Hepatocellular adenoma (HCA) subtypes are considered as risk factors for malignant transformation; thus, an accurate diagnosis is important. We report a case of resected HCA previously diagnosed as unclassified HCA using immunohistochemistry, subsequently discovered to harbor a mutation in exon 3 of the beta (β)-catenin gene using deoxyribonucleic acid (DNA) sequencing.The patient was a 26-year-old woman who was referred to our hospital because of a 150-mm tumor in the right lobe of the liver. Considering the possibility of malignancy, we performed right lobe hepatectomy. Based on the histopathological and immunohistochemical findings, the tumor was diagnosed as an unclassified HCA. Next, we performed sequencing of DNA isolated from the tumor and identified a mutation in exon 3 of β-catenin, suggesting that the tumor contained an activating mutation of the β-catenin gene.β-Catenin mutations in HCA cannot be detected by immunohistochemistry alone, and molecular analysis is required to accurately diagnose and evaluate its prognosis. Hepatocellular adenoma (HCA) is a rare, benign tumor of the liver, often observed in young women taking oral contraceptives, with an estimated incidence of 3 per 1,000,000 per year . ImmunohHerein, we report a case of UHCA that was demonstrated to have β-catenin mutation and discuss the significance of the collaboration between immunohistochemistry and molecular analysis in the diagnosis of HCA.18F-fluorodeoxyglucose positron-emission tomography showed a slightly high 2-fluoro-2-deoxy-d-glucose uptake in the tumor  ; alanine phosphatase, 14 U/L ; gamma-glutamyl transpeptidase, 260 IU/L ; total bilirubin, 0.7 mg/dL ; carcinoembryonic antigen, 0.9 ng/mL ; carbohydrate antigen 19–9, 9.0 U/mL ; alpha-fetoprotein, 1.1 ng/mL ; and protein induced by vitamin K absence or antagonist II, 192 mAU/mL . An indocyanine green retention rate at 15 min was 4.2% with Child–Pugh grade A. Enhanced computed tomography showed a 150-mm-diameter enhanced tumor on the right hepatic lobe (Fig. The postoperative course was good, and the patient was discharged 13 days after the surgery. No recurrence has been observed for 1 year.UHCA accounts for 10% of all HCAs and has not been characterized by a specific phenotype, radiological features, or genetic mutations . The sigB-HCA accounts for 7–14% of HCA and is characterized by activating mutations of the CTNNB1 gene  and are Several mutations have been identified in the CTNNB1 gene in HCA, leading to the activation of β‐catenin. Large in‐frame exon 3 deletions and point mutations occurring at the D32‐S37 residues cause a strong β‐catenin signal due to the direct disruption of the β‐TrCP binding site, consequently suppressing its ubiquitination and proteasomal degradation . Male seIn our patient, although the tumor had a large deletion of exon 3, including the β‐TrCP binding site, it did not show nuclear β-catenin staining, leading to UHCA diagnosis. Because both tumor and non-tumor sections showed similar membranous β-catenin staining, it was not suggested that the large deletion of CTNNB1 gene caused the loss of β-catenin antigenicity.Regarding cases with discordance between GS and β-catenin staining, the tumors are speculated to have activation of an alternative pathway causing GS expression . AustinaAbnormal GS expression is considered to be a useful marker for b-HCA, even in the absence of aberrant nuclear β-catenin staining . In HCC,We performed hepatectomy for HCA, and β-catenin mutation was detected using PCR and direct DNA sequencing. It is necessary to perform mutational analysis in cases with positive GS staining.Additional file 1: Figure S1. Immunohistochemistry findings of β-catenin in non-tumor tissue. β-Catenin is positive in the cellular membrane and negative in the nucleus.


Marine-derived bioactive peptides have shown potential bone health promoting effects. Although various marine-derived bioactive peptides have potential nutraceutical or pharmaceutical properties, only a few of them are commercially available. This study presented an osteogenic mechanism of blue mussel-derived peptides PIISVYWK and FSVVPSPK as potential bone health promoting agents in human bone marrow-derived mesenchymal stem cells (hBMMSCs). Alkaline phosphatase (ALP) activity and mineralization were stimulated using PIISVYWK and FSVVPSPK as early and late markers of osteogenesis in a concentration-dependent manner. Western blot and RT-qPCR results revealed that PIISVYWK and FSVVPSPK increased osteoblast differentiation of hBMMSCs by activating canonical Wnt/β-catenin signaling-related proteins and mRNAs. Immunofluorescence images confirmed nuclear translocation of β-catenin in osteogenic differentiation. Treatment with the pharmacological inhibitor DKK-1 blocked PIISVYWK- and FSVVPSPK-induced ALP activity and mineralization, as well as mRNA expression of the canonical Wnt/β-catenin signaling pathway in hBMMSC differentiation into osteoblasts. These findings suggested that PIISVYWK and FSVVPSPK promoted the canonical Wnt/β-catenin signaling pathway in osteogenesis of hBMMSCs. Blue mussel-derived PIISVYWK and FSVVPSPK might help develop peptide-based therapeutic agents for bone-related diseases. Osteoporosis is one of the most common diseases caused by an imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. The aging population is more prone to osteoporosis as a result of bone resorption rates being higher than bone formation rates . AntiresMarine organisms are recognized as a potential source of bioactive compounds. Due to a unique environment, bioactive compounds in marine organisms show potential health benefits, including anticancer, antioxidant, anti-inflammatory, antimicrobial, and antihypertensive activities ,17,18,19The effects of PIISVYWK and FSVVPSPK on hBMMSC viability were determined using a 3--2,5-diphenyltetrazolium bromide (MTT) assay. No effects were observed in concentrations up to 100 μM (data not shown). To investigate the effects of PIISVYWK and FSVVPSPK on hBMMSC differentiation into osteoblasts, hBMMSCs were treated with osteogenic medium (OM) in the presence or absence of PIISVYWK and FSVVPSPK (0~100 μM). After incubation for 7 days, ALP activity as an early stage marker in osteoblast differentiation was first determined. As depicted in The canonical Wnt/β-catenin signaling pathway is known to be involved in osteoblast differentiation of MSCs . To undeTo test whether PIISVYWK and FSVVPSPK induced osteoblast differentiation by activating the Wnt/β-catenin signaling pathway, cells were treated with DKK-1 (100 ng/mL), an antagonist of Wnt signaling, for 1 h prior to exposing the peptides. On day 7, ALP activity with and without DKK-1 in the presence of the peptides was determined. As shown in The effect of DKK-1 on mineralization was also examined. On day 21, the degree of mineralization with and without DKK-1 in the presence of the peptides was determined . ResultsBone formation is a complicated process that includes osteoblast proliferation, differentiation and, ultimately, formation of mineralized bone matrix comprising COL1A1 fibers. ALP is a good indicator of bone formation and is a major regulator of phosphate supplementation in bone mineralization . ALP is Considerable studies have shown that the Wnt signaling pathway is involved in regulation of bone formation and MSC differentiation into osteoblasts ,7,32. WnTo investigate the role of Wnt/β-catenin in hBMMSC differentiation with PIISVYWK and FSVVPSPK, we used DKK-1, an antagonist of the canonical Wnt/β-catenin signaling. DKK-1 can bind to LRP5/6, thereby preventing the interaction between Wnt ligands and their receptors for signal transduction. Overexpression of DKK-1 downregulated endogenous β-catenin and ALP activity in osteoblasts and, ultimately, DDK-1 inhibited bone formation . In thisBlue mussel-derived peptides (PIISVYWK and FSVVPSPK) were chemically synthesized and supplied by Peptron Inc. .2 at 37 °C. When the cells were 80–90% confluent, osteoblastic differentiation was induced by changing to an osteogenic medium . To test the effects of PIISVYWK and FSVVPSPK on osteoblastic differentiation, cells were treated with OM in the presence of peptides. All media were changed every 2 days.Human bone marrow-derived mesenchymal stem cells  were obtained from the American Type Culture Collection . hBMMSCs were cultured in Dulbecco’s Modified Eagle’s Medium  supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin  in a humidified incubator with 5% COAfter treatment, MTT solution (0.5 mg/mL) was added to the cells, incubated for 4 h for the metabolism of MTT to formazan crystals, and insoluble formazan crystals were dissolved in dimethyl sulfoxide (DMSO). Absorbance was measured at 540 nm .ALP activity was measured using staining and spectrophotometry. At day 7 of culturing, the cells were washed with PBS and then fixed with 10% formalin for 5 min at room temperature. Then, the cells were rinsed with PBS three times and stained with NBT/BCIP substrate solution  in incubation for 10 min at 37 °C. The cells were washed with deionized water after aspirating the staining solution.To measure ALP activity, at day 7 of culturing the cells were washed and lysed with sodium carbonate buffer  supplemented with 1.0% triton X-100. ALP activity in the supernatant of the lysate was measured according to our previous method [At day 21 of culturing, the cells were rinsed with deionized water four times and fixed with 70% ethanol for 1 h at 4 °C. Finally, the cells were stained with 2% ARS (pH 4.2) for 15 min with gentle agitation at room temperature, followed by washing in deionized water. The calcium deposit level was observed using a light microscope. The deposit was dissolved in 10% cetylpyridium chloride in 10 mM sodium phosphate buffer (pH 7.0) for 15 min, and absorbance was measured at 562 nm .TM BCA Protein Assay Kit . Protein was separated on SDS-PAGE and transferred to a nitrocellulose membrane, followed by blocking with 5% skim milk in tris-buffered saline containing 0.1% Tween 20 (TBS-T) for 1 h at room temperature. The membrane was incubated overnight at 4 °C with primary antibodies as follows: β-Actin  and lamin B , Wnt1 , Wnt3a , RUNX2 , Osterix , GSK3β , COL1A1 , cyclin D1 , and β-catenin . The membrane was rinsed with TBS-T and incubated with a horseradish-peroxidase-conjugated secondary antibody  for 2 h at room temperature. Finally, the membrane was detected using an ECL kit (Pierce Biotechnology).After 4 or 7 days of culturing, the cells were washed with ice-cold PBS and lysed with a RIPA buffer containing a protease inhibitor . For β-catenin analysis, cytosolic and nuclear fractions were prepared using a nuclear extraction kit  according to the manufacturer’s instructions. Protein concentration was determined using a Pierce-ΔΔCT method. β-actin was amplified as an internal control.After 4 or 7 days of culturing, total RNA was isolated using a TRIzol reagent  according to the manufacturer’s instructions. cDNA was synthesized using a cDNA synthesis kit  according to the manufacturer’s instructions. RT-qPCR was performed using a Magnetic Induction Cycler with a QuantiSpeed SYBR kit . The sequences of primers are listed in Cells were grown on glass coverslips and incubated with PIISVYWK and FSVVPSPK for 4 days. The cells were rinsed with ice-cold PBS twice and fixed with 3.7% paraformaldehyde in PBS for 15 min, followed by permeabilization with 0.1% Triton X-100 in PBS for 15 min. The cells were blocked with 2% bovine serum albumin for 60 min and then incubated with a β-catenin antibody conjugated with Alexa Flour 488  in 0.1% BSA for 3 h at room temperature, followed by staining with Hoechst 33342 (2 μg/mL) for 15 min. The cells were washed with PBS three times and viewed on a fluorescence microscope .p < 0.05 and ** p < 0.01, or #p < 0.05 and ##p < 0.01, were considered statistically significant.The data were analyzed using Sigma Plot 12.0  and presented as mean ± standard deviation (S.D). Statistical analysis was performed using one-way analysis of variance. Values of * In conclusion, this study demonstrated that blue mussel-derived antioxidant peptides PIISVYWK and FSVVPSPK promoted osteoblast differentiation by increasing ALP activity and the level of mineralization in hBMMSCs. The two peptides also increased osteoblastic differentiation markers, such as Runx2, OSX, and COL1A1. The mechanistic study revealed that the two peptides increased Wnt molecule signaling by transcriptional and translational regulation. In addition, pharmacological approaches suggested that the two peptide-mediated Wnt/β-catenin signaling pathway was involved in osteoblast differentiation of hBMMSCs. These findings may be helpful for new insights into peptide-mediated osteogenesis and developing peptide-based therapeutic agents for bone-related diseases.


