Cellular temperature and pH govern many cellular physiologies, especially of cancer cells. Besides, attaining higher cellular temperature plays key role in therapeutic efficacy of hyperthermia treatment of cancer. This requires bio-compatible, non-toxic and sensitive probe with dual sensing ability to detect temperature and pH variations. In this regard, fluorescence based nano-sensors for cancer studies play an important role. Therefore, a facile green synthesis of orange carbon nano-dots (CND) with high quantum yield of 90% was achieved and its application as dual nano-sensor for imaging intracellular temperature and pH was explored. CND was synthesized from readily available, bio-compatible citric acid and rhodamine 6G hydrazide using solvent-free and simple heating technique requiring purification by dialysis. Although the particle size of 19 nm (which is quite large for CND) was observed yet CND exhibits no surface defects leading to decrease in photoluminescence (PL). On the contrary, very high fluorescence was observed along with good photo-stability. Temperature and pH dependent fluorescence studies show linearity in fluorescence intensity which was replicated in breast cancer cells. In addition, molecular nature of PL of CND was established using pH dependent fluorescence study. Together, the current investigation showed synthesis of highly fluorescent orange CND, which acts as a sensitive bio-imaging probe: an optical nano-thermal or nano-pH sensor for cancer-related studies. Intracellular pH and temperature values regulate pathological and physiological processes and disruptive changes of such parameters3 are indicators of functional disorder such as cancer4. Assessment of cellular pH and temperature are essential in cancer metastasis and hyperthermia treatment studies and this is possible if temperature and pH fluctuations in cancer cells are evaluated with sensitive probes such as non-invasive or non-destructive optical nano-sensors. Thus, fluorescent carbon nanodots (CNDs)5 are ideal nano-probes due to their bio-compatibility, non-toxicity, chemical and photo-stability which makes them suitable for various applications such as in-vivo animal bio-imaging, light emitting diodes, optical sensing and photo-catalysis8. The CNDs, which comprises mostly carbon, are typically less than 10 nm in diameter and can be produced by breaking down larger carbon structures (top-down approach) or by building from smaller carbon precursors (bottom-up approach).The latter process involves either combustion/thermal or supported synthesis/microwave methods/solvothermal process during which the CNDs are formed from molecular precursors. The primary challenge in the synthesis of CND by bottom-up processes is the size-control of products of variable polydispersity and properties9. The secondary challenge is attainment of high fluorescence of CND which is generally governed by quantum and physio-chemical processes10. In order to achieve conjugated sp2-domain conducive for orange and red emissions, the size of the particle needs to be increased. However, large sized particles usually contain huge surface defects, leading to non-efficient emissions11. Quantum yield (QY) exceeding 50% have seldom been reported for large CNDs in the orange-red regime and hence improvement for their development for widespread applications is warranted12. Therefore, rhodamine based fluorescence moieties appear to be particularly attractive due to their excellent spectroscopic properties, high fluorescence quantum yield and high photostability13. Generally, the spirolactam ring of rhodamine-based fluorescence probe is non-fluorescent and colorless in neutral or alkaline medium, whereas ring opening leads to strong fluorescence emission and pink coloration either on account of chelation to metal or at low pH or acidic conditions  forming strongly luminescent molecular fluorophore, though mostly emitting in the blue region. Thus in our present study, to achieve remarkable enhancement of the fluorescence in CNDs, citric acid was covalently functionalized with rhodamine 6G  hydrazide. Since spirolactam ring of Rh6G hydrazide opens in acidic conditions hence, this moiety present in CND has the ability to sense pH changes. Moreover, fluorophores display molecular flexibility at elevated temperature which affects fluorescence intensity and therefore, temperature changes can also be detected using variations occurring in PL intensity. However, there are few reports of a bio-compatible, water soluble, non-toxic fluorescent nano-sensor which has the ability to detect either thermal or pH fluctuations inside cells19. Simultaneous detections of intracellular temperature and pH is a challenging task since intracellular pH is a varying parameter and photo-luminescent materials usually change their optical property such as emission intensity depending on environment. Thus luminescent sensors having high sensitivity towards temperature and low sensitivity towards pH are highly desirable. This can be achieved by synthesizing photo-luminescent materials having high quantum yield (QY) which can make temperature sensitivity more compared with pH sensitivity20. Hence Rh6G hydrazide and citric acid based CND fulfils the conditions required for a dual nano-sensor to detect intracellular temperature and/or pH for cancer studies owing to its high QY of 90%. In the past decades, technologies were developed extensively to non-invasively measure intra- and/or extracellular pH in cancer21. Quantitative imaging of distribution of acidity helps in understanding the role of tumor microenvironment in cancer progression and metastasis, which is associated with switching metabolism to glycolysis in cancer cells for generating acidic environment. The CND developed in the current investigation is helpful in measuring variation in pH in cancer cells under fixed temperature and also in measuring fluctuations in temperature in cancer cells at constant pH.Variations in temperature and pH are markers of cellular events23 from Rh6G dye by refluxing it with hydrazine hydrate for 6 h in methanol and characterized by 1H NMR . CNDs were prepared from citric acid and Rh6G hydrazide by heating them at 200 °C in a heating mantle   and as prepared dialysate without further purification was used for all subsequent experiments and analysis. The CNDs were characterized using NMR Fig. , 13C NMRNMR Fig.  and FT-INMR Fig. , elementNMR Fig.  and EnerNMR Fig. E, S7. ThM) Figs. , TransmiM) Figs. , ScanninM) Figs.  and zetaM) Figs. . The parM) Figs. A. Wang eges Fig. B. Duringges Fig. B,C was a31. The zeta potential of CND in ethanol was + 1.2 mV indicating positive surface potential of CND. The development of positive charge can be attributed to protonation of the amide and/or amine groups generated from Rh6G hydrazide. However, this result is in sharp contrast with the one obtained by Qu et al.11 where the zeta potentials of the orange CND were found to be − 17.3 and − 35.7 mV respectively. This further implies the absence of negative carboxylate groups resulting due to amide linkages between citric acid and Rh6G hydrazide. The stability of any colloidal system can be attributed to any of the two factors namely steric stabilization due to presence of large groups or existence of charged functional groups which leads to stabilization of the system as a result of electrostatic repulsion33. Thus, the synthesized CND is sterically stabilized due to existence of large groups like Rh6G hydrazide moiety covalently linked with citric acid. This can be easily confirmed by the insignificant change in time dependent absorbance value of the system under consideration  and wide angle X-ray scattering experiments Fig.  revealedion Fig. A,B. Furtion Fig. C. No subThe UV–Vis absorption and excitation spectrum Fig. A,B exhib35, the emission spectra of the current variant of CNDs were observed constantly at higher wavelength of 550 nm which is similar to observations made by Qu et al.11 where the CND exhibited strong orange luminescence with excitation independent PL in ethanol. The quantum yield of CND from citric acid and Rh6G hydrazide was found to be 0.90 . It was found that CND exhibited better photo-stability as compared to Rh6G dye  in normal cells is lower than extracellular pH (pHe = 7.3–7.4). In contrast, cancer cells have higher values of pHi (7.12–7.25) and lower pHe (6.2–6.9). Reversal in the pH gradient assists in the progression of cancer such that elevated pHi allows cancer cell proliferation41. During short duration of hyperthermia (30–60 min), it is expected to have no effect on the intracellular pHi, which requires long term switching effects of glycolysis. In this regard, incubation of CND with MCF-7 (breast adenocarcinoma) led to rapid uptake and cytoplasmic accumulation of CND, as evident from the high orange fluorescence of CND  and MCF-7 cells respectively. IC50 value in CHO cells was 1133 µg/mL while in MCF-7 cells; IC50 of 1177 µg/mL was observed  improve the outcomes of chemotherapy and radiotherapy of cancerCND Fig. , signifiCND Fig.  was asseCND Fig.  and S20 CND Fig. . To evalved Fig. .Figure 4This is the first report of highly fluorescent (QY of 90%) Rh6G and citric acid based CND, achieved from a facile and solvent-free synthesis route. The synthesized CND was easily purified using dialysis and the dialysate was further studied for its photo-physical and particle size properties and bio-imaging application. The CND behaves as a molecular fluorophore which is susceptible to temperature variations and the spirolactam ring present in the CND is pre-disposed to changes in pH. The dual sensor attributes for temperature and pH sensing makes CND ideal as a bio-imaging agent for cancer related studies. The results clearly show that CND is non-toxic to both normal and cancer cells. Together, synthesized CND can be used as a sensitive bio-imaging probe to measure intracellular temperature and pH with no or minimum toxicity and excellent biocompatibility.−1. 