Due to the presence of both epoxy and double carbon–carbon pendant groups, one can apply two distinct mechanisms: (i) cationic ring-opening polymerization or (ii) free-radical polymerization to crosslink polymer chains. Synthesized epoxy (meth)acrylate prepolymers were further employed to formulate photocurable coating compositions. Hence, when cationic photoinitiators were applied, polyether-type polymer chains with pending acrylate or methacrylate groups were formed. In the case of free-radical polymerization, epoxy (meth)acrylates certainly formed a poly(meth)acrylate backbone with pending epoxy groups. Further, photopolymerization behavior and properties of cured coatings were investigated regarding some structural factors and parameters. Moreover, reaction rate coefficients of photo-cross-linking by both cationic ring-opening and free-radical photopolymerization of the received epoxy (meth)acrylate resins were determined via real-time infrared spectroscopy (RT-IR). Lastly, basic physicomechanical properties, such as tack-free time, hardness, adhesion, gloss, and yellowness index of cured coatings, were evaluated.In this work, a series of novel multifunctional epoxy (meth)acrylate resins based on a low-viscosity aliphatic triepoxide triglycidyl ether of trimethylolethane (TMETGE) and acrylic acid (AA) or methacrylic acid (MMA) have been synthesized. Thanks to the performed modification, the obtained prepolymers have both epoxides as well as carbon–carbon double bonds and differ in their amount. The obtained results indicate that the carboxyl-epoxide addition esterification occurs in the presence of a catalyst (triphenylphosphine) at a temperature of 90 °C, whilst the required degree of conversion can be achieved simply by varying both the reagents ratio and reaction time. The structure of synthesized copolymers was confirmed by spectroscopic analyses (FT-IR,  Photopolymerization is a method that allows the transformation of a liquid polymerizable composition into a solid in the blink of an eye simply through its exposure to electromagnetic radiation (most often UV). The global UV-curable resins and formulated products market  was valued at USD 4.27 billion in 2019 and is projected to reach USD 8.66 billion by 2027 . As a reThe global market of UV-curable resins is divided into acrylated epoxies, acrylated polyesters, acrylated urethanes, acrylated silicones, and others. The largest share contains epoxy acrylates, which are readily used in UV-curable paints and varnishes . Epoxy aMore and more attention is also paid to so-called hybrid monomers having different types of polymerizable groups in their structure . They alThe polymers containing reactive pending groups, such as the ones obtained from the hybrid resins, can be applied for the creation of graft copolymers by copolymerization of the pending groups with appropriate monofunctional monomers or can be crosslinked further just by the application of an appropriate complementary initiator .In this study, novel multifunctional epoxy acrylate resins were synthesized, and the effects of the functionality of end-capping groups, such as epoxy or (meth)acrylate groups, on UV-curing behavior and physicochemical properties were investigated. To examine the effects of the amount of epoxy or (meth)acrylate groups, the new epoxy (meth)acrylate resins were synthesized by the addition of acrylic or methacrylic acid to triepoxide triglycidyl ether of trimethylolethane (TMETGE), taking into account the different molar ratio of acid to epoxy groups. Four new trifunctional epoxy (meth)acrylate resins were obtained, which contained at least one epoxy group and at least one acrylate group. The structure of the obtained resins was confirmed by FT-IR and NMR methods. The synthesized resins were investigated as compositions with a cationic or radical photoinitiator. After making the thin polymeric film, UV-curing behavior was investigated using two different methods (Fourier transform infrared (FT-IR), and photo-differential scanning calorimetry (photo-DSC)), then the conversion and the curing rate were compared. The tack-free time, pendulum hardness adhesion, gloss, and yellowness index were measured to evaluate the physicochemical properties of cured coatings.The industrial-grade, low-viscosity aliphatic triepoxidetriglycidyl ether of trimethylolethane (Erisys GE-31), abbreviated to TMETGE, was purchased from Huntsman, . Acrylic acid (AA) and methacrylic acid (MAA), both stabilized with hydroquinone with a purity of 99.5%, was supplied by Acros Organics . Triphenylphosphine (PPh3), Apollo Scientific, Bredbury, UK, was used as a catalyst in the reaction between TMETGE and AA or MAA, while hydroquinone  was used as a polymerization inhibitor. All chemicals were employed as received.The following titration reagents and indicators were used: glacial acetic acid, toluene, potassium hydroxide standard solution 0.1 M in ethanol (KOH) and crystal violet purchased from Chempur ; chloroform from PPH Stanlab ; ethyl alcohol from Avantor ; tetraethylammonium bromide provided by Acros Organics ; perchloric acid standard solution 0.1 M in glacial acetic acid supplied by Fischer Chemicals AG ; phenolophtalein 1% in ethyl alcohol solution from Eurochem BGD . All chemicals were analytical grade and were used as received.Epoxy acrylates (EAs) were obtained by the addition of acrylic or methacrylic acid to the triglycidyl ether of trimethylolethane. The synthesis conditions were established on the basis of previous works on the synthesis of epox(met)yacrylates by esterification reaction of diglicydyl ether of bisphenol-A  or epoxy−1, at room temperature.The infrared spectra were acquired with a Thermo Nicolet 380 FT-IR spectrometer . Sixteen scans were averaged for each sample in the range of 4000–400 cm1H NMR and 13C NMR. 1H-NMR (400.13 MHz) and 13C-NMR (100.62 MHz) spectra were recorded in CDCl3 on a Bruker Avance III HD 400 spectrometer . TMS was used as an internal standard.The prepared compounds were identified by m2/m1) × 100%, where m1 is the weight of the EA sample; m2 is the residual weight of the sample after heating.The nonvolatile matter content (NV) was evaluated thermogravimetrically using a Moisture Analyzer MAX 60/NP , according to ISO 3251:2019 standard. The analysis was performed at 140 °C for 30 min. The sample weight was approximately 1 g. NV (%) = , V1 is the volume of the KOH solution used to neutralize the resin solution (mL), V2 is the volume of the KOH solution used for the blank test (mL), and c is a concentration of KOH solution (mol/L).Partial acid values (PAVs) were determined by colorimetric titration according to EN ISO 2114:2000 standard. The EA samples were dissolved in a mixture of solvents , and then titrated with a standard KOH solution (0.1 N) in the presence of phenolphthalein. As the endpoint of the titration, the color change of the solution into pink (lasting from 20 to 30 s) was assumed. PAVs (mgKOH/g) were calculated using the following formula:The partial acid value of solid resin was calculated using the following equation:s0 is the initial value of PAVs (mg KOH/g).The PAVs values were used to estimate methacrylic acid conversion (MAAC) according to the following equation:m—the mass of the EA sample (g), V0—the volume of the perchloric acid solution used in the blank determination (mL), V1—the volume of the perchloric acid solution used during titration (mL), t—the temperature of the perchloric acid solution during the EE determination and the blank test (°C), st—the temperature of the chloric acid solution during titer adjustment (°C), and c—concentration of a perchloric acid solution (mol/L).Epoxy equivalent (EE) was determined by means of colorimetric titration, according to EN ISO 3001:1999 standard. EAs samples were dissolved in chloroform and then glacial acetic acid and tetraethylammonium bromide solution were added. The titration was carried out with the standard solution of chloric(VII) acid (0.1 N) to achieve a stable green color in the presence of crystal violet. EE (g/mol), was calculated according to the equation:0 is the initial value of EE (g/mol) and EEmeasured—EE value at a specific time.Epoxy group conversion (EGC) was estimated from EE measurements via the following formula:The viscosity tests were carried out using a cone-plate viscometer LAMY RM-100 plus CP 2000.2 to dryness.The coating compositions have been formulated using epoxy resin or synthesized epoxy acrylates and 3 wt.% PI (bis(dodecylphenyl)iodoniumhexaflouroantimonate in propylene carbonat, Deuteron UV 1240, Deuteron as a cationic photoinitiator or ethyl-phenyl phosphinate, Omnirad TPOL, IGM Resins, as a radical photoinitiator. The maximum absorption characteristics of the cationic photoinitiator (Deuteron 1240) are between 220–250 nm, and the radical photoinitiator is at the wavelength 274, 290, and 370 nm, respectively. The components were stirred together under dark conditions until a homogeneous mixture was obtained. Subsequently, the curing solution was applied to the glass substrates by means of a gap applicator (120 µm). The polymeric film was cured under a light source  at room temperature and irradiated under UV light with an intensity of 200 mW/cm2; Excelitas Technologies, Waltham, MA, USA), providing a first hint about the photoreactivity of the obtained systems. A polymerization solution was composed of epoxy (meth)acrylate resin and 3 wt.% of cationic or radical photoinitiator.The UV-curing process of epoxy (meth)acrylates was isothermally monitored (25 °C) in a nitrogen atmosphere for 15 min by means of a photo-DSC apparatus  equipped with the Omnicure S2000 UV light emitter  was used to determine the conversion of epoxide groups of acrylic double bonds. More importantly, this spectroscopic technique permits in situ monitoring of the chemical processes by mimicking the disappearance of the characteristic bonds of the reactive monomer subjected to UV exposure [2) and an IR analyzing light beam. The absorbance change of the epoxide group (C–O) and acrylate double bond (C=C) peak area was correlated to the extent of polymerization. The degree of conversion (DC) can be expressed by the following relations: DC (%) = (A0 − At)·100/A0, where A0 is the initial peak area before irradiation and At is the peak area at time t. The photopolymerization rate (Rp) was calculated by the following relations: Rp = dDC/dt, where t is the time of irradiation [Fourier transform infrared spectra (FT-IR) were obtained on a Nicolet iS5 instrument . The resolution is 4 cmexposure . The mixadiation .The following tests were performed in order to evaluate the mechanical properties of the cured coatings: tack-free time, pendulum hardness test, adhesion, gloss, and yellowness index. Tack-free time was measured as a surface cure time according to ISO 9117. This is the time at which the coating is deemed to have properly adhered and has achieved the final technical parameters. The hardness of coatings was tested using Persoz pendulum hardness on the glass substrate  according to ISO 1522 standard. Adhesion to glass substrate was evaluated according to PN-EN ISO 2409 . Gloss was measured by spectrometer GLS  according to ASTM D523. Yellowness Index is a number calculated from spectrophotometric data that describes the change in color of test samples. This parameter was measured according to ASTM E313 using precision colorimeter NH-145 .