Copernicia prunifera palm tree, known as carnauba fibers, was performed. The tensile properties for the composite reinforced with 40 vol% of carnauba fibers showed an increase (40%) in the tensile strength and (69%) for the elastic modulus. All composites presented superior elongation values in comparison to neat epoxy. Izod impact tests complemented by fibers/matrix interfacial strength evaluation by pullout test and Fourier transformed infrared (FTIR) analysis revealed for the first time a significant reinforcement effect (> 9 times) caused by the carnauba fiber to polymer matrix. Additional thermogravimetric analysis (TG/DTG) showed the onset of thermal degradation for the composites (326 ~ 306 °C), which represents a better thermal stability than the plain carnauba fiber (267 °C) but slightly lower than that of the neat epoxy (342 °C). Differential scanning calorimetry (DSC) disclosed an endothermic peak at 63 °C for the neat epoxy associated with the glass transition temperature (Tg). DSC endothermic peaks for the composites, between 73 to 103 °C, and for the plain carnauba fibers, 107 °C, are attributed to moisture release. Dynamic mechanical analysis confirms Tg of 64 °C for the neat epoxy and slightly higher composite values (82–84 °C) due to the carnauba fiber interference with the epoxy macromolecular chain mobility. Both by its higher impact resistance and thermal behavior, the novel carnauba fibers epoxy composites might be considered a viable substitute for commonly used glass fiber composites.A basic characterization of novel epoxy matrix composites incorporated with up to 40 vol% of processed leaf fibers from the  Sustainable action to mitigate worldwide pollution and climate changes are promoting the use of natural materials in the substitution for synthetic ones. A typical example is the use of fibers extracted from plants replacing glass fibers as reinforcement in polymer matrix composites ,2,3. IndCopernicia prunifera palm tree, endemic in the northeastern of Brazil where it is referred to as canaubeira. Copernicia prunifera is the carnauba wax worldwide commercialized in the form of yellow-brown flakes [A surging in the interest for polymer composites incorporated with natural fibers in past decades motivated a significant number of research works ,13,14,15n flakes . This wan flakes . After rTo our knowledge, a single research work on carnauba fiber incorporated polymer composites has been published so far. Melo et al.  investigAn important point to be noted in Therefore, in the present work the possible use of carnauba fiber as reinforcement of epoxy composites is for the first time investigated regarding the tensile properties, impact resistance by Izod tests, as well as fiber/matrix interfacial strength obtained by pullout tests. The Fourier transform infrared (FTIR) spectroscopy and thermal analysis contribute to characterize the limits for engineering applications of these composites, which are expected to be established in the future ongoing works by our research group.3.Carnauba green leaves with stalks, illustrated in The material used as matrix for the composite plates was a commercial epoxy resin type ether, diglycidyl bisphenol A (DGEBA), hardened with triethylene tetramine (TETA), associated with a stoichiometric ratio of 13 parts of hardener to 100 parts of resin. The resin manufacturer was Dow Chemical (São Paulo), supplied and distributed by Epoxy Fiber (Rio de Janeiro), both in Brazil.3 was adapted to a hydraulic press  with maximum load capacity of 30 tons.The fibers shown in 3 was used for the DGEBA-TETA epoxy resin [3 for the carnauba fibers [In order to fabricate the composite plate, continuous and aligned carnauba fibers were precisely hand lay-up along the mold’s 15 cm greater dimension. The amount of fiber for each plate corresponds to its defined volume fraction in the composite. Still fluid epoxy resin DGEBA-TETA was carefully poured into the mold avoiding fibers movement. The system was then sealed and subjected to a pressure of 5 tons for 24 h. xy resin  and 1.34a fibers . ComposiFor the tensile tests, four specimens of each group were cut from the composite plates, following the dimensions required by the ASTM D3039 standard , 150 × 1Izod impact tests were carried out according to the ASTM D256-10 standard . Notched1) is assumed if the value of F  is higher than the critical Fc (tabulated), thus concluding that at the significance level of 5% there is a difference between the averages of the treatments applied. Given this information, the use of the Tukey test, known as the honestly significant difference test (HSD), becomes necessary. The objective is to quantitatively assess each of the percentages of fibers used.The analysis of variance (ANOVA) was applied, using the F test, in order to assess whether there was a significant difference between the results obtained for Izod impact energy. The 95% confidence level was adopted for the tests, where the alternative hypothesis . From the results, it is possible to reject or not the hypothesis of equality between the averages compared through the lower significant difference (LSD), according to:By using this methodology, it was possible to quantitatively determine in a comparative way the influence of the volumetric fraction of the carnauba fibers applied in the production of the composites. The fracture surfaces of the Izod specimens together with the pullout specimens were analyzed by scanning electron microscopy (SEM) in a Quanta FEG 250 FEI microscope, operated with secondary electrons at 10 KV.−1 and the data generated were treated using the equipment data analysis program. The respective transmittance (%) spectra were generated as a function of the wave number (cm−1).The Fourier transform infrared spectroscopy (FTIR) analysis was performed in a Spectrum Two Perkin Elmer  equipment. The fibers and composites were ground in the required powder condition to produce the sample tablets. The samples were scanned from 4000 to 450 cmFor thermogravimetric analysis, both composites and fibers were crushed and allocated in a platinum crucible introduced in a Shimadzu model TG-50  equipment operating with nitrogen atmosphere with a heating rate of 10 °C/min in a temperature range of 25 to 600 °C. The TG/DTG analysis followed the ASTM E1131 standard .For differential scanning calorimetry (DSC) analysis the composites and fibers were crushed and placed in an aluminum crucible. A Shimadzu model DSC-60A Plus  equipment was used. The DSC analysis was carried out under nitrogen atmosphere with a flow rate of 50 mL/min, heating rate of 10 °C/min, in the temperature range of 25 to 600 °C.Dynamic mechanical analysis was carried out in order to identify important parameters such as glass transition temperature, as well as to investigate the viscoelastic behavior of composites. Specimens with 0, 10, 20, 30, and 40 vol% of carnauba fibers were fabricated according to ASTM D4065 standard  and the cL) of the carnauba fiber in relation to the epoxy matrix. The upper straight line with the lowest slope represents the linear adjustment between the maximum tensile stress values in pullout observed in the fiber embedded length range of 7.5 to 30 mm. A stress value of approximately 65 to 110 MPa, was found, which is relatively lower as compared to the range of tensile strength, 205–264 MPa, shown for the carnauba fiber in The results observed in The linear adjustments applied to the pullout stresses of the carnauba fiber/epoxy are represented by Equation (2) with higher slope curve and Equation (3) with lower slope curve.cL = 6.79 mm, the fibers were removed (pulled out) from the matrix, while for a higher value, an almost constant linear relation of the tensile strength with the embedded length, occurs around 65–110 MPa. In this case, when the maximum pullout stress is reached, the fiber breaks without being removed from the epoxy matrix. The interfacial resistance of the carnauba fiber in relation to the epoxy matrix was evaluated by the equation of Kelly and Tyson [d” is the equivalent diameter of the fiber and fσ is the tensile strength of the fiber. With the value of cL = 6.79 mm it was possible to calculate the value of iτ through Equation (4), considered as the interfacial shear resistance between the fibers and the matrix [It is important to note that, for embedding values lower than nd Tyson .(4)τi=de matrix . The aveiτ = 3.69 MPa) herein obtained is relatively higher than the epoxy-coconut fiber interaction (iτ = 1.42 MPa) and very close to that for the epoxy-PALF fiber interaction (iτ = 4.93 MPa) reported by Da Luz et al. [The obtained value is considered relatively low. This fact may be a consequence of the hydrophilic nature of the fibers, which contributes to the reduction of the interfacial interaction between the fiber/matrix. However, the value  = 2.496. Therefore, the volume fraction of carnauba fibers in the composites has a direct effect on the Izod impact energy.With the data obtained from the ANOVA presented in In order to verify which fraction of fibers presented the best results of Izod impact energy, the Tukey test was applied to compare performances with a 95% confidence level. The lower significant difference (LSD) was found as 34.04. The comparison data between the averages of Izod impact energy between the volume fractions of carnauba fibers are shown in Based on the results in The increase in impact energy with the increase amount of carnauba fibers in the composite may be related to the fracture mechanisms acting for composites of 20, 30, and 40 vol% of fibers. In order to confirm and better understand the evolution of the fracture mechanisms acting on the tested materials, SEM images of the fracture surfaces of the specimens are shown in In this figure, it is possible to identify several fracture mechanisms acting on the composites. In For the composites with 20 vol% of fibers, The incorporation of carnauba fibers in composites can be directly linked to the increase in the impact energy presented by these materials. This fact is associated with the effect of carnauba fibers as a means of interruption or deviation of the crack’s propagation direction, which contribute to a greater rupture surface area. Therefore, this causes an increase in the absorbed impact energy . The impThe FTIR spectra are presented in −1, which is attributed to the extension of the OH bond [−1 are attributed to CH2 vibrations, related to molecules present in cellulose and hemicellulose [−1, −1 can be attributed to vibrations in aromatic rings [−1 refers to the stretching of the CO bonds of phenolic groups present in the fibers constituents. The alcohols present in the constitution of cellulose show vibrations of deformation associated with the OH bond and generally appear around 1360 cm−1 [The FTIR spectrum for the carnauba fiber shown in  OH bond . Bands aellulose . The banlose −1, ,47. The ic rings , and in 360 cm−1 .