More importantly, BSA DC gel displayed non-swelling properties while maintaining high strength in DI water, pH = 3.0, and pH = 10.0. Moreover, BSA DC gel also demonstrated large hysteresis, rapid self-recovery, and excellent fatigue resistance properties. It is believed that our BSA DC gel can potentially be applied in biomedical fields.Natural protein-based hydrogels possess excellent biocompatibility; however, most of them are weak or brittle. In the present work, high strength hybrid dual-crosslinking BSA gels (BSA DC gels), which have both chemical cross-linking and physical cross-linking, were fabricated by a facile photoreaction-heating process. BSA DC gels showed high transparency (light transmittance of ~90%) and high strength. At optimal conditions, BSA DC gel exhibited high compressive strength (σ Because of excellent biocompatibility, natural protein hydrogels have attracted a great deal of attention and have been used in a wide range of biological and biomedical fields  hydrogels. For example, Numata et al.  reportedBovine serum albumin (BSA) is one of the typical natural globulin proteins with a large number of functional groups, which provide many possibilities for chemical cross-linking of BSA to form covalent BSA gels. It is well-known that tyrosine groups can form dityrosine bonds via enzymatic reaction, and these play an important role in the mechanical properties of natural materials  of 37.81 ± 2.61 MPa and tensile strength  of 0.62 ± 0.078 MPa, showing it to be much stronger than physically cross-linked BSA gel  and chemically cross-linked BSA gel . More importantly, BSA DC gel displayed non-swelling properties while maintaining high performance in DI water, pH = 3.0, and pH = 10.0. Owing to the presence of physical interactions, BSA DC gel also demonstrated large hysteresis, rapid self-recovery and excellent fatigue resistance properties.Herein, high-strength hybrid dual-crosslinking BSA gels (BSA DC gels) were fabricated by covalent cross-linking of BSA via dityrosine bonds followed by heat-induced denaturation of protein to form physical cross-linkings between BSA molecules. BSA is selected in the present work because it has plenty of tyrosine groups, low cost, high water solubility, and good biocompatibility. BSA DC gels showed high transparency and high strength. Under optimal conditions, BSA DC gel exhibited high compressive strength . As shown in 2, BSA DC gel sustained its weight without bending; however, BSA PC gel and BSA CC gel were bent because of their weight. Compressive and tensile tests were conducted to evaluate the mechanical properties of BSA DC gels. From c) of 37.81 ± 2.61 MPa, which was 12 and 15 times larger than BSA PC gel (3.10 ± 0.06 MPa) and BSA CC gel (2.49 ± 0.29 MPa) (f) was 0.62 ± 0.078 MPa. In contrast, the σf values of BSA PC gel and BSA CC gel were 0.06 ± 0.008 and 0.25 ± 0.04 MPa, respectively. BSA DC gel also showed a higher tensile modulus (2939 ± 289 kPa), which was the reason that BSA DC gel could sustain its weight without bending. Statistical analysis was also performed (p < 0.01), indicating that hybrid cross-linking is an effective strategy for improving the mechanical properties of BSA gels.Owing to the hybrid cross-linking network structure, BSA DC gel possessed high strength. Three cylindrical samples (8.5 × 10 mm) of BSA DC gel could withstand 5 kg weight without breakage a1. As sh.29 MPa) , respect.29 MPa) : the tenerformed . BSA DC BSA), Ru(II) concentration [CRu(II)], exposure time, heating temperature, and heating time, on the compressive properties of BSA DC gels were also investigated, and the results are summarized in BSA, i.e., σc and Ec increased from 0.54 ± 0.03 to 34.73 ± 2.92 MPa and from 292 ± 17 to 1,320 ± 25 kPa, respectively. At CBSA=100 mg/mL, BSA DC gel was too week to test. Compared to the gel at CBSA = 300 mg/mL, the σc value of the gel at CBSA = 400 mg/mL was significantly increased from 3.22 ± 0.61 to 26.00 ± 2.10 MPa. The highest value reached was 34.73 ± 2.92 MPa at CBSA = 500 mg/mL. The presence of Ru(II) was also important for achieving high-strength BSA DC gels. Without Ru(II), σc was only 3.10 ± 0.06 MPa. However, after adding a very small amount of Ru(II) (CRu(II) = 200 μM), σc reached up to 23.45 ± 1.37 MPa. The compressive properties of BSA DC gel were the best at CRu(II) = 600 μM (σc of 34.73 ± 2.92 MPa and Ec of 1320 ± 25 kPa). Unfortunately, the compressive properties deteriorated if CRu(II) >600 μM, probably because of excessive cross-linking of dityrosine. From BSA of 500 mg/mL, CRu(II) of 600 μM, exposure time of 4 h, heating temperature of 80°C, and heating time of 10 min, which achieved σc of 37.81 ± 2.61 MPa and Ec of 1515 ± 43 kPa.The effects of various parameters, such as BSA concentration (CG′), however, G′ deceased along with shear strain as γ > 1%. In contrast, the loss modulus (G″) was perpetually increased with an increase in shear strain. Clearly, at γ ≤ 1%, G′ of BSA DC gel was 4,457 Pa, which was greater than those of BSA PC gel (2167 Pa) and BSA CC gel (627 Pa). We estimated the effective network chain density (N1) of the gels by Ge = N1RT, where Ge, R, and T are the equilibrium shear modulus, gas constant, and absolute temperature, respectively. Taking the room temperature (~25°C) as the reference temperature, the estimated N1 values were 172, 85, and 24 mol/m3 for BSA DC gel, BSA PC gel, and BSA CC gel, respectively. Distinctly, the results indicate that BSA DC gel demonstrates much higher cross-linking density compared to the other two gels, which was consistent with the morphological results in N1 for BSA DC gel increased 7.2 times, inferring that the physical cross-linkings induced by heat denaturation of BSA were dominant in BSA DC gel. In G′ and G″ increased as ω increased, indicating that they displayed viscoelasticity. In addition, G′ was always larger than G″ for the three BSA gels, implying that they exhibit elastic properties under small deformation (γ = 0.1%).Rheological tests were also conducted to evaluate the viscoelastic properties of BSA gels. As shown in c values of BSA DC gel in DI water, pH = 3.0, pH = 10.0, and 20 mM SDS solution were 35.85 ± 5.66, 23.60 ± 3.21, 30.27 ± 5.17, and 17.34 ± 2.23 MPa  and toughness recovery (based on Uhys), were defined as in our previous reports  was purchased from Aladdin (Shanghai), and Bovine serum albumin  and tris ruthenium(II) dichloride [Ru(II)] were purchased from Sigma-Aldrich Inc. All materials were used without further purification.oC for testing. BSA CC gels were synthesized via photoreaction without heating, while BSA PC gels were fabricated via heating pure BSA solution [without APS or Ru(II)] at 80°C for 10 min.BSA DC gels were prepared by a photoreaction-heating process as reported. Briefly, BSA (500 mg/mL), APS (300 mM), and Ru(II) (600 μM) were first dissolved into DI water. The precursor solution obtained was then injected into a cylindrical mold (D = 8.5 mm) and was exposed to white light (at a power of 8 W) for 4 h. The gel was then heated in a water bath (60–90°C) for 0–30 min. After cooling to room temperature, BSA DC gels were stored at 4c) was defined as F/A0, where F is the force applied to the sample and A0 is the initial cross-sectional area of the sample. The compressive strain was estimated as h/h0, where h is the height of deformed gel and h0 is the original height. The uniaxial tests of BSA DC gels (thickness = 1 mm and length = 60 mm) were pulled up at a constant rate of 100 mm/min. The tensile stress and strain were measured as F′/A0′ and l/l0, where F′ is the force applied to the sample, 0 is the original length. The compressive and tensile moduli were calculated from the slope of the linear region of the stress-strain curves. At least three independent experiments were conducted. The errors were calculated via Excel. The arithmetic mean value of the data was calculated first, and then the standard deviation (S.D.) was calculated. Statistical analysis was done by one-way analysis of variance (ANOVA) and LSD's multiple comparison post-test with a 99.9% confidence interval among the applied treatments (p < 0.01). Cyclic loading tests of gels were also performed by the abovementioned machine at 5 mm/min with maximum strain from 10 to 80%. Successive loading-unloading tests were done on the same gel specimen. For self-recovery tests, the samples were recovered at different temperatures (RT to 80°C) for various times (0–60 min). The dissipated energies were calculated via the area between loading-unloading curves. The recovery rates were defined as the rate of dissipated energy  or elastic modulus (Et) to the first loading cycle . Stiffness recovery was defined as the ratio of the elastic modulus at various times or temperatures to that of the first loading cycle, while toughness recovery was evaluated as the ratio of dissipated energies at various times or temperatures to those of the first loading cycle at a maximum compressive strain of 50%.Compressive and tensile tests were carried out with a WSM-10 kN machine (Changchun Zhineng Instrument Company). For compressive tests, the cylindrical gel samples  were conducted at 5 mm/min. The compressive stress  were firstly weighed and then immersed in different solutions at 25°C. After a predetermined time, the swollen hydrogels were taken out and weighed again. The swelling ratio (SR) was obtained from the following equation: SR = mScanning electron microscope (SEM) tests were performed using a MERLIN Compact instrument  at a voltage of 15 KV. All the gel samples were frozen and fractured in liquid nitrogen, and they were then dried in a lyophilizer. Before the tests, the samples were coated with a thin layer of gold. Fourier transform infrared spectra (FT-IR) of the BSA hydrogel powders were obtained with a Bruker Tensor 27. The BSA gel sheet was cut into a disk for rheological tests . Rheological tests were carried out on a rheometer  via the plate-plate mode. Amplitude sweep tests were carried out at ω of 10 rad/s and 25°C. Frequency sweep tests were performed at a strain of 0.1%, ω of 0.1–100 rad/s, and at 25°C. The ultraviolet spectrum of BSA DC gel was recorded on a double-beam UV-visible spectrophotometer .The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The manuscript was prepared by QC and GS, and the gels were synthesized by SL, LZ, and QW. The tests were cooperated and finished by SL, ZL, CT, and HS. The data were analyzed by JY, GQ, and GS.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