−1 [−1 [−1 [−1, thereby suggesting that ester bonds are formed due to the reaction of carboxyl groups of AA and epoxide groups of TMETGE. This is in good agreement with previous studies reporting the shift of the C=O band position to a higher value of wavenumber after the esterification reaction [−1 progressively decreases, whilst the intensity of broadband at 3600–3200 cm−1 slightly increases, suggesting the consumption of epoxy group (due to epoxide ring-opening) and evolution of additional –O–H groups’, respectively.In order to gain some insight into the reaction course, we performed an FT-IR spectroscopy kinetics study on the model epoxy-acrylate system. A mixture of TMETGE, AA, catalyst, and reaction inhibitor were heated to 90 °C and FT-IR spectra were collected every 10 min for a total of 60 min  ~1725 cm−1, C=O stretching vibrations and (iii) ~3600–3200 cm−1, stretching vibrations of -O-H groups’ , titration (PAVs and EE) along with nonvolatile matter content (NV) investigations were performed. The spectra of reaction substrates, TMETGE-AA and TMETGE-MAA prepolymers, are shown in el of −1 . The var1H and 13C NMR spectra, the results of which are illustrated in 1H NMR spectra of substrates (acrylic acid or methacrylic acid and triglycidyl ether of trimethylolethane) and obtained products (using an equimolar amount and with twice the molar excess of acrylic acid or methacrylic acid). In 2 = occurring in the range of 5.87–5.98 ppm and 6.45–6.53 ppm) in acrylates, (CH2 = occurring in the range of 5.60–5.69 ppm and 6.14–6.26 ppm) in methacrylates and the epoxy group 2.61–2.62 ppm (–CH2-) and 2.79–2.80 ppm (–CH–), respectively. 1H NMR spectrum, which shows the appearance of new signals as a result of the reaction of the ring-opening of the epoxide . These signals are assigned to the protons of the –CH(OH) (~4.2 ppm) and CH2O group (~4.0 and ~4.2 ppm), respectively. At the same time, signals from the vinyl group and epoxy groups are visible.In order to confirm the triepoxidetriglycidyl ether of trimethylolethane etherification modification with AA and MAA, the obtained epoxy (meth)acrylic resins were characterized by 13C NMR spectra, which showed the appearance of additional signals (C and D) from the CH2O group carbons (~65.5 ppm) and –CH(OH) (~70.0 ppm). The 13C NMR spectra of both the substrates and the obtained products are shown in 2 = occurring in the range of 133.17–131.36 in acrylates, 126.01–127.86 ppm and 135.74–135.98 ppm, respectively, in methacrylates, and 44.04–44.08 ppm (–CH2–) and 50.87–50.94 ppm (–CH–), respectively, in the epoxy group.Obtaining the desired structures was confirmed also by recording The kinetics of photopolymerization is useful in understanding the rate and degree of curing. It is well known that acrylates and methacrylates do not polymerize by a cationic polymerization mechanism, while epoxy monomers do not polymerize by free-radical means. Hence, when cationic photoinitiators were applied, polyether-type polymer chains with pending (meth)acrylate groups were formed. In the case of free-radical polymerization, epoxy (meth)acrylates certainly formed a polymethacrylate backbone with pending epoxy groups .p) and conversion of epoxy (meth)acrylates containing different amounts of epoxy, acrylate, or methacrylate groups curing via the cationic or radical process were investigated, as shown in −1, which is due to C–O stretching in the epoxy ring. Simultaneously, the extent of double bond reactions was determined by the peak areas of the double bond peak at 1635 cm−1.Through the RT-IR method, the curves of the photopolymerization rate acryl groups in place of epoxy groups resulted in a reduction of the photopolymerization rate and lower conversion. This is related not only to the reduction in the number of epoxy groups but also to the increase in the viscosity of the system, forming polyether-type polymer chains with pending (meth)acrylate groups. It can be seen that the photopolymerization rate of all of the processes by the cationic mechanism increases to 5 min, while the rate of radical processes experiences a steep increase in the first 2 min and then slows down. Thus, the rate of radical polymerization and formed a polymethacrylate backbone with pending epoxy groups is much higher than the cationic one, which could be attributed to the increase in the decomposition rate of the radical photoinitiator, leading to a larger number of primary radicals in the system at the beginning of the reaction. The higher initiation rate of radical photopolymerization may also be related to the absorption characteristics of the radical photoinitiator used, which completely match the emission characteristics of the UV lamp, especially for the highest intensity bands. In addition, the significant increase in unsaturated double bond conversion might be caused by a temporary excess of free volume due to the high amount of primary radicals. It is well known that, during the reaction of the UV-curing system, the mobility of the polymer chain and the diffusion capacity of the active radicals have a great influence on the conversion efficiency . Epoxy  and kinetic of photopolymerization on the cured films, the basic performance of coatings with different epoxy and acrylic group content was explored, as shown in 1H NMR, 13C NMR). It has been observed that the carboxyl-epoxide addition esterification proceeds in the presence of the catalyst, at a temperature of 90 °C. Moreover, one can easily control the degree of epoxy group conversion by varying the molar ratio of TMETGE to AA/MAA and the reaction time. Prepolymers developed herein are characterized by relatively low viscosity ranging from ~1100 mPa·s to ~4200 mPa·s, limited acid value (<20 mgKOH/g), and, most importantly, are capable of producing coatings by two different photopolymerization mechanisms: cationic and radical. The photoinitiated cationic or radical polymerization of the prepolymers was monitored by means of real-time infrared spectroscopy. It has been shown that cationic polymerization proceeds slower than radical polymerization and will lead to a lower degree of reaction of photoreactive functional groups. This may result from the difference in the rate of initiation rate of the photoinitiators used, the progressive stage of propagation, the viscosity of the system, the content of functional groups, and their reactivity. Moreover, the steric effect between the molecules caused by the presence of methyl groups in methacrylates made it difficult to move the segments, limiting the diffusion or migration of molecules. As a consequence, the efficiency of molecular polymerization is limited. This may also be related to the formation of different types of polymers (polyether or methacrylate backbone), differences in specific mobility of polymer chains, as well as reduced diffusion of active centers. As expected, the tack-free time is the shortest for the cationic process in resins containing the highest content of epoxy groups and for the radical process in resins containing the highest content of acrylate double bonds. The latter can be directly related to the photopolymerization rate of these systems. It has been shown that, despite the same backbone of the prepolymer molecules, cationic photopolymerization leads to coatings with better physicomechanical properties, i.e., hardness, adhesion, and gloss. This is probably related to the formation of a tightly cross-linked polyether structure.In this paper, a series of epoxy (met)acrylate prepolymers containing both epoxide and carbon-carbon double bond were successfully synthesized by means of an addition reaction of acrylic or methacrylic acid to the ether of trimethylolethane (TMETGE). The reaction course was monitored through titration methods . The structures of prepolymers after the modifications were unambiguously confirmed using spectroscopic methods (FT-IR,


Hyperactivation of Wnt/β-catenin signaling has been reported in hepatocellular carcinoma (HCC). However, the mechanisms underlying the hyperactivation of Wnt/β-catenin signaling are incompletely understood. In this study, Pantothenate kinase 1 (PANK1) is shown to be a negative regulator of Wnt/β-catenin signaling. Downregulation of PANK1 in HCC correlates with clinical features. Knockdown of PANK1 promotes the proliferation, growth and invasion of HCC cells, while overexpression of PANK1 inhibits the proliferation, growth, invasion and tumorigenicity of HCC cells. Mechanistically, PANK1 binds to CK1α, exerts protein kinase activity and cooperates with CK1α to phosphorylate N-terminal serine and threonine residues in β-catenin both in vitro and in vivo. Additionally, the expression levels of PANK1 and β-catenin can be used to predict the prognosis of HCC. Collectively, the results of this study highlight the crucial roles of PANK1 protein kinase activity in inhibiting Wnt/β-catenin signaling, suggesting that PANK1 is a potential therapeutic target for HCC. Hepatocellular carcinoma (HCC) is one of the most common malignancies The Wnt/β-catenin signaling pathway is abnormally activated in HCC Approximately 20-30% of HCC patients harbor constitutively activating mutations in β-catenin, and Axin was deleted in approximately 15-20% of patients The PANK family has four members: PANK1, PANK2, PANK3 and PANK4 In the present study, the expression pattern and functions of PANK1 in HCC were investigated, and the regulation of the Wnt/β-catenin signaling pathway by PANK1 was studied.2, 37°C). Cell transfection was performed using Lipofectamine 8000 according to the manual. The Wnt3a conditional medium was prepared from the L-Wnt3a cells according to