−1 band shown in The increase in the volume fraction of fibers in the composite causes the variation in the relative absorption of the 1737 cmAs shown in The results in g) of the epoxy resin, as highlighted in g of the epoxy matrix. This may explain the increase in the endothermic peaks temperature (75–103 °C) with the increase in the amount of fibers in the composite, in spite of the well-known interference of NLFs with the polymer matrix crystalline arrangement [The increase of the volume fraction of carnauba in the epoxy matrix causes the displacement of the endothermic peak (63 °C) observed in the DSC curve for the neat epoxy. The composites endothermic peaks in angement ,51.The E’ results in g [g for the neat epoxy obtained from DSC (63 °C) is close to that from DMA tan δ (74 °C). As for the Tg of the composites (82–84 °C), they are slightly higher due to the carnauba fiber interference with the mobility of epoxy macromolecular chains [The E” peak is related to the structural relaxation and might be assigned to Tg . A similr chains .Pullout tests provided a critical embedded length of 6.79 mm for the carnauba fiber in the DGEBA/TETA epoxy resin with an interface shear strength value of 3.69 MPa, which is comparable to those presented by other NLFs.Epoxy composites reinforced with carnauba fibers showed higher tensile strength values (37.8–40.9 MPa) than the neat epoxy (29.3 MPa), characterizing a reinforcement effect. There was also a tendency of increasing the composites elastic modulus (2.29 to 2.80 GPa) as the volume fraction increases, which was attributed to the higher stiffness of the carnauba fiber. The total strain (1.41.5%) did not show any significant variation between the different carnauba fiber composites, but is superior to the neat epoxy resin (1.1%).Izod impact tests revealed an increase in impact energy with the volume fraction of carnauba fibers incorporated in the epoxy resin. The maximum value obtained for the Izod impact energy of 201.9 J/m, for the percentage of 40 vol% carnauba fibers is more than nine times that of the neat epoxy of 21.5 J/m. The ANOVA confirmed the highest Izod impact energy results for the epoxy composites with 40 vol% carnauba fiber and, together with the tensile properties, demonstrated for the first time the carnauba fiber reinforcement effect.The SEM micrographs of the fracture surface revealed an evolution in the fracture mechanisms with an increase in the composite volume fraction of carnauba fibers, going from totally brittle neat epoxy (0%) to ductile-brittle for the 40 vol%. It was also possible to identify several active mechanisms.3 band, thus being able to make a relationship with the intensity presented by the band and the concentration of fibers in the composite.The FTIR analysis showed expected results, with bands referring to molecular vibrations of functional groups belonging to the basic constituents of NLFs, such as cellulose, hemicellulose, and lignin. The FTIR of the composites showed a variation in the relative intensity of the 1737 cmThe TG analysis of the composites displayed an intermediate behavior on the thermal stability of the composites, when relating the thermal behavior of the neat epoxy resin and the plain carnauba fiber. The composites, on average, showed significant loss of mass above 300 °C. The increase in the volume fraction of carnauba fibers in the composite leads to a reduction in the initial degradation temperature and temperature of maximum degradation rate.g) of the epoxy matrix.The DSC curves disclosed an increase in the temperature of the endothermic peak observed in the neat resin, with the increase in the composite volume fraction of carnauba fibers. This is mainly attributed to the moisture release from the carnauba fiber and a possible contribution to the glass transition temperature  in comparison with that (64 °C) for the neat epoxy.DMA results confirm the carnauba fiber reinforcement effect associated with an improvement in the storage modulus, as compared to the neat epoxy. Moreover, the TAccording to the mechanical and thermal properties presented by the carnauba fiber epoxy composites, their reinforcement effect and thermal stability above 300 °C make them viable substitutes for other epoxy composites, especially those reinforced with glass fiber.


Riboflavin transporter deficiency (RTD) is a childhood-onset neurodegenerative disorder characterized by sensorineural deafness and motor neuron degeneration. Since riboflavin plays key functions in biological oxidation-reduction reactions, energy metabolism pathways involving flavoproteins are affected in RTD. We recently generated induced pluripotent stem cell (iPSC) lines from affected individuals as an in vitro model of the disease and documented mitochondrial impairment in these cells, dramatically impacting cell redox status. This work extends our study to motor neurons (MNs), i.e., the cell type most affected in patients with RTD. Altered intracellular distribution of mitochondria was detected by confocal microscopic analysis (following immunofluorescence for superoxide dismutase 2 (SOD2), as a dual mitochondrial and antioxidant marker), and βIII-Tubulin, as a neuronal marker. We demonstrate significantly lower SOD2 levels in RTD MNs, as compared to their healthy counterparts. Mitochondrial ultrastructural abnormalities were also assessed by focused ion beam/scanning electron microscopy. Moreover, we investigated the effects of combination treatment using riboflavin and N-acetylcysteine, which is a widely employed antioxidant. Overall, our findings further support the potential of patient-specific RTD models and provide evidence of mitochondrial alterations in RTD-related iPSC-derived MNs—emphasizing oxidative stress involvement in this rare disease. We also provide new clues for possible therapeutic strategies aimed at correcting mitochondrial defects, based on the use of antioxidants. Riboflavin transporter deficiency (RTD), formerly known as Brown-Vialetto-Van Laere syndrome, is a rare autosomal-recessive motor neuron disease. Typical clinical features include peripheral and cranial neuropathy, muscle weakness, sensory loss, diaphragmatic paralysis, and respiratory insufficiency, and multiple cranial nerve deficits, such as sensorineural hearing loss, bulbar symptoms, and loss of vision, due to optic atrophy ,2. ChildSLC52A2 (encoding RFVT2) and SLC52A3 (encoding RFVT3) were demonstrated as causative factors for RTD  retinoic acid was added to the medium, which was changed every day. NeuroCult has also supplemented with [2 μM] Dorsomorphin and [3 ng/mL] Activin A, from day 17. Finally, [10 ng/mL] BDNF, [2 ng/mL] GNDF, [400 μM], dbcAMP and [200 μM] ascorbic acid, were added to cell medium from day 24. Human iPSCs were obtained from healthy subjects and RTD patients, carrying reported . SpecifiFor confocal analysis, cells underwent our previously described double immunofluorescence protocol , using tCtrl and RTD MNs were plated in a chamber slide . Some samples were treated with 1 µM RF  and 100 µM N-acetylcysteine amide (NAC)  overnight.Treated and untreated cells were fixed and embedded in epoxy resin, according to Colasuonno and coll. . ElectroImageJ (NIH) software was used to quantify immunofluorescence intensity, while statistical analysis was performed using Prism software . A minimum of 300 cells/sample per experiment, were analyzed for fluorescence intensity levels, then a parametric t-test was used, since a normal distribution was assumed.For mitochondrial quantitative analyses, three samples/cell culture type and 10 cells/sample per experiment, were examined, by manually counting regular and altered mitochondria of Ctrl and RTD MNs, and their percentage was calculated. Two-way ANOVA was used to analyze differences among genetic conditions and treatments, followed by a Bonferroni post-hoc test. p-value of 0.05 or less was considered statistically significant (*), while ** indicate p-value equal to or lower than 0.01.The results are presented as means ± SD of n ≥ 3 independent experiments. A Confocal analyses of RTD and Ctrl iPSCs-derived MNs were performed after immunofluorescence, using SOD2 as a dual marker for mitochondrial number and for an antioxidant response, and βIII-Tubulin (βIII-TUB) as a neuronal differentiation marker. While the distribution of βIII-TUB in Ctrl cells highlights long neuritic processes, forming an intricate network, in RTD MNs, this neuronal marker reveals the presence of shorter neurites . Such mocristae  of the number of damaged mitochondria in patients’ MNs after RF + NAC treatment , a glutathione (GSH) precursor, could exert a protective effect when administered with RF. As shown by electron microscopic images e–h, thisreatment i.Riboflavin (RF) and its derivatives, FMN and FAD, play a crucial role in essential cellular processes, including mitochondrial energy metabolism, stress responses, vitamin, and cofactor biogenesis, ensuring the catalytic activity and folding/stability of flavoenzymes ,12. RF iIn the present study, we took advantage of iPSC potential, to differentiate MNs. Hypothesizing the involvement of oxidative stress of mitochondrial origin in RTD MNs, we investigated whether the same alterations demonstrated in iPSCs derived from RTD patients , were ma2−. Scavenging ability, in total agreement with data obtained in undifferentiated cells from the same patients [Overall, our data on patient-specific MNs emphasize the involvement of mitochondrial alterations in RTD pathogenesis . Indeed,patients , likely patients ,8. Alterpatients ,21,22,23patients . cristae and the presence of vacuoles in the mitochondrial matrix. Since mitochondrial dysfunction is known to trigger mitophagy, as a quality control mechanism, we looked for autophagic vacuoles. However, RTD cells failed to show an appreciable number of double-membrane limited vacuoles, containing intact or partially digested organelles. Differently, in healthy MNs, we detected numerous autophagosomes and lysosomes, suggesting active autophagic processes, as expected for an intensely active cell type. Possible involvement of insufficient/defective systems of repair/removal of damaged organelles in RTD pathogenesis has been suggested [Ultrastructural results correlate well with confocal data. Indeed, FIB/SEM analysis shows in RTD MNs remarkable mitochondrial dysmorphologies, strongly suggesting their dysfunction ,11. Alteuggested ,24, and We also explored novel therapeutic approaches by combining RF supplementation—known to partially ameliorate RTD phenotype—with the antioxidant, namely, N-acetylcysteine (NAC), which is widely employed for free radical scavenging and glutathione replenishment. Indeed, this combined treatment proved successful, in improving patients’ cell morphology, particularly as regards mitochondrial ultrastructural features. This result is consistent with work in progress at our laboratories showing that such combined treatment results in increased neurite length in RTD MNs , possiblHuman iPSCs represent an innovative model for numerous clinical studies, opening new perspectives to research and applications in the field of rare neurodegenerative disorders, which are generally difficult to study, due to the lack of models recapitulating onset and progression of the pathology. In this context, our recent  and pres


Arginine methyltransferase 5 (PRMT5) is involved in a variety of cancers. We used bioinformatics analysis to investigate PRMT5 overexpression in bladder urothelial cancer (BUC) and its clinical significance. We also conducted molecular biology experiments to investigate the effect of PRMT5 on the phenotype of BUC cells in vitro and in vivo. PRMT5 was found to be upregulated in BUC tissue in the Oncomine and The Cancer Genome Atlas databases. We validated the results from these databases in a cohort of BUC samples. Kaplan-Meier and Cox multivariate analyses demonstrated that PRMT5 upregulation is an independent prognostic risk factor for BUC. The in vitro and in vivo phenotypic experiments found that downregulated expression of PRMT5 in BUC cells inhibits BUC cell proliferation and aggression. In addition, gene set enrichment analysis demonstrated that PRMT5 knockdown leads to cell cycle G1/S arrest, deactivation of Akt, and mTOR phosphorylation in BUC cells. These results suggest that PRMT5 could be used as a potential molecular marker for BUC in the future. Bladder urothelial cancer (BUC) is one of the most common genitourinary carcinomas in China . It is aProtein arginine methyltransferase 5 (PRMT5) is the predominant type II methyltransferase and is involved in numerous cellular and biological processes, such as Golgi homeostasis, nucleic acid metabolism, transcriptional regulation, cell cycle regulation, and ribosome biosynthesis , 12. PRMFew diagnostic and prognostic genetic markers have been verified in BUC. Based on previous studies, this study was designed to detect PRMT5 gene expression in BUC and to assess its clinical significance. In addition, we studied the effect of PRMT5 on the proliferation and aggression of BUC.We performed bioinformatics analysis in three BUC data sets from the Oncomine database and found that PRMT5 was upregulated in superficial or infiltrating BUC tissue compared with nontumor bladder tissues –1D. On fvs 58.2%, respectively; P = 0.0106). The Kaplan-Meier curves also demonstrate poorer overall survival of patients with high PRMT5 expression, compared with those with low expression, with MIBC (T2-4) (P = 0.0360), absence of lymph node metastasis (P = 0.0298), and high-grade tumors  was conducted using GSE133624. GSEA enrichment plots show a significant positive correlation between cell cycle and mTOR signaling pathway and PRMT5 in BUC , 6B. To Hypophosphorylated retinoblastoma (Rb) protein is a crucial regulator of cell transit from the G1 to S phase –20. We iSubsequently, we tested the impact of PRMT5 on the PI3K/Akt/mTOR pathway in BUC cells. Western blot analysis demonstrated that PRMT5 knockdown led to downregulation of PI3K, which deactivated Akt and mTOR phosphorylation. Conversely, PRMT5 upregulation promoted PI3K, resulting in activation of Akt and mTOR phosphorylation BUC is the seventh most common cancer in men worldwide. The TNM staging system is based on the extent of disease at presentation and clinicopathology and provides clinical prognostic indicators for BUC. However, patients with the same TNM stage and undergoing the same treatment display considerable variability in recurrence and survival . Many BUMany arginine and lysine methyltransferases have been reported in cancers. Arginine methylation is an important regulator of biological function, tumorigenesis, and tumor progression , 14. PRMPRMT5 is a ubiquitous and evolutionarily conserved protein, but it may be involved in proliferation, migration, and invasion of cancer. PRMT5 upregulation results in increased proliferation and anchorage-independent colony growth, whereas cellular proliferation and colony formation in cancer are significantly inhibited by PRMT5 knockdown , 32–34. Distorted activity of proteins that affect the cell cycle induces abnormal proliferation, resulting in malignant transformation. We demonstrated that PRMT5 facilitated the progression of cells through the G1 to S phase, in accordance with the bioinformatics analysis. PRMT5 mediated the repression of p27 and the activation of Rb in BUC. Furthermore, our data showed that PRMT5 boosted the activity of the mTOR signaling pathway in BUC through upregulation of PI3K and Akt. Specifically, PRMT5 inhibition markedly impaired the PI3K/Akt/ mTOR pathway. PRMT5 may affect BUC tumorigenesis through this pathway. However, the relationship between PRMT5 and the PI3K/Akt/mTOR signaling pathway needs further research.In summary, we demonstrated that PRMT5 upregulation in BUC is correlated with a poor prognosis and BUC proliferation, migration, and invasion. In addition, PRMT5 affects BUC tumor progression by distorting the Rb and p27 proteins involved in the cell cycle and facilitating the PI3K/Akt/mTOR signaling pathway. PRMT5 inhibitors may be a potential therapeutic approach that offers promising perspectives of BUC in the future.2.J82, UMUC, RT4, Biu87, TCCsup, 5637, and T24 bladder cancer cells from the American Type Culture Collection were cultured in RPMI 1640 medium or DMEM  with 10% fetal bovine serum . All of cells were cultivated in an atmosphere at 37°C with 5% CORNA isolated with Trizol (Invitrogen) from the samples was quantified using an ultramicrospectrophotometer , and it was reverse transcribed into cDNA using PrimeScriptTM Master Mix . Then qRT-PCR was run on a Light-Cycler 480 instrument . The relative quantitative value was calculated using the 2-ΔΔCt method. The final result was recorded as fold change. Every sample was performed with three replicates.All protein samples were extracted from the clinical bladder tissues and BUC cell lines using RIPA buffer  and proteinase inhibitor cocktail . The concentration of the protein was quantified using the BCA protein assay . Western blots were implemented in accordance with the standard methods . ImmunohTo knock down the expression of PRMT5, two PRMT5 small-interfering RNAs (siRNAs) and the cognate control siRNA were synthesized by Gene Pharma . Then, 20 μm of siRNA was transfected into the indicated cells in six-well plates through the Lipofectamine 2000 Reagent (Invitrogen) according to the manufacturer’s instructions. The target sequences of PRMT5 for constructing the siRNAs are as follows: siRNA1, 5′-CCAGUUUGAGAUGCCUUAUT TAUAAGGCAUCUCAAACUGGGC-3′; siRNA2, 5′-GUUUCAAGAGGGAGUUCAUTTAUGAACUCCCUCUUGAAACGC-3′. Western blot was used to verify the effect of gene silencing 48 hours after transfection.The indicated cells were sowed in each well at a density of 2,000 cells on 96-well plates. We examined the cell growth every 24 hours with CCK-8  after incubation for 30 minutes at 37°C. According to the reference wavelength, the absorbance value was detected at 450 nm.The indicated cells were sowed in each well at a density of 500 cells on six-well plates. After cultivation for 7 days, the colonies were stained with crystal violet and visualized with a microscope.5-Ethynyl-20-deoxyuridine (EdU), a more specific and sensitive method, was mainly used as the marker of cellular replication activity . For an 4 cells/well) were plated on the upper chamber of 24-well plates in RPMI 1640 without serum. Then 20% formaldehyde was used to fix the invading cells, and crystal violet was used for staining at 24 hours after seeding.Biu87 and T24 cells  according to the standard protocol. Stained cells were tested using a flow cytometer .https://www.oncomine.org) and The Cancer Genome Atlas . One hundred thirty-two clinical BUC samples from Chinese patients from 2002 to 2016 were collected at the Sun Yat-Sen University Cancer Center. The clinical and pathological information collected included age, gender, surgery, tumor size, TNM stage, differentiation grade, vital status, and follow-up time, and tumors were classified according to the guidelines of the European Association of Urology [PRMT5 expression data and detailed clinical information of BUC patients were first ascertained from the data sets of the Oncomine database  and GraphPad Prism 6.0 , the mRNA levels of PRMT5 in BUC tumor tissues were compared with those in adjacent nontumor tissues using a paired student’s The Ethical Committee of Sun Yat-Sen University Cancer Center provided permission to use the clinical samples. All patients provided written consent for the clinical samples and data to be used for scientific research. The animal experiment was approved by the Animal Ethical and Welfare Committee of Sun Yat-Sen University and abided by the Guide for the Care and Use of Laboratory Animals.