1H and 13C NMR was recorded in VARIAN 500 MHz and Bruker 300 MHz instrument. The SCXRD data were collected from a single crystal at 298(2) K on a XtaLAB Synergy, Dualflex, HyPix four-circle diffractometer with a micro-focus sealed X-ray tube using mirror as monochromator and a HyPix detector. Elemental analysis was carried out using Eurovector Instruments and Software EuroEA3000 CHNS-O elemental analyzer instrument. Scanning electron microscopy was performed using SEM-Carl Zeiss, Germany coupled with Dispersive X-ray spectroscopy (EDX)-INCA Energy 250, Oxford Instruments. Sample was prepared on a clean glass slide and sputtered with gold using standard methods. The scanning of images was performed at an accelerating voltage of 10 keV and EDX spectra were obtained between 0 and 20 keV.Infrared spectroscopy was carried out in Bruker TENSOR III instrument. The measurement was made by attenuated total reflection method with frequency range of 4000–500 cm1H NMR : δ 7.78–7.73 , 7.49–7.44 , 6.95–6.91 , 6.27 , 6.10 , 3.13 , 1.87 , 1.20 .13C NMR : 14.2, 17.1, 37.5, 65.1, 96.0, 105.1, 117.9, 122.2, 123.5, 127.1, 128.1, 129.5, 132.4, 147.4, 151.4, 152.2, 165.4.Rh6G  was dissolved in methanol (50 mL) and to the stirred solution hydrazine hydrate  was added. The reaction mixture was refluxed for 6 h and then allowed to cool to room temperature. To the resulting solution, ethyl acetate was added and washed with 33 mL of NaOH (1 M) solution twice. The obtained Rh6G hydrazide was dried over MgSO4 and purified using silica gel chromatography (25% MeOH: 75% CHCl3). Recrystallization was done in chloroform and methanol solvent system and colorless crystals were obtained  which was characterized by FT-IR, NMR Fig. , 13C NMRNMR Fig.  and singNMR Fig. 22,23.FT-1H NMR : δ 6.90 (s), 6.81 , 6.28 (s), 5.89 , 5.74 (s), 3.31(s), 2.94–2.76 (m), 2.14 (s), 2.05 , 1.36–1.22 (m).13C NMR : 9.8, 11.4, 20.5, 20.6, 20.9, 27.14, 33.83, 36.6, 38.4, 42.1, 43.9, 74.3, 121.9, 122.5, 129.2, 129.6, 130.6, 130.7, 136.3, 147.1, 147.7, 150.8, 166.1, 168.1, 168.2, 168.5, 169.8, 172.6, 172.8, 173.6, 173.6, 175.0, 176.9.Citric acid 3 g) was heated at 200 °C to give a colorless melt in which Rh6G hydrazide (0.01 g) was added resulting in immediate color change from colorless to purple. The resulting liquid was heated at 200 °C for ten minutes, allowed to cool down to room temperature and dissolved in 50 mL of ethanol which gave an orange-colored fluorescent solution. The solution was put in a dialysis bag and dialyzed for 6 h. The resulting dialyzed solution was characterized by NMR Fig. , 13C NMRNMR Fig. , FT-IR : δ 7.06 (s), 6.90 (s), 6.73 (s), 6.39 (s), 6.28–6.24 (m), 5.88 (s), 5.74 (s), 3.88 (s), 3.65–3.61 (m), 3.07–2.76 (m), 2.16 (s), 1.30 (m).13C NMR : 24.3, 31.5, 34.7, 38.4, 39.8, 41.9, 44.0, 74.3, 128.4, 129.0, 129.4, 132.7, 136.5, 139.2, 165.7, 169.3, 171.1, 173.6, 176.9.Citric acid (3 g) was heated at 350 °C for fifteen minutes to give a colorless melt which turned into sticky yellow compound. The product was allowed to cool down to room temperature and dissolved in 50 mL ethanol which gave a pale yellow solution. The solution was put in dialysis bag and dialyzed for 6 h. The resulting dialyzed solution was characterized by UV–VIS and Fluorescence spectroscopy Figs.  and S19,NMR Fig. , 13C NMRNMR Fig. , FT-IR . All data were integrated and a multi-scan absorption correction was applied using CrysAlis PRO24. The structure was solved by direct method using OLEX and refined by full-matrix least-squares method against F2 by SHELXL-2017/126. Hydrogen atoms were placed in idealized positions and were set riding on the respective parent atoms. All non-hydrogen atoms were refined with anisotropic thermal parameters. Crystallographic data (including structure factors) for the structure reported in this paper have been deposited with the Cambridge Crystallographic Data Centre (CCDC No. 2051200). Oakridge thermal ellipsoid plot (ORTEP) was employed for the final data presentation and structure plots27. NTMDT Ntegra Atomic Force Microscope was used for AFM. About 10 µL of the CND solution was spotted on to freshly cleaved mica surface and allowed to air dry in laminar air flow for about 25–30 min. This mica adhered CND sample was attached to sapphire substrate and placed on the sample holder of NTMDT Ntegra Atomic Force Microscope. The mica surface was scanned in semi contact mode with NSG11 golden silicon probe tip using a 10-micron scanner under universal head. Measurements across X–Y–Z axis were measured using the NTMDT NOVA 1.1.01780 software. Arbitrary height measurement tool of this software was used to measure height of 50 randomly selected CNDs as described earlier28.The data were collected from a single crystal at 298(2) K on a XtaLAB Synergy, Dualflex, HyPix four-circle diffractometer with a micro-focus sealed X-ray tube using mirror as monochromator and a HyPix detector. The diffractometer was equipped with a low temperature device and used Cu Kα, λ = 0.1542 nm) operated at 40 kV and 50 mA. The sample was placed in paste cell and thermostated using Peltier controlled sample holder. The scattering intensities were monitored by using a two dimensional (2D) charged-coupled device (CCD) detector (pixel size 24 micron) to span q (momentum transfer) range of 0.15 to 5 nm−1. A semi-transparent beam stop was employed to measure transmittance and zero q position. The 2D SAXS images were processed into one-dimensional (1D) scattering profiles and were collected for transmission and background scattering using standard protocols in SAXSquant  software. Ensemble averaged microstructural analysis of the carbon dots was carried out by SAXS. Fig. p  weighted by the distribution function, as given by the equationv(R) represents the volume weighted particle size distribution, K is a constant depending on the instrument geometry, Δρ is the scattering contrast given by difference in the electron density of matrix and particle, and Vp is the particle volume. Figure 43.Transmission Electron Microscopy was carried out in LIBRA 200FE TEM instrument. TEM samples were prepared by evaporating 10 µL of sample solution on carbon coated  Cu grids purchased from TED PELLA Inc. Small angle X-ray scattering (SAXS) was done in Anton Paar SAXSpace instrument which uses line collimated monochromatic X-ray source (Cu Wide angle XRD X-ray scattering (WAXS) was done in Rigaku Smart Lab (9 W) Wide angle XRD instrument. The experiment was performed with a scan from 5 to 80° with 3° per minute at 6 W power. The zeta-potential measurement was carried out in Zetasizer nanoseries, Malvern Instruments. A UV–Vis spectrophotometer  was used to carry all the ground-state absorption measurements in an optical quartz cell of path length 1 cm. The steady-state (SS) fluorescence measurements for the studied systems were carried out using spectrofluorometer  where sample solutions were taken in a 10 mm × 10 mm quartz cuvette and excited with 515 nm steady light beam. In the temperature dependent study, the sample solutions were heated using a temperature controller  having accuracy of ± 0.5 °C. The temperature was increased in a stepwise manner, changing in the steps of 5 °C, and allowing the solution to equilibriate at each set temperature for about 10 min, before taking the measurements. The adjustment of the pH was done using dilute solutions of NaOH and HCl and measured using edge blu Bluetooth smart pH meter-HI 2202. All the photo-physical studies were carried out at a concentration of ~ 0.3 mg/mL of CND. Photo-degradation studies were carried out using Nd: YAG nanosecond laser (M/s Ekspla) with pulse duration 6 ns, repetition rate 10 Hz at 532 nm wavelength emission. Dialysis purification was done in MilliQ water using Spectra/Por Molecular Porous Membrane Tubing membrane MWCO:12-14000. Rhodamine 6G, citric acid and hydrazine hydrate were purchased from Sigma-Aldrich. Quantum Yield experiment was carried out using relative quantum yield method. Rhodamine 6G was taken as a standard (QY = 0.95 at 505 nm). The measurements of the CND sample were carried out in ethanol and QY obtained was 90% at 505 nm.2. MCF-7 cells  were seeded in 35-mm imaging dish (genetix) and incubated overnight. The cells were gently washed with DMEM media and maintained at respective temperatures for 10 min, followed by imaging. Cellular imaging was performed in live-cell mode of LSM 780 confocal microscope (Zeiss) with inbuilt temperature control module . T-PMT was used to capture bright field images of the cells. The lambda scanning for fluorescence emission spectra of the intracellular CND was carried out with confocal microscope (Zeiss LSM 780). All chemicals and reagents like MTT -2,5-diphenyltetrazolium bromide), sodium acetate, sodium chloride, sodium bicarbonate, Dimethylsulfoxide (DMSO), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), DMEM media were procured from Sigma chemical Inc. (USA). Fetal Bovine Serum (FBS) was purchased from GIBCO Laboratories, USA. Polar star Omega Plate Reader Spectrophotometer-BMG LABTECH, Germany was used to measure spectrometric reading. The IC50 values were calculated using graphpad prism 6. MTT -2, 5-diphenyltetrazolium bromide) assay, a colorimetric assay was employed to measure cellular metabolite activity and hence the cell viability. Viable cells that contain NAD(P)H-dependent oxidoreductase enzymes reduce the MTT reagent to formazan, to give a deep purple color. For MTT cytotoxicity assay, MCF-7 (Human breast adenocarcinoma cell line) and CHO (Chinese Hamster ovary cell line) cells were cultured in DMEM medium containing 10% FBS and grown up to 60–70% confluence at 37 °C and 5% CO2 in an incubator. After trypsinization, 7 × 103 cells were seeded in 96 well tissue culture plates and allowed to grow monolayer after incubation overnight at 37 °C and 5% CO2. MCF-7 and CHO cells were treated with different concentrations of carbon nanodots  and incubated for 18 h. Vehicle control was set up wherein cells were treated with formulation without carbon nanodots. Control cells were incubated in cell culture medium. All treatments were carried out in quadruplicate (n = 4). After 24 h, MTT solution ) was added to each well for dissolving the formazan crystals. The plate was kept on plate shaker in dark. After 30 min, absorbance was measured at 570 nm that corresponds to viability of cells. Absorbance at 600 nm was used as a reference wavelength. The percentage of dead cells was calculated as  of vehicle control sample − OD of treated sample/OD of control sample] * 100) and represented as mean ± Standard Deviation (SD) (n = 4). This was plotted against different log concentrations of the CND formulation and half maximal inhibitory concentration (IC50) was calculated. MCF-7 cells were incubated with 1075.6 µg/mL of CND for 24 h for its internalization. After 24 h, cells were washed with PBS, pH 7.4 and acid stripping buffer , pH 3.5) alternatively twice before suspending cells in 1 mL PBS, pH 7.4. This procedure was carried out to remove supernatant and membrane bound CND in the cells after internalization. The photoluminescence intensity (PL) was measured at excitation 544 nm and emission 590 nm, with 10 °C increase in temperature from 25 to 45 °C.MCF-7 (human breast cancer) and MCF-10 A cell lines were purchased from ECACC (Europe) while CHO cells were purchased from National Centre for Cell Sciences, Pune, India and maintained in high glucose containing Dulbecco’s Modified Eagle Medium (DMEM)  at 37 °C under 5% COSupplementary Information.