the method from the ATCC.HCC cell lines , normal liver cells (HHL-5) and 293T were all obtained from the Cell Bank of the Chinese Academy of Sciences. The PVTT cells were established as described previously The HCC tissues and paired adjacent tissues came from the Shanghai Eastern Hepatobiliary Surgery Hospital, Second Military Medical University. All tissue samples were collected with the informed consent of the patients, and were used to study the expression level of PANK1. The clinical features of 72 HCC patients were listed in The CDS sequence of PANK1 was inserted into the pLVX-IRES-puro vector. The shRNA targeting PANK1 was designed with reference to the information on the Sigma website and cloned into the pLKO.1-puro vector. The shRNA sequence was listed in TRIzol (Invitrogen) was used to extract RNA, and then 1μg of RNA was reversely transcribed into cDNA using the PrimeScript™ RT kit (Takara) according to the instructions. The SYBR Green kit and CFX96 real-time fluorescent quantitative PCR detection system  were used for qPCR. Taking 18S as internal reference, the Ct (18S-PANK1) method was used to calculate the expression of the target gene. See The cells were cleaned twice with PBS, and lysed on ice in the RIPA Lysis buffer, which contained a protease inhibitor and a phosphatase inhibitor. The supernatant was collected after the cell lysis was centrifuged. Then the protein concentration was quantified using the BCA protein detection kit. Equal amounts of protein were taken for SDS-PAGE. After separation, the protein was transferred onto the PVDF membrane and incubated with a primary antibody at 4°C overnight. Then, they were incubated with the HRP-conjugated secondary antibody for 1-2h. The immune signal was detected with a chemiluminescence reagent , and analyzed with Image Lab software. The primary antibodies used in this experiment were as follows: PANK1 , tubulin , GSK3β , Flag , Myc , β-catenin , Phospho-β-Catenin , and Ubiquitin , CK1α .The tissue arrays were obtained from the Shanghai Eastern Hepatobiliary Surgery Hospital, Second Military Medical University. The clinical features of the tissue array were listed in In order to measure the interaction between exogenous PANK1 and CK1α, the tagged PANK1 and CK1α plasmids were transferred into 293T cells. 48h after transfection, the cells were lysed with an IP lysis buffer . The supernatant was collected after centrifugation. Flag-beads  or Myc-beads  were added to the supernatant for incubation overnight at 4°C. The next day, the beads were washed 3 times in wash buffer (50mM Tris-HCl (pH 8.0), 150mM NaCl, 1% NP-40), with 1×loading buffer added, and heated at 100°C for 5 min. Then, the supernatant was taken for western blot analysis.To detect whether there was any interaction between the endogenously expressed PANK1 and CK1α in the HCC cells, after the cells were treated, an IP lysis buffer containing protease and phosphatase inhibitor was used for lysis. Equal amounts of protein were taken, and 0.25μg of PANK1 antibody was added for incubation overnight at 4°C. The next day, 40μL of Protein A/G beads  was added for incubation overnight at 4°C. The beads were washed 3 times in the wash buffer, then 1×loading buffer was added for western blotting analysis.Cells with 50% confluence were seeded into a 12-well plate. Then, the cells underwent co-transfection with 0.1μg of expression vector, 0.05μg of reporter plasmid, and 0.02μg of Renilla luciferase. 24h after the transfection, the cells were incubated with lithium chloride (LiCl) or Wnt3a for 8h. Then, the dual-luciferase reporter assay system  was used to test the reporter gene activity. The experiment was repeated three times.Flag-PANK1 and HA-GSK3β were transfected into PVTT cells. 48h later, Flag-PANK1 and HA-GSK3β, were purified by CO-IP, and competitively eluted with Flag-tag polypeptide, obtaining Flag-PANK1 and HA-GSK3β protein. IPTG was used to induce the expression of GST-β-catenin (N-terminus) and GST-CK1α. Briefly, the E. Coli were collected, and the IP lysis buffer was used for resuspension and lysis. Then, they were sonicated at 20% power for 9s, and treated in an ice bath for 1min. The operation was repeated 3 times. Then, the solution was centrifuged  for 10min, and the supernatant was collected. 20μL of GST beads  was added and incubated with the supernatant at 4°C for 2 hours. The beads were then washed 3 times in the wash buffer. 10 mM ATP , 10×kinase buffers , Flag-PANK1 and HA-GSK3β were added, followed by the reaction at 32°C for 45 min. At the end of the reaction, the loading buffer was added, and the western blot analysis was performed.The stable cell line was treated with MG132 at a final concentration of 20μM for 10 hours. The cells were lysed in an IP lysis buffer, the supernatant was collected and then 0.5μg of β-catenin antibody was added for incubation overnight at 4°C. The next day, 40μL of Protein A/G beads  was added and incubated with the supernatant for 4 hours at 4°C. The beads were washed 3 times in the wash buffer, and then 1×loading buffer was added for 5min of heating at 100°C. The supernatant was taken for western blot analysis.6 control cells and Huh7 cells with overexpressed PANK1 were injected subcutaneously at each point. The tumor volume was calculated according to the following formula: volume = (length×width2)/2. The mice were killed in the 7th week after the start of the experiment to harvest the tumors.All work related to animals was approved by the Animal Ethics Committee of the Central South University. Nude mice aged 4-6 weeks were used, with 4 in each group. 4×103 cells) was added to each well and placed in a constant temperature incubator  for 10-14d. 5 fields of view were selected randomly under the microscope for colony counting.When the confluence reached 60-70%, the cells were digested, and a cell suspension was prepared. Lower-layer gel  40% 1.25% Agar) was prepared. 400μL of gel was added to each well in the 24-well plate. The gel was placed in an incubator at 37°C. The gel was solidified for later use. Upper-layer gel  was prepared and mixed evenly with the cell suspension. 400μl . The next day, the old medium was replaced with a fresh medium containing 10% CCK8, which was placed in the incubator for 2h of incubation to measure its absorbance at 450nm. Tests were performed on days 1, 3, 5 and 7.The cells were seeded into a 96-well plate, with 1×104 cells in each well. The cells were measured using the Cell-Light EdU Apollo567 In Vitro Kit , while a fluorescent microscope was used to take photographs for analysis.The cells were seeded into a 96-well plate, with 1×10In previous study To study the role of PANK1 in the progression of HCC, Flag-tagged PANK1 was overexpressed in four HCC cell lines  Figure A. Cell gTo study the role of endogenous PANK1 in HCC cells, PANK1 expression was knocked down by shRNA in Huh7 and QGY-7701 cells Figure A. ConsisSince it was during kinase library screening that PANK1 was discovered to be a potential kinase effective in inhibiting the Wnt/β-catenin signaling pathway, the regulation of PANK1 on the Wnt/β-catenin signaling pathway was investigated. Overexpression of PANK1 in 293T cells and Huh7 cells significantly inhibited the activation of the Topflash reporter, suggesting that PANK1 inhibits the transcriptional activity of β-catenin Figure A. FurtheAxin2 is a target gene of the Wnt/β-catenin signaling pathway. PANK1 overexpression reduced the mRNA level of Axin2, while the mRNA level of Axin2 was increased by knocking down the expression of PANK1 Figure F-G. NextAs discovered above, PANK1 affects the stability of β-catenin. Then, whether PANK1 expression affected the degradation of β-catenin via the ubiquitination pathway was explored. Phosphorylation of the N-terminal serine/threonine residues of β-catenin is essential for its ubiquitination Phosphorylation of the serine/threonine residues at the N-terminus of β-catenin occurs in the β-catenin destruction complex. Next, we examined the interactions between PANK1 and the components of the β-catenin destruction complex. Exogenous Flag-PANK1 overexpressed in 293T cells interacted with Myc-CK1α Figure C. HoweveTo explore the functions of PANK1 in the tumorigenicity of HCC cells, the tumorigenicity of Huh7 cells with the overexpression of PANK1 and control cells was examined in the nude mice. Overexpression of PANK1 significantly impaired the tumorigenicity of Huh7 cells, which was demonstrated by size, growth curve and weight of the xenograft Figure A-C. MoreTo further investigate whether PANK1 suppresses the malignant phenotype of HCC cells by inhibiting Wnt/β-catenin signaling, constitutively active β-catenin (T41A) was overexpressed in cells with overexpression of PANK1. As shown in Figure To further explore the correlation between the expression of PANK1 and β-catenin, the protein levels of PANK1 and β-catenin in the HCC tissue array were examined. In the HCC tissue array, the level of β-catenin protein was increased in the HCC tissues, as PANK1 expression was downregulated in the HCC tissues Figure A.Subsequently, the effect of PANK1 and β-catenin expression on the prognosis of HCC patients was analyzed. The results showed that among patients with high expression of β-catenin, the overall survival times of those with high expression of PANK1 were significantly longer than those of patients with low PANK1 expression Figure B. FurthePANK1 is the rate-limiting enzyme in the synthesis of CoA The serine/threonine kinase activity of PANK1 is one of the important findings of this study. Existing reports show that PANK1 uses pantothenic acid as substrate, and catalyzes the rate-limiting reaction in de novo synthesis of CoA For example, PGAM1 interacts with the phospholipase WIP1 and therefore retains it in the cytoplasm to prevent it from dephosphorylating ATM, thus promoting DNA repair In the present study, the in vitro kinase assay showed that PANK1 and CK1α cooperate to phosphorylate the N-terminal serine/threonine residues of β-catenin. This effect is similar to the synergistic effect of GSK3β and CK1α PANK1 activity is inhibited by metabolites such as CoASH, acetyl-CoA, malonyl-CoA and long-chain acetyl-CoA In conclusion, the present study reveals that PANK1 expression is downregulated in HCC, which promotes the progression of HCC by activating the Wnt/β-catenin signaling pathway. This finding suggests that HCC may also be treated by enzymatically activating PANK1.Supplementary figures and tables.Click here for additional data file.