STAT3 depletion resulted in regulation of nuclear translocation of nuclear factor (NF)-κB subunits p50 and p65 and IκBα/Akt/PI3K signaling. Moreover, we found that one Src family kinase, Lyn kinase, was phosphorylated in STAT3 knockout macrophages. In addition to using pharmacological inhibition of NF-κB, we found out that STAT3KO activation of NF-κB subunit p50 and p65 and expression of iNOS was significantly inhibited; furthermore, Akt tyrosine kinase inhibitors also inhibited iNOS and COX-2 gene expression during early time points of LPS stimulation, demonstrating an NF-κB- Akt-dependent mechanism. On the other hand, iNOS expression was downregulated after prolonged treatment with LPS. Activation of NF-κB signaling was also suppressed, and consequently, nitric oxide (NO) production and cell invasion were repressed. Overall, our data indicate that STAT3 differentially regulates early- and late-phase TLR4-mediated inflammatory responses.Toll-like receptor 4 (TLR4) signaling is an important therapeutic target to manage lipopolysaccharide (LPS)-induced inflammation. The transcription factor signal transducer and activator of transcription 3 (STAT3) has been identified as an important regulator of various immune-related diseases and has generated interest as a therapeutic target. Here, we investigated the time-dependent roles of STAT3 in LPS-stimulated RAW264.7 macrophages. STAT3 inhibition induced expression of the pro-inflammatory genes  The innate immune response, including inflammatory cytokine production, immune cell recruitment, and phagocytosis by macrophages and neutrophils, plays critical role in host defense during the early stages of infection . InflammPrimary phagocytes, also known as macrophages, contribute significantly to host defense and regulation of inflammation . MacrophMycobacterium tuberculosis infection pyrimidine) and staurosporine (STS) were purchased from Merck (Germany). Fetal bovine serum (FBS) and Roswell Park Memorial Institute (RPMI) 1640 were obtained from Gibco . RAW264.7 cells, a BALB/c-derived murine macrophage cell line (ATCC No. TIB-71). Antibodies to phospho-specific and total protein of p50, p65, IκBα, IKKα/β Catalog No.#2694) ERK, JNK, Akt, STAT3, Syk, Src, Lyn, p85, β-actin, and lamin A/C were obtained from Cell Signaling .LPS (A CRISPR-CAS9 approach was used for STAT3 deletion. Guide RNA was designed according to a CRISPR designing tool (dna20.com). A STAT3-targeted sequence  was chosen. The sgRNA was cloned within a lentiCRISPRv2 vector . Transfection and puromycin (1–3 μg/mL) selection were completed using a previously established protocol . To prep2. For each experiment RAW264.7 and STAT3 knockout cells were harvested with a cell scraper.RAW264.7 and STAT3KO macrophages were cultured in RPMI 1640 supplemented with 10% heat inactivated FBS and antibiotics (penicillin and streptomycin) and were maintained at 37 °C in 5% CO6 cells/mL) cultured with or without LPS stimulation were plated on six-well plates ad were wounded by manual scratching with a 200 μL pipette tip. Subsequently, cells were washed with PBS and incubated at 37 °C in complete media. At the indicated time points, phase-contrast images at specific wound sites were obtained.Cell migration was assessed by wound-healing assays. Briefly, confluent RAW264.7 and STAT3 knockout cells  served as the chemoattractant in the bottom chamber. After incubation for 72 h at 37 °C in 5% CO2, the cells that had not penetrated the filter were completely removed with a cotton swab, and the cells that had migrated to the lower surface of the filter were fixed, stained, and counted in five randomly selected microscopic fields (100×) per filter.The ability of cells to invade through Matrigel-coated filters was determined using 24-well plates with 8-mm pore polycarbonate membranes either uncoated (for migration) or pre-coated with Matrigel Basement Membrane Matrix . RAW264.7 and STAT3 knockout cells were seeded at a density of 5 × 106 cells/mL) were seeded on 96-well plate and incubated for 18 h and then treated with LPS (1 μg/mL) for 24 h. After 24 h, 100 μL of the cell supernatant and 100 μL of Griess reagent were mixed and absorbance was measured at 540 nm using spectrophotometer.RAW264.7 and STAT3KO cells (1 × 106 cells/mL), cells were incubated with or without LPS for 24 h. The cytotoxicity of each inhibitor was evaluated using the MTT assay as previously reported [After overnight pre-incubation of RAW264.7 and STAT3 knockout cells (1 × 10reported . Three hreported  and the 6 cells/mL) were incubated with LPS (1 μg/mL) for various durations, and total RNA was isolated using TRIzol Reagent  according to the manufacturer instructions. RNA samples were then quantified, and one microgram of total RNA was reverse transcribed with M-MuLv Reverse Transcriptase  according to the manufacturer instructions. mRNA expression was than studied by real-time PCR using SYBR-premix Ex Taq according to the manufacturer instructions . Reactions were performed in real-time using a thermal cycler . Results represents the mean values obtained from three independent experiments. Full description of the primer sequence used are in RAW264.7 and STAT3 knockout (1 × 106 cells/mL) were incubated with or without LPS for 0–60 min. The cells were then harvested and washed with ice-cold PBS. The pellet was then lysed with ice-cold lysis buffer containing . Total cell lysates were centrifuged at 12,000 rpm for 5 min at 4 °C and stored at −20 °C until use. Nuclear cell lysates were prepared as previously described [2, 0.1 mM EDTA, 1 mM DTT, 0.1 mM PMSF, 2 μg/mL leupeptin, and 2 μg/mL aprotinin). 25 μL of 10% NP-40 was added to the lysates, and the lysates were vigorously vortexed. The lysates were then subjected to centrifugation at 14,000 rpm for 0.5 min at 4 °C, and the supernatant was removed. Pellets were repeatedly washed with 400 μL of lysis buffer and then 50 μL of extraction buffer . The solution was stored at 4 °C for 20 min and centrifuged at 14,000 rpm at 4 °C for 5 min. The supernatant was transferred to fresh tubes and stored at −80 °C until required for testing.RAW264.7 and STAT3 knockout cells  using running buffer . The proteins were transferred by electroblotting to a polyvinylidene difuoride membrane using transfer buffer . Membranes were blocked using 3% bovine serum albumin (BSA) in TBST at room temperature for 1 h, followed by incubation with specific primary antibodies for 1 h at room temperature or overnight at 4 °C. After primary antibody incubation, the membranes were washed with 0.1% TBST  three times for 10 min each, incubated with HRP-linked secondary antibodies with 3% BSA for 1 h at room temperature, and washed with 0.1% TBST three times for 10 min each. Phosphorylated and total forms of p65, p50, Src, p85, AKT, IKKα/β, IκBα, Lyn, p85, β-actin, iNOS, COX-2, and lamin A/C were visualized using an ECL reagent . For calp values of less than 0.05 were considered statistically significant.Data are presented as mean ± standard deviation calculated from three replicates. Comparisons between multiple time points were analyzed by using an analysis of variance and Scheffé’s post-hoc test, as well as the Mann–Whitney test, and significance was analyzed by the log-rank test. In all the tests,


Among the various Mn1-xNixCO3 electrodes, the Mn0.75Ni0.25CO3 electrode exhibited the higher specific capacitance (364 F g−1 at 1 A g−1) with capacity retention of 96% after 7500 cycles at 5 A g−1. Moreover, the assembled solid-state asymmetric supercapacitor based on Mn0.75Ni0.25CO3//graphene nanosheets performed a high specific capacity of 46 F g−1 and energy density of 25 Wh kg−1 at a power density of 499 W kg−1 along with high capacity retention of 87.7% after 7500 cycles. The improved electrochemical performances are mainly owing to the intrinsic conductivity and electrochemical activity of MnCO3 after Mn1-xNixCO3  with appropriate Ni concentration. This study highlights the potentiality of the Mn0.75Ni0.25CO3//GNS asymmetric supercapacitor device for promising energy storage applications.Long–term cycling performance of electrodes for application in supercapcitor has received large research interest in recent years. Ultra-stable Mn Interest on the development of low cost, high energy density and power density electrochemical energy storage systems compatible with nuclear, solar and wind energy resources are growing tremendously particularly on supercapacitors and batteries3, former one satisfy all the requirements. Whereas, conventional capacitors deliver high power, but lower energy density which instigates the search for long-term cyclic stability, operating safety, environmental friendly, low cost and can deliver high power and energy density supercapacitors7. The prominent features of supercapacitors are long-term cyclic stability, operating safety, environment friendly, and can deliver high power density. The supercapacitors can be classified in to two types such as; (i) EDLC  and (ii) pseudocapacitors which is differentiated by their charge-storage mechanism9. In EDLC charge is stored by rapid adsorption/ desorption at the electrode/electrolyte interface, there is no redox reaction occurs in the electrode materials but the specific capacitance is very lower which depends on the electrode active surface area, whereas the surface of the electrode materials will undergo a reversible redox (Faradaic) reaction in the pseudocapacitors during the charge – discharge processes particularly in the metal oxide or conducting polymer electrode materials. EDLC offers lower capacitance which necessitate to identify a suitable pseudocapacitor materials  that can able to store more charges for longer period without noticeable energy loss11.In the 212. XH2O is a well-known pseudocapacitor with good performance and have high specific capacitance (760 F g−1) with enhanced cycle-life. However, the low porosity, toxicity, less abundance of its raw materials and rapid decrease in power density at high charge- discharge current limit, restrict its glassine applications13. A large number of electrochemical studies are focused in finding a low-cost metal oxide as a replacement for RuO2. Recently, enormous effort has been focused on transition metal compounds containing nickel, cobalt, manganese with different morphologies. Wang and his co-workers synthesized interlinked multiphase Fe-doped MnO2 nanostructures with electrochemical properties14. Chen et al. have synthesized NiCo2O4@NiWO3 nanowire arrays that can serves as an electroactive material for super capacitors and it delivers a high capacity retention and long-term cycle stability15. Zhang et al. fabricated 3D Co3O4–Ni3(VO4)2 heterostructured nanorods on nickel foam possessing improved electrochemical properties for supercapacitor electrodes and exhibits good comprehensive electrochemical performance16. These transitional electrode materials with high specific surface area have been widely used as pseudocapacitor applications19. In general, aforementioned reports illustrate the use of transition metal oxides, sulphides and hydroxides as the supercapacitors electrode materials, only a few electrodes with metal carbonates are reported26. However, to the best of our knowledge, first time we have reported the synthesis of Mn0.75Ni0.25CO3 nano/sub-microspheres and applied as electrode materials for solid-state asymmetric supercapacitor (ASC) applications, since it is environment friendly, and can be easily fabricated with capacitance performance.The transition metal oxides have received great attention towards pseudocapacitor materials owing to their variable oxidation states and ease of preparation. Among the various metal oxides, RuO1-xNixCO3  nano/sub-microspheres. The crystalline structure, functional groups and morphology of the as-prepared materials were characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and transmission