Musa fruits in the body, which represent correlations with their constituent micronutrient factors and dietary polyphenolic constituents such as minerals, vitamin members, anthocyanins, lutein, α-,β- carotenes, neoxanthins and cryptoxanthins, epi- and gallo catechins, catecholamines, 3-carboxycoumarin, β-sitosterol, monoterpenoids, with series of analytical approaches for the various identified compounds being highlighted therein. Derivative value-products from the compartments (flesh and peel) of Musa fruits are equally highlighted, bringing forth the biomedicinal and nutritional relevance, including the potentials of Musa species in dietary diversification approaches.Nutritional quality and the well-being of the body system are directly linked aspects of human survival. From the unborn foetus to adulthood, the need for sustainable access to micronutrient-rich foods is pertinent and the global consumption of banana and plantain fruits, in effect, contributes to the alleviation of the scourge of malnutrition. This review is particularly aimed at evaluating the pharmacological dimensions through the biological mechanisms of  Micronutrient deficiency has developed into a key issue in developing and third world nations, with more than two billion people affected globally and expectant mothers and children being the majorly affected group ,2. InfanMusa is found in the Musaceae family. Other members of the family are the Ensete and Musella [Musa paradisiaca (plantain); Musa sinensis (banana). A comprehensive taxonomic classification of the genus Musa is shown in The genus  Musella . They arMusa plants range from two to nine metres in height (cultivated) and 10–15 m (wild), consisting of the false stem (pseudostem), corm, foliar including the flowering part.The M. sinensis L.) is a tree-like, perennial herb. The term banana was developed out of “banan” of Arab origin  [M. paradisiaca L.), similar to the unripe banana, is larger, has a starchy flesh and can be used in the unripe and cooked forms. It is a tropical staple, ranked the tenth most important in the world and contains more starch and less sugar than a banana. The mature, yellow variety can be peeled as is typically done with banana.Banana (slation) . It was slation) . The plaMusa genus and its herbaceous members, including the fruits they bear, have flowers that are medicinally useful in the treatment of bronchitis, dysentery  and ulcers. Diabetic patients are traditionally served cooked flowers, while the astringent sap is usually applied for conditions such as hysteria, epileptic seizures, fever, leprosy and diarrhoea [Musa fruit peels compared to a number of fruit peels such as Mangifera indica, Carica papaya, Citrus sinensis, Ananas comosus, Malus domestica, Citrullus lanatus and Punica granatum [The Banana family, broadly used to describe the iarrhoea . It is aiarrhoea . Banana iarrhoea . It alsoiarrhoea , and coniarrhoea . In sub-iarrhoea , anthocyiarrhoea  and antiiarrhoea ,35. Studiarrhoea ,37. Planiarrhoea . Banana iarrhoea ,39. Studgranatum . A detaiMusa fruits as shown in They broadly encompass trace elements, vitamins and mineral elements key to regular cellular and molecular functioning, which makes them of significance to human health in spite of their small requirement levels ,42,43. MZinc, structurally depicted in Dietary zinc is involved in the synthesis and secretion processes of insulin within the β-cells of the pancreas, on the basis of the β-cell viability role of zinc in an animal model , while iDietary zinc acts as a neurological transmitter in the brain and is functionally key in cognition, learning and memory  and actiDietary boron, structurally shown in Iron, depicted structurally in Manganese deficiency is not quite pronounced in humans as opposed to animals . However9) enables red blood cell production (erythropoiesis). It enhances immunity levels and biosynthesizes nucleic acids, nerve tissues  and proteins [Folate  is thought to be implicated in the differentiation and functionality of immune cells by regulating the oxidization of fatty acid [Riboflavin (Vitamin Btty acid . Dietarytty acid . Riboflatty acid . Its str6), structurally shown in Pyridoxine (Vitamin B1) is centrally important in nerve functioning and energy generation from carbohydrates [Thiamine , shown in Niacin  in the body . Other wα-tocopherol has been implicated in non-antioxidant mechanisms such as modulation of cell functions , inhibitCatechins, shown in Coumarins have anti-inflammatory and antioxidant activity , they arPhytosterols inhibit the absorption of cholesterol by depleting the collection of metabolizable cholesterol, thereby lowering the risk of cardiovascular disease ,178,179.Terpenoids are anti-inflammatory and immunomodulatory in action  with antAnthocyanins are neuroprotective, antiobesity and antidiabetic ,192. TheCatecholamines trigger biological reactions and increased energy metabolism levels ,200, whiCan banana peels be eaten? This non-conventional issue has been addressed and the point is that indeed, banana peels can be eaten. Reference  noted thMusa genus are ranked fourth in export value after wheat, rice, and corn (4.5 to 5.0) billion United States Dollars (USD) per year from 1998 to 2000. According to the Food and Agricultural Organization (FAO), banana cultivation is evident in more than 130 nations and on more than 5.5 million hectares of land. A worldwide output running to 145 million tons has also been recorded [Musa species are one of the globally most important staple food crops and arguably most popular international trade fruits, based on the Food and Agricultural Organization reports [Banana is an important subtropical and tropical fruit, which is generally cultivated in subsistence or small scale and economic or large scale across the globe as reported by . Accordirecorded . In esse reports . Reports reports  assert t reports . Plantai reports .The global banana supply chain is a complex one, much dependent on several collaborations of parties. Most of the banana export stems from Ecuador, Guatemala and Costa Rica as of the year 2016. However, only about 15 to 20% of banana produced globally, end up traded in the global market. More than 1000 banana varieties are produced and consumed globally. However, Cavendish banana, accounting for 47% of global production recovers quickly from natural disaster shocks. An estimated 50 billion tonnes of Cavendish are produced yearly around the globe, and predominantly supplied to the United States of America and Europe, because they are indeed better suited to international  trade and possess more resilience to global travels. Precise production figures can prove a little difficult to pin down, especially because the cultivation of banana plants is majorly done by small scale farmers and trade in the informal sector. However, the available data point to the fact that between the years 2000 and 2017, the global production rate rose at about 3.2%, reaching 114 million tonnes by 2017 from 67 million tonnes in the year 2000, with Asia, the Americas and Africa being the chief producing regions . BananasMusa species. These bioactive constituents inevitably confer a series of potential pharmacological values against oxidative damage, which helps in preventing genetic damage or DNA compromise, enhancement of immunological competence, neurological functioning and lowered risk of cardiovascular disorder. It is thus clear that banana and plantain fruit peels, as much as the flesh compartments, have a host of nutritionally and pharmacologically significant values to human nutrition, health and dietary quality. This consequently places banana and plantain peels in good stead as potential natural products and functional food options, adding credence to the need for their increased dietary utility. Summarily, given the activities described in this review, these primary and secondary metabolites exhibit properties that justify more research by the scientific community, to drive preclinical and clinical studies leading to the development of new drugs. The pharmacological insights in this review indicate the potential of Musa fruits as key elements in driving further preclinical research, in a bid to achieve natural product-based drug development from the fruits of Musa genus.This review identified and discussed a spectrum of micronutritional factors and dietary bioactive compounds in the fruit compartments (pulp and peel) of


Pure titanium T328H wires of industrial grade II were employed as a work material. No adhesion or galling of metallic titanium was detected on the contact interface between this β-SiC die and titanium work, even after this continuous forging process, up to a reduction in thickness by 70%. SEM (Scanning Electron Microscopy) and EDX (Electron Dispersive X-ray spectroscopy) were utilized to analyze this contact interface. A very thin titanium oxide layer was in situ formed in the radial direction from the center of the contact interface. Isolated carbon from β-SiC agglomerated and distributed in dots at the center of the initial contact interface. Raman spectroscopy was utilized, yielding the discovery that this carbon is unbound as a free carbon or not bound in SiC or TiC and that intermediate titanium oxides are formed with TiO Pure titanium has been utilized for metallic parts and tool components in various medical and biomedical applications due to its well-defined bio-compatibility . HoweverThick SiC (Silicon Carbide) coating was selected as a die material for upsetting the pure titanium wires and bars ,9. After2 debris. These two mechanisms are responsible for the galling-free cold forging of titanium and titanium alloys with a high reduction and lower friction.In the present paper, these β-SiC-coated SiC punch and core-die are prepared for dry cold forging of pure titanium T328H wires of industrial grade II, up to a reduction in diameter by 70%. This high reduction forging becomes an essential step to shape the blank plates for glass-frames from titanium wires and bars in a single shot [The SiC-coated SiC punch and core-die was prepared for dry cold forging experiments with a high reduction in thickness. These punch and die were fixed into the upper and lower die sets. A CNC stamping system was utilized for this experiment with the specified loading sequence. During loading, the power to stroke relationship was in situ monitored to describe the dry forging behavior, up to the reduction in wire diameter by 70%. Thick β-SiC coating was deposited onto the sintered SiC substrate by thermal CVD  process at 1500 K. The coating thickness was 4 mm. Both the punch head and core die were polished and cleansed by the ultrasonic cleaner . This SiC-coated SiC substrate was directly utilized as a punch, as shown in m), held for 1 s and then moved up to the original position at the same velocity. This δm was varied for each reduction in wire diameter. No interface delamination occurred before or after cold forging. To be discussed later, the estimated maximum forging stress in local is 10 GPa, lower than the compressive strength of 30 GPa for SiC coating.These SiC-coated SiC punch and core die were fixed into the upper and lower die sets, respectively, as shown in A pure titanium T328H wire in the industrial grade II was employed as a work material in the following forging experiments. Its diameter was 0.98 mm and its length was 10 mm. The chemical composition of this T328H wire consists of hydrogen by 0.0012 mass %, oxygen by 0.097 mass %, nitrogen by 0.007 mass %, iron by 0.042 mass %, carbon by 0.007 mass % and titanium for balance.SEM–EDX  and a three-dimensional profilometer were utilized to analyze the contact interface condition between the SiC coating and the titanium work. In particular, two regions were selected in the contact interface to describe the difference in the titanium oxide debris film formation and in the carbon isolation from the SiC coating. Raman spectroscopy  was used to analyze the binding state of silicon, carbon, titanium and oxygen on the contact interface.Dry cold forging with high reduction in thickness is performed to investigate the material flow during loading. The power–stroke relationship is in situ monitored to describe the flattening process from a circular wire to a thin plate. The surface area extension rate is estimated and employed as a parameter to analyze the formation of the fresh surface in this flattening behavior. SEM–EDX analyses as well as Raman spectroscopic analyses were utilized to describe the titanium oxide debris film formation on the contact interface and to investigate the role of free carbon agglomerates from the SiC coating on the solid lubrication under dry cold forging with a high reduction.A circular pure titanium wire was forged in dry and cold conditions, up to each specified reduction in thickness. During this forging process, the wire was placed at the micro-groove in the cavity of SiC-coated SiC die.When using the SKD11 punch and core die, the bulging process dominates this upsetting behavior of the titanium wire because of significant friction on the contact interface. As seen in both 0); e.g., e = A/A0. A is estimated for simplicity by A = 2 (W + H) × L for the current flattened work width (W) and height (H) during cold forging. A0 is defined by πd × L for the diameter (d) and length (L) of the wire before forging.The surface area extension ratio (e) is employed as a parameter to describe this flattening process under the assumption that the work material flow is in the plain strain condition. Then, e is defined by the ratio of the current side surface area (A) of forged wire to the original side surface area of circular wire  were selected for Raman spectroscopy analysis; e.g., A1 was located in the b-zone in 2, in the a-zone and A3, near the b-zone. The distance among A1–A3 was constant by 5 βm. As in the literature [−1 and 1600 cm−1, respectively. This proves that a-zones in Raman spectroscopy was employed to locally characterize the carbon agglomerates as well as the titanium oxide thin films on the contact interface. terature ,16,17,18terature , two bro−1 and 950 cm−1, respectively, with weak intensities reveal that β-SiC punch surface coexists with a tribofilm. Compared to high intensity peaks at β = 800 cm−1 and 950 cm−1 just outside of the contact interface, their peak intensities are much smaller. This implies that the contact interface on the SiC coating punch is mainly covered by the tribofilm as well as the carbon dots. Other peaks than these two include a broad peak detected around Λ = 200~600 cm−1. If TiO2 were present as a titanium oxide in the interface, the narrow peaks could be detected at Λ = 400 cm−1 and 600 cm−2, respectively. No detection of narrow peaks implies that titanium oxide film consists of various binding states in the titanium - oxygen system from TiO to TiO2 through the intermediate Magneli phase oxides. In particular, other broad peaks at Λ = 150 and 250 cm−1 suggest that the intermediate titanium oxides are formed