The respiratory tract is a vital, intricate system for several important biological processes including mucociliary clearance, airway conductance, and gas exchange. The Wnt signaling pathway plays several crucial and indispensable roles across lung biology in multiple contexts. This review highlights the progress made in characterizing the role of Wnt signaling across several disciplines in lung biology, including development, homeostasis, regeneration following injury, in vitro directed differentiation efforts, and disease progression. We further note uncharted directions in the field that may illuminate important biology. The discoveries made collectively advance our understanding of Wnt signaling in lung biology and have the potential to inform therapeutic advancements for lung diseases. Cody Aros, Carla Pantoja, and Brigitte Gomperts review the key role of Wnt signaling in all aspects of lung development, repair, and disease progression. They provide an overview of recent research findings and highlight where research is needed to further elucidate mechanisms of action, with the aim of improving disease treatments. The advent of single-cell sequencing and other technologies continues to increase this number and contributes to the striking complexity of the lung. At its most proximal portion, the cartilaginous conducting airways harbor a pseudostratified mucociliary epithelium that plays a vital role in host defense. Inhaled harmful particles, pathogenic organisms, and debris are expectorated from the airways via a process known as mucociliary clearance (MCC). Inhaled contents are first entrapped by a layer of mucus, produced by goblet cells, that are then transported proximally by the unidirectional beating of cilia from terminal bronchioles to the trachea2. The coordinated removal of debris by ciliated cells and mucus-producing goblet cells is facilitated by lubrication from a periciliary water layer. The concerted undertaken by these multiple cell types together comprise the mucociliary escalator2. The bronchioles, also referred to as the conducting airways, are additionally important for moving gases to and from the distal lung, which contain the alveolar sacs necessary for gas exchange. Atmospheric oxygen undergoes exchange for blood carbon dioxide, a process that promotes cellular respiration for all tissues of the body.The lung is a structurally and functionally intricate organ, with over 40 different, known cell types3. R-spondin ligands are cysteine-rich glycoproteins that bind to their cognate leucine-rich repeat-containing G-protein coupled receptor (LGR) LGR4/5/6 receptors and E3 ubiquitin ligases ring finger protein (RNF)43/ZNRF3 via their furin-like domains5. In the absence of R-spondins, RNF43/ZNR43 ubiquitinate the Frizzled receptors that targets them for degradation /β-catenin signaling pathway plays an instrumental role in stem cell self-renewal across several tissue epitheliaion Fig. . As suchion Fig. 3. These ein Fig. .Fig. 1Ov7. Wnt ligand then binds to LRP5/6 and appropriate Frizzled co-receptor on the same or neighboring cell. Moreover, R-spondins can potentiate and amplify Wnt signaling by forming complexes with their cognate LGR pair and binding to RNF43/ZNR43 to prevent Frizzled receptor ubiquitination. Together, these events allow for a cascade of molecular events that results in disassembly of the cytoplasmic destruction complex  and encounters Porcupine, an ER-resident protein that palmitoylates N-terminal cysteine residues of Wnt proteins. This lipid tail modification has been shown to be necessary for Wnt ligand secretionlex Fig. . β-catenlex Fig. . In thislex Fig. . The can8. A second arm of non-canonical Wnt signaling lies in Wnt-Frizzled binding that triggers Phospholipase C and downstream Ca2+ activity for regulation of cell migration and fate decisions8. It is important to note that Porcupine palmitoylates all 19 mammalian Wnt ligands and is therefore necessary for their secretion, including those that partake in non-canonical signaling.In contrast, non-canonical Wnt signaling is often thought of as the β-catenin-independent pathway. One arm of this pathway regulates planar cell polarity (PCP), during which Frizzled receptors trigger downstream activation of RhoA and Rac GTPases that promote cytoskeletal remodelingOver the past several years, much research has been put forth toward carefully dissecting the nuanced role of Wnt signaling across several disciplines pertaining to lung biology. This review aims to highlight the major contributions made to our current understanding of the Wnt signaling pathway in lung and airway development, its role in proximal and distal airway homeostasis and relevant niche biology, as well as its role in directed differentiation efforts of induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) to the lung lineage, in organoid culture models, and its perturbations in disease states.12. To briefly summarize murine lung development, during the embryonic stage, the ventral anterior foregut endoderm (AFE) expresses the transcription factor Nkx2.1 at mouse embryonic day (E) 9.0 as a sign of the specification to promote initial lung budding. From E9.5–E12.5, two lung buds with high Nkx2.1 expression and a proximal portion with low Nkx2.1 that later forms the trachea emerge concomitantly with tracheo-esophageal septation. From E12.5–E16.5 during the pseudoglandular stage, the lung buds undergo a period of branching morphogenesis to form the lung tree and terminal bronchioles. Upon completion of the canalicular and saccular stages of development  day 4), the terminal bronchioles narrow and begin to form epithelial sacs. These structures later form fully mature alveolar structures for gas exchange by P21 during the alveolarization phase9. In contrast, alveolarization begins pre-partum during human lung development and continues postnatally into childhood13.Detailed staging and mechanisms of both human and murine lung development have been well reviewed by others15. Studies assessing the expression levels of various Wnt/β-catenin signaling members and activity reporters in these aforementioned compartments during lung development have identified contrasting findings17. However, a myriad of studies collectively demonstrates that the developing lung mesenchyme displays several highly regulated interactions with the lung endoderm in both mouse and human that together coordinate normal lung organogenesis9, many of which are Wnt-mediated.Coordinated development of the conducting and distal airways is a vital process during which both the epithelial and mesenchymal compartments play integral roles. The developing lung endodermal buds penetrate the splanchnic mesoderm and mesothelium around E9.5. The developing distal lung then acquires four distinct layers, each with its own unique anatomical, cellular, morphologic, and molecular profiles: endoderm (epithelium), subepithelial mesoderm (mesenchyme), submesothelial mesoderm (mesenchyme), and mesothelium. Transient amplifying submesothelial cells give rise to a parabronchial smooth muscle cell (PSMC) progenitor population. This cell population then migrates more proximally around the bronchi and differentiates into smooth muscle cells (SMCs)18. These cells are important for the vital epithelial–mesenchymal interactions that occur during lung development.Wnt signaling plays a role in some of the earliest stages of cardiopulmonary specification. Wnt2+ Gli1+ Isl1+ cells comprise the multipotent cardiopulmonary mesoderm progenitors (CPPs) that orchestrate heart and lung development. Lineage tracing of Wnt2+ CPPs at E8.5 demonstrates their capacity to generate the cardiac inflow tract and pulmonary mesoderm cell lineage by E17.520. Canonical Wnt2/2b ligands are spatiotemporally regulated by Hox5 genes during this stage, with notable mesodermal expression near the ventral aspect of the anterior foregut between E9.0 and E10.521  is prominently expressed in the developing stomach mesenchyme where it orchestrates endodermal differentiation, its expression throughout the dorsal foregut mesenchyme and mainstem bronchi has been shown to inhibit endodermal Wnt signaling to promote thoracic foregut specification26  signaling that, in turn, facilitates differentiation of immature platelet-derived growth factor receptor (Pdgfr)α/β+ smooth muscle cells by regulating the expression of myocardin and Mrtf-B, critical transcription factors in myogenesis26 Fig. . Mesencht27 Fig. .28. Consistent with this, R-spondin 2 (Rspo2) facilitates normal embryonic lung growth and the start of branching morphogenesis by potentiating Wnt/β-catenin signaling29. A recent study identified, however, that Rspo2 antagonizes RNF43 and zinc and ring finger (ZNRF)3 to regulate limb and lung formation in xenopus4. These efforts resulted in a paradigm shift, demonstrating that Rspo2 can potentiate Wnt signaling in the absence of LGR30. Interestingly, while Rspo2 drives branching morphogenesis earlier in development, it has no role in the differentiation of epithelial or mesenchymal cell types29. This suggests functional redundancy in the varying upstream regulators of Wnt signaling in embryonic lung development.Constitutive activation of β-catenin in surfactant protein C (Sftpc)+ cells drives distal conducting airway dilatation concomitant with no differentiation to either the secretory or ciliated cell fate31  and Fgf10 signaling, respectively34. Mesenchymal Wnt5a is primarily responsible for tracheal elongation and cartilage ring formation via Ror2-dependent mechanisms in the dorsal tracheal mesenchyme33.Several Wnt ligands have been well characterized in the pseudoglandular stage. Wnt5a is diffusely expressed in both the epithelium and mesenchyme as early as E12.031 Fig. . Its exp20. Epithelial- or mesothelial-derived FGF9 triggers activation of mesenchymal FGFR1/2 at E13.514. Fgfr1/2 then activates submesothelial Wnt2a to converge on β-catenin signaling that, in turn, promotes mesenchymal cell proliferation that is important for organ growth14 , a cargo protein involved in trafficking lipid-modified Wnt from the Golgi to the cell surface. Epithelial deletion of Wls also impairs tracheal development, branching morphogenesis, and proximo-distal patterning as early as E12.540. Further, in cultured lung explants, canonical Wnt signaling inhibitor dickkopf1 (Dkk1) inhibits lung tree branching and mesenchymal differentiation by decreasing distal mesenchymal Wnt2a, Pdgfrα, fibronectin, and alpha-smooth muscle actin expression20.As with the embryonic phase, tight regulation of canonical Wnt signaling activity is also important in the pseudoglandular stage. Notum, a deacylase that removes Wnt lipid modifications and therefore inhibits Wnt secretion, is critical for tracheal development41. In addition, transcription factor Gata binding factor 6 (Gata6) activates non-canonical receptor Frizzled (Fzd2), which inhibits canonical Wnt signaling to constrain bronchioalveolar stem cell expansion during development42. The loss of Gata6 in Sftpc+ lung epithelium leads to dilated airways and neonatal death42.Inhibition of canonical Wnt signaling in the pseudoglandular stage is also achieved by