electron microscopy (TEM) studies. The electrochemical studies show that the Mn0.75Ni0.25CO3 nano/sub-microspheres exhibits a higher specific capacitance of 364 F g−1 at a current density of 1 A g−1, high rate capability and superior cyclic stability. In addition, the solid-state ASC with Mn0.75Ni0.25CO3//GNS configuration was fabricated device using Mn0.75Ni0.25CO3 nano/sub-microspheres and graphene nanosheets (GNS) as positive and negative electrodes, respectively. The solid-state ASC Mn0.75Ni0.25CO3//GNS device exhibits a high energy density of 25 Wh kg−1 with a power density of 499 W kg−1. The fabricated Mn0.75Ni0.25CO3//GNS configuration is a potential system for commercial applications.In this work, we have adopted a facile template-free co-precipitation method assisted with sodium bicarbonate to synthesize the shape-controlled Mn1-xNixCO3  nano/sub-microspheres. As can be seen from the spectra, the MnCO3, Mn0.80Ni0.20Co3, Mn0.75Ni0.25CO3 and Mn0.70Ni0.30CO3 nano/sub-microspheres samples show the diffraction peaks of (012), (104), (110), (113), (202), (018), (116), (122) and (300) planes which are corresponding to the reflection located at 2Ɵ values of 24.25, 31.36, 37.52, 41.42, 45.18, 51.48, 51.68, 59.18 and 67.70 respectively27. All the diffraction peaks are related to the pure rhombohedral phase of MnCO3 structure with the space group of R3-c (JCPDS card No. 44-1472) and the peak sharpness and broadness show the highly crystalline and nano/sub-micron nature of the particles. The MnCO3, Mn0.80Ni0.20Co3, Mn0.75Ni0.25CO3 and Mn0.70Ni0.30CO3 nano/sub-microspheres samples does not shown any new peak associated with other phases such as Mn(OH)2, MnNi(OH)2, Ni(OH)2, NiCO3 or metallic Mn and Ni based phases in the samples. As the Ni content increases, the peak intensity decreases due to the generation of charge imbalance arises by the Ni ratio. The average crystallite size of MnCO3, Mn0.80Ni0.20Co3, Mn0.75Ni0.25CO3 and Mn0.70Ni0.30CO3 nano/sub-microspheres samples was calculated by using the Debye-Scherere’s equation which are 70 nm 65 nm 50 nm and 43 nm respectively28. The data shows that the presence of Ni ions in Mn0.80Ni0.20Co3, Mn0.75Ni0.25CO3 and Mn0.70Ni0.30CO3 prevented the growth of crystal grains and slows down the motion of a grain boundary due to the interruption on a movement of the grain boundaries by Zener pinning29. The smaller crystallite size and phase pure of the Mn0.75Ni0.25CO3 nano/sub-microspheres are expected to offer high electron transport at electrode/electrolyte interface for high power applications.The X-ray diffraction (XRD) analysis was employed for the crystallite and phase structural characterization of the samples. Figure 3, Mn0.80Ni0.20Co3, Mn0.75Ni0.25CO3 and Mn0.70Ni0.30CO3 nano/sub-microspheres samples were characterized by the FT-IR spectroscopy in the range of 4000–450 cm−1 and the respective spectra are depicted in Figure 3 nano/sub-microspheres still contain water molecules since OH and CO2 molecules have the property of chemisorption on to the MnCO3 surface when they are exposed to the atmospheres. The Mn1-xNixCO3 samples displayed a broad peak at ~3450 cm−1, which is attributed to the O-H stretching vibration and a band at ~1623 represents the bending vibration of water molecules present in the Mn1-xNixCO3 nano/sub-microspheres. These two vibrational modes show the residual water and hydroxyl groups on the nano/sub-microspheres. The broadness of the peak at 3458 cm−1 is decreased slowly on Ni indicating the water molecules adsorption diminished. The band in the range of 450–750 cm−1 is attributed to the MnCO3 rhombohedral sites; MnCO3 stretching vibration peak appeared at ~530 is the characteristic peak for the Mn-C-O (MnCO-Mn2+ with CO32−) and the other peaks at 1026 cm−1 is attributed to the C-O stretching vibration of CO32− ion and the 2353 cm−1 peak is related to carbon dioxide31. The intensity of characteristic peak for the Mn-C-O appeared at ~530 cm−1 is increasing on the addition of 20% Ni, then on the 25% addition, the peak stabilized and the other peak at 414 cm−1 is decreasing and increasing by the consequent additions, these characteristics are the indication of the of the formation of Mn0.75Ni0.25CO3 homogenously. These FT-IR spectra as well as the XRD patterns indicate that the framework and Ni does not interfere with the MnCO3 structure.The Fourier transform infrared spectroscopy (FT-IR) is usually employed as an additional probe to find the organic and inorganic functional species present in the samples. The as-prepare MnCO3, Mn0.80Ni0.20Co3, Mn0.75Ni0.25CO3 and Mn0.70Ni0.30CO3 nano/sub-microspheres were investigated by scanning electron microscopy presented in Figure 3, Mn0.80Ni0.20Co3, Mn0.75Ni0.25CO3 and Mn0.70Ni0.30CO3 nano/sub-microspheres are highly uniform and homogenously distributed with an average diameter size of 430 to 470 nm. The formation of nano/sub-microspheres morphology is possible in the co-precipitation method as it takes place in the carbonate medium (digestion of carbonate solution with a base and the precipitation process simultaneously). Under these circumstances, the carbonate solution controls the particles agglomeration and leads to the formation of thermodynamically stable sphere shape of MnCO3, Mn0.80Ni0.20Co3, Mn0.75Ni0.25CO3 and Mn0.70Ni0.30CO3 nano/sub-microspheres. The smaller Ni2+ ion (70 pm) with the bigger (81 pm) Mn2+ ion lattice, contracts the crystals, thus smaller crystallites are formed XRD, FT-IR and SEM results are corroborated one another. Surface roughness is also increases with increasing the Ni2+ level, which attracts more electrolyte and expected higher capacitance and faster exchange of e- on its surface.The morphology of the as-prepared MnCO3 and Mn0.75Ni0.25CO3 nano/sub-microspheres at different magnifications are shown in Figure 3) and 450 nm (Mn0.75Ni0.25CO3). Figure 3 and Mn0.75Ni0.25CO3 nano/sub-microspheres, demonstrate that the lattice distance is 7.4 Å (MnCO3) and 7.2 Å (Mn0.75Ni0.25CO3) agrees well with (104) planes and the inset images of both are the MnCO3 and Mn0.75Ni0.25CO3 well well-defined spots arranged in circular rings confirms the poly crystalline formations. Both the nanosphere surface possesses pores, which can offer an effective electron and ion transport consequently supports for the improved electrochemical performance for the supercapacitor applications.The TEM micrographs for MnCO0.75Ni0.25CO3 was determined using X-ray photoelectron spectroscopy (XPS) technique shown in Figure 3/2 and Mn 2p1/2 respectively, which coincide with the Mn2+ state of Mn in the Mn0.75Ni0.25CO3 samples33. The convolution of Ni 2p peaks at binding energy positions of 855.2 eV and 873.1 eV attributed to the Ni 2p3/2 and Ni 2p1/2 respectively and other two satellite (shake-up process) peaks appeared at 862.5 eV and 880.2 eV are correspond to the Ni 2p3/2 and Ni 2p1/2 respectively, as shown in Figure 2+ state in the Mn0.75Ni0.25CO3 samples35. The high resolution spectra of C 1 s was convolute into three binding energy peaks and shown in Figure 38. The O 1 s spectra , i is the constant current (A), ∆t is the discharge time (s), m is the total mass of the active material, and ∆v is the potential window (V)39. In our charge - discharge studies, the specific capacitance contribution from the nickel foam current collector was ignored. The specific capacity value of MnCO3, Mn0.80Ni0.20CO3, Mn0.75Ni0.25CO3 and Mn0.70Ni0.30CO3 nano/sub-microspheres electrodes is 280, 295, 364 and 315 F g−1 respectively at 1 A g−1. Higher specific capacitance value was obtained for Mn0.75Ni0.25CO3 electrode owing to its higher surface area and stabilized structure. Whereas, at 30% Ni, disintegration (Mn0.70Ni0.30CO3) of crystal structure reduces the capacitance. The specific capacitance of Mn1-xNixCO3  nano/sub-microspheres electrodes with different current densities are presented in the Figure 0.75Ni0.25CO3 exhibits higher specific capacitance than the other fabricated electrodes in this work and also with Mn0.75Ni0.25CO3 previously reported values  measurements are further evaluated in the potential window range between 0.0 V and 1.0 V to assess the performance of various nano/sub-microspheres electrodes at 1 A g1 Figure . The sup1 Figure . The cha0.75Ni0.25CO3 nano/sub-microspheres electrode  is higher than that of pristine MnCO3 (280 F g−1 at 1 A g−1) in 1 M of Na2SO4 electrolyte. The specific capacity retention of the Mn0.75Ni0.25CO3 is slowly decreased (280 F g−1) by increasing the current density and retained about 78% at 9 A g−1 current density. Whereas, the MnCO3 nano/sub-microspheres electrode exhibits only 66% specific capacitance retention of at 9 A g−1. The specific capacitance of Mn based metal oxides electrodes are shown in Table 0.75Ni0.25CO3 nano/sub-microspheres.The specific capacitance of the Mn0.75Ni0.25CO3 nano/sub-microspheres electrode was measured over 7500 continuous charge-discharge cycles at a specific current of 5 A g−1 in 1 M of Na2SO4 electrolyte  of the MnCO3, Mn0.80Ni0.20CO3, Mn0.75Ni0.25CO3 and Mn0.70Ni0.30CO3 nano/sub-microspheres electrodes is 17.59, 6.45, 5.51 and 9.81 ohm, respectively. Evidently, the lower resistance value is obtained for Mn0.75Ni0.25CO3 nano/sub-microspheres electrode, which result better electrochemical performance. The fast transfer of charged species between the electrode and electrolyte is confirmed by the lower Rct values of the Mn0.75Ni0.25CO3 nano/sub-microspheres electrode, which could lead to good electrochemical performance. The synergism between Ni and Mn give rise to good specific capacitance and cycle life40.The electrochemical impedance spectra (EIS) of the MnCO0.75Ni0.25CO3 nano/sub-microspheres, we assembled solid-state ASC device using Mn0.75Ni0.25CO3 nano/sub-microspheres as the positive electrode, graphene nanosheets as the negative electrode and PVA- Na2SO4 as the solid-state electrolyte , specific capacitance (F g−1), discharge potential (V), power density (kW kg−1) and discharge time (s), respectively41. The energy density of the fabricated ASC decreases from 25 to 12.22 W h kg−1 as the power density increases from 499 to 15840 W kg−1 (The ASC device of Mn0.75Ni0.25CO3//GNS can be used to light the red-light emitting diode (LED) after being charged . These results reveal that our Mn0.75Ni0.25CO3//GNS solid-state ASC device possess a great potential for practical applications. The high energy and power density of solid-state ASC applications electrode materials are due to the uniform nano/sub-microspheres morphology as cathode and more surface area of GNS as an anode material that lead to more accessible to smaller electrolyte ions and favourable for the fast diffusion of the electrolyte ions at the surface of electrode materials. Meanwhile, the solid-state ASC electrode materials prevent the loss of capacity retention during the repetitive incorporation/extraction process. The highlighted solid-state ASC device (Mn0.75Ni0.25CO3//GNS) is novel, cost effective, easy to fabricate and possesses an excellent potential for energy and power density devices.The galvanostatic charge-discharge (GCD) curves at different current densities are shown in Figure y Figure . Further1 Figure , testifyt