together with TiO2 debris particles in the tribofilm.Two narrow peaks at Λ = 800 cm1 reveals that b-zone on the contact interface mainly consists of a titanium oxide film on the SiC coating with less free carbons. The Raman spectra at A2 and A3 show that the a-zone mainly consists of the free carbon dots with less formation of titanium oxide film. This Raman spectroscopy proves that free carbon agglomerates are formed on the contact interface away from the titanium oxide tribo-film..The Raman spectrum at ARaman spectroscopy as well as SEM–EDX analyses prove that the free carbon agglomerates with the size of 10 m work as a solid lubricant to prevent the SiC coating punch from adhesion to the titanium fresh surface and to lower the friction on the interface even at its center. The Magneli-phase intermediate titanium oxide thin tribofilms also play as a lubricious layer on the contact interface.Dry cold forging with a higher reduction in thickness than 50% is necessary for near-net shaping of titanium wires or bars for medical parts or tools. This shaping is also accompanied with a higher surface extension area ratio than 1.5. This implies that a fresh surface comes out of the inside of the titanium work and contacts with the die surfaces with the risk of its adhesion or galling to tools and dies. The present dry cold upsetting with the use of SiC-coated SiC punch and core-die proves that cold dry forging with a reduction higher than 50% is put into practice with low friction and without galling. This significant difference in cold forging performance from the conventional process with the use of tool steel and WC (Co) dies is attributed to the formation of titanium oxide tribofilm as well as the solid lubrication by the free carbon agglomerates.2 debris particles splash from the deforming work surface, deposit onto the die surfaces and lock the flattening process in forging [2. As reported in [In the conventional forging, hard TiO forging . The titorted in ,20, thesAs had been studied in ,12,14, nThe reduction in thickness in cold forging with the use of conventional dies is strictly limited to be less than 30% so that hot incremental forging processes with thermal annealing are necessary to shape the circular wire and bar to thin plates for further near-net shaping. When using the SiC coating punch and core-die, the wire can be shaped into a flat plat by a single-shot cold forging. This saving of processing and production time is attractive to practical operations in forging. In addition, the forged titanium plate has fresh and smooth surfaces ready to be directly shaped into the products by the successive steps in fine forging. SiC-coated tools are also useful to be anti-galling even for use in these forging steps. In addition, the tribofilms and carbon agglomerates in x (1 < x < 2) as well as TiO2; this TiOx film works as a lubricious thin layer to reduce the friction by its elasto-plastic flow. In addition, the unbound carbon agglomerates are also formed only at the center of interface which experiences high stress transients during forging. Owing to this in situ formed solid lubricant, the SiC coating surface is prevented from galling by the titanium fresh surface even under high stress states as in forging. This in situ solid lubrication has a close relationship to the carbon supersaturation in the CVD-coated SiC microstructure. Further precise analyses are necessary to describe the effect of stress transients on the carbon isolation from the SiC coating.SiC-coated SiC die technology is an anti-galling solution for dry cold forging of titanium work with a high reduction in thickness. No metallic titanium transfer was detected even after continuous forging up to the reduction in thickness by 70%. In situ formed tribofilm on the contact interface of the SiC coating on the titanium work is responsible for this anti-galling forging. As analyzed by SEM–EDX and Raman spectroscopy, the titanium oxide film is in situ formed on the SiC coating in the radial direction from the center of initial contact interface with the flattening of forged titanium work. This film consists of the Magneli-phase intermediate titanium oxides TiOIn the present study, the sintered SiC block was utilized as a substrate for the SiC CVD coating. Other substrate materials than monolithic SiC are available as a die system for cold and hot forging processes in each application. The graphitic carbon and carbides are candidate materials for CVD SiC coating to yield an anti-galling die system. The warm or hot forging behavior when the holding temperature is less than 673 K, is nearly the same as this cold forging process. Toward the high temperature forging, the die substrate material is appropriately selected to be free from severe oxidation.


Optimizing neurofeedback (NF) and brain–computer interface (BCI) implementations constitutes a challenge across many fields and has so far been addressed by, among others, advancing signal processing methods or predicting the user’s control ability from neurophysiological or psychological measures. In comparison, how context factors influence NF/BCI performance is largely unexplored. We here investigate whether a competitive multi-user condition leads to better NF/BCI performance than a single-user condition. We implemented a foot motor imagery (MI) NF with mobile electroencephalography (EEG). Twenty-five healthy, young participants steered a humanoid robot in a single-user condition and in a competitive multi-user race condition using a second humanoid robot and a pseudo competitor. NF was based on 8–30 Hz relative event-related desynchronization (ERD) over sensorimotor areas. There was no significant difference between the ERD during the competitive multi-user condition and the single-user condition but considerable inter-individual differences regarding which condition yielded a stronger ERD. Notably, the stronger condition could be predicted from the participants’ MI-induced ERD obtained before the NF blocks. Our findings may contribute to enhance the performance of NF/BCI implementations and highlight the necessity of individualizing context factors. Advances in health technology such as neurofeedback (NF) or brain–computer interfaces (BCIs) can improve the quality of life for individuals with neurological or psychiatric conditions. While the underlying closed-loop framework may be identical for NF and BCIs, the focus of NF is on improving health conditions, and that of BCIs is on steering technology . Both NFMI refers to mentally simulated movements that are not succeeded by overt motor output . The neuMany MI-based BCI and NF paradigms involve recording the neurophysiological signal of interest by means of electroencephalography (EEG) . In the Some EEG studies already touched upon the combination of gaming and MI BCI/NF paradigms by introducing single- and multi-user conditions in collaborative and competitive modes . For inHere we investigated how social context affects MI NF ERD. To this end, a multi-user mobile EEG MI paradigm with an engaging robotic NF was implemented. Participants underwent a single-user condition and a competitive multi-user condition of steering a humanoid robot as far as possible. We tested whether the competitive multi-user condition leads to better performance than the single-user condition. Post hoc, we additionally explored whether some participants performed better in the single-user condition while others performed better in the competitive multi-user condition, with better performance indicated by a stronger relative ERD. We further tested whether specific EEG and behavioral parameters would allow us to predict the social condition in which a participant had the stronger relative ERD. Predictors were ERD in the absence of NF, general performance motivation, and general control beliefs in dealing with technology.M ± SD: 24.93 years ± 2.95). The study protocol was approved by the Commission for Research Impact Assessment and Ethics of the University of Oldenburg.A total of 27 healthy right-handed young adults participated in this study. All participants reported normal or corrected-to-normal vision, had no history of neurological and psychiatric disorders, and had no prior experience with neurofeedback or MI paradigms. Each participant signed an institutionally approved consent form prior to the experiment. Two data sets were excluded from further analyses, one due to technical issues during the experiment (unstable connection between computer and robot) and one because of non-compliance with task instructions (systematic movements during the MI phase). Consequently, the analysis comprised 25 participants  aged between 19 and 30 years  in front of them. The participant and robot faced in the same direction. Partitions were used to restrict the participants’ fields of view cf. A. ParticAll participants performed four experimental blocks. Each block comprised 40 trials. In the first block, foot movements miming walking were physically executed while participants sat on the chair. In the following three blocks, participants remained seated and were instructed to imagine the kinaesthetic sensation of the executed foot movement from a first-person perspective. Between the second and the third block, the channel with the strongest relative ERD was selected for online NF  or participants steered their robot in a race (competitive multi-user) against a pretended opponent (CZ). The order of conditions was counter-balanced across participants. The pretended opponent was only in the room during the block running the competitive multi-user condition. The opponent wore the same EEG equipment as the participant and was barefoot. Other than the pretended opponent, the second robot was in the room in all blocks. The robot of the opponent was computer-controlled and walked each of the possible distances  for one-quarter of the 40 trials in pseudorandomized order. This resulted in a total distance of 150 cm. Ten out of 25 participants won against the pretended opponent in the competitive multi-user condition, that is, their robot covered a greater distance than the opponent’s, while 15 participants lost the race. Participants were asked to assess their state motivation on a visual analog scale ranging from “not motivated at all” to “highly motivated” before each experimental block. After each of the three MI blocks, participants rated their MI experience in the previous block on 5-point Likert scales. Ratings were given for easiness of MI  and vividness of MI . The scales also allowed for ratings in between scale intervals. After the main experiment, participants completed questionnaires about performance motivation in general   and geneEEG data were acquired using a wireless, head-mounted EEG system . The system features a sampling rate of 500 Hz, a resolution of 24 bits, and a bandwidth from DC to 250 Hz. EEG data were collected from 24 scalp sites using sintered Ag/AgCl electrodes with FCz as ground and AFz as reference , and A is the power at time point t, with t = 0 indicating the onset of MI [EEG data were analyzed online to provide NF. Online analysis consisted of three parts. The first part was performed after the second experimental block  to determine the channel over sensorimotor areas with the strongest relative ERD using MATLAB  and the EEGLAB toolbox . The secet of MI . For eacFor the second part, the raw data of the selected channel were filtered between 8 and 30 Hz and segmented into baseline intervals ranging from −3.5 s to −1.5 s and MI intervals from 0.5 s to 4.5 s within OpenViBE Version 0.17.1. Segments were spaced to avoid interference of task initialization and expectancy effects with the baseline interval. The resulting intervals were subdivided into 1 s time bins overlapping by 0.9375 s. The logarithmic power of the band-pass-filtered 1 s time windows represented the features for linear discriminant analysis (LDA) classification using sevenfold cross-validation . Based oThe third part of the online analysis constituted the actual NF delivery during the two MI NF blocks. Incoming data of the selected channel were 8–30 Hz band-pass filtered in OpenViBE Version 0.17.1. Features were extracted as described above and classified. The single trial classifier output was then compared to the individual thresholds derived from the training data in the second part of the analysis. MI NF classification outputs corresponding to the range below the first quartile of the training classification distribution led to a robot walking distance of 7.5 cm (3 steps), between the first quartile and the second quartile (median) to a walking distance of 5 cm (2 steps), and between the median and the third quartile to a distance of 2.5 cm (1 step), while classifier outputs out of these ranges led to no movement of the robot. SD = 0.08%) for the training, 63% (SD = 0.09%) for the single-user condition, and 64% (SD = 0.12%) for the competitive multi-user condition. With 40 trials per condition, online classification accuracies were not significantly above the chance level of 62.5% [The online classification accuracy was on average 62% (of 62.5% .https://sccn.ucsd.edu/wiki/EEGLAB_Extensions) with upper and lower boundaries of two standard deviations from the mean across all channels separately for all conditions . Identified channels were spherically interpolated. Following this, data were segmented from −7 s to 9 s relative to the start of each trial. Artefactual epochs as indicated by the joint probability and kurtosis  within each of the experimental blocks were discarded from further analyses. Additionally, epochs containing values identified as outliers (data points outside 1.5 times the interquartile range above the upper quartile) were removed . The relative ERD was calculated for every channel and every MI block  separately following the same procedure as described for online EEG analysis. In contrast to the online analysis, not a single channel but a region of interest (ROI) comprising CZ, CP1, CPz, and CP2 was used for offline analysis . The EEG raw data of all four conditions were high-pass filtered at 1 Hz  and subsequently low-pass filtered at 40 Hz . Filtered data were segmented into consecutive non-overlapping time intervals of 1 s. Segments containing artifacts were rejected . Remaining data were submitted to the extended infomax independent component analysis (ICA)  to estimysis cf. C. For stt-test between single and competitive multi-user conditions was conducted. Prior to statistical analyses, the effects of experimental block order were examined using Fisher’s exact test. To test for the statistical significance of MI NF condition differences in relative ERD, a paired competitive-gain, while positive ΔERD led to an assignment to the group single-gain  were conducted in total between each group’s training condition ERD and MI NF ERD .The finding that a significant difference between the competitive multi-user condition and the single condition was absent in the data was followed up by an exploration