activation of non-canonical Wnt signaling. Epithelial expression of the Cadherin EGF LAG seven-pass G-type receptor 1 (Celsr1) and PCP protein 2 (Vangl2) proteins involved with PCP axis promote branching morphogenesis, as mice with mutations in either of these proteins display misshapen lungs and fewer, more narrow airway lumens35. These findings stand in contrast to those found by another group identifying normal, preserved cell differentiation from Wnt7b−/− mouse lungs37. Others report that β-catenin in Sftpc-expressing lung endodermal progenitor cells is necessary for the formation of terminal alveolar saccules, as its loss results in a proximalized lung with enlarged bronchial tubes as early as E13.544. Ectopic Dkk1 expression phenocopies these findings, likely through activation of Fgf10-Fgfr2 signaling44. During this stage, β-catenin is sufficient but not necessary for Sftpc+ lung endodermal progenitor cell proliferation45. Constitutive activation of β-catenin decreases differentiation of distal pulmonary cell types and instead promotes ectopic expression of gut and intestinal cell lineages45. This is accomplished by β-catenin binding to the N-myc, Bmp4, and Fgf promoters44  cells that contribute to the budding alveolus47  inhibition, thereby contributing to epithelial cell dysplasia and goblet cell hyperplasias47 Fig. . In cont30? Under what circumstances is R-spondin interaction with its cognate LGR required for distinct phases of development?While all of these studies have proven instrumental in allowing us to define the role of Wnt signaling throughout the varying stages of lung development, much remains to be learned. It is apparent that the phases of lung development are interdependent on each other, rendering it difficult to interpret the biology that is specifically responsible for a given phase in isolation from other phases of development. It is critical that we begin to further dissect the dynamic nature of niche interactions during distinct phases of development. As such, development of novel tools to achieve this level of granularity and isolation will yield more nuanced insight of not only Wnt signaling, but other signaling cascades, in lung developmental biology. In addition, the advent of in vitro organoid systems has and will continue to allow for us to better understand human airway and lung development specifically. At the molecular level, much remains to be understood about the dynamic receptor–ligand interactions. The seminal work of Szenker-Ravi et al. and Lebensohn et al. raises the question: how does Rspo2 mechanistically potentiate Wnt signaling in the absence of LGR receptors51.Submucosal glands (SMGs) are tubuloacinar structures of the submucosal region of the airway that are contiguous with the surface airway epithelium (SAE) by connecting ducts and play a critical role in mucous secretions that facilitate MCC, a function that is vital for host defense. SMGs contain several cell types including myoepithelial cells (MECs), ciliated cells, secretory cells, and basal cells. It was recently observed that SMGs also harbor glandular progenitors and that these cell types act as a reserve stem cell population to facilitate proximal airway regeneration following severe injury52. In contrast, mice have primitive SMGs that are restricted to only the most proximal tip of the trachea. As such, ferrets and pigs have recently emerged as a more tractable model system to study SMG biology, as their SMGs develop postnatally and localize throughout the proximal airway and are therefore more similar to what is observed in humans51.In humans, SMGs localize throughout the trachea and bronchi of the proximal airway. However, in diseases such as cystic fibrosis, SMGs have been reported to extend more distally into the bronchioles of humans in addition to being more hyperplastic and hypertrophicLef1 localization at the invaginating tips of the SMG buds in early development53. Using human, ferret, and mouse model systems, Lymphoid enhancer-binding factor 1 (LEF1) was then shown to be necessary but not sufficient for SMG morphogenesis52 despite no reportedly appreciable changes in β-catenin nuclear localization54. Sox2 and Sox17 each function to suppress Wnt/β-catenin-mediated activation of the Lef1 promoter58, indicating that these signaling axes are key regulators of SMG morphogenesis.Early studies in ferret airways offered insight into 59. Further, Wnt3a is sufficient to promote glandular progenitor proliferation. Wnt-active cells reside near label-retaining cells of the SMG and were thought to be a regulatory component of the stem cell niche for repair59. This was later confirmed in a follow-up study that illustrated the capacity of MECs to migrate to the SAE and contribute to the repair process by giving rise to several differentiated cell types of the airway51. Further, Lef1 overexpression is sufficient to induce cell movement and migration programs that promotes MEC commitment to differentiated lineages of the proximal airway48  cells, and ciliated cells. Under the majority of circumstances, the basal cell is the resident adult stem cell responsible for giving rise to differentiated progeny. However, because the trachea of mice has a slow turnover rate of approximately one cell every 7–11 days, injury repair models are often employed to understand the cellular dynamics of stem cell fate decisions63. In addition, as described above, Wnt signals facilitate MEC contribution to proximal airway regeneration following severe injury48. At the cellular level, a recent group identified that a subset of Pdgfrα+ cells in the intercartilaginous zone are transiently, dynamically activated to secrete Wnt ligand that signals to the airway epithelium to promote its proliferation64. During differentiation, β-catenin activity is increased in air–liquid interface (ALI) cultures, a culturing system used to model airway epithelial remodeling, and enhances specification for the ciliated cell lineage61. In vivo, this phase is also driven by Wnt secretion from K5+ ABSCs that are necessary for differentiation to the ciliated cell fate64.The development of an understanding for how Wnt signaling modulates proximal airway homeostasis and regeneration remains in its infancy. It is known that basal cell β-catenin signaling is dynamically activated and necessary for the proliferative phase in murine injury repair models65. In contrast, high levels of Wnt signaling drive ABSC hyperproliferation concomitant with a phosphorylated form of nuclear β-catenin at Y489 (p-β-cateninY489) and abolishment of differentiation to the ciliated cell fate65  houses the Scgb1a1+ Sftpc+ Sca1+ bronchoalveolar stem cell (BASC)71. Further, Sftpc+ ATII cells in the alveolar region undergo self-renewal and give rise to ATI cells72. Most recently, a family of stem/progenitor cells referred to as the lineage-negative epithelial progenitors (LNEPs) have been characterized as quiescent at homeostasis but are mobilized under injurious conditions to regenerate the lungs74. Although the intricate complexity of the identity of these stem cell progenitors continues to be an area of current investigation, Wnt signaling plays critical roles in several of these regional compartments in regeneration.As the trachea branches and forms the distal lung, there are appreciable changes in the cell types that act as stem or progenitor cells in homeostasis and regeneration that are spatially defined. In the mouse bronchiole, Scgb1a1+ Scgb3a2+ club cells, Scgb1a1− Upka3+ club cells, and Cgrp+ neuroendocrine cells can all act as stem/progenitor cells in either homeostatic or regenerative conditions75, several subsequent findings indicate a highly important and intricately regulated role for this pathway at the cellular level in repair.From a cell biology perspective, several of the aforementioned identified progenitor populations or non-stem supporting niche cells employ Wnt signaling in bronchiolar, bronchioalveolar, or alveolar regeneration. Although an early study reported that β-catenin in club cells is dispensable for bronchiolar epithelial repairFgf10 expression77 + LNEPs toward an ATII-like rather than K5+ cell fate by inhibiting Notch and hypoxia signaling following influenza infection80  via Activin A, a Nodal-like TGFβ family member known to induce an endodermal fate91, the first report of lung-directed differentiation was described in 2011. Green et al. first illustrated accurate specification of FOXA2+ SOX2+ AFE via inhibition of BMP and TGFβ signaling in Activin A-induced definitive endoderm92  of this lineage. Wnt activation via GSK3β inhibition was identified as necessary for BMP4-dependent generation of NKX2.1-expressing lung endoderm progenitors   additionally requires low BMP signaling96.To address this concern, Wong et al. optimized the stepwise differentiation of iPSCs-derived lung endoderm into proximal lineage-specific epithelial cells through dynamic additions of various growth factors targeting major developmental pathways including Wnt signalingT+) Fig. 95. Impor97  and subsequently added back to promote cellular proliferation and subsequent generation of ATII cells103. In addition, the same group also utilized knowledge gained from Nkx2.1+ murine lung epithelial progenitors in vivo to efficiently generate proximalized or distalized airway epithelial cells104. Inspired by prior studies, Wnt3a was removed from the generation of the definitive endoderm and instead was included in a “distalization” media on sorted NKX2.1+ reporter cells106 , bronchopulmonary dysplasia (BPD), and chronic obstructive pulmonary disease (COPD) among others.119. However, early studies using β-catenin gain-of-function animals crossed with Nkx2.1 tissue-specific Cre recombinase mice developed tracheal and bronchial polyps that contain an undifferentiated epithelium that surrounds a mesenchymal core120, implicating a role for the pathway in carcinogenesis. Subsequently, a myriad of other studies has demonstrated its key role in lung tumor burden progression as well as chemoresistance, as described below.Lung cancer kills more people than breast, colon, and prostate cancers combined, with an overall 5-year survival rate that remains 18%. Although mutations in the Wnt/β-catenin signaling pathway are common in malignancies in several tissue types, including colon and intestine, they remain rare in lung cancerKrasG12D mutations accelerates disease progression121. Further, KrasG12D;p53fl/fl;Prkci (KPI) lung adenocarcinoma (LADC) mouse model tumors arise from Tm4sf1 + Axin2+ ATII cells whereas KrasG12D;p53fl/fl (KP) tumors arise from BASCs122. In addition, KPI tumors are reliant on higher levels of Wnt/β-catenin signaling for their expansion, a signature that is also reflected in human patient tumors122.NSCLC is the most common histologically defined subtype of lung cancer, accounting for 85–90% of all lung cancers, and for which the most is known with respect to Wnt signaling. While activated Wnt/β-catenin signaling alone cannot drive tumorigenesis of the bronchiolar epithelium, its activation in the setting of 121. Indeed, a subset of LADCs uniquely display Wnt target gene transcriptional profiles that are