There was a significant variation in MCs’ physical structure and surface morphology, which was evaluated using energy dispersive X-ray analysis (EDX), X-ray diffractometer (XRD), high-resolution transmission electron microscopy (HR-TEM), and scanning electron microscope (SEM). The anti-proliferative activity of MCs was examined by MTT assay and DAPI staining using human colorectal carcinoma cells (HCT-116), human cervical cancer cells (HeLa), and a non-cancerous cell line—human embryonic kidney cells (HEK-293). Post 72 h treatment, MCs caused a dose dependent inhibition of growth and proliferation of HCT-116 and HeLa cells. Conversely, no cytotoxic effect was observed on HEK-293 cells. The anti-fungal action was assessed by the colony forming units (CFU) technique and SEM, resulting in the survival rate of Candida albicans as 20%, with severe morphogenesis, on treatment with MCs x = 1.0. These findings suggest that newly engineered microspheres have the potential for pharmaceutical importance, in terms of infectious diseases and anti-cancer therapy.The current study offers an efficient design of novel nanoparticle microspheres (MCs) using a hydrothermal approach. The Co Nanomaterials are promising materials for various therapeutic applications, especially in diagnosing and treating diverse types of cancers and infectious diseases. Metal-based treatments are known to be an attractive research field in medicinal chemistry. Although platinum-based therapy is currently used, it is accompanied by resistance, deterrent side effects and a deficit of selectivity, forcing researchers to find more safe metals . For exaPreviously, it has been shown that combining two or more nanoparticles is an effective strategy to synthesize nanocomposites for targeted drug delivery and anti-cancer treatment ,28,29,302O4 microspheres have the capability to attach to negatively charged bacterial pathogens  [E. coli, and immobilize it on their surface with an efficiency of 90% [xFe3−xO4  microspheres for magnetic targeting and microwave heating, that can therefore be used for targeted and controllable drug delivery [The microsphere structure has attracted more attention due to low density, good dispersion, high specific surface area, high surface activity, strong permeability, good stability, super paramagnetic, etc. ,32,33,34E. coli) . Others y of 90%  (Huang ey of 90% . Taking y of 90% . Likewisdelivery .0.5Ni0.5GaxFe2−xO4 (0.0 ≤ x ≤ 1.0) MCs by hydrothermal approach. The MCs structure and morphology were evaluated by using XRD, EDX, TEM, and SEM techniques. The impact of Co0.5Ni0.5GaxFe2−xO4 (0.0 ≤ x ≤ 1.0) MCs was examined using colon cancer HCT-116 and cervical cancer HeLa by using MTT assay. In addition, the anti-fungal effects of Co0.5Ni0.5GaxFe2−xO4 (0.0 ≤ x ≤ 1.0) MCs were also evaluated on Candida cells.In the present study, the elements such as cobalt, nickel, gallium, and iron oxide were combined, and gallium was substituted with cobalt and nickel, to prepare Co2O and C2H5OH (ethanol), respectively, and then dried at 60 °C. For the synthesis of Co0.5Ni0.5GaxFe2−xO4 (0.0 ≤ x ≤ 1.0) MCs, the stoichiometric amount of carbon sphere template and nitrate salts of Fe, Co, Ni, and Ga were dissolved in 50 mL of deionized water under continuous stirring, followed by sonication for approximately 30 min. Afterwards, the homogeneous solution was transferred into a Teflon-lined stainless steel autoclave and sealed to heat at 180 °C for 10 h. The final products were washed with distilled water and ethanol and dried at 60 °C. Finally, the solid product was heated at 500 °C for 4 h to get the Co0.5Ni0.5GaxFe2−xO4 (0.0 ≤ x ≤ 1.0) MCs. The characterization of synthesized nanomaterials was done as per the protocol by Almessiere et al. [Carbon microspheres were prepared hydrothermally using 1M glucose aqueous solution in a Teflon-lined autoclave, at 180 °C for 10 h. The black solid was obtained by centrifuging, washed many times with deionized H0.5Ni0.5GaxFe2−xO4 (0.0 ≤ x ≤ 1.0) MCs on cell viability and cell proliferation. A non-cancer cell line, such as embryonic kidney cells (HEK-293), was used as control cell line and to examine the specificity of the MCs. As per the previously described method [2 incubator for 4 h. Later, the cell culture media was replaced with DMSO (1%), and the plate was examined at a wavelength of 570 nm by using a Plate reader . The percentage of cell viability was calculated for the statistical analysis [Human colorectal carcinoma (HCT-116) and human cervical cells (HeLa) were taken for the study of the impact of Cod method ,41, the analysis ,43.0.5Ni0.5GaxFe2−xO4 (0.0 ≤ x ≤ 1.0) MCs (50 µg/mL) were added. Seventy-two hours post-treatment, both groups were exposed to paraformaldehyde and then washed with phosphate-buffered saline (PBS). Cells were then stained with DAPI for 5 min, and examined by using confocal scanning microscope  [DAPI  staining assay was done to examine cancer cell DNA. The cells were divided into two groups: the control group, in which no MCs were added, and the experimental group where CoGermany) ,43. The Candida albicans ATCC14053 (yeast) was chosen for the anti-fungal assay of the prepared Co0.5Ni0.5GaxFe2−xO4 (0.0 ≤ x ≤ 1.0) MCs. C. albicans was aerobically grown using Sabouraud broth (SDB), at 28 ± 2 °C for 24 h with shaking. Subsequently, the cell density was adjusted to approximately 107 CFU/mL. For the nanomaterial sample preparation, a concentration of 20 mg/mL of synthesized MCs was used after sonication for 10 to 15 min, to get a suspended solution.C. albicans was added to the MCs solution and incubated in a rotary shaker for 48 h at 28 ± 2 °C. Untreated C. albicans was used as a control. On the completion of the incubation period, 100 µL of culture was plated out onto the SDA plates and incubated as mentioned above. On the other day, the plates were observed and recorded for the number of colonies on each plate. Each colony counted is taken to have emerged from a single viable Candida cell. The plates having colonies above 400 were considered as a lawn culture. The survival percentage of C. albicans was calculated by the formula S% = (A/B) × 100  [To demonstrate the anti-candida activity of MCs, colony-forming units (CFU) were used to evaluate the viable microbial cell concentrations in a sample, post treatment. Briefly, sterile tubes containing different concentrations of nanomaterial in SDB, were sonicated for 10 to 15 min to get the suspended solution. An adjusted inoculum of control) . The funcontrol) . The dat0.5Ni0.5GaxFe2−xO4 (0.0 ≤ x ≤ 1.0) MCs was offered through X-ray diffractometer, as seen in 0.5Ni0.5GaxFe2−xO4  MCs. The samples showed an accumulation of the cubic shape particles. The elemental composition (EDX) of Co0.5Ni0.5GaxFe2−xO4 (x = 0.2) MCs is shown in The structure analysis of Co0.5Ni0.5GaxFe2−xO4 (0.0 ≤ x ≤ 1.0) MCs on both colon cancer (HCT-116) and cervical cancer (HeLa) cells was examined. The cell viability assay confirmed a significant decrease in the cell viability after the treatments of Co0.5Ni0.5GaxFe2−xO4 (0.0 ≤ x ≤ 1.0) MCs. The treatments of Co0.5Ni0.5GaxFe2−xO4 MCs (0.0 ≤ x ≤ 1.0) showed inhibitory action on cancer cell growth and proliferation. The calculated inhibitory concentration (IC50) of MCs was 25 µg/mL to 59 µg/mL for HCT-116 and 35 µg/mL to 60 µg/mL for HeLa cells. The impact of MCs on non-cancerous cells (HEK-293) was also tested. There was a decrease in the cancer viability, but the percentage of the decrease was not statistically significant (0.5Ni0.5GaxFe2−xO4 (0.0 ≤ x ≤ 1.0) MCs possess an inhibitory effect on HCT-116 and HeLa cells compared to HEK-293 cells. This is the first study that demonstrates the inhibitory effect on cell viability of synthesized Co0.5Ni0.5GaxFe2−xO4 (0.0 ≤ x ≤ 1.0) MCs against HCT-116 and HeLa cells.The anti-proliferative impact of Conificant . Based oWe previously reported the impact of different nanomaterials on colon and breast cancer cells ,46,47. T0.5Ni0.5GaxFe2−xO4 (0.0 ≤ x ≤ 1.0) MCs (50 µg/mL) caused a significant decrease in the number  (The treatment of Cols/well) B–G of Hels/well) A. The de1.0) MCs  µg/mL caCandida in the inoculated media added with nanomaterial was investigated as the potential of the synthesized microspheres. After plating the harvested treated culture on SDA plates for 24 h, the existing colonies of Candida were observed. The survival rate of Candida is shown in Candida was reduced with the increasing ratio of microspheres, i.e., the maximum effect was shown by x = 1.0 with a survival rate of 20%, followed by 0.8 and 0.6, with 57 and 75%, respectively. Other ratios were not found to be significantly useful and showed an approximately 100% survival rate, that was similar to control, i.e., the untreated Candida. As shown in Candida). This indicates that the increased ratios of microspheres had an impact on the growth of the organism, where maximum growth reduction was seen with samples x = 1.0, x = 0.8, and x = 0.6. Using one-way ANOVA, the variation in the survival rates of C. albicans when using a different concentration of nanomaterial was found to be significant (p < 0.001).The anti-candida action of MCs was assessed by CFU technique. The varying concentration of test material was taken into consideration. The survival percentage of C. albicans caused by the treatment of MCs was also studied by using SEM. The image  microspheres (MCs) were prepared using the hydrothermal approach, and the structures were confirmed using XRD, EDX, HR-TEM, TEM, and SEM techniques. The anti-proliferative impact of Co0.5Ni0.5GaxFe2−xO4 (0.0 ≤ x ≤ 1.0) MCs was examined by using MTT assay that showed a dose-dependent inhibition of cancer cells . Conversely, no cytotoxic effect was observed on the normal cell line (HEK-293). The treatment of Co0.5Ni0.5GaxFe2−xO4 (0.0 ≤ x ≤ 1.0) MCs also caused nuclear DNA disintegration in the cancer cells, as revealed by DAPI staining. Besides, the anti-fungal action was assessed by the CFU technique, and the survival rate of C. albicans was found to be reduced with the increasing ratio of Co0.5Ni0.5GaxFe2−xO4 (0.0 ≤ x ≤ 1.0) MCs. These findings suggest that the synthesized Co0.5Ni0.5GaxFe2−xO4 (0.0 ≤ x ≤ 1.0) MCs possess potential anti-fungal and anti-cancer capabilities, to be considered for potential pharmaceutical applications.The Co