Figure , these t1-xNixCO3  nano/sub-microspheres via facile co-precipitation method using sodium bicarbonate as the precipitating agent for the first time. The microscopic and X-ray diffraction results reveal that the synthesized rhombohedral Mn1-xNixCO3 particles are homogeneously dispersed crystalline nano/sub-microspheres morphologies with 430–470 nm size. Galvanostatic charge-discharge result showed high specific capacity of 364 F g−1 offered by Mn0.75Ni0.25CO3 nano/sub-microspheres electrode which is 23.07% higher capacity than the capacity offered by MnCO3 in 1 M of Na2SO4 electrolyte. The Mn0.75Ni0.25CO3 nano/sub-microspheres electrode is to be one of the promising electrodes for long-term cycle stability as it exhibited stabilized performance at 5 A g−1 for 7500 cycles. Further, the as-fabricated Mn0.75Ni0.25CO3//GNS device displayed a power density of 499 W kg−1 at high specific energy density of 25 Wh kg−1, as well as the solid-state ASC capacitance retention of 87.7% was delivered even after 7500 cycles, which is an additional benefit, and suitable for the commercial applications. The Mn0.75Ni0.25CO3 nano/sub-microspheres materials further provides a new pathway for the new Li-ion anode, sensor and photocatalyst applications.In conclusion, we have reported the facile template-free synthesis of Mn4.6H2O), Manganese sulphate tetrahydrate (MnSO4.4H2O), sodium bicarbonate (NaHCO3), Sodium sulfate (Na2SO4), Acetylene black, Polyvinylidene fluoride and N-Methyl-2-Pyrrolidone were procured from Sigma-Aldrich, India. Ethanol was purchased from SRL Pvt. Ltd, India. All the purchased chemicals and regents were in analytical grade and used as received without any further purifications.Nickel sulphate hexahydrate  and nickel sulphate (0.25 mmol) were dissolved in double distiller water (70 mL) and sodium bicarbonate (10 mmol) was dissolved separately in double distiller water (70 mL). A 7 mL of ethanol was then added to the MnSO4 and NiSO4 solutions with constant stirring. After its complete dispersion, the NaHCO3 solution was added to the above mixture at room temperature. After 5 min, the reaction solution was turned to green colour. This indicated that the initial formation of Mn0.75Ni0.25CO3 nano/sub-microspheres and mixture was to continuously stirred for 3 hours at room temperature to form Mn0.75Ni0.25CO3 nano/sub-microspheres. The Mn0.75Ni0.25CO3 nano/sub-microspheres formed were separated by filtration and washed several times with ultrapure water and ethanol to remove impurities. Finally, Mn0.75Ni0.25CO3 nano/sub-microspheres were dried at 120 °C for 12 h to remove the adsorbed water molecules on the surface of the nano/sub-microspheres. The same procedure was followed for the synthesis of Mn1-xNixCO3  nano/sub-microspheres.Mn1-xNixCO3 nano/sub-microspheres were examined using X-ray diffraction (XRD) measurements using a PAN Analytical X’ per PRO Model X-ray diffractometer with Cu Kα radiation (α = 1.5418 Å) from 10–80 °, Fourier transform infrared (FT-IR) spectral analyses were performed using a Nicolet Avater 370 with KBr pellet technique from 450 to 4000 cm−1. The morphology and surface nature of Mn1-xNixCO3 nano/sub-microspheres were characterized using scanning electron microscopy using JEOL-JSM and transmission electron microscopy (TEM) taken on a JEOL/JEM 2100. X-ray photoelectron spectroscopy (Carl Zeiss) was carried out in ultra-high vacuum with Al kα line (1486.6 eV) as an exciting source to analyse the surface chemistry and valence state of Mn0.75Ni0.25CO3 sample20.The structural, functional and morphologies of Mn1-xNixCO3  nano/sub-microspheres electrodes the cyclic voltammetry (CV), galvanostatic charge-discharge (GCD) and electrochemical impedance spectroscopy studies with three electrode system used. The working electrode was prepared by mixing the synthesized Mn1-xNixCO3 nano/sub-microspheres materials , conductive materials (acetylene black-20%), binder (polyvinylidene fluoride (PVDF-10%)) and a few drops of n-methyl-2-pyrrolidone (NMP) was used as the solvent. The active material (1.5 mg) was coated on a nickel foam substrate (1 cm ×1 cm) and was dried at 90 °C for 10 h. This served as the working electrode, saturated calomel (SCE) and a platinum wire was used as the reference and counter electrode, respectively. The electrode evaluated in 1 M of Na2SO4 aqueous solutions electrolyte at different scan rate and current densities.To evaluate the electrochemical properties of the synthesized Mn0.75Ni0.25CO3 nano/sub-microspheres were used as the positive electrode (1.2 mg) and the graphene nanosheet (GNS) (3.87 mg) was used as the negative electrode, which was prepared by pasting of 20% acetylene black and 10% of PVDF in NMP slurry on a nickel foam. The solid-state electrolyte was prepared by the addition of 2 g PVA powder into 20 ml of deionized water under vigorous stirring at 95 °C, a clear solution was obtained. Later, 1 g of Na2SO4 was added to the above clear solution under stirring for 30 min to form PVA- Na2SO4 solid -state gel. The positive (Mn0.75Ni0.25CO3 nano/sub-microspheres) and negative (GNS) electrode and separator were dipped into the PVA- Na2SO4 solid-state solution for 5 min, taken out, assembled and kept under vacuum desicater to form solid-state ASC. For the optimal mass ratio between the positive and negative electrodes (m+/m) was obtained from the maximum capacitance and operating potential window, which was calculated by using the following charge balance equation42,m is the mass, c is the specific capacitance and v is the operating voltage range. The electrochemical performances in both configurations such as in three electrode and in the two electrode systems were carried out using the CHI 660D electrochemical work station.The constructed solid-state ASC device was measured with a two-electrode system, including two slices of nickel foam (2 ×1 cm) as current collectors and a cellulosic paper as a separator. In the two-electrode system, the MnSupplementary Information.


There is an increasing application of moderately hypofractionated radiotherapy for prostate cancer. We presented our outcomes and treatment-related toxicities with moderately hypofractionated (67.5 Gy in 25 fractions) radiotherapy for a group of advanced prostate cancer patients from China.From November 2006 to December 2018, 246 consecutive patients with prostate cancer confined to the pelvis were treated with moderately hypofractionated radiotherapy (67.5 Gy in 25 fractions). 97.6% of the patients received a different duration of androgen deprivation therapy. Failure-free survival (FFS), prostate cancer-specific survival (PCSS), overall survival (OS), and cumulative grade ≥ 2 late toxicity were evaluated using the Kaplan–Meier actuarial method. Prognostic factors for FFS, PCSS, and OS were analyzed.P = 0.003). Overall, 5- and 10-year PCSS rates were 95.7% (95% CI 93.0–98.5%) and 88.2% (95% CI 82.8–93.8%). Prostate cancer-specific mortality rates for the high-risk, locally advanced, and N1 groups were 4.0%, 8.2%, and 23.8%, respectively (P < 0.001). Overall, 5- and 10-year actuarial OS rates were 92.4% (95% CI 88.8–96.1%) and 72.7% (95% CI 64.8–81.5%). High level prostate-specific antigen and positive N stage were significantly associated with worse FFS (P < 0.05). Advanced T stage and positive N stage emerged as worse predictors of PCSS (P < 0.05). Advanced age, T stage, and positive N stage were the only factors that were significantly associated with worse OS (P < 0.05). The 5-year cumulative incidence rate of grade ≥ 2 late GU and GI toxicity was 17.8% (95% CI 12.5–22.7%) and 23.4% (95% CI 17.7–28.7%), respectively.The median follow-up time was 74 months (range: 6–150 months). For all patients, the 5- and 10-year FFS rates were 80.0% (95% CI: 74.7–85.7%) and 63.5% (95% CI 55.4–72.8%). The failure rates for the intermediate, high-risk, locally advanced, and N1 groups were 6.1%, 13.0%, 18.4%, and 35.7%, respectively (Moderately hypofractionated radiotherapy (67.5 Gy in 25 fractions) for this predominantly high-risk, locally advanced, or N1 in Chinese patients demonstrates encouraging long-term outcomes and acceptable toxicity. This fractionation schedule deserves further evaluation in similar populations. With a rapidly aging population and changing lifestyles, the incidence of prostate cancer in China has increased from 4.0 per 100,000 to 20.0 per 100,000 between 1990 and 2017 . HoweverIn the last decade, by using of contemporary high precision treatment delivery techniques such as image-guided three-dimensional conformal radiotherapy, intensive-modulated radiotherapy (IMRT), and proton therapy, the irradiation dose of target volume has been increased, the local control rate, and disease-free survival rate have been improved, while the normal surrounding tissue sparing has been enhanced –6. EvideHowever, there are less data on long-term outcomes for patients treated with hypofractionated radiotherapy in China, especially for a relatively higher risk Chinese cohort. We previously reported preliminary results using moderately hypofractionated IMRT (67.5 Gy delivered at 2.7 Gy/fraction) for pelvic-confined prostate cancer from our center . This tiFrom November 2006 to December 2018, 246 consecutive patients with prostate cancer confined to the pelvis were treated with moderately hypofractionated radiotherapy (67.5 Gy in 25 fractions) at our institution. Patients with biopsy-proven adenocarcinoma of the prostate, stage T1-4N0-1M0, according to the American Joint Committee on Cancer staging system, with any Gleason score (GS), any PSA level, and with a World Health Organization performance status of 0–1 were selected for this study. Pre-treatment evaluation consisted of a complete medical history, digital rectal examination, bone scan, chest radiograph, computed tomography (CT) of the abdomen and pelvis, magnetic resonance imaging (MRI) of the prostate and blood work including serum PSA and testosterone, and liver and renal function tests. Risk stratification was performed using modified National Comprehensive Cancer Network (NCCN) 2017 criteria.All patients were instructed to empty the rectum, fill the bladder, and drink 1000 ml of water 1 h before the CT simulation and each treatment fraction. Patients were immobilized in the supine position using thermoplastic pelvic fixation. CT scans were performed from the superior border of the fourth vertebra to 5 cm inferior to the ischial tuberosities, with 3/5 mm slice thickness and spacing. To facilitate clinical target volume (CTV) and critical normal structures delineation, intravenous and oral contrast were administered. The CT images were transferred to a Pinnacle planning system  for contouring and treatment planning.The CTV comprised the prostate alone for low-risk patients and the prostate and proximal 1.5–2.0 cm seminal vesicles for intermediate-risk patients. In high-risk and locally-advanced (T3b-T4) patients, CTV1 included the prostate and proximal 2.0–2.5 cm seminal vesicles, and CTV2 included the pelvic lymph nodes if its involvement probability ≥ 30% (estimated from Roach’s equation: LN% = (2/3) PSA + (GS − 6) × 10) . For N1 The prescribed dose was 67.5 Gy in 25 