associated with and drive metastatic potential to brain and bone123. Specifically, TCF1, TCF4, LEF1, and HOXB9 are key transcription factors that mediate cancer cell metastatic behavior123.NSCLC tumors with constitutively active β-catenin induce a more distal embryonic lung phenotype that also represses E-cadherin, consistent with a role for Wnt/β-catenin signaling in EMT-like phenotypic processes that may facilitate metastasis to distant sites124. Human premalignant lesions and squamous lung cancer samples also display increased stromal and epithelial Porcupine as well as nuclear p-β-cateninY489 65, further elucidating the Wnt signaling niche networks that are targetable with small molecule inhibitors.While many efforts have mechanistically dissected the molecular biology of Wnt signaling within the Wnt-responsive cancer cells, it is also important to recognize the source of Wnt ligand that triggers these downstream signaling and cellular events. Recent work has identified the presence of Wnt-producing (Porcupine+) epithelial cells that are closely apposed to Wnt-responding (LGR5+) epithelial cells in LADC120. The Wnt pathway is also implicated in the biology underpinning chemoresistance, as negative regulators of the pathway, APC and CHD8, were mutated in one-third of relapsed patients relative to their primary lesion samples prior to chemotherapy treatment125. In vitro APC knockdown studies conferred activated Wnt signaling and subsequent chemoresistance in SCLC cell lines125. Future studies in both NSCLC and SCLC should investigate the cellular heterogeneity of the transcriptional profiles of Wnt production and responsiveness in the context of chemoresistance biology.SCLC is a rare, yet aggressive disease characterized by tumors with neuroendocrine features, expressing markers such as Chromogranin A, Synaptophysin, UCHL1, ASCL1, and NEUROD1. Although initially responsive to chemotherapy, resistant disease rapidly emerges, accounting for its high mortality rate. Tracheal and bronchial polyps with constitutive activation of β-catenin contain higher levels of neuroendocrine marker Uchl1126, specifically p-β-cateninY489 127. Subsequent studies further elucidated a pathogenic role of the pathway in the disease process that is associated with poor prognosis128. Specifically, Wnt/β-catenin signaling is activated in humans with IPF and mouse models of the disease in both the mesenchyme as well as epithelium132. Further, administration of various Wnt signaling inhibitors attenuates the fibrotic phenotype both in vitro and in vivo133.IPF is a rare, progressive disease marked by chronic lung scarring that results in poor lung function and for which there is currently no therapy to halt or reverse the fibrosis. The role of Wnt signaling in IPF remains convoluted, although studies have observed nuclear β-catenin in bronchiolar lesions, alveolar structures, and fibrotic foci129. Further, LRP5 promotes β-catenin driven fibrosis by, in part, increasing TGFβ abundance128. LRP5 also acts to regulate the differentiation of alveolar macrophages and promote persistence of fibrosis134. In addition, the TGFβ pathway activates canonical Wnt signaling in human fibroblasts130 as well as induces extracellular vesicle secretion and subsequent release of Wnt5a ligand135. Together, these events trigger fibroblast proliferation and the fibrotic response. These findings also suggest multiple nodes of crosstalk between Wnt and TGFβ signaling cascades in IPF that warrants further investigation.Wnt target gene Wnt-inducible signaling protein 1 is responsible, in part, for the fibrotic phenotype and decreased survival observed in animal models136, suggesting the presence of an intricate epithelial-mesenchymal niche that parallels work identifying its functionality in normal injury repair of the distal lung83. Once internalized, the Wnt-responsive ATII cell secretes IL1β, which may then act as a profibrotic cytokine137.At the single-cell level, Wnt-producing cells are distinct from Wnt-responsive cells in humans with IPF138. Taken together, these findings suggest complex, multi-faceted roles for Wnt/β-catenin signaling in IPF prevention, pathogenesis, persistence, and resolution.In contrast, some have purported a protective role for β-catenin in the alveolar epithelium in bleomycin mouse models of fibrosis by promoting wound healing139. BPD also shares a common downstream activated, phosphorylated form of β-catenin at Y489 that is also observed in IPF140, though much remains to be explored in this disease context. COPD also displays perturbed non-canonical Wnt signaling biology, as pulmonary fibroblasts secrete Wnt5a to inhibit alveolar canonical Wnt/β-catenin signaling, thereby preventing epithelial repair141. Recent work has also begun to think about the role of Wnt signaling in the context of aging as well. Lehmann et al. reported that aged ATII cells exhibit increase senescence that is driven by activation of Wnt/β-catenin signaling and is associated with profibrotic changes142.Bronchopulmonary dysplasia (BPD) is a rare pediatric lung disease characterized by abnormal distal lung development, declined lung function, increased inflammation, and increased fibrosis. BPD is often seen in premature infants or neonates that have received mechanical ventilation. In vitro and in vivo models of BPD employing hyperoxia injuries demonstrate reliance on mesenchymal Wnt5a on disease phenotypes83. The success of this lies, in part, because its generated understanding of the Wnt-producing  cells and the Wnt-responsive (Axin2+) cells via single-cell sequencing studies. This approach should be adopted in the context of disease processes, as it could hold particular promise with disease processes with known intermediates such as the malignant transformation underpinning lung squamous cell carcinoma. There also exists a plethora of outstanding questions largely underexplored within the sphere of aging. For example, how do stem cell niches throughout the airway and lung change with aging, both at the cellular and molecular levels? And how, if at all, do the alterations in Wnt signaling that occur in aging relate to distinct pathophysiology of disease processes?The work of Nabhan et al. has paved the way for understanding the role of single-cell niches within distal lung regenerationTaken together, it is clear that Wnt signaling plays a major role in lung development, lung repair and regeneration, and the progression of many lung diseases. The rapid technological advances in the fields of molecular and cellular biology are greatly facilitating the study of Wnt signaling in lung biology. Advancing our knowledge on the exact mechanisms of Wnt signaling in the lung will allow for the development of more Wnt pathway targeted therapies that will hopefully lead to a therapeutic benefit for patients with lung diseases.


However, the wrinkled morphology of GO and FGO improves the nanoplatelet-matrix interlocking mechanism, which produces a significant improvement in the out-of-plane shear modulus (increased by 2 orders of magnitudes). The influence of the nanoplatelet content and aspect ratio on the mechanical response of the nanocomposites has also been determined in this study. Generally, the predicted mechanical response of the bulk nanocomposite materials demonstrates an improvement with increasing nanoplatelet content and aspect ratio. The results show good agreement with experimental data available from the literature.The impact on the mechanical properties of an epoxy resin reinforced with pristine graphene nanoplatelets (GNP), highly concentrated graphene oxide (GO), and functionalized graphene oxide (FGO) has been investigated in this study. Molecular dynamics (MD) using a reactive force field (ReaxFF) has been employed in predicting the effective mechanical properties of the interphase region of the three nanocomposite materials at the nanoscale level. A systematic computational approach to simulate the reinforcing nanoplatelets and probe their influence on the mechanical properties of the epoxy matrix is established. The modeling results indicate a significant degradation of the in-plane elastic Young’s (decreased by ~89%) and shear (decreased by ~72.5%) moduli of the nanocomposite when introducing large amounts of oxygen and functional groups to the robust sp The success of using advanced composite materials as an alternative for traditional metallic alloys represents a great milestone in the aerospace industry ,2,3. HowThe one-atom-thick and defect-free sheet of graphene, which was discovered in 2004, is the strongest ever tested material ,14. The 2 hexagonal structure into a weaker sp3 structure, experimental studies have shown that it can improve the mechanical and other engineering properties of the produced nanocomposite [While the graphene nanoplatelet waviness/wrinkling is found to enhance the interlocking mechanism with the polymer matrix, graphene nanoplatelet agglomeration, which is triggered by the noncovalent van der Waals (vdW) force and the π-conjugation effect, is found to hinder graphene nanoplatelet dispersion in the polymer matrix ,22,23,24omposite ,32,33,34omposite ,36,37,38The aspect ratio of the filler used to reinforce the polymer matrix represents another essential factor that can greatly affect the properties of the composite. For polymer matrix reinforced with microscale fibers; such as carbon fibers (CF) and fiberglass, an infinite aspect ratio is commonly used for the fiber unless it is chopped. However, for the nanoscale fillers or particles, such as graphene nanoplatelets and carbon nanotubes, the situation is different as these nanofillers are produced with a wide range of aspect ratios ,40,41,42Although there are a wide range of experimental characterization tools that provide information about the structure and properties of nanocomposites, there is information that they cannot easily provide. For example, intermolecular interaction energy, interphase region characteristics, and molecular-level mechanical failure behavior cannot be fully resolved utilizing the available experimental tools. For this reason, computational simulation techniques have been widely integrated with experiments to provide a more comprehensive understanding of material structure and behavior at the atomic scale. Molecular dynamics (MD) simulation is one of the most powerful computational tools that can be utilized to predict properties of different types of materials. Polymers and their nanocomposite materials have been broadly investigated with MD modeling to predict molecular structure and physical behavior under different types of mechanical loadings and thermal conditions ,46,47,48As MD modeling simulations can be computationally expensive for studying multiple case studies considering the wide range of composite material parameters, fast and efficient microscale computational approaches represent the typical tools for this purpose ,50. MD mDue to the limitations in computational resources used for MD modeling, computational studies are usually constrained to a few nanoseconds for type MD models of organic materials (a few thousand atoms). This could lead to significant uncertainty