Tebuconazole (TEB), a triazole fungicide, is frequently applied to agriculture for the increase of food production. Although TEB causes liver toxicity, its effects on cellular lipid accumulation are rarely investigated. Therefore, this study aimed to study the effects of TEB on lipid metabolism and accumulation in HepG2 cells. HepG2 cells were exposed to 0–320 µM TEB for 1–24 h. TEB -treated cells showed lipid accumulation. Further, TEB  increased the nuclear translocation of peroxisome proliferator-activated receptors and the expression of lipid uptake and oxidation-related markers such as cluster of differentiation 36, fatty acid transport protein (FATP) 2, FATP5, and carnitine palmitoyltransferase 1. Oxidative stress levels in TEB-treated cells  were higher, compared to those in the control. TEB  also induced the loss of mitochondrial membrane potential and lower levels of microsomal triglyceride transfer protein in the cells. Thus, TEB can induce lipid accumulation by altering the expression of lipid-metabolizing molecules and can therefore impair lipid metabolism. Our data suggest that human exposure to TEB may be a risk factor for non-alcoholic fatty liver disease. Tebuconazole (TEB), a triazole fungicide, is often applied to agriculture for the increases of crop yield and food production . TEB actde novo lipogenesis, oxidation of fatty acids, and transport of lipids in very low-density lipoproteins (VLDL) [Ingested TEB residues are absorbed in the gastrointestinal tract and are mainly metabolized to TEB-1-hydroxy and TEB-carboxylic acid via CYP-mediated oxidation in the liver for excretion into urine and feces . Howevers (VLDL) . Howevers (VLDL) .NAFLD is defined as fat accumulation of no less than 5% in the liver without secondary factors, such as alcohol or drugs . The preTaken together, despite growing concerns regarding the adverse effects of TEB residues in drinking water and food on human health, the effects of TEB on hepatic lipid accumulation and lipid metabolism have not been entirely elucidated. Thus, this study aimed to examine the effect of TEB on NAFLD development and progression, particularly lipid accumulation and lipid metabolism-related mechanisms in HepG2 cells.TEB was purchased from Sigma-Aldrich . Dulbecco’s modified Eagle’s medium, fetal bovine serum, penicillin/streptomycin, and 0.05% trypsin and 0.53 mM ethylenediaminetetraacetic acid solution were obtained from Welgene Inc. . Phosphate-buffered saline (PBS) was purchased from Lonza . N-acetylcysteine (NAC) and dimethyl sulfoxide (DMSO) were obtained from Amresco . Lipofermata was purchased by Cayman Chemical Co. . The Pierce bicinchoninic acid protein assay kit was provided by Sigma-Aldrich . Nitrocellulose membranes and enhanced chemiluminescence reagents were supplied by GE Healthcare Bio-Sciences  and Thermo Fisher Scientific , respectively.2 incubator containing 5% CO2 at 37 °C. The culture medium was changed every 2 days, and the cells were sub-cultured into 96-well plates, 6-well plates, and T-25 flasks  when they reached 80–90% confluency using 0.05% trypsin and 0.53 mM ethylenediaminetetraacetic acid. For the experiment, TEB was dissolved in DMSO at a concentration of 40 mM. The cells were treated with 0–320 µM TEB or vehicle (DMSO) control for 1–24 h. The concentration range of TEB for cell treatment was chosen from previous studies [Human liver HepG2 cells  were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in CO studies ,18.n = 3 wells/group). The media were replaced with 100 µL of fresh media and were added with 10 µL MTT solution (5 mg/mL in PBS). Subsequently, the cells were incubated in a CO2 incubator containing 5% CO2 at 37 °C for 3 h. After discarding 90 µL MTT solution, 180 µL acidic isopropanol was added into each well, and the plate was placed at 37 °C for 1 h. The optical density (OD) was measured at 570 nm and 630 nm using an Epoch spectrophotometer , and the percentage of cell viability was calculated with the following Formula (1):An MTT assay was conducted to evaluate cell viability using 3--2,5-diphenyl-2H-tetrazolium bromide , as described previously . HepG2 w® non-radioactive cytotoxicity assay kit . HepG2 cells were cultured in a 96-well plate and exposed to various concentrations of TEB  or DMSO for 24 h (n = 3 wells/group). To measure the maximum LDH release, 10× lysis solution was supplemented to the wells about 45 min before the end of the TEB treatment. Then, 50 µL of supernatants were transferred into a new 96-well plate. The 96-well plate was incubated at 25 °C for 30 min after adding the CytoTox 96® reagent of 50 µL to each well. The 50 µL of stop solution was then added to each well. The OD was measured at 490 nm using a spectrophotometer, and the percentage of LDH release was calculated with the following Formula (2):An LDH release assay was conducted to evaluate the cell membrane integrity using the Cytox 96n = 3 wells/group). The cells were fixed with 10% formalin at 25 °C for 1 h, and then rinsed with 60% isopropanol. Oil Red O staining solution (1 mL) was added to each well. After incubation for 10 min, the cells were washed thrice with deionized distilled water for the removal of the unbound dye. The stained areas were imaged and captured using a Nikon Eclipse Ti2-U and Nikon Eclipse Ts2R camera, respectively . The quantified levels of captured images were assessed using ImageJ software version 1.52a .Intracellular triglycerides and cholesterol esters were determined using Oil Red O . HepG2 cells were grown in 6-well plates and treated with 20, 40, and 80 µM TEB or DMSO for 24 h with or without NAC pretreatment (5 mM in PBS) for 1 h (n = 3 wells/group). The cells were washed thrice with PBS and lysed using a hypotonic buffer containing 20 mM Tris (pH 7.4), 10 mM sodium chloride, 3 mM magnesium chloride, and protease inhibitor cocktail . The cell lysates were centrifuged at 600× g at 4 °C for 10 min after adding Triton X-100 to the cell lysates for the extraction of cytosolic proteins. The nuclear proteins were extracted using a cell extraction buffer containing 100 mM Tris (pH 7.4), 1 mM ethylenediaminetetraacetic acid, 2 mM sodium orthovanadate, 100 mM sodium chloride, 1 mM ethylene glycol-bis(β-aminoethyl ether)-N,N, N′N′-tetra acetic acid, 10% glycerol, 10% sodium dodecyl sulfate, 1 mM sodium fluoride, 0.5% sodium deoxycholate, 1% Triton X-100, 20 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 1 mM aprotinin, and 1 mM leupeptin. After resuspending the pellets using the cell extraction buffer, the samples were centrifuged at 14,000× g at 4 °C for 30 min. The protein concentrations were then analyzed using a bicinchoninic acid protein assay kit, and the samples were used for Western blotting.Protein levels of nuclear PPARγ and α were evaluated by nuclear fractionation. HepG2 cells were grown in 6-well plates and then treated with 20, 40, and 80 µM TEB or DMSO for 1 and 12 h (n = 3 wells/group). The cells were lysed using a radioimmunoprecipitation assay buffer . The cell lysates were then centrifuged at 18,000× g at 4 °C for 20 min. The protein concentrations were analyzed using a bicinchoninic acid protein assay kit. The protein samples (20 µg) were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The -separated proteins were transferred onto a nitrocellulose membrane. After that, 3% nonfat milk buffer was used for blocking the membrane for 1.5 h at 25 °C. The membrane was incubated with the primary antibodies: anti-PPARα , anti-PPARγ , anti-cluster of differentiation 36 (CD36) , anti-fatty acid transporter protein (FATP) 2 , anti-microsomal triglyceride transfer protein (MTTP) , anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) , and anti-Lamin B  at 4 °C from overnight to 3 days. After washing thrice with Tris-buffered saline with Tween 20, secondary antibodies  and donkey anti-goat IgG  conjugated with horseradish peroxidase) were incubated for 1.5 h at 25 °C. Subsequently, the membrane visualized using enhanced chemiluminescence detection reagents. The quantified levels of protein expression were assessed using ImageJ software version 1.52a .Protein levels of the lipid metabolism associated cell signaling pathways were measured using Western blotting, as described previously . HepG2 cn = 3 wells/group). RNA samples from the cells were extracted using TRIzol reagent , and total RNA (2 µg) was reverse transcribed using the TOPscript RT DryMIX kit  to synthesize cDNA. Gene expression levels were analyzed using an RT-PCR system  and 2× RT-PCR Smart mix . The thermal cycling conditions were as follows: 95 °C, 15 min and then 60 cycles of denaturation , annealing , and extension . The 2−ΔΔCt method was used to quantify gene expression levels. GAPDH gene expression levels were used as a control for normalization. The primers  were designed using the AmplifX software version 1.7.0   and NAC (5 mM in PBS) for 1 h (il 2021) .n = 3 wells/group). After treatment with 10 µM DCFDA and DHE, the cells were incubated at 37 °C for 30 min in a 5% CO2 incubator. After washing thrice with PBS, green fluorescence (DCFDA-positive) and red fluorescence (DHE-positive) areas were imaged using a Nikon Eclipse Ti2-U and Nikon Elipse Ts2R camera. Fluorescence intensities were quantified using ImageJ software version 1.52a .Intracellular hydrogen peroxide and superoxide anion levels were evaluated using 2′,7′-dichlorofluorescin diacetate  and dihydroethidium . Briefly, HepG2 cells were cultured in 6-well plates and exposed to 20, 40, and 80 µM TEB or DMSO for 24 h with or without pretreatment with Lipofermata (20 µM in DMSO) for 1 h  and exposed to 20, 40, and 80 µM TEB or DMSO for 24 h with or without pretreatment with Lipofermata (20 µM in DMSO) (n = 3 wells/group). The JC-10 dye-loading solution of 50 µL was used to stain the cells, with incubation for 45 min in the dark. Assay buffer B (50 µL) was then added to each well, and the integrity of the MMP was evaluated as the ratio of the fluorescence of JC-10 aggregates (Ex/Em = 490/525 nm) and monomeric JC-10 (Ex/Em = 540/590 nm). The intensity of fluorescence was analyzed using a SpectraMax Gemini EM microplate reader , and the percentage of MMP loss was calculated as follows (3):MMP (ΔΨm) was estimated using the JC-10 mitochondrial membrane potential assay kit , according to the instruction of the manufacturer. HepG2 were grown in Corningt-test. Statistical significance was defined at p < 0.05.Data are presented as mean ± standard error of the mean. The data were analyzed using SPSS-PASW statistics software version 18.0 for Windows . The significance was analyzed using a one-way ANOVA with post hoc Dunnett’s test and Student’s p > 0.05) (p < 0.01). In addition, no significant difference was observed in cell viability and LDH release between the control (no treatment) and vehicle control (DMSO) (p > 0.05). Therefore, TEB concentrations of 20, 40, and 80 µM, which showed no cytotoxicity, were used for subsequent experiments.HepG2 cells exposed to TEB for 24 h did not show alterations in cell viability or LDH release at concentrations up to 80 µM ( > 0.05) A,B. Howep < 0.01)  showed prominent lipid accumulation, compared to the control A. Furthe < 0.01) B.p < 0.05) (p < 0.05) (p < 0.05)  significantly increased the translocation of PPARγ from the cytosol to the nucleus, compared with the control cells ( < 0.05) A. Furthe < 0.05) B. TEB al < 0.05) C.p < 0.05) (p < 0.05) (p < 0.05).Cells exposed to 40 and 80 µM TEB for 12 h showed significantly increased nuclear translocation of PPARα, compared to the control cells ( < 0.05) D. In add < 0.05) E. Howevep < 0.05) (p < 0.05) (p < 0.05).HepG2 cells exposed to 20, 40, and 80 µM for 24 h showed 2.4-, 3.5-, and 8.0-fold higher levels of intracellular hydrogen peroxide than the control ( < 0.05) A,B. Intr < 0.05) A,C. Howep < 0.05) (p < 0.05).The treatment of cells with TEB (40 and 80 µM for 24 h) markedly increased the loss of MMP, compared to that in the control cells ( < 0.05) A. Howevep < 0.05) (p < 0.05) (p < 0.05)  showed decreased protein and mRNA levels of MTTP, compared to the control ( < 0.05) B,C. Howe < 0.05) B,C. More < 0.05) D,E.Despite increasing concerns about pesticide residues in food products and water, various pesticides have been widely used to control plant diseases and improve crop yield. A previous study showed that insecticide exposure impaired lipid metabolism, thereby resulting in the development of NAFLD . MoreoveNAFLD development is initiated by the accumulation of lipids in the liver due to the imbalance between lipid uptake and export. Therefore, we first evaluated lipid accumulation at concentrations of up to 80 µM TEB, which did not affect the cell viability and membrane integrity of HepG2 cells. Our results indicated that TEB-treated cells had significantly higher lipid accumulation. Furthermore, TEB-treated cells showed increased protein and gene expression levels of CD36 and FATP with the nuclear translocation of PPARγ. According to a previous study, TEB induced triglyceride accumulation in HepaRG cells through the pregnane X receptor ,26. The Fatty acids that enter the cytoplasm through facilitation by CD36 and FATP undergo oxidation to generate energy . Fatty aLipid export is the only means for the decrease of liver lipid content in addition to mitochondrial β-oxidation . HoweverCollectively, our data demonstrated that TEB exposure can induce lipid accumulation in HepG2 cells by increasing lipid uptake, generating oxidative stress via excessive activation of mitochondrial β-oxidation, and impairing lipid export. Cellular oxidative stress through mitochondrial β-oxidation was identified as a major pathway in the disruption of lipid metabolism. Although further in vivo studies are required to elucidate the effect of TEB on lipid accumulation and lipid metabolism, our data suggest that TEB exposure in humans can be a risk factor for NAFLD development and progression.