fractions for PTV/PTV1, which was equivalent to a total dose of 81 Gy in 2 Gy/fraction, using an α/β ratio of 1.5 , 20. TheADT was administered at the discretion of the treating physician and consisted of surgical castration (n = 12) or luteinizing hormone-releasing hormone agonists combined with antiandrogens. Low-risk patients received no hormone therapy; intermediate-risk patients received 2–3 months of neoadjuvant hormone therapy, followed by concurrent then adjuvant therapy totaling 4–6 months ; high-risk, locally advanced, and N1 patients were given 4–6 months hormone therapy neoadjuvantly and continued for up to 24–36 months. The median duration of androgen suppression for the intermediate-risk group was 10 months (range: 4–142 months), and 36 months (range: 1–154 months) for high-risk, locally advanced, and N1 patients.Patients were evaluated at regular intervals after treatment . The follow-up evaluation included patient symptom assessment at each visit, physical examination, serum PSA measurements, and imaging studies when necessary. During each patient interview, gastrointestinal (GI) and genitourinary (GU) toxicity were assessed and graded according to Common Terminology Criteria for Adverse Events, version 4. Late toxicity was defined as any toxicity documented 3 months after the end of radiotherapy. The cumulative worst-grade toxicity was documented for each patient.All patients who were unable to follow-up were censored at their last follow-up visit. Biochemical failure was defined according to the Radiation Therapy Oncology Group and American Society for Radiation Oncology’s Phoenix definition as nadir plus 2.0 ng/ml . ClinicaP < 0.1 in univariate analysis were included in multivariate analysis. A P value of < 0.05 was considered statistically significant. In all statistical analysis, the P values were two-sided. Statistical analyses were performed using R version 3.6.1.FFS, prostate cancer-specific survival (PCSS), overall survival (OS), and incidence probabilities of cumulative grade ≥ 2 late toxicity were evaluated by the Kaplan–Meier actuarial method. Univariate and multivariate Cox-regression analyses were done to assess the prognostic factors for FFS, PCSS and OS, including age at diagnosis, pre-ADT PSA level, biopsy GS, clinical T-stage, and clinical N-stage. In the univariate analysis, the difference between groups was compared by using the log-rank test. The variables with Baseline characteristics of patient and treatment are shown in Table P = 0.003) . 19 patients (7.7%) had biochemical failures alone, 21 patients (8.5%) had biochemically detected and clinically radiographic evidence of distant failure, and 13 patients had bone metastases. No patient developed a clinical in-field relapse. The median time to failure was 33.5 months (range: 3–96 months). For the entire cohort, the 5- and 10-year FFS rates were 80.0% (95% CI 74.7–85.7%) and 63.5% (95% CI 55.4–72.8%)   had died, and 18 patients (7.3%) had died from prostate cancer. The median time to death was 70 months (range: 10–113 months). The 5- and 10-year PCSS rates were 95.7% (95% CI 93.0–98.5%) and 88.2% (95% CI 82.8–93.8%) . Advanced T-stage, positive N-stage, and higher biopsy GS were associated with lower PCSS in univariate analysis . Older age and positive N-stage were associated with lower OS in univariate analysis .In univariate analysis, early T-stage, negative N-stage, and decreasing baseline PSA were significant predictors of prolonged FFS  and positive N-stage were significantly associated with worse FFS. Advanced T-stage (T4 vs T1–2) and positive N-stage emerged as significant predictors of worse PCSS. Finally, advanced age, T-stage (T4 vs T1–2), and positive N-stage were the only factors that were significantly associated with worse OS (Table P = 0.51).Toxicity data were available for all patients. Late GU and GI toxicities are summarized in Table P = 0.0056).With respect to GI toxicity, 54 patients experienced grade ≥ 2 late GI toxicity, commonly hematochezia, frequent defecation, and fecal incontinence, with a 5-year cumulative incidence rate of 23.4% (95% CI 17.7–28.7%)  Chinese prostate cancer patients with predominantly high-risk, locally advanced, and N1 diseases; (2) similar treatment strategy; (3) mature follow-up.Given the excellent therapeutic outcomes, both in terms of disease control and incidence rates of toxicity, the adoption of alternative modern, dose-escalated fractionation regimens necessitates careful clinical validation prior to widespread implementation. This is particularly important for those with high-grade disease as it has been hypothesized that the α/β ratio for such disease may be higher than that for low-grade prostate cancer . HoweverThere are several phase III randomized trials comparing standard versus moderate hypofractionated radiation therapy using escalated doses. Some researchers recommended hypofractionated radiotherapy as a new standard of care for localized prostate cancer, while others could not confirm that hypofractionation was non-inferior for cumulative late toxicity compared with standard fractionation , 26, 27.The HYPRO trial enrolled 804 intermediate- or high-risk patients, and randomly assigned them to receive either standard fractionation with 39 fractions of 2 Gy  or hypofractionation with 19 fractions of 3.4 Gy  , 27. 67%P = 0.745). The overall incidences of grade ≥ 2 late GU and GI reactions for hypofractionated IMRT were 44.9% and 18.1%, respectively. There were no statistically significant differences in late toxicity between the two arms. Recently, Abu-Gheida et al. [Our finding is more consistent with those reported from the same fraction dose of 2.7 Gy prostate radiotherapy combined with ADT. Pollack et al.  conducteIn our study, 77.7% of the patients enrolled were high-risk or more advanced. Nevertheless, this fractionation schedule (67.5 Gy in 25 fractions) still achieved promising results, with the 5- and 10-year FFS of 80.0% and 63.5%, and the 5- and 10-year PCSS rates of 95.7% and 88.2%. Also, there were no clinical in-field recurrence cases in our group, and the clinical failure presented as distant metastasis, indicating that this dose segmentation scheme was reasonable for the control of local prostate lesions, although our total dose was lower than that reported by Pollack et al. . In thisCompared with the above reports, severe late GU and GI toxicity events in our study were also low, and eased over time in line with the results from other studies , 28, 29.18F-Choline-PET/CT was a promising diagnostic tool in the definition of clinical stage and decision-making strategy of treatment volumes when integrated with conventional staging imaging [18F-Choline-PET/CT was not available in our hospital. Furthermore, toxicity outcomes were physician reported rather than patient reported. In addition, modern daily image-guided delivery techniques were selectively used in our patients. Finally, the median follow-up time was relatively short, and the sample size was relatively small for prostate cancer.There are several limitations to our study. This is a single-institution, single-arm, retrospective study involving a Chinese population, ranging from low risk to N1 disease. As a result, target volume and ADT duration varied greatly. Secondly, N1 disease was detected using imaging (CT and/or MRI) and was not histologically confirmed.  imaging . But 18FModerately hypofractionated radiotherapy (67.5 Gy in 25 fractions) for this predominantly high-risk, locally advanced, or N1 Chinese patients demonstrates encouraging long-term outcomes and an acceptable incidence of toxicity. This fractionation schedule deserves further evaluation in similar populations.


Brilliant cresyl blue (BCB) staining is used to select developmentally competent cumulus-oocyte complexes (COCs) for in vitro maturation (IVM). However, limited attention has been paid to what drives the higher developmental competence of BCB+ COCs. Sonic hedgehog signaling (SHH) is an important signaling pathway for ovarian follicular development and oocyte maturation. Therefore, this study investigated the effect of oocyte quality assessed by BCB staining on cumulus cell expansion, oocyte nuclear maturation, subsequent embryo development, apoptosis levels, and SHH signaling protein expression, in porcine COCs. After IVM, BCB+ COCs exhibited a significantly higher proportion of complete cumulus cell expansion and metaphase II rate in oocytes than BCB- COCs. After in vitro fertilization, the BCB+ group showed a significantly higher monospermy rate, fertilization efficiency, percentage of cleavage and blastocyst formation, with a higher total cell number and a lower apoptosis in blastocysts as compared with the BCB- group. Furthermore, significantly lower apoptosis levels and a higher expression of SHH-signaling proteins in COCs were observed, before and after IVM. In conclusion, high-quality oocytes had a greater potential to expand their surrounding cumulus cells with active SHH signaling and a lower apoptosis. This could provide COCs with a proper environment for maturation, thereby leading to a better subsequent embryo development. The in vitro maturation (IVM) of immature oocytes is an important assisted reproductive technology (ART) capable of generating mature oocytes that can develop to term . DespiteBrilliant cresyl blue (BCB) has been used to select more competent oocytes prior to IVM in various species, including pigs, mice, goats, cattle, and buffaloes . In 1993Cumulus cell expansion is an essential condition for oocyte maturation and the acquisition of developmental competence . A suffiSHH signaling is involved in the development of the ovary  and is aConsidering the interactive cellular milieu in which oocytes grow and the role of surrounding somatic cells, such as cumulus cells, it was thought that differential levels of cumulus cell expansion and SHH signaling would be observed between BCB- and BCB+ cumulus-oocyte complexes (COCs), after IVM. Therefore, we aimed to determine whether BCB+ COCs have more potential to expand cumulus cells with active SHH signaling to provide COCs with a proper environment for maturation. In this study, pig oocytes were used as a model of low-quality (BCB-) and high-quality (BCB+) oocytes. We investigated the cumulus cell expansion, nuclear maturation of oocytes, subsequent development of parthenogenetic activation (PA) and in vitro fertilization (IVF) embryos, apoptosis levels, and SHH signaling in COCs between BCB- and BCB+ porcine COCs.p < 0.05) proportion of COCs exhibiting a complete cumulus cell expansion  and lower (p < 0.05) proportions of degrees 1, 2, and 3 as compared with BCB- COCs  (p < 0.05) MII rate (95.2 ± 0.8% vs. 65.7 ± 2.3%) and lower (p < 0.05) degeneration and immature rates as compared with the BCB- group .Before IVM, COCs were classified as BCB- or BCB+, according to their coloration, following their exposure to BCB staining a,b and sctively) c,d. In tctively) e,f, the p < 0.05) cleavage and blastocyst formation rates after PA as compared with the BCB- group  (p < 0.05) total cell number (41.0 ± 0.9 vs. 33.4 ± 2.8), including the trophectoderm (TE) cell number (34.8 ± 0.9 vs. 27.7 ± 