of individual differences. To this end, participants were grouped based on their ERD in the MI NF blocks. For the grouping, the relative ERD of the single condition was subtracted from that of the competitive multi-user condition (ΔERD) for each participant. Negative values reflect a stronger relative ERD in the competitive multi-user condition while positive values indicate a stronger relative ERD in the single condition. Negative ΔERD led to an assignment to the group gain cf. B. The fisingle-gain, competitive-gain) with different questionnaire data as the dependent variable were conducted. In order to evaluate the evidence for the interaction condition × group only, the model containing both main effects and interaction effect was compared to the model with only the main effects . Moreover, Bayesian independent-sample t-tests were performed to characterize both groups regarding their questionnaire data .Further, to test whether the groups differed in their state motivation, MI intensity, or MI easiness ratings for training, single, and competitive multi-user conditions, three separate (2 × 3) Bayesian mixed ANOVAs with within-subject factor condition  and between-subject factor group (two levels: train), as well as individual scores of the LMI [To identify predictors of the condition with a stronger ERD, a multiple linear regression with forward selection was conducted to predict ΔERD based on relative ERD derived from the training block (ERD the LMI  and KUT  the LMI  assessmed) for t-tests. All Frequentist statistics were calculated as implemented in RStudio  [All numerical values are reported as mean ± SD except where otherwise stated. Effect sizes are reported as Cohen’s d (_001905) . All Bay_001905) .t-test for the ERD of both conditions. We did not find a significant effect . In other words, our data do not provide evidence for the hypothesis that the competitive multi-user condition induces a stronger ERD than the single condition.The time-course and topography of MI-induced relative ERD for both MI NF conditions are shown in single-gain, while stronger relative ERD in the competitive multi-user condition compared to the single condition led to an assignment to the group competitive-gain. The final group sizes were 16 subjects in single-gain and 9 subjects in competitive-gain. Prior to further analyses, the effects of experimental block order were examined via Fisher’s exact test. Order had no significant effect on which condition had an ERD gain  or on the outcome (win/lose) of the competitive multi-user condition . Additionally, a Fisher’s exact test showed that gender had no effect on which condition had an ERD gain .The absence of a significant difference between the competitive multi-user condition and the single condition was followed up by an exploration of interindividual differences in the condition that induced the stronger ERD. To this end, we divided the sample into two groups based on the relative ERD in the single and competitive multi-user conditions, see t-tests were conducted. For the single-gain group we found very strong evidence of a difference between training and single conditions (BF = 46.0) but only anecdotal evidence for no difference between training and competitive multi-user conditions (BF = 0.46). For the competitive-gain group there was moderate evidence for no difference between single and training conditions (BF = 0.33) and weak evidence for no difference between competitive multi-user and training conditions (BF = 0.94). The time-course and topography of MI-induced relative ERD for both groups are shown in single-gain group and 65% ± 0.08 for the training, 65% ± 0.07 for the single, and 69% ± 0.09 for the competitive multi-user conditions for the competitive-gain group.The online classification accuracies were, on average, 59% ± 0.08 for the training, 62% ± 0.1 for the single, and 60% ± 0.12 for the competitive multi-user conditions for the single-gain and stronger ERD for the competitive multi-user condition for group competitive-gain, are paralleled by differences in state motivation, MI easiness, or MI intensity, we conducted three (2 × 3) Bayesian mixed ANOVAs including the within-subject factor of condition  and the between-subject factor of group  with state motivation, MI easiness, or MI intensity as the dependent variable. In all three cases the comparison of the model containing main effects and the interaction effect with the model containing only the main effects provided moderate evidence against the interaction  and their beliefs in dealing with technology . To further characterize the two groups, we compared their general performance motivation  and general beliefs in dealing with technology  by means of two Bayesian independent sample single-gain, six participants preferred the single condition, six preferred the competitive multi-user condition, and four participants had no preferred condition. In the group competitive-gain, two participants preferred the single condition, two preferred the competitive multi-user condition, four participants had no preferred condition, and one participant would have preferred a collaborative condition. Thus, no clear pattern emerged for ERD gain and subjectively preferred social condition. As regards the second aspect, i.e., the influence of the robotic NF, in the group single-gain, three participants felt negatively influenced by the robotic NF, six evaluated the influence as neutral, and seven as positive. In the group competitive-gain one participant felt negatively influenced by the robotic NF, four evaluated the influence as neutral, and four as positive.Participants were also asked which social condition they subjectively preferred and how the robotic NF influenced them. In the group train. We performed a multiple linear regression with forward selection to predict ΔERD, i.e., the difference in relative ERD between single and competitive multi-user conditions, based on LMI-K, KUT scores, and ERDtrain. A significant regression equation was found for ERDtrain as predictor , with an adjusted R2 of 0.255. The participants’ predicted ΔERD was equal to 5.91 + 0.41ERDtrain, where ERDtrain is measured in percent. ΔERD increased 0.41 percent for each percent of ERDtrain. ERDtrain was the only significant predictor of ΔERD. This result indicates that subjects with a stronger relative ERD in the training block had a stronger relative ERD in the competitive multi-user compared to the single condition . Participants with a negative ΔERD had a stronger relative ERD in the competitive multi-user condition compared to the single condition (group competitive-gain in pink); participants with a positive ΔERD had a stronger relative ERD in the single condition compared to the competitive multi-user condition (group competitive-gain in azure). The black line corresponds to the regression line of the significant regression equation with ERDtrain as the single predictor (see text for details). The grey area indicates the 95% confidence interval. Scatter plot of ΔERD and ERDsingle-gain group, relative ERD was significantly stronger for the single compared to the training condition, but not stronger for the competitive multi-user compared to the training condition. For the competitive-gain group, none of the MI NF conditions showed a stronger relative ERD compared to the training condition. We further tested whether the condition with the stronger relative ERD could be predicted by EEG and/or questionnaire data collected a priori. The only significant predictor emerging from this analysis was EEG, that is, the relative ERD magnitude in the training block. In the present study, we aimed to investigate whether social context affects MI NF ERD by comparing single and competitive multi-user conditions. Specifically, we tested whether the competitive multi-user condition leads to better performance than the single-user condition. We did not find a significant difference between the single and competitive multi-user conditions. We further explored the idea that despite the absence of an overall condition effect, participants’ MI NF ERD differs across social context, and that inter-individual differences exist regarding the direction of the difference. Participants were split into two groups based on the MI NF condition with the larger ERD gain. For the single-gain had a stronger ERD in the single than in the competitive multi-user condition, group competitive-gain showed the opposite pattern. The significantly stronger ERD in the single compared to the training condition for the group single-gain indicates that for these users, the consideration of social context of an MI NF can boost NF performance. That is, because for participants with lower performance during training, the social context significantly affects their MI ERD, those participants’ MI NF performance is likely to benefit from a single condition compared to a competitive multi-user condition. As already pointed out by Bonnet and colleagues, it is conceivable that these individuals may be distracted by the additional feedback generated by their counterparts or their counterparts in general [We did not find a significant difference in relative ERD between single and competitive multi-user conditions in this study. This is in line with results reported by Bonnet and colleagues, who did not find significant differences between single and competitive as well as collaborative multi-user conditions in classification accuracies . To expl general , or the  general . One rea general  and indi general  of MI NFNotably, the social condition related to a stronger ERD did not consistently match the self-reported preference among participants. This discrepancy between neurophysiological data and self-report is in line with findings by Bonnet and colleagues . They alcompetitive-gain. In contrast, the ERD of the weaker social condition was very comparable to the training block ERD for both groups. Future research will show whether this pattern emerged by incidence or whether it is systematic.The focus of this study was on ERD differences between single and competitive multi-user conditions. However, an interesting pattern emerged when additionally considering ERD in the training block, that is, when no NF is given. Here, on a descriptive level, both groups had less pronounced ERD in the training condition than in their stronger social condition, even though the difference was not significant for the group train) in the prediction. We found that ERDtrain predicted the condition with stronger ERD, while test scores made no significant contribution to prediction. Since collecting training data is a prerequisite for most NF setups, this means that a prediction of the social context resulting in a stronger ERD may be possible with only little additional effort. However, whether the regression equation derived from the present data set generalizes to new, online scenarios remains to be tested. Based on the descriptively stronger ERD in the stronger social condition when compared to the training condition and the similarity between the ERD in the training condition and weaker social condition, we further aimed at predicting the MI NF condition associated with a stronger ERD. We included scores indicating general control beliefs in dealing with technology (KUT) and general performance motivation (LMI-K) and based on the relative ERD obtained from the training block . The diFinally, another aspect is that the robotic NF was delivered in a delayed manner to prevent participants from being disturbed or stopping to perform MI before the end of the MI phase. As simulated in a study by Oblak and colleagues, NF performance may be related to an interplay of feedback timing (continuous/intermittent) and self-regulatory mechanisms (cognitive/automatic) , promptiSocial context constitutes a promising aspect of NF/BCI practice and may help us to enhance NF/BCI performance. Which of the two investigated social contexts generated the stronger ERD differed between individuals; thus, no significant difference between competitive multi-user condition and single condition was found. However, the social context that yielded a stronger ERD could be predicted from the training ERD. Collecting training data is a prerequisite for most NF setups. This result therefore indicates that for the optimization of future MI NF interventions—in particular, those that can operate in single and multi-user mode or, more generally, in individual and group therapy setups—it should be possible to predict the social condition best suited for NF from routinely collected EEG data. To conclude, it seems equally promising and challenging to increase MI NF performance on individual levels , e.g., b


Dear Editor,We have carefully read the Letter to the Editor by Pang and Chin related to our paper entitled “Food for bone: evidence for a role for delta-tocotrienol in the physiological control of osteoblast migration”  publishedietary tocotrienol level has not been shown to correlate with bone health, probably due to the absence of a reliable dietary questionnaire that could assess the tocotrienol intake”, since Shen and colleagues (2018) have reported that a 12 week annatto-derived tocotrienol supplementation, previously used to examine the effects of tocotrienol on bone turnover, resulted in a significant increase in serum delta-tocotrienol levels in postmenopausal osteoporotic women [The paper from Shen and colleagues 2018 [We agree with the Authors that several PI3K inhibitors are available. However, LY294002 is widely used as inhibitor of PI3k (see 172 papers in Medline from 2010 to 2020). Its strong effect in inhibiting PI3kinase action is well established . The autδ-TT treatment increased β-catenin transcriptional activity as detected by luciferase assay. Indeed, the plasmid coding for TCF-LEF-RE is activated only after the translocation and the binding of β-catenin.δ-TT treatment increased the gene expression of β-catenin target genes such as BMP2 and OC.Pretreatment with procaine, a local anesthetic drug, previously reported to inhibit the Wnt/β-catenin pathway ,15, prevAkt phosphorylation is involved in the regulation of several signaling pathways and transcriptional networks controlling osteoblast function . In our As reported by the authors, procaine in addition to an inhibitory action on the Wnt signaling pathway, exerts a DNA-methyltransferase inhibitory activity. This effect has been detected in colon cancer cells  not in pThe authors wrongly reported that we have previously shown that δ-TT increases MC3T3E1-cell proliferation. In this paper, we have shown the effect of δ-TT on cell viability by using MTT assay , which iWe have some issues regarding the points raised by the authors.As far as the use of NONIDET-P40 (is correct), it is widely considered a nonionic, nondenaturing detergent , used inConsidering the common interest with the authors in the studies regarding the effects of natural compound on bone cell activities, we hope for a future fruitful collaboration.