The facial markers included inferior facial angle (IFA), maxilla-nasion-mandible (MNM) angle, facial maxillary angle (FMA) and profile line (PL) distance. These markers were measured through ViewPoint 6 software by two experienced sonographers.Three hundred and eighty fetuses were selected. The ICCs (95 % CI) of intra-operator 1 reproducibility of IFA, MNM angle, FMA, PL distance were 0.944 (0.886 ~ 0.973), 0.804 (0.629 ~ 0.902), 0.834 (0.68 ~ 0.918) and 0.935 (0.868 ~ 0.969), respectively. The ICCs (95 % CI) of intra-operator 2 reproducibility of IFA, MNM angle, FMA, PL distance were 0.931 (0.857 ~ 0.967), 0.809 (0.637 ~ 0.904), 0.786 (0.600 ~ 0.892) and 0.906 (0.813 ~ 0.954), respectively. The ICCs (95 % CI) of inter-operator reproducibility of IFA, MNM angle, FMA, PL distance were 0.885 (0.663 ~ 0.953), 0.829 (0.672 ~ 0.915), 0.77 (0.511 ~ 0.891) and 0.844 (0.68 ~ 0.925), respectively. The average ± SD of IFA, MNM angle, FMA and PL distance were 80.2°±7.25°, 4.17°±1.19°, 75.36°±5.31°, 2.78 ± 0.54 mm, respectively. IFA and PL distance significantly decreased with CRL, while MNM angle and FMA significantly increased with CRL.It was feasible to measure fetal facial markers during the first trimester. In Chinese population, the reference ranges of IFA, MNM angle, FMA and PL distance were 80.2°±7.25°, 4.17°±1.19°, 75.36°±5.31°, 2.78 ± 0.54 mm, respectively, and the measurements were found to correlate with CRL. Actually most facial structures of the fetus have been differentiated during the first trimester [Fetal facial malformations mainly include cleft lip and palate (CLP), micrognathia, maxillary dysplasia, and absence of nasal bone, which are closely related to some chromosomal abnormalities or genetic syndrome , 2. Withrimester , so it irimester . The gui Images of the first trimester ultrasound screening peformed in the Affiliated Suzhou Hospital of Nanjing Medical University between August 2017 and July 2019, which best met inclusion criteria and have high recognizable structure, were retrospectively selected. The pregnancy outcome was followed-up by the Suzhou Maternal-children health care system.+6 weeks’ gestation of the Fetal Medicine Foundation (FMF). The forehead, nasal bone, palate, mandible, upper lip, lower lip and other structures should be clearly displayed in this section.The inclusion criteria were as follows: (1) both parents of the fetus were Chinese; (2) pregnancies with significant maternal complications were excluded; (3) singleton pregnancy; (4) fetuses with normal ultrasound findings and normal follow-up outcomes; (5) the selected two-dimensional ultrasound (2D-US) images were the standard mid-sagittal section for measuring nuchal translucency (NT) thickness, which met the standardized protocol at 11 ~ 13 The study was approved by the Ethics Committee of Suzhou Municipal Hospital.A Philips Affiniti70 and a GE Voluson E10 four-dimensional (4D) color ultrasound machines were utilized in this study; each was equipped with a convex probe of C9-2 and C5-1, with the frequency of 2 ~ 9 MHz and 1 ~ 5 MHz, respectively. The images obtained by transabdominal ultrasound examination were imported into the ultrasound workstation software, ViewPoint 6 in DICOM  format. In addition, fetal facial markers were measured through ViewPoint 6.IFA  was defiFetal facial markers  were measured through ViewPoint 6 software by two experienced sonographers, who had obtained the FMF certification for NT scan. The average value of each marker was taken after three measurements. Generally speaking, it takes every 1.5-2 min to measure each of the four markers once, and every 5-6 min for three times.The analysis was performed by SPSS21.0  and Graphpad Prism8.0. The intraclass correlation coefficient (ICC) and Bland-Altman analysis  were useAmong 3520 fetuses who underwent the first trimester ultrasound screening, 380 were selected because they met the inclusion criteria. The successful rate of measuring all four facial markers is almost 100 %. The maternal age was 28 (4) years old. The CRL was 66 (10) mm, and the NT thickness was 1.80 (0.5) mm. The distribution of fetal cases in each gestational week is shown in Fig. Thirty ultrasonographic images in fetal facial mid-sagittal section were randomly selected from 380 fetuses that met the inclusion criteria to evaluate the reproducibility and feasibility of the measurement. The ICCs (95 % CI) of intra-operator 1 reproducibility of IFA, MNM angle, FMA, PL distance were 0.944 (0.886 ~ 0.973), 0.804 (0.629 ~ 0.902), 0.834 (0.68 ~ 0.918) and 0.935 (0.868 ~ 0.969), respectively. The ICCs (95 % CI) of intra-operator 2 reproducibility of IFA, MNM angle, FMA, PL distance were 0.931 (0.857 ~ 0.967), 0.809 (0.637 ~ 0.904), 0.786 (0.600 ~ 0.892) and 0.906 (0.813 ~ 0.954), respectively. The ICCs (95 % CI) of inter-operator reproducibility of IFA, MNM angle, FMA, PL distance were 0.885 (0.663 ~ 0.953), 0.829 (0.672 ~ 0.915), 0.77 (0.511 ~ 0.891) and 0.844 (0.68 ~ 0.925), respectively.Table p < 0.001). IFA was Gaussian distributed, and its reference range was 65.99°~94.41° (mean ± 1.96SD).The measurement range of IFA was 55.9°~107.89° (80.2°±7.25°). IFA had significant negative correlation to CRL during the first trimester . The MNM angle was Gaussian distributed, and the reference range of the MNM angle was 1.84°~6.50° (mean ± 1.96SD).The measurement range of MNM angle was 1.66°~9.21° (4.17°±1.19°). The MNM angle had significant positive correlation to CRL during the first trimester . FMA was Gaussian distributed, and the reference range was 64.95°~85.77° (mean ± 1.96SD).The measurement range of FMA was 56.29°~89.59° (75.36°±5.31°). FMA depended significantly on CRL . The PL distance was Gaussian distributed, and its reference range was 1.72 ~ 3.84mm (mean ± 1.96SD).The measurement range of PL distance was 1.53 ~ 4.37mm (2.78 ± 0.54mm). The PL distance decreased with CRL , is the most common chromosomal abnormality and is accompanied by different degrees of mid-facial hypoplasia and skin edema , 13. ComWith the rapid development of science and technology, NIPT  has become the first choice for screening chromosomal aneuploidy due to its non-invasiveness, high sensitivity, and high specificity . To someThe embryonic development of facial bones has its main characteristics. The maxilla and mandible begin to ossify from 8 weeks onward . The maxIn 2002, Rotten et al.  studied The MNM angle could reflect the relative position of the maxilla and mandible, further to evaluate fetal facial profile. De Jong-Pleij et al.  reportedFMA can directly reflect the relative position of the maxilla and mandible to evaluate fetal facial profile. In order to avoid the influence of the curvature of the vomer, Lu et al.  used theDe Jong-Pleij et al.  showed tThere were some limitations in our study. Firstly, it is a retrospective study, and the static image selected from the first trimester ultrasound screening was to measure the NT thickness rather than observe facial abnormalities. Secondly, the same stored ultrasound image was used by the two operators to assess the inter-operator reproducibility, and thus accounted at least partly for the high ICC. Thirdly, facial abnormalities with or without aneuploidies were not included. In addition, all parameters were measured on 2D images, without the use of 3D reconstructed techniques. Some research , 14 show+ 6 weeks’ gestation). It’s feasible to measure these markers during the first trimester. The reference range of each marker was obtained through large sample data, and these markers were significantly related to CRL. If the measurement is not in this range, micrognathia and other facial abnormalities should be highly suspected. On this basis, further studies will be carried out to investigative the detection rate and false positive rate of these markers for facial abnormalities with or without aneuploidies. However, their clinical application shall be cautious. A large multi-center prospective cohort and enough positive cases are required.Intra-operator and inter-operator reproducibility for facial profile markers  were good during the first trimester (11 ~ 13


The studied patients were categorized into five groups based on eGFR, i.e., the relatively stable (RS), fast decline (FD), modest decline (MD), modest increase (MI), and fast increase (FI) groups.A total of 2,599 patients with T2D were enrolled if they were registered in the Diabetes Sharecare Information System, were aged 18–75 years, and had 2–3 HbA2 for patients in the FD, MD, RS, MI, and FI groups, respectively. Up to 1,153 (44.4%) subjects experienced an eGFR decline of ≥3.5 ml/min per 1.73 m2, including 821 (31.6%) FD subjects and 332 (12.8%) MD subjects. A decreased trend was found between the eGFR change and HbA1c decrease category, even after multivariable adjustment. In general, an eGFR FD was frequently found in patients who had an HbA1c reduction of ≥3.00% and a baseline HbA1c ≥8.0%; alternatively, such a result was also observed for a urinary albumin-to-creatinine ratio (UACR) of 30.0–300.0 mg/g, regardless of a diabetes duration of <10.0 or ≥10.0 years, or in patients who had an HbA1c reduction of ≥1.00% accompanied by hyperfiltration.The median eGFR changes from baseline were −22.14, −6.44, 0.00, 6.32, and 20.00 ml/min per 1.73 m1c reduction, independent of UACR stage, diabetes duration, and hyperfiltration. Sustained monitoring and cautious interpretation of the HbA1c and eGFR changes will be needed in clinical practice.Some patients with T2D experienced an eGFR FD or MD during the ≤12-month follow-up period. A significant downward trend in eGFR change was demonstrated alongside an HbA This DSIS was developed for diabetic patients’ registration, follow-up, and preservation of clinical and biochemical measurements. Only patients who provided oral consent for inclusion in the study were allowed to be registered. RDCHs began using DSIS in 2016. All data were aggregated for each person after registration (baseline) and during each follow-up visit. The analysis was based on individuals with a documented T2D onset and clinical visits at the baseline measurement and second/third follow-ups. The Declaration of Helsinki guidelines were followed while conducting this