2.7) (p < 0.05) number and percentage of apoptotic cells in blastocysts as compared with the BCB- group  (The subsequent development of PA embryos derived from BCB- and BCB+ oocytes was investigated. The BCB+ group showed a significantly higher (ctively) g,h. In a7 ± 2.7) i,j, and ctively) k,l.p < 0.05) monospermy and fertilization efficiency as compared with the BCB- group  (p < 0.05) cleavage and blastocyst formation rates after IVF as compared with the BCB- group  (p < 0.05) total cell number (61.0 ± 1.4 vs. 42.1 ± 2.8), including the number of cells in the inner cell mass (ICM) and the number of TEs  (p < 0.05) percentage of apoptotic cells in blastocysts as compared with the BCB- group (8.3 ± 0.7% vs. 13.6 ± 1.8%) , the BCB+ group exhibited a significantly higher (ctively) a,b. On dctively) c,d. In actively) e,f, and  ± 1.8%) g,h.p < 0.05) percentage of apoptotic cells in COCs as compared with the BCB- group (0.4 ± 0.1% vs. 2.4 ± 0.5%) (p < 0.05) percentage of apoptotic cells in COCs as compared with the BCB- group (1.3 ± 0.1% vs. 4.3 ± 0.6%) (p < 0.05) percentage of apoptotic cells in COCs as compared with the BCB- group (0.9 ± 0.1% vs. 4.7 ± 1.0%)  a,b. Afte ± 0.6%) c,d. Afte ± 1.0%) e,f.p < 0.05) expression levels of SHH-signaling proteins, including SHH , PTCH1 , and GLI1  in both cumulus cells and oocytes as compared with the BCB- group (p < 0.05) expression levels of SHH-signaling proteins, including PTCH1  and GLI1  in both cumulus cells and oocytes as compared with the BCB- group , unless otherwise stated.Porcine ovaries were collected from prepubertal gilts at a local abattoir and transported to the laboratory in physiological saline at 32–35 °C. The COCs were aspirated from superficial ovarian follicles (3–6 mm diameter) using a disposable 10 mL syringe with an 18-gauge needle and allowed to settle as sediment in 50 mL conical tubes at 37 °C for 5 min. The supernatant was removed, and the sediment was washed three times with Tyrode’s Albumin Lactate Pyruvate-HEPES medium. Only COCs with at least three uniform layers of compact cumulus cells and a homogeneous cytoplasm were used for the experiments.Immediately after the COCs were isolated, they were incubated in a Dulbecco’s phosphate-buffered saline  supplemented with a 0.4% bovine serum albumin (BSA) (PB1) medium containing 26 μM of BCB for 90 min at 38.5 °C in humidified air. After incubation, the COCs were washed three times in a PB1 medium and classified into the following two groups, depending on the coloration of the cytoplasm: oocytes without blue coloration (BCB- group) and oocytes with blue coloration (BCB+ group). After classification, the COCs were washed twice in an IVM medium and subjected to IVM.2 in 95% humidified air. After 21–22 h of maturing the culture with hormones, the COCs were washed twice in a fresh hormone-free IVM medium and cultured in a hormone-free IVM medium for an additional 21–22 h.The IVM medium was composed of a tissue culture medium (TCM)-199 supplemented with 10% porcine follicular fluid, 10 ng/mL of β-mercaptoethanol, 0.57 mM of cysteine, 10 ng/mL of epidermal growth factor, 10 IU/mL of pregnant mare serum gonadotropin , and 10 IU/mL of human chorionic gonadotropin . Approximately 50 COCs were matured in 500 μL of the IVM medium in a four-well multi-dish  at 38.5 °C with 5% COA total of 1063 COCs was used in seven independent replicates. After 44 h of IVM, the degree of cumulus cell expansion was assessed by microscopic examination, as previously described . BrieflyA total of 1043 oocytes was used in seven independent replicates. After 44 h of IVM, the COCs were denuded by gently pipetting with 0.1% hyaluronidase in a PB1 medium and washed three times in a PB1 medium. The denuded oocytes were evaluated under a microscope  and classified as immature (without a first polar body extrusion), degenerate , or metaphase II (MII) (with a first polar body extrusion) .2·2H2O, 0.1 mM of MgSO4·7H2O, and 0.01% polyvinyl alcohol (PVA). For activation, the MII oocytes were placed between electrodes covered with an activation medium in a chamber connected to an Electro Cell Fusion Generator . The oocytes were activated with a single direct current (DC) pulse of 1.1 kV/cm for 50 μs. The electrically-activated oocytes were cultured in a porcine zygote medium-3 (PZM-3) supplemented with 2 mM of 6-dimethylaminopurine and 5 μg/mL of cytochalasin B for 4 h at 38.5 °C in a humidified atmosphere of 5% CO2. After 4 h, the activated oocytes were washed and cultured in PZM-3 at 38.5 °C in 5% CO2 in air for 6 days.A total of 509 oocytes was used in five independent replicates. For PA, the COCs were denuded by gently pipetting with 0.1% hyaluronidase, after 44 h of IVM culturing, washed three times in a PB1 medium, and gradually equilibrated in an activation medium consisting of 0.28 M of mannitol, 0.5 mM of HEPES, 0.1 mM of CaCl2 in air. To prepare the spermatozoa using the swim-up method before fertilization, the semen sample was kept at 17 °C for 1–3 days and washed three times with a sperm washing medium (DPBS (Gibco) supplemented with 1 mg/mL of BSA, 100 µg/mL of penicillin G, and 75 µg/mL of streptomycin sulfate). After washing, 2 mL of the sperm washing medium was gently added to the spermatozoa pellet and incubated for 15 min at 38.5 °C in 5% CO2 in air. After incubation, the supernatant was washed with mTBM and resuspended with 1 mL of mTBM. The initial sperm concentration of the supernatant was determined by computer-assisted sperm analysis (CASA) and diluted in order to obtain 3.75 × 106 spermatozoa/mL. Then, 2 µL of diluted spermatozoa was added to a 48 µL droplet of mTBM containing 10–15 oocytes, giving a final concentration of 1.5 × 105 spermatozoa/mL. The oocytes were co-incubated with the spermatozoa for 6 h at 38.5 °C in 5% CO2 in air. After 6 h, oocytes were stripped by gentle pipetting and transferred to PZM-3 for culturing at 38.5 °C in 5% CO2 in air. To count the pronuclei, fertilized embryos, at 10 h after insemination, were washed three times with DPBS supplemented with 0.1% PVA (DPBS-PVA) and fixed with 4% paraformaldehyde overnight at 4 °C. Then, they were washed three times in DPBS-PVA and mounted on glass slides with 2 µg/mL of a Vectashield mounting medium using 4′,6-diamidino-2-phenylindole (DAPI) . The pronuclei were observed under UV light using an epifluorescence microscope .A total of 181 oocytes was used in four independent replicates to evaluate the embryo development after IVF. A total of 108 oocytes was used in three independent replicates to evaluate the number of pronuclei. For IVF, the COCs, after 44 h of IVM culturing, were denuded by gently pipetting with 0.1% hyaluronidase in a PB1 medium. Then, they were washed three times in a modified Tris-buffered medium (mTBM) containing 2.5 mM of caffeine sodium benzoate and 1 mg/mL of BSA. Next, 10–15 oocytes were placed into 48 µL droplets of an IVF medium under mineral oil pre-equilibrated at 38.5 °C in 5% COIn total, 24, 42, and 28 COCs were sampled at different time points  and used in three independent replicates. A total of 18 PA-derived and 30 IVF-derived blastocysts was used in four independent replicates. To evaluate the apoptotic cells in COCs or blastocysts, a TUNEL assay was performed using an in situ cell death detection kit . The COCs or blastocysts were washed three times in DPBS-PVA and fixed in 4% paraformaldehyde overnight at 4 °C. Fixed COCs or blastocysts were permeabilized in DPBS containing 1% Triton X-100 at room temperature for 60 min. Subsequently, COCs or blastocysts were washed three times with DPBS-PVA and incubated with fluorescein-conjugated dUTP and terminal deoxynucleotidyl transferase for 1 h at 38.5 °C. After incubation, the COCs or blastocysts were washed three times with DPBS-PVA and mounted on clean glass slides with 2 µg/mL of Vectashield mounting medium using DAPI (Vector Laboratories). DAPI-labeled or TUNEL-positive nuclei were observed under a fluorescence microscope. COCs were observed under a laser-scanning confocal fluorescence microscope . Blastocysts were observed under a fluorescence microscope . Apoptotic cell numbers per COC or blastocyst were judged by counting the nuclei with blue (DAPI) and green (TUNEL) signals. To calculate the percentage of apoptosis in COCs, TUNEL-positive and DAPI-positive cumulus cells were counted using the ImageJ software , and the percentage of TUNEL-positive cells, relative to the total number of cumulus cells (DAPI-positive) in the same COC, was calculated.For SHH staining in COCs, in total, 34 and 32 COCs were sampled at different time points  and used in three independent replicates. For PTCH1 staining in COCs, in total, 30 and 22 COCs were sampled at different time points  and used in three independent replicates. For SMO staining in COCs, in total, 40 and 24 COCs were sampled at different time points  and used in three independent replicates. For GLI1 staining in COCs, in total, 30 and 34 COCs were sampled at different time points  and used in three independent replicates. For Caudal-type homeobox 2 (CDX2) staining in blastocysts, a total of 18 PA-derived and 20 IVF-derived blastocysts was used in four independent replicates. The COCs or blastocysts were fixed in 4% paraformaldehyde overnight at 4 °C and washed three times in DPBS-PVA for 10 min each. For membrane permeabilization, the fixed COCs or blastocysts were incubated in DPBS containing 1% Triton X-100 for 1 h at room temperature. Subsequently, the COCs or blastocysts were washed three times in DPBS-PVA and stored in DPBS-PVA supplemented with 1% BSA (DPBS-PVA-BSA) at 4 °C overnight for blocking. To stain the SHH-signaling proteins in COCs, the COCs were incubated overnight at 4 °C with mouse monoclonal primary antibodies against SHH , PTCH1 , SMO , and GLI1 . For CDX2 staining, blastocysts were additionally blocked with 10% normal goat serum for 1 h. Next, blastocysts were incubated overnight at 4 °C with mouse monoclonal primary antibody against CDX2 . Subsequently, the COCs or blastocysts were washed three times in DPBS-PVA-BSA for 10 min each and incubated for 1 h at room temperature with conjugated secondary antibody, Alexa Fluor 488-labeled goat anti-mouse IgG (1:200 in DPBS-PVA-BSA). After the COCs or blastocysts were washed three times in DPBS-PVA-BSA for 10 min each, they were mounted with 2 µg/mL of the Vectashield mounting medium using DAPI (Vector Laboratories) on clean glass slides. COCs were observed under a laser scanning confocal fluorescent microscope . Blastocysts were observed under a fluorescence microscope .U-test for data with a non-normal distribution  to determine the differences between the experimental groups. Data are expressed as the mean ± standard error of the mean. Differences at p < 0.05 were considered statistically significant.Statistical analyses were performed using Sigmastat software . All data were tested for normality and homoscedasticity, and then subjected to a Mann–Whitney


