “Py8”. The glucose concentrationwas found to be higher in “Py1” (30.5 g/L) than in theother cultivars. The highest level of sucrose was found in “Py8”(32.4 g/L) followed by “Py4” (30.2 g/L), “Py12”(28.8 g/L), and “Py6” (28.2 g/L). “Py12”showed the highest sorbitol content of 24.5 g/L among the studiedpear juices. Xylose was detected in all the studied pear juices attrace amounts, ranging from 0.3 to 1.9 g/L.The main nutrients and taste componentsof fruit juices, sugars,and organic acids contribute to the main soluble contents and thesensory characteristics of pear juices, such as sweetness, sourness,and bitterness.42 Similar results were also found in the currentstudy; the malic acid concentrations in the studied pear juices rangedfrom 1.8 (“Kru”) to 8.6 g/L (“Py3”). Thecontent levels of quinic acid and citric acid were mainly dependenton the pear cultivars. Quinic acid was the second most abundant organicacid in most of the studied pear juices, whereas citric acid was thesecond most abundant organic acid in certain pear cultivars, suchas “Py11” and “Py12” derived from “RumnajaKedrina × Pakurlan Päärynä”. A previousstudy also demonstrated that citric acid was the second most abundantorganic acid in “Dangshan” pear juices.41 For the minor organic acids, all the studied juices werequantified with similar contents of succinic acid (0.2–0.5g/L) and ascorbic acid (0.2–0.7 g/L).Succinic acid, malicacid, quinic acid, citric acid, and ascorbic acid were the main organicacids identified from the pear juice samples 4. The to3.543 However, most of those compounds were detected in trace amounts,and only a fraction of key volatile metabolites (depending on theirquantitative abundance and olfactory thresholds) were reported toplay important roles in pear juices to provide pleasant fruity aroma.43 Thus, it is important to identify and quantifythe major volatile metabolites in the studied pear juices. The keyvolatile metabolites were detected in the studied pear juices in thecurrent study, including five esters, four alcohols, three aldehydes,and one volatile acid -2-hexenal, also showedrelatively high amounts among the studied pear juices. The (E)-2-hexenal concentration varied dramatically from 0.3μg/L in “Py9” to 6.6 μg/L in “Py8”.In addition, the concentration of acetic acid varied among juices,ranging from 2.8 (“Py3”) to 8.3 μg/L (“Py2”).All the volatile compounds were cultivar dependent in this study.For other volatile compounds, acetaldehyde wasthe most abundantaldehyde in the studied juices 5. The co3.6O-caffeoylquinic acid (17),5-O-caffeoylquinic acid (15), caffeoyl N-tryptophan (13), caffeoylhexose (30), coumaroylquinic acid isomerII (22), syringic acid hexoside II (9), procyanidin dimer B2 (6),(+)-catechin (5), and succinic acid (46). In contrast, cultivars “Con”,“Cla”, “Py13”, and “Py12”were located on the negative side of PC1 due to the higher amountsof major volatile compounds, primarily n-propyl acetate(54), butyl acetate (53), hexan-1-ol (59), butan-1-ol (58), and acetaldehyde(60). The pear selections “Py4”, “Py5”,“Py6”, “Py7”, “Py8”, and“Py11” were located in the middle part of the PCA plot.The PCA results also showed a varietal effect, which was in oppositedirections between 5-O-caffeoylquinic acid (15),caffeic acid (16), 4-O-caffeoylquinic acid (17),and malic acid (47) on the one side (positive) and volatile compoundsbutyl acetate (53), n-propyl acetate (54), ethanol(57), and butan-1-ol (58) on the other side of the plot (negative).In addition, the Pearson correlation coefficient heatmap  revealed a significant bivariate connectionbetween these compound variables in pear juices, which was supportedby hierarchical co-clustering of the samples. The high contents ofhydroxycinnamic acids in pear cultivars “Py1”, “Py2”,“Py3”, “Py10”, and “Sto”indicate high natural antioxidative and antimicrobial capacities,potentially protecting from natural harms by constituting a secondaryreactive oxygen species (ROS) scavenging system in plants.45 Various studies have investigated the positive correlation betweenthe phenolic concentrations and antioxidant activities for human nutrition,providing with anticancer, anti-inflammatory, antimicrobial, and antidiabeticactivities.47 In contrast, pear breeding selections“Py12” and “Py13” together with the commercialcultivars “Con” and “Cla” were correlatedclosely with the aforementioned volatile compound variables, and thusthe cultivars may be considered as putative dessert pears. Generally,dessert pears have more pleasant flavors due to the relatively highamounts of attractive volatile compound and less phenolic compounds.Therefore, understanding the biosynthesis of the potential flavor-activecompounds and their interactions are required to more efficientlydevelop cultivars for different purposes.To assess the overall cultivar differencesin the chemical compositions of pear juices, all the data (77 X-variableswith 51 samples) regarding the phenolic compounds, major volatilecompounds, sugars, and organic acids were analyzed using the PCA 1A model.Y-data, n = 3) in the chemical compositions were analyzed usingPLS-DA ,these three groups were separated well from each other. Overall, theputative perry pears were located on the negative side along Factor1, with higher contents of total quantified hydroxybenzoic acids,total quantified hydroxycinnamic acids, and total quantified organicacids. In contrast, putative dessert pear group was located on thepositive side along Factor 1. Interestingly, the full-sib pear selectionsof breeding program with the same parental cultivars (Py1-Py5) weredivided into perry or dessert groups, result not fully unexpectedby the complex inheritance and low or moderate heritability of thesetraits in pear.28 Moreover, the putative dessert/perry pear group wasseparated well from the other samples along Factor 2 and was locatedon the negative side of Factor 2. However, “Py12” (putativeperry pears) was located close to the putative dessert pears in thePLS-DA model, and similar results were observed in the PCA model  according to their tentativeuse determined by breeders. Currently, “Sto”, “Py1”,“Py3”, “Py5”, “Py10”, and“Py12” are grouped as putative perry pears; “Py6”,“Py7”, “Py8”, and “Py11”are grouped as putative multiuse dessert/perry pears; and “Py2”,“Py4”, “Py9”, “Py13”, “Kru”,“Con”, and “Cla” are grouped as putativedessert pears. The differences among the putative dessert pear group,putative perry pear group, and putative dessert/perry pear group -catechin,and succinic acid. However, as exceptions, juices made from “Py12”,putative perry type (determined by breeders) contained high volatilemetabolites, whereas “Py2” (putative dessert pear) containedhigh phenolic compounds. The study provides a theoretical basis forproduct development to promote the utilization of local pear cultivarsdeveloped and grown in Finland. The potential of using the breedingselections in perry making deserves more investigation in the future.In conclusion, phenolic compounds, physiological characteristics,and other chemical compounds  were investigated comprehensively in pear juices madefrom 17 pear cultivars, including 13 pear breeding selections, 2 testcultivars, and 2 commercial dessert pears. A total of 39 phenoliccompounds were identified and quantified in the 17 studied pear cultivars.The genetic background effect on the phenolic profiles of pear juicesis complex, and the chemical compositions of the breeding selectionswith the same parental cultivars varied dramatically from cultivarto cultivar. In general, the putative dessert group contained higheramounts of major volatile metabolites, primarily as