in the predicted material properties due to the small statistical sampling of the molecular structure and the presence of significant thermal fluctuations at non-zero temperatures. The use of multiple model samples (replicates) for MD simulation can help to capture the variance in predicted properties due to these factors. The force field selection is another critical factor used for accurate predictions in MD modeling. As MD modeling with a reactive force field requires significant simulation times, most computational studies have been constrained to the use of fixed-bond force fields, which are less computationally expensive. However, reactive force fields have been shown to accurately predict mechanical properties of epoxy-based materials ,48,55.The aim of this study is to develop a material-designing computational approach for graphene nanoplatelet/epoxy nanocomposites. Various graphene nanoplatelet types are considered: pristine (GNP), functionalized with oxygen groups (GO), and further functionalized with amine groups (FGO). The adopted approach involves a multiscale analysis procedure to relate molecular structure to bulk level mechanical properties of the composite materials. For the nanoscale level, MD modeling is used to establish the molecular structure and load-transfer capability of the interphase region between GNP, GO, or FGO and epoxy. The load transfer is quantified using interfacial interaction energy, interfacial adhesion, nanoplatelet wrinkling, and mechanical interlocking. A reactive force field is used to more accurately simulate the nonlinear force-distance relationship  of covalent bonds. At the micro and macro-levels, micromechanics is used to predict the effect of the nanofiller functionalization, content, and aspect ratio on the mechanical response of the nanocomposite material. The outcomes of the modeling approach were compared with several experimental studies in the literature.For the nanoscale computational work, MD simulations were performed using the open-source LAMMPS software package  ,57. The Three unique nanocomposites were considered in this study to address the functionalization effect on the mechanical properties of the material. Simulated epoxy unit cells reinforced with GNP, GO, or FGO were created using LAMMPS. The size of each MD model was carefully assigned to only capture the graphene and surrounding epoxy interphase, not the bulk epoxy, as discussed previously ,55. FiveEPON 828  as the epoxy resin, and the EPIKURE curing Agent W  as the hardener, were simulated as the epoxy matrix in this study. One EPON 828 molecule (49 atoms) and one DETDA molecule (31 atoms) were individually created  via the x-axis and ~47.7 Å along y-axis, as shown in The “lattice” command was used in a LAMMPS script to create a single layer of GNP. The lattice parameters for the hexagonal atomic structure of a pristine sheet of graphene were taken from Gray et al. . A squar2 bonds into the weaker sp3 structure. In other words, the sp3/sp2 bond ratio  in the GNP oxidized with a specific amount of epoxide groups is double of that if the oxygens were presented in the hydroxyl form. Generally, the interfacial interaction is highly dependent on the functional group size, chemical composition, and molecular configuration [For the GO MD model, the core-level spectra of the oxygen content (Ols = 39.35 at%) was assumed to be 50% of epoxide (–O–) and 50% of hydroxyl (–OH) functional groups as there is no detailed information about the chemical composition of the oxygens in the reported data. It is important to note that each oxygen group has a unique effect on the GNP structure as each epoxide oxygen atom forms two covalent bonds with two carbon atoms in the GNP, while each hydroxyl oxygen atom forms only one covalent bond with GNP. This implies that epoxide groups have a greater detrimental effect on the robust structure of the GNP by turning a greater number of spguration ,64. Figuguration .2), 20.8% amide groups (O=C–NH2), and 20.1% graphitic nitrogen (–N–). These values were taken from curve fitting of the Nls spectra of aminated A21-GO performed by Park et al. [For the FGO MD model, the core-level spectra of the oxygen content Ols = 31.67 at%) was assumed to be 50% epoxide (–O–) and 50% hydroxyl (-OH) functional groups. The core-level spectra of the nitrogen content (Nls = 4.35 at%) was assumed to be 59.1% amine groups  were simulated with continuous lateral edges (periodic boundary conditions). The presence of carboxyl and carbonyl as functional groups in GO and FGO was ignored because they are more likely to exist in low concentrations at the edges and in defected regions (open rings or voids) in the graphene nanoplatelets ,16. Five2 structure. The functionalization degree can be used to specify the sp3/sp2 ratio, which is a measure of the graphene nanoplatelet purity and its structural robustness. The lower the sp3/sp2 ratio, the more pristine and planar the graphene nanoplatelets are. 3/sp2 ratio for the simulated nanoplatelets. While pristine GNP has a fully intact sp2 structure, GO and FGO exhibit sp3/sp2 of 0.973 and 0.798, respectively. This indicates that GO has the highest functionalization degree; however, both GO and FGO are almost completely covered with functional groups.Both GO and FGO MD models shown in To determine the effect of functionalization on the graphene nanoplatelet structural integrity, the three nanoplatelet types were subjected to simulated uniaxial tensile deformations along the zigzag-axis. To understand how the functional groups can help in improving the graphene nanoplatelet dispersion, the following MD simulations were performed. The modeling procedure is inspired by the Hummers method  for larg2 structural integrity  and temperature ,25,26,273). Each densifying simulation had a unique velocity seed number to maintain a random and unique distribution of the epoxy monomers for each MD replicate. All the densifying simulations were performed with the NPT  ensemble with the Nose/Hoover barostat along the lateral direction at 300 K. All lateral surfaces  of MD simulation boxes were maintained at 1 atm pressure. Hence, the nanoplatelets were free to adjust their lateral size under the normal compression from the epoxy monomers. To maintain a thermodynamically equilibrated molecular structure once the densification was complete, the systems were subjected to 1 ns of MD simulation with the NVT  ensemble followed by a molecular minimization (MM) simulation. The simulations involved the use of the Nose/Hoover thermostat with a temperature ramped up to 600 K, then ramped down to 300 K.The MD models of the unpolymerized and low-density epoxy monomers were combined with GNP, GO, and FGO MD models to form MD unit cells of the nanocomposites. The lateral size of each MD unit cell was governed by the equilibrated lateral size of the nanoplatelets. As five unique MD replicates of each nanoplatelet were already created, five MD replicates of GNP/, GO/, and FGO/epoxy nanocomposite unit cells were consequently created. The total number of atoms in the GNP/, GO/, and FGO/epoxy MD models were 7028, 7841, and 7811 atoms, respectively. This combining process involved MD simulation settings to maintain the center of mass (COM) of the nanoplatelets at the center of the simulation box. In addition, each MD model was subjected to slow densification (size reduction) simulation steps for 2.5 ns along the normal axis to the plane of the nanoplatelets to densify the epoxy monomers to the bulk level density  with the parameterization of Liu et al.  were perz-axis. The COM point of each nanocomposite MD model was selected at the origin  of the x, y, and z coordinates. The average simulation box size of the GNP/epoxy MD models was (47.55 ± 0.03)x Å × (47.67 ± 0.04)y Å × (29.68 ± 0.30)z Å. Likewise, the average simulation box size of the GO/epoxy MD model was (42.38 ± 1.97)x Å × (43.41 ± 0.83)y Å × (38.42 ± 2.35)z Å, and the average size of the FGO/epoxy models was (43.39 ± 1.16)x Å × (42.42 ± 0.83)y Å × (38.51 ± 1.16)z Å. For all three nanocomposite types, the model size was sufficiently large enough to capture the entire interphase region, which was previously shown to be 10 Å wide [s was 47. ± 0.03x 3 at 4 Å from the GNP) followed by a density drop to ~1 g/cm3 and a gradual rise to the bulk density at ~10 Å from center  in which the interfacial interaction between GNP and epoxy diminishes with distance from the GNP.The mass density distribution of the GNP/epoxy MD model exhibits a clearly defined profile reflected about the COM. There are four distinct regions that can be recognized from the mass density profile of the GNP/epoxy MD model: (i) the large spike at the center, which represents the flat GNP atoms; (ii) the gap region between the GNP and the epoxy, which is caused by the repulsive portion of the van der Waals forces exerted by the GNP and epoxy atoms ; (iii) ts region ); and , which decreases with distance to the bulk density at ~10 Å from center; and (ii) the bulk epoxy region (~1.2 g/cm3) in which the interfacial interaction between the nanoplatelet and epoxy diminishes with distance away from GO or FGO nanoplatelets. According to the density profile analysis and MD models shown in For the GO/epoxy and FGO/epoxy nanocomposite models, two distinct regions can be recognized from their mass density profiles: (i) the interphase region, which includes the diffuse nanoplatelets (GO or FGO), and the adjacent highly interacted epoxy region with a density of ~2 g/cm2 structure due to the potential presence of vacancies, defects, and functional groups can also increase the waviness degree [In most experimental observations, graphene nanoplatelets exhibit one to several stacked-nanosheets with different sizes and morphologies having planar or wrinkled structures with sharp or folded edges ,39,70. Ts degree ,71. It hs degree ,72. Therx-axis, and 47.65 Å along y-axis) of an equilibrated planar GNP using ReaxFF. x-axis (WFx) and y-axis (WFy) calculated for all replicates of GO/epoxy and FGO/epoxy MD models. The overall WF is calculated as the mean value of the averaged values over WFx and WFy. It is noteworthy to mention that all equilibrated GNPs exhibit an overall WF of ~1, which indicates a relatively flat GNP morphology in the GNP/epoxy MD models, as shown in The waviness (wrinkling) factor (WF) associated with graphene nanoplatelets has been defined as the ratio of the direct distance between the two ends of a wrinkled graphene nanoplatelet  . The WF The properties of nanocomposites are typically evaluated with respect to the amount of the nanofiller used in reinforcing the polymer matrix. The nanofiller amount can be represented by either its weight fraction (wt%) or volume fraction (vol%). The typical goal of designing nanocomposites is to improve properties with the lowest amount of reinforcing nanofiller material. Excessive amounts of the nanofiller can make processing difficult (increased resin viscosity and filler agglomeration) and produce detrimental effects on the nanocomposite, such as increasing the weight and the number of critical micro-defects. Thus, it is necessary to study the nanocomposite properties