study. The research was approved by the Ethics Committee of Beijing Ruijing Diabetes Hospital. Due to the nature of the study , patient consent was not required.1c and eGFR values at the 12-month follow-up visits. Patients were excluded if they had a history of T1D, gestational and/or secondary diabetes, a malignant tumor, blindness, dialysis, diabetic foot ulcers, other kidney diseases; no medical records of baseline HbA1c, eGFR, or body mass index (BMI) and eGFR values <30.00 or ≥200 ml/min per 1.73 m2 at the baseline or follow-up visits; or uncontrolled blood pressure , a triglyceride (TG) level ≥9 mmol/L   or third records of HbAs blood) , and a Us blood) Figure 11c measurements were tested with high-performance liquid chromatography using HA-8180  and MQ-2000PT  chromatograph, standardized according to the Diabetes Control and Complications Trial (1c and serum creatinine were obtained from the laboratory database from the time of cohort entry to the second (if no third) record or third follow-up exam. BMI was categorized as not overweight/obese: <24.0 kg/m2 and overweight/obese: BMI ≥24.0 kg/m2. The target LDL-C was <2.60 mmol/L; target blood pressure (BP) was <130/80 mmHg  , where eGFR change <−10.00 ml/min per 1.73 m2; a moderate decline (MD) group, where eGFR change was −10.00 to −3.50 ml/min per 1.73 m2; a moderate increase (MI) group, where eGFR change was 3.50–10.00 ml/min per 1.73 m2; a fast increase (FI) group, where eGFR change was >10.00 ml/min per 1.73 m2  18–49 years, eGFR >120 ml/min per 1.73 m2; (2) 50–59 years, eGFR >110 ml/min per 1.73 m2; (3) 60–69 years, eGFR >100 ml/min per 1.73 m2; (4) 70–75 years, eGFR >90 ml/min per 1.73 m2 (The simplified (Chinese) MDRD equation was used to calculate the eGFR as follows: MDRD = 175× serum creatinine (mg/dl)  female) . Accordi 1.73 m2 . The CKDincluded . The hyp 1.73 m2 .Medication information was obtained on the registration date and during follow-up visits. Treatment for hyperglycemia was categorized into two groups, i.e., a non-insulin  and insulin (insulin only or insulin plus other management) usage. Whether the patients used sodium-glucose cotransporter-2 inhibitors (SGLT-2i), GLP-1RA, and angiotensin-converting enzyme inhibitors (ACEIs)/angiotensin receptor blockers (ARBs) was also categorized.1c changes were determined. Subgroup analyses were conducted according to baseline HbA1c category (HbA1c <8.0 and ≥8.0%), baseline hyperfiltration (yes or no), baseline BMI category (BMI <24.0 and ≥24.0 kg/m2), and diabetes duration (<10.0 and ≥10.0 years). Multivariable-adjusted general linear models  were reported separately. A two-sided P-value of <0.05 was considered to be statistically significant. All analyses were performed using the SPSS Statistics 22.0 software.Patient characteristics were summarized using mean and standard deviation and median and interquartile range or numbers (percentages). Additionally, 95% confidence intervals were reported. One‐way analysis of variance tests were used for normally distributed continuous variables, Kruskal–Wallis tests were used for non-normally distributed continuous variables, and Pearson’s chi‐square tests were used for categorical variables. Interactions with HbAThe final analytic cohort comprised 2,599 individuals with T2D, with a median age at entry of 59.7 years, diabetes duration of 8.2 years, and follow-up of 8 months. The proportion of patients that had a history of hypertension, dyslipidemia, diabetic retinopathy (DR), or DKD was 37.9%, 33.5%, 14.7%, and 9.7%, respectively Table 1.2 for patients in the FD, MD, RS, MI, and FI groups. Up to 1,153 (44.4%) of the study subjects experienced an eGFR decline of ≥3.5 ml/min per 1.73 m2, including 821 (31.6%) FD subjects and 332 (12.8%) MD subjects. The baseline eGFR, HbA1c, the age-adjusted prevalence of hyperfiltration decreased from group FD to FI, while HbA1c change increased gradually from group FD to RS (P < 0.001)  Table 2.1c decreasing groups . Patients who showed an HbA1c change of ≤−3.00% had the largest decline in eGFR (−12.61 ml/min per 1.73 m2) (1c <8.0% or ≥8.0%) of the association between HbA1c reduction ranges and eGFR change, no significant association was found when baseline HbA1c <8.0%; however, a significant association was found when baseline HbA1c ≥8.0% (P < 0.001). The HbA1c reduction played an important role in the eGFR decrease in subjects with hyperfiltration. The decline of eGFR reached −21.25 ml/min per 1.73 m2 when HbA1c reduced ≥3.00%. This association was not found in the subgroup with no hyperfiltration (P = 0.076). When comparing groups of baseline UACR stages of A1 (<30 mg/g) and A2 (30–300 mg/g) and groups of diabetes duration of <10.0 and ≥10.0 years, a significant association was found between HbA1c reduction range and eGFR change (P < 0.001). In general, eGFR FD was frequently found in patients with a baseline HbA1c ≥8.0% and an HbA1c change of ≤−3.00% or in patients with hyperfiltration and an HbA1c change of ≤−1.00% and patients with UACR stage A2 and an HbA1c change of ≤−3.00%. Regardless of a diabetes duration <10.0 or ≥10.0 years, eGFR FD was frequently found in patients with an HbA1c change of ≤−3.00% (A decreased trend in eGFR change was found among the HbA1.73 m2) Table 4.2 (16% reduction) after HbA1c dropped from 10% to 7% and increased significantly toward the baseline values along with HbA1c control deterioration in patients with T1D , in participants with prediabetes and was associated with significantly increased odds ratios (ORs) of hyperfiltration (1c levels and a 2.0 mmol/L increase in 2-h post-load glucose (2hPG) were associated with a 17% and 61.4% higher risk of hyperfiltration, respectively , however, followed by a slower rate of loss .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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Several reports have suggested that hippocampal PPARα is implicated in antidepressant responses. Here we speculate that hippocampal PPARα may participate in the antidepressant mechanism of vortioxetine. In this study, chronic unpredictable mild stress (CUMS), chronic social defeat stress (CSDS), behavioral tests, the western blotting and adenovirus associated virus (AAV)-mediated gene knockdown methods were used together. It was found that vortioxetine administration significantly reversed the inhibitory actions of both CUMS and CSDS on the hippocampal PPARα expression. Pharmacological blockade of PPARα notably prevented the antidepressant actions of vortioxetine in the CUMS and CSDS models. Moreover, genetic knockdown of PPARα in the hippocampus also significantly blocked the protecting effects of vortioxetine against both CUMS and CSDS. Therefore, the antidepressant effects of vortioxetine in mice require hippocampal PPARα.As a well-known multimodal-acting antidepressant, vortioxetine is thought to aim at several serotonin (5-HT) receptors and the 5-HT transporter. However, recently more and more proteins besides 5-HT are being reported to participate in the antidepressant mechanism of vortioxetine. As a widely known nuclear hormone receptor, peroxisome proliferator activated receptor  People who have major depressive disorder (MDD) experience various psychological symptoms including feeling of hopelessness, sadness, lack of motivation, and difficulties to concentrate . Due to Peroxisome proliferator activated receptor α (PPARα) is widely distributed in both the peripheral and central nervous system tissues . Early i3, 5-HT7 and 5-HT1D receptors, activate 5-HT1B and 5-HT1A receptors, and inhibit 5-HT transporter (SERT)  . Vortioxr (SERT) . Althoug3; bedding replacement twice a week). The behavioral experiments were carried out during the light phase. For animal sacrifice, all mice were anaesthetized using carbon dioxide and then killed by cervical dislocation. The experiment procedures involving animals and their care were conducted in accordance with the ARRIVE guidelines  were bought from SLAC Laboratory Animal Co., Ltd. . Adult male and female CD1 mice (50 weeks old) were got from the experimental animal center of Nantong University. Before use, the experimental mice were housed in groups (5 per cage) for 1 week to allow acclimatization. The experimental mice were kept at 23–25°C under a 12:12 h light/dark cycle with free access to food and water . GW6471 was provided by Tocris . The vehicle for vortioxetine, GW6471 and MK886 was 1% DMSO in 0.9% saline. The dosages of vortioxetine (10 mg/kg), GW6471 (1 mg/kg) and MK886 (3 mg/kg) in this study were determined according to published reports . All druAs we previously described, in brieAs we previously described , brieflyIn brief, the mice were individually placed in a transparent glass tank  for 6 min. A stopwatch was used to record the duration of immobility for each mouse during the last 4 min. The water was replaced after each trial. The immobility of each mouse was defined as it was floating in the water without struggling or having only slight movements to keep its nose above the water. This test was recorded with the observer unaware of the experimental grouping.In brief, the tail tip of each mouse was individually glued to a rail 60 cm above the floor, and hung for 6 min. The immobility (completely motionless) duration of each mouse during the 6-min period was recorded. This test was recorded with the observer unaware of the experimental grouping.The test C57BL/6J mice were individually housed and allowed to drink 1% sucrose solution and pure water freely. Before the test, a process of sucrose preference training was performed. Each test mouse was given one bottle of pure water and one bottle of 1% sucrose solution for 2 days, during which the positions of two bottles was exchanged every 6 h to avoid side preference. On third day, the test mice were deprived of food and water for 18 h. On fourth day, each mouse was given two pre-weighed bottles for 6 h. After that, two bottles were weighed again. The sucrose preference index was calculated as a percentage of the consumed sucrose solution relative to the total amount of liquid intake.This test comprises two trials for 5 min each. Briefly, an experimental C57BL/6J mouse was placed into a white plastic box (50 × 50 × 45 cm) containing an