We isolated rat BMSCs and transfected them with sclerostin gene (SOST) overexpressing or knockdown constructs and assessed osteogenic induction in the presence or absence of ICA. Sclerostin significantly inhibited BMSC proliferation and osteogenic differentiation, whereas the presence of ICA not only increased the number of viable BMSCs but also enhanced ALP activity and formation of calcium nodules during osteogenic induction. In addition, the osteogenic genes including Runx2, β-catenin, and c-myc as well as antioxidant factors  were downregulated by sclerostin and restored by ICA treatment. Mechanistically, ICA exerted these effects by activating the Wnt/β-catenin pathway. In conclusion, ICA can promote the proliferation and osteogenic differentiation of BMSCs in situ and therefore may enhance the therapeutic efficiency of BMSC transplantation in OP.Osteoporosis (OP) is a metabolic disease characterized by decreased bone mass and increased risk of fragility fractures, which significantly reduces the quality of life. Stem cell-based therapies, especially using bone marrow mesenchymal stem cells (BMSCs), are a promising strategy for treating OP. Nevertheless, the survival and differentiation rates of the transplanted BMSCs are low, which limits their therapeutic efficiency. Icariin (ICA) is a traditional Chinese medicine formulation that is prescribed for tonifying the kidneys. It also promotes the proliferation and osteogenic differentiation of BMSCs, although the specific mechanism remains unclear. Based on our previous research, we hypothesized that ICA promotes bone formation via the sclerostin/Wnt/ Osteoporosis (OP) is a common skeletal disease characterized by reduced bone mineral density (BMD), bone microstructure deterioration, and an increased risk of fragility fractures , 2. The SOST gene that is secreted by bone cells [β-catenin pathway [SOST-/- mice show accelerated osteoblast differentiation via β-catenin activation [SOST gene may enhance osteogenic differentiation of BMSCs and promote bone renewal.Recent studies show that bone marrow mesenchymal stem cells (BMSCs) can repair bone defects in several animal models  and are ne cells  and inhi pathway . The SOStivation , which sHerba Epimedii, which is used to treat renal disorders and prevent bone loss [β-catenin signaling pathway [β-catenin signaling pathway. To this end, we isolated rat BMSCs and transfected them with SOST overexpression or knockdown constructs and treated them with ICA during osteogenic induction.Icariin  rats weighing 200–300 g were purchased from the Experimental Animal Center of Sun Yat-sen University  2016-0029). Animal experiments were performed in accordance with the guidelines of the Regulations of the People's Republic of China on the Administration of Experimental Animals and approved by the Animal Ethics Committee of the Second Hospital Affiliated with Guangzhou Medical University.β-catenin antibody was purchased from GeneTex  and the antibodies targeting GSK-β and p-GSK-β from GST . Secondary antibodies were obtained from Abcam .Dulbecco minimum essential medium (DMEM), fetal bovine serum (FBS), potassium phosphate buffer saline (PBS), and penicillin-streptomycin were purchased from Hyclone . Icariin  and dimethyl sulfoxide (DMSO) were obtained from Sigma . The alkaline phosphatase (ALP) staining kit (BCIP/NBT kit) was obtained from Beyotime Biotechnology  and the ALP activity measurement kit from Nanjing Jiancheng Biotech . Anti-2 flask and cultured at 37°C under 5% CO2 [The animals were euthanized by cervical dislocation, and the tibia and femurs were removed. The bone marrow cavities were flushed repeatedly with 10% FBS-supplemented DMEM using a sterile 10 ml syringe to prepare a single cell suspension. The cells were seeded into a 25 cmr 5% CO2 . The med6 cells/ml) were incubated with phycoerythrin-labeled anti-CD90, anti-CD44, anti-CD31, or anti-CD34 antibodies at room temperature in the dark. After washing twice with PBS at 800 rpm for 5 minutes, the cells were acquired by flow cytometry [BMSCs . The serum-free medium was replaced after 8 hours with complete medium. The cells were cultured for 48 hours, and the supernatant was collected and concentrated. The virus titer was measured, and the transduction rate was measured by PCR [The d by PCR .SOST overexpression or shRNA lentiviruses and cultured in osteogenic induction medium with or without 0.1 μM ICA. Nontransfected and normal controls were also included. After predetermined time points, osteogenic differentiation was assessed by specific tests.The BMSCs were transduced with the 4 per well, and 10 μl CCK-8 reagent  was added to each well after 24, 48, and 72 h of culture. After incubating for 2 hours, the absorbance was measured at 450 nm using a microplate reader .BMSCs were seeded into 96-well plates at the density of 1 × 10The suitable cells were fixed with 10% formaldehyde for 15 minutes, rinsed with distilled water for 30 seconds, and stained with NBT-BCIP solution  at 37°C in the dark for 15 minutes . After wThe differentiated BMSCs were transferred to a six-well plate and fixed with 2 ml 4% neutral formaldehyde solution for 30 min. After rinsing twice with PBS, 1 ml Alizarin red solution was added to each well, and the cells were stained for 3–5 min. The cells were rinsed 2–3 times and observed under a microscope.Total RNA was extracted using Trizol reagent  and quantified using a Thermo Scientific Microplate Reader . RT-qPCR was conducted using SYBR Green qPCR SuperMix  on the ABI PRISM® 7500 Sequence Detection System . The primer sequences are listed in β-catenin, anti-GSK-3β, and anti-p-GSK-3β primary antibodies at 4°C, followed by secondary antibody (1 : 2000 Southern Biotech) at room temperature for 1 hour. GAPDH  was used as the internal control [Western blotting was performed according to standard protocols . Briefly control . The post-test was used to compare different groups. P values less than 0.05 were considered statistically significant.Data are presented as mean ± standard deviation (SD). Statistical analyses were performed using SPSS version 16.0 software . One-way analysis of variance (ANOVA) or Student's The initial BMSC cultures exhibited adherent cells, colonies, and floating cells , A, and μM ICA significantly increased proliferation of the BMSCs compared to the untreated controls. Osteogenic differentiation of the BMSCs was assessed by ALP staining and activity and Alizarin red staining. ICA significantly enhanced ALP levels and activity in the BMSCs compared to the control . Furthermore, ICA activated the Wnt/β-catenin pathway in SOST-overexpressing BMSCs. Thus, ICA may promote osteogenic differentiation of BMSCs by activating the sclerostin/Wnt/β-catenin signaling pathway.The incidence of osteoporotic fractures is steadily increasing due to a globally aging population and is associated with reduced quality of life as well as significant socioeconomic burden . TherefoSOST-/- mice display accelerated bone formation, as well as increased bone mass and strength [in vitro. Sclerostin overexpression significantly inhibited the proliferation of rBMSCs and downregulated osteogenic factors, which was neutralized by ICA, indicating a novel regulatory pathway of osteogenic differentiation.Sclerostin is secreted by bone cells and inhibits differentiation of osteoblasts . SOST-/-strength . Furtherstrength . To the β-catenin pathway and mediates cell proliferation. Indo et al. [The transcription factor Runx2 is necessary for osteoblast differentiation and bone formation and is an early indicator of the same . Previouo et al.  reportedOxidative stress plays an important role in osteoporosis  by accelβ-catenin signaling pathway mediates BMSC proliferation and osteoblast differentiation. Following interaction of the Wnt glycoprotein with frizzled (FZD) and low-density lipoprotein receptor-related protein 5/6 (LRP5/6) [β (p-GSK3β), resulting in the cytosolic accumulation of β-catenin and nuclear translocation, eventually leading to the transcriptional activation of the target genes [β-catenin and p-GSK-3β, which were increased by ICA treatment. Thus, ICA promotes osteogenesis via the sclerostin/Wnt/β-catenin signaling pathway (schematic diagram shown in The Wnt/(LRP5/6) , axin iset genes . Sclerosβ-catenin pathway. It can potentially enhance the survival and differentiation of transplanted BMSCs in situ and improve the efficacy of BMSC-based regenerative therapy for OP.In summary, ICA promotes osteogenic differentiation of BMSCs by inhibiting the action of sclerostin by activating the Wnt/