Herein, we describe interfacially-assembled [7]helicene films that were deposited on graphene monolayer using the Langmuir-Schaefer deposition by utilizing the interactions of nonplanar (helicene) and planar (graphene) π–π interactions as functional antifouling coatings. Bacterial adhesion of Staphylococcus aureus on helicene—graphene films was noticeably lower than that on bare graphene, up to 96.8% reductions in bacterial adhesion. The promising bacterial antifouling characteristics of helicene films was attributed to the unique molecular geometry of helicene, i.e., nano-helix, which can hinder the nanoscale bacterial docking processes on a surface. We envision that helicene—graphene films may eventually be used as protective coatings against bacterial antifouling on the electronic components of clinical and biomedical devices. Helicenes are an intriguing class of materials involving ortho-condensation of benzene or similar aromatic rings to form polycyclic aromatic nanoarchitectures with nonplanar, helical (screw-shaped) geometries . In spitThe presence of the nonplanar π–groups of helicene has been utilized for binding and self-assembly of helicenes on metals via metal–π interactions and on carbon nanomaterials via π–π interactions ,9,10. FeStaphylococcus aureus (S. aureus) is a bacterial pathogen commonly responsible for various types of human infections such as skin, bone, bloodstream, lung and respiratory tract infections, and device-related infections helicene  in anhydrous dichloromethane via bath-sonication for 10 min at room temperature (23 °C). Then, 5 µL of helicene solution was spread at the air-water interfaces of a Langmuir trough  . The surThe films were characterized by an atomic force microscope  via the tapping mode at room temperature under atmospheric conditions. The measurements were performed with a silicon tip , which has a radius of curvature of 7 nm, a spring constant of 9 N/m, and a resonant frequency of 150 kHz, at a scan rate of 0.5 Hz. The wetting behavior of water on the prepared films was determined under static conditions via the sessile drop technique. In these experiments, 5 µL of Milli-Q water was dropped on the surface of interest, then there was a 1 min wait for the dissipation of surface waves-induced by the impact of placement. The image of the water droplet was taken by a high-resolution camera  and analyzed through ImageJ software , Bethesda, MD, USA) via Low-Bond Axisymmetric Drop Shape Analysis (LBADSA) with three replicate .S. aureus (ATCC 13565) was prepared as described in previous studies helicene demonstrated a repulsion behavior with a gradually steeper slope as the degree of lateral confinement increases . Above aial film . To inveThe amplitude error is an extremely sensitive mode of AFM for determining the shape of the topographical features of surfaces involving different phases. The static contact angle of water was 82.3° ± 1.3° on GR, indicating close to hydrophobic behavior, while it was 85.8° ± 2.5°, 96.3° ± 1.3°, and 106.7° ± 2.0° for Gr-H0.5, Gr-H5, and Gr-H15, respectively . Since tS. aureus with GR and helicene-deposited samples were investigated using SEM. As shown in S. aureus on GR, GR-H0.5, GR-H5, and GR-H15 samples were 5.94 ± 0.07 log10 cell/cm2 (8.8 ± 1.4 × 105 cell/mL), 5.67 ± 0.17 log10 cell/cm2 (5.0 ± 2.0 × 105 cell/mL), 5.27 ± 0.07 log10 cell/cm2 (1.8 ± 0.3 × 105 cell/mL), and 4.29 ± 0.46 log10 cell/cm2 (2.8 ± 2.1 × 104 cell/mL), respectively. It is important to note that with SEM counting, it is not always possible to identify bacterial multilayers helicene hinders the bacterial docking processes on the surface. Overall, this work reports a novel property of helicene-based materials: the ability of bacterial antifouling. This characteristic may be exploited in emerging coatings for biomedical devices.The chiral compound with helical nanoarchitecture, [7]helicene, was deposited on graphene monolayers via the Langmuir-Schaefer method. The areal density of interfacially-assembled domains increased with increasing surface pressure, reaching a fully packed geometry at a surface pressure of 15 mN. Such helicene coated graphene films demonstrated notable ability to inhibit the bacterial adhesion, up to 96.8% (31-fold) reduction compared to bare graphene monolayers upon 4-h drop-cast inoculation with


BioRoot RCS caused a significant reduction in viable counts of the biofilms compared to AH Plus and the control (p < 0.05), while no significant difference could be observed compared to TotalFill BC and Tubli-seal (p > 0.05). CLSM analysis showed that BioRoot RCS and TotalFill BC exhibited significant biofilm inhibition compared to Tubli-seal, AH Plus and the control (p < 0.05). BioRoot RCS presented with the highest microbial killing, followed by TotalFill BC and Tubli-seal. Alkalizing activity was seen from the onset by BioRoot RCS, TotalFill BC and AH Plus. After 28 days, BioRoot RCS demonstrated the highest pH in HBSS (pH > 12). Conclusions: Calcium silicate sealers exhibited effective antimicrobial properties. This was demonstrated by superior biofilm inhibition capacity and microbial killing, with strong alkalizing activity compared to epoxy-based and zinc oxide-eugenol-based sealers.Purpose: This study compared the antimicrobial efficacy of calcium silicate sealers  and conventional sealers  against planktonic bacteria and a nutrient-stressed multispecies biofilm. Methods: Antimicrobial properties of freshly mixed sealers were investigated using the direct contact test (DCT) and a nutrient-stressed multispecies biofilm comprised of five endodontic strains. Antimicrobial activity was determined using quantitative viable counts and confocal laser scanning microscopy (CLSM) analysis with live/dead staining. The pH of the sealers was analysed over a period of 28 days in Hanks Balanced Salt Solution (HBSS). Analysis of variance (ANOVA) with Tukey tests and the Kruskal–Wallis test were used for data analysis with a significance of 5%. Results: All endodontic sealers exhibited significant antimicrobial activity against planktonic bacteria ( The primary aetiology of endodontic disease is the microbial infection of the root canal system ,2. MultiThe aim of this study was to investigate in vitro the antimicrobial activity of calcium silicate and conventional endodontic sealers against planktonic microbes and the aforementioned nutrient-stressed multispecies biofilm model. The secondary aim was to analyse the pH value of the sealers over a period of 28 days.For the purposes of this study, two commercially available calcium silicate sealers, Total Fill BC  (Group 1) and BioRoot RCS  (Group 2), were used along with AH Plus  (Group 3), an epoxy-based sealer, and Tubli-seal  (Group4), a zinc oxide-eugenol based sealer. The chemical composition of the sealers is shown in P. acnes), Actinomyces radicidentis (A. radicidentis), Staphylococcus epidermidis (S. epidermidis), Streptococcus mitis/oralis  recovered from root canals of teeth with refractory endodontic infections and Enterococcus faecalis  strain OMGS 3202  values of total biovolumes (μm3/μm2) and mean percentages of red (dead), green (live) and orange (unknown) biovolumes. The images were reconstructed using ImageJ software and analysed using bioImage_L [For this analysis, the nutrient-stressed multispecies biofilm was grown on the surface of the sealer discs as mentioned in oImage_L .n = 12, 3 per group with the experiment performed in triplicates), 200 μL was placed at the base of individual wells of a sterile 12-well flat bottom plate  using a 1-mL sterile syringe. 10 mL of HBSS  was immediately added to each well and stored at 37 °C. The HBSS was replaced at each endpoint . The collected HBSS solution was transferred to a glass vial  and vortexed for one minute. Determination of the HBSS solution pH was performed at room temperature using a pH meter  and a temperature compensated pH electrode  previously calibrated at five points  using buffer solutions . The mean and standard deviation of each were calculated.Of each freshly prepared sealer (3/μm2). The overall analysis was performed with SPSS software . In all tests, the level of statistical significance was set at p < 0.05.A normality test (Kolmogorov–Smirnov) was performed for the examination of data distribution. Analysis of variation (ANOVA) with post-hoc Tukey test was used for data analysis and comparison of the quantifiable viable counts amongst all groups of the DCT and multispecies biofilm inhibition test. The Kruskal–Wallis (nonparametric) test was performed for data analysis of total biovolumes  at the start of DCT was 3 × 108 mL (9.47 log10 CFU mL−1). After seven days of growth, the log10 CFU values were increased E. faecalis (10.59 ± 0.14); A. radicidentis (10.55 ± 0.12); P. acnes (10.37 ± 0.09); S. epidermidis (10.28 ± 0.31) and S. mitis/oralis (10.22 ± 0.29).The results of the DCT are shown in p < 0.05). At T2 (24 h), all sealer groups showed a reduction of viable counts, but there were no significant differences between the sealer groups (p > 0.05). At T3 (168 h), BioRoot RCS (2.74 ± 0.21) presented the lowest value of detectable viable counts and was significantly more effective in microbial killing than Tubli-seal (4.47 ± 0.23) (p < 0.05). However, there was no significant difference when compared to TotalFill BC (3.12 ± 0.15) and AH Plus (3.36 ± 0.36) (p > 0.05).At T1 (1 h), AH plus (5.98 ± 0.68) exhibited a significant reduction of detectable viable counts when compared to TotalFill BC (7.71 ± 0.02) and BioRoot RCS (7.36 ± 0.32) (A. radicidentis at T1 (p < 0.05), but no significant differences were found among the groups (p > 0.05). At T2, AH Plus (3.77 ± 0.32), BioRoot RCS (4.21 ± 0.24) and TotalFill (4.77 ± 0.05) showed significant microbial killing compared to Tubli-seal (6.06 ± 0.04). All sealers presented lower detectable viable counts at T3 following the order AH plus (3.31 ± 0.71) > BioRoot RCS (3.96 ± 0.09) > TotalFill BC (4.54 ± 0.12) > Tubli-seal (5.22 ± 0.19) when compared to the positive control group (p < 0.05), but no significant differences were present between the sealer groups (p > 0.05).All sealers presented a significant reduction of detectable viable counts of P. acnes (p < 0.05). There was no significant difference between BioRoot RCS (5.26 ± 0.28) and Tubli-seal (4.69 ± 0.29) (p > 0.05), but both sealer groups exhibited enhanced microbial action compared to TotalFill BC (7.75 ± 0.09) and AH Plus (6.63 ± 0.06), with the latter being more effective than the former (p < 0.05). At T2, the detectable viable counts for Tubli-seal were the lowest (3.56 ± 1.01), but with no significant difference against the rest of the sealer groups (p > 0.05). At T3, BioRoot RCS (2.72 ± 0.11) and Tubli-seal (3.14 ± 0.49) presented similar reduction in viable counts (p > 0.05), and no difference was present when Tubli-seal was compared with AH Plus (4.40 ± 1.21) and TotalFill BC (4.53 ± 0.09). BioRoot was more effective than TotalFill BC (p < 0.05).At T1, all sealers presented a reduction of detectable viable counts of S. epidermidis. Tubli-seal exhibited limited microbial killing efficacy, which was not statistically significant from the positive control group (p > 0.05). At T2, AH Plus (2.97 ± 0.12) and BioRoot RCS (3.54 ± 0.60) presented lower detectable viable counts and more enhanced microbial action than Tubli-seal (6.29 ± 0.74) and TotalFill BC (5.22 ± 0.11) (p < 0.05). At T3, AH Plus (2.13 ± 0.07), BioRoot RCS (2.44 ± 0.23) and Tubli-seal (3.79 ± 0.43) presented significantly lower detectable viable counts than TotalFill BC (5.50 ± 0.10) (p < 0.05).At T1, BioRoot RCS (5.27 ± 0.18), TotalFill BC (6.13 ± 0.13) and AH Plus (6.23 ± 0.16) presented significant reduction of detectable viable counts of S. mitis/oralis control group (p < 0.05). Tubli-seal (4.30 ± 0.47) presented similar reduction in viable detectable counts compared with BioRoot RCS (4.73 ± 0.25), but enhanced microbial killing efficacy compared to TotalFill BC (5.77 ± 0.18) and AH Plus (5.11 ± 0.57) (p < 0.05). BioRoot RCS was more effective than TotalFill BC (p < 0.05). At T2, Tubli-seal (3.53 ± 0.14) and BioRoot RCS (4.03 ± 0.28) exhibited enhanced microbial killing compared to TotalFill BC (5.39 ± 0.03) (p < 0.05), but no significance difference when compared to AH Plus (4.39 ± 0.77). At T3, BioRoot RCS (3.01 ± 0.08) and Tubli-seal (3.34 ± 0.05) presented enhanced microbial killing compared to AH Plus (3.84 ± 0.22) and TotalFill BC (4.56 ± 0.25) (p < 0.05). There was no significant difference between BioRoot RCS and Tubli-seal (p > 0.05).All sealers presented a significant reduction at T1 when compared to the 10 CFU, compared to AH Plus (10.04 ± 0.22) and the control (10.4 ± 0.50). BioRoot RCS group presented the lowest log10 CFU counts, which was statistically significant compared to AH Plus and the control (p < 0.05). No significant difference was observed between BioRoot RCS, Tubli-seal and TotalFill BC (p > 0.05). Negative control group specimens presented nondetectable viable counts.The results of the biofilm inhibition test are shown in p < 0.05). The lowest mean total biovolume was detectable in TotalFill BC discs, followed by BioRoot RCS. This was significantly less compared to AH Plus and Tubli-seal (p < 0.05). BioRoot RCS presented enhanced microbial killing, with the highest mean percentage of 46%, followed by TotalFill BC and Tubliseal with 29% and 25% red (dead) biovolume, respectively. AH Plus showed a mean percentage of 89% green (live) biovolume. Negative control group specimens presented no viable biovolumes.The effects of endodontic sealers on the inhibition of biofilm growth are presented in The pH values of the endodontic sealers tested after 15, 30, 60, 120 min and 1, 3, 7, 14, 28 days are shown in P. acnes, A. radicidentis and S. mitis/oralis and a nutrient-stressed multispecies biofilm model. Considering the relevance of multispecies biofilms in endodontic infections, the advantages of using this model is that it represents the in vivo microbiological environment of the root canal system as closely as possible and permits well-controlled, standardised experimental conditions, enabling complete enumeration of microbial counts. A limitation of the aforementioned model, adapted for this study, was not utilizing substrates of dentine for biofilm growth. However, the use of a dentine model has disadvantages, such as complexities in retrieving the remaining microbial cells adhered on the substrate and deleterious effects on the collagen fibres due to sterilisation of the dentine, which may lead to poorer adhesion of the biofilms.The antimicrobial efficacy of calcium silicate sealers has been evaluated using the DCT and single species biofilm models ,16,17,18In the present study, all sealers demonstrated significant antimicrobial activity against the five planktonic bacteria analysed in the DCT. Previous studies evaluating the antimicrobial efficacy of calcium silicate sealers using DCT have reported a pronounced killing effect against planktonic bacteria ,14,17. TSubstantive long-term antimicrobial effectiveness of calcium silicate sealers demonstrated by the multispecies biofilm test may be positively correlated with calcium ion release  and the The biological and physiochemical properties of calcium silicate sealers depend on their engineered chemical composition and their setting reactions, which consist of a hydration reaction followed by a precipitation reaction. There are many factors which affect these reactions, such as the inclusion of additives, namely calcium phosphate and calcium chloride, that may alter the properties of calcium silicates .In this study, freshly mixed AH Plus demonstrated high microbial killing but negligible antimicrobial efficacy after setting. The antimicrobial properties of AH Plus might be associated with its alkalinity, with the presence of bisphenol A diglycidyl ether or with the elution of unpolymerized components such as amines released during the setting stage ,37. TublWithin limitations of this study, freshly prepared endodontic sealers exhibited antimicrobial activity against planktonic bacteria. Calcium silicate sealers showed effective biofilm inhibition capacity and microbial killing, with strong alkalizing activity compared to epoxy-based and zinc oxide-eugenol-based sealers.


To study the associations between fatty liver (FL), insulin resistance, myocardial high-energy-phosphate (HEP) and glucose metabolism, and heart function in patients with uncomplicated type 2 diabetes mellitus (T2DM).1H-MRS of the liver for the assessment of liver fat, myocardial 31P-MRS for assessment of myocardial HEP metabolism and MRI to determine left ventricular function were performed. Furthermore, a hyperinsulinemic, euglycemic clamp was performed to establish whole body insulin sensitivity. Moreover, PET with H215O (fasting conditions) and [18F]-2-fluoro-2-deoxy-D-glucose (clamp conditions) were used to determine myocardial blood flow (MBF) and myocardial metabolic rate of glucose uptake (MMRglu) in a subgroup of 28 patients.We studied 35 T2DM patients (Mean ± SD Hba1c = 7.0 ± 0.8%) without coronary artery disease or heart failure, as determined by echocardiography. 2, p < 0.05), reduced whole body insulin sensitivity (0.45 ± 0.48 vs 0.74 ± 0.44 (mg/kg·min)/(pmol/L), p < 0.05), and reduced MMRglu , as compared with patients without FL, while MBF was not different. The ratio of phosphocreatine over adenosine triphosphate, a marker of myocardial HEP metabolism, was reduced in patients with FL , also after adjustment for BMI, and correlated to MMRglu . LV systolic and diastolic function were not statistically significantly different. Figure 1Patients with FL  showed increased body mass index (29.5 ± 3.2 vs 27.2 ± 2.8 kg/mFatty liver in patients with uncomplicated T2DM is associated with decreased myocardial HEP metabolism. In addition myocardial HEP metabolism is modulated by myocardial glucose uptake.


Pterodon, in nanostructured lipid carriers (NLCs), a second generation of lipid nanoparticles which act as new controlled drug delivery system (DDS). Encapsulation was performed by hot high-pressure homogenization (HPH) technique and the samples were then analyzed by dynamic light scattering (DLS) for mean average size and polydispersity index (PI) and by electrophoretic light scattering (ELS) for zeta potential (ZP), immediately after production and after 24 h of storage at 4 °C. An optimal sucupira-loaded NLC was found to consist of 0.5% (m/V) sucupira oil, 4.5% (m/V) of Kollivax® GMS II and 1.425% (m/V) of TPGS (formulation no. 6) characterized by a mean particle size ranging from 148.1 ± 0.9815 nm (0 h) to 159.3 ± 9.539 nm (at 24 h), a PI from 0.274 ± 0.029 (0 h) to 0.305 ± 0.028 (24 h) and a ZP from −0.00236 ± 0.147 mV (at 0 h) to 0.125 ± 0.162 (at 24 h). The encapsulation efficiency and loading capacity were 99.98% and 9.6%, respectively. The optimized formulation followed a modified release profile fitting the first order kinetics, over a period of 8 h. In vitro cytotoxicity studies were performed against Caco-2 cell lines, for which the cell viability above 90% confirmed the non-cytotoxic profile of both blank and sucupira oil-loaded NLC.Essential oils are odorant liquid oily products consisting of a complex mixture of volatile compounds obtained from a plant raw material. They have been increasingly proven to act as potential natural agents in the treatment of several human conditions, including diabetes mellitus (DM). DM is a metabolic disorder characterized by chronic hyperglycemia closely related to carbohydrate, protein and fat metabolism disturbances. In order to explore novel approaches for the management of DM our group proposes the encapsulation of sucupira essential oil, obtained from the fruits of the Brazilian plants of the genus  Essential oils (EOs) are odorant liquid oily products, consisting of a complex mixture of lipophilic and volatile compounds obtained from a plant raw material by one of three distinct procedures: Dry distillation, driving by steam of water or suitable mechanical method without heating ,3,4,5,6.Pterodon, commonly known as “faveira”, “sucupira”, “sucupira-branca”, “fava-de-sucupira”, or “sucupira-lisa” and has been recognized as promising agent for the management of DM and its complications . Th. Th67]. R2 of 0.9829, the closest to 1. The first order release kinetics describes that variations in concentration over time only depends on the concentration of the sucupira oil inside the particles and is translated by the following equation: k is the first-order release constant. By integration, the logarithmic equation is obtained:t. A modified release has been obtained which is not governed by the physicochemical properties of the carriers. These results were expected as NLC are not made of solid lipid only [From the profiles shown in pid only . Indeed,Blank NLC  and formulation no. 6 was subject of cytotoxicity analysis against Caco-2 cells .d-α-tocopheryl polyethylene glycol succinate, or vitamin E TPGS, has been approved by the Food and Drug Administration as a safe ingredient and is commonly used in pharmaceutical dosage forms [The human intestinal Caco-2 cell line, a cell line derived from a colorectal adenocarcinoma, has a epithelial like-morphology and is commonly used as in vitro model for cytotoxicity screening of formulations intended for oral administration. In this work, the cell viability results, was tested in two time-points and using empty (blank) and loaded NLC, as recommended by Silva et al. ,69. Resuge forms . Alpha-tge forms . It is ige forms ) as cell® GMS II and of TPGS. A 22 factorial design has been successfully used to determine the best combination of solid lipid and surfactant. An optimal sucupira oil-loaded NLC should consist of 0.5% (m/V) sucupira essential oil, 4.5% (m/V) of Kollivax® GMS II and 1.425% (m/V) of TPGS, for which a mean particle size ranging from 148.1 ± 0.1 nm (0 h) to 159.3 ± 9.5 nm (at 24 h), a PI from 0.274 ± 0.029 (0 h) to 0.305 ± 0.028 (24 h) and a ZP from −0.00236 ± 0.147 mV (at 0 h) to 0.125 ± 0.162 (at 24 h) was recorded. The release profile followed the first order kinetics over the course of 8 h. For the optimized formulation an EE of 99.98% and LC of 9.6% were obtained. The particles also showed no cytotoxic effect when tested against Caco-2 cell line, up to 48 h. Further studies are planned to evaluate the interest of sucupira oil-NLC as an innovative anti-hyperglycemic formulation in a diabetic animal model. This work described the successful loading of sucupira oil in NLC composed of a new combination of Kollivax