empty wire mesh cage (9 × 9 × 10 cm) positioned against the wall and allowed to explore for 5 min (target absent). Then, the C57BL/6J mouse was returned to its home cage for 1 min, while an unfamiliar male CD1 mouse was placed inside the wire mesh cage. Next, the C57BL/6J mouse was placed into the box again and allowed to explore for another 5 min (target present). The duration of time spent in the interaction zone (5 cm wide area around the wire mesh cage) for each trial was recorded by an investigator blind to the groups. The equipment was cleaned with 70% ethanol and dried between trials.This method was performed according to previous studies with slight modifications . After s12 TU/ml before use. The sequences for PPARα-shRNA and Scrambled-shRNA were 5′-AGAAATTCTTACCTGTGAA-3′ and 5′-TTCTCCGAACGTGTCACGT-3′ respectively  has been described in our previous study . In brieectively .p < 0.05 was considered statistically significant.The SPSS Statistics 26.0 software was adopted for statistical analyses. Multiple group comparisons were performed using two-way ANONA followed by Bonferroni’s test. All data are expressed as means ± S.E.M, and a value of p < 0.01; Vortioxetine, F = 20.874, p < 0.01; Interaction, F = 1.073, p = 0.307] and TST  but also the down-regulating effects of CUMS on the sucrose preference  of mice (n = 10). Similarly, p < 0.01; Vortioxetine, F = 23.576, p < 0.01; Interaction, F = 0.950, p = 0.336] and TST  but also the down-regulating effects of CSDS on the sucrose preference  and social interaction  of mice (n = 10). Thus, the antidepressant actions of vortioxetine were confirmed in mice.The antidepressant actions of vortioxetine were first examined in the CUMS and CSDS models. As shown in p < 0.01; Vortioxetine, F = 32.494, p < 0.01; Interaction, F = 38.035, p < 0.01] (n = 5). Similarly, p < 0.01; Vortioxetine, F = 23.372, p < 0.01; Interaction, F = 12.285, p < 0.01] on the level of PPARα protein in the hippocampus (n = 5). Moreover, vortioxetine administration produced none effects on the hippocampal PPARα expression in naive control mice (n = 5). The protein expression of hippocampal β-actin was nearly the same between all groups (n = 5).Then, we did western blotting to evaluate the effects of vortioxetine on the protein expression of hippocampal PPARα. The results are displayed in Taken together, the antidepressant actions of vortioxetine in rodent models may involve PPARα in the hippocampus.Then, GW6471 and MK886, two selective antagonists of PPARα, were adopted to investigate whether the antidepressant effects of vortioxetine in mice require PPARα. Therefore, mice subjected to chronic stress were co-treated with vortioxetine and PPARα antagonists for 2 weeks. Afterwards, behavioral tests and western blotting detection were performed.p < 0.05; Vortioxetine, F = 12.951, p < 0.01; Interaction, F = 1.985, p = 0.167], TST  and sucrose preference test , these ameliorating effects were notably attenuated by GW6471 co-administration (n = 10). Similarly, p < 0.05; Vortioxetine, F = 8.723, p < 0.01; Interaction, F = 3.025, p = 0.091], TST , sucrose preference test  and social interaction test , these reversal actions were notably attenuated by GW6471 co-administration (n = 10). Meanwhile, p < 0.01; Vortioxetine, F = 20.484, p < 0.01; Interaction, F = 3.615, p = 0.075] and CSDS  on hippocampal PPARα (n = 5), consistent with the behavioral results.The GW6471 results are summarized in p < 0.05; Vortioxetine, F = 10.759, p < 0.01; Interaction, F = 3.225, p = 0.084], TST  and sucrose preference test , these ameliorating effects were notably blocked by MK886 co-treatment (n = 10). Also, p < 0.01; Vortioxetine, F = 11.059, p < 0.01; Interaction, F = 1.922, p = 0.171], TST , sucrose preference test  and social interaction test , these reversal actions were notably blocked by MK886 co-treatment (n = 10). Moreover, p < 0.05; Vortioxetine, F = 7.156, p < 0.05; Interaction, F = 0.757, p = 0.397] and CSDS  on hippocampal PPARα(n = 5), consistent with the behavioral results.The MK886 results are summarized in Collectively, pharmacological inhibition of PPARα attenuated the protecting effects of vortioxetine against the CUMS and CSDS models of depression.Furthermore, AAV-PPARα-shRNA was used to selectively knockdown PPARα expression. The silencing efficacy of AAV-PPARα-shRNA has been demonstrated in p < 0.01; Vortioxetine, F = 19.936, p < 0.01; Interaction, F = 2.410, p = 0.129; p < 0.01; Vortioxetine, F = 11.903, p < 0.01; Interaction, F = 3.494, p = 0.070; n = 10). Additionally, genetic knockdown of hippocampal PPARα significantly prevented the increase induced by vortioxetine administration in the sucrose preference of mice exposed to CUMS  and CSDS-exposed  mice (n = 5), consistent with the behavioral results.The results of the CUMS experiments are displayed in In summary, combined with the above results involving PPARα antagonists, it can be concluded that the antidepressant mechanism of vortioxetine require hippocampal PPARα.So far there are a variety of available pharmacological options in clinical practice for treating MDD, mainly including monoamine oxidase inhibitors (MAOIs), tricyclic antidepressants (TCAs), SSRIs, SNRIs and some other antidepressants . Current guidelines for depression recommend SSRIs over SNRIs, MAOIs and TCAs due to their favorable adverse effect profile . SSRIs h3, 5-HT7 and 5-HT1D, a partial agonist at 5-HT1B, an agonist at 5-HT1A and an inhibitor of SERT, the antidepressant mechanism of vortioxetine is actually not fully understood. It is unclear if and how these modulating actions of vortioxetine on the 5-HT receptors and SERT contribute to its antidepressant effects (in vivo evidence suggesting that vortioxetine induces antidepressant effects in mice models of depression by significantly promoting the hippocampal PPARα expression. Our study extends the knowledge of vortioxetine’s pharmacological effects and further highlights the role of PPARα in depression.Although proved to be an antagonist at 5-HT effects . There i effects . Here, oet al., Lu et al. and Sun et al., (et al., (et al., (et al., (2+/calmodulin-dependent kinase α. Moreover, BDNF is critical for LTP, neuroplasticity and neuronal activity (et al., (Due to the recent marketing authorization, molecular studies on the pharmacological actions of vortioxetine are limited until now. For example, Yu  et al.,  all repo(et al.,  showed t(et al.,  indicate(et al.,  demonstractivity . From th(et al.,  reported(et al., . We have(et al., . In addi(et al., . However(et al., .et al. showed that CSDS did not influence mice behaviors in the FST and TST, in contrary to our results (As to by which way vortioxetine increases the expression of hippocampal PPARα, one possibility is that like WY14643, fenofibrate and gemfibrozil, vortioxetine directly binds and activates PPARα, functioning as a PPARα agonist. To validate this possibility, further in-depth studies involving time-resolved FRET, electrospray ionization MS and in silico structural analysis are needed . It is a results . AnotherIn addition to PPARα, there are a lot of other proteins that not only control BDNF biosynthesis and neuroplasticity but also are implicated in depression, such as salt-inducible kinase 2, glycogen synthase kinase 3β and mammalian target of rapamycin . These p


P. gingivalis. CPP solution from MCPM, DCPD, and TTCP was prepared. Four concentrations of ε-polylysine(ε-PL) solutions  were prepared. Dentin discs were prepared from recently extracted human third molars. Dentin discs were incubated with P. gingivalis (ATCC 33277) bacterial suspension (ca. 105 bacteria) containing Brain Heart Infusion medium supplemented with 0.1 g/mL Vitamin K, 0.5 mg/mL hemin, 0.4 g/mL L-cysteine in anaerobic jars (37 °C) for 7 days to allow for biofilm formation. P. g–infected dentin specimens were randomly divided into four groups: CPP + 0.125% ε-PL, CPP + 0.25% ε-PL, CPP + 0.5% ε-PL, CPP + 1% ε-PL. On each dentin specimen, CPP solution was applied followed by polylysine solution with microbrush and immersed in artificial saliva. Precipitate formation, antibacterial effects, and occlusion of dentinal tubules were characterized in vitro over up to 72 h using scanning electron microscopy. ε-PL showed 34.97% to 61.19% growth inhibition levels against P. gingivalis (P. g) after 24 h of incubation. On P. g-infected dentin specimens, DCPD + 0.25% ε-PL, and DCPD + 0.5% ε-PL groups showed complete bacterial inhibition and 78.6% and 98.1% dentin tubule occlusion, respectively (p < 0.001). The longitudinal analysis on fractured dentin samples in DCPD and TTCP groups revealed deeply penetrated hydroxyapatite-like crystal formations in dentinal tubules after 72 h of incubation in artificial saliva.In this study, ε-polylysine and calcium phosphate precipitation (CPP) methods were employed to induce antibacterial effects and dentin tubule occlusion. Antibacterial effects of ε-polylysine were evaluated with broth dilution assay against  Streptomyces albulus  (MCPM), dicalcium phosphate dihydrate [(CaHPO4) 2H2O] (DCPD) and tetracalcium phosphate [Ca4(PO4)2O] (TTCP) in 0.001 mol/L phosphoric acid (H3PO4) in room temperature. The ε-polylysine stock solution containing 25% ε-polylysine was obtained from Chisso Co., Tokyo, Japan. Four concentrations of ε-polylysine (ε-PL) solutions  were prepared by diluting stock 25% ε-polylysine in double distilled water. The crystalline phases of the calcium phosphate powders mixed with different concentrations of ε-polylysine were determined using X-ray diffraction .Calcium phosphate precipitate (CPP) solution was prepared by dissolving monocalcium monohydrate [Ca  cultures were grown anaerobically in Brain Heart Infusion (BHI) broth  supplemented with 0.1 g/mL vitamin K, 0.4 g/mL L-cysteine and 0.5 mg/mL hemin at 37 °C. The antibacterial efficacy of ε-polylysine was determined using broth dilution assay. ε-Polylysine was added to the starting inoculum of bacterial suspension with 109 bacterial cells to obtain four concentrations of ε-polylysine solutions  when diluted in BHI broth. The test solutions were incubated anaerobically at 37 °C up to 24 h. Periodically, 100 µL was collected and serial dilution was performed in PBS and inoculated on BHI with 5% sheep blood agar plates. The plates were incubated anaerobically at 10% CO2 for 2 days and colony forming units (CFU) were counted. The experiments were performed in triplicate. The antibacterial efficacy of ε-polylysine against P. gingivalis was determined by growth inhibition level. Growth inhibition level (%) was calculated by the formula; Pc is control bacterial population and Pt is bacterial population in the test solution. Bacterial populations from the same time period were considered.P. gingivalis, in vitro P. gingivalis, infected dentin disc models are used. The dentin specimens were obtained from the cervical area (4 m × 4 m × 1 m) of recently extracted human third molar teeth after removing the crown and root sections with diamond saw . The dentin discs were polished with 600–1000 grit silicon carbide paper to remove the cementum, soaked in 1 M acetic acid for 30 s, rinsed with ddH2O and autoclaved for 20 min at 121 °C. The dentin side placed up, the discs were placed in 24-well plates and 2 mL bacterial suspension containing P. gingivalis (105 CFU/mL) was inoculated into the each well. The wells were incubated anaerobically for 7 days at 37 °C. The BHI medium was changed every two days. The P. g-infected dentin discs were rinsed in PBS (2×) before proceeding for a treatment.To stimulate dentin hypersensitivity and evaluate the antibacterial effects of ε-polylysine on P. g-infected dentin were allocated into five groups: CPP + 0.125% ε-PL, CPP + 0.25% ε-PL, CPP + 0.5% ε-PL, CPP + 1% ε-PL, negative control (untreated). On each dentin specimen CPP solution was applied by microbrush followed by ε-polylysine application with microbrush. The treated P. g—dentin specimens were then proceeded for surface SEM analysis. Another subgroup of P. g—infected dentin specimens were prepared for dentin tubule longitudinal analysis. The dentin specimens were treated with CPP and ε-polylysine applications and immersed in artificial saliva at 37 °C for 72 h. Artificial saliva was prepared from 20 mM HEPES, 1.5 mM CaCl2, 0.9 mM KH2PO4, 130 mM KCl, 1 mM NaN3 and pH was adjusted with 1 M KOH at 7.0. Artificial saliva was replenished every day (24 h). The specimens were rinsed with ddH2O and fractured with chisel to expose longitudinal dentinal tubules. The dentin discs were fixed, dehydrated, and sputter-coated with Au-Pd on the fractured site. Scanning electron microscopy  was carried out to assess the bacterial inhibition and crystal formation on the surface and fractured sites of the dentin specimens.p-values < 0.05 were set for statistical significance.The mean tubule diameter in the dentin tubules was obtained by measuring 20 tubules on each disc. The tubule diameter and occluded area was measured to evaluate the change in tubule diameter before and after treatment using ImageJ software ). Data are presented as the mean ± standard deviation (SD). Statistical analyses between groups in differences in tubule occlusion percentage were calculated by ANOVA test. The Calcium phosphate compounds followed by ε-polylysine application showed excellent antibacterial and dentin tubule occluding effects in vitro. Calcium phosphate precipitation method combined with natural antibacterial polypeptide showed promising results for dentin hypersensitivity treatment. Considering the limitation of this study, various oral diseases like periodontitis are strongly associated with dysbiotic microbial communities. Multispecies biofilm model should be tested in this study. Further research should include acid resistance of the occlusion crystals and long term ε-PL bacterial inhibition effect.


Amanita hemibapha subsp. javanica (Amanitaceae) is an edible Korean wild mushroom. A. hemibapha subsp. javanica is often confused with A. subjunquillea, known as the East Asian death cap, which is potentially fatal when ingested. This study aimed to conduct the first chemical investigation of A. hemibapha subsp. javanica, which resulted in the isolation of seven fatty acid derivatives (1–7) and three steroids (8–10) from the MeOH extract of its fruiting bodies, and their structures were determined by comparing their NMR spectroscopic data with those previously reported, along with the data from LC/MS. Compound 1 was reported previously without the identification of its absolute configuration; its structure, including the absolute configuration was confirmed for the first time, in this study, by using 1H NMR and its fragmentation patterns in MS/MS data, and LC/MS analysis. A recently developed method using competing enantioselective acylation (CEA) coupled with LC/MS analysis was applied for determining the absolute configuration of compound 1, which revealed the 11S-configuration. In the anti-Helicobacter pylori activity test, compound 3 showed antibacterial activity against H. pylori strain 51 with 38.0% inhibition, comparable to that of quercetin (34.4% inhibition) as a positive control. Specifically, compound 4 displayed the most potent antibacterial activity against H. pylori strain 51 with 80.5% inhibition at the final concentration of 100 μm with a MIC50 value of 72 μm. These findings suggested that the active compound 4 is a natural antibiotic that may be used in the development of novel antibiotics against H. pylori. In addition, the first chemical investigation of A. hemibapha subsp. javanica revealed that this mushroom can serve as a promising natural source for the bioactive natural products. Amanita are the most well-known psychoactive and poisonous basidiomycete fungi  indicated that the oxygenated methine could be located next to the olefinic proton, and the olefinic protons were determined to have a trans-form. The overall 1H NMR data showed that the Compound 1 was a fatty acid derivative [2 of compound 1 yielded main fragment ions at m/z 297.2 [M − H]− and 155.1 [C10H19O]− (1 was determined to be (E)-11-hydroxy-9-octadecenoic acid, which has been reported previously without determining its absolute configuration [Compound HR-ESIMS . The IR mpound 1  showed s10H19O]− . Thus, t1, a recently developed chemical-derivative method, competing enantioselective acylation (CEA) coupled with LC/MS analysis − ; 1H NMR : δ 5.59 , 5.40 , 3.94 , 2.24 , 2.03 , 1.59 , 1.51 , 1.24-1.44 , 0.89   .1 was prepared by dissolving 0.1 mg of sample in 200 μL MeOH. The solution was further diluted with MeOH, filtered through a 0.45 μm hydrophobic PTFE filter, and analyzed using LC/MS/MS, Agilent 1290 Infinity II series with a 6545 LC/Q-TOF mass spectrometer . The analysis was conducted by injecting 1 μL of the sample using an Agilent Eclipse Plus C18 RRHD  set at 30 °C. The mobile phase consisting of formic acid in H2O (0.1% (v/v)) (A) and formic acid in acetonitrile (0.1% (v/v)) (B) was delivered at a flow rate of 0.3 mL/min by applying the following programmed gradient elution: 0–3.0 min, 10% (B); 3.0–10.0 min, 10–100% (B); 10.0–12.0 min, 100% (B); 12.0–15.0 min, 10% (B). The MS system was equipped with an ESI source and operated in both negative and positive ionization modes with a data acquisition range from m/z 100 to 600.Stock solution of compound S- and R-HBTM . Compound 1  was transferred to two transparent and capped 5 mL vials at room temperature, and DMF (90 μL) was added as the organic solvent for the CEA reaction. Both S- and R-HBTM  were added, and N,N-diisopropylethylamine  was successively transferred. Propionic anhydride  was added to start the CEA reaction. After 10 min, 2 μL aliquots from each reaction were acquired for LC/MS analysis and quenched with 98 μL of MeOH to obtain a total volume of 100 μL.Parallel reactions for the CEA reaction were performed as reported by Lee et al. , using S18, 4.6 × 100 mm, 3.5 μm, flow rate: 0.3 mL/min), and full scans in positive- and negative-ion modes (scan range m/z 100−1000) were applied to identify the desired acylated derivative. The mobile phase consisted of 0.1% (v/v) formic acid in distilled water (A) or acetonitrile (B) with a gradient solvent system as follows: 10%−100% B for 10 min, 100% B (isocratic) for 5 min, and then 10% B (isocratic) for 5 min for the post-run washing procedure of the column. The reaction rate catalyzed by both S- and R-HBTM was determined by measuring the peak areas of the acylated derivatives.An aliquot (5 μL) of the sample (100 μL) acquired from each parallel reaction was directly injected into the LC/MS and analyzed using an analytical Kinetex C18 100 Å column (Phenomenex)  supplemented with 10% horse serum . The culture conditions were 37 °C, 100% humidity, and 10% CO2 for 2–3 days.A clinical strain of 8 cfu/mL and twenty microliters of two-fold diluted samples and controls were added to each well of a 6-well plate containing Brucella broth medium  supplemented with 10% horse serum. The final volume was brought to 2 mL. After 24 h of incubation, bacterial growth was evaluated by measuring the optical density at 600 nm on spectrophotometer . MIC50 and MIC90 values were defined as the lowest concentrations of samples at which bacterial growth was inhibited by 50% and 90%, respectively, and were calculated using GraphPad Version 5.01 . All of the values were obtained from three independent experiments.Minimal inhibitory concentrations (MICs) were determined by broth dilution method previously reported . Twenty A. hemibapha subsp. javanica, an edible mushroom among Amanita species, led to the isolation and identification of seven fatty acid derivatives (1–7) and three steroids (8–10). The structure of compound 1 (amanitahemic acid A), including its absolute configuration, was elucidated using 1H NMR, MS/MS fragmentation data, and the application of CEA reaction coupled with LC/MS. In the anti-H. pylori activity test, compound 3 showed antibacterial activity against H. pylori strain 51 with 38.0% inhibition, comparable to that of quercetin (34.4% inhibition). Specifically, compound 4 displayed the most potent antibacterial activity against H. pylori strain 51 with 80.5% inhibition at the final concentration of 100 μm with the MIC50 value of 72 μm. Based on these findings, we conclude that compound 4 could be used to develop novel antibiotics against H. pylori.In this study, the first chemical investigation of the fruiting bodies of