at different levels of nanofiller reinforcement.In this work, both wt% and vol% values were calculated for the GNP, GO, and FGO nanoplatelets in their nanocomposite MD models. For the wt% evaluation, a simple LAMMPS script was performed on each MD model to obtain the ratio of the partial weight of the nanoplatelet to the overall weight of the nanocomposite. The “group” command in LAMMPS was utilized to specify the atom types that were used to determine the partial weight for each nanoplatelet. For the vol% evaluation, an acceptable estimation can be made for the GNP utilizing the MD simulation box volume and its mass density distribution in The interfacial interaction energy (l (PEMD) ,55:(1)IIous work . The 4GNAccordingly, the presence of functional and/or oxygen groups on the GNP surface resulted in a significant improvement in the x- and y-directions to predict the in-plane elastic modulus z-direction were used to predict the out-of-plane elastic modulus xy-, xz-, and yz-planes were performed on each MD sample. While the deformation simulations in xy-plane were used to predict the in-plane shear modulus xz- and yz-planes were used to predict the out-of-plane shear modulus 8 s−1, which resulted in a maximum engineering strain of 5%.To predict the effective mechanical properties, the MD models were subjected to six deformation MD simulations. As performed previously , each ofThe Micromechanics Analysis Code based on the Generalized Method of Cells (MAC/GMC) ,76,77 waIn order to predict the effective mechanical properties of the nanoplatelet/epoxy nanocomposites, a multiscale approach was utilized to incorporate the local interphase properties of the MD models a into thNP/RUC) can be calculated using the following expression:NP/MD represents the GNP, GO, or FGO volume fraction within the MD model that is given in MD/RUC represents the volume fraction of the MD model within the RUC that can be adjusted in the MAC/GMC script. A similar approach can be used to specify the nanofiller wt% within the epoxy matrix (wt% NP/RUC) according to the following expression:NP/MD represents the weight percent of GNP, GO, or FGO within the MD model that is given in MD/RUC represents the weight percent of the MD model within the RUC that can be evaluated using the following expression:3. The bulk epoxy volume fraction within the RUC (volB/RUC) can be determined fromFor the nanofiller content, it was shown previously ,54,55 thThe nanoplatelet aspect ratio –(10) provide the flexibility to predict the effective mechanical properties of the nanocomposites at different nanofiller contents and aspect ratios without the need for running additional MD simulations. It is important to note that the wrinkle phenomenon becomes more significant as the nanoplatelet aspect ratio increases. Researching this issue is computationally expensive with MD modelling. Fortunately, MAC/GMC allows users to control the nanoplatelets size by adjusting the size of the fiber subcell in the RUC. This helps to extend the effect of the nanoplatelet wrinkling as the aspect ratio increases.After running the first MAC/GMC script utilizing the RUC settings illustrated in The predicted mechanical properties of the nanoplatelet/epoxy nanocomposites are described in this section. Comparison with experimental data from the literature is provided for modeling validation. The nanoplatelet/epoxy composite predictions can be divided into two levels: first, the localized interphase MD predictions, and second, the bulk nanocomposite predictions.Referring to 2 structure of the GNP due to the functionalization, as shown above in opG is also partially due to the additional covalent bonds formed between the nanoplatelet and epoxy matrix. For the Poisson contractions, the in-plane Poisson’s ratio  can be used to justify the current results. Considering the discrepancies between nanoplatelet type and the force field used in the MD modeling and predictions, the predicted mechanical properties in this study show a reasonable agreement with those predicted by Hadden et al. [The results shown in n et al.   from Chong et al. [g et al.  indicatelus (Em) b,c allowlus (Em) c. HoweveZaman et al.  performe3 to infinite. The mean modulus value from experiment and the predicted modulus of FGO/epoxy are in excellent agreement around an aspect ratio of 350.z et al. . Even thAmong several types of graphene-based nanocomposites tested by Chong et al. , the XG-The predicted moduli from GNP/epoxy and 4GNP/epoxy models shown in To some extent, the judgment used in justifying the results shown in 4) is more than 100%. However, the reinforcing effect of GNP plateaus at aspect ratios greater than 104 as indicated in 2, and it becomes significant within a 102–104 aspect ratio span. This trend can be clearly observed at high GNP contents. At a lower levels of GNP dispersion (4GNP/epoxy), the bulk nanocomposite response exhibits a similar trend to the bulk GNP/epoxy, as shown in The above discussion indicates that the multi-scale modeling approached used herein agrees with experimental findings and is thus experimentally validated. The proceeding discussion is focused on a detailed understanding of the individual effects of nanoplatelet content, aspect ratio, and functionalization on the mechanical properties. For the ideal case with perfect GNP dispersion in the matrix, the elastic  is ~9% and ~10%, respectively. However, for the FGO/epoxy mechanical response shown in 4) is ~10% and ~11%, respectively. The reinforcing effect of GO and FGO plateaus at aspect ratios greater than 103 as indicated in 1 and improves within the 101–103 aspect ratio span. This trend can be clearly observed at high GO or FGO contents.Referring to Considering 4. Both the GO/epoxy and FGO/epoxy systems exhibit a nearly identical response with the FGO/epoxy slightly higher. For those systems, increasing the aspect ratio results in a very small improvement in the mechanical response. This can be mainly attributed to the detrimental effect of the high concentration of oxygen introduced to the GNP as discussed above. The mechanical response of the GO and FGO systems is comparable with the bulk 4GNP/epoxy response at aspect ratio values below 102. The results indicate that the GNP/epoxy composite response can be assumed to be the ideal case or the upper bound of the mechanical response. The FGO/epoxy or GO/epoxy systems can be assumed to be the lower bound of the mechanical response. That is, GNP nanoplatelets with lower oxygen concentrations are expected to exhibit an intermediate mechanical response.Regardless of the broad goal, which is to reduce the time and effort to design and manufacture epoxy-based nanocomposite materials using computational modeling, this work provides important information about using functionalized graphene nanoplatelets to reinforce epoxy matrices. The main advantages of using functional groups in GO and FGO is the increase in the interfacial binding and the nanoplatelet wrinkling and surface roughness, which improves the mechanical interlocking between the nanoplatelets and the epoxy matrix. In addition, the process of surface functionalization of the nanoplatelets helps to increase the interlayer spacing between graphene nanosheets in the graphene nanoparticles. This has the effect to improve and maintain the dispersion of the nanoplatelets for two reasons. First, functionalization reduces the nanoplatelet restacking as the pi-pi stacking effect is much lower than with pristine nanoplatelets. Second, interfacial binding between the nanoplatelets and the epoxy matrix helps to preserve the nanoplatelet dispersion level. However, there is a tradeoff effect of nanoplatelets surface functionalization since excessive functional groups degrade the nanoplatelet strength, which in turn limits its reinforcing function.2 structure to the more compliant sp3 structure. As a result, FGO/epoxy and GO/epoxy MD models demonstrate a less stiff mechanical response relative to GNP/epoxy. For instance, introducing oxygen groups to the GNP resulted in in-plane elastic and shear moduli of the GO/epoxy composite less than that for the GNP/epoxy system by 89.25% and 72.42%, respectively. On the other hand, a drastic improvement in the interfacial interaction energy was observed when introducing functional and/or oxygen groups to the GNP. More specifically, the interfacial interaction energy of the FGO/epoxy system surpassed that for GO/epoxy and GNP/epoxy by 113.6% and 573.6%, respectively. The surface roughness and wrinkled morphology of the GO and FGO systems along with high interfacial interaction energy were found to improve the interlocking mechanism with the hosting matrix.A multiscale computational study has been performed to assess the mechanical performance of epoxy composites reinforced with functionalized graphene nanoplatelets. A single layer of GNP was initially modeled using MD to represent the pristine nanoplatelet of graphene and then modified to create a highly concentrated graphene oxide (GO) nanoplatelet and functionalized graphene oxide (FGO) nanoplatelet. The three nanoplatelets were used to reinforce the epoxy matrix (DGEBA-DETDA epoxy system) and generate three MD models of nanoplatelet/epoxy nanocomposites. These MD models were used to predict the localized mechanical properties of the interphase region and to address the interaction energy between each of the nanoplatelets and the hosting matrix. The outcome at the nanoscale indicated a significant degradation in the reinforcing effect of the GO and FGO systems relative to the pristine GNP system. The weakness in the mechanical performance of GO and FGO originates from the alteration in the carbon lattice structure of GNP from its robust spThe predicted mechanical properties at the nanoscale level were then further analyzed using a micromechanics approach to predict the mechanical response of the nanocomposites at the bulk level for various nanoplatelet content and aspect ratio values. Despite the diversity in the factors, which could potentially affect the mechanical response of the nanocomposites, such as the nanoplatelet dispersion/agglomeration, content, aspect ratio, and their chemical composition, the predictions were in good agreement with experimental results available from the literature. At the bulk level, the optimal mechanical response was observed for the bulk GNP/epoxy nanocomposite. This can be attributed to the perfect dispersion of the strong GNP within the hosting matrix. Thus, its mechanical response can be assumed as the upper bound limit. However, the mechanical response of GO/epoxy and FGO/epoxy can be assumed as the lower bound limit. Any other cases of GNP with oxygen concentration lower than that used herein are expected to register a mechanical response between the designated upper and lower bounds. The predictions indicate that the bulk GO/epoxy and FGO/epoxy can produce a comparable mechanical response to the bulk GNP/epoxy at low nanoplatelet content and aspect ratio values. At larger nanoplatelet contents and aspect ratios, however, the detrimental effect of the oxygen and functional groups on the